poster abstracts

Transcription

poster abstracts

Monday 22 June – Poster session
Rhizosphere Microbiome
5
Branching out: towards a trait-based understanding of fungal ecology in the
rhizosphere
Carlos A. Aguilar-Trigueros*1, Stefan Hempel1, Jeff R. Powell2, Ian C Anderson3, Janis
Antonovics4, Joana Bergmann1, Timothy R Cavagnaro5, Baodong Cheng6, Miranda M
Hart7, John Klironomos7, Jana S Petermann1, Erik Verbruggen8, Stavros D Veresoglou1,
Matthias C Rillig1
1
Freie Universität Berlin, Germany, 2Univeristy of Western Sydney, Australia, 3University
of Western Sydney, Australia, 4Univeristy of Virginia, USA, 5University of
Adelaide, Australia, 6Chinese Academy of Science, China, 7University of British
Columbia, Canada, 8University of Antwerp, Netherlands
Rhizosphere fungal ecology lags behind in the use of traits (i.e. phenotypic characteristics) to
understand ecological phenomena. We argue this is a missed opportunity and that the
selection and systematic collection of trait data throughout the fungal kingdom will reap
major benefits in ecological and evolutionary understanding of fungi. To develop our
argument, we first employ plant trait examples to show the power of trait-based approaches
in understanding ecological phenomena such as identifying species allocation resource
patterns, inferring community assembly and understanding diversity-ecosystem functioning
relationships. Second, we discuss ecologically relevant traits in fungi (focusing on rootassociated fungi) that could be used to answer such ecological phenomena and can be
measured on a large proportion of the fungal kingdom. Third, we identify major challenges
and opportunities for widespread, coordinated collection and sharing of fungal trait data, for
which we solicit input from the community of researchers. The view that we propose has the
potential to allow mycologists to contribute considerably more influential studies in the area
of fungal ecology and evolution, as has been demonstrated by comparable earlier efforts by
plant ecologists. This represent a change of paradigm, from community profiling efforts
through massive sequencing tools, to a more mechanistic understanding of fungal ecology in
the rhizosphere.
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The relationship between strigolactones, symbiotic fungi and drought tolerance
in rice
Beatriz Andreo Jimenez*1, Carolien Ruyter-Spira1, Philippe Vandenkoornhuyse2,
Amandine Le Van2, Marie Duhamel2, Harro Bouwmeester1
1
Wageningen University, Netherlands, 2Rennes University, France
Abiotic stresses, such as drought, are the primary causes of yield reduction in rice. One of the
responses of plants to cope with drought is optimization of their root system architecture.
Root architecture is controlled by the joint interaction of several plant hormones. Recently, it
has been shown that strigolactones also play an important role in this process. Strigolactones
are also known to stimulate the beneficial symbiosis with arburscular mycorrhizal (AM) fungi,
which is also of importance during periods of water limitation because these fungi provide
plants not only with nutrients but also with water.
1
Rhizosphere Microbiome 1
One of the objectives of our study is to monitor how a group of rice lines that differ in
drought tolerance respond to drought with respect to strigolactone levels and endophytic
fungi/AMF community structure. For this purpose we selected two well-known rice indica
varieties, which historically have been selected under different cultivation managements: Apo
(non-flooded conditions, drought tolerant) and IR64 (flooded conditions, drought sensitive).
Interestingly, strigolactones levels in roots from the drought tolerant variety are higher when
compared to the drought sensitive variety. This difference correlates with their tillering
behavior, being lowest for Apo. Furthermore, SSU rRNA gene amplicons mass sequencing
was used to analyze the entire endophytic and AM fungal community associated with these
two varieties under field conditions (plants were grown in an upland field in the Philippines).
In both rice genotypes, drought clearly affects endophyte community structure. These
preliminary results may suggest a role for strigolactones in plant adaptation to water deficit
through their effect on AMF symbiosis and plant architecture.
The hypothesis that wet cultivation practices have led to more drought sensitive plants,
having a lower endophytic and AM fungal association, higher tiller number and lower
strigolactone levels will further be explored in a larger set of rice ecotypes.
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Rhizobacterial community structure in Mahikeng rhizospheric soil and
associated plant growth promoting potential
Olubukola Oluranti Babalola*
North-West University, South Africa
Denaturing gradient gel electrophoresis (DGGE) profiles of Mahikeng soil can indicate
dominant soil bacterial types and Plant Growth Promoting Rhizo-Bacteria (PGPR) can
stimulate the growth of the host plant. The aforementioned were examined in relation to nine
rhizospheric soils. Rhizobacteria with PGPR traits were selected for use in pot experiments on
tomato and spinach.
The rhizobacterial isolates tested were found to produce ammonia; several of them produced
indole acetic acid (IAA; 38%) and hydrogen cyanide (HCN; 38%). Also exhibited are 1aminocyclopropane-1-carboxylate (ACC) deaminase activity (48%), phosphate solubilisation
(48%) and antifungal activity (21%) against test pathogen Fusarium solani. All the HCNproducing bacteria belong to the genus Bacillus. B. amyloliquefaciens indicated high
cyanogenic potential compared to other strains. The treatment of both crops with the
bacterial inoculants promoted plant growth in terms of increased shoot length at P<0.05. B.
amyloliquefaciens MR16 had significantly higher growth at P<0.05 compared to the
uninoculated control treatment. DNA revealed some percentage identity with yet uncultured
Bacillus sp. (94%), Rubrobacter sp. (90%), Rhizobiales bacterium (95%), and soil bacterium
(87%) besides the culturable B. megaterium (97%) and Cohnella sp. (84%).
PGPR can be used to make reliable and accessible products such as biofertilizers for farmers.
Metagenomics holds the promise to reveal several important questions regarding the
unculturable fraction of the rhizosphere community.
2
8
Comparative analysis of autochthonous and zymogenous bacteria in Mahikeng
Agricultural Ecosystem
Olubukola Oluranti Babalola*, Keokeditswe Raven Motsewabangwe
North-West University, South Africa
Soil is a complex biological system and it is difficult to determine the composition of
microbial communities within it. Autochthonous and zymogenous soil bacteria collected in
Mahikeng, South Africa were investigated. Compounded nutrient agar and soil extract agar
used for isolation had glucose amendments of 0.0 to 2.0 g. Media containing 0.0 to 0.01 g of
glucose were considered as having poor carbon substrate amendment. Media that contained
glucose at a mass of 0.25 to 2.00 g were considered as having rich carbon substrate
amendment.
Soil analysis revealed between 6.5 and 7.0 average pH, 20.25 kg/acre of nitrogen, 14.50
kg/acre of phosphorus and 75 kg/acre of potassium. A great variety of bacterial species were
isolated from poor media including species such as Agrobacterium tumefaciens,
Planomicrobium sp, Cronobacter turicensis, Enterobacter hormaechei, Bacillus pumilis as well
as B. subtilis. Bacillus species isolated from rich media were B. subtilis, a bacterial species that
functions as a fungicide in the roots of plants. B. mojavensis, a unique group of bacteria that
form endophytic associations with plants for the prevention of diseases, B. pumilis and, B.
aryabhattai, species that colonise plant root systems and portray antibacterial and antifungal
properties, Arthrobacter globiformis and A. ramosus species has the ability to degrade soil
pesticides as well as pollutants such as hexavalent chromium as well as Rhizobium sp. bacteria
that colonize plant cells within root nodules where they convert atmospheric nitrogen to
ammonia and then provide organic nitrogenous compounds to the plant.
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Microbes From Inner Space: I. Comparison of biological soil suppression
potential against invertebrate pests across ten New Zealand pastoral soils
Nigel Bell*1, Katharine Adam1, Damien Fleetwood2, Gabriela Burch3, Richard Johnson3,
Alison Popay3, Faith Mtandavari3, Rhys Jones3, Vanessa Cave3
1
AgResearch Ltd, New Zealand, 2Biotelliga Ltd, New Zealand, 3AgResearch, New Zealand
White clover (Trifolium repens) is the key legume component of New Zealand pastoral
agriculture due to the high quality feed and nitrogen inputs it provides. There are a number
of invertebrate pests which constrain white clover growth in New Zealand soils and our work
is investigating rhizosphere or endosphere associated microbial controls for these pests. The
degree of suppressiveness of ten soils from across New Zealand to added Meloidogyne hapla
nematodes and Costelytra zealandica scarab larvae was measured in untreated soil and, in a
second experiment, a comparison of plant growth between untreated and irradiated soil
carried out on five of those soils using Pyronota sp. as the scarab larvae. As expected, of the
ten soils most showed no suppressive activity against these pests but, promisingly, two
showed activity against M. hapla and two against C. zealandica larvae. Suppression was
expressed as significantly reduced galling/ mm root by nematodes and significantly reduced
survival of scarab larvae compared to the other soils. Soil irradiation reduced white clover
shoot growth by ca 60–70% in two soils, demonstrating the importance of the microbial flora
in these soils. In a further two soils there was no significant effect of irradiation on plant
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growth, while in one soil there was a ca 16× increase in plant growth after irradiation, likely
due to pathogen release as evidenced by a significant reduction in root rotting. Lack of
consistent changes in soil macronutrients and pH post-irradiation suggest these were only
partially responsible for plant responses. Rhizosphere and endosphere microbes responsible
for pest suppressive and plant growth responses are being elucidated via next-gen
sequencing and results will be detailed in an aligned presentation (Johnson et al, this
volume), as will potential for beneficial microbes to be carried endophytically in white clover
seed (Monk et al, this volume).
10
Release of secondary plant metabolites in soil as mediated by arbuscular
mycorrhizal fungi in response to Fusarium oxysporum
Fernando Monroy1, Benedetta Cangelosi1, Paolo Curir1, Valeria Bianciotto2, Roberto
Borriello*2
1
Consiglio per la Ricerca e l'Analisi dell'Economia Agraria (CRA-FSO), Italy, 2Institute of
Plant Protection (IPP-CNR), Italy
The production of root exudates containing defensive compounds able to inhibit soilborne
microbial pathogens has been proposed as one of the mechanisms involved in plant
protection by arbuscular mycorrhizal fungi (AMF). However, there is little evidence of the
induced release of such compounds in the soil and of their direct or indirect effects on
pathogenic microorganisms. We carried out a full-factorial experiment under greenhouse
conditions to check for the release of secondary metabolites by two cultivars of Ranunculus
asiaticus in soils inoculated with a mix of three AMF species (Rhizophagus intraradices,
Funelliformis mosseae, Glomus sp.) and the vascular wilt fungus Fusarium oxysporum f. sp.
ranunculi. Chemical analyses of the rhizosphere soil revealed the presence of significant
amounts of three secondary metabolites, namely syringic acid, ferulic acid and isorutin,
released only by the mycorrhized plants challenged with F. oxysporum. Although the
production of these compounds has been previously recorded in mycorrhized plants, we
found that none of them had a direct inhibitory effect on F. oxysporum growth. Additional
inoculation tests carried out with syringic acid showed a persistence of less than one day for
this compound in non-sterilized soil, and the formation of syringic-derived decomposition
products. These results suggest that rhizosphere microorganisms are biochemically involved
in the defense response induced by AMF to F. oxysporum.
11
Characterization of the microbiome involved in the Nitrogen cycle from
Kavango soils of Namibia using qPCR- and omics- based methods
Claudia Sofía Burbano*, Thomas Hurek, Barbara Reinhold-Hurek
Department of Microbe-Plant Interactions, University of Bremen, Germany
Nitrogen is the biggest limiting factor in plant productivity in the savanna ecosystems of
southern Africa. It resides in the soil organic matter and its microbial mineralization is critical
for plant nutrition and soil productivity. To learn about the relation between soil management
practices and the microbial nitrogen cycle in the savanna soils, we are studying
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key transformations processes of it on soils from the Kavango region of Namibia. Different
land use strategies are included from two types of soils.
Quantitative analyses on key functional marker genes for nitrification, denitrification and
nitrogen fixation were done by qPCR and were targeted at the DNA (microbial population) and
RNA (actual transcription activity) level. Microorganisms involved in those processes were
present in all type of soils. At the functional level nitrification from Archaea was the most
prominent process. Transcripts from nitrogen fixation were not detected in most soils
studied.
To characterize the structure and activity of the soil microbiome without a bias introduced by
PCR we applied metagenomic and metatranscriptomic approaches to selected soils from the
different land uses. We obtained five metagenomes and two metatranscriptomes using
Illumina (MiSeq) paired-end sequencing technology. We obtained ~3 and ~15 Million
sequences for all the metagenomes and metatranscriptomes, respectively.
Our initial metagenome taxonomic analysis showed that bacterial sequences are
predominant in all soils. They are affiliated to 16 phyla with Actinobacteria and Proteobacteria
representing the more prevalent ones. Archaeal sequences were also present in the soils but
in low abundances. The taxonomic analysis of the metatranscriptomes together with all the
functional annotation will also be presented.
The omics- and qPCR-based approach will give a better understanding of how the nitrogen
cycling microbial communities behave under different land uses and will give a clue on how
they contribute to the agricultural productivity of Namibian savanna soils.
12
Characterization of beneficial microorganisms isolated from the rhizosphere of
saffron
Imane Chamkhi*, Jamal Aurag, Laila Sbabou
Mohammed V University, Faculty of Sciences, Morocco
Saffron or Crocus sativus L., is a local product or terroir product with high added value, which
is included in the national Green Morocco Plan (PMV= Plan Maroc Vert) with a fixed goal to
increase three times its production by 2020. The production of this spice is not exceeding
presently 3 tons per year and still rivalled in the international market. This production is
limited to the region of Taliouine-Taznakht (area <600 ha) in the south of Morocco.
Furthermore, saffron fields show a decline in production from the 6th year of use and require
regular renewal. The project aims the conception of microbial biofertilizers for saffron to
improve plants yield and quality.
A first year experiments was carried and 90 microorganisms were isolated from rhizosphere
sampled from Taliouine-Taznakht, on specific media. Isolates were screened for their plants
growth promoting abilities (phosphate solubilisation, auxin and siderophores production).
The isolates were identified using the 16S rDNA gene sequencing. Six genera were identified
(Rahnella, Pseudomonas, Variovorax, Delftia, Bacillus and Luteibacter).
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The best three isolates, with higher levels of biological activities, were selected and used for
the conception of saffron biofertilizers. In situ and ex situ trials (field and greenhouse
conditions) are ongoing.
13
The function of the root cap in shaping the microbiome of Arabidopsis
rhizosphere
Xu Cheng*, Rene Geurts, Carolien Franken, Ben Scheres, Ton Bisseling
Wageningen University, Netherlands
Microbial communities play a pivotal role in the functioning of plants by influencing their
physiology and development. In the rhizosphere, plant roots release low molecular weight
compounds (amino acids, organic acids and sugars), mucilage and proteins. These
rhizodeposits are a major driving force in the regulation of microbial diversity and activity on
plant roots, and also affect plant to modulate the rhizosphere microbiome for selecting
beneficial microorganisms. However, to what extend the root cap contributes to this root
exudate is not clear. As part of the maturation of root cap cells they detach from the root
after which they stay alive for some time in the soil. The availability of Arabidopsis mutants
that make fewer or immature root cap cells provides good opportunities to determine the
role of the root cap in shaping the microbiome. Previous studies showed that a loss of
function mutation in FEZ results in a reduced number of columella and lateral root cap layers.
SOMBRERO (SMB) BEARSKIN1 (BRN1) and BRN2 regulate the cellular maturation of root cap
cells. Lateral root cap cells fail to detach from the root in smb-3 mutant, columella cells fail to
detach in brn1-1/brn2-1 double mutants and cells fail to mature in all parts of the root cap in
the smb-3/brn1-1/brn2-1 mutant. To reveal the function of root cap in shaping plant
rhizospheric microbiome, in this study, we grew Arabidopsis Columbia wild type and smb-3,
fez-2, brn1-1/brn2-1 and smb-3/brn1-1/brn2-1 mutants under controlled condition. Total
genomic DNA was isolated from the rhizosphere and microbial compositions were analyzed
by using 16S rDNA amplicom sequencing with Miseq250 platform. The role of the root cap
on shaping the microbiome will be discussed.
14
The influence of protist grazing on corn rhizodeposition and recalcitrant litter
decomposition
Fionn Clissmann*1, Sebastian Löppmann2, Anna Gunina2, Johanna Pausch2, Yakov
Kuzyakov2, Robert Köller3, Michael Bonlowski1
1
University of Cologne, Germany, 2University of
Göttingen, Germany, 3Forschungszentrum Jülich, Germany
Plants provide carbon (C) to the C limited microbial community by means of root exudates
and thereby increase decomposition and N release from recalcitrant litter in soil, the so called
plant `priming` effect. Protists have also been found to increase N release from the microbial
community, through the ingestion and destruction of bacterial cells and excretion of
ammonia. This boosts plant growth, increasing exudate release, leading to further microbial
decomposition and N release, the so called `microbial loop`.
6
To investigate the contribution of protists to the plant `priming` effect we isotopicaly labelled
both C channels, organic litter (13C, 15N) and root exudates (14C) in corn (Zea mays)-planted
microcosms and duplicated all treatments with an added model protist (Acanthamoebae
castellani). Plants were transferred into soil after initial fluxes of easily available C had
subsided from the parent soil and mixed 13C, 15N recalcitrant litter (Lolium perenne root
material) allowing for a elucidation of root exudation induced increases in microbial
decomposition and N release. CO2 released from the soil was trapped throughout the
experimental duration and plants were pulse labelled with 14C three days before harvest. 14C
content in the shoot, root and microbial biomass and CO2, phospholipid fatty acid and
microbial biomass 13C content, total DNA and enzymatic activity were analysed.
First results include a higher 14C activity detected in the microbial biomass in the presence of
protozoa and increased DNA content in litter amended and planted soil in the presence of
protozoa.This indicates that the added protozoa significantly boosted plant C allocation to
the soil, potentially speeding the decomposition of recalcitrant soil litter.
15
Impact of benzoxazinoids in root exudates of maize on interactions with
rhizosphere bacteria and arbuscular mycorrhizal fungi
T.E. Anne Cotton*1, Steve Rolfe1, Duncan Cameron1, Pierre Pétriacq1, Georg Jander2,
Matthias Erb3, Jurriaan Ton1
1
University of Sheffield, United Kingdom, 2Boyce Thompson Institute for Plant
Research, United States, 3University of Bern, Switzerland
Interactions between plant roots and soil microbes are important determinants of plant
growth and health. Plants can modify their rhizosphere by the exudation of primary and
secondary metabolites. Benzoxazinoids (BXs) are tryptophan-derived secondary metabolites
of maize and other grasses that contribute to aboveground resistance against pests and
diseases. When exuded from roots, BXs can act as belowground recruitment signals for
beneficial soil microbes, such as Pseudomonas putida, and root-feeding pests, such as the
corn rootworm (Diabrotica virgifera). Plant growth promoting rhizobacteria and arbuscular
mycorrhizal (AM) fungi have also been reported to alter BX levels in roots. Hence, BXs are
emerging as important regulators of belowground plant-biotic interactions. However, little is
known about the exact mechanisms by which BXs are secreted by roots, their degradation
pathways in the rhizosphere, and their impacts on rhizosphere microbial communities. The
European research consortium BENZEX will address these shortfalls in our current knowledge.
Within this consortium, we will use maize plants with mutations in three BX biosynthesis
genes (Bx1, Bx2 and Bx6), in conjunction with molecular methods, such as high throughput
sequencing, to examine the effects of BXs on agricultural rhizosphere bacterial communities.
In addition, we will examine the impact of AM fungi on BX exudation as a possible driver of
mycorrhizosphere development and biocontrol of belowground pests and examine the
impact of BXs on mycorrhizal development. Our results will both enhance our fundamental
understanding of soil-microbial interactions and provide valuable insights into the potential
application of soil microbes in control strategies for sustainable agricultural production.
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16
Combined effects of compost and arbuscular mycorrhizal inoculation on soil
fertility, microbial community structure and plant growth
Vincenza Cozzolino*1, Vincenza Cozzolino1, Hiarhi Monda1, Vincenzo Di Meo1,
Riccardo Spaccini2, Alessandro Piccolo1
1
University of Naples Federico II, Italy, 2University of Naples, Italy
Increasing the sustainability of cropping systems requires management strategies that
involve the beneficial activity of rhizosphere microorganisms and the recycling of organic
wastes. Limited information are available on the combined application of arbuscular
mycorrhizal fungi (AMF) inoculants and composted organic wastes on soil microbial
community structure and their effects on crop productivity and soil fertility.
Therefore, a field study was conducted to evaluate the effects of commercial inoculation (CI)
and the application of composted municipal organic wastes on physical-chemical and
biological parameters. Three fertilizer treatments were compared: 1. inorganic fertilizer (NPK),
2. compost (CM), 3. half inorganic fertilizer dose plus compost (HCM). Maize plants in all
treatments were inoculated (CI+) or uninoculated (CI-). The effects on maize growth, soil
physical-chemical properties (soil aggregation, N, C total and available P), mycorrhizal fungi
growth (root colonization, C16:1ω5 phospholipid-PLFA and neutral-NLFA fatty acid content)
and soil microbial community structure (PLFA profiles) were determined in soil and plant
samples.
CI+ maize treatments increased AMF root colonization, yield and P uptake in NPK and CM
treatments, in respect to CI- plots, although the largest yield were obtained with NPK. On the
contrary, application of compost reduced AMF growth as indicated by the decline of root
colonization and NLFA C16: 1ω5 content. Compost increased total PLFA and altered soil
microbial community structure, by decreasing Gram-positive to Gram-negative bacteria ratio
and enhancing fungal PLFA, as compared to NPK. In combination with CI treatments, our
compost favored soil macroaggregates formation and enhanced total C, N and available in
both CM and HCM treatments, by improving structural and soil fertility parameters, whereas
its related yields were not comparable to NPK treatments and depressed AMF growth.
Alteration of soil microbial community promoted activity of an antagonistic microflora,
thereby hindering the relationship between AMF and plants and compromising plant growth.
17
The rhizosphere microbiome of seagrasses and their role in sulfur processes
Catarina Cúcio*1, Aschwin Engelen2, Gerard Muyzer1
1
Institute for Biodiversity and Ecosystem Dynamics, University of
Amsterdam, Netherlands, 2Centre of Marine Sciences, University of Algarve, Portugal
Seagrass meadows grow in coastal, biogeochemically active sediments. These productive
ecosystems are distributed from tropical to temperate areas and are dominated by
seagrasses, a group of marine angiosperms that is often threatened by the presence of
hydrogen sulfide, a phytotoxic gas produced by belowground bacterial communities. The
seagrass rhizosphere is still poorly described, however the occurrence of die-off events
caused by high levels of sulfide stresses the need to understand these plants and bacterial
communities present in their rhizosphere – rhizobiome. Using Next Generation Sequencing of
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16S rDNA amplicons, we described the bacterial communities of three North Atlantic
seagrasses, Zostera marina, Z. noltii and Cymodocea nodosa. Comparative analysis revealed
that the rhizobiome of seagrasses is strongly influenced by the plant and significantly differs
from the communities present in their surrounding environment (i.e., bulk sediment and
seawater). However, we found that the rhizobiomes of the different seagrass species did not
vary at a local scale, but were significantly different between plants from different
geographical locations. The core rhizobiome of seagrasses is mainly composed of OTUs
related to bacteria known for being involved in the sulfur cycle (sulfate reduction and sulfur
oxidation), and for their ability to fix nitrogen. Moreover, our results point to a niche
differentiation of sulfur bacteria, in which sulfide oxidation is performed by different taxa.
These results are a strong indication of the importance of sulfur bacteria in seagrass ecology.
18
The selection on sugarcane rhizosphere upon the Pseudomonas community
Lucas Dantas Lopes*, Michele de Cássia Pereira e Silva, Luana Bresciani, Fernando Dini
Andreote
University of Sao Paulo, Brazil
The knowledge on interactions between plants and key microbial groups is essential for a
better exploration of rhizosphere interactions though an efficient and sustainable plant
production system. Here we targeted the selection exerted by sugarcane (an important crop
for sugar and bioethanol production) rhizosphere upon the community of Pseudomonas spp.,
a well-known bacterial group involved in plant growth promotion. Six randomly selected
plants were sampled in a cultivation field, and bulk soil was contrasted with rhizosphere
(manually separated by selecting soil highly adhered to the maximum of 1 mm or the roots
vicinity). The responsiveness of Pseudomonas spp. to the sugarcane rhizosphere was
determined by culture independent analyses, where quantitation results (copies of the 16S
rRNA gene per g of soil) showed that Pseudomonas is significantly enriched in the
rhizosphere (p < 0.01), shifting from 2.5x104 in the bulk soil to 8.7x105 in the rhizosphere. A
NMDS constructed by using the OTU table, from a sequence-based analysis of the 16S rDNA
performed by Illumina, indicated a clear distinction between the structures of Pseudomonas
spp. communities in bulk soil and rhizosphere. We gained insights on the intra-genus
variations by cultivation of 34 isolates of Pseudomonas in Pseudomonas Agar Base medium
(21 from bulk soil and 13 from rhizosphere). The 16SrDNA sequencing revealed the presence
of three groups; two in both environments (P. fluorescens/P. koreensis and P.
plecoglossicida/P. monteilii), while the group affiliated to P. putida was exclusively found in
the rhizosphere. BOX-PCR analysis indicated a deeper variation on genome contents of these
isolates, clustering 7 genotypes (some exclusive in soils or rhizosphere composing 23 BOX
profiles). Our final target will be to determine whether these variations are commonly
observed among the Pseudomonas cells in soils, or if the genomic re-arrangements are
important on rhizosphere-succeeded isolates.
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19
Microbial eco-compatible strategies for improving wheat quality traits and
rhizospheric soil sustainability (MIC-CERES)
Cindy Dasilva*1, Nathalia Arias Rojas1, Daniel Garçia Seco de Herrera1, Eddy Ngonkeu2,
Valentina Fiorilli3, Florence Wisniewski-Dyé4, Diégane Diouf5, Candida Vannini6, Valeria
Terzi7, Jacques Le Gouis8, Aboubacry Kane5, Madiama Cisse9, Marcella Bracale6, Paola
Bonfante3, Ralf Koebnik1, Lionel Moulin1
1
IRD, UMR Interactions Plant Microorganisms Environment, France, 2Institute of
Agricultural Research for Development & University Yaoundé, Cameroon, 3University of
Torino, Dpt. Life Science and Systems Biology, Italy, 4University Claude Bernard Lyon I,
UMR-CNRS 5557 Microbial Ecology Laboratory, France, 5University Cheikh Anta Diop,
Common laboratory of Microbiology (IRD/UCAD/ISRA), Senegal, 6University of Insubria,
Dpt. Biotechnology and Life Science, Italy, 7CRA-GPG, Genomics Research
Centre, Italy, 8INRA/UBP, UMR 1095, Génétique, Diversité et Ecophysiologie des
Céréales, France, 9Senegal Institute of Agricultural Research, Common laboratory of
Microbiology (IRD/UCAD/ISRA), Senegal
Wheat (Triticum aestivum L. subsp. aestivum) is one of the most important sources for food,
animal fodder and industrial raw materials. However these past years, world-wide wheat
production has not met demand, largely due to the adverse effects of climate change, soil
salinization, and limited water availability. Here, we present the project MIC-CERES (20142016) funded by Agropolis (France) and Cariplo (Italy) Fondations, with seven groups from
four countries (France, Italy, Senegal, and Cameroon), and in collaboration with the
International Maize and Wheat Improvement Center (CIMMYT) and INRA (France). The goal
of this project is to characterize the response of wheat to colonisation by arbuscular
mycorrhizal fungi and symbiotic beneficial bacteria, with a view to using them as natural
biofertilizers and bioprotectors in integrated strategies for wheat cultivation. Our main
objectives are: 1) determine structure, diversity and activity of the microbiome associated to
the roots of wheat by a barcoding approach of ribosomal markers with Illumina MiSeq,
coupled with a culturable approach to isolate plant growth-promoting bacteria. Several
varieties of wheat are tested on soils with different characteristics (from four countries) in
order to understand the impact of both soil origin and plant variety on the microbiota
associated to wheat roots; and 2) characterize the molecular wheat responses to beneficial
and harmful microbes by both transcriptomic and proteomic analysis to better understand
the wheat-microbes interactions. Wheat will be challenged with a mycorhizal fungi,
Azospirillum brasilense, Burkholderia graminis or Xanthomonas translucens in single or
multiple inoculations. We will present the first data on the culturable approach on the wheat
plant-growth promoting bacteria.
20
Effect of biochar amendment on the rhizosphere microbiome of lettuce and
strawberry
Jane Debode*, Caroline De Tender, Pieter Cremelie, Bart Vandecasteele, Martine Maes
Institute for Agricultural and Fisheries Research (ILVO), Plant Sciences Unit, Belgium
Biochar, a solid coproduct of biomass pyrolysis, can be used as soil amendment. It thereby
has potential to help mitigate climate change, as it permanently sequesters carbon from the
atmosphere and it may be beneficial for agricultural crops. We investigate the effects of
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biochar soil amendment on plant health and rhizosphere microbiology (FP7-Fertiplus
project).
For 2-3 months, lettuce was grown in field soil and strawberry was grown in peat, each
amended with 0, 1 or 3% biochar produced from holm oak. Changes in chemical
soil/substrate and plant properties were measured. The associated changes in the
rhizosphere microbiome were studied using phospholipid fatty acid (PLFA) analysis and
amplicon sequencing of the bacterial V3-V4 region of the 16SrDNA.
Biochar amendment had a different effect on the lettuce-soil versus the strawberry-peat
rhizosphere. For the lettuce-soil rhizosphere, PLFA analysis showed that the 3% biochar
application increased the population of arbuscular mycorrhizal fungi, whereas no significant
shifts could be observed in the bacterial microbiome based on both PLFA and 16S analysis.
For the strawberry-peat rhizosphere, PLFA analysis showed a significant clustering of the 3%
biochar application as compared to the 0% and 1% applications. This was confirmed by the
16S analysis, which dedicated the shift to the reduction in relative abundance of the
Proteobacteria, mainly Burkholderia species. This microbial information was combined with
plant and substrate properties, showing a relationship between strawberry rhizobiome,
biochar application rate, plant growth, plant resistance to Botrytis cinerea and substrate
moisture and water-soluble phosphorus content.
In sum, PLFA analysis and 16S amplicon sequencing showed that biochar incorporation can
have an influence on the rhizosphere microbiome, associated with induced plant growth and
health. This influence was more pronounced in the peat substrate than in soil, which is a
more diverse and complex environment and is thus probably less sensitive to disturbance.
21
Mollicutes-related endobacteria in basal fungi: genetic determinants of the
fungal microbiome
Alessandro Desirò*1, Martin Bidartondo2, Paola Bonfante3, Gregory Bonito1
1
Department of Plant, Soil, Microbial Sciences, Michigan State University, USA, 2Imperial
College London and Royal Botanic Gardens, Kew, United Kingdom, 3Department of Life
Sciences and Systems Biology, University of Turin, Italy
Fungi are often associated with microorganisms such as bacteria, which colonize their outer
surface or, in some fungal taxa, live in their cytoplasm as endobacteria. An example of
endobacterium-fungus interaction is represented by arbuscular mycorrhizal fungi
(Glomeromycota), which are important component of the root and rhizosphere microbiome.
Two types of endobacteria are known in arbuscular mycorrhizal fungi: a β-proteobacterium
called Candidatus Glomeribacter gigasporarum, and a coccoid bacterium which represents an
undescribed taxon of Mollicutes-related endobacteria. Interestingly, the two bacterial
populations have also been simultaneously detected in some Glomeromycota spores hosting
a newly described fungal microbiota. However, some evidence indicates that other fungi
belonging to Mucoromycotina contain endobacteria, e.g. an endobacterium closely related to
Candidatus Glomeribacter gigasporarum has been identified in Mortierella elongata, while
endobacteria similar to Mollicutes-related endobacteria have been morphologically detected
in Endogone flammicorona.
11
Here we investigate i) bryophytes-associated fungi, and ii) Endogone fruiting bodies in order
to identify their endobacteria. Molecular analyses revealed the presence of Mollicutes-related
endobacteria both in bryophytes-associated fungi and Endogone fruiting bodies. The partial
sequencing of the 16S rRNA gene on the MiSeq platform shed light on the variability of
Mollicutes-related endobacteria dwelling in fruiting bodies. Phylogenetic reconstructions
placed the Mollicutes-related endobacteria sequences retrieved from Endogone with the
ones from Glomeromycota, forming, however, a separate new clade.
These results confirm previous morphological studies on Endogone and allow to identify
Mollicutes-related endobacteria in bryophytes-associated fungi and Endogone fruiting
bodies. The presence of exclusive Mollicutes-related endobacteria phylotypes, both in
Glomeromycota and Endogone, suggests that they may be useful markers of fungal presence.
The presence of Mollicutes-related endobacteria in a group of basal fungi, Endogone, close
to, but potentially more ancient than arbuscular mycorrhizal fungi, opens new questions
concerning the relationships between Mucoromycotina and Glomeromycota, and the origin
and evolution of this little known group of endobacteria.
22
Landscape to rhizosphere: large and small-scale drivers of Pseudomonas
community structure in New Zealand pastoral soils
Bryony Dignam*1, Leo Condron2, Maureen O'Callaghan3, George Kowalchuk4, Jos
Raaijmakers5, Steve Wakelin3
1
AgResearch Ltd and Bio-Protection Research Centre, Lincoln University, New
Zealand, 2Bio-Protection Research Centre, Lincoln University, New Zealand, 3AgResearch
Ltd, New Zealand, 4Utrecht University, Netherlands, 5Netherlands Institute of
Ecology, Netherlands
Pseudomonas species are ubiquitous in soils and contribute towards suppression of various
soil-borne phytopathogens. This study aims to identify opportunities to manage
Pseudomonas communities as ‘biological resources’ for sustainable agricultural systems.
Sequential investigations were performed across spatial scales to identify edaphic,
environmental and agricultural management factors affecting the composition of
Pseudomonas communities in New Zealand pastoral soils.
A country-wide survey of 50 soils showed that farm management practises were more
dominant than geographic or environmental properties, with farm intensification and soil
properties (notably organic matter quality (C:P ratio)) associated with Pseudomonas
community structure. Soil and plant-specific influences were experimentally tested among
‘brown’ and ‘recent’ soils, each planted to ryegrass, clover and kale. No direct influence of soil
order was evident. However there were differences in community structure among individual
soils (P=0.001), driven by soil pH and E.C., and to a lesser extent, among plant species. Thus,
physicochemical properties were stronger drivers of Pseudomonas communities than plantbased selection.
To determine the influence of selection at the soil-root interface, a single soil was planted to
three pasture plant species and Pseudomonas communities profiled in the bulk soil,
12
rhizosphere and rhizoplane compartments. There were significant differences between plant
species (P=0.005), but more strongly among compartments (P=0.001). Surprisingly, all bulk
and rhizosphere communities were similar; the majority of variation among Pseudomonas
communities occurred at the rhizoplane (P=0.001). Ryegrass rhizoplane communities were
highly dissimilar to those of the other two plant species.
Our findings show that soil properties are stronger drivers of Pseudomonas communities than
plant-based selective pressures at larger spatial scales. However, at the small spatial scales of
the root-soil interface plant influences were observed especially in the rhizoplane. Ongoing
work is determining whether ‘disease suppressive’ functional components of the
Pseudomonas communities (i.e. functional genes) are driven by similar factors to the
phylogenetic component.
23
Cultivation of bacteria from the sugarcane rhizosphere and the role of roots
exudates on its development
Danielle Gonçalves dos Santos, Simone Raposo Cotta, Fernando Dini Andreote*
University of São Paulo, Brazil
The sugarcane (S. officinarum) is a perennial gramineous used for bioethanol production, a
renewable energy source. Studies related to the improvement of cultivation conditions are of
great importance. It is known that the rhizosphere is an environment that hosts an intimate
interaction between plants and their respective microbiomes, being it mediated by the roots
exudates. This study aimed to understand the effect of plant exudation on composition and
behavior of microbiome through cultivation-dependent methodologies. Soil and rhizosphere
samples from a sugarcane field were used for bacteria cultivation, followed by the genetic
(BOX-PCR and partial sequencing of the 16S rRNA gene) and metabolic (BIOLOG®)
characterization of isolates. Results demonstrated higher numbers of culturable bacteria in
rhizosphere when compared to soil samples, with the prevalent affiliation of isolates to the
phylum Proteobacteria (especially with the classes Gammaproteobacteria and
Betaproteobacteria). The BOX-PCR results showed a great genetic diversity, even when
isolates affiliated to the same taxa are compared. In counterpart, the analysis of the 16S rRNA
gene sequence indicated that several isolates preserve high similarities in the ribosomal gene.
The metabolic profiling results corroborated with the BOX-PCR data, which isolates highly
correlated in the taxonomical analyses presented distinct capacities to use the carbon sources
that were tested. At the end, this metabolic diversity was evidenced by the distinct behavior
of isolates belonging to the same genera, but isolated from soils or rhizosphere samples,
when cultivated in the presence of roots exudates. In general, this study demonstrated that
sugarcane plants can influence the behavior of bacterial communities present in soils. It can
also be indicated the individuality of components of the rhizosphere microbiome, with
distinct behavior regardless to the taxonomical affiliation of isolates.
13
24
Long-term fertilization influenced the dynamics of rhizosphere microorganisms
in maize
Ivica Djalovic*1, Yinglong Chen2, Nastasija Mrkovacki1, Dragana Bjelic1
1
Institute of Field and Vegetable Crops, Serbia, 2School of Earth and Environment, and
UWA Institute of Agriculture, University of Western Australia, Australia
The aim of this study was to investigate dynamics of microorganisms in the rhizosphere of
maize in long-term monoculture in dependance of inoculation with azotobacter. The study
treatments were: 1. Ø (control treatment without mineral or organic fertilizers); 2. Ø (control
traetment with inoculation); 3. NPK (fertilized with mineral fertilizer); 4. NPK + AC (fertilized
with mineral fertilizer with inoculation); 5. NPK + M (fertilized with manure and mineral
fertilizer); 6. NPK + M + AC (fertilized with manure and mineral fertilizer with inoculation); 7.
NPK + CR (plowing crop residues - maize and mineral fertilizer); 8. NPK+ CR + AC (plowing
crop residues – maize and mineral fertilizer with inoculation). Inoculation was done with
liquid culture of Azotobacter chroococcum (mixture of three strains). Soil samples were taken
for microbiological analysis at three dates (20th May, 7th June and 2nd August). The application
of differetiated fertilization and the succesive stages of plant development found a reflection
in the changes of the number of the studied soil microorganisms. At the first sampling period
significantly higher total number was obtained in treatments 2, 4 and 5 in relation to the
other treatments. At the second period treatment 6 had the largest total microbial number,
and significantly higher number compared to treatments 1, 5 and 7 was achieved in
treatments 2, 3, 4 and 8. At the third period only treatments 5 and 6 had significantly higher
total number compared to other treatments. The total number of microorganisms decreased
during the growing season, except at the second sampling period in NPK + M + AC
treatment. At the first period of sampling the highest total microbial number was recorded,
while the lowest values were obtained at the third period. NPK + M + AC treatment positively
affected the total number of microorganisms.
25
Factors determining the composition of rhizosphere and root-inhabiting
bacterial communities of Arabis alpina
Nina Dombrowski*1, Klaus Schläppi2, George Coupland1, Paul Schulze-Lefert1
1
Max Planck Institute for Plant Breeding Research, Germany, 2Agroscope, Switzerland
Plants growing in soil associate with a plethora of microorganisms, including nematodes,
fungi and bacteria. Bacteria can colonize the rhizosphere and roots of plants and influence
plant growth via hormone modulation, nutrient mobilization and suppression of defense
responses. However, little is known about the structure and functions of bacterial consortia
living in association with plant roots. To address this gap, we used the arctic-alpine plant
Arabis alpina as a model to unravel factors influencing community structure. To determine
the influence of plant residence time in soil and plant development on community
composition, A. alpina wild-type and pep1 mutant plants were grown under controlled
environmental conditions for seven months. The pep1 mutant does not require a
vernalization treatment to flower and allows a direct comparison of a flowering and nonflowering plant at the same time point. This enabled us to disentangle the influence of plant
residence time in soil and plant development on community structure. Profiling of the 16S
rRNA gene by Illumina sequencing identified a distinctive taxonomic structure of bacterial
14
communities within the soil, rhizosphere and root compartments. Additionally, the taxonomic
composition of bacterial communities was only partially stable, for example Bacteroidetes
were outcompeted over time. Notably, plant developmental stage did not influence
community structure. To validate these observations and to assess the importance of soil
type and environmental conditions, we harvested A. alpina roots at a natural site in the
French Alps and profiled bacterial communities. We could confirm the absence of an effect of
plant developmental stage on community assembly. In addition, we found that soil type and
environmental conditions induce pronounced shifts in rhizosphere and root-inhabiting
bacterial communities. Surprisingly, ~30% of those communities were stable across soil types
and environmental conditions, allowing us to identify a shared bacterial microbiota, which
might contribute bacterial functions for plant growth.
26
Intercropping is an effective way to control fusarium wilt of faba bean and
improve rhizosphere microbe diversity
Yan Dong*1, Li Tang1, Yi Zheng2
1
Yunnan Agricultural University, China, 2Southwest Forestry University, China
Fusarium wilt of faba bean is one of the most widespread and destructive diseases that
reduce faba bean yield and quality. As a soil-borne fungal disease, fusarium wilt is difficult to
control with conventional chemicals in field production. Intercropping has been practiced for
a long history in crop production in China, and widely accepted as a sustainable practice due
to its yield advantage and disease suppression.
Field experiment was conducted to examine the effects of wheat and faba bean intercropping
on faba bean yield, rhizosphere microecology and occurrence of faba bean fusarium wilt.
Results showed that aboveground dry weight，grain yield and 100-seed weight of faba bean
significantly increased by 99.11%, 44.29% and 12.17%, fusarium wilt incidence and disease
index of faba bean significantly decreased by 20% and 30.4% respectively as intercropped
with wheat.
The Biolog analysis with ECO plate could interpret the metabolic functions diversity of
rhizosphere microbial community, results showed that intercropping significantly increased
the average well color development (AWCD) of faba bean, the AWCD value of intercropped
faba bean increased by 82.7% in comparison with that of monocropped. Functional diversity
index of Shannon index (H) and substrate richness (S) in Biolog ECO microplates were
significantly higher with intercropping than that with monocropping, substrate richness of
intercropped faba bean increased by 30.3%.
Intercropping changed the microbial community diversity, the activity of invertase, urease
and catalase in rhizosphere of faba bean. It is suggested that wheat and faba bean
intercropping could improve rhizosphere soil micro eco-environment and control the soilborne disease caused by faba bean continuous cultivation.
15
27
Influence of primextra on rhizosphere microorganisms of cowpea
Fatuyi Ekundayo*, Adebanke Adedeji
Federal University of Technology, Akure, Nigeria
Fungicides are known to either be stimulatory or inhibitory to microorganisms that are
essential for plant growth. This present investigation was therefore aimed at determining the
effects of primextra at four different concentrations on the rhizosphere microorganisms of
cowpea. A field experiment was carried out at the back of Crop, Soil and Pest Management
Department, Federal University of Technology, Akure, Ondo State, Nigeria for a period of 30
days. Cowpea seeds were planted and after 14 days, four different concentrations (0.00, 0.50,
1.00, 3.00) of primextra were added. After experimental trial, rhizosphere microorganisms
were isolated using Wakes A and B agar. The ability of the isolated microorganisms to
solubilise tricalcium phosphate was evaluated on Pivoskaya’s medium. The microbial isolates
were also screened for phosphatases by standard techniques. There was decrease in the
bacterial and fungal counts with increase in the concentration of primextra. The bacterial
isolates obtained from primextra treated soils include Bacillus subtilis, B. cereus, Klebsiella
pneumoniae, Proteus mirabilis, Escherichia coli, Serratia marcescens, Staphylococcus aureus
and Micrococcus luteus while the fungal isolates were Aspergillus niger, Penicillium italicum.
The isolated microorganisms were able to solubilize tricalcium phosphate in varying
degrees.The concentration of solubilized tricalcium phosphate ranged from 97.87 to
559.47µ/mg. Bacillus subtilis, B. cereus, Klebsiella pneumoniae, P. mirabilis, Serratia
marcescens had high phosphatase activity while E. coli, S. aureus low phosphatise activity. The
microorganisms with high phosphatase activity could be inoculated into the rhizosphere soil
of cowpea in primextra treated soil.
28
Effects of bio-effectors on the growth and rhizosphere microbiome of tomato
plants grown in low phosphorus soil
Namis Eltlbany*1, Guoch Ding2, Mohamed Baklawa1, Kornelia Smalla1
1
Julius Kühn-Institut, Federal Research Centre for Cultivated Plants (JKI), Institute for
Epidemiology and Pathogen Diagnostics, Braunschweig, Germany, 2College of Resources
and Environmental Science, China Agricultural University, China
Bio-effectors are a viable microorganism which directly or indirectly affect plant performance
and can contribute to a reduced fertilizer and pesticide use in crop production based on
biological mechanisms interfering with soil-plant-microbe interactions. Low phosphorus
availability limits plant growth in many soils and is a common constraint to agricultural
productivity. Greenhouse experiment was conducted aiming to study the effect of four bioeffectors (B1: Trichoderma harzianum T-22; B2: Pseudomonas sp.; B3: Bacillus
amyloliquefaciens FB01, B4: Pseudomonas jessenii RU47) on the growth of tomato plants in a
phosphorus limited soil and their effects on the indigenous rhizosphere bacterial community
compared to non-inoculated plants (B0). At each sampling time the rhizosphere competence
and colonization patterns of the bio-effectors were monitored in rhizosphere samples using
colony forming units counts. Effects on the bacterial community composition were
determined using denaturing gradient gel electrophoresis (DGGE) and pyrosequencing of
16S rRNA gene fragments amplified from total bulk and rhizosphere community DNA. All
bacterial bio-effectors had a good rhizocompetence and promoted the growth of tomato
16
with B3 and B4 showing the best activity. The UPGMA analysis for DGGE and amplicon
sequences showed significant differences between the rhizosphere bacterial community
composition of B0 and the inoculated samples. Amplicon sequencing allowed us not only to
reveal bacterial genera with significantly increased or decreased relative abundance in the
tomato rhizosphere compared to the bulk soil dominant genera enriched in the rhizosphere
but also responders to the inoculation. B4 became a dominant population and caused strong
but transient bacterial community changes. At the last sampling time a significantly increased
relative abundance of Bacteroidetes and Betaproteobacteria was observed for all bio-effector
treatments and for Lysobacter for all bacterial inoculants. Our data showed that inoculants
cause major but often transient shifts in the bacterial community which might also contribute
to the PGP effects observed.
29
Exudates of arbuscular mycorrhizal hyphae trigger phytin mineralization and P
turnover in the hyphosphere
Fei Wang*, Ning Shi, Rongfeng Jiang, Fusuo Zhang, Gu Feng
China Agricultural University, China
The aim of this study was to elucidate a direct pathway for the translocation of
photoassimilated carbon from maize plants to extraradical mycelium-associated phosphate
solubilizing bacteria (PSB) in the hyphosphere, and in turn, these PSB mediate the
mineralization and turnover of organic P. The flux of photosynthesized C from maize to
bacteria associated with extraradical hyphae of AM fungus was traced using stable isotope
probing (SIP). The microbes in the hyphosphere soil receiving 13C from maize were assessed
through T-RFLP analysis with stable isotope probing (DNA-SIP). The bacteria actively
assimilating C derived from pulse-labeled maize plants were Massilia aurea
(Oxalobacteraceae), Streptomyces spp. (Streptomycetaceae) and Pseudomonas alcaligenes
(Pseudomonadaceae). In turn, PSB interacted with AM fungus in the hyphosphere chamber to
mediate a marked decline in the organic P concentration and an increase in the microbial
biomass phosphorus concentration in hyphosphere soil but did not contribute to the uptake
of P by maize. These results provide the first in situ demonstration of the pathway underlying
the carbon flux from plants to the AM mycelium-associated PSB and that the PSB rewards the
acceleration of phytin mineralization and turnover with carbohydrates.
30
Structure of fungal and bacterial communities in a boreal forest podzol and their
response to nitrogen fertilisation
Roger Finlay*, Srisailam Marupakula, Juan Santos-González, Shahid Mahmood
SLU, Uppsala BioCenter, Sweden
Symbiotic ectomycorrhizal fungi form diverse communities in boreal forests, mobilising
nutrients from organic polymers with different degrees of recalcitrance and from mineral
substrates that are weathered at different rates. These fungi may also sequester
photosynthetically-derived carbon in the soil, but the different roles of individual taxa and
their location within the soil profile are still poorly understood. Structure and activity of soil
microbial communities are influenced by both atmospheric nitrogen deposition and
17
applications of fertiliser, but detailed knowledge of the community dynamics of these
responses is still lacking. In this study we investigated fungal and bacterial community
structure in different horizons of an 80 year old pine forest at Lamborn, Sweden, using highthroughput sequencing. Samples were taken in three replicate plots of two treatments. Half
the plots were unfertilised, the other plots had been fertilised with 150 kg N ha-1 16 months
prior to sampling. Ten replicate cores were taken from each plot, divided into O, E and B
horizons and pooled prior to DNA extraction, PCR and pyrosequencing of both soil and root
fungi. Sequences were quality filtered and clustered using the bioinformatics pipeline SCATA
for fungi and the RDP pipeline for bacteria. A total of 807 fungal OTUs N>2 reads (496 OTUs
with N>5 reads) was found in the soil samples with corresponding figures of 546 and 350
OTUs in the root samples. Fungal community structure in soil differed significantly between
soil horizons but not between N treatments. Total numbers of OTUs declined significantly
down the soil profile and were higher for soil DNA in N+ treatments in the O horizon than in
the corresponding N- treatment. The horizon effect on OTUs from roots was not significant
but there was a significant decrease in the number of root-associated OTUs in response to N.
31
Structure and successional changes in communities of bacteria, archaea and
fungi colonising granitic rock surfaces in a boreal forest
Roger Finlay*, Shahid Mahmood
SLU, Uppsala BioCenter, Sweden
In boreal forest ecosystems, bedrock outcrops and boulders are generally covered by morlayer soil, with distinct mycelial mats at the interface between the rock surface and mor-layer.
Using such rocks as model systems, we are studying microbial diversity and patterns of
colonisation in relation to biogeochemical weathering processes that lead to mobilisation of
mineral nutrients essential for plant growth and ecosystem function. The nutrients released
from the rocks are utilised by the microbial community, taken up directly by roots or
transported to roots via mycelia of ectomycorrhizal fungi forming symbiotic associations with
tree roots. We hypothesise that both microbial diversity and abundance will be greater on
rocks receiving higher amounts of recently fixed photosynthetic carbon (to produce
weathering agents such as organic acids, or siderophores) compared to bare rocks. To test
this we sampled rock surfaces that were either: 1) bare or colonised only by lichens, 2)
colonised by mosses or 3) colonised by tree roots and fungal mycelia. We analysed
communities of bacteria, archaea and fungi using high-throughput 454-pyrosequencing.
Significant differences were found between fungal and bacterial communities colonising the
three rock-types. Lichen forming fungi were dominant on bare rocks while rocks with tree
roots were dominated by ectomycorrhizal fungi and also exhibited higher fungal diversity
than the bare rocks or rocks with mosses. A decline in abundance of lichens from bare to
ectomycorrhizal rocks suggests that lichens may have facilitated the colonisation of mosses
and subsequently ectomycorrhizal mycelia and tree roots during the course of boreal
ecosystem development. Rocks with mosses and ectomycorrhizal roots had higher numbers
of bacterial sequences than the bare rocks. Extremely low abundance of archaea on rocks
suggests that they probably have little or no direct role in biological weathering. However
crenarchaeota appear to have a specific association with rocks colonised by mosses.
18
32
Phytophthora diversity in Bavarian oak forests
Carmen Morales Rodríguez, Wolfgang Oßwald, Frank Fleischman*
Technische Universität München, Germany
Phytophthora species are considered to be devastating pathogens of many herbaceous and
woody plants. Up to now, about 140 species or taxa has been described, most of them cause
soil born root diseases. However, recent studies in different forest ecosystems using next
generation sequencing techniques reviled an unexpected high diversity of Phytophthora
species in various soils without plants showing severe disease symptoms, questioning the
ecological relevance of Phytophthora species as pathogens in their natural habitats.
In Bavarian (South Germany) oak forest a decline symptom of mature trees can be observed.
As cause for this dieback a complex of edaphic and climatic factors, root pathogens mainly of
the genera Phytophthora and Armillaria, mildew leaf disease and most of all herbivory is
discussed. The most frequently isolated Phytophthora species in central European oak forest
(Quercus robur or Quercus petrea) are P. plurivora and P. quercina, and their occurrence can
be correlated with above ground dieback symptoms. In a two year survey using MiSeq-ITS1amplicon sequencing we characterize the Phytophthora microbiome of oak roots in 15
symptomatic and asymptomatic oak stand, to elucidate the possible contribution of
Phytophthora in this decline complex. The first year results will be presented.
33
Investigation on the interactions between soil nutrient availability, rhizosphere
microbiome, as well as plant growth and development – a pot experiment
Davide Francioli*, Elke Schulz, Witoon Purahong, François Buscot, Thomas Reitz
Helmholtz Centre for Environmental Research - UFZ, Germany
The composition and functioning of the rhizosphere microbiome is directly affected by the
nutrient content of the soil. Besides this, the availability of nutrients also strongly influences
the structure and performance of plant communities, which in addition affects the microbial
community structure and accompanying microbial traits due to difference in the quantity
and/or the quality of rhizodeposition. The composition and functioning of the rhizosphere
microbiome is, in turn, one of the major driving factors in plant-soil feedback interactions and
directly influences plant growth and development. In order to determine the role of soil
microorganisms for plant nutrient acquisition at different nutrient levels we set up a pot
experiment. The soil for this study was taken from a long-term agricultural experiment, where
various fertilization regimes were consistently applied for the last 113 years. The soil
microbial communities, related to four different fertilization regimes (no fertilization, mineral
fertilization, organic fertilization, and combined mineral and organic fertilization), were
analysed revealing significant differences in their structure and activity. For the pot
experiment the four soils were gamma sterilized, and subsequently cross re-inoculated in a
factorial design with the four distinct microbial communities of the studied soils. In the pots
common grassland species were sown including the grasses Poa annua and Bromus mollis
and the herbs Viola arvensis and Matricaria inodora. We examined several phenotypic traits
of the plants and we performed fingerprinting analysis of the soil microbial community at
several time points of the experiment to identify the microbial key actors and processes
determining plant growth and development under the different nutrient levels. The results
19
indicated the soil nutrient content as the main driver in plant growth. In addition, notable
shifts in microbial community composition were observed within the rhizosphere microbiome
at specific plant growth stages and/or different soil nutrient levels.
34
A novel isolation procedure to isolate bacteria from Penicillium bilaii hyphae
Behnoush Ghodsalavi*, Ole Nybroe, Stefan Olsson, Mette Haubjerg Nicolaisen
University of Copenhagen, Denmark
Deficiency of plant-available forms of phosphorus limit plant production in many soils. Plants
are generally assisted by microbial associations, where fungi have major importance for
phosphate solubilization and may function as biofertilizers. For mycorrhiza fungi, which may
also improve plant growth, it is known that their hyphae are colonized by bacteria, which
include helper bacteria with positive impact on fungal growth and performance. The nonmycorrhiza fungi Penicillium bilaii has been shown to increase plant-available phosphorus,
and thereby improve plant growth. However, it remains unknown if comparable relationships
exist between P. bilaii and its hyphae associated bacteria. The objective of the current study
was to characterize bacteria associating with P. bilaii hyphae in a close-to-natural soil
system. We initially established a novel microcosm system to mimic the natural habitat for
fungal-bacterial interaction in the soil. Hyphal growth of P. bilaii was established on glass
cover slides that were placed in mesh bags and transferred to soil. After incubation for 8
days, the presence of hyphae-associated bacteria was confirmed by SYBR Green staining and
fluorescence microscopy. Quantification of culturable bacteria from washed, colonized cover
slips versus un-colonized controls showed more than 102 fold difference supporting the
notion that bacteria isolated from colonized cover slips were indeed hyphae-associated. A
strain collection of 100 hyphae associated isolates was established. By universally primedPCR finger printing, the isolates could be assigned to 25 different groups. Representative
isolates were subjected to 16S rRNA gene sequencing, which revealed that the hyphaassociated bacteria primarily belonged to the genera Bacillus and Pseudomonas. Future
studies will test if growth and activity of P. bilaii can be improved by hyphae associated
helper bacteria in order to improve plant growth.
35
Effects of inoculation with Serratia proteamaculans S4 on structure and
allocation of plant-derived C in root and rhizosphere communities of oilseed
rape
Konstantia Gkarmiri*, Shahid Mahmood, Sadhna Alström, Nils Högberg, Roger Finlay
Swedish University of Agricultural Sciences, Sweden
The rhizosphere is an active and dynamic niche where plant-derived carbon supports
microbial growth. Rhizosphere communities can be source of high biodiversity but there are
few studies of how this may be affected by applications of biological control agents. Serratia
spp. have demonstrated plant growth promoting and antagonistic effects, and should
compete more successfully than indigenous communities for plant-derived carbon and alter
C allocation patterns to root and rhizospheric microbial communities. A greenhouse study,
using field soil, was performed to identify bacterial and fungal communities actively
20
assimilating plant-derived C in the root and rhizosphere of oilseed rape plants following
inoculation with Serratia bacteria and the fungal phytopathogen Verticillium longisporum.
Oilseed rape seedlings were inoculated with Serratia proteamaculans S4 and were sown in
soil artificially inoculated with Verticillium. Factorial combinations of Serratia and Verticillium
inoculation were used, as well as additional plant-free soil treatments. Plants were grown for
four weeks. Continuous labelling for 6 hours was initiated 4 weeks after the seeds were sown
in pots. The first harvest took place 24h prior to labelling (SIP t0). Following 13CO2 injection, 4
time points were decided for harvesting. No morphological differences or difference in shoot
or root dry weight was observed between the treatments. However total 13C enrichment in
rhizospheric microorganisms was different between the different treatments suggesting a
treatment effect on carbon allocation below-ground, either in the level of microorganisms or
due to increased root exudation because of stress. The community structure associated with
the roots and rhizosphere was examined using 454 pyrosequencing and RNA-based stable
isotope probing (SIP) of 13C-labelled 16S rRNA fragments was used to identify taxa actively
involved in the assimilation of root-derived carbon. The main focus of this study was to
improve our understanding of the relationship between plants, indigenous microbial
communities and externally applied biocontrol bacteria.
36
Comparative root microbiome studies of a Verticillum longisporum resistant and
susceptible rapeseed line
Stefanie Glaeser*, Christian Obermeier, Ebru Cevik, Nima Haghighi, Rod Snowdon,
Peter Kämpfer
Justus-Liebig-University Giessen, Germany
Agricultural plants harbor a high diversity of microbes that colonizing endophytic
compartments of roots. Among those are several antagonistic bacteria, which protect plants
against root pathogenic fungi. Root exudation pattern and other so far unknown plantderived factors strongly affect the root colonization by these specifically adapted microbes.
We investigated bacterial root-endophytic communities of two contrasting oilseed rape
double haploid lines from a cross of a susceptible and a resistant parent. One of the rapeseed
lines is resistant, the other susceptible to Verticillium longisporum infection. We compared
the microbiome of both lines after two weeks of growth in soil and sand to determine if
specific root microbiomes can be linked to resistance or susceptibility against Verticillium.
Roots were harvested (a pool of 6 plants per treatment), the rhizosphere was removed and
roots further purified by sonication and washing. Genomic DNA was extracted from roots to
compare endophytic bacterial communities by 16S rRNA gene amplicon Illumina sequencing.
Bacterial communities showed higher similarities between the two rapeseed lines grown either
in soil or sand than for the same rapeseed lines after growth in sand and soil. However,
specific endophytic bacterial communities were developed for both rapeseed lines. In
summary 37 bacterial phyla or candidates phyla were detected. The most abundant phyla in
all samples were the Proteobacteria, with 60-62% relative abundance if rapeseed lines were
grown in soil and >54% if grown in sand. Second and third most abundant phyla after growth
in soil were Actinobacteria (6 to 9%) and Bacteroidetes (7 to 10%). A strong rapeseed line
21
specific effect was determined after growth in sand with an unexpected high abundance of
Planctomycetales (20%) in the Verticillium resistant rapeseed line.
Our data gave a first insight in rapeseed line specific microbiomes which may contribute to
the obtained resistance against Verticillium.
37
Influence of rhizosphere, root and P soil on fungal and bacterial communities
associated with maize genotypes with contrasting P use efficiency
Eliane Gomes*1, Ubiraci Lana1, Bangzhou Zhang2, Christiane Oliveira1, Lauro
Guimarães1, James Tiedje2
1
Embrapa Maize and Sorghum, Brazil, 2Michigan State University, USA
Maize (Zea mays L.) represents one of the main economic crops for food and energy in the
world, and its associated microbial communities have been intensively investigated using
different approaches. However, low-resolution profiling methods often make it difficult to
understand the complicated microbial communities. Using Illumina MiSeq high-throughput
sequencing, we analyzed the bacterial and fungal communities from washed roots
(endosphere) and from the rhizosphere soil both originated from the same root sample of
two maize genotypes with contrasting phosphorus (P) use efficiency cultivated in two
different P soil concentrations. A total of 2.588.087 and 2.516.453 reads were identified from
16S-V4 (bacteria) and ITS (fungi) rRNA gene regions, respectively, and all taxonomic
classifications were assigned using the naïve Bayesian algorithm developed for the RDP
Classifier. We observed higher Chao 1 richness and Shannon–Weaver diversity indices in the
rhizosphere than inside the roots, suggesting that endosphere has a specific microbial
community. The predominant bacteria in rhizosphere were from the phyla Proteobacteria
(63.9%), Actinobacteria (8.4%), and Bacteroidetes (6.7%), while the main phyla inside the roots
were Proteobacteria (50.5%), Tenericutes (15.8%) and Bacteroides (13.4%). The soil P level
also influenced the bacterial community distribution since members of Burkholderia and
Ralstonia genera were enriched in the low P rhizosphere while Pseudomonas and Leclercia
were predominant in the high P rhizosphere soils. Considering the fungal communities,
Ascomycota (85.7%) was the dominant phylum, followed by Basidiomycota (9.5%) and
Glomeromycota (3.0%) in the rhizosphere and endosphere in both P conditions.
Glomeromycota phylum (mycorrhizal fungi) was represented at 1% in the rhizosphere and at
5% in the endosphere, and was especially abundant in the low P soil. In general, we conclude
that rather than maize genotypes, the overall patterns of soil microbial diversity was
influenced by the rhizosphere and endosphere habitats followed by the soil P concentration.
38
Microbial community dynamics of growing media in soilless culture systems
Oliver Grunert*1, Emma Hernandezsanabria1, Marie-Christine Van Labeke2, Dirk
Reheul2, Maaike Perneel3, Nico Boon4
1
Laboratory of Microbial Ecology and Technology (LabMET), Ghent
University, Belgium, 2Department of Plant Production, Ghent University, Coupure Links
653, Belgium, 3Peltracom, Belgium, 4Laboratory of Microbial Ecology and Technology
(LabMET), Ghent University, Coupure Links 653, Belgium
22
The hairy roots syndrome caused by Agrobacterium rhizogenes is an increasing problem in
greenhouse horticulture in Spain, France, Germany, UK, Ireland, Poland, Canada, USA, Mexico,
the Netherlands and Belgium. Current control measures are limited to removal of excessive
leaves from the plants and the plastic cover from the slabs. There is a lack of effective control
strategies and the search for alternative strategies also has been stimulated by public
concerns about the adverse effects of biocides on the environment and human health.
Natural disease-suppressive soils and probably horticultural growing media and its microbial
community can effectively protect plants against soilborne pathogens. In this study, we
compared the physicochemical and microbial community characteristics of an mineral and
organic horticultural growing medium in relation to the presence of the plant pathogen
Agrobacterium rhizogenes. We aimed to identify how microbial and environmental
interactions influenced the development and spread of this disease in a soilless cultivation
system. In this study we hypothesised that there is a fundamental difference of a young
(mineral) and an old (organic) growing medium-associated microbial community in its
general suppressiveness against Agrobacterium rhizogenes causing the hairy roots syndrome
and resulting according to the definition of Baker and Cook in little or no visible damage to
the plant, although the pathogen may persist in the growing medium. Multivariate statistical
analysis performed to assess the characteristics of each growing medium revealed the key
variables impacting the microbial community, such as potassium and ammonia
concentrations. High throughput sequencing analysis of the bacterial abundance of the
communities present in organic substrate showed significant interactions among
Methylophilaceae and Actinobacteridae with A. rhizogenes. Our results clarified the complex
bacterial relationships in horticultural growing media. Knowledge regarding these
relationships may be used to develop strategies to control the hairy roots syndrome.
39
Microbes in root-soil interfaces: colonization patterns in oxic/anoxic microenvironments of wetland rice
Hannes Schmidt*1, Thilo Eickhorst2
1
University of Vienna, Department of Microbiology and Ecosystem Science, Division of
Microbial Ecology, Austria, 2University of Bremen, Soil Microbial Ecology, Germany
In paddy soils, the presence of numerous electron acceptors and root-derived substances in
oxic/anoxic micro-environments such as the rhizosphere may attract, stimulate, or inhibit soil
microorganisms. The intensity of influence of the plant on microbes and vice versa is likely to
be greatest in close vicinity to the root surface and may decrease with distance from the
rhizoplane. It is therefore essential to detect sites of microbial rhizosphere colonization, and
thus to identify potential areas of microbe-plant interaction.
A 16S rRNA-targeted approach including PCR-based fingerprinting and CARD-FISH was
applied to identify, localize, quantify, and visualize microbial cells associated with roots
(rhizoplane, rhizosphere) and selected soil compartments (bulk soil, oxidized layer). With
emphasis on microbial rhizoplane communities, the spatial distribution of archaea, bacteria,
and relevant microbial populations was investigated.
The growth stage of rice and the distance to the root surface strongly influenced microbial
colonization. Patchy distribution patterns of microbes observed at early stages turned into
23
more uniform colonization with increasing plant age. Accordingly, the highest colonization
densities shifted from the tip to more mature regions of rice roots and were found at
flowering stage. β-Proteobacteria became predominant in the oxygenated rhizosphere that
were affiliated with lithoautotrophic iron-oxidizing bacteria. They were further visualized to
densely colonize areas of pronounced iron coatings indicating an active role in the biotic
formation of Fe-plaque on rice roots. The co-localization of oxygen-sensitive methanogenic
archaea with oxygen-dependent methanotrophic bacteria on the rhizoplane indicated the
formation of micro-sites of contrasting oxic/anoxic conditions within short distance.
Beneficial effects of a close association with root surfaces were indicated by proportionally
higher numbers of methane-oxidizing bacteria on the rhizoplane compared to the
rhizosphere.
The presented data provides essential quantitative and spatio-temporal information on
microbial rhizosphere colonization and thus may ideally complement studies on microbial
diversity as analyzed via high-throughput sequencing.
40
Taxonomic and functional diversity of the microbiome of agricultural plants and
its relation to the rhizosphere effect
Julie Hernandez-Salmeron*1, Eduardo Valencia1, Gabriel Moreno-Hagelsieb2, Gustavo
Santoyo1
1
Universidad Michoacana de San Nicolas de Hidalgo, Mexico, 2Wilfrid Laurier
University, Canada
Plants rhizosphere is the microenvironment that connects free life microorganisms in soil with
the plant, through the flow of rhizodeposits by the roots. Each plant species has a differential
effect on the way they select or set a microbiome in its particular rhizosphere, whose main
function is to promote plant growth and phyto-pathogen antagonism. In this study, we
hypothesized that after growing different plant species in the same soil with the same
repertoire of microorganisms they will diferencially select and promote a microbiome specific
to the plant genotype, we selected plants of alimentary importance for our country: wheat,
maize, sorghum and beans. The in vitro experiment was conducted under controlled
conditions of temperature and light in a growth chamber using the same soil type where the
plants were grown, so that the only variable was attributed to the plant genotype.
Rhizospheric and bulk soil was removed after four weeks of plants growth, from which DNA
was extracted and metagenomic bacterial diversity was initially screened using DGGE, these
results showed a clear variation in the diversity of microorganisms in each plant species.
Therefore, metagenomic DNA was purified and sent to pyrosequencing to analyze genetic
functional diversity in each plant rhizosphere. We expect to obtain sequences in the following
months and complete the in silico analysis of metagenomes by using MEGAN to show the
results in this event. Diversity of culturable microorganisms was also analyzed by RAPD
fingerprinting, where the highest haplotype percentage was found in sorghum plants,
followed by wheat, and corn rhizosphere.
This study would allow us to deepen more about the factors that influence the structure of
bacterial communities in the rhizosphere and infer ecological functions relevant during plantmicroorganism interaction.
24
41
Activity profiling of extra-radicular enzymes in urban trees
Josef Valentin Herrmann*, Ullrich Gilge
Bavarian State Institute for Viticulture and Horticulture, Germany
Urban trees grow in a stressful, unnatural environment. Limited tree pits restrict their rooting
zone, degraded, compacted soil disturbs water and air balance. Multiple pollutants as well as
frequent mechanical damage affect the tree vitality. Additionally, climate change has
worsened these demanding conditions. Under such adverse circumstances mycorrhizal
associations play an even more significant role for plant health.
To select urban trees for the future, a long-term project was started in 2010. Different tree
species were planted at three sites with differing climate conditions. Representing different
mycorrhizal preferences, some species (Carpinus betulus, Fraxinus pennsylvanica ’Summit’,
Magnolia kobus, Ostrya carpinifolia, Parrotia persica, Quercus cerris, Tilia tomentosa ’Brabant’)
were chosen to study extraradicular enzymes relevant to tree nutrition and connected to
mycorrhization. At planting half of the trees were inoculated with a commercial
mycorrhizaproduct (INOQ).
Since 2011 root sampling has been performed twice a year. To measure the activity of 8
hydrolytic and oxidative enzymes (cellulose and hemicellulose degrading xylosidase,
glucoronidase, cellobiohydrolase, β-glucoronidase, chitin degrading Nacetylglucosaminidase, phosphate mobilizing phosphomonoesterase, protein degrading
leucinaminopeptidase) a rapid and sensitive fluorometric microplate multiple enzymatic test
was performed on single root tips. In addition, each root tip was microanalyzed for
mycorrhizal colonization.
The data revealed a high diversity of activity profiles. The pattern showed a tree species
specificity may be representing the species specific mycorrhization. In addition, a seasonality
could be detected with higher activities in spring compared to summer and autumn. A
difference in enzymatic profiles between inoculated and non-inoculated trees could not be
observed. Comparing mycorrhizal and non mycorrhizal root tips significant (Mann-Whitney
Rank Sum Test) alterations were detected with some enzymes displaying higher and some
lower activities. Correlation analysis to substrate soil, soil condition and planting site are
under way.
42
Impact of microbial species loss on rhizosphere microbiome assembly
Gera Hol*, Wietse de Boer, Wim van der Putten
Netherlands Institute of Ecology, Netherlands
Plant growth may respond positively or negatively to the soil microbial community, depending
on the balance between pathogens and mutualists. Growing plants repeatedly in the same soil
may result in increase of pathogens (negative plant soil feedback) or increase of mutualists
(positive plant-soil feedback). While the concept of plant-soil feedback is well- known, there is
a gap in the knowledge of microbial community development during feedback, in particular
with regards to the role of less abundant species. Loss of microbes is expected to affect plant
growth when the balance between pathogens and mutualists is
25
disturbed. Here we tested to what extent loss of rare microbes will affect the development of
plant-soil feedback. Inoculation of sterilized soils with serial diluted suspensions from two soil
origins and subsequent incubation resulted in soils with similar microbial biomass but
differences in microbial community. Pyrosequencing of 16S rRNA was used to track how
dilution and incubation affected microbial community composition directly after incubation
and after one or two cycles of plant growth. Dilution decreased species richness and similarity
as compared to the original field soil. Growing plants on the inoculated soils once or twice
increased similarity of the bacterial community to that of the field soil. While the most diluted
communities showed the least overlap with the original field soil, the similarity between
replicates was high. Also the similarity between microbial communities after one and two
growing cycles was highest in the most diluted community. On basis of the sequence data
and literature we identified potential pathogens and mutualists of the host plant Triticum
aestivum and followed how those bacteria species reacted to a second growing cycle with the
same host plant. We conclude that loss of species will make microbial community dynamics
more predictable.
43
Hybrid rice stimulates greater abundance of AOB relative to AOA in the
rhizosphere
Qaiser Hussain*1, Genxing Pan2
1
Pir Mehr Ali Shah Arid Agriculture University, Pakistan, 2Institute of Resource,
Ecosystem and Environment of Agriculture, Nanjing Agricultural University, China
It is well documented that rice paddy soils have activities of both AOB and AOA, but impact of
hybrid rice on the ecology of ammonia oxidizers (AOB & AOA) is still poorly understood under
field condition. We hypothesized that hybrid rice with the trait of vigorous root system
releases more oxygen that can influence the structure and abundance of AOB and AOA in the
rhizophere.
A field experiment was initiated to assess the structure and abundance of ammonia oxidizers
in the rhizosphere of hybrid and conventional rice cultivars at three growing stages.
Rhizosphere samples were collected at selected growth stages for each rice cultivar: 45 days
after planting (tillering stage-S1, 81 days after planting (grain filling stage-S2) and 107 days
after planting (ripening stage-S3). PCR-DGGE and real-time PCR approaches were used to
evaluate the structures and relative abundances of ammonia oxidizers (AOA and AOB) by
targeting a functional gene fragment coding ammonia monooxygenase (amoA). Moreover,
important band retrieved from DGGE gel were analyzed using sequencing and phylogenetic
analysis.
Hybrid rice promoted 53%, 30% and 23% higher rhizosphere amoA (AOB) gene abundance
relative to conventional cultivar at S1, S2 and S3 stages, respectively. Though, rice cultivars
showed no considerable effect on the amoA (AOA) gene abundance. Principal component
analyses (PCA) of PCR-DGGE profile revealed a profound shift of AOB community structure
between two cultivars across particular growing stages. However, rice cultivars did not
significantly influence the structure of archaeal-amoA (AOA) in rhizosphere showing the
higher stability of AOA communities. Diversity of AOA was limited to uncultured
Crenarchaeote, whereas AOB analyses revealed several distinct operational taxonomic units
26
markedly resemble to Nitrosospira sp. in the rhizosphere of hybrid cultivar compared to
conventional cultivar. Hybrid rice can select from a particular group of AOB in the rhizosphere
relative to AOA throughout growth periods.
44
Effects of different organic fertilizers on soil rhizospheric microbial functional
diversity of greenhouse chrysanthemum
Rong Jiang*1, Zhi Xu2, Li Tang2
1
College of Resources and Environmental Science,Yunnan Agricultural
University, China, 2College of Resources and Environmental Science, Yunnan
Agricultural University, China
Organic fertilization can affect microbial community in rhizosphere soils which is thought to
be responsible for biological processes that are necessary for maintaining a healthy soil.
However, its efficacy of different organic fertilizers on soil microbial community structure in
highly intensive flower industry under greenhouse condition had little been elucidated.Field
experiment was conducted to study the effects of applying different organic fertilizers and
their combination with 20% reduced chemical fertilizers on the microbes in rhizosphere soil
of chrysanthemum. As compared with conventional chemical fertilization (100% chemical
fertilizers, CF), applying bio-organic fertilizer in combination with 20% reduced chemical
fertilization (BIO) significantly increased the number of bacterial by 101.2%, actinomycetes by
84.3% and the total microbial number by 100.1% in the rhizospheric soil, and decreased the
fungi number by 64.2%, while applying refined organic fertilizer in combination with 20%
reduced chemical fertilization (OF) only decreased the fungi number by 30.4%. As compared
with CF, the BIO treatment increased the rhizospheric microbial diversity of Shannon index
(H) and McIntash index (U) of chrysanthemum by 8.6% and 21.6%, respectively, and the OF
treatment showed no significant difference. OF and BIO could significantly increased the
carbohydrates utilization ability by 13.3% and 15.8%, the phenolic carbon source utilization
ability increased by 13.0% and 36.5%,repectively. The BIO also increased the capacity of
amino acids significantly. The principal component analysis (PCA) demonstrated that, BIO
significantly affected the soil microbial community structure, mainly depending on the
utilization of carbohydrates, amino acids and carboxylic acids. Under 20% chemical fertilizers
reduction during the full growth stage, applying bio-organic fertilizer (BIO) increased the
stem length and stem diameter significantly, showed the increasing of the flower diameter
and stalk length, yield and dry matter, improved the flower quality of chrysanthemum.
45
Plant growth promoting rhizobacterial efficacy in cowpea (Vigna unguiculata
(L.) walp.)
Kannan Kanthaiah*, Velu Rajesh Kannan
Bharathidasan University, India
Plant growth promoting rhizobacteria (PGPR) are known to influence the growth of the plant
by various plant growth promoting activities such as Indole Acetic Acid (IAA) production,
phosphate solubilization activity, ammonia production (NH3), nitrogen fixation, siderophore
production and hydrogen cyanide production (HCN). In search for the potential PGPR from
27
the Rhizosphere soil of cowpea, a total of 16 rhizobacteria (B1-B6 & D1-D9) were isolated. By
comparing the results two potential rhizobacteria (B6 & D4) have been selected for the
further evaluation of Plant Growth Promoting (PGP) activity. These isolates biochemically
characterized, B6 and D4 confirmed as Pseudomonas and Bacillus by 16SrRNA gene
sequencing. Their ability of IAA production ranging from (95 and 220.5 µg/ml) respectively,
they showed positive results to ammonia, nitrogen fixation, hydrogen cyanide production
and siderophore production. Their phosphate solubilizing ability ranging from 16 mm and
their solubilization efficiency (SE) ranging from 200 and 155 and their Solubilization index (SI)
3.0 and 2.5 respectively. In vitro and in vivo nursery field studies revealed that 100% seed
germination rate using Bacillus safensis and 92% for Pseudomonas aeruginosa showing
promosing results. The study showed the characters of benefits to use these organisms as
biofertilizers for sustainable agriculture to improve crop yield.
46
Pseudomonas species in the secretion of auxin
Nazanin Khakipour*1, Kazem Khavazi2, Abdolreza Akhgar3
1
Islamic Azad University- Savadkooh branch, Iran, 2Soil and Water Institute, Iran, 3Vali-e
Asr university of Rafsanjan, Iran
P.putida and P.fluorescence stimulate plant growth and crop yield can be increased. One of
the important characteristics of fluorescent Pseudomonads bacteria produce organic
compounds in concentrations too low to be able to control physiological processes.
Secretion of growth regulators as one of the most important mechanisms of plant growth is
active. The purpose of this study was to compare different strains of bacteria P.putida and
P.fluorescence secrete a variety of auxin and it was under the same conditions. For this
purpose, 23strains P.fluorescence and 27 strains P. putida randomly of Microbial bank was
prepared.
The IAA strains P.fluorescens of zero to 31.6milligrams per liter respectively. Of the 23 strains,
7 strains did not produce IAA. The amount of IAM at P.fluorescens strains was from zero to
16.12 mg per liter respectively. Of the 23 strains did not produce 2 strain IAM. The ILA also
from zero to 7.2 milligrams per liter respectively. IBA did not generate any of the strains
studied P.fluorescens. Among different strains P.fluorescens this study the prevalence of
strains capable of producing IAA, IAM, ILA and IBA 8,15,10 and zero, respectively. The IAA
strains P.putida of zero to 24.8 milligrams per liter .7 strains of 27 strains did not produced
IAA . The amount of IAM at P.putida strains was from zero to a maximum of 17.2 milligrams
per liter respectively. Of the 27 strains did not produce 6 strain IAM. The ILA also from zero to
10 milligrams per liter respectively. Of the 27 strains, 8 strains did not produce ILA. P.putida
strains studied did not generate any of the IBA. P.putida among different strains of the study,
the prevalence of strains capable of producing IAA, IAM, ILA and IBA 11, 13, 12 and zero,
respectively.
28
47
Microbial diversity analysis of the rhizosphere of tomato cultivars that are
resistant or susceptible to bacterial wilt
Seon-Woo Lee1, Hyun-Gi Kong*1, Min-Jung Kwak2, Eun Joo Jung1, Ju Yeon Song2,
Jihyun Kim2
1
Dong-A University, South Korea, 2Yonsei University, South Korea
Bacterial wilt caused by Ralstonia solanacearum is lethal wilt resulting in significant yield loss
on many Solanacea plants. Bacterial wilt resistance in several resistant plants is controlled by
quantitative trait loci and also by rhizosphere conditions probably by microbial composition.
In this study, we investigated the microbial community structure of tomato rhizosphere using
two different tomato cultivars, Hawaii7996 as a bacterial wilt resistant cultivar and
Moneymaker as a bacterial wilt susceptible cultivar. We cultivated tomato cultivars under
greenhouse condition with field soils at Dong-A Agricultural experimental station. Microbial
communities of tomato rhizosphere of both cultivars were analyzed by amplifying 16S rRNA
genes from those rhizosphere soil and pyrosequencing of the amplified clones through GSFLX 454 sequencer. Microbial community structure was analyzed using MG-RAST system. The
most abundant microbial phylum was proteobacteria both in bulk soil and in tomato
rhizosphere soil. Compared to bulk soils, the proportions of the phyla such as Bacteroidetes
and Firmicutes were significantly increased in tomato rhizosphere, while that of phylum
Acidobacteria was decreased in tomato rhizosphere. Especially, bacterial members in class
Flavobacteria were highly abundant in tomato rhizosphere. Comparison of microbial
communities of tomato rhizosphere revealed that rhizosphere microbiome between
Hawaii7996 and Moneymaker was distinguishable. The tomato rhizosphere showed the
similar pattern of microbial diversity for two consecutive years. Our result suggested that the
unique microbial community forms in tomato rhizosphere in a cultivar specific manner.
48
Selection of rhizobial and rhizospheric isolates associated to chickpea (Cicer
arietinum) for promoting its culture in region dedicated for grain culture
Saadia Laabas*, Zineb faiza Boukhatem, Tsaki Hassini, Abdelkader Bekki
University of Oran 1 Ahmed Benbella, Algeria, Algeria
Consumption of chickpea (Cicer arietinum L.) holds a special place in Algerian culture but its
production, however, is far from to be sufficient for the population needs. It has been
attempted to introduce this legume in wilaya of Tissemsilt (Algeria), in rotation with cereal
crops. This study aims to establish the best microbiological approach to improve the legume
performance. Strains isolated from Cicer arietinum L. rhizospheric soil from five selected sites
were screened for their plant growth promoting (PGPR) potential under controlled conditions,
for IAA production and P solubilization ability. In a second step, were selected native rhizobial
strains with high nitrogen-fixing potential. Then the single effect of PGPR isolates was
compared to their combination with rhizobial inoculant on plant growth, under greenhouse
conditions.
29
49
Effect of phosphorus fertilization on bacterial microbiome in microsites of
ryegrass rhizosphere
Lorena Lagos*1, Oscar Navarrete2, Fumito Maruyama3, María de la Luz Mora1, Milko
Jorquera1
1
Universidad de La Frontera. Center of Plant, Soil Interaction and Natural Resources
Biotechnology, Chile, 2Universidad de La Frontera, Chile, 3Kyoto University. Section of
Microbiology,Graduate School of Medicine, Japan
The structure and diversity of microbial communities along the rhizosphere differ in their
composition, activity and abundance according to diverse biotic and abiotic factors, such as
root exudates, plant species, type of soil, agronomic practices like fertilization. In the present
report we analyzed the effect of P fertilization on rhizobacterial composition in microsites of
Lolium perenne grown in a volcanic soil (Andisol) characterized by low-P available. Lolium
perenne grown in rhizotrons were separately fertilized with inorganic (phosphate; KH2PO4;
300 mg P kg-1 of soil) and organic (phytate; C6H18O24P6 and H2O; 2.5 mM (825 mg phytate
kg-1 of soil) P sources. Total DNA samples were extracted from two microsites of the
rhizosphere (root tip and mature zone). Control without fertilization and bulk soil were also
analyzed. The bacteria DNA sequences were analyzed by pyrosequencing of 16S ribosomal
RNA (rRNA) genes followed for a silico analysis by MOTHUR. Our results revealed according to
the alpha diversity analysis that in all rhizosphere samples there was a higher relative
abundance of the Proteobacteria (45%; alpha- and Betaproteobacteria) followed by
Actinobacteria (17%), Cloroflexi (12%) and Acidobacteria (11%). In contrast, in bulk soil more
relative abundance groups were Proteobacteria (68%; alpha- and gammaproteobacteria) and
Firmicutes (21%, Bacilli). On the other hand, beta diversity analyses indicated difference in
ocurrence (presence/absence) based on index of Jaccard for microsites of the rhizosphere
and bulk soil. Moreover, Yue and Clayton theta index indicated similarity for bacterial
community of microsites of the rhizosphere. The analyses were evaluated at level of species
and classes 0.03 and 0.10 OTUs, respectively. The present study showed that P fertilization
induced changes in the composition of bacterial communities at microsite level in the
rhizosphere of Lolium perenne.
50
Host-based microbial recruitment at the aquatic rhizosphere
Yong Jian Lee*, Sanjay Swarup
National University of Singapore, Singapore
The aquatic rhizosphere is a region around the roots of aquatic plants. Many studies focusing
on terrestrial rhizosphere have led to a good understanding of the interactions between the
roots, its exudates and its associated rhizobacteria. The rhizosphere of free-floating roots,
however, is a different habitat that poses several additional challenges, including rapid
diffusion rates of signals and nutrient molecules, which are further influenced by the
hydrodynamic forces. These can lead to rapid diffusion and complicates the studying of
diffusible factors from both plant and/or rhizobacterial origins. These plant systems are being
increasingly used for self purification of water bodies to provide sustainable solution. A better
understanding of these processes will help in improving their performance for ecological
engineering of freshwater systems. The same principles can also be used to improve the yield
of hydroponic cultures.
30
We have begun to unravel the complexity of rhizobacterial communities associated with
aquatic plants. Using fluorescence in-situ hybridization (FISH) and Illumina Miseq Next
Generation Sequencing of 16S amplicons and metagenomic DNA, we investigated the rootassociated microbial community of established P. amaryllifolius grown in four different water
bodies. The community structure of rhizobacteria from plants grown in these water bodies
are highly similar. The top three phyla belonged to Proteobacteria, Bacteriocedes and
Actinobacteria, as validated by FISH analyses. This suggests that the rhizosphere have an
innate ability to attract and recruit rhizobacterial communities, possibly through the
metabolic compounds secreted through root exudation. The selection pressure through plant
host is higher compared to environmental pressures that are different between the two water
sources. In comparison with the terrestrial rhizosphere, the aquatic rhizosphere microbiome
seems more specialised and has a high influence by the host. We are using these findings to
further understand the role of microbes in the performance of freshwater aquatic plants.
51
Motility in atrazine-degrading Arthrobacter bacteria and its possible ecological
role
Chengyun Li*1, Dmitry Bazhanov1, Hongmei Li2, Jishun Li1, Hetong Yang2
1
Key Laboratory for Applied Microbiology of Shandong Province, Biotechnology Center
of Shandong Academy of Sciences, China, 2Biology Institute of Shandong Academy of
Sciences, China
Bacteria of the genus Arthrobacter are known as ubiquitous and most frequently isolated soil
inhabitants. It is believed that the prevalence of Arthrobacter spp. is due to their resistance to
stressful conditions. No behavioral mechanisms contributing to the survival of Arthrobacter
bacteria in soil were so far known.
Based on the results of ERIC-PCR genotyping and phylogenetic analysis of directly isolated
atrazine degraders, we found that Arthrobacter spp. prevailed in the root-associated
communities of atrazine-degrading bacteria both in industrial and agricultural soils.
Genetically similar A. ureafaciens bacteria were predominant atrazine degraders in the maize
rhizosphere at geographically distant agricultural sites. Regardless their geographical origin,
atrazine-degrading A. ureafaciens strains were motile, spread along fungal hyphae and
colonized plant roots after seed inoculation. By characterizing more than 100 atrazinedegrading strains, we revealed that active motility was exhibited by representatives of various
phylogenetic groups of the genus Arthrobacter. Some strains spread in semisolid agar and on
the surface of soft agar, suggesting various types of motility. Our data suggest that active
motility is widely distributed among Arthrobacter bacteria and can play an important role in
their rhizosphere competence and soil survival. Some atrazine-degrading strains we have
isolated can be considered promising model bacteria for studying root colonization by
Arthrobacter spp. Mechanisms of the active motility possessed by Arthrobacter bacteria and
their role in the root colonization will be further investigated.
31
52
Effect of Bio-organic fertilizer on bacterial wilt and rhizospheric soil microbes of
flue-cured tobacco
Zhang Yunwei, Tang Li*, Xu Zhi, Li Yanhong, Song Jianqun
Yunnan Agricultural University, China
Two years field experiments were carried out to study the effects of refined organic fertilizer
and bio-organic fertilizer, both combined with 20% reduced chemical fertilizer on flue-cured
tobacco yield and quality, resistance to bacterial wilt and soil microbes. The effects of
different organic fertilizers on soil microbial quantity, pathogen number, and microbial
functional diversity were studied by means of traditional enumeration methods and BIOLOG
technology.
Under reduction of 20% chemical fertilizers, the effects of applying organic fertilizer (OF) on
tobacco yield and quality was not significantly, but applying bio-organic fertilizer (BIO)
significantly improved tobacco yield, output value and the proportion of high grade tobacco
leaves by 12%, 25% and 27%, respectively. Compared with conventional chemical fertilization
(CF, 100% NPK chemical fertilizers), both treatments applying either organic fertilizer (OF), or
bio-organic fertilizer (BIO), decreased the bacterial wilt, increased the bacterial number and
improved the microbial activity significantly. Compared with CF, the disease incidence and
the disease index of bacterial wilt by BIO treatment were reduced 20%~32.1% and
36.2%~40.9%.
Compared with OF, BIO treatment decreased the disease incidence of bacterial wilt by
11.1%~13.7% and the disease index of BIO were significantly reduced by 20.9%~31.1%,
respectively. BIO had 70.5%~171.7% higher bacteria counts and 2~4 times higher
actnomyces counts than OF. Compared with OF, The fungi number and pathogen number of
BIO treatment were decreased by 64% ~74.3% and 55.8%~66.7%, respectively.
Biolog ECO results showed that, AWCD data which indicate the soil microbial activity varied
significantly in order from highest to lowest of BIO>OF>CF. The same trend (BIO>OF>CF)
was found when functional diversity was expressed as Shannon’s diversity index (H),
McIntosh’s index and Simpson’s index. Compared with CF, the utilization rate of carboxylic
acids, amino acids and phenols of BIO were increased by 30%~32%, 27.2%~39.7% and
77.2%~117.8%, respectively.
53
Rhizosphere bacteria constitute a principal influential force on the outcome of
cotton replanted diseases
Xiaogang Li*, Xingxiang Wang
Institute of Soil Science, Chinese Academy of Sciences, China
Cotton yield and quality are seriously compromised by consecutive cropping of cotton in the
primary cotton regions of China. The primary hypothesis of this study was that the
rhizobacterial community would constitute a principal influential force for governing cotton
replant disease. To assess this possibility, plant growth, disease resistance, and the effect of
root exudates on the development of Fusarium oxysporum and on rhizobacterial community
composition were evaluated. The experiment was conducted in two different replanted soils,
32
one in use for four years and the other in use for 15 years with a fallow soil as the control.
The replanted soil significantly influenced plant growth and rhizosphere-secreting
characteristics with a lower resistance to Fusarium wilt. Also, a bioassay revealed a loss of
spore germination and sporulation of F. oxysporum in cotton root exudates from replanted
soils compared to those from the control soil, and MiSeq sequencing of 16S rRNA gene
amplicons demonstrated clear variations in the rhizobacterial community of cotton grown in
replanted soils and in the control soil. Specifically, the Gammaproteobacteria and
Betaproteobacteria, including Xanthomonadaceae, Comamonadaceae, Oxalobacteraceae, and
Caulobacteraceae known to comprise strains with plant-beneficial and disease-suppressive
properties, were identified as the most dynamic taxa associated with cotton health in the
control soil, while fewer bacterial taxa (Flavobacteriaceae, Streptomycetaceae,
Sphingomonadaceae) were associated with cotton-replanted soils, possibly representing
greater cotton root damage in replanted soil. Collectively, these results suggest that the
complex phenomenon of cotton replant disease may not simply be ascribed to
accumulations of pathogens or a single group, but is most likely associated with the
rhizobacterial community recruited by cotton for its protection against pathogen infections.
54
The consecutive monoculture problems of Pseudostellaria heterophylla and its
control strategy
Wenxiong Lin*, Linkun Wu, Hongmiao Wu, Jun Chen, Xianjin Qin, Yongpo Zhao,
Juanying Wang
Fujian Agriculture and Forestry University, China
The consecutive monoculture problems are one of the big issues in modern agriculture. In this
study, Pseudostellaria heterophylla, an important medicinal plant was used as experimental
material. Based on our previous study, the changes in the rhizosphere microbial community
under consecutive monoculture were studied through the approaches of modern rhizosphere
biology. The results showed that P. heterophylla consecutive monoculture altered the
rhizospheric microbial composition with fewer beneficial microorganisms and more
pathogenic microbes. The qRT-PCR analysis confirmed that beneficial microbes including the
Pseudomonas, Bacillus subtilis, etc. decreased with the increasing years of monoculture, the
reverse was true in case of the pathogenic microbes such as Salmonella sp., Fusarium
oxysporum, etc.. Further study found the mixtures of phenolic acids in the same ratio as
determined in P. heterophylla root exudates could significantly promote the growth of the
specific pathogenic fungi but inhibit the beneficial microbes. Some pathogenic microbes,
such as Salmonella sp. showed a chemotaxis response to several root exudates. The sequent
test showed these pathogens could cause serious wilt disease on P. heterophylla seedlings
and led to the up-regulation of the genes involving in calcium signaling transduction system
and energy production, which explains in part the underlying mechanism of declines in yield
and quality of plants in response to consecutive monoculture stress. Accordingly, different
control strategies were used, suggesting that the specific microbial fertilizers and some
cropping sequences such as paddy-upland rotation patterns could effectively remediate the
imbalanced soil microbial community, in turn leading to increased yield of replanted P.
heterophylla. It is therefore concluded that the plant-microbe interactions play important
roles in consecutive monoculture problems of P. heterophylla, and to develop a specific
33
biofertilizer with a good combination of beneficial microbes to the disease-conducive soil is
an efficient way to overcome the soil-borne disease of replanted P. heterophylla.
55
Characterization of bacterial communities from halophytes growing in Khewra
Salt Ranges by culture-dependent and metagenomic approaches
Salma Mukhtar, Samina Mehnaz, Kauser Malik*
Forman Christian College (A Chartered University), Pakistan
The microbial diversity in the rhizosphere of halophytes can provide information regarding
various mechanisms enabling the plants to survive under such an extreme condition of
abiotic stress. Only 1-3% of the soil microbial population is culturable. DNA-based methods
have been developed to have an insight into the remaining fraction of soil microflora. This
study, investigated the distribution of culturable and non culturable bacteria in the
rhizosphere of halophytes. Culturable bacterial isolates were characterized morphologically,
biochemically and through sequencing of 16S rRNA gene. These were identified as strains of
different species of Bacillus, Pseudomonas, Staphylococcus, Micrococcus, Planococcus, Vibrio,
Burkholderia, Klebsiella, and Kocuria. Based on salt tolerance, fifty five strains were selected.
Plasmid curing of isolates was done to study the effect of plasmid conferring salt tolerance.
These plasmids were isolated, transferred into E. coli and growth response of original and
transformed E. coli strains was compared at 1.5 to 4 M concentration of NaCl. Almost all of
these, showed optimum growth at 1- 3.5 M NaCl. Cured isolates did not grow in halophilic
medium but grew well on LB medium. Most of the transformed E. coli strains grew up to 3M
NaCl concentration.
Culture independent diversity was studied by PCR and sequencing of 16S rRNA gene.
Metagenomic analysis from rhizosphere showed that 40% bacteria were uncultured and
unclassified. Firmicutes, Proteobacteria, Acidobacteria, Bacteriodetes, Plantomycetes,
Cyanobacteria, Thermotogae and Choroflexi were predominant groups. Results revealed a
wide diversity of culturable and non-culturable halophilic bacteria in the rhizosphere of
halophytes.
56
Metagenomic analysis of the rhizosphere microbiome of the common bean
resistant to Fusarium oxysporum
Lucas William Mendes*1, Rodrigo Mendes2, Siu Mui Tsai1
1
University of Sao Paulo, Brazil, 2EMBRAPA Environment, Brazil
The rhizosphere microbiome plays a key role in the functioning of the host plant, influencing
its physiology and development. It has been suggested that plants use mechanisms present
in the rhizosphere microbiome to fend off infections, such as fungal diseases. This work
aimed to assess the microbial community inhabiting the common bean rhizosphere in order
to identify potential groups related to the suppression of the soil-borne pathogen Fusarium
oxysporum. Therefore, using shotgun metagenomic sequencing (Illumina Miseq), we
investigated the phylogenetic and potential functional diversity of microbial communities
colonizing the rhizosphere of four cultivars of common bean with different levels of
34
resistance to the fungus, ranging from high susceptibility to resistant. Quantitative PCR of
total bacteria in rhizosphere samples showed an increase of 16S rRNA copy number with the
increase of resistance to the fungus. Mesocosms experiments, including four common bean
cultivars cultivated in Amazonian Dark Earth and three replicates, were conducted in
greenhouse conditions and we obtained over than 12 million metagenomic sequences. The
overall microbial diversity did not present significant variations across common bean
cultivars. From the classified sequences, 97.4% were affiliated to Bacteria and 1.48% to
Archaea. Proteobacteria represented the most abundant phyla (41.7%), followed by
Actinobacteria (29.4%), Firmicutes (5.9%) and Acidobacteria (4.1%). The microbial communities
structure were different between bulk soil and rhizosphere samples. Comparing all bean
cultivars, the resistant one showed an overrepresentation of the phyla Spirochaetes,
Nitrospirae and Euryarchaeota. The resistant bean cultivar presented high number of
sequences affiliated to the genus Bacillus. Interestingly, the resistant and moderately resistant
cultivars, presented high proportion of sequences related to bacteriocin, a narrow spectrum
antibiotics. Preliminary analysis showed that different common bean cultivars could select
differential microbial groups in the rhizosphere environment. Further analysis will search for
bacterial groups potentially related to the fungal antagonism.
57
Improving the recovery outcomes of the critically endangered Wollemi pine: is
success determined by soil microbes?
Jessica Mowle*1, Ian Anderson1, Brajesh Singh1, Steve Clarke2, Catherine Offord3, Jeff
Powell1
1
Hawkesbury Institute for the Environment, University of Western
Sydney, Australia, 2University of Western Sydney, Australia, 3Science and Conservation,
Royal Botanic Gardens and Domain Trust, Australia
Wollemi pine (Wollemia nobilis W. Jones, K. Hill & J. Allen) is a monotypic species, of which
fewer than 100 trees are known in the wild. The Wollemi pine Recovery Team has proposed
translocation as a conservation strategy to establish 'back‐ups' to the wild population;
however, knowledge regarding the environmental/ biotic requirements of individuals planted
in new environments is limited. One of the most important limitations to the introduction or
reintroduction of tree species is the presence of suitable microbial partners. Plants in novel
environments will encounter fewer co-evolved mutualists. Wollemi pine grows on shallow
soils of poor nutrient status and high acidity and is likely to be highly dependent on
mycorrhizal fungi, which have been observed associated with the roots of Wollemi pine, and
bacteria that contribute to nutrient cycling. A plant-soil feedback experiment was undertaken
to estimate the effect of soil microbial communities on seedling growth and survival, under
semi-realistic conditions. We found that microbial communities associated with soils under
Wollemi pine in the wild differed from those under neighbouring species. We also found that
Wollemi pine seedlings were slightly larger at 5 months when interacting with their own
microbes than with microbes associated with these neighbours, suggesting that generalist
pathogens and/or a lack of host-specific beneficial associations may be an important factor
limiting Wollemi pine recruitment in the wild.
35
58
Bioinoculants versus chemical fertilizers: Assessment of rhizospheric microbial
communities in pigeonpea (Cajanuns cajan) under field condition
Shilpi Sharma*1, Richa Sharma2, Preeti Chopra3, Vijay Pooniya4, Virendra Swarup
Bisaria2, Karivaradharajan Swarnalakshmi3
1
Indian Institute of Technology Delhi, India, 2Dept of Biochem Engg and Biotechnology,
IIT Delhi, India, 3Division of Microbiology, Indian Agricultural Research
Institute, India, 4Division of Agronomy, Indian Agricultural Research Institute, India
In today’s agriculture for environmental sustainability there is an increased emphasis on
employing eco-friendly agricultural amendments like bioinoculants. While there are various
reports on their performance with respect to enhancement of crop’s growth and yield under
field conditions, a largely ignored aspect has been their impact on rhizospheric microflora.
Application of plant growth promoting microbes in numbers larger than what are naturally
present in the rhizosphere is bound to exert, at least a transient, effect on the resident
microflora, thereby affecting crucial soil processes. Hence, the present study was undertaken
to assess the effects (target and non–target) of plant growth promoting bioinoculants,
viz. Bacillus sp., Pseudomonas sp. and Azotobacter sp. (individually and in different
combinations) in pigeon pea. The effects of bioinoculants were compared with recommended
dose of chemical fertilizers. Together with measuring plant growth parameters and soil
nutrient status, cultivation–dependent (enumeration on specific media) and –independent
[qPCR and denaturing gradient gel electrophoresis (DGGE)] tools were employed to
characterise the structural and functional diversity of rhizospheric microflora.
The triple inoculation proved to be the best compared to other treatments of bioinoculants in
terms of promotion of plant's growth, with results comparable to that of chemical fertilizers.
The same trend was also observed with respect to nitrogen, phosphorous and potassium
uptake at vegetative stage. A marked rhizosphere effect could be observed for all treatments
as compared to uninoculated control. Plant’s growth stage exerted a more pronounced
impact on culturable microbial diversity compared to the treatments. However, both qPCR
and DGGE revealed the non-target effects of the bioinoculants on abundance of 16S rRNA
gene and genes involved in various steps of nitrogen cycle.
With the triple inoculation’s efficiency being comparable to that of chemical fertilizers, and no
detrimental effect observed on rhizospheric microflora, the combination can be termed as
ecologically "safe” for agricultural application.
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Signaling
61
The interkingdom volatile signal indole promotes root development by
interfering with auxin signalling
Aurélien Bailly*1, Ulrike Groenhagen2, Stefan Schulz2, Markus Geisler3, Leo Eberl4, Laure
Weisskopf1
1
Agroscope, Switzerland, 2Technische Universität Braunschweig, Germany, 3University of
Fribourg, Switzerland, 4University of Zurich, Switzerland
The plant rhizosphere attracts and hosts complex microbial communities in a dynamic
environment where rapid and targeted communication between soil microbes and plants is
an important asset for the survival of both partners. Bacteria produce volatile organic
compounds (VOCs) that mediate communication with plants. Although some bacterial VOCs
that promote plant growth have been identified, their underlying mechanism of action is
unknown. Here we demonstrate that indole, which was identified using a screen for
Arabidopsis growth promotion by VOCs from soil-borne bacteria, is a potent plant-growth
modulator. Beyond indole-to-auxin conversion, its prominent role in increasing the plant
secondary root network is mediated by interfering with the auxin signalling machinery. Using
auxin reporter lines and classic auxin physiological and transport assays we show that the
indole-derived signal invades the plant body, reaches zones of auxin activity and acts in a
polar auxin transport-dependent bimodal mechanism to trigger differential cellular auxin
responses. Our results suggest that indole, alongside its importance as a bacterial signal
molecule, can serve as a remote messenger to manipulate plant growth and development.
Therefore VOCs-mediated shifts in the plant hormonal balance may represent an effective
mechanism by which bacteria directly modify their ecological niche to their advantage. Better
understanding of inter-kingdom volatile communications in soil may prove useful for
designing cost-effective sustainable agricultural strategies.
62
Fluorescent pseudomonad injectisomes and manipulation of plant defenses:
biocontrol versus pathogenic rhizosphere agents
Dorian Bergeau*1, Xavier Latour2, Sylvie Mazurier3, Marie-Laure Follet-Gueye4,
Abderrakib Zahid4, Jean-Claude Mollet4, Nicole Orange5, Marc Feuilloley5, Philippe
Lemanceau3, Maïté Vicré-Gibouin4, Xavier Latour5
1
Normandie Université, Laboratoire de Microbiologie Signaux et Microenvironnement EA 4312 & IRIB, France, 2Normandy University (University of Rouen), France, 3INRA
Dijon, UMR 1347 Agroécologie, France, 4Normandie Université, Laboratoire de
Glycobiologie et Matrice Extracellulaire Végétale - EA 4358, France, 5Normandie
Université, Laboratoire de Microbiologie Signaux et Microenvironnement - EA
4312, France
Fluorescent pseudomonads are called ‘rhizobacteria’ since their density and activities are
strongly stimulated and most elevated in the plant rhizosphere. They have a broad capability
to adapt to fluctuating environments, for example by exchanging signaling molecules with
the host. Among them, Pseudomonas syringae pathovars are major microbial pathogens that
can cause diseases on economically important plants. During the parasitic phase, P. syringae
37
Signaling
forms a type-III secretion system (T3SS) i.e. a long and flexible pilus involved in cell-to-cell
communication. This structure is essential for the injection of multiple effector proteins into
the plant cell, to suppress plant innate immune defenses, to manipulate hormone signaling
and to elicit cell death.
In contrast, numerous P. fluorescens strains are mentioned to improve plant health and are
considered as biocontrol agents of soilborne diseases. They synthesize secondary
metabolites, which are implied in antagonistic activity against various phytopathogens, affect
the plant physiology by producing growth substances, and elicit defense reactions of the
host plant (induced systemic resistance). Recently, several plant P. fluorescens isolates were
found to carry T3SS genes although their role is unknown. De facto, the structure of P.
fluorescens T3SS and its potential role in virulence or in beneficial interaction with eukaryotic
hosts are also ignored. The objective of this work is to investigate the T3SS morphology by
transmission electron microscopy and the plant immune response induced by a biocontrol P.
fluorescens strain in comparison with a phytopathogenic P. syringae strain. Induction of T3SS
genes by exudates analogous were also compared for these strains.
63
Nitric oxide is involved in phosphate deficiency-induced stomatal closure in
maize
Lingyun Cheng*, Ying Wang, Jianbo Shen
Phosphorus is an essential element required for plant growth, and P deficiency could
decrease plant water use. Nitric oxide (NO) is emerging as an important messenger molecule
involved in many important physiological processes in plants. We hypothesized that NO is
involved in phosphate deficiency-induced stomatal closure in maize. In this work, we studied
the effect of NO donor sodium nitroprusside (SNP) and NO scavengers methylene blue (MB)
on the response of stomatal behavior of maize (Zea mays L.) grown at different phosphate
treatments. In contrast to phosphate-sufficient treatment, long-term phosphate starvation
and short-term deprivation treatments strikingly induced the NO accumulation in lateral root
tips of maize, as wells as the reduction of stomatal conductance and stomatal aperture of
leaves. After short-term phosphate deprivation, detached maize leaves that were pretreated
with MB restored the stomatal conductance and stomatal aperture to the level found in
phosphate-sufficient leaves. Coincidently, exogenous supply of SNP to phosphate-sufficient
leaves was able to induce a 50% closure of stomata in leaves. In parallel experiments,
applying SNP or MB to the root medium regulated the stomatal behavior in a similar way as
applying to the detached leaves. Taken together, these results suggest that NO might confer
the stomatal closure in response to phosphate starvation.
38
Signaling
64
Indole acetic acid biosynthesis and nitric oxide metabolism crosstalk in
Azospirillum brasilense SM
Vatsala Koul*, Alok Adholeya, Mandira Kochar
The Energy and Resources Institute, India
Plant growth promoting (PGP) rhizobacteria, Azospirillum brasilense SM releases
phytohormone-Indole-3-acetic acid (IAA) and other plant growth regulators into the
rhizosphere which enhances plant development. A crosstalk between IAA and
gasotransmitter, Nitric oxide (NO) is speculated which may further benefit the plants. To
corroborate this, mutant strains over-expressing essential NO metabolism genes i.e. nitrous
oxide reductase (nosZ), nitrous oxide reductase regulator (nosR) and nitric oxide reductase
(norB) were generated. The production of NO was established by fluorescence assay using
NO-specific probe: 4,5- diamino-fluorescein diacetate (DAF-2DA) and NO scavenger: 2-(4carboxyphenyl)-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). Increased release of
NO was observed by all the mutants in the presence of Tryptophan (IAA substrate) and IAA.
Quantitative IAA estimation suggested that nosR and norB influences regulation of IAA
biosynthesis in A. brasilense SM. The NO quencher, inhibitor and donor reduced or blocked
IAA biosynthesis in wild type and mutants, emphasizing a common regulatory role of these
molecules in IAA biosynthesis. Surface colonization of strain SM on sorghum roots was
established by electron microscopy and improved plant development was observed with the
mutants, possibly mediated by increased NO and IAA levels. Expression studies by qPCR
revealed that apart from Arginine, Tryptophan is able to induce expression of NO metabolism
genes. IAA biosynthesis gene, indole-3-pyruvate decarboxylase (ipdC) was influenced
positively by Tryptophan but negatively by Arginine. These results impress some shared
signalling mechanism or potential crosstalk involving IAA and NO in strain SM.
65
Plant activating proteins from microbes (PAPM) as plant health determinants
Sivakumar Uthandi, Tamilarasi Mani*
Tamil Nadu Agricultural University, India
In order to feed the estimated global population of 9 billion in the year 2050, agricultural
yields will have to increase by 70 to 100% which can be achieved by improved understanding
of plant-microbe interactions to secure nutrients, deter pathogens and resist environmental
stress. While plants recognize and respond to environmental signals, including pathogen
infection, an incompatible interaction will not only restrict pathogen proliferation by causing
hypersensitive response (HR) but also leads to a systemic acquired resistance (SAR) that
prevent subsequent infections by other pathogens. HR responses are exerted by secreted
protein from the Gram negative plant pathogenic bacteria through type III secretory system
which acts as an elicitor of plant defence. The hrp (hypersensitive response & pathogenicity)
genes encode elicitor proteins (harpin like proteins) and induce resistance in a variety of
plants against a broad array of pathogens and enhancing plant growth.In the present study,
Xanthomonas axonopodis pv. dieffenbachiae which cause blight disease in ornamental plants
was cultured in Hrp inducing media. The cell-free extracellular proteins upon concentration
were infiltrated onto tobacco leaves and the response was monitored until visible necrosis
like HR. Further, Hydrogen peroxide (H2O2) detected at the site of infiltration suggesting the
activation of signal transduction for the production of reactive oxygen species (ROS)
39
Signaling
scavenging enzymes. Consequently, ROS scavenging enzymes including super oxide
dismutase, polyphenol oxidase and catalases were measured. In addition, callose deposition
on leaves suggests that HR was mediated by the applied proteins. To identify the proteins
responsible for HR, the secretome of X. axonopodis pv. dieffenbachiae under apoplast fluid
based induction was analysed and a unique protein having molecular weight around 26 kDa
was detected. Further confirmation of an indicator of SAR signalling pathway, the nonexpresser of pathogenicity related1 (npr1) gene regulation and over expression of gene
responsible for PAPM are underway.
66
Investigating mechanisms for vermicompost promotion of tomato growth in
organic production systems
Juana Munoz Ucros*
Cornell University, United States
One of the main limitations of conventional farming is nutrient management, where crops
tend to be nutrient limited and animal production operations result in excess nutrients. In this
proposed work we intend to understand the mechanisms by which vermicompost (VC) of dairy
farm waste can provide plant growth promotion to horticultural crops as an alternative to
synthetic fertilizers, optimizing food production and nutrient recycling while reducing waste.
Tomato transplants will be grown in the greenhouse amended with VC that has been
autoclaved, non-autoclaved, and autoclaved plus re-inoculated with the microbial community
found naturally in the VC. Plants will be monitored weekly for changes in germination and
growth parameters, and plant gene expression. Soil samples will be collected to evaluate soil
exoenzyme activity, available N, and microbial community composition. The results of this
work will contribute to the understanding of the mechanisms by which VC amendments
promote plant growth.
67
Root exudate- induced influences on surface properties and plant infectivity of
slow and fast growing strains nodulating Glycine max.L
Satish Naik*, David Biate, Annu Kumari, Lakkineni Vital, Kiran K Reddy, Rajesh Kumar,
Kannapalli Annapurna
Indian Agricultural Research Institute, India
Root exudates have been implicated as playing an important role in mediating recognition
and specificity in the Rhizobium–legume nitrogen-fixing symbiosis. To consolidate this
hypothesis, we characterized the root exudate profiles of five soybean genotypes for organic
acids, amino acids and flavonoids. High nodulating soybean genotypes showed propanoic
acid, benzenedicarxylic acid, D-threo-Pentonic acid –methyl glutaconic acid where as the low
nodulating genotypes showed distinct presence of octadecanoic acid. Among amino acids
high amounts of glutamic acid and aspartic acids were found in high and low nodulating
genotypes respectively. We also studied the influence of these exudates on the surface
properties, protein profiles and plant infectivity of slow and fast growing rhizobial strains
nodulating soybean. Soybean root exudates were collected from plants grown under
hydroponic conditions for 7 days under 16h and 8h day/night cycle, concentrated 10 fold,
40
Signaling
filter sterilized and tested on the slow growing Bradyrhizobium strain KAS1 and the fast
growing Rhizobium strain DS-1. Root exudates increased the growth yield of the two strains
in the presence of a carbon source but the strains were unable to utilize root exudates as sole
carbon or energy source. Exposure to root exudates altered Congo-red dye binding, which
indicated changes in the bacterial surface properties at the fatty acid level. Fourier transform
infrared spectroscopy (FTIR) confirmed fatty acid changes and revealed further carbohydrate
changes. Root exudates –induced changes in surface components of the strains may
contribute to successful root colonization and nodule formation with subsequent plant
growth promotion.
68
EcfE, a master regulator of pea root attachment and colonization of Rhizobium
leguminosarum bv viciae 3841
Vinoy Ramachandran*1, Eduardo Balsanelli2, Alison East2, Andrew McNally2,
Karunakaran Ramakrishnan2, Philip Poole1
1
University of Oxford, United Kingdom, 2John Innes Centre, United Kingdom
Attachment and colonization of legume roots by rhizobia is one of the very early and critical
steps in symbiosis. Legumes tightly regulate attachment and colonization by very specific
time-dependent signals and the ability to perceive and respond to such signals by rhizobia is
a requirement for successful colonization.
From our earlier rhizosphere transcriptomics studies, we identified a three-gene operon
coding for lipoprotein (LppE), ECF (EcfE) and anti-ECF (AsfE) sigma factors to be highly and
specifically transcribed during root colonization and weakly in lab culture, only when
supplemented with phenylalanine. Current work, attempts to characterize the role of these
genes in root attachment and colonization by taking advantage of cutting-edge lux
luminescence assays, confocal imaging, RNA-seq and ChIP-seq.
In-planta root colonization assays with wild-type harboring lppE promoter lux fusion showed
activation of lppE operon specifically on pea root elongation zone, emphasizing an important
role of this operon in early stages of Rhizobia-legume interaction. Further in-vitro induction
studies of lppE promoter lux fusion by phenylalanine in different mutant background
demonstrated that EcfE regulates this operon.
In-vitro root attachment assay showed that, lppE mutant were severely attenuated in root
attachment whereas asfE (EcfE over-expressing strain) mutant showed a hyper-attachment
phenotype. In contrast, in-planta colonization assay showed that lppE and ecfE mutants were
able to colonize pea roots as wild-type whereas asfE mutant failed to colonize. Together
these results emphasize the necessity of temporal regulation of specific genes in the
attachment regulon to allow rhizobia to spread and colonize.
To identify the further genes regulated by EcfE, we performed microarray analysis of asfE
mutant, which showed elevated expression of several EcfE target genes, unraveling a complex
hierarchical regulatory network governing pea root attachment and colonization. Together,
this suggests that EcfE is a master regulator of pea root attachment and colonization.
41
Signaling
69
Influence of type VI secretion system (T6SS) on rhizosphere competence of a
rhizobacterium
Alain Sarniguet*, Muriel Marchi, Charline Lecomte, Morgane Boutin, Anne-Yvonne
Guillerm-Erckelboudt, Lionel Lebreton, Kévin Gazengel, Stéphanie Daval, Alain
Sarniguet
INRA, France
Rhizosphere competence of rhizobacteria is governed by multiple mechanisms enabling
surface colonization. Protein secretion systems such as type VI secretion system (T6SS) may
be involved in such rhizobacterial life traits. The role of one T6SS in the ability of the
rhizobacterial Pseudomonas fluorescens Pf29Arp strain to colonize different environments
was investigated. A deletion mutant of the T6SS-IV cluster was obtained and compared to the
wild type. This ΔT6SS-IV mutant displayed distinct colony morphology and cell
rearrangement, suggesting a role of T6SS-VI genes in phenotype variability. However, the
ΔT6SS-IV mutant was a better colonizer of wheat rhizosphere than the wild type strain
whereas the deletion did not affect the bacterial survival in non-sterile bulk soil. Some of the
T6SS-IV genes are involved in colony morphology and in rhizosphere colonization abilities of
the P. fluorescens Pf29Arp.
70
Early root responses to beneficial rhizobacteria and elicitors
Ioannis Stringlis*, Christos Zamioudis, Corné Pieterse
Utrecht University, Plant-Microbe Interactions, Netherlands
To defend themselves against pathogens, plants have evolved a sophisticated immune
system that recognizes conserved components of microbial origin, collectively referred to as
Microbe-Associated Molecular Patterns (MAMPs). Although, innate immune signaling in
leaves has been studied in depth, information available on MAMP-triggered immunity in the
roots is limited. Root colonization by selected strains of non-pathogenic rhizobacteria
triggers an induced systemic resistance (ISR) in diverse plant species that is effective against a
broad range of pathogens. Here, by employing Affymetrix ATH1 whole-genome
transcriptional profiling and Gene Ontology (GO) analysis, we demonstrate that the
transcriptome of Arabidopsis roots two days after bacterization by the reference strain
Pseudomonas fluorescens WCS417 is associated with immune responses commonly activated
upon perception of MAMPs, including metabolic and cellular defense responses. Aiming to
further characterize early events upon perception of microbial signals and dissect signaling
mechanisms underpinning MAMP-triggered immunity locally in roots, we treated Arabidopsis
roots with fungal and bacterial elicitors and P. fluorescens WCS417. Application of the wellstudied elicitors flg22 and chitin resulted in a transient upregulation of the MAMP-responsive
genes MYB51 and CYP71A12, indicating that roots actively respond to immune elicitors of
bacterial and fungal origin. However, P. fluorescens WCS417 caused weak upregulation of
these genes that returned earlier back to basal levels, suggesting that initial host immune
responses become suppressed, possibly to accommodate colonization of the beneficial.
Amongst various hormonal marker-genes that we have tested for their responsiveness to
immune-elicitors, we observed induction of ethylene and auxin responsive genes, pointing to
a role of ethylene-mediated signaling and auxin distribution in MAMP-triggered immunity in
roots. An RNA-sequencing experiment following this initial findings will provide us with a
42
Signaling
better understanding on the molecular mechanisms and the timing of the responses involved
in early steps of root-microbes interactions.
71
Expanding the repertoire of mRNA targets controlled by the post-transcriptional
Gac/Rsm cascade in Pseudomonas protegens CHA0
Patricio Sobrero, Claudio Valverde*
Universidad Nacional de Quilmes, Argentina
The rhizospheric strain Pseudomonas protegens CHA0 regulates the expression of several
traits involved in biocontrol of phytopathogenic fungi through a well characterized network
that includes an extracellular unknown autoinducer-like signal, the GacS/A two component
system, two RNA-binding proteins (e.g., RsmA/E) and three small non-coding regulatory
RNAs (RsmX, RsmY and RsmZ). The Gac/Rsm pathway orchestrates the induction of cyanide
and antibiotics production, and of several extracellular hydrolytic enzymes. Based on
structural and sequence requirements of the interacting elements of the cascade (proteins
and RNAs) it is anticipated that expression of novel uncharacterized genes/operons may be
subject to regulation by the Gac/Rsm system, and thus contribute to the ecological fitness
and biocontrol activity of strain CHA0. In order to get a broader insight into the regulon of
the Gac/Rsm network, we carried out an in silico prediction of novel Gac/Rsm targets by
inspecting the genome sequence of P. protegens strain CHA0 for the presence of RsmEbinding sites near or at their cognate ribosome binding site within the untranslated region
(UTR) of all annotated genes. The output list was seized according to local secondary
structure folding and prediction of correct RNA-RsmE residue contacts, and by conservation
of the putative binding site among related pseudomonads. Among a set of 45 identified
putative novel Gac/Rsm targets, a subset of candidates was experimentally tested by means
of translational ‘lacZ reporter fusions in the genetic background of Gac/Rsm mutant strains.
We present evidences of translational regulation by Gac/Rsm of the following mRNA genes:
1) PFLCHA0_c21910, encoding a LuxR-like transcriptional factor that is physically associated
with a cyclic lipopeptide biosynthetic operon in different pseudomonads; 2) dprA
(PFLCHA0_c00250), encoding a putative DNA protecting protein; 3) pksP (PFLCHA0_c02690),
encoding a putative acyl carrier protein. Our results broaden the repertoire of genes/operons
under the control of the post-transcriptional Gac/Rsm system.
72
Root cell type-specific gene expression in response to plant growth-promoting
rhizobacteria
Eline H. Verbon*1, Christos Zamioudis1, Louisa M. Liberman2, Philip N. Benfey2, Corné
M.J. Pieterse1
1
Utrecht University, Netherlands, 2Duke University, USA
Plant growth-promoting rhizobacteria are known to promote growth and to induce systemic
resistance against pathogen attack in a wide variety of crop species, indicating that these
bacteria can be used as natural fertilizers and pesticides. To maximize the agricultural
potential of the bacteria, a detailed understanding of the molecular mechanisms underlying
both growth promotion and systemic resistance is essential. In the shoot, the plant
43
Signaling
endogenous jasmonic acid and ethylene defense signaling pathways are generally activated
to induce systemic resistance. In the root no signaling pathways are known that translate root
colonization into a signal that primes the shoot defense responses. Previous experiments
have shown that the transcription factor MYB72 is upregulated in the root upon colonization
by beneficial Pseudomonas spp. and is essential for the induction of systemic resistance. The
upregulation of MYB72 depends on an increase in photosynthesis in the shoot. Interestingly,
MYB72 is also involved in the iron deficiency response. Our aim is to uncover root specific
gene regulatory networks activated upon root colonization and to determine the role of
MYB72 in these networks. We used fluorescent activated cell sorting to enrich for vascular,
endodermal, cortical or epidermal cell populations of fluorescently tagged roots grown in
either sterile or colonized conditions. Next, we sequenced the RNA libraries of each of these
cell types. Analysis of these transcriptomes will allow us to investigate the molecular details of
the early root responses elicited upon colonization by beneficial rhizobacteria. These
responses will subsequently be tested to determine whether they are involved in the
induction of systemic resistance or promotion of plant growth.
73
When signaling gets smelly: bacterial volatiles and their effects on rhizosphere
inhabitants
Laure Weisskopf*
Agroscope, Switzerland
In addition to soluble molecules, bacteria emit a wide range of volatile organic compounds,
which have raised increasing interest over the last decade. An overview of our recent work on
volatile-mediated effects of bacteria on plants, fungi, oomycetes and bacteria themselves will
be presented. Particular emphasis will be made on the protective effect of bacterial volatiles
against disease-causing organisms such as the late blight causing agent Phytophthora
infestans. Detailed profiling of the volatiles emitted by a collection of newly isolated
rhizosphere bacterial strains revealed high chemical diversity even within closely related taxa.
These volatile signals had target-specific effects and some of them, mostly long chain alkenes
and sulphur compounds, induced drastic growth inhibition and physiological perturbations in
the disease-causing agents. In bacteria-bacteria interactions, we have shown that bacteria are
able to discriminate between their own volatiles and those of other, distantly related species.
Future challenges, such as the need to investigate volatile emission in the natural
environment of the bacterial strains, or to elucidate the cues triggering volatile emission in
rhizosphere bacteria, will be highlighted. The potential of applying these volatile signals or
their bacterial producers for sustainable crop enhancement and protection in the field will be
discussed.
44
Signaling
74
Hijacking common mycorrhizal networks for defense signal transfer and
underground interplant communication
Song Yuanyuan*1, Wen Xiong Lin1, Suzanne W. Simard2, Ren Sen Zeng1
1
Fujian Agriculture and Forestry University, China, 2Department of Forest and
Conservation Sciences, University of British Columbia, Canada
How plants can communicate each other is an intriguing scientific question. Although
increasing evidences show existence of plant-plant communication, the vast majority of the
studies conducted so far mainly focus on induced volatile-mediated communication.
Mycorrhizae are ubiquitous plant-fungus symbiosis in land ecosystems. Common mycorrhizal
networks (CMNs) link multiple plants together in ecosystems, and ecological significance of
plant-to-plant carbon and other nutrient movement through CMNs is well addressed. We
hypothesized that plants can hijack CMNs as an underground conduit for transferring
induced defense signals and interplant communication. We established CMN between two
tomato plants in pots with mycorrhizal fungus Funneliformis mosseae, challenged a ‘donor’
plant with either a caterpillar or a pathogen, and investigated defense responses and
resistance in neighbouring CMN-connected ‘receiver’ plants. After CMN establishment
caterpillar infestation or pathogen infection on the ‘donor’ plant led to increased resistance
and activities of putative defensive enzymes, induction of defense-related genes and
activation of jasmonate (JA) pathway in the ‘receiver’ plant. However, use of a JA biosynthesis
defective mutant spr2 as ‘donor’ plants resulted in no induction of defense responses and no
change in resistance in ‘receiver’ plants, suggesting that JA signaling is required for CMNmediated interplant communication. Our results suggest that plants are able to hijack CMNs
for induced defense signal transfer and interplant defense communication.
45
Signaling
Rhizosphere and Climate Change
75
Above- and belowground plant responses to long- and short-term frost
manipulations
Gesche Blume-Werry*1, Juergen Kreyling2, Hjalmar Laudon3, Ann Milbau1
1
Climate Impacts Research Centre, Umeå University, Sweden, 2University
Greifswald, Germany, 3SLU Umeå, Sweden
Boreal forests make up one third of the world’s forested areas and store 30% of the global
terrestrial carbon pool. They are shaped by distinct seasonality with a persistent snow cover
during wintertime. However, with decreasing extent and duration of snow cover with climate
change, soils will experience more severe frost which has been shown to affect soil carbon and
nutrient fluxes. Vegetation responses, in particular root mortality, have been proposed as
possible mechanisms.
We directly compared short- (1 year) and long-term (11 years) effects of an absent snow
cover, and thereof resulting severe air and soil frost, on above- and belowground growth,
phenology and biomass of vegetation in a Norway Spruce forest in northern Sweden.
In the long-term treatment vascular plants had reduced shoot growth, their cover had
decreased by 93% and root biomass had decreased by 40% compared to the control. In the
short-term manipulation, the same effects were seen as trends, yet were less substantial and
thus not significant. However, the short-term manipulation resulted in severe visible frost
damage of in average 30% on shoots of understory dwarf shrubs and delayed their spring
phenology by over a week, compared to the control. Surprisingly, this was not present in the
long-term manipulation, which could indicate either an adaptation of the vegetation to the
long lasting severe frost, or a survival of only those individuals that can cope with these
extreme conditions. Data on root growth, mortality and turnover, measured with
minirhizotrons, are currently being analyzed and will be incorporated in the final
presentation.
In general, we show that snow removal significantly affects boreal vegetation both aboveand belowground with implications for soil carbon and nutrient cycling and that some of the
more significant and far-reaching ecosystem effects were only seen in the long-term
experiment.
46
76
Phosphate depletion in Olea europea L. rhizosphere related to mycorrhizal
colonization in different climates
Malika Boudiaf Nait Kaci*1, Louisa Bouhired2, Sophia Mouas Bourbia3, Noria Smail
Saadoun3, Arezki Derridj4
1
Laboratoire Ressources Naturelles Université Mouloud Mammeri, Algeria, 2Université
Houari Boumediene, Algeria, 3Laboratoire Ressources Naturelles, Département des
Sciences Agronomiques, Faculté des Sciences Biologiques et Agronomiques Université M.
Mammeri, Algeria, 4Laboratoire d’Ecologie, Département des Sciences Agronomiques,
Faculté des Sciences Biologiques et Agronomiques UMMTO, Algeria
In Algeria, olive groves are widespread due to their socioeconomic importance and their
rusticity. However, since several decades these groves have been progressively neglected in
profit of more yielding cultures. Such policies led to a deterioration of agroecosystems for
which currently attempting to restore function. Olea europea L., a perennial plant is able to
form arbuscular mycorrhizae. The aim of this work was to study the root induced chemical
changes occurring in the rhizosphere of olive that can influence the dynamic of phosphorus.
Bulk, rhizospheric soils, roots and leaves were collected in three orchards in summer, from
sub-humid, semi-arid to arid regions of Algeria. P-available was obtained by the method of
Olsen. The rhizosphere soil showed a significantly higher concentration of organic-carbon
and total nitrogen. The concentrations of all phosphorus fractions in the rhizospheric soil
were significantly lower than those in bulk soil. A P-available deficiency was measured in bulk
and rhizospheric soil and confirmed by P-foliar. The uptake of phosphate by root induced a
depletion of all P fractions in the rhizosphere. Philips and Hayman’s technic applied to the
root samples showed in the cells of roots characteristic structures of arbuscular mycorrhizae
like hyphal coils, arbuscules and vesicles. Arbuscular mycorrhiza is a widespread symbiotic
association between plants and fungal microsymbionts that supports plant development
under nutrient-limiting and various stress conditions. However their frequency is higher in the
root sample of arid orchard. Olive trees surveyed in the three climates showed heavy AM
colonization, indicating a high mycorrhizal dependency of olive groves in this environment.
77
Effects of plant-growth-promoting rhizobacteria on barley plants under
different CO2 and water regimes
Olga Calvo*, Ndubuisi Chimelue Nwabufo, Andreas Fangmeier
University of Hohenheim. Institute of Landscape and Plant Ecology, Germany
Rising CO2 concentrations in combination with drought stress are likely to influence not only
aboveground growth, but also belowground plant processes. Plant-growth-promoting
rhizobacteria (PGPR) colonize the rhizosphere of many plant species and confer beneficial
effects under environmental stresses. Furthermore, root exudates play a role in interactions
between plant roots and other organisms present in the rhizosphere.
Only few reports have been published on PGPR as elicitors of tolerance to abiotic stresses,
such as drought. Furthermore, little is known about the influence of environmental factors on
root exudation patterns. Therefore, this study was conducted in order to investigate the effect
of two commercially available PGPRs on the growth and root exudation of barley (Hordeum
vulgare L.) under different CO2 and water treatments.
47
In a growth chamber experiment climatic conditions of a field site close to Stuttgart were
simulated. Barley plants were grown in pots filled with sand and exposed to ambient (380
ppm) or elevated (550 ppm) CO2. Plants received the normal daily amount of rainfall in the
region of Stuttgart or 33% less. Plants were harvested at the stem elongation growth stage
and when the inflorescences emerged. At both dates, data were collected on above and
belowground variables. In addition, analyses of water use efficiency, stomatal conductance,
SPAD values and composition of root exudates were performed. Preliminary results showed
significant effects of the factors and their interactions on some of the measured variables.
In the context of food security and agricultural sustainability, further studies with other crop
plants are needed to demonstrate whether PGPR cause a range of crops to be tolerant to
environmental stresses improving crop production.
78
Drought effects on rhizodeposition and ecological implications
Alberto Canarini*, Andrew Merchant, Feike Dijkstra
The University of Sydney, Australia
Drought intensity is predicted to increase in the next decades across many areas of the world.
This phenomenon will affect plant production and consequently rhizodeposition with
potentially large ecological impacts on soil carbon (C) and nutrient cycling. However, the
direction and magnitude of these impacts are still unclear.
Here we investigate the effect of drought on rhizodeposition and the connection with C
stabilization and nitrogen (N) cycling. We combine stable isotope techniques with
chromatography and mass spectrometry analyses in order to understand how drought affects
the allocation of C into different soil pools and the nature of compounds invested
belowground. We investigate wheat, soybean and sunflower as species of major agroeconomical value due to their wide spread use and potential to cause large ecological
impacts.
Results illustrate that plants are able to allocate substantial amounts of C belowground
through rhizodeposition, which have the potential to increase the amount of mineral
associated C (a relatively stable form of soil C), most likely through microbial interactions
(indicated by correlation between plant derived C found in microbial biomass and the stable
C pool, R2 = 0.54, P < 0.001). Organic compounds obtained from phloem sap, root tissues
and exudates collection will then be discussed with reference to the effect of drought on the
abundance of these compounds. Samples are being processed using a recently developed
method for liquid chromatography – mass spectrometry targeting major metabolites in
plants (sugars, amino- and organic-acids), important for different soil processes.
These results provide an improved understanding of biochemical exchange in the plant-soil
system in a future of reduced water availability for agro-ecosystems.
48
79
Characterizing root traits for edaphic stress adaptation
Yinglong Chen*1, Yan Fang1, Suiqi Zhang1, Kadambot Siddique2, Zed Rengel3
1
Institute of Soil and Water Conservation, Northwest A&F University, and Chinese
Academy of Sciences and Ministry of Water Resources, China, 2The UWA Institute of
Agriculture, The University of Western Australia, Australia, 3The UWA Institute of
Agriculture, and School of Earth and Environment, The University of Western
Australia, Australia
Sustainable crop production is challenged by the climate changes with likely increased
production limitation and uncertainty in the future. Edaphic stresses, such as drought and
low-fertility soils, are the main factors restricting crop production in many counties. Selecting
and breeding cultivars with root architecture traits efficient in water and nutrient use
becomes an important breeding strategy aiming for increased crop adaptation to edaphic
stress. A serial of phenotyping experiments involving novel semi-hydroponic platform, soilfilled columns and rhizotrons were carried out to study phenotypic variability in root traits and
root responses to water and phosphorus deficits in narrow-leafed lupin. Large variability
among the tested genotypes was observed with 21 (out of 38) root traits had greater
coefficient of variation values than 0.5. Multivariate root traits were constructed with Principal
Components Analysis resulted in 9 components with eigenvalues greater than one.
Observations confirmed the inability of this species in forming functional mycorrhizas nor
cluster roots under any phosphorus (P) status. Significant variations were observed among
genotypes in root architecture and distribution in response to drought and low-P stresses.
Placing P fertiliser deeper enhanced root growth and P-use efficiency. Plants produced
greater amounts of carboxylates in the rhizosphere when plants grown in low P environment.
Localised P patches had significant impacts on root architecture and exudation compared to
uniform P application. Alteration of root distribution and architecture and changes in
rhizosphere exudation of carboxylates were important strategies for efficient P acquisition in
low P soil. Our study provides detailed description of the phenotypic variability in root
architecture and the insight into the mechanistic responses of root traits influencing water
and nutrient acquisition in low-fertility drying soils.
80
Microbial invasions - a complex interplay between soil properties and biology
Renata Slavikova1, Jan Jansa2, Muhammad Ali1, Emmanuel Frossard1, Hannes
Gamper*1
1
ETH Zurich, Agricultural Sciences, Switzerland, 2The Czech Academy of Sciences,
Institute of Microbiology, Czech Republic
Biological invasions are greatly facilitated by global traveling and trade and are widely
recognized as potential threads to local biodiversity and ecosystem functioning. Some
introduced species can be devastating. Yet, research has mainly focused on plants and
animals. Microbial dispersal, except for crop and human pathogens and parasites, received
much less attention so far, despite its potentially important consequences for soil and plant
functioning, due to microbe-soil adaptation. Arbuscular mycorrhizal fungi (AMF) mediate
elemental fluxes and plant productivity via assisting plants with mineral nutrient acquisition
and protecting them from abiotic and biotic stresses. Here we report on the findings of a pot
experiment where we simulated, based on a cross-factorial design, gradients of a major biotic
49
and abiotic perturbation event by mixing two soils from geographically well-separated, but
climatically similar sites in Switzerland. We mixed the native living soils in nine different
proportions and established control treatments in which one or both soils had their native
AMF removed by gamma irradiation. Other native soil microbes smaller than the propagules
of AMF were added back. The experiment so far revealed strong impairments to the growth
of Plantago lanceolata when the two soils were alive, as compared to their sterilized
counterparts. These impairments were to a great extent rectified by addition of just only 5%
of foreign sterilized soil. The mechanistic basis behind this phenomenon will be elaborated
based on combined elemental flux and microbial community analyses, using stable (13C, 15N)
and radioactive (33P) tracers and molecular genetic fingerprinting.
81
Community assembly processes of N2O-reducing prokaryotes in the rhizosphere
- effect of edaphic factors and plant species
Daniel R. H. Graf*, Christopher M. Jones, Ming Zhao, Sara Hallin
Swedish University of Agricultural Sciences, Department of Microbiology, Sweden
Nitrous oxide (N2O) is the dominant ozone depleting substance and a potent greenhouse
gas. Nearly 70% of global N2O-emissions can be attributed to terrestrial ecosystems, of which
45% originate from agricultural land. The rhizosphere is known as a hotspot for prokaryotes
producing and reducing N2O. The only enzyme known to reduce N2O to nitrogen gas (N2) is
the N2O-reductase (N2OR), which is encoded by two variants of the nosZ gene, clade I and
clade II, the latter of which has been shown to be correlated with a higher N2O sink capacity.
To determine the effect of plant species and soil type on the diversity, structure, and
functioning of N2O reducing communities in the rhizosphere and on the root surface, we
conducted a pot experiment growing sunflower (Helianthus anuus) and barley (Hordeum
vulgare) in a clay and a silt soil. We observed higher potential N2O production rates in
rhizosphere in the clay soil compared to the silt soil, and rates were higher when planted with
barley than sunflower. In agreement, potential N2O production measurements on washed
roots revealed a significantly higher production rate by attached microbial communities or
endosymbionts of barley roots as opposed to sunflower roots. Using quantitative PCR, we
demonstrate that low N2O production correlates with low nosZ clade I/clade II abundance
ratios in the rhizosphere soil. The ratios overall were more dissimilar between the soils than
between the plants, even on the roots, indicating a stronger effect of soil type than plant on
the community assembly of N2O-reducing prokaryotes in the rhizosphere. Amplicon
sequencing of both nosZ clades from rhizosphere soil and root surfaces was performed and
additional analyses will be presented on the relative influence of soil and plant species on
diversity and composition of N2O reducing communities in the different compartments.
50
82
The Bryosphere as a regulator of peatland carbon balance in response to climate
warming
Vincent Jassey*1, Constant Signarbieux2, Alexandre Buttler3, Bjorn Robroek2
1
Research Institute WSL, Switzerland, 2School of Architecture, Civil and Environmental
Engineering (ENAC), Ecole Polytechnique Fédérale de Lausanne EPFL, Ecological Systems
Laboratory (ECOS), Switzerland, 3Swiss Federal Institute for Forest, Snow and Landscape
Research (WSL), Switzerland
In analogy to the rhizosphere, the bryosphere describes the interactions between bryophytes
and their associated organisms and forms an important transitional zone mediating
aboveground-belowground linkages in northern ecosystems such as peatlands. In particular,
Sphagnum mosses are important players for ecological processes in peatlands, especially for
carbon (C) cycle. For example, Sphagnum contributes substantially to aboveground leaf area
and biomass, and therefore plays an important role in C assimilation. Furthermore, this moss
is tightly linked to a wide range of microorganisms and microfauna, which interact and form a
complex food web responsible of C cycling. Hence, Sphagnum-bryosphere can influence both
primary production and decomposition processes in peatlands.
Using a five-year field warming experiment in a Sphagnum-dominated peatland, we studied
the response of the Sphagnum-bryosphere to temperature increase. We showed that the
response of Sphagnum-bryosphere affects a series of peatland functions with implications for
C recycling. We found that a moderate warming (ca. +1°C) exacerbated the effect of drought
on Sphagnum-photosynthesis. While rate of Sphagnum-photosynthesis declined by 70%
compared to wet conditions with drought alone, it became negative under the combined
effect of drought and warming, indicating a carbon loss. In parallel, we found that warming
strongly decreased the biomass of microbial predators (-50%) after 5 years of warming,
especially mixotrophic predators (i.e. organisms combining autotrophy and heterotrophy).
Such decrease of mixotrophs, in turn, modified the structure of the microbial food web and
shifted the microbial food web towards a bottom-up control. This implies potentially a faster
C turnover, and enhanced organic matter decomposition. Interestingly, our findings also
highlighted an important role for mixotrophs declining to overall Sphagnum-photosynthesis,
which could explain the differences we found between controls and warmed plots.
Overall, our findings suggest that warming-effects on Sphagnum-bryosphere will alter the
carbon cycle in peatlands.
83
Use of light-use efficiency functions to describe CO2 uptake at a semi-arid site,
role of leaf-area index and leaf density
Georgia Koerber*1, Wayne Meyer2
1
University of Adelaide, Australia, 2Adelaide University, Australia
The Calperum-Chowilla OzFlux site (34°00.163S 140°35.261E) was established in July 2010
located about 20 km north-west of Renmark in South Australia within an extensive semi-arid
Mallee woodland. Eddy covariance flux of carbon dioxide and water from a 20 m tower were
recorded during the height of the millennium drought, its subsequent breaking during 2011
and then a wild fire in January 2014.
51
During 2011 the perennial vegetation gradually increased in leaf area and hence sequestered
carbon With future projections of increased fluctuations in temperature and rainfall events in
Autumn, we want to assess the role of perennials and the soil in carbon sequestering that is
likely to have a long residence time.
Monthly leaf area index (LAI) using digital cover photography, leaf mass per area, thickness,
soil respiration and above ground biomass additions and carbon budgets have been
constructed splitting 24 hours into night and day using a shortwave radiation threshold of 10
W m-2. Nighttime flux filtered by insufficient turbulence and corrected to match daytime
temperatures was subtracted from daytime flux to estimate photosynthesis. Light use
efficiency curves were solved for the light compensation point and then respiration in the
light and dark as described by the “Kok effect”.
With improved estimates of night and day respiration and photosynthesis we regressed
ecosystem respiration against monthly LAI. We hypothesize the y-intercept is plant
independent respiration or heterotrophic soil respiration. With this component we will be able
to derive a more accurate estimate of this ecosystems accumulation or loss of carbon. We
want to track this with prevailing environmental conditions such as water and
temperature/seasonality and compare this with other ecosystems. We also want to see if
changes in leaf area or leaf characteristics play a part in continual adjustment and adaptation
to the variable environmental controllers, especially water.
84
Deciphering transcriptomic profiles and Verticillium wilt development in olive
cultivars under different scenarios of climate change
Blanca B Landa*1, Miguel Montes-Borrego1, Carmen Beuzón2, Juan A. Navas-Cortés1
1
Institute for Sustainable Agriculture-CSIC, Spain, 2University of Málaga, Spain
Plant disease epidemics result from interactions of a susceptible host, a virulent pathogen and
conducive environment. Shifts in any component can change disease expression to still
unknown directions. Verticillium wilt (VW) of olive caused by the soilborne fungus Verticillium
dahliae (Vd) is of major concern for olive industry in the Mediterranean basin.
We carried out experiments using olive cvs. Picual and Arbequina, which grew in soil infested
by the defoliating (D) or non-defoliating (ND) Vd pathotypes at 20, 24 and 28ºC and CO2 levels
of 386, 550 and 750 ppm, representative of current and future SRES-IPCC A2 and B2 scenarios
for southern Spain. Surface response models quantified the combined effects of temperature
and CO2 on VW intensity and plant growth, demonstrating a differential effect of biotic and
abiotic factors. Optimum VW development occurred at 20-24°C and current CO2, being faster
and severe in 'Picual'/D. Raising CO2 delayed VW and Vd infection.
The transcriptomic profile of olive cultivars in response to Vd infection and the three climate
SRES scenarios was assessed using a 12-plex microarray of 37,449 olive unigenes (OLEAGEN
project). Main differences in the expression profiles were due to olive genotype, followed by
the climatic conditions at the three SRES scenarios and in a lesser extent by Vd infection.
Thus, plants growing at current climate showed higher number of differentially expressed
genes compared to plants growing at SRES-IPCC A2 and B2 scenarios with a general trend to
52
decrease the number of significantly transcribed genes as temperature and CO2 increased.
Furthermore, a higher number of genes (up to 10x) were expressed differentially in
'Arbequina' than in 'Picual' in response to environmental changes as well as to infection by
the D pathotype with a low number of genes being common for both cultivars.
85
Root exudates can control soil N dynamics
Adrian Langarica Fuentes*1, Susan Mitchell1, Marta Manrubia Freixa2, Tim Daniell1
1
The James Hutton Institute, United Kingdom, 2Netherlands Institute of Ecology (NIOOKNAW), Netherlands
Agriculture represents the dominant source of the potent greenhouse gas nitrous oxide
(N2O). This is largely due to the conversion of added nitrogen-based fertiliser through the
action of the microbial nitrogen cycle in soil. Denitrification represents the main source of
nitrous oxide emission and is a carbon driven process, as it maintains respiration under low
oxygen conditions with nitrogen oxides acting as alternative electron acceptors. Initial
experiments using barley plants (Hordeum vulgare) indicated that different cultivars support
significant variation in N2O emission from denitrification from associated soil and that these
effects are connected to root exudation difference rather than direct interaction or litter
effects. In this study, a controlled microcosm experiment was designed to explore the
interaction between root exudation and denitrification in terms of N2O production and
community dynamics. Different quantities of artificial root exudates were added daily to soil
at three different water regimes. N2O emissions were measured and community dynamics
were assessed using T-RFLP and real time PCR. Results suggest that N2O emissions are driven
by nitrous oxide reductase activity (consumption) rather than nitrite reductase activity
(production). Soil condition, through differential aerobic status and variability, also appeared
to have a marked effect both on flux through denitrification and community dynamics.
Further work is underway to dissect the role of exudation quality in driving the alteration in
nitrogen cycle flux and the end product of denitrification.
86
Impact of chemical fertilization and organic amendment on carbon and nitrogen
lability between rhiz. and non-rhiz. ecosystems
Ibrahim Ortas*1, Gulistan Köskeroglu2, Refik Islam3
1
Cukurova Universitey, Turkey, 2Ç.Ü, Turkey, 3Ohio University, United States
Soil carbon and nitrogen stoichiometry is important for evaluating soil organic matter quality.
Our study was conducted to evaluate the impact of chemical fertilization and various organic
amendments on biological, chemical and physical C and N pools to assess soil C and N
dynamics under wheat (Triticum aestivum L.) - corn (Zea mays L.) rotation in semi-arid
Mediterranean climate of south-western Turkey. Treatments included control, chemical
fertilization (NPK), compost, animal manure, and compost+mycorrhizal inoculations. Soil
samples were randomly collected from 0 to 15 and 15 to 30 cm depth and analyzed for
rhizosphere and non-rhizosphere total organic carbon (TOC) and nitrogen (TN), extractable
phosphorus (EP), active carbon (AC), particulate organic C (POC) and nitrogen (PON), and
soluble carbon (SC) fractions to calculate C and N lability and management indices (CMI).
53
Results showed that the organic amendments such as compost and manure applications
significantly increased TOC, TN, EP, AC, POC, PON and SC as compared with the chemical
fertilization and control, respectively. In 0-15 cm depth AC in control treatment was 2.37 mg
kg-1, however in mineral fertilizer, manure, compost and compost +mycorrhiza treated soils
252, 272, 302, and 274 mg kg-1 respectively. Similar repose was got in non-rhizosphere soil.
The C and lability was higher in the rhizosphere than in the non-rhizosphere ecosystems. Our
results suggest that higher C and N lability is associated with organic amendments to
improve soil quality for crop production.
87
Plant parasitic nematodes attraction by root exudates of range expanding plant
species and native congeners
Julio Carlos Pereira da Silva*, Rutger Wilschut, Wim H. van der Putten
NIOO-KNAW, Netherlands
Global climate changes enable many plant species to shift their ranges to higher latitudes and
altitudes. Range expansion leads to novel interactions with a non-co-evolved multitrophic
belowground community in the new range. It is possible that the novel traits of rangeexpanding plant species make them unattractive, toxic or even not recognized as a new food
source to the enemies in the invasive range, resulting in reduced herbivore pressure. This
novelty, however, may go at the expense of indirect control of enemies by higher trophic
level organisms, as these will not recognize the cues emitted by the novel plant species. So, it
may be an advantage of range-expanding plant species if novel enemies do not recognize
and respond to chemical signals of host, but it will be a disadvantage when the enemies
recognize the host plants, but their natural enemies not.
Our study makes a first step in unravelling bottom-effects on root-feeding nematodes in the
rhizosphere of range-expanding plant species and comparing this to congeneric plant
species that are native in the invaded range. We present pilot results of two such pairs of
congeneric plant species and two types of root-feeding nematodes (ectoparasitic or
endoparasitic species). We performed experiments on petri dishes and in a greenhouse in
order to study root attraction of ectoparasitic and endoparasitic nematodes. Our hypotheses
are that (1) nematodes are more attracted by native plant species than by related range
expanders and (2) ectoparasitic nematodes, which have less specific relations with the roots
than endoparasitic nematodes, will show a less strong preference for native plants than
endoparasites.
88
The role of plants in methane flux of upland soils and their influence on archaeal
community composition in the rhizosphere
Nadine Praeg*, Paul Illmer
University of Innsbruck, Institute of Microbiology, Austria
Methane is an important greenhouse gas that is produced and consumed in soils by
microorganisms. Until recently, studies of the contribution of plants to the global methane
flux have focused on the role of plants as conduits for soil-borne methane emissions from
54
wetlands, but pretty barely examined to date are the influence of plants on methane flux and
the presence of methanogenic and methanotrophic microorganisms in aerobic upland soils.
The present study used soils from grassland sites from siliceous and calcareous bedrock
located in Northern Tyrol, Austria at ~750 m a.s.l. Besides in situ methane measurements and
profound soil microbiology analyses, lab-scale gas measurements with two different sitetypical grassland plants were conducted at different temperatures. Gas samples were
withdrawn and subsequently analyzed via gas chromatography. To characterize the archaeal
community structure in bulk and rhizosphere soil of the respective plants and soil sites, nextgeneration sequencing was conducted.
In our investigations we could prove a distinct influence of plants on net methane emissions
out of upland soils. Our data proved not only a clear influence of plants on methane flux in
comparison to uncovered soil but between the investigated plants as well. On a lab-scale
basis temperature was a crucial factor that influenced the effect of plants on methane fluxes
significantly. Community analyses revealed clear differences within the archaeal community
of the investigated rhizosphere and bulk soils. Furthermore, the two plant species led to
different archaeal community shifts within the soils and the shifts were different between
siliceous and calcareous bedrock. The study emphasizes the need to better resolve the
influence of plants on the methane cycle and its involved microorganisms.
89
Drought affects spatial distribution of enzyme activities in the rhizosphere
Baharsadat Razavidezfuly*1, Muhammad Sanaullah2, Evgenia Blagodatskaya2, Yakov
Kuzyakov2
1
George-August-University of Göttingen, Germany, 2University of Göttingen
/Agricultural Soil Science, Germany
Due to high inputs of easily degradable organic compounds from the roots, the rhizosphere
is a very important and dynamic hotspot of microbial activity in soil. Consequently, the
enzyme activities in the rhizosphere are a footprint of complex plant-microbial interactions
and may reflect functional response to climate changes.
We modified the in situ soil zymography for identification and localization of hotspots of βglucosidase activity in the rhizosphere of maize under drought stress (30% of field capacity).
Zymographic images showed highlighted spots of β-glucosidase activity along the roots. The
β-glucosidase activity was especially high at root tips and was much stronger under drought
as compared with optimal moisture (70% of field capacity). The high β-glucosidase activity
under drought was confirmed by enzyme assay based on fluorogenically labelled substrates
applied to the root exudates. The activity of β-glucosidase of root exudates (produced by
roots and root-associated microorganisms) was significantly higher by drought stressed
plants as compared with optimal moisture. In contrast, the β-glucosidase activity in
destructively sampled rhizosphere soil was lower under drought stress compared with optimal
moisture. Without roots, drought did not affect β-glucosidase activity in soil. Consequently,
the release of organic compounds (such as mucilage) by roots under drought increased βglucosidase activity in the rhizosphere. Thus, the zymography visualized the distribution of βglucosidase activity and allowed assessment of consequences at the root55
soil interface under drought. Furthermore, coupling of zymography and enzyme assays in the
rhizosphere and non-rhizosphere soil enabled not only the precise mapping of the twodimensional distribution of enzyme activities, but also allowed quantitative assessment of soil
depth imaged on the zymograms.
90
Does fungal community matter to drought tolerance of cork oak (Quercus suber
L.)?
Francisca Reis*1, Paula Baptista2, Rui Tavares1, Teresa Lino-Neto1
1
Biosystems & Integrative Sciences Institute (BioISI), Plant Functional Biology Center
(CBFP), University of Minho, Portugal, 2Mountain Research Center (CIMO), Polytechnic
Institute of Bragança, Portugal
Mediterranean forests are one of the biodiversity “hotspots”. The cork oak (Quercus suber L.)
forest (“montado”) is an unique and emblematic resource for Portugal, both social and
economic manner, protected by EU (Habitats Directive 92/43/EEC). Portugal is the greatest
cork producer with 50% of cork extraction worldwide. Nowadays, cork oak faces a severe
global climate change and a reduction in water availability is expected for the near future,
which is expected to decrease cork oak growth and productivity. Plant benefits from many
symbiotic relations that occur between microorganisms and roots that are able to enhance
nutrient and water supply. However, root colonization efficiency and fungal community
structures are dependent on environmental conditions, such as water availability. The main
goal of this work was to evaluate the relationship between fungal abundance and diversity
conferred by different drought scenarios. The effect of drought in cork oak fungal ecosystem
was accessed by studying cork oak field trees in 7 different Portuguese forests. Five different
locations (Gerês, Macedo de Cavaleiros, Vimeiro, Grândola and Moura) were evaluated
according to a gradient of water-availability. Samples from extreme conditions, the driest
(Moura) and wettest (Gerês) places were sampled in two different sites. Soil samples (35)
were analysed by ITS barcoding on ECM tips and metabarcoding using Illumina platform. The
obtained results will help to understand not only the importance of fungi to drought
tolerance in cork oak forest, but also which are the main fungal colonizers.
91
Combined effects of climate change induced shifts in the plant community and
drought on the structure and functioning of the rhizosphere microbiome
Thomas Reitz*1, Mika Tarkka2, Jessica Gutknecht3, Tesfaye Wubet1, Francois Buscot1,
Erik Welk4, Alexandra Weigelt5
1
Helmholtz Centre for Environmental Research, Germany, 2HelmholtzCentre for
Environmental Research, Germany, 3University of Minnesota, United States, 4MartinLuther-University Halle, Germany, 5University of Leipzig, Germany
The rhizosphere microbiome and its functioning are directly affected by plant root exudates.
Given the fact that these exudates differ between plant species, climate change induced shifts
in the abundance of plant species changes the structural and functional composition of the
microbial soil community. Besides this, increasing drought is expected to directly (due to
reduced soil moisture) and indirectly (due to stress-induced, altered plant root exudates
56
production) affect the soil microbiome. In order to study the combined effects of drought and
altered plant community composition on the functioning of the rhizosphere community, we
performed a common garden experiment. Two plant pools were defined and sown on
experimental plots. The first pool included plant species that are predominantly distributed in
southwestern Europe and, in the context of climate change, may potentially become more
dominant in central Europe. The second pool included species mainly distributed in
northeastern Europe, which are predicted to become increasingly displaced in central Europe.
To simulate drought half of the plots were roofed for five weeks in early and late summer.
After each roofing period we determined different plant root traits as well as structural and
functional responses of the rhizosphere community. We observed drought-induced,
morphological changes in the root structure whereas stronger effects occurred after the
second roofing period. Microbial activity indices (nitrification potential and soil enzymes) as
well as selected activities of isolated microorganisms were generally reduced by drought, but
the extent of the reduction was related to the plant community, indicating different buffering
capacities against drought stress. In terms of phosphate solubilization, contrasting responses
to drought of bacteria and fungi suggest functional redundancies in the rhizosphere
microbiome. In conclusion, plant root morphology was strongly affected by drought.
Microbial traits were also highly affected by drought but these changes were possibly
mediated through plant community responses.
92
Influence of climate change and heavy metal on the plant-microbe interactions
Sukamal Sarkar*, Krishnendu Ray, Hirak Banerjee
Bidhan Chandra Krishi Viswavidyalaya (State Agricultural University), India
The various biotic and abiotic stress factors can affect the growth and development of crops.
Particularly, the change of climatic condition and also heavy metal stress like lead (Pb),
arsenic (As), cadmium (Cd), copper (Cu) etc. influence various plant physiological and
metabolic process as well as development and yield of the crops. The change of climate
especially the elevated atmospheric CO2 condition enhance the biomass production and also
metal ion accumulation in most of the plants and help plants to support greater microbial
populations and protect the microorganisms against the impacts of heavy metals. Besides,
the indirect effects of climate change on the function as well as structure of plant roots,
diversity and activity of rhizosphere-microbes would lead to altered metal bio-availability in
soils and thus affect plant growth. However, the effects of rising of global temperature,
drought or combined climatic stress on the growth of plant and metal accumulation vary
substantially across physical, chemical and also biological properties of the environment (e.g.,
soil pH, heavy metal type and its bio-available concentrations, microbial diversity, and
interactive effects of climatic factors). Overall, direct and indirect effects of climate change on
heavy metal mobility in soils may further hinder the ability of plants to adapt and make them
more susceptible to stress. In this study we have discussed how the various climatic
parameters including atmospheric CO2, temperature and drought influence the plant–metal
interaction in polluted soils. The effects of climate change and heavy metals on plant–
microbe interaction, heavy metal phytoremediation and safety of food have also been
narrated. This review shows how the plant–metal interaction responds to changing climatic
57
condition and how is it beneficial in selecting crops that would be able to produce more
yields without accumulating toxic heavy metals for future food security.
93
Influence of drought on the antioxidative stress defense and root exudation of
european grassland species
Peter Schröder*1, Christian Huber2
1
Helmholtz Zentrum Muenchen GmbH, Germany, 2Helmholtz Zentrum
Muenchen, Germany
Extreme weather situations like prolonged periods of drought as well as heavy precipitation
within short periods of time as a consequence of climate change have an impact on our
terrestrial ecosystems. It´s likely that these conditions will not only affect the vegetation but
also plants associated rhizosphere bacteria and fungi. Grassland vegetation fulfills a large
number of ecosystem functions that need to be preserved. With its high biodiversity, it forms
natural habitats for animals and bird species. It serves as an important source for animal
fodder, protects the environment from erosion and leakage of nutrients as well as it
maintains our countryside and landscapes.
Plants actively influence the chemical properties of the rhizosphere by exudating a variety of
compounds via their root system. Many of these compounds, mostly low molecular weight
organic acids, alter distinct processes within rhizospheres that might have an impact on the
interactions between different plants species as well as the interactions between plants,
microbes and fungi.
In this study we investigated the influence of drought on the antioxidative stress defense
system of typical European grassland species (Plantago lanceolata, Lotus corniculatus). For
collection and analysis of root exudates we introduce a rhizotron–microsuction cup–system
that allows for a continuous and noninvasive collection of exudate samples while growing the
plants in a natural soil system. Analysis of exudates was done by a combination of HPLCUV/VIS, ion chromatography and LC MS/MS techniques.
Analysis of different biomarkers (e.g. stress enzyme activities, thiol contents) suggests the
formation of reactive oxygen species in water stressed plants. Qualitative analysis of root
exudates revealed the occurrence of different phenolic and aliphatic acids with the potential
to affect rhizosphere processes and its biota.
94
The complex roles of lignin and suberin in plant micronutrient uptake
Tânia S. Serra*1, Kasra Talebi1, Ikram Blilou2, Mark G. M. Aarts1
1
Wageningen University, Laboratory of Genetics, Netherlands, 2Wageningen University,
Plant Developmental Biology Laboratory, Netherlands
Roots are specialized tissues with a central function in water and mineral nutrient uptake
from the soil, to support the progressive plant growth and development. Radial transport
across the root to the vascular system occurs through the apoplastic, symplastic or
transmembrane pathway. To regulate the non-selective apoplastic transport, a ring-like
58
structure composed of lignin, named Casparian strip (CS), surrounds the endodermis cells. As
the root develops, transmembrane transport is blocked through the deposition of suberin at
the endodermal layers. The specific mechanism underlying the generation of this diffusional
barriers is not fully known, but a few studies show that defects in the barrier composition and
structure can have major effects in transport of water and solutes. Thus, understanding the
biosynthesis and function of such barriers will allow the future manipulation of root systems
in important crops, to improve water and nutrient uptakes. To assess the role of CS/lignin and
suberin in plant adaptation to mineral stress we selected several Arabidopsis mutants affected
in the composition and/or structure of these polymers, such as esb1, ralph and horst. The root
and shoot phenotype are studied under both deficiency and excess concentrations of zinc,
copper, manganese and iron, as well as toxic amounts of cadmium. By integrating the mineral
stress-response in the different root properties of each mutant, we will further understand
how root structure affects metals metabolism. In addition, analysis of root structure in the
natural heavy metal hyperaccumulator Noccaea caerulescens showed additional cell layer
adjacent to the endodermal cells displaying lignin characteristics.
Previous studies showed that several genes involved in lignin and suberin metabolism are upregulated in N. caerulescens compared to Arabidopsis plants. Thus, modifications of the root
structure may account for the extraordinary ability of N. caerulescens to tolerate and
accumulate high metal concentrations.
95
Hypoxia induces changes in root maintenance costs of Prunus rootstocks
Guillermo Toro*1, Paula Pimentel1, Manuel Pinto2
1
Centre for Advanced Studies in Fruit, Chile, 2Institute of Agricultural Research, Chile
Low oxygen in the rhizosphere (hypoxia) induces alterations in root respiration. Under
hypoxia, the regulation of the energy requirements by roots seems to be necessary to cope
with the energy unbalanced produced by hypoxia. Maintenance requirements can be one of
them, however no much studies have been done on this matter particularly in rootstocks. In
this work, we investigated whether the hypoxia tolerance in Prunus rootstock is associated to
changes in the maintenance costs.
Three Prunus rootstocks with contrasting tolerance to hypoxia: Marianna 2624 (tolerant),
CAB6P (semi-tolerant) and Mazzard F12/1 (sensitive) were grown in nutrient solution and
exposed to normal (normoxia: 7.8 mg O2 L-1) and low (hypoxia: 0.5 mg O2 L-1) oxygen
concentration. Hypoxia treatment was achieved by bubbling gaseous nitrogen into the
nutrient solution (0.1% agar). The root respiration components were obtained by multiple
regression analysis of the root oxygen evolution and the root biomass accumulation.
Under hypoxia, tolerant Prunus rootstock showed a significant decrease in root maintenance
respiration from 1.8 mmol O2 g-1DW d-1 under normoxia to 0.7 mmol O2 g-1DW d-1 under
hypoxia (60% reduction). This reduction in maintenance costs was paralleled by an increase of
the ion uptake respiration from 5.6 mmol O2 mmol-1N under normoxia to 9.4 mmol O2 mmol1
N under hypoxia (70% increase). In this case no changes were observed in growth respiration,
which in average was 7.8 mmol O2 g-1DW in both oxygen conditions. In the sensitive rootstock
the maintenance respiration increased from 2.3 mmol O2 g-1DW d-1 under normoxia to 3.8
mmol O2 g-1DW d-1 under hypoxia (65% increase). But the growth respiration,
59
decreased to 1.8 mmol O2 g-1DW under hypoxia (65% reduction). In this case, no differences
were observed in ion uptake respiration.
These results showed that sensitivity to hypoxia in Prunus rootstock is related to the
respiratory maintenance costs.
96
Climate warming effects on plant belowground biomass allocation in tundra
Peng Wang*1, Monique Heijmans1, Liesje Mommer1, Frank Berendse1, Trofim
Maximov2
1
Wageningen University, Netherlands, 2The Institute for Biological Problems of
Cryolithozone, Siberian Branch of the Russian Academy of Sciences, Russia
Climate warming is known to have large impacts on tundra ecosystems. Increases in
aboveground plant biomass in response to climate warming in the Arctic have been widely
reported. However, little is known about the belowground.
In order to improve our understanding of the climate warming effects on the belowground
part of tundra vegetation, we did both a meta-analysis and a field experiment. We collected
belowground biomass and annual temperature data from 38 published studies which were
performed in tundra vegetation. Aboveground biomass increased with increasing
temperature, as earlier observed. However, the belowground temperature response was
significantly lower than aboveground. Moreover, belowground temperature response
differed between vegetation types, as in shrub dominated vegetation belowground biomass
significantly increased with temperature, but in graminoid dominated vegetation it did not
change. These findings have important implications for the C and nutrient cycling in tundra
ecosystems as we expect that a shift in vegetation types will occur in the future warmer
conditions.
We also performed a soil warming experiment in northeastern Siberia by using heating
cables connected to solar panels. The experiment lasted for 5 summers and the warming
treatment significantly increased soil temperature and active layer thickness during the
growing season. We will present aboveground and belowground biomass results for
deciduous shrubs, evergreen shrubs and graminoids harvested at the end of the growing
season of 2014. We expect that the warming treatment changed the biomass allocation of
different plant functional types along soil depth and thus affected the competitive
relationships.
97
Impacts of atmospheric CO2 on root exudation chemistry and rhizospherecolonizing bacteria
Alex Williams*, Pierre Pétriacq, T.E. Anne Cotton, David Beerling, Jurriaan Ton
University of Sheffield, United Kingdom
Atmospheric CO2 concentrations have fluctuated in historic times to present day and are set
to increase due to anthropogenic emissions. Little is known about the effects of altered CO2
60
levels on interactions with beneficial root-colonizing microbes in relation to the chemical
composition of root exudates. This study aims to address the impact of low (200ppm),
ambient (400 ppm) and high CO2 (1200 ppm) on root exudation chemistry and root-microbe
interactions in the model plant species Arabidopsis thaliana. Our data indicate that CO2
influences root colonisation activity of the rhizobacterial strains Pseudomonas putida KT2440
and Pseudomonas fluorescens WCS417r, and these effects are associated with changes in root
exudation chemistry. Additionally, previous data indicates that there is a positive relationship
between CO2 concentrations and ontological factors that influence the speed at which plants
develop. We have therefore considered the effects of developmental stage of the plant on
root colonisation and exudation chemistry. Further investigations aim to reveal the
physiological mechanisms and ecological consequences of these responses, as well as the
impact of atmospheric CO2 on global rhizosphere communities.
98
Comprehensive analysis of rhizosphere effect in top and deep horizons of
podzolic soil under spruce trees
Ilya Yevdokimov*1, Alla Larionova1, Tatiana Sokolova2, Inna Tolpeshta2
1
Institute of Physicochemical and Biological Problems in Soil Science RAS, Russian
Federation, 2Faculty of Soil Science, Moscow State University, Russian Federation
Global warming is known to affect the carbon cycle in forest ecosystems through the
intensification of soil organic matter (SOM) decomposition. It is well established that
rhizodeposits stimulate CO2 efflux from the topsoil, whereas the role of rhizosphere effect in
deep horizons in C and nutrients cycling has not been sufficiently estimated. We
hypothesized that the rhizosphere effect is significantly different in top and deep soil
horizons.
C turnover rate and nutrients stoichiometry were determined in rhizosphere and bulk soil
sampled from the experimental plots with podzolic soil (Albeluvisol) under spruce trees (Picea
abies L.) in the Central Forest Reserve (Tver region, Russia). The rhizosphere factors (Rf)
expressed as a ratio of soil characteristics in rhizosphere to that in bulk soil were determined in
the top AEL (3 – 15 cm) and deep EL horizons (15 – 46 cm).
We subdivided soil characteristics into 2 groups according to Rf value. The group I with 1.05
< Rf < 1.5 includes SOM, acidity, available N, P and exchangeable K. The group II with Rf > 1.5
includes microbial biomass, basal respiration and SOM turnover rate. Increased SOM turnover
rate is connected with better SOM quality in rhizosphere soil as evidenced by the increase in
polysaccharides portion in rhizosphere SOM revealed by 13C-MAS-NMR spectroscopy. C:N:P
stoichiometry in microbial biomass might also affect the SOM susceptibility to
decomposition; C:N ratio in rhizosphere microbial biomass was higher than that in bulk soil,
while C:P displayed the opposite tendency.
The most drastic differences between top and deep rhizosphere were found in basal
respiration and SOM turnover rates. Rf for SOM turnover in top soil was about 1.5, while in
the deep soil horizon it was as high as 6. Thus, deep rhizosphere was found to be the more
pronounced hot spot of biological activity than top one.
61
Metabolomics
100
Genomic, metabolomic and functional characterization of beneficial
Burkholderia species from natural disease suppressive soils
Victor J Carrion*1, Desalegn W. Etalo2, Viviane Cordovez2, Kazuki Fujiwara3, Irene de
Bruijn2, Victor de Jager2, Jos M. Raaijmakers2
1
Netherlands Institute of Ecology (NIOO-KNAW), Netherlands, 2Netherlands Institute of
Ecology (NIOO-KNAW), Netherlands, 3National Agriculture and food Research
Organization, Japan
Disease-suppressive soils are ecosystems in which crop plants suffer less from specific
diseases than expected owing to the activities of antagonistic rhizosphere microorganisms.
For most disease-suppressive soils, however, the beneficial microbes and underlying
mechanisms involved in pathogen control are largely unknown. In previous studies, we
identified key bacterial taxa involved in suppression of the fungal root pathogen Rhizoctonia
solani by PhyloChip-based metagenomics of the rhizosphere microbiome of sugar beet
seedlings. Members of the Proteobacteria, Firmicutes, and Actinobacteria were found to be
consistently associated with disease suppression. Here we focus on the β-Proteobacteria,
specifically on the Burkholderia genus. We isolated approximately 50 Burkholderia strains
from the rhizosphere of sugar beet seedlings grown in a soil suppressive to R. solani. Based
on MLST sequencing, these isolates were classified as B. caledonica, B. graminis, B. hospita, B.
pyrrocinia and B. terricola. These five Burkholderia species showed different activities. B.
graminis inhibited R. solani via volatile organic compounds (VOCs), whereas B. caledonica,
hospita, pyrrocinia and terricola showed antifungal, antioomycete activity and antibacterial
activity. Strains of all five species exhibited in planta activity against R. solani. Comparative
VOCs profiling of all five species revealed that sulphur-containing compounds were unique
for B. graminis. By comparative genomics of the five fully sequenced species/strains, three
unique gene clusters were identified in the B. graminis genome that are most likely involved
in the biosynthesis of these sulphur-containing VOCs. Comparative genomics also revealed
the presence of four non-ribosomal peptide synthetase gene clusters in B. caledonica,
hospita, pyrrocinia and terricola that may contribute to the observed broad-spectrum
antimicrobial activities. Mutagenesis, cloning, heterologous expression and chemical analyses
are ongoing to resolve the functions of these novel gene clusters identified in the genomes
of the five Burkholderia species from disease suppressive soil.
62
Metabolomics
101
Mining rhizobacteria-induced metabolome reprograming in plants
Desalegn Etalo*1, Judith van de Mortel2, Je Seung Jeon1, Ric de Vos3, Henk Gude4,
Thierry Janssen5, Jos Raaijmakers1
1
Department of Microbial Ecology, Netherlands Institute of Ecology, NIOOKNAW, Netherlands, 2HAS University of Applied Sciences, Netherlands, 3Plant Research
International, Wageningen University and Research Centre, Netherlands, 4Flower Bulbs,
Applied Plant Research, Wageningen University & Research
Centre, Netherlands, 5MicroLifeSoilutions, Netherlands
The colonization of plant roots, stem, leaves or fruits by beneficial microorganisms can
protect plants from diseases, promote growth and enhance yield/biomass. Application of the
beneficial rhizobacterium Pseudomonas fluorescens strain SS101 to roots of Arabidopsis
thaliana resulted in a distinct shift in the plant transcriptome as compared to non-treated
plants, with approximately 1150 plant genes differentially regulated. Gene set enrichment
analysis (GSEA) revealed that genes involved in sulfur metabolism were among the most
overrepresented gene clusters that were up-regulated in plants treated with the beneficial
rhizobacterium. Furthermore, the upregulated gene category included also genes involved in
sugar metabolism and auxin biosynthesis. In line with the transcriptome data, the
metabolome analyses showed that sulfur containing metabolites were among the metabolites
that accumulated in Arabidopsis plants treated with the rhizobacterium. Based on the
observed transcriptome and metabolome changes associated to sulfur metabolism in
Arabidopsis, experiments are ongoing to boost the production of specific compounds,
including sulforaphane, in other cruciferous plants like broccoli. Furthermore, root, bulb and
sprout samples obtained from two daffodil cultivars (Carlton, Geranium) treated with
rhizobacterial strain SS101 were subjected to untargeted metabolomics. Our analyses of these
bulb crops indicated that specific groups of metabolites like spermidine-conjugates were
induced only in the sprout of both cultivars that were treated with the rhizobacteria.
Collectively, these results show that specific rhizobacteria can (re)program the plant
metabolome.
103
Discriminating disease suppressive soils for cereal pathogen Rhizoctonia solani
AG-8 using metabolomics
Helen Hayden*, Simone Rochfort, Vilnis Ezernieks, Pauline Mele
Department of Environment & Primary Industries, Australia
Metabolomic analyses were used to investigate possible functional mechanisms for disease
suppression in soils known to be suppressive to the fungus Rhizoctonia solani AG-8, which
infects cereal crops. Disease suppression refers to a lack of disease manifestation even in the
presence of the pathogen, host plant and favourable environmental conditions. Currently the
only way to identify fields with disease suppression of R. solani AG-8 is by doing glasshousebased pot trials. Soil samples were collected from two adjacent fields, one known to have a
high disease suppression and the other low disease suppression resulting in infected cereal
crops. Samples were collected at different times throughout the cropping cycle over two
years and analysed for their metabolite profiles. Differentiation of the high and low
suppression fields was carried out using multivariate analyses of liquid chromatography mass
spectrometry (LC-MS) data, acquired in both the positive and negative ionisation modes, and
63
Metabolomics
nuclear magnetic resonance (NMR) data. Several peaks were significantly more abundant in
the high suppression soil for the positive and negative ionisation modes. Potential LC-MS
biomarkers for the high suppression soil were identified and corroborated by analysing two
years of soil samples. The structure of these LC-MS biomarkers was elucidated using accurate
mass data and MS fragmentation spectrum information. Analyses of the NMR loadings plots
identified soils with high suppression have a greater abundance of polyols and terpenes. Two
dimension NMR identified sugar biomarkers in the high suppression soils. Metabolite
biomarkers with high abundance in disease suppressive soils were shown to match standards
of macrocarpals, which are phloroglucinol containing compounds. Suppression of Rhizoctonia
solani AG-8 may be occurring through an antibiotic mode of action as shown for soils
suppressive to take all disease of cereals (Gaeumannomyces graminis var. tritici) or through
microbial effects upon the plant-pathogen interaction.
104
Effects of microbial signaling molecules on the growth and secondary
metabolism of Arabidopsis thaliana
Katharina Sklorz*, Michael Bonkowski
University of Cologne, Zoological Institute, Germany
All plants are simultaneously colonized by a multitude of microorganisms of very different
taxonomic affiliation. It is still uncertain how plants orchestrate the complex interaction with
the surrounding microbiome, but it is clear that specific signaling molecules must exist that
mediate the communication between plant species and their root microbes.
We investigated the role of bacterial autoinducers (N-Acyl-Homoserine Lactones, AHL´s),
which are employed in quorum sensing systems in various gram negative bacteria, on root
growth and the secondary metabolism of Arabidopsis thaliana. We will discuss the observed
patterns in the light of plant defense and interkingdom communication.
106
Presence of a growth-promoting endophyte affects the composition of plant
secondary metabolites and root exudates in Arabidopsis thaliana
Katja Witzel*1, Nadine Strehmel2, Susanne Baldermann1, Susanne Neugart1, Dierk
Scheel2, Monika Schreiner1, Rita Grosch1, Silke Ruppel1
1
Leibniz Institute of Vegetable and Ornamental Crops, Germany, 2Leibniz Institute of
Plant Biochemistry, Germany
We have isolated the strain Kosakonia radicincitans (DSM 16656) from the phyllosphere of
winter wheat under temperate conditions. Growth promotion of root and shoot, along with
increased yield, was conferred by inoculation of different crop and model plant species.
Endophytic plant growth promoting bacteria are discussed to impact significantly plant
physiology, but regulatory pathways and biochemical alterations are still unclear. Therefore,
transcriptome and metabolome investigations were conducted under controlled conditions
to gain a better understanding. A global transcriptome analysis of Arabidopsis thaliana plants
inoculation with Kosakonia radicincitans identified an over-representation of genes involved in
secondary plant metabolism. Profiling the glucosinolate, carotenoid and phenylpropanoid
64
Metabolomics
content of leaves and roots revealed a specific response to endophytic colonization. As some
secondary plant metabolites are known to act as signaling molecules in the rhizosphere, such
as flavonoids and coumarins, root exudates of control and inoculated plants were collected
and analyzed. More than 50 primary and secondary metabolism compounds were
differentially enriched when plants were colonized by K. radicincitans. The results and
possible implications of these analyses on plant-endophyte interactions are discussed.
65
Metabolomics
Root Endophytes
107
Consortia of ACC deaminase-producing bacteria (both endophytic and
rhizospheric) isolated from avocado plants mitigate salt stress of wheat plants
Patricio Barra*1, Nitza Inostroza1, Maria de la Luz Mora1, David Crowley2, Milko
Jorquera1
1
Universidad de La Frontera, Chile, 2University of California Riverside, USA
Plants growing under salinity stress conditions increase ethylene production, which triggers
inhibition of root elongation. Bacterial enzyme 1-aminocyclopropane-1-carboxylate
deaminase (ACCD) cleave the ethylene precursor, 1-aminocyclopropane-1-carboxylate, thus
decreasing ethylene levels and consequently their detrimental effects. Some bacterial strains
also produce phytohormone indol acetic acid (IAA), which increases cell elongation.
Therefore, we hypothesize that ACCD- and IAA-producing bacteria (both endophytic and
rhizospheric bacteria) isolated from avocado plants are able to mitigate salt stress effects of
wheat plants; and we also hypothesize than bacterial location (both endophytic and
rhizospheric), as well as, the levels of ACCD and IAA production by bacteria have different
effects on salt stress response of wheat plants.
Twelve bacterial strains were isolated from avocado plants, and four bacterial consortia were
formulated, each composed of three strains, as follows: 1) Endophytic bacteria with higher
ACCD and IAA production; 2) Endophytic bacteria with lower ACCD and IAA production and
3) Rhizobacteria with higher ACCD and IAA production; 4) Rhizobacteria with lower ACCD
and IAA production. Wheat seeds were inoculated with the bacterial consortia, and then
seeds were grown under salt stress conditions. Length, dry weight and superoxide dismutase
(SOD) activity of wheat shoot and roots were determined.
The results showed that at lower levels of bacterial IAA and ACCD production, the endophytic
bacteria were more efficient than rhizobacteria consortia mitigating salt stress effects.
Between rhizobacteria consortia, only those with higher production were able to promote the
growth of stressed plants. Both endophytic bacteria and rhizobacteria consortia with higher
production were able to increase SOD activity. Finally, bacterial strains isolated from avocado
plants mitigate plant stress and therefore have the potential to be used as commercial
inoculum of avocado plants.
108
Stenotrophomonas rhizophila SPA P69: deep insights into an endophytic stress
protecting agent
Gabriele Berg*, Henry Müller
Graz University of Technology, Austria
Stenotrophomonas rhizophila is able to promote plant growth of many crops and to protect
roots against biotic and a-biotic stresses. We studied mechanisms associated with osmotic
stress using transcriptomic and microscopic approaches. In response to salt or root extracts,
the transcriptome of S. rhizophila SPA P69 (syn. DSM14405T) changed drastically. We found a
notably similar response for several functional gene groups responsible for general stress
66
Root Endophytes
protection, energy production, and cell motility. However, unique changes in the
transcriptome were also observed: the negative regulation of flagella-coding genes together
with the up-regulation of the genes responsible for biofilm formation and alginate
biosynthesis were identified as a single mechanism of S. rhizophila against salt shock.
However, production and excretion of glucosylglycerol (GG) were found as a remarkable
mechanism for the stress protection of this strain. For S. rhizophila treated with root exudates,
the shift from the planktonic lifestyle to a sessile one was measured as expressed in the
down-regulation of flagellar-driven motility. These findings fit well with the observed positive
regulation of host colonization genes and microscopic images that show different
colonization patterns of oilseed rape roots. Spermidine, described as a plant growth
regulator, was also newly identified as a protector against stress. In addition to both the
changes in life style and energy metabolism, phytohormons, and osmoprotectants were also
found to play a key role in stress protection. Risk assessment studies reveal no health risks.
This is mainly because SPA P69 is unable to growth at the human body temperature, 37°C
due to the absence of heat shock genes and a temperature-regulated suicide mechanism.
Taken together SPA P69 is a promising endophytic stress protecting agent (SPA) ready for
commercial applications.
109
Better efficiency of biofertilizers in combination with N2-fixing endophytes in
low quality sandy soil
Borbala Biro*, Zita Szalai, Tamás Kocsis, Zsolt Kotroczó
Corvinus University of Budapest, Hungary
Biofertilizer and/or soilconditioner products are containing very often the various types of the
Nitrogen-fixing and P-mobilising microorganisms, as only one or 2-3 types of species
components. Question arises if single or combined products are more efficient and what is
the main driving force for the efficiency in a low quality sandy soil?
Tomato Solanum lycopersicon Mill. ’Mobil’ was used in a field experiment, inoculated with
Trichoderma harzianum T-22 and TDM, Hungarian Trichoderma inoculums, including also of
Azotobacter and Azospirillum N2-fixers in one product. There were patentkali and calcinit
fertilizers applied at 1200 kg/ha doses also to the soil. Inoculation treatment was performed
twice during the vegetation, at the sowing and at the time of the plantation of tomato
seedlings. Growth of tomato, shoot and root biomass was assessed and general soil
characterization, including physical-chemical parameters, the MPN counts of some beneficial
physiological groups in tomato rhizosphere, outside and inside the plants. Results were
evaluated by statistical probes.
The beneficial effect was realised with the combined tomato inoculation in comparison with
the only Trichoderma strain application. Effect of Trichoderma TDM inoculation was improved
with free-living and associative Nitrogen-fixers and thus provided better nutrition. The
relatively high P-content (430 mg/kg) was found in the slightly humous sandy soil, where the
N2-fixing microbes might able to improve the N,P, K ratio for the more efficient tomato
growth. Plant microbe interaction is largely dependent on the soil nutrient-status and the
balance among main nutritive elements. In this process the multifunctional inoculums might
providing greater plant-growth promotion beyond the single strain effects.
67
Root Endophytes
110
A survey of bacterial root endophytes associated with vegetation at a bitumen
impacted site
Natalie Blain*1, Bobbi Helgason2, James Germida1
1
University of Saskatchewan, Canada, 2Agriculture and Agri-Food Canada, Canada
Bacterial root endophytes can help alleviate plant stress and promote plant growth, and the
potential use of these endophytes to assist land reclamation is receiving increased interest.
Bitumen is a heavy oil that influences plant growth due to its hydrocarbon composition. The
Bitumount provincial historical site is considered the location of two of the world’s first oil
sands (bitumen) extraction plants. Operations at Bitumount began in 1923 and ceased
completely in 1958. Through natural reclamation, vegetation has re-colonized the area
including hardened bitumen. The Bitumount site offers a unique opportunity to study plantmicrobe associations that have co-adapted to this stressed environment. Sampling locations
were established in June 2014, and plant and soil samples collected. A total of 6 different
plant species were identified and sampled based on their abundance at each sampling
location. They included: Bromus inermis, Equisetum spp., Agropyron trachycaulum, Poa
pratensis, Fabaceae spp. and Fragaria virginiana. Soil samples were analyzed for hydrocarbon
content, which ranged from 330 to 24,700 mg kg-1 throughout the site. Culture dependent
and independent methods were used to characterize bacterial root endophytes. The number
of culturable root endophytes varied significantly (p<0.05) throughout the site and between
plant species, with values ranging from 4.3 to 6.3 log CFU g-1 of fresh roots. Phylogenetic
identification of isolated endophytes and Illumina MiSeq bacterial profiling revealed diverse
communities associated with different plant types. The function of these endophytes is
currently under investigation.
111
Desert root endophytes enhance the growth of Arabidopsis thaliana under salt
conditions
Ameerah Bokhari*, Juan S. Ramirez, Axel de Zelicourt, Feras F. Lafi, Maged Saad,
Intikhab Alam, Vladimir Bajic, Heribert Hirt
Center for Desert Agriculture, King Abdullah University of Science and Technology
(KAUST), Saudi Arabia
Understanding the communication and regulation between host plants and endophytic plant
growth promoting bacteria might lead to improved agricultural outputs. Here, our aim is to
study root endophytes from desert pioneer plants that ensures plant growth in different
extreme conditions. As part of the DARWIN21 project (http://www.darwin21.net) we isolated
endophytic bacteria from desert plants in Saudi Arabia, Jordan and Pakistan. We used
cultivation-based techniques followed by molecular characterization via sequencing 16S
rRNA genes. Our in-house screening assay was developed to select strains for inducing plant
growth promotion and/or abiotic stress tolerance on the model plant Arabidopsis thaliana. To
date we have isolated 984 bacteria which belong to four phyla, Proteobacteria, Firmicutes,
Actinobacteria and Bacteroidetes. Our screening assay revealed several bacterial strains which
promote the growth of Arabidopsis thaliana under salt stress conditions. So far, we have
sequenced the genomes of nine of these endophytes and we are currently sequencing
several more distinct bacterial strains that display the latter properties. Our comparative
genomic, transcriptomic and proteomic approach should help to unravel the mechanisms of
68
Root Endophytes
conferring salt stress tolerance to plants. As a future goal we aim to create a catalog of
microbial strains that can help to improve crop resistance to different biotic and abiotic
stresses.
112
Bacterial diversity of native Acacia sp. nodular endophytes in Tamanrasset
Zineb Faiza Boukhatem*1, Chahinez Merabet Merabet1, Abdelkader Bekki Bekki1, Sonia
Sekkour Sekkour1, Odile Domergue Domergue2, Antoine Galiana Galiana3
1
Oran 1 Ahmed Ben Bella University, Algeria, 2INRA, UMR LSTM, France, 3CIRAD, UMR
LSTM, France
Tamanrasset located in the heart of the Sahara desert in southeastern Algeria, is the only
region where are localized five indigenous species of Acacia: A. ehrenbergiana Hayne, A.
nilotica (L.) Delile, A. seyal Delile, A. tortilis (Forssk.) Hayne, and A. laeta Delile, these rustic
leguminous trees are surviving under extremely harsh conditions of drought in dry oueds.
Some preliminary observations led us to investigate the diversity of nodular endophytes
which is poorly documented especially those associated with Acacia species. Among 79
isolates purified from nodules of the five Acacia sp., 24 representative strains were
characterized and identified as belonging to nine bacterial genera, namely: Paenibacillus,
Ochrobactrum, Stenotrophomonas, Pseudomonas, Microbacterium, Rhizobium, Agrobacterium,
Brevibacillus,and Advenella. The isolates of these nodular endophytes revealed a strong
tolerance profile to salinity and high temperatures accordingly to their natural ability to
survive to harsh edaphoclimatic conditions. PCA confirmed that no correlation existed
between bacterial tolerance to maximum growth temperature and depth of sampling. On the
other hand, there was no relationship either between in vitro tolerances of rhizobial strains to
NaCl concentration and high temperature and the corresponding edaphoclimatic
characteristics of the sampling sites.
113
Bacterial root endophytes associated with agricultural crops in Saskatchewan,
Canada
Jorge Cordero Elvia*1, Renato de Freitas2, James Germida2
1
University of Saskatchewan, Canada, 2University of Saskatchewan / Department of Soil
Science, Canada
Endophytes influence growth and productivity of crops in agricultural ecosystems, and are an
important component of the plant-microbiome. Because of the potential benefits of
endophytes, it is important to investigate their diversity and function in different plant
species and how edaphic factors influence this relationship. This study investigated the
diversity of bacterial root endophytes associated with wheat (Triticum aestivum), canola
(Brassica napus), lentil (Lens culinaris) and field pea (Pisum sativum) grown in three
Chernozem (Orthic Brown Chernozem Calcic Kastanozem, Orthic Dark Brown Calcic
Chernozem and Orthic Black Calcic Chernozem) soils in Saskatchewan, Canada. These soils
differed mainly in organic matter content, pH and texture. Culturable bacterial root
endophytes (n=298) were isolated using non-selective culture media and identified by
Sanger sequencing. In addition, endophyte community profiles were analysed using culture
69
Root Endophytes
independent denaturing gradient gel electrophoresis (DGGE). Culture dependent and
independent techniques indicated that some endophyte communities varied depending on
crop and soil types. Analysis of culturable bacteria by crops revealed that the predominant
genus in canola was Stenotrophomonas, whereas Bacillus and Rhizobium were dominant in
pea. The most common genera in lentil were Bacillus and Pantoea, whereas Xanthomonas
was predominant in wheat. Culture independent DGGE analyses revealed that band patterns
were distinct for each crop, with wheat yielding the largest number of endophyte bands.
Similarly, soil type influenced endophytic community structure as demonstrated by higher
number of culturable endophytes and DGGE bands in the Orthic Brown Chernozem Calcic
Kastanozem, followed by Orthic Dark Brown Calcic Chernozem and Orthic Black Calcic
Chernozem. Our results suggest that crops select specific bacterial endophyte species, but
soil properties also influence their community structure.
114
Endophytic protists in plant roots
Michael Bonkowski, Kenneth Dumack*
University of Cologne, Germany
Protist root pathogens such as Plasmodiophora brassicae the agent of club root disease in
Brassicaceae, or Spongospora subterranea the agent of powdery scab in potatoes are well
known to phytopathologists. However, scarce knowledge exists on the identity of protists
that colonize roots without causing visible disease symptoms.
We conducted a systematic screening for root-colonizing amoeboid protists in soil and
isolated a new species of the Viridiraptoridae (Glissomonadida) from an agricultural field in
Göttingen, Germany. Viridiraptoridae have been recently described as limnic protist predators
of filamentous algae, but environmental sequencing studies suggested that they were also
common and highly diverse in terrestrial habitats.
To investigate the feeding preferences of our isolate, we offered bacteria, fungi, algae,
nematodes, and sterile plant seedlings as a possible food sources. The protists multiplied
exclusively, but strongly, in roots of various plants like Arabidopsis thaliana and Lolium
perenne, and to a lesser extend in dead nematodes. To overcome the plant defense system
endophytic protozoa must possess highly adapted signalling mechanisms. Deciphering these
interactions may strongly improve our understanding of interactions of plant roots with
protozoan pathogens.
70
Root Endophytes
115
Potato endophytic compartments - a hidden reservoir for promising biocontrol
agents against Ralstonia solanacearum
Tarek Elsayed*1, Stéphane Compant2, Rita Grosch3, Kornelia Smalla1
1
Julius Kühn-Institut – Federal Research Centre for Cultivated Plants (JKI), Institute for
Epidemiology and Pathogen Diagnostics, Germany, 2Austrian Institute of
Technology, Austria, 3Leibniz Institute of Vegetable and Ornamental Crops, Department
of plant Health, Germany
Ralstonia solanacearum (Rs, biovar2, race3) is an epidemic soil-borne phytopathogen.
Endophytic antagonists might be a solution to control this pathogen. In this study, we
assessed the diversity and abundance of naturally occurring bacterial antagonists of Rs, in
nine potato microenvironments across three soil types. A total of 2016 isolates was screened,
and 215 out of 480 isolates with in vitro antagonistic activity were further characterized. BOXPCR showed similar genotypes in different microenvironments and soils. Endophytic
compartments had the highest proportion of antagonists, and most of these antagonists
were affiliated to Pseudomonas. Potential modes of action were in vitro tested and phosphate
solubilization and siderophore production were dominant traits. Rhizocompetence of 11 green
fluorescent protein (gfp) tagged antagonists, representing all microenvironments, was tested
in a greenhouse experiment with tomato. Plate counts revealed good rhizosphere
competence. A selection of seven antagonists was tested for biocontrol activity against Rs
(B3B) in a new greenhouse experiment. Five different consortia, each consisting of three
antagonists were prepared (P. chlororaphis, P. brassicacearum in addition to one variable
isolate). Based on the wilting symptoms, P. koreensis and P. helmanticensis showed a good
biocontrol activity. Confocal laser scanning microscopy (CLSM) confirmed the colonization of
root tips. Plate counts and qPCR showed good rhizosphere competence and a reduction in Rs
population on the rhizosphere. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA
gene fragments amplified from TC-DNA showed pronounced shifts in bacterial communities.
UPGMA analysis revealed distinct clusters based on the inoculated isolates and the severity of
infection. P. koreensis and P. helmanticensis showed a good colonization and biological control
activity and, in addition, plant growth promotion, suggesting the future use of these isolates
as biocontrol agents against bacterial wilt in regions where Rs is endemic.
116
Effect of Azospirillum brasilense strains on Lotus japonicus root growth and
development
Estrella Elvira-Ramirez*1, Simona Radutoiu2, Dorthe B. Jensen3
1
Aarhus university, Denmark, 2Aarhus Univeristy, Denmark, 3Aarhus
University, Denmark
Azospirillum brasilense, a nitrogen-fixing bacteria, is broadly present in soils around the
world. Strains of this bacterium can live isolated in the soil or in the rizosphere of a wide plant
families. The beneficial effects of Azospirillum on some crops, such as maize or wheat are well
documented and have been mainly associated with the increase of root system and
therefore, the enlarged area available for mineral uptake. The beneficial effect of Azospirillum
on crops proved to be, however from time to time inconsistent, and environment dependent.
On the other hand, the molecular mechanisms behind the beneficial or less favourable
interactions with the different hosts are largely unknown.
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Root Endophytes
We set to better understand the molecular basis of the interaction between Azospirillum and
the model legume host, Lotus japonicus. Specific growth conditions in which Azospirillum has
a detrimental effect on the legume host have been identified. We are currently using such
defined conditions to better understand the molecular mechanism involved in the different
responses mounted by the two interacting partner leading to contrasting responses in the
host. We will present here our progress on this study based on Lotus mutants analyses and
host gene expression investigations.
117
Characterization of free-living and endophytic diazotrophs from rice plants
grown on different type of Uruguayan soils
Lucia Ferrando*1, Andrea Martinez2, Ana Fernández Scavino3
1
Universidad de la República- Uruguay/ School of Chemistry, Uruguay, 2School of
Chemistry- Universidad de la Republilca, Uruguay, 3School of Chemistry- Universidad de
la República, Uruguay
The soils used for agriculture suffer important changes over the course of the successive
cycles of crops, affecting fertility and soil biodiversity. The great contribution of endophytic
bacteria to plant growth promotion is well known, in particular, for diazotrophs. Endophytes
mainly come from the soil, so soils with different characteristics could determine or affect
microbial communities established in plant tissues.
The abundance, diversity, and composition of diazotrophic communities from seven
Uruguayan soils and endophytic communities established in roots from rice plants grown in
them under controlled conditions were addressed using the nifH as marker gene.
The abundance of nifH gene in leaves was similar among those from different type of soil and
a hundred-fold lower than in rice roots or soils. Rice leaves seem to support a numerically
stable and independent population poorly affected by soil type. On the other hand, the nifH
copy number present in soils was similar to the retrieved from roots. The most fertile soil
presented significant higher nifH abundance. Diazotrophic communities present in the soils
were significantly more diverse than those established inside rice roots, according to the
Diversity Indexes obtained. However, Clustering analysis from data obtained by T-RFLP of
nifH gene revealed that diazotrophic communities from different soils presented a greater
similarity among them (45%) than root endophytic communities (20%). Pyrosequencing of
nifH gene is being performed for a better understanding of these communities.
Diazotrophic populations inhabiting rice roots would be similarly abundant than the ones
from soils. However, diazotrophic communities from soil were more diverse and
homogeneous among the different soils than the communities established in rice roots.
These results suggest that rice plant could be playing an important role in the selection of a
restricted group of diazotrophic species which effectively colonize the root achieving to
establish themselves as endophytes.
72
Root Endophytes
118
Microbispora spp. are the dominant actinobacterial endophytes of Australian
rice plants
Christopher Franco*1, Fitri Widiantini2
1
Flinders University, Australia, 2Flinders University, Australia
Microbispora spp. were detected as the predominant endophytic actinobacterial population
making up 85% of the two hundred and sixty four endophytic actinobacteria isolates
obtained from rice plants grown in Yanco, over two consecutives rice growing seasons,
whereas they constituted less than 2% of the 287 actinobacteria isolated from the source rice
paddy soils. The results were confirmed by culture-independent methods.
The Microbispora isolates were categorised into 16 different morphological groups that were
morphologically dissimilar to the nearest neighbour type strains based on 16S rRNA gene
and gyrB gene sequence similarity. The Microbispora isolates differentiated further based on
BOX-PCR fingerprinting. At least 5 strains demonstrated a distinct relationship between each
other as well as with the type strains. Further polyphasic studies were able to characterise
their novelty.
Further confirmation of these unexpected findings was achieved by growing the original and
3 other types of rice cultivars (Langi, Doongara, Amaroo and Nipponbare) in 4 different soils
which included the original rice paddy soils. Approximately 94% of the total isolates were
Microbispora-like isolates.
Challenge experiments were carried out with the rice endophytes Streptomyces sp. R32 and
Saccharothrix sp. OS6 added as seeding coatings on rice seeds prior to sowing, or adding
these cultures as seed drenches onto rice seed sown in rice paddy soils. In all these
experiments Microbispora spp. constituted more than 60% of all endophytes isolated from
these plants at 6 weeks after sowing.
These results revealed that the rice plants growing in Australia preferentially harbour a high
degree of diverse Microbispora.
119
Endophytic bacterial communities in different tissues of mountain sorrel (Oxyria
digyna)
Cindy Given*, Riitta Nissinen
University of Jyväskylä, Finland
Endophytic bacteria live inside the plants without harming them, and can provide benefits for
their hosts. Endophytes have been studied especially in the crop plants, but very little is
known about endophytes from extreme environments like the Arctic.
Mountain sorrel (Oxyria digyna) is used in this study as a model plant. It is a non-mycorrhizal,
arcto-alpine species, that can grow in very low nutrient soils and is a typical pioneer plant
species. In this study, we want to understand the acquisition and functioning of endophytic
community in different tissues of O. digyna.
73
Root Endophytes
In 2013, 80 endophyte-free micropropagated O. digyna plantlets were planted in the field site
on fell Jehkas, in Kilpisjärvi (69°’N; 20°50’E), northwestern Finland. Half of the plants were
harvested after 30 days (summer) and the rest were left to over-winter, and harvested in
spring 2014. Endophytic bacterial communities in leaves and roots were analyzed by
community amplicon sequencing. Additionally, the culturable endophytic bacteria were
isolated from the summer samples. The isolates were identified by 16s rRNA gene
sequencing and cross-compared with the molecular data.
Most of our isolates were closely related to bacteria isolated from other cold environments.
Several bacterial species showed a tight association with the host, as they had been
previously isolated from seeds and vegetative tissues of O. digyna. The preliminary results
showed that Pseudomonas sp. and Microbacterium sp. were the dominant genera in both
leaves and roots. Some genera, however, were tissue-specific: Sphingomonas spp. were found
only from leaves and Flavobacterium spp. were isolated only from roots. Strains representing
the dominant endophyte groups in different tissues were phenotypically profiled in order to
detect putative tissue specific and plant beneficial traits.
120
Is the root colonizing endophyte Acremonium strictum an ericoid mycorrhizal
fungus?
Gisela Grunewaldt-Stöcker*, Henning von Alten
Leibniz University of Hannover/ Institute of Horticultural Production Systems/ Section
Phytomedicine, Germany
Acremonium strictum was associated with roots of several ericaceous plants and thus
regarded as a potential mycorrhizal fungus. However, its mycorrhizal traits were not proven
yet.
A. strictum is an endophyte without visible effects on the host plant, non-systemic and
restricted to the root system, with multifunctional traits, and thus can be classified as type II.
Entomologists revealed its antagonistic feature to affect herbivorous insects in feeding
behaviour and development by altering phytosterols in the host plants. In our previous
investigations on flax and tomato it could induce disease resistance towards Fusarium-wilts.
Moreover, in our microscopic studies on host-fungus interactions with wild-type and gfptransformants we found hyphal structures of A. strictum in the rhizodermal cells of
greenhouse cultured flax plants and later in hair roots of inoculated Rhododendron plantlets
in sterile liquid culture with striking similarity to hyphal coils and colonizing mycelia of ericoid
mycorrhizal fungi. Therefore the hypothesis of the mycorrhizal nature of A. strictum was
revived.
Structures bearing resemblance to ericoid mycorrhiza at a low colonization level were shown
by confocal laser scanning microscopy of fuchsine acid-stained hair roots of inoculated
Rhododendron spec.. Real progress to verify or reject our hypothesis was achieved by using
the fluorochrome dye FUN-1® in unfixed tissue to observe the vitality status of the host cells
in colonized hair roots. In inoculation trials with in vitro raised mycorrhiza-free
Rhododendron plants - tested in sterile liquid and in greenhouse pot culture - A. strictum was
never observed in living epidermal or cortex cells. As compared to the mycorrhizal fungi
74
Root Endophytes
Oidiodendron maius and Rhizoscyphus ericae that invaded metabolic active host cells, A.
strictum was found with elongated or coiled hyphae only in disordered or visibly dead cells
and in the apoplast. This corroborates its non-mycorrhizal endophytic attribute.
121
Density and diversity of endophytic bacteria associated with Salicornia europaea
L. and Aster tripolium L. in saline soils
Katarzyna Hrynkiewicz*1, Silke Ruppel2
1
Nicolaus Copernicus University, Poland, 2Leibniz-Institute of Vegetable- and
Ornamental Crops Grossbeeren/Erfurt e.V., Germany
Worldwide salt affected soils cover 800 million hectares (6% of the land in total) with still
increasing areas, expanding the scale of this phenomenon. Plants evolved a wide range of
adaptation mechanisms, which allow them to grow and develop in environments of high soil
salinity. We expect a halophyte specific selection of the plant microbiome in respect to the
biological atmospheric nitrogen fixing community. To elucidate the question if halophytic
plants retrain specific diazotrophic bacterial communities we evaluated S. europaea L.
(herbaceae) and Aster tripolium L. growing at two saline sites in central Poland: a salty
meadow in the vicinity of a soda factory (anthropogenic salinity) and an area affected with
natural brine (landscape park).
Leaves, stems and roots were investigated separately. Total bacterial 16S rDNA- and nifHgene copy numbers as well as phylogenetic bacterial strain comparison showed both a plant
species specificity and soil salinity impact on the plant microbiome composition.
Higher bacterial density and diversity was detectable at test sites with higher salinity and
generally numbers of diazotrophic bacteria increased in the following order: leaves > shoots ³
roots. The ratio of nifH-gene to 16S rDNA-gene was higher at naturally saline test site (0.6)
compare to the test site with anthropogenic salinity (0.4). Most of isolated diazotrophs were
identified to the phylum Proteobacteria and Actinobacteria.
122
Reaction of perennial ryegrass (Lolium perenne L.)/Glomus spp. symbiotic
association on infection by Fusarium poae and Rhizoctonia solani
Malgorzata Jeske*, Dariusz Panka, Marcin Juda, Karol Lisiecki, Katarzyna Koczwara,
Monika Młynska
UTP University of Science and Technology, Poland
Perennial ryegrass is one of the most important grasses in Poland, used for pasture and for
turf. It is often attacked by numerous pathogens, which can decrease yield and its quality.
Use of fungicides for plants protection against diseases is often limited. The other way of
protection of perennial ryegrass can be use of arbuscular mycorrhizal fungi (AMF) of the
Glomus genus. These fungi belong to the most commonly occurring soil microorganisms of
the world and are associated with at last 80% of plants of the Earth. Arbuscular mycorrhizal
fungi increase the root absorptive area and hence the plant nutrition. Additionally,
AMF increase the tolerance of plants to heavy metals, water stresses, as well as pathogenic
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Root Endophytes
fungi. The exact mechanism of higher resistance of AMF infected plant is yet not fully
understood, but it can be assumed that PR (Pathogenesis-related) proteins have a significant
role. Detailed experiments in controlled conditions were conducted to determine the impact
of AMF on induction of specific defense mechanism, including production of PR proteins:
chitinases and β-1,3-glucanases in perennial ryegrass. Severity of plant’s infection was
estimated 2, 4, 6, and 8 days after inoculation with F. poae and R. solani. The level of
chitinases and glucanases in plants was determined 0, 2, 4, 6 and 8 days after infection with
use of Abeles et al. (1970) method with modifications. The specific activity of chitinases and
glucanases in the extracts was expressed as moles of reducing sugars released in one minute
of incubation per one milligram of total protein in the extract. Occurrence of Glomus spp.
affected the amount of chitinases and β-1,3-glucanases in perennial ryegrass. The amount of
enzymes was also dependent on the time after infection. Results indicate the role of PR
proteins in resistance of plants exposed to infection.
123
Genetic analysis of root endophytic Pseudomonas putida bp25 and chemoprofiling of its antimicrobial volatile organic compounds
Aundy Kumar*1, Neelam Sheoran1, Agisha Valiya Nadakkakath2, Vibhuti Munjal1, Aditi
Kundu3, Kesavan Subaharan4, Vibina Venugopal4, Suseelabhai Rajamma2, Santhosh
Eapen2
1
Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, India, 2Division
of Crop Protection, ICAR-Indian Institute of Spices Research, India, 3Division of
Agricultural Chemicals, ICAR-Indian Agricultural Research Institute, India, 4Division of
Crop Protection, ICAR-Central Plantation Crops Research Institute, India
Black pepper (Piper nigrum) associated bacterium BP25 was isolated from root endosphere of
apparently healthy cultivar, Panniyur-5 that protected black pepper plants against
Phytophthora capsici and Radopholus similis -the major production constraints. The bacterium
was genetically characterized and mechanisms of its antagonistic action against major plant
pathogens elucidated. The polyphasic phenotypic and genotypic analysis revealed its identity
as a strain of Pseudomonas putida (PpBP25). Multi Locus Sequence Typing revealed that the
bacterium shared gene sequences with several other isolates representing diverse habitats.
Tissue localization assays exploiting green fluorescence protein expression clearly indicated
that PpBP25::gfp endophytically colonized not only its host -black pepper, but also other
plants such as edible ginger (Zingiber officinale) and a model plant, Arabidopsis thaliana.
Coupled with PpBP25 colonies enumerated from internal plant tissues four weeks post
inoculation indicated its stable establishment and persistence in the plant system. Strikingly,
the bacterium inhibited broad range of economically significant plant pathogens
representing diverse taxonomic groups such as Phytophthora capsici; Pythium myriotylum;
Giberella moniliformis; Rhizoctonia solani; Athelia rolfsii; Colletotrichum gloeosporioides;
Magnaporthe oryzae; Ralstonia solanacearum; Xanthomonas axonopodis pv. punicae;
Xanthomonas oryzae pv. oryzae; and plant parasitic nematode, Radopholus similis by its
volatile organic compounds (VOCs). Gas Chromatography/Mass Spectrometry (GC/MS) based
chemical profiling revealed presence of Heneicosane; Tetratetracontane; Pyrrolo [1, 2-a]
pyrazine-1, 4-dione, hexahydro-3-(2-methylpropyl); Tetracosyl heptafluorobutyrate; 1,-3Eicosene, (E)-; 1-Heneicosanol; Octadecyl trifluoroacetate and 1-Pentadecene in PpBP25
76
Root Endophytes
metabolites. Dynamic head space GC/MS analysis of airborne volatiles indicated the presence
of many aromatic compounds such as Dimethyl trisulfide; Dimethyl disulphide; Pyrazine, 2,5dimethyl-; Pyrazine, methyl-; Pyrazine, 2-ethyl-5-methyl-; Pyrazine, 2-Ethyl-3,6-dimethyl;
Heptamethyl-2-nonene; β-Naphthol; Octadecyl vinyl ether; Tetradecane, 2,6,10-trimethyl;
Cyclobutene, 2-propenylidene-; Heptamethyl-1-nonene; 1,8-Nonadien-3-ol; Octadecanal,2bromo; Isoamyl alcohol; and 1-Undecene. The work paved way for profiling and consequent
identification of several broad spectrum anti-oomycetes, antifungal, antibacterial and
nematicidal VOCs in black pepper root endophytic bacterium for next generation plant
disease management.
124
Evaluation of ACC deaminase-producing endophytic bacteria isolated from
organic products by mung bean assay
Hiroaki Matsuoka*1, Yoshinari Ohwaki1, Junko Terakado-Tonooka2, Fukuyo Tanaka
Tanaka1
1
National Agriculture and Food Research Organization Agricultural Research
Center, Japan, 2Saga University, Japan
Bacterial endophytes have been associated with the growth promotion of various crops. In
particular, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-producing bacteria have
received widespread attention because of its ability to improve the stress tolerance of plants
by lowering ethylene level under environmental stress. We supposed that organic crops
which are exposed to frequent attacks of pathogen and pest may establish the beneficial
interaction with bacteria with such property. In this study, therefore, we isolate and evaluate
ACC deaminase-producing bacteria from organic crops in order to find new candidates of
biocontrol agents in practical use for cultivated crops.
Bacterial strains were isolated from the fruit or root of organic crops including carrot, aroid,
turnip, sweet pepper, and apple by using Dworkin–Foster minimal-salt medium with ACC as a
sole nitrogen source. The isolated strains were further selected from amplifying the ACC
deaminase synthase (acdS) gene by colony PCR. Moreover, the strains possessing acdS genes
were examined for enzyme activity by measuring the production of α-ketobutyrate from ACC
，and bioassay with growth-response of mung bean in the presence of ACC under
gnotobiotic condition.
Twenty-four strains were found to have acdS genes in all the 80 strains isolated on the
medium containing ACC. Of those, 10 strains were observed ACC deaminase activity by
enzyme assay. Among them, 4 strains were shown to promote the shoot elongation of mung
bean in the bioassay. Interestingly, the ACC deaminase activity in the isolated strains showed
different trends with growth response of mung bean. Our data indicate that selected bacteria
in organic crops produce ACC deaminase and have potential for plant growth promotion. We
will also report the results of inoculation of selected 3 strains into cultivated crop.
77
Root Endophytes
125
Influence of plant host species on the root associated microbial community and
functional redundancy of the root microbiome under phosphate starvation
Tatiana Mucyn*1, N. W. Breakfield2, G. Castrillo1, S. H. Paredes1, S. M. Yourstone1, C.
Hunter1, J. L. Dangle1
1
University of North Carolina, Chapel Hill, NC, USA, 2NewLeaf Symbiotics, St Louis,
MO, USA
Plants harbor a specific and complex microbiota at the surface of the root (rhizosphere) and
within the root (endophytic compartment) which influences plant health and productivity.
Various factors such as soil type, abiotic/biotic stress, host developmental stage, and host
species shape the root microbiome. The extent to which the host selects its root microbial
community remains poorly understood. To determine the influence of plant host species on
the assembling of both the rhizospheric and endophytic microbiome, we are comparing the
well-studied bacterial root bacterial microbiome of Arabidopsis thaliana Col-0, with those of
two monocot species Brachypodium distachyon Bd21 and Setaria viridis A10-1 from plants
grown in the same wild soil, using high-throughput bacterial 16S rDNA, as well as fungal
internal transcribed spacer 2 profiling. We are characterizing both core and species-specific
root microbiomes and are exploring the hypothesis that plants more closely related to each
other share more similar microbial communities. The Dangl laboratory has isolated ~600
bacterial strains from the rhizoplane and endophytic compartments of Arabidopsis thaliana
and is evaluating the effect of these isolates on plant health under various nutrient starvation
stresses. This bacterial collection is currently being tested on Setaria viridis focusing on the
identification of isolates that rescue phosphate starvation stress to establish to whether the
core microbiome may also present functional conservation across species. We are in parallel
establishing a collection of bacterial strains isolated from the root of Setaria viridis to refine
our characterization of both the core and potential species-specific microbiome.
126
ACC deaminase-expressing endophyte increases plant tolerance to FD
phytoplasma infection
Elisa Gamalero1, Elisa Bona1, Giorgia Novello*1, Cristina Marzachì2, Luciana Galetto2,
Flavio Veratti2, Nadia Massa1, Bernard Glick3, Simone Cantamessa1, Giovanni
D'Agostino4, Graziella Berta1
1
Università del Piemonte Orientale, DiSIT, Italy, 2CNR, Italy, 3University of
Waterloo, Canada, 4Mybasol srl, Italy
Flavescence dorée (FD) is an epidemic yellows disease of grapevine caused by a phytoplasma
(FDP), living in the phloem for which there is currently no cure. We have previously shown
that treatment of plants with rhizobacteria leads to mitigation of phytoplasma-induced
disease. The alleviation of a variety of disease symptoms by rhizobacteria may involve the
reduction of the stress-related plant ethylene via the 1-aminocyclopropane-1-carboxylate
(ACC) deaminase.
We tested whether the endophytic bacteria Pseudomonas migulae 8R6, able to synthesize
ACC deaminase, can limit the damages induced by FDP. Since FD-infected grapevines show
symptoms the year after infection or later, the FDP host plant periwinkle was used as model.
The use of a mutant of strain 8R6, lacking the ability to produce ACC deaminase, may
78
Root Endophytes
demonstrate the involvement of ethylene in the development of FDP symptoms in the plant.
The strain 8R6 colonized the internal tissue of periwinkle, thus confirming its endophytic
aptitude and significantly reduced the number of symptomatic plants (53% vs. 93%). A lower
number of symptomatic plants was also detected in plants inoculated with the 8R6 mutant
compared to controls (73% vs 93%) thus suggesting that ACC deaminase is not the only
mechanisms involved in the increased tolerance to FD infection. Quantification of
phytoplasma inside the leaves was not affected by both bacterial strains. However, FD
phytoplasma title was under the quantification threshold in 38% of plants inoculated with the
strain 8R6. This value was more than what recorded for FD infected plants (14%). ACC
deaminase activity seems to be involved, as FD phytoplasma population was under the
quantification threshold in 8% of the plants inoculated with the mutant. In conclusion, ACC
deaminase activity of P. migulae 8R6 might help the plant to regulate the level of the stressrelated hormone ethylene, potentially leading to improved tolerance to phytoplasma
infection.
127
The roles of root colonizing bacterial endophytes on suppression of Fusarium
oxysporum f. sp. cucumerinum and growth promotion of cucumber plants
Hatice Ozaktan*, Ayşe Gul, Bi̇ rsen Çakir, Lalehan Yolageldi, Mustafa Akbaba, Sahika
Akat
Ege University, Turkey
The aim of this study was to find that endophytic bacterial (EB) isolates obtained from healthy
cucumber plant tissues could promote the plant growth and marketable fruit yield and
suppression of soilborne plant pathogen of cucumber plants caused by Fusarium oxysporum
f. sp. cucumerinum (FOC). Cucumber plants with treated EB as seed coating and soil drenching
(108–109 CFU ml-1). were transplanted to peat inoculated with FOC spore suspension (105 spore
ml-1
). According to growth chamber test results, 38% of tested EB strains exhibited the disease
reduction between 30 to 60 %, comparing to only FOC inoculated plants. In the next step of
this research, EB strains applied as seed coating and soil drenching inhibited the Fusarial wilt
development at the rate of 49 to 52% compared to only FOC inoculated cucumber plants for
two months growing period in soilless growing system. Moreover, EB treatments, without
being any disease pressure, increased the total marketable fruit yield at the rate of 5 to 28%
compared to non-treated cucumber plants for 2 months growing period in soilless cultivation.
The population density of some EB strains in the root tissues was determined as
approximately 103-105 CFU plant g-1 75 days after seed bacterization. It was determined that
colonization rate of EB strains in the root tissues was higher than shoot tissues. So, the
application of EB as seed coating and soil drenching was recorded as effective and practical
techniques in terms of biological control, growth promotion of cucumber plants and root
colonization of bacteria inside the plant tissues.
79
Root Endophytes
128
Siderophore production and iron phosphate solubilization by root endophytic
bacteria isolated from maize
Vitoria Palhares*1, Ivanildo Marriel2, Eliane Gomes2, Ubiraci Lana2, Crisia Abreu3, Cássia
Almeida1, Christiane Oliveira2
1
UniFEMM, Brazil, 2Embrapa Mayze and Sorghum, Brazil, 3Federal University of Minas
Gerais, Brazil
Tropical soils generally exhibit acidic condition with predominant phosphate immobilized in
insoluble forms of iron (P-Fe). Many soil microorganisms solubilize P from P-Fe by
siderophores and organic acids production. The microorganisms that live in the rhizosphere
and in the interior of plants (facultative endophytic) with potential for phosphate
solubilization are important to enhance the production of the maize crop in order to reduce
the use of soluble fertilizers. The objective of this work was to isolate efficient facultative
endophytic bacteria solubilizing iron phosphates and evaluate their production of
siderophores. Maize plants were harvested at flowering stage and 113 bacteria were isolated
from roots (54.9%), leaves (20.4%) and sap (24.8%). Fifty-eighty selected strains, most root
endophytic, were evaluated in liquid culture medium containing iron phosphate, after 9 days
of incubation. Soluble phosphorus was determined using modified ammonium molybdate
method. The production of siderophores was through inoculation of microorganisms in solid
medium containing the indicator cromoazurol. Strains were identified based in the 16S rDNA
gene. Maize plants had a high diversity of endophytic bacteria solubilizing iron phosphate
and siderophore producing. One strain of Pantoea and two of Bacillus showed the highest P
solubilization, releasing 68.7; 64.1 and 64.08 mgP.L-1, respectively. The siderophore produced
by 65% of the evaluated microorganisms was the type carboxylate, and the most strains
producing siderophores are efficient in solubilizing P associated with iron. The use these
facultative endophytic solubilizing microorganisms as inoculant may be considered a
promising strategy in environmental and economic terms for maize production.
129
Effect of GFP-labeled Paenibacillus polymyxa on growth of agricultural crops
Kiran Preet Padda*, Akshit Puri, Christopher Chanway
University of British Columbia, Canada
Paenibacillus polymyxa strain P2b-2R, previously isolated from internal stem tissue of a
naturally regenerating pine seedling, fixes nitrogen in association with lodgepole pine
seedlings and promotes their growth. This strain has been shown to colonize lodgepole pine
seedling tissues endophytically using a green fluorescent protein (GFP)-labelled derivative of
the wild type.
We wanted to see if the GFP labelled derivative of P2b-2R would fix N and promote growth
of agricultural crops such as corn and canola in ways similar to the wild type strain.
We inoculated corn and canola seeds with wild type P2b-2R or the GFP labelled derivative
and grew seedlings for 40 days in an N-limited soil mix. Seedlings were harvested 20, 30 and
40 days after inoculation and evaluated for biological nitrogen fixation and growth
promotion.
80
Root Endophytes
Seedlings inoculated with the GFP labelled P2b-2R strain derived small amounts of N from
the atmosphere (up to 17%) but were 40% taller and accumulated 70% more biomass than
those treated with wild type P2b-2R. We conclude that GFP modification of strain P2b-2R
resulted in a significant enhancement of the wild type’s growth promotion efficacy on corn
and canola and facilitated some in situ biological nitrogen fixation with these two crop
species.
130
Next generation sequencing analysis of soil and plant associated fungal
assemblages in sub-Arctic sand dune ecosystem
Anbu Poosakkannu*, Riitta Nissinen, Minna-Maarit Kytöviita
University of Jyväskylä, Finland
Our research focusses on plant associated microbial communities in the sub-arctic inland
primary successional site, located in an Aeolian sand dune area in subarctic Northern
Fennoscandia (68° 29' N). One of the very few species capable of colonizing these Arctic sand
dunes and enabling ecosystem restoration is the grass Deschampsia flexuosa. In the study
site, the early successional stage is characterized by D. flexuosa growing as monoculture in
the blow-out areas. Late successional stage is mountain birch forest vegetation with
continuous ground cover vegetation composed of abundant D. flexuosa together with other
plants and under the cover of mountain birch trees.
This goal of this study was to assess the community composition of fungal assemblages in
soil (bulk and rhizosphere) and in the internal tissues (endophytes) of D. flexuosa growing in
the two different successional stages. Endophytic fungal studies in cold environment
especially in arctic are very limited, although they can improve host nutrient acquisition and
protect the plants from different stresses.
Deschampsia flexuosa (leaf and root), rhizosphere and bulk soil samples were collected from
four different blow-out areas between 150 and 2250 meters apart. We collected eight
biological replicates (two samples per blow out area and successional stage). Soil and plant
associated fungal community structure was studied using Next generation Ion torrent
sequencing of partial internal transcribed region (ITS) amplicons. Mothur based
bioinformatics analysis of ITS sequences revealed 99217 quality-filtered sequences which are
abundant (OTUs with less than 10 sequences were removed) that were separated into 2781
species-level OTUs. Of fungal taxa, Ascomycota (67.1%), Basidiomycota (18.9 %),
encompassed the largest proportion of OTUs. The phylum Ascomycota was the predominant
phylum identified and present in all samples followed by Basidomycota, Zygomycota and
Glomeromycota. Endophytic samples are dominated by phylum Ascomycota. The
compositional variation was mainly accounted for by successional stages.
81
Root Endophytes
131
Can an endophyte isolated from lodgepole pine trees reside inside agricultural
crops and fix N?
Akshit Puri*, Kiran Preet Padda, Christopher Chanway
University of British Columbia, Canada
Several Paenibacillus strains that were able to fix nitrogen were isolated from extracts of
surface-sterilized lodgepole pine seedling and tree tissues. One strain, Paenibacillus Polymyxa
P2b-2R, was found to fix high amounts of nitrogen when reintroduced to the gymnosperms,
lodgepole pine and western red cedar.
We wanted to determine if Paenibacillus polymyxa P2b-2R could colonize, fix N and promote
the growth of important agricultural crops such as corn and canola.
We inoculated corn and canola seeds with P. polymyxa strain P2b-2R and grew seedlings for
40-60 days. Corn seedlings were harvested 10, 20 and 30 days after inoculation and canola
seedlings were harvested 20, 40 and 60 days after inoculation for evaluation of endophytic
and rhizospheric colonization. Seedlings were also evaluated for biological nitrogen fixation
and growth promotion at these harvest intervals.
P2b-2R colonized rhizosphere as well as inside tissues of root (endophytically). Corn and
canola seedling growth was promoted significantly by inoculation with P2b-2R with an
increase of up to 35% in height and 30% in biomass from the controls. P2b-2R also provided
more than 20% of foliar nitrogen. These results suggest that Paenibacillus polymyxa P2b-2R
has a broad range of plant hosts and is able to fix N and promote the growth of at least
certain agricultural crops.
132
Maize roots endophytic bacteria and their potential as plant growth promoting
and biological control agents
João A. C. Vieira1, Natalia L. S Alves1, Christiane A. O. Paiva2, Vera Lúcia Santos*1
1
Laboratory of Applied Microbiology, Microbiology Department, Institute of Biological
Science, Federal University of Minas Gerais, Brazil, 2Embrapa Milho e Sorgo Empresa
Brasileira de Pesquisa Agropecuária (Embrapa), Brazil
Studies have shown that many endophytic microorganisms, which lives asymptomatically
within plant causing no signs of harm to the host, may work as plant-growth promoters
and/or biocontrol agents which has made them valuable for agriculture for improving crop
performance. In this work, endophytic bacteria were isolated from roots of Pioneer 30F35
Herculex hybrid corn crops, cultivated with and without phosphorus fertilization. After
identification by partial sequencing of the 16S rDNA gene, a total of 80 bacteria was
evaluated regarding solubilization of inorganic phosphate (CaPO4), antagonizing bacterial
(Bacillus subtilis and Pantoea ananatis) and phytopathogenic fungal (Fusarium verticillioides
and Coletotrichum graminicola) growth, mineralization of phytate and production of IAA
(indole-3-acetic acid). There was an equal distribution of the isolates from Actinobacteria,
Firmicutes and Proteobacteria clades, and only one isolate (Flavobacterium acidificum RT3B41) of Bacteroidetes phylum was found. The strains were grouped into 26 genera, which the
82
Root Endophytes
most frequent were Bacillus, Leuconostoc, Pseudomonas, Serratia and Enterobacter. From the
total, 32 isolates were able to solubilize inorganic phosphate (dosages between 10 and 527
mg/l) and 45 showed production of IAA (4.5 to 111 μg/ml). In the plate antagonism test, 10
isolates inhibited the growth of B. subtilis, 6 of the gram negative pathogen P. ananatis, 11 of
the fungus C. graminicola and 4 of F. verticillioides. In plate qualitative tests, 52 isolates also
had the ability to mineralize phytate. This work demonstrates the enormous potential
application of these isolates, which must still be confirmed by in vivo and field tests.
133
Development of a bacterial cell enrichment method for the analysis of the
endophytic microbiota in sugarcane stems
Stefan Schwab*1, Carlos dos Santos2, Daniel de Souza2, Dayana Rosa2, José Baldani1
1
Embrapa Agrobiologia, Brazil, 2Universidade Federal Rural do Rio de Janeiro, Brazil
Sugarcane is an important culture in Brazil, with high economic support and social relevance.
Sugarcane plants are rich in endophytic bacteria, which can promote and modulate plant
growth through diverse mechanisms, such as phytohormone production, antagonistic activity
against phytopathogens, and improvement of nutrient utilization by the plant; however, most
bacteria are yet uncultivable. Therefore, cultivation-independent strategies are essential to
better understand their genetic diversity and functional characteristics. Metagenomic surveys
of endophytic microbiota currently represent a challenge, due to the low number of bacterial
cells in relation to the host plant. In this work a bacterial cell enrichment procedure was
established, cultivation-independently, from the inner tissues of the base of sugarcane stems.
Results showed that the enriched material contained bacterial cells that are colony-forming,
and can be visualized by bright-field microscopy or Gram stain test. DNA content analysis of
the enriched material revealed efficient elimination of plant DNA, and results of PCR and
ARDRA showed that bacterial DNA is predominant. Preliminary results of 16S rDNA
sequencing revealed the presence of alpha-, beta- and gamma-Proteobacteria. In order to
obtain a deeper insight of the bacterial taxa, 16S rDNA sequencing of the enriched material on
Illumina platform is being conducted, and results will be presented. The developed
method may allow, in the future, accessing the sugarcane endophytic microbiome, and reveal
bacterial genetic resources with agrobiotechnological applications.
134
Indirect stimulation of the vegetative growth of ‘Elkat’ strawberry plants by the
bacterium Pantoea sp. (N52AD)
Pawel Trzciński*, Lidia Sas Paszt, Edyta Derkowska, Michał Przybył
The Research Institute of Horticulture, Poland
The most important problems of agriculture are: increasing the abundance of macro- and
microelements in the soil, maintaining adequate moisture levels, and protection from
diseases and pests. Excessive use of fertilizers (mineral and manure) and pesticides may
reduce soil fertility and biodiversity, and contaminate surface and ground waters. One way of
reducing the use of fertilizers and chemical plant protection products is to employ beneficial
microorganisms which can decrease the number of pathogens in the soil, increase the
availability of macro- and microelements to plants, and improve the resistance of plants to
83
Root Endophytes
stress caused by, for example, drought. Among the most common and universal
microorganisms used are arbuscular mycorrhizal fungi. By colonizing the roots of the host
plant, these fungi increase the uptake of water, macro- and microelements by the plant and
protect it from soil-borne pathogens. The degree of the symbiosis is affected by some
microorganisms, for example, mycorrhiza helper bacteria. Application of such bacteria can
improve the colonization of plant roots by autochthonous mycorrhizal fungi or those present
in commercial products, thus allowing plants to make a more efficient use of nutrients and
consequently contributing to a reduction in the amounts of the fertilizers and pesticides
used.
The aim of this study was to evaluate the impact of the bacteria from the genus Pantoea
(strain N52AD) on the degree of colonization of strawberry roots by mycorrhizal fungi and on
the vegetative growth of strawberry plants. Frigo plantlets of the cultivar Elkat were planted
in pots filled with the soil collected from an experimental field in Dąbrowice. In the
experiments conducted in 2012-2013 in greenhouse conditions, the plants inoculated with
the N52AD strain were colonized by arbuscular mycorrhizal fungi to a greater extent and
produced a greater mass of leaves than the control plants (non-inoculated with N52AD
strain).
135
Improvement of soil fertility and plant production in the Democratic Republic of
Congo by implementation of arbuscular mycorrhizal fungi and Sebacinales
Jolien Venneman*, Danny Vereecke, Geert Haesaert
Department of Applied Biosciences, Faculty of Bioscience Engineering, University
Ghent, Belgium
A growing world population is associated with an increased demand for food. This trend is
most pronounced in developing countries where the availability of fertile arable land is
becoming rather limited. An important reason is the typical physical and chemical properties
of tropical soils, e.g. P-fixation and low cation exchange capacity (CEC), in addition to soil
degradation caused by unsustainable agricultural practices.
Since the access to mineral fertilizers is very restricted in tropical regions, the mentioned
problems should be addressed through the application of an integrated soil fertility
management. This includes the use of sustainable cultivation techniques in combination with
adapted plant genetic material, micro-doses of chemical fertilizers, and maximal amounts of
organic matter. In our research, we particularly focus on the implementation of growth
promoting micro-organisms, which are isolated from soil and plant root samples collected in
the Democratic Republic of Congo, region of Kisangani. Arbuscular mycorrhizal fungi and
basidiomycetes belonging to the order Sebacinales are two groups of root endophytes that
attracted our attention. To reveal the mechanisms behind the plant-fungus interactions we
combine molecular diversity analysis with functional tests. In addition, we plan to set up onfarm based systems for mass production of inoculum in Kisangani in order to provide local
farmers with the knowledge on how to maintain the beneficial indigenous micro-organisms.
84
Root Endophytes
136
Monitoring and quantifying Bacillus mycoides in the potato rhizosphere
Yanglei Yi*, Jan Spoelder, Marielle van den Esker, Oscar Kuipers
University of Groningen, Netherlands
Bacillus mycoides is a rod-shaped soil bacterium belonging to the B. cereus species-group. On
agar plates, a fungus-like shape forms resulting from cells linked end to end. B. mycoides is
reported to have plant growth-promoting effects on sugar beet, cucurbits and tobacco. B.
mycoides is believed to be an important bacterium for potato growth as well, due to its
abundance in potato rhizosphere and endosphere without causing visible signs of infection.
Unraveling mechanisms mediating plant host-endophytes recognition, establishment and
colonization dynamics requires a reliable method to monitor these processes. Visualization of
cells in the rhizo-/endo-sphere using fluorescent protein (FP) as a marker has been adopted
by many researchers to study plant-microbe interactions. However, many laboratories
struggle with incorporating foreign DNA into Gram-positive bacteria including some wild
isolates of Bacillus. Here, we describe a method to transform the microbe by electroporation
with a plasmid encoding a constitutive promoter FP, resulting in fluorescent cells.
Combination of quantitative and qualitative data achieved by both molecular and microscopy
methods are probably the best choice to monitor endo- and rhizo-bacteria, with the
advantages of each technique complementing the drawbacks of the other.
85
Root Endophytes
Root Development
137
Architectural analysis of date palm root systems (Phoenix dactylifera L.)
Mimoun Asma*1, Stokes Alexia2, Rey Hervé3, Lecoustre René3, Jourdan Christophe4,
Bennaceur Malika1
1
Oran 1 University, Algeria, 2INRA, Montpellier, France, 3AMAP/CIRAD
BIOS, France, 4CIRAD-Eco&Sols, France
Architectural traits were used to describe the structure and development of the date palm
(Phoenix dactylifera L.), root system. To characterize root system architecture, two parallel
experiments were conducted: one in a rhizotron whereby root growth was measured over
time and one in a nursery bags from which roots were sampled regularly. These roots were
sectioned and examined under a light microscope in order to determine how the anatomical
structure of roots changes as they age. Double staining was performed on roots at different
ages and from diverse root zones. The topology (arrangement of axes relative to each other)
and typology (classification of different root axes based morphological and functional
criteria) of root systems was measured in the first rhizotrons. Root diameter and root
elongation were measured regularly on seedlings grown from seed (0 to 6 months).
RhizoDigit software (©CIRAD) was used to digitize root observations and to compile data for
statistical analyses. Results showed that during this developmental period, the root system
architectural unit comprises seven root types having distinct characteristics and spread over
three different topological orders (primary, secondary and tertiary). The establishment of
different types of root axes evolves over time and depends on the root category. Root
growth was variable depending on the root type and branching order of the diameter
according to its class. There was an absence of both radical mortality and the arrest in growth
of short secondary roots during this period. The anatomies of the radical and adventitious
roots were typical of monocotyledonous roots. Radical anatomy also had a characteristic
spatial and temporal development during the juvenile stage. We will use these results to
develop three-dimensional models for use in agricultural management.
139
Root architecture phenotyping of different quinoa (Chenopodium quinoa Willd)
accessions
José Correa*1, Phil Pstrong2, Francisco Pinto2, Kurt Ruf1, Iván Matus1, Kerstin Nagel2,
Fabio Fiorani2, Manuel Pinto1
1
Instituto de Investigaciones Agropecuarias, Chile, Chile, 2Forschungszentrum
Jülich, Germany
Quinoa has received an increasing attention because of its nutritional value, adaptability to
different environmental constraints, and its vast biodiversity. All these crop aspects are
relevant for global food security. Root system architecture plays an important role in
determining the ability of a plant to explore the soil and foraging for water and nutrients.
These aspects linked to root architecture are not yet well characterized in quinoa accessions
and genetically diverse panels. According to that, two pilot experiments were carried out to
evaluate the applicability of a rhizotron system for root assessment in quinoa. Plants were
grown under greenhouse conditions in rhizotrons at IBG2: Plant Sciences at
86
Root Development
Forschungszentrum Jülich GmbH, Germany. The first experiment focused to establish whether
the visible root traits at the interface of the rhizoboxes correlate with root and shoot traits
measured destructively and the second one to analyze the effect of water stress on root
architectural parameters. To represent the diversity of quinoa, three Chilean wild ecotypes
and three commercial varieties graciously supplied by Wageningen breeding program, were
used. During the second experiment the water stress treatment was performed on the more
vigorous ecotypes. The following traits were measured: length of primary, lateral and tertiary
roots; root system depth, width and area; and root dry weight. Correlations and differences
among ecotypes for all traits were found. In addition, the effect of water stress on root
architecture was mainly manifested by a decrease of root system length, width, laterals, and
area. In conclusion, there are relationships between traits measured in rhizotrons and traits of
the plant/root system allowing analyzing the variation observed in the plant/root system and
providing details about the effect of water stress on root development. These findings
indicate that protocols for screening germplasm in pre-breeding scenarios could be
developed specifically for quinoa.
141
First root allometric model for shifting cultivation systems in PDR Lao
Iain McNicol1, Nicholas J Berry1, Thilde B Bruun2, Andreas de Neergaard*2, Casey Ryan1
1
University of Edinburgh, Scotland, 2University of Copenhagen, Denmark
Shifting cultivation remains an important land use across of Southeast Asia and other parts of
the tropics. This practice creates complex mosaic landscapes with regrowing fallows of
various age interspersed with active fields and patches of mature forest. Quantifying root
carbon stocks in these secondary forests is limited by the availability of reliable allometric
models for these systems, inhibiting the development of policies aimed at reducing the
intensification of shifting cultivation systems (REDD+). We developed new allometric models
for prediction of both tree stem and root biomass in shifting cultivation systems based on a
destructive harvest of 150 trees from Luang Prabang Province, Laos PDR. This study is the
first to develop allometric models of root biomass for shifting cultivation landscapes, which
we hypothesised would be a major carbon pool given that resprouting, and associated high
root biomass, is a common physiological trait. Tree height was less important for estimating
root biomass with “diameter only” models performing best. Resprouting trees exhibited
significantly greater root biomass compared to trees growing from seed. Our best-fit
allometric models were subsequently applied to 12 nearby plots which span a
chronosequence of fallows to calculate the impact of accounting for resprouting allometry on
forest biomass. We found that root biomass stocks (Mg/ha) were around 58% (22 - 85%)
higher after accounting for resprouting, resulting in an average 9.5% (4 - 13%) increase in
total biomass stocks, thus demonstrating the need to correctly account for re-sprouting trees
in shifting cultivation fallows. Our analysis suggests that using our models will substantially
improve the accuracy of tropical estimates of tree biomass and its distribution among
different pools in shifting cultivation fallows. We also find that models fit using non-linear
regression provide equally good fits to the data compared to the traditional approach of logtransforming biomass data.
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145
Root dynamics of two grapevine cultivars differing in their hydraulic behavior
Jhonathan Ephrath*1, Brian Hoefgen2, Uri Hochberg2, Shimon Rachmilevitch2
1
Ben Gurion University of the Negev, Jacob Blaustien Inst. for Desert
Research, Israel, 2Blaustein Institutes for Desert Research, French Associates Institute for
Biotechnology and Agriculture of Drylands, Ben Gurion University of the Negev, Israel
The interaction between scion and rootstock in grafted grapevines is extensively investigated.
Nonetheless, the effect of canopy on root morphology has not been widely tested in field
conditions. We tested root morphology in response to two irrigation regimes for two
hydraulically different cultivars Shiraz (SH) and Cabernet Sauvignon (CS) grafted onto the
same rootstock, 140 Ruggeri. These two grapevine cultivars have different aboveground
hydraulic behavior (SH near-anisohydric and CS near-isohydric), and it is assumed that these
differences may also cause belowground differences.
This research was conducted in a four year old experimental vineyard at Ramat Negev, Israel.
The grapevines underwent different irrigation regimes (50% and 25% of crop
evapotranspiration). Aboveground and belowground physiological parameters were
measured for a period of 16 months. Transparent tubes were installed to a depth of 180 cm
at distances of 25 and 75 cm from the trunk. Root photographs were taken during different
developmental stages of the year using a minirhizotron.
Our results indicated that scion hydraulic behavior affected root dynamics. Larger differences
were seen in SH than compared to CS in response to the irrigation treatment. It seems that
SH invested more growth directly below the trunk, while CS roots were more evenly
distributed throughout the soil profile.
146
Modelling of root dynamics in split-root rhizoslides reveals strong selective root
placement of maize in response to nitrogen
Dina in 't Zandt*1, Chantal Le Marié2, Norbert Kirchgessner2, Eric J.W. Visser1, Andreas
Hund2
1
Radboud University Nijmegen, Netherlands, 2Swiss Federal Institute of Technology
Zurich, Switzerland
The plant’s root system is highly plastic, and can respond to environmental stimuli such as
high nitrogen in patches. A root generally responds to a nitrogen patch by selective
placement of new lateral roots within the patch to increase nitrogen uptake efficiency. This is
a desirable trait in breeding programs, since it may decrease NO3 - leaching and N2O
emission. Roots of maize (Zea mays L.) were grown without nitrogen in split-root rhizoslides,
a system that enables non-destructive root measurements via direct imaging of the root
system. Half of the root system was subjected to high nitrogen after 15 days, and root growth
was traced for a subsequent 15 days and modelled. The elongation rates of crown axile roots
on the nitrogen-treated side followed a logistic increase to a 5.3 cm d-1 maximum, 95% of
which was reached within 4 days. On the untreated side, axile root elongation dropped
linearly to 1.2 cm d-1 within 6.4 days and stayed constant thereafter. Twice as many lateral
roots were formed on the crown axis on the nitrogen side compared to the untreated side.
Furthermore, the elongation rates of laterals exposed to high nitrogen increased linearly with
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most of the roots reaching an asymptote approximately 8 days after start of the nitrogen
treatment. The laterals without nitrogen did not elongate. Furthermore, it was shown that the
crown root system had a greater influence on shoot performance than the seminal root
system 15 days after nitrogen application.
147
Effect of phenanthrene exposure on apoplastic barriers formation in maize
Pierre Leglize*, Joan Dupuy, Quentin Vincent, Ivan Zelko, Christian Mustin, Stéphanie
Ouvrard, Thibault Sterckeman
Université de Lorraine, France
Management of Polycyclic Aromatic Hydrocarbons (PAH) contaminated soils through
phytoremediation process is promising in controlled conditions. However, in situ experiments
were not so successful due to plant growth limitation induced by PAH toxicity. Improving
revegetation of contaminated site appears therefore crucial in phytoremediation application
but requires a better understanding of contaminant’s impact on plant functioning. PAH
exposure affects plant physiology such as mineral nutrition. Because the nutrient uptake is
linked with the maturation of exodermis and endodermis, we focused on (i) the effect of PAH
on the suberin lamellae formation and (ii) the localization of phenanthrene in root. Maize
plants were grown in contaminated sand (50 and 150 mg PHE kg-1 dry sand) for 10 or 20
days. Epi-fluorescence microscopic observation of root sections was used to assess PHE
localization within the root and maturation of exodermis and endodermis by Fluorol yellow
088 detection of suberin.
For 10 days of cultivation, suberization of exodermis and endodermis of maize exposed to
PHE was more extensive and PHE were only observed within suberized exodermis. This could
be related to an inducer effect of PHE on the deposition of hydrophobic compounds within
exodermis, which may act as barrier against PHE penetration. However after 20 days of
exposure, exodermis and endodermis of non-exposed roots were totally suberized whereas
PHE-exposed roots where less suberized. After 20 days, PHE seemed to inhibit root
maturation, which could be caused by its toxicity as it is related with the plant biomass
reduction. Furthermore, PHE patches were located only within suberized exodermis and
endodermis, which may therefore act as retention zone where the hydrophobic PHE
accumulates during its radial transport.
148
Responses of root development and zinc uptake to heterogeneous soil water
distribution in lupin and cucumber
Huifang Ma*1, Michael Wassilios Evangelou1, Peter Vontobel2, Rainer Schulin1
1
ETH Zurich, Institute of Terrestrial Ecosystems, Switzerland, 2Paul Scherrer
Institute, Switzerland
The distribution of moisture is typically heterogeneous in soil even at the scale of a root
system, and plant root systems are known to adapt to such heterogeneity. In this study we
investigated how root growth allocation of two common crop plants, white lupin (Lupinus
albus) and cucumber (Cucumis sativus L.), responded to lateral heterogeneity in soil moisture
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over time with neutron radiography and how this affected zinc (Zn) uptake in a low-Zn sandy
soil with patchy Zn distribution. Single plants were grown in Al-containers (inner size: 27 cm
length × 27 cm height × 1.2 cm thickness). To one lateral section of each container 20 mg
Zn/kg were added. Soil water heterogeneity was achieved by mixing coarse sand into the soil
in one lateral section of the respective containers, either on the same (cis) side as or on the
side opposite (trans) to the Zn-enrichment, while no sand was added to control containers.
The same amount of water was added to all containers. As a result the soil water content
decreased in the order: sections without sand in heterogeneous containers > control
containers > sections with sand in heterogeneous containers. In lupin, root growth (primary
root length, cluster root number and length, cluster root length/total root length, and root
dry weight) was reduced in the heterogeneity treatments, with fewer roots being produced in
sand than in no-sand sections, while Zn uptake was not affected. In cucumber, the
heterogeneity treatment increased root growth (root length density and total dry weight) and
shoot Zn uptake. Lupin roots responded already after 6 days to the heterogeneity treatment,
cucumber roots only after 35 days.
149
Effect of drought on surface and deep root dynamics of teak (Tectona grandis)
and rubber (Hevea brasiliensis) trees in mainland South East Asia
Jean-Luc Maeght*1, Corentin Clément2, Santimaitree Gonkhanmdee3, Oloth
Sengtaheuanghoung4, Alexia Stokes5, Alain Pierret6
1
UMR Iess Paris ; IRD, France, 2Iess Paris IRD Nafri, Lao People's Democratic
Republic, 3Faculty of Agriculture, Thailand, 4NAFRI, Lao People's Democratic
Republic, 5INRA UMR-AMAP, France, 6UMR Iess Paris ; IRD - NAFRI, Lao People's
Democratic Republic
We present the main results of two experiments conducted in a teak (Tectona grandis) tree
plantation in Northern Laos and a rubber (Hevea brasiliensis) tree plantation in North East
Thailand. We aimed at documenting the response of fine roots to seasonal and induced
drought and quantify the corresponding carbon stocks.
Fine root growth of 20-year old teak trees and 18-year old rubber trees were monitored at
monthly intervals over 2 and 3 years, respectively. Observations were made using root
windows installed at 50 cm depth increments, to a depth of 4.5 m. A rainfall exclusion
experiment was performed in the teak plantation. Destructive samples were used to quantify
fine root-related carbon stocks.
In the case of teak, fine roots were found to a depth of 12 m. In both species, root dynamics
is linked to transient changes in water availability. This work shows that deep fine roots are
organs of foremost functional importance and we demonstrate their instrumental role in
deep-water extraction during critical dry periods. The presence of significant amounts of
deep roots in the soil profile further indicates that they should be taken into account when
quantifying long-term carbon storage.
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150
The characteristics of root cell components of plants adapted to acidic soil
Eriko Maejima*1, Toshihiro Watanabe1, Tadao Wagatsuma2, Mitsuru Osaki1
1
Research faculty of Agriculture, Hokkaido University, Japan, 2Faculty of Agriculture,
Yamagata University, Japan
Melastoma malabathricum and Melaleuca cajuputi naturally grow in highly acidic soil in
tropical and subtropical zones. They are both reported to be highly aluminum tolerant, but
their tolerance mechanisms still remain to be explained. Melastoma malabathricum
accumulates high concentration of aluminum, whereas Melaleuca cajuputi accumulates low
concentration of aluminum. Plasma membrane and cell wall compositions, organic acids
released from root tips, and/or phenolics in root cells have often been reported to affect
aluminum tolerance and aluminum accumulation in roots. In this study, we focused on woody
species in Melastomataceae and Myrtaceae and investigated their root cell components
possibly responsible for aluminum tolerance and/or accumulation.
Melastoma malabathricum (Melastomataceae), Tibouchina urvilleana (Melastomataceae), and
Melaleuca cajuputi (Myrtaceae) were grown in hydroponic culture and transferred to an
aluminum treatment solution (phosphorus-free nutrient solution containing 0 or 500 μM
AlCl3) for 1 week. Aluminum concentration in roots and leaves, lipid and cell wall
composition, and organic acid and phenolics concentrations in roots were determined. We
also investigated rice (Oryza sativa L.), an aluminum-tolerant crop, for comparison.
Aluminum concentration in Melastoma malabathricum and Tibouchina urvilleana was higher
than that in Melaleuca cajuputi. Melastoma malabathricum and Melaleuca cajuputi contained
much lower proportion of phospholipid in root cells than that in rice, suggesting that the
lipid composition in the plasma membrane in root cells is involved in aluminum tolerance
mechanisms in such highly tolerant plants. Moreover, all woody plants investigated contained
the highest concentration of phenolics compared with that in rice. Although Melaleuca
cajuputi contained highest concentration of phenolics in roots, the extracted phenolics did
not have the capacity to chelate aluminum. In contrast, phenolics extracted from Melastoma
malabathricum formed a stable chelate with aluminum. These results suggest that phenolics
in roots are likely to be involved in the detoxification of aluminum in roots of Melastoma
malabathricum.
151
Root system of crops under different soil management systems in sugarcane
field reform
Gustavo Mateus*1, Felipe Giglio Bernardoni2, Denizart Bolonhezi2, Rafael Müller3,
Carlos Alexandre Costa Crusciol3, Humberto Sampaio Araújo2
1
São Paulo Agency of Agribusiness Technology, Brazil, 2São Paulo Agency of
Agribusiness Technology - APTA, Brazil, 3Department of Crop Science, College of
Agricultural Sciences - FCA, Sao Paulo State University - UNESP, Brazil
Sustainable agricultural production systems that improve the quality of soil and water and
reduce the emission of greenhouse gases, are essential in modern agriculture. This work was
developed in the São Paulo Agency of Agribusiness Tecnhology, Andradina, São Paulo, Brazil.
The aim of this study was to evaluate the effect soil management systems and different
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rotation system in sugarcane field reform on the root dry matter and root length of rotated
crops. The experimental design was randomized blocks in split plots with four replications.
The plots treatments consisted of three soil management systems, (conventional tillage,
minimum tillage and no-tillage). The split plot consisted of four comercial crops, corn,
peanut, sorghum and soybeans, a choice of green manure (Crotalaria juncea + jack beans),
and fallow system. Roots were sampled in the flowering plants. No significant interactions
were found among the soil management systems and different crops rotation in sugarcane
field reform, for all variables. This can be explained by the dry spell and high temperatures
occurring during crop development, which allowed the species presented similar behavior for
the root dry matter and root length. Thus the volume of soil explored by the roots was the
similar to the species regardless of the tillage system. The yield of root dry matter was
observed values of 2.053, 2.496 and 1.195 g m-3 in the layers 0-10, 10-20 and 20-40 cm,
respectively. Regardless of soil management systems and rotated crops was found that 80.7%
of the root system concentrated at between the layers 0-20 cm.
152
Rooting of jujube (Ziziphus jujube mill) Li variety cuttings using some root
promoting micro-organism and plant growth regulators
Nabil Omar*
Agricultural Research Centre, ARC, Egypt
This study was conducted at the experimental nursery of the Horticulture Research Institute,
Giza, Egypt during 2008 and 2009 seasons. Semi-hard wood cuttings were taken from mature
15 years old trees of jujube (Ziziphus Jujube Mill) Li. Roots treatments inoculated with some
plant growth promoting rhizobacteria strains (PGPR) 1- Bacillus polymyxa, 2- Bacillus
circulans, 3- Bacillus megaterium, 4- Bacillus pasteurii, 5- Pseudomonas fluorescens, 6- Yeast
strain (Saccharomyces cerevisiae), and mixed inoculants from previous PGPR strains. In
addition, indole butyric acid (1000 and 2000 ppm), naphthalene acetic acid (1000 and 2000
ppm) were also tested as compared with untreated (control).
The obtained results showed that, the effect of Bacillus megaterium as PGPR resulted in the
highest significant rooting percentage (60% and 50%). On the contrary, the lowest significant
effect of treatments was found as a result of naphthalene acetic acid at 1000 ppm and
control during the two seasons of the study. Histological studies revealed that the callus
originated from the cambial and phlom parenchyma cells below the cork cells from these
protrusions the adventitious roots were developed.
In conclusion the use of PGPR offers an attractive way to replace chemical fertilizer and
supplements most of the isolates result in a significant increase in plant height, root length,
and dry matter production of shoot and root of plants.
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153
Effect of Streptomyces sp. isolated from acidic cultures of minerals in root
development in environments polluted with mercury
Sara Liz Pacheco Huerta*, Jasmin Elena Hurtado Custodio, Patricia Sheen Cortavarria
Universidad Peruana Cayetano Heredia, Peru
Two strains of Streptomyces sp. (E1 and K2) and one strain of Streptomyces variabilis (K1A)
were isolated from acidic cultures of mineral ores from Peruvian highlands. Molecular and
phenotypic identification of the isolate strains was performed. The isolates were also
evaluated to determine their resistance to metals, growth at different concentrations of
sodium chloride, growth to different pH and exposed to different temperatures. The
Streptomyces sp. strains (E1 and K2) were able to grow in 100 ppm of mercury and
Streptomyces variabilis (K1A) was able to grow in 50 ppm of mercury.
The three strains were evaluated to determine their ability to contribute to the development
of roots in Lactuca sativa and Medicago sativa when exposed to mercury. L. sativa showed
regular root development in 10 ppm mercury. However, when treated with Streptomyces sp
(E1 and K2) and Streptomyces variabilis (K1A), L.sativa showed a remarkable root
development in length in up to 50 ppm of mercury. Moreover, M.sativa has the ability to
tolerate concentrations of mercury up to 100 ppm allowing a significant root development. In
presence of Streptomyces sp (E1 and K2) the root development was in up to 200 ppm of
mercury. The results obtained in this study allowed us to identify the ability of these strains to
contribute to the development of plants in contaminated areas and therefore allow the
restoration of polluted environment.
154
Chili pepper growth and drought tolerance are regulated by Bacillus vallismortis
strain EXTN-1 producing volatile organic compounds
Kyungseok Park*, Sarnalee Dutta
National Academy of Agricultural Science, South Korea
The plant growth-promoting rhizobacteria (PGPR), especially Pseudomonas spp. and Bacillus
spp., colonize roots of monocots and dicots, and directly or indirectly promote plant growth
and elicit induced systemic resistance, among which Bacillus vallismortis strain EXTN-1 plays a
pivotal role in enhancing induced systemic resistance (ISR) against multiple pathogens in a
variety of crops. Despite of accumulating evidence the role of EXTN-1, the precise underlying
mechanism of volatile organic compounds (VOCs) emitted by EXTN-1 has not been
conclusively studied. In this study, in order to elucidate the potential physiological role of
VOCs from EXTN-1 we investigated the plant growth and development in response to VOCs.
Any phenotypic changes were not observed in aerial parts of VOCs-exposed chili pepper
plants. However, root architecture was significantly altered in which increased root biomass
was obtained in VOCs-exposed plants. Interestingly, a combination treatment between EXTN1 and VOCs from EXTN-1 had a negative effect on pepper growth. In addition, root
colonization rate in rhizosphere was higher in VOCs-exposed plants, relative to control plants.
These results suggest that increased population density of Bacillus may positively modulate
the VOCs-mediated plant growth promotion. Intriguingly, improved drought tolerance was
exhibited in plants exposed to VOCs derived from EXTN-1. Our data clearly propose that
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VOCs from EXTN-1 contribute to augmentation of plant growth and that VOCs are normally
required for abiotic stress tolerance.
155
Response of plant roots and mycorrhizal fungi to soil hypoxia
Yasmine Piñuela Samaniego*1, Peter Železnik2, Hojka Kraigher2, Irena Maček1
1
University of Ljubljana, Biotechnical Faculty, Slovenia, 2Slovenian Forestry
Institute, Slovenia
Root growth and colonization with plant symbiotic arbuscular mycorrhizal (AM) fungi was
measured in barnyard grass seedlings exposed to geological CO2 and hypoxia. Barnyard grass
(Echinochloa crus-galli (L.) Beauv.) is an annual C4 grass and a cosmopolitan weed. It has been
shown to be tolerant to soil hypoxia and flooding. Our experiment was conducted in the area
of natural CO2 springs (mofettes) in Slovenia, and was designed to follow the growth of
Echinochloa in three different CO2 soil exposures: control (<1 % CO2), medium CO2 and high
CO2 exposed plot with peak CO2 concentrations 7.7 ± 2.0 % CO2 and 34.5 ± 6.7 % CO2,
respectively. For each location, holes in the ground were dug and were filled with
homogenized soil from a control site. In June 2014, 288 root ingrowth soil cores where
installed. Individual cores were sampled in August, September and November 2014. Samples
were soaked in water and sieved through sieves of different sizes (mesh size 2 mm and 2 µm).
Roots where scanned in water on an optical scanner and root parameters were measured
with WinRHIZO® (Regent Instruments Inc., CA) software. Subsequently, the roots were dried
in an oven and weighed. Additionally, root colonization with AM fungi was assessed.
Preliminary analyses of root biomass show a distinct decreasing trend in root biomass from
August to November. At the control site, root biomass was significantly higher compared to
the medium and high CO2 exposed site in the August sampling. In samples collected later in
the year significant differences were found only between root biomass at control and high
CO2 exposed plot. We found colonization with AM fungi in all the treatments. Interestingly,
despite reduced plant growth and lower root biomass in CO2 exposed sites, in the most
extreme conditions high levels of root fungal colonization were found.
156
Local and systemic effects of metals on root growth: towards revealing
molecular mechanisms and optimizing growth
Tony Remans*, Stefanie De Smet, Ann Cuypers, Jaco Vangronsveld
Hasselt University, Centre for Environmental Sciences, Belgium
Diffuse contamination by excess metals affects large areas worldwide, and using these areas to
produce biomass may bring them back into sustainable use. However, plants unavoidably take
up excess metals and are subjected to their toxic effects. Molecular mechanisms of metal
uptake, sequestration and detoxification have been intensively investigated. Root placement
in the soil is another important factor determining metal uptake, given the heterogeneous
distribution of metal contamination in the soil, but little is known on the molecular
mechanisms of interference of metals with intrinsic root developmental pathways.
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We use Arabidopsis thaliana as a model and investigate local and systemic effects of metal
exposure on root architecture when plants are heterogeneously exposed in split-root vertical
agar plates. As local effects, cadmium (Cd) and copper (Cu) stimulated the outgrowth of
lateral roots, but the elongation of these lateral roots was much stronger inhibited by Cd
than by Cu. Excess zinc (Zn) inhibited the emergence of primordia and meristem activation.
Systemic effects of metals on root development may either inhibit or stimulate the
colonization of less-contaminated soil patches. A systemic inhibitory effect on primary and
lateral root growth was observed when part of the root system was exposed to excess Zn, but
not when exposed to Cd or Cu. These observations indicate the existence of metal-specific
local and systemic interference with intrinsic root developmental pathways. Hormone
production and signaling mutants are being studied to start identifying underlying molecular
mechanisms. Knowledge on molecular mechanisms of growth-activating and –inhibiting
effects of metals can support strategies for improving plant growth to the purposes of
phytoremediation or safe biomass production, for which root development in contaminated
zones is desired or should be avoided, respectively.
157
The evaluation of root system architecture in rice plant using the data of root
distribution
Ryosuke Tajima*, Toyoaki Ito, Masanori Saito
Tohoku University, Japan
Root system architecture has been shown to be important in agricultural systems. It is
extremely difficult to identify the parameters of root system architecture, such as root
elongation rate, from field experiments. Here, we tested to determine the parameters using
the data of vertical root distribution in the field with root modeling. We evaluated five rice
lines, Hitomebore (a modern lowland cultivar in Japan), IR8 (a semi-dwarf lowland rice),
IRAT109 (an upland rice), NERICA3(an interspecific hybrid rice) and IR65564-44-51 (a lowland
rice, New Plant Type), cultivated in flooded condition. At harvesting, one soil monolith (10 cm
diameter) was taken from each plot to a depth of 10 cm. Then, the monolith was divided to
five layers (0-2 cm, 2-4 cm, 4-6 cm, 6-8 cm and 8-10 cm depth) and each root length density
was measured. In addition, we measured the panicle number, the nodal root number and the
minimum value of initial angles in nodal roots of each plant, which can be easily measured. In
these results, the panicle and nodal root numbers of Hitomebore and IR8 were much larger
than those of the other lines. The minimum values of initial angles in nodal roots of IRAT109,
NERICA3 and IR65564-44-51 were larger than those of Hitomebore and IR8. In the vertical
root distribution, IR8 had a shallowest root system in the five lines, while the deep-root ratio
(the percentage of the deep part in the total) of IRAT 109 was highest. Using these datasets,
we determined an average root elongation rate and gravitropism of each plant with root
modeling and nonlinear optimization. Both of the root elongation rate and the gravitropism
of Hitomebore were predicted to be higher than that of IR65564-44-51, although the
prediction was changed depending on initial parameters.
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158
Effect of soil type on the beech fine root distribution
Marie-Pierre Turpault*1, Christophe Calvaruso1, Stéphane Uroz1, Paul-Olivier Redon2,
Gil Kirchen1
1
INRA, France, 2Andra, France
Different parameters as climate, tree age, stand management and soil properties can affect
the distribution of tree roots in soil. In the experimental site of Montiers (north-east of
France) managed by INRA and Andra, all these parameters are constant including a
dominance of beech (Fagus sylvatica L.), except for the soil properties. Indeed soils
significantly differ from alocrisol-brunisol, bruni-calcisol to rendisol. The alocrisol-brunisol is
deep (> 2 m) and acidic while the rendisol is shallow (<30 cm) and alkaline. Two methods
were used to quantify the fine roots (< 2 mm diameter) at different depths in these three
soils: the root mass and the root impact methods. The root mass method consists in the
collect of a same volume of soil at different soil depths. The roots are manually extracted
from the total volume of soil and weighed. For each soil depth, the density of roots in the soil
is estimated. The root impact method corresponds to the use of a calibrated grid placed in
the soil pit and was used to count the number of root impacts in each square, to obtain ten
measures of root density by soil thickness of 10 cm.
Our analyses revealed that despite its shallowness, the rendisol presents a significantly higher
quantity of fine roots compared to that in the alocrisol-brunisol. Furthermore, roots colonize
the calcareous rock beyond two-meter deep, potentially by dissolving the limestone (root
imprints observed). In the rendisol, which is comparatively to the other studied soils the lessdeep and the most sensitive to water deficits and to P and K deficiencies, trees seem to
strongly invest in the development of fine roots in the soil horizons (higher density), but also
surprisingly in the calcareous rock (for the same depth, higher density in the rock compared
to that in the alocrisol-brunisol).
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Tuesday 23 June – Poster session
Nutrient Acquisition
2
Iron deficiency-induced root exudation of coumarins in Prunus rootstocks
grown at high pH
Ana Alvarez-Fernandez*, Yolanda Gogorcena, Javier Abadia, Anunciacion Abadia
Aula Dei Experimental Station-CSIC, Spain
Plant Fe deficiency is a problem in calcareous soils, due to the low solubility of Fe(III)
hydroxide. Peach trees are among the fruit crop species most sensitive to Fe-deficiency.
Prunus rootstocks differ widely in their susceptibility to Fe deficiency. Phenolics root secretion
is associated to Fe deficiency tolerance in different plant species. Recent studies have shown
that the fluorescent coumarin scopoletin along with other coumarins are exudated by the
roots of the model species Arabidopsis and tomato in response to Fe deficiency. However, no
information on Fe deficiency-induced root phenolics secretion has been reported for any fruit
tree species so far. In the present work, two Prunus rootstocks (GF677 and Cadaman) with
different tolerance to lime-induced chlorosis were grown for two weeks in half-strength
Hoagland solution buffered at 7.5 pH, including either 0 (Fe-deficient plants) or 90 μM Fe(III)–
EDDHA (Fe-sufficient plants). Iron deficiency induced the root secretion of fluorescent
compounds in both genotypes, and leaf chlorosis was less intense in the Fe-efficient
rootstock (GF677) than in the Fe-inefficient one (Cadaman). Root extracts and exudates were
analyzed by liquid chromatography coupled to mass spectrometry. Several glycosides of
different coumarins accumulated in GF677 roots in response to Fe deficiency. Also, several
coumarins and coumarin derivatives were secreted in response to Fe deficiency by GF677
roots. The concentration of these compounds in root extracts and exudates of Fe-deficient
Cadaman plants were lower than those from GF 677 roots. Phenolics removal from growth
medium of Fe-deficient plants, by circulating the nutrient solution through a C18 resin
column, led to a more advanced leaf chlorosis in GF677 but not in Cadaman. This supports
that the root secretion of phenolics by Fe-efficient Prunus rootstocks could play an important
role in Fe deficiency tolerance at high pH.
3
Rhizosphere P is affected by N sources applied to corn
Elialdo Alves de Souza*1, Camila da Silva Grassmann2, Janaina Matias Pereira da Silva2,
Sara Isa Vieira Kuchta de Almeida2, Ciro Antônio Rosolem2
1
São Paulo State Universtiy - Department of Crop Science, Brazil, 2São Paulo State
University - Department of Crop Science, Brazil
Nitrogen (N) source can affect rhizosphere activity and phosphorus (P) uptake in
neutral/alkaline soils. The aim of this work was to investigate the impact of ammonium and
nitrate-N on P availability in corn rhizosphere in an acidic soil. Corn plants were grown in 12 L
pots with soil or in plant growth containers to build a root mat. Nitrogen was applied at 240
mg kg-1 (first experiment) or 120 mg kg-1 (second experiment) as calcium nitrate and
ammonium sulfate (plus dicianodiamide-DCD), combined with 0, 40, 80, 120 and 160 mg kg-1
of P doses. Corn was grown and clipped 45 days after emergence. In the first experiment
adhering soil (rhizosphere soil) was separated from roots by gently shaking the soil off the
roots, and in the second experiment the soil was sliced at 0-0.5; 0.5-1.0; 1.0-2.0; 2.0-3.0; 3.097
4.0; 4.0-5.0; 5.0-10.0 mm from the root mat. Then Labile-P was determined using anion
exchange resin (AER Pi) and 0.5 M sodium bicarbonate (BIC Pi and BIC Po). In presence of
ammonium-N corn responded to higher P rates, with higher dry matter yields with 120 and
160 mg kg-1 of P. However, root dry matter was higher when nitrate-N was applied, resulting
in higher root/shoot ratio. Ammonium-N resulted in lower labile-P in rhizosphere soil up to
80 mg kg-1 of P. Lower values of Labile-P were found from 0 to 0.5 mm from the root mat, but
from 0.5 to 10.0 cm labil P was generally higher with nitrate-N. Ammonium-N increases P
availability, corn growth and response to high P fertilization rates in acidic soils.
4
Phosphatase activity and phosphorus lability in the rhizospheric region of
sugarcane genotypes
Bruna Arruda*, Marcos Rodrigues, Gabriel Novoletti, Paulo Sergio Pavinato
Esalq/USP, Brazil
Phosphorus (P) is easily adsorbed on the soil solid phase, reducing its availability. When
inorganic P (Pi) availability is low in the soil, plants and microorganisms exude organic
compounds that can mineralize the organic P phase (Po) by phosphatases activity, supplying
in this way plant demands. This study aimed to evaluate the dynamic of P in the rhizosphere
of four distinct sugarcane genotypes under low and high P levels. The experiment was
conducted in greenhouse at Department of Soil Science - ESALQ – USP, Piracicaba - Brazil, in
a sandy soil following a randomized block design with four replications. It was compared the
genotypes most used in Brazil: i) RB92-579; ii) RB85-5156; iii) RB86-7515; iv) RB96-6928 and
also no plants (witness), with and without P fertilizer application (STP). At harvest, the soil
adhered to the root system (rhizospheric soil) was sampled for analysis. When without plants,
the bulk soil was sampled to compare to the rhizospheric one. It was determined the
phosphatase enzyme activity in the soil freshly sampled and the fractions of labile P by
Hedley´s procedure. The data were statistically analysed and means compared by LSD test at
5%. In the witness there was no difference between P fertilization in phosphatase activity, but
in the rhizospheric soil of the four sugarcane genotypes when fertilized it was observed higher
phosphatase activity than without fertilizer. No differences between genotypes or witness
were observed without P fertilization. However, when P fertilized without plants it has
presented a lower enzyme activity, 218 mg kg-1 h-1, much lower compared to the rhizospheric
soil of the genotypes cultivated. Between genotypes, RB96-6928 had the lowest activity, 245
mg kg-1 h-1 compared to the other ones, whose mean was 280 mg kg-1 h-1 and do not show
any difference between them.
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5
Effect of zinc solubilizing bacteria on growth promotion and zinc nutrition of
wheat
Azadeh Bapiri*1, Ahmad Asgharzadeh2, Hesam Mojallali3, Kazem Khavazi2, Ebrahim
Pazira3
1
Department of Soil Science, Savadkooh Branch, Islamic Azad University,
Savadkooh, Iran, 2Department of Soil Microbiology, Soil and Water Research Institute,
Tehran, Iran, 3Department of Soil Science, Science and Research Branch, Islamic Azad
University, Tehran, Iran
Plant growth promoting rhizobacteria (PGPR) are the soil bacteria inhabiting around/on the
root surface and are directly or indirectly involved in promoting plant growth and
development via production and secretion of various regulatory chemicals in the vicinity of
rhizosphere. One of the most important direct mechanisms is the increase of nutritional
elements availability specially zinc in rhizosphere. Zinc deficiency is generally universalized in
agricultural fields especially in calcareous soils. In this research, the solubilizing ability of one
low-soluble zinc compound (ZnCO3) was studied by 11 Fluorescent Pseudomonads strains in
a greenhouse experiment. The plant growth promotory properties such as Zn solubilization
of the isolates were checked in a previous study. These isolates were showed effective in
significantly increasing the root dry weight (97%), shoot dry weight (8.70%), number of tillers
(9%), length panicle (4.25%) and the total Zn uptake (44.75%) as compared with the uninoculated control. The PGPR colonized wheat plants were more efficient in acquiring Zn from
either added or indigenous source, than non-colonized plants. One of the hopefully methods
to zinc deficiency remedy is to apply bacteria which are ability to soluble low- soluble and
insoluble compounds of this element.
6
Role of native microorganisms in shoot biomass and p uptake of maize in low
phosphorus soil
Pratapbhanu Singh Bhadoria*, Ishita Paul Paul
Indian Institute of Technology, Kharagpur, India
To study the phosphate solubilizing microorganisms from maize rhizosphere in low P soil was
carried out. Among several strains grown on Pikovskaya media modified with ferric
phosphate, a pigmented, Gram negative strain of bacteria was isolated. A second pot culture
was run in which factorial combinations of inocula from screened phosphate solubilizing
bacteria (PSB) and vesicular arbuscular mycorrhiza (VAM) were applied in presence and
absence of 100 mg phosphorus per kg soil. Samples of 21 days after sowing showed that PSB
treatment led to 12% increase in shoot dry weight over control when aided by phosphorus
application. On the other hand, interaction between VAM treatment and phosphorus
application enhanced shoot phosphorus content 33% more than only phosphorus
application. Without P fertilizer, PSB and VAM individually enhanced shoot weight by 94% and
49% respectively. When compared at 39 DAS, individual treatments with VAM and PSB gave
186% and 74% increase in shoot dry weight unaided by applied phosphorus while interactions
between VAM and PSB in joint application with phosphorus gave 32% increase in shoot
weight and 26% increase in shoot phosphorus concentration over only phosphorus
application. Results showed that shoot weight and shoot phosphorus concentration were
significantly enhanced at each additional interaction level. Determination of soil fractions of
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aluminium, iron and calcium bound phosphorus led to the inference that the bacterial strain
mobilized unavailable phosphorus and locked up excess soluble phosphorus in rhizosphere
and slowly released it in soluble form as depletion zones formed round the roots; while the
VAM fungi rapidly dissolved any mineral phosphorus to maintain steady uptake of
phosphorus. The results suggest that microbial inhabitants of the soil favorably affect crop
growth in the long term. This study recommends supplementation of phosphorus fertilizers
applied to acid soils with dual inoculation of VAM and PSB.
7
Effect of different nitrogen sources on nitrogen dynamics, mineral elements and
microbial communities in rhizosphere soil of different plant species
Qingnan Chu*1, Toshihiro Watanabe2, Zhimin Sha3, Mitsuru Osaki2
1
Faculty of Agriculture, Hokkaido University, Japan, 2Hokkaido
University, Japan, 3Shanghai Jiaotong University, China
Little is known with respect to the variation of microbial metabolism, minerals and
metabolites in rhizosphere of different plant species in response to different nitrogen
sources. Soybean is able to perform nitrogen fixation and sorghum can utilize organic
nitrogen efficiently. We therefore conducted the rhizobox experiment to investigate the
effect of different nitrogen sources (ammonium sulfate, manure, without nitrogen fertilizer)
on rhizosphere environment in soybean and sorghum. Plant roots and rhizosphere soil were
separated from bulk soil with a nylon cloth (300 mesh, wire diameter 30μm) and different soil
fractions moving laterally away from root compartment were analyzed for mineral elements,
metabolites and microbial community. For plant growth, ammonium-source caused
significant decrease of biomass and nitrogen accumulation in soybean nodules but no
significant difference was detected for biomass or nitrogen concentration in other soybean
organs. Manure application facilitated nitrogen accumulation in sorghum stems. Fertilization
without nitrogen resulted in the significant biomass reduction in sorghum roots and leaves,
nitrogen concentration in leaves. The Biolog EcoplateTM was analyzed for characterizing
microbial communities in soil. By principle component analysis using the Biolog data, 55.6%
of total variance was revealed by the first principle component and 22.4% by the second
principle component. Two principle components both differentiate the effect on microbial
communities in rhizosphere of soybean but did not between different nitrogen sources in
sorghum rhizosphere. More efficient carbon source consumption activity excluding amino
acid and malic acid was detected in soybean rhizosphere receiving manure application. The
distribution of mineral elements, metabolites, microbial carbon source utilization, and their
interactions in rhizosphere environment of soybean and sorghum under the influence of
different nitrogen sources will be discussed.
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8
Phosphorus efflux rates from roots of Trifolium repens, T. uniflorum and T.
repens × T. uniflorum interspecific hybrid clovers
Jim Crush*, Shirley Nichols
AgResearch, New Zealand
Phosphorus efflux rates are six times higher in white clover (Trifolium repens) than in perennial
ryegrass, and it has been suggested that this may contribute to the poor competitive ability
for phosphorus of clover grown with ryegrass. Recent research has shown that some T. repens
× Trifolium uniflorum interspecific hybrids backcrossed to T. repens can grow better at low to
intermediate levels of soil phosphorus, than both of the parental species. T. uniflorum, a close,
wild relative of white clover demonstrates adaptation to low soil phosphorus and intermittent
phosphorus enrichment. No information exists on phosphorus efflux rates from T. uniflorum
roots. The ecology of the species suggests these may be low and, if so, this may contribute to
the observed improved growth of T. repens × T. uniflorum interspecific hybrids when soil
phosphorus is strongly limiting for white clover.
Phosphorus efflux rates from roots were measured on hydroponically grown plants of two
white clover parental cultivars, two T. uniflorum seedlines, and six T. repens × T. uniflorum
hybrids that had been back-crossed to the two white clover cultivars. Efflux of phosphorus
was measurable for every clover type. One T. uniflorum line had a significantly (P<0.05) lower
phosphorus efflux rate than the white clover cultivars (3.87 µg phosphorus g-1 root dry
weight h-1 compared with 7.73 or 6.55 µg phosphorus) but the second T. uniflorum line did
not differ from the white clovers. Efflux rates in the hybrid lines varied widely and included
the highest, and lowest, values recorded. On average less than 0.4% of root phosphorus was
lost over the four hour assay, suggesting that variation in phosphorus efflux rates should be a
low priority target in breeding for improved phosphorus use efficiency in white clover.
9
Solubilization of inorganic phosphorus from biochar by phosphate-solubilizing
microorganisms
Aikaterini Efthymiou*, Mette Grønlund, Nelly Sophie Raymond, Dorette Sophie MüllerStöver, Iver Jakobsen
Global reserves of rock phosphate for fertilizer production are declining and this coincides
with the generation of phosphorus rich organic wastes. There is a need for strategies to
efficiently recycle the phosphorus in waste products. Organic wastes such as sewage sludge
are rich in phosphorus and can be pyrolized, which ensures energy recovery, improved
hygiene, volume reduction and a biochar product that is a potentially valuable phosphorus
fertilizer. The use of phosphate-solubilizing microorganisms is a promising biotechnological
strategy for the management of soil phosphorus resources, however, there is limited
knowledge regarding their ability to solubilize phosphorus from recycling products.
In this study, 14 phosphate solubilizing rhizosphere bacteria and fungi are screened for their
ability to solubilize inorganic phosphate from sludge biochar, rock phosphate and Ca3(PO4)2
in vitro and the mechanisms of solubilization are being evaluated. Selected Penicillium spp.
and Pseudomonas spp. strains result in higher solubilisation of phosphorus in the biochar.
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The production of organic acids appears to be the main mechanism for the solubilization of
inorganic phosphates and we are currently investigating how the production pattern varies
with the combination of microorganism, carbon source and phosphate source. Furthermore,
the selected strains are being evaluated for their ability to improve the plant availability of
the biochar-phosphorus in pot experiments.
10
Effect of Thiobacillus on nutrient uptake and quality and quantity characteristics
of two varieties of turnips forage (Brassica rapa L.)
Seyed MohammadReza Ehteshami*1, Neda Moazeni2
1
University of Guilan, Iran, 2Islamic Azad University, Iran
In order to investigate the effect of Thiobacillus bacteria on nutrient uptake and quality and
quantity characteristics of the forage turnips (Brassica rapa L.), an experiment conducted as
factorial arrangement with randomized complete block design with three replications at the
research farm of Rice Research Institute Experimental. Investigated Factors include sulfur
powder (use or non-use), sulfur-oxidizing bacteria (Thiobacillus use or non-use), Phosphorous
fertilizer (use or non-use) and two varieties of forage turnips (white purple and white globe).
Results showed that combined application of bio-fertilizer, phosphorus and sulfur powder
increased LAI and morphological indices during the growing season. The highest of
Chlorophyll a that is the most important role in the biomass production, abserred in the
integrated treatment of sulfur powder Thiobacillus + cultivar. The highest of quality
parameters, including dry matter digestibility, total ash and water-soluble carbohydrates in
shoots and tubers of forage turnip obtained in sulfur powder + bacteria Thiobacillus +
cultivar.The highest forage yield (tons / ha) obserred in integrated application of powdered
sulfur + Thiobacillus + cultivar. Thiobacillus bacteria increases the oxidation of sulfur and
sulfate reducing soil acidity and soil result in increased production, thus increasing the
absorption of some nutrients, particulary phosphorus, is seeking. totally, taking into account
the results of all evaluated traits, seems to Using a combination of powdered sulfur +
Thiobacillus bacteria + phosphorus fertilizer and the chemical Fertilizers way for reducing
consumption, Improve soil and improve plant nutrition and moving toward sustainable
agriculture.
11
Potentials of rumen digesta as a source of organic manure in Owerri Imo
Southeast Nigeria
Ibiam Ikwuo Ekpe*1, S.C Asagwara Asagwara1, Emmanuel Nwannebuife Ogbodo2,
Bethel Uzoho1, Samuel Okere1, Nnaemeka Anyanwu1, Maureen Nwaigwe3, Agatha
Elem Ekpe4
1
Federal University of Technology, Nigeria, 2Ebonyi State University,, Nigeria, 3Alvan
Ikoku Federal College of Education, Nigeria, 4Ministry of Lands and Housing,Ebonyi
State, Nigeria
An assessment of the potentials of rumen digesta as a source of organic manure in Owerri
zone in Imo state was studied. the identification of all the Abattoirs in the city, the species of
animals slaughtered, the number of each of the ruminant animal species slaughtered per
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day/month and per year, total rumen digesta yield and possible economic returns from sale
of bagged digesta per annum. The results revealed that a total of 65,160 cow, 28080 goat
and 13320 sheep were slaughtered annually with a corresponding total rumen digesta yield
of 2,952,720kg. When this quantity is processed and bagged into 50kg sizes and sold at
₦600.00/bag, a total financial return of ₦35,432,640.00K was possible. Rumen digesta after
decomposition has the ability to replenish soil nutrients, provide cementing agents that bind
soil particles together, increase soil moisture content, increase infiltration rate and general
increase in crop yield. The quantity of rumen digesta produced in Owerri zone in Imo State is
high. One can run a rumen digester enterprise by bagging and selling rumen digesta. The
environment will be better, cleaner and healthier while at the same time increasing crop yield
and providing employment opportunities to many youths.
12
Actinobacteria with phosphate solubilizing activity in tropical soils
Marcela Franco-Correa*1, Luis Daniel Prada-Salcedo2, Claudia Liliana Cuervo Patiño3,
Jose Salvador Montaña Lara3
1
Pontificia Universidad Javeriana, Colombia, 2UNIDIA, Colombia, 3Pontificia Universidad
Javeriana/UNIDIA, Colombia
The evaluation of biofertilizer products are expected to have the capacity to solubilizing
nutrients, for this reason the aim of this work was to isolate actinobacteria able to release
soluble phosphate in vitro and identify organic acids associated with this activity. We isolated
57 strains of actinobacteria, to screening the isolates of actinobacteria we used two qualitative
assays to determine the efficiency of solubilization by measuring the halo of hydrolysis in a
Pikovskaya’s agar plate. A second assay was performed on broth with NBRIP medium
containing BPB following the protocol of Mehta and Nautiyal (2001). Finally to measure the
release soluble phosphate by actinobacteria was quantified using insoluble Ca3(PO4)2 or AlPO4
as sole sources of P. Ten of these strains demonstrated by qualitative and quantitative
assessments have a high and significant phosphorus solubilizing activity. Molecular
identification was realized for the best isolates, revealed that belong to the genus
Streptomyces, (T3A, T1C, T1H, T1J, T3C, P3E, F1A, F2A, V1E and V2B). Addition of new records
for the country of the genera Kitasatospora (L3A) and Streptacidiphilus (M2A) was reported.
The strain T3A have a very good activity with both sources of phosphate Ca3(PO4)2 or AlPO4,
with activities of 500 mgP/l and 14 mgP/l respectively. The phosphorus solubilizing activity of
this strain is associated to the release of organic acids, this was evidenced by a pH decrease,
used of the sugar source in the medium and the presence of organic acids like gluconic acid,
citric acid and oxalic acid identified by HPLC.
13
Transcriptomic data reveal new insights in Si effects on iron acquisition of rice
Martin Hinrichs*1, Manfred K. Schenk2
1
Leibniz Universität Hannover, Germany, 2Institute of plant nutrition, Germany
Silicon is one of the most abundant elements in the earth crust. Many plants are able to
accumulate Si with several beneficial effects. One of these effects is the earlier formation of
Casparian Bands (CB), as shown in rice, maize and onion roots. In rice it was demonstrated
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that this anatomical change resulted in a decreased radial oxygen loss. Investigations of the
molecular background of the enhanced CB formation by means of a custom made microarray
with 265 genes putatively involved in CB formation resulted in the identification of twelve
differentially regulated genes. To get a full insight in Si- induced genes a 45k gene chip
approach was used. The experimental design consisted of four biological repeats and two
technical repeats of rice plants grown in nutrient solution in a growth chamber. Sections of 26 cm behind the root tip of adventitious rice roots grown with Si (30 mg/l) and without Si (>3
mg/l) were harvested for RNA extraction.
The transcriptomic data revealed regulation of genes related to the phenylpropanoidpathway. Surprisingly, also an up regulation of iron acquisition strategy II related genes like
OsIRO2, OsNAS2, TOM1 or OsHRZ1 was observed. The Fe-concentration in shoot dry matter
was reduced by half in Si fed plants. This shortage of Fe caused by Si supply was reflected in
the transcriptomic data. Reason for reduced iron uptake could be a lower Fe flux into the
apoplasm, because of enhanced CB development.
14
Performance and yield of maize in response to grinded-sawdust application
Chris Ifeanyi Igbozuruike*1, Oliver Opara_nadi2
1
Abia State University, Uturu, Nigeria, 2Abia State University, Nigeria
The effect of grinded sawdust on the performance of maize was carried out in 2013 at Federal
University of Technology (FUTO) Owerri, Imo state Nigeria. Grinded sawdust was either
applied single and in composite with other organic amendments. The treatments were T1 =
control, T2= sawdust not grinded, T3=grinded sawdust, T4 =grinded sawdust + cow dung,
T5= grinded sawdust + poultry droppings, T6 =grinded sawdust + pig waste, T7=grinded
sawdust + NPK 10: 10: 10, T8=Cow dung alone, T9 =poultry manure alone, T10= pig waste
alone, T11= NPK 10: 10: 10: alone. Both in single and composite applications, treatments were
applied 10tons/ha except where NPK was involved which was applied npk
100kg/ha and sawdust 5tons/ha. These were laid out in randomized complete block design
(RCBD). Growth and yield Parameters were taken. Grinded sawdust increased the growth and
yield of maize relative to the control (T1) with highest effect occurring in T7 followed by T11,
T5, T6, T4, T9, T8,T10, T3,T2. There were yield advantages of about 50 % where grinded
sawdust were involved over 40 % of noninvolvement. Also about 35 % yield advantages
occured where grinded sawdust were involved over no-grinded sawdust (05 %). Comparative
assessment of the seasons saw residual growth and yield highest where grinded sawdust
were involved, and also had yield advantages of 70 % (residual) to against 50 % (first season).
The results were discussed in light of the nutritive ability of the various components in
mixture.
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15
Investigation of the molecular mechanisms of phosphate starvation-induced
growth of root hairs in Brassica carinata
Thomas W. Kirchner*, Kim L. Rössig, Timo Lauterbach, Manfred K. Schenk
Gottfried Wilhelm Leibniz Universität Hannover, Institute of Plant Nutrition, Germany
The deficiency of inorganic P induces long root hairs in Brassica carinata cv. Bale, but not in
cv. Bacho. The aim of the present study was the identification and characterization of genes,
which are responsible for this different reaction. Therefore, root tips with a length of two
centimeter were harvested from Bale and Bacho plants, grown in nutrient solutions under
sufficient (1 mM) and deficient (0 mM) P conditions, respectively. Besides root hair length,
genome-wide expression profiles of Bale and Bacho were compared under both conditions
by Massive Analysis of cDNA Ends (MACE). Genes, which were differentially expressed in Bale,
but not in Bacho, were defined as candidate genes. After validation of the expression of these
genes by qRT-PCR, the genes were further characterized. To investigate, if these genes are
involved in rather local or systemic signaling pathways, their expression was determined in
samples obtained from a split-root experiment. Furthermore, nutrient specificity of gene
expression was explored in potassium and nitrogen deficient plants.
16
Nutrient depletion from rhizosphere solution by maize grown in soil with longterm compost amendment
Leandro Bortolon1, John Kovar*2, Michael Thompson3, Clesio Gianello4
1
EMBRAPA, Brazil, 2USDA Agricultural Research Service, USA, 3Iowa State
University, USA, 4Federal University of Rio Grande do Sul, Brazil
Improved understanding of rhizosphere chemistry will enhance our ability to model nutrient
dynamics and on a broader scale, to develop effective management strategies for applied
plant nutrients. With a controlled-climate study, we evaluated in situ changes in macronutrient concentrations in the rhizosphere of juvenile maize (Zea mays L.) grown in soil (fineloamy, mixed, superactive, mesic, Typic Hapludoll) amended with cattle (Bos taurus L.) manure
compost. Soil was collected from two depths within a 10-yr. no-till study, so that nutrient
stratification could be investigated. Maize seedlings at the two-leaf stage were transplanted
into mini-rhizotrons, and grown for five days. Moist soil (550 g dry weight) collected from the
0-5 cm layer was placed in one compartment of a vertically divided minirhizotron, and soil
from the 5-10 cm layer was placed in the other compartment. Micro- capillaries (15 per
chamber) were used to collect rhizosphere and bulk soil solution at 24-h
intervals. Samples were analyzed for pH and phosphorus (P), potassium (K), calcium (Ca), and
magnesium (Mg) concentrations. Shoot dry matter production was similar in the two soil
materials (0.65 g/plant in the 0-5 cm layer and 0.60 g/plant in the 5-10 cm layer), suggesting
that nutrient availability was not limiting. Phosphorus concentrations in solution were similar
in the two soil layers, but were lower at the root surface. Potassium concentrations were
higher in the surface soil material, but did not change with distance from the root surface.
Solution pH and Ca and Mg concentrations did not differ among the soil layers and distances
from the root surface. From a practical perspective, results indicate that significant differences
in rhizosphere solution chemistry can develop quickly. From a crop production point of view,
the use of compost can be a means to reduce the inputs of mineral P and K fertilizer.
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17
Characterizing the variation of Brassica napus root system architecture: an in
vitro screening approach
László Kupcsik*, Hugues de Gernier, Christian Hermans
Université libre de Bruxelles / LPGMP, Belgium
Agricultural productivity relies quantitatively on nitrogen fertilization, which in excess is
detrimental to the environment. Hence, ameliorating the nitrogen use efficiency of crops is
an important approach for breeding future in order to reduce nitrate leaching and nitrous
oxide emission via denitrification. We study Brassica napus (oilseed rape) which has small
recovery of applied N in the seed. Our goal is to ameliorate its nitrogen use efficiency by
redesigning a more branched root system that explores a larger soil volume for better N
capture. As a first step, we examined the existing root trait variation between cultivars.
We screened 57 cultivars from the Belgian Catalogue of Agricultural Plant Varieties upon in
vitro culture at two N supplies (0.01 vs. 10 mM nitrate). After one week treatment, organ
biomass and multiple root traits were measured. These outcome variables were interrelated,
so principal component analysis was used to reduce data complexity.
We showed that the first two principal components were responsible for 55% of the total
variation. The primary source of variation (45%) was root system ramification, which consisted
of length and number of lateral roots, and root biomass. The second most important
component (10%) reflected the primary root morphology: its length and the position of the
lateral root zone. These two principal components clearly separated certain groups of
cultivars, and were also influenced by the nitrate availability in the culture medium.
These results demonstrate that the most important difference between the surveyed cultivars
lies indeed in the lateral expansion of their root system. The method described here also
enables us to select contrasting genotypes, and thereby to investigate the genetic regulation
of root system architecture in Brassica napus.
18
Maize nutrient uptake, rhizosphere pH and root architecture affected by soil
compaction and application of biogas-digestate
Daniela Piaz Barbosa Leal*1, Stephan Blossfeld2, Nicolai Jablonowski2
1
Forschungszentrum Jülich (IBG-2: Plant Sciences), Germany, 2Forschungszentrum
Jülich, Germany
Application of biogas-residues as fertilizers may promote enhanced cycling of nutrient
resources. Plants obtain water and mineral nutrients from the soil due their capacity to
develop extensive root systems. However, soil compaction may restrict deep root growth.
Thus, we evaluated the effects of the biogas-digestate applied as a fertilizer on root
architecture, rhizosphere pH, nutrient uptake and biomass development of maize growth
when subjected to soil compaction, in comparison to NPK fertilizer application and noncompacted soil condition. Experimental factors were: fertilizers - biogas-digestate (40 Mg ha1
), NPK (equivalent amount) and biogas-digestate (20 Mg ha-1) + NPK (equivalent amount);
and soil compaction or non-compaction. The upper compacted layer (25 cm) received an
equivalent pressure of 1.2 kg cm2. The biogas-digestate (elemental composition: 41.1% C,
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3.2% N, 1.5% P and 3.8% K) was composed of maize silage as the major feedstock, and minor
amounts of chicken manure. Fertilizers were mixed into the soil (50%v. arable field soil +
50%v. peat substrate). Plants were grown for 28 days under greenhouse conditions (16h per
day of light period, day/night temperature of 22°C/17°C and 60% of humidity). At harvest,
the measurements included: shoot and root dry mass and nutrients content (C, N, P and K);
leaf area; root architecture; and soil elemental analyses. Rhizosphere pH was measured with
planar optodes along the experimental period. Preliminary results showed variation of the
rhizosphere pH along the experimental period for all treatments. The lowest rhizosphere pH
(4.2) was observed for the biogas-digestate + NPK/soil compaction treatment and the
highest rhizosphere pH (5.3) was observed for the NPK/soil compaction treatment. Similar
shoot and root biomass and nutrient uptake were observed between the treatments. These
findings can enhance our understanding of rhizosphere pH dynamics and root architecture
subjected to soil compaction aiming to improve soil fertilization practices.
19
Dynamics of competitive N and P uptake between intercropped wheat and faba
bean in a calcareous soil
Chunjie Li*, Haigang Li, Fusuo Zhang
Center for Resources, Environment and Food Security (CREFS), China Agricultural
University, China
Cereal/legume intercropping is well known to be complementary and facilitative on N and P
uptake because of N2 fixation and P mobilized by intercropped legumes. However, the
dynamics of N and P uptake by intercropped plants during the co-growth period were rarely
documented in a N- and P-deficient soil. The aim of present study is to reveal these dynamics.
Wheat and faba bean were grown isolated or together in pots contained 2.0 kg N- and Pdeficient soil. Biomass, N and P content, carboxylates and phosphatases activity were
measured in 13 destructive samplings. Data of biomass and N, P content were fitted to the
logistic model to derive the cumulative biomass and N, P uptake curves and relative
interaction index (RII). There was gradually increased competition for N and P uptake by
intercropped wheat and faba bean along with growing time. In terms of dynamic competitive
N uptake, the RII of wheat (from -0.06 to –0.26) was always higher than that of faba bean
(from -0.10 to -0.20) during the whole growth stage, which may resulted from soil N
depletion for wheat but faba bean has the ability to fix N2 from atmosphere (39 mg N/plant).
Although competition for P uptake was observed between intercropped wheat and faba
bean, the RII of faba bean (from -0.13 to -0.22) was hardly changed while the RII of wheat
increased from -0.12 to -0.33 and decreased to -0.10 in the final sampling. From the analysis
of rhizosphere processes, faba bean has higher ability to exudate carboxylates and acid
phosphatase activity, which is helpful for it to adapt to the depletion of soil P and facilitate P
uptake of wheat. This study provided experimental evidence about the dynamic N and P
uptake by intercropped plants and the relationship with rhizosphere process.
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20
Physiological and biochemical responses of olive (Olea europea L.) to aluminum
Adamo Domenico Rombolà1, Enrico Maria Valentino Liquori*2, Michele Di Foggia2,
Gianfranco Filippini2, Annamaria Pisi2, Sergio Bonora2
1
University of Bologna - Department of Agricoltural Sciences, Italy, 2University of
Bologna, Italy
Crops native of alkaline-calcareous soils (e.g. barley) are more often cultivated on acid soils
with high levels of soluble aluminum, which can cause heavy adverse effects on root growth
and mineral nutrition. The aim of this work was to investigate the response mechanisms of
olive to the presence of aluminum (Al) in the growing medium. Micro-propagated plants (cv
Leccino) were grown in hydroponics with a 1/4 Hoagland solution adjusted to pH5. After a
week of acclimation, plants were subjected, at the same pH, to 5 different Al concentrations:
0, 50, 250, 500 and 1000 µM. pH of the nutrient solution, stem and root length and number
were daily measured. Nutrient solutions were renewed every 4 days. During the experiment,
root exudates were collected and analyzed for organic acids and phenols (HPLC) and their
complexes with Al (IR and RAMAN). After 1 month (end of the experiment), root apexes were
excised and analyzed by SEM. Noteworthy, root and shoot growth occurred even at the
highest Al concentration. Plants were able to increase the pH of nutrient solution. At SEM, the
root epidermal layer of plants exposed to the highest Al concentration looked more compact,
less defined and the morphology of the cells in cross section was altered. In contrast, root
epidermis of low aluminum-treated plants was more preserved and very similar to that of
controls. The main organic acids detected in root exudates were oxalic and formic. Their
concentration resulted higher in 500 and 1000 µM Al treatments. Spectroscopic measures
conducted on exhausted nutrient solutions showed the formation of Al-complexes, mainly
under the form of Al-oxalate.
21
Morphological variation is the main adaptive response in maize roots to low
phosphorus availability
Haitao Liu*1, Chunjian Li2
1
China Agricultural University, China, 2Department of Plant Nutrition, China
Different plants show different adaptive responses in roots to phosphorus (P) deficiency,
include morphological and physiological traits. To clarify the adaptive mechanisms of maize
root to low P availability, hydroponic experiments under P-sufficient (HP, 250 µM) and Pdeficient (LP, 1 µM) conditions were conducted, with two legumes (Vicia faba L. and Lupinus
albus L.) as references. The results showed that LP treatment increased root biomass, root to
shoot dry weight ratio (R/S), and total root length (TRL) of maize and faba bean with more
dramatic change in maize than in faba bean in R/S and TRL. However, the same treatment for
12 d did not cause any variation in shoot and root growth in white lupin. The measured root
exudation of carboxylates including malate and citrate and acid phosphatase (APase) activity
on maize root surface showed no significant difference, while dramatically increased in both
legumes when they grew under LP, compared with that under HP. Rhizospheric alkalization
(agar technique) or net H+ influx (ion-selective electrode technique) on maize root surface
was observed regardless of P levels. By contrary, increased rhizospheric acidification or net H+
efflux on root surface of both legumes were determined under LP compared with that under
108
HP. P inflow rate (mg P m-1 root length d-1) and P uptake efficiency (mg P m-1 root length) in
legumes were remarkable high than maize under LP. However, P use efficiency (mg dry
weight mg-1 P) of maize compared with both legumes was much high. The results suggest
that differing from the physiological-type and morpho-physiological-type response in white
and faba bean respectively, morphological variation is the main adaptive response to low
phosphorus availability.
22
Relationship between phosphorous deficiency tolerance and rhizosphere
management in Vicia genus
Wissal M'sehli*, Haythem Mhadbi
Centre de Biotechnologie de Borj Cedria CBBC, Tunisia
Plants resort many adaptive strategies in response to abiotic environmental stresses such as
soil phosphorous deficiency. These adaptive mechanisms include changes in the rhizosphere
processes. Objective of the present study was to assess inter and intra-specific variations in
Vicia genus to P deficiency response and to elucidate the implication of rhizospheric
processes on phosphorus (P) deficiency tolerance.
Experiments in controlled condition for Vicia sativa and two Tunisia Vicia faba varieties (Locale
and Saber 2) were conducted. Seedlings were growing hydroponically under three treatments:
+ P (360 mM); - P (5 mM); + P +Bicarbonate (360 mM + 10 mM NaHCO3). It was revealed that
P deficiency (-P and + bicarbonate treatments) restricted the whole plant RGR (the relative
growth rate) in both species of vicia, particularly in – P treatment. This depressive effect was
more pronounced in Vicia sativa (- 66.6%) than in Vicia Faba (var. Locale: -23.8%; var. Saber 2:
- 44.8%). In our experiments, an increase in the root/shoot P content, the P acquisition and
use efficiency was found in the two varieties of Vicia faba compared to Vicia sativa. The
highest increase was found in the tolerant cultivar Locale both at low P levels and in the
presence of bicarbonate.
The rhizosphere management underling the differential responses of the vicia species and
cultivars to P deficiency were explored. Exposure to P-deficient medium led to an increase in
root biomass and the intracellular root phosphatase activity. This enhancement was more
pronounced in the tolerant cultivar Locale. In our study, we proposed that root-induced
acidification of the rhizosphere by proton release and the exudation of phosphatases acids as
a mechanism of facilitation P acquisition in deficient medium. Our results showed that these
two mechanisms were more pronounced in the tolerant cultivar Locale.
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23
The ability of beech seedlings (Fagus sylvatica) to shape their rhizosphere in
order to cope with low phosphorus availability
Sonia Meller*1, Beat Frey1, Emmanuel Frossard2, Marie Spohn3, Jörg Luster1
1
Swiss Federal Institute for Forest, Snow and Landscape Research
WSL, Switzerland, 2Swiss Federal Institute of Technology ETH, Switzerland, 3Bayreuth
University, Germany
The German priority research program SPP 1685 aims to better understand the adaptation of
forest ecosystems to low availability of nutrients, in particular P. The leading hypothesis is
that an ecosystem facing depletion of mineral P sources appears to decrease P losses by a
more effective P recycling. Within this program we address the question to what extent
beech (Fagus sylvatica) is able to enhance the mobilisation of specific P sources in its
rhizosphere. We hypothesize that direct adaptation mechanisms such as root exudation of
carboxylates are governed by the internal demand of P status – and thus the overall
availability of soil P - rather than by the actual relative availability of organic and inorganic P.
Thus, P uptake efficiency could be enhanced, by investing carbon to stimulate the growth of
P mobilising soil microbes that are better adapted to local nutrient availability.
Beech seedlings were grown in different acid forest soils using rhizoboxes. Specific visually
identified microzones, e.g. the rhizosphere around a root tip, were assessed for potential P
mobilisation. We analysed P in soil solution („available“ soil P fractions), P mobilising
substances / mechanisms (phosphatase activity, filter collection of carboxylates, pH changes
using optodes) and composition of microbial communities (amplicon sequencing using
Illumina MiSeq). In this presentation we will show the results from a first experiment with
one-year old beech seedlings originating from a soil poor in mineral P growing in a soil
relatively rich in both mineral and organic P. Preliminary data show lower phosphatase
activity and pH around root tips and elongation zones of non-mycorrhizal long roots than in
bulk soil. This may indicate that beech seedlings are not suffering from P limitation. This
interpretation will be evaluated using data on rhizosphere modifications by roots and on
phosphatase activity in plant cells as indicator of P status.
24
Different root exudation patterns affect the mobilization of Fe from soil
minerals
Rebeka Fijan1, Roberto Terzano2, Concetta Eliana Gattullo2, Youry Pii3, Luca Medici4,
Stefano Cesco1, Tanja Mimmo*1
1
Free University of Bolzano, Italy, 2University of Bari, Italy, 3Free University of
Verona, Italy, 4CNR, Italy
Plants can cope with Fe deficiency by either acidifying the rhizosphere and enhancing the
ferric chelate reductase activity (strategy I) or by releasing high affinity complexing
compounds known as phytosiderophores (strategy II). In this research, tomato, barley and
cucumber plants were grown hydroponically in an Fe free (–Fe) nutrient solution using the
RHIZOtest. Fe-deficient (–Fe) plants were grown on a calcareous agricultural soil and root.
Root exudates were determined quali-quantitatively using HPLC and colorimetric analyses.
The accumulation of nutrients in plant tissues was measured by ICP-OES. Soil mineral
modifications were assessed by XRD and SEM-EDX.
110
Root exudates obtained by the hydroponic system were higher than those obtained from the
soil extracts; plant uptake and adsorption by soil particles could be, at least in part,
responsible of this result. After a 6-day soil contact the plants show a visible recovery from Fe
deficiency symptoms at leaf level suggesting the efficacy, of root exudate release in the
mobilization of Fe into soluble soil forms and its uptake. Moreover, significant soil mineral
modifications were observed.
At last, organic ligands released in Fe-deficient soil conditions show very complex exudation
patterns both in monocotyledonous and dicotyledonous plants. For instance, barley plants
besides the dominant release of phytosiderophores also significant amounts of amino acids
and traces of organic acids were found. The results of this research will enable to better
understand the soil influence on the release of root exudates, the dynamics of mineral
weathering in the rhizosphere and the capability of plant species to mobilize and take up
poorly soluble Fe forms.
25
Potential of rhizospheric and endophytic microorganism to enhance P
availability from organic and inorganic phosphate and phytohormones
production
Christiane Oliveira*1, Eliane Gomes1, Ivanildo Marriel1, Maycon Oliveira1, Ubiraci Lana1,
Vitoria Palhares1, Astrid Beinhauer2, Ralf Greiner2
1
Embrapa Mayze and Sorghum, Brazil, 2Max-Rubner-Research Institute, Germany
Rhizosphere microorganisms contribute significantly to solubilization of inorganic and organic
forms of phosphorus fixed in soil, but only a few endophytic microorganisms are known in
this respect. Since the amount of organic phosphorus in soil with no-tillage farming is
increased and use of organic fonts, microorganisms efficient in the mineralization of
organic P are more promising as plants inoculants. Apart from the P solubilizing abilities, the
phosphate solubilizing microorganisms have the ability to produce plant growth hormones
and enzymes for mineralization of organic phosphorus. The aim of this work was to
investigate the potential of phosphate solubilization and mineralization of rhizosphere and
endophytic microorganisms in order to select bacteria as plants inoculants. The production of
phytase and phytohormone auxin indole-acetic acid (IAA) were also evaluated. Nine
rhizobacteria from Embrapa collection and 113 endophytic bacteria isolated from maize
roots, leaves and sap were evaluated for calcium and phytate phosphate bio-solubilization.
The soluble P produced was quantified by a modified ammonium molybdate method after
3,6 and 9 days of bacterial growth. The most efficient phosphate solubilizing bacteria were
identified based in the 16S rDNA sequence and evaluated for phytase and IAA production. IAA
production was evaluated in a liquid culture medium supplemented with tryptophan. The
rhizobacteria Bacillus subtillis strain B70 showed the highest calcium-P (167.8 mg P.mL-1) and
phytate-P releasing (93,79 mg P.ml-1). The best intracellular phytase producer were identified
as Pantoea sp. 1931 (85 mU.ml-1) and the above mentioned B. subtillis B70 (64mU.mL-1). The
highest IAA production (150 µg.mL-1) was observed with the above mentioned Pantoea sp.
1931 and a non-identified endophytic bacteria (isolate 1913). It was concluded that
bacterium from corn rhizosphere and endophytic microbiome have promising features of
bio-solubilization and plant promotion of growth. Furthermore, Bacillus isolates were
identified as promising candidates for future inoculation studies in plants.
111
26
Response of maize mycorrhizas to green manure
David Ortiz*, John Larsen
Universidad Nacional Autónoma de México, Mexico
Green manure crops are employed in most plant production systems, but still limited
information is available on the effects of green manure on important plant growth promoting
microorganisms such as arbuscular mycorrhizal fungi. In the present study the response of
maize mycorrhizas to different types of green manure crops was examined in a greenhouse
pot experiment. The experiment had a fully factorial design with two main factors: 1) Green
manure with four levels (without, rape, oat, vetch) and 2) Mineral fertilization with two levels
(without and with). All eight treatments had four replicates giving a total of thirty two
experimental units. Green manure crops were grown in 3 liter pots with an agricultural soil
low in P (4 ppm) mixed with sand (1:1, w/w) for six weeks and the green manure mixed in to
the soil and left for eight weeks to decompose. Maize hybrid seeds were then sown in the soil
with the different types of green manure and mineral fertilizer treatments and the maize
plants were harvested after another ten-week period. Variables measured included plant
growth performance (shoot and root dry weight), shoot nutrient content (N and P) and
arbuscular mycorrhiza fungus root colonization. The green manures oat and rape reduced
maize plant growth performance as well as total N and P shoot content, except for vetch,
which on the contrary increased maize plant growth and total N and P shoot content in
treatments without mineral fertilization. Oat and rape green manures also reduced the
arbuscular mycorrhizal fungus root colonization with 20% and 49% respectively, whereas
vetch had no effect on mycorrhiza formation. In conclusion, green manure crops not only
affect growth and nutrition of maize, but also maize mycorrhiza formation, which seems to be
important to consider when planning crop rotation programs.
27
Rhizospheric labile phosphorus fractions affected by phosphate source and filter
cake
Paulo Pavinato*, Bruna Arruda, Aline Santos, Marcos Rodrigues
University of São Paulo/College of Agriculture Luiz de Queiroz, Brazil
In tropical soils the adsorption of phosphorus (P) is higher onto Fe/Al oxides, reducing plant
availability. In this way, the soil mass exploitation by the root system becomes much more
important in rhizospheric region for adequate plant nutrition. This study aimed to evaluate
the dynamic of labile and moderately P in the soil sugarcane rhizosphere under sources of
phosphate and filter cake. The experiment was conducted in a sandy soil under greenhouse
conditions at Department of Soil Science, ESALQ – USP, Piracicaba-SP-Brazil, in randomized
blocks with four replications. The treatments followed a factorial design of 2x3: a) with and
without filter cake (10 t DM ha-1) and b) no P fertilizer; soluble phosphate and rock phosphate
(both at the rate of 180 mg soluble P2O5 kg-1 soil). Soil samples were obtained from the
rhizospheric soil, 0-2 mm from the rhizoplane (root/soil separation system), using PVC
columns with a horizontal mesh for root exclusion, in a tube with 0.15 m diameter and 0.30 m
tall. These samples were collected 45 days after seedlings planting. It was determined in
laboratory the labile P fractions by anion exchange resin (PAER) and bicarbonate (PBIC)
extractors, and moderately labile fractions by NaOH and HCl. The data was analysed and
means were compared by Tukey test at 5%. The average of PAER for all treatments was 10.0
112
mg kg-1 and PiBIC was 27.9 mg kg-1. There was a significant interaction between P source and
filter cake use for both PAER and PBIC. Both fractions under soluble phosphate presented the
highest values of labile P, regardless the absence or presence of filter cake. Without P
fertilization and under rock phosphate, the presence of filter cake increased both labile P
fractions. Other fractions will be presented and discussed at the conference.
28
Urea and nitrate: a reciprocal interaction affecting nitrogen acquisition by maize
plants
Laura Zanin1, Anita Zamboni2, Nicola Tomasi1, Zeno Varanini2, Stefano Cesco3, Roberto
Pinton*1
1
University of Udine - Dip. Scienze Agrarie e Ambientali, Italy, 2University of Verona Dip. Biotecnologie, Italy, 3University of Bolzano - Faculty of Science and
Technology, Italy
Although urea (U) and nitrate (Ni) are the two main nitrogen forms applied as fertilizers in
agriculture and occur concomitantly in soils, the reciprocal influence of these two nitrogen
sources on the mechanisms of their acquisition is poorly understood. In the present work, the
molecular and physiological aspects of U and Ni uptake were investigated in maize, a crop
plant consuming large amounts of nitrogen.
Morphological investigation allowed comparing the root architecture of plants fed for 1 week
with U, Ni or U+Ni. Under U and U+Ni treatments, the presence of U in the external solution
promoted the development of the root system showing an extensive proliferation and
elongation of the roots, more than under Ni alone. This stimulatory effect might allow plants
to increase the volume of the rhizospheric soil explored by the roots promoting nutrient
acquisition.
The capacity of maize plants to acquire urea and nitrate was evaluated by net-uptake assay at
time intervals during 24 hours of root exposure to the two sources. Like Ni, also U is taken up
by maize roots by an inducible high-affinity transport system. However, the induction of Ni
and U high-affinity transport mechanisms were reciprocally limited when both sources were
present in the root-external solution.
Root transcriptomic analyses revealed that the simultaneous presence of U and Ni promoted
the activation of both the plastidial GS2/GOGAT cycle and a cytosolic nitrogen-assimilation
pathway (cytosolic glutamine synthetase and asparagine synthetase). Furthermore, the
induction of the aromatic amino-acid synthesis, that would sustain the production of
secondary metabolites, was observed. Based on these results, the transcriptional modulation
induced by the presence of both nitrogen sources would determine an increase in nitrogen
metabolism promoting a more efficient assimilation of taken-up nitrogen.
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29
Do nitrogen-fixing plants show higher root phosphatase activity on phosphoruspoor soils
Guochen Kenny Png*1, Patrick Hayes1, Hans Lambers1, Benjamin Turner2, Etienne
Laliberté3
1
The University of Western Australia, Australia, 2Smithsonian Tropical Research
Institute, Panama, 3Institut de recherche biologie végétale, Canada
Symbiotic nitrogen fixation in nitrogen-fixing plants may enhance plant performance on
nitrogen-poor soils, but may not be favoured on phosphorus-poor soils, due to its high
phosphorus costs. Yet surprisingly, nitrogen-fixing species are abundant in ecosystems with
nitrogen-rich soils such as lowland tropical rainforests, where phosphorus is likely to limit
plant growth. A prominent hypothesis seeking to explain this paradox is that nitrogen-fixing
plants have a greater ability to acquire organic phosphorus through higher root phosphatase
activity. However, evidence to support this hypothesis remains limited. We measured
extracellular root phosphomonoesterase activity from 18 species of nitrogen-fixing (including
legumes and two non-legume Allocasuarina species) and non-nitrogen-fixing species along a
soil age gradient in Western Australia that shows a ~40-fold decline in total soil phosphorus
from the youngest to the oldest soils, leading to some of the most phosphorus-impoverished
soils found in any terrestrial ecosystem. In support of the hypothesis, we found that nitrogenfixing legumes had higher phosphomonoesterase activity than co-occurring non-legumes on
all sites, and that the difference in phosphomonoesterase activity between legumes and nonlegumes increased with declining soil phosphorus concentration. However,
phosphomonoesterase activities of both nitrogen-fixing Allocasuarina species (which form
associations with Frankia) were consistently low across all soils which do not support the
hypothesis. We conclude that the high root phosphatase activity of legumes on phosphoruspoor soils is likely a phylogenetically conserved trait that is not necessarily linked to their
nitrogen-fixing ability.
30
Mobilization of trace elements by phytosiderophores in contaminated soils
Markus Puschenreiter*1, Barbara Gruber1, Eva Oburger1, Walter W. Wenzel1, Yvonne
Schindlegger1, Stephan Hann1, Walter D.C. Schenkeveld2, Stephan M. Kraemer2
1
University of Natural Resources and Life Sciences Vienna, Austria, 2University of
Vienna, Austria
Phytosiderophores (PS) are a group of root exudates released by grass species (Poaceae) for
the mobilization of iron. PS have high complex stabilities with iron, but also with other trace
elements, such as Zn, Cu and Ni. In trace element-contaminated soils, the PS-induced
mobilization rate of Fe might be reduced by the presence of competing elements. A rhizotest
experiment has been conducted with wheat exposed to four different contaminated soils.
Three soils were enriched by Zn, Cd, and Pb due to atmospheric deposition in the vicinity of
metal smelters, whereas the fourth soil was a serpentine soil naturally enriched by nickel.
Prior to the soil contact period, the wheat plants were grown in Fe-deficient or normal
conditions. In addition, a trace element mobilization experiment was carried out by adding
different PS concentrations to an extraction solution. An increase of root exudation and of PS
release was found for Fe-deficient wheat. Due to the enhanced PS concentrations in the
rhizosphere, also the mobilization of other elements than Fe was increased, leading to higher
114
Zn and Cu concentrations in wheat shoots. Ni concentrations were higher in Fe-deficient
wheat exposed to the serpentine soil. No changes were found for Cd and Pb. Likewise, in the
extraction experiment Cd and Pb were hardly mobilized, whereas a significant solubilisation
was found for Fe, Zn, Cu and Ni, in particular for PS concentrations of 100 and 1000 µMol/L in
the extraction solution. Our results show that grass species grown on trace element-enriched
soils might accumulate pollutants under conditions of Fe deficiency.
31
IPNM vis-à-vis soil-plant-water environment on diversified wetland crops for
yield stability and quality - sustainable for rural livelihoods in India
Anandamoy Puste*1, Kalyan Jana2, Krishnendu Ray2, Dorashila Nagarjuna2
1
Bidhan Chandra Krishi Viswavidyalaya (State Agricultural University), India, 2Bidhan
Chandra Krishi Viswavidyalaya, India
The science ‘IPNM (integrated plant nutrient management)’ combines & supply of all key,
macro and micronutrients in proper doses, resulted good growth, production potentiality and
quality of produces, more effective in developing country like India. With this significance
importance, case studies on IPNM at farmers’ field during 2009 - 2012 on diversified valuable
wetland crops [water chestnut, makhana, Cyrtosperma chamissomis, water-lily] under wetland
ecosystem were undertaken in the different agro-zones. Major and other essential plant
nutrients were combined and balanced through organic and inorganic sources including
spray [F1 - Control, (farmer’s practice), F2 - F4 consisting of FYM/vermicompost/neem oilcake
+ NPK (60:30:30 kg ha–1), F5 - F7 composed of F2 - F4 along with Zn (chelamin) spray at 20, 40,
60 DAT (days after transplanting) + NPK spray at 60 DAT] and FYM @ 3.0 t and
vermicompost and neem oilcake was used @ 0.2 t ha–1 and Zn + NPK @ 0.5% each],
respectively. Root rhizosphere (growth, proliferations and establishment) as well as food
quality were analyzed following standard methods. Results shows that yields of water
chestnut, seeds of makhana, lati or stolons of Cyrtosperma and flower-stalks of water-lily
(11.06, 2.98, 37.43 and 12.07 t ha–1, respectively) were remarkably influenced and highest by
IPNM (F7) with optimum and balance form of plant nutrients that favours soil-plant-water
interrelations including its quality (carbohydrates, starch, sugar, protein, minerals). Among
various organic sources, neem oilcake performed better applied with proper dose and in
time. From the experimental results pertaining to the field performance, it may be concluded
that this vast unused wetlands, particularly in north-eastern part of the country may be
utilized for aquatic crops, more precisely with balanced and economic sources of plant
nutrients for crop productivity, making congenial soil-plant environment and sustainability to
the farming communities in the regions.
115
32
Yield improvement, indigenous nutrient supply, and nutrient use efficiency of
hybrid maize cultivars in an inceptisol of West Bengal, India
Krishnendu Ray*1, Hirak Banerjee2, Anandamoy Puste2, Sukamal Sarkar2
1
Bidhan Chandra Krishi Viswavidyalaya (State Agricultural University), India, 2Bidhan
Chandra Krishi Viswavidyalaya, India
Indian soil has greater diversity from poor to humus-rich, somewhere more scarce due to
acute nutrient loss, even from inherent soil sources due to prevalence of sub-tropical to
tropical environment. In this context, site-specific nutrient management (SSNM) has a pivotal
role for supplying balanced and in-time nutrients for plant growth and development. Plant
based approach for nutrient requirement determination requires omission plot technique
(OPT), which facilitates development of decision support tool (DST) for implementation of
SSNM at field levels and formulates fertilizer guidelines with a clear estimation of indigenous
nutrient supply (INS). Present study was conducted during consecutive winter seasons of
2012-2014 to develop SSNM package for hybrid maize at the new alluvial soils of West
Bengal, India. Target yield was fixed using the Nutrient Expert (NE) software. The experiment
was laid out in strip-plot design with three hybrid maize cultivars (V1: P-3522, V2: P-3396 and
V3: Rajkumar) in the horizontal strips and nine different fertilizer doses [F1: 50% RDF; F2: 75%
RDF; F3: 100% RDF (Recommended dose of fertilizer 200:60:60 kg N: P2O5: K2O / ha is the
State recommendation for hybrid maize); F4: 125% RDF; F5: 150% RDF; F6: 100% of P2O5 and
K2O (N omission); F7: 100% of N and K2O (P omission); F8: 100% of N and P2O5 (K omission);
F9: Absolute control (NPK omission)] in vertical strips replicated thrice. Nitrogen proved to be
most limiting nutrient followed by K for P-3522 and P-3396, and P for Rajkumar. Results
showed that the indigenous supply of N and P was highest for P-3396; however, P-3522
witnessed highest indigenous K supply. Application of 100% NPK (RDF) showed significantly
higher growth, yield attributes and yields of the cultivars over omission plots, thus suggesting
higher use efficiency of nutrients owing to better root plastic responses in the nutrient-rich
plots.
33
Modelling nutrient uptake by roots of crops in fixed and variable volume of soil
Juan Carlos Reginato*1, Jorge Luis Blengino1, Domingo Alberto Tarzia2
1
Departamento de Física, Facultad de Ciencias Exactas Físico-Químicas y Naturales,
Universidad Nacional de Río Cuarto, Argentina, 2Departamento de Matemática–
CONICET, Facultad de Ciencias Empresariales, Universidad Austral, Argentina
Nutrient uptake by roots of crops in fixed (pots) and variable soil volume (field conditions) is
modelled through a moving boundary problem. Thus, we evaluate the nutrient uptake for
different situations and compare results with those obtained by 1D and 3D fixed boundary
models. Nutrient uptake model is formulated through a one-dimensional moving boundary
problem, both in the case of fixed soil volume fixed as the variable soil volume. To compute
influxes on root surface, the problem is solved by immobilisation of the domain and
application of the adaptive finite element method (FEM). To estimate the cumulative nutrient
uptake a generalised and verified formula is used. The results obtained show that fixed and
moving boundary models produce similar results for ions without limitations of availability,
low variation of root density and low Peclet numbers. For low concentrations, large variations
of root density and low numbers of Peclet the moving boundary produces better predictions
116
particularly for K. For P the moving boundary produces better predictions only at low
concentrations being these predictions comparable to the obtained by 3D-dimensional
architectural models. Obtained improvements are mainly due to three factors: the use of a
generalised formula for the cumulative nutrient uptake, the use of a same dynamics to obtain
influxes and the cumulative uptake in the moving boundary model, and the use the finite
element method. Moreover, only our model satisfies the mass balance. Finally, in the light of
these findings, conclusions drawn by previous papers could be reinterpreted.
34
Modelling nutrient uptake by roots of crops in fixed and variable volume of soil
Juan Carlos Reginato*1, Jorge Luis Blengino1, Domingo Alberto Tarzia2
1
Universidad Nacional de Rio Cuarto, Argentina, 2Universidad Austral, Argentina
Nutrient uptake by roots of crops in fixed (pots) and variable soil volume (field conditions) is
modelled through a moving boundary problem. Thus, we evaluate the nutrient uptake for
different situations and compare results with those obtained by 1D and 3D fixed boundary
models. Nutrient uptake model is formulated through a one-dimensional moving boundary
problem, both in the case of fixed soil volume fixed as the variable soil volume. To compute
influxes on root surface, the problem is solved by immobilisation of the domain and
application of the adaptive finite element method (FEM). To estimate the cumulative nutrient
uptake a generalised and verified formula is used. The results obtained show that fixed and
moving boundary models produce similar results for ions without limitations of availability,
low variation of root density and low Peclet numbers. For low concentrations, large variations
of root density and low numbers of Peclet the moving boundary produces better predictions
particularly for K. For P the moving boundary produces better predictions only at low
concentrations being these predictions comparable to the obtained by 3D-dimensional
architectural models. Obtained improvements are mainly due to three factors: the use of a
same dynamics to obtain influxes and the cumulative uptake in the moving boundary model,
the use of a generalised formula for the cumulative nutrient uptake and the use the finite
element method. Moreover, only our model satisfies the mass balance. Finally, in the light of
these findings, conclusions drawn by previous papers could be reinterpreted.
35
Can Fe-chelating bacteria increase bioavailable Fe-content in lentil (Lens
culinaris L.)?
Mortuza Reza*, Fran Walley, Renato de Freitas
University of Saskatchewan, Canada
Iron (Fe) deficiency is the most common human nutrient deficiency worldwide, particularly in
developing countries. Legumes including lentil (Lens culinaris L.) are an important and
relatively inexpensive source of protein and are consumed worldwide. Iron bio-fortification of
legumes can be an effective approach to increase Fe-bioavailability in areas affected by
human Fe deficiencies. The objective of this study is to obtain natural Fe-chelating bacteria
associated with lentil roots and assess their potential to improve crop Fe uptake. Lentil (cv.
CDC Milestone) was grown in a growth chamber using soils collected from eight locations in
Saskatchewan, Canada. Iron chelating bacteria (n=553) were isolated from the rhizosphere
117
(n=271) and endorhizosphere (n=282) using CAS selective medium and in vitro siderophore
production was subsequently assessed in a Fe-free M9 minimal medium. Additionally,
siderophore concentration was measured in cell free supernatants using a SideroTecTM Assay
kit. Preliminary results indicate that selected isolates of both rhizosphere and
endorhizosphere bacteria produced siderophores in concentrations ranging from 0.04 to
119.54 µg∙mL-1, although this population represented less than 1% of the total culturable
heterotrophs. Of these, 14 isolates exhibited relatively high siderophore production i.e., >50
µg∙mL-1. These putative Fe-chelating bacteria isolates will be further identified using Sanger
sequencing. Our results demonstrate that Fe-chelating bacteria colonize both the rhizosphere
and endorhizosphere of lentil and likely play a role in the iron nutrition of the host plant.
Studies designed to assess the effect of Fe-chelating bacteria on iron acquisition of lentil are
currently in progress.
36
Potassium dynamics in ruzigrass rhizosphere as affected by K rates
Ciro Rosolem*, Amanda Gomes
São Paulo State University, Brazil
Ruzigrass (Brachiaria ruzisiensis) has been used in Brazil as a cover crop because it has a
vigorous root system and is efficient in cycling nutrients in the system. It has been shown to
take up soil K considered as non-exchangeable. This study assessed exchangeable and nonexchangeable K dynamics in the rhizosphere of ruzigrass as affected by long term K
fertilization. Samples were taken from an 11-year field experiment where plots have been
fertilized with a total of zero to 1980 kg ha-1 of K2O as KCl. The soil is a Rhodic Hapludox with
210 g Kg-1 of clay, 78 % of kaolinite and less than 3 % of gibbsite. Ruzigrass was grown for 48
days in pots divided in half by a nylon screen. The top half was filled with sand and the
bottom half with soil from each field plot. The soil from the bottom was sliced and analyzed
for exchangeable (resin) and non-exchangeable (hot HNO3) K. Potassic fertilization had no
effect on ruzigrass dry matter yields, but K uptake responded linearly to K rates. Rhizosphere
exchangeable K was not affected in K absence. With 60 Kg ha-1 per year, K was depleted in
the rhizosphere, which was increased with 120 Kg ha-1 of K per year or more. Nonexchangeable K was higher than in bulk soil where K fertilizer was applied. The K considered
as non-exchangeable is affected by K fertilization and plays a role in ruzigrass nutrition. The
use of high rates of K in a sandy loam rich in kaolinite results in a higher proportion of K
transported in soil by mass flow, avoiding K depletion in the rhizosphere of ruzigrass. If K rate
is high enough there is accumulation of the nutrient in the rhizosphere both in exchangeable
and non-exchangeable forms.
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37
Rock phosphate solubilization by rhizobacteria and endophytic associated with
maize (Zea mays L.)
Ubiana Silva1, Luiz Freitas1, Julliane Medeiros2, Christiane Oliveira3, Sara Cuadros2,
Eliane Gomes3, Vera Santos*1
1
Laboratory of Applied Microbiology, Microbiology Department, Institute of Biological
Science, Federal University of Minas Gerais, Brazil, 2René Rachou Research
Center, Brazil, 3National Research Center Mayze and Sorghum, Brazil
Phosphate fertilizers are largely used for improving soil fertility. However, the use of less
reactive phosphates requires the activity of phosphate solubilizing microorganisms. Some
bacteria possess the ability to solubilize phosphate and increase the bioavailability of this
nutrient in the soil-plant system. The aim of this work was to isolate and to characterize the
phosphate solubilization potential of rhizobacteria and endophytic bacteria from Zea mays.
Rhizobacteria were isolated from Zea mays rhizosphere soil cutivated without phosphate
fertilizer and with Araxá rock phosphate. Thirty five bacteria obtained were evaluated for
phosphate solubilization of rock Araxá jointly 10 endophytic bacteria obtained from
collection of National Research Center mayze and Sorghum. Solubilized phosphate was
quantified by molybdenum blue method at 880 nm after 72 h. The pH medium was also
measured. Strains were identified based in the 16S rDNA gene. Furthermore, was evaluated
the organic acid production for strains more efficient. The results were subjected to variance
analysis and compared by Scott-Knott tests (P<0.05). Pantoea ananatis and Pantoea sp.
showed major phosphate solubilization capacity, releasing 75 mg L -1. Other strains of the
Enterobacter and Klebsiella genera, released 54 until 69 mg L -1, but some strains these
genera showed also low potential for solubilization. Lower values of pH and acid production
occurred in the samples with more phosphate released. It was found citric, acetic and latic
acids. Citric acid was the most produced by efficient strains, aproximated 120 mg L -1. Thus,
the phosphate solubilization capacity was influenced by strain evaluated. Moreover, the acid
production certainly contributed to the dissolution of phosphate from rock Araxá, as has
been reported in several experiments.
38
A window for iron uptake in Strategy II iron acquisition
Walter Schenkeveld*1, Martin Walter1, Eva Oburger2, Yvonne Schindlegger2, Stephan
Hann2, Markus Puschenreiter2, Stephan Kraemer1
1
University of Vienna, Austria, 2Boku, Austria
Graminaceous plants (grasses including staple crops like wheat and barley) exude so-called
phytosiderophores (PS) into the rhizosphere in a diurnal pulse release for acquiring the
essential micronutrient iron (Fe). Phytosiderophores are chelating agents that are able to bind
and solubilize soil-Fe and make it available for plant uptake. When PS are released, they
participate in rhizosphere processes including adsorption, degradation and complexation of Fe
and competing metals.
In a series of batch interaction experiments with a calcareous clay soil from Santomera
(Spain) to which the PS 2’-deoxymugineic acid (DMA) was added at various concentrations,
the influence of rhizosphere processes on Fe mobilization was explored. It was found that
these processes draw up a time and concentration window, during which DMA increases the
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Fe concentration above background levels: the window for Fe uptake. This size of the window
proved to strongly depend on the DMA concentration. The window was further constrained
in time by biodegradation and metal competition (particularly with copper (Cu)).
Our work shows that the extent to which PS are able to provide plants with Fe depends on
environmental and rhizosphere soil conditions, which affect thermodynamically and
kinetically controlled processes. The influence of these conditions on the success or failure of
Strategy II Fe acquisition can be interpreted by means of the window concept. In this context,
it will be discussed how soil parameters including temperature, rhizosphere pH, electrolyte
type and concentration, and abundance of soil reactive surfaces and competing metals affect
the effectiveness of Strategy II Fe acquisition.
39
Biostimulant and PGPR treatment efficacy for P-solubilisation and winter wheat
growth in large pot trial
Shekhar Sharma*1, Chris Selby1, David Nelson1, Graham McCollum2, Eugene
Carmichael2, Fiona Clarke2, J R Rao2
1
Agri-Food Biosciences Institute, United Kingdom, 2AFBI, United Kingdom
The efficacy of phosphorus (P)-solubilising bacterial soil treatments comprising commercial
formulations ABiTEP Bacillus simplex + FZB42 Bacillus amyloliquefaciens, foliar biostimulant
Alga Vyt, Agreges and P solubiliser bacteria were evaluated. The treatments were assessed for
their effects on growth and grain yield of winter wheat grown on either a regularly cultivated
soil or a nutritionally poorer soil that has not received fertiliser for several years. Seventy litre
pots were filled with each soil type, and winter wheat seeds were sown to a depth of 1.5 cm
in open air. The soil P solubilising bacterial preparations were applied as a 20 ml spray to the
soil surface of each pot at the two leaf stage and later on fully grown plants. The AFBI-P
solubiliser was an equal mixture comprising two Rahnella aquatalis and one Burkholderia spp.
selected from low input soils. The foliar biostimulant Alga Vyt (1.5 g/l) was applied weekly, 4
times. Soil samples were collected for analysis. The addition of bacteria and treatment with
foliar biostimulant did not exhibit apparent growth/development enhancement of wheat
plants. A total count of P-solubilisers at the start and end of trial remained unchanged.
However, closer scrutiny of the results indicated that wheat matured marginally faster on the
standard input soil compared to the low input soil without increasing ultimate grain yield.
Such subtle interactions between soil type and the biostimulants on plant growth and
development that were often not recognised by standard plant analyses prompted us to
study P-solubiliser activities in the soil/root rhizosphere. A protocol using an anion exchange
membrane technique demonstrated that membrane matrix yielded better soil P-recovery than
traditional direct soil chemical extraction methods and may impact the final analyses for
validation of results.
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40
Rhizosphere interactions – Competition and environment regulate plant
nitrogen acquisition
Judy Simon*
University of Konstanz, Germany
Plants have developed different mechanisms to optimise the utilisation of limited N resources,
for example by N uptake via mycorrhiza symbionts and / or acquisition of organic N sources.
However, the complex interactions between different vegetation components, mycorrhizal
fungi, and free-living soil microorganisms with regard to the competition for N in the
rhizosphere are currently only little understood. Thus, the research presented here provides
new insights into the understanding of the processes involved in the regulation of
belowground competition for N between the different players in temperate forest
ecosystems (i.e. seedlings vs. adult trees, different woody species, tree vs. soil
microorganisms). For example, competition for N is regulated by N source (organic vs.
inorganic), signals in the rhizosphere (i.e. nitric oxide and/or carbon dioxide as well as
rhizodeposition) and shifts depending on environmental factors (i.e. soil N availability).
Furthermore, plants acquire different N forms (i.e. organic, inorganic) when growing in
competition. In addition, plants do not have to rely on the support of soil microorganisms,
such as mycorrhizal fungi, to gain access to organic nitrogen but could have a competitive
advantage over other plant species via exudation of proteases to obtain organic nitrogen
directly from the humus layer, thus forgoing the microbial loop of nitrogen turnover
processes.
41
Simultaneous use of gypsum and legume crops reduces soil pH and alters soil
carbon on rhizosphere of calcareous alkaline soils
Ehsan Tavakkoli*1, Pichu Rengasamy2, Glenn McDonald2
1
NSW Department of Primary Industries, Wagga Wagga Agricultural
Institute, Australia, 2Waite Research Institute, School of Agriculture Food and Wine, The
University of Adelaide, Australia
Large areas of the world’s arable land have alkaline soil and in the southern cropping region
of Australia alkaline sodic soils, often with high concentrations of carbonate salts, are
widespread. High pH presents many nutritional challenges to crop production that influence
both direct and indirectly, the accumulation of soil organic C. The use of legumes in
conjunction with the supplying additional Ca to soils has the potential to lower pH on highly
alkaline soils. To examine the potential to lower pH, two short-term rotation experiments at
South Australia and Victoria were established on alkaline calcareous soils. In addition an
experiment was conducted to investigate plant and gypsum effects on the chemistry of the
rhizosphere in a growth chamber experiment The experiments examined the effects of
legume biomass and gypsum application on soil pH, soil C and productivity of wheat grown
in the following year. Applying 2.5 t/ha of gypsum reduced soil pH by between 0.2 and 0.4
units to a depth of 30 cm after 12 months. The changes in pH were associated with a decline
in dissolved organic C at both sites and with higher organic C. Biomass production by
legumes did not affect these soil properties. We also used C (1s) near-edge x-ray absorption
fine structure (NEXAFS) and synchrotron-based Fourier transform infrared (FTIR) spectroscopy
to investigate the C and N speciation in the rhizosphere of alkaline soils. Together, this
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information is not only useful in terms of understanding the mechanisms responsible for SOC
retention/dissolution but also in terms of nutrient bioavailability, as this is strongly related to
SOC speciation. In this presentation, analyses of SOM chemistry in relation to its speciation
and stability and implications for sequestration and nutrient cycling will be discussed.
42
Zinc biosorption mediated by exopolysaccharide in plant growth promoting
Pseudomonas fluorescens Psd
Anamika Upadhyay*1, Mandira Kochar2, Manchikatla Venkat Rajam3, Sheela Srivastava3
1
Department of genetics, University of Delhi South Campus, India, 2TERI Deakin
Nanobiotechnology Centre, The Energy and Resources Institute
(TERI),, India, 3Department of Genetics, University of Delhi South Campus, India
Zinc is an essential trace element required for the growth and development of all organisms
including bacteria, but may exert toxic effects at higher concentrations. Extracellular
biosorption is the mechanism of zinc resistance in the plant growth promoting bacterium,
Pseudomonas fluorescens strain Psd. Our study aims to identify the key players involved in
Zn2+ biosorption and their effect on the plant-growth promoting potential of the strain. The
increased Zn2+ accumulation by the strain was accompanied by an increase in the various
plant-growth promoting parameters like siderophore, phenazine production and phosphatesolubilization. IAA production, however, was found to decrease with increasing Zn2+
concentrations. Higher Zn2+ accumulation also led to increase in total exopolysaccharide
content. The compositions of the exopolysaccharides were measured using different
biochemical assays and FT-IR spectroscopy. Expression analysis was carried out for genes
responsible for biosynthesis of two of the important exopolysaccharides, alginate (alg8) and
psl (psl). Quantitative RT-PCR analysis revealed an increase in expression of alg8 with
increased Zn2+ accumulation by the strain, whereas pslA expression levels remained
unchanged. Biofilm formation was also found to be increased with enhanced
exopolysaccharide biosynthesis.
Our study explores the property of plant-growth promoting rhizobacteria to secrete
exopolysaccharides under metal-rich/contaminated environments, which aids in bacterial
survival as well as its attachment to the root surface ensuring better plant-rhizobacterial
association.
43
Exudation mechanisms in strawberry plants as affected by iron and phosphorus
deficiency
Fabio Valentinuzzi*, Youry Pii, Stefano Cesco, Tanja Mimmo
Free University of Bozen-Bolzano, Italy
Strawberries are among berries a very popular fruit, especially for their beneficial effects for
human health. However, their bioactive compound content is strictly related to the nutritional
status of the plant and might be affected by nutritional disorders. To overcome the nutrient
shortages plants evolved different mechanisms, which often involve the release of low
molecular weight compounds known as root exudates. The biochemical and molecular
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mechanisms underlying root exudation and its regulation are yet still poorly known, in
particular in woody plants. The aim of this work was therefore to characterize the pattern of
root exudation of strawberry plants grown either in phosphorous (P) or iron (Fe) deficiency. In
addition, phylogenetic analyses were carried out to identify the sequences of putative genes
involved in the release of organic acids and protons. This is the first study that shows the
release of citric acid from strawberry roots. In particular, this release significantly increased
with time being +250% and +300% higher in Fe and P deficient plants, respectively,
compared to the control. Furthermore, concomitantly, a significant acidification of the growth
medium was observed in both treatments. Phylogenetic analyses allowed the identification of
five proteins which clustered in the MATE sub-family characterized for the transport of citrate
in response to Fe and aluminum stresses. Moreover, also putative strawberry PM H+-ATPases
were identified.
In conclusion the response differs in function of the nutritional disorder (Fe vs P) and the
extent of organic acids release together with the extrusion of protons depends on plant
growth stage and treatment. These results will allow managing agronomical practices
towards a more sustainable agriculture.
44
Arbuscular mycorrhizal fungi increase plant phosphate uptake from phytate
Xinxin Wang*1, Ellis Hoffland1, Gu Feng2, Thomas Kuyper1
1
Wageningen University, Netherlands, 2China Agricultural University, China
Phytate constitutes the largest pool of soil organic phosphorus (P). Increased P nutrition of
arbuscular mycorrhizal plants after phytate addition has been repeatedly reported; however,
earlier studies assessed acid phosphatase rather than phytase as an indication of mycorrhizal
fungi-mediated phytate use. Earlier studies also disregarded the potential effect of phytate
addition on mobilization of mineral P, due to competitive adsorption of phytate and
concomitant desorption of phosphate ions on metal (hydr-)oxides. We investigated the effect
of mycorrhizal hyphae-mediated phytase activity on phytate mineralization and subsequent P
transfer to the host plant. Two maize (Zea mays L.) genotypes, non-inoculated or inoculated
with the AM fungi Funneliformis mosseae or Claroideoglomus etunicatum, were grown in twocompartment rhizoboxes. The soil in the hyphal compartment was supplemented with 20,
100 and 200 mg P kg-1 soil as calcium phytate. We measured activity of phytase and acid
phosphatase in the hyphal compartment, hyphal length density, P uptake and plant biomass.
Our results showed: (1) phytate addition increased phytase and acid phosphatase activity, and
resulted in larger P uptake and plant biomass; (2) increases in P uptake and biomass
were correlated with phytase but not with acid phosphatase; (3) lower phytate addition rate
increased, but higher rates decreased hyphal length density. We calculated that at the higher
rate, competitive desorption of phosphate is substantial, providing an explanation for the
decrease in hyphal length density at high phytate addition P. We conclude that P from
phytate can be a major source for arbuscular mycorrhizal plants.
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45
Growth condition and P mobilizing properties of cluster roots of Helicia
cochinchinensis (Proteaceae) at Miyajima Island, Japan
Jun Wasaki*, Taiki Yamauchi, Jin Takahashi, Hayato Maruyama, Shinji Uchida, Seiji
Mukai, Hiromi Tsubota
Hiroshima University, Japan
Some specific plants form bottle brush-like root structures, so-called cluster roots, during P
deficiency. The family Proteaceae are well known group for cluster root-forming plants.
Helicia cochinchinensis Lour. is only one species present in Japan among the Proteaceae, and
is distributed in the range from southwest Japan to the Indochina Peninsula. It is still
unknown what properties of soils distributing H. cochinchinensis and whether this species
forms cluster roots. The aim of this study is to investigate the growth conditions,
morphological properties of roots, and phosphate mobilizing abilities of H. cochinchinensis
growing in Miyajima Island, Hiroshima, Japan. Available P in soils growing H. cochinchinensis
grew was very poor; ranged 0.46 – 3.7 mg-P/kg-soil (Olsen-P). We found cluster roots from P
deficit H. cochinchinensis in natural and hydroponic conditions. It was shown that acid
phosphatase activity was increased in the rhizosphere soil. Activity staining revealed a higher
activity of acid phosphatase in half-part from root tip of each rootlet. Strong decrease of pH
in the rhizosphere of matured cluster roots was also shown, suggesting that the cluster root
enhanced organic acid exudation like other Proteaceae plants. P concentration of leaves of H.
cochinchinensis was low level; those of mature and senesced leaves ranged 0.34 - 0.69 mgP/gDW and 0.15 - 0.29 mg-P/gDW, respectively. This range was some extent high than that
of other Proteaceae plants growing under very low P soils in Western Australia, but it was
very low level among general plant species. It was concluded that the P mobilizing capacity
of cluster roots and the tolerance to low P content supported the growth of H.
cochinchinensis under low P soil at Miyajima Island.
46
Ionomic variation in plant species growing in various soil environments
Toshihiro Watanabe*1, Yoshinobu Kusumoto2, Sayaka Morita2, Tomoyo Koyanagi2,
Mitsuru Osaki1, Syuntaro Hiradate2
1
Laboratory of Plant Nutrition, Research Faculty of Agriculture, Hokkaido
University, Japan, 2National Institute for Agro-Environmental Sciences (NIAES), Japan
Ionomics is the study of all metal, metalloid, and nonmetal accumulation in living organisms.
Plant ionomics has been applied to various types of study in the last decade. For example,
many ionomics studies have been conducted to investigate the relationship between the
ionome and genome in model plants, such as Arabidopsis thaliana or Lotus japonicus. In our
previous report of a broad survey of various plant species, we investigated higher-level
phylogenetic effects influencing the leaf composition of a wide range of elements. However,
the effect of changes in the rhizosphere environment on the plant ionome in different species
has hardly been investigated. Therefore, in the present study, we analyzed concentrations of
25 elements in leaves of various plant species growing under different soil environments to
evaluate the ionomic variation among different soil environments and to compare the
variation among different plant species. Twenty-three herbaceous species were sampled at
80 sites in Shiozuka highland, Japan. Concentrations of 25 elements in leaves were
determined by inductively coupled plasma mass spectrometry. Chemical properties of soils,
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including the ammonium acetate- or water-extractable concentrations of the same 25
elements, at 80 sites were also analyzed. The coefficients of variation in concentration of each
element among different sampling sites varied among plant species, particularly in nonessential elements, which showed higher coefficients of variation than those in essential ones.
No significant correlations were observed between concentration in leaf and extractable
concentrations in soil in most elements. However, in some elements, a positive correlation
was observed under some phylogenetic influence. With respect to the correlation between the
vegetation cover rate and extractable concentration of each element in soil, Miscanthus
sinensis showed significant positive correlations for aluminum, sodium, and several heavy
metal elements and a negative correlation for phosphorus, possibly associated with its higher
adaptation capacity to acidic soil.
47
Tree roots: plastic in architectural traits, but not in morphological and biotic
traits
Monique Weemstra*1, Frank Sterck1, Eric Visser2, Thomas Kuyper1, Frits Mohren1, Liesje
Mommer1
1
Wageningen University, Netherlands, 2Radboud University Nijmegen, Netherlands
Functional traits are important drivers of resource acquisition and growth of trees. Yet,
knowledge on plastic responses in belowground traits is still limited, impeding our
understanding of relationships between root traits, nutrient acquisition, and tree growth. This
study is among the first to examine plasticity in root functional traits of large forest trees. We
compared root traits of beech and Norway spruce trees on a nutrient-rich clay soil and on a
nutrient-poor sandy soil in the Netherlands. We hypothesized that trees adjust their roots to
nutrient-poor conditions relative to rich soils, by 1) increasing the size of their root system, 2)
changing their root morphology, and 3) investing more in mycorrhizal symbioses.
Trees of both species had a higher root length density and higher root growth rates at the
poor than at the rich soils. Root morphology (specific root length and root tissue density)
however, did not differ between soils. Mycorrhizal mycelium abundance was also larger at the
sandy soil, but this difference was not significant when correcting for the higher root length
present. On the root tips, however, we observed a larger number of mycorrhizal exploration
types that form mycelia on the sandy soil, while we only found mycorrhizal exploration types
that do not make mycelia on the clay soil.
Our study demonstrates that on poor soils, trees adapt their roots by enhancing the length of
their root system, and not by changing their root morphology. The larger number of
mycorrhizal mycelia on the poor soils seemed partly driven by larger root length, but the
larger abundance of mycorrhizal types that form mycelia may point at increased tree
investments in mycorrhizal fungi that are carbon-costly but more efficient in soil exploration
and resource uptake.
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48
Root morphological traits and rhizosphere processes of maize with increasing
shoot P concentration on a calcareous soil
Zhihui Wen*, Jianbo Shen, Haigang Li
China Agricultural University, Department of Plant Nutrition, China
The modifications of root growth and rhizosphere processes in maize (Zea mays L.) induced
by phosphorus (P) deficiency were well documented in previous studies. However, the
continuous changes of these modifications with increasing shoot P concentration have not
been investigated. We hypothesized that the changes in modifications of root growth differ
from rhizosphere processes with increasing shoot P concentration. A broad-gradient shoot P
concentration of maize (0.96-3.96 mg g-1) was founded in a pot experiment by application of
a series of P fertilizer rates (0, 2.5, 5, 10, 25, 50, 75, 150, 300, 600 and 1200 mg P kg-1 soil) on a
calcareous soil. Root morphology and rhizosphere processes were determined after 28 day
growth.
Maize reached to maximum shoot biomass when shoot P concentration was 2-3 mg g-1,
above which biomass did not increase any more, and below which biomass reduced because
of P deficiency. Total root-length (TRL), specific root-length (SRL) and the proportion of fine
root (diameter < 0.2 mm) to total root-length (PFR) showed the opposite patterns. TRL, SRL
and PFR of maize maintained a low level at P sufficient status (>2-3 mg g-1), and these root
parameters showed an increasing trend with decreasing P supply until shoot P reduced to
1.1-1.3 mg g-1, below which these traits exhibited a rapid decrease. In contrast, rhizosphere
acidification, acid-phosphatase activity and carboxylates accumulation in rhizosphere were
enhanced with increasing shoot P concentration, and had no reduction even if shoot P
concentration reached up to 4.96 mg g-1 that was seldom found in field. In conclusions, we
observed the modification trajectory of root morphological traits differs from rhizosphere
processes of maize with increasing shoot P concentration, indicating that maize had a strong
root morphological alteration in response to P deficiency compared to root exudation in the
rhizosphere.
49
Improving phosphorus acquisition efficiency in rice: The role of mycorrhizal
versus plant-induced uptake mechanisms
Matthias Wissuwa*1, Lidia Campos Soriano2, Blanca San Segundo2, Stephanie WattsWilliams3, Asako Mori1, Erina Shimamura1, Vincent Pujol1, Adam Price4
1
JIRCAS, Japan, 2CRAG, Spain, 3Monash University, Australia, 4University of
Aberdeen, United Kingdom
Phosphorus (P) is a limited resource and price hikes have made P fertilizers increasingly
costly. Breeding P-efficient rice varieties has therefore become an objective in many national
rice breeding programs. We previously identified the major P uptake QTL Pup1, and showed
that the underlying gene OsPSTOL1 improves P uptake via higher root growth. Here we
report on the identification of novel P uptake donors and QTLs for enhanced P acquisition
efficiency (PAE), the capacity for improved P uptake per given unit root size. Field
experiments on a P fixing soil identified sets of rice genotypes contrasting for total P uptake
and PAE. High PAE genotypes took up as much as 40 µg P m-2 root surface area compared to
an average of 18 µg P m-2. To examine whether arbuscular mycorrhizae (AM) are associated
126
with PAE we used three AM detection methods: a common staining method, quantification of
AM-specific DNA within rice roots, and detection of OsPT11 gene expression (OsPT11 being a
P transporter specifically expressed upon infection of rice roots by AM). Root samples taken
from the field did show OsPT11 expression in all genotypes, possibly indicating P transfer
from fungus to plant occurring very commonly. The staining and DNA methods also detected
the presence of AM in all roots but colonization rates varied from 10-70 % between
genotypes. However, this variation was not associated with genotypic differences in P uptake.
Additional pot experiments in soil-sand mixtures with rock-phosphate or phytate as main P
sources confirmed genotypic differences in P uptake. Together these results suggest plant
induced changes in the rhizosphere rather than associations with AM to be underlying causes
of superior PAE. Whether this rhizosphere effect is related to root exudation and direct P
solubilization or to indirect effects via beneficial changes in soil microbial populations is
discussed.
50
Effect of different levels of organic manure and chemical fertilizer inputs on
crop yield and soil nutrient content in winter wheat-summer maize rotation
system
Zhang Xiaoning*, Liu Ruili, Zhang Hongyan
Since the autumn of 2007, a field experiment was carried out in Quzhou Experimental Station
of China Agricultural University to compare the different influences of organic manure (OM)
and chemical fertilizer (CF) fertilization on crop yield and soil nutrient content in winter wheat
(WW) - summer maize(SM) rotation system. There were one CK treatment, four OM
treatments and four CF treatments in the experiment. The OM treatments were fertilized by
cattle manure of 3, 6, 9 and 12t DM/crop/year/ha, respectively. By contrast, the CF treatments
were designed with the same NPK input as that in the four OM treatments, respectively. The
preliminary results showed that the 7 year averaged WW grain yields in all the OM
treatments were significantly lower than that in the CF treatments at the same NPK input
level. At the same NPK input levels, the 7 year averaged SM grain yield in the OM treatments
of 3 and 6 t DM/ha/growing season were significantly lower than that of CF treatments,
although the 7 year average grain yield in the OM of 9 and 12 t DM OM/ha/growing season
were the same as that in CF treatments.The yield differences between OM and CF treatments
at the same NPK input level reduced gradually with planting years. After 7 year rotation, soil
organic matter content increased with the increase of OM inputs but did not changed with
the increase of CF input. Olsen-P, available K and Nmin content in soil increased with the
increasing of both OM and CF inputs. Generally, the Olsen-P content in OM treatments were
higher than that in CF treatments while Nmin content in CF treatment were higher than that
in OM treatments with the same NPK input level.
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51
Effect of phytase adsorbed on montmorillonite on the root activity of Malus
hupehensis and soil enzymes and microorganism in root-zone
Hongqiang Yang*, Pingping Yang
Shandong Agricultural University, China
Phytate is rich in phosphorus, but it cannot easily be absorbed by the root directly. Phytase
can catalytic the release of phosphorus in phytate, but it is easily inactivated directly applied
into the soil. Montmorillonite has good adsorption properties of enzyme and other organic
matter. In this experiment, the phytase adsorbed on montmorillonite (PAM) was prepared by
the liquid phytase sprayed onto montmorillonite and added it to the soil of potted Malus
hupehensis seedlings.
The result show that the montmorillonite can effectively adsorb the phytase and delay the
decline of phytase activity in soil, the activity of soil phytase by PAM treated was 18.97%
higher than that by liquid phytase treated on the 50th day after two kinds of phytase added.
The activity of soil phosphatase, urease and dehydrogenase, the quantity of soil
microorganisms, the content of available phosphorus in root-zone soil, and the root activity
of Malus hupehensis seedlings were all increased significantly after applying PAM and liquid
phytase to the soil, and all of which increasing with the increase of the applying dosage. With
the time prolonged, the activity of soil phytase became stable after dropping rapidly, the
activity of soil phosphatase rose gradually in the mass, and the content of available
phosphorus declined slowly after rising fast. In addition, PAM suggested more effective and
longer validity than that of liquid phytase.
52
Iron deficiency in Zea mays: transcriptomic changes and acquisition of different
Fe-sources
Laura Zanin*1, Silvia Venuti1, Nicola Tomasi1, Anita Zamboni2, Stefano Cesco3, Zeno
Varanini2, Roberto Pinton1
1
University of Udine - Dip. Scienze Agrarie e Ambientali, Italy, 2University of Verona Dip. Biotecnologie, Italy, 3University of Bolzano - Faculty of Science and
Technology, Italy
Plants react to iron (Fe) deficiency using different adaptive strategies. Under limited Fe
availability maize, a model species for Strategy II plants, improves Fe acquisition through the
release of phytosiderophores (PS) into the rhizosphere and the subsequent uptake of the FePS complex into root cells. Aim of the present work was to study, at physiological and
transcriptional level, the Fe-deficient response in a commercial maize hybrid widely grown in
Europe.
Eleven-days-old maize plants (Zea mays L., PR33T56) were fed for 6 days with a nutrient
solution containing 0 (Fe-deficient) or 100 μM Fe (Fe-sufficient). At the end of the growing
period, visible symptoms of Fe deficiency were observed in the interveinal yellowing of young
leaves and proliferation of lateral roots and root hairs, with increase in the diameter of the
sub-apical root zone. Transcriptomic analysis on root tissues (NimbleGen microarray)
revealed that the Fe-deficiency modulated the expression levels of 376 genes (289 up- and
87 down-regulated, respectively). As expected, roots of Fe-deficient maize plants
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overexpressed genes involved in the synthesis and release of 2’-deoxymugineic acid (the
main PS released by maize roots). Moreover, a strong modulation of those genes involved in
the regulatory aspects, Fe translocation, root morphological modification, in primary
metabolic pathways and in hormonal metabolism were induced by the nutritional stress.
The capacity of maize plants to respond to Fe-deficiency was further evaluated exposing roots
to soluble or poorly soluble Fe-sources for up to 24 hours. Real time RT-PCR analyses and 59Fe
uptake experiments showed that the mechanisms involved in Fe acquisition were induced by
the nutritional stress; while the pathways involved in the translocation and distribution of the
micronutrient within the plant were not yet activated in Fe-deficient plants.
53
The influence of phosphorus sources on the growth and rhizosphere soil
characteristics of two wheat (Triticum aestivum L.) genotypes
Shuxiang Zhang*, Xiaoyng zhan
Institute of Agricultural Resources and Regional Planning, Chinese Academy of
Agricultural Sciences, China
A rhizobox experiment was conducted to investigate the effects of phosphorus (P) sources on
the rhizosphere soil characteristics of two wheat genotypes, namely Xiaoyan54 (P-efficient)
and Jing411 (P-inefficient), which were colonized with arbuscular mycorrhizal fungus and
grown on Cumulic Haplustoll. The four P sources included a control (no P), OP (organic P: Naphytate), IP (KH2PO4) and OPIP (Na-phytate plus KH2PO4). The results showed that when no P
was added, the shoot biomass for Xiaoyan54 was significantly higher than Jing 411 by 28%.
The rhizosphere soil acid phosphatase activity was significantly depressed when inorganic P
was added; the acid phosphatase activity for Xiaoyan54 was higher than that of Jing411 in
most of the layers for all the P sources. Inorganic and organic P fertilizer applications did not
significantly impact the soil pH relative to the control, and the pH did not lead to significant
variations between the two genotypes. In conclusion, significantly greater soil acid
phosphatase activity but not acidification promoted greater shoot biomass in Xiaoyan54 than
Jing411 when no P was added. The rhizosphere soil pH was not affected whether choosing
different wheat genotypes or various P sources, but the greater acid phosphatase activity was
found in the control and OP treatment compared with IP and OPIP, meanwhile the acid
phosphatase activity of Xiaoyan54 was higher compared with Jing411 for all the P sources.
The response mechanism of rhizosphere soil acidification is different from that of acid
phosphatase exudation to different P sources application under arbuscular mycorrhizal
fungus inoculation.
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54
Positive interactions between a P-mobilizing (Embothrium coccineum) and a Nfixing species (Sophora microphylla) on volcanic soils in Southern South America
Alejandra Zúñiga-Feest*1, Susana Valle2, Angela Bustos-Salazar2, Mabel Delgado2,
Frida Piper3
1
Instituto de Ciencias Ambientales y Evolutivas, Universidad Austral de
Chile, Chile, 2Universidad Austral de Chile, Chile, 3Centro de Investigación en
Ecosistemas de la Patagonia, Chile
Young volcanic soils are frequently found in Southern South America. These soils have
limited availability of phosphorus (P) and nitrogen (N), however some species have
developed root physiological strategies for P and N acquisition; Sophora microphylla
(Fabaceae) is a tree with symbiotic N-fixing bacteria, and Embothrium coccineum (Proteaceae)
is a tree with cluster roots (CR) which exude high rates of carboxylates (malate and citrate)
improving their P acquisition. It have been proposed that P-mobilizing species improve
nutrient acquisition to neighbors; however, scarce evidence exists for Southern South
America environments. We studied the interaction between E. coccineum and S. microphylla
to test if both species growing together have higher growth and better nutritional status than
growing separately. Seedlings of both species were grown on volcanic or alluvial soils (with or
without P and N limitations, respectively), for one growing season, in pots and watering at
field capacity, at full sun light conditions in Valdivia (40°S), Chile. We evaluated
photosynthesis rate, growth, biomass distribution, and N and P content in plants and in
rhizospheric and non-rhizospheric soils. Both species grew less in volcanic than alluvial soil,
without differences between “growing together” or “separately”. E. coccineum showed higher
photosynthesis rate in alluvial soil, higher size of individual cluster roots in volcanic soil and
higher cluster roots biomass distribution in alluvial soil, when it grew together. Higher P
concentration was observed in roots of S. microphylla growing together. E. coccineum
showed higher N and P content growing together than separately in alluvial soil. The
rhizospheric soil P availability increased in pots of E. coccineum, in both separate and
combined treatments. These results imply that P-movilizing and N-fixing species grown in a
combined system could lead to facilitation.
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Human Pathogens in the Rhizosphere
55
First pictures and key features support a role of Pseudomonas fluorescens
injectisomes in interactions with rhizosphere or clinical hosts
Dorian Bergeau*1, Xavier Latour2, Sylvie Mazurier3, Marie-Laure Follet-Gueye4,
Victorien Decoin1, Annabelle Merieau1, Nicole Orange5, Marc Feuilloley5, Philippe
Lemanceau3, Maïté Vicré-Gibouin4, Xavier Latour5
1
Normandie Université, Laboratoire de Microbiologie Signaux et Microenvironnement EA 4312 & IRIB, France, 2Normandy University (University of Rouen), France, 3INRA
Dijon, UMR 1347 Agroécologie, France, 4Normandie Université, Laboratoire de
Glycobiologie et Matrice Extracellulaire Végétale - EA 4358, France, 5Normandie
Université, Laboratoire de Microbiologie Signaux et Microenvironnement - EA
4312, France
Many plant and animal pathogenic Gram negative bacteria use a type III secretion system
(T3SS) as a molecular syringe to inject effector proteins directly into the host cell. Two
different classes of T3SSs are well described in Pseudomonas, a bacterial genus in which
members are frequently encountered in the rhizosphere: (i) a short needle involved in
translocation of exotoxins by the human pathogen Pseudomonas aeruginosa and, (ii) a long
and flexible pilus able to pierce the thick plant cell wall carried by phytopathogenic P.
syringae.
T3SSs were detected more recently in the species P. fluorescens, including in (i) strains
considered as saprotrophic or as plant growth-promoting rhizobacteria, known as devoid of
pathogenic potential, and (ii) strains associated with clinical cases (blood bacteremia, cystic
fibrosis). The role of these secretion systems in P. fluorescens strains is still unclear. In order to
elucidate the nature of the interactions mediated by P. fluorescens T3SSs, the structure of the
secretion system was apprehended in representative strains by transmission electron
microscopy in comparison with P. aeruginosa and P. syringae T3SSs. For these strains, ability to
interact with animal and plant cells, and expression of T3SS genes by qPCR were also studied.
The results together with ecological data reveal potential interactions mediated by T3SS with
rhizospheric partners, noteworthy at the level of myccorhizosphere, and with animal cells.
These results support the hypothesis that P. fluorescens T3SSs would be implied in
interactions with human host, thereby promoting opportunistic infections, but also with fungi
leading to beneficial effects on the plant. To the best of our knowledge, we unveil here the
first electron micrographs of T3SS apparatus observed within the P. fluorescens species.
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56
Factors influencing the fate of human pathogens in the plant environment
Eva Fornefeld*, Kornelia Smalla
Julius Kühn-Institut, Federal Research Centre for Cultivated Plants (JKI), Institute for
Epidemiology and Pathogen Diagnostics, Germany
Diseases caused by human pathogens (HP) on vegetables are increasingly reported. However,
so far knowledge is scarce about factors influencing the persistence of HP in the plant
environment. In this study, we analysed the influence of sludge, preadaptation of HP and
presence of nematodes on the survival of HP and their internalisation into plants. Therefore,
two greenhouse experiments with lettuce plants (Lactuca sativa ‘Tizian’) and Salmonella
enterica serovar Typhimurium LT2 were performed to analyse the effects of the presence of
sludge, preadaptation of HP and coinoculation of plant-parasitic nematodes. Preadaptation
of HP was simulated by cultivation under non-optimal conditions in a self-made culture
medium. The first experiment included six different treatments: soil amended with sludge or
not and each inoculated with Salmonella, preadapted Salmonella or no inoculum. Soil was
sampled regularly, and numbers of Salmonella were monitored using culture-dependent and
-independent methods. Salmonella counts in soil decreased overall from about 106 to 103 per
g dry soil within five weeks. Direct plating showed significantly higher numbers of Salmonella
in the treatment with preadapted Salmonella without sludge compared to the other
treatments from 10 days post inoculation (dpi). Significant differences were confirmed using
qPCR for 14 and 21 dpi. Salmonella was detected in soils up to 175 dpi using PCR-Southern
blot hybridisation and enrichment culture and subsequent plating. In a second greenhouse
experiment, the presence of the plant-parasitic nematodes Meloidogyne hapla and
Pratylenchus penetrans did not lead to internalisation of Salmonella through lettuce roots.
Despite a rapid decline of Salmonella in soil our data showed a long-term survival at low
abundance. Preadaptation promoted the survival in soil while the presence of sludge reduced
the survival. The presence of nematodes did not seem to promote internalisation of
Salmonella into lettuce roots.
57
Transcriptomic analysis of enterohaemorrhagic Escherichia coli O157:H7 in
response to plant extracts
Robert Jackson*1, Louise Birse2, Ian Toth3, Carol Wagstaff1, Simon Andrews1, Yannick
Rossez3, Nicola Holden3
1
University of Reading, United Kingdom, 2James Hutton Institute and University of
Reading, United Kingdom, 3James Hutton Institute, United Kingdom
Enterohaemorrhagic Escherichia coli (EHEC) are a group of food and contact-borne
pathogens responsible for haemorrhagic colitis. The bacteria can be transmitted by
contaminated meat, but importantly, also by plants and have been involved in many largescale produce associated outbreaks. The bacteria can use plants as a secondary host, where
they associate with both the leaves and the roots. Colonisation in the roots & rhizosphere of
plants is thought to be the main habitat for colonisation. Global gene expression changes of
EHEC O157:H7 strain Sakai were measured in response to plant extracts such as leaf lysates,
root exudates and leaf cell wall polysaccharides from spinach and lettuce. A significant
change in expression of 17% of genes on exposure to leaf lysates of spinach was determined
by microarray. A more selected response was seen to spinach leaf cell wall polysaccharides
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with only a 1.5% change. In contrast, when exposed to lettuce leaf cell wall polysaccharides a
higher change of 4.8% was seen, indicating plant species-specific responses from the
bacteria. The different metabolic pathways induced upon exposure to polysaccharides of the
different plant species involved the utilization of plant-specific polysaccharides. As well as
this, several pathways were induced that are involved in different aspects of adaptation of the
bacteria to the plant environment in many of conditions tested. Expression of selected
differentially regulated genes was validated in planta by qPCR, and targeted for functional
analysis by mutagenesis. These results provide insight to the colonisation of different plants
by human pathogenic EHEC and provide the foundation for further work towards developing
strategies to mitigate pathogen contamination of food produce.
58
T3SS and virulence markers highlight similarities and differences between
human and plant-associated Pseudomonas fluorescens related isolates
Sylvie Mazurier1, Annabelle Merieau2, Dorian Bergeau2, Victorien Decoin2, Daniel
Sperandio2, Alexandre Crépin2, Corinne Barbey2, Katy Jeannot3, Maïté Vicré-Gibouin2,
Patrick Plésiat3, Xavier Latour2, Philippe Lemanceau*1
1
INRA, France, 2Normandie Université, France, 3Université de Franche-Comté, France
Opportunistic bacterial pathogens are a major concern for human health since they are able
to cause or aggravate serious clinical conditions. Among these bacteria, isolates related to
the species Pseudomonas fluorescens remain little known although they appear to be
regularly found associated with bacteremia or in the respiratory tract of patients suffering
from cystic fibrosis. Meanwhile, knowledge on environmental P. fluorescens related isolates is
increasing, especially that on their ability to interact with eukaryotic hosts via Type III
Secretion Systems (T3SS). Plant-associated and clinical isolates were compared for a series of
genotypic and phenotypic traits and two bacterial groups were delineated. Isolates of the first
group only included clinical isolates from blood infections and were showed on the basis of
16S rRNA phylogeny to belong to the P. putida complex and not to P. fluorescens as initially
identified in hospitals. T3SS sequences of these isolates were highly conserved and belonged
to the Ysc-T3SS family known to include P. aeruginosa. The presence Ysc-T3SS genes in
clinical isolates belonging to the P. putida complex, described here for the first time, is
proposed to be used for the identification of these bacteria which are potential human
pathogens as supported by virulence assay using an amoeba. The second group included
plant-associated and clinical isolates from patients suffering from cystic fibrosis. They all
belonged to P. fluorescens and harbored T3SS genes of Hrp1-T3SS family which is commonly
found in plant-associated P. fluorescens. Clinical and plant-associated isolates of this group
could be differentiated neither on their 16S rRNA and T3SS gene phylogenies, nor on their
virulence, some plant-associated isolates expressing a positive response in virulence assay.
Therefore, no identification test could be proposed to discriminate clinical and environmental
isolates in this second group commonly found in the rhizosphere.
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59
Investigation of the colonization of the root system of Festuca arundinacea by
Listeria monocytogenes
Pascal Piveteau*1, Gilles Boussemart1, Laurent Gal2, Dominique Garmyn1
1
Université de Bourgogne, France, 2AgroSup, France
Listeria monocytogenes is a ubiquitous opportunistic human pathogen responsible for
listeriosis, a life-threatening food-borne disease. L. monocytogenes is detected in many
habitats spanning from the farm environment (soil, vegetation) to the food industry
(foodstuff, drains, working surfaces) and the gastrointestinal tract of animals and humans.
Contamination of raw farm products such as vegetables and crops is a major problem as a
source of contamination of raw and processed foods. A better understanding of the
adaptation of L. monocytogenes to soil and plants is clearly necessary. In the present study,
we investigated the rhizosphere as a potential habitat of L. monocytogenes. Soil mesocosm
experiments clearly demonstrated that the presence of Festuca arundinacea improved
survival of L. monocytogenes. This prompted us to investigate colonisation of the root system
of this monocotyledon by L. monocytogenes. Plants were grown aseptically on Hoagland
plates and incubated in plant culture cabinets. The root system was inoculated with L.
monocytogenes after 7 days of growth of the seedlings. Growth was observed and L.
monocytogenes reached around 2x106 CFU/root after 4 days of incubation. The population
then stabilised until the end of the experiment, 7 days after inoculation. Organisation of the
listerial cells was followed in situ by confocal microscopy during colonisation. Plantassociated biofilms were observed during colonisation of the root system. We further
investigated intrinsic factors that could facilitate root colonization. Interestingly, InlA and InlB,
two surface proteins of the internalin family were involved during root-associated growth of
L. monocytogenes. These two surface proteins are known virulence factors required for
adhesion to enterocytes during infection of the mammalian host. Further evidence points out
to a global reshaping of the physiology of L. monocytogenes during plant-associated growth.
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Below and Above Ground Interactions
61
The effect of grazing on root morphology and mycorrhiza formation in
simulated ryegrass (Lolium rigidum) pasture
Lynette Abbott*1, Jing-Wei Fan2, Zakaria Solaiman1, Bede Mickan1, Yan-Lei Du2, FengMin Li2
1
University of Western Australia, Australia, 2Institute of Arid Agroecology, Lanzhou
University, China
Defoliation of shoot tissue by grazing can alter the structure and function of grassland
ecosystems. Grazing modifies morphology of plant root systems. There are contradictory
reports of impacts of defoliation on colonization of roots by arbuscular mycorrhizal (AM)
fungi. Defoliation has been shown to both decrease and increase mycorrhiza formation when
assessed as % root length colonized by AM fungi. We investigated mycorrhiza formation in
simulated pasture swards of ryegrass (Lolium rigidum cv. Wimmera) grown for 6 months in a
glasshouse in response to three defoliation regimes. Plants were sampled 5 times during this
period. Our objective was to determine effects of moderate and severe defoliation. The
correspondence of several root traits (root length, root mass and specific root length) to
nutrient uptake, rhizosphere soluble carbon and mycorrhiza formation was evaluated in
response to defoliation. We hypothesized that (i) shoot growth would be reduced in the
severe defoliation treatment and less so with moderate defoliation; (ii) root biomass and root
length would be reduced by both defoliation treatments, (iii) defoliation would increase
rhizosphere soluble carbon, and (iv) defoliation intensity would alter the relative abundance
of AM fungi in roots. Both shoot and root biomass decreased with defoliation intensity and
remained stable from 87 days after sowing, but both continually increased in non-defoliated
plants. Defoliation had a major impact on root traits. The proportion of roots colonized by
AM fungi (measured as % root length colonized) increased with defoliation severity. In
contrast, the length of root that was colonized by AM fungi was reduced by defoliation.
Soluble carbon concentration and microbial biomass carbon in rhizosphere soil were reduced
by defoliation intensity. Root length colonized by AM fungi, rather than % root length
colonized by AM fungi, was more closely related to soluble carbon and soil nutrient status of
rhizosphere soil.
62
Biotic plant-soil feedback effects on germination and seedling growth of
agricultural and semi-natural plant species
Janna Barel*, Jingjue Wang, Gerlinde De Deyn
Plant-soil feedbacks co-determine plant productivity and vegetation dynamics and are
increasingly being studied. However, studies on germination and seedling growth are
underrepresented. Moreover, comparisons between agro-ecosystem and natural grassland
plant-soil feedbacks are rare although they could offer valuable mechanistic insights.
We aimed to understand biotic plant-soil feedback effects on germination rate and success,
relative growth rate and biomass production. We studied four agricultural plants and four
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grassland congeners (respectively Lolium perenne, Vicia sativa, Raphanus sativus, Cichorium
endivia and Festuca rubra, Vicia cracca, Raphanus raphanistrum, Cichorium intybus).
In a climate cabinet we tested soil legacy effects on germination and whether crops germinate
better than their semi-natural counterparts. In a greenhouse, we verified if crops grow faster,
and whether soil legacy left by conspecific plants would suppress plant productivity more than
soil legacy of heterospecific plants. Overall, crops were expected to respond more negatively
to conspecific soil conditioning than semi-natural species, and more positive to heterospecific
conditioning.
As hypothesized, crops germinated faster with a higher success rate than semi-natural plants.
However, soil legacy didn’t affect germination. Relative growth rate differed between plant
species but not between agricultural and semi-natural species. Soil legacy effects on biomass
production gave contrasting results: agricultural species responded similarly to soil
conditioning, with soil legacies of C. endivia and L. perenne promoting plant growth in
comparison to unconditioned soil. While, semi-natural plant productivity was driven by the
identity of the response plant.
Overall our results demonstrate that soil legacy effects on subsequent plant performance can
depend on both the identity of the preceding species generating the legacy and on the
species that responds to the legacy. In agro-ecosystems preceding species play a main role,
while the responding species are important determinants in the semi-natural system. This
indicates that the two systems don’t seem to mirror each other’s functioning.
63
Can plant growth-promoting rhizobacteria mitigate P-starvation stress in
Brachypodium distachyon?
Caroline Baudson*, Benjamin M. Delory, Patrick du Jardin, Pierre Delaplace
University of Liège, Gembloux Agro-Bio Tech, Belgium
Phosphorus (P) is a macronutrient essential to plants but mainly present in unavailable forms
in soils. Although it is abundant, P is often a limiting factor for crop productivity due to its
poor mobility. While the fossil P reserves are diminishing, new strategies allowing a better
exploitation of the soil P resources have to be developed. In this regard, biotic interactions
occurring between plant roots and soil microorganisms are increasingly considered. Among
those microorganisms, beneficial rhizospheric bacteria (called plant growth-promoting
rhizobacteria, PGPR), are able to mitigate P deficiency stress in plants by several mechanisms
(e.g. solubilization and mineralization of unavailable P forms, modification of root
morphogenesis). In this context, the current research project aims at studying the effects of
three PGPR strains (Bacillus subtilis AP305-GB03, Pseudomonas fluorescens Pf29Arp and
Azotobacter vinelandii A60 - F08 19) on Brachypodium distachyon (L.) Beauv. Bd21 response
to P deficiency, in an ex-vitro co-cultivation context. The first step of the project consisted in
investigating Bd21 response to P deficiency and allowed the identification of contrasted P
conditions, from strong stress to optimal growth conditions. Based on these results, P limiting
conditions are used to reveal potential mitigation effects of the selected PGPR strains on P
deficiency stress in Bd21. Plants and bacteria are co-cultivated in sand with different P
supplies. Biomass production and allocation, root system architecture parameters and P
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content will be measured in Bd21. This study will help us to find out if there is a beneficial
bacterial activity improving P availability, P uptake capacity and plant development in our
experimental conditions. The presented results will be discussed from fundamental and
applied viewpoint.
64
A new methodology for assessing root amino acid exudation in soil and
interactions with the rhizosphere
Hélène Bobille*1, Anis L. Limami2, Richard Robins3, Gaétan le Floch4, Joëlle Fustec1
1
Ecole Supérieure d'Agriculture (LEVA), France, 2University of Angers,
IRHS, France, 3University of Nantes, CEISAM, France, 4University of Brest, LUBEM, France
Legumes release substantial amounts of nitrogen into soil via rhizodeposition and constitute
a sustainable source of nitrogen incorporation in cropping systems. In addition, the exudation
of small molecular weight compounds such as amino acids may significantly contribute to
defining the composition of rhizospheric microbial communities. Our aim is to unravel the
adaptive response of legumes to changes in the rhizosphere in terms of amino
acids exudates, since root exudate composition is likely to be influenced by biotic and abiotic
factors. However, studying exudation from plants grown in unsterilized soil is challenging due
to rapid uptake of exudated molecules by the microorganisms or their sorption on soil
mineral particles. In the present work, we have developed a new methodology that allows us
to establish relationships between exudated free amino acids in the rhizosphere, the
physiological status of the plant, the substrate type and the microflora. Medicago truncatula
plants were grown in controlled conditions, either in unsterilized soil, or in sterilized sand and
amino acids were extracted in formic acid (2 mM). Biomass and diversity of the microbial
communities were also characterized. Results on the legume rhizosphere showed that the
presence of a plant substantially modifies the total amino acid content of the rhizospheric
soil. The highest content of amino acids was observed at the end of the vegetative stage.
There was a great diversity in the amino acid profile in rhizospheric sterilized sand: the major
amino acids were glycine, alanine, glutamate and serine. In rhizospheric unsterilized soil there
was less diversity in amino acids profile with asparagine representing 38% of the total. These
results highlight that it is crucial to work with unsterilized soil in order to obtain reliable
information on the dynamics of amino acids in the rhizosphere in relation to plant physiology
and diversity of the soil microbial communities.
65
Sugarcane root system affected by insecticide and plant growth regulator
Denizart Bolonhezi*1, Adriano Mastro2, Caio Santili2, Tais Lima da Silva3, José Custódio
de Souza4, Eglairto Veloso de Carvalho4, Edmar Veloso de Carvalho4, Roberto Botelho
Ferraz Branco4
1
APTA -IAC, Brazil, 2Syngenta Company, Brazil, 3Moura Lacerda
University, Brazil, 4APTA, Brazil
In Brazil the sugarcane crop is cultivated in almost 9.2 million hectares, which 60% of
plantations are concentrated in Sao Paulo state. Nowadays, the intense mechanization
increases the soil compaction; consequently the sugarcane root system is affected. Some
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insecticides and plant growth regulator normally used in commercial field could help to
improve the root characteristics in sugarcane crop. The objective of this research was to
quantify the effect of Moddus® (trinexapac) and Actara® (thiametoxam) on the sugarcane
root characteristics in three different field conditions. The trials were installed in three
different soils, located at Colorado Mill (Miguelopolis city) and Maringa Mill (Araraquara city),
both in Oxisoil and Noble Mill (Catanduva city) under Ultisol. Roots were sampled with probe,
in two dates defined by the layers down to a depth of 1,0 m. The evaluations were done 30
(effect of trinexapac) and 180 (thiamethoxan) days after harvesting of the third ratoon,
respectively December 2012 and April 2013. After washing, sieving and cleaning 2800
samples collected, the images of roots were analysed by Safira® software in order to
determine the diameter, area and volume. After that samples were dried to know the dry
biomass. Results showed that the association of Moddus® and Actara® increased the dry
biomass of roots, the area and root length, respectively in 2,3 Mg ha-1, 60 m2 and 8 m per
trenches. It could be concluded that, the application of trinexapac associated with
thiamethoxam improve the sugarcane root system, consequently the influence of water stress
could be reduced as well as the nutrient uptake.
66
Soil bacterial and fungal community composition along a plant species richness
gradient
Sigrid Dassen*1, Henk Martens1,2, George Kowalchuk1,3, Wim van der Putten1,2,
Gerlinde De Deyn1,2
1
Netherlands Institute of Ecology, Netherlands, 2Wageningen University and Research
Centre, Netherlands, 3Utrecht University, Netherlands
Abundance and diversity of microbes in soil is extremely large, yet their distribution is not
totally random. Several studies have shown how particular microbial groups respond to
increasing plant species richness but it remains unclear how the soil microbial community, in
terms of microbial richness, identity and abundance changes in response to plant species
richness and composition. We hypothesised that 1) the fungal community is more responsive
to variation in plant communities than the bacterial community, 2) α-diversity (richness within
communities) of soil microbes increases and 3) β-diversity (turnover of species between
communities) decreases with plant species richness. To test these hypotheses we sampled
soil from the long-term Jena biodiversity experiment comprising a plant species richness
gradient of 1, 2, 4, 8, 16, and 60 grassland species, replicated in 4 blocks. We used 454pyrosequencing of 16S and 18S to identify the soil microbial community composition.
Most bacterial species occurred in few plots and at low relative abundance, while a minority
of species were highly abundant and occurred in nearly all plots. In contrast several fungal
species occurred in only few plots and at high relative abundance, indicating that fungal
species respond stronger to the local plant community than bacteria, confirming the first
hypothesis. α-diversity of bacteria and fungi was highly variable across the plant
communities, but variation could not be explained by plant species richness, rejecting the
second hypothesis. β-diversity of bacteria, but not fungi, decreased with increasing plant
species richness, partly accepting the third hypothesis. Additionally, the variation in α- and βdiversity of bacteria and fungi could partly be explained by plant functional group identity
(grasses, legumes, small and tall herbs). Lowest α-diversity observations are mainly from
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grass dominated plots, and regarding β-diversity it was found that legume monocultures
cause homogeneity in both the bacterial and fungal community composition.
67
Effect of plant cover diversity on soil organisms: a preliminary study
Baptiste Drut*1, Nathalie Cassagne1, Mario Cannavacciuolo1, Gaëtan Lefloch2, Joëlle
Fustec1
1
LUNAM Université Angers - Groupe ESA - LEVA, France, 2LUBEM, Université de
Bretagne Occidentale, France
In low input agrosystems, positive interactions between diversified plant cover and soil
organism communities could be powerful tools to increase yields. The aim of this study was
to assess how a genetic and/or specific plant cover diversification affects the performance of
Triticum aestivum L. and soil organisms. In a greenhouse experiment, we investigated the
effects of plant diversity on the rhizospheric fungal biomass and the nematofauna. We also
tested whether genetic or specific diversity has an effect on wheat biomass and nitrogen
status.
Plants were sown in mesocosms filled with soil added with five endogeic earthworms. We
tested three levels of plant diversity: single wheat genotype, mixture of three wheat
genotypes and the three genotypes mixed with Trifolium hybridum L. Nitrogen concentration
in roots and shoots were measured and the percentage of legume nitrogen derived from
biological ﬁxation was determined using the 15N natural abundance method.
Within wheat-clover association, the performance of wheat (biomass, nitrogen content) was
improved by higher N availability thanks to complementarity effects. Indeed, the nitrogen
content of clover shoots was mainly due to atmospheric nitrogen fixation. The fungal
biomass tended to be higher in wheat mixture than in single wheat. The gradient of plant
diversity positively increased the abundance of bacterial-feeding nematodes especially the
family of Rhabditidae. The abundance of phytophagous nematodes (Meloidogynidae) in each
plant cover modality increased in time.
Overall, these results suggest that plant cover diversity influences soil organisms and wheat
performance. Root systems of several wheat genotypes seem to favor fungal biomass. Plant
cover diversity increases especially opportunistic bacterial-feeding nematodes abundance
indicating a higher bacterial biomass. Abundance of phytophagous is related with higher
density of plant cover during the experiment. Finally, plant diversity seems to influence soil
nematode community structure however more information is needed.
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68
Monitoring the impact of Bt maize events on the rhizosphere microbial
communities cultivated in Brazil
Ubiraci Lana, Eliane Gomes*, Fernando Santos, Lucas Silva, Christiane Oliveira,
Fernando Valicente
Embrapa Maize and Sorghum, Brazil
Genetically modified (GM) maize expressing the insecticidal protein from Bacillus
thuringiensis (Bt) has been commercially cultivated in Brazil since 2008. The monitoring of
transgenic plants after commercial release should be done in order to assess and evaluate
possible environmental effects on non-target organisms. Microorganisms in soil can be into
contact with Bt proteins when they are released from root exudates of Bt maize or from
decomposing plant tissue, and the impact of these proteins on the composition of the rootassociated microbiota is still poorly understood. The objective of this study is to evaluate the
impact of commercial events of transgenic maize expressing Bt proteins on the rhizosphere
soil bacterial community. To this end, the transgenic maize MON 810, Bt 11, Herculex, MON
89034, VTPro II and Viptera, their respective isogenics treated and not treated with chemical
insecticides were assessed using Biolog EcoPlates™ and DGGE (Denaturing Gradient Gel
Electrophoresis). The rhizosphere soil was collected at 30 and 60 days after germination in
two environments (Sete Lagoas and Janaúba - MG), for three consecutive years (2010/2011,
2011/2012 and 2012/2013), under field conditions. The activity and metabolic diversity
evaluated using the Biolog EcoPlates™, after 72 h of incubation, showed no differences
between transgenic and non-transgenic maize. The values of Shannon diversity index
demonstrated a high level of diversity in the rhizosphere bacterial community for all the
treatments. However, the statistical analysis indicated no significant differences among
transgenic, isogenic counterparts and samples sprayed with chemical insecticide. The DGGE
analysis also did not show difference between transgenic and non-transgenic treatments, but
the samples were grouped by environment, showing the importance of soil modulating the
microbial community. In conclusion, these results demonstrated that the events of maize Bt
cultivated in Brazil showed no significant impact on the soil bacterial community structures
based on the parameters studied.
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Using a whole plant crop model to assess phosphate fertiliser use in barley
James Heppell*1, Sevil Payvandi1, Peter Talboys2, Konstantinos Zygalakis1, David
Langton3, Roger Sylvester-Bradley4, Robin Walker5, Davey Jones2, Tiina Roose1
1
University of Southampton, United Kingdom, 2Bangor University, United
Kingdom, 3Agrii, United Kingdom, 4ADAS, United Kingdom, 5Scotland's Rural
College, United Kingdom
The role of phosphorus (P) is essential to the agricultural world providing a valuable nutrient
for crop production. Its future scarcity, along with escalating concerns over climate change,
could greatly harm global food security. In order to improve P efficiency in farming, we have
developed a whole plant crop model that allows us to assess a range of P fertiliser and soil
cultivation strategies. The model estimates plant P uptake and leaf mass throughout the crop
life cycle, given uncertain climate conditions. Two barley field trials are used to validate the
model and enable a forecast for other possible fertiliser and soil cultivation strategies.
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The whole plant crop model combines an above ground leaf model (to estimate leaf mass)
with our existing below ground root and soil model (to estimate plant P uptake). The leaf
model includes the role of photosynthesis to estimate carbon mass stored by the plant.
Carbon mass in conjunction with temperature is used to set the root growth rate.
With the addition of the leaf model we achieve a better estimate of two sets of barley field
trial data for leaf mass and plant P uptake, compared with just the root and soil model alone.
There is a positive feedback effect from the two models i.e., an increase in plant P uptake
from the root system increases carbon production via photosynthesis in the leaves, and vice
versa.
The whole plant crop model is sensitive to the initial state of P and its distribution within the
soil profile; experimental parameters which are rarely measured in the field. The combination
of modelling and experimental data provides useful agricultural predictions for site specific
locations.
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Long-term management effects on root biomass and carbon rhizodeposition of
field grown maize
Juliane Hirte*, Jens Leifeld, Hans-Rudolf Oberholzer, Jochen Mayer
Agroscope ISS, Switzerland
Below ground carbon (C) inputs, i.e. dead roots and C rhizodeposition, by agricultural plants
into the soil are an important variable in soil C modelling and are mostly estimated from
above ground biomass. Agricultural management practices affect above ground biomass
considerably; however, their effects on below ground C inputs are only poorly understood.
Our aims are therefore to determine management effects on below/above ground C ratios,
total root biomass, C rhizodeposition, and vertical root distribution of field grown maize. We
hypothesise that, with increasing management intensity, (i) total root biomass is not affected
but (ii) below/above ground C ratios, C rhizodeposition, and deep root biomass decrease.
In 2013, we conducted field experiments with maize on two Swiss long-term trials (“DOK”,
Basel and “ZOFE”, Zurich) covering seven different management treatments. Four individual
plants per treatment were grown in microplots and pulse-labelled with 13C-CO2 in weekly
intervals throughout the growing season. After harvest, the microplot soil was sampled in
three layers to 0.75m depth, coarse- and fine root biomasses were determined by picking
and wet sieving, and all samples were analysed for their δ13C values.
Preliminary results reveal a decrease of below/above ground C ratios with increasing
management intensity on both sites and range from, on average, 0.38 to 0.22 on the “DOK”
site and 0.32 to 0.18 on the “ZOFE” site (trends only). Total root biomasses and C
rhizodeposition do not differ between treatments on both sites. While the proportion of
subsoil (0.25-0.75m) root biomass of the total (0-0.75m) root biomass decreases with
increasing management intensity on the “DOK” site, this trend is not visible on the “ZOFE”
site. These findings show that below ground C inputs cannot easily be estimated from above
ground biomass and input data for soil C models should be differentiated according to the
management system.
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71
Small but important fraction of DOM: exudation of plant dominants in swamp
forest
Eva Kastovska*1, Tomas Picek1, Keith Edwards1, Jakub Borovec2, Hana Santruckova1
1
University of South Bohemia, Faculty of Science, Czech Republic, 2iology Centre of the
Academy of Sciences of the Czech Republic, Czech Republic
Swamp forest is a common type of peatland, its herbal floor typically co-dominated by
Sphagnum mosses, Eriophorum vaginatum and Vaccinium myrtillus distributed in patches
tracking the differences in microtopography and hydrology of the terrain. As with other
peatlands, swamp forests are characterized by formation of high amounts of dissolved
organic matter (DOM). In the DOM, mostly derived from accumulated soil organic matter of
low biodegradability, regular daily input of fresh plant-derived compounds (rhizodeposition)
can represent the key C and energy source for soil microorganisms and form the most
reactive part of DOM.
Therefore, we aimed to quantify and compare the “exudation rate” from roots of Eriophorum
and Vaccinium and stalks of Sphagnum girgensohnii (sampling of exudates from living roots
of plants and fresh moss in intact peat monoliths), quality of the released compounds (C and
N content, size exclusion chromatography and HPLC) and contribution of the released
compounds to DOM and their biodegradability (13CO2 pulse-labeling of plants placed in fresh
natural DOM).
We found that plant-derived compounds formed only 2-3% of the DOM within the rooting
zone, with the exudation rate largest for Sphagnum and lowest for Vaccinium (per m2 of the
particular vegetation cover). Sphagnum “exudates” were of very different quality than in the
other two species, poorer for simple compounds but relatively rich in oligosaccharides, had
larger C/N ratio and lower biodegradability. The difference in the quality of the compounds
released from Sphagnum and the other two herbal dominants suggests that the patchy
distribution of vegetation in the understory of the swamp forest ecosystem could significantly
contribute to the high spatial diversity of the soil microbial community and of the rates of C
and N processing in its upper soil layer.
72
Carbon input into soil originating from fine root and foliage litter in two
Norway spruce forests
Jaana Leppälammi-Kujansuu*1, Lasse Aro2, Maija Salemaa2, Karna Hansson3, Dan Kleja
Berggren4, Heljä-Sisko Helmisaari1
1
University of Helsinki, Finland, 2Natural Resources Institute Finland, Finland, 3INRA,
Centre de Nancy, Biogéochimie des Écosystèmes Forestiers, France, 4Swedish University
of Agricultural Sciences, Sweden
Fine root litter forms a significant carbon input to soil. Knowledge of the quantity of fine root
litter is scarce – but highly valued in C budget calculations. We quantified fine root litter
production in two Finnish Norway spruce stands and compared it to foliage litter. The stands
differed in their geographical location and environmental conditions. Finally, we added data
from four previously published spruce stands in Sweden for determining the above- and
belowground litter ratios along latitudinal and soil fertility gradients.
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The amount of belowground litter was estimated from fine root turnover with the
minirhizotron method and biomass from soil cores. Foliage litter was collected with litter
traps and ground vegetation was sampled.
In the less fertile, northern stand, the fine root biomass per stand basal area was almost
double and the tree fine root lifespan longer than in the southern stand. The annual
production of tree foliage litter was less than in the southern stand, but the total amount of
litter (including trees and understorey, above- and belowground) was similar at both sites, as
was the ratio between the above- and belowground litter production. The increased
contribution of understorey vegetation towards low fertility sites was clearly seen when
regressing the aboveground:belowground litter production -ratio of the Finnish and Swedish
sites against the C:N-ratio of the organic layer in the sites.
In boreal forest soil, the input of carbon via fine roots at least equals to foliage litterfall and
the share of understory vegetation is also substantial. This shift in the litter production
pattern from above to belowground in the least fertile sites may have an impact on litter C
quality and soil C storage and should be studied further.
73
Rhizosphere adaptive strategies of three plant species of high-mountain
environment under periglacial conditions (Majella Massif, central Italy)
Luisa Massaccesi*1, Gian Maria Niccolò Benucci1, Giovanni Gigliotti2, Mauro De
Feudis1, Stefania Cocco3, Giuseppe Corti3, Alberto Agnelli1
1
Department of Agricultural, Food and Environmental Sciences, University of Perugia,
Italy, Italy, 2Department of Civil and Environmental Engineering, University of Perugia,
Italy, Italy, 3Department of Agricultural, Food and Environmental Sciences, Polytechnic
University of Marche, Italy, Italy
The rhizosphere is a highly dynamic interface for chemical, physical and biological
interactions between plants and soil. As rhizosphere processes have been widely investigated,
little is known about the rhizosphere adaptative strategies of pioneer plants in soils of
periglacial environment. The rhizosphere processes in these severe and nutrient-poor
environments may provide a key in the prediction of plant growth and health, soil quality and
nutrients availability.
We selected three plants (Helianthemum nummularium (L.) Mill. subsp. grandiflorum (Scop.),
Dryas octopetala (L.), and Silene acaulis (L.) Jacq. subsp. cenisia (Vierh.) P. Foum) that sparsely
occupy deglaciated areas of the Majella Massif (central Apennines, Italy), with the aim to
assess how they overcome harsh pedoclimatic and nutritional conditions, we investigated
physico-chemical properties together with microbial community structure, abundance and
activity for both rhizosphere and bulk soil. The three plant considered in this study have
shown different adaptative strategies addressed to force changes in soil and nutrient uptake.
Helianthemum induces a strong rhizosphere impact through a synergistic effect between the
roots activity and a well-adapted rhizosphere microbial community. For Dryas, which does
not foster a microbial community structure specifically designed for its rhizosphere, an
intense consumption of the energetic resources supplied by the plant is needed to make
nutrients available. The scarce rhizosphere effect produced by Silene suggested that also the
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root morphology influences the intensity of the soil changes induced by roots and associated
microorganisms. In the case of Silene, the ability to colonize harsh environments may be
mostly linked to the shape and functions of its canopy rather than to a functional rhizosphere
effect.
This study showed that the activity of the roots and associated microbial community are
decisive in modifying the soil properties, so to create a suitable environment where plants are
able to grow even under a hostile climate.
74
Linking below ground and above ground phenology of Hybrid Walnut in
temperate agroforestry systems
Awaz Mohamed*1, Yogan Monnier1, Sylvie-Annabel Sabatier2, Jean-Luc Maeght3,
Christophe Jourdan4, Alexia Stokes1, Merlin Ramel1
1
INRA, UMR AMAP Montpellier, France, 2CIRAD, UMR AMAP Montpellier, France, 3UMR
Iees Paris, Institut de Recherche pour le Développement (IRD) c/o UMR AMAP, CIRAD,
Montpellier, France, 4CIRAD UMR Eco&Sols Montpellier, France
Climate models predict that an increase in atmospheric CO2 concentration, precipitation and
temperature could affect many biological phenomena and increase the frequency and
magnitude of extreme weather events. Root and shoot phenology could be strongly
influenced by the variations in soil water content, soil and air temperatures. Changes in plant
phenology are considered to be a very sensitive and observable indicator of plant responses
to climate change. In contrast, very little is known about the relationship between shoot and
root phenology especially in the natural soil environment. Knowledge about this relationship
is needed to understand the response of vegetation to climate changes which will give us a
better understanding of ecosystem structure and function. The objective of this study is to
quantify the dynamics of root and shoot growth of Hybrid Walnut (Juglans regia x nigra) in
three temperate agroforests, along a climatic gradient of precipitation and temperature. We
also regularly measured soil carbon, nitrogen and phosphorus concentrations. Fine root
growth dynamics were measured every 3 weeks in rhizotrons and minirhizotrons. The results
of this project will provide data allowing a better understanding and assessment of spatial
and temporal impacts of climate on vegetation.
75
Bacterial volatile organic compound that hampers plant growth
Jun Murata*, Takehiro Watanabe, Hajime Komura
Suntory Foundation for Life Sciences, Japan
Selected soil microbes have been shown to trigger alteration of plant growth by releasing a
blend of volatile chemicals called volatile organic compounds. Examples of such bacterial
strains include a plant growth promoting rhizobacteria Bacillus subtilis GB03 which is known
to release 2,3-butanediol that enhances growth of Arabidopsis seedlings. However, it is
largely unknown whether such bioactivity of volatile organic compounds can be found only in
specialized bacterial strains. Moreover, molecular mechanism of how such volatile organic
compounds are recognized by plants still remains elusive. Here we show that the growth of
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Arabidopsis seedlings can be interfered by volatile organic compounds that were released
from an experimental strain of Bacillus spp. in a distance-dependent manner. Similar effects
upon plant growth were observed when either E. coli or Agrobacterium instead of Bacillus
was used as a source of volatile organic compounds, suggesting that the active compound is
conserved among various bacterial species. Notably, while Arabidopsis seedlings grown in a
position relatively closer to Bacillus showed severe growth retardation, the seedlings grown
in a position distal to Bacillus showed rather enhanced growth in their above-ground parts
compared to control seedlings without Bacillus co-culture. Through activity-guided
purification approach, we identified isovaleric acid as a key volatile that exhibits plant growth
retardation activity. Authentic isovaleric acid was capable of inhibiting the growth of
Arabidopsis seedlings as in the case of Bacillus volatiles. Bioassay experiments using 14Clabeled isovaleric acid showed that isovaleric acid was incorporated into leaf and root tissues
of Arabidopsis seedlings in a manner dependent on the distance from the source of the
volatile. These data provide novel insights into the molecular basis of how soil microbes
interfere with the growth of plants.
76
Resilience of ectomycorrhizal communities under drought conditions
Karin Pritsch*1, Uwe Geppert1, René Kerner1, Franz Buegger1, Martina Peter2, Joerg
Luster2, Arthur Gessler2, Frank Hagedorn2, Marcus Schaub2, Rolf Siegwolf3, Matthias
Arend2
1
Helmholtz Zentrum München, Germany, 2WSL, Switzerland, 3Paul Scherrer
Institut, Switzerland
Prolonged drought periods are predicted in most future climate scenarios. Especially for
long-lived tree species, genetic adaptation of the plant may be hindered by long generation
times. Instead, mycorrhizal symbionts may mitigate drought stress by improving the nutrient
and water status of plants. Different mycorrhizal fungi are ecologically beneficial to the plant
to very varying extents.
We present results from the drought experiment ‘BuKlim’ performed over 3 years in the
model ecosystems of WSL (CH). The aim was to study mechanisms of resilience of the whole
plants including their ectomycorrhizosphere after strong drought events and upon
rewatering. For this purpose, young beech trees (Fagus sylvatica) were 13CO2 labelled at the
end of the drought period and after rewatering. We then followed the time course of
allocation of 13C through the plant to the ectomycorrhizosphere and into the soil.
Aboveground reactions of trees such as reduced carbon uptake during drought and high
compensation of photosynthesis upon rewatering were reflected by an enhanced 13C
allocation into below-ground parts. A wide range of 13C accumulation was detected in
different ectomycorrhizal exploration types indicating which species were resistant and
resilient under severe drought conditions.
Overall, our results show plastic reactions at the whole root-system level, a strong link
between above and below-ground reactions, and different mechanisms that may contribute
to the observed resilience of ectomycorrhizal communities under extended drought periods.
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77
Pseudomonas sp. P482 shows broad spectrum of antagonistic activity towards
plant pathogenic bacteria
Sylwia Jafra, Dorota Krzyzanowska, Adam Ossowicki, Magdalena Rajewska*,
Magdalena Jablonska
University of Gdansk, Poland
Plant-associated Pseudomonas produce large numbers of biologically-active secondary
metabolites. Many of them were demonstrated to have antimicrobial properties allowing the
bacteria to survive in a competitive environment. Pseudomonas sp. P482, a tomato
rhizosphere isolate, displays antagonistic activity towards a broad spectrum of plant
pathogenic bacteria, including pectinolytic plant pathogens – from Pectobacterium and
Dickeya genera - which cause important economic losses in potato production worldwide.
Spreading of these pathogens calls for development of an efficient and environmentallyfriendly control strategy based on natural compounds and microorganisms.
The aims of this study were to verify the potential of the Pseudomonas sp. P482 strain to
antagonize bacterial plant pathogens and to analyze the genetic background of this strain’s
antimicrobial activity by genome data mining approach.
To investigate whether the Pseudomonas sp. P482 could be employed as a biological control
agent against soft rot, in planta tests on potato tubers and chicory leaves, involving coinoculation of plant tissue with bacterial mixtures, were applied. Additionally, the ability of
P482 to colonize the roots of soil-grown potato plants was tested. Genome sequencing of
P482 was performed, followed by bioinformatics analysis with the antiSMASH and BAGEL3
platforms in order to determine the ‘candidate’ genes potentially involved in the
antimicrobial activity of P482.
In planta analyses revealed that although P482 inhibits growth of bacterial pathogens in a
plate assay, the biocontrol effect is pathogen-specific. The P482 is a moderate potato root
colonizer and thus can be tested for its biocontrol potential on this host in a greenhouse or
field setup. A draft genome sequence of P482 has been obtained and annotated. The BAGEL3
analysis yielded no putative bacteriocins. The antiSMASH analysis revealed five biosynthetic
clusters encoding genes potentially involved in the synthesis of antimicrobial factors.
Selected genes from these clusters were further investigated.
78
Aboveground insect infestation attenuates belowground Agrobacterium
Choong-Min Ryu*1, Geun Cheol Song1, Soohyun Lee1, Jaehwa Hong1, Hye Kyung
Choi1, Gun Hyong Hong1, Dong-Won Bae2, Kirankumar S. Mysore3, Yong-Soon Park1
1
KRIBB, South Korea, 2Gyeongsang National University, South Korea, 3The Samuel
Roberts Noble Foundation, United States
Agrobacterium tumefaciens causes crown gall disease. Although Agrobacterium can be
popularly used for genetic engineering, the influence of aboveground insect infestation on
Agrobacterium induced gall formation has not been investigated. Nicotiana benthamiana
leaves were exposed to a sucking insect whitefly infestation and benzothiadiazole (BTH) for 7
days, and these exposed
146
plants were inoculated with a tumorigenic Agrobacterium strain. We evaluated both in planta
and in vitro, how whitefly infestation affects crown gall disease. Whitefly infested plants
exhibited at least a 2-fold reduction in gall formation on both stem and crown root. Silencing
of isochorismate
synthase 1 (ICS1), required for salicylic acid (SA) synthesis, compromised gall formation
indicating an involvement of SA in whitefly-derived plant defence against Agrobacterium.
Endogenous SA content was augmented in whitefly infested plants upon Agrobacterium
inoculation. In addition, SA level was three times higher in root exudates from whitefly
infested plants. As a consequence, Agrobacterium-mediated transformation of roots of
whitefly infested plants was clearly inhibited when compared to control plants. These results
suggest that aboveground whitefly infestation elicits systemic defence responses throughout
the plant. Our findings provide new insights into insect-mediated leaf-root intra
communication and a framework to understand interactions between three organisms,
whitefly, N. benthamiana and Agrobacterium.
79
Influence of bacterial N-acyl-homoserine-lactones on growth and defense in
barley and yam bean
Peter Schröder*1, Christine Götz-Rösch2, Tina Sieper3, Anton Hartmann2, Agnes
Fekete4
1
Helmholtz Zentrum Muenchen GmbH, Germany, 2Helmholtz Zentrum Muenchen
Gmbh, Germany, 3Helmholtz Zentrum Muenchen, Germany, 4Universität
Würzburg, Germany
Bacteria communicate with each other and sense their environment in a population density
dependent mechanism known as quorum sensing (QS). N-acyl-homoserine lactones (AHLs)
are the QS signalling compounds of Gram-negative bacteria which are frequent colonizers of
rhizospheres. While cross-kingdom signalling and AHL-dependent gene expression in plants
has been confirmed, the responses of enzyme activities in the eukaryotic host upon AHLs are
mainly unknown. We investigated the influence of three different AHLs, namely N-hexanoyl(C6-HSL), Noctanoyl-(C8-HSL) and N-decanoyl- homoserine lactone (C10-HSL) on two
agricultural crop plants. The AHL-effects on Hordeum vulgare (L.) as an example of a
monocotyledonous crop and on the less known tropical leguminous crop plant Pachyrhizus
erosus (L) were compared. While plant growth and pigment contents in both plants were less
influenced, AHL treatment triggered tissue- and compound-specific changes in the activity of
important detoxification enzymes. The activity of dehydroascorbate reductase (DHAR) in
barley shoots after C10-HSL treatment for instance increased up to 384% of control plant
levels, whereas superoxide dismutase (SOD) activity in barley roots was decreased down to
23% of control levels upon C6-HSL treatment. Other detoxification enzymes reacted similarly
within this range, with interesting clusters of positive or negative answers towards the stress.
In general, changes on the enzyme level were more severe in barley than in yam bean. This
might be due to the different abilities of the plants to degrade AHLs to metabolites such as
the hydroxy- or keto-form of the parent signalling compound.
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80
Effects of silver nanoparticles on soil microorganisms and maize biomass are
linked in the rhizosphere
Wouter Sillen*1, Sofie Thijs1, Nele Weyens1, Jason White2, Jaco Vangronsveld1
1
Hasselt University, Belgium, 2Connecticut Agricultural Experiment Station, United States
Silver nanoparticles form part of an industrial revolution that has two faces. On the one hand,
nanotechnology has developed into an industry that is capable of making use in the most
efficient and targeted way of the well-known anti-microbial properties of silver. On the other
hand, it are the increasing quantities in which silver nanoparticles are produced and applied,
as well as the special characteristics caused by their nanodimensions, that have led to the
development of the study of nanotoxicology because earlier toxicology studies of bulk silver
are not applicable. Silver nanoparticles therefore are highly promising and useful, mainly for
combatting microorganisms, but are also a significant reason for concern as they exert their
toxicity in natural environments to non-target microbes and plants. Agricultural crops like
maize (Zea mays sp.) in particular are likely subjects of silver nanoparticle exposure, as these
particles may end up in the agro-ecosystem during manufacturing, incorporation into
products, and use of nanoparticle-containing products.
Maize growing in hydroponics responds to silver nanoparticle exposure by producing less
biomass, while exposure to realistic concentrations in natural soil often leads to an increase in
maize biomass. Because of silver’s well-known antimicrobial properties, microbial
communities associated with maize are expected to be important mediators in this outcome,
although very few studies have investigated the effects of silver nanoparticles in combined
plant-microbial systems. Our results confirm the existence of a strong link between the
responses of maize and the microbial communities in its rhizosphere to silver nanoparticles.
The specific conditions that shape the rhizosphere also alter the effects of silver nanoparticles
on microbial communities, compared to the bulk soil. The link between plant and rhizobiome
response highlights the role of rhizosphere microorganisms in the welfare of their host plant
and its response to contaminants, especially those that strongly target microorganisms.
81
Root exudate cocktails: the link between plant diversity and soil
microorganisms?
Katja Steinauer*1, Julia Friese2, Nico Eisenhauer1
1
German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig,
University of Leipzig, Germany, 2Friedrich Schiller University Jena, Institute of
Ecology, Germany
Higher plant diversity is often associated with higher soil microbial biomass, which is
assumed to be – at least partly – due to higher root exudate diversity. However, little is
known about the quantity and diversity of root exudates shaping their specific soil microbial
community. In this experiment we tested whether higher root exudate diversity enhances soil
microbial biomass in a plant diversity gradient. We set up a plant diversity gradient ranging
from monocultures to 2- and 3-species mixtures in microcosms using functionally dissimilar
plant species (grass: Anthoxanthum odoratum, small herb: Plantago lanceolata, tall herb:
Centaurea jacea) in soil of the Jena Experiment, Germany. After one month of plant growth,
exudate cocktails were added to the microcosms combining the most common sugars (e.g.,
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glucose), organic acids (e.g., citric acid), and amino acids (e.g., alanine). In total, we used four
different exudate cocktails, two exudate diversity levels (low and high), two nutrient
availability levels (carbon and nitrogen), and a control with only water addition. We
hypothesized the addition of a diverse exudate cocktail to offset significant plant diversity
effects on soil microbial biomass. After two months, we found a significant increase of soil
microbial biomass with increasing plant diversity in the control treatment. Microbial biomass
in the low exudate diversity treatment increased, though not significantly, with increasing
plant diversity. As hypothesized, microbial biomass was at similar levels across all plant
diversity levels in the high exudate diversity treatment, because microbial biomass increased
particularly at low plant diversity. Furthermore, microbial biomass was significantly higher in
the carbon treatment than in the nitrogen-rich exudate treatments. This corresponds to
previous findings of carbon-limited soil microbial communities at the field site of the Jena
Experiment. Our findings provide the first experimental evidence that root exudate diversity
represents a crucial link between plant diversity and soil microorganisms.
82
Changes in the root metabolism of Arabidopsis thaliana during mutualistic
association with a beneficial rhizobacterium
Nadine Strehmel*1, Sylvia Krueger1, Paul Schulze-Lefert2, Dierk Scheel1
1
Leibniz Institute of Plant Biochemistry, Germany, 2Max Planck Institute for Plant
Breeding Research, Germany
The narrow zone between the root and its surrounding soil is defined as the rhizosphere.
Here, hundreds of thousands of bacteria can be found, exhibiting a beneficial, a deleterious
or no effect on the performance of the plant. In general, bacteria colonizing the plant root
and promoting plant growth are referred to as plant growth-promoting rhizobacteria (PGPR).
Arabidopsis thaliana was cocultivated with Rhizobium leguminosarum under sterile conditions
in a growth cabinet, which resulted in an increased production of root and shoot dry and
fresh weight. In addition, changes in root morphology were observed, as roots of inoculated
plants were longer and exhibited more lateral roots than those of control plants. To
understand the underlying metabolic processes during this mutualism in the rhizosphere,
Arabidopsis thaliana was cocultivated for a defined time period with a plant growth
promoting strain of Rhizobium leguminosarum on agar plates. Finally, roots were analyzed for
changes in semipolar secondary metabolism following a non-targeted metabolite profiling
approach. Comprehensive analysis revealed a decline of major substance classes such as
glucosinolates and their degradation products, flavonoids and coumarins. In contrast,
signaling metabolites and defense compounds, phenylpropanoids as well as oligolignols
increased during cocultivation. To examine whether these compounds are essential for this
interaction, cocultivation studies were conducted in a hydroponic system and samples were
analyzed for changes in secondary metabolism. This revealed that phenylpropanoids and
oligolignols showed a contrasting behavior in root tissue and the corresponding root
exudates.
These studies are essential to understand microbial community dynamics in the rhizosphere
and thus contribute to the understanding of rhizosphere biology.
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83
Screening for ISR-inducing Trichoderma strains
Christine Vos*1, Katrijn Raymaekers2, Kaat De Cremer2, Barbara De Coninck2, Kemal
Kazan3, Bruno Cammue2
1
KU Leuven and CSIRO Australia, Australia, 2KU Leuven, Belgium, 3CSIRO Agriculture
Flagship, Australia
The genus Trichoderma constitutes a promising collection of potential biocontrol organisms
(BCOs), reducing plant disease either via direct interaction with plant pathogens and/or
indirectly through induced systemic resistance (ISR). The ISR capacity is mostly investigated by
performing classical disease assays in which physical contact between the BCO and pathogen
is avoided, however, such tests are very labour, time‐ and space-consuming. The discovery of
general ISR markers could therefore greatly facilitate the search for novel or more efficient
BCOs. We reported that application of Trichoderma hamatum T382 to Arabidopsis thaliana
roots resulted in ISR against leaf infection by the necrotrophic pathogen Botrytis cinerea and
performed a genome-wide analysis of ISR-related leaf gene expression, both before and after
B. cinerea infection. In addition, we recently completed a similar microarray analysis with
tomato replacing A. thaliana in the tripartite interaction. Based on the comparison of the
transcriptomic analyses in both plants, a series of orthologous genes
up-regulated in both tripartite systems was selected for their potential as general markers for
Trichoderma-induced ISR. We developed a screening system based on pMarker-GUS lines,
allowing fast and effective visual detection of ISR-inducing Trichoderma spp. The potential of
the pMarker-GUS lines as a screening tool is demonstrated by the clear correlation between
the percentage of disease reduction and the degree of staining of the pMarker-GUS lines. In
this presentation we will describe our screening assay and cover the most recent advances
that we have made with this system. Hereby we will focus on our ongoing characterization of
Trichoderma isolates with so-far unknown ISR capacity, as well as on the extrapolation of our
discoveries to tomato. In addition, we will address the question whether the screening system
can also be used for BCOs that do not belong to the genus Trichoderma.
84
The critical level of soil available P for the highest grain yield and the lowest
AMF colonization is identical in field-grown maize
Chao Wang*, Chunjian Li
The concentration of soil available phosphorus (P) is important for both plant growth and
colonization of arbusular-mycorrhizal fungi (AMF). A critical level of soil P for getting the
highest maize grain yield is reported in many studies, whilst that for AM colonization in the
field condition is not clear. Here a two-year field experiment (2013 and 2014) using maize at
six P application rates from 0 to 300 kg ha-1 on a long-term experiment site was conducted at
the Shangzhuang Experimental Station (40o8′20″N, 116o10′47″E), China Agricultural
University. The soil is a typical Ustochrept with a silty loam texture, mesic soil temperature
regime, and mixed mineralogy. Shoot, root and soil samples in 0-20 (topsoil) and 20-40
(subsoil) cm soil layers were collected at five growth stages over the whole growth period.
The results demonstrated that soil Olsen P in the topsoil layer, rather than in the subsoil layer,
increased with the increased application rate of P-fertilizers. The shoot biomass, grain yield,
shoot P content and concentration increased and reached the highest values when the
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amount of P fertilizers increased from 0 to 75 kg ha-1, and then kept constant, in spite of
further increases in P-fertilizer input. By contrast, the AMF colonization rate onto topsoil-layer
maize roots decreased with the increased amount of P-fertilizers, and reached the lowest value
at the P application rate of 75 kg ha-1, and kept unchanged regardless of the further input of
P-fertilizers. The measured Olsen-P concentration corresponding to the P fertilizer rate of 75
kg ha-1 was 6-10 mg kg-1 in both years. The results suggest that the shoot P concentration
decided the AMF colonization rate on maize roots in the field. However, the
AM colonization rate on subsoil-layer maize roots distributed kept relatively constant at
different P application rates.
85
Inoculation of arbuscular mycorrhizal fungi and Gaeumannomyces graminisvar
causes asymmetric competition between wheat and faba bean in intercropping
system
Guangzhou Wang*, Haigang Li, Fusuo Zhang, Junling Zhang
The mechanistic understanding of the effect of soil microorganisms to overyielding in
intercropping systems can be instructive in optimizing the productivity and ecosystem
service. To date the influence of soil microbes on interspecific and intraspecific interactions,
and consequently the system productivity is not well understood. In this study, we inoculated
arbuscular mycorrhizal fungi (AMF) of Funneliformis mosseae or a mix of six species (Glomus
etunicatum, Funneliformis mosseae, Rhizophagus intraradices, Glomus versiforme, Glomus
etumicatum and Glomus aggregatum) 10 days prior to the inoculation of wheat take-all
fungus of Gaeumannomyces graminisvar. tritici (Ggt) to wheat and faba bean in mono and
poly-culture to explore their respective and combined effects on plant growth, nutrient
uptake and competitive relationships between the two plant species. No overyielding was
observed. The dry biomass, N and P uptake by wheat in monoculture decreased significantly
by 18.7%, 20.7% and 26.5% respectively when inoculated with Ggt. When intercropped with
faba bean, the competitive disadvantage was aggravated and wheat biomass, N and P uptake
decreases by 31.8%, 39.6% and 40.2% respectively. By contrast, inoculation of Ggt improved
the dry biomass, N and P uptake of intercropped faba bean compared to those of mono
plants. Neither the growth of both plant species nor the disease severity of wheat was
significantly affected by the inoculation of one or mix AMF. However, inoculation of Ggt
reduced colonization percentage in wheat roots but not faba bean roots in both mono and
poly-culture treatments. Whereas inoculation of AMF enhanced P uptake of wheat and faba
bean whether inoculating with Ggt or not. Our results show that soil pathogen leads to
asymmetric competition between wheat and faba bean in intercropping system, and the
beneficial effect of AMF on plant P uptake does not offset the detrimental effects of soil
pathogen on the host plant.
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86
Above and belowground productivity of experimental grassland communities in
the field during initial assembly
Emanuela W. A. Weidlich*1, Philipp von Gillhaussen2, Vicky M. Temperton1
1
Forschungszentrum Jülich, Germany, 2University of Bayreuth - Department of
Disturbance Ecology, Germany
The order of arrival of different species can change the trajectory of a community as it
assembles. This is known as a priority effect, where the plants that first arrive affect the
further development of the system to such an extent that their impact on the system is still
detectable many years after their arrival. The more we understand community assembly, the
more information we will have on how to return a degraded area to a functioning ecosystem.
A large scale field experiment, called Priority Effect was established in 2012 to test whether
we could create priority effects by sowing high and low diversity mixtures and changing the
sequence in which plant functional groups arrived in the system. We measured above and
below ground productivity, soil chemistry and root productivity using the ingrowth core
method. In the first year plots with legumes sown first were more productive in aboveground
biomass than grasses or forbs sown first, but not more than the control sown at the same
time. Belowground we found opposite patterns of productivity: legume-first plots were less
productive belowground versus grasses-first as most productive. These results indicate
different biomass allocation patterns to above and belowground plant parts depending on
which functional group arrived first. We repeated the in-growth core method to assess root
biomass and turnover in 2014 and will see whether the belowground priority effect remains
or have disappeared with time. Following the development of the species and productivity in
this experiment will help us to understand the mechanisms of priority effects in grassland
communities in order to use this knowledge in improving grassland restoration as well
ensuring a certain level of productivity.
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Induced systemic resistance in Arabidopsis against Pseudomonas syringae pv.
tomato by disease suppressive soils
David Weller*1, Johan A. van Pelt2, Corné M. J. Pieterse2, Peter A. H. M. Bakker2
1
USDA-ARS, United States, 2Utrecht University, Netherlands
Two-week-old Arabidopsis thaliana ecotype Col-0 seedlings were transferred into an
autoclaved sand-soil mixture amended with 10 or 20% (weight/weight) soil that is
suppressive to either take-all or Rhizoctonia root rot of wheat from fields in Washington
State USA. These soils contain population sizes greater than 105 colony forming units per
gram of wheat root of 2,4-diacetylphloroglucinol or phenazine-1-carboxylic acid-producing
pseudomonads, respectively. Plants were challenge-inoculated with Pseudomonas syringae
pv. tomato three weeks later. Both suppressive soils induced resistance in Arabidopsis against
P. syringae similar to the well-studied Pseudomonas strains WCS417r, Q2-87 (produces 2,4diacetylphloroglucinol) and 2-79 (produces phenazine-1-carboxylic acid). Pasteurization of
the suppressive soils before adding them into the sand-soil mixture eliminated 2,4diacetylphloroglucinol and phenazine-1-carboxylic acid-producing pseudomonads from the
Arabidopsis rhizosphere and significantly reduced induced systemic resistance activity.
However, population sizes of total aerobic culturable bacteria were similar in the rhizosphere
of plants grown in the mixes with pasteurized and raw suppressive soils. This is the first report
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of induced systemic resistance activity by take-all and Rhizoctonia suppressive soils and the
ability of phenazine-1-carboxylic acid to induce resistance.
88
Insights into bacterial communities associated with apple replant disease soil
revealed by 16S rRNA gene amplicon sequencing
Bunlong Yim*1, Traud Winkelmann2, Guo-Chun Ding3, Kornelia Smalla4
1
Leibniz University of Hannover, Germany, 2Leibniz Universität Hannover – Institute of
Horticultural Production Systems, Section of Woody Plant and Propagation Physiology,
Germany, 3College of Resources and Environmental Sciences, China Agricultural
University, China, 4Julius Kühn-Institut – Federal Research Centre for Cultivated Plants
(JKI), Institute for Epidemiology and Pathogen Diagnostics, Germany
Apple replant disease (ARD) severely affects apple production worldwide. The possible causes
are parasitic nematodes, accumulation of soil borne pathogenic microorganisms or absence
of antagonists and beneficial microorganisms, and toxic effects from decomposition of crop
residues. The aim of this study was to reveal bacterial communities associated with ARD soils
and to analyze the effects of soil treatments with heat and gamma irradiation on plant
growth and bacterial community composition and diversity in two different soil types. The
results of a biotest with micropropagated apple rootstock M26 plants showed that the plant
growth in replant disease soils from two nurseries (Kle and Alv) was reduced in ARD soils
(control) compared to soils either treated at 50 oC (H50) or with gamma irradiation (Gamma).
Soil attached to roots of the plants after eight weeks of the biotest experiment was collected
and submitted to soil total community DNA (TC-DNA) extraction. The TC-DNA was subjected
to 454-pyrosequencing of amplified 16S RNA gene fragments. The differences in plant
growth were associated with differences in the bacterial communities. The two soil types with
different culture practices and management showed different bacterial community
composition and diversity. In both soils, the treatments led to an increased abundance of
Proteobacteria (Gamma), Firmicutes (H50), and Bacterioidetes (Gamma), whereas members of
Acidobacteria (H50, Gamma) and Actinobacteria (Gamma) decreased. Despite of the soil type
specific responses a few genera were recorded as responders to the treatments in both soils.
Significantly increased relative abundance was recorded for Streptomyces, Bacillus,
Paenibacillus and Sphingomonas in the H50 treatments while Mucilaginibacter, Devosia and
Rhodanobacter had an increased relative abundance in the Gamma treatments of both soils.
The increased abundance of potential degraders of phenolic compounds might contribute
the improved plant growth in the H50 and Gamma treatments.
89
Linking shoot and root characteristics with soil microbial communities to better
understand the influence of agroforests on soil structure
Monnier Yogan*1, Amandine Erktan1, Luis Merino-Martin1, Catherine Roumet2, Yves Le
Bissonnais3, Alexia Stokes1
1
INRA, UMR AMAP, France, 2CNRS, UMR CEFE,, France, 3INRA, UMR LISAH, France
In European agricultural landscapes, conventional farming is often described as a factor
incrementing soil erosion. Recent policies have been encouraging agroforestry practices that
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are believed to provide a number of ecosystem services and improve biodiversity. However
research is still required to determine the best plantation and management practices,
depending on land use, climate, topography and characteristics of species used. Our project
investigates above and belowground interactions in crop/forest systems and changes that
occur in the rhizosphere of different cropping systems. Eight field-sites representative of
different agroforests with distinct ages and pedoclimatic characteristics were selected
throughout France. On each site, measurements were performed to assess the influence of
the cultivated tree row, the distance to trees, and the perennial plant cover on shoot
morphology, litter cover, soil aggregate stability, root traits, soil microbial activity and
microbial metabolic diversity. Preliminary results show that in a number of agroforests, soil
aggregate stability is greater along the tree row because root density is higher. However, the
magnitude of these effects seems to be related to the pedological characteristics of the
agroforest. Results will allow to determine how above and belowground characteristics of
plant communities contribute to soil aggregate stability. This research will provide
stakeholders and agroforesters with data to determine the best soil management strategies
to protect against erosion with regard to above and below traits of cultivated trees.
90
A new conceptual framework show that plant genotype and soil N availability
modify both plant nutritional strategies and the associated rhizosphere
microbiome
Anouk Zancarini*, Christophe Mougel, Anne-Sophie Voisin, Marion Prudent,
Christophe Salon, Nathalie Munier-Jolain
INRA, France
In the context of sustainable agriculture, it is desirable to lower the input of mineral fertilizers.
Therefore, we need to select ‘new’ genotypes that are both adapted to ‘low fertilizer inputs’
and more efficient in nutrient use. A better understanding of plant-microbe interactions
under low input of fertilizers is now needed. Nevertheless, in microbial ecology, conceptual
frameworks or models are used to analyse plant-microbe interactions but plant phenotype is
currently viewed as a “black box”. We developed a new conceptual framework to study plantmicrobe interactions using a multidisciplinary approach combining Microbial Ecology and
Plant Ecophysiology.
The links among plant genotype, soil nitrogen availability and the plant associated
rhizosphere microbiome were assessed at both structural and functional level. On the one
hand, the influence of both Medicago truncatula genotype and soil nitrogen availability on
the genetic structure of the soil microbiome was determined by DNA fingerprint and 454
pyrosequencing. On the other hand, the different nutritional strategies of the plant-microbe
interactions were evaluated using an ecophysiological framework.
We observed that nitrogen availability affected rhizosphere bacterial communities only in
presence of the plant. Furthermore, we demonstrated the existence of a strong Medicago
truncatula genotype x nitrogen availability effect on the rhizosphere bacterial communities.
Finally, the nutritional strategies of the plant varied greatly in response to a modification of
nitrogen availability. Three contrasted structural and functional adaptive responses of plantmicrobe interactions to nitrogen availability were thus identified.
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With recent developments in statistics and high throughput genotyping, this new conceptual
framework is now used in a genome-wide association studies (GWAS) to identify plant
genetic determinants that may be linked to the selection of more beneficial microbiome for
plant under low input of fertilizers.
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92
Comparisons of two root water uptake models based on field observations
Gaochao Cai*1, Jan Vanderborght1, Valentin Couvreur2, Harry Vereecken1
1
Agrosphere, Institute of Bio- and Geosciences (IBG-3), Forschungszentrum
Jülich, Germany, 2Department of Land, Air andWater Resources, University of California,
Davis, California, USA
Accurate estimation of water requirements and efficient water management of crops requires
appropriate mathematic descriptions of water flow. Modeling root water uptake is an
important approach to evaluate water movement in the soil-root system. However, dynamic
root distribution and a physically based concept to describe water uptake from soil profiles
with vertical variations in soil water availability are often not taken into consideration.
Therefore, we incorporated observations of soil moisture and root development within two
different modeling frameworks and compared the simulated root water uptake. To do so, we
carried out field experiments where soil water content, soil water potential, and root growth
were monitored non-invasively for three water treatments: sheltered, rain-fed, and irrigated.
Root growth of winter wheat was measured by using 7-m long horizontally installed
minirhizotubes at six depths with three replicates per treatment. The observed data were
interpreted by Feddes model and an upscaled model based on hydraulic architecture
approach that considers compensatory root water uptake and hydraulic conductance in the
coupled soil root system.
Results showed that root water uptake simulated by Feddes model was affected by root
distributions, whereas it was influenced by the conductance of the root system when using
upscaled model with the same soil hydraulic properties and boundary conditions. Moreover,
root water uptake simulated by the two models tended to deviate in dry and wet conditions
due to different water stress functions. The upscaled model showed a promising optional
approach for simulations of root water uptake by using 1-D spatial discretization to represent
soil water dynamics in soil root system. To validate simulated root water uptake by these
models, additional measurements, such as sap flow, might be helpful for the predictions of
water requirement of different crops.
93
Mathematical modelling for water movement in ridged plant systems
Simon Duncan*1, Tiina Roose1, Paul Sweeney2
1
University of Southampton, United Kingdom, 2Syngenta Ltd., United Kingdom
In arable farming a widely used practice of growing crops is the use of row production. The
potato is traditionally grown in a ridge and furrow system in which the ground’s surface is
adapted to form a periodic series of ridges and furrows to allow water flow through the field
and provide water to the plants. The model used compares the saturation levels in two
different ground geometries for the growth of a potato crop, these geometries are the ridge
and furrow system and a flat ground structure.
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The water movement model is based on an adapted Richards equation. The model includes a
function of water uptake via root activity in each of the geometries that is responsible for
reducing the saturation in the root region. The boundary conditions for the model consist of
a flux boundary condition on the ground’s surface to allow the inflow of water, a pressure
boundary condition at the base of the soil geometry of pressure value pF2 and finally two
zero flux boundaries at the sides of the geometries.
We determined that the saturation levels in each of the geometry systems remains similar
over long periods of time. However we determine there is a difference in the amount of water
uptake by the plants in the two structures. We found that the ridge and furrow system has a
greater uptake of water when compared to the flat geometry.
We observed the formation of dry regions in the area of root activity for each of the two
geometries and determined that there is a larger amount of water uptake by the potato
plants in the ridge and furrow system.
94
Seeking water: Rooting depth and drought tolerance of Zea mays genotypes
driven by nutrient patches and subsoil fertilization
Christian Fritz*1, Eric Visser2, Philippe Hinsinger3, Claude Doussan4, Hans de Kroon2,
Dina in 't Zand2, Annette Bérard4, Florian Wichern1
1
Rhine-Waal University of Applied Sciences, Germany, 2Radboud
University, Netherlands, 3INRA Montpellier, France, 4INRA Avignon, France
Water deficiency upon drought is a major constrain on yields. Increasing rooting depth and
the soil volume explored by lateral roots seem effective mechanism to prevent water stress in
cereals. This research reports the effects of vertical fertilizer placement on root distribution,
water and nutrient acquisition of a selection of genotypes (6) in Zea mays.
Experiments were conducted at the Phytotron facility in Nijmegen (NL) using 70 cm deep
containers equipped with mini-rhizotrons and drip irrigators over 2 growing seasons (full
factorial design experiments). We mixed nutrients into the topsoil (0-30 cm) or subsoil (35-70
cm) at field rates. Additionally, nutrients were added as patches (15 vs. 40 cm deep). Drought
was simulated by mid-season drying out of the topsoil without affecting subsoil water supply.
Trials were complemented by pot experiments manipulating nitrate patches only. Maize
genotypes were selected from the EURoot maize panel.
We found that rooting depth of maize was strongly dependent on vertical nutrient
availability. Fertilization of subsoil increased averaged rooting depth by 3 to 5 times with
highest root densities found nutrient patches (280 m.l-1). Improved rooting depth was mainly
due to increased root biomass allocation at relatively stable specific root length (~190 g.m-1).
Genotypes, however, could differ strongly in root allocation response to nutrients.
Plants receiving subsoil fertilization under drought showed higher rates (18%) of
photosynthesis and transpiration. Subsoil fertilized plants acquired significantly more water
from deep soil layers especially at the height of drought. Interestingly, water use efficiency
was little affected by vertical fertilizer placement or concentrations. We observed a trade-off
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between early-stage vigour and late-season drought tolerance in treatments receiving subsoil
fertilizers.
Nutrient placement plays a dominant role in root system architecture and consequently water
uptake. Drought tolerance may be further improved by a better understanding of genotype
specific root-soil-nutrient interaction.
95
Imaging of root water uptake by MRI in combination with tracer motion
Sabina Haber-Pohlmeier*1, Andreas Pohlmeier2, Jan Vanderborght2
1
RWTH Aachen University, ITMC, Germany, 2Research Center Jülich, Germany
Flow processes in natural porous media are often too slow to be monitored by direct flow
imaging, therefore the visualization of such fluxes is best performed by tracer tracking. While
T1 reducing contrast agents are well known in medical diagnostics, their usefulness in natural
porous media is not yet well explored. As pointed out in a preceding study, GdDTPA is most
convenient since it is very stable and does not adsorb at the soil matrix. Furthermore, it´s
specific relaxivity in the liquid phase is sufficiently high to yield good contrast when used in a
strongly T1 weighted pulse sequence. Here, we use a simple procedure for the quantification
of tracer concentration in saturated and unsaturated natural porous media, where the
reduction of relaxation times by desaturation is compensated for by a reference
measurement. The procedure is applied to examples from natural porous media, which are
decisive bottlenecks in the water flow from soil to the atmosphere: Root water uptake and
evaporation from top soil.
While plant roots take up water from the surrounding soil, GdDTPA is first enriched in the
neighborhood of some roots, indicating their activity. But the tracer is also enriched in the
immediate, some mm thick, layer around the root, the so-called rhizosphere, which appears
dark in conventional MRI sequences. Although this layer has been frequently interpreted as
water depletion zone, the enrichment of GdDTPA there proves the high permeability for
water and solutes.
96
Linking mesoscopic to macroscopic scale modeling of water and osmotic
stresses on root water uptake
Helena Jorda Guerra*1, Adi Perelman2, Naftali Lazarovitch2, Jan Vanderborght3
1
KU Leuven, Belgium, 2Ben-Gurion University of the Negev, Israel, 3Forschungszentrum
Jülich GmbH, Germany
Current soil-hydrological models predict the effect of water and salt stresses on macroscopic
root water uptake by using so-called transpiration reduction functions. However, the use of
these functions is restricted to the environmental conditions at which they were empirically
developed.
This research intends to develop a macroscopic reduction function based on biophysical
knowledge. Simulation experiments are conducted for a range of atmospheric conditions, soil
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and plant properties, irrigation water quality and scheduling using a 3-D physically-based
model that resolves flow and transport to individual root segments and that couples flow in
the soil and root system. The effect of salt concentrations on root water uptake is accounted
for by including osmotic water potential gradients between the solution at the soil root
interface and the root xylem sap in the hydraulic gradient between the soil and root.
Simulation experiments are performed in a soil volume around a single root segment. We
discuss how the simulation setup can be defined so as to represent: (i) certain root system
characteristics, (ii) plant transpiration rate, (iii) leaching fraction of the irrigation, (iii) salinity of
the irrigation water, and (iv) water potential threshold for stress onset. The output of these
simulation experiments gives a first insight in the effect of salinity on transpiration and on the
relation between the bulk salinity, which is used in macroscopic salt stress functions of
models that do not resolve processes at the root segment scale, and the salinity at the soilroot interface, which determines the actual root water uptake. In a next step, simulations
considering the whole root architecture will be conducted to evaluate how the outcome of
the single root simulation experiments can be upscaled to the whole root system scale.
97
Variability of some important Tunisian Chickpea genotypes (Cicer arietinum L.)
response to drought stress
Abdelmajid Krouma*1, Chedly Abdelly1, Tatsuhito Fujimura2
1
Centre of Biotechnology, Tunisia, 2School of Life and Environmental sciences, Japan
Scarcity of water is a severe environmental constraint to plant productivity. Drought-induced
loss in crop yield probably exceeds losses from all other causes, since both the severity and
duration of the stress are critical. In the current study, a glasshouse experiment was
conducted to assess the effects of a progressive drought stress on plant growth,
photosynthetic activity and water relations parameters in three Tunisian chickpea genotypes
(Cicer arietinum L.). These genotypes were largely cultivated in Tunisia for their diverse uses,
Amdoun with big size seeds shown tolerant to salt stress, Chetoui with small size seeds
shown sensitive to salt stress and Kesseb with medium size seeds. A progressive drought
stress was applied during 16 days of water starvation. A close relationship between plant
growth, photosynthesis, leaf water status and osmotic adjustment was found. In comparison
to Chetoui and Kesseb, Amdoun showed the highest plant growth and photosynthetic
activity, and an important osmotic adjustment under drought stress.
98
Influence of soil nitrate concentration on plant root water uptake
Sophie Maloteau*1, Jan Diels2, Sarah Garré3
1
Université de Liège, Gembloux Agro-Bio Tech, Belgium, 2KULeuven, Department of
Earth and Environmental Sciences, Belgium, Belgium, 3Université de Liège, Gembloux
Agro-Bio Tech, Belgium, Belgium
Many real world and modelling experiments, seeking for a quantification of root water
uptake pathways, were conducted under simplified conditions. However, we know that plants
are able to regulate their hydraulic conductivity coarsely by increasing root growth in certain
areas (long term regulation) and finely by changing the conductivity of specific parts of the
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root system along the water flow pathway (short term regulation). This regulation is rarely
taken into account in models, and its impact at larger scales has never been studied. Novel
experimental and modelling techniques must be developed in order to simultaneously
monitor root system characteristics, soil solution concentrations and flow rates along the soilplant continuum in a real soil medium.
In this context, we will study the influence of nitrate on the hydraulic conductivity of plants
root systems. First, we will adapt an existing root water uptake model (R-SWMS), by adding a
nitrate-dependent hydraulic root architecture. Next, we will conduct a destructive experiment
to quantify how much water and nitrate is taken up by the plant at different development
stages, depending on the nitrate concentration in the root zone. As the measures are realized
at different times of the experiment on similar plants, short term regulation of plants water
uptake can be distinguished from long term regulation. Finally, a second experiment will be
prepared to monitor temporal changes in local root water uptake and transpiration upon
locally increased nitrate concentration. Plants will be grown in two-dimensional rhizotrons so
that they can be used for neutron radiography imaging of the soil-root and root-root water
fluxes upon locally increased nitrate concentrations. The combined information on root
system architecture, transpiration rate and local root water uptake will then be used in an
inverse modelling to obtain root hydraulic conductivities and describe how nitrate can affect
it.
99
Minimize the tillage operation improve the root water and nutrient uptakes
Rajesh Nallaiah*
Tamil Nadu Agricultural University, India
As farm tractors and field equipment become larger and heavier, there is a growing concern
about soil compaction. Soil compaction can be associated with a majority of field operations
that are often performed when soils are wet and more susceptible to compaction. Heavy
equipment and tillage implements can cause damage to the soil structure. Soil structure is
important because it determines the ability of a soil to hold and conduct water, nutrients, and
air necessary for plant root activity. Now a day all the field tillage operation utilized heavy
loaded machines only. Compare the traditional and modern tillage operation soil compact
more in modern tillage operation. The plant response to subsoil compaction, as with surface
compaction, depends on the crop, soil conditions, and the climatic conditions in a particular
year. If plants are already stressed for water, subsoil compaction may add to the stress by
limiting the growth of plant roots to additional water. If plants are growing in soils that have
aeration problems due to high water content, subsoil compaction will slow drainage and
could result in an anaerobic root environment that limits nutrient uptake. it does observe the
available water and nutrient uptakes with soil and plant relationship. Recently aware of the
minimum tillage operation it’s minimized the soil compact condition compare the maximum
tillage operation condition. The short duration crops or shallow rooted crops mostly affected
the soil compact. Shallow root systems crop very sensitive the water and nutrient uptake its
will affect soil compact condition. If they are given the needful tillage operation based on
crop condition its will get good root growth easy to uptake the available moisture and
nutrient.
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100
A novel 3-D imaging of water on its passage through the rhizosphere in plantsoil systems
Sascha Oswald*1, Christian Tötzke2, Nicole Rudolph-Mohr2, Sabina Haber-Pohlmeier3,
Andreas Pohlmeier4, Eberhard Lehmann5
1
University of Potsdam, Germany, 2University of Potsdam, Institute of Earth and
Environmental Science, Germany, 3RWTH Aachen, Institute for Technical and
Macromolecular Chemistry, Germany, 4Forschungszentrum Jülich IBG-3, Germany, 5Paul
Scherrer Institute, Germany
We use a combination of neutron tomography (NT), fluorescence imaging and magnetic
resonance imaging (MRI) to capture the dynamic behaviour in the root – soil interface in situ.
This complementary approach provides detailed information about water distribution as well
as oxygen or pH patterns within the rhizosphere. The big advantage is that all these
measurements are non-invasive and allow a spatially highly-resolved mapping of the
rhizosphere throughout the root systems of young plants, in 2-D and 3-D, and dynamic
changes during hours and days.
Recently, we started to combine NT with MRI to study the dynamic behaviour at the root–soil
interface. The high spatial resolution of NT and its sensitivity for water can be exploited for 3D analysis of root morphology and detailed mapping of water content at the root-soil
interface and surrounding soil, while MRI can yield complementary information, that is what
fraction of water placed in smaller or larger pores or bound in mucilage. The hypothesis is
that beyond this it is possible to use special tracers to investigate the fluxes of water through
soil, rhizosphere and roots, working with both imaging methods on the same samples.
With NT we explored if these tracers pass the rhizosphere and to which degree its
concentrations and fate can be detected. The samples were especially constructed to allow
MRI in parallel and some of them were also imaged via MRI. The results suggest that by NT
the root system can be identified well, its morphology retrieved and related to the
concentration changes of the tracer during water uptake, while MRI can work with the same
samples at least to image the root system. A detailed analysis of spatial differences in water
and tracer passage is on its way and will be complemented by additional MRI measurements
and information from fluorescence imaging.
101
Simulating root trait evolution in a functional structural plant model: effect of
spatial and temporal water availability
Pieter Poot*, Michael Renton
University of Western Australia, Australia
Functional-structural plant models (FSPMs) represent the interaction between a plants’
structure, the processes occurring within that structure and the surrounding environment.
They usually model the dynamic development of the plant structure with explicit topology
and geometry, as well as the dynamic processes occurring within and between the structure
and its environment. We developed a FSPM that captures the key processes involved in the
growth and development of root structures and that can be used to evaluate optimal
structural rooting strategies. The model is flexible enough to allow a wide range of strategies
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to be represented and explored but simple enough to be linked with an evolutionary
optimization algorithm without becoming computationally infeasible. In recently published
work we applied this novel approach to a case study habitat with a spatially and temporally
heterogeneous distribution of water: perennial plants occurring on shallow soils in seasonally
dry environments that depend on cracks in the underlying rock to access more permanent
water. The model successfully simulated the evolutionary dynamics of root structures leading
to improved fitness under two different fitness criteria and predicted the evolution of several
root traits in a direction that has been observed for plants confined to these habitats. Here
we will further refine our approach by incorporating more realistic assumptions in the FSPM,
the evolutionary algorithm and the fitness criteria. We will show how the structure of optimal
root systems and specific root traits change as dependent on the spatial accessibility of soil
water (i.e. the frequency of cracks in rocks or pores in compacted soils), and the frequency
and amount of rainfall during the wet season. Results will increase our understanding of the
evolution of root structures and root traits and will be highly relevant in the context of our
changing climate.
102
Modelling root exploration of biopores in soil
Katrin Huber1, Glyn Bengough2, Jan Vanderborght1, Mathieu Javaux1, Magdalena
Landl1, Harry Vereecken1, Andrea Schnepf*1
1
Agrosphere (IBG 3), Forschungszentrum Jülich GmbH, Germany, 2The James Hutton
Institute, United Kingdom
Biopores are soil macropores formed by biological activity that enable plant roots to reach
water reservoirs at depth. Here we present an extension of the R-SWMS model for water and
solute transport in the soil-plant system in which roots are able to grow in pre-existing
biopores in the soil.
Root growth is based on vector addition and influenced by the local soil parameters, e.g.
penetrometer resistance or nutrient availability, around a growing root tip. Water uptake by
roots growing inside air-filled macro pores occurs only from the soil matrix and depends on
the contact area between roots and biopore walls.
Root architecture and growth velocity are shown to depend on plant growth parameters, in
particular tropisms, as well as soil parameters, especially the penetrometer resistance and the
inclination of macro pores. This has implications on root water uptake patterns that can be
beneficial for plants in drought periods.
As biopores are often coated with nutrient rich material, this modelling approach can also be
used to investigate the benefits of biopores for plant nutrient uptake.
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Water Relations
103
Effect of mucilage on rhizosphere hydraulic properties: a new experimental
approach
Nico Schultze*1, Eva Kröner2, Andrea Carminati2, Doris Vetterlein1
1
Helmholtz Centre for Environmental Research GmbH - UFZ, Germany, 2Georg-August
University of Goettingen, Germany
Roots are hypothesized to alter rhizosphere hydraulic properties by release of mucilage. This
mechanism is expected to have strong implications for root water uptake under drought
conditions. Direct measurement of rhizosphere hydraulic properties is hindered by the
dynamic nature of the components involved; root hydraulics change with ontology; mucilage
production, composition and diffusion are not constant; soil water content changes. An
experimental approach was developed which enables to simultaneously measure hydraulic
conductivity and water retention curve around artificial roots covered with mucilage or
modified model substances mimicking individual properties of mucilage. The system
accounts for the radial geometry of root water uptake.
The set-up consists of a soil-filled cylinder connected via a ceramic plate to a water supply for
establishing initial soil water content. An artificial root with known and constant hydraulic
properties is placed in the center of the soil cylinder and connected to a pump. A
microtensiometer is inserted half way between the cylinder wall and the artificial root for soil
matric potential measurement. The installation is placed on a weighing cell for determination
of changes in soil water content. The experiment is modelled solving a modified version of
the Richards’ equation that includes mucilage dynamics. The functions to be fitted are soil
matric potential and water outflow from the artificial roots in analogy to classical multistep
outflow experiments. Fitting parameters are the van Genuchten-Mualem parameters of the
soil and how mucilage modifies them.
First results will be presented for homogeneous distribution of gel throughout the soil
cylinder versus gel localized just around the artificial root.
104
Numerical study on the impact of rhizosphere processes on root water uptake
Nimrod Schwartz*1, Eva Kroener2, Andrea Carminati2, Mathieu Javaux1
1
Catholic University of Louvain, Belgium, 2Gottingen University, Germany
For many years, the rhizosphere is known as a unique soil environment with different
physical, biological and chemical properties than those of the bulk soil. Indeed, recent studies
showed that root exudates, in particular mucilage, alter the hydraulic properties of the soil and
that induce non-equilibrium water dynamics in the rhizosphere during drying and
wetting cycles. While there are experimental evidences and simplified 1D model for those
concepts, an integrated model that couples rhizosphere processes with a detailed decsription
water flow in soil and roots is absent. Therefore, the objective of this work was to develop a
3D physical model of water flow in the soil-plant continuum that takes in consideration root
architecture and rhizosphere specific properties. To achieve this objective, we coupled a
detailed model of water flow in soil and root system (R-SWMS) with a model of the
rhizosphere hydraulic properties.
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Water Relations
We simulate wetting and drying cycles and examine the water content distribution around
roots and the onset of plant stress. We also performed a scenario analysis in which the
impact of different rhizosphere processes on water flow and root water uptake was
examined. For the wetting process, the model predicted that after infiltration the water
content in the rhizosphere remained lower than in the bulk soil (non-equilibrium), but over
time water infiltrated into the rhizosphere and eventually the water content in the
rhizosphere became higher than in the bulk soil. During drying, the high water holding
capacity of the rhizosphere and the non-equilibrium between water content and water
potential delayed the onset of stress. These results are in qualitative agreement with the
available experimental data on water dynamics in the rhizosphere and suggest an important
role of the rhizosphere in modulating plant water stress during drying and wetting cycles.
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Water Relations
Root-Root Interactions
105
Unraveling the positive biodiversity-productivity relationship: linking plant
community productivity to root trait diversity
Lisette Bakker*, Liesje Mommer, Frank Berendse, Jasper van Ruijven
The positive relationship between plant biodiversity and community productivity is well
established. However, our knowledge about the mechanisms underlying this positive
relationship is still limited. The consensus is that the positive effects on productivity are
driven by the functional diversity of the community, i.e. the diversity in functional
characteristics ('traits') among species, rather than species richness per se. Communities with
a higher diversity in traits are expected to better explore the available resources in space and
time, a concept called resource partitioning. However, experimental evidence for specific trait
combinations underlying resource partitioning is scarce. This study aims to elucidate the link
between trait diversity and the productivity of plant communities. Our aims are 1) to establish
the relationship between trait diversity and community productivity, and 2) to identify those
traits that best predict productivity. In a common garden experiment in Wageningen, the
Netherlands, 16 grassland species are grown in monocultures, 4-species mixtures differing in
trait diversity, and 16-species mixtures. Here, we focus on the results from the first growing
season. Complementarity effects, indicating resource partitioning, did occur but differed
greatly between communities (ranging from + 101% to – 48%). Trait diversity of the
communities is calculated based on measurements in the greenhouse and existing trait
databases, and linked to community productivity. In contrast to most previous studies, we
explicitly incorporate belowground traits in our approach, as the resource partitioning is
expected to occur predominantly in the soil. However, diversity in belowground traits such as
rooting depth and specific root length could not explain complementarity effects. Instead,
positive complementarity effects were associated with the presence of specific species. The
implications of these findings for biodiversity-ecosystem functioning research will be
discussed.
106
Spatial heterogeneity of plant-soil feedback affects root interactions and
interspecific competition
Hans de Kroon*1, Marloes Hendriks1, Janneke Ravenek1, Wim H. van der Putten2, Liesje
Mommer3
1
Radboud University, Netherlands, 2Netherlands Institute of
Ecology, Netherlands, 3Wageningen University, Netherlands
Soils are inherently heterogeneous. At a small scales, availability of water and nutrients may
differ by an order of magnitude. Spatial variation in soil microbial communities is likely to be
large as well, as plant species have been shown to accumulate species-specific soil
communities in their vicinity. While responses of roots to nutrient heterogeneity have been
studied for decades we hardly know how plants respond to soil biotic heterogeneity. Here we
report on an experiment in which we combined a plant-soil feedback approach with a
competition experiment, involving two grass and two forb species from temperate
165
grasslands. We subjected pairs of plants of the same or different species to patches of soil
that were previous conditioned by the same or different species. This was repeated in all
possible combinations, resulting in 100 different treatments. This design allowed us to
examine root interactions as a function of soil type and soil heterogeneity.
All species suffered negative soil feedback, but the strength was species-specific, reflected by
decreased root growth in own soil compared to foreign soil. Interestingly, the competitively
superior species also suffered the strongest negative plant-soil feedback. Root competition
was affected by soil conditioning and interacted with soil biotic heterogeneity. Specifically, in
heterogeneous soils, substantial reduction in root growth by the superior competitor in
patches with own soil provided opportunities for inferior competitors belowground. Our
results suggest that plants do not need to perish due to their own soil enemies, but can
escape on small spatial scales to better resorts in terms of plant-soil feedback. Negative
plant-soil feedback effects are considered an important mechanism of species coexistence
and plant community productivity. Small-scale responses of root systems to soil biotic
heterogeneity may be an important factor contributing to these effects that has hitherto
been overlooked.
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Start from scratch: how a cereal and a legume create spatial heterogeneities and
hotspots of soil microbial activities in the rhizosphere
Philippe Hinsinger1, Sarah Placella1, Josiane Abadie1, Camille Cros1, Gabrielle Daudin1,
Esther Guillot*2, Agnès Robin3, Claire Marsden4, Naoise Nunan5
1
INRA, France, 2IRD, France, 3CIRAD, France, 4SupAgro, France, 5CNRS, France
Rhizosphere processes are known to generate gradients of biological, chemical and physical
properties in the soil as a function of the distance to root surface. This has been rather well
documented, while micro-heterogeneities of such properties along roots have been little
evidenced and quantified. Sampling at the appropriate (millimetric or less) scale is a
methodological challenge, and many of the gradients data have been obtained with
microcosms designed to simplify the geometry of the rhizosphere. Here we report of the use
of rhizoboxes with 2D access to the soil profile, in which we mapped acid phosphatase
activity in situ in chickpea and durum wheat, either grown alone or intercropped, as well as in
rhizoboxes containing bare soil (control treatment without plant). This enzymatic activity was
actually monitored over 5 weeks with a non-destructive technique based on the use of a
membrane sorbing the enzymes, that was applied once a week against the soil profile for a
collection period of 3.5 hours. The soil had been sampled in the nill treatment of a long-term
fertilizer trial that had not received phosphorus application over 40 years, and thus had very
low phosphorus availability. It was sieved and packed so that the spatial heterogeneity in
each rhizobox was minimal at the start of the experiment. The presence of roots of both plant
species was clearly responsible for the increased heterogeneity of acid phosphatase activity
observed relative to bare soil. Greater acid phosphatase activity was generally observed near
roots, especially chickpea roots and root nodules. The greatest activities were found in the
vicinity of the younger parts of the roots (apices and elongation zones). While there was a
clear difference between the two plant species, there was no significant effect of
intercropping at the fine spatial scale of investigation reported in the present study.
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108
Root exudate effects on humic acids and the consequences for belowground
plant species interactions
Diederik Keuskamp*1, Rob Comans1, Liesje Mommer2, Ellis Hoffland1
1
Department of Soil Quality, Wageningen University, Netherlands, 2Nature Conservation
and Plant Ecology, Wageningen University, Netherlands
Humic acids are supramolecules that are the major organic components of most soils. They
have been shown to induce plant species specific (root)growth responses, such as lateral root
formation and higher (relative) root biomass. These root responses depend on the molecular
size of the HA. Interestingly, organic acids are known to destabilize HA into smaller fractions.
Since root exudates contain several organic acids, such as citric acid or phenolic acids, root
exudates may be of extreme relevance for the HA induced growth responses. The quantity
and quality of these root exudates are known to be plant species specific. This could mean
that the root exudates of a plant can affect the growth of neighbouring plants via their
interaction with HA, especially when these neighbouring plants are of another species.
Mommer and co-workers showed root aggregation for the grass species Anthoxantum
odoratum when in interspecific interaction. In our research project we test the hypothesis
whether this root aggregation could be (partly) explained by humic acids (HA) and its
interaction with root exudates. We investigate this interaction by growing A. odoratum and
Leucanthemum vulgare seedlings in a mixed culture and under the sterile conditions of agar
plates. These growth conditions enable us to identify changes in root development
(independent of different microbial and/or soil effects) and allow us to add HA at different
concentrations to test the effects of these HA. We will also make use of HA pre-treated with
root exudates or organic acids, trying to mimic the growth responses in monocultures of
each of the species. On the poster we will present the preliminary results of our ongoing
research.
109
Effects of different wheat varieties intercropped with faba bean on root
morphology of wheat
Wang Yuyun, Ren Jiabing, Tang Li*
Yunnan Agricultural University, China
Wheat and faba bean intercropping is one of the important intercropping systems in China.
The effectiveness of this intercropping system on grain yield increasing, nutrients acquisition
improvement and disease control has been demonstrated. But the genotypes difference of
intercropping has been seldom studied.
A field experiment was conducted to investigate the effect of intercropping on wheat root
morphologyalong the soil depth. Three wheat varieties YM-47, YM-42 and MY-29, with
different resistances to wheat powdery mildew were used by monocropping or intercropping
with faba bean.
Results showed that, wheat and faba bean intercropping significantly increased the wheat
root length density and root surface area, but decreased the root average diameter.
Compared with monocropping wheat, the root length density of intercropped YM-47 and
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YM-42 were increased by 225.8% and 301.7%, the root surface area were greater by 235.9%
and 236.5% respectively at 0-10cm soil layer, but the intercropped MY-29 showed no
significant difference with that of monocropping. There was no significant difference of root
length density at 10-20 cm layer between intercropping and monocropping. But the root
length density and root surface area of wheat were lower at 20-30 cm layer under
intercropping. Compared with monocropping wheat, No significant difference was found at
0-10 cm layer, but the root average diameter in intercropping decreased by 12%, 16% and
11% at 10-20 cm and by 32%, 12% and 14% at 20-30 cm soil layers of YM-47, YM-42 and
MY-29, respectively.
The root length density, root average diameter and root surface area showed significant
positive correlation with soil available P content. Wheat and faba bean intercropping
significantly increased wheat yield and showed significant intercropping advantage. The LER
was 1.20, 1.14 and 1.01 for wheat YM-47, YM-42 and MY-29, respectively.
In conclusion, The intercropping advantages were influenced by intercropped wheat varieties
in YM-47>YM-42> MY-29.
110
Plant nitrophily drives plant productivity and plant-microbial interactions
Barbara Pivato*1, Hugues Busset2, Florence Deau2, Annick Matejicek2, Philippe
Lemanceau2, Laurent Philippot2, Delphine Moreau2
1
UMR Agrécologie - INRA Dijon, France, 2INRA, UMR1347 Agroécologie, France
The interest for plant interactions, especially between crop and weed species, in agricultural
situations has increased with the pressure to reduce the use of chemical inputs. Thus, a better
knowledge is required in plant traits mediating the issue of plant competition and how this
competition is impacted by the environment.
Nitrophily is a plant trait referring to plant habitat, ranging from oligotrophic species mostly
found in soils with low N content to nitrophilic species better adapted to soils with high N
content.
We hypothesize that nitrophily impacts (i) the issue of plant-plant interactions, through
competition for N and light and (ii) plant associations-rhizospheric microbial community
interactions. We can expect that microbial community from nitrophilic/oligotrophic roots
association is (i) different from community associated to the same plants cultivated alone,
and (ii) differently impacted according to the association type.
To test these hypotheses, three nitrophilic and two oligotrophic graminaceous species (one
crop plant and four weeds) were cultivated in mono- or bi-specific associations in a soil
supplemented or not with N. The issue of the plant-plant interactions was assessed by
checking their productivity through biomass measures. The impact of the different plant
combinations on total bacterial communities was characterized by A-RISA (AutomatedRibosomal Intergenic Spacer Analysis) fingerprinting.
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The biomass was significantly higher in nitrophilic than in oligotrophic species when grown in
N supplemented soil. Bi-specific associations showed different root biomass compared to
monocultures in both N conditions.
Genetic structure of rhizospheric bacterial community differed according to the N level. In
higher N availability, bacterial communities structures were very similar, whereas in N limiting
conditions, the rhizosphere of bi-specific associations harbored different bacterial
communities in comparison to monocultures.
These results suggest that nitrophilic plant trait is a driver of plant productivity via plant-plant
competition, and this affects rhizospheric microbial community in N limiting conditions.
111
From pots to plots: Hierarchical trait-based prediction of population biomass in
a mesic grassland
Thomas Schröder-Georgi*1, Christian Wirth2, Karin Nadrowski3, Sebastian T. Meyer4,
Liesje Mommer5
1
University Leipzig, Biology, Germany, 2University of Leipzig, Institute of Biology /
German Centre for Integrative Biodiversity Research (iDiv), Germany, 3University of
Leipzig, Institute of Biology, Germany, 4Technische Universität München, Department of
Ecology and Ecosystemmanagement, Germany, 5Wageningen University, Nature
Conservation and Plant Ecology group, Netherlands
Plant functional traits are powerful tools when it comes to the prediction of plant performance
and ecosystem functioning. For a long time ecologists focused on aboveground traits but
widely ignored root traits because their sampling and measurement is expensive and labourintensive. A number of newer studies have shown that root traits link not only to
belowground (e.g. N cycling) but also to aboveground functions (e.g. biomass production).
So far it has never been tested, how the importance of root traits for plant performance
depends on the hierarchical level (individual vs. population). We hypothesized that root traits
are more important on the population level than on the individual level, due to an increase in
process complexity.
We used univariate and multiple regression analyses to test the importance of 35 root-, leafand stature traits for the prediction of individual and population biomass production of 59
European grassland species.
We found that traits of all three clusters (root, leaf and stature) correlate with both individual
and monoculture biomass. The most parsimonious multiple regression model significantly
improved for both, individual and monoculture biomass, when root traits are included.
However, whereas root traits improved the individual biomass model by only 3% they
improved the monoculture biomass model by 28%.
Root traits are important for both, individual and population performance but the
significance of root traits increases with increasing hierarchical level.
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112
Research on rhizosphere soil phosphorus fractions and availability in a maizesoybean relay intercropping system
Chun Song*1, Xia Xiao2, Lu Mao2, Min Xu2, Taiwen Yong3, Wenyu Yang3
1
Sichuan Agricultural University, China, 2Institute of Ecological and Environmental
Sciences, College of Resources and Environment, Sichuan Agricultural University,
China, China, 3Key Laboratory of Crop Ecophysiology and Farming System in Southwest
China, College of Agronomy, Sichuan Agricultural University, China, China
Intercropping is a traditional and sustainable cultivation model based on ecology principle of
plant-plant interaction and plant-soil feedback between different biological species. However,
the knowledge of crop rhizosphere soil phosphorus forms transformation and availability
under intercropping system is still limited. This research focused on the effect of root
interaction on crop aboveground biomass, rhizosphere soil phosphorus availability in a
maize-soybean relay intercropping system by treated maize and soybean root system with
root separation by polyvinyl chloride (PVC) clapboards or without root separation in PVC
boxes. Two seedlings of maize and four seeds of soybean were planted per PVC box, and the
distances between maize and soybean plants were 60cm. Soluble chemical reagents of
CO(NH2)2, KH2PO4 and K2SO4 were applied as N, P and K fertilizer. We collected plant and soil
samples in maize silking and maturity stages, and soybean branching, early flowering and
maturity stages separately, and then determined the crop aboveground biomass and
rhizosphere soil phosphorus fractions, to clarify the mechanism of rhizosphere soil
phosphorus fractions transformation and bioavailability in maize-soybean relay intercropping
system. The plant biomass data showed that soybean aboveground biomass were
significantly higher in treatments without root separation. The phosphorus fractionation
results showed that treatments without root separation significantly improved the
rhizosphere soil NaHCO3-Po and NaHCO3-Pi content in maize silking stage, whereas
decreased NaHCO3-Po content in soybean branching stage, and the NaHCO3-Pi content in
soybean early flowering stage. These results indicated that maize had stronger competition
for phosphorus uptake than soybean during maize and soybean co-growth stage. Residue P
content of maize rhizosohere soil in maturity stage considerably higher than soybean
maturity stage, which indicated that soybean had higher soil phosphorus use efficiency. In a
word, maize-soybean relay intercropping can improve soil phosphorus use efficiency by root
interaction, so as to improve crop yield.
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113
Root priority effects: Are root-root interactions in grasslands significantly
affected by who interacts with whom and who arrives first?
Vicky Temperton*1, Philipp von Gillhaussen2, Marco Brendel3, Marc Faget4, Stephan
Blossfeld3
1
Plant Sciences (IBG-2), Forschungszentrum Jülich & Leuphana University
Lüneburg, Germany, 2Bayreuth University, Germany, 3Plant Sciences (IBG-2),
Forschungszentrum Jülich, Germany, 4University of Louvain, Germany
Whilst we all know that the timing of interactions between plants has important implications
for plant performance, we still know relatively little about how community assembly (either
aboveground or belowground) is affected by who arrives first. In a large field experiment we
tested the possible role of priority effects of by sowing either grasses, legumes or nonlegume forbs five weeks before the other two functional groups, at the same time as testing
effects of sowing a low or a high diversity seed mixture. Intriguingly, the species richness of
the mixture did not have much of an effect on aboveground biomass, whereas the arrival
time of each functional group had a very significant effect on aboveground as well as
belowground productivity. When legumes were sown before forbs and grasses, community
root productivity was significantly lower during the first growing season than when grasses
were sown first. A repeat measurement of root turnover using in-growth cores in 2014 (data
pending) will show whether this pattern was stable and created a priority effect underground
or not.
Which species interact can also affect rhizosphere pH. In a controlled experiment combining
maize with bean, we measured rhizosphere pH and found that if these two species were
grown together variation in pH was lower than when each species was growing alone. We
also tested whether there were species-specific differences in the extent to which grass
species experienced nitrogen facilitation when interacting with the legume Trifolium repens
and this also in presence of an invasive non-legume forb Senecio inaequidens. The grass
Holcus lanatus benefitted the most from growing near the legume, but pots with Festuca
pratensis in had highest root biomass and less pronounced facilitation effects. Overall our
data show that plant root performance is clearly affected by the identity and functional traits
of the neighbouring plants.
114
Characteristics of phenolic acids exuded by roots in wheat and faba bean
intercropping
Jingxiu Xiao*1, Yi Zheng2, Li Tang1
1
Yunnan Agricultural University, China, 2Southwest Forestry University,Yunnan
Phenolic acids play important role in rhizosphere process, and few works have been cited on
the effects of intercropping on phenlic acids exudation. Wheat and faba bean is an important
planting pattern in southwest of China due to its significant yield advantages and diseases
resistance. Hydroponic culture was conducted and the exudates form the roots were
collected and examined by HPLC to investigate the effects of wheat and faba bean
intercropping (W//F) on the phenolic acids exudation. The results showed that the three
171
phenolic acids were identified in root exudates: ρ-hydroxybenzoic, vanillic and syringic acid in
both mono and intercropping. Intercropping decreased total amounts of phenolic acids in
root exudates by 39.0%-70.1% and 37.56 -57.79% respectively, in comparison with that of
mono cropping wheat (MW) and faba bean (MF). The maximum amount of phenolic acids
exuded by root was found on 35d after transplanting in both mono and intercropping
treatments, and exudation rate tended to decrease with crop growth from35d to 85d. During
whole crops growth stages, intercropping significant decreased exudation rate of ρhydroxybenzoic and syringic acid by 41.06%-100% and 37.89%-64.35% respectively in
comparison with that of MW. In W//F, vanillic and ρ-hydroxybenzoic acids were not detected
from root exudates on 35d and 55d after transplanting respectively, which were detected till
to85d in mono cropped wheat and faba bean.
172
Root Turnover
115
Root decomposition along a grassland plant diversity gradient
Hongmei Chen*1, Arthur Gessler2, Hans de Kroon3, Michael Scherer-Lorenzen4, Liesje
Mommer5, Christian Wirth1, Alexandra Weigelt1
1
University of Leipzig, Germany, 2Swiss Federal Institute for Forest, Snow and Landscape
Research WSL, Switzerland, 3Radboud University, Netherlands, 4University of
Freiburg, Germany, 5Wageningen University, Netherlands
Decomposition of plant litter is a key process for carbon cycling and nutrient availability in
ecosystems. However, we know very little about decomposition of root compared to leaf
litter, especially in the light of biodiversity-ecosystem functioning relationships. To elucidate
how plant diversity affects root decomposition in grassland and to disentangle the effects of
root litter quality from environmental factors (including both abiotic and biotic aspects), three
decomposition experiments using a litter-bag approach were conducted at the Jena
Experiment, Germany, on 80 plots differing in species richness (1, 2, 4, 8, 16 species): 1)
decomposition of plot-specific roots in their origin plots to assess plant diversity effects; 2)
decomposition of plot-specific roots in a common-garden plot to assess root litter quality
effects; and 3) decomposition of standard roots (from Lolium perenne) in all plots to assess
general environmental effects. The litter bags were installed in April 2014 and retrieved 1, 2
and 4 months afterwards to trace the decomposition progress. Mass loss was determined as
a measure of decomposition rate.
With increasing plant diversity, decomposition of plot-specific roots decreased both in their
origin plots and in the common-garden plot, while decomposition of standard roots did not
change along the plant diversity gradient. These patterns remained constant over time.
Moreover, in the common-garden plot, there was a negative effect of the presence of grasses
on root decomposition, which increased along the diversity gradient. Our results suggest: 1)
that the overall effect of plant diversity on root decomposition is negative; 2) that this effect
is primarily due to changing root litter quality rather than environmental factors; and 3) that
grasses have a substantially higher negative effect on root litter quality and thus
decomposition compared with other functional groups.
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Root Turnover
116
Fertilization and root presence of permanent grassland have no effect on
microbial biomass and mineralization
Christoph Knoblauch*1, Conor Watson2, Nicole Wrage-Mönnig3, Rolf Becker4, Clara
Berendonk5, Florian Wichern2
1
Rhine-Waal University of Applied Sciences, Faculty of Life Sciences, Germany, 2Faculty
of Life Sciences, Rhine-Waal University of Applied Sciences, Germany, 3Faculty of
Agricultural and Environmental Sciences, University of Rostock, Germany, 4Faculty of
Communication and Environment, Rhine-Waal University of Applied Sciences, KampLintfort, Germany, 5Chamber of Agriculture North Rhine-Westphalia, Kleve, Germany
The release of nitrogen (N) from soil organic matter (SOM) and plant residues is an important
source of available N in permanent grassland. Added inorganic fertilizer can result in an
alteration of root biomass and therefore also influence the input and turnover of SOM.
However, there is a lack of knowledge about interactions of inorganic fertilization and
mineralization in grassland soils. The objectives of the current study were to (i) measure the
carbon (C) and N mineralization of grassland soils, (ii) to quantify microbial biomass (MB) and
(iii) to monitor the effect of roots on C and N mineralization. We initiated a short-term
incubation experiment with varying organic and inorganic fertilizer rates and the presence or
absence of course roots and measured the effects on C and N mineralization and MB. Soil
was taken from a fertilizer field trial on permanent grassland (Lolium perenne L.) and visible
roots were removed manually. During incubation, we measured soil respiration at least once
a week for a period of 35 days. Inorganic N and MB were determined at the beginning and
end of the experiment. The presence of roots did not significantly affect the mineralization
processes within the contrasting fertilizer rates. However, inorganic N tended to increase with
a higher application rate of the two fertilizer types. Neither cumulative soil respiration nor MB
was affected by fertilizer type or application rate. Yet, we determined a hysteresis effect of
the pre-equalized water content on soil respiration. Especially at the beginning, water content
concealed the importance of fertilizer rate in terms of soil respiration. In conclusion, the
heterogeneity of the taken samples within each fertilizer treatment predominated over the
amount and type of fertilizer or presence and absence of roots.
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Long live the roots! Characterizing root lifespan in a grassland biodiversity
experiment
Natalie Oram*, Liesje Mommer, Frank Berendse, Jasper van Ruijven
Nature Conservation and Plant Ecology, Wageningen University and Research
Centre, Netherlands
Since the discovery of the positive plant diversity-productivity relationship in grasslands, the
mechanisms underlying this relationship have become central research questions. It is wellknown that greater plant species diversity results in higher standing root biomass. However,
whether this is the result of increased root production or decreased root mortality is largely
unknown. Yet, understanding the dynamics of root biomass is crucial with respect to carbon
and nutrient cycling. Increased root production could lead to enhanced organic matter inputs
into the soil, whereas increased root lifespan may actually slow down these inputs, leading to
different soil C and N dynamics in the long term. An increasingly positive plant-soil feedback,
as well as changes in microclimate and nutrient uptake that parallel high plant diversity may
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Root Turnover
lead to a more favourable environment for roots. We hypothesize that root lifespan will be
positively correlated with species richness; greater root lifespan in diverse plots will
contribute to the observed increase in standing root biomass. We test this hypothesis in a
long term experimental grassland biodiversity experiment in Jena. We aim to quantify root
lifespan in a novel and more precise way using repeated imaging of roots through
minirhizotrons and quantification of root decomposition with a litterbag experiment. Root
morphology and standing root biomass will also be measured. Imaging started in March,
2015, one year after the minirhizotrons were installed. Preliminary results will be presented.
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Effect of nitrogen form, pH and plant species in the mobilization and acquisition
of P from a recycled phosphorus fertilizer
Ana Alejandra Robles Aguilar*1, Vicky M. Temperton2, Stefan Blossfeld2, Nicolai David
Jablonowski2
1
Forschungszentrum Jülich GmbH, Germany, 2Forschungszentrum Jülich GmbH, Institute
für Bio- und Geowissenschaften, Germany
World phosphorus resources are limited. Therefore recycling of phosphorus from waste
materials is important, and struvite (MgNH4PO46H2O) is a common precipitate recovered
from waste water treatments or during anaerobic digestion of manure. Our approach is to
evaluate how mobilization of phosphorus may differ when two different species (narrow–
leaved Lupin and Maize) are grown in an acidic or alkaline sand with phosphorus added as
either struvite or as superphosphate. Nitrogen was applied as ammonium or nitrate as an
important factor that could affect phosphorus availability by changing soil pH. The
parameters to evaluate the mobilization of phosphorus from struvite were phosphorus
uptake, phosphorus present in the soil and comparison of plant performance within the
different treatments.
Lupines are capable of symbiotically fixing atmospheric nitrogen, as well as to release
phosphate-mobilizing carboxylates. These two traits make lupines good candidates
forstudying nutrient mobilization in the rhizosphere. In order to observe if citrate (mimicking
root exudates) was able to make the P from the struvite more available compared with water,
our study also included a flushing experiment with citrate in columns filled with two different
sands of acidic and alkaline pH mixed with struvite. We predict that lupine growing on
alkaline sand will have better access to phosphorus in struvite (than on acidic sand) due to its
ability to acidify the rhizosphere via exudation of carboxylates. Thus we expect lupine to have
higher biomass when growing on alkaline sand with an ammonium supply.
119
Plant presence reduces root decomposition rate of non-legume species
Sirgi Saar*1, Marina Semchenko1, Janna M Barel2, Gerlinde DeDeyn2
1
Tartu University, Estonia, 2Wageningen University, Netherlands
Litter nutrient concentrations are important traits for litter decomposition, which generally
proceeds faster with increased nutrient concentrations in the litter. However, the presence of
living roots can affect decomposition rates due to a priming effect by releasing root exudates
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Root Turnover
which can give saprotrophic microbes an energy boost, enabling them to degrade the litter
faster. To test whether and how plant presence affects decomposition of roots with different
traits, we used dead roots of seven species (3 grasses, 3 legumes, 1 forb) as litter material and
measured the litter mass loss after 8 weeks of incubation in soil with or without a white clover
(Trifolium repens) plant. We expected that the decomposition rate would increase in the
presence of a living plant, especially in the case of nitrogen-rich litter, because root exudates
can provide a source of easily available carbon and stimulate microbial activity while
subsequent nitrogen release from the litter could further enhance microbial activity and thus
decomposition. On the contrary, we found that the decomposition rate of grass and forb
roots decreased in the presence of living roots, while the decomposition rate of legume roots
was not significantly affected by the presence of a living plant. Our results show that root
decomposition rate can be slowed down in the presence of a living plant, but that this effect
depends on the properties of the decomposing roots, with reduction being pronounced in
root litter poor in N and P but not in the relatively nutrient-rich legume root litters.
120
Consequences of plant kin recognition for microbial soil feedback and root
decomposition
Marina Semchenko*1, Anu Lepik2, Sirgi Saar2
1
University of Manchester, United Kingdom, 2Department of Botany, University of
Tartu, Estonia
Recent studies have shown that plants are able to detect the genetic identity of their
neighbours. Some species proliferate roots when grown next to unrelated individuals but
apparently avoid direct competition with kin by reducing root growth. Neighbour recognition
can be mediated by root exudates and involve complex changes in root growth and
morphology. The consequences of such behaviour for ecosystem functioning are as yet
unknown. In this study, we examined the consequences of kin recognition for microbial soil
feedback and root decomposition. In the conditioning stage, plants of Deschampsia
caespitosa were grown in groups of either siblings or unrelated individuals from the same
population. In the feedback stage, new seedlings were planted into soil conditioned by either
siblings or non-siblings. Conditioned soil included dead roots, allowing estimation of root
litter decomposition during the feedback stage of the experiment.
We found that microbial soil feedback for seedlings at early stages of development tended to
be more negative on soil previously occupied by siblings. However, soil feedback did not differ
between sibling and non-sibling soils at later stages of development. We also found evidence
for significantly slower decomposition of root litter obtained from plants grown in groups of
siblings compared with groups of unrelated individuals. Roots of plants that grew with siblings
also had a significantly higher C:N ratio. This may explain slower root decomposition and
indicate increased investment into pathogen defence at the expense of competitive ability
when interacting with kin.
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Root Turnover
121
Phosphorus availability and its interaction with plant belowground carbon,
nitrogen and phosphorus input into Oxisol
Pierre Stevenel*1, Astrid Oberson1, Samuel Abiven2, Idupulapati M. Rao3, Emmanuel
Frossard1
1
ETHZ - Institute of Agricultural Sciences, Switzerland, 2University of Zürich Department of Geography, Switzerland, 3International Center for Tropical
Agriculture, Colombia
Phosphorus (P) deficiency is an issue for a large part of tropical soils worldwide. Low P
availability in Oxisols is mainly due to strong P sorption and often limits plant growth. This
feature affects the carbon (C) cycle by influencing its capture by plants, and the input of
belowground C contained in roots and rhizodeposition. Moreover, low P availability
influences nitrogen (N) input in soil by limiting symbiotic N2 fixation by legumes. Additionally,
plants have developed strategies to cope with P deficiency, such as root system extension or
exudation enhancement, which also determine belowground organic matter input. However,
a quantitative understanding of combined root and rhizodeposition C and N inputs in
response to P availability has not yet been studied. In this project, we want to investigate the
role of P availability on the regulation of belowground C, N and P inputs, and its impact on
the turnover of these inputs in soil nutrient pools over time.
Experiments will be performed with two tropical plant species: the legume Stylosanthes
guianensis and the grass Brachiaria decumbens. Plants will be grown in an Oxisol along a
gradient of plant available P. A promising tri-isotopes (13C, 15N, 33P) labeling method will be
used to quantify belowground C, N, P inputs. The isotopic composition and isotope recovery
in shoots, roots and root free soil will be determined. Following this experiment and after
removing the aboveground biomass, nutrient turnover will be studied in soils incubated
during three months. The incorporation of plant belowground input of C, N and P into soil
pools over time will be determined with the isotopic composition.
This project will provide a better understanding of the influence of P availability on C and N
inputs to soil but also strong foundations on the long term storage of those nutrients in soils.
122
Root activity and dormancy in forest communities along an elevational gradient
Yan Wang*1, Zhun Mao2, John Kim3, Christophe Jourdan4, Herve Ray4, Alexia Stokes3
1
Université Montpellier 2, France, 2c IRSTEA, UR EMGR, 2 Rue de la Papeterie, BP 76,
38402 Saint-Martin-d’Hères Cedex, France, France, 3INRA, UMR AMAP, Boulevard de la
Lironde, 34398 Montpellier Cedex 5, France, France, 4CIRAD, UMR Eco&Sols – Ecologie
Fonctionnelle & Biogéochimie des Sols & Agroécosystèmes (Montpellier SupAgroCIRAD-INRA-IRD), 2 Place Viala, 34060 Montpellier, France, France
Belowground processes in plant communities drive the sequestration of carbon into soil, but
how these processes are modified by climate remains largely unexplained. Elevational
gradients are unique tools for studying the responses of communities to climatic variability in
situ.
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Root Turnover
We installed rhizotrons in contrasting forest communities at 1400 m, 1700 m and 2000 m. We
measured the number of growing roots and elongation every month over 4 years. Results
showed that mean daily root elongation rate (RER) was driven by soil temperature between
0-8°C, outside which extreme soil temperatures perturbed growth. RER peaked in springtime,
and a smaller peak was sometimes observed in autumn. Cumulated root length was not
significantly different between altitudes, although the growing season was significantly
shorter at 2000 m. However, when winter snow cover was > 6 months, root growth was
severely limited the following year. Root longevity was dependent on altitude and the season
in which roots were initiated; roots emitted in the autumn lived significantly longer than
those initiated in springtime. Root diameter was a significant factor explaining much of the
variability. Few differences in root demography between contrasting plant communities
within each altitude were found, and were explained by air or soil temperature.
We conclude that at high altitudes and when extreme abiotic events are not restrictive to
carbon supply, more resources are invested belowground in a shorter period of time,
revealing a plastic response to climatic variables within a community.
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Root Turnover
BIOFECTOR
123
Single and tripartite biofectors for tomato (var. Mobil) upscaled from pots to
plots
Borbala Biro*, Zita Szalai, Anita Dudás, Tamás Gáspár, Heléna Wass-Matics, Zsolt
Kotroczó
Corvinus University of Budapest, Hungary
Living and non-living bioeffectors are beneficial treatments of organic and sustainable
agricultural practices. It is a question if single or 2-3-types of microbes in one particular
product is the most necessary or how to successfully upscaling the beneficial effects from
pots to the fields?
Tomato Solanum lycopersicon Mill. ’Mobil’ was used in the experiment. Bioeffector products,
as BE1: Trichoderma harzianum T-22 and BE-4: Hungarian Trichoderma TDM, including
Azotobacter and Azospirillum N2-fixers, BE2: Pseudomonas sp., BE3: Bacillus amyloliquefaciens
Rhizovital 42 F1 were applied on the bases of the supplier’s recommendations. The plants
were grown in 2.5 kg soil/pot in 4 replicates. TSP (0,62g), Rock-phosphate RP (1,59g),
Patentkali (1,67g) and Calcinit (1,46g) were applied at the pots and 1200 kg/ha from both at
the field. BE application was performed both at sawing and at the time of plantation. Growth
of tomato, shoot and root biomass was assessed and general soil characterization, including
MPN counts of bioeffectors in tomato rhizosphere. Results were evaluated by statistical
probes.
Upscaling was resulting the similar beneficial effect at Bacillus (BE3) bioeffector, with greater
variability and less improvement at the field. Shoot biomass production was less variable
parameter than the root or fruit biomass, more particularly also among the rather changeable
field condition. Combined tripartite microbial inoculations seem to have better beneficial
influence, then the only single bioeffector treatments in general. Effect of BE1 Trichoderma
inoculation was improved for instance in combination with free-living and associative
Nitrogen-fixers, due to the relatively high P-content (430 mg/kg) of the slightly humous
sandy soil. Plant microbe interaction is largely dependent on the soil nutrient-status and the
balance among nutritive elements.
124
Soil application of seaweed extracts and micronutrients to improve the cold
stress tolerance in maize during early growth
Klara Bradacova*1, Nino Weber2, Markus Weinmann2, Günter Neumann2
1
University of Hohenheim, Institute of Crop Science (340h), Germany, 2University
Hohenheim, Institute of Crop Science (340h), Germany
Low soil temperature in spring is a major constraint for cultivation of tropical crops in
temperate climates, associated with impaired seedling development, inhibition of root
growth and activity.
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In this study, we tested the potential of commercial bio-effector products such as seaweed
extracts and rhizobacteria (strains of Bacillus and Pseudomonas) with plant growth promoting
potential to improve the tolerance of maize to low root zone temperatures during early
growth. Maize (v. Colisee) was cultivated in pots containing 2 kg of a fully fertilized silty-loam
soil (pH 6.8). In order to control the root zone temperature (RZT), pots were installed in a
cooling system. After germination at 22 °C, the cold stress phase (12-14 °C) started at 14 days
after sowing to simulate a cold-period in spring. Bioeffectors were supplied close to the
plants with a dosage of 109 CFU kg-1 soil for bacteria and 0.017 g kg-1 soil for seaweed
extracts at 0, 2 and 4 weeks after sowing.
In two independent experiments, positive BE effects on plant growth and particularly on root
development at low RZT were exclusively detected for seaweed extracts with high Zn/Mn
contents and similar growth promotions were induced by sole application of Zn and Mn in
comparable amounts. This finding suggests that also the seaweed extracts were mainly acting
via improved Zn and Mn supply to the plants. Assays of different oxidative stress markers
showed that the beneficial effect of Zn/Mn treatments was associated with increased
superoxide dismutase (SOD) acitivity in the root tissue, playing a key role in antioxidative
defense mechanisms. In this context, Zn/Mn supplementations may effectively improve cold
stress tolerance of maize by their function as enzymatic co-factors particularly for SOD.
Accordingly, formation of necrotic leaves in cold-stressed plants was associated with a low
Zn-nutritional status.
125
The combination of humic substances and inoculation with different
microorganisms enhances the growth of maize (Zea mays L.)
Vincenza Cozzolino*, Hiarhi Monda, Daniele Todisco, Davide Savy, Giovanni Vinci,
Alessandro Piccolo
University of Naples Federico II, Italy
Plant biostimulants, or bioeffectors (BEs), include diverse substances and rhizosphere
microorganisms that enhance plant growth, contributing to improve fertilizer use efficiency,
enhancing nutrient uptake, and increasing root health through competition with root
pathogens. The well-known direct effect of humic substances (HS) on root growth has
produced an expanding market for HS extracted from various composted green wastes, that
represent a more sustainable source than those obtained from expensive fossil matrices, such
as lignites or peats.
The aim of this work was to evaluate the ability of these new biotechnological tools to
promote the growth of maize plants in soil with low P availability. A pot trial was performed
with the following BE treatments: 1. B0 (no inoculation), 2. B2, Pseudomonas sp plant growth
promoting bacteria (Proradix), B3, Bacillus amyiloliquefaciens PGP (Rhizovital 42); MYC
Funneliformis mosseae and Rhizophagus irregularis, commercial inoculum of arbuscular
mycorrhizal fungi (Aegis); HA, humic acid (HA) from composted artichoke wastes. We
evaluated the benefits of each BE alone or in combination. The P addition to soil comprised:
P0 (no P addition), and CM (composted cow and buffalo manure at 50 mg P/kg soil). The
impact of P fertilization, inoculation and HS were evaluated on biomass production and P and
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N content. The plants were grown in pots filled with 5 kg of substrate, prepared using an
alkaline clay-loam soil and harvested 8 weeks after sowing.
The benefits of the combined action of compost, microorganisms and HS were evident.
Inoculation with the bioeffectors enhanced plant growth, as well as N and P content in
shoots, especially in treatments where each microorganisms were mixed with HA. This new
generation of biological products based on HS and selected beneficial microorganisms
provides opportunities for an effective biological support of a sustainable crop production.
126
Combinations of rhizosphere-competent fungal and bacterial isolates improve
nutrient acquisition and growth in maize and tomato
Joerg Geistlinger*1, Markus Weinmann2, Borbala Biro3, Alessandro Piccolo4, Shekhar
Sharma5, Rainer Borriss6
1
Anhalt University of Applied Sciences, Germany, 2University of
Hohenheim, Germany, 3Corvinus University of Budapest, Hungary, 4University of Naples
Federico II, Italy, 5Agri-Food & Biosciences Institute, United Kingdom, 6ABiTep
GmbH, Germany
BIOFECTOR (7th EU Framework Programme, grant agreement 312117) works on “an improved
understanding and utilization of biological processes supporting soil fertility”, essentially on
the soil microbiome and bioactive natural compounds interacting with the root system
and/or mutual or symbiotic microbes, termed bio-effectors (“BEs”). Here, we report on the
results of Work Package 2: BE combinations and synergisms. Ten partners from 5 EU countries
agreed on a corporate standardized experimental design for greenhouse and field trials with
tomato and maize as model crops. Local soils received basal fertilization (low in P) and were
enriched with standard BEs (BE1= Trichoderma harzianum (TRIANUM-P™), BE2=
Pseudomonas spec. (PRORADIX™), BE3= Bacillus amyloliquefaciens (RHIZOVITAL™)),
combinations thereof and in combination with newly developed fungal and bacterial strains
as well as extraction products from seaweed and compost. Main findings after 2 years of
research were in maize: (1) combining standard BEs1-3 in a loamy top soil with low P
availability has no positive effects, but growth-promotion was achieved by combining BE2
with seaweed extracts. (2) BEs1-3 have no effects on cold stress induced necrotic leaf areas
but necrosis could be completely avoided by combining Zn/Mn micronutrients with seaweed
extracts. (3) Composted cow manure with the combination of BE3 and humic acid extracted
from compost yielded 65% more dry weight biomass. (4) Cultivar/strain-specific effects
between different maize cultivars and Trichoderma strains were confirmed by qPCR. In
tomato (1) the combination of Trichoderma with associated N-fixing bacteria had positive
effects on yield, (2) the combination of Trichoderma and Gram+ bacteria (Bacilli) doubled
yield, (3) early application of Piriformospora indica (Sebacinales) caused positive seedling
development and (4) co-colonization of tomato roots by different Trichoderma strains was
proven by molecular marker application. Currently new combinatorial BE-products are
developed on the bases of Trichoderma, seaweed extracts rich in micronutrients, Bacilli and
N-fixing bacteria.
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127
Influence of bioeffectors application on maize growth and yield
Martin Kulhanek*, Pavel Tlustoš, Jiří Balík, Zlata Holečková, Jindřich Černý
Czech university of life sciences in Prague, Czech Republic
Phosphorus will become probably the limiting nutrient in near future. The sources of suitable
rock phosphates for phosphorus fertilizer production are restricted. Therefore, the
improvement of phosphorus acquisition from less available soil forms is needed. One of
these ways is the bioeffectors (BEs) application. The aim of this study was to confirm, that the
BEs have a positive influence on soil phosphorus mobilisation leading to better P uptake by
plants and their higher yields and quality. In this study, the data from pot experiment were
evaluated. Three BEs – Trianum (Trichoderma harzianum), RhizoVital (Bacillus
amyloliquefaciens) and Proradix (Pseoudomonas sp.) in different combinations with rock
phosphate and triple superphosphate were tested on maize plants. The soils from two sites
(Humpolec and Lukavec) with low P level and diluted with sand (2:1 soil:sand) were used for
the experiment. Plant height, shoot and root dry mass yield, and P plant contents were
measured. The average plant height was significantly higher at all treatments fertilized with
triple superphosphate in first two months of the experiment. Thereafter, at harvest after 15
weeks of growth, significant differences among plant heights disappeared. The above ground
biomass dry weight was significantly higher at the treatments fertilized with triple
superphosphate only at Lukavec site. The significant influence of BEs on plant height and
above ground dry weight was not confirmed. The root dry weight was slightly, but not
significantly higher at the treatments without P fertilization. On the contrary, the lowest root
dry mass weight was measured at the triple superphosphate treatments. From the obtained
data is possible to conclude, that used bio-effectors did not significantly influenced the plant
height and root and shoot dry biomass amount weight as well.
128
Effect of bio-effective microorganisms on early Maize root development and P
uptake under low temperature
Jonas Duus Stevens Lekfeldt*, Beatriz Gomez Muñoz, Jakob Magid, Lars Stoumann
Jensen, Andreas de Neergaard
Maize is a rapidly expanding crop in Northern Europe, but emergence and establishment can
be challenged by low soil temperatures in early spring. Microbial inoculants have been
demonstrated to stimulate root development, and may help overcoming initial water and
nutrient stress, caused by poor root development. However, if soil nutrient levels are low,
early investment of plant biomass in root development may not be a successful strategy from
a nutrient acquisition point of view. We tested the ability of selected microorganisms with
known root stimulation potential (Penicillium sp; Bacillus sp) and humic acids on maize root
development in soils of low and medium P availability at 10 and 16 degrees. The soil used
was sandy loam from a long-term depletion trial, one with very low P availability, as well as a
moderate P treatment, fertilized with animal manure for the past 10 year. 25% sand was
mixed into the soils to enable root re-isolation at harvest, and avoid water-logging.
Plants were destructively harvested after 5 and 8 weeks, and analysed for shoot and root
biomass, plant P in root, shoot and remaining in the seed. Root architecture parameters from
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entire isolated roots were assessed using WinRhizo software. Soil extractable P and pH was
measured at planting and harvest.
Results from the ongoing trial will be presented at the conference.
129
Microbial bio-effectors combined with manure proliferate plant growth of
tomato: investigations on the modes of action
Zhifang Li*1, Justus Riemann1, Angelika Lüthi1, Nino Weber1, Markus Weinmann1,
Gheorghe Poşta2, Günter Neumann1
1
University Hohenheim, Institute of Crop Science (340h), Germany, 2Banat’s University of
Agricultural Sciences and Veterinary Medicine, Faculty of Horticulture and
Forestry, Romania
When tested under practice conditions, commercial bio-effector preparations induced strong
growth improvements (80 % increase in plant height) of tomato seedlings grown on a
substrate with composted cow-manure (45 %), garden soil (30 %), peat (15 %), and sand (10
%), but without mineral fertilizers. To study the modes of action how these products based
on (Bacillus strains FZB42 and R41, Pseudomonas sp. DSMZ 13134 and Penicillium sp. PK 112)
isolates could improve plant growth, pot experiments under controlled conditions were
conducted.
The question whether the plant growth-promoting effect was due to mobilization of
sparingly available mineral nutrients from the humified manure was studied in comparative
tests where the portion of manure was replaced by an increased share of peat in the
substrate while adding mineral nutrients in easily available forms or not. The role of manure
as a carbon source supporting the establishment and activity of the introduced microbial
strains was tested through substitution by alternative carbon sources. Root morphological
characteritics and colonization by arbuscular mycorrhizal (AM) fungi were assessed to detect
if an improved spatial acquisition of mineral nutrients could explain the beneficial effects of
the bio-effectors.
Contrary to the strong effects observed under practice conditions, weak or no growth
responses to the bio-effector treatments were found in plants under controlled conditions.
However, the Pseudomonas treatment induced a significant increase in root hair length. The
biomass production of tomato plants grown on the substrate with manure raised by 9 % in
response to the combined application of the Penicillium preparation with an AM inoculum
(Glomus intraradices). Plant growth-promotion by microbial bio-effectors, therefore, the
combination of diverse modes of action seems to be involved, whose relative importance
may vary depending on the environmental conditions.
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BIOFECTOR
130
The effect of biomass ashes and bioeffectors on soil solution composition
Filip Mercl*, Václav Tejnecký, Petra Hubová, Pavel Tlustoš, Pavla Ochecová, Jiřina
Száková
Czech University of Life Sciences in Prague, Czech Republic
Biomass ash, the waste material coming from biomass combustion, is generated in large
quantities worldwide. Mineral nutrients, bound in ash, are usually poorly plant-available,
therefore there is an effort to enhance their availability using Bioeffectors (BEs). Objectives of
this study was to i. assess the influence of biomass ash on soil solution composition and ii.
investigate BEs ability to enhance availability of nutrients from ash in soil-plant system.
In the pot experiment, plants of wheat (Triticum aestivum L.) and maize (Zea mays L.) were
grown in BE inoculated mixture of soil and biomass ash under outdoor precipitationcontrolled conditions. Two different types of ash were used (straw and wood ash), and two
different BEs were tested (BE3 - Rhizovital® and BE4 – Bacto_Prof). Soil solution was sampled
using suction cups (MacroRhizons, Rhizosphere Research Products B.V., Netherlands) from
the root zone of plants five times during the vegetation period. In samples of soil solution,
concentrations of phosphorus (P) and major low-molecular-mass organic acid (LMMOA)
anions (acetate, formate, lactate, oxalate, pyruvate) were determined. The yield of grain and
their P content were also determined.
Results showed different influence of ashes on soil solution composition. Application of straw
ash significantly increased P concentrations in soil solution throughout the vegetation period.
Both types of ashes did not differ in the influence on the grain yield, but straw ash significantly
increased P content in wheat grains. We observed no significant influence of tested BEs on
parameters listed above. Changes in relative and total amount of individual LMMOAs after ash
addition were registered. BE4 significantly changed oxalate content in soil solution for one
treatment (wheat + straw ash) in early stage of plant development (27 days after planting).
131
Humic, fulvic and water-soluble organic fractions from different compost
structure-bioactivity relationship revealed by chemical-biological
characterization
Hiarhi Monda*, Vincenza Cozzolino, Riccardo Spaccini, Alessandro Piccolo
The influence of humified organic matter on soil fertility has led to a growing interest in the
use of humic substances (HS) as soil amendments, revealing their ability to positively affect
biochemical processes underlying plant growth and nutrition. An ecologically based approach
to farm management consist in the use of compost, recognized as a natural method for
recycling organic wastes, and widely used as soil conditioner and fertilizer. In this study we
combine the bright side of both technologies by comparing three different organic extracts,
humic acids (HA), fulvic acids (FA) and water-soluble organic matter (WOM), from
composts made out of different biomass wastes: tomato (C1), artichoke (CYN), and artichokefennel (CYN+F). We conducted bioactivity assay on maize seedling with the humic extracts,
which were thoroughly characterized. All the extracts generally increased the whole plant
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biomass and positive effects were observed on root length and chlorophyll content. The
molecular composition assessed by CPMAS-NMR spectroscopy and pyrolysis-GC-MS of all
original composts appeared generally similar, whereas that of the HA and FA extracts varied
somewhat as by hydrophobic and hydrophilic components. However, all HA and FA extracts
were generally low in aromatic compounds, while they were abundant in biolabile
compounds, such as carbohydrates and lipids. The hydrophobic/hydrophilic ratio seemed to
play an important role in determining their bioactivity. Moreover, the biological assay pointed
out that the larger the content of carbohydrates and sugar-like compounds, as well as nonlignin aromatic components, and the smaller the amount of phenols groups, the greater was
the biological/chemical effects exerted by HS. These results confirm the role of humified
organic matter in stimulating plant growth, especially when the content of potentially
bioavailable hydrophilic components is predominant.
132
Supplementation of silicon in maize (Zea mays) to improve P acquisition and
AMF symbiosis efficiency
Narges Moradtalab*1, Markus Weinmann2, Roghieh Hajiboland3, Günter Neumann2
1
University of Hohenheim, Institute of Crop Science (340h), Germany, 2University
Hohenheim, Institute of Crop Science (340h), Germany, 3University of Tabriz, Iran
Phosphorus (P) deficiency is a major limiting factor for plant growth and development. Based
on preliminary observations in strawberry (see: Moradtalab et al., this issue), we hypothesized
that an optimum level of silicon (Si) supplementation improves arbuscular mycorrhizal (AM)
colonization and P acquisition in maize.
In this work, the effects of Si supplementation (as K2SiO3) and a commercial silicone fertiliser
(Actisil, Yara, ) on AMF colonization, symbiosis efficiency and P availability were studied in
maize (Zea mays L. cv. Colisee) plants grown in organic farming soil under greenhouse
conditions with additional AM inoculation. Concentrations of 6, 100, 1000 and 10000 mg
Si/kg soil (as K2SiO3 by adding to the soil) and 6 mg Si/kg soil of commercial Actisil were
applied to the 3 L pots. Application of Actisil was performed either as start application or by
weekly irrigation. No P fertilizer was added to the soil within first 4 weeks but after
appearance of stronger deficiency symptoms, a moderate P fertilisation was performed with
50 mg P kg-1 soil.
Si-supplied plants showed less P deficiency symptoms and growth stimulation especially at
application rates of 6 and 100 mg kg-1 as compared with plants without Si and 1000 and
10000 mg/kg Si. Application of 10000 mg kg-1 Si exerted detrimental effects on plant growth
and the plants died after 5 weeks application. Start application with Actisil was more effective
than weekly fertigation. Our results indicated an optimal level of Si application at 6 mg Si/kg
soil. Evaluation of AM colonisation is currently on the way.
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133
Effects of silicon supplementation in drought-stressed strawberry plants in the
presence or absence of arbuscular mycorrhizal fungi
Narges Moradtalab*1, Roghieh Hajiboland2, Markus Weinmann3, Günter Neumann3
1
University of Hohenheim, Institute of Crop Science (340h), Germany, 2University of
Tabriz, Iran, 3University Hohenheim, Institute of Crop Science (340h), Germany
Drought stress seriously affects plant growth and development. In this work the effects of
silicone (Si) supplementation (3 mM, as Na2SiO3) were studied in strawberry (Fragaria ×
ananassa var. Parus) plants.
Three levels of irrigation (field capacity, 0.75 field capacity, 0.35 field capacity) and two levels
of inoculation with arbuscular mycorrhizal fungi (G. Clarum) (‒AMF, +AMF) were applied in a
pot experiment and plants were cultivated for 6 weeks under growth chamber conditions.
Plant dry matter production and protein content decreased in drought-stressed plants
associated with significant reduction of net photosynthesis. In response to drought, soluble
carbohydrates and proline were accumulated in the leaves up to 2 and 10 folds, respectively.
Application of Si and AMF significantly increased shoot and root dry weight and relative
water content not only in drought-stressed but also in well-watered plants. Both Si and AMF
treatments significantly increased leaf photosynthesis rate, protein and relative water content
and decreased leaf accumulation of osmolytes and malondialdehyde. Combined Si and AMF
treatments exerted stronger effects on the improvement of physiological and biochemical
parameters of drought-stressed plants than single applications, associated with significantly
higher AMF root colonization of +Si treated plants. Our results suggest a beneficial effect of Si
on yield, photosynthesis and water relation parameters as well as AMF responsiveness in
strawberry plants.
134
Improved P acquisition in crops by inoculation with P-solubilising
microorganisms - vision or reality?
Guenter Neumann*1, Mira Kuhlmann2, Nicole Probst3, Mehdi Nkebiwe2
1
University of Hohenheim, Germany, 2University of Hohenheim, Institute of Crop Science
(340h), Germany, 3nicole.probst.88@gmail.com, Germany
Mobilisation of sparingly-soluble phosphates via pH modifications of the growth medium
and release of chelating metabolites are widespread mechanisms for P acquisition in many
plant growth-promoting microorganisms (PGPMs) cultivated on artificial media and is
promoted also as a measure to improve P acquisition in crops. However, within the
BIOFECTOR-Project, more than ten experiments in eight countries with three different crops
and nine P-solubilising microorganisms failed to show any crop benefit via acquisition of
sparingly-soluble P sources. Therefore, this study aimed to characterise critical factors,
determining microbial mobilisation of sparingly-soluble P sources in the rhizosphere.
Maize was used as test crop with low adaptive potential for root-induced P-solubilisation. A
calcareous Loess sub-soil, extremely low in available P (2-3 ppm) and organic matter (0.1%)
but rich in acid-soluble Ca-P was supplied (1) with or (2) without rock phosphate fertilisation
and (3) with soluble P as positive control. Plants inoculated with 6 different P-solubilising
microorganisms (Pseudomonas sp. Poradix®, Pseudomonas sp., Paenibacillus mucilaginosus,
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Bacillus subtilis, Streptomyces spp., Penicillium sp.) were cultivated on this substrate, ensuring
that plant P-acquisition in treatments (1 and (2) was only possible after microbial Ca-P
solubilisation. However, all microbial inoculants failed to stimulate P-acquisition of the test
plants and even exerted inhibitory effects on plant growth. Also simultaneous fertigation with
a mixture of glucose and glycine had only marginal effects on crop performance, suggesting
that limitation of root exudates as C and N source was not a critical factor. However,
successful P acquisition and plant growth promotion was observed after diluting a similar
growth substrate with 75 % (w/w) washed quartz sand, drastically lowering the pH buffering
capacity and the CaCO3 concentration for unspecific binding of P-solubilising chelators.
These findings suggest that the soil-buffering capacity may be a major constraint which limits
the P-solubilising potential of many PGPMs.
135
Placement of Pseudomonas sp. PRORADIX around NH4+-based fertilizer depots
in maize stimulates root exploitation of the fertilizer depot
Peteh Mehdi Nkebiwe*1, Markus Weinmann2, Günter Neumann2, Torsten Müller3
1
University of Hohenheim, Plant Nutrition: Fertilisation and Soil Matter Dynamics
(340i), Germany, 2University of Hohenheim, Plant Nutrition: Nutritional Crop Physiology
(340 h), Germany, 3University of Hohenheim, Plant Nutrition: Fertilisation and Soil
Matter Dynamics (340 i), Germany
Combining fertilizer placement and inoculation with plant growth-promoting rhizobacteria
that stimulate root-growth and solubilize phosphates may be a strategy to improve root
exploitation of fertilizer depots. Moreover, root colonization by such rhizobacteria may be
enhanced in the depot zone with intense root development as a consequence of increased
availability of root exudates induced by localized supply of root-attracting nutrients like
ammonium.
After confirming that the rhizobacteria strain Proradix®WP (Pseudomonas sp. DSMZ13134,
Sourcon Padena, Germany) grows normally at high ammonium concentrations (50 mM) in
the presence or absence of the nitrification inhibitor 3,4-Dimethylpyrazole phosphate, we
investigated the effect of placing concentrated ammonium sulfate (64 mg N ml-1) with 3,4Dimethylpyrazole phosphate as a fertilizer depot in combination with PRORADIX as inoculant
on growth-stimulation of maize roots (Zea mays L.), rhizosphere pH changes, root
colonization by PRORADIX, nitrogen (N) and phosphorous (P) uptake in a rhizobox
experiment on a calcareous Loess subsoil, pH 7.6 and in a field experiment on silty loam, pH
7.1. In the rhizobox experiment, increased root length densities around the NH 4+-depot were
recorded 8 weeks after sowing, associated with intense root surface and rhizosphere
acidification by 2 pH units and to higher N and P uptake per plant compared to the control
variant with homogenous NO3 - fertilization. This may be attributed to improved spatial
nutrient acquisition and to mobilization of acid-soluble calcium-phosphates, which are
usually formed in neutral to alkaline carbonate-rich soils. Particularly in the depot zone, a
high root colonization density of Pseudomonas was detected by culturing root extracts on a
selective nutrient medium. In the field, root length density was doubled in the ammoniumdepot zone inoculated with PRORADIX at 11 weeks after sowing, as a first indication of
improved root exploitation of the fertilizer depot, induced by simultaneous rhizobacteria
inoculation under field conditions.
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136
No matter whether dead or alive? An investigation of bio-effector application in
tomato and maize experiments under phosphorus limited soil conditions
Dinah Reinhardt*1, Marie Spohn2, Sven Marhan1, Namis Eltlbany3, Kornelia Smalla3,
Ellen Kandeler1
1
University of Hohenheim/Institute of Soil Science and Land
Evaluation, Germany, 2University of Bayreuth/Department of Soil
Ecology, Germany, 3Julius Kühn-Institute/Institute for Epidemiology and Pathogen
Diagnostics, Germany
Bio-effectors are viable organisms and active natural compounds that are able to promote
plant growth and health. This ability is enabled, for instance, by mobilizing sparingly soluble
mineral nutrients like phosphorus. Particularly plant growth promoting rhizobacteria (PGPR),
such as strains from genera Pseudomonas, Bacillus and Rhizobium are among the most
powerful phosphate mobilizers but the functional mechanisms are still poorly understood. In
the frame of the BioFector project we focus on the effects of phosphorus solubilizing
rhizobacteria. We hypothesize: (I) Plant growth promoting effects of PGPR are based on an
increasing phosphatase activity (especially acid phosphatase) and here we assume a higher
activity in rhizosphere than in bulk soil; (II) PGPR colonization will be displayed in
phosphatase activity hotspots. Two rhizobox experiments (tomato and maize) under
phosphorus limited soil conditions were performed using Pseudomonas jessenii as the bioeffector. Experiments comprised five control treatments, including inoculation with native soil
microorganisms and dead Pseudomonas jessenii cells, respectively. During plant cultivation a
soil in situ zymography for determination of spatial and temporal phosphatase activity was
conducted. Results showed a significant plant growth promoting effect, but contrary to our
expectation, in treatments with living as well as dead Pseudomonas jessenii cells. In both
treatments we observed yields that nearly corresponds to the optimally phosphorus fertilized
plants; 79% in plant height and 83% in biomass, for example. First evaluation of zymography
indicates a higher acid phosphatase activity in rhizosphere than in the bulk soil across all
treatments. A particularly high activity was revealed in maize, especially on root tip surface.
Additional analyses will identify whether the improved plant growth is based on an increasing
nutrient availability and uptake. In addition, we will analyse the cell components of lysed P.
jessenii to identify potential plant growth promoting compounds.
137
Max-Res TGA and FTIR evaluation of plant growth promoting rhizobacteria
David Nelson, Eugene Carmichael, Graham McCollum, JR Rao, Gary Lyons, Shekhar
Sharma*
Agri-Food Biosciences Institute, United Kingdom
Analysis of bacterial biomass provides valuable information on the cell contents and
structural differences in cell wall polysaccharides. The objective is to develop rapid methods
for evaluating plant growth promoting rhizobacteria (PGPR) by Fourier Transform Infrared
spectroscopy (FTIR) and thermogravimetric analysis (TGA) maximum resolution (Max-Res)
protocol.
Bacterial strains (BM- Bacillus mucilaginosus, FBZ42- B. amyloliquefaciens A, RLH – B. subtilis
S, 4.2 – Burkhoweria sp, H2.6/RC2.5 – Rahnella aquatalis) were inoculated in potato dextrose
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broth for 4 days and incubated on a rotary shaker at 160 rpm for 48 h at 25oC. After removing
the supernatants by centrifugation at 6000g for 10 min, the bacterial pellets were washed
three times with distilled water and lyophilized. FTIR spectra of the samples were obtained
using a diamond ATR. TGA was carried out at a heating rate of 20oC/min in nitrogen. TGA
heating rates for maximum resolution (Max Res) analysis were programmed at
20oC / min when no weight loss was detected and a heating rate of 5oC / min on detection of
weight loss.
The six isolates can be distinguished from the differences in infrared functional groups and
TGA composition data of the isolates. Max-Res analysis of the isolates showed compositional
differences between the six isolates as indicated by differences in the weight loss steps.
138
Development of Quality Assurance protocols for biostimulant products
Shekhar Sharma*, Eugene Carmichael, Eugene Conlon, Trevor Martin, Chris Selby
Plant biostimulants are usually formulated using a range of components including macroalgae, animal or plant proteins, amino acids, minerals and plants extracts containing a wide
range of trace compounds. The objective is to develop quality assurance (QA) protocols for
evaluating physico-chemical characteristics of biostimulants.
Six formulations (Rygex, Algavyt, Ryzoset, Manek, Ecoryg and Algavyt Zn/Mn – Agriges
products) containing algal/plant extracts, humic and amino acids, lipids and inorganic
components were assessed for particle size distribution, zeta potential and auxin activity. The
particle size range of biostimulant products can be reduced by employing a wet mill and
micro-fluidiser.
The particle sizes of Rygex, Algavyt, Manek and Ecoryg were less than Rizoset and Algavyt
Zn/Mn. Among the six products, Algavyt and Manek showed the highest zeta potential (ZP)
values. The formulations were assessed for auxin activity using a mung bean rooting bioassay
(cytokinin bioassay result not presented). Measurements of total bean roots per cutting and
root length demonstrated the presence of significant auxin activities in some formulations
(e.g. Rygex and Ecoryg). The gross differences in the particle size, ZP and composition of the
products could be used for developing formulations to improve field performance and
stability during transportation and storage.
139
Field performance of barley treated with two biostimulant products
Trevor Martin, Fiona Clarke, Chris Selby, Eugene Conlon, JR Rao, Shekhar Sharma*
Two types of arable fields were selected on the basis of their nutrient status: (a) standard
input field – regularly applied with inorganic fertiliser to maintain the soil nutrient status for
good plant growth and (b) low input field - not treated with fertiliser during the past ten
years, with the exception of nutrient input grazing animal waste. The objective is to evaluate
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BIOFECTOR
if certain biological extracts can influence the yield of spring barley in either a standard input
soil or a soil of low nutritional value.
Two formulations of biostimulants supplied by Bioatlantis, Ireland (A039F249 and D002G138)
were assessed for biological activity and evaluated as foliar treatments. Spring barley was
sown at both sites (standard nutrient input soil and low input soil) in randomised blocks.
Treatment applications at three rates were applied during the growth of the crop. Mung bean
bioassays was carried out to monitor growth hormone activities of the products to ascertain
their biostimulant potential. Yields and 1000 grain weights were assessed at harvest.
The bioassay of the products using mung bean root extension data showed that two
products (A039F249 and D002G138) are biologically active. The application of D002G138
increased the number of mung bean roots, while both products at concentrations 1 and 5
gml-1 initiated greater root length. Field trials with spring barley at the two concentrations
showed differing levels of chlorophyll between sites but no significant differences in either
the yield of grain or the 1000 grain weight.
140
Response of cold and heat stressed Arabidopsis and wheat plants on the
expression of salicylic acid and jasmonic acid
Colin Fleming, Thomas Fleming, Colin McRoberts, Stewart Floyd, Shekhar Sharma*
The impact of cold and heat stresses on the productivity of arable crops is expected to be a
major factor worldwide in reducing yields by 10 to 30% . This study was aimed at developing
tools to monitor key biotic and abiotic stress related plant hormones such as salicylic acid
and jasmonic acid in wheat and Arabidopsis plants. The objective is to monitor salicylic acid
(SA) and jasmonic acid (JA) expression in Arabidopsis and wheat plants in response to cold
and heat stress.
Heat and cold stresses (4oC and 38oC for 18 hrs) were applied to plants in growth cabinets. SA
and JA levels were measured directly using Q-TOF-LC/MS. Total mRNA was extracted and
cDNA produced before RT-PCR, targeting the PR1 SA pathway gene and MYC2 JA pathway
gene.
The conclusions are (a) heat and cold stress significantly affected the concentration of SA and
JA in wheat and Arabidopsis plants (b) heat and cold stress changed expression levels of wheat
genes in the SA and JA pathways and (c) these models can be used to monitor the effects of
stress modifying materials on monocotyledons and dicotyledons.
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141
Evaluation of anion exchange membrane for monitoring of phosphate
solubilisation in soil types
Chris Selby, Eugene Carmichael, Eugene Conlon, Shekhar Sharma*, JR Rao
It is relatively easy to identify soil bacteria capable of solubilising phosphorous by plating
them on microbiological media containing an insoluble phosphate (P) and observing rings of
media clearing around active colonies. The objective is to estimate phosphorous
solubilisation in soils using an anion exchange membrane (AEM) protocol.
A method was evaluated to confirm if these organisms including AFBI P solubilisers and
Porodix are equally active in agricultural soils. The method involved using sections of AEM
(1.5 x 4 cm) to act as “artificial roots” by placing them in soils for a given period then
recovering them to assay (molybdate reactive P) captured PO4 3- ions.
The conclusions are (a) AEMs can be used to assess the availability of P to plants in
agricultural soils and give a good correlation with estimates found using conventional
chemical analyses of soils; (b) AEMs can be used to distinguish P availabilities between soils
and effects of supplementation of soils with various P salts; (c) No significant increased P
solubilization occurred with either the AFBI P solubilisers or Porodix regardless of P
supplementation or soil type and (d) Future work will focus on why the bacterial inoculae do
not appear to be solubilising P in soils. In the first instance this will involve providing
additional supplements such as increased organic matter or organic compounds common in
root exudates (eg glutamic and aspartic acids and their amines) to provide the bacteria with a
more readily available energy source.
142
Evaluation of ryegrass extracts for plant bio-stimulant activities
Chris Selby, Eugene Carmichael, Eugene Conlon, Shekhar Sharma*
Plant fibres are generally regarded as being the major product of the bio-refining of grasses.
However, this process also produces large volumes of aqueous extract that is potentially
useful to the agricultural and horticultural industries. Here we compare the plant defence
elicitor activity (French bean cell suspension cultures) and auxin-like activity (mung bean
hypocotyl rooting test) of extracts produced from high pressure screw-pressing with those
prepared by homogenising foliage in water for. The objective is to evaluate if ryegrass
extracts have the potential to act as crop bio-stimulants by assessing their biological activity
in a series of bioassays.
Two growth hormone (auxin and cytokinin) bioassays were used to compare screw-pressed
juice with extracts prepared by homogenising foliage with water. In both assays, all six
extracts were equalised to the lowest sugar content sample, as assayed using the anthrone
reagent, by dilution with water.
Extracts of perennial ryegrass contained potent elicitors of plant defence that caused rapid
and intense browning of cultured French bean cell suspensions. This is indicative of the
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synthesis of phenolic compounds, many of which have phytoalexin activity. Extracts produced
by screw-pressing were marginally but significantly more active than those produced by
homogenisation of tissues in water. This was illustrated by the significant interaction between
extraction method and treatment concentration. Differences between ryegrass varieties were
small and insignificant, particularly if attempts were made to equalise sample strengths using
a measure such as sugar concentration. Root induction (a) and growth (b) in hypocotyl
cuttings of mung bean indicate that the ryegrass extracts are rich in “auxin-like” or auxin cofactor activity. Studies are in progress to characterise the extract components responsible for
the biological activities reported here.
143
Quantitative tracing of two Pseudomonas strains in the roots and rhizoplane of
maize, as related to their plant growth-promoting effect in contrasting soils
Carla Mosimann1, Thomas Oberhänsli1, Dominik Ziegler2, Dinah Reinhardt3, Ellen
Kandeler3, Thomas Boller4, Paul Mäder1, Cécile Thonar*1
1
FiBL (Research Institute of Organic Agriculture), Switzerland, 2Mabritec
AG, Switzerland, 3University of Hohenheim, Institute of Soil Science and Land
Evaluation, Germany, 4Zürich-Basel Plant Science Center, University of
Basel, Switzerland
Plant growth-promoting rhizobacteria (PGPR) are able to facilitate plant nutrient acquisition
and can act as biocontrol agents by suppressing soil-borne diseases. Efficient strains can be
formulated as microbial inoculants and their successful use for field application often requires
a certain ability of the strains to survive in the soil where they are inoculated. In this respect,
there is a need to create tools enabling the tracing of inoculated PGPR which can also serve
to monitor their spread in space and time.
In this poster we describe the development and application of a molecular method allowing
the quantitative detection of two Pseudomonas strains (Pseudomonas fluorescens Pf153 and
Pseudomonas sp. DSMZ 13134) contained in commercial formulations. The method is based
on a Taqman qPCR assay targeting two polymorphic regions of the bacterial genome in order
to ensure the specificity of the detection. Inoculation experiments with maize were conducted
in three contrasted soils. Eight weeks after planting and inoculation, Pf153 could still be
detected and its persistence in root and rhizoplane was shown to be the best in the organic
soil (versus the two other conventional soils). On the other side, DSMZ 13134 could not be
detected anymore after 8 weeks but its persistence after 4 weeks in root and rhizoplane
samples was equally good in the three tested soils. Combined with the plant responses, our
data indicate that the persistence of the two strains cannot clearly explain the measured plant
biomass and nutrient acquisition (nitrogen and phosphorus) due to inoculation of the two
strains. The discrepancy observed between the level of colonization by the strains and their
effects on plant indicate that more research is needed to elucidate the mechanisms and
conditions leading to successful application of PGPR. The developed tools will definitively
contribute to this objective.
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144
Metabolites Profiling of Zea mays inoculated with the Bacillus
amyloliquefaciens as Bioeffector
Vincenza Cozzolino, Giovanni Vinci*, Hiarhi Monda, Daniele Todisco, Alessandro
Piccolo
Bacillus amyloliquefaciens represents the largely studied plant growth-promoting
rhizobacteria (PGPR) that competitively colonize plant roots and can act as either
biofertilizers or antagonist biopesticides or both. PGPR enhance growth and yield of cereals
through mechanisms that include production of growth stimulating phytohormones,
solubilization and mobilization of phosphate and induction of plant systemic resistance to
pathogens. For this reasons, PGPR represent biofertilizers well recognized as efficient tool for
sustainable and safe agriculture.
In this study, we investigated the effects of Bacillus amyiloliquefaciens when inoculated on
plants of Zea mays under different P amendments. In order to improve our understanding we
determined the metabolic profiling followed by multivariate analysis. Gas chromatography–
mass spectrometry (GC-MS) was employed to screen potential differences among metabolic
leaf extracts. A number of 56 primary polar metabolites, comprising amino acids, organic
acids, sugars, phenolic acids, amino sugars, sugar alcohols, and 7 unknown compounds, were
identified. The PCA showed a robust reproducibility among five replicates, revealed a
significant separation between control and treatments with Bacillus a. Moreover, the different
P amendments (no P addition, triple super phosphate, rock phosphate, composted cow
manure, composted horse manure) showed a substantial difference in plants metabolic
profiles. A significant variance from control was found for plants undergone to both Bacillus
a. inoculation and compost treatment, whereby the difference in metabolic profile was
accounted by a large abundance of sugars and organic acids metabolites.
These results indicated that the different P amendments and the presence of Bacillus
amiloliqefaciens strongly influenced the presence and distribution of primary metabolites.
However, more detailed analyses of the metabolome (secondary metabolites) will better
elucidate how Bacillus a. affect the metabolic pathways and consequently the performance of
maize plant.
145
Bioeffectors promoting Bioeffectors - Seaweed extracts as prebiotics for plant
growth-promoting rhizobacteria?
Nino Weber*1, Markus Weinmann1, Peteh Mehdi Nkebiwe2, Kristin Dietel3, Sarah
Symanczik4, Günter Neumann1, Uwe Ludewig1
1
University Hohenheim, Institute of Crop Science (340h), Germany, 2University
Hohenheim, Institute of Crop Science (340i), Germany, 3ABiTEP
GmbH, Germany, 4FiBL, Switzerland
A major constraint for a successful practical application of plant growth-promoting
microorganisms is the limited reproducibility of the desired effects, depending on often
unknown external factors. One important determinant for effective application of microbial
bioeffectors is their rhizosphere competence and effective root colonization of the host plant,
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frequently promoted by repeated inoculations with high inoculum densities. However, under
practical conditions, this approach is not always economic. Using the prebiotic potential of
various natural compounds such as seaweed extracts to stimulate microbial growth may be
an alternative strategy.
Our in vitro studies with bacterial suspensions of low CFU showed highly prebiotic potential of
selected seaweed extracts on strains of Bacillus amyloliquefaciens and Pseudomonas sp.
“Proradix” which could not be attributed to carbon source effects as demonstrated by control
treatments with glucose.
In a pot experiment with maize, combinations of Proradix® and seaweed extracts led to
significantly higher plant biomass compared to the untreated control, even on a well
fertilized field soil. Based on these results, promising product combinations were tested in a
field experiment with maize on a silty loam (pH 6.8) and fertilization according to farmers
practice. For tracing of the bacterial strains in the field selective plating assays and RT-qPCR
with specific primers were employed. Plant sampling at four weeks after sowing revealed no
effect of the selected seaweed extracts (Superfifty, Algafect) either on root colonization of
Proradix or on the total cell number of B. amyloliquefaciens. However, the proportion of
Bacillus spores was increased by Algafect. No significant effects on biomass production and
final grain yield of maize were observed. The results suggest that seaweed extracts may exert
prebiotic effects on plant growth-promoting bacteria but the characterization of conditions
determining positive interactions with the host plant still needs further investigation.
146
Strategies for bio-effector application to improve the growth and mineral
nutrition of maize under field conditions
Markus Weinmann*1, Mehdi Peteh Nkebiwe2, Nino Weber1, Klara Bradacova1, George
Fora3, Torsten Müller2, Günter Neumann1
1
University Hohenheim, Institute of Crop Science (340h), Germany, 2University
Hohenheim, Institute of Crop Science (340i), Germany, 3Banat’s University of
Agricultural Sciences and Veterinary Medicine, Romania
For many bio-effectors, such as plant growth-promoting microorganisms, the ability to
support the mineral nutrition of plants has been proven under controlled conditions.
Mobilization of mineral nutrients, stimulation of root growth and activity, and beneficial
interactions with other microorganisms are among the most noticeable modes of action.
These properties could be effectively utilized for the development of more environmentally
friendly and sustainable plant nutrition strategies that are less dependent on mineral
fertilizers. A critical task is to meet the demand of high yielding crops for phosphate and
other essential minerals that are sparingly supplied by the soil or from alternative fertilizers.
However, the agricultural implementation of such approaches is still limited and this is likely
due to a lack of appropriate application techniques ensuring the functioning of bio-effectors
under adverse environmental conditions.
In the present work, commercial preparations of bacterial (Bacillus strains FZB42 and R41,
ABiTEP GmbH, Berlin, Germany; Pseudomonas sp. DSMZ 13134, Sourcon Padena GmbH & Co.
KG, Tübingen, Germany) and fungal (Penicillium sp. PK 112, Bayer CropScience Biologics
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GmbH, Malchow/Poel, Germany) bio-effectors were tested with different application methods
to improve the phosphorus nutrition of field grown maize.
No significant improvements in growth or mineral nutritional status of the plants were found
when the bio-effectors were applied by seed incrustation, broadcast distribution with soil
incorporation, or spray application on top of the plants. These findings suggest that the
application of these microorganisms as discrete measures is not decisive for the productivity
of maize on fertile soils. Advanced application strategies, such as the combination of bioeffectors with fertilizer placement and/or the use of recycling fertilizers are proposed to
provide viable alternatives to the current practice of mineral fertilization.
147
Microbial bio-effectors combined with manure proliferate plant growth of
tomato: investigations on the modes of action
Zhifang Li1, Justus Riemann1, Angelika Lüthi1, Nino Weber1, Markus Weinmann*1,
Gheorghe Posta2, Günter Neumann1
1
University Hohenheim, Institute of Crop Science (340h), Germany, 2Banat’s University of
Agricultural Sciences and Veterinary Medicine, Faculty of Horticulture and
Forestry, Romania
When tested under practice conditions, commercial bio-effector preparations induced strong
growth improvements (80 % increase in plant height) during the early development of
tomato seedlings grown on a substrate prepared with composted cow manure (45 %),
garden soil (30 %), peat (15 %), and sand (10 %), but without mineral fertilizers. To study the
modes of action how these products based on bacterial (Bacillus strains FZB42 and R41,
Pseudomonas sp. DSMZ 13134) and fungal (Penicillium sp. PK 112) isolates could improve
plant growth, pot experiments under controlled conditions were conducted.
The question whether the plant growth-promoting effect was due to mobilization of
sparingly available mineral nutrients from the humified manure was studied in comparative
tests where the portion of manure was replaced by an increased share of peat in the
substrate while adding mineral nutrients in easily available forms or not. The role of manure
as a carbon source supporting the establishment and activity of the introduced microbial
strains was tested through substitution by alternative carbon sources. Root morphological
characteritics and colonization by arbuscular mycorrhizal (AM) fungi were assessed to detect
if an improved spatial acquisition of mineral nutrients could explain the beneficial effects of
the bio-effectors.
Contrary to the strong effects observed under practice conditions, weak or no growth
responses to the bio-effector treatments were found in plants under controlled conditions.
However, the Pseudomonas treatment induced a significant increase in root hair length. The
biomass production of tomato plants grown on the substrate with manure raised by 9 % in
response to the combined application of the Penicillium preparation with an AM inoculum
(Glomus intraradices). In plant growth-promotion by microbial bio-effectors, therefore, the
combination of diverse modes of action seems to be involved, whose relative importance
may vary depending on the environmental conditions.
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Food-web Interactions
148
Carbon flow and enzyme activities during protozoan grazing in rhizosphere and
detritusphere
Sebastian Löppmann*1, Fionn Clissmann2, Anna Gunina1, Johanna Pausch1, Robert
Koller3, Michael Bonkowski2, Yakov Kuzyakov1
1
University of Göttingen, Büsgen-Institute, Germany, 2University of Cologne, Institute of
Zoology, Germany, 3Forschungszentrum Jülich, Institute of Bio- and
Geosciences, Germany
The differences in complexity and accessibility of plant provided carbon (C) sources for the
soil food web result in two major decomposition pathways based on 1) root and shoot litter,
and 2) rhizodeposits (especially exudates). The amount and quality of substrates entering the
soil mainly controls microbial processes in the rhizosphere and detritusphere. Furthermore,
soil fauna has important functions in regulating microbial activity and enzymatic substrate
utilization.
A triple-labeling (13C, 14C and 15N) experiment was conducted focusing on the identification of
C resources (rhizodeposited C by 14C and root litter by 13C) that fuel microbial-protozoan
interactions in two soil hotspots: rhizosphere and detritusphere.
Soil was taken from an arable field planted with maize, autoclaved and re-inoculated with a
microbial community extracted from the same soil. The following treatments were
established: 1) no addition of plant C, 2) addition of sterilized 13C /15N labeled maize root
litter, representing detritusphere 3) 14CO2 pulse labeling of growing maize plants,
representing rhizosphere.
To determine the effects of faunal grazing on nutrients (by 15N) release each treatment was
setup with and without amoeba. Enzyme kinetics was implemented as indicator for microbial
activity and 13C flux to the microbial pool was determined by 13C-PLFA analysis. We
analyzed14C, 13C and 15N in the microbial biomass, soil, plant, root and CO2. Additionally,
microbial biomass was assigned by dsDNA extraction.
Between 65-89% of soil released 14CO2 was respired by roots and microorganisms in the first
3 h after the 14CO2 pulse. First results showed higher 14C activity in the microbial biomass in
presence of protozoa. The dsDNA-extracted microbial biomass was significantly higher in
litter amended and planted soil compared to the unplanted control. Furthermore, the DNA
contents were increased in the presence of protozoa. Clear effects of enzymatic affinity to the
specific substrate were elucidated during protozoan grazing.
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149
Litter decomposition and home-field advantage of range-expanding plant
species
Marta Manrubia Freixa*, Ciska Veen, Wim van der Putten
The current climate warming enables many native plants to expand ranges to higher altitudes
and latitudes. Plants develop in close interaction with soil organisms in a direct (e.g. via
pathogenesis) and indirect way (e.g. via the detritus food web). During range shifts, these
specific plant-soil interactions might become temporally disrupted since soil organisms have
limited dispersal capacity. Consequently, range-expanding plants are expected to benefit
from the release of soil-borne pathogens and suffer from losing positive interactions with
specialized decomposer organisms. The present research aims to study local specialization by
decomposers of range-shifting plant species along latitudinal gradients and determine
whether specialization results in ‘home field advantage’.
We tested the hypothesis that soil microbial communities from the native range have higher
affinity with litter of range-expanding plants than communities in the new range. We
collected senesced leaves and soil of two range-expanding plants (Centaurea stoebe and
Lactuca serriola) and a native congener (Centaurea jacea) in the native and expansion range.
We set up a 97-day microcosm incubation experiment for each plant species. Litter was
reciprocally transplanted to all soils within and between ranges and we measured CO2 fluxes
over the incubation period. Soil heterotrophic respiration response to litter addition was used
as a proxy for decomposition activity.
We found that soil and litter origin had the strongest effect on soil heterotrophic respiration
for all 3 plant species, whereas no local specialization effect was observed in litter transplants
within or between ranges. High within-range variability suggested that litter decomposition
controls operate at a highly local scale.
150
A root-feeding insect in the shallow soil zone significantly alters yield, mortality,
and size structure of Lolium perenne populations
Tomonori Tsunoda*1, Naoki Kachi2, Jun-Ichirou Suzuki2
1
Tokyo Metropolitan University / Yokohama National University, Japan, 2Tokyo
Metropolitan University, Japan
We evaluated the effects of vertical distributions of a larva of a root-feeding insect, Anomala
cuprea, on the yield, mortality, and size structure of Lolium perenne populations. We
conducted a growth experiment with a two-way factorial randomized block design with nine
replications. Factors included plant density (36 plants per pot, and six plants per pot) and
vertical distribution of A. cuprea. Pot soil was divided into three distinct vertical zones (top,
middle and bottom) with a wire gauze, and each pot received a larval insect into one zone.
There were two additional treatments, one with a larva but without a partition and one
without an insect. Yield of L. perenne significantly decreased in the treatments with herbivory,
with the largest decrease in the top feeding zone, i.e. the shallowest soil. Yield was also
significantly reduced at low plant density, but was more affected by herbivory. Plant mortality
occurred only when the herbivore was in the top feeding zone. Plant density did not
significantly alter the number of dead plants. At low plant density, the shoot biomass of the
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three largest plants significantly decreased with a larval herbivore, but the three smallest
plants not. Thus, the standard deviation in shoot biomass significantly decreased under
herbivory. At high plant density, shoot biomass was not significantly affected by herbivory,
irrespective of plant ranks. The standard deviation in the shoots was smaller at high density
than at low density. In contrast with aboveground herbivory, which is known to increase size
variance in plant populations, the root-feeding of an insect in shallow soil decreased the size
variance in a sparse plant population.
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Top-down and bottom-up control of generalist root-feeding nematodes in the
rhizospheres of range-expanding plant species and native congeners
Rutger Wilschut*, Stefan Geisen, Wim van der Putten
As a consequence of climate warming many plant species are expanding their range to higher
latitudes and altitudes, thereby leaving behind their rhizosphere soil organisms. It is unknown
how new rhizosphere communities become assembled in the new range. Generally, rangeexpanding plant species are less negatively affected by soil communities in the new range
than congeneric native plant species. This may be due to a lower amount of herbivores and
pathogens that are able to attack the range expanding plants in the new range, either
because of a lack of co-evolutionary history or by stronger direct or indirect defense
mechanisms.
We study how specific multitrophic interactions develop in the rhizospheres of rangeexpanding plant species. In a greenhouse experiment we exposed two range-expanding plant
species and their congeneric natives to different generalist root-feeding nematodes and
inoculated microbial communities that may contain antagonists of the nematodes.
Subsequently, we examined if nematode population growth differed between rangeexpanding and native plant species and if top-down control of nematodes by the microbial
communities was plant species-dependent. We tested the hypotheses that 1) if top-down
control of root-feeding nematodes is plant-mediated, this will be stronger in native species
than in range-expanders, as native plants have a shared evolutionary history with the local
soil community and 2) range-expanding plants are better defended against generalist rootfeeding nematodes than congeneric natives, because of strong direct defense mechanisms.
Our results suggest that these defense mechanisms are species-specific, rather than
depending on range shift capacity.
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Engineering the Rhizosphere
152
Severe rhizosphere oxygenation optimising the carbon footprint of
Paludiculture // (Sustainble biomass production on peat soils)
Christian Fritz*1, Eric Visser2, Alfons Smolders3, Leon Lamers2, Florian Wichern1, Bikila
Dullo4, Theo Elzenga5, Veronica Pancotto6, Ab Grootjans5
1
Rhine-Waal University of Applied Sciences, Germany, 2Radboud
University, Netherlands, 3B-Ware Research Center, Netherlands, 4Addis Abeba
University, Ethiopia, 5Rijksuniversiteit, Netherlands, 6CADIC, Argentina
Water-logging is a major threat to plant health and causes substantial methane emissions
from crops and natural vegetation. Flood adapted plant species influence soil processes by
releasing root exudates and degradable litter. This can increase methane production and
nutrient availability. In contrast, little is known to what extent root derived oxygen caps
methane and nutrient release. Our research tested which growth conditions are necessary to
maintain oxygen release at sufficient rates to dominate rhizosphere processes.
Root-methane-soil interactions were studied by comparing methane emissions, stock and
oxygen availability in depth profiles below stands of cushion plants and paludicrops. We
followed rhizosphere nutrient-availability along depth profiles in Ethopia, Argentina and the
Netherlands. We investigated nutrient cycling by 15N field experiments and N:P ratios of plant
organs.
Cushion plants, Eriocaulon schimperi and Astelia pumila, formed extensive root systems up to
150 cm deep. Root biomass (3590 g.DW.m-²) dominated the belowground biomass of
cushion plants but resulted in a higher nutrient demand. Roots of paludicrops, Typha spec.
and Phragmites spec., were in general shallow (< 70 cm) and root proliferation was sensitive
to vertical nutrient availability. In contrast, soils surrounding cushion plant roots were
depleted in methane and plant-available nutrients. Main finding is that methane emissions
were then cut to zero high root densities promoting soil oxygen (> 1.5 mg.l-1). High soil N:P
ratios and low temperatures seemed major controls on root density and rhizosphere
oxygenation. Increase in shallow root length was associated with higher 15N recycling
Our study shows that roots of cushion plants and biomass crops can dominate
biogeochemical processes at the ecosystem scale given certain growth conditions. Oxygen
release from the dense root biomass was sufficient to cause a thorough soil oxidation. Soil
nutrient ratios seem promising in reducing greenhouse gas emissions from water-logged
fields by tuning the root system architecture.
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153
Biocontrol practices alter rhizosphere community structure in pepper and
suppress Phytophthora blight
Lori Hoagland*, Dan Egel, Natasha Cerruti, Jyothi Thimapuram, Clayton Colling, Ketaki
Bhide
Purdue University, United States
Phytophthora blight has become one of the most serious threats to the vegetable industry in
the Midwest U.S. Controlling Phytophthora capsici, the pathogen responsible for Phytophthora
blight is difficult because it has a broad host range, spreads rapidly under ideal environmental
conditions, and produces resilient spores that can survive indefinitely in soil. There is also
insufficient resistance in crop varieties, and P. capsici is now resistant to many fungicides.
Biocontrol practices that are thought to suppress soil-borne pathogens via changes in soil
microbial communities could help control Phytophthora blight, though the mechanisms are
not well understood limiting the effective deployment of these practices. In this study, we
evaluated the potential for various cover crop species grown alone, or in combination with a
biochar amendment made from hardwood forest species to suppress Phytophthora blight in
two naturally infested field soils. Changes in the rhizosphere community structure of a
subsequently planted pepper crop were quantified with semi- selective media and 16S
sequencing performed by Illumina MiSeq and analyzed using QIMME. A wheat cover crop and
biochar, alone and in combination, increased pepper root and shoot biomass, and increased
the abundance of Pseudomonas fluorescens in the pepper rhizosphere in both soils. In the low
organic matter soil, P. capsici abundance in the pepper rhizosphere was reduced most by
treatments that received the wheat cover crop, whereas in the high organic matter soil P.
capsici was reduced most by treatments that received biochar. These results indicate that
including a wheat cover crop and applying biochar can improve pepper growth in soil
infested with P. capsici, though the suppression mechanisms and most effective biocontrol
strategy could vary given soil type. Results of on-going sequencing analyses will provide
additional insights to the potential mechanisms regulating P. capsici suppression and
enhanced pepper growth observed in this study.
154
Beneficial interaction between tomato plant and Flavobacterium strains isolated
from tomato rhizosphere
Seon-Woo Lee*1, Soo Yeon Choi1, Eun Joo Jung1, Ju Yeon Song2, Jihyun Kim2
1
Dong-A University, South Korea, 2Yonsei Unversity, South Korea
Plant and microbe interaction in rhizosphere may influence various plant functions such as
plant growth, plant development and tolerance to stresses. Microbial community analysis of
tomato rhizosphere from our study revealed that members of phylum Bacteroidetes were
abundant in tomato rhizosphere compared to bulk soil. We hypothesized that those bacteria
dominating tomato rhizosphere contribute to the plant function. A pair of specific primer
targeting those bacteria in class Flavobacteria was designed and used to select bacterial
isolates in a member of the class Flavobacteria by colony PCR of bacterial colonies derived
from tomato plant rhizosphere. A number of bacteria were isolated and examined for
phenotypes relevant to plant-beneficial interaction, such as biofilm formation, seedling
growth promotion, and antimicrobial activities. Five strains were selected for further study
because they showed plant-beneficial effects. One of the isolates, F. daejeonensis RCH33,
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showed the delayed occurrence of bacterial wilt in tomato plants, when RCH33 strain was
treated in tomato rhizosphere prior to the bacterial wilt pathogen Ralstonia solanacearum
inoculation by soil-soaking method. Genomes were analyzed for all five strains in
Flavobacterium and revealed that they harbor genes for plant growth promotion, such as
auxin biosynthesis and ethylene removal, and for secondary metabolites which may be
involved in beneficial effect to plants. However, one of novel species of Flavobacterium TCH32 did not contain any known genes for beneficial interaction with plant, while plant growth
promotion effect and the capability of biofilm formation were remarkable. We are under
investigation of bacterial gene expression in planta for these Flavobacterium strains.
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The right set of circumstances – Selecting microbial bio-effectors for
applications in alternative fertilisation systems – the BIOFECTOR project
Guenter Neumann*
University of Hohenheim, Germany
Practical application of plant growth-promoting microorganisms (PGPMs) as so-called biofertilisers to improve growth and nutrient acquisition of crops is frequently biased by low
reproducibility of the desired effects. Various environmental factors including biotic and
abiotic stresses, competition with the indigenous microflora and genotypic differences in host
compatibility may limit the rhizosphere competence, the survival and thus the efficiency of
the microbial inoculants. BIOFECTOR is a collaborative project, located within the 7th EU
Framework Programme with the aim to select suitable microbial partners for applications in
various alternative fertilisation systems. It is expected that a strategic combination of
microbial strains adapted to the culture conditions characteristic for the respective
fertilisation systems will reduce the variability of host plant responses and thus the efficiency
of the plant-microbial interaction.
First promising results but also drawbacks and open questions are summarised with respect
to PGPM use for improved nutrient acquisition from organic recycling fertilisers, in fertiliser
placement strategies and mobilisation of sparingly soluble nutrients.
156
The engineered rhizosphere bacterium help plant sense a hazards chemical
Choong-Min Ryu*, Hae-Ran Lee, Soohyun Lee
KRIBB, South Korea
Synthetic biology is a new field of biological engineering that generates new biological
modules and synthesizes novel pathways to reprogram organisms. It is valuable to the
development of reporter plants to provide a rapid, low-cost, and in situ monitoring of
environmental hazards and plant diseases. The reporter plants are also useful for the
ecological risk assessment of industrial chemicals. We attempted to develop a reporter plant
that senses hazardous aromatic compounds such as toluene. Bacterial two-component signal
transduction system such as TodST of a rhizosphere bacteria Pseudomonas putida is useful to
construct artificial genetic regulatory module for synthetic biology. For developing toluene
sensing reporter plant, we exploited root-rhizosphere bacteria (rhizobacteria) interaction.
First, we generated P. putida KT2440 to produce indole-3-acetic acid (IAA) depending on
201
toluene concentrations through TodST system. Secondly, we manipulate Arabidopsis and
tobacco plant to elicit RNAi of the magnesium chelatase (ChlH) gene induced by
concentration dependent manner of IAA produced by P. putida under presence of toluene.
Collectively, we provide new two-step chemical sensing system which turn over signals from
chemical sensing rhizobacteria to plant indicator.
157
Plant-Sediment Microbial Fuel Cells: electron transfer mechanisms and their
energy efficiencies
David Strik*, Koen Wetser, Cees Buisman
Wetlands can be used to generated electricity by means of Plant-Sediment-Microbial Fuel
Cells (PSMFC). In this since 2008 emerging technology, solar energy is converted into
electricity. Basically the technology consists of: 1) an anode which is placed in the vicinity of
plants roots to take-up electrons; 2) an electric circuit to harvest energy out of released
electrons.; and 3) a cathode placed at an oxygen rich environment to deliver electrons for
oxygen reduction. Experimental work supports that electrochemical active bacteria are
responsible for catalysis of anodic oxidation and cathodic reduction reactions.
The PSMFC provides a new solid electron acceptor and donor in the wetland. Plants and
sediments both provide a wide pool of (potential) electron donors (e.g. exudates, lysates,
sulfide) and electron acceptors (e.g. oxygen, metals, nitrate, sulfate, carbon dioxide). Electron
donors can be converted and transferred via different direct and indirect mechanisms. E.g.
micro-organisms can oxidise acetate and directly deliver electrons to the anode. Also,
microbial or plant-based mediators can be used to transfer electrons from micro-organisms to
the solid electrode. Based on thermodynamics and actual conditions, one can calculate the
energetically most attractive route for electron transfer at different sites like root tips and
root-turn over locations.
Aim of this study was to determine (novel) theoretical pathways of electron transfer
mechanisms in the Plant-Sediment Microbial Fuel Cells and reveal their energy efficiency.
With this work we present the working principles of novel electron transfer pathways and
provide in-sights on effective placement of anodes and cathodes. Past experimental results
were linked to the outcome of the model. By further experimental elucidation of the
proposed model, the actual processes and efficiencies in Plant-Sediment Microbial Fuel Cells
can be quantified.
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158
Rhizogenesis: Exploring the physical development of the emerging root:soil
interface
Jonathan Helliwell1, Craig Sturrock*1, Sacha Mooney1, Anthony Millar2, Richard
Whalley3
1
University of Nottingham, United Kingdom, 2John Innes Centre, United
Kingdom, 3Rothamsted Research, United Kingdom
The rhizosphere is a distinct zone of soil directly influenced by a plant root, with all below
ground resources passing through this dynamic zone prior to capture by plants. Therefore
the physical nature of the interface between the rhizosphere and the bulk soil is crucial for
plant development. It is well known that the soil microbial community play a significant role
in the evolution of the rhizosphere and some studies have shown that it is structurally a
different environment to the bulk soil. However how this evolution or genesis is influenced by
the underlying soil physical properties and how this interacts with different plant species is
less well understood. Examining the undisturbed rhizosphere has represented a major
obstacle to research, due to its microscopic size and often fragile nature. Here we have
employed X-ray Computed Tomography (CT) to successfully map the physical architecture of
the developing rhizosphere in natural soils for the first time. We compared the temporal
changes to the intact porous structure of the rhizosphere during the emergence of a
developing root system, by assessing changes to the soil porous architecture across a range
of soil textures and plant species. Our results indicate the physical zone of influence of a root
at an early stage is more localised than previously thought (at the µm rather than mm scale).
Soil porosity increases at the immediate root surface due to localised crack formation in both
fine and coarse textured soils. As such the soil porous architecture at the root interface is
enhanced and not compacted as previously considered. This 'rhizosphere structure' and
associated dynamics have important consequences for several important root-soil processes
including water uptake efficiency and gaseous exchange between individual aggregates and
subsequently our efforts to model their behaviour.
159
Effects of crop rotation on pathogen-suppressive activity and shifts in antibiotic
activity of soilborne actinomycete communities
Patricia Vaz*1, Nora Altier2, Carlos Pérez3, Linda Kinkel4
1
INIA- Las Brujas, Uruguay, 2Sección Bioinsumos, INIA- Las Brujas, Uruguay, 3Facultad
de Agronomía, Universidad de la República, Uruguay, 4Department of Plant Pathology,
University of Minnesota, United States
Indigenous soil microbes have intrinsic potential to protect plants from pathogens, yet our
understanding of the factors that determine the dynamics of antagonistic populations remains
limited. Bacterial antibiotic production may change in the proximity of another organism, thus
not only the ability of the bacterial community to produce antibiotics but also interactions
with neighbors are likely to determine community antagonistic potential. We explored the
relationships between crop rotation and edaphic characteristics with community antagonistic
activity, and with induction of changes in inhibition among sympatric actinomycetes. Soil
samples were taken from an experiment established 13 years earlier, with
10 treatments in a randomized block design (n = 3 blocks). Bacterial, actinomycete, and
inhibitor densities were measured for all plots. Mean inhibition of actinomycetes against
203
target organisms was also determined. Three plots were selected for isolation of a random
collection of sympatric actinomycetes using a 1 cm diameter soil corer. Ten isolates from
each community were tested for their abilities to induce changes in inhibition among all
possible sympatric isolate pairs. Soils having different rotation histories had significantly
different bacterial, actinomycete and inhibitor densities. Soil communities from longer
rotations had significantly higher microbial densities than communities from shorter
rotations. Moreover, communities with higher inhibitor densities had better inhibitors. Soil
nitrate, organic matter and soluble potassium were significantly correlated with microbial
community characteristics. Inhibition among sympatric isolates was both increased and
decreased by the presence of a partner. However, no significant differences in the
frequencies of such shifts were observed among communities. Our work suggests that
agronomic practices that contribute to increasing total bacterial densities are likely to
enhance the potential for microbial communities to suppress plant pathogens. In this study,
longer rotations, with higher plant diversities over time, were most effective in achieving high
bacterial densities and strong inhibitory populations in soil.
160
Managing replant disease of apple and sweet cherry with compost
Tristan Watson*1, Louise Nelson1, Tom Forge2
1
The University of British Columbia - Okanagan Campus, Canada, 2Agriculture and
Agri-Food Canada, Canada
Replant disease refers to the poor growth of fruit trees planted into soil previously used for
tree-fruit production. The plant-parasitic nematode, Pratylenchus penetrans, and a diverse
array of root-associated fungi have been implicated in replant disease worldwide. Recent
restrictions on soil fumigants have generated interest in alternative management strategies,
particularly those associated with promotion of beneficial microorganisms in the rhizosphere.
This study aimed to investigate: (1) the impacts of amending soil with biocontrol agents and
composts on plant growth and recovery of root pathogens, and (2) differences in N
mineralization between two different composts. A greenhouse pot experiment was
performed, consisting of apple and sweet cherry seedlings grown in old apple orchard soil
amended with either a biocontrol agent, an agricultural or municipal waste compost, granular
chemical fumigant (Basamid®), or nothing (untreated control). After 19 weeks growth, the
abundance of P. penetrans was determined for each pot, and colonization by root-associated
fungi assessed through isolation and molecular identification (ITS region). Differences in soil
microbial communities were determined using Biolog EcoPlatesTM combined with real-time
PCR analyses of total bacteria. Differential nutrient mineralization was assessed using freeliving nematode community indices of nutrient enrichment as well as analysis of NO3-N
content. Fumigation and compost amendments decreased the abundance of P. penetrans
and colonization by root-associated fungi, however only fumigation and agricultural waste
compost improved root volume. Fumigation and compost amendments selected for distinct
microbial communities, with agricultural waste compost supporting a greater abundance of
total bacteria relative to non-compost amended treatments. Similarly, agricultural waste
compost also supported a greater nematode enrichment index, lower channel index, and
higher soil NO3-N content relative to municipal waste compost, suggesting enhanced Nmineralization through the bacterial decomposition pathway. Overall, agricultural waste
204
compost shows potential as a non-fumigant option for control of replant disease impacting
apple and sweet cherry.
205
Wednesday 24 June – Poster session
Symbiosis
1
Effect of Sinorhizobium sp. and phosphorus on growth, nodulation, and
nitrogen fixation of fenugreek (Trigonella foenum-graecum L.)
Abdolreza Akhgar*, Sara Larki
Vali-e-Asr University of Rafsanjan, Iran
The Legume-rhizobia symbiosis is the basis of biological nitrogen fixation and improving soil
fertility. Inoculation of legumes with rhizobia is an important practice to maximize biological
N2 fixation capacity in legume crops. Phosphorus is also an essential element for Rhizobia to
convert atmospheric nitrogen into an ammonium form usable by plants. In this study, a
greenhouse experiment was carried out to investigate effects of simultaneous application of
Sinorhizobium sp. isolates and phosphorus levels on growth, nodulation, and nitrogen
fixation of fenugreek. The experiment studied four Sinorhizobium isolates combined with four
phosphorus levels (0, 20, 40 and 60 mg kg-1soil). The Sinorhizobium isolates were isolated
from root nodules of fenugreek grown in farms located in Khuzestan and Kerman Province.
The greenhouse experiment showed that simultaneous application of Sinorhizobium and
phosphorus significantly increased shoot dry weight and nodule number and significantly
enhanced N, P, Ca, Mg, Fe and Mn uptake.
2
Plant Growth Promoting Rhizobacteria (PGPR) to benefit root architecture and
nutrition of nursery crops
Shelby Berg*, Stephen Mudge, Susanne Schmidt, Yun Kit Yeoh
The University of Queensland, Australia
A healthy root system is fundamental for plant growth and development. Plant growth
promoting rhizobacteria (PGPR) form complex symbiotic relationships with plants and benefit
plant growth in the rhizosphere, on the root surface, within intercellular spaces and root cells.
Here we investigated microbes for their ability to act as PGPR and colonise root systems of
young maize and tomato plants in the presence of native soil biota. A number of putative
PGPR isolates with strong phytate degrading and phosphorus solubilising activities were
identified of which two isolates (an Enterobacter sp. and Burkholderia sp.) were selected for
further testing. Tomato and maize seedlings inoculated with only Burkholderia or both
Enterobacter and Burkholderia, cultivated in low P soil did not show improved plant growth.
However Enterobacter inoculation led to increased shoot and root biomass, root surface area,
branching and lateral root growth of both maize and tomato seedlings. Such changes in root
architecture are desirable for nursery container production whereby roots inoculated with
PGPR remain confined within the pot and do not proliferate from the base. These results also
suggest that the Enterobacter strain, although originating from the sugarcane rhizosphere,
has a wider host range. We are currently using genetically-engineered Enterobacter
expressing green fluorescent protein (GFP) to study microbial colonisation of root systems
and survival post-inoculation. The effects of Enterobacter inoculation on pre-existing
rhizosphere microbial populations is also being analysed using bacterial community profiling
technologies. We discuss the findings in context of formulating efficient PGPR inoculums to
specifically target crops of interest for growth promotion.
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Symbiosis
3
Nutritional value of tomato and strawberry fruits is affected by plant inoculation
with soil bacteria
Elisa Bona*1, Elisa Gamalero2, Guido Lingua2, Simone Cantamessa2, Nadia Massa2,
Valeria Todeschini2, Paola Manassero2, Giorgia Novello2, Andrea Copetta2, Giovanni
D'Agostino3, Graziella Berta2
1
Università del Piemonte Orientale, Italy, 2Università del Piemonte Orientale,
DiSIT, Italy, 3Mybasol, srl, Italy
Soil bacteria can stimulate plant growth; however, little information is available about the
impact of these bacteria on the nutritional value of fruits. Therefore, we considered two
economically relevant crops: tomato for the evidence of reduced risk of cancer and cardiovascular diseases and strawberry because they are an important source of antioxidants and
anti-inflammatory phytonutrients.
Tomato plants were inoculated with Pseudomonas sp. 19Fv1T, or Pseudomonas fluorescens C7
and grown in open field condition. Strawberry plants were inoculated with P. fluorescens Pf4
or Pseudomonas spp. 5Vm1K and grown in pot under glasshouse condition. Both plant species
were subjected to reduced fertilization. The impact of the bacterial strains on the fruit
nutritional value was assessed by measuring the concentration of soluble sugars, organic
acids, vitamins, anthocyanin, lycopene and b-carotene together with nitrate and nitrite
amount in fruits.
The size and biomass of tomato fruits were unaffected by the bacterial strains. However, the
sugar content was modulated by the microorganisms (i.e. fructose increase and glucose
reduction). 19Fv1T induced an enhancement of malic, tartaric, ascorbic and glutamic acids,
beta carotene and a reduction of oxalic acid in the fruits compared to controls. The strain C7
induced reduction of the glutamic, oxalic and ascorbic acid, and decrease of nitrite level in
fruits compared to those produced by uninoculated plants.
Both strawberry fruit size and yield were increased by the bacterial. The concentrations of
sugars were differently affected according to the microorganism. Both bacteria enhanced the
amount of ascorbic acid in fruits, but only Pf4 increased folic acid concentration.
Fruit yield and quality can be improved by plant inoculation with soil bacteria; this can have a
real world application, leading to economical, ecological and human health benefits in
relation to the reduced chemical input and to the increased quality of the fruits.
4
Roots and nodules bacterial diversity of Scorpiurus muricatus in western Algeria
Zoulikha Bouchiba*1, Zineb faiza Boukhatem1, Zohra Ighilhariz1, Mustapha MIsbah el
Idrissi2, Abdelkader Bekki1
1
University of Oran 1 Ahmed Benbella, Algeria, Algeria, 2University Mohamed V
Agdal,Rabat - Morocco, Morocco
Algeria is facing chronic forage deficit, in order to improve the production and contribute to
increasing animal production for sustainable development, the use of spontaneous forage
legumes of pastoral interest as Scorpiurus muricatus ssp. sulcatus seems interesting. The
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Symbiosis
diversity of microorganisms associated with this plant is very little studied worldwide. The
purpose of this study is to investigate the genotypic characteristics of microorganisms
associated with this plant. Fifty nodular and root bacterial strains respectively were isolated
from plants sampled in six regions in western Algeria. Nodular isolates are grouped into
different clusters according to profiles obtained by repPCR by ERIC and BOX primers. NodC
amplification allowed us to select renodulant strains. Nodular and root endophytes are
studied by PCR-RFLP of DNAr16s and compared with references strains, a representative of
each group is sequenced. The endophytes ability to solubilize phosphorus and IAA
production is determined.
5
Biological Nitrogen Fixation Book Volume I and II
Frans J. de Bruijn*
INRA/CNRS Laboratory of Plant Microbe Interactions, France
Nitrogen is arguably the most important nutrient required by plants. However, the availability
of nitrogen is limited in many soils and although the earth’s atmosphere consists of 78.1%
nitrogen gas (N2) plants are unable to use this form of nitrogen. To compensate, modern
agriculture has been highly reliant on industrial nitrogen fertilizers to achieve maximum crop
productivity. However, a great deal of fossil fuel is required for the production and delivery of
nitrogen fertilizer. Moreover carbon dioxide (CO2) which is released during fossil fuel
combustion contributes to the greenhouse effect. Biological nitrogen fixation is one
alternative to nitrogen fertilizer. It is carried out by prokaryotes using an enzyme complex
called nitrogenase and results in atmospheric N2 being reduced into a form of nitrogen
diazotrophic organisms and plants are able to use (ammonia). It is this process and its major
players which will be discussed in the “Biological Nitrogen Fixation” Books. The best known
and most extensively studied example of biological nitrogen fixation is the symbiotic
interaction between nitrogen fixing “rhizobia” and legume plants. Here the rhizobia induce
the formation of specialized structures (“nodules”) on the roots of the legume plant and fix
nitrogen which is directly assimilated by the host plant;. It is this symbiotic interaction which
will be highlighted in the Book. While legumes are important as major food and feed crops,
cereals such as wheat, mays and rice) do not have this symbiotic nitrogen fixing interaction
with rhizobia. It has thus been a “holy grail” to transfer the ability to fix nitrogen to the
cereals and different timely approaches towards this goal are also discussed in the Books.
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Symbiosis
6
Phosphorus use efficiency in N2-fixing legume-rhizobia symbioses
Jean Jacques Drevon*1, J. Abadie1, L. Amenc1, A. Bargaz2, O. Dommergue3, M. Lazali4,
M. Zamanallah5
1
INRA Ecologie Fonctionnelle & Biogéochimie des Sols &
Agroécosystèmes, France, 2Swedish University of Agricultural Sciences, Department of
Biosystems and Technology, Sweden, 3Laboratoire des Symbioses Tropicales et
Méditerranéennes, Campus International de Baillarguet, France, 4Université de Khemis
Miliana, Algeria, 5CIMMYT, Southern Africa Regional Office, Zimbabwe
Low phophorus availability in about 40% of the world’s arable land limits crop yield, most
particularly for leguminous crops when their growth depends upon symbiotic N2-fixation
(SNF). Therefore, our work aims to increase the phosphorus use efficiency (PUE) for SNF, and
its contribution to a more effective coupling between the P and N bio-geochemical cycles.
Myo-inositol hexakisphosphate (phytate) constitutes the main source of organic P in soils, but
is unavailable to plants. Phytases are the only phosphatases able to hydrolyse phytate
efficiently into inorganic Pi, thus increasing P bio-availability for plants. In this work we
demonstrate the existence of phytase activity in root-nodules, and show the expression of a
purple acid phytase within nodule inner-cortex by in situ RT-PCR. Also histidine acid- and bpropeller- phytases (HAP and BPP) were found among legume-nodulating bacteria, i.e.
rhizobia, with major expression in nodule infected-cells. Moreover, phosphoenol pyruvate
phosphatase and trehalose 6P phosphatase were discovered in nodules with puzzling
localization. The nodular expression of all these genes and the enzymatic activity of their
products increased significantly under P-deficiency, and varied among recombinant inbred
lines of Phaseolus vulgaris that are contrasting in their PUE for SNF. It is concluded that the
differential expression of bacterial and plant phosphatase-genes in nodules offers a new
understanding of the N2-fixing legume physiology and its specific P requirements. The
potential of these phosphatase-genes for a virtuous circle of P and N soil-fertility will be
addressed in relation with the interdisciplinary research strategy of the FABATROPIMED
federative project of Agropolis Montpellier.
7
Diagnosis of common bean inoculation needs and selection of effective bacterial
biofertilizers adapted to soil conditions of the Skhirate region in Morocco
Imane El Attar*1, Jamal Aurag2, Imane Thami Alami3, Mohamed El Khadir3
1
Laboratory of Microbiology and Molecular Biology (LMBM), Faculty of Science,
Mohammed V University, Morocco, 2Laboratory of Microbiology and Molecular Biology
(LMBM, LMI), Faculty of Science, Mohammed V University, Morocco, 3National Institute
for Agricultural Research (INRA), Morocco
The Skhirate region (North-West part of Morocco) provides about 20% of of common bean
production in the country under an intensive production system based on the uncontrolled
use of chemical fertilizers. In this context our research project aims to reduce the amount of
fertilizers used during the cultivation of common bean causing negative environmental
impacts. For this purpose, we plan to develop bacterial biofertilizers that can contribute
efficiently to the nutrition of common bean and its growth by replacing, partially, the intakes
of mineral fertilizers. This approach is based on the establishment of a sustainable
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agrobiological system for the nutrition of common bean using mostly symbiotic nitrogen
fixation and inorganic phosphate solubilization.
During the growing season of common bean, a general survey was carried out in the Skhirate
region in order to diagnose the nodulation status of this legume and determine if there is a
need for inoculation with bacterial inocula. Common bean nodulation was found to be very
low or even absent in most of the visited fields, thus we decided to carry out additional
surveys in other production areas in Morocco (Beni Mellal, Moulay Bouselham and Berkane).
Nodules collected during these surveys have enabled the creation of a collection of 143
bacterial isolates which were subjected to a physiological characterization, especially their
tolerance to salt and pH stresses and their potential plant growth promoting activities (PGP
activities). The results of this characterization revealed a large number of isolates with
interesting PGP activities and tolerance to both types of stress studied.
The symbiotic properties of these isolates are being characterized under controlled
conditions. The strains able to establish a symbiotic relationship with the host plant
(Phaseolus vulgaris) will be identified by sequencing the 16S rRNA gene.
8
Does mycorrhizal phosphate uptake influence the root-associated microbiome?
Nina Gerlach*, Martin Willmann, Eva Koebke, Alexander van Burgeler, Marcel Bucher
University of Cologne, Botanical Institute, Cologne Biocenter, Cluster of Excellence on
Plant Sciences (CEPLAS), Germany
The plant microbiome is a key determinant of plant growth and fitness. Arbuscular
mycorrhizal fungi are part of the fungal microbiome of most terrestrial plants and function as
an extension of the root system facilitating bi-directional exchange of soil-born nutrients and
plant-derived carbon between both symbionts.
Maize plants colonized by arbuscular mycorrhizal fungus Rhizophagus irregularis exhibited
enhanced growth under nutrient-deficient conditions. Systemic metabolic alterations
included anthocyanin and lipid metabolism likely in response to an improved P-status of
mycorrhizal maize leaves. An overall increase in leaf C versus N metabolism highlighted
changes in metabolic fluxes. A parallel induction of defense gene expression and
accumulation of secondary metabolites suggested priming of mycorrhizal maize leaves.
Phosphate uptake in plants is mediated by transporters of the Pht1 protein family. A pht1;6
transposon insertional maize mutant is strongly impaired in mycorrhizal phosphate uptake
and thus exhibited reduced biomass accumulation in agricultural soil poorly fertilized with
phosphate. Arbuscular mycorrhizal fungal colonization of isolated pht1;6 plants was strongly
diminished. However, when cultivated together with wild type plants, fungal colonization of
pht1;6 was restored but mutant roots exhibited increased arbuscule degeneration and
formation of strongly septated stunted arbuscules.
In consideration of the role of Pht1;6 in maize root and shoot physiology, our current interest
is on the impact of Pht1;6 transporter activity on the root-associated fungal microflora
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Symbiosis
including mycorrhizal but also other fungi. To this end, the fungal microbiome of pht1;6,
heterozygous and wild type plants was analyzed by a PCR-based automated ribosomal
intergenic spacer analysis. Trans-complementation assays highlighted the impact of
mycorrhizal neighboring plants on the mutant root-associated microbiome. Moreover the
taxonomic structure of fungal assemblages as a function of Pht1;6 will be resolved by a
culture-independent molecular approach.
9
Endofungal bacteria in plant symbiotic fungi
Anton Hartmann*1, Dan Li1, Michael Rothballer1, Jochen Blom2, Peter Kämpfer3,
Stefanie P. Glaeser3, Karl-Heinz Kogel4
1
Helmholtz Zentrum München, German Research Center for Environmental Health,
Department Environmental Sciences, Research Unit Microbe-Plant
Interactions, Germany, 2Bioinformatics and Systems Biology, Justus Liebig
University, Germany, 3Institute of Applied Microbiology, Research Centre for BioSystems,
Land Use and Nutrition, Justus Liebig University, Germany, 4Institute of Phytopathology,
Research Centre for BioSystems, Land Use and Nutrition, Justus Liebig
University, Germany
Endofungal bacteria occur in some ectomycorrhizal as well as arbuscular mycorrhizal fungi; a
mycorrhizal helper function was attributed to these bacteria. In the order of Sebacinales
(Basidiomycota), also endofungal bacteria have been demonstrated. In many cases, the
endofungal bacteria are not cultivated yet, but in the case of Piriformospora indica, the
endofungal bacterium Rhizobium radiobacter F4 (RrF4) was cultured and its genome and
physiology could be investigated. A better understanding of the role of R. radiobacter in this
tripartite plant-fungus-bacterium symbiosis is of high relevance, because P. indica has a wide
range of plant beneficial applications in agriculture.
The genome of RrF4 has been sequenced by second generation pyro-sequencer (454 GS FLX
Titanium). It is organized in a circular and a linear chromosome and two plasmids (pTiF4 and
pAtF4) in the same manner as in R. radiobacter C58 (formerly Agrobacterium tumefaciens).
The average amino acid identity (AAI) of RrF4 to biovar I strains (e.g. C58) is 99.8%. While
RrF4 and C58 showed a high degree of similarity based on the circular and linear
chromosomes, the plasmids were more diverse. In addition to some rearrangements between
the two plasmids, RfF4 most importantly lacks – in contrast to C58 - the complete transferred
DNA (T-DNA) region and some adjacent genes belonging to the nopalin catabolic region.
Like in C58, strain RrF4 contains the structural gene traI for N-acylhomoserine lactone (AHL)synthesis and traR for the AHL-receptor traR. Detailed chemical analysis showed that RrF4
produces a range of oxo- and hydroxyl-AHLs with C8-, C10-, or C12 alkyl side chains. These
autoinducer signaling molecules are able to induce systemic resistance in different plant
hosts as well as having plant growth promoting effects. We therefore hypothesize that the
endofungal bacterium RrF4 of P. indica contributes to its plant growth promoting action in
the tripartite plant-fungal-bacterium symbiosis.
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10
An investigation on the colonisation of bacterial endophytes in oilseed rape,
grown in the presence of entomopathogenic nematodes
Mary Jo Hurley*, Dina Brazil, Thomais Kakouli-Duarte
Institute of Technology Carlow, Ireland
Pathogenic microorganisms and pests are a predominant threat in agriculture worldwide and
control of these has relied heavily on chemical pesticides. The overuse of pesticides has been
directly related to the intensification of agriculture over previous decades, but with
substantial environmental costs. The overall aim of this project focuses on the biocontrol of
economically important insects utilising a combination of entomopathogenic nematodes and
bacterial endophytes, to help reduce pesticide input in the environment.
Both entomopathogenic nematodes and endophytic bacteria have widespread applications
as biocontrol agents, however the potential of synergism between these nematodes and
endophytic bacteria has yet to be explored. Investigating the effects of various endophytic
bacteria on nematode biology and behaviour will not only contribute to knowledge on these
but also increase nematode efficiency and predictability within a dynamic environment.
Results are presented here from experiments designed to (a) investigate the effects of
bacterial endophytes on nematode virulence and (b) examine the potential of increased
bacterial-plant colonisation in the presence of entomopathogenic nematodes. Nematode
dose response experiments were carried out to determine negative or stimulatory effects of
endophytes on nematode infectivity. In all bacterial treatments Heterorhabditis bacteriophora
infected in lower numbers than Steinernema feltiae and Steinernema carpocapsae. Following
bacterial exposure fewer S. feltiae (e-nema) infective juveniles were recovered, when
compared to the control. Moreover, for all nematodes species fewer numbers were recovered
from insects exposed to bacterial strain L228. Bacterial colonisation of oilseed rape was
determined via bacterial plate counts and the polymerase chain reaction, using green
fluorescent protein specific primers. To date gfp labelled bacteria have been isolated from
root, rhizosphere and stem samples, in the presence and absence of nematodes. This work is
currently on-going.
11
Symbiotically fixed N substitutes fertilizer N by enhancing C and N assimilation
during reproductive stages of soybean
Jian Jin*1, Yansheng Li2, Xiaobing Liu2, Guanghua Wang2, Zhenhua Yu2, Mathesius
Ulrike3, Judong Liu2, Herbert Stephen4
1
Northeast Institute of Geography and Agroecology, Chinese Academy of
Sciences, China, 2Key Laboratory of Mollisols Agroecology, Northeast Institute of
Geography and Agroecology, Chinese Academy of Sciences, China, 3Division of Plant
Science, Research School of Biology, The Australian National
University, Australia, 4Stockbridge School of Agriculture, University of
Massachusetts, USA
Excessive fertilizer has been commonly applied in the soybean cropping system in fertile
Mollisols in northeast China. It is necessary to understand the mechanisms of how reducing N
fertilizer application impacts the plant N acquisition and remobilization, which is associated
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with photosynthetic C assimilation and seed yield. Thus, the aim of this study was to
investigate the origin of plant N, i.e. N2 fixation-, fertilizer- and soil-derived N under two
different N application levels, and the subsequent influence on C assimilation. A pot
experiment was conducted with soybean grown in a Mollisol supplied with 5 (N5) or 100
(N100) mg N kg-1 soil. Nitrogen was applied as 19.83% of 15N atom excess in urea before
sowing, and 13CO2 labeling was performed at the R5 (initial seed-filling) stage. Plants were
harvested at R5 and R8 (full maturity) stages to determine the 15N and 13C abundance in plant
tissues. Seed yield and N content were not affected by different N rates. The symbiotically
fixed N accounted for 64% of seed N in N5, while fertilizer-derived N dominated seed N in
N100, resulting in 58% of seed N. The proportion of soil-derived N in seed showed no
difference between the two N treatments. The enhanced N2 fixation in N5 significantly
increased the assimilation of N and C during the seed-filling period compared to N100. The
nodule density (nodule number per unit root length) and the amount of photosynthetically
fixed 13C in roots in N5 were greater than those in N100. These results indicate that
prolonging N2 fixation to increase assimilated N during the seed-filling period is likely to
meet N demand for maintaining soybean yield when fertilizer N supply is reduced. More
allocation of photosynthetic C to roots and enhanced nodulation would greatly contribute to
the alteration of N acquisition pattern.
12
Selection of specific Rhizobium leguminosarum genotypes by different Fabeae
legume hosts: A Pool-Seq mesocosm study
Beatriz Jorrin*1, Amalia Soenens1, Juan Imperial2
1
Centro de Biotecnolgia y Genomica de Plantas. Universidad Politecnica de
Madrid, Spain, 2Centro de Biotecnolgia y Genomica de Plantas. Universidad Politecnica
de Madrid. Centro Superior de Investigaciones Cientificas, Spain
Rhizobium leguminosarum bv viciae can establish effective symbioses with members of the
Fabeae legume tribe (Pisum, Lathryrus, Lens and Vicia). Previous studies have suggested that
different Fabeae select specific genotypes of rhizobia from those available in soil. We
extended these observations at the genomic level by applying a Pool-Seq analysis to isolates
selected from a soil population by pea, lentil, fava and vetch plants, and showed that plantselected sub-populations differ at the single nucleotide polymorphism level.
The nature and extent of plant-specific genotypic preference were further studied by
performing controlled mesocosm assays. Sterilized seedlings were planted in soil:vermiculite
1:1, and 21-day roots were collected, sterilized superficially, homogenized and centrifuged at
low speed. This supernatant was used as inoculant for the next cycle of selection in the same
soil with new seedlings. After 5 cycles, roots were collected, and rhizobia (25 from each host)
isolated from nodules. Each of the isolates was grown separately, all cultures from the same
host were pooled, and their pooled DNA was isolated and subjected to Pool-Seq analysis.
Sequence reads were aligned against the R. leguminosarum bv viciae 3841 reference genome
and both coverage and polymorphism analyses were performed for specific regions. No
genotypic selection by the host was observed in the rDNA region, whereas increased
genotypic selection was very clear along the mesocosm experiment for the nod cluster, but
only in the pea and vetch subpopulations. In contrast, no further polymorphism profile
changes were observed in the nod region for the fava and lentil subpopulations after the first
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Symbiosis
round of plant host selection. These results suggest the existence of differences in plant host
selection of rhizobial genotypes along the mesocosm experiment for different hosts and
genomic regions.
13
Effect of different biochars on the establishment of the symbiosis between
Rhizophagus irregularis and leek grown in peat-based substrate
Vicky Levesque*1, Hani Antoun1, Martine Dorais2, Noura Ziadi2, Martin Trépanier3
1
Laval University, Canada, 2Agriculture and Agri-Food Canada, Canada, 3Premier
Tech, Canada
Arbuscular mycorrhizae are affected by the use of peat-based substrates and inhibition of
mycorrhization was observed with Allium species. We hypothesized that the use of biochar
could alleviate the observed inhibition of mycorrhization of leek transplants grown in a
soilless mix containing 75 to 85% sphagnum peat moss and inoculated with Rhizophagus
irregularis. Since biochar physicochemical properties are influenced by feedstock nature and
the pyrolysis temperature used, five different biochars were produced (maple bark 400˚C,
550˚C and 700˚C, pine chips 700˚C and willow chips 400˚C) and applied at three different rates
(5, 10 and 15% in volume). The experiment was realized during nine weeks in a greenhouse
with 5x3 factorial design for biochar types and rates. A control (0% of biochar) was also
included for comparison purpose. Biochars were added to potting mix and inoculated with
monoxenic culture of Rhizophagus irregularis DAOM 197198. Leek (Allium ampeloprasum var.
Lancelot) was used as host plant. The effect of biochar amendment on leek root colonization
by mycorrhizae, plant biomass and phosphorus uptake were evaluated at the seventh leaf
stage. Results indicated that the type of biochar and the level of amendment have an effect
on root colonization by mycorrhizal fungi. Indeed, in comparison to the control, the addition
of 15% of maple biochar increased (ρ < 0.05) the percentage of root colonization (ranging
from 23% to 46%). Nevertheless, the results have shown a
reduction (ρ < 0.05) in shoot dry matter biomass and phosphorus uptake with maple biochar.
Observed decreases could be attributed to the early stage of plant development. In
conclusion, biochar could alleviate peat inhibition in soilless mix and improve mycorrhization,
which will produce more robust leek transplants.
14
Nitrogen transfer in soybean/maize intercropping system inoculated arbuscular
mycorrhizal fungi and rhizobium
Shumin Li*1, Aiyuan Zhang2, Lingbo Meng3, Xiaoguang Han2, Fei Wang2, Dejiang
Wang2
1
Northeast Agricultural University, China, 2Resource and Environmental College,
Northeast Agricultural University, China, 3Department of Life Science, Harbin
University, China
The tripartite symbiosis between legumes, rhizobia and mycorrhizal fungi are generally
considered to be beneficial for nitrogen (N) uptake of legumes, but the facilitation of the
symbiosis in legume/non-legume intercropping system is not clear.Therefore, the aims of the
research are 1) to certify if the dual inoculation can facilitate the N uptake and N transfer in
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Symbiosis
maize/soybean intercropping system, 2) to calculate how much N will be transferred from
soybean to maize. A pot experiment with different root separations (solid barrier,
mesh(30µm) barrier and no barrier) was conducted and 15N isotopic tracing method was used
to calculate how much N transferred from soybean to maize inoculated
arbuscularmycorrhizal fungi (AMF) and rhizobium in soybean (Glycine max L.cv. Dongnong
No.42)/maize (Zea mays L.cv.DongnongNo.48) intercropping system. In comparison with the
inoculatingGlomus mosseae(G.m.), Rhizobium SH212 and no inoculation, both inoculating
Glomus mosseaeand Rhizobium SH212 increased the N uptake of soybean by 28.73%, 39.62%
and 93.07% in solid barrier system. N uptake of maize inoculated both Glomus mosseae and
rhizobium was 1.20, 1.28 and 1.67 times greater than that ofinoculating Glomus mosseae,
Rhizobium SH212 and no inoculationrespectively. In addition, the amount of N transferred
from soybean to maize of dual inoculation with mesh barrier was 7.25 mg, 7.01 mg and 11.21
mg greater than that ofinoculating Glomus mosseae, Rhizobium SH212 and no inoculation,
and similarly 6.40 mg, 7.58 mg and 12.46 mg increased in no barrier system. Inoculating both
AMF and rhizobium in soybean/maize intercropping system could improve the N fixation
efficiency of soybean, and promote N transfer from soybean to maize, which result in the
improvement of yield advantage in legume/non-legume intercropping.
15
Insect vectors efficiently convey complex endophytic communities across
grapevine plants
Sebastian Lòpez-Fernàndez, Valerio Mazzoni, Pier Luigi Bianchedi, Ilaria Pertot, Andrea
Campisano*
Fondazione Edmund Mach, Italy
Microbial endophytes colonize the inner tissues of plants. It is commonly held that most
endophytes invade the host tissues through the roots or through discontinuities on the plant
surface, including wounds and stomata. Microorganisms can also be transferred through root
anastomoses, as it occurs for instance with some pathogenic mollicutes, such as the
phytoplasmas. Some insects able to penetrate the plant surface are also vectors of
phytoplasmas. Very little is known about the ability of such vectors to harbour and transfer
other microorganisms.
To unravel the ecological role of insects for endophytic microorganisms, we used freshly
hatched nymphs of the sap-feeding leafhopper Scaphoideus titanus (vector) to transport
microorganisms across grapevine plants. We used adult, greenhouse-grown (donor) plants
with an established endophytic fauna, and micropropagated (acceptor) grapevines hosting no
detectable bacteria. We used 454 pyrosequencing of the bacterial 16S rDNA gene to estimate
the composition of bacterial endophytic communities in donor plants, vector insects and
acceptor plants, and to track microbial communities along the insect-plant-microbe network.
After contact with the vector, acceptor plants were colonized by a complex endophytic
community dominated by Proteobacteria, highly similar to that present on donor plants.
Interestingly, a similar bacterial community, but with a higher ratio of firmicutes, was found
on S. titanus. Insects feeding only on acceptor plants transferred an entirely different bacterial
community dominated by Actinobacteria, where the opportunistic human pathogen
Mycobacterium abscessus played a major role. Despite the fact that insects dwelled mostly on
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Symbiosis
plant stems, the bacterial communities in plant roots resembled more closely those inside
and on insects, when compared with above-ground plants.
We establish here for the first time the potential of insect vectors to transfer entire bacterial
communities across plants. We also define the probiotic role of plants and microbial
endophytes in establishing microbial communities in plant-feeding insects.
16
Inoculation with selected strains of Azospirillum spp. replaces nitrogen in pearl
millet (Pennisetum glaucum (L.)) in Brazilian cerrado soil
Ivanildo Evodio Marriel*1, Izabelle Melo2, José A. Rodrigues1, Christiane A Oliveira1,
Eliane A. Gomes1, Francisco A Souza1
1
Embrapa Maize and Sorghum, Brazil, 2UFSJ, Brazil
The cultivation of pearl millet has received increasing attention as an alternative crop for
mulch formation in no-till systems and as forage for livestock production, mainly in the
Brazilian Cerrado fields. There are evidences of the benefic contribution of the inoculation of
diazotrophic bacteria to agronomic crops as a source of nitrogen (N), including pearl millet,
but there is no strains recommended for this crop in the country. Here we report the effect of
inoculation of Azospirillum strains on pearl millet (BRS 1501) growth and nitrogen fertilizer
replacement. We evaluated 20 Azospirillum strains (CMS01 to 20) under three doses of N (0,
30 and 60 kg N.ha-1 on field condition at the Municipality of Sete Lagoas, MG State, Brazil.
The crop was cultivated in plots at the field; the experimental design was randomized blocks
with four replications. A basic fertilization was applied at sowing and consisted of 40 kg ha-1
of urea, 400 kg.ha-1 of superphosphate, 100 kg ha-1 of potassium chloride and 20 kg ha-1 FTE.
At the flowering stage, plants were collected for determination of dry matter accumulation,
concentration and content of macronutrients in the shoots. There were significant differences
(p <0.05) between treatments for the variables analyzed, except magnesium content. The dry
matter accumulation ranged from 163 to 367g for 3 plants and N content between 3.1 to
6.84g for 3 plants. The strains CMS 7 and 11 provided shoots growth and nitrogen uptake
similar to those observed with 60 kg / ha of N. Our results also suggested the role of these
bacteria as plant growth promoters. We highly the importance of screening of several
different strains to obtain promising bacteria for inoculant formulation, seeking replacement
of the nitrogen in the pearl millet crop.
17
Microbes from Inner Space: II. Diversity of seed inhabiting endophytes in white
clover (Trifolium repens) across continents and their plant beneficial potential
Jana Monk*1, Richard Johnson2, Rhys Jones3, Katharine Adam3, Damien Fleetwood4,
Nigel Bell3
1
AgResearch Ltd, New Zealand, 2AgResearch Ltd, Grasslands Research Centre, New
Zealand, 3AgResearch Ltd, Ruakura Research Centre, New Zealand, 4Biotelliga Ltd,
Institute for Innovation in Biotechnology, New Zealand
Endophytes are microbes that can be found inside plant tissues, where they can live
commensally or execute beneficial functions for the host. It is believed that every plant
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Symbiosis
species harbors endophytes, and seeds of many plant species have been reported inhabiting
endophytes. This study investigates whether seed endophytes in white clover are conserved
at a global scale and what beneficial traits are associated with the culturable fraction. Seed
from 18 countries across the globe was provided by the Margot Forde Forage Germplasm
Centre (New Zealand). The culturable microbial fraction as well as DNA was extracted from
seed followed by identification of endophytic microbes and DGGE fingerprinting analysis. No
fungal endophytes were detected in clover seed. DGGE analysis of Gammaproteobacteria
showed that the seed community composition varied across samples with only a few shared
bands. The richness of bands was low with only 2 to 5 bands per seed sample. In contrast,
DGGE fingerprints of Betaproteobacteria showed greater level of richess and some common
bands across most samples. Gammaproteobacteria dominated the culturable fraction with
Pantoea spp., Erwinia spp. and Pseudomonas spp. prevalent. Around 200 isolates are currently
being evaluated for their ability to influence rhizosphere functions such as producing auxins,
catabolizing the precursor of ethylene (ACC), solubilizing phosphorus, sequestering iron and
antagonizing pathogens. For isolates with desirable effects on clover plant growth, in-depth
analysis of the interactions with the host will be performed to determine the spatial
distribution within the host plant and whether the endophyte(s) can be transmitted to new
seed following artificial inoculation. The most promising isolates will be used to test for
effects on root pathogens and invertebrate root pests.
18
Comparative effects of TRP and TSP on the development of two multi-purpose
trees in the presence of mycorrhizae
Sacko Ousmane*1, Kane Aboubacry2, Sanon Kadidia Bibata3, Yattara Inamoud Ibny1
1
Faculty of Sciences and Technics, University of Sciences, Technics and Technologies of
Bamako, Mali, 2Common Laboratory of Microbiology IRD / ISRA /
UCAD, Senegal, 3INERA, Burkina Faso
West African soils are structurally deficient in nitrogen (N) and phosphorus (P). P is one of the
nutrients that affect agricultural and forestry production. Its supply is mainly provided
through the provision of agricultural inputs.
There are large deposits of rock phosphate (RP) in most countries of West Africa and among
these deposits, Tilemsi rock phosphate (TRP) coming from northern Mali, is one of the best
and most receptive. Unfortunately, it is underutilized because of its slow dissolution
compared with triple superphosphate (TSP) which cost is very expensive for farmers.
Mycorrhizal fungi allow to plants which they are associated a better absorption of water and
phosphorus (P).
Experiments were conducted under greenhouse and laboratory to highlight the effects of the
TRP on development, growth and nutrition of two multipurpose trees, Gliricidia sepium and
Sesbania sesban, inoculated with an endomycorrhizal fungus (Glomus aggregatum). The
effects of the TRP were compared to those of TSP used as fertilizer of reference. The soilof
Nioro (Senegal) was used as the culture substrate for the trees.
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Symbiosis
The results showed that the TRP has been the preferential phosphate; used at the rate of 75
mg P.kg-1, it was better used by S. sesban in which the height, biomass, and the absorption
of the major elements (N, P, K) have been improved, especially in the presence of mycorrhizal
fungus.
19
Expression analysis of two novel lipid transfer proteins during the symbiotic
interaction between Medicago truncatula and Sinorhizobium meliloti
Chiara Santi1, Barbara Molesini1, Youry Pii*2, Tiziana Pandolfini1
1
University of Verona, Italy, 2Free University of Bolzano, Italy
Lipid transfer proteins are small basic proteins that constitute a large family characterized by
the ability to transfer phospholipids between a donor and an acceptor membrane. MtN5 is a
non-specific lipid transfer protein expressed at a very early phase of the rhizobial symbiosis in
the epidermal cells, root hairs and root nodules. By manipulating MtN5 expression in the
roots, we demonstrated that this rhizobial protein positively regulates the nodulation
process. To get more information about MtN5 function in the symbiosis we have now
obtained stably transformed plants overexpressing and silencing MtN5. MtN5-silenced plants
were impaired in nodulation, showing a 40% reduction in the number of nodules compared
with wild type plants, while transgenic plants overexpressing MtN5 developed 34% more
nodules with respect to wild type plants. These evidences indicate a crucial role for MtN5 in
controlling bacterial invasion and nodule organogenesis in M. truncatula.
We have identified two novel putative lipid transfer proteins of Medicago truncatula,
Medtr3g055250 and Medtr7g052640, showing high similarity to MtN5. We have observed that
the expression pattern of these lipid transfer proteins is modulated during the rhizobial
symbiosis. In roots bearing nodule primordia (at 6 days post inoculation) the transcript level
of Medtr7g052640 was almost 6 fold higher than in non-inoculated roots. At 14 days post
inoculation, both genes were highly expressed in the root nodules. These data suggest that
the different M. truncatula lipid transfer proteins might be involved in the regulation of root
nodule development.
20
Microbial improvement of compost and biochar products by combination with
arbuscular and ericoid mycorrhizal fungi
Areg Poghosyan*, Henning von Alten
Leibniz University Hanover, Germany
The EU funded project REFERTIL has the mission to transform organic waste from a costly
disposal process into an income generating activity. The output products will be safe,
economical and standardized compost and biochar products containing phosphorous and
nitrogen that can be economically and beneficially used by farmers. Within this framework
LUH has the objective to combine inocula of arbuscular and ericoid mycorrhizal fungi with
biochars and composts. The joined application shall combine the positive effects on plant
nutrition and soil properties the latter have with the known beneficial effects of mycorrhizal
fungi.
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Symbiosis
Isolates of arbuscular mycorrhizal fungi (AMF) and ericoid mycorrhizal fungi (ERMF) can be
produced in vitro or in living plants and be used to inoculate crops. In the REFERTIL project
LUH has shown that these isolates can be successfully applied together with biochar made
from plant material or animal bones (ABC) and with composts. Aim was the microbial
improvement of these soil amendments and organic fertilizers. The AMF isolates differed in
the amplitude of their effects; they transported P from ABC to the plant roots and improved
plant growth in combination with composts. The mixed application of biochars and composts
together with AMF on expanded clay as carrier material is problem-free from a technological
point of view. AMF/compost mixtures can even be stored for a certain time. That is of
importance for the practical use of the micro-biologically improved new products.
Aim of the combined application together with ERMF was the microbial improvement of the
composts with the objective of a combined or synergistic suppression of the soil-borne
disease caused by the Oomycet Phytophthora cinnamomi. The ERMF isolates in combination
with composts were able to reduce the disease.
21
Functional diversity of the arbuscular mycorrhizal symbiosis
Sabine Ravnskov*1, John Larsen2
1
Aarhus University/Department of Agroecology, Denmark, 2Universidad Nacional
Autónoma de México/Laboratorio Agroecologia, Mexico
Arbuscular mycorrhiza is known to alter growth and nutritional status of plants, and to
enhance plant tolerance to pathogens. However, functional diversity of the arbuscular
mycorrhizal symbiosis has been revealed in studies focusing either on plant phosphorus
uptake or on plant tolerance to pathogens. The objective of the present study was to
examine the functional diversity of arbuscular mycorrhiza in symbioses between three
arbuscular mycorrhizal fungi, Funneliformis mosseae, Rhizophagus irregularis and
Claroideoglomus claroideum and six varieties of Cucumis sativus (cucumber) with respect to
plant growth and nutritional status, and to the tolerance of cucumber cv Tiffany to Pythium
ultimum. The results showed a phenotypic variation of each of the 24 combinations of
plant/fungal genotypes involved in the arbuscular mycorrhizal symbioses both with respect
to root colonization by the arbuscular mycorrhizal fungi, to plant growth, to nutrient
composition in shoots and to plant tolerance to the pathogen, but the functional diversity
between the measured parameters varied and was not correlated. As the measured functions
are related e.g. by the uptake of phosphorus could be influenced by uptake of ions of other
nutrients, growth of plants could be influenced by the nutrient uptake by the plant, and both
plant growth and nutrient content is determinant for the quality of the plant as habitat and
carbon source for the pathogen, functional diversity of arbuscular mycorrhizal symbiosis
seems to be more complex than earlier understood. This underlines that arbuscular
mycorrhizal fungi are an integrated part of plant functioning across different plant functional
parameters, and it calls for more research within the complex functional diversity of the
arbuscular mycorrhizal symbiosis.
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22
Drought tolerance of rhizobia from nodules of Indigofera argentea
Guiling Ren*, Leo van Overbeek, Henk Franssen, Mirjam Schilder, Jan Verver
Rhizobium bacteria are able to form nitrogen fixing root nodules on legume plants. In
agriculture rhizobia are used as inoculum to obtain optimal nodulation on the root system of
legume crops. Seed inoculation with effective strains of rhizobia provide the nitrogen
requirements of the legume to achieve increased yields. However, rhizobia are sensitive to
desiccation, and inoculum have a short half-life during seed coating and subsequently
storage. We questioned whether phenotypic variation in desiccation tolerance can be
observed in different rhizobial species. How rhizobia in crucial dry desert environment keep
active in inducing nodule formation in desert legume. Up to date, all literatures investigating
the activity of desiccated rhizobia in the laboratory has used broth-grown, planktonic cells. In
this study, a diverse range of 78 rhizobia strains from desert soils were isolated. And we
developed a quantitative bioassay to illustrate desiccation tolerance on planktonic cells as
well as biofilm-formed colony cells. By comparing fitness of desert isolates to elite strains that
are used as inoculum under low humidity conditions, we found that colony formation
improves the fitness of rhizobia dramatically when compared to bacteria grown in liquid
cultures. Bacteria inside a micro-colony survive as long as 4 weeks at 28% relative humidity,
whereas individual bacteria that were grown in a liquid culture do so for only 1-2 days. We
noted a difference in colony structure of desert rhizobia when compared to elite rhizobial
strains, as colonies formed by dessert strains are more compact and the extracellular matrix
undergoes different physical changes upon rehydration. This difference in colony structure
may contribute to the fitness of the bacterium under drought stress conditions. Our results
demonstrate that the micro-colony structure protects rhizobia during drought stress. This
finding may be exploited to improve rhizobial inoculum used in agriculture.
23
Deciphering the role of plant growth-promoting rhizobacteria in the tolerance
of Spartina densiflora to physicochemical properties of marshes soils
Ignacio D Rodriguez-Llorente*1, Enrique Mateos-Naranjo1, Mesa Jenifer1, Alfonso
Pérez-Martín2, Miguel A Caviedes1, Eloisa Pajuelo1
1
University of Sevilla, Spain, 2IRNAS-CSIC, Spain
In the salt marshes of the SW Spain, Spartina densiflora grows on contaminated sediments
under unfavourable physicochemical conditions. 22 different cultivable bacterial strains were
isolated from the rhizosphere of S. densiflora grown in those soils. 70% of the strains showed
one or more plant growth promoting (PGP) properties, including phosphate solubilisation
and siderophores or indolacetic acid production. Among them, a bacterial consortium,
composed by Aeromonas aquariorum SDT13, Pseudomonas composti SDT3 and Bacillus sp.
SDT14, was selected for further research. A glasshouse experiment was designed to
investigate the role of this consortium in plant growth and physiological tolerance to the
physicochemical properties of marshes soils. Plants of S. densiflora were randomly assigned
to two inoculation treatments (with and without inoculation) in combination with two soil
types with different physicochemical characteristics (from Piedras and Odiel marshes, SW
Spain) for 50 days. Plant responses were examined using growth analysis, combined with
measurements of gas exchange, efficiency of PSII biochemistry, total content of
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Symbiosis
photosynthetic pigments and leaf water content. In addition, the accumulation of nutrients
and trace elements in roots and leaves were determined. The results confirm that inoculation
improved growth of S. densiflora through a beneficial effect on its photochemical apparatus
due to its impact on chlorophyll concentration. This growth enhancement happened under
both soil conditions and was mainly reflected in a greater length and diameter of roots.
Moreover, this consortium was able to stimulate ions uptake in roots and leaves. Rhizospheric
bacteria appear to play a significant role in S. densiflora growth response and tolerance to the
physicochemical properties of soils, through diverse protective effects on the photosynthetic
apparatus, water-use efficiency and mineral nutrient balance.
24
The high efficiency species arbuscular mycorrhizal fungi play the main function
for plant growth in symbiont
Ning Shi*, Gu Feng
College of Resources and Environmental Sciences, China Agricultural University, China
Different species Arbuscular mycorrhizal fungi (AMF) would affect each other when they lived
together in one root system. This study used two experiment designs to determine the
interaction of AMF on contribution for Maize. One pots experiment was randomized in
complete with three AMF species (Rhizophagus intraradices, Funneliformis mosseaeand
Gigaspora margarita) either separately or in mixtures and two harvest times. The real-time
PCR was used to determine the composition of AMF communities in roots by the number of
large ribosomal subunit (LSU) genes. The other was used the root compartment design and
32
P labeled to explain the P uptake contribution for plant of three species AMF when they
existed in one strip root. The fungi communities were advantage for symbionts although the
fierce competition happened between the different species fungi. R. intraradices significantly
promote the growth and had the highest radioactivity of 32P, but with less amount of LSU in
the communities, it was "high efficiency" fungi. Gig. Margarita and F. mosseae emerged
benefit for plant at 8 week but with larger number of LSU at single inoculation, and were
decreased significantly when combined with R. intraradices, they were "low efficiency" fungi.
Used the real-time PCR and 32P labeled, direct evidence is provided for competition among
species within the AMF community existed in one single root system.
25
Mycorrhiza as a biotic elicitor in in vitro developed dual root culture system of
Ocimum basilicum
Shivani Srivastava*1, David Cahill2, Alok Adholeya3
1
TERI, India, India, 2Deakin University, Australia, 3TERI, India
Arbuscular mycorrhizal fungi (AMF) are the most abundantly found symbionts of the plants.
They colonize plant roots which are rich source of secondary metabolites having medicinal
importance. Rosmarinic acid is a caffeic ester derivative obtained from Ocimum basilicum
(Sweet basil) and is used as an anti-inflammatory, antiviral and anti-cancer agent widely. This
study reports selection of best cultivar of Ocimum basilicum and development of an in vitro
dual culture system between pRi derived transformed roots of Ocimum basilicum and
CMCCROC3 for the production of mycorrhiza and rosmarinic acid. Developed co-culture
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Symbiosis
system was studied to provide insights into interaction mechanism between transformed roots
and AM fungi. The developmental stages were studied by light, confocal and SEM
microscopy.Potential of mycorrhiza as a biotic elicitor in comparison to non-mycorrhized root
cultures was also studied. It was found that mycorrhization significantly affected RA level
shown by HPLC and enzyme activity studies. Detailed results obtained through these studies
will be presented and discussed.
26
Population genetic diversity and structure of symbiotic rhizobia nodulating
lentil (Lens culinaris) in Morocco
Kaoutar Taha*1, El bekkay Berraho2, Gilles Béna3, Jamal Aurag2
1
Laboratoire de Microbiologie et de Biologie Moléculaire - Laboratoire Mixte
International (LMBM-LMI), Faculté des Sciences, Université Mohammed
V, Morocco, 2Laboratoire de Microbiologie et de Biologie Moléculaire - Laboratoire Mixte
International (LMBM-LMI) - Faculty of science, Mohamed V
University, Morocco, 3Laboratoire des Interactions Plantes Microorganismes
Environnement, IRD, France
Lentil (Lens culinaris L.) is a food legume appreciated for its richness in protein, vitamins and
minerals and also for its ability to fertilize soil. In Morocco, cultivated area by this culture is
ranked in third position among food legumes, after bean and chickpea; however the mean
yields remains low.
To improve the production of this crop, it is necessary to identify the best bacterial symbiotic
partners to use as inoculums. To attain this objective, both nodules and soils were sampled
from the four major Moroccan producing regions: Rabat Zemmour Zaer, Chaouia Werdigha,
Azilal and the Eastern region. A collection of 400 isolates was constituted from root nodules
collected either directly from the field or by trapping in the laboratory. Isolates were
characterized by sequencing housekeeping genes (partial 16S rRNA, recA and gln II) and the
nodulation nodC gene.
The analysis based on the 16S rRNA gene sequencing showed that all our isolates belong to
the Rhizobium genus, with a genetic similarity with R. leguminosarum specie ranging from 97
to 99%. Maximum likelihood phylogenetic trees constructed based on analysis of recA and
gln II genes showed that we recovered 21 distinct multilocus haplotypes most of which are
clustered with isolates of R. leguminosarum bv. vicia recovered previously at a high frequency
in Eastern and Central Europe.
The nodC gene analysis also revealed a high diversity, with 34 different haplotypes where 10
of them include 186 isolates over the 228 sequenced. 41% of our isolates are closely related
to strains previously identified in Mediterranean countries such as Tunisia, Morocco and
Spain; others being related to strains isolated from different regions of the world.
Phenotypic and symbiotic characterizations of isolates are in progress in order to select the
most effective strain to be used as inoculums in Moroccan soil and climatic conditions.
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27
Streptomyces-elicited actions of rhizosphere microbes and pedunculate oak
Mika Tarkka*1, Florence Kurth2, Markus Bönn2, Lasse Feldhahn2, Sylvie Herrmann3,
Liliane Ruess4, Silvia Schrey5, François Buscot2
1
Helmholtz-Centre for Environmental Research, Germany, 2Dept. Soil Ecology,
Helmholtz-Centre for Environmental Research, Germany, 3Dept. Community Ecology,
Helmholtz-Centre for Environmental Research, Germany, 4Institute of Biology, Ecology
Group, Humboldt-Universität zu Berlin, Germany, 5IMIT-Dept. Physiological Ecology of
Plants, University of Tübingen, Germany
The establishment of mycorrhizal symbiosis can be stimulated by bacterial isolates termed
Mycorrhiza Helper Bacteria (MHB). It has been suggested that certain MHB do not only
promote mycorrhization but also reduce damage by phytopathogens. Using a culture system
in which the mycorrhizosphere isolate MHB Streptomyces sp. strain AcH 505 is grown in a
soil-vermiculate substrate with a defined microbial starting community and pedunculate oak
as plant host (www.trophinoak.de), we investigated the interactions of AcH 505 with
microorganisms, nematodes and oak.
Using specific primers, we observed that the AcH 505 PCR signal increased in the presence of
the mycelium of the mycorrhizal fungus Piloderma croceum. Oak rhizosphere inoculation with
AcH 505 counteracted the damage of the oak by the root parasitic nematode Pratylenchus
penetrans. This antagonistic effect of AcH 505 was linked to shifts in the rhizosphere
microbial community fostering fungi.
Using plant RNA sequencing, we estimated how the oak responds to AcH 505. In the
Streptomyces treatment, oak genes encoding microbe-associated pattern recognition-related
leucine-rich repeat receptor protein kinases and xyloglucan cell wall transglycolases/
hydrolases were up-regulated in both roots and leaves. Co-inoculation of the rhizospheres
with the mycorrhizal fungus Piloderma croceum attenuated AcH 505-elicited defense gene
expression. In contrast, the defense gene expression in oak leaves was, in part, enhanced
upon the challenge with oak powdery mildew Microsphaera (Erysiphe) alphitoides, and this
response resulted in reduced powdery mildew symptoms, indicative of priming of plant
defenses by the bacterium.
Our studies suggest that the mycorrhizal fungus can enhance MHB growth, and that AcH 505
treatment affects the structure of the microbial community. It offers novel insights into the
mechanisms of priming by actinobacteria and highlights the diversity of services provided by
this Streptomyces strain.
28
Exudation of acid phosphatase from extraradical hyphae of arbuscular
mycorrhizal fungus Rhizophagus clarus
Keitaro Tawaraya*1, Takumi Sato1, Tatsuhiro Ezawa2, Weiguo Cheng1
1
Yamagata University, Japan, 2Hokkaido University, Japan
Arbuscular mycorrhizal (AM) fungi enhance uptake of available phosphorus (P) from soil. The
mechanism underlying this P uptake enhanced by AM fungi is the increase in surface area for
absorption of available P. Little is known about utilization of unavailable P by AM fungi. We
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Symbiosis
investigated whether extraradical hyphae of AM fungi exude ACP and whether ACP activity
responds to phosphorus condition. Sterilized Andosol was packed in pots that were
separated into the mycorrhizal and hyphal compartments with a nylon net of 30 μm pore
size. Seeds of Allium fistulosum L. were inoculated or uninoculated with the AM fungus
Rhizophagus clarus in P fertilized soil (0, 0.15, 0.30, and 0.50 g P2O5 g-1 soil). Mullite ceramic
tubes were buried in the soil of each compartment, and soil solution was collected. A.
fistulosum L. and Linum usitatissimum L. inoculated with R. clarus were grown in sand culture
and in vitro monoxenic culture, respectively. The soil solution, hyphae extracts, root extract,
and root exudates were subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) analysis. Shoot P concentration, shoot P content, and shoot dry
weight were higher in the inoculated treatment than in the uninoculated treatment. Activity
staining of the gel revealed that ACP activity at 187 kDa was observed in the soil solution in
the inoculation treatment and hyphal extract collected from sand culture and in vitro
monoxenic culture, but neither in the exudate of non-mycorrhizal roots grown in the
hydroponic culture nor in the root extracts irrespective of mycorrhizal status. ACP activity in
soil solution of inoculated treatment was higher in P0 than in P3 but there was no deference
between P0 and P3 in uninoculated treatment. These findings suggest that extraradical
hyphae of AM fungi exudes ACP into the hyphosphere and its activity is induced by Pdeficiency of host plant.
29
Ectomycorrhizal and soil enzyme activity profiles at the tree line
Lixia Wang*1, Burenjargal Otgonsuren2, Douglas Godbold1
1
University of Natural Resources and Life Sciences, Vienna/forest ecology
institute, Austria, 2Mongolian University of Life Sciences/School of
Agroecology, Mongolia
At a tree line site (1700m) and a lower elevation site (1100m) in the Austrian Alps, the
ectomycorrhizal community structure was determined on Picea abies and Pinus mugo. The
activity of a number of enzymes was determined on two of the dominant ectomycorrhizas for
each tree species. In soil, the activity of a range of enzymes was determined from soil under
the tree species and under Rhododendron hirsutum.
The ectomycorrhizal community structure differed between Pinus mugo and Picea abies at
the higher elevation site, but also between the higher and lower elevation Picea abies sites. At
the higher altitude Picea abies site, the ectomycorrhizal community was dominated by
Cortinarius sp, whereas at the lower elevation site the community was dominated by Russula.
In Pinus mugo the dominant species were Amanita muscaria and Russula orchroleuca.
In soil, the activity of the enzymes protease, exoglucanase, phenol oxidase, glucosidase,
phosphatase, peroxidase were highest at the lower altitude Picea abies site. At the high
altitude site, with the exception of phosphatase, the enzyme activities increased in the order
Picea abies > Pinus mugo> Rhododendron hirsutum. In roots, the enzyme activities of βglucosidase, N-acetyl-β-D-glucosaminidase, acid phosphatase, leucine aminopeptidase were
higher in mycorrhizal root tips than non-mycorrhizal tips. Between ectomycorrhizal species,
the activity of acid phosphatase, averaged for all species of mycorrhizas investigated, was
higher in both tree species at the high elevation site, compared to Picea abies at the lower
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Symbiosis
elevation. No clear relationship was shown between enzyme activity in the soil and that of the
dominant ectomycorrhizal species, and also between the enzyme activity and rate of net
nitrogen mineralization.
30
Modelling resource allocation in the legume - rhizobium symbiosis
Annet Westhoek*1, Neil Dalchau2, Lindsay A. Turnbull3
1
University of Oxford, United Kingdom, 2Computational Science Laboratory, Microsoft
Research, United Kingdom, 3University of Oxford, Department of Plant Sciences, United
Kingdom
The symbiosis between legumes and the nitrogen-fixing bacteria, rhizobia, is of major global
importance, with symbiotic nitrogen fixation accounting for a third of the total nitrogen input
in agricultural systems. Increasing demands for protein are only likely to increase reliance on
legumes, which already form a key source of protein in human diets.
Rhizobia provide legumes with nitrogen in return for carbon in the form of photosynthates. It
is well established that investment by legumes in rhizobial symbionts depends both on the
external nitrogen environment and on the rhizobial strain. However, we lack a quantitative
understanding of how legumes allocate resources to symbionts of different nitrogen-fixing
efficiency under a wide range of external nitrogen concentrations.
In this study, we develop a model which investigates how a plant can maximise its fitness
through optimising its resource allocation. The model simulates the growth of a plant
infected with one or more rhizobial strains. We assume that growth is limited either by
carbon or by nitrogen. The plant obtains carbon via investment in leaves while nitrogen is
obtained by direct uptake via roots (which depends on the soil nitrogen concentration)
and/or via nitrogen fixation in nodules (which depends on the nitrogen fixation rate of
rhizobial strains). We then use the model to test alternative hypotheses about resource
allocation strategies under different conditions. In particular we investigate whether the
optimal allocation strategy depends on the assumptions made about the way inorganic
nitrogen is supplied in soil.
The model will be able to answer questions such as: 1) under what conditions investment in
inefficient strains could be beneficial for legume growth; 2) and what would be optimal
allocation for maximum growth?
31
Symbiotic effectiveness and host range of indigenous Rhizobia isolates
nodulating Sesbania sesban
Wassie Haile Woldeyohannes*1, Elias Dogiso Dagne2
1
Hawassa University, Ethiopia, 2Bureau of Agriculture, Ethiopia
Exploitation of biological N-fixation as a cheap and sustainable source of N for plants
requires identification of Rhizobia strain capable of inducing nodulation in plants and
effective in fixing atmospheric nitrogen. Experiments were conducted to evaluate symbiotic
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Symbiosis
effectiveness of forty indigenous Rhizobia isolates on Sesbania sesban and to determine their
host-ranges. Each isolate was grown on YEMB for 3-5 days in the Laboratory and inoculated
to pre-germinated Sesbania seeds in modified Leonard Jars. -Ve and +ve-N control
treatments were also included. The experiment was laid out in CR design with three
replications. Data on nodulation, plant growth and yield parameters; and plant tissue N
content were collected and subjected to ANOVA. Cluster analysis of data was also done. The
results revealed that all isolates except AC100e induced nodulation on S. sesban. The isolates
varied significantly in their effects on nodulation and plant growth parameters, biomass yield
and tissue N contents of S. sesban. Accordingly, 70% of the isolates produced significantly
higher growth and biomass yield of the plant than that produced in –N control. But, only 25%
of the isolates produced growth and biomass yield of the plant similar to that produced in
+N treatment. Cluster analysis data revealed that the 40 isolates were grouped in to six
clusters. Those isolates in cluster-VI were AC50b, AC51C, AC61a, AC61d and AC100c resulted
in the highest yield and N content of S. sesban and were best. The results of cross inoculation
study revealed that out of 10 isolates tested only 40 and 90% of the isolates induced
nodulation and growth on soybean and haricot bean respectively. It is concluded that there is
a high potential to isolate infective and effective Rhizobia strains capable of inducing
nodulation and N-fixation in S. sesban, Soybean and haricot bean from indigenous sources in
Ethiopia.
32
Arbuscular mycorrhizal fungal hyphal exudates prime bacterium mediated
phytate mineralization in hyphosphere
Lin Zhang*1, Minggang Xu2, Yu Liu3, Fusuo Zhang1, Angela Hodge4, Gu Feng1
1
China Agricultural University, China, 2Chinese Academy of Agricultural
Sciences, China, 3Zhejiang University, China, 4University of York, United Kingdom
Ecology and evolutionary biology seeks to understand how cooperative strategies evolve and
are maintained in species networks. Here, we focus on the three-partner relationship between
plants, arbuscular mycorrhizal fungi (AMF) and hyphosphere bacteria to ask if the interaction
between AMF and bacteria can pay back an essential resource (in this case phosphorus) to
their associated host plant by consuming plant derived carbon (C). A microcosm and two
Petri plate experiments which separate the plant roots, AMF hyphae and bacteria were
conducted to demonstrate the direct effects of hyphal exudates on the growth and activity of
bacteria in organic phosphorus (P) mobilization and, the reciprocal impact of the bacteria on
growth and activity of the AMF in P uptake and transfer to the plant. Results showed that
AMF released substantial C to the environment, triggering bacterial growth and activity
resulting in enhanced organic P mineralization and turnover. While, in return, bacteria
enhanced AMF hyphal proliferation which in turn, resulted in enhanced capture of the
available P released. Under low soil C:P conditions, the AM fungi-bacteria interaction
improved aboveground plant P nutrition. Our results suggest a C-P tradeoff occurs in plantAMF-bacteria systems. AMF and bacteria share the photosynthate of the plant, and as
reciprocation, the AMF -bacteria interaction repays the plant with P by jointly mobilizing soil
organic P forms and P acquisition for the plant through division of labour between these two
microbial groups.
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33
Specificity and resilience of the arbuscular mycorrhizal fungal community in
intensive agroecosystems
Jiachao Zhou*, Xiaojing Wang, Yang Chen, Gu Feng
The composition of arbuscular mycorrhizal (AM) fungi communities can have a large effect on
the performance of their plant hosts. The dynamic changes of AMF in ecosystem is hotspot
recently. It is generally believed that the growth period of plants influenced the AMF
community composition obviously. In agroecosystem, whether AMF communities can keep
stable and be helpful for crop growth under traditional fertilization and pesticide is yet
unresolved.
We have carried out indoor pot and field test, to study the effect od benomyl and N
fertilization on AMF communities and crop development. Changes in the community were
characterized by root colonization, cloning, sequencing, tRFLP and DNA copy. While we used
biomass and shoot P to measure crop development.
Benomyl inhibited AM fungi infection while N fertilization increased abundance of AMF,
some new batchs of fungi emerged after nitrogen added AMF diversity after nitrogen added.
Community composition changed at different development stages of crop growth. The study
found that after using benomyl G. intraradices appeard, the reason might be that G.
intraradices can produce rich exogenous hyphae network and has strong restorability, thus
adapt to benomyl.
In vegetative growth period, AMF abundance was larger than reproductive period and
different groups of AMF respond to differennt period altered. In vegetative period,
mycorrhizal colonization rate was positive correlation with shoot biomass, nitrogen and
phosphorus absorption. During reproductive growth stage, mycelium density was positive
correlation with phosphorus absorption. In addition, during reproductive period mycorrhizal
colonization has linear negative correlation with nitrogen nutrient absorption. In maturation
phase, mycelium density had certain relevance with corn biomass, grain yield and harvest
index.
AM fungi in this study differed greatly in their response to perturbation and can be helpful to
plant growth. In agroecosystems, AMF communities was important to crop even if apply
traditional fertilization and pesticide.
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Towards Application
36
ToT (transfer of technology) needs for promoting farmers on IPNM in
agricultural production system in Indian sub-continent
Manas Mohan Adhikary*, Anandamoy Puste, Kalyan Jana
Bidhan Chandra Krishi Viswavidyalaya (State Agricultural University), India
Modernizing agriculture is essential for meeting challenges of reducing hunger and poverty,
increasing & sustaining productivity. Agriculture is the backbone of growth for developing
countries like India, may achieve how well and how fast be able to manage a rural
transformation (ToT) on integrated plant nutrient management (IPNM). Keeping this, field
research on IPNM was emphasized during consecutive summer seasons at farmers’ field in
different agro-zones of this sub-tropics. Improvised field demonstrations were undertaken on
rice cv. Satabdi (IET 4786) in medium (S1) and medium-lowland situation (S2) to standardize
production trend with IPNM (both organic and inorganic sources), which comparable with
control and farmer’s practice [T1 - Control, T2 - Farmer’s practice, applied generally N:P2O5:K2O
@ 50:30:30 as inorganic sources, T3 - N:P2O5:K2O @ 120:60:60 as 100% inorganic and T4 and
T5 - 25% of N through FYM (farm yard manure) as organic, and 25% of N through green
manuring + 75% of N:P2O5:K2O @ 120:60:60 kg ha-1, respectively as inorganic sources].
Results showed that grain yield of rice were significantly increased with IPNM in balanced
form over farmers & control plot. Among organic sources, highest yield exhibited with green
manuring along (25% N) with 75% of N, P2O5 and K2O through fertilizers. Gaining yield of rice
is almost double in respect to farmers’ practice of the zones (Cluster I to IV). It reveals that
benefit-cost ratio had gone in favour of highest productivity of rice grain (3.58 t ha-1), using
maximum and balanced use of plant nutrients (IPNM), which is more compatible with the
nutrients available to the crop plants and this was economically viable (B-C ratio 2.28) to the
rural farming communities, it may be concluded that ToT field-based technological
intervention on IPNM is imperative for boosting up crop productivity and promoting
livelihoods of the rural farming community.
37
Combined use of Kosakonia radicincitans and Trichoderma harzianum in the
northern and southern hemisphere: First results
Beatrice Berger*1, Hernan Paillan Legüe2, Eduardo Donoso3, Silke Ruppel1
1
Leibniz Institute of Vegetable and Ornamental Crops, Germany, 2Universidad de
Talca, Chile, 3Bio Insumos Nativa, Chile
Microorganisms originating from nature are promising candidates to keep high yield gain
and quality standards of agricultural products. However, successful use of microorganisms in
bio-economy presupposes elucidation and understanding of microbial behavior in various
environmental niches. Our bilateral project, funded by the BMBF (Project Management
Jülich), between Germany (IGZ, Grossbeeren) and Chile (University of Talca) aims to study
mechanisms of plant-microbe interaction using the plant growth-promoting bacteria
Kosakonia radicincitans (German partner) and the fungal antagonist Trichoderma harzianum
strain Queule (Chilean partner) in interaction with various horticultural plants. In cooperation
with our Chilean partners we investigate the microbial mechanisms on the plant when grown
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under different nitrogen- and phosphor nutrition regimens, salinity stress under natural
occurring soil conditions in the southern and northern hemisphere. In detail, we will measure
changes in primary and secondary plant metabolism, moreover the nutrient up-take by the
plant and we will evaluate the growth -promoting potential of bacteria and fungi as well as
their colonization ability. Our results are intended to support the use of microbial products
worldwide. Here we present first results on the combination of K. radicincitans and T.
harzianum in greenhouse and field studies.
38
Efficacy evaluation of seed coated plant – root associated plant growth
promoting rhizobacteria in microcosm system
Rita Choudhary*, Alok Adholeya
The Energy and Resources Institute (TERI), India
Plant growth promoting Rhizobacteria (PGPR) are the important group of microorganisms
which play a major role in stimulating plant growth through mobilizing nutrients in soils,
producing numerous plant growth regulators, protecting plants from phytopathogens by
controlling or inhibiting them and by improving soil fertility. This study was conducted with a
view to isolate bacteria associated with roots of wheat, maize and soybean from different
locations of Punjab, Rajasthan and Madhya Pradesh, India. A total of 130 bacterial isolates
were screened biochemically for their plant growth promoting traits like phosphate
solubilization, production of Indole Acetic acid (IAA), hydrogen cyanide (HCN) and
siderophore. It was found that 58.5% of them showed IAA production, 22% showed
phosphate solubilization, 48.3% siderophore production, 70% showed HCN production,
whereas 9.7 % isolates showed all the plant growth promoting characteristics. Under
polyhouse conditions, the efficacy of these biochemically characterized isolates was
assessed. Formulations were prepared and an uniform <0.1mm thickness one layer coating
was done in NIKLAS (W5/0.1) seed coating machine for 30 seconds on maize seeds. Results
amply proved that by using plant beneficial rhizobacteria and by delivering viable number of
cells on seed surface, promote better germination, enhanced dry weight, plant height and
root length. Thus, these isolates have the potential not only to be used as biological seed
coating but are also effective in decreasing the global dependency on hazardous agriculture
chemicals which destabilize the soil health.
39
Soil protection against concentrated flow erosion with Arabidopsis roots
S. De Baets*1, T. Denbigh2, T. Liverpool3, I. V. Chenchiah3, B. Higgins3, T. A. Quine1, C.
Grierson2
1
College of Life and Environmental Science, University of Exeter, United
Kingdom, 2Biological Sciences, University of Bristol, United Kingdom, 3Department of
Mathematics, University of Bristol, United Kingdom
Soil resources are under more pressure than at any time in human history. The current extent
of global soil degradation, of which soil erosion and soil salinization are the dominant
processes, is estimated at ~2 × 109 ha. Soil erosion is responsible for a global contemporary
agricultural sediment flux on cropland of about 22 Pg year-1 and an additional approximately
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11 Pg year-1 is mobilized on pasture- and rangelands. This corresponds with a total
agricultural soil organic carbon erosion rate and hence soil fertility loss of 0.47 to 0.61 Pg C
year−1. Due to increased rainfall intensity, climate change will lead to a significant increase of
soil loss by 2050. Soil protection measures are therefore of great importance for future soil
conservation.
Plant roots have proven to be very effective in stabilizing the soil and protecting the soil
against erosion. However, no clear insights are yet obtained into the root traits that are
responsible for root-soil cohesion in order to better select the best species for soil protection.
Research using Arabidopsis mutants has made great progress towards explaining how root
systems are generated by growth, branching, and responses to gravity, producing mutants
that affect root traits. In this study, the relative importance of root traits (e.g. root hairs, ratio
primary/lateral roots) will be ranked by comparing the performance of selected Arabidopsis
mutants in root-soil cohesion assays. Our first results show that wild type Arabidopsis (Col-0)
is very effective in reducing concentrated flow erosion rates compared to other previously
tested soil protection crops and that other mutants, containing less root hairs or having less
laterals, are less effective. The results of this study can be used to rank root traits in order of
importance for reducing erosion and to define molecular markers to look for in crop plants,
optimizing soil protection.
40
Screening of biological and chemical treatments to control blackleg disease in
potato
Mout De Vrieze*1, Laure Weisskopf1, Santiago Schaerer1, Wolfgang Vogt2, Brice
Dupuis1
1
Agroscope, Switzerland, 2Sourcon Padena, Germany
In Switzerland, blackleg caused by Dickeya spp. in potato is the main cause of seed potato lot
rejection during field inspections for seed potato certification. In order to elaborate a control
strategy against this disease, nine Pseudomonas strains were evaluated for their potential to
reduce the development of blackleg symptoms on potato plants in the field. Their inhibitory
capacities were compared to those obtained for several biological and chemical treatment
products including disinfectants, plant extracts, elicitors, essential oil and fertilizer. Initially, all
candidate strains and products were tested in vitro for their ability to inhibit D. dianthicola’s
growth and pectinolytic activity. The three best strains and the two best candidates of each of
the other product categories were then selected for a greenhouse trial. A field trial was also
conducted for the three selected Pseudomonas strains and completed with the already
commercialized biological and chemical products. In both trials, treatment with the
candidates consisted of tuber treatments, as well as an additional soil treatment for the
antagonistic bacteria. Experiments performed in vitro and on potato tubers revealed that all
nine strains, as well as the tested disinfectant, essential oil and two plant extracts, were
capable of inhibiting the growth of Dickeya dianthicola. In the greenhouse, tuber and soil
treatment with the bacteria did not lead to symptom reduction. The disinfectant sodium
hypochlorite showed the most promising results, reducing the number of rotten stems per
plant by 67 % in comparison with untreated plants, while the elicitors Bion® and chitosan
showed slight symptom reduction, with reductions of rotten stems per plant of 20 and 15 %
respectively. However, in the field, the best protection was obtained with the bacterial
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antagonist Pseudomonas DSMZ 13134, the active ingredient of Proradix®, which reduced
disease incidence by 40 % in comparison with untreated plots.
41
Identification of a new cheap carrier for rhizobium inoculant
Soumaya Tounsi Hammami1, Sana Dhane Fitouri*1, Salah Rezgui1, May Granier2, Faysal
Benjeddi1
1
INAT, Tunisia, 2ATAE, Tunisia
Production and quality of rhizobial inoculants in several developing countries are frequently
limited that are attributed to inaccessibility of suitable carriers. Experiments were conducted
to evaluate the potential of a new affordable and widely available carrier material in Tunisia.
The carrier is a compost produced from the mixture of two native plant species: Arundo
donax L and Medicago arborea L.
Survival of Rhizobium sullae RSU9 was monitored over a period of 6 months at 4 ° C in sterile
and non-sterile conditions. Compost maintained rhizobial population during this period.
There was over than 109 and 107 rhizobia per gram of inoculant, in sterilized and unsterilized
conditions. Throughout the storage period, compost maintained rhizobial population similar
or higher than that observed in peat independently of the production conditions .
The effect of this carrier material on nodulation and growth of Sulla coronarium L. plants was
also investigated in glasshouse experiment. The sterilized compost showed the highest
number and dry weight of nodules, respectively 30 nodules per plant and 0.13 mg per plant.
It increased also shoot dry weight (0.31 gram per plant) compared to sterilized peat (0.25
gram per plant).
Compost can produce high quality, inexpensive inoculants, maintaining high bacterial
populations for at least 6 months. Further testing of this carrier material should be confirmed
to assess its efficiency with other rhizobial species or other microorganisms and under field
conditions.
42
Overcoming barriers for the microbial suppression of Rhizoctonia root rot on
wheat
Christopher Franco*1, Stephen Barnett2, Sophia Xue Lian Zhao3, Ross Ballard2
1
Flinders University, Australia, 2South Australian Research and Development
Institute, Australia, 3Flinders University, Australia
Root rot caused by Rhizoctonia solani AG8 is the major fungal root disease of cereals in low
to medium rainfall areas in southern Australia. The application of microbial inoculants is
being explored as a potential control option, because chemical and agronomic control
options have had limited success. Attempts at obtaining strains effective in the field have
been incosistent due to a lack of competence of the selected strains in the rhizosphere, and
because the strain taken into commercial development do not always possess optimal traits.
Success was achieved though collaboration of mycologists and bacteriologists (including
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actinobacteriologists) in testing a wide range (>2300) from a number of sources including the
ecto and endorhizosphere of native plants, cereals, legumes and included endophytes and
rhizosphere colonising microorganisms. The first phase of the high-throughput in planta
screening consisted of field soil with added R. solani in 50 ml tubes and test strains added as
a suspension directly to seeds. The 4.3 % of strains that reduced disease symptoms were then
screened in more rigorous disease bioassays and characterised for properties important for
the commercial development for such products. The 43 effective strains represented a
diversity of microbial genotypes that included fungi and 4 phyla of bacteria-Actinobacteria,
Firmicutes, Proteobacteria and Bacteroidetes. Further rigorous evaluation of minimum
effective inoculum reduced the number to 6 strains for field trails. A range of inoculum
delivery methods –from seed coating, drenches at different locations around the seed at
sowing- and testing methods to account for the patchiness of the disease- resulted in the
selection of three strains which were effective in a range of soil types in more than a single
season.
This holistic approach coupled with a team that understood each class of microorganism
showed how inoculants can be developed to be effective in the rhizosphere.
43
The effect of nitrogen fertilization on rooting patterns and nitrogen recovery of
catch crops
Dina in 't Zandt*1, Alison Arico2, David Lehnert2, Christian Fritz1, Florian Wichern2
1
Radboud University Nijmegen, Netherlands, 2Rhine-Waal University of Applied
Sciences, Germany
In agriculture, nitrogen is typically applied in excess to increase crop yield. However, crops
only take up 30-50% of the applied nitrogen leaving large amounts of nitrogen behind
causing nitrate leaching and subsequent pollution of ground, surface and coastal waters. To
reduce nitrate leaching, residual nitrogen can be immobilized in plant biomass by cultivating
so called catch crops after harvesting the main crop. The objective of our study was to
establish the relationship between root distribution and residual nitrogen immobilization by
catch crops. In addition, we studied the effect of nitrogen addition to stimulate plant growth
and nitrogen uptake. Since soil microorganisms compete with plants for nitrogen and
immobilize nitrogen, microbial biomasses were also estimated.
In a pot experiment, three catch crops, Raphanus sativus oleiformis L., Brassica rapa oleifera L.
and Phacelia tanacetifolia Benth., were grown in a loamy sand soil at three fertilizer levels: 0
kg ha-1, 40 kg ha-1 and 80 kg nitrogen ha-1. Catch crops decreased nitrate concentrations up
to tenfold compared to soils without catch crops. Interestingly, Brassica produced almost
twice as many roots as Raphanus, but recovered comparable amounts of nitrate. Phacelia, on
the other hand, captured a smaller fraction of the applied nitrogen, which was consistent with
a decrease in root length and an increase in root diameter. For both Brassica and Raphanus,
leftover nitrogen was comparable between all three nitrogen levels indicating that these
plants respond to higher nitrogen availability by increasing their nitrogen uptake.
Furthermore, nitrogen fertilization increased microbial carbon, but not nitrogen biomass,
whereas catch crop cultivation increased only microbial nitrogen biomass. In conclusion,
catch crops did not only contribute to reduced nitrogen losses by nitrogen uptake, but also
by stimulating microbial nitrogen immobilization.
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44
Effects of microbial fertilizer on soil chemical and biological properties of soil
cultivates with wheat, barley and corn plant in different climatic conditions
Nurgül Kitir*, Meti̇ n Turan
Yeditepe University, Turkey
This study was conducted to determine effectiveness of microbial fertilizers on soil chemical,
biological properties and biodiversity of soil cultivated with wheat, barley and corn plant
under different climatic conditions in Turkey. The Microbial Fertilizer was applicate in field
conditions to wheat, barley and corn plant 30 L/ha. Each treatment were conducted three
region that Kayseri, Urfa and Erzurum in Turkey. Plant and soil samples were taken at the end
of the growing period each plant and each region. Some soil enzymes such as acid and
alkaline phosphate, urease and dehydrogenase, amino acid exhausted from soil and plant
roots, and macro and micronutrients elements of plant, soil microbial type and amount of the
microorganism in the soil rhizospere were determined. The results obtained have shown that
amino acids, plant and soil enzymes exhausted from root parts significantly affected the
LifeBac NP microbial fertilizer. Results show that efficiency of LifeBac NP microbial fertilizer
was affected to plant species and region climatic conditions. All plant species and region that
studied LifeBac NP microbial fertilizer increased yield and yield parameters, and this
increasing value has been very significantly as statical.
45
Development of an atlas of fine roots of European tree species
Tanja Mrak*, Jožica Gričar, Hojka Kraigher
Slovenian Forestry Institute, Slovenia
Tree fine roots are an active component of belowground carbon cycle because of their fast
turnover rates, and the exchange sites for nutrients and water. In this part of the rhizosphere,
multiple interactions with various soil organisms occur, mycorrhizas being one of the central
for the functioning of the forest ecosystems. Identification of tree fine roots is needed to
elucidate the role of different tree species in belowground functional traits. Furthermore, tree
root identification may serve in the field of cultural heritage protection as tree roots may
cause structural damages to historical buildings. Since the molecular tools for identification
of tree fine roots are costly and do not allow for quantification of species occurrence, the
identification with anatomical-morphological approaches is a good alternative, especially for
routine work. In our study ten temperate European tree species were investigated and for
each species 3-5 individuals sampled. Roots of 5, 3 and 1 mm in diameter, as well as the most
distal fine roots, were embedded into paraffin, longitudinal and radial sections prepared and
studied with a light microscope. Morphology of fine roots was observed under the dissecting
microscope and photographed. Compared to the stem wood of the same species, root wood
differs in several characteristics, such as wedging growth rings, pattern of porosity, smaller size
of vessels etc. Some characteristics not present in stem wood occur in fine roots – e.g. central
channel in Abies alba Mill. roots. As the bark and primary tissues represents a high proportion
of tissues in roots of smaller diameter, their anatomical characteristics can be
used for identification purposes. The most important morphological characters for
identification are the colour and texture of the bark, pattern of ramification and type of
mycorrhiza.
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46
Analysis of secondary metabolites produced by different strains of Pseudomonas
chlororaphis isolated from halophytes, mesophytes and xerophytes
Salma Mukhtar*1, I Shahid1, Muhammad R2, Deeba Baig1, Rahman Saleem3, Kauser
Malik1
1
Forman Christian College (A Chartered University), Pakistan, 2Pakistan Council of
Science and Technology, Pakistan, 3School of Science and Engineering, LUMS, Pakistan
Several bacterial strains have been isolated from plants growing in diverse environments, such
as halophytes, mesophytes and xerophytes (paragrass, sugarcane, cotton, cactus), of Pakistan.
Among these, eight isolates were identified as strains of Pseudomonas chlororaphis subspecies
chlororaphis and P. chlororaphis subspecies aurantiaca, based on 16SrRNA gene sequence.
Four strains, RP4 (paragrass), ARS38 (cotton), FS2 (cactus) and PB-St2 (sugarcane),
representing isolates from three different habitats, were selected for detailed study. These
strains were screened for phosphate solubilization, indole acetic acid production, activity
against plant pathogens, phenazine O (phzO) and pyrrolnitrin A (prnA) genes. All strains were
positive for antifungal activity, indole acetic acid production, phzO and prnA genes.
Secondary metabolites produced by these strains were analyzed and compared with each
other by using MS technique. Phenazines, cyclic lipopeptides, homserine lactones, pyoverdin,
pyrolnitrin and derivatives of lahorenoic acid have been detected in variable amount in these
strains. Screening of these compounds against fungal pathogens is going on. These strains
have great potential to be used as biocontrol agent due to phenazines and antibiotic
production.
47
Studies on the bio-ecological characteristics and control methods of melon
necrotic spot nepovirus (MNSV) and its soil fungal vector, Olpidium spp.
Jin-woo Park*1, Kyung-Seok Park2, Se-weon Lee3
1
Agricultural Microbiology Division, National Academy of Agricultural
Science(NAAS), South Korea, 2National Academy of Agricultural Science(NAAS), South
Korea, 3Technology Cooperation Bureau, South Korea
Melon necrotic spot nepovirus(MNSV) transmitted by seed and fungal vector Olpidium spp.
in soil, is a most serious viral disease on melon. This study aimed to analyze the biological
characteristics of MNSV and the impact of soil environment on the outbreak of a disease, as
well as to select chemical control agents against Olpidium spp. which transmits MNSV. For
the detection of MNSV in plant and Olpidium spp. in soil, this study used the real-time PCR,
and genetic analysis of capsid protein gene of MNSV and resting spore ITS region of
Olpidium were performed, respectively. Based on the result, it was found that among MNSV
128 isolates, the homogeny of 34 isolates whose pathogenicity is strong was more than 96%
while 88~95% variations were confirmed over 84 isolates whose pathogenicity was weak. In
addition, the genetically homology was found to be higher related to Spanish and Israeli
strain than Japanese strain. Genetically analysis result showed that the Olpidium isolated from
MNSV occurring areas was identified to be Olpidium blassicae. As a result of the analysis of
the correlation between MNSV outbreak and in-plastic house environmental factors, it was
found that MNSV occurrence was severe in soil with alkalinity of more than 7.5 while
moisture in soil had no big impact on MNSV occurrence. The results of the study showed that
the proper soil temperature for MNSV outbreak was 25°C. The result of survey on the density
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of Olpidium spp. which transmits MNSV from the surface soil to 45cm on a 15cm unit by six
regions was found to be high at the layer near the surface soil of the region with high
Olpidium spp. From the seven kinds of agricultural chemicals selected for fungal vector
control, the Benomyl and Chlorothalonil water-dispersible powders were found to have high
control effect.
48
The interactions between plant, microorganism and soil affect Fe acquisition in
cucumber plants
Youry Pii*1, Alexander Penn1, Concetta Eliana Gattullo2, Ignazio Allegretta2, Roberto
Terzano2, Carmine Crecchio2, Tanja Mimmo1, Stefano Cesco1
1
Free University of Bolzano, Italy, 2University of Bari, Italy
Plants have evolved two different strategies (Strategy I and II) to cope with Fe shortage,
based on the exudation of organic and inorganic compounds to favor its mobilization and
the root uptake.
The role of the soil biotic component in the nutritional processes in the rhiszophere needs to
be elucidated, since plants inoculated with PGPR showed an increased content of nutrients
and a stronger resistance to abiotic stresses.
The aim of the present work is the evaluation of the physiological effects, induced by
Azospirillum brasilense in a calcareous soil on cucumber plants.
Plants were grown in hydroponic Fe deficient solution followed by a 7-day period of contact
with the A. brasilense-inoculated calcareous soil. At sampling, biometrics measurements,
quali-quantitative analyses of root exudates and analyses of the nutrients content in plant
tissues were carried out. Variations in soil mineralogy were assessed by X-ray powder
diffraction (XRPD).
Our results showed that A. brasilense facilitates plant growth in calcareous soils due to an
enhanced recovery from the micronutrient deficiency. A. brasilense increases most likely the
Fe availability within the rhizosphere by a) affecting the solubilisation of Fe thanks to the
siderophore release and b) up and down-regulating the exudation activity of plants with an
effect also on its molecular complexity. Further studies are needed to better understand and
highlight the interactions between these two mechanisms and microorganisms. In particular,
the present study shed light for the first time on two AAs, namely Gly and Glu, which could
be involved in the plant-microorganism-soil interaction for the retrieval of Fe within a
calcareous soil. XRPD analysis revealed a slight decrease of calcite and an increase of smectite
under Fe-deficiency conditions.
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49
Growth and nutrient uptake of sugarcane inoculated with diazotrophs
Veronica Reis*1, Valfredo Chaves2, Willian Pereira2
1
Embrapa Agrobiologia, Brazil, 2Universidade Federal Rural do Rio de Janeiro, Brazil
Sugarcane is a important crop for Brazilian economy by the production of sugar, ethanol and
energy. Technologies that favor growth and better use of nutrients using diazotrophic plant
growth promoting bacteria (PGPB) can reduce costs, environment impact and improve crop
yield. The aim of this work was to evaluate the effects of inoculation with selected PGPB in
the sugarcane varieties RB867515 and IACSP95-5000.
Germination was measure during 40 days in a sterile substrate sand/vermiculite and also
growth and biomass accumulation was measured in pots containing a mixture of sand/soil
during 50 days. Treatments used: uninnoculated control and inoculation with G.
diazotrophicus (Gd - strain PAL-5T); H. rubrisubalbicans (Hr - HCC103); H. seropedicae (Hs HRC54); A. amazonense (Aa - CBAMc) and B. tropica (Bt - PPe8T) applied in as a mixture or
individually by immersion using a sett composed of a single stem node. The assays used a
randomized block experimental design with 8 replications.
In general inoculation with PGPR improved germination, biomass accumulation and nutrient
uptake specially phosphorus and potassium but the response was dependent by the variety
and strain used, showing that plant-bacteria interaction was modulated by plant and bacteria
genotypes.
the magnitude of inoculation has a different response in each variety but the single
inoculation of Gd, Hr and Hs presented better results in the two sugarcane genotypes tested.
50
Enhancing the effective use of rhizobium inoculants by legume growers in
southeastern Australia
Maarten Ryder*1, Judith Rathjen1, Matthew Denton1, Ross Ballard2
1
School of Agriculture, Food and Wine, University of Adelaide, Australia, 2South
Australian Research and Development Institute, Australia
Legumes growers in Australia commonly use rhizobium inoculants. However farmers can be
unsure whether or how frequently inoculants should be used on a particular field. A
nodulation assessment guide has been made available (online at
http://www.agwine.adelaide.edu.au/research/farming/legumes-nitrogen/legumeinoculation/) that guides grain legume growers in determining whether or not the inoculation
of a crop has been successful. Preliminary surveys have been conducted in several farming
regions of southeastern Australia to test this approach. Although nodulation levels have often
been rated as adequate, poor nodulation of an inoculated crop should be investigated, and a
troubleshooting guide is being developed to help solve problems with nodulation. Poor
nodulation of an uninoculated crop implies that inoculation is advisable in future, to help
improve nodulation and nitrogen fixation rates. Farmers are also often uncertain about any
negative effects of mixing inoculant with other treatments at sowing, for example fertilizers,
trace elements or pesticides. The compatibility of faba bean rhizobia (R. leguminosarum bv.
viciae WSM1455) with a liquid zinc preparation used by farmers for improved plant nutrition
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was tested. When mixed in proportions used by the farmer, the zinc sulphate preparation
(final pH approx pH 3.2) was found to kill 80% of rhizobia within 10 minutes and no rhizobia
were detected after 2 h of incubation. This result clearly shows an incompatibility between
current farm practice and rhizobium survival, and has led to a change in method. Further data
on the impact of Zn, low pH and water quality on survival of rhizobia at sowing time will be
presented.
51
Two volatile organic compounds trigger plant self-defense against a bacterial
pathogen and a sucking insect in cucumber under open field conditions
Choong-Min Ryu, Geun Cheol Song*
KRIBB, South Korea
Systemic acquired resistance (SAR) is a plant self-defense mechanism against a broad-range
of pathogens and insect pests. Among chemical SAR triggers, plant and bacterial volatiles are
promising candidates for use in pest management, as these volatiles are highly effective,
inexpensive, and can be employed at relatively low concentrations compared with
agrochemicals. However, such volatiles have some drawbacks, including the high evaporation
rate of these compounds after application in the open field, their negative effects on plant
growth, and their inconsistent levels of effectiveness. Here, we demonstrate the effectiveness
of volatile organic compound (VOC)-mediated induced resistance against both the bacterial
angular leaf spot pathogen, Pseudononas syringae pv. lachrymans, and the sucking insect
aphid, Myzus persicae, in the open field. Using the VOCs 3-pentanol and 2-butanone where
fruit yields increased gave unexpectedly, a significant increase in the number of ladybird
beetles, Coccinella septempunctata, a natural enemy of aphids. The defense-related gene
CsLOX was induced by VOC treatment, indicating that triggering the oxylipin pathway in
response to the emission of green leaf volatiles can recruit the natural enemy of aphids.
These results demonstrate that VOCs may help prevent plant disease and insect damage by
eliciting induced resistance, even in open fields.
52
From seed to whole plant: seed defense priming by rhizobacteria and its
determinant dipeptide
Choong-Min Ryu, Geun Cheol Song*, Hye Khung Choi
KRIBB, South Korea
Seed priming is a technique to be controlled hydration and drying of seeds resulting in more
rapid germination when are re-imbibed. Defense priming that has been induced by pretreatment of certain beneficial microbes and natural/synthetic compounds can enhance
defense responses more rapidly or aggressively to biotic- or abiotic stresses. In this study, we
were newly developed a immerged technology of the two priming methods referred to as
“seed defense priming (SDP)” that is by seed priming with its supernatant (secreted
metabolites) from root-associated Bacillus spp. in order to induction of systemic resistance
(ISR) seven after transplanting to field. Seed defense priming mediated by bacterial
supernatants from the 1800 strains of Bacillus spp. isolated from various soil samples in
South Korea were tested ISR against Pseudomonas syringae pv. lachrymans (PSL) in cucumber
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seedlings. Symptom development on the SDP with strains PB69 and 1628 was reduced 40 and
28% respectively in vitro. Under field condition, pretreated pepper plants were assessed their
disease severity by infiltration of Xanthomonas axonopodis pv. vesicatoria (Xav) at 20, 30, and
40 days post-transplanting (DAT). SDP by Bacillus spp. strains PB69 and 1628 elicited ISR as
compared to control treatment at 20 and 30 but not 40 DAT. Cyclo (leu - pro) was isolated by
various column chromatography and NMR spectra from culture filtrates of PB69. SDP by cyclo
(leu - pro) (0.1 ppm) induced systemic resistance in cucumber plant. Our results indicate that
seed defense priming triggered by bacterial supernatants can be de novo method to induced
ISR even under field condition and cyclo (leu - pro) involves in the activator of plant defense
reactions, leading to induced resistance against PSL in cucumber.
53
Is thinking about a below-ground landscape useful for scaling up understanding
to practice?
Elizabeth Stockdale*
Newcastle University, United Kingdom
Below-ground processes result from the interaction of soil habitats and their associated
populations where the structure, composition and flows between these components are
critical in defining the outcome and rate of the processes observed at the soil scale. Soil can
be conceptualised as a series of linked habitats, including the rhizosphere, rather than a
single habitat for soil organisms.
Habitat types in soil:
Resources (places): root, root surface (rhizoplane), rhizosphere, organic matter (litter to old
humus), mineral surfaces.
Pores (spaces): storage (Air/water filled), transmission (AIR filled) and residual (WATER filled).
A multi-habitat (landscape) conceptualisation has been shown to provide a useful
representation of soil faunal populations. However, application of landscape ecology
approaches to below-ground ecology is not easy.
Above-ground landscape ecology is moving away from a simple patch-matrix view of
landscape and consequently connectivity is considered as an aggregate property of the
structural configuration of the landscape elements. Habitat characteristics must be defined
from across a range of scales and pattern prediction is complex and multifactorial –
interaction between access to resources and refuge from predators.
Habitat elements defined below-ground should clearly differ in quality; in the Table each has
distinctive physical and chemical characteristics together with distinguishable communities of
soil organisms. The context and connectivity of these elements are then key. The plant or
plant community integrates across the diversity of below-ground ecosystem functioning via
the roots and rhizosphere; in some way this role can be compared to that of the top predator
in above ground systems.
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This conceptual model is applied to an analysis of the impacts of agricultural management on
the size, activity and diversity of soil organisms and highlights the key role of the rhizosphere
in mediating plant-soil interactions and their resilience.
54
Innovative biochar (hydrothermal carbonization) as an additive to soil-substrate
for sustainable plant production
Maren Stollberg*1, Thorsten Kraska2, Ralf Pude2, Guido Dericks3, Ulrich Schurr1, Arnd
Kuhn1
1
Forschungszentrum Jülich, Germany, 2Friedrich-Wilhelms Universität
Bonn, Germany, 3Grenol GmbH, Germany
Just as natives peoples thousands of years ago started the story about “Terra preta”, an
anthropogenic fertile tropical soil, we would like to imitate this phenomenon today using
hydrothermal carbonization (HTC-biochar).
The advantage of HTC-biochar is the possible use of all kinds of wet organic material as a
parent material. We used digestate originating from biogas production which was converted
to charcoal in a reactor at 200°C and 20 bar within 6 hours. The better-known pyrolysisbiochar instead is based on dry woody material only.
In first pre-experiments we found a depression of Lactuca sativa var. crispa. growth after
adding HTC-Biochar to field soil to increase carbon content. Therefore our aim was to remove
any growth-reducing substances (e.g. aromatic compounds) by different extra treatments of
the HTC-Biochar. The biochar treatments were (1) drying at 80°C, drying and washing with (2)
water or (3) with 10% Ethanol solution.
5% HTC-Biochar (dry matter) was mixed with a silty loamy soil (field-soil, 40 soil-points,
sieving at 2 mm) in pots and either Zea maize, Lactuca sativa var. crispa. or Brassica rapa
subsp. pekinensis were grown on this substrate for 14-49 days. We performed a sequence of
3 - 4 harvests per species.
Growth parameters such as dry matter, leaf area and minerals were measured. Zea maize
showed after 14 days a negative effect on plant growth with untreated HTC-Biochar and also
of the pyrolysis variant. The biomass of these variants was increased up to 20% compared to
the control variant (pure soil) after 28 and 35 days. All HTC-Biochar treatments in comparison
to untreated HTC-Biochar -as well as compared to pure soil- had negative effects on plant
biomass.
Therefore our chosen extra treatment of HTC-Biochar did not lead to an improvement in
plant growth. Our assumption is that we lost mineral nutrients during these treatments.
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55
Tracing of Pseudomonas inoculants in root and rhizosphere samples
Carla Mosimann, Sarah Symanczik, Thomas Oberhänsli, Paul Mäder, Cécile Thonar*
FiBL (Research Institute of Organic Agriculture), Switzerland
Plant growth-promoting rhizobacteria (PGPR) are able to facilitate plant nutrient acquisition
and can act as biocontrol agents by suppressing soil-borne diseases. Efficient strains can be
formulated as microbial inoculants and their successful use for field application often requires
a certain ability of persistence in the soil where they are inoculated. In this respect, there is a
need to create tools enabling the tracing of inoculated PGPR which can also serve to monitor
their spread in space and time.
Here we report the development and application of a molecular method allowing the
quantitative detection of two Pseudomonas strains contained in commercial formulations. The
method is based on a Taqman qPCR assay targeting two polymorphic regions of the bacterial
genome in order to ensure the specificity of the detection. The assays have been used with
several samples (root or rhizosphere DNA) originating from various pot and field experiments
with maize as host plant. The first results achieved in pot experiments indicate that for one
strain the survival is influenced by the soil management (organic versus conventional) and
that in general the high abundance of native Pseudomonas strains will not prevent a
reasonable persistence of the inoculated Pseudomonas strains. In field conditions, the
method has shown that the strain survival was improved when inoculated in combination
with compost amendments.
More results using these detection tools will be presented and will highlight the set of
conditions (e.g. soil, inoculation techniques and frequency, co-inoculation or combination
with other amendments) associated with reasonable persistence of inoculated PGPR.
56
Altered carbon and nitrogen cycling in soils following biochar application
Tess van de Voorde*1, Simon Jeffery1, T. Martijn Bezemer2, Jan Willem Van Groenigen1,
Liesje Mommer1
1
Wageningen University, Netherlands, 2NIOO-KNAW, Netherlands
Biochar, pyrolysed biomass, is being widely promoted as a means to improve soil quality,
sequester carbon, and improve soil-based ecosystem services. However, large knowledge
gaps remain and the majority of research has been performed in managed agricultural land
or controlled pot experiments. We aimed to study the effects of biochar amendment under
semi-natural conditions. To do so we utilise a field experiment set up in 2011 in a nature
restoration area near Ede, The Netherlands.
Biochar was produced from cuttings collected from a local semi-natural grassland and
pyrolyzed at 400oC or 600oC. The field experiment consisted of 4 treatments in 6 replicate
blocks, resulting in 24 plots in total. The four treatments are: incorporation of biochar
produced at 400oC, biochar produced at 600oC, incorporation of the non-pyrolyzed cuttings
from which the biochar was produced, and a control treatment in which no material was
incorporated (Control). Biochar and residue were applied at a rate of 10 ton/ha and mixed
through the top soil layer (~10 cm).
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Three years later, in autumn 2014, we collected soil samples in all plots, which were used for a
series of experiments to investigate the functioning of the soil microbial community,
focussing especially on carbon (C) and nitrogen (N) cycling. We hypothesised that microbial
communities exposed to biochar (1) differ in their ability to utilise a range of carbon
substrates, and that (2) these communities will be better at decomposing more recalcitrant
substrates. Overall, net N mineralisation measurements, a denitrification inhibition assay,
substrate induced respiration experiments and a MicroRespTM assay (both using multiple Csubstrates), showed increased CO2 production in the soils that received biochar and hay
residue, and reduced N2O production in the biochar amended soils, as compared to the noamendment control. We will link these findings to functional gene activities using GeoChip
5.0.
57
Root traits and aboveground yield of silage maize varieties under field
conditions
Nick Van Eekeren*1, Natalie Oram2, Joachim Deru1
1
Louis Bolk Institute, Netherlands, 2Nature Conservation and Plant Ecology, Wageningen
University and Research Centre, Netherlands
Drought tolerance of agricultural crops is critically important in the face of water shortages
caused by climate change and competition for drinking water. Silage maize is a major fodder
crop for the dairy industry on dry sandy soils in The Netherlands. Root architectural traits play
a key role in improving drought tolerance of crops. Our objective was to study root
architecture of silage maize in relation to aboveground production under field conditions. An
experiment with 14 commercial maize varieties was carried out on a sandy soil in Loosbroek,
The Netherlands. The experiment was managed according to common agricultural practice.
At harvest, aboveground nitrogen content (g N / kg dry matter) and yield (kg N / ha) were
measured along with other yield components (dry matter, starch, calculated feeding value).
Root traits were quantified in two ways: 1) using ‘shovelomics’: a practical in-field
measurement of brace and crown root number, angle, and branching; 2) harvested brace and
crown roots were dried, weighed and scanned. Root length was determined with image
software, discerning lateral from main roots. Aboveground, results show that nitrogen
content differed between varieties and nitrogen yield did not. In root architecture, we found
differences in crown root branching and root weight between varieties but not in root
biomass or angle. Variety effects were also found in the proportion of lateral root length in
total root length. Aboveground nitrogen yields were significantly positively correlated with
root traits: total root length (R2 = 0.29), lateral root length (0.28) and crown root branching
(0.32). There was no correlation between root traits and nitrogen content. Equivalent
responses were found for the other yield components. Our results show that root traits that
are quickly assessed in the field could be used in maize breeding to improve drought
tolerance without affecting yield components.
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58
Root properties of dike vegetation and their effects on concentrated flow
erosion
Wouter Vannoppen*1, Jean Poesen1, Sarah De Baets2, Matthias Vanmaercke1, Patrik
Peeters3, Bart Vandevoorde4
1
KU Leuven, Belgium, 2University of Exeter, United Kingdom, 3Flanders Hydraulics
Research, Belgium, 4Research Institute for Nature and Forest, Belgium
The predicted climate change and the associated sea level rise are major challenges for the
near future. The Scheldt basin will be exposed to an increased risk of flooding due to wave
overtopping. To safeguard the land from dike breakthrough both above-ground and belowground biomass are important to keep the erosion resistance of the dikes sufficiently high.
Plant roots are more effective in reducing soil erosion by concentrated flow compared to plant
shoots. Therefore, the main goal of this study is to determine the erosion-reducing potential
of the belowground biomass of dike vegetation types. Root properties of five dike vegetation
types were studied: i.e. species-rich grassland (SR), species-rich grassland dominated by
Arrhenatherum elatius (SRAe), grassland dominated by Arrhenatherum elatius (Ae), grassland
dominated by Arrhenatherum elatius with few nettles (AeN) and nettle- dominated vegetation
(NDV). The erosion-reducing effect (RSD) was estimated using a Hill curve model linking RSD
to root length density (RLD, km/m³) based on an analysis of a global database on the erosionreducing potential of plant roots. Results based on the measured RLD in the topsoil (0-5cm)
indicate that erosion rates would be reduced by more than 80% compared to a bare soil for
dike vegetation types without nettles (129 < RLD < 235 km/m³). Dike vegetation types with
nettles (22 < RLD < 58 km/m³) were less effective in controlling erosion due to concentrated
flow. The degree of overgrowth of grassland by nettles, which have thicker roots and therefore
a lower RLD, explains this pattern. To maintain a high resistance of the topsoil against
concentrated flow erosion it is important to avoid the overgrowth of grassland by nettles
through an effective management of the dike vegetation. Preventing the enrichment of the
topsoil by nutrients is therefore an important measure.
59
How do plant roots affect rill and gully erosion?
Wouter Vannoppen*1, Jean Poesen1, Matthias Vanmaercke1, Sarah De Baets2
1
KU Leuven, Belgium, 2University of Exeter, United Kingdom
An important ecosystem service of plant roots is their effectiveness in controlling
concentrated flow erosion rates. However an extrapolation of model results from individual
case studies to estimate the erosion-reducing potential of plant roots is not reliable as rootsoil interactions in different types of environments, with differences in both root and soil
characteristics are not yet fully understood. The objectives of this study were therefore: i) to
provide a state of the art on studies quantifying the erosion-reducing potential of plant roots
in controlling concentrated flow erosion rates; and ii) to explore the overall trends in erosion
reduction as a function of root density (RD, kg/m³) and root length density (RLD, km/m³),
root system architecture and soil texture, based on a meta-analysis of published research
results. We therefore compiled a dataset of measured relative soil detachment rates (RSD) for
RD and RLD. The decreases in RSD as a function of RD and RLD could be best described by a
Hill curve model. Taking into account root system architecture and soil texture improved the
model accuracy, especially for root length density. Fibrous root systems are in general more
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effective in controlling soil erosion by concentrated flow as they have a larger root-soil
contact. As there was little variation in soil types among the collected data, the effect of soil
texture on the erosion-reducing potential of plant roots could not be proved. The remaining
unexplained variance is attributed to measuring errors and differences in experimental set
ups (e.g. plant species tested, soil characteristics) that could not be explicitly accounted for.
By taking these uncertainties into account we were able to establish relationships to assess
the likely erosion-reducing effects of plant roots which are reliable irrespective of differences
in root and soil characteristics.
60
Mixtures of maize genotypes produce more biomass than monocultures when
mycorrhizal
Xinxin Wang*1, Ellis Hoffland1, Gu Feng2, Thomas Kuyper1
1
Wageningen University, Netherlands, 2China Agricultural University, China
Arbuscular mycorrhizal fungi can play a key role in enhancing plant productivity in
multispecies natural ecosystems; however, their role in enhancing crop productivity and P
uptake efficiency in single species genotype mixtures is hardly known. Therefore, we grew
monocultures (one genotype) and mixtures of two genotypes of maize in low P soils in a
greenhouse (with Funneliformis mosseae) and in a field experiment with an unidentified
species mixture. We measured P uptake, hyphal length density and plant biomass. Genotype
mixtures showed overyielding and enhanced P-uptake when mycorrhizal but not when nonmycorrhizal. The increase in relative yield total and P uptake was largely due to
complementarity effects, and not to enhanced competitive ability of the larger genotype.
Genotype mixing increased hyphal length density. The effects occurred both in the
greenhouse and in the field. Our results suggest that genotype mixing increases the extent
and activity of the mycorrhizal network. So mixing maize genotypes may be beneficial for
enhancing productivity and P uptake efficiency.
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Rhizo Remediation and Fate of Pollutants
62
On-site PAH removal and microbial diversity during six-years monitoring under
plant-assisted attenuation
Thierry Beguiristain*1, Amélia Bourceret1, Aurélie Cébron1, Noële Raoult2, Pierre Faure1,
Emilie Tisserant3, Pascal Poupin1, Pascale Bauda1, Corinne Leyval1
1
LIEC UMR 7360 CNRS-Université de Lorraine, France, 2GISFI, France, 3IAM UMR1136,
INRA, France
During the XXth century, the intensive coal and steel industries contributed to the chronic
pollution of soils. Their dismantling left large areas of wasteland soils highly contaminated by
recalcitrant organic compounds such as polycyclic aromatic hydrocarbons (PAH). We are
interested in studying the potential for soil bioremediation using plants assisted
rhizodegradation combining the potential of plant with those of rhizospheric
microorganisms.
Our objectives were to evaluate the fate of PAH and characterize the microbial diversity of an
aged contaminated soil using on-site natural attenuation in experimental plots. A 24 plots
devices with 6 different treatments in 4 replicates allowed the comparison between bare soil
and different rhizospheric soils (alfalfa, naturally colonising vegetation) during 6 years
monitoring. Soil characteristics and fate of pollutants were analysed together with microbial
parameters. Our work is one of the first to characterize the microbial diversity in such aged
PAH-contaminated soil. The impact of plant rhizosphere on both bacterial and fungal
densities and diversities was assessed using qPCR and pyrosequencing.
During this 6 year period of time an increase of pH and C/N was observed and a 50 % PAH
removal was estimated. Plants had a limited impact on these soil parameters. On the
contrary, plants favoured a higher density and originally increased the diversity of
microorganisms. The bacterial community was dominated by Proteobacteria, Actinobacteria
and Bacteroidetes and the fungal community was mainly represented by Ascomycota.
Moreover some OTUs were foster by plants, such as members of Arthrobacter, Fusarium,
Bionectria, Acremonium genera. Similarly, the plant rhizosphere seemed to favour the PAHdegrading bacteria belonging to the Actinobacteria as shown by qPCR targeting PAH-ring
hydroxylating dioxygenases. Plants seemed to favour the PAH-degrading functional
community but, in such aged contaminated soils, the main limiting factor to their activity
remained the low PAH-bioavailability.
63
Dark-septate endophytic fungi: a solution for trees on metal-contaminated
sites?
Damien Blaudez*1, Charlotte Berthelot1, Julie Foulon2, Michel Chalot2, Corinne Leyval1
1
Université de Lorraine, France, 2Université de Franche-Comté, France
Dark-septate endophytes (DSE) are conidial or sterile ascomycetous fungi that colonize living
plant roots without causing apparent negative effects. DSE comprise a heterogeneous group
244
of root associated endophytic fungi, characterized by melanized inter- and intra-cellular
running hyphae and microsclerotia within the epidermis and/or the cortex of plant roots.
High tolerance of DSE to metal pollution and their relatively high abundance in contaminated
habitats suggest that DSE might have an important function for host survival in these
extreme conditions.
Fungal endophytes could affect heavy metal uptake of their host plants and increase plant
metal tolerance. Therefore, in the context of phytoremediation assisted by symbiotic fungi,
we have first isolated a set of DSE strains from poplar roots from metal-polluted sites. A
wide-range screening of the strains was performed to select the best promising symbionts.
Fungal isolates were identified as members of the Phialophora, Cadophora, Leptodontidium,
and Exophiala genera. They were characterized for their plant growth promoting abilities,
through different tests such as production of indol-3 acetic acid (AIA), release of volatile
organic compounds or production of antifungal compounds. Metal tolerance of the fungal
isolates was also studied under axenic conditions.
For the most promising strains, an inoculation experiment was performed to monitor the
effects of the fungi on the growth of poplar and birch on metal-contaminated soils.
Moreover, we also investigated the interaction between DSE and endomycorrhizal fungi
through dual inoculations of host plants.
64
Degradation of iprodione, vinclozolin and propanil by an Arthrobacter spp.
strain isolated from a pristine rhizospheric soil
Marco Campos*1, Dimitrios Karpouzas2, Maria Cristina Diez3
1
Universidad de la Frontera, Chile, 2Department of Biochemistry and Biotechnology,
University of Thessaly., Greece, 3Chemical Engineering Department, Universidad de La
Frontera., Chile
Biological degradation constitutes the major processes controlling the dissipation of
pesticides in soil. In this way, rhizodegradation in cooperation with pesticide-degrading
bacteria could be a suitable tool to avoid point source contamination by iprodione,
vinclozolin and propanil pesticides. We aimed to test the degradation of iprodione,
vinclozolin and propanil by a rhizospheric bacteria strain isolated from a pristine grassland.
Rhizospheric soil samples collected from ryegrass (Lolium perenne) grassland with and
without previous exposure to pesticides were used for the bacterial isolation. After several
enrichment in mineral soil medium (MSM) amended with iprodione, the isolation of an
effective iprodione-degrading culture (C2.7) was reached from the pristine soil enrichments.
Molecular fingerprinting revealed that C2.7 was composed of two strains identified via
cloning as Arthrobacter spp. (C1) and Achromobacter spp. (C2). Degradation studies with the
purified strains C1, C2 and their combination in minimal and rich media showed that C1 was
the key iprodione-degrader, whereas C2 was only able to slowly co-metabolize iprodione.
After that, C1 capacity to degrade pesticides of similar chemical structure to iprodione
(vinclozolin, procymidone, propanil, diuron and isoproturon) was investigated. This strain
completely degraded vinclozolin, a chemical analogue of iprodione belonging to the
dicarboxamide family, in 20 days, and this was accompanied by bacterial growth and the
245
formation of 3,5-dichloraniline. In contrast, this only partially degraded propanil with the
production of small amounts of 3,4-DCA, while no degradation of procymidone (the other
member of the dicarboxamide fungicides family) diuron and isoproturon were observed.
These results provided first evidence that the degradation of iprodione proceeds via cleavage
of the carboxamide bond common in iprodione and vinclozoline. Additionally, degradation of
propanil provide significant information about the degrading versatility of our Arthrobacter
spp. strain for future application in rhizodegrading systems.
65
Short-term effect of plants on the identity, abundance and activity of
phenanthrene-degrading microorganisms slows down bioremediation in a
contaminated soil
François Thomas, Aurelie Cebron*
LIEC, UMR7360 CNRS-Universite de Lorraine, France
Microbial degradation is a promising soil remediation strategy for polycyclic aromatic
hydrocarbons (PAHs) frequently polluting post-industrial environments, but the effect of
plants on PAH dissipation rates and overall microbial community diversity and activity, is still
unclear. Here, we compared the short-term dynamics of pollution, microbial communities
and PAH-degraders in bare or ryegrass-vegetated aged-contaminated soil, using
phenanthrene as a model PAH. Actively growing roots were allowed to colonize
phenanthrene-recontaminated soil for 2 to 10 days in compartmented microcosms. Samples
were collected from rhizospheric soil directly adherent to roots, bulk soil from vegetated
microcosms and bare soil from non-vegetated controls. Phenanthrene concentrations were
significantly lower after 10 days in bare soil than in rhizospheric or bulk vegetated soils.
Measurements of total dissolved organic carbon, organic acids and carbohydrates showed
that root exudation provided labile substrates that might be preferentially consumed instead
of phenanthrene, therefore impeding its dissipation. Although the abundance of 16S rRNA
genes and transcripts increased throughout the time course for both Bacteria and Archaea,
these communities were more active in rhizospheric than in bulk vegetated soil after 8 days.
In contrast, while plants favored the abundance of PAH-degrading genes compared to bare
soil, their transcription level was similar in all conditions except after 10 days where the
Actinobacteria activity was enhanced in bulk vegetated soil. To specifically target
metabolically active PAH-degraders, similar microcosms were spiked with 13C-labelled
phenanthrene in a stable isotope probing experiment. After 10 days, 16S rRNA gene
fingerprinting revealed that 13C-phenanthrene was metabolized by different bacterial taxa
depending on the presence of plants. Metagenomic characterization of the 13C-DNA fractions
is underway. Together with the analysis of variations in total and active microbial community
composition over the 10 days, it will provide an unprecedented view of the effect of plants on
the identity of PAH-degraders in aged-contaminated soils.
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66
Characterizing zinc tolerance genes in Suillus luteus, an ectomycorrhizal fungus
with properties promising for use in phytostabilization applications
Laura Coninx*1, Joske Ruytinx2, Michiel Op De Beeck3, Vangronsveld Jaco1, Colpaert
Jan1
1
Universiteit Hasselt, Belgium, 2INRA, France, 3Lund University, Sweden
Pyrometallurgical industry and mining activities have led to the contamination of vast areas
with heavy metals. In these areas, biodiversity of plants and micro-organisms is often greatly
reduced. Surviving organisms are subjected to a high selection pressure for metal tolerance,
often resulting in the evolution of metal tolerant ecotypes of plants, fungi and bacteria. Zn
contamination in the northern part of Limburg (Belgium), has led to the evolution of Zn
tolerant ecotypes of Suillus luteus (L.) Roussel, an ectomycorrhizal basidiomycete that forms
symbiotic associations with Pinus sylvestris L. These Zn tolerant ecotypes thrive in heavily
contaminated soils and in the meantime protect their hosts from metal toxicity. This
protective feature combined with the fact that S. luteus is a pioneer species common to sandy
soils in temperate climate regions make S. luteus a suitable candidate for use in
phytostabilization applications. However, to fully exploit the potential of such applications, a
better understanding of the Zn tolerance mechanism is crucial. Previous investigations have
shown that the basis of the tolerance trait is a mechanism promoting Zn efflux. Yet, since Zn
is an essential nutrient, many homeostatic pathways are expected to be involved in
maintaining an optimal Zn concentration in all cell compartments. This makes it a challenging
task to characterize the tolerance mechanism. Hence, we first focused on establishing which
Zn homeostatic pathways are present in S. luteus. Here we report 7 Cation Diffusion
Facilitator proteins and 4 Zrt- Irt- like Proteins that have been identified in the S. luteus
genome. Further characterization of these proteins and their response to increased zinc
concentrations in zinc tolerant and sensitive isolates may lead to a better characterization of
the zinc tolerance mechanism in S. luteus.
67
Bacterial resources for assisted phytostabilization of acid mine drainageaffected mountain stream bank
Anna Corsini*1, Sarah Zecchin1, Milena Colombo1, Nicoletta Guerrieri2, Giorgio
Lucchini1, Gian Attilio Sacchi1, Lucia Cavalca1
1
University of Milano, Italy, 2CNR-ISE, Italy
Several areas of the Italian Alps have an arsenic content that exceeds the Italian law limit (20
mg kg-1, D.Lgs 152/2006), due to mineralogy of bedrock and to mining activities. Rio Rosso
mountain stream (Anzasca Valley, Piedmont), is affected by arsenic (1015 mg kg-1) and iron
(22 g kg-1) contaminated sediments that leach from an abandoned gold mine. In view of a
bacterial-assisted phytostabilization action, rhizospheric bacteria and root endophytes of
liverwort, fern and willow inhabiting stream bank were isolated and characterized. A total of
240 colonies were isolated and screened for arsenic-related features and plant growpromoting traits. Most of rhizobacteria and endophytes were resistant up to 7500 mg L-1
arsenate (21 and 20 respectively) and up to 750 mg L-1 arsenite (17 and 24 respectively),
regardless plant species. More than 80% of the isolates were capable to reduce 75 mg L-1 of
arsenate, while arsenite oxidation rarely occurred (<5% of the isolates), reflecting the
prevalence of As(V) in the sediments. The endophytes of fern and willow mostly exhibited
247
indole acetic acid and exopolysaccharide production, while rhizobacteria primarily possessed
P solubilizing activity. This evidenced that the two different plant compartments exerted an
effect on plant grow-promoting traits. These were homogenously distributed among
rhizobacteria and endophytes of liverwort.
A super imposed selective pressure was exerted by arsenic on the plants of the mountain
stream bank, thus resulting in the distribution of arsenic resistance in plant rhizobiomes. The
bacterial resources characterized in the present study will be exploited for the development
of a bacterial-assisted phytostabilization of the arsenic contaminated stream.
68
Evaluation of the bioremediating ability of bacterial endophytes using
nematodes as bioindicators in bioenergy crop
Aoife Egan*, Thomais Kakouli-Duarte
Institute of Technology Carlow, Ireland
Nematodes are representative of their habitat, they respond quickly to disturbance in the soil
composition and are therefore well suited as bio-indicators for environmental monitoring.
Bioenergy crops, hosting certain bacterial endophytes, can be both utilised for bioenergy
production and in bioremediation efforts, if they are grown on contaminated land. The work
presented here utilises nematodes as indicators of soil health to evaluate the effectiveness of
the bioremediation process of oilseed rape. This work is currently ongoing. The plants were
grown in nickel contaminated soil in the presence of endophytic bacteria. Two approaches
were adopted to evaluate nematodes as indicators of bioremediation efficiency: (1) Nematode
assemblages in the rhizosphere of the bioenergy crops were characterised morphologically. In
addition, a molecular analysis was performed using polymerase chain reaction of the 18S
gene, followed by denaturing gradient gel electrophoresis. So far, results in the control
samples (nickel free soil) were indicative of favourable conditions free from stresses. The most
commonly occurring coloniser persister value was 4, with a maturity index of 4. The Shannon
wiener index varied from 1.73-2.54 and the Simpson index varied from
0.80-0.99. (2) Transgenic Caenorhabditis elegans biosensors are utilised to assess the nickel
bioremediating capacity of the bacterial endophytes. Both approaches aim to indicate the
level of soil health and evaluate the phytoremediating capacity of oilseed rape in the
presence of endophytic bacteria. The ability of nematodes as indicators of soil health in this
system will be discussed.
69
Impact of diesel on the symbiotic partners Medicago truncatula/Rhizophagus
irregularis in in vitro conditions
Mónica Garcés*1, Maryline Calonne2, Stephan Declerck2
1
Université catholique de Louvain UCL, Belgium, 2Université catholique de
Louvain, Belgium
The tropical rain forest of Ecuador located in the Amazon region, in particular the Yasuni
Reserve, is a hotspot of diversity. It also contains a major reservoir of oil. Since 1970, the oil
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industry development has become a serious threat to the environment, resulting in pollution
of soil. The remediation of polluted soils by crude oil has become a priority for Ecuador.
Currently, there is an increased interest in phytoremediation methods assisted by arbuscular
mycorrhizal fungi (AMF). Indeed, these fungi colonize the majority of plant roots and are
considered essential for the survival of many plants in polluted environments.
In the present study, we investigated in monoxenic culture conditions the direct impact of oil
on the symbiotic partners. The host plant Medicago truncatula and the AMF Rhizophagus
irregularis MUCL 41833 were cultivated in absence or in presence of diesel which was added
to the culture medium at the concentrations of 0.05; 0.1; 0.5 and 1%.
The results demonstrated that the fungal spore germination as well as plant growth were
inhibited in the presence of the two highest concentrations of diesel. As a consequence, this
beneficial association could be hampered under such polluted conditions. These preliminary
results open the door to investigate the mechanisms behind this inhibition. The in vitro
system also allows the study of the impact of diesel on one of the major function of the
mycorrhizal symbiosis, i.e. the nutrient transport from the fungus to the roots.
70
Ecopiling: A combined rhizoremediation and passive biopiling system for
remediating hydrocarbon impacted soil
Kieran Germaine*1, Xuemei Liu2, David Ryan1, David Dowling1
1
Institute of Technology, Carlow, Ireland, 2MicroGen Biotech Ltd, Ireland
Remediation of large tracks of hydrocarbon contaminated land can be labour intensive and
extremely expensive remediation projects. Biopiling is an ex situ bioremediation technology
that has been extensively used for remediating a wide range of petrochemical contaminants
in soils. Biopiling involves the heaping of contaminated soils into piles and stimulating the
biodegrading activity of microbial populations by creating near optimum growth conditions.
Rhizoremediation is another very successful bioremediation technique and involves the use
of plants and their associated microbiomes to degrade, sequester or bioaccumulation
pollutants from contaminated soil and water. The objective of this study was to investigate
the effectiveness of a combined phytoremediation/biopiling system to remediated
hydrocarbon impacted industrial soils. The effectiveness of the Ecopiling process was
investigated at two former industrial sites. One site was a food manufacturing site and held
~4800m3 of soil contaminated with waste engine oil. The second site was a former wood
preservation facility with soil heavily contaminated with creosote. At both sites the
contaminated soil was amended with chemical fertilisers, inoculated with petroleum or
polycyclic aromatic hydrocarbon degrading bacterial consortia and used to construct passive
biopiles. Finally, a phyto-cap of Perennial rye grass (Lolium perenne) and White Clover
(Trifolium repens) was sown on the soil surface. Monitoring of important physico-chemical
parameters and hydrocarbon concentrations were carried out at regular intervals throughout
the trials. The Ecopile system is a multi-factorial bioremediation process involving biostimulation, bio-augmentation and phytoremediation. One of the key advantages to this
system is the reduced costs of the remediation process, as once constructed, there is little
additional costs in terms of labour and maintenance (although the longer process time may
249
incur additional monitoring costs). The other major advantage is that the aesthetics and
many ecological functions are rapidly restored to the site, with concurrent bioremediation
processes taking place within the piles.
71
Impact of aerobic-anaerobic interfaces established in the rice rhizosphere on the
fractional distribution of pentachlorophenol and the microbial community
Malik Hayat*
COMSATS University, Pakistan
Rhizoremediation is an emerging technology for remediating organic pollutants such as
pentachlorophenol (PCP) in soils. Wetland plants like rice contain aerenchyma tissue, which
channel air transports from the leaves to the roots, and uniquely develop aerobic-anaerobic
interfaces in the rhizosphere. To investigate the rice rhizosphere effects on the dynamic
changes of various extractable fractions of PCP and the microbial community, a glasshouse
experiment was conducted by using a rhizobox in which rice seedlings were grown for 45
days. The soil was spiked with 20 and 45mg kg-1 PCP. Soil in the rhizobox was divided into
five compartments at various distances from the roots. Sequential PCP extractions were
conducted with three extractants: CaCl2 (0.01molL-1), butanol (99%), and DCM (99%).
Butanol extractable form of PCP showed a significant difference in the rhizosphere at 3mm
distance from the roots at both PCP levels. Thirty four phospholipid fatty acids (PLFAs）in the
rice rhizosphere were identified in soils given the two different PCP concentrations. The total
soil PLFAs concentration in the planted soils ranged from 29 to 52 nmol g-1. The highest
concentration of PLFAs was at 3 mm distance from the roots at both PCP concentrations. The
results suggested that the aerobic-anaerobic interface established by the root in the
rhizosphere of rice and microbial community in the rice rhizospher sheowed significant role
in the dissipation of PCP.
72
Improving phytoremediation of chlorendic acid by exploiting plant-associated
microorganisms
Inge Jambon*, Francois Rineau, Nele Weyens, Robert Carleer, Jaco Vangronsveld
Hasselt University / Centre for Environmental Sciences, Belgium
The possibility to remediate an industrial site contaminated with chlorendic acid, a fire
retardant and expected carcinogen, by applying phytoremediation using poplar (Populus
deltoides x (trichocarpa x deltoides) cv. Grimminge) is investigated. To improve
phytoremediation efficiency, plant-associated (including rhizospheric and endophytic)
microorganisms can be exploited. Bacteria can use organic pollutants as a carbon source and
metabolize them to a greater extent than plants. Moreover they possess plant growthpromoting characteristics. Fungi can produce extracellular enzymes that act on a broad array
of organic compounds, thereby degrading them. However, no biological degraders were yet
described for chlorendic acid. Therefore, the aim of this research is to isolate chlorendic aciddegrading plant-associated microorganisms, which can later be applied together with poplar,
to obtain an efficient phytoremediation of chlorendic acid whereby the plants bring the
contaminant into close contact with the degrading microorganisms.
250
To find suitable microorganisms, the cultivable plant-associated bacterial and fungal
community of the chlorendic acid-contaminated site was isolated and screened for their
degradation capacity. Of the 75 isolated fungi, 4 were able to significantly lower the
concentration of chlorendic acid after 2 weeks, one even to 29% of its original concentration.
After selective enrichment of a soil sample, 1 bacterial consortium was found to significantly
decrease the concentration of chlorendic acid, but only to 94% of its original concentration.
The role of the isolated fungi in the degradation of chlorendic acid therefore seems more
important than that of the bacteria. However, plant-associated bacteria can still improve
phytoremediation by promoting plant growth. Therefore, the isolated bacteria are screened
for different plant growth-promoting characteristics, after which a consortium will be
selected, consisting of chlorendic acid-degrading fungi and plant growth-promoting
bacteria. The effect of inoculation of poplar with this consortium on the phytoremediation
efficiency will then be evaluated in a greenhouse experiment.
73
Influence of silicon on phytotoxicity and uptake of antimony(V) in maize
Miroslava Vaculikova1, Ying Ji*2, Susan Tandy2, Rainer Schulin2
1
Slovak Academy of Sciences, Slovakia, 2ETH Zürich, Switzerland
With the increasing antimony emissions on a worldwide scale, Sb-polluted soil are receiving
more and more attention. Of particular concern is the potential transfer of this toxic element
into the human food chain, as the mechanisms of Sb uptake by plants are not sufficiently
understood. On the other hand, silicon (Si) has been reported to protect plants from various
types of stress including heavy metal toxicity. Thus, we addressed the question how Si would
affect the uptake and phytotoxicity of Sb.
In a hydroponic experiment, we investigated how Sb(V) at concentration varying between 0
and 30 mg L-1 affected root growth and anatomy of maize seedlings in presence and
absences of 70 mg L-1 Si. Increasing Sb concentration led to a decrease in root length, root
surface and aerenchyma. The addition of Si diminished these effects. It also increased the
distance from root tip at which the Casparian bands formed, possibly explaining why Si
increased the uptake of Sb into the shoots at low to medium Sb concentrations (5 to 10 mg
L-1). The Casparian bands are known to interrupt the apoplastic pathway from the root cortex
to inner root cylinder and thus inhibit the transfer of solutes in the apoplast from entering
the xylem and being translocated into shoots. At the highest Sb concentration (30 mg L-1), at
which the plants showed the most severe toxicity symptoms, the addition of Si greatly reduced
Sb uptake into the shoots. The mechanism behind this is unknown and needs further
research.
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74
Root elongation responses of a drought tolerant shrub species Acacia chisholmii
to elevated concentrations of cobalt and nickel in solution
Sebla Kabas*1, Longbin Huang1, Peter Kopittke2
1
The University of Queensland / Sustainable Minerals Institute, Australia, 2The University
of Queensland / School of Agriculture and Food Sciences, Australia
Sulfidic base metal mine tailings such as the Cu/Pb-Zn tailings at Mount Isa Mines, Australia,
contain elevated concentrations of copper (Cu), cobalt (Co), nickel (Ni), lead (Pb), and zinc
(Zn), their phytostabilization requires introduction of tolerant native plant species. Acacia
chisholmii, a drought tolerant shrub native to north-eastern Australia, has been found to
colonize weathered Cu/Pb-Zn tailings under semi-arid climatic conditions. Despite past
focuses on Cu, Pb and Zn-tolerance in plants, little attention has been paid to the toxicity of
Co and Ni, which are present at high concentrations (ca.14 and 512 (mM) respectively) in
pore water of the tailings. The present study investigates the growth responses of A.
chisholmii to elevated Co and Ni concentrations in solution examining root growth and plant
metal accumulation/distribution, initially under laboratory conditions.
Young (5 days) and old (3 months) seedlings were treated with different Co, Ni or Co+Ni
concentrations. Root elongation was limited at 25 µM in the young after 2 day exposure and
growth reduction was observed at 60 µM in the old after 7 day. Individual exposure of young
seedlings to the same concentrations of soluble Ni and Co showed similar root growth
reductions, but the seedlings accumulated ca. 4 times more Ni (418 µg g-1 d.w.) than Co (114
µg g-1 d.w.). In 60 µM Co-only treatment, Co concentration in the roots of the old seedlings
was up to 472 µg g-1 d.w.), but low as 28 µg Co g-1 d.w. in the shoots. This tentatively
suggested Co exclusion in this species. Nevertheless, in the Co+Ni mixture, the Ni competed
against Co uptake, resulting in lowered Co bioconcentration factor ([metal]root/[metal]solution).
In conclusion, A. chisholmii showed Co/Ni-exclusion behaviour compatible with the
phytostabilization purposes. Further investigations are required to elucidate differential
rhizosphere mechanisms involved in the Co/Ni root uptake.
76
Influence of biochar and compost on microbial community in Technosols
Ali Kanso*1, Catherine Sirguey2, Emile Benizri2, Antoine El-Semrani3, Ahmad Kobeissi3,
Guillaume Echevarria2
1
Universite de Lorraine, France, 2Université de Lorraine, France, 3Lebanese
University, Lebanon
The LORVER project, supported by Lorraine Region (France) and European Union (FEDER),
aims to create industrial chains for plant biomass production, from derelict lands and
industrial by-products. In this work, phytoextraction was used as a sustainable restoration
technology allowing metals recovery from constructed Technosols with industrial soil and byproducts. The effect on the microbiological and soil biochemical properties, which are known
to play a key role in soil functioning and fertility, was assessed.
Technosols were constructed according three formulations: a Biocentre® treated soil
artificially contaminated in trace elements by additions of a mix of industrial sludges (BTS),
252
BTS amended with biochar (B2TS), and BTS amended with a green waste compost (BTSC).
Planted (two hyperaccumulators plants: Noccaea caerulescens and Alyssum murale) and nonplanted soils were transferred to a growth chamber for three months. Measured parameters
were: soil physicochemical properties, plant growth, microbial biomass carbon (MBC) and
nitrogen (MBN), soil enzyme activities and bacterial and fungal genetic structure.
MBC and MBN were significantly higher in BTSC soils without any plant effect, whereas MBN
was significantly lower in cultivated B2TS soils. Mean microbiological biomass C:N ratios were
not significantly different among non-cultivated Technosols and reached 19.7. In planted BTS
and B2TS soils, this value significantly increased up to 41.5 and 106 respectively whereas it
was not affected in cultivated BTSC soils. Fluorescein Diacetate and enzyme (urease,
phosphatase and beta-glucosidase) activities were systematically higher in BTSC soils when
measured per soil mass unit, whereas urease and phosphatase activities were higher in B2TS
soils when values were normalized to MBC.
Technosols may have a low potential for microbial activity due to soil nutrient microbial
limitations. If biochar strengthened nutrient limitations, green waste compost alleviated them.
Compost amendments could thus improve Technosols fertility and microbial activities linked
with biogeochemical process at both structural and functional levels.
77
Rhizoremediation of soils contaminated with petrogenic hydrocarbons using
Australian native plants
Navjot Kaur*, Paul Greenwood, Andy Whiteley, Suman George, Megan Ryan
University of Western Australia, Australia
Most industries require hydrocarbon-based fuels; however routine activities such as
transportation, storage and refilling pose considerable spillage risk on land. We conducted a
glasshouse experiment to examine the mechanism of breakdown of persistent hydrocarbons
by native Australian plants, and associated microbial communities, in crude oil contaminated
soil. A concentrated mix of oil 12% (w/w) and white sand was aged for 6 months in the
glasshouse. The mix of soil and sand, or sand only (control), was added to dried, sieved (2
mm), low-nutrient field soil. Fresh soil was then applied to achieve 1% oil contamination.
Eight species (7 native, 1 exotic) belonging to five families were grown for 3 months. There
were 5 replicates of each species/treatment combination. We hypothesized that the level of
oil biodegradation would depend on the populations of degrader communities selected by
the plant species on the basis of type and/or amount of low molecular weight root exudates
released under contamination stress. At harvest, total plant biomass, root area and plant
height differed little between oil-contaminated and control pots. Community-level
physiological profiling of bulk soil showed microbial communities from contaminated soil
had an increased response for carbohydrates and carboxylates. The response was
significantly higher for Viminaria juncea and Lolium perenne, possibly due to higher root
mass. Unplanted controls were most effective in reducing soil total aliphatic and aromatic
content, by 71% and 65%, respectively, which was marginally better than for Chloris truncata,
Iseilema vaginiflorum, and V. juncea (aromatic) and Acacia holosericea. The least effective
species were Trachymene pilosa, Rhagodia preissii and Rhodanthe chlorocephala. The results
suggest a competitive interaction among plants and primary microbial communities. Total
253
root exudates will be measured by high-pressure liquid chromatography and molecular
techniques will be applied to understand quantitative changes in microbial communities after
oil contamination.
78
Rhizosphere competence of atrazine-degrading Arthrobacter bacteria
Dmitry Bazhanov1, Chengyun Li*1, Hongmei Li2, Jishun Li1, Hetong Yang2
1
Key Laboratory for Applied Microbiology of Shandong Province, Biotechnology Center
of Shandong Academy of Sciences, China, 2Biology Institute of Shandong Academy of
Sciences, China
The occurrence and diversity of atrazine degraders in the rhizosphere of cogon grass and
common reed growing near an atrazine factory and in the rhizosphere of maize from
geographically distant fields were investigated. Atrazine degrading bacteria were isolated by
direct plating on the specially developed SM agar. ERIC-PCR genotyping of the isolates
followed by 16S rRNA gene phylogenetic analysis revealed a variety of atrazine-degrading
Arthrobacter spp. in the rhizosphere of cogon grass and common reed, with a strong
prevalence of one genomospecies related to the cluster comprising A. aurescens, A. ilicis, and
A. nitroguajacolicus. Genetically similar A. ureafaciens bacteria which occurred as minor
inhabitants of cogon grass roots in the industrial soil were predominant atrazine degraders in
the maize rhizosphere at all the agricultural sites. The genetic structure of atrazine degrading
communities of heavily contaminated industrial soils appeared to be governed mainly by the
contamination rate. More complex factors seemed to influence the survival of atrazine
degrading A. ureafaciens in agricultural soils exposed to low rates of atrazine. We found that
the atrazine degrading strains of A. ureafaciens exhibited active movement in liquid and
semisolid agar media, spread on the surface of solid agar along fungal hyphae and
competitively colonized roots of maize, wheat and alfalfa after seed inoculation. The atrazine
was not required for the root colonization. However, the highest population densities of the
root-associated atrazine degrading A. ureafaciens were detected in the atrazinesupplemented soil. The root colonization resulted in the atrazine degradation and protection
of the plants from atrazine injury. Our results demonstrate rhizosphere competence of
atrazine-degrading Arthrobacter bacteria and their potential for use in rhizoremediation of
polluted soils.
79
Ranking of mechanisms governing the phytoavailability of cadmium in
agricultural soils by using a model analysis
Zhongbing Lin1, André Schneider2, Thibault Sterckeman2, Christophe Nguyen*2
1
INRA UMR 1391 ISPA, France, 2INRA, France
This study aimed at determining the influential mechanisms governing the phytoavailability
of the toxic trace element cadmium (Cd) in agricultural soils using a mechanistic model.
We built a phytoavailability model simulating the Cd2+ uptake by 1 cm² root surface and
including the transport by diffusion and convection, the kinetics of sorption/desorption and
of complexation with the dissolved soil organic matter. The model variables were ranked by
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performing a sensitivity analysis where a 30-days uptake of Cd2+ was simulated for 100 000
combinations of the parameters chosen within ranges consistent with the French agricultural
soils.
Generally, the most influential variable was the initial concentration of Cd2+, which was low,
making the root capacity for absorbing Cd2+ of little influence. The convection was almost
always negligible and the diffusion was the dominant process of Cd phytoavailability. The soil
impedance factor, the water content and more particularly the soil buffer power for Cd2+
were therefore the following most influential variables. The kinetics of adsorption and
desorption were of little Influence. The Cd-complex was shown to generally contribute little
to the uptake due to a strong kinetic limitation for the dissociation. Consequently, the
dominant process buffering the Cd2+ concentration at the root surface was desorption.
Significant complex contributions were observed for very labile complex and high water flux
toward the root surface.
On the whole, the simulated uptake correlated very well with a simplified model considering
only the transport and sorption at equilibrium of Cd2+. This model relied on the total soil Cd
concentration, the soil pH, the soil organic carbon content, the concentrations of calcium and
dissolved organic matter, and the partitioning of the dissolved organic matter between fulvic
and humic acids; these variables determined the soil/solution partitioning of Cd2+.
80
Characterization of bacteria isolated from heavy metals contaminated soils of
the region of Oujda (Morocco)
Malika Oubohssaine*1, Laila SBABOU2, Jamal Aurag2
1
Faculty of Sciences, Mohammed V University, Morocco, 2Faculty of Sciences, Morocco
After stoppage of mines activities, most of the exploitations are abandoned and the soils are
heavily loaded with heavy metals. Toxic particles are transported in the ambient areas,
causing a serious imbalance in the biogeochemical cycles and important changes in the
functioning of ecosystems. These contaminated habitats are considered extreme regarding
the abundant and chronic presence of heavy metals,. This combination of environmental
conditions allows the development of pioneer microorganisms possessing the necessary
biological mechanisms conferring them tolerance or resistance to toxic concentrations of
metals and facilitating of the installation and colonization of plant species in these hostile
environments.
It is therefore interesting to study the diversity of this particular microflora and its interaction
with the local plants metallophytes, in order to use them in the development of
phytoremediation strategies for metalliferous sites. For this purpose rhizobacteria from metal
contaminated soils will be selected and inoculated to Hedysarum spinosissimum plants under
metal stress conditions, in order to study the usefulness of this biotechnological approach in
phytoremediation programs.For the achievement of this objective, we have first realized a
collection of 700 bacteria isolated from the rhizospheric and bare soils collected from three
mining zones of Oujda (Sidi Boubker, Oued El Heimer and Touissit). The tolerance of the
isolates to heavy metals (Pb, Zn, As) has been studied and multi-resistant bacteria to different
concentrations of these metals has been identified. Qualitative and quantitative studies of
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potential plant growth promoting activities permitted the identification of 47 rock phosphate
solubilizing isolates , 145 auxin producing strains and 54 siderophores producing bacteria .
Sequencing the 16S rDNA gene of the most interesting bacteria allowed their identification at
the genus and / or species levels. It was found that they belong to different families and
clusters of bacteria and they are dominated by the genus Bacillus.
81
Effect of nitrogen addition on rhizospheric Zn, Pb and Cd mobility and plant
uptake in contaminated technosol
Bashar Qasim*1, Mikael Motelica-Heino2, Domenico Morabito3, Sylvain Bourgerie3,
Arnaud Gauthier4
1
Université d'Orléans/ ISTO, France, 2ISTO, France, 3LBLGC, France, 4Université Lille
1, France
Mining and smelting of metal ores activities have increased the spread and occurrence of
potential toxic elements such as metal(loid)s in soils. However, they can be readily absorbed
and enter into the food chain, causing serious threats to human health.
Our study aimed at reporting the effect of the addition of two nitrogen form (NH 4+ and
NO3−) on rhizospheric soil pore water pH, dissolved organic carbon concentration, potential
toxic elements concentrations in SPW and their uptake by Populus euramericana Dorskamp
grown in contaminated soils.
Soil samples were taken from metallophyte grassland contaminated with Zn, Pb and Cd
located at Mortagne – du –Nord (North France).
A plant growth experiment with poplar woody stem cuttings was conducted with forty-five
pots (3 soil samples × 3 treatments × 5 replicates for each treatment) for 35 days. The
experiment consisted of two nitrogen treatments (NH4Cl and KNO3) and an untreated control
soil (without fertilizer) was included for comparison.
For all studied soils, rhizospheric pH decreased and increased gradually with NH 4+ and NO3–
addition respectively, whilst, it decreased initially then increased at the end of the experiment
for the untreated control soil. Dissolved organic concentrations increased gradually with time
during the cultivation period. An increase in the rhizospheric total dissolved concentrations of
Zn, Pb and Cd was observed with NH4 + addition associated with the lowest pH, whereas the
opposite was observed with the addition of NO3 –. Nitrogen form addition enhanced plant
roots metal uptake compared to plant shoots uptake for all studied metals.
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82
Assessment of ecosystem services rendered by agromining of nickeliferous soils:
association of leguminous and hyperaccumulator
Ramez Saad*1, Guillaume Echevarria1, Ahmad Kobeissi2, Emile Benizri1
1
Université de Lorraine - UMR 1120 Sols Environnement INRA, France, 2Université
Libanaise, Faculté des Sciences 1, Laboratoire Biologie Végétale et
Environnement, Lebanon
The objectives of Agromining are to decontaminate soils by extracting metals with high
economic importance with the use of hyperaccumulators and then to improve their
agronomic value for traditional agricultural use.
Nevertheless, some hyperaccumulator species grow slowly and produce little biomass
because of the soil’s low fertility, which could limit their use for phytoextraction, in terms of
quantities exctracted.
Thus, it is necessary to improve phytoremediation by the implementation of new and
appropriate cropping systems dedicated to Agromining. To attain this goal, we propose to
identify the effect of the combination of plants (hyperaccumulator and non-accumulator, i.e.
legume) on the efficiency of phytoextraction (biomass production, remediation and
restoration of soil quality). Indeed, legumes have shown to increase the soil-N pool which
Brassica could benefit from. Intercropping or co-cropping with legumes has the potential to
combine high economic performance and low environmental impact by reducing the amount
of fertilizer supplied.
Our aims are to evaluate the efficiency of associating Lens culinaris with Alyssum murale on an
ultramafic Ni-rich soil in order to enhance biomass production and to achieve better Ni
phytoextraction. However, it is known that abiotic stress, originating from the presence of
metals in soil, causes the production of ethylene in plants, which in turn affects the
development of roots and nodule formation in legumes. So, the purpose of the present study
is to test if Lens can make nodules and if these are functional when it grows on Ni-rich soil.
The soil used in this experiment naturally contains Rhizobium and was artificially
contaminated with different concentrations (0 to 90 mg Ni.kg-1 soil) of nickel sulfate. Natural
15
N abundance will be used to estimate the percentage of plant N derived from N2 fixation
(%Ndfa) and to estimate the capacity of Lens to grow and form functional nodules under Ni
contamination.
83
A meta-model to predict the phytoavailability of cadmium in French agricultural
soils
André Schneider*1, Zhongbing Lin2, Thibault Sterckeman3, Christophe Nguyen1
1
INRA, Biogeochemistry of Trace Elements, UMR 1391 ISPA, France, 2School of Water
Resources and Hydropower Engineering, Wuhan University, China, 3INRA, Laboratoire
Sols et Environnement, UMR 1120, France
Predicting the phytoavailability of toxic trace elements such as cadmium (M) in soils is an
important issue for crop quality. A set of partial differential equations can be derived for
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simulating the root influx of M2+, the transport by diffusion and convection, the kinetics of
sorption/desorption, and the kinetics of complexation in solution, for the free ionic species
(M2+), for a mean ligand, and for the complex. The aim of this study was to develop a simple
meta-model that would be easier to handle than these partial differential equations.
d and are out of equilibrium in a reaction layer of thickness µ < d; in this reaction layer the
concentration of the complex is assumed constant, and the association rate of the complex is
neglected. Under these assumptions and for two extreme behaviors of the complex, i.e. when
complex is totally labile (µ=0) or totally inert (µ=d), the analytical equation for calculating the
concentration of trace element at the root surface, and therefore the uptake, was derived. For
intermediate complex lability (0 <µ < d), the uptake was obtained by linear interpolation of
the uptake between µ=0 and µ=d. The meta-model was evaluated for cadmium in the
context of French agricultural soils.
For a dataset of about 105 simulations covering the variability of soil characteristics, the metamodel estimates of the uptake fitted very well the values obtained by the numerical
integration of the partial differential equations (r²=0.996). Indeed, for 95% of the simulations,
the values of the relative error of prediction ranged between -14 and 27%, and the mean
absolute relative error of prediction is quite excellent (5.9%).
84
Phenanthrene-induced alterations in Noccaea caerulescens roots and
consequences for metal phytoextraction
Ivan Zelko1, Catherine Sirguey*1, Stéphanie Ouvrard2
1
Université de Lorraine - UMR 1120 LSE, France, 2INRA - UMR 1120 LSE, France
Industrial decline in recent decades has led to the emergence of thousands of acres of
brownfields in North-East France. Some of them are multi-contaminated by heavy metals and
organic pollutants and their remediation need to address both problems. A combined
decontamination using simultaneous phytoextraction of metals and rhizodegradation of
organic pollutants appears a promising solution. However, organic pollutants may adversely
affect hyperaccumulator plants growth and extraction activity thus limiting their use in this
context. The aim of this work was to study root morphological and structural features of the
heavy-metal hyperaccumulator Noccaea caerulescens exposed to phenanthrene (PHE) in
combination with cadmium (Cd).
Seedlings of N. caerulescens were cultivated, during one week in a climatic chamber, on a
nutritive agar medium non supplemented (control) or supplemented with 2 mM PHE and/or
5 µM Cd. At the end of the culture, we assessed plant growth and production parameters
(elongation, biomass and plant water content), root architecture (WhinRhizo® software).
Root structural features (suberin lamellae and peri-endodermal layer) were observed using
epifluorescence microscopy.
The tested PHE concentration caused serious inhibition (about 40%) of growth and biomass
production of both roots and shoots, while Cd had no significant adverse effect. Moreover,
Cd moderately impacted root architecture whereas PHE strongly reduced (about 50%) total
root length and surface. Additionally, PHE promoted lateral root formation and inhibited root
258
hair elongation. Moreover, endodermal cells with suberin lamellae appeared closer to the root
apex when exposed to PHE compared to control and Cd treatments. This can be explained by
modified lateral root formation. The stage with well-developed suberin lamellae was not
influenced by PHE. However peri-endodermal layer development was impaired in PHE-treated
plants.
These morphological and structural root modifications in response to PHE exposition might
in turn limit Cd phytoextraction by N. caerulescens in co-contaminated soils.
85
Nodulation and growth of Fabaceae in mine spoils as influenced by inoculation
of resident rhizobacteria displaying multi-trait phytostimulation properties
Souhir Soussou*1, Brigitte Brunel1, Marjorie Pervent1, Diederik van Tuinen2, JeanClaude Cleyet-Marel1, Ezekiel Baudoin1
1
Tropical and Mediterranean Symbiosis Laboratory, France, 2UMR1347
AGROECOLOGIE, France
Heavy metals (HM) extraction by metallurgical activities generates highly toxic mine spoils
that still contain noxious levels of HM. To limit further HM dissemination by leaching and
eolian transport, rubble mines can be phytostabilised by engineering a dense and perennial
plant cover. In particular, autochtonous legume ecotypes spontaneously growing in severely
HM-polluted areas are attractive candidates in such vegetalisation scenarios owing to their
tolerance mechanisms and their organic nitrogen-carbon inputs in such nutrient-depleted
substrates. In this background, inoculations of rhizobia and plant growth promoting
rhizobacteria (PGPR), isolated from the rhizosphere of such metallophytes growing in mine
spoils, are to be tested so as to facilitate the development of legumes.
Here, we report on the rhizosphere of Anthyllis vulneraria ssp. carpatica, a local legume
ecotype grown in highly Zn-Pb-Cd contaminated mine ponds (St. Laurent-le-Minier, France),
as a reservoir of diverse fluorescent Pseudomonas species displaying multi-trait
phytostimulation properties. 11% of the strains harboured the acdS gene and were ACCdeaminase positive. Development and nodulation patterns of the metallophyte legumes A.
vulneraria ssp. carpatica and Lotus corniculatus grown in the same mine substratum were
observed following individual inoculations of 11 selected PGPR strains together with the
relevant rhizobium strains. Plant development was highly dependent on the identity of both
metallophyte and PGPR strain. A single strain proved the most efficient on both legumes and
two strains systematically induced a strong inhibition of plant development, irrespectively of
their in vitro PGPR potentials.
These data suggest that successfull inoculations of resident PGPR strains as a tool to facilitate
resident metallophyte development in mine tailings first rely on the integration of the
metallophyte-PGPR genotypes interaction.
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86
A radio-isotopic dilution technique for functional characterisation of the
associations between organic and inorganic contaminants and natural
nanoparticles
Ehsan Tavakkoli*1, Albert Juhasz2, Enzo Lombi2
1
NSW Department of Primary Industries, Wagga Wagga Agricultural
Institute, Australia, 2Centre for Environmental Risk Assessment and Remediation,
University of South Australia, Building X, Mawson Lakes Campus, Australia
Despite evidence that the fate and behaviour of organic and inorganic contaminants are
influenced by their interactions with water-dispersible naturally occurring soil colloids, our
understanding of the mobility and bioavailability of contaminant–colloid associations has
been hampered by the limitations of common operationally defined analytical techniques. In
this paper, an isotopic dilution method was developed to quantify the isotopically
exchangeable and non-exchangeable forms of zinc, phosphorus and phenanthrene in filtered
soil-water extracts. In addition, the effect of filter size on the determination of contaminant
exchangeability was investigated. The results showed that the isotopically non-exchangeable
Zn and P in filtered soil-water extracts respectively ranged between 5 and 60 % and 10 and
50 % and was associated with water-dispersible colloids. Filter pore size had a significant
effect on Zn and P exchangeability. Whereas the <0.1-µm filtrates contained isotopically
exchangeable Zn and P fractions equal to the total Zn and P concentrations (i.e. 100 %
isotopically exchangeable Zn and P), the filtrates obtained from larger filter sizes (0.22, 0.45
and 0.7 µm) contained increasing proportions of non-exchangeable Zn and P. Our findings
also suggest that the exchangeability of phenanthrene in sodium tetraborate is controlled by
both inorganic and organic colloids, while in aqueous solutions inorganic colloids play the
dominant role (even though coating of these by organic matter cannot be excluded).
87
Regulation and stabilisation of physical, chemical and microbiological alteration
processes by plants’ exudates and antioxidants in Callovo-Oxfordian argillite
Dimitri Ubersfeld*1, Corinne Leyval1, Paul-Olivier Redon2, Christian Mustin1
1
LIEC UMR 7360 CNRS-Université de Lorraine, France, 2Andra, Direction Recherche et
Développement, Centre de Meuse/Haute-Marne, Route départementale 960, France
This study is focused on vegetalisation of Callovo-Oxfordian clay rock stockpiles excavated
from the underground Andra research laboratory (CMHM, Bure, France). The aim was to
understand the effect of planting hardy and resilient plants (i.e. lavender) on the physical and
chemical weathering processes (erosion, oxidation, leaching) of clay materials, made of
smectites, sulphides and carbonates. A bioweathering experiment was conducted on sieved
and ground clay material samples inoculated with a microbial inoculum coming from the
site’s topsoil. Percolation devices were watered once a week and incubated at 28°C for three
months. Plant effect was simulated by the addition of antioxidant molecules (linalool and
thymol) and synthetic root exudates to eluents. Both physico-chemical and biological
transformations of clay materials were assessed by quantifying metal amounts in leachates.
Bacterial density was estimated by DNA extraction from clay material and quantification of
16S rDNA gene copy number. Microbial inoculation and exudate addition lead to a significant
pH decrease of the leachates and an important leaching of Al, As, Ca and Fe. With antioxidant
inputs, the lixiviation of most important metals (Fe, S…) was slightly lower and the
260
bacterial density concomitantly and significantly decreased with thymol. In an opposite way,
exudates stimulated the growth of microflora. In conclusion, these results motivate the use of
hardy plants producing antioxidants (essential oils) for phytostabilisation of clayed excavated
residues.
88
Effect of glyphosate on rhizosphere microbial communities of Cortaderia
selloana plants
Mikel Anza1, Lur Epelde1, Unai Artetxe2, Julen Urra*1, Jose Maria Becerril2, Carlos
Garbisu1
1
Neiker-Tecnalia, Spain, 2University of the Basque Country, Spain
In the north of the Iberian Peninsula, Cortaderia selloana plants have invaded not only human
degraded areas but also ecosystems with high ecological value such as marshes, dunes and
riparian forests. As a consequence, in many areas, the high proliferation of C. selloana has
become a matter of great ecological concern, as few strategies are available for its control. In
our region, the control of this invasive species is attempted by spot treatment with a postemergence application of glyphosate (a non-selective systemic herbicide).
The main objective of this work was to determine the impact of glyphosate treatment on
rhizosphere microbial communities of C. selloana plants. To achieve this goal, a microcosm
pot experiment was established with previously grown C. selloana seedlings. After two
months of growth, pots were treated with glyphosate. Three rhizosphere soil samplings were
carried out: just before treatment, and one and two months after treatment. A variety of
parameters that provide information on the abundance, activity and diversity of soil microbial
communities (ergosterol concentration, enzyme activities, basal respiration, community-level
physiological profiles, etc.) were determined in these rhizosphere soil samples.
After one month of treatment, our results indicate a negative impact of glyphosate on soil
microbial communities. In the longer term (after two months of glyphosate application), this
impact was more than reversed with a significant increase in the values of the microbial
parameters determined here. Most importantly, glyphosate-treated soil kept its capacity to
properly support plant growth (in particular, the growth of Festuca rubra plants; in our region,
this species is a good candidate for revegetation after glyphosate-treatment of C. selloana
plants). Finally, soil microbial properties have proven to be good indicators of soil ecosystem
health.
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89
Spatial pattern of bacterial diversity in a site with mixed and uneven
contamination, and assessment of rhizoremediation potential
F. Mapelli1, L. Vergani*1, R. Marasco2, B. Chouaia1, M. Fusi2, A. Di Guardo3, G. Raspa4, E.
Zanardini3, C. Morosini3, S. Armiraglio5, S. Anelli6, P. Nastasio6, V.m. Sale6, D.
Daffonchio1, S. Borin1
1
Defens, University of Milan, Italy, 2King Abdullah University of Science and
Technology, Saudi Arabia, 3DSAT, University of Insubria, Italy, 4DCEME, University of
Rome La Sapienza, Italy, 5Municipality of Brescia, Museum of Natural
Sciences, Italy, 6ERSAF, Italy
The SIN Caffaro is a large polluted site of national priority located in the Northern Italy,
originated by the activities of the former Caffaro s.p.a. chemical factory. The soil in the site
presents a mixed contamination of halogenated Persistent Organic Pollutants and heavy
metals in variable concentrations, uneven distributed in the area and often exceeding the
safety values. The use of plants to extract and modify the pollutants (phytoremediation)
together with root associated microbes to i) degrade or modify the pollutants
(rhizoremediation) or ii) support plant growth (plant growth promotion, PGP) has recently
arose as a promising approach for bioremediation.
In this context, we collected soil samples from three different areas in the site to a depth of 1
meter, which were chemically and microbiologically characterized. A DNA-based
fingerprinting approach was applied to describe the bacterial community’s structure, which
proved to be significantly different according to the area and depth of collection. We also
tested the influence of selected environmental parameters, showing that the concentration of
different classes of pollutants was significantly related to the microbiome structure.
Furthermore, the rhizosphere of three autochthonous plant species was collected in the most
contaminated area of the site. The overall bacterial community was studied by 16S rRNA
pyrosequencing and a collection of bacterial strains was in parallel established and tested for
PGP potential. The results showed that the rhizosphere-dwelling microbiome was highly
similar between the plant species, in terms of both phylogenetic diversity and PGP potential,
confirming the existence of a strong selective pressure given by the pollution profile rather
than the plant species.
Overall, this work highlighted the occurrence of distribution patterns in bacterial populations
related to gradients in soil pollution, and showed the intrinsic potential of the highly
contaminated soils at the Caffaro site for rhizoremediation potential.
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Quantitative and qualitative synthesis of siderophores by Streptomyces sp.
strains in the presence of Cd2+
Michał Złoch*, Dominika Thiem, Sonia Szymańska, Renata Gadzała-Kopciuch,
Katarzyna Hrynkiewicz
Nicolaus Copernicus University, Poland
Microbial siderophores are low-molecular mass chelators which act as solubilizing agents for
iron from minerals or organic compounds under conditions of iron limitation. Additionally,
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they can also form stable complexes with other heavy metals like Cd, Cu, Pb, Al or Zn.
Moreover, stimulating effect of heavy metals on siderophore biosynthesis by some bacteria,
which may indicate involvement of heavy metals in regulation of siderophore biosynthesis
pathways, was suggested.
We hypothesized that: (1) production of siderophores by 5 rhizosphere Streptomyces sp.
strains can be stimulated or inhibited by increasing concentration of Cd2+ in the medium and
(2) diversity of siderophores depend on the concentrations of Cd2+ in the medium (0, 0.5, 1, 2
and 3 mM Cd2+). In the analysis we used a spectrophotometric chemical assays, high
performance liquid chromatography (HPLC) as well combined techniques - LC-Q-TOF/MS.
We have revealed relevant differences between investigated strains both in type and amount
of synthesized siderophores under Cd2+supplementation. In case of two investigated strains
increasing concentration of Cd2+ in the substrate stimulated synthesis of hydroxamic,
catechol and phenolic siderophores. In general, presence of Cd2+ stimulated synthesis of
siderophores compare to the control variant (not supplemented with Cd2+), however effect of
metal concentration differed between investigated strains. HPLC analysis allowed for
detection and identification of ferrioxamine B in culture medium of 4 studied strains.
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New Methods and Concepts in the Rhizosphere
91
High-throughput amplicon sequencing for microbe identification in complex
colony communities of culture collections
Jaderson Armanhi*1, Laura Migliorini de Araujo1, Rafael Soares Correa de Souza1,
Natalia Verza Ferreira1, Vagner Katsumi Okura1, Beatriz Jorrín Rubio2, Juan Imperial2,
Paulo Arruda1
1
State University of Campinas (UNICAMP), Brazil, 2Centro de Biotecnología y Genómica
de Plantas (CBGP), Campus de Montegancedo, Universidad Politécnica de Madrid, Spain
Construction of a microorganism culture collections isolated from environment can be timeconsuming since individual colonies containing single microbes might demand several
streaking steps for colony purification. In addition, the pursuing of pure colonies may restricts
important insights on mutual association. Potentially, in extreme cases, microorganisms in
consortia might depend strictly on beneficial microbe-microbe interactions. Here we present
a method for construction and identification of a collection of microorganisms and consortia
of microorganisms isolated by a single step of plating and colony picking. Complex microbial
mixtures isolated from sugarcane roots and stalks were plated in defined media at proper
dilution to obtain colonies. Colonies were picked, grown in 96-deepwell plates and stored at 80 °C. This collection comprises over five thousand wells containing individual
microorganisms or consortia. In order to identify these microorganisms we have developed a
method to sequence pools of amplicons in 96-well plates using barcodes to tag plates, rows
and columns in two PCR steps. Pooled plates can be sequenced into a single run of PacBio
and delivers high quality amplicons relying on circular consensus sequence (CCS) for error
corrections. Together, the multi-tagging system and PacBio platform allows the identification
of microbe composition of each well, regardless if it contains pure or consortia colonies.
Results will be discussed in the context of cost-effective alternatives for large-scale
identification of individuals in complex colony communities by sequencing pools of
multiplexed 96-well plates.
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Combination of X-ray micro tomography and soil solution studies to analyse root
system development and soil chemistry in situ as a response to different Nforms
Sebastian Blaser*1, Doris Vetterlein1, Enrico Thiel2
1
Helmholtz Centre for Environmental Research - UFZ, Germany, 2SKW Stickstoffwerke
Piesteritz GmbH, Agricultural Application Research, Germany
Only 30-50% of the applied N fertilizers are captured by crops. Therefore increasing both, N
use efficiency and crop production is a major challenge for sustainable agriculture. Urea is the
most widely used N fertilizer. Urea undergoes rapid hydrolisation applied at the soil, after
which ammonium is oxidized to nitrate. Due to the use of nitrification inhibitors, the
importance of NH4+ as N source has increased. NH 4+ is more protected against nitrification
creating benefits for environment and plant growth.
264
Application of urea granules with or without inhibitors is expected to result in a spatially
heterogeneous and temporally dynamic distribution of different N-forms in the soil matrix.
Plasticity of root growth can be observed when roots are exposed to localized sources of
nitrogen. Two general ways of response seem to be common including systemic repression
of lateral root growth by high N status of the plant and local stimulation by initiation and
elongation or inhibition of lateral root growth by availability of NO3 or NH4. These effects are
related to the nutritional and signalling effect of the respective ion but dynamics are difficult
to observe due to the opaque nature of soil.
The aim of this work is to verify hypotheses, derived from studies with flow-through-systems
or gel plates, for soil systems, using X-ray micro tomography to visualize and characterize
dynamic root system development in soil as a response to different N-forms in situ. These
analyses are combined with soil chemical studies in the same temporal resolution. Micro
suction cups are installed to extract soil solution with known distance to roots and localized
fertilizer. This setup is applied to answer the questions when, where and why do
modifications of root system architecture occur in relation to soil chemical and N-nutritional
conditions.
93
Microdialysis – an alternative approach to identifying plant-available nitrogen in
soils
Scott Buckley*, Richard Brackin, Susanne Schmidt
The University of Queensland, School of Agriculture and Food Sciences, Australia
Given the importance of nitrogen (N) availability in controlling N acquisition in plants and
microbes, estimating soluble and exchangeable N forms in soil is vital to understanding these
processes. However, we have relied on extraction methods that severely disrupt the soil
environment. Microdialysis offers an alternative method of sampling soil N with minimal
disturbance, but it is unclear whether this method better represents plant-available N in soil.
We evaluated microdialysis and conventional salt (KCl) and water extraction methods in the
context of a microcosm experiment, sampling N release (NO3 -, NH4+ and amino acids) from
sugarcane litter (high C:N ratio) and soybean litter (low C:N ratio) decomposition over 30
days. Microbial activity (microbial biomass-N, protease activity, CO2 respiration) was also
measured to assess microbial response to varying litter quality, and to predict N cycling
processes. Diffusive fluxes (measured via microdialysis) provided a high-resolution snapshot
of N availability at day 30, highlighting N cycling processes that were insufficiently resolved
using salt or water extractions. Patterns of N immobilisation were observed in sugarcane
treatments, whereas significant N mineralisation was found in soybean treatments. Each
pattern increased with litter concentration, and was consistent with observed microbial
activity to differing qualities of litter (microbial biomass-N, protease activity and CO2
respiration being greatest in soybean treatments). Such patterns were not apparent in KCl or
water extractions, both showing uniform N concentrations and N species across treatments,
with the exception of highly amended soybean treatments. These findings challenge the
effectiveness of conventional soil extraction techniques to sufficiently estimate plantavailable N, and to resolve N cycling processes in soil environments.
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94
Water repellency and percolation across the rhizosphere
Mohsen Zarebanadkouki, Eva Kroener, Andrea Carminati*
Georg-August University of Goettingen, Germany
The ability of plants to extract water from the soil is influenced by the hydraulic conductivity
of roots and their rhizosphere. Recent experiments showed that the rhizosphere turned water
repellent after drying and it remained dry after rewetting of the bulk soils. Our objective was
to investigate whether and in what conditions rhizosphere water repellency is a limit to root
water uptake.
We used neutron radiography to trace the transport of deuterated water (D2O) in the roots of
lupins experiencing local soil drying. The soil was partitioned in nine compartments. We let
one of the upper compartments dry, while keeping the other compartments wet. We
injected D2O in different locations. The radiographs showed that root water uptake in the soil
region that was let dry and then irrigated was 4-8 times smaller than in the wet soil regions.
In a parallel experiment, we monitored the rehydration of lupin roots that were irrigated after
severe drying. Based on measurements of root swelling and xylem pressure, we found that the
hydraulic conductivity of the rhizosphere was the limiting factor for root rehydration.
Our hypothesis is that rhizosphere water repellency was caused by mucilage exuded by roots.
We measured capillary rise in soils with varying mucilage concentration. We found that below
a critical mucilage concentration water could easily cross the soil, while above the critical
concentration water could no longer percolate through the soil. The critical mucilage
concentration depended on soil particle radius. The results were well reproduced by a new
pore-scale model of water percolation through the rhizosphere.
Our studies suggest that the rhizosphere is near the percolation threshold, where small
variations in mucilage concentration sensitively alter the soil hydraulic conductivity. It is
tempting to conclude that mucilage exudation is a plant mechanism to efficiently control the
rhizosphere conductivity and water uptake.
95
Effect of root exudates on soil mineralogy: possible implications on nutrient
mobilization
Concetta Eliana Gattullo*1, Ignazio Allegretta1, Giovanni Cuccovillo1, Luca Medici2,
Tanja Mimmo3, Youri Pii4, Stefano Cesco4, Nicola Tomasi5, Roberto Pinton5, Roberto
Terzano1
1
Department of Soil, Plant and Food Sciences - University of Bari, Italy, 2Institute of
Methodologies for Environmental Analysis, CNR, Italy, 33Faculty of Science and
Technology, Free University of Bolzano, Italy, 4Faculty of Science and Technology, Free
University of Bolzano, Italy, 5Department of Agricultural and Environmental Sciences University of Udine, Italy
Plants release in the rhizosphere flavonoids and organic acids which exert multifunctional
roles. In a recent study, we observed that the flavonoid rutin, alone or combined with
genistein or organic acids (citrate, malate, oxalate), usually present in plant exudates,
mobilized Fe from a calcareous soil with a great efficiency. However, the effects of these
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exudates on soil mineralogy, especially on Fe-bearing minerals and clays, remained
unexplored.
This work aims at evaluating the changes in the mineralogical composition of a calcareous
soil after 24h-treatment with an aqueous solution of rutin (35 µM), alone or combined with
other flavonoids (10 µM) or organic acids (1 mM). After centrifugation, the solid fraction was
dried, homogenized with corundum (internal standard) and analysed by X-ray powder
diffraction (XRPD), using the Rietveld method for mineralogical quantification.
XRPD analysis of the natural soil revealed the presence of calcite (57.0%), illite (17.9%),
smectite (5%), quartz (4.2%), heulandite (2.8%), and amorphous phases (13%). No Fe-oxides
and oxyhydroxides were detected. In all the treatments with rutin, the sum of illite and
smectite considerably increased, compared to the natural soil, especially when rutin was
combined with organic acids or genistein. Conversely, the amorphous residue decreased. It
can be assumed that the increase of illite+smectite was caused by the transformation of the
amorphous phases into clay minerals. Rutin and its combinations with organics possibly
mobilized Fe from soil along with other cations which could then find suitable conditions to
recrystallize as new forming clay minerals. Na, K, and Ca could enter in the interlayer regions
of phyllosilicates, while Al and Si could induce the formation of new sheets. Although the
variation in soil mineralogy could not be easily related to Fe mobilization, it was evident that
some treatments producing the higher Fe solubilization showed also the higher increase of
the illite+smectite fraction.
96
A microfluidic device for imaging and asymmetric perfusion of Arabidopsis
roots
Claire Stanley1, Jagriti Shrivastava2, Rik Brugman2, Dirk van Swaay1, Andrew deMello1,
Guido Grossmann*2
1
ETH Zürich, Institute for Chemical and Bioengineering, Germany, 2Universität
Heidelberg / Centre for Organismal Studies, Germany
Plant roots are highly sensitive to changing environmental conditions such as water and
nutrient availability, biotic and abiotic stresses. Long-distance communication from root to
shoot has recently been reported to be specific for salt stress perceived by roots. Open
questions include how and why certain stimuli cause local, cell-autonomous responses, whilst
others result in cell-cell communication and coordinated responses by tissues, organs or the
entire plant.
To understand how roots sense and process information about their environment we need
tools that allow live imaging of roots and provide precise control over the root
microenvironment. Over the past years, a number of microfluidic devices have been
developed to cultivate and perfuse Arabidopsis roots at the microscope. These devices have
substantially advanced experimental access to roots and some devices, e.g. the RootChip,
allowed pulsed treatments. So far, in devices where roots grow in channels it has been
difficult to locally apply treatments only to selected regions of the root, as the organ was
usually perfused as a whole. Moreover, due to roots bending within observation chambers,
an even perfusion was often challenging to achieve, thus affecting reproducibility of the
treatments.
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Here we present a new imaging and perfusion device for Arabidopsis roots that provides
guidance of the root tip and centering of the root within the chamber to allow symmetric or
asymmetric perfusion of the root. By applying treatments specifically to one side of the root
we are able to distinguish between cell-autonomous responses and coordinated responses to
changing environmental conditions.
97
Screening for allelochemical stress on cytoplasmic protein synthesis pattern of
selected plants
Aasifa Gulzar*, M.B. Siddiqui, Shazia Bi
Aligarh Muslim University, India
Determining the mode of action of allelochemicals is one of the challenging aspects in
allelopathic studies. Recently, allelochemicals have been proposed to cause differential
protein expression in target tissue and inhibit the early growth. Phenolic acids, one of the
common allelochemicals emitted from rhizosphere soil is known for its growth-inhibitory
activity. The aim of the present study was to determine the inhibitory effect of Phenolic acids
on seedling growth, dry biomass, total protein content and expression levels of proteins.
Effects of allelochemicals on early seedling growth and dry biomass were studied in three test
species, Glycine max Willd., Lycopersicon esculentum L. and Lantana camara L. Total protein
content and their differential expression were studied in the leaves of test species.
Rhizosphere soil was analysed for the detection of phenolic acids.
The root length, shoot length and dry biomass were significantly reduced in rhizosphere soil
as compared to control soil. SD-PAGE analysis showed different protein expression in three
species on exposure to rhizosphere soil. Protein banding pattern not only differ between the
control soil and rhizosphere soil, but also among test species. The protein content was
increased in Glycine max, and esculentum while it was decresed in L. camara in response to
rhizosphere soil. Rhizosphere soils contained significantly higher amount of phytotoxic
phenolics as the putative allelochemicals, which were ferulic acid, vanillic acid, p-coumaric
acid and benzoic acid.
The study concluded that C. procera roots and rhizosphere soil exerted allelopathic effect on
test species by releasing water-soluble phenolic acids as putative allelochemicals in soil which
will act as a structural substitute for synthetic herbicides.
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98
Quantification of C- and N- rhizodeposition of peas under field conditions
Anke Hupe*1, Florian Wichern2, Hannes Schulz3, Franziska Näther3, Rainer Georg
Jörgensen1
1
University of Kassel, Soil Biology and Plant Nutrition, Germany, 2Rhine-Waal University
of Applied Sciences, Faculty of Life Sciences, Germany, 3University of Kassel, Department
of Organic Farming and Cropping, Germany
Quantification of the C- and N- rhizodeposition under field conditions is difficult as labelling
plants with stable or radioactive isotopes and separating labelled and unlabeled roots is
difficult and often creates unnatural conditions. As a consequence experiments are often
done under controlled conditions. To estimate a realistic amount of C and Nrhizodeposition, experiments have to be conducted under field conditions without
influencing the root system or the water and nutrient budget. The presentation will focus on
the results of a study, which quantified the C- and N- rhizodeposition of peas in a field
experiment in 2013. In particular, the spatial and temporal distribution of rhizodeposition and
their transfer into different soil compartments was measured. Furthermore, the percentage of
the microbial biomass C and N deriving from rhizodeposition was quantified. For this, the pea
(Pisum sativum cv. Santana) was labeled fortnightly (beginning at BBCH 13) with a solution of
2% 13C-glucose (99 atom%) and 0.5% 15N urea (95 atom%) using the cotton wick method.
Sampling took place on four dates depending on the growth stage (beginning at BBCH59).
Soil samples were taken at 0-30 and 30-60 cm depth. The sampling of the soil always took
place in three defined sectors of the microplots (directly on the plant; between two plants in
the row; between two rows). In order to calculate the complete root biomass and
rhizodeposits of one pea plant and to reduce errors, the weighted mean of the three sectors
in 0-30 cm and 30-60 cm depth was calculated.
In the presentation the following hypotheses are discussed:
1. the amount of C and N rhizodeposition under field conditions is substantially higher
compared to pot experiments;
2. the development of rhizodeposition depends on the development of above-ground
plant biomass.
99
Density-based approaches to interface models and data in root biology
Dimitris Kalogiros*1, Michael Adu2, Xavier Draye3, Mariya Ptashnyk4, Glyn Bengough4,
Lionel Dupuy2
1
University of Dundee / The James Hutton Institute, Scotland, 2The James Hutton
Institute, Scotland, 3Earth and Life Institute, Université Catholique de
Louvain, Belgium, 4University of Dundee, Scotland
Understanding the development of root system architectures is difficult, because they consist
of numerous interconnected roots which develop in parallel in response to many intrinsic and
environmental cues. Root system architecture models have proved useful to deal with that
complexity, yet they suffer from the lack of quality growth data for roots grown in soils.
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We present recent advances in the construction, calibration and validation of root system
architecture models. Mathematical models use density distributions and time-delay partial
differential equations to describe root developmental mechanisms. We also present methods
to estimate root density distribution from experimental image data (kernel-based estimators)
and test different optimisation algorithms to parameterise the mathematical model on such
data. Results show that density-based models can be parameterised accurately from
experimental density distribution function, paving the way for new approaches to understand
growth responses to environmental cues. Plants develop diverse root system architectures to
capture soil water and nutrients.
This study shows that density-based models provide a simpler link with experimental data,
thereby complementing existent root system architecture models and setting the stage for
characterisation of root properties and investigation of root functions.
100
Application of laser capture microdissection for assessing pyoverdine synthesis
in planta by endophytic bacteria
Laure Avoscan1, Christine Arnould2, Aline Bonnotte3, Jeannine Lherminier2, Philippe
Lemanceau*1, Sylvie Mazurier1
1
INRA/UMR Agroécologie, France, 2INRA/UMR Agroécologie/Plateforme DimaCell,
Centre de Microscopie INRA, France, 3Université de Bourgogne/UMR
Agroécologie/Plateforme DimaCell, Centre de Microscopie INRA, France
Previous studies have shown that some isolates of fluorescent pseudomonads are able to
colonize root tissues and are detected in root intercellular spaces and root cells. These
bacteria are also known to scavenge iron from the soil and from the rhizosphere
environment, thanks to the synthesis of secondary metabolites called siderophores.
Meanwhile a bacterial siderophore produced by Pseudomonas fluorescens C7R12
(pyoverdine) has been shown to be incorporated in planta and to contribute to plant iron
nutrition. Taken together, these observations raise the question of whether siderophores may
be produced in planta by endophytic bacterial cells. To address these questions, laser capture
microdissection (LCM) and gene expression studies have been combined. More precisely, the
model plant Medicago truncatula has been grown axenically under controlled conditions of
iron supply and inoculated or not with P. fluorescens C7R12. Firstly, root zones colonized by
the bacteria have been described by transmission and scanning microscopy observations.
Then, labeling and fixation procedures for microscopy have been adapted to allow the
selective laser capture microdissection of endophytic bacterial cells and nucleic acid
microextraction prototocols were adjusted to extract RNAs from the dissected samples. PCR
primers targeting genes of P. fluorescens C7R12 implied in the pyoverdine synthesis were
designed and RT-qPCR protocols were developed to compare the level of expression of these
genes ex planta and in planta. The results of this comparison will be presented together with
further plans to evaluate the influence of the pyoverdine on the plant iron homeostasis.
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101
Plant water-uptake effects on salt distribution at the root-soil interface
Adi Perelman*1, Helena Jorda Guerra2, Jan Vanderborght3, Andreas Pohlmeier2,
Shimon Rachmilevitch4, Naftali Lazarovitch4
1
Ben-Gurion University of the Negev, Israel, 2Forschungszentrum
Jülich, Germany, 3Forschungszentrum Jülich, KU Leuven, Belgium, 4Ben-Gurion
University of the Nege, Israel
In recent years, increased salinization of soils, along with the depletion of available water, has
become a major threat for agriculture. The rhizosphere plays an important role in connecting
processes at the soil-plant- atmosphere interface. A better understanding of these
connections and their mutual influences can help in improving crop yield. New technologies
provide better tools with which to study root structure and function in non-destructive ways.
These techniques also provide a refine resolution regarding processes occurring where the
roots meet the soil, e.g., water uptake and salt accumulation. Bell peppers are considered to
be sodium excluders, whereas carrots take up sodium. Therefore, we hypothesize that salt
accumulation around the roots will create a distinction between them under the same
irrigation regime. The initial results from rhizoslides (capillary paper growth system) show the
salt concentration gradient is decreasing with distance from the root, compared with the bulk
that remained more stable. At the microscopic scale, magnetic resonance imaging (MRI) will
be implemented to observe root structures, water content and sodium concentration
distributions around single roots. At the macroscopic scale, root systems and solute and
water distributions within the root zone of plants grown in lysimeters will be monitored using
a combination of electrical resistivity tomography (ERT) and local soil sensors. Data will be
used to calibrate a model that is expected to predict root water uptake in saline soils for
different climatic conditions and different soil water availabilities. Sensitivity analyses from a
simulation study with this detailed model for different soil salinities, irrigation regimes and
weather scenarios will be presented.
102
Dissecting root secretion using novel lux biosensors
Joshua Roworth*, Philip Poole
University of Oxford, United Kingdom
Plant roots secrete a complex mixture of metabolites, which are key to determining the
structure of the microbial community (microbiome) in the rhizosphere. The microbiome in
turn can alter plant growth both positively (plant growth promotion) or negatively
(pathogenesis).
Lux biosensors were developed in Rhizobium leguminosarum biovar viciae 3841, which
respond to metabolites secreted by plant roots both specifically (e.g. phenylalanine) and
generally (e.g. expression is dependent on available carbon). By using a highly sensitive,
cooled CCD camera these biosensors can be used for non-invasive, temporal and spatial
mapping of metabolite secretion. Using this novel system it is possible to screen plant
mutant libraries of rice for both increased and decreased metabolite secretion. Once
secretory mutants have been identified the nature of a mutation can be determined (e.g.
increased formate secretion) and phenotypic characterisation of the effects these mutations
have on plant growth, productivity and rhizosphere colonisation can be assessed.
271
Identifying the roles that key metabolites play in plant health and the rhizosphere is an
important step in understanding how plants manipulate the rhizosphere to their advantage.
Identifying metabolites that play a key role in the rhizosphere are key targets for genetic
manipulation to aid in plant growth, health and productivity. In the case of rice, this in the
future will hopefully lead to increased yields in one of the most important food crops.
103
Fungi as food source for rhizosphere bacteria
Max-Bernhard Rudnick*1, Wietse de Boer2, Hans van Veen2
1
Leibniz Institute of Vegetable and Ornamental Crops (IGZ), Germany, 2Netherlands
Institute of Ecology (NIOO-KNAW), Netherlands
Fungi and bacteria are primary consumers of plant-derived organic compounds and,
therefore, considered as basal members of soil food webs. Trophic interactions among these
microorganisms could, however, induce shifts in food web energy flows.
Given increasing evidence for a prominent role of fungi as primary consumers of root
exudates, we propose that fungus-derived carbon may be an important resource for
rhizosphere bacteria.
To test this assumption, two common rhizosphere-inhabiting saprotrophic fungi, Trichoderma
harzianum and Mucor hiemalis, and a plant-pathogenic fungus, Rhizoctonia solani, were
confronted in microcosm systems with bacterial communities extracted from the
rhizospheres of two plant species, Carex arenaria and Festuca rubra.
Bacteria that associated with hyphae were screened for their ability to feed on growing fungal
hyphae. This screening revealed a widespread ability of rhizosphere bacteria to feed on
energy resources provided by living fungi. The identity of the fungi had a strong effect on the
composition of these mycophagous bacteria, whereas the effect of rhizosphere origin (sedge
versus grass) was small.
Our results suggest that secondary consumption of fungal resources instead of primary
consumption of root exudates can be an important nutritional strategy among rhizosphere
bacteria. Food web models should take this possibility into account as this has important
consequences for carbon fluxes with more carbon dioxide released by microbes and less
microbial carbon available for the soil animal food web.
Another, interesting finding is that several of the “fungus-feeding” bacteria appeared to be
closely related to plant-pathogenic bacteria.
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104
Use of high throughput sequencing to study oomycete communities in soil and
roots
Rumakanta Sapkota*, Mogens Nicolaisen
Aarhus University, Denmark
Many of the plant diseases caused by oomycetes such as cavity spot and damping off involve
a complex of several species emphasizing the need to use a community approach when
studying these organisms. Despite the economically importance of plant pathogens such as
Phytophthora and Pythium, we have limited understanding of the diversity of oomycetes in
symptomatic plant tissue as well as in root zones. The aim of this study was to improve and
validate techniques for using high throughput sequencing as a tool for studying oomycete
communities. Primer sets ITS4, ITS6 and ITS7 that have been used earlier in similar studies but
with limited success, were used in this study with an improved protocol. Our result shows
that the proportion of retrieved oomycete sequences dramatically increased, mainly by
increasing the annealing temperature during PCR. The optimized protocol was validated
using mock communities, DNA extracted from carrot tissue samples with symptoms of
Pythium infection and soil samples collected from agricultural fields. Sequence data from
Pythium and Phytophthora mock communities showed that our strategy successfully detected
all included species. Taxonomic assignments of operational taxonomic units from
symptomatic lesions in carrot resulted in 94% of the reads belonging to oomycetes with a
dominance of species of Pythium that are known to be involved in causing cavity spot.
Moreover, soil samples showed that 95% of the sequences could be assigned to oomycetes
including Pythium, Aphanomyces, Peronospora, Saprolegnia and Phytophthora. A high
proportion of oomycete reads was consistently present in all symptomatic lesions and soil
samples showing the versatility of the strategy and thus demonstrating the usefulness of the
method in plant and soil DNA background.
105
Quantifying and comparing AM fungi biomass in three soils colonized by maize
using signature fatty acids and microscopic methods
Mahaveer P Sharma*1, Jeffrey S Buyer2
1
ICAR-Directorate of Soybean Research, India, 2USDA-ARS, SASL, BARC-West, United
States
The arbuscular mycorrhizal (AM) fungi association of the Phylum Glomeromycota is one of
the most common associations colonizing the roots of more than 80% terrestrial plants. AM
fungi are well-known plant symbionts which provide many benefits to plants such as
enhanced growth, mineral nutrition particularly phosphorus and zinc uptake, protection
against soil pathogens, and improved response to moisture stress. There is increased interest
in the area of mass production and application of AM fungi which necessitates selection of
efficient AM strains. Selection of superior AM candidates for mass production and growth
promotion is being done based on quantification of AM fungi, hence the quantification of AM
fungal biomass in soil and roots is essential to every experimental setup involving the AMFplant interaction. Quantification of mycorrhizal biomass and root colonization has
traditionally been performed by root staining and microscopic examination methods which
are time-consuming, laborious and rely on utilizing specific stains to visualize the AM-roots
273
and then quantify based on person’s skills in microscopic observations and often lacks
reproducibility of quantified data by different laboratories.
In this study we compared two microscopic methods, spore counts and root colonization, to
three biomarker methods: neutral and phospholipid fatty acids (16:1ω5 PLFA and NLFA), and
16:w5 ester-linked fatty acid analysis in three different soils raised on maize. Irrespective of
soil type, consistently the 16:1ω5 ester-linked fatty acid (EL-FAME) was found to be positively
correlated both with spore count and root colonization whereas neutral lipids correlated well
only with spore count.
Overall, analysis of 16:1ω5 ester-linked fatty acid was the most useful biomarker for assessing
AMF biomass, while 16:1ω5 neutral lipid fatty acid analysis was useful for estimating
mycorrhizal spore counts.
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Rhizosphere Microbiome
107
Effects of four plant functional types on rhizosphere bacterial community
composition in a subarctic Sphagnum peatland
Sylvain Monteux*1, James T. Weedon2, Frida Keuper3, Ellen Dorrepaal4
1
Climate Impacts Research Centre, Department of Ecology and Environmental Sciences,
Umeå University, Sweden, 2University of Antwerp, Department of
Biology, Belgium, 3French National Institute for Agricultural Research,
Laon, France, 4Climate Impacts Research Center, Department of Ecology and
Environmental Sciences, Umeå University, Sweden
Northern peatlands store about one third of the world’s soil organic carbon, the future
stability of which is a global concern that remains poorly understood. Bacterial communities
play a major role in decomposition, and can be affected by many environmental factors. Of
these, rhizodeposition is particularly interesting as its possible effects on bacterial community
structures and priming effects could greatly affect the rate of soil organic carbon
decomposition.
While effects of hydrology and microclimate on microbial communities have recently been
investigated in peatlands, little is known about the impacts of rhizodeposits from different
vascular plants. We therefore investigated whether bacterial communities are affected by
differential rhizodeposition by vascular plant species in Sphagnum peatlands. We
hypothesized that plant species belonging to different plant functional types with different
rhizodeposits harbour different bacterial communities in their rhizosphere than those
observed in bulk Sphagnum peat. Alternatively, it may be that Sphagnum peat composition is
sufficiently homogeneous to overcome rhizodeposits effects.
To test this hypothesis we sampled Sphagnum peat soils from a subarctic peatland in
northern Sweden (close to Abisko, 68°21′N). We compared Sphagnum peat from areas with
low vascular plant cover (bulk peat) with the rhizosphere of four common species of vascular
plants representing four functional types: sedges (Eriophorum vaginatum), forbs (Rubus
chamaemorus), deciduous shrubs (Betula nana) and evergreen shrubs (Empetrum nigrum).
We present bacterial communities’ profiles obtained through Illumina sequencing of 16S
rRNA gene amplicons and test the extent to which community composition in bulk peat
differs from those in the rhizosphere, and whether plant species from different functional
types harbour distinct bacterial communities in their rhizosphere.
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Plant traits related to nitrogen uptake influence plant-microbe competition
Delphine Moreau*, Barbara Pivato, David Bru, Hugues Busset, Florence Deau, Céline
Faivre, Annick Matejicek, Florence Strbik, Laurent Philippot, Christophe Mougel
INRA, France
Plant species is an important driver of soil microbial communities. However how plant
functional traits are shaping these communities has received less attention while linking plant
and microbial traits is crucial for better understanding plant-microbes interactions. Our
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objective was to determine how plant-microbes interactions were affected by plant traits.
Specifically we analyzed how interactions between plant species and microbes involved in
nitrogen-cycling were affected by plant traits related to nitrogen nutrition in interaction with
soil-nitrogen availability. Eleven plant species, selected along an oligotrophic-nitrophilic
gradient, were grown individually in a nitrogen-poor soil with two levels of nitrate availability.
Plant traits for carbon and nitrogen nutrition were measured and the genetic structure and
abundance of rhizosphere microbial communities, specifically the ammonia oxidizer and
nitrate reducer guilds, were analyzed. The structure of the bacterial community in the
rhizosphere differed between plant species and these differences depended on nitrogen
availability. The results suggest that the rate of nitrogen uptake per unit of root biomass and
per day is a key plant trait, explaining why the effect of nitrogen availability on the structure
of bacterial community depends on the plant species. We also showed that the abundance of
nitrate reducing bacteria always decreased with increasing nitrogen uptake per unit of root
biomass per day, indicating that there was competition for nitrate between plants and nitrate
reducing bacteria. This study demonstrates that nitrate reducing microorganisms may be
adversely affected by plants with a high nitrogen uptake rate. Our work puts forward the role
of traits related to nitrogen in plant-microbe interactions whereas carbon is commonly
considered as the main driver. It also suggests that plant traits related to ecophysiological
processes, such as the nitrogen uptake rate, are more relevant for understanding plantmicrobe interactions than composite traits, such as nitrophily, related to a number of
ecophysiological processes.
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Soil microbial diversity effects on primary production and symbiotic interactions
Christophe Mougel*1, Clémentine Lepinay2, Julie Aubert3, Sébastien Terrat4, PierreAlain Maron2, Christophe Salon2, Thierry Rigaud5
1
INRA, France, 2INRA Agroécologie, France, 3INRA Mathématiques et Informatique
Appliquées, France, 4INRA Agroécologie & Plateforme GenoSol, France, 5UMR CNRS
6282 Biogéosciences, France
The consequences of biodiversity losses on ecosystem functioning are now frequently
addressed in ecology and biological conservation, mainly because of the importance of
ecosystem functioning in providing goods and services to human activities. Most studies on
the consequences of biodiversity losses are on plants. However, the importance of telluric
microorganisms linked together by trophic exchanges with plants, that sustain all ecosystems
through primary production, is known. However, the role of soil microbial diversity for
primary production remains controversial.
We test the hypothesis that decreased soil microbial diversity may impact negatively plant
primary production and symbiotic associations. For this, we manipulated the soil microbial
diversity by experimental erosion, and measured the changes in microbial community
diversity using massive sequencing. We then measured the effect of diversity loss on
plant/symbionts association and on plant growth and fitness. The results showed that the
impact of microbial diversity on plants depends on their reliance on symbionts. On the whole,
Medicago. truncatula was negatively affected by erosion, Brachypodium distachyon was
positively affected and there was no significant effect on Arabidopsis thaliana.
These results are of interest to predict the consequences of soil microbial diversity loss, on
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crop plants productivity under low nutrient inputs, according to microbial communities and
symbiotic interactions.
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Taxonomic and metabolic diversity of Pseudomonas spp. fluorescents isolated
from the rhizosphere of the southern Algerian palm groves
Ben Oussaid Nacera*1, Benchabane Messaoud2, Phillipe Thonart3
1
University Blida 1, Departement of Biology, Algeria, 2University Blida1, Departement of
Agronomy, Algeria, 3University of Liege, Walloon Center for Industrial Biology, Belgium
Pseudomonas spp. fluorescents characterized by genetic and phenotypic diversity in relation
to their positive impact on the functioning of the rhizosphere, exerting direct and / or indirect
actions on the development of the plant. However, the exploitation of microbial resources
occupies a prominent place in applications preservation and restoration of soil fertility. The
effectiveness of some strains of Pseudomonas spp. fluorescents organic plant protection has
been demonstrated in numerous studies in experimental conditions and practices. The
intended benefits are essentially on biocontrol of plant pathogens, the nutritional status of
the plant and its defensive natural abilities. Our work presents the characterization of a
collection of strains of Pseudomonas spp. fluorescent, isolated from the rhizosphere of
different palm of Bechar, Ghardaia and Ouargla province southern of Algeria and their
potential use as biological means. The selection of effective isolates was carried out on the
basis of exhibition of substantial involved in the processes of biocontrol and phytostimulation
(siderophores, HCN, phenazines, AIA...). In addition to the basic bacteriological tests
(morphological, physiological and biochemical tests, Biolog), 16S rRNA was amplified by PCR.
Sequencing was performed using an ABI Prism® 3130XL Genetic Analyzer (Applied
Biosystems, Foster City, CA, USA) and the phylogenetic analysis was performed using the
software package BioNumerics (Applied Maths, Belgium).). Phenotypic and genotypic
identifications showed that the predominant species is P. fluorescens (50%), in addition to the
presence of P.putida, P.chlororaphis, P.aureofasciens and intermediate strains. The results
indicate the presence of a taxonomical diversity and the particular metabolic faculties in the
group of Pseudomonas spp. fluorescent and particularly in type species P. fluorescens and P.
putida. The selection of microbial taxa, in palm groves, can be evoked.
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Diversity and abundance of genes for the synthesis of osmoprotectans in the
rhizosphere of the halophyte Arthrocnemun macrostachyum
Salvadora Navarro-Torre*1, José María Barcia-Piedras1, Eloisa Pajuelo1, Miguel Ángel
Caviedes1, Susana Redondo-Gómez1, María Camacho2, Ignacio D. Rodríguez-Llorente1
1
University of Seville, Spain, 2IFAPA, Centro Las Torres-Tomejil, Spain
Arthrocnemum macrostachyum is a halophyte naturally growing in salt marshes and salt
steppes of the south of Spain. Our interest is focused in the utility of this plant and the
microorganisms colonizing its rizosphere to desalinate coastal agricultural soils. In this
context, the aim of this work was to study the diversity of bacteria able to produce
osmoprotectants related with salt tolerance (ectoine and betaine) in the rhizosphere of the
plant. Based on their salt tolerance and requirements, several halophilic and halotolerant
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bacteria were isolated and identified. All of them showed the presence of ectC (ectoine
synthase) and betB (betaine aldehyde dehydrogenase) genes. Three of this isolates (Cs16b,
Cs25 and Hvs18) have been identified as halophilic strains, being Hvs18 able to tolerate up to
20% NaCl. Cs16b, Cs25 and the halotolerant strain named Hv16, have been described as
novel species within the genera Microbulbifer, Labrenzia and Kokuria, respectively, indicating
that rhizosphere of halophyte plants are an excellent ecosystem to isolate new
halophilic/tolerant species. In order to study the biodiversity of bacteria with ectC and betB
genes, an internal fragment of each was amplified from total soil DNA isolated from the
rhizosphere of A. macrostachyum growing in two different soils. Amplified ectC fragment was
cloned into pGEM-T easy vector and 100 positive clones were sequenced. 70% of the
sequences corresponded to ectC and at least 17 different sequences were identified. On the
other hand, betB fragment was used use as template for a DGGE analysis of the abundance
and diversity of betB in those rhizospheres. Our results show a great biodiversity of bacteria
harboring genes for the synthesis of osmoprotectants, most of them described for the first
time in this work.
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Rhizosphere bacterial communities from wheat field in Brazil and the occurrence
of wheat blast
Vanessa Nessner Kavamura*1, Ana Gabriele Barbosa-Casteliani2, Rodrigo Taketani1,
Angelo Sussel3, Itamar Melo1
1
Embrapa Environment, Brazil, 2University of São Paulo, Brazil, 3Embrapa
Cerrados, Brazil
Brazil is the second largest wheat producer in South America. However, wheat blast disease
outbreaks caused by Magnaporthe grisea result in high yield losses. Recent studies have
shown that this fungus has the ability to infect through the roots as well as the foliar system.
Understanding the changes in the structure of rhizosphere microbial communities of wheat
plants in areas with different levels of infection might help unveil which groups of
microorganisms could be acting to supress M. grisea. Rhizosphere and bulk soil samples were
collected from a wheat field in Distrito Federal (DF), with disease levels of 10% and 100%.
DNA was extracted and bacterial community profiling by 16S rRNA (V6-V7) amplicon
sequencing was achieved through the Ion Torrent platform. Analyzes were performed using
QIIME, PAST and STAMP software. Nonmetric multidimensional scaling (NMDS) plots
revealed three major groups (stress value=0.1218), one comprised bulk soil samples and the
other two were made up of rhizosphere samples, which were separated by disease incidence.
Similarity percentage (SIMPER) analysis revealed that bacterial communities from rhizosphere
samples were more different from each other (overall dissimilarity=96.16%) than the
equivalent bulk soil samples (overall dissimilarity=88.12%). Besides, at class level, STAMP
analysis revealed that Cytophagia, Deltaproteobacteria and Rubrobacteria are more abundant
in both bulk soil and rhizosphere samples with higher incidence of disease (P<0.05). Future
work will comprise more samplings to obtain a better understanding of the structure of
bacterial communities associated with wheat in different fields from Brazil, with different
levels of disease incidence. This, combined with isolation of antagonists to M. grisea, might
help in the development of biological control methods.
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113
Microbiome of Vitis vinifera cv. Pinot Nero in two vineyards with different soil
managements
Giorgia Novello*, Elisa Gamalero, Elisa Bona, Lara Boatti, Flavio Mignone, Patrizia
Cesaro, Nadia Massa, Guido Lingua, Graziella Berta
Università del Piemonte Orientale, DiSIT, Italy
Soil is a complex microhabitat where microorganisms regulate plant productivity and the
maintenance of biogeochemical cycles. Due to the release of carbon-rich exudates
(rhizodeposition), bacterial densities increase in the rhizosphere. The rhizosphere is a site of
intense interactions among the plant roots, the soil and the microflora. The composition, the
activity and the density of microbial cells living in the rhizosphere is stimulated by the
rhizodeposition: as a result, the community structure of the rhizosphere is different by that
found in bulk soil. Both the density and the biodiversity of bacteria in soil are affected by the
soil condition and by its management.
The bulk soil and the rhizospheric soil of the grapevines were sampled before and after grape
production. DNA was extracted from 1 g of soil (bulk soil and rhizospheric soil) using the
UltraClean™ kit and amplified with primers for the V1 and V4 regions of 16S rDNA according
to the methods optimized for 454 Roche pyrosequencing.
Preliminary data indicated that the sampling time is an important factor regulating
biodiversity. Actinobacteria was the dominant group followed by Sphingobacteriales. Among
Proteobacteria, alpha Proteobacteria were prevalent with the Rhizobiales as the most
representative group. Burkholderiales was the most prevalent among the beta
Proteobacteria, while members of gamma Proteobacteria were not detected in bulk soil. The
abundance of firmicutes was low in all sites; however, the species Desulfosporinus meridiei
was dominant among the firmicutes found in the bulk soil.
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Molecular identification and functional diversity of microorganisms isolated
from the rhizosphere of plants grown in a suppressive soil substrate
Iakovos Pantelides*, Stella Papageorgiou, Αnastasis Antoniou, Eleftheria Demetriou,
Maria-Dimitra Tsolakidou, Dimitris Tsaltas
Cyprus University of Technology, Cyprus
In this study we investigated the effectiveness of a compost amendment, consisting of
recycled plant material, against the wilt pathogens of tomato Verticillium dahliae and
Fusarium oxysporum f. sp. lycopersici. The plants grown in sterile compost (SC) mix exhibited
significantly more severe symptoms and fungal biomass in their vascular tissues compared to
the plants grown in non-sterile compost (NSC) substrate. Plant height, total leaf surface and
fresh weight of tomato plants grown in the NSC mix were significantly more compared to the
plants grown in the SC substrate. Therefore, the microorganisms inhabiting the compost are
likely responsible for the suppressive properties observed and may act as growth promotion
inducers. In order to identify the microorganisms responsible for these phenotypes we first
characterized the compost’s microbial population and then we identified the microbes that
colonized the rhizosphere of tomato plants grown in the NSC substrate. 132 bacterial and 79
fungal compost isolates and 143 bacterial and 50 fungal rhizosphere isolates were identified
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by sequencing of the 16S and ITS region respectively. It was found that the incidence of the
rhizosphere microorganisms was different compared to that of the compost. The isolated
rhizobacteria were evaluated for their biological activity against 4 soilborne pathogens (V.
dahliae, F. oxysporum f. sp. lycopersici, F. oxysporum f. sp.radicis- lycopersici and Sclerotinia
sclerotiorum) by the dual culture technique and tested for hydrogen cyanide and indole-3acetic acid production, phosphate solubilization, protease, chitinase, pectinase and cellulose
activity. The present study revealed a high degree of functional and genetic diversity among
the microorganisms isolated from the rhizosphere of plants grown in a suppressive soil.
These isolates are considered to play a vital role in disease suppression and plant growth
promotion due to their potential of producing an array of antifungal metabolites, hormones
and plant growth promoting enzymes.
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Bacterial inoculation on maize crops seen under invasion ecology perspectives –
is the best invader the best plant growth promoter?
Pedro Beschoren da Costa*1, Samanta Bolzan de Campos2, Paul Dirksen2, André
Dresseno1, Volker Wendisch2, Luciane Passaglia1
1
Universidade Federal do Rio Grande do Sul, Brazil, 2Bielefeld University, Germany
Bacterial inoculants applied to crops are very important for sustainable agriculture, but might
not be always effective under field conditions. As interaction between the inoculants and the
native microbial community plays a key role in effective root colonization and plant growth
promotion, classical invasion ecology might be applied for inoculants on crops to improve
microbial management effectiveness. In this work, we apply hypothesis based on invasion
ecology to crop fields under bacterial inoculation. We evaluate the associated microbial
community composition of inoculated and non-inoculated plants to test if: (I) an inoculant
with a higher invasion potential will provide a higher plant growth promotion effect;(II) if
lower diversity/resource ratio in the environment facilitates invasion. To do this, we analyzed
3 different maize crops locations in south Brazil, which received the same 4 bacterial
inoculants (one Azospirillum, one Achromobacter, and two Pseudomonas). Metagenomic
samples were taken from bulk soil immediately before planting, and from rhizospheric soil of
10 day old plants. The V4 region of the 16S rDNA gene was then sequenced on the MiSeq
platform. Principal Coordinate Analysis shows that samples clustered by the 3 locations.
Principal Component Analysis, based on the Similarity Percentage test results of the controltreatment pairs, was used as a proxy for inoculant invasion ability. Data from this approach
suggests that 2 inoculants may induce very different shifts in the community upon
inoculation, acting differently in different locations. These different shifts that suggest higher
invasion ability, however, were not associated to greater crop productivity. The
diversity/resource ratios are still to be calculated, so invasion difficulty across environments
must yet be estimated. Our preliminary conclusion is that it is not possible to predict the
most effective inoculants by looking at shifts on the rhizospheric community 10 days after
planting – what would be extremely useful in field trials.
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116
Soil microbiomes are different in healthy and declined Quercus suber (cork oak)
Diogo Pinho*1, Cristina Barroso1, Paula Gomes1, Miguel Pinheiro1, Conceição Santos
Silva2, Conceição Egas1
1
Next Generation Sequencing Unit of CNC/Biocant, Portugal, 2Coruche Forest
Landowners Association (APFC), Portugal
Cork oak is an evergreen tree of utmost importance in Portugal because of the social and
economic value of cork and also in the preservation of forest biodiversity. However, cork oak
forests have been declining, due to a complex series of biotic and abiotic factors. The
recognition of plant-associated microbiomes as key determinants of plant health and
productivity led us to deeply characterize the cork oak soil microbial community and test the
hypothesis that a specific soil microbial community is associated with Quercus suber health.
The bacterial and fungal soil communities were surveyed by pyrosequencing and the
microbial community associated with healthy and diseased cork oaks compared.
Results unveiled a rich bacterial and fungal soil community in the cork oak forest, with
Proteobacteria and Basidiomycota as the most abundant phyla. When compared, soils from
healthy cork oaks were significantly enriched in ectomycorrhizal fungi while saprobes were
enhanced in soils from diseased trees (p<0.05). Interestingly, the healthy cork oaks fungal
community was dominated by a single ectomycorrhiza, Cortinarius, while several saprobes
and other ectomycorrhiza prevailed in soils from declined cork oaks. A global comparison of
the bacterial and fungal communities was performed by Principal Coordinates Analysis and
significant differences were observed between the tree health conditions (RANOSIM=0.344,
p=0.002; SIMPER analysis: group dissimilarity of 43.7%), with Cortinarius, Steroidobacter and
Mycobacterium as the largest fungal and bacterial contributors for the differences.
This study provided a deep description of the soil microbiome of cork oak rhizosphere,
supporting the hypothesis that tree health condition has a specific microbiome associated. In
the future, the combination of rhizosphere microbiome characterization with Q. suber
genomic resources (transcriptome and genome) will enhance the understanding of cork oak
biology, and promote strategies leading to improved resistance to biotic and abiotic stresses,
growth promotion and increased cork production.
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Changes in rhizosphere microbial community association with different parental
types of Arabidopsis thaliana MAGIC lines
Carla Porges*1, Tesfaye Wubet1, Jörg Overmann2, Boyke Bunk2, Guillaume Lentendu1,
Johannes Sirkorski2, Francois Buscot1
1
Centre for Environmental Research UFZ, Germany, 2Leibniz Institute DSMZ, Germany
Plants can interact with microbes through changes in the composition and concentration of
root exudates. The composition of bacterial communities in the rhizosphere and within the
roots are distinct between plants, developmental stages and even ecotypes and differ from
the microbial community in bulk soil. However, while studying the microbial community in
the rhizosphere of A. thaliana most works are focusing on the bacteria, disregarding the
fungal part.
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In this study, which is part of the joint project “Chemical Communication in the Rhizosphere”,
19 parental lines of the model plant species A. thaliana were cultivated in pots under
controlled phytochamber conditions in two different soils. When the plants had reached an
early flowering stage rhizosphere soil and bulk soil were collected for DNA extraction.
Afterwards the genomic DNA of the rhizosphere of the 19 parental lines was analyzed for
potential bacterial communities by paired end Illumina 16S rDNA sequencing.
We found that the main driver of the rhizosphere bacterial communities was the soil origin.
Furthermore the presence of plants on the soil as well as the accession type had an influence
on the rhizosphere bacterial community due to the release of root exudates. Those
differences where significant in relative abundance and richness. In addition we identified a
dominance of the Alphaproteobacteria in the rhizosphere among the parental lines.
Further investigation, including sequencing of the rhizosphere fungal community via paired
end Illumina sequencing targeting the ITS2 region, will aim to elucidate the selection effect of
A. thaliana accessions on fungal taxa.
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Effect of plant domestication on the rhizosphere microbiome of common bean
(Phaseolus vulgaris)
Juan Esteban Pérez Jaramillo*1, Rodrigo Mendes2, Jos M. Raaijmakers1
1
Netherlands Institute of Ecology, Netherlands, 2Brazilian Agricultural Research
Corporation, Embrapa Environment, Brazil
Plant domestication was a pivotal achievement for human civilization and subsequent plant
improvement increased crop productivity and quality. However, domestication also caused a
strong reduction in the genetic diversity of modern cultivars compared to their wild relatives.
It is known that plants rely, in part, on the rhizosphere microbial community for growth,
development and tolerance to (a)biotic stresses. Hence, plant domestication events may have
adversely affected recruitment of and interactions with their beneficial microbial partners. In
here, we investigated the bacterial diversity of the rhizosphere of two wild relatives, three
landraces and three modern cultivars of common bean (Phaseolus vulgaris). These different
lines belong to the Mesoamerican bean gene pool of Colombia and were selected amongst
more than 37,000 accessions kept in the Genetic Resources Program of the International
Centre for Tropical Agriculture (CIAT, Colombia). The eight accessions were grown in both
native and in agricultural soils collected in the province of Antioquia (Colombia). At different
plant growth stages, DNA was extracted from rhizospheric soil and bacterial taxonomic
diversity was analysed by metagenomic sequencing of the V3-V4 region of the 16S rRNA.
Our approach of going “back to the roots” using native soils together with wild relatives
provides new fundamental insights in host genotype-mediated recruitment of beneficial
microbes and in the functional and metabolic potential of the rhizosphere microbiome of
native soils and wild relatives of modern crop cultivars.
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119
Description of tomato root colonization by Bacillus subtilis EA-CB0575 using
Fluorescence in situ hybridization (FISH) and Scanning Electron Microscope
(SEM)
Luisa Fernanda Posada Uribe*1, Valeska Villegas Escobar2, Javier Correa Álvarez2,
Magally Romero T.3, Luz Estela Gonzáles de Bashan4, Yoav Bashan4
1
Universidad EAFIT Medellin, Colombia, 2Universidad EAFIT
Medellín, Colombia, 3Universidad Nacional de Colombia, Colombia, 4CIBNOR
México, Mexico
Different Bacillus species have potential applications on important agricultural crops as Plant
Growth Promoting Bacteria (PGPB). Studying PGPB colonization is an important subject to
understand plant-microbe interactions; nevertheless, few studies have been conducted with
Gram positive bacteria. Techniques such as culture-based detection, PCR (polymerase chain
reaction), gfp insertion, immunodetection, FISH (Fluorescent in situ hybridization), have been
used for detecting colonization of microorganisms in plant roots or rhizospheric area. FISH
combines precision of molecular techniques, visual information of microscopy, and does not
generate genetic instability issues with transformed cells, therefore it is highly recommended
for Gram positive colonization studies.
Bacillus subtilis strain EA-CB0575 isolated from the rhizosphere of a banana plant in Urabá,
Colombia was reported as PGPB of different crops such as pepper, coffee, maize and banana.
In this study, the colonization of Bacillus subtilis EA-CB0575 was evaluated on tomato roots
using FISH and SEM techniques and the effect on plant growth promotion was also
determined. Probes Bs575-FAM and Bs575-Cy3 were designed based on 16s rDNA sequence
and their specificity was evaluated with different strains of Bacillus genus and other different
genus. It was determined that Bs575 probe was specific to Bacillus subtilis species, stable in
time and it was found in roots of tomato grown in MS medium, Hoagland nutrient solution
and arid soil. Additionally, when using the probe to determine the colonization patterns of
the strain in tomato roots, it was found that B. subtilis EA-CB0575 forms microcolonies at the
intersection between main and secondary roots after one month of inoculation. Moreover,
exogenous application of this strain to tomato plants promoted plant length and total dry
weight with significant statistical differences respect to control without bacteria.
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Effect of tillage systems on the prevalence of antifungal genes implicated in
fungal disease suppression of wheat within the microbiome of winter oilseed
rape
Ridhdhi Rathore*1, Kieran Germaine1, Dermot Forristal2, John Spink2, David Dowling1
1
Institute of Technology Carlow, Ireland, 2Teagasc Crop Research Centre, Oak Park,
Carlow, Ireland
Plants grow in intimate association with complex microbial communities, which live in the
rhizosphere, endosphere and phyllosphere. The relationships between this microbiota and its
host plant can vary from pathogenic to beneficial. This microbiota has been shown to
promote plant growth and productivity, improve carbon sequestration and enhance
phytoremediation of pollutants. In addition, some plant associated microorganisms play an
important role as biocontrol agents against pathogens.
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Winter oilseed rape (wOSR) is very important crop in Europe for biodiesel and edible seed oil
production. This crop is widely used as a break crop for Wheat to reduce ‘take all’ a major
fungal disease. Conventional cultivation of Wheat requires the use of chemical fungicides to
reduce losses resulting from this disease while significantly increasing the cost of crop
production. Since the crop production sector is facing a major profitability problem due to
continuing high input costs in its cultivation, novel technologies and practices are being
investigated to help reduce these costs.
The implementation of conservation tillage practices can significantly reduce the cost of
cultivation and has huge environmental benefits associated with it. However, there is
currently very limited knowledge on how conservation tillage practices impact the microbial
communities associated with wOSR. We have investigated the impact that conservation and
conventional tillage practices have on the prevalence of bacterial genes involved in fungal
disease suppression within the microbiome associated with OSR. The prevalence of genes
involved in two of these antifungal systems (PhlD and PhzB) was determined using qPCR on
DNA extractions isolated from rotational trials of Wheat and wOSR.
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Effect of Azospirillum inoculation on the abundance and genetic diversity of key
phytobeneficial microbial functional groups in the maize rhizosphere
Sébastien Renoud*, Jordan Vacheron, Jacqueline Haurat, Claire Prigent-Combaret,
Laurent Legendre, Yvan Moënne-Loccoz, Daniel Muller
UMR CNRS 5557 Microbial Ecology, France
Cereal seed inoculation with plant growth-promoting rhizobacteria (PGPR) can lead to
enhanced root system development, thereby allowing seedlings to rapidly explore deeper
soil layers and gain access to larger supplies of water and mineral nutrients. In addition, PGPR
inoculation can modify rhizomicrobial community structure, at least in the first stages of plant
development, which may result from inoculant effects on root physiology and
rhizodeposition, as well as more direct inoculant interactions with other rhizosphere
microorganisms. These microbial interactions between PGPR inoculants and the indigenous
microbiota may, in turn, have an impact on root and rhizosphere functioning, but this is
hardly documented. Microbial functioning of the rhizosphere relies on individual functions
carried out by functional groups e.g. nitrogen fixers, phytohormone producers, etc., which
often are comprised of multiple taxa, with taxa-specific contributions to a given ecological
function. Therefore, it is not feasible to infer the impact of PGPR inoculation on rhizosphererelevant microbial functional groups based on our knowledge of inoculant impact on the
taxonomic composition of the rhizomicrobial community. To address this issue, we assessed
the effect of seed inoculation with the phytostimulatory PGPR Azospirillum lipoferum CRT1 on
the size and diversity of microbial functional groups driving N dynamics or promoting plant
growth in the rhizosphere of field-grown maize. The first results with quantitative PCR
showed that inoculation resulted in small reductions in the size of microbial groups involved
in plant hormonal modification in the three fields studied. Sequencing data will be shown in
order to evaluate whether diversity modifications also took place in the maize rhizosphere.
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122
Dynamics of the microbial community structure in the rhizosphere of narrowleafed lupin and tomato as related to nitrogen form provided
Ana Alejandra Robles Aguilar*1, Oliver Grunert2, Vicky Temperton1, Stephan
Blossfeld1, Dirk Reheul3, Emma Hernandez-Sanabria2, Nico Boon2, Nicolai
Jablonowski1
1
Forschungszentrum Jülich GmbH, Institute für Bio- und
Geowissenschaften, Germany, 2Laboratory of Microbial Ecology and Technology
(LabMET), Ghent University, Belgium, 3Department of Plant Production, Ghent
University, Belgium
Ammonia-oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) are the most
important organisms responsible for ammonia and nitrite oxidation in agricultural
ecosystems and growing media. Ammonia and nitrite oxidation are critical steps in the soil
nitrogen cycle and can be affected by the application of mineral fertilizers or organic
fertilizers. The microbial community and structure associated with the growing medium will
also be influenced by the plant species identity. The functionality of the microbial community
has a major impact on the nutrient turnover and will finally influence plant performance. In
our study, we used two different plant species from different functional groups. Lupin, a
legume that makes a nitrogen-fixing symbiosis with the microbial community and tomato,
both with the availability to exudate nutrient mobilizing acids. We studied plant performance
in rhizotrons (a phentotyping system for imaging roots), including an optical method (planar
optodes) for non-invasive, quantitative and high-resolution imaging of pH dynamics in the
rhizosphere and adjacent medium. The horticultural growing medium was supplemented
with organic-derived nitrogen or ammonium derived from struvite. The possible differences
in the root structure between treatments is compared with the total root length. Destructive
growing medium sampling and high throughput sequencing analysis of the bacterial
abundance of the communities present in the rhizosphere and the bulk soil will be used in
order to study the growing medium-associated microbial community structure, and this will
be related to pH changes in the rhizosphere and the bulk soil. Our hypothesis is that the
growing medium-associated microbial community structure changes depending on the
nitrogen form provided, the plant species used, the rhizosphere and bulk soil. We expect a
higher abundance of bacteria in the treatment with organic fertilizer and a higher abundance
of AOB and NOB in the rhizosphere in comparison to the bulk soil.
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Use of 14C labelled model exudates to evaluate plant species effects on carbon
use efficiency in rhizosphere soil
Harriet Robson*1, Tom Sizmur1, Andrew Neal2, Liz Shaw1
1
University of Reading, United Kingdom, 2Rothamsted Research, United Kingdom
Rhizodeposition, defined as the flow of organic carbon from plant roots to soil, plays an
important role in the soil carbon cycle: it represents a major flux of photosynthetic C from
plant to soil and also helps shape the structure of the rhizosphere microbial communities that
are responsible for the rhizodeposit decomposition. Growing evidence suggests that belowground plant inputs make a large contribution to the stable soil organic carbon pool which is
composed mainly of organic carbon that has been processed by the microbial biomass rather
than selectively preserved plant litter C. Therefore, it follows that the efficiency by which
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rhizosphere microbial communities process rhizodeposited carbon to microbial products (as
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opposed to CO2; i.e. the carbon use efficiency) will be important for the long-term
stabilisation potential of plant-derived C in the rhizosphere. Here we examine how carbon
use efficiency in rhizosphere soil varies with respect to plant species with a focus on both
legume and non-legume grassland species. We determine carbon use efficiency through
tracking the fate of 14 C-labelled model exudates (glucose, succinate, cysteine, salicylic acid)
in sampled rhizosphere soil and examine the relationships with rhizosphere microbial
community structure. This poster presents results from this ongoing experiment.
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Effects of bacterial inoculation on sugarcane growth, physiology and assembly
of rhizosphere bacterial community
Mauricio Rocha Dimitrov*1, Raquel de Paula Freitas2, Adriana Parada Dias da Silveira2,
Eiko E. Kuramae1
1
Netherlands Institute of Ecology, Netherlands, 2Instituto Agronômico, Brazil
Sugarcane is one of the most important agricultural crops in the world, being source of
sugar, renewable energy (biofuel) and biomaterials. Brazil is the largest sugarcane producer in
the world and the leader in production of bio-ethanol, a renewable energy source that has
been presented as a great potential replacement for gasoline. However, to maximize
production, high rates of fertilizers are often applied to the field, which may lead to nutrient
leaching, increase in greenhouse gases emissions and reduction in biodiversity. Therefore, a
key challenge is to intensify agriculture practices while minimizing harmful environmental
effects of intensive fertilization. The use of beneficial bacteria, such as Plant Growth
Promoting Bacteria (PGPB), has been shown to enhance plant growth and health in controlled
conditions, even with low levels of fertilization. Effects of six bacterial isolates, which had been
previously isolated from sugarcane stalks, on sugarcane growth (shoot and root dry mass),
physiology (total sugar, sucrose, starch and nitrate concentration, as well as activity of
enzymes related to the nitrogen cycle) and assembly of rhizosphere bacterial community were
assessed in greenhouse conditions for six weeks. Bacterial isolates and controls were tested in
two different treatments regarding nitrogen content (0 and 350 mg N kg-1 of soil). All six
isolates promoted a significant increase on shoot and root dry mass in both nitrogen
treatments, when compared to controls that did not receive any inoculation. Results of
physiological endpoints as well as determination of rhizosphere bacterial
community are currently under analysis. Such results will contribute to a better understanding
of the effects promoted by bacterial isolates used here on sugarcane plants. Furthermore,
result may provide a glimpse of the sugarcane rhizosphere microbiome, as well as indicate
whether previous inoculation of beneficial bacteria may affect the formation of rhizosphere
bacterial community.
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Microbial community of Bacillus in the rhizosphere, an alternative to improve
growth and plant health
Ligia Sanchez*, Lucia Corrales, Graciela Lancheros, Yesid Ariza, Estefania Castañeda,
Lina Mendez, Andres Zarate, Paola Barrera, Adriana Diaz, Solange Benitez, Cristian
Layton, Luisa Monroy, Edna Maldonado, Jenny Celis, Milena Cordero
Colegio Mayor de Cundinamarca University, Colombia
In Colombia, agriculture plays an important role in the development of the country,
geographical position, biological diversity and climatic zones, related to the variety of plants
as food for its people and open markets throughout the world. Factors such as technological
development, cultural roots and the use of agrochemicals have not allowed high production
rates, reason why universities and research centers are focusing their projects towards
environmentally friendly alternatives that enable the farmer improve the growth and health
their crops. The research group has dedicated its efforts to determine the functional potential
from bacteria of the genus Bacillus from indigenous rhizosphere. The aim of this study was to
determine if some native gram positive bacilli from rhizosphere of Ornithogallum umbelatum,
Thymus vulgaris and Rosmarinus officinalis had capacity to fix nitrogen, solubilize phosphate
and produce antibiotics to promote growth and development in plants. The methodology
included functional tests with agar Ashby, Pikovskaya, evidence of antagonism against
Fusarium sp, and coding presence of genes for several antibiotics. Taking into account the
results, it was obtained the most effective antibiotic against the fungus by aerobic
fermentation. Finally, the Bacillus was genotyped as potential biochemical antibiotic. It was
found that several bacilli grew in culture media Ashby; most microorganisms showed
phosphate solubilization and in vitro antagonism against Fusarium sp. All bacillus showed the
presence of some gene encoding antibiotic. Even though, one of the rods showed presence
of the secondary metabolite Iturin A. They were identified by 16s all bacillus Biochemical
potential. It was concluded that the genus Bacillus can be a good alternative for growth and
development in plants by their functional versatility and be an environmental friendly option.
Currently, a master student of research group is developing a project to microencapsulate
bacilli and use it as biofertilizer.
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Seasonal observation of prokaryotic and root exudate diversity in
pneumatophore rhizosphere microhabitat (PRM) of Avicennia marina in Vellar
estuarine
Dinesh Sanka Loganatha Chetti*, Sundararaman Muthuraman
Bharathidasan University, India
Pneumatophores are aerial roots originating from the primary roots of mangrove plants. It
produces numerous fine roots, and creates a zone around them, called as the
pneumatophore rhizosphere microhabitat (PRM). Metagenomic DNA was isolated from PRM
associated composite soil samples (n=2) of Avicennia marina, Vellar estuarine with the aim of
studying the prokaryotic diversity and root exudates during different seasons. The variable
regions of 16s rRNA, V5-V9 (bacteria) and V3-V5 (archaea) regions were amplified from
metagenomic DNA. The amplicons were sequenced using a Tag Encoded FLX Amplicon
Sequencer (TEFAP). The predominant phylum for bacterial domain was Proteobacteria,
irrespective of the seasons. Cyanobacteria was dominant during the monsoon, whereas
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Acidobacteria was dominant in summer. In archaea domain, Thaumarchaeota phylum was
dominant in both seasons, while Crenearcheota was higher in summer and Euryarcheota
dominant in monsoon. Thermoprotei (class) was three times higher in summer compared to
monsoon. Highest bacterial species richness and diversity was observed in summer at 97%
sequence similarity. Meanwhile, highest archaeal richness and diversity was observed in
monsoon at 97% sequence similarity. Ethanolic extracts (70% v/v) of pneumatophores were
identified using gas chromatography-mass spectrometry. Fifteen metabolites during
monsoon, fifty two metabolites during summer and twenty one common to both the seasons
were observed. PCoA analysis revealed the effect of seasons by placement seasons at
opposite ends for archaeal and bacterial phyla. Prokaryotic community structure variation
and differential expression of metabolites observed in this study might be attributed to
prevailing conditions during seasons. To the best of our knowledge, it is identified that this is
the first study to reveal the PRM prokaryotic diversity and metabolites. Further studies
needed to affirm the role of these root exudates in shaping prokaryotic diversity in PRM.
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Abundance and functional traits of rhizosphere yeasts in maize
Marcela Sarabia*1, Cecilia Madrigal2, Yazmin Carreon3, Sylvia Fernandez Pavia3, John
Larsen2
1
Universidad Nacional Autonoma de Mexico, Mexico, 2Universidad Autonoma de
Mexico, Mexico, 3Universidad Michoacana de San Nicolas de Hidalgo, Mexico
Yeasts are common soil inhabitants, but little is known about their ecological functions in this
environment. In this study we characterized natural populations of yeasts in the maize
rhizosphere in nine maize fields with contrasting production systems (low, medium and high
input) and soil physico-chemical characteristics (pH, CEC, Base saturation, N, P, K, Ca, Mg, Cu,
Na, organic matter and texture). Rhizosphere soil samples were collected at the vegetative,
flowering and senescence plant growth stages from which a culture collection of 190 isolates
was obtained. Pure yeast isolates were examined for different traits in relation to plant
nutrition (P solubilization) and root health (biocontrol against the root pathogens Pythium sp.
and Fusarium verticilloides). The results showed that yeasts were abundant (104 - 105 CFU g-1
soil) and diverse (10 morphotypes) in all maize fields during the complete growing cycle. The
highest abundance of yeasts was obtained in fields without use of agrochemicals during the
florescence plant growth stage. On the other hand, the agrosystem managed intensively with
high input of agrochemials presented the highest number of morphotypes. Among the soil
physico-chemical characteristics examined the yeast population density correlated positively
and negatively with organic matter and percentage of clay, respectively. In terms of function,
P solubilization seems to be a common trait in rhizosphere yeasts (45% of the 190 yeast
isolates examined solubilized CaPO4 in in-vitro assay), which in some cases coincided with
plant growth promotion in corresponding plant bioassays. Another interesting trait of
rhizosphere yeasts was a strong antagonistic potential against the maize root pathogens
Pythium sp. and Fusarium verticilloides as examined in terms of in-vitro confrontation tests
and plant bioassays. In conclusion, yeasts seem to be a common part of the maize
rhizosphere microbiome with important functional traits related to improved plant nutrition
and root health.
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SYMBIO BANK - the collection of beneficial soil microorganisms
Lidia Sas Paszt*, Paweł Trzciński, Beata Sumorok, Anna Lisek, Edyta Derkowska,
Sławomir Głuszek, Eligio Malusa, Krzysztof Weszczak, Michał Przybył, Mateusz Frąc
The Research Institute of Horticulture, Poland
An important part of the project called EcoTechProduct, which is carried out at the Research
Institute of Horticulture in Skierniewice (Poland), is to establish and maintain a Bank of
Symbiotic Microorganisms, called SYMBIO BANK. The collected material of isolated spores of
mycorrhizal fungi and PGPR bacteria comes from organic orchards and plantations in Central
Poland, the Bieszczady and Białowieża areas. Results of studies to date have shown that there
are large differences in the occurrence of mycorrhizal fungi depending on the species and
plant cultivation method.
Plant-soil microorganisms can modulate the uptake of mineral nutrients through feedback
processes that reflect plant responses to environmental conditions. The intimate interrelation
between the root and symbiotic arbuscular mycorrhizal fungi and the resulting enhancement
in the uptake of N and P by the plant are further expanded by the interactions between the
fungus and bacteria present in both the rhizosphere and mycorrhizosphere. Numerous
species of plant growth promoting bacteria form biofilm when colonizing roots, which can
affect bio-geochemical processes and can result in increased availability of poorly available
mineral nutrients. The soil biotic communities are formed by several kinds of microorganisms
that can live symbiotically or in association with roots. Four major groups of microorganisms
are considered as beneficial to plants: arbuscular mycorrhizal fungi, plant growth promoting
rhizobacteria, nitrogen-fixing rhizobia, which are usually not considered to be PGPR, and
microbial biocontrol agents, which are composed of bacteria, yeasts and fungi.
The collection in SYMBIO BANK contains over 53 thousands of arbuscular mycorrhizal fungi
spores isolated from the soil of the different plant species and over 1500 isolates of
rhizosperic bacteria and filamentous fungi. The most effective strains and species of
microorganisms will be registered in Poland as bacterial and mycorrhizal inocula to be used
in fruit production and in phytoremediation.
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Characterization of plant growth promoting activities of bacteria in the
rhizosphere of barley and tomato
Marina Scagliola*1, Patrizia Ricciuti1, Youry Pii2, Tanja Mimmo2, Stefano Cesco2,
Carmine Crecchio1
1
University of Bari "A. Moro", Italy, 2Free University of Bolzano, Italy
Plant growth promoting rhizobacteria might be an alternative to chemical fertilizers and
pesticides as they influence several mechanisms such as enhancing nutrient uptake, plant
pathogens suppression and phytohormone production.
This work aims at isolating and characterizing rhizobacteria and their beneficial activities like
siderophores and indole 3-acetic acid (IAA) production and phosphate solubilization.
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Microorganisms were isolated from rhizosphere of barley and tomato, which were grown in
the RhizoTest-System first hydroponically (either in a full nutrient solution or in an iron
deficient solution) and then in soil. Two hundred isolates for each soil sample were screened
and eighty isolates were selected on the basis of their ability to produce siderophores.
Siderophores were further purified by chromatographic analysis and characterized by
spectrophotometric analysis.
To investigate their capability to produce IAA, isolates were tested by colorimetric method
using Salkowski reagent; the presence of IAA was confirmed and quantified by HPLC. Their
capability to solubilize phosphates was evalutated on Pikovskaya agar; quantitative
estimation was performed in liquid medium by spectrophotometric method using ascorbic
acid.
Isolates were also characterized by molecular analysis. A region of the 16S rRNA gene of
about 1 kb was amplified and sequenced from both ends. Sequences were aligned by BLASTn
to 16S rRNA sequences available on NCBI. Molecular evolutionary and phylogenetic analysis
was performed using Seaview 4; phylogenetic trees were constructed according to the
maximum likelihood method. Phylogenetic analysis of the selected population was not able
to differentiate the microbiome evolved by different plant species in function of the
nutritional status (Fe deficiency vs sufficiency).
Forty-seven isolates, mostly belonging to genus of Pseudomonas, Azotobacter and Rhizobium,
exhibited high levels of the three activities simultaneously.
Further studies are needed to characterize the role of the selected microorganisms on the
mechanisms underlying the nutrient acquisition in plants.
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Exploring the mycorrhizosphere of urban trees
Angelika Schartl*1, Josef Valentin Herrmann1, Arthur Schuessler2
1
Bavarian State Institute for Viticulture and Horticulture, Germany, 2SYMPLANTA
Laboratory, Ghana
Urban trees grow in an unnatural environment. Limited tree pits restrict their rooting zone,
degraded, compacted soil disturbs water and air balance. Multiple pollutants as well as
frequent mechanical damage affect the tree vitality. Additionally, climate change has
worsened these stressful conditions. Under such adverse circumstances mycorrhizal
associations play an even more significant role for plant health.
To select urban trees for the future, a long-term project was started in 2010. Different tree
species were planted at three sites with differing climate conditions. Some, representing
different mycorrhizal preferences, were chosen (Carpinus betulus, Fraxinus pennsylvanica
’Summit’, Magnolia kobus, Ostrya carpinifolia, Parrotia persica, Quercus cerris, Tilia tomentosa
’Brabant’) for root monitoring, especially to scrutinize their mycorrhization status. At planting
half of the trees were inoculated with a commercial mycorrhiza product (INOQ).
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Since 2011 root monitoring has been performed twice a year. Besides microanalysis of
mycorrhizal fungi, molecular biological analyses were performed for species identification by
BLAST against the UNITE and NCBI databases, including a deep sequencing by SYMPLANTA
in 2012.
In all trees tested both ecto- and endo mycorrhiza could be detected, except for Magnolia,
where only endomycorrhiza was present. The frequency was always higher than 50% and
displayed seasonal changes. Intensity of endomycorrhiza colonization averaged 15%. A
difference between inoculated and non-inoculated trees could not be detected.
Sequence analyses revealed a complex community of mycorrhiza on all trees with individual
patterns. Nevertheless, some characteristic associations between tree and fungal species
could be detected. Conventional sequencing rarely identified endomycorrhizal fungi. A
connection of mycorrhizal patterns to nursery origin and/or cultivation site has to be
discussed.
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Effect of plant genotype and soil type on the rhizosphere microbiome structure
of Sorghum bicolor (L.) Moench and Strigolactone production
Thiago Roberto Schlemper*, Eiko Kuramae, Jos Raaijmakers, Hans van Veen
Netherlands Institute of Ecology - NIOO-KNAW, Netherlands
In terrestrial systems, rhizosphere microbes are important players in plant growth, plant
health and nutrient acquisition. Here we determined if different Sorghum bicolor cultivars
select different microbial assemblages in the rhizosphere. Originated from Africa, S. bicolor is
a valuable staple crop mainly used for feed and food. S. bicolor is the major cereal crop in the
semi-arid regions of the world, in particular in sub-Saharan Africa. To determine the effect of
the Sorghum genotype on the rhizosphere microbiome structure, a greenhouse experiment
was conducted with 7 different cultivars, planted in two different soils. Bulk soil and
rhizosphere samples were taken at 4 different time points representative of different
developmental stages of Sorghum. The rhizosphere and bulk soil samples were subjected to
total genomic DNA extraction and the bacterial/archaeal communities were assessed by 16S
rRNA gene sequencing and the fungal community by 18S rRNA gene sequencing. We will
present results on soil - and host - genotype specific effects on the rhizosphere microbiome
of S. bicolor and will provide insight into the correlation of microbiome structure and
Strigolactone exudate profiles.
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Diversity and spatial distribution of diazotrophs associated with microenvironments of wetland rice
Hannes Schmidt*, Dagmar Wöbken
University of Vienna, Department of Microbiology and Ecosystem Science, Division of
Microbial Ecology, Austria
Rice is one of the world’s most important crop plants. The production is strongly limited by
nitrogen (N), which is typically supplied by industrial fertilizers that are costly and hazardous
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to the environment. It is known that Biological Nitrogen Fixation through N2-fixing bacteria
and archaea (diazotrophs) can alleviate the N-shortage in rice cultivation. However, our
knowledge on the micro-sites of N2 fixation, as well as the diversity and in situ N2 fixation
activity of diazotrophs in the soil-microbe-plant interface (i.e. rhizosphere) of flooded rice
fields is still rudimentary.
A greenhouse study was performed to identify key factors that control rice-diazotroph
association and related N2 fixation activities. Paddy soils were cultivated with two genotypes
of wetland rice. Samples were taken from the bulk soil, the root-associated rhizosphere soil,
the rhizoplane, and the endosphere at flowering stage of rice plant development. These
samples were subjected to functional assays and various molecular biological techniques
including high-throughput sequencing to identify the diazotroph community.
Based on Illumina sequencing of 16S rRNA and nifH genes and transcripts, we will present
first insights into the diversity of bacterial/diazotroph communities with emphasis on
assessing the potential influence of (a) the soil microbial “seed bank” or (b) plant genotype in
shaping the microbiomes in each micro-environment. These data will be combined with N2
fixation activity measurements as assessed by incubation assays using acetylene-enriched
atmospheres. The localization of selected diazotrophs on the rhizoplane of rice roots via
fluorescence in situ hybridization and confocal laser scanning microscopy is currently
underway to identify areas of potential N-transfer between diazotrophs and rice roots.
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Evaluation of strategies for the separation of root-associated microbial
communities
Tim Richter-Heitmann1, Thilo Eickhorst2, Michael W. Friedrich1, Hannes Schmidt*3
1
University of Bremen, Microbial Ecophysiology, Germany, 2University of Bremen, Soil
Microbial Ecology, Germany, 3University of Vienna, Department of Microbiology and
Ecosystem Science, Division of Microbial Ecology, Germany
The analysis of plant-microbe interactions has emerged as an important topic in rhizosphere
research. Based on comparative studies applying high-throughput sequencing, it has been
postulated that plants may shape distinct root microbiomes consisting of microbial
populations attracted from the soil seed bank. A main premise for the value of such
investigations is that microorganisms living near, on, and inside roots were well separated.We
applied different protocols to plant roots of two soil-grown plant species (Oryza sativa, Vicia
faba) and assessed their potential to detach rhizoplane colonizing cells by washing,
sonication, and treatment with NaOCl. Sonication was performed with (i) sonication bath and
(ii) sonication probe at low and high intensity, respectively. The number of microbial cells
attached to the rhizoplane after each treatment was evaluated via SYBR Green-staining and
fluorescence microscopy. Accordingly, cell numbers of rhizoplane-detached cells and the
estimated level of plant cell destruction were recorded. Community fingerprinting and next
generation sequencing of root samples is applied to assess the effect of each treatment on
the composition of microbial populations associated with roots.
First results showed that washing alone yielded in the majority of cells still being attached to
root surfaces. The highest amount of cells detached from rice roots was found with the
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sonication bath system at highest energy. However, the intensity of plant cell destruction
increased as well, leading to a potential release of endophytes into the rhizoplane
compartment. Our results also suggest that presence of root hairs - as found in Vicia faba interfered with all procedures as the numbers of cells detached from the rhizoplane did not
increase with increasing intensity of the applied treatments.
Hence, we conclude that strategies to separate root associated bacterial communities need
to be carefully evaluated for each plant genotype, which can be easily performed by
fluorescence microscopy.
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A fragrant neighborhood: The role of volatiles in the formation of bacterial
communities in soil
Kristin Schulz*1, Wietse De Boer2, Paolina Garbeva1
1
Netherlands Institute of Ecology, Netherlands, 2Netherlands Institute of Ecology,
Soil bacterial species living in the rhizosphere interact in numerous and versatile ways. It is
increasingly recognized that volatile organic compounds (VOCs) produced by bacteria can
function as bioactive growth-promoting or growth-inhibiting agents as well as info-chemicals
for inter- and intra-specific communication. Due to their physical properties, VOCs can act on
a wide scale in soil and, consequently, may play a key role in interspecific bacterial
interactions and in the development of microbial soil communities. Soil microcosm
experiments with bacterial model strains growing on artificial root exudates are performed to
assess (1) the influence of volatiles on the formation of bacterial communities and (2) the
ability of VOCs to stimulate non-active bacteria. Pilot microcosm experiments with
monocultures and mixture of bacterial isolates (Burkholderia, Dyella, Janthinobacterium,
Pseudomonas, and Paenibacillus) revealed that each strain produced a different set of
volatiles and that the production of different volatile compounds was triggered by
interspecific interactions in the mixture. Interestingly, the poorly-growing Gram-positive
Paenibacillus strain had a strong effect on the growth-performance of the other strains as
well as on the production of volatiles in the mixture.
We are currently testing how trapping (removal) of microbial volatiles from the soil
microcosms will affect the development of the microbial community. Furthermore, we are
testing if VOCs produced by an active bacterial community can stimulate the growth of nonactive (starving) bacteria.
In conclusion, with the obtained results we aim to reveal new insights into the ecological role
of volatiles in microbial interactions and community dynamics in soil.
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Deciphering plant growth promoting rhizobacteria from rice rhizosphere soil
and plant growth promotion in rice
Lalan Sharma*, Dipak T. Nagrale, Dhananjaya P. Singh
Indian Council of Agricultural Research - National Bureau of Agriculturally Important
Microorganisms, India
Sampling of rhizospheric soil samples have been done of different rice varieties of IndoGangetic plain of Uttar Pradesh. Soil samples have analysed for physico-chemical properties
like pH, electric conductivity and organic carbon. It recorded that soil pH ranges 7.0 to 8.4,
electric conductivity ranges 1.3 to 1.8ds/m and organic carbon in soil medium to medium
high and free living rhizospheric bacteria were isolated by using different inoculation
techniques on various culture media. 143 rhizobacteria have isolated and characterized by
Gram staining, Streo-microscopically, Scanning Electron Microscope and plant growth
promoting attributes like HCN production, Siderophore production, and Phoshphate
solubilisation. 16s rDNA amplification has been done by using universal primers and also
phenolic chromatogram has prepared by High Performance Liquid Chromatography for
secondary metabolites detection. Seed bio-priming has done with potential rhizobacterial
isolates using Gnotobiotic system. The data are recorded and calculated that some isolates
either individually or in consortium form like MAU143, MRT84 and MRT92 are promising
microbial inoculants to enhance rice seed vigour and antioxidants properties in rice seedlings.
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Characterisation of medium-term selection of Rhizobium leguminosarum bv
viciae populations by different legume plant hosts
Amalia Soenens Martinez de Murguia*1, Beatriz Jorrin1, Juan Imperial2
1
Universidad Politécnica de Madrid, Spain, 2Universidad Politécnica de Madrid / Centro
Superior de Investigaciones Cientificas, Spain
Rhizobium leguminosarum bv viciae is a nitrogen-fixing soil bacterium able to establish
specific root-nodule symbioses with legumes of four different genera: Pisum, Vicia, Lens and
Lathyrus. Available evidence suggests that although Rhizobium leguminosarum bv viciae
strains are able to nodulate the four plant genera, plant hosts select specific rhizobial
genotypes from those present in the soil. This has been shown previously in our laboratory at
the genomic level following a population genomics approach (Pool-Seq). Pool genomic
sequences from 100 isolates from each of four plant species: P. sativum, L. culinaris, V. faba
and V. sativa show different specific profiles at the single nucleotide polymorphism (SNP) level
for relevant genes. In this work we characterised the extent of plant host selection on
Rhizobium leguminosarum bv viciae genotypes both in nodules and in the soil. For that
purpose we proceed to do a mesocosm experiment, where we started with a wellcharacterised soil population, applied plant host selection and those rhizobia selected by the
different legume hosts: P. sativum, L. culinaris, V. faba and V. sativa were used as the
inoculum for the next plant growth. This was done for 5 cycles. Direct soil and nodule
individual strains from each of these mesocosm studies have been isolated and initially tested
for specific rhizobial genes (glnII and fnrN) and symbiotic genes (nodC and nifH). The selected
populations were further characterised by means of Sanger sequencing of both the rpoB
phylogenetic marker gene and the symbiotic genes nodC and nifH. The distribution and
composition of the different rhizobial populations showed changes for each legume host
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during the mesocosm study. We hypothesize that these changes mimic those occurring
under real field conditions and should help reveal the underlying bases of rhizobial genotype
selection by the plant host.
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Changes of the bacterial community in the rhizosphere of wild soybean (Glycine
soja) at different growth stages
Youngmi Lee1, Jae-Hyung Ahn1, Hang-Yeon Weon1, Jung-Hoon Yoon2, Jaekyeong
Song*1
1
Agricultural Microbiology Division, National Academy of Agricultural Science,
RDA, South Korea, 2Department of Food Science and Biotechnology, Sungkyunkwan
University, South Korea
We analyzed bacterial community of rhizosphere at four growth stages of soybean, Glycine
soja using pyrosequencing based on the 16S rRNA genes. In the bulk soil and the rhizosphere
of wild soybean the predominant phylum was Proteobacteria (32-42%) and followed by
Firmicutes (7-27%), Actinobacteria (7-12%) and Bacteroidetes (3-16%). The rhizosphere of wild
soybean had higher abundance of the phyla Proteobacteria and Bacteroidetes than bulk soils.
In the class level, Gammaproteobacteria was much more abundant in bulk soils, while
Alphaproteobacteria and Betaproteobacteria were more abundant in the rhizosphere of wild
soybean. The predominant genus was Bacillus in the rhizosphere of the wild soybean,
followed by Flavobacterium. There were some changes in bacterial community structure of the
rhizosphere of wild soybean during growth unlike those of bulk soils. Of which, the
abundance of the genus Bacillus changed markedly between growth stages, full seed stage
and maturity stage of wild soybean, indicating that Bacillus responds preferentially to change
of materials such as root exudates of wild soybean.
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Defining the microbiome of a sugarcane plant
Rafael Souza*1, Vagner Katsumi Okura1, Nuria Lozano García2, Jaderson Silveira Leite
Armanhi1, Laura Migliorini de Araujo1, Natalia Verza Ferreira1, Manuel GonzálezGuerrero2, Homayoun Bagheri3, Marcio José da Silva1, Juan Imperial1, Paulo Arruda1
1
State University of Campinas (UNICAMP), Brazil, 2Centro de Biotecnología y Genômica
de Plantas (CBGP), Campus de Montegancedo, Universidad Politécnica de
Madrid, Spain, 3Repsol Technology Center, Spain
Studies on beneficial microorganisms associated to crops have traditionally used culturebased techniques, which are known to sample only a minute portion of the microbial
community. Sugarcane has been a target of such researches, since there are repeated
evidences of a microbial community supporting plant development and sustaining high
yields under conditions of little or no fertilization. However, none study has taken into
consideration the diversity or the qualitative/quantitative microbial communities in the
biology of the plant. We intend to unravel the microbial community associated to sugarcane
plants cultivated for four cuts without fertilization. Exophytic and endophytic microbial
communities were evaluated in root, stalk, leaf and young shoot. We also accessed the
microbial communities of bulk soil. Illumina platform was used to sequence 16S and ITS
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amplicons. Our data revealed that factors driving the community composition differ between
prokaryote and fungi. Although the microbial diversity is huge for any tissue, a core
microbiome comprised of less than 1% of the diversity represents 65% to 96% relative
abundance of the total microbial diversity. Interestingly, the most abundant microbial groups
are not the ones that have been isolated and evaluated over the past decades in sugarcane.
The data open a new window for the investigation of untargeted microbes potentially
beneficial for sugarcane growth and development.
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Deciphering the cross-talk between host genotype, microbiota structure, and
plant growth in barley
Rodrigo Alegria Terrazas*1, Katharin Balbirnie2, Eric Paterson3, Elisabeth Baggs4, Davide
Bulgarelli2
1
The University of Dundee at The James Hutton Institute, United Kingdom, 2University of
Dundee, United Kingdom, 3The James Hutton Institute, United Kingdom, 4University of
Aberdeen, United Kingdom
The rhizosphere microbiota is a potential resource of plant probiotic functions. However, its
deployment in agriculture is impaired by the limited knowledge of the mechanisms
regulating plant-microbiota interactions.
Wild barley (Hordeum vulgare subp. spontaneum) populations from marginal soil areas are
considered a genetic resource for crop improvement. To investigate the contribution of the
wild barley-microbiota interactions to plant growth, we have grown 14 wild populations,
representing the three major ecotypes in Israel (i.e., ‘north’, ‘coast’ and ‘desert’), and a
modern variety in a reference agricultural soil under greenhouse conditions. At early stem
elongation, rhizosphere specimens were collected and stem dry weight measured. We
adopted an llumina MiSeq protocol to generate high-resolution 16S rRNA gene profiles of
the rhizosphere and unplanted soil controls. We used QIIME to quality-filter the reads and to
identify Operational Taxonomic Units (OTUs) at a 97% sequence similarity. Consistent with
previous investigations, we observed a taxonomically coherent barley microbiota, dominated
by members of the phyla Proteobacteria and Bacteroidetes, whose enrichment discriminates
rhizosphere and soil profiles. Beta diversity calculation revealed distinct host-dependent
substructures of the rhizosphere microbiota. In particular, the microbiota retrieved from the
desert ecotype accessions clustered separately compared with those from other ecotypes and
the modern variety. Non-parametric statistical analyses and supervised learning classification
indicated that the recruitment of members of the families Comamonadaceae,
Cytophagaceae, Hyphomicrobiaceae and Micrococcaceae support the observed
diversification, several of which have previously been reported as plant growth promoting
rhizobacteria. Remarkably, host-dependent microbiota profiles correlated with a biomass
gradient among the tested plants, underlying a potential cross-talk among the host
genotype, the establishment of substructures of the microbiota and plant growth. To further
explore these relationships, we are currently investigating the contribution of the barley
rhizosphere microbiota to plant adaptation to soil limiting nutrient supplies.
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140
Rhizobacterial community structure and function in a dryland agroecosystem
Linda Thomashow*1, James Parejko2, Melissa LeTourneau3, Olga Mavrodi4, Dmitri
Mavrodi4, Robert Bonsall3, David Weller5
1
USDA-ARS Washington State University, United States, 2University of Wisconsin La
Crosse, United States, 3Washington State University, United States, 4Southern Mississippi
State University, United States, 5USDA-ARS, Washington State University, United States
Certain members of the Pseudomonas fluorescens species complex produce phenazine
antibiotics inhibitory to soilborne root pathogens. Included among these bacteria are the
model strain 2-79 and representatives of three other Pseudomonas species that produce
phenazine-1-carboxylic acid (PCA) and comprise up to 10% of the culturable rhizobacteria on
the roots of wheat grown in dryland soils of the inland Pacific Northwest, USA. PCA
production on roots of field-grown spring wheat occurred mainly during the first half of the
growing season but remained detectable for at least 130 days after planting. In vitro, the
compound persisted with a half-life of 3 or 4 days in dry or moist soils, respectively, and
remained detectable throughout the 20-day experiment. These data indicate that most PCA
synthesis in the field occurs early in the season, but suggest that some synthesis continues to
occur even as soil water potentials approach as low as -400 to -500 kPa. We suggest that PCA
producers (Phz+) survive desiccation on roots in biofilms. PCA influenced biofilm formation by
some, but not all, Phz+ strains from dryland wheat and its impact varied with matric and
osmotic stress levels. Structural differences also were observed in colony biofilms grown from
strains of different species under control and stress conditions, and distinct differences were
observed between some wild-type Phz+ strains and their Phz- mutants.
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High-throuput detection of plant pathogens using next generation sequencing
Andrzej Tkacz*, Philip Poole
Plant Sciences, University of Oxford, United Kingdom
The soil microbial community has an enormous impact on crop yields. There has been
extensive research conducted using cultivation and environmental DNA and RNA sequencing
methods. However, only recently with the advent of the new high-throughput sequencing
methods are we starting to understand the delicate structure of the soil microbiota, both in
its spatial and temporal scale. Here we present our recent findings on the soil community
inter- and intra-kingdom structure caused by the rhizosphere influence of different plants.
We are detecting pathogen build-up in the soil of plants grown in monoculture. Using
selected plant species and secretion mutants, we are correlating pathogen invasion of the
rhizosphere with plant host genetics. We are especially interested in wheat lines and their
progenitors, rice secretion mutant lines and signalling mutants of Medicago.
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143
Comparison of the diversity, structure and function of the fungi and bacterial
rhizosphere community of main rice production areas from Colombia (SA)
Daniel Uribe*1, Giovanna Landazabal2, Catalina Camelo2, Javier Vanegas3
1
Universidad Nacional de Colombia/ Biotechnology institute, Colombia, 2Universidad
Nacional de Colombia/ Biotechnology Institute, Colombia, 3Universidad Nacional de
Colombia / Biotechnology Institute, Colombia
The deterioration of agricultural soils, mainly represented by the erosion, loss of fertility and
environmental contamination with different types of substances, has resulted in decrease of
productivity and sustainability of many agroecosystems. This phenomenon, has increased the
interest to assess the impact of agricultural practices on soil microbial communities, in
relation to soil ecological function in specific agroecosystems. Studies carried out for this
purpose, have shown that the activity, diversity and structure of microbial communities are
affected by different management practices and land use. In Colombia, the intensive
cultivation of rice, the third largest crop in terms of planted area, has led to a progressive
deterioration of the soil, which is reflected in the reduction in rice production levels,
especially in Tolima and Meta, the two largest rice production areas of Colombia. This study
compared the structure, diversity and enzymatic activity of rhizosphere bacterial and fungal
communities of 16 rice crop farms, from Tolima and Meta, using denaturing gradient gel
electrophoresis. Sixteen farms located in four areas contrasting in terms of fertility, physicalchemical characteristics and the rice production system in terms of water management were
studied. Cluster and principal component analysis (PCA), showed that the structure, diversity
and function of rhizosphere bacterial and fungal communities were influenced by the
physical and chemical properties of soils and rice production system. Most bacterial and
fungal OTUS identified belongs to unculturable species.
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Rhizosphere bacterial community composition of the native potato Solanum
tuberosum group phureja associated with nitrogen and phosphorus cycle
Daniel Uribe*1, Nathalia Florez-Zapata2, Anyela Rodriguez2
1
Universidad Nacional de Colombia/ Biotechnology institute, Colombia, 2Universidad
Nacional de Colombia/ Biotechnology Institute, Colombia
Soils where Solanum tuberosum group phureja is cultivated are characterized as being highly
soil phosphate binders. Besides, it is well known the important role of nitrogen in agricultural
production. Therefore, to meet the nutritional demands of the crop, high phosphorous and
nitrogen based fertilizers are applied, which increase production costs, as well as undesirable
environmental effects. Because of that, some farmers apply organic amendments as an
alternative to ameliorate such negative effects. However, the effects of such practices, as well
as the soil type on S. tuberosum group phureja P an N related microbial communities are still
unknown. The aim of this study was to analyze the edaphic communities related to
phosphorous and nitrogen metabolism in spatially independent rhizosphere soil samples
from S. tuberosum group phureja crops, contrasting in terms of soil structure and fertilization
strategy. Such analysis was done through a polyphasic approach, which includes physicalchemical soil analyses, enzimatic activity quantifications, and culture-dependent and
independent microbial communities’ composition analysis, in order to identify the factors
that regulate P and N cycling in this ecosystem. Statistical significant differences were found
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in microbial counts, community composition and enzimatic activities in the four soil samples.
Besides, differences were found in terms of the effect of soil type on the structure and
function of N and P associated bacteria, suggesting differences in the susceptibility of those
two functional groups to the soil physical-chemical environment.
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Forest tree species shape their soil microbiome
Stephane Uroz*1, Oger Phil2, Tisserand Emilie1, Cébron Aurélie3, Turpault Marie-Pierre1,
Marc Buée1, Wietse De Boer4, Johan H Leveau5, Pascale Frey-Klett1
1
INRA, France, 2CNRS, France, 3CNRS/Univ.
Lorraine, France, 4NIOO, Netherlands, 5University of California, USA
The impact of plant species on the soil microbial communities and the physico-chemical
characteristics of the soil begins to be well documented especially for non-perennial plants.
However, our understanding of the diversity and structure of the tree-associated microbial
communities as well as the of tree species effect remains limited. Here, we investigated the
archaeal, bacterial and fungal communities in replicate soil samples, using 16S rRNA, 18S
rRNA and fungal ITS sequences, in the long term experimental site of Breuil-Chenue. We
showed significant difference in abundance, composition and structure of the microbial
communities associated to two contrasted tree species, Fagus sativa and Picea abies
developed on the same soil. Our results highlighted a strong host effect on the soil microbial
communities, with a stronger effect on the fungal communities. Although the
pyrosequencing approach showed a limited rhizosphere effect, quantitative PCR revealed a
significant enrichment of specific bacterial genera known for their ability to weather minerals
in the tree root vicinity. At last, co-occurrence analysis revealed very different networks below
the two tree species, suggesting a modification of the structure and abundance of the
microbial communities, but also modification of the interactions established between
microorganisms. In all the microbial communities considered we observed a host effect with
variable intensity, suggesting that the tree host shapes its soil microbiome.
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Organic amendment effects on lettuce production and rhizosphere soil
Julen Urra*1, Carlos Garbisu1, Jose Maria Becerril2, Unai Artetxe2, Fernando Blanco1,
Iker Martin1, Mikel Anza1, Iker Mijangos1
1
Neiker-Tecnalia, Spain, 2University of the Basque Country, Spain
Intensive farming practices, and in particular the utilization of synthetic chemical fertilizers,
lead to the degradation of the environment, including the soil resource. In consequence,
there is growing interest in the development of sustainable agricultural practices which
protect the integrity of the soil ecosystem while producing healthy and abundant crops. In
this respect, fertilization with organic amendments not only provides a wide range of
nutrients but also improves soil physicochemical properties and, most relevantly, might have
a beneficial effect on soil microbial communities.
Here, the effect of organic amendments from chicken and horse manure (i.e., fresh manure,
compost, Bokashi compost), as well as the effect of a liquid amendment obtained from forest
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leaf litter and Biovin®, on lettuce production and rhizosphere soil was studied under
controlled conditions in a growth chamber. The effect of organic amendments on lettuce
rhizosphere soil was investigated through the determination of a variety of physicochemical
(pH, organic matter, nutrient concentrations, cation exchange capacity, texture, etc.) and
microbial properties. Pertaining to these latter microbial properties, we determine a great
deal of parameters which provide information on the biomass (e.g., microbial biomass C,
substrate-induced respiration, RT-qPCR for bacteria and fungi), activity (e.g., RNA/DNA ratio,
basal respiration, potentially mineralizable N, and enzyme activities such as β-glucosidase,
urease, alkaline phosphatase and arylsulphatase) and diversity of soil microbial communities.
The application of organic amendments resulted in optimal lettuce yields while stimulating
the activity of soil microbial communities.
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Microbiomics of cormosphere and rhizosphere of Crocus sativus, Saffron
Jyoti Vakhlu*, Deepika Trakroo, Sheetal Ambardar
University Of Jammu, India
Microbial associations with the roots have been explored in a great detail but other
belowground plant parts like corm, bulb and onion etc have been ignored so far. Crocus
sativus, Saffron, is world’s costliest spice and has interesting corm-root cycle. Our laboratory
for the first time has initiated the study on the microbial (bacterial and fungal) association of
below ground parts of Crocus sativus i.e. corm and root and comparison of their specific
associations. The study was initiated by cataloguing the bacterial diversity during vegetative
stage and fungal diversity during flowering stage by cultivation-independent metagenomic
approach, involving deep sequencing of phylogenetically relevant genes amplicons i.e 16S
rRNA gene for bacteria and ITS for fungi.
Bacterial diversity of cormosphere was high as compared to rhizosphere which was also
represented by higher Alpha diversity in cormosphere (11) than in rhizosphere (9).
Proteobacteria was the dominant bacterial phylum in both rhizosphere and cormosphere with
greater abundance in cormosphere. In rhizosphere Lutteibacter (10.4%) was found to be
predominant genus followed by Rhizobium (6.77%) and Sphingomonas (5.68 %). However
Chryseobacterium (7.6%; Bacteriodetes phylum) was abundant genus in cormosphere
followed by Sphingomonas (5.5%) and Burkholderia (3.1%).
In fungi, Zycomycota was the dominant phylum in both rhizosphere and cormosphere but its
relative abundance was higher in cormosphere (54%) than in rhizosphere (43%). The Shannon
alpha diversity was found 6 and 7 for cormosphere and rhizosphere, respectively, indicating
higher number of fungal species in rhizosphere than cormosphere. At genus level Rhizopus
(Zygomycota) was dominant in rhizosphere. In cormosphere though Zycomycota was
dominant phylum but Cryptococcus (Basidiomycota) was dominant genus.
In this preliminary investigation there is clear indication of organ specific microbial–
association. However what is the influence of such association, especially microbe–corm
association is matter of further investigation.
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148
In depth exploration of the rhizosphere and endosphere of maize
Tom Viaene*1, Raul Tito2, Hilde Nelissen1, Jeroen Raes2, Sofie Goormachtig1
1
VIB, UGENT, Belgium, 2VIB, KU Leuven, Belgium
We have initiated an in-depth characterization of the microbiome composition in the
rhizosphere and endosphere of maize in a Flemish soil type. Using 16S rRNA pyrosequencing
on the Illumina Miseq platform, relative abundance values of the different bacterial phyla
show a gradual transition from bulk via rhizosphere to endosphere samples, with the
endosphere being most different. At the family level, we are able to identify bacterial families
that are significantly upregulated in the endosphere (and/or) rhizosphere, compared to bulk
soil samples. These are families (e.g. Rhizobiaceae, Comamonadaceae and
Streptomycetaceae) to which well-known plant growth promoting bacteria belong. To
understand the functionality of the microbiome data, we have developed two plant growth
promoting assays in maize to evaluate the effect of selected microbes on maize growth.
These assays are currently used to screen plant growth promoting effects of individual corn
rhizosphere bacteria.
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Impact of soil heat treatment on bacterial community reassembly in the
rhizosphere
Menno van der Voort1, Rodrigo Mendes*2, Jos M. Raaijmakers3
1
Wageningen University, Netherlands, 2Embrapa Environment, Brazil, 3Netherlands
Institute of Ecology (NIOO-KNAW), Netherlands
The rhizosphere microbiome offers a range of ecosystem services to the plant, including
nutrient acquisition, tolerance to abiotic stress and protection against diseases. Here we
studied how heat treatment of soil disturbs the reassembly of the bacterial community in the
rhizosphere and how this affects tolerance to pathogen infection. Using PhyloChip-based
community profiling, we assessed the impact of 1-hour heat treatments of 50ºC or 80ºC on
the bacterial community composition in the rhizosphere of sugar beet seedlings grown in a
soil that is naturally suppressive to the soil-borne fungus Rhizoctonia solani. The heat
disturbance caused significant increase of alpha diversity and led to a partial (50ºC) or
complete (80ºC) loss of protection against fungal infection. The bacterial families Bacillaceae,
Comamonadaceae, Paenibacillaceae and Alcaligenaceae showed a significant increase in
relative abundance with increasing temperatures. The Pseudomonadaceae and
Burkholderiaceae showed higher abundance only when the soil was heat-treated at 80ºC.
Conversely, the bacterial families Streptomycetaceae, Micrococcaceae, Solibacteraceae and
Mycobacteriaceae showed a reduction in relative abundance when the soil was heat-treated at
80ºC. Based on these results, we propose a reassembly model where bacterial groups that are
most heat-tolerant and with high growth rates increase in relative abundance after heat
disturbance, while temperature-sensitive and slow growing bacteria have a disadvantage. The
results also point to a potential role of slow growing bacterial families from Actinobacteria and
Acidobacteria phyla in protection of plants against fungal infection. With this study we
showed that heat disturbance in soil results in a rearranged rhizosphere bacterial community,
which in turn leads to changes in the ecosystem services of the soil.
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150
The potato microbiome and its potential impact on late blight resistance
Mout De Vrieze*, Aurélien Bailly, Christian Ahrens, Tomke Musa, Laure Weisskopf
Agroscope, Switzerland
Late blight caused by the oomycete Phytophthora infestans is a major threat for potato
production worldwide. In organic farming, control is based on the use of copper-based
fungicides, which are the only efficient control products currently available. However, due its
toxicity towards the environment, the use of copper compounds for agricultural purposes is
being questioned. In Europe, the current approval expires in 2018 and the EU aspires at
reducing the use of copper in the future. The need to develop alternative organic control
methods is therefore evident. In natural and agro-ecosystems, plant roots and shoots are
colonized by a diverse community of microorganisms. In the model plant Arabidopsis
thaliana, a protective role of this plant’s microbiome against a number of phytopathogens
has been demonstrated. However, the composition of the potato leaf microbiome is so far
unknown, as is its putative role in protecting the plant against pathogens such as P. infestans.
In this project, the microbiome of non-infected and P. infestans-infected potato plants of
various cultivars will be characterized using next-generation sequencing techniques with the
aim to reveal its composition and the shifts occurring when the plants are infected with P.
infestans. Furthermore, the genomes of selected Pseudomonas strains recently isolated from
field-grown potatoes and showing antagonistic potential against P. infestans will be
sequenced to identify the genomic determinants of the anti-Phytophthora activity. These
findings should provide better understanding of the mechanisms involved in the antiPhytophtora activity of the bacteria and reveal how these mechanisms are regulated at the
molecular level. The ultimate goal of this study is to improve our use of bacteria as biocontrol
agents through a deeper understanding of their metabolic possibilities and of their needs.
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Exploring the root-associated bacterial community turnover along a primary
succession in salt marsh
Miao Wang*, Joana Salles
University of Groningen, Netherlands
The root-associated microbiome represents the bridge between soil and plant hosts, being
influenced by biotic and abiotic factors. Although soil characteristics provide a stronger
selective force than plant species, i.e. the same plant species growing in different soils select
for distinct bacterial communities, these comparisons are mostly performed in soils with
distinct characteristics, origins and from different environments. Primary succession fields
provide a perfect setting to test the relative influence of soil and plant type on plantassociate microbiome, as the same plant species grows along a gradient of soil development,
under similar environmental conditions. Here, we used an undisturbed salt marsh
chronosequence, spanning more than 100 years of primary succession, to study the bacterial
communities associated with the soil, rhizosphere and the root endopshere of Limmonium
vulgare using 454-pyrosequencing. Our hypotheses were that soil characteristics would
determine community composition and that selective force exert by the endosphere would
be stronger. The soil and rhizosphere bacterial communities were phylogenetically more
diverse than those in endosphere, which could be explained by plant “filtration”. Moreover,
this diversity remained stable over the chronosequence. Multivariate analyses showed that
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endopshere communities were distinct from the others and did not cluster by soil age.
Conversely, rhizosphere and soil-associated communities were similar and grouped
according to succession stage, confirming our first hypothesis for rhizosphere communities.
The variation in the bacterial community assembly, analyzed by co-occurrence network
matrices, indicated bacterial species turnover along the succession to be the highest in the
endosphere and lowest in the rhizosphere. Contrary to the perspective of more stable
community composition in the endosphere due to less competition compared to the soil or
rhizosphere, we showed that the rhizosphere performed higher selective force than
endosphere, the latter being under the control of stochastic mechanisms and colonized by
passenger endophytes.
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Dilution of microbial soil community selects for K-strategists (serial dilution
approach)
Simone Weidner*, George Kowalchuk, Alexandre Jousset, Joost Keuskamp
Utrecht University, Netherlands
Soils are hot spots of diverse microbial communities. Soil microbial communities are
important for plants in terms of being the species pool from which the plant recruits its
rhizosphere microbes. However, there is not much known about how possible diversity loss
might be linked to changes in microbial community structure.
We created a diversity gradient in a microbial soil community using a serial dilution approach.
Briefly, the microbial community from a natural soil was extracted, diluted, and γ irradiated
soil was inoculated with these microbial communities. Although it is known that dilution
decreases the diversity by out-selecting rare microbes, much less is known about structural
changes in the soil microbial community which result from competitive interactions during the
regrowth period after inoculation. In order to secure equal microbial biomass across the
treatments, soils were incubated in the dark at 20°C for nine weeks before first analyses.
Potential growth rates of the communities were determined via substrate induced growth
respiration and used as proxy for the dominance of r vs K-selected species. Biolog plates were
used to assess whether changes in community structure affected carbon resource use
patterns.
This study shows that dilution causes a bias towards r-selected species upon regrowth with
moderate dilutions, whereas strong dilutions lead to K-selected communities. We suggest
that the connectivity during the regrowth period determines the dominance of K vs rselected species after the regrowth period. Detected changes in carbon resource use patterns
across dilution might result in effects of root exudate composition on the community and
recruitment of rhizosphere microbes.
As a next step we will investigate how these changes in community structure affect plants’
recruitment of certain microbes from bulk soil to the rhizosphere. Focus will be lying on
effects of dilution on the role of the recruited microbial community for plant protection.
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153
Analysis of community structure of metabolically active bacteria in a rice field
subjected to long-term fertilization practices
Jae-Hyung Ahn, Min-Young Choi, Jaekyeong Song, Hang-Yeon Weon*
Agricultural Microbiology Division, National Academy of Agricultural Science,
RDA, South Korea
To estimate the effect of long-term fertilization on metabolically active bacterial communities
in a rice field, RNA was extracted from endosphere (rice root), rhizosphere, and bulk soil that
had been subjected to different fertilization regimes for 59 years and the 16S rRNAs were
analyzed using the pyrosequencing method. The richness and diversity of metabolically active
bacteria were higher in bulk soil than in the endosphere and rhizosphere, and showed no
significant difference between non-fertilized and fertilized plots. Weighted UniFrac analysis
showed that each compartment had characteristic bacterial communities and that the effect
of long-term fertilization on the structure of bacterial community was more pronounced in
bulk soil than in the endosphere and rhizosphere. The 16S rRNAs affiliated with
Alphaproteobacteria and Firmicutes were more abundant in the endosphere than in bulk soil
while those affiliated with Chloroflexi and Acidobacteria were more abundant in bulk soil than
in the endosphere. Several dominant operational taxonomic units (clustered at a 97%
similarity cut-off) showed different frequencies between non-fertilized and fertilized plots,
suggesting that the fertilization affected their activities in the rice field.
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Metagenomic insight into the plant-microbe rhizosphere interaction under
Rehmannia glutinosa consecutive monoculture regime
Linkun Wu*, Juanying Wang, Hongmiao Wu, Jun Chen, Xianjin Qin, Wenxiong Lin
Fujian Agriculture and Forestry University, China
Under consecutive monoculture regime the biomass and quality of Rehmannia glutinosa, an
important Chinese medicinal plant suffers from significant decline. The objective was to
evaluate the response of soil bacterial and fungal communities to consecutive monoculture
using omics technique and assess the positive and negative effects of root exudates on the
key microbes associated with consecutive monoculture problems. The results showed that
consecutive monoculture led to a great shift in rhizospheric microbial community. In details,
consecutive monoculture led to significant reduction of populations assigned to genus
Pseudomonas. However, Fusarium oxysporum or Fusarium sp. belonging to Ascomycota was
significantly higher in consecutively monocultured soil than in newly-planted soil. Real-time
PCR assay confirmed the reduction in abundance of Pseudomonas sp. and an increase in F.
oxysporum in consecutively monocultured soil. Furthermore, the relative abundance of
Pseudomonas sp. with the antagonistic activity against F. oxysporum was greatly lower in
consecutively monocultured soil than in newly-planted soil. We aslo found that phenolic
compounds mixture in an ratio as detected in the soil could greatly promote the growth of
pathogenic F. oxysporum, but inhibit the beneficial antagonistic bacteria. The isolated hostspecific F. oxysporum caused wilt disease on the tissue culture seedlings of R. glutinosa. This
study demonstrated that consecutive monoculture resulted in the alteration of rhizospheric
microbial composition with fewer microorganisms providing beneficial functions and more
microorganisms with pathogenicity, which is mediated by root exudates and had a negative
impact on R. glutinosa growth and development.
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155
Selection of microbial community by plant and soil from diverse inocula
Yan Yan*1, Eiko Kuramae1, Peter Klinkhamer2, Hans van Veen1
1
Netherlands Institute of Ecology, Netherlands, 2Leiden University, Netherlands
The experimental approach applied is based on the assumption the biodiversity of the
microbial community in soil can be altered by inoculation of diluted suspensions in a
sterilized soil. Although this method has frequently been used earlier, little is known how the
assemblage of bacterial communities in soil and rhizosphere proceeds after inoculation of
more or less diluted suspensions. For that purpose, serial dilutions of a soil suspension were
made, and re-inoculated into the original soil previously sterilized by γ-irradiation. We
determined the structure of the microbial communities by using 454-pyrosequencing of the
16S rRNA gene.
Upon dilution, the community diversity at the species level in the suspensions reduced
dramatically. The structure of microbial community in soil was changed drastically after
incubation as compared to the structure of the community in the inoculum suspension.
Although the microbial community was more homogenous in the rhizosphere, differences
between the original bulk soil and rhizosphere soil for all dilutions were only apparent at the
genus or species level. The selection power by soil after incubation was stronger than that by
the plant. Assemblage processes in soil of the high-diluted community followed clearly a
niche type model, but the assemblage in the rhizosphere appeared to be a neutral, random,
selection process. Network analysis showed a more complex system in the rhizosphere than
in the bulk soil, which, in contrast to the assemblage rules models, indicates the importance
of plant related mechanisms operating in the assembly of microbial communities in the
rhizopshere.
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Evasion and suppression of root immunity by beneficial microbes
Ke Yu*1, Roeland Berendsen1, Chiel Pel2, Corné Pieterse1, Peter Bakker1
1
Plant-Microbe Interactions, Utrecht University, Netherlands, 2University of
Toulouse, France
Plants can detect pathogenic microbes by recognizing microbe-associated molecular
patterns (MAMPs) through cell-surface pattern-recognition receptors (PRRs), which can
induce MAMP-triggered immunity in hosts. Plant-beneficial microbes living in the
rhizosphere possess similar MAMPs, however, this does not lead to massive activation of
plant defenses. This indicates that these beneficial microbes actively evade or suppress local
immune responses. Plants recognize flagellin as a MAMP through the PRR FLAGELLIN
SENSING 2. For pathogenic pseudomonads it was shown that an alkaline protease A
(AprA) that degrades flagellin monomers, limits recognition and thus virulence. The plant
growth promoting rhizobacteria Pseudomonas simiae WCS417 and P. capeferrum WCS358
can both suppress the immune responses in Arabidopsis roots elicited by flagellin. An AprA
homolog was also identified in the genome of WCS417. However the genome of WCS358 did
not comprise an AprA ortholog, suggesting WCS358 deploys a different strategy to suppress
the root immune responses. Site-directed mutagenesis and transposon mutant library
screening have been initiated to explore mechanisms involved in the evasion and
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suppression of root immune responses elicited by MAMPs during plant-beneficial microbe
interaction.
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Pyrosequencing reveals the structure and functional traits in a coastal halophyte
rhizosphere microbiome
Zhilin Yuan*1, Yuan Qin2, Yicun Chen2, Rusty Rodriguez3, Regina Redman3, Chulong
Zhang4, Fucheng Lin4
1
Chinese Academy of Forestry, China, 2Institute of Subtropical Forestry, Chinese
Academy of Forestry, China, 3Adaptive Symbiotic Technologies, United States, 4Zhejiang
University, China
Soil and root-associated microbes are always, if not all, of great importance for improving
host fitness. Current knowledge of microbiome in salt circumstances, however, is largely
lacking. As a step in this direction, the microbial community capable of prospering in the bulk
soils (BS), rhizosphere soils (RS), root endosphere (R) of Suaeda salsa, a widely distributed
halophyte in China's east coast, was investigated using platform of 454 pyrosequencing.
Rarefraction curves documented an impressive decreasing trend of α-diversity from soils to
endophytic compartments. Proteobacteria was the abundant phyla in all communities. The
genus Acinetobacter dominated the roots. Many groups of bacteria showed high
phylogenetic affinities to known halo-tolerant species. We used membership and phylogenybased Venn diagram to reveal the core bacterial colonizers. In addition, the relatively high
abundance of unclassified fungal and bacterial OTUs was prevalent at different taxonomic
levels, indicating the novelty of microbes in saline environment. For fungi, OTUs assigned to
Pleosporales were frequently found. Beauveria bassiana, Leptosphaeria sp., Retroconis sp.,
Monosporascus sp. were heavily enriched in BS, RS, R respectively. All of them displayed a
moderate to high level of genetic diversity. We hypothesize that such apparent phylogenetic
conservatism (redundancy) would be responsible for plants to be well adapted to salt stress.
Tajima's D test and the star-like structure of haplotype networks further revealed the
population expansion of above taxa. Beyond this, Montagnulacea sp. and Monosporascus sp.,
much more common in roots than BS and RS, were also easily isolated, and inoculation test
confirmed their ability to confer beneficial effects to plants under organic nitrogen or salt
stress condition. Overall, our data yields some evidence that rhizosphere microbiome of S.
salsa readily evolved to enable plants to survive in adverse conditions and opens new avenue
for generating symbiotically-modified salt-tolerant plants.
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Plant breeding and its effect on the rhizosphere microbiome - a sugar beet
example
Christin Zachow*1, Henry Müller1, Ralf Tilcher2, Gabriele Berg1
1
Graz University of Technology, Austria, 2KWS SAAT AG, Germany
Worldwide crop yields are lost due to abiotic and biotic stresses like drought, salinity, pests
and diseases. Breeding of stress-tolerant and pathogen-resistant cultivars has a long history
and approaches targeting plant beneficial microbes are developing. We used omics and
microscopic technologies analysing a naturally composed microbiome as seed treatment for
sugar beet plants against abiotic stresses and the late root rot caused by the soilborne
pathogen Rhizoctonia solani. Two sugar beet cultivars (BERETTA/JENNA) were used, which are
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characterized by R. solani sensitiveness/tolerance and by different abundance, enrichment
and interaction of a beneficial, sugar beet-specific Pseudomonas genotype. In the
Rhizoctonia-tolerant cultivar JENNA, the Pseudomonas genotype was enhanced in seed,
endorhiza and rhizosphere independent of the surrounding soil as example of naturally
occurring biocontrol. In contrast, the Rhizoctonia-sensitive cultivar BERETTA contains no
detectable number of this genotype. One strain of the genotype was compared to other
Pseudomonas strains of the same clade and characterized in detail; the genome information
of the endophyte P. poae RE*1-1-14 explains the endophytic lifestyle and the antagonistic
effect in 191 unique genes. Additionally, we analysed the impact of genetic variation in ten
cultivars on the composition of the microbiota. The overall results will contribute to
integrated management strategies in modern sustainable agriculture.
159
Iron-reducing bacteria in rice rhizosphere contribute to arsenic mobilization
under flooded conditions
Sarah Zecchin*1, Anna Corsini1, Raffaella Zanchi1, Maria Martin2, Gian Maria Beone3,
Marco Romani4, Lucia Cavalca1
1
University of Milano, Italy, 2University of Torino, Italy, 3Università Cattolica del Sacro
Cuore, Italy, 4Ente Nazionale Risi, Italy
Rice is among the crops mostly affected by arsenic contamination. Whereas in arsenic
contaminated soils (> 40 mg kg-1) rice farming contributes to population exposure, in rice
field soils with low arsenic content agronomic conditions represent an issue for arsenic
bioavailability Iron-reducing bacterial populations (Geobacteraceae and Shewanellaceae)
inhabiting rice rhizosphere were assessed in box plots (total arsenic 18.4 mg kg-1) cultivated
with different water regimes, in order to evidence their role in the release of iron and
consequently arsenic from root ferric iron plaques. Geobacteraceae and Shewanellaceae 16S
rRNA genes significantly increased in rice rhizosphere from the order of 105 to 106 copies (g
dry weight)-1 under flooding, while no significant increase occurred in aerobic rice.
Fluorescence in situ hybridization in rhizoplane evidenced a similar trend related to the active
populations. This increase was concomitant with the release of arsenic in soil solution under
continuous flooding: from 1.40 μg L-1 to 190 μg L-1 over the cropping cycle, whereas in
aerobic rice no significant release occurred. Analogously, ferrous iron increased from 0.75 mg
L-1 to 51.1 mg L-1 and dissolved organic C from 8.9 to 79.9 mg L-1 until late reproductive
stage. In aerobic rice soluble iron remained almost negligible and dissolved organic C never
exceeded 30 mg L-1. Arsenic content in rice grains was 237 μg kg-1 under flooding and 4.67
μg kg-1 in aerobic rice, thus reflecting this solubilisation trend. These outcomes evidence that
iron-reducing bacteria promote the arsenic release from iron plaques under flooded
conditions in non-contaminated rice fields thus contributing to grain contamination.
308

poster abstracts

Transcription

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