Instructions to authors for writing an extended abstract to be

6. SLOVENSKI SIMPOZIJ O RASTLINSKI BIOLOGIJI
z mednarodno
udeležbo
6. SLOVENSKI
SIMPOZIJ O
Fakulteta
za kmetijstvo in biosistemske
vede
RASTLINSKI
BIOLOGIJI
11. - 12. september 2014, Hoče
z mednarodno udeležbo
th za kmetijstvo in biosistemske vede
Fakulteta
11. - 12. september 2014, Hoče
6 SLOVENIAN SYMPOSIUM ON PLANT BIOLOGY
with international participation
Faculty of Agriculture and Life Science
September 11 - 12, 2014, Hoče, Slovenia
KNJIGA POVZETKOV
BOOK OF ABSTRACTS
Uredniki / Edited by:
Jasna Dolenc Koce
Tine Grebenc
Andreja Urbanek Krajnc
Izdalo / Issued by:
Slovensko društvo za biologijo rastlin
Slovenian Society of Plant Biology
Oblikovanje / Designed by:
Jasna Dolenc Koce
CIP - Kataložni zapis o publikaciji
Narodna in univerzitetna knjižnica, Ljubljana
581(082)(0.034.2)
SLOVENSKI simpozij o rastlinski biologiji z mednarodno udeležbo (6 ; 2014 ; Hoče)
Knjiga povzetkov [Elektronski vir] = Book of abstracts / 6. slovenski simpozij o
rastlinski biologiji z mednarodno udeležbo,11.-12. september 2014, Hoče = 6th
Slovenian Symposium on Plant Biology with International Participation, September 1112, 2014, Hoče, Slovenia] ; [organizator] Slovensko društvo za biologijo rastlin =
[organized by] Slovenian Society of Plant Biology ; [uredniki Jasna Dolenc Koce, Tine
Grebenc, Andreja Urbanek Krajnc]. - Ljubljana : Slovensko društvo za biologijo rastlin =
Slovenian Society of Plant Biology, 2014
ISBN 978-961-91014-4-5
1. Dolenc Koce, Jasna 2. Slovensko društvo za biologijo rastlin
275284224
KNJIGA POVZETKOV
ORGANIZACIJSKI ODBOR
Tine GREBENC, predsednik / President
Andreja URBANEK-KRAJNC, tajnica / Secretary
Aleš KLADNIK, blagajnik / Treasurer
Jasna DOLENC KOCE, program / Programme
Tina DEMŠAR, stik s pokrovitelji / Contact wit sponsors
Matevž LIKAR, spletna stran / Web page
NASLOV ORGANIZACIJSKEGA ODBORA
Organizacijski odbor 6. slovenskega simpozija o rastlinski biologiji /
Organizing committee of the 6th Slovenian Symposium on Plant Biology
Slovenian Forestry Institute (dr. Tine Grebenc)
Večna pot 2
SI-1000 LJUBLJANA
E-mail: info@plantslo.org
http://www.plantslo.org
I
POKROVITELJI
Zahvaljujemo se naslednjim pokroviteljem, ki so s svojo finančno pomočjo omogočili izvedbo
simpozija.
Mikro+Polo d.o.o.
Fakulteta za kmetijstvo in biosistemske vede Univerze v Mariboru
Nacionalni inštitut za biologijo
Biotehniška fakulteta Univerze v Ljubljani
Kemomed d.o.o.
Mediline d.o.o.
Mettler Toledo d.o.o.
Omega d.o.o.
KRKA, tovarna zdravil, d.d.
Lotrič d.o.o.
CHEMASS d.o.o.
Kambič Laboratorijska oprema d.o.o.
II
SPLOŠNE INFORMACIJE
Simpozij se bo začel v četrtek, 11. septembra 2014 ob 9.00 uri in končal v petek, 12. septembra 2014 popoldan.
Simpozij bo potekal na Fakulteti za kmetijstvo in biosistemske vede, Pivola 10, 2311 Hoče pri Mariboru
(Lat.:46.505466; Lon.:15.622579)
Recepcija bo odprta vsak dan od 8.00 do 12.00 ure.
Oblike prispevkov:
 vabljeno predavanje (IL)
 kratko predavanje (SL)
 poster (P)
Predavatelji bodo imeli za svojo predstavitev osebni računalnik (Windows 7 in Office 2013) in ozvočenje (1x
fiksni mikrofon, 3x prenosni mikrofon). Predavatelje prosimo, da svoj material oddajo operaterju 15 minut
pred začetkom prve sekcije dneva.
Posterji bodo razstavljeni v osrednjem hodniku Fakultete za kmetijstvo in biosistemske vede (1. nadstropje
novogradnje). Avtorje prosimo, da posterje obesijo na začetku simpozija in odstranijo po zaključku, da bodo na
ogled celoten čas simpozija. Prosimo, da je v času, ki je v programu predviden za predstavitev, ob posterju
navzoč vsaj eden od avtorjev.
Udeleženci simpozija imajo možnost objave znanstvenega članka po opravljeni recenziji v naslednjih revijah:
 Acta biologica Slovenica (http://bijh-s.zrc-sazu.si/ABS/EN/ABS/Index.asp)
 Acta agriculturae Slovenica (http://aas.bf.uni-lj.si/index-en.htm)
 Acta silvae et Ligni (http://www.forestry.bf.uni-lj.si/)
Kotizacija vključuje udeležbo na predavanjih, knjigo povzetkov, osvežitve med odmori, kosilo in slavnostno
večerjo.
III
The symposium will start on Thursday, September 11th 2014 at 9.00 a.m. and will close on Friday, September
12th 2014 in the afternoon.
The Symposium will be held at the Faculty of Agriculture and Life Sciences, Pivola 10, SI-2311 Hoče near
Maribor, Slovenia (Lat.:46.505466; Lon.:15.622579).
The Symposium reception desk will be open every day from 8.00 to 12.00.
Contributions will be presented as:
 invited lecture (IL)
 short lecture (SL)
 poster (P)
Facilities for projecting slides (personal computer equipped with Windows 7 in Office 2013) and audio system
will be available. The speakers are kindly requested to deliver their presentation material at least 15 minutes
prior the beginning of the first session of the day at the operator's desk.
Posters will be displayed in the main corridor (1st floor) of the Faculty of Agriculture and Life Sciences. Authors
are kindly requested to mount their posters at the beginning of the Symposium and to dismount them after
the last session in order to present their work throughout the period of the Symposium. At least one of the
authors is requested to be available for discussion during the corresponding poster session (indicated in the
program).
Scientific papers can be submitted for the publication in following peer reviewed journals:
 Acta biologica Slovenica (http://bijh-s.zrc-sazu.si/ABS/EN/ABS/Index.asp)
 Acta agriculturae Slovenica (http://aas.bf.uni-lj.si/index-en.htm)
 Acta silvae et Ligni (http://www.forestry.bf.uni-lj.si/)
Registration fee includes admission to all scientific sessions, book of abstracts, refreshments during breaks,
lunch and conference dinner.
IV
URNIK
11.9.2014
Otvoritev
12.9.2014
9:00
Sept 11, 2014
Opening
Moderne tehnologije
Sept 12, 2014
Modern technologies
9:30
Sistemska in molekularna
biologija rastlin
10:00
Rastlinska biotehnologija
Systems and molecular
plant biology
10:30
odmor
Plant biotechnology
break
odmor
11:00
break
11:30
Sistemska in molekularna
biologija rastlin
Struktura in funkcija
rastlin
12:00
postri
12:30
kosilo
13:00
kosilo
Systems and molecular
plant biology
Structure and function
poster session
lunch
lunch
13:30
14:00
postri
poster session
Interakcije rastlin
z drugimi organizmi
14:30
Plant interactions
with other organisms
15:00
Interakcije rastlin z okoljem
15:30
Plant interactions
with environment
odmor
break
16:00
odmor
Interakcije rastlin
z drugimi organizmi
break
Plant interactions
with other organisms
Plant interactions
with environment
Undergraduate session
16:30
17:00
Interakcije rastlin z okoljem
Dodiplomska sekcija
17:30
Zaključek
Večerja
18:00
18:30
V
Closing
Conference dinner
PROGRAM SIMPOZIJA
Četrtek, 11. 9. 2014 / Thursday, September 11th 2014
08.00 – 09.00
Registracija / Registration
09.00 - 09.30
Otvoritev / Opening ceremony
1
Sistemska in molekularna biologija rastlin
Predsedujoči / Chair: P. Tijskens, K. Gruden
09.30 - 10.00
10.00 - 10.30
10.30 - 10.45
M. Naseem: A systems biology perspective on cytokinins crosstalk to plant immune networks
(IL-1)
S. Pollmann: Induced auxin overexpression reveals a link between auxin signaling and
lignification through the induction of ethylene biosynthesis and several cell wall modifying
enzymes (IL-2)
N. Gozukirmizi: Sukkula retrotransposon insertion polymorphisms in barley calli (SL-1)
10.45 - 11.15
11.15 - 11.45
11.45 - 12.15
12.15 - 12.30
12.30 - 12.45
12.45 - 13.00
13.00 – 13.15
Odmor / Break
S. F. Elena: A systems biology approach to the evolution of virus-plant interactions (IL-3)
Š. Baebler: Digging potato molecular responses to virus infection (IL-4)
D. Dobnik: Suitability of wild potato relatives for functional analysis of potato genes using virus
induced gene silencing (SL-2)
A. Coll Rius: Functional analysis of an Ethylene Response Factor involved in potato defence
response to PVY (SL-3)
A. Lazar: Involvement of potato (Solanum tuberosum L.) MKK6 in response to Potato virus Y
(SL-4)
S. Suomalainen: Using a magnetic particle system in plant NA purification (presentation of
ThermoFisher Scientific)
VI
13.15 - 14.15
14.15 - 15.00
2A
Kosilo / Lunch
Poster sekcija / Poster session
(vse sekcije / all sessions)
Interakcije rastlin z okoljem in drugimi organizmi
Predsedujoči / Chair: H. Pfanz, D. Vodnik
15.00 - 15.30
15.30 - 15.45
15.45 - 16.00
16.15 - 16.30
M. Müller: Dynamic compartment specific changes in glutathione and ascorbate levels in
plants under different environmental conditions (IL-1)
S. Al Sayegh Petkovšek: Risk assessment of receptor organisms: analysis of intake of metals
through food chain (SL-1)
Z. Jeran: Biomonitoring of temporal and spatial patterns of metal deposition in Slovenia during
the last twenty years (SL-2)
I. Maček: Soil abiotic factors determine arbuscular mycorrhizal fungal communities in hypoxic
soils at natural CO2 springs (mofettes) (SL-3)
16.15 - 16.45
16.45 - 17.15
17.15 - 17.30
17.30 - 17.45
17.45 - 18.00
18.00 - 18.15
Odmor / Break
J. Šuštar-Vozlič: Abiotic stress in agricultural plants: Phaseolus sp. and drought (IL-2)
R. Povše: Physiological studies of drought stress in hop (Humulus lupulus L.) (SL-4)
A. Golob: UV radiation and selenium addition affects silicon accumulation in buckwheat plants
(SL-5)
T. Trošt Sedej: Growth in conifer and deciduous trees under enhanced UV-B radiation (SL-6)
K. Eler: The functioning of two karst ecosystems revealed by eddy-covariance measurements
(SL-7)
18.30 - Slavnostna večerja / Conference dinner
VII
Petek, 12. 9. 2014 / Friday, September 12th 2014
3
Moderne tehnologije in Rastlinska biotehnologija
Predsedujoči / Chair: S. Pollman, J. Šuštar Vozlič
09.00 - 09.30
09.30 - 10.00
10.00 – 10.30
10.30 - 10.45
10.45 - 11.00
L. M. M. Tijskens: Green mathematics: Benefits of including biological variation in your data
analysis (IL-1)
M. Kokalj: Infrared spectroscopy in plant science (IL-2)
D. Eichert: 3D Investigations of morphological and structural parameters in plants by
Synchrotron Radiation X-ray micro Computed-Tomography (IL-3)
J. Ambrožič-Dolinšek: Cryopreservation by encapsulation-dehydration affects the morphology
and regeneration capacity of endemic Hladnikia pastinacifolia (Apiaceae) (SL-1)
B. Bohanec: Report on the progress of development of biotechnological approaches for
breeding pumpkins (Cucurbita pepo L.) (SL-2)
11.00 – 11.30
4
Odmor / Break
Struktura in funkcija rastlin
Predsedujoči / Chair: D. Eichert, A. Kladnik
11.30 – 12.00
12.00 – 12.15
12.15 – 12.30
12.30 - 13.00
H. Pfanz: Non-foliar photosynthesis - existence in and necessity for plants (IL-1)
M. Chersicola: Assymetry of molecular processes during abscission in tomato (Solanum
lycopersicum L.) (SL-1)
T. Mrak: Arsenate and phosphate relationships during the uptake in lichens (SL-2)
Poster sekcija / Poster session
(vse sekcije / all sessions)
13.00 - 14.00
Kosilo / Lunch
VIII
2B
Interakcije rastlin z okoljem in drugimi organizmi
Predsedujoči / Chair: M. Mueller, M. Naseem, J. Dolenc Koce
14.00 - 14.30
14.30 – 14.45
14.45 – 15.00
15.00 – 15.15
15.15 – 15.30
15.30 – 15.45
M. Likar: Distribution and diversity of arbuscular mycorrhizal fungi in grapevines from
production vineyards along the eastern Adriatic coast (IL-1)
M. Flajšman: Testing of Verticillium albo-atrum virulence on different plant hosts and
development of a transformation system for Verticillium albo-atrum gene knock-out (SL-1)
V. Progar: Transcriptional analysis of differential gene expression in hop following infection
with Verticillium albo-atrum (SL-2)
T. Pokorn: Viroid-derived small RNA (vd-sRNA) target identification in hop (SL-3)
D. Kutnjak: What can "plant defence molecules" tell us about population structure of plant
virus? (SL-4)
M. Pompe-Novak: Morphology and gene expression in lesions in compatible and incompatible
potato – potato virus Y interaction (SL-5)
15.45 - 16.15
16.15 – 16.30
16.30 – 16.45
16.45 – 17.00
17.00 - 18.00
Odmor / Break
M. Petek: Potato Virus Y infection hinders potato defence response and renders plants more
vulnerable to Colorado potato beetle attack (SL-6)
M. Rupar: Fluorescently labelled Potato virus Y (PVY): in vivo tracking and measuring the rate
of spread of PVY in the host plants (SL-7)
J. Lamovšek: Pathogenic Agrobacterium tumefaciens antagonises root-knot nematode
Meloidogyne ethiopica reproduction on tomato roots (SL-8)
Dodiplomska sekcija / Undergraduate session
Predsedujoči / Chair: M. Kokalj, S. Elena, T. Grebenc
Kratka predstavitev posterjev / Short presentation of posters
Ocenjevanje / Evaluation
18.00 - 18.30
Zaključek / Closing
Razglasitev rezultatov Dodiplomske sekcije / Announcement of the results in the Undergraduate session
IX
KAZALO
Sistemska in molekularna biologija rastlin
Plant systems biology and molecular plant biology
1
Interakcije rastlin z okoljem in drugimi organizmi
Plant interactions with environment and other organisms
14
Moderne tehnologije in Rastlinska biotehnologija
Modern technologies and Plant biotechnology
33
Struktura in funkcija rastlin
Structure and function of plants
45
Interakcije rastlin z okoljem in drugimi organizmi
Plant interactions with environment and other organisms
53
Dodiplomska sekcija
Undergraduate session
70
Indeks avtorjev
Author indeks
82
Seznam udeležencev
List of participants
86
X
1 Sistemska in molekularna
biologija rastlin
1
A systems biology perspective on cytokinins crosstalk to plant immune networks
1
IL-1
Muhammad Naseem*, Thomas Dandekar
Functional Genomics and Systems Biology group, Department of Bioinformatics, Biocenter, Am Hubland, D-97074
Wuerzburg, Germany
* corresponding author (muhammad@biozentrum.uni-wuerzburg.de)
Molecular genetic studies have helped to elucidate a multistep two-component cytokinin (CK) signalling
system in Arabidopsis. The importance of CK signalling in controlling plant growth and development has
thoroughly been investigated. The efforts highlighting its implications in plant immunity are lagging
behind. Higher plant CK levels and signalling correlate with increased plant resistance against various
pathogens. However, the underlying molecular mechanisms whereby CK signalling modulates plant
immune networks are not fully understood.
To investigate mechanisms how CK responses culminate in protecting plants from infection with model
pathogen Pst DC3000, we mapped high quality Arabidopsis Protein-Protein Interactions (Y2H-PPI). A
dynamic of change to the basal network is given by transcriptome data after treatment of Arabidopsis
with Pst DC3000. Nodes of the cellular interactome enriched in immune functions and their interacting
partners are organized into sub-networks. According to our analysis, seventeen proteins of CK canonical
pathway are involved in immune network. Based on topological analyses, nodes connectivity and
network modularity, we identified functional hubs in our sub-networks. Two of the CK signalling proteins
AHK2 (receptor) and ARR14 (type-B response regulator) are among the top ten most connected hubs.
Besides, we also conducted analyses on modulated CKs conditions such as deletion of type-A ARRs as well
as exogenous CK application. These analyses culminate in a functional core module where nodes of SA
mediated defense and Cytokinins pathway actively participate. Furthermore, we established a Boolean
model of plant immune and pathogen virulence specific proteins. Model analyses and dynamic
simulations for various hormone types with and without pathogen infection manifested diverse immune
responses. Taken together, our integrated systems biology approaches are highly instrumental in
deciphering hormonal crosstalk in plant immunity.
2
Induced auxin overexpression reveals a link between auxin signaling and lignification
through the induction of ethylene biosynthesis and several cell wall modifying
enzymes
Marta-Marina Pérez Alonso, Daniel Jesús García Martínez, Stephan Pollmann*
Centro de Biotecnología y Genómica de Plantas (UPM – INIA), Campus de Montegancedo, 28223 Pozuelo de Alarcón
(Madrid), Autovía M-40 km 38, Spain
* corresponding author (stephan.pollmann@upm.es)
Auxin is associated with the regulation of virtually every aspect of plant growth and development. Many
previous genetic and biochemical studies revealed that, among the proposed routes for the production of
auxin, the indole-3-pyruvic acid (IPyA) pathway is the main source for indole-3-acetic acid (IAA) in
plants. The IPyA pathway involves the action of 2 classes of enzymes, tryptophan-pyruvate
aminotransferases (TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA 1)/TRYPTOPHAN
AMINOTRANSFERASE RELATED (TAR)) and flavin monooxygenases (YUCCA). Both enzyme classes
appear to be encoded by small gene families in Arabidopsis consisting of 5 and 11 members, respectively.
We recently showed that it is possible to induce transcript accumulation of 2 YUCCA genes, YUC8 and
YUC9, by methyl jasmonate treatment (Hentrich et al. 2013a). Both gene products were demonstrated to
contribute to auxin biosynthesis in planta. Furthermore, we reported that the overexpression of YUC8 as
well as YUC9 led to strong lignification of plant aerial tissues likely due to the up-regulation of ethylene
biosynthesis (Hentrich et al. 2013b). New transcriptomic and metabolomic evidence indicates that this
abnormally strong secondary growth is not only linked to increased levels of ethylene production, but
also to substantial changes in the expression level of a subclass of cell wall modulating enzymes. Those
enzymes play a role in cell wall softening and consequently in the modification of the secondary cell wall
composition.
Hentrich M., Böttcher C., Düchting P., Cheng Y., Zhao Y., Berkowitz O., Masle J., Medina J., Pollmann S. (2013a). Plant Journal 74
(4): 626-637.
Hentrich M., Sánchez-Parra B., Pérez Alonso M. M., Carrasco Loba V., Carrillo L., Vicente Carbajosa J., Medina J., Pollmann S.
(2013b). Plant Signaling & Behavior 8 (11): e26363.
3
1
IL-2
A systems biology approach to the evolution of virus-plant interactions
1
IL-3
Santiago F. Elena*
Instituto de Biología Molecular y Celular de Plantas (CSIC-UPV), Campus UPV CPI 8E, Ingeniero Fausto Elio s/n,
46022 València, Spain; The Santa Fe Institute, 1399 Hyde Park Road, Santa Fe, NM 87501, USA
* corresponding author (sfelena@ibmcp.upv.es)
Understanding the mechanisms by which viruses overcome host defenses in order to proliferate has been
a challenging problem owing to the multiplicity of factors and complexity of interactions involved. The
advent of genomic techniques has opened the possibility to grasp a global picture of the interaction. We
used Arabidopsis thaliana to identify and compare genes that are differentially regulated upon infection
with seven distinct plant viruses (Rodrigo et al. 2012). In a first approach, we established lists of genes
differentially affected by each virus and compared their involvement in biological functions and metabolic
processes. We found that phylogenetically-related viruses significantly alter the expression of similar
genes and that viruses that naturally infect Brassicaceae display a greater overlap in the lists of altered
genes. In a second approach, virus-manipulated genes were contextualized using transcriptional and
protein-protein interaction networks of A. thaliana. This method uncovered a general mode of action of
plant viruses, in which perturbations preferentially affect genes that are highly connected, central and
organized in modules.
Rodrigo G., Carrera J., Ruiz-Ferrer V., Del Toro F. J., Llave C., Voinnet O., Elena, S. F. (2012). PLoS ONE 7, e40526.
4
Digging potato molecular responses to virus infection
1
IL-4
Špela Baebler*
National Institute of Biology, Večna pot 111, Ljubljana, Slovenia
*corresponding author (spela.baebler@nib.si)
Potato (Solanum tuberosum L.) is most widely grown tuber crop and the fourth most important food crop
with a continuous progression of the world production of tubers that is challenged by changing
environment and new emerging diseases. Understanding its biology by using systems biology tools can
contribute to plant breeding and development of efficient agricultural practices. However, the
exploitation of data obtained by high-throughput wet lab methods is hindered by dispersed data and
unavailability of potato specific functional annotation and visualization tools. The genome of diploid
potato was sequenced three years ago and enabled better integration of the existing data.
To combine existing information on potato genes and enable data integration from various datasets, we
have merged information from two potato gene models and two sets of potato unigens to 35609 gene
paralog groups. For easier data interpretation we have adapted plant-specific MapMan ontology and
designed a centrally maintained database and a web interface GoMapMan (www.gompaman.org, Ramšak
et al., 2013) for browsing, searching and editing the ontology and gene annotations. Within the ontology,
genes of the model species Arabidopsis and 4 crop species (potato, tomato, tobacco and rice) are
connected trough ortholog groups.
Developed tools have allowed us translation of existing knowledge on model and other crop species to
potato as well as better visualization and interpretation of high-throughput experimental data on potato Potato virus Y interaction.
Ramšak Ž., Baebler Š., Rotter A., Korbar M., Mozetič I., Usadel B., Gruden K. (2014). Nucleic acids research 42, D1167–75.
5
Sukkula retrotransposon insertion polymorphisms in barley calli
Nermin Gozukirmizi*, Gonul Kartal-Alacam, Sibel Yilmaz, Sevgi Marakli
Istanbul University, Faculty of Science, Department of Molecular Biology and Genetics, 34134, Vezneciler, IstanbulTURKEY
*corresponding author (nermin@istanbul.edu.tr)
Barley has economical value and it is an important model plant for genetic studies. Transposons cover
more than 80% of barley genome. More than 40 retrotransposons were characterized in barley genome.
Some of them are autonomous but some are not. This type of transposons replicates via RNA and moves
in the genome. As a result of these movements, mutations and genome enlargements are occurred.
Somaclonal variations are spontaneously occurred variations in tissue culture conditions. These
variations could be produced by genetic and/or epigenetic mechanisms and result in problems in gen
transfer applications. In this study, mature barley embryos (Hordeum vulgare cv. Tokak 157/37) were
cultured for callus formation. Sukkula (a non-autonomous retrotransposon) polymorphism was
investigated in calli with different culturing time (40 and 80 day-old) which was derived from the same
embryo in MS medium supplemented with 3 mg/l Dicamba. Polymorphisms were detected using InterRetrotransposon Amplified Polymorphism (IRAP) and Inter Primer Binding Site Amplification (iPBS)
techniques with two different Sukkula-specific primers. Non-cultured 5 mature embryos were used as
control. While up to 14% polymorphism rates were calculated from non-cultured embryos, 61% and 0%
polymorphism rates were observed with IRAP analyses in 40 and 80 day-old calli, respectively. iPBS
analyses revealed polymorphism ratio up to 29% in non-cultured materials, and up to 58% and 70% in
40 and 80 day-old calli materials, respectively. These findings show that tissue culture conditions and
culturing time cause insertional activation of some barley retrotransposons. These stress induced
activation might be responsible for development of somaclonal variations.
6
1
SL-1
Suitability of wild potato relatives for functional analysis of potato genes using virus
induced gene silencing
1,
1
2
1
1
David Dobnik *, Maja Jamnik , Vivianne G.A.A. Vleeshouwers , Kristina Gruden , Jana Žel
Department of biotechnology and systems biology, National institute of biology, Večna pot 111, 1000 Ljubljana,
Slovenia
2 Wageningen UR Plant Breeding, Wageningen University and Research Centre, P.O. Box 386, 6700 AJ, Wageningen,
The Netherlands
* corresponding author (david.dobnik@nib.si)
1
Potato is the fourth most important food crop worldwide. It is susceptible to many pathogens, where
potato virus Y (PVY) is one of the most important ones. To better understand the interaction between
potato and PVY several “omics” studies were already performed, resulting in a plethora of interesting
genes. However, when it comes to the confirmation of importance of individual genes by the means of
functional analysis, the results are rather scarce, as the cultivated potatoes are not the optimal model
organisms. To provide the scientific community with the most suitable system for functional analyses of
genes from potato using virus induced gene silencing (VIGS), we have screened large panel of wild potato
relatives for their susceptibility to VIGS. Based on a visual marker (silencing of PDS) we have selected 15
appropriate Solanum species for further evaluation. We have challenged the selected wild potato relatives
with PVY and followed the spreading of the virus. To check whether the empty silencing vector (based on
Tobacco rattle virus) has an effect on PVY spreading, we performed VIGS with empty vector and then
inoculated the same plants with PVY. Finally we challenged the selected wild potato relatives with PVY
labelled with GFP (PVY-GFP). Based on the results of these experiments the most appropriate candidate
for VIGS experiments was selected. To confirm that the selected species is suitable for VIGS studies of
potato genes, we have silenced some of the selected genes from potato and observed the effect on the
viral spreading with the use of PVY-GFP. Our results have shown that the selected system is appropriate
for functional analysis of genes from potato.
7
1
SL-2
Functional analysis of an Ethylene Response Factor involved in potato defence
response to PVY
Anna Coll*, Ana Lazar, David Dobnik, Katja Stare, Špela Baebler, Kristina Gruden
Department of Biotechnology and Systems Biology, National Institute of Biology, Ljubljana, Slovenia
* corresponding author (anna.coll@nib.si)
Potato (Solanum tuberosum L.) is the world’s most widely grown tuber crop and potato virus Y (PVY) is
one of the major potato pathogen causing severe crop loss in different areas worldwide. To better
understand the potato defence response against PVY we studied the role of ethylene response factor (ERF)
genes since they function as important plant-specific transcription factors in regulating biotic and abiotic
stress response.
A potato ERF gene, StERF, that was identified and selected based on previous transcriptomics
experiments performed in our group has been characterised and functionally analysed in this study.
Expression patterns of the gene in hypersensitive resistance (HR) potato cultivar infected with PVY
pointed to its importance as a signalling component in potato defence response. Localisation studies
showed that StERF strongly accumulated in cell nucleus after PVY infection. In addition, using virusinduced gene silencing (VIGS) we demonstrated that PVY systemic spread is delayed in StERF silenced
plants. We further examined the potential hormonal signalling involved in the expression of StERF gene
and demonstrated that our gene integrates several signalling pathways.
Taken together our results suggested the importance of SotubERF in potato defence mechanisms against
PVY and its involvement in the ethylene signalling pathway. Therefore the data contributes to better
understand the complex network of plant defence signalling pathways.
8
1
SL-3
Involvement of potato (Solanum tuberosum L.) MKK6 in response to Potato virus Y
Ana Lazar*, Anna Coll, David Dobnik, Špela Baebler, Jana Žel, Kristina Gruden
Department of biotechnology and systems biology, National institute of biology, Ljubljana, Slovenia
* corresponding author (ana.lazar@nib.si)
Mitogen-activated protein kinase (MAPK) cascades have crucial roles in the regulation of plant
development and in plant responses to stress. Plant recognition of pathogen-associated molecular
patterns or pathogen-derived effector proteins has been shown to trigger activation of several MAPKs.
This then controls defence responses, including synthesis and/or signalling of defence hormones and
activation of defence related genes. The MAPK cascade genes are highly complex and interconnected, and
thus the precise signalling mechanisms in specific plant–pathogen interactions are still not known.
Here we investigated the MAPK signalling network involved in immune responses of potato (Solanum
tuberosum L.) to Potato virus Y, an important potato pathogen worldwide. Sequence analysis was
performed to identify the complete MAPK kinase (MKK) family in potato, and to identify those regulated
in the hypersensitive resistance response to Potato virus Y infection. Arabidopsis has 10 MKK family
members, of which we identified five in potato and tomato (Solanum lycopersicum L.), and eight in
Nicotiana benthamiana. Among these, StMKK6 is the most strongly regulated gene in response to Potato
virus Y. The salicylic acid treatment revealed that StMKK6 is regulated by the hormone which is in
agreement with the salicylic acid-regulated domains found in the StMKK6 promoter. The involvement of
StMKK6 in potato defence response was confirmed by localisation studies, where StMKK6 accumulated
strongly only in Potato-virus-Y-infected plants, and almost exclusively in the cell nucleus. Using a yeast
two-hybrid method, we identified three StMKK6 targets downstream in the MAPK cascade: StMAPK4_2,
StMAPK6 and StMAPK13. These data together provide further insight into the StMKK6 signalling module
and its involvement in plant defence.
9
1
SL-4
Using a magnetic particle system in plant NA purification
1
SL-5
Suomalainen Sini*
Thermo Fisher Scientific, Vantaa, Finland
* corresponding author (sini.suomalainen@thermofisher.com)
Sample preparation is often a limiting step for different kind of genomics studies. By using magnetic
particles for nucleic acid isolation, it is possible to speed up the purification process and achieve better
results from variety of sample types. In this presentation there will be an overview to the magnetic
particle purification and examples how to enhance the process by using KingFisher magnetic particle
system. The KingFisher system is an open platform allowing the user to choose any available magnetic
particle purification reagents from different manufacturers. There are instruments available for low
throughput research laboratories as well as routine testing laboratories needing higher throughput. The
high-throughput extraction of plant nucleic acids is very important for several applications like the plant
breeding, quality control of seeds and other molecular screening tests of plants. With the KingFisher
system, highly pure DNA can be obtained from fresh, frozen, dried or lyophilized plant samples. Magnetic
particle purification is not only efficient and reproducible, but also a gentle way to isolate good quality
nucleic acids, also intact RNA from fresh and frozen plant samples.
10
Genome-specific distinction between Brassica oleracea L. and Sinapis arvensis L.
seeds in seed lots
Barbara Pipan*, Romana Rutar, Darja Vouk, Vladimir Meglič
Agricultural institute of Slovenia, Hacquetova ulica 17, SI-1000 Ljubljana
* corresponding author (Barbara.pipan@kis.si)
Diverse Brassicaceae family includes economically important Brassica L., Sinapis L. and Raphanus L.
species. Brassica oleracea L. (CC genome) and Sinapis arvensis L. (SS genome; also known as Brassica
kaber (DC.) L.C. Wheeler) are closely related diploid species (2n=18). B. oleracea is widely used as
vegetable crop; S. arvensis is mostly appeared as weedy/wild plant with a series of superior traits, since is
becoming an important germplasm resource in Brassicaceae breeding schemes. Close relationsip
between B. oleracea and S. arvensis is reflected in a high level of morphological, genetic and karyotypic
similarity. Consequently, it is difficult to distinguish these two species, especially when analyzing seed
purity in seed testing laboratories. The problem lies primarily in seed identification, where seeds of S.
arvensis represent impurities in B. oleracea seed lots. The method to distinguish between these two
species according to the seed coat morphology, using binocular at low magnification (x40), is not reliable.
The fast and cost effective method is required to provide accurate information for species identification.
Molecular markers, such as SSRs (Short Sequence Repeats), could be readily applied due to their
informative nature.
To distinguish between B. oleracea and S. arvensis species on molecular level, we developed fast and
reliable SSR method using genome-specific markers. According to Pipan et al. (2013), we tested 15 highly
informative SSR markers, originating from different Brassica species. DNA was extracted from
germinated seedlings of morphologically identified S. arvensis or B. oleracea seeds used for a seed purity
test. Reference DNA (internal resource) from both observed species was included in the study. Two
markers (Na12-C08 and Ra2-E12) were proven to be genome-specific due to their successful
amplification in B. oleracea only, but not in any of the S. arvensis samples. The third marker (Ra2-A01)
was amplified in all samples, but in a different allele forms that distinguished B. oleracea (at ~ 150 bp
length) and S. arvensis (at ~ 180 bp length) genomes. Moreover, this locus could also be applied for intraspecific differentiation within S. arvensis genome.
The use of genome-specific markers (Na12-C08, Ra2-E12 and Ra2-A01) in seed testing laboratories
would provide a fast, reliable and cost-effective tool for qualitative genetic screening and identification of
S. arvensis seeds as impurities in B. oleracea seed lots or vice versa.
Pipan B., Šuštar-Vozlič J., Meglič, V. (2013). Genetika 45 (2), 309-327.
11
1
P-1
Plant pathogens detection by simultaneous multiplex isothermal amplification and
detection of RNA and DNA targets
1
1,2
1
David Dobnik* , Dany Morisset , Rok Lenarčič , Maja Ravnikar
1
Department of biotechnology and systems biology, National institute of biology, Večna pot 111, 1000 Ljubljana,
Slovenia
2 Current address: CropDesign N.V., Technologiepark 21C, 9052 Gent (Zwijnaarde), Belgium
* corresponding author (david.dobnik@nib.si)
1
The impact of climate change and expansion of trade networks is expected to cause an emergence of new
plant pathogens in places, where they have not been present before. Reliance on sometimes lengthy
laboratory testing can cause significant delays in preventive actions. A relatively small number of
detection tools is currently available for field use with many having serious shortcomings in terms of
sensitivity, complexity or cost. Thus, there is a real need for rapid, simple and robust detection methods
that can be used in the field to allow early and simultaneously detection of several plant pathogens.
Molecular detection methods for plant pathogens mostly rely on amplification of specific genetic
elements. To reduce the costs and time needed for analyses, the development should be focused to
detection methods allowing multiplex amplification and detection. The genetic material of pathogens can
be carried in RNA or DNA form, therefore the method for their multiplex detection should also enable
amplification and detection of both in parallel. We developed an isothermal nucleic-acid-sequence–based
amplification [NASBA]-implemented microarray analysis (NAIMA) suitable for simultaneous multiplex
amplification of RNA and DNA targets and their detection on ArrayTubes™. The method was
demonstrated to be very sensitive and specific for detection of two economically important quarantine
plant pathogens, Potato Spindle Tuber Viroid (RNA target) and Ralstonia solanacearum (DNA target). The
advantage of the NAIMA method is that it is isothermal and therefore no expensive apparatus is needed
for work, which makes it also suitable for the determination of plant pathogens directly in the field. The
method could also be applicable to any domain where there is need to detect RNA and DNA pathogens or
other targets simultaneously.
12
1
P-2
Interactions between components of the plant defence response and viral proteins
Živa Ramšak*1, Tjaša Stare1, Tjaša Lukan1, Dragana Miljković2, Kristina Gruden1
1 Department
of Biotechnology and Systems Biology, National Institute of Biology, Ljubljana, Slovenia
Department of Knowledge Technologies, Jožef Stefan Institute, Ljubljana, Slovenia
* corresponding author (ziva.ramsak@nib.si)
2
Infection of a plant by a pathogen initiates a complex interaction between both players involved, leading
to changes in the complex signalling network, which result in gene activity changes and reprogramming
of the cell metabolism. A systems biology approach was adopted for the purpose of modelling complex
biological processes in order to understand the mechanisms and dynamics involved in potato plant
defense following the infection with potato virus Y (PVY).
A qualitative model of potato plant defence signalling network (PDS) was constructed (Miljkovic et al.,
2012), describing the biosynthesis and signal transduction pathways for three crucial phytohormones
involved in plant defence: salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). The prior knowledge
from literature was expanded with information on the viral and plant component interactions, proteinprotein interactions and protein-DNA interactions in plant Arabidopsis.
The resulting robust qualitative model offers new insights into the plant-virus interaction by expanding
the knowledge on critical components of plant defence signalling, thus producing novel hypotheses to be
tested in the wet lab. First efforts of dynamical modelling of the selected ethylene model sub-component
were already performed, indicating the importance of protein degradation efficiency to obtain
biologically relevant representations of network responsiveness.
Miljkovic, D., Stare, T., Mozetic, I., Podpecan, V., Petek, M., Witek, K., Dermastia, M., Lavrac, N., Gruden, K. (2012). PloS ONE, 7(12),
e51822. doi:10.1371/journal.pone.0051822
13
1
P-3
2 A Interakcije rastlin z okoljem
in drugimi organizmi
14
Dynamic compartment specific changes in glutathione and ascorbate levels in plants
under different environmental conditions
Maria Müller*, Bernd Zechmann
Institute of Plant Sciences, University of Graz, Schubertstraße 51, Graz, Austria
* corresponding author (maria.mueller@uni-graz.at)
Ascobate and glutathione are the most important antioxidants in plants. They are involved in the
detoxification of reactive oxygen species (ROS), redox signaling, in the modulation of defense gene
expression and they are important for the regulation of enzymatic activities. For these reasons levels of
ascorbate and glutathione are often used as stress markers in plants. Nevertheless, most studies in the
past focused on measuring ascorbate and glutathione in whole tissues and organs rather than in single
cells and organelles. Considering that stress situations affect cell compartments differently and that
ascorbate and glutathione metabolism is highly compartment specific we applied this method on different
plant species during abiotic and biotic stress situations to gain thorough knowledge about the subcellular
distribution of ascorbate and glutathione in plants and on the importance of these antioxidants in certain
cell compartments during stress situations.
Acknowledgement: This work was supported by the Austrian Science Fund.
15
2A
IL-1
Abiotic stress in agricultural plants: Phaseolus sp. and drought
Jelka Šuštar-Vozlič1*, Marko Maras1, Tanja Zadražnik1, Marjetka Kidrič2, Mateja Zupin1, Jaka
Razinger1, Dominik Vodnik3, Vladimir Meglič1
1
Agricultural Institute of Slovenia, Hacquetova ulica 17, 1000 Ljubljana Slovenia
Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia
3 Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia
* corresponding author (jelka.vozlic@kis.si)
2
Different abiotic factors such as drought, flood, extreme temperatures, high salinity and others affect
plant growth and crop productivity. Drought contributes to considerable yield losses in different crops,
including common bean (Phaseolus vulgaris L.). The response of common bean to drought is not well
characterized yet. The aim of our research has therefore been oriented towards obtaining a deeper
insight into the molecular and morpho-physiological adaptation of bean plants to drought.
Differential gene expression study was conducted on leaves of eight common bean genotypes at different
levels of dehydration. The results revealed the alteration of 15 drought-responsive transcripts. The
expression profiles of selected genes were studied also in different bean species. The mode of expression
was found consistent within Phaseolus spp. A proteomic approach was used to identify droughtresponsive proteins in leaves of two cultivars differing in the response to drought, Tiber and more
sensitive Starozagorski čern. The identified proteins were divided into different functional categories that
include metabolic proteins, cellular defence/stress proteins, proteins involved in reactions of
photosynthesis and proteins with unknown function. Analysis of stem proteome of cultivar Tiber under
drought stress using in-gel stable isotope labelling revealed proteins grouped into several functional
groups. By studying proteases we have shown that there are several serine endopeptidases and
aminopeptidases in leaves of common bean which are differently affected by drought. A protease from
leaves of cultivar Zorin has been studied in details; characterization at the protein and gene level led to its
classification as a new plant subtilisin-like serine protease, named PvSLP2. Genetic characterization of
cvs. Tiber and Starozagorski čern and 82 recombinant inbred lines (RILs) using molecular markers has
been launched recently. The main aim is to establish genetic map of bean genome, which will be used in
identification of markers linked to the quantitative trait loci involved in drought response of common
bean. Several hundred microsatellites have been included in the characterization and different
physiological parameters were measured in parental cultivars and RILs in order to define differences of
their response to drought.
The integrated knowledge of the genomic, proteomic and physiological processes during drought
response will contribute to the basic understanding of drought tolerance, not only in bean but also in
other plants, and will offer novel opportunities for bean management in different environments.
16
2A
IL-2
Risk assessment of receptor organisms: analysis of intake of metals through food
chain
1
1
2
Samar Al Sayegh Petkovšek *, Nataša Kopušar , Davorin Tome , Boris Kryštufek
3
1 ERICo
Velenje, Inštitut za ekološke raziskave d.o.o., Koroška 58, 3320 Velenje
inštitut za biologijo, Večna pot 111, 1000 Ljubljana
3 Prirodoslovni muzej, Prešernova 20, 1000 Ljubljana
* corresponding author (samar.petkovsek@erico.si)
2 Nacionalni
On the basis of the results for heavy metal levels in soil, in herbs and in tissues of receptor organisms
(small mammal species, passerine birds) obtained in the period 2011-2014 in differently polluted areas
in Slovenia, the following conclusions can be made. (a) The soil in the vicinity of the former smelter
(Žerjav) is heavily polluted with Pb, Cd, Zn and As, and burdened with Mo and Hg. The latter is reflected in
increased levels of metals in herbs (Cd, Pb, Zn, Mo, Ni) and in earthworms (Pb, Cd, Zn). Particularly
problematic is Cd, which accumulates in herbs (Sesleria albicans Kit. Ex Schultes) such as in earthworms.
Pb accumulates only in earthworms, but the intake of this element in the herbs is quite large, despite its
relatively poor mobility (BCF: 0.08 to 0.50), due to the extremely high levels of Pb in soil. Therefore, these
two dietary sources (herbs, earthworms) could be the most significant route of Pb and Cd exposure for
receptor organisms. To a much lesser extent this applies to fruits and seeds of tree and shrubs species
(Cornus sanguinea L., Sorbus aria (L.) Crantz, Acer pseudoplatanus L. and Pinus sylvestris L.). The mean Pb
and Cd concentrations in liver of small mammals species (Microtus agrestis (Linnaeus, 1761)), Myodes
glareolus (Schreber, 1780), Apodemus flavicollis (Melchior, 1834)), reached effect concentrations in a
significant proportion of the livers of the small mammal specimens (Pb: 40%, Cd: 67%). These findings
indicate that the majority of small mammals trapped in the area of the former lead smelter are at risk of
toxicological effects due to the very high uptake of Pb and Cd in the organism. (b) On the contrary, Pd and
Cd concentrations in livers of small mammals from the other study areas (vicinity of the largest Slovenian
thermal power plant (Veliki Vrh) and along the main road (Črnova)) were comparable with reference
values and considerably lower than effect concentrations. The smallest levels of pollutants in all types of
samples were found in soil, plant and animal samples from reference location (The Logar Valley). In
general, these differences were statistically significant. (c) Pb and Hg in the tail feathers of passerine birds
from polluted study areas were statistically significant higher in comparison with reference area. (d)
Hazard Quotient (HQ) higher than 1 (existence of risk) was calculated for Myodes glareolus, sampled in
the vicinity of the lead smelter, if the proportion of the roots in its diet is at least 20% and for Apodemus
flavicollis, collected from the same study area, if the proportion of the earthworms in its diet is at least
10%.
17
2A
SL-1
Biomonitoring of temporal and spatial patterns of metal deposition in Slovenia
during the last twenty years
1
1
Zvonka Jeran *, Darja Mazej , Mitja Skudnik
2
1Department
of Enivironmental Sciences, “Jožef Stefan” Institute, Jamova 39, 1000 Ljubljana, Slovenia
Forestry Institute, Večna pot 2, 1000 Ljubljana, Slovenia
* corresponding author (zvonka.jeran@ijs.si)
2Slovenian
Several country wide monitoring surveys were performed since 1992 in Slovenia in which two types of
biomonitoring organisms were used to assess metal and nitrogen air pollution. In 1992 and 2001
epiphytic lichen Hypogymnia physodes was collected and analysed for more than 20 elements, however in
1995 also moss Hypnum cupressiforme was for the first time used in our country as a monitor organism as
a part of European Moss Survey within the UN-ECE ICP-Vegetation. Moss monitoring was since then
regularly repeated at 5 years intervals. Moss samples were always collected in the period between the
middle of June and the end of August at the regular grid of 16 x 16 km or 16 x 8 km (2010), though the
number of plots at which samples were taken differed among the surveys, namely from 30 in 1995 to 102
plots in 2010, of which 32 plots were the same in all the last three surveys. Over the 15 years there was
also a change in methods for elemental determination, namely since 2005 ICP-MS was used instead of
Instrumental neutron activation analysis. All together 17 elements were determined: As, Cd, Cr, Co, Cu, Fe,
Mo, Ni, Pb, Sb, Se, Sr, V, Ti and Zn. For Hg determination cold vapour AAS (2005) or direct measurement
using the DMA method (2010) was used. Also since 2001 nitrogen and sulphur were analysed using the
CNS method.
Although there were slight modifications in moss sampling over the 15 year period, the spatial pattern of
elements and their temporal trends well reflect the overall pollution in the country, as well as also the
natural background. Similarly to the majority of European countries, a decrease of element
concentrations was observed in Slovenia since 1995, which was most probably due to economic factors
since some of the heavy polluters have shut down or modernized their technology to decrease emissions.
Another reason for the great decline as observed in median levels of all elements on a national scale in
2010 in comparison to 2005 might lie in the fact that in 2010 special attention was devoted to sampling
and testing the influence of environmental parameters on element levels in mosses. The spatial pattern of
levels of anthropogenically derived elements especially Pb, Cd, Mo, Zn, Sb and nitrogen in Slovenia can be
explained on the one hand by the population density and the locations of the main pollution sources and
on the other hand, by transboundary air transport of pollutants from neighbouring countries, mainly as a
consequence of meteorological conditions (wind, precipitation).
18
2A
SL-2
Soil abiotic factors determine arbuscular mycorrhizal fungal communities in hypoxic
soils at natural CO2 springs (mofettes)
1
1
2
Irena Maček *, Nataša Šibanc , Alex J. Dumbrell , Thorunn Helgason
3
Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1001 Ljubljana, Slovenia
School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK
3 Department od Biology, Wentworth Way, University of York, York YO10 5DD, UK
* corresponding author (irena.macek@bf.uni-lj.si)
1
2
Natural CO2 springs (mofettes) are specific and extreme ecosystems, characterized by high soil CO 2 and
reduced soil O2 concentrations. A locally hypoxic environment is established that is similar to those found
in waterlogged or flooded soils. In mofettes, geological CO2 exhalations result in a long-term abiotic
selection pressure affecting local soil biota. The characterization of arbuscular mycorrhizal (AM) fungal
communities from plant roots sampled in the Stavešinci mofette area (Slovenia), confirmed the presence
of apparently unique AM fungal assemblages across a range of soil CO 2 concentrations. We examined
whether AM fungal taxa abundant in locations with high soil CO2 concentrations are local specialists or
widespread generalists able to tolerate a range of different soil conditions. To answer this, a high
throughput sequencing approach (Roche 454 sequencing) was used to see if those taxa present in areas
with high soil CO2 concentrations are also found in low abundance among AM fungal communities from
soils with ambient CO2 concentrations. In addition to hypoxia, we investigated other abiotic soil factors
(e.g. soil pH) driving changes in AM fungal communities under the extreme environmental conditions
found in mofette sites. Results show that some fungi are more strongly associated with local variations in
the soil environment, particularly hypoxia, than with other abiotic soil characteristics or distributions of
their host plants.
19
2A
SL-3
Physiological studies of drought stress in hop (Humulus lupulus L.)
Rozalija Povše1*, Dominik Vodnik2, Stanislav Mandelc2, Branka Javornik2, Andreja Čerenak1
Inštitut za hmeljarstvo in pivovarstvo Slovenije, Cesta Žalskega tabora 2, 3310 Žalec
Biotehniška fakulteta, Jamnikarjeva 101, 1000 Ljubljana
* corresponding author (zala.povse@ihps.si)
1
2
Frequent droughts, irregular precipitation patterns and limited sources of water dictate plant breeding
programmes oriented to the selection of crops with higher tolerance to water stress. Combined use of
omic approaches (transcriptomics, proteomics) and conventional physiological techniques (gas exchange
techniques, fluorescence, water potential measurements) enables a better insight into the response of
plants, which is crucial for selecting targeted properties of drought tolerant cultivars.
This approach was used in a study with two hop cultivars, Aurora (tolerant) and Savinjski golding
(susceptible). Two pot experiments were run in two consecutive seasons. Measurements and sampling of
leaves were done at different stages of developing drought, documented by soil water content, relative
water content in leaves and leaf water potential measurement. In our contribution we will present results
on stomatal and non-stomatal inhibition of photosynthesis, differences in water use efficiency and
photochemical efficiency of the tested cultivars, and the preliminary results of differential proteomics.
20
2A
SL-4
UV radiation and selenium addition affects silicon accumulation in buckwheat plants
Aleksandra Golob1*, Vekoslava Stibilj2, Ivan Kreft3, Mateja Germ1
Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia
Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenija
3 Slovenian Forestry Institute, Večna pot 2, SI-1000 Ljubljana, Slovenia
*corresponding author (aleksandra.golob@bf.uni-lj.si)
1
2
UV radiation affects organisms, especially plants, on different levels. UV radiation is consequently a
carrier of information about the environment of plants. However, when exposed to enhanced doses of UV
radiation, plants can be damaged because of the enhanced production of reactive oxygen species which
cause oxidative stress to plants. Selenium (Se) is one of biologically essential elements for animals and
humans, needed for activity of antioxidative enzymes, such as glutathione peroxidase and thioredoxin
reductase. Se has not been classified as an essential element for plants, but it has been considered as
beneficial. It has been demonstrated that Se can increase the tolerance of plants to UV-induced oxidative
stress and promotes the growth of plants subjected to high-energy radiation. In higher concentrations Se
has toxic effect. Silicon (Si) is second most abundant element in Earth crust. Si has an important role in
stress mitigation, through increased production of antioxidants and its binding to, and co-precipiataion
with metal ions. We investigated the effect of different levels of UV radiation and foliar spraying with Se
on biochemical and physiological traits as well as Si accumulation in leaves of Fagopyrum tataricum and
Fagopyrum hybridum. Experimental plants were exposed outdoors to four treatments regarding the
quantity of UV radiation (ambient or reduced) and added Se (naturally accessible or foliarly treated with
Na selenate). The content of pigments and UV absorbing compounds, transpiration rate, photochemical
efficiency of PS II, respiratory potential and the biomass of plant parts were measured. UV treatment had
no effect on pigment content, but enhanced potential photochemical efficiency at the early stage of
development of both buckwheat species. Addition of Se increased effective photochemical efficiency and
transpiration, while decreased potential photochemical efficiency. In condition of reduced UV radiation
addition of Se decrease respiratory potential, while increased it under ambient UV conditions. Just the
opposite effect was observed for the mass of the seeds. Preliminary results showed that addition of Se
significantly increased accumulation of Si in leaves of both buckwheat species. In Tartary buckwheat UV
radiation had positive effect on Si accumulation, while in hybrid buckwheat there were no differences
between UV treatments.
21
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SL-5
Growth in conifer and deciduous trees under enhanced UV-B radiation
Tadeja Trošt Sedej1*, Dušan Rupar1, Alenka Gaberščik1
1
Department of Biology, Biotehnical Faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia
* corresponding author (tadeja.trostsedej@bf.uni-lj.si)
Trees, as plants in general, possess diverse systems which respond to UV-B radiation. The results
available in the literature for conifer and deciduous trees indicate that UV-B radiation may affect those
tree groups differently according to their physiology, biochemistry and morphology, to needle and leaf
development stage, to interaction with environmental conditions and multilevel interactions between all
those factors. We compared strategies in Norway spruce (Picea abies (L.) Karst.) and European beech
(Fagus sylvatica L.) for coping with enhanced UV-B radiation. The two tree species were exposed
outdoors to enhanced UV-B for three years during which time, selected parameters were monitored
(Trošt Sedej & Gaberščik 2008, Trošt Sedej & Rupar 2013). Spruce and beech exhibited great variability in
the amounts of chlorophyll, methanol-soluble UV-B and UV-A absorbing compounds, and optimum
quantum yield of photosystem II. The effects of UV-B radiation varied with needle and leaf development
stage and interaction with environmental conditions. Enhanced UV-B radiation triggered responses in
both trees and a reduced negative effect of UV-B radiation on spruce photochemical efficiency was
observed during prolonged drought. The results show high UV-B tolerance of both tree species and
indicate the complexity of plant response to UV-B, involving multilevel interactions with environmental
factors.
Trošt Sedej, T., Gaberščik, A. (2008). The effects of enhanced UV-B radiation on physiological activity and growth of Norway
spruce planted outdoors over 5 years. Trees 22 (4), 423-435.
Trošt Sedej, T., Rupar, D. (20013). Deciduous and evergreen tree responses to enhanced UV-B treatment during three years. Acta
Biologica Slovenica 56 (2) 35-50.
22
2A
SL-6
The functioning of two karst ecosystems revealed by eddy-covariance measurements
2A
Klemen Eler1*, Mitja Ferlan2, Giorgio Alberti3, Alessandro PERESSOTTI3, Primož Simončič2, Dominik
Vodnik1
SL-7
University of Ljubljana, Biotechnical Faculty, Jamnikarjeva 101, Ljubljana, Slovenia
Slovenian Forestry Institute, Večna pot 2, Ljubljana, Slovenia
3 Department of Agricultural and Environmental Sciences, University of Udine, Udine, Italy
* corresponding author (klemen.eler@bf.uni-lj.si)
1
2
Since 1980's the eddy covariance measurements of trace gasses, latent and sensible heat have made their
way in ecosystem research, providing many valuable information on yearly, seasonal and daily
cumulatives of carbon (C) and water vapour exchange of different ecosystems of the world. Moreover,
this key ecosystem research method greatly increased our understanding of environmental regulation of
carbon emission and consumption rates which is inevitable input information to predict global change
effects of different ecosystems and to designate a proper management.
In this contribution carbon and water vapour exchanges of a spatially heterogeneous tree-shrubgrassland mosaic developed on former semi-dry calcareous grassland in the sub-Mediterranean region of
Slovenia were studied and compared with the nearby grassland still in use as a pasture. Yearly and
seasonal net ecosystem exchange and evapotranspiration of both ecosystems were examined together
with their environmental controls (soil water content, air temperature, light availability) during the
period August 2008 - December 2012.
Results showed significant differences in yearly cumulatives, seasonal phenology of C assimilation and
drought resistance between the investigated ecosystems. The occurrence of drought seemed to largely
govern ecosystem functioning and productivity. By identifying the drought periods objectively using the
evapotranspiration response at different soil water content values it was shown that more than the
severity of drought the duration of the longest yearly drought affected the estimated yearly gross primary
productivity. The functioning of ecosystems was also compared with respect to ecosystem-level water
use efficiency, rain use efficiency and light use efficiency in different environmental conditions and
phenological phase.
23
Change of phenolic compounds pattern in Nicotiana megalosiphon as a response to
abiotic and biotic stress
Saša Likić*, Gordana Rusak
University of Zagreb, Faculty of Science, Department of Biology, Marulicev trg 9a, 10000 Zagreb, Croatia
* corresponding author (sasa.likic@biol.pmf.hr)
Quantitative and qualitative changes of phenolic compounds were compared in untreated, wounded,
Cucumber mosaic virus (CMV) infected and in Cucumber mosaic virus containing satellite RNA (CMVsat)
infected Nicotiana megalosiphon plants. Significant changes in total phenolics (TP) content were analysed
at five different time intervals: immediately after inoculation, 2, 4, 7 and 14 days after inoculation. The
highest decrease of TP was detected in wounded while the highest increase of TP was detected in both
CMV and CMVsat infected plants. Defence response to wounding in investigated plants resulted in a
significant decrease of detected phenolics content compared to untreated plants. On the other hand,
significant increase in the content of detected phenolics in infected plants compared to wounded plants
was detected. Nevertheless, the content od phenolics in CMVsat infected plants was significantly higher
compared to the content in CMV infected plants. Depending on treatment and on time interval, significant
increase or decrease of quercetin or kaempferol was detected in investigated species. Observed changes
of phenolics suggest distinct responses to wounding and virus infection, which could probably be
involved in activating different defence response to a specific stress.
24
2A
P-1
Phenols, photosynthetic pigments and leaf morphology as biomarkers of
environmental influences on Posidonia oceanica (L.) Delile
1
1
2
3
1
Željka Vidaković-Cifrek *, Mirta Tkalec , Jasna Dolenc Koce , Donat Petricioli , Adam Cvrtila , Jelena
Katanović1, Janja Maltar1, Mateja Smiljanec1, Nina Zupanič2, Tatjana Bakran-Petricioli1
1University
of Zagreb, Faculty of Science, Department of Biology, Rooseveltov trg 6, HR-10000 Zagreb, Croatia;
of Ljubljana, Biotechnical Faculty, Department of Biology, Večna pot 111, SI-1000 Ljubljana, Slovenia;
3D.I.I.V. Ltd., for marine, freshwater and subterranean ecology, Obala Petra Lorinija bb, HR-23281 Sali, Croatia
* corresponding author (zeljka.vidakovic-cifrek@biol.pmf.hr)
2University
The endemic Mediterranean seagrass Posidonia oceanica (L.) Delile is a very important benthic primary
producer. Even though its meadows have multiple ecological functions they have been modified and
destroyed due to a changing environment and an increased human impact. As a slow-growing and longliving species sensitive to environmental changes, P. oceanica is a good bioindicator. In this work,
phenolic compounds (total phenolics, flavonoids and anthocyanins), photosynthetic pigments and leaf
morphology were evaluated in intermediate leaves of P. oceanica. The samples were collected on 2 (or 3)
and 10 m of depth, in summer and winter on three locations of the Zadar coastal area: Zadarski kanal
(ZK), Brbišćica Bay (B) and Zaglav Port (ZA) on island Dugi otok. The aim was to find correlation among
measured parameters, environmental conditions and the primary production of the seagrass meadow
and to estimate the suitability of parameters to be used as bioindicators. Plants collected in ZK and B at
10 m depth had higher portion of flavonoids in total phenols than those taken at 2 (3) m. Also, higher
values were noticed in winter. In samples from ZA the portion of flavonoids was almost the same
regardless of depth and season. On the other hand, anthocyanin portion was the highest in summer at 2
(3) m of depth. The ratio of chlorophyll a and chlorophyll b was higher in winter in plants grown at both
depths and all locations. The ratio of carotenoids and total chlorophylls was mainly 0.20-0.23, except in
ZK in summer at 2 m with significantly lower value (0.16). Similar results were obtained for ZA samples.
In morphological study mesophyll width was between 74% and 88% of total leaf thickness. Plants at all
three locations had thicker mesophyll in winter and at 2 (3) m depth but the differences were not always
significant. Principal component analysis showed that samples grouped mostly due to season and depth.
Two deviations were noticed, namely samples in ZA at 3 m and in winter were closer to ZA samples
collected at 10 m. Also, samples from ZK at 2 m and in summer differed from other samples due to higher
primary production which could be connected with human impact.
25
2A
P-2
Temperature affects reproductive traits in olive tree (Olea europaea L.) – flowering
timing and pollen performance
1
1
Gabriela Vuletin Selak *, Slavko Perica , Smiljana Goreta Ban
2
1 Institute
for Adriatic Crops, Put Duilova 11, 21 000 Split, Croatia
of Agriculture and Tourism Poreč, K. Hugues 8, 52 440 Poreč, Croatia
* corresponding author (Gabriela.Vuletin.Selak@krs.hr)
2 Institute
The reproductive phase has proven to be one of the most temperature sensitive stages in plants.
Relatively high or low temperatures are often correlated with low fertilization percentages and low fruit
setting. The interest in the effect of temperature on reproductive processes is increasing because the
global rise in temperature has affected plant populations by inducing a shift in several phenological traits,
such as dormancy release and flowering time. In the Mediterranean basin, olive flowers between April
and June which is later than most other tree species. This later date allows olive to meet the requirements
of an ample accumulation of heat units during the growing season. Six olive cultivars (‘Lastovka’,
‘Leccino’, ‘Levantinka’, ‘Oblica’, ‘Pendolino’ and ‘Itrana’) were employed in field experiment during three
years to determine the effect of accumulated temperatures preceding the flowering periods on the
beginning of flowering. The flowering periods were assessed by visual examination of eight trees per
cultivar. The average flowering date (AFD) was calculated as the number of days from 1 January until the
start of the flowering period. The AFD was determined for each experimental year using flowering dates
of all tested cultivars. The number of chilling units (CU) was calculated using the daily maximal and
minimal air temperatures. The heat accumulation start dates were the first days after the chilling
requirement had been met and continued until the start of flowering season. Heat units were expressed in
growing degree-days (GDD). The effect of temperature on pollen germination and pollen tube growth was
evaluated using in vitro system. Pollen performance has been tested under different temperatures (15,
22±2, 30, 35 and 40 ºC) to evaluate the sensitivity of the microgametophyte to temperature extremes.
The olive tree can be used as a sensitive biological indicator of climatic changes since the yearly
temperature variations control the timing of the flowering period. High correlations have been found
between values of AFD and those of CU and GDD. Moreover, simultaneous flowering of different cultivars
is important in achieving a high fruit set after cross-pollination. The flowering periods of most of the
studied cultivars overlapped to some degree. Pollen germination in vitro was enhanced when pollen
grains were exposed to higher temperatures up to 35 ºC; however germination failed at 40 ºC. In most of
the studied cultivars, the longest pollen tubes were developed at 22±2 ºC and the pollen tube length
decreased with higher incubation temperatures. Temperature significantly affected flowering timing and
pollen performance in olive cultivars and therefore participates in regulation of final fruit set and yield.
26
2A
P-3
Examination of physiological response of common bean (Phaseolus vulgaris L.) to
drought stress
1
1
1
2
3
Mateja Zupin *, Marko Maras , Jelka Šuštar Vozlič , Marjetka Kidrič , Dominik Vodnik , Jaka
Razinger4, Vladimir Meglič1
1Agricultural
Institute of Slovenia, Crop Science Department, Slovenia
Stefan Institute, Department of Biochemistry and Molecular Biology, Slovenia
3University of Ljubljana, Biotechnical faculty, Department of Agronomy, Slovenia
4Agricultural Institute of Slovenia, Plant Protection Department, Slovenia
*corresponding autor (mateja.zupin@kis.si)
2Jožef
Common bean is nutritionally very important legume plant that exhibits sensitivity to drought which
affects its growth and yield. Plants developed mechanisms in adapting to drought, which are expressed by
changes of gene expression and functional protein content, together with responses at physiological and
morphological levels. Identification of changes in response to drought in different genotypes will provide
markers essential for introduction and implementation of marker assisted selection in breeding for
greater drought tolerance.
To establish differences between plants subjected to water withdrawal and normally watered plants of
common bean, the parental cultivars, drought tolerant Tiber and drought susceptible Starozagorski čern,
were grown in a growth chamber under controlled environmental conditions. Plants were watered daily
to the same pot weight. After three weeks the half of plants were stressed by withholding irrigation.
Physiological parameters were measured on third trifoliate leaves at different drought stages. The
hydration state of leaves was defined by their relative water content (RWC), water potential (Ψw) and
different photosynthetic parameters. Detached leaf samples are kept at -80°C for further analyses.
In water stressed plants RWC dropped to 50% compared to control plants. Due to the reduction of water
potential, the duration of the leaf stomata closure was increased as confirmed by the results of
photosynthetic and other physiological parameters. Other differences in response to drought between the
cultivars were identified, which is of great importance for further study of genetic factors governing
drought response by means of QTL mapping.
27
2A
P-4
Analyses of salt stress alleviation with homobrassinosteroid in germinating barley
roots
Sevgi Marakli*, Nermin Gozukirmizi
Istanbul University, Faculty of Science, Department of Molecular Biology and Genetics, 34134, Vezneciler, IstanbulTURKEY
* corresponding author (sevgi.marakli@istanbul.edu.tr)
Effects of Homobrassinolide (HBR) (0.5-1 μM HBR) on root germination and cell division of barley
(Hordeum vulgare L. cv. Hilal) roots grown under different salt concentrations (150 mM and 250 mM) for
48 and 72 h were investigated with their controls. Increased salt concentration caused negative effect on
primary root and seminal root lengths as compared to control (1.8 ± 0.6 and 2.4 ± 0.4, 48 h and 72 h,
respectively), the lengths of primary root decreased in 150 mM salt concentration (1 ± 0.4 and 1.5 ± 0.6,
48 h and 72 h, respectively) and 250 mM (0.5 ± 0.2 and 0.9 ± 0.2, 48 h and 72 h, respectively) salt
concentration. On the other hand, HBR (0.5-1 μM HBR) applications enhanced primary root lengths,
seminal root lengths and number of roots from one seed compared to controls and only salt-treated
samples. Salt + HBR treatments showed varying results depending on concentration and timing. Mitotic
activity in control barley roots was 20.4% and 23.6% (48 h and 72 h respectively) of the cells. Salt stress
reduced mitotic index (8.3% and 5.6% in 150 mM salt treated and 5.5% and 3.6% in 250 mM salt treated
samples for 48 h and 72 h). Moreover, mitotic index of HBR application was higher than both control and
salt treated samples. Increasing concentration of HBR alleviated salt stress effectively but their effect
could be seen after long time (1 μM application showed more mitotic index for 150 mM salt and 250 mM
salt application both at 48 h and 72 h). Roots treated with HBR showed more mitotic activity, mitotic
abnormalities and significant enlargements at the root tips. In addition, salt + HBR applications
stimulated root lengths and showed more mitotic activity and mitotic abnormalities when compared to
only salt-treated samples. Depending on concentration and timing, HBR hormone alleviates salt stress on
germinating barley roots.
28
2A
P-5
Polytrichaceous mosses are resistant to short termed salt stress
2A
Marija Rajčić, Vesna Nerić, Milorad Vujičić, Marko Sabovljević, Aneta Sabovljević*
P-6
Institute of Botany and Garden, Faculty of Biology, University of Belgrade, Takovska 43, 11000 Belgrade, Serbia
* corresponding author (aneta@bio.bg.ac.rs)
Bryophytes are among the earliest land plants. They have a high degree of abiotic stress tolerance, which
helps them to survive in the harsh environment.
The aim of this study was to investigate the effects of different salt concentrations (5mM - 500mM NaCl)
on biomass and photosynthetic pigment (chlorophylls and carotenoids) content in selected bryophyte
species (Atrichum undulatum (German and Serbian genotype) and Polytrichum formosum (German
genotype). These species were cultured for 4 days in vitro, at the half strength Murashige and Skoog (MS)
medium enriched with different NaCl concentrations and subsequently 24 days at the basal MS half
strength medium.
At higher concentrations of NaCl, total chlorophyll content is decreasing. Index of multiplication at lower
NaCl concentrations is linearly decreasing. Survival of plants (all three genotypes) is 100% up to 100mM
NaCl. At higher concentration of NaCl (200-500 mM) there is linearly decrease of the survival rate.
Chlorophyll content increased up to 200 mM NaCl and carotenoid content up to 300 mM NaCl, both in
comparison to the untreated control group.
Biomass production in both species, decreased in the presence of higher concentrations of salt, compared
to biomass production of control groups.
Although unexpected, both species and three genotypes showed resistance to salt stress.
29
Rain simulation with heavy metal deposition: effects of exposure duration and lead
concentration on survival and development of the moss Hypnum cupressiforme
1
2
1
1
1
3
Marko Sabovljević *, Mitja Skudnik , Milorad Vujičić , Jovana Pantović , Nada Nikolić , Zvonka Jeran ,
Franc Batič4, Aneta Sabovljević1
1Institute
of Botany and Botanical Garden, Faculty of Biology, University of Belgrade, 11000 Belgrade, Serbia
Forestry Institute, Department of Forest and Landscape Planning and Monitoring, Večna pot 2, 1000
Ljubljana, Slovenia
3”Jožef Stefan” Institute, Department of Environmental Sciences, Jamova 39, 1000 Ljubljana, Slovenia
4Biotechnical Faculty, Universiy of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia
* corresponding author (marko@bio.bg.ac.rs)
2Slovenian
The moss Hypnum cupressiforme Hedw. is widely used as a model-moss in study of atmospheric
deposition of contaminants. Many European-wide heavy metal deposition measurements in mosses as
biomonitors were done on this ubiquitous moss. However, up to date there was no experimentation on
this moss in controlled axenical conditions. With the aim to control the effects of rain duration and lead
concentration in simulated rain on mosses we established in vitro culture of moss Hypnum cupressiforme.
This pleurocarpous moss was grown in controlled light and temperature conditions. Axenically
propagated plants over a few years in laboratory conditions were used as start material for further
experimentation. Forty explants (each of 10 mm long) per each exposure period and concentration were
exposed to simulated rain for 0.5, 2 and 24 hours, and the rain contained 0, 50, 150, 300 μM Pb-acetate as
a carrier of lead. After exposure to simulated rain the plants were grown for eight weeks on half strength
Murashige and Skoog basal solid medium prior to developmental features and survival measurements.
The results obtained clearly showed that the duration of exposure to the simulated rain and the increase
of Pb concentration decreased the regeneration of the 10 mm start explants both in secondary protonema
production and index of multiplication. Survival also decreased with duration of exposure and increase of
Pb concentration. The lethal concentrations were those above 700 μM (tested sequently up to 5mM) of
Pb-acetate in any duration of simulated rain tested. Further experiments of simulated rain to chlorophyll
production and other pigment contents, as well as synergism with other heavy metals and the repetition
of subsequent simulated rains followed by the intermediate time of moss recovery are ongoing.
30
2A
P-7
The effect of nickel stress on snake's head fritillary (Fritillaria meleagris L.)
2A
Valerija Vujčić1*, Marija Petrić2, Sandra Radić Brkanac1, Slađana Jevremović2, Angelina Subotić2,
Branka Pevalek-Kozlina1
P-8
1 Department
2 Institute
of Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, Zagreb, Croatia
for Biological Research "Siniša Stanković", University of Belgrade, Bulevar despota Stefana 142, Belgrade,
Serbia
* corresponding author (vvujcic©biol.pmf.hr)
Snake's head fritillary (Fritillaria meleagris L.) is an endangered and protected plant species in the family
Liliaceae mainly inhabiting moist grasslands. As a result of negative anthropogenic impacts such as
ameliorization and urbanization, the habitats rapidly disappear and/or are affected by pollutants such as
heavy metals. The aim of the study is to estimate level of sensitivity and/or resistance of snake's head
fritillary to nickel (Ni), a metabolically important microelement though also toxic when present at
excessive amounts in soil. Plants cultivated in vitro were subjected to increasing Ni concentrations (0-5
mM) for four weeks. Relative growth rate (RGR) and certain indicators of oxidative stress - soluble
proteins, malondialdehyde content and antioxidant enzyme activities were measured. A decrease of RGR
based on fresh weight and protein content was noted in response to growing Ni concentrations whereas
malondialdehyde (indicator of lipid peroxidation) content increased. Antioxidant response was observed
under Ni stress, with large increases in catalase and peroxidases. The results suggest induction of
oxidative stress under Ni toxicity, but also demonstrate capacity of F. meleagris to upregulate its
antioxidative defense, thus indicating certain degree of plant's tolerance to Ni exposure.
31
Analysis of stem proteome of common bean under drought using in-gel stable
isotope labeling
1
1
Tanja Zadražnik *, Jelka Šuštar-Vozlič , Wolfgang Egge-Jacobsen
2
1 Agricultural
Institute of Slovenia, 1000 Ljubljana, Slovenia
of Oslo, Department of Molecular Biosciences, Oslo, Norway
* corresponding author (Tanja.Zadraznik@kis.si)
2 University
Drought is an abiotic stress that strongly influences plant growth, development and productivity. By
proteomics study it is possible to identify the complex mechanism of water-stress response. The aim of
this research was the analysis of common bean (Phaseolus vulgaris L.) stem proteome changes under
stress conditions by applying a water deficit treatment. Quantitative proteomics using in-gel stable
isotope labeling of stem proteins in combination with mass spectrometry was used for the analysis. Mass
spectrometry data were analyzed using MaxQuant software, where quantification and presentation of the
results were performed. In total, 45 protein groups were found to change significantly in abundance
between control and stressed samples. The quantified proteins were grouped into several functional
groups, mainly into energy metabolism, photosynthesis, proteolysis, synthesis and proteins related to
ROS, defence and stress. Mixed abundance profiles of proteins involved in energy metabolism and
photosynthesis showed different range of proteins involved in the complex regulation and production of
different metabolites within energy metabolism and reaction of photosynthesis. Heat shock protein
showed the highest increase in abundance among all 45 proteins, suggesting its role in protecting plants
against stress by re-establishing normal protein conformation and thus cellular homeostasis. Proteins
involved in protein synthesis were all increased in abundance under drought. Increased levels of protein
synthesis are important in order to restore the damaged proteins for full restoration of the plant cell's
metabolic activities and general growth. A decrease in abundance of protein tubulin, a classical
cytoskeleton-related protein was noticed. This indicate the involvement of cell size adjustment as one of
the cellular adaptive strategies to drought stress. Also other important proteins were quantified and were
included in a wide range of biological pathways that are involved in plant protection. The research
provided the basic insight into the molecular regulatory mechanism of stem proteins in common bean
under drought.
32
2A
P-9
3 Moderne tehnologije in
Rastlinska biotehnologija
33
Green mathematics: Benefits of including biological variation in your data analysis.
L.M.M. Tijskens 1,*, Rob E. Schouten 1, Tatjana Unuk 2, Marjan Simčič 3
Wageningen University, Horticulture and Product Physiology, The Netherlands
University Maribor, Faculty of Agriculture and Life Sciences, Slovenia
3 University of Ljubljana, Biotechnical Faculty, Slovenia
* corresponding author (Pol.Tijskens@wur.nl)
1
2
Variation exists in all samples of natural products. Sometimes the variation is small, often it is large. To
avoid interference from this biological variation, the main effort in developing handling procedures in
horticulture, is to try to make variation in production during growth as small as possible while
maximising the yield. The still existing variation is dealt with in various postharvest sorting and grading
systems as used in packing houses. That works most of the time pretty well for commercial purposes. So,
this technology is applied for the last 100 to 150 years quite successfully.
This strategy, however, does intrinsically not invite to study the basic rules that govern this biological
variation and prevents thereby a more basic understanding of its sources and dynamic behaviour in time.
It therefore also prevents efforts of turning that omnipresent nuisance into an advantage. Understanding
the dynamics of biological variation (also in QTLs and other genetic information), will open complete new
alleys for optimising production and distribution of horticultural products. Knowledge on variation in
product properties, well in advance of harvest time, enables a more suitable use of product classes in the
postharvest supply chain.
In this paper, the methodology, statistical as well as mathematical, of including differences between
individuals in a batch of products will be explained (Tijskens et al. 2005) and highlighted (Tijskens et al
2003), and the benefits in terms of reliability of data interpretation will be indicated based on a number
of examples, ranging from colour and size development in the apple orchard in Slovenia.
Tijskens L.M.M. Konopacki P., Simčič M. (2003). Postharvest Biology & Technology 27/1, 15-25.
Tijskens L.M.M., Heuvelink E., Schouten R.E., Lana M.M., van Kooten O. (2005). Acta Horticulturae 687, 39-46
Groups. Fourth Edition, Springer, Berlin: 506 pp.
34
3
IL-1
3
Infrared spectroscopy in plant science
IL-2
Meta Kokalj*, Samo Kreft
University of Ljubljana, Faculty of pharmacy, Aškerčeva 7, 1000 Ljubljana, Slovenia
* corresponding author (meta.kokalj@ffa.uni-lj.si)
Infrared spectroscopy (IR) has been applied in plant material identification, in assessing quantity of
certain compounds and also imaging. IR is fast, easy to use and does not use organic solvents. In order to
apply IR to plant samples it is necessary to combine it with chemometrical methods. Infrared spectra are
a very large and complex sets of data therefore the analysis is very complex. However once the optimal
model for given data is established, the use is very simple and can be automatized (Bunaciu A. A. 2011,
Gad H. A. 2011).
The analysis with IR is composed of three main steps: spectra measurement, spectra pre-treatment and
chemometrical analysis. Each of these steps has to be optimized case-bycase since the properties of the
samples and the goal of analysis can be very different. In different modes of spectra measurement it is
important to take in account from which part of the sample (the surface or the inside) the spectra are
collected, (Kokalj M., Kolar J., et al. 2011). With the pretreatments of the spectra important information
can be emphasized or, if inappropriate technique is used, important information can be lost (Kokalj M.,
Rihtarič M., et al. 2011). Since the infrared spectra are a complex set of data it is also important to
carefully choose the mathematical and statistical methods for their interpretation (Kokalj M.,2010). There
are no general rules, so optimal conditions have to be found for each specific case.
Bunaciu A: A., Aboul-Enenin H. Y., Fleschin S. (2011). Applied Spectroscopy Reviews 46, 251-260.
Gad H. A., El-Ahmady S. H., Abou-Shoer M., Al-Azizi M. M. (2011). Phytochemical analysis 24, 1-24.
Kokalj M., Kolar J., Trafela T., Kreft S. (2011). Phytochemical analysis 22, 541-546.
Kokalj M., Rihtarič M., Kreft S. (2011). Chemometrics and Intelligent Laboratory Systems 108, 154-161.
Kokalj M., Strgulc Krajšek S., Kreft S. (2010). Journal of Chemometrics 24, 611-616.
35
3D Investigations of morphological and structural parameters in plants by
Synchrotron Radiation X-ray micro Computed-Tomography
Diane Eichert*
ELETTRA – Sincrotrone Trieste, S.S. 14 Km 163.5 in Area Science Park, 34149 Basovizza, Trieste, Italy
*corresdponding author (diane.eichert@elettra.eu)
Microscopic analysis is the first step of investigation in many fields of science, but its involvement in
botany and plant science in general is a long term tradition, as witness the precise descriptions of the first
botanical drawings and the variety of details which were successively reported through though time.
Imaging is used to reveal the organization of biological or physiological structures, and, if possible, their
relation with their functions and dynamics. A plethoric of techniques is nowadays at the disposal of the
plant physiologist to address fundamental questions in structural biology of living organism, from optical
to electron or X-ray transmission microscopy, UV or radiation induced X-ray fluorescence. In addition,
technical progresses have extended the range of assessment of structural information from pure
microscopy (2D) to tomography (3D).
Among them, X-ray micro Computed Tomography (-CT) is a relatively new investigation tool which
potential still needs to be fully apprehended and its application further developed in plant science.
In particular, synchrotron radiation (SR) based -CT offered the possibility to study non-destructively,
with a micron-scale resolution “large” object, in such revealing its details at various levels of organization
(organ, tissue, cellular, sub-cellular).
Based on differences in the local attenuation of the X-rays within the specimen, and on powerful
reconstruction algorithms taking advantage of both the transmission and the phase contrast modalities,
SR -CT provides 3D information on the morphology and structural characteristics without the need of
any sample preparation procedures (cutting, staining). Moreover, the radiation dose can be monitored
and threshold to avoid potential damage and the sample environment can be customized (cryo-stage,
furnace, liquids, etc.) to probe various in-situ characteristics and dynamics. Some key features related to
morphological, structural, physiological and performance related traits, such as quantitative
measurements of voids/air cavities/pores, interconnections, inter-relationships or densities within
tissues or cells can be visualised and monitored.
In this presentation will be highlighted some results obtained at the Syrmep beamline [Tromba et al,
2010], Elettra – Sincrotrone Trieste on plant science studies on wood, leaves, and in particular seeds.
Tromba G., Longo R., Abrami A., Arfelli F., Astolfo A., Bregant P., Brun F., Casarin K., Chenda V., Dreossi D., Hola M., Kaiser J.,
Mancini L., Menk R.H., Quai E., Quaia E., Rigon L., Rokvic T.,Sodini N., Sanabor D., Schultke E., Tonutti M., Vascotto A., Zanconati F.,
Cova M., Castelli E. (2010). AIP Conference Proceedings 1266, 18-23.
36
3
IL-3
Cryopreservation by encapsulation-dehydration affects the morphology and
regeneration capacity of endemic Hladnikia pastinacifolia (Apiaceae)
2
2
1
Terezija Ciringer , Mitja Kaligarič , Jana Ambrožič-Dolinšek *
University of Maribor, Faculty of Education and Faculty of Natural Sciences and Mathematics, Koroška cesta 160,
2000 Maribor, Slovenia
2 University of Maribor, Faculty of Natural Sciences and Mathematics, Koroška cesta 160, 2000 Maribor, Slovenia
* corresponding author (jana.ambrozic@um.si)
1
The objectives of our work were optimization of cryopreservation by encapsulation-dehydration and
determination of the effect of this procedure on the morphology and regeneration capacity of in vitro
culture of Hladnikia pastinacifolia Rhcb., a monotypic endemic genus with an extremely narrow
distribution area (Slovenia). The morphology and the regeneration capacity of in vitro plant material,
originating from cryopreserved and non-cryopreserved shoots and regularly transferred to a fresh MS
multiplication medium, were evaluated one year after cryopreservation treatment. After one year of
continuous propagation the cryopreserved plant material behaves like non-cryopreserved one and
developed morphologically similar shoots and roots. The differences between both materials were seen
in regeneration capacity and its line dependant. The cryopreserved plant material developed more shoots
and slightly smaller number of roots.
Acknowledgements: This research was supported by the Slovene Ministry of Higher Education, Science
and Technology within the programme Research to Ensure Food Safety and Health (Grant No. P1-0164).
37
3
SL-1
Report on the progress of development of biotechnological approaches for breeding
pumpkins (Cucurbita pepo L.)
1
1
2
1
1
1
Kristina Košmrlj , Jana Murovec , Aleš Kladnik , Tomaž Krumpestar , Ester Stajič , Borut Bohanec *
Agronomy Dept., Biotechnical Faculty, University of Ljubljana, 1000 Ljubljana, Jamnikarjeva 101, Slovenia
Dept. of Biology, Biotechnical Faculty, University of Ljubljana, 1000 Ljubljana, Jamnikarjeva 101, Slovenia
* corresponding author (borut.bohanec@bf.uni-lj.si)
1
2
Our attempts were to improve biotechnological procedures to accelerate breeding of cultivated
pumpkins, in particular of two types - the styrian oil pumpkins and the zucchini types. For styrian oil
pumpkins we screened available germplasm for the presence of partial absence of seed coat using
scanning electron microscopy. Using this method absence of different seed coat layers was clearly noted
while the remaining layers were distinctly pronounced A new completely hull-less seed type was
described for the first time. Later our attempts were focused on the development of a parthenogenetic
haploid induction protocol based on pollination with soft X-ray irradiated pollen. Several factors affecting
haploid production were tested such as genotype of mother plants and pollen donor, irradiation dose and
season. Data such as fruit set, embryo formation and its ploidy were scored. A limited number of haploids
were obtained along with diploid, triploid and tetraploid plants. Using SSR marker analysis the
heterozygous status of diploid regenerants was clearly confirmed, but no spontaneously doubled haploid
plants were found. Several other approaches were studied to increase frequency of haploid induction. In
vitro pollen germination protocols were optimized and used for measurement of irradiation dose on
pollen grains. Exposure of pollen to irradiation at high air humidity was found superior to normal
treatment in room air. Additionally, the application of plant growth regulators (4-CPPU or 2,4-D) in
weekly intervals after pollination was tested. Other procedures that are in progress based on hard X-ray
treatments and in vitro pollination will be reported. In additional approaches we studied adventitious
somatic regeneration as a tool to induce somaclonal variation. Following optimization of media used for
regeneration from mature cotyledons fusaric acid (FA) was added to provide selection advantage to
putative somaclones in order to select for Fusarium resistance. Surprisingly, FA at low concentrations (5
mg/L) stimulated regeneration but at higher concentrations (10 and 20 mg/L) induced genome doubling.
We also documented that the status of initial status of endopolyploidy of explant tissues was related to
the regeneration ability.
38
3
SL-2
Detection and quantification of GMOs with multiplex droplet digital PCR
Dejan Štebih*, David Dobnik, Tina Demšar, Jana Žel
Department of biotechnology and systems biology, National institute of biology, Ljubljana, Slovenia
* corresponding author (dejan.stebih@nib.si)
Use of genetically modified organisms (GMOs) in the world is increasing and there are also more and
more GMO lines available. Monitoring and control of food, feed and seed for GMOs is therefore becoming
more and more difficult, because samples have to be checked for more GMO lines.
Quantitative real-time Polymerase Chain Reaction (qPCR) is currently the most common technology for
detection and quantification of GMOs. However it is sensitive to the inhibitors and higher multiplexing
together with quantification is very difficult.
Droplet digital PCR (ddPCR) has been shown to be very efficient for detection and quantification of single
GMO line in a singleplex and duplex reaction, and, with few samples analysed in parallel, also cost
comparable to currently used technology of qPCR. Costs are also reduced due to the fact that no standard
curve from reference material is needed. ddPCR was also shown to be very robust and more resistant
against the PCR inhibition effect.
To reduce the costs and increase the throughput, multiplex ddPCR was developed. Fourplex ddPCR
detected and quantified three GMO lines and endogene in single reaction thus reducing cost and time
needed.
39
3
P-1
Interspecific hybridization in genetic breeding of ˝Styrian green gold˝ - oil pumpkins
Andreja Urbanek Krajnc*, Andreja Sober, Ignac Janžekovic, Anton Ivančič
Faculty of Agriculture and Life Sciences, University of Maribor, Slovenia
* corresponding author (andreja.urbanek@um.si)
The currently grown thin coated pumpkin varieties belong to Cucurbita pepo and originated from the
same variety called Styrian oil pumpkin (Cucurbita pepo subsp. pepo var. styriaca) which appeared as an
accidental natural mutation in the late 19th century. The mutated plants were characterized by thin
coated seeds which facilitated the production of pumpkin seed oil because the time-consuming removal
of the seed coat was no longer needed. The thin coated seed is characterized by one or two amorphous
layers that lack lignification of the cell walls (collapsed epidermis and aerenchyma), whereas the
chlorenchyma has more cell layers than some thick coated types, which leads to its dark green color. The
major problems in cultivation of oil pumpkins are viral diseases, extremely high temperatures and
prolonged drought which may cause severe economic losses. The Slovenian breeding program is based on
a modified recurrent selection and includes materials derived from complex interspecific crosses
combining three cultivated pumpkin species (C. argyrosperma, C. moschata and C. pepo) and one wild (C.
okeechobeensis). The range of adaptation for these cucurbit species includes tropical and subtropical
regions as well as temperate areas. We considered three crucial selection criteria: hull-less seed (non
lignified seed coat), bushy growth (plants with permanently short stems) and light (white, light ocre or
light yellow) fruit exterior, in order to make oil pumkins more tolerant to higher levels of solar radiation
and higher temperatures. The interspecific crosses of pumpkins are generally not simple and the success
is usually low due to strict reproductive isolation between most of the parental species. Normal fertility of
the resulting progenies was restored by intercrossings, self-fertilization, and backcrosses which involved
thin coated genotypes of C. pepo as recurrent parents. The aim of the investigation was to evaluate
chemical composition of seeds of the existing breeding materials originating from different interspecific
crosses. Interspecific hybridization was found to be very useful for expanding the variation range of
almost all analyzed chemical substances, including tocopherols, pigments, ascorbate and thiols. The
expanded variation ranges of most of the traits will enable breeders to conduct more rigorous selection
aimed at the creation of highly productive and high quality cultivars, with the desired composition of
antioxidants.
40
3
P-2
Asymbiotic germination of yellow slipper orchids (Cypripedium calceolus L.) from
diversely ripe seeds
1
2
Marko Frank , Zlata Luthar *
Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia
Nusdorferjeva 11, 1000 Ljubljana, Slovenia
* corresponding author (zlata.luthar@bf.uni-lj.si)
1
2
A yellow lady’s orchid (Cypripedium calceolus L.) is the only European species of the genus Cypripedium.
Despite the fact that the sites stretch across large part of Europe, it is today in certain areas, because of
the land use methods, almost impossible to find (Cribb, 1977). Reproduction in nature is slow and poorly
studied. In horticultural production asymbiotic propagation has been shown to be convenient and
inexpensive method to meet the demand for these rare perennials. The purpose of the research was to
investigate the germination and further development of immature seeds, which were collected 35-65
days after hand pollination, and of ripe seeds, where cold centrifuge has been used to rupture the testa
between seeds disinfection. The seeds were inoculated on a commercial medium produced by Sigma
P6668 in full 27.3 g/L (S) and half 13.65 g/L (S ½) concentration and on simple culture medium based on
amino acid (M) (Hicks, 2000). In the case of disinfection with 2% dichloroisocyanuric acid, we eliminated
only 27.4% of petri dishes with seeds. The immature seeds produced the best germination when seed
capsules were removed in the period of 40 to 54 days after the pollination, while the ripe seeds
germinated poorly or did not germinated at all. The results of germination show that there are no
statistically significant differences between different mediums (p = 0.86). Significant differences were
apparent in further development of protocorms, where the best development of immature seeds was on
medium S and S ½. In case of ripe seeds, further development appeared only on medium S ½ and M. The
results showed that the use of cold centrifuge can soften the testa and enable access of water into the
embryo. However seeds also contain several other mechanisms that prevent germination.
Cribb P. (1997). The genus Cypripedium. Oregon, Timber Press: 301 pp.
Hicks A.J. (2000). Asymbiotic tehnique of Orchids seed germination. Chandler, The Seedbank Project: 134 pp.
41
3
P-3
Effects of pH value and activated charcoal on microspore culture in rutabaga
(Brassica napus var. napobrassica )
Katarina Rudolf Pilih*, Zlata Luthar, Borut Bohanec
Biotechnical faculty, Jamnikarjeva 101, 1000 Ljubljana, Slovenia
* corresponding author (katarina.rudolfpilih@bf.uni-lj.si)
Rutabaga (Brassica napus var. napobrassica) is a root vegetable that originated as a cross between the
cabbage and the turnip. It is an allopolyploid. The roots are prepared for human food in a variety of ways,
and also leaves can be eaten as a leaf vegetable. Although rutabaga (Brassica napus L. var. napobrassica) is
a popular crop, especially in North Europe and North America, its sprouts are a new kind of vegetable.
The biological activities of rutabaga are antiproliferative and proapoptotic to tumor cells. These findings
strongly advocate the application of rutabaga sprouts as functional food (Pasko et al., 2013). We decided
to investigate responsiveness of rutabaga (cv. Hoffmanova rumena) to microspore embryogenesis. This is
the first step in the development of new variety with better morphological and biochemical
characteristics. We used liquid growth medium (NLN) without growth regulators with pH values 6.0 or
6.4 and with activated charcoal in two concentrations (0.02% and 0.1%). From six tested plants four of
them were responsive to haploid induction (~0.5 – 8 embryos/flower bud). The number of induced
embryos was higher on the medium with pH 6.4 and supplemented with charcoal. The embryos were
treated with ABA and then dried for one month. After this period the embryos will be placed on
germination medium B5. If embryos were not dessicated, the germinated plants were vitrified. According
to preliminary results a new experiment with several varieties of rutabaga will be performed in order to
get high number of doubled haploids for further analysis.
Pasko P, Bukowska-Strakova K, Gdula-Argasinska J, Tyszka-Czochara M.(2013). J Med Food 16(8), 749-759
42
3
P-4
Androgenesis in tobacco (Nicotiana tabacum L.) – effect of cold stress on anther and
microspore culture
1
1
1
1
Edith Stabentheiner *, Regina Willfurth , Christof Winkler-Hermaden , Andreas Oberländer , Andreas
Zimmermann2, Tobias Eisenberg2
Institute of Plant Sciences, University of Graz, Schubertstrasse 51, 8010 Graz, Austria;
Institute of Molecular Biosciences, University of Graz, Humboldstraße 50, 8010 Graz, Austria
* corresponding author (edith.stabentheiner@uni-graz.at)
1
2
Male microspores can be reprogrammed to shift from the gametophytic to the embryogenic pathway.
This developmental reprogramming, called “embryogenesis” or “androgenesis”, needs stress as an
indispensable trigger. Cold stress is widely used for this stress pre-treatment (Shariatpanahi et al. 2006).
Responsive microspores dedifferentiate; the cells start dividing, form multicellular structures, and an
embryo or undifferentiated callus develops. Gametic embryogenesis is a valuable tool in plant breeding,
since it is a rapid method for developing homozygous lines (Germana 2011), as well as in basic research
studying developmental reprogramming.
The influence of cold stress on reprogramming of microspores was examined in tobacco (Nicotiana
tabacum L.). Young flowers with microspores in the late uninucleate to early binucleate stage were
exposed to low temperatures (4°C, 8°C) and embryo and callus formation was followed in anther and
microspore culture. Fluorescence dyes (FDA, SNARF1, DRAQ5, FM4-64) were used to characterize the
microspores at different developmental stages. Fluorescence assisted cell sorting (FACS) was tested to
separate gametophytic and embryogenic microspores.
A cold treatment of 5-10 days positively influenced microspore reprogramming. A pre-treatment of the
buds at 8°C resulted in embryogenesis and the formation of a high amount of plants. In contrast, pollen
maturation, reduced embryo formation and an increased formation of undifferentiated calli was observed
when buds were pre-treated at 4°C. Callus formation could not be observed in anther cultures.
Microspore and anther culture are reliable systems and valuably complement one another in studying the
reprogramming of microspores towards embryogenesis.
The support from NAWI Graz is gratefully acknowledged.
Shariatpanahi M.E., Bal U., Heberle-Bors E., Touraev A. (2006) Physiologia Plantarum 127, 519-534.
Germana M.A. (2011) Plant Cell Report 30, 839-857.
43
3
P-5
A large-scale production and microwave-assisted extraction of shikimic acid from the
aerial parts of Sicyos angulatus (Cucurbitaceae) in Korea
1,2
1
3
3
1
Chae Sun Na , A Ram Kim , Yong Ho Lee , Sun Hee Hong , Tae Wan Kim *
Institute of Ecological Phytochemistry, Department of Plant Life and Environmental Science, Hankyong National
University, Anseong 456-749, Gyeonggi-do, Republic of Korea
2
Institute of Botany, University of Innsbruck, Sternwartestraße 15, A-6020 Innsbruck, Austria
3
Seed Bank of Wild Resource Plants, school of Life Sciences and Biotechnology, Korea University, Seoul 136-701,
Republic of Korea
* corresponding author (taewkim@hknu.ac.kr)
1
Shikimic acid is the starting material for the commercial synthesis of oseltamivir (Tamiflu ®), which is the
anti-flu drug. This work was conducted to determine the optimal conditions for large-scale production of
shikimic acid from Sicyos angulatus (Cucurbitaceae). Whole plants of S. angulatus were treated with
glyphosate (N-phosphonomethyl glycine) at a lethal dosage of 0.39, 0.77 and 1.54 grams active ingredient
(g a.i.) a-1 and stems, leaves and fruits were collected every day for 6 days after treatment. Extraction of
shikimic acid in distilled water was conducted by microwave-assisted extraction (MWAE), which is a
rapid and effective method for recovering shikimic acid. Shikimic acid content of all plant tissues
significantly increased at least four-fold after glyphosate treatment and in stems even up to 90-fold. The
highest shikimic acid content of stems and fruits were obtained at 1.54 g a.i. a-1 of glyphosate dosage and
6 days after treatment (DAT), whereas shikimic acid of leaves was not changed significantly by the
glyphosate dosage and harvest time. In conclusion, the optimal conditions for large-scale production of
shikimic acid, as the starting material for the commercial synthesis of oseltamivir (Tamiflu ®), were 1.54 g
a.i. a-1 of glyphosate dosage and 6 DAT of harvest time.
Acknowledgements
This work was supported by a grant (code-402-122-003) from the Ministry of Environment, Republic of Korea.
44
3
P-6
4 Struktura in funkcija rastlin
45
Non-foliar photosynthesis - Existence in and necessity for plants
4
IL-1
Hardy Pfanz*
Institute of Applied Botany, University of Duisburg-Essen, Campus Essen, Germany
*corresponding author (hardy.pfanz@uni-due.de)
Not only leaves are green. Nearly all plant organs can contain chlorophyll when exposed to light.
Chlorophyll in a viable chloroplast is a prerequisite for a functional photosynthesis. That means that
green fruits like cucumber, bell pepper, apple and pear are performing photosynthesis at least for a
certain period. The green parts of a flower (pistil, stamina, sometimes petals) can do the same. In some
Equisetum species, even spores are “functional green”. Twigs and branches and sometimes even old stems
(e.g. 150 years in Fagus sylvatica) perform photosynthesis when illuminated. Some semi-desert trees like
Parkinsonia aculeata or Cercidium floridum (Palo verde) lose all their leaves during drought periods and
photo-reduce up to 75% of their annual carbon gain with their pure limbs. And roots are no exception.
Tree roots emerging from a forest soil may photosynthesize to some extent, whereas roots of some
epiphytic orchid species (ghost orchid, Epipogium aphyllum, Dendrophylax spec.) have to wholly
compensate the missing stems and leaves.
But, why and when is non-foliar photosynthesis important for plant survival? Non-foliar photosynthetic
carbon reduction may compensate for carbohydrate production (pathogenic defoliation, insect pests),
may be used in times of water shortage (deserts) or low temperatures (winter) or may just be used as an
oxygen provider to avoid hypoxia (wood photosynthesis). It may also allow pollen, spores or seeds to
survive the period until germination or leaf development. But also in wound-healing processes corticular
photosynthesis may play a decisive role.
Aschan G, Wittmann C, Pfanz H (2001). Trees 15: 431-437
Pfanz H (1999) Phyton 39: 29-33
Pfanz H, Aschan G (2001) In Esser K, Lüttge U, Kadereit JW, Beyschlag W (Hrsg.). Progress in Botany 62: 477-510
Pfanz H, Aschan G, Langenfeld-Heyser R, Wittmann C, Loose M (2002) Naturwissenschaften 89, 147-167
46
Assymetry of molecular processes during abscission in tomato (Solanum
lycopersicum L.)
1
2
3
1
Marko Chersicola *, Aleš Kladnik , Amnon Lers , Marina Dermastia
Department of Biotechnology and Systems Biology, National Institute of Biology, Slovenia
Department of Biology, Biotechnical Faculty, University of Ljubljana, Slovenia
3 Department of Postharvest Science of Fresh Produce, Agricultural Research Organization, Volcani Center, Israel
* corresponding author (Marko.Chersicola@nib.si)
1
2
Abscission is a highly regulated process in which various organs, including leaves, flowers and fruits, are
separated from the mother plant as a natural stage of plant development. Abscission occurs specifically in
the abscission zone (AZ) tissue. We have shown at the gene expression and cellular level that various
abscission related processes occur asymmetrically between the AZ proximal and distal sides [1, 2].
However, the asymmetry confirmed by the immunolocalization studies has been demonstrated only for
LX ribonuclease (LX). Moreover, the transgenic plants with inhibited LX show delayed leaf abscission, but
it is currently not known if LX localizes in a similar manner than in the wild type plants. In this study we
show the asymmetric immunolocalization of several enzymes involved in the abscission of tomato in the
wild type and in LX inhibited lines as well. Specifically we followed the localization of LX,
polygalacturonases that break down the middle lamella between the abscission zone cells, and ACC
oxidase that catalyses the final step in the biosynthesis of ethylene. In addition, the expression of various
genes involved in the abscission process was evaluated by qPCR both in the wildtype and LX inhibited
lines. The findings of this study further elucidate the asymmetry of molecular processes in abscission
with a potential use in biotechnology for the controlled abscission of fruits and manipulation of
abscission time.
[1] Bar-Dror et al. (2011), Plant cell 23, 4146.
[2] Dermastia et al. (2012), Plant signaling & behavior 7, 1106.
47
4
SL-1
Arsenate and phosphate relationships during the uptake in lichens
Tanja Mrak1*, Zdenka Šlejkovec2, Zvonka Jeran2, Franc Batič3
1 Slovenian
Forestry Institute, Večna pot 2, 1000 Ljubljana,
Jožef Stefan, Jamova cesta 39, 1000 Ljubljana,
3 University of Ljubljana, Biotechnical faculty, Jamnikarjeva 101, 1000 Ljubljana
* corresponding author (tanja.mrak@gozdis.si)
2 Institute
Arsenate, As(V), the most common form of arsenic in oxic environments, is characterized by almost
identical chemical properties as phosphate (O’Day 2006) and is taken up into the cells via the phosphate
transporters (Rosen 2002, Meharg & Hartley-Whitaker 2002, Maciaszczyk-Dziubinska et al. 2012).
Typical As(V) uptake curve in lichens consists of two phases: in the first phase As(V) is quickly taken up,
while in the second phase it is excreted out of the thalli in the form of arsenite, As(III) (Mrak et al. 2008).
It was shown that lichens are able to take up phosphate from very diluted solutions (Ahmadjian 1993).
The presence of the second anion in water solution can inhibit or stimulate the uptake of the first anion,
but sometimes more complex changes may occur (Nieboer et al. 1984). To test the effect of phosphate on
As(V) uptake, two species of lichens were tested, epiphytic Hypogymnia physodes (L.) Nyl. and terricolous
Cladonia furcata (Huds.) Schrad. In both species we observed the inhibitory effect of phosphate on As(V)
uptake. The influence was stronger for C. furcata. The influence was less pronounced when lower molar
ratios of As(V) to phosphate were used and also when lower As(V) concentrations were used. Extremely
high concentrations of phosphate had a very strong inhibitory effect on the uptake of As(V) which also
influenced the shape of the uptake curve – the uptake curve lost its excretory part.
Ahmadjian V. (1993). The lichen symbiosis. John Wiley & Sons, New York: 250 pp.
Maciaszczyk-Dziubinska E., Wawrzycka D., Wysocki R. (2012). International Journal of Molecular Sciences 13, 3527-3548
Meharg A. A., Harley-Whitaker J. (2002). New Phytologist 154: 29-43.
Mrak T., Šlejkovec Z., Jeran Z., Jaćimović R., Kastelec D. (2008). Environmental Pollution 151, 300-307.
Nieboer E., Padovan D., Lavoie P. (1984). New Phytologist 96: 83-93.
O’Day P. A. (2006). Elements 2, 77-83.
Rosen B. P. (2002). FEBS Letters 529, 86-92.
48
4
SL-2
Towards fine root identification key of common tree species
Tanja Mrak*, Peter Železnik, Jožica Gričar, Hojka Kraigher
Slovenian Forestry Institute, Večna pot 2, 1000 Ljubljana
* corresponding author (tanja.mrak@gozdis.si)
Fine roots (defined as roots thinner than 2 mm) comprise less than 2 % of the tree biomass in temperate
and boreal forest stands, but their role in the formation of belowground carbon pools is of great
importance because of their short turnover rates (Brunner & Godbold 2007). Quantitative analyses of
species’ root distribution may reveal belowground carbon allocation patterns and competition
relationships (Rewald et al. 2012). Since anatomy of the roots can differ significantly from the anatomy of
stem, specialized root identification keys are needed (Rewald et al. 2012). Besides anatomical
characteristics, morphology of the fine root system e.g. diameter of lateral branches, branching pattern,
colour, texture of the root bark or epidermis (Pregitzer et al. 2002) may also be valuable for
determination purposes. The most valuable root identification key of Cutler et al. (1987) is sold out and
hard to obtain, while the comprehensive atlas of Kutschera & Lichtenegger (2002) is missing
identification key and information on typical characteristics of the presented species are difficult to find.
There is practically no identification key or atlas that would present morphological characteristics of fine
roots. In the frame of EUFORINNO project (REGPOT no. 315982), we are investigating both anatomical
and morphological approaches on ten common tree species to specify the most useful identification
characteristics. These characteristics will be used to prepare an identification key for the studied species
and if possible, extended to other woody plants.
Brunner I. and Godbold D. (2007). Journal of Forest Research 12 (2), 78-82.
Cutler D. F., Rudall P. J., Gasson P. E., Gale R. M. O. (1987). Root identification manual of trees and shrubs. A guide to the anatomy
of roots of trees and shrubs hardy in Britain and northern Europe. First Edition. Chapman and Hall, London: 245 pp.
Kutschera L. and Lichtenegger E. (2002). Wurzelatlas mitteleuropäischer Waldbäume und Sträucher. Ersteauflage, Stocker, Graz:
604 pp.
Pregitzer K. S., DeForest J. L., Burton A. J., Allen M. F., Ruess R. W., Hendrick R. L. (2002). Ecological Monographs 72 (2), 293–309
Rewald B., Meinen C., Trockenbrodt M., Ephrath J. E., Rachmilevitch S. (2012). Plant Soil 359, 165-182
49
4
P-1
Can the structure of dormant cambium and the widths of phloem and xylem
increments be used as indicators for tree vitality?
1
1
2
2
3
Jožica Gričar , Špela Jagodic , Bogoslav Šefc , Jelena Trajković , Klemen Eler *
1Slovenian
Forestry Institute, Department of Yield and Silviculture, Ljubljana, Slovenia
of Forestry, University of Zagreb, Zagreb, Croatia
3Biotechnical Faculty, Department of Agronomy, University of Ljubljana, Ljubljana, Slovenia
*corresponding author (Klemen.eler@bf.uni-lj.si)
2Faculty
In Slovenia and Croatia, oaks (Quercus robur L. and Q. sessiliflora Salisb.) are economically and
ecologically very important tree species, representing 7 and 22 %, respectively, of the entire wood stock.
As in many European countries, a trend of decreasing vitality of Q. robur in the form of defoliation and
reduced height and girth increment has been observed at most sites in recent decades.
We investigated the structure and width of the dormant cambium and of the increments of phloem and
xylem of Quercus robur to estimate their potential as indicators for tree vitality. The samples were taken
from three woodlands, two in Slovenia (KRA and MUS) and one in Croatia (KOB), with reported tree
decline. In December 2009, microcores containing inner phloem, cambium and outer xylem were
collected from living trees at 1.3 m above ground. The tissues were fixed, embedded in paraffin, cut on a
rotary microtome, and stained with safranine/ astra blue water mixture. The cross-sections were
examined with light microscope and image analysis system.
The number of dormant cells seems to reflect the initial capacity of the cambium to accomplish cell
division. With the exception of two trees at KRA, cell production was always higher on the xylem side
than on the phloem side. The annual phloem increments were narrower, less variable among trees and
with clear lower and upper limits. With increased cambial cell productivity, the share of the xylem in the
total annual radial increment increased following a curvilinear function. In trees with an annual radial
increment > 3.5 mm, the xylem size represented more than 90 % of the total radial growth. The
anatomical variables analyzed show that the most limiting environmental conditions seem to prevail at
KRA, whereas the conditions at MUS seem to be most favorable in terms of radial growth. The width and
structure of xylem and phloem increments, the number of dormant cambial cells and their interrelationships can provide additional information on the vitality of oaks.
50
4
P-2
Ionome of wheat (Triticum aestivum), common and tartary buckwheat (Fagopyrum
esculentum, Fagopyrum tataricum) grains from a conventional production field
1
1
1
1
2
2
Lea Orožen , Matevž Likar , Katarina Vogel-Mikuš , Paula Pongrac , Marijan Nečemer , Peter Kump ,
Marjana Regvar1*
1 University
of Ljubljana, Biotechnical Faculty, Jamnikarjeva 101, SI-1000 Ljubljana
Jožef Stefan, Jamova 39, SI-1000 Ljubljana
* corresponding author (marjana.regvar@bf.uni-lj.si)
2 Institut
Main factors affecting mineral composition (ionome) of grains are soil mineral availability,
bioaccumulation capacity of plant species and plant-microbe interactions. We investigated the feasibility
of grain biofortification with two commercial organic fertilizer treatments (Symbivit; S) and Bioaktiv; B)
in wheat (Triticum aestivum L.), common buckwheat (Fagopyrum esculentum Moench), and tartary
buckwheat (F. tataricum Gaertn.), grown at the same field under conventional grain production. Grain
element concentrations were measured using energy dispersive X-ray fluorescence spectrometry, while
soil samples were analysed using total reflection X-ray fluorescence spectrometry (Vogel-Mikuš et al.
2006). Root fungal colonization parameters were estimated according to Trouvelot et al. (1986).
Bray-Curtis (1957) dissimilarity indexes of grain mineral composition were tested by permutational
analysis of variance (perMANOVA; Anderson 2001). Results confirm plant species (p0.001) and
interaction of fungal colonisation and plant species (p0.001) affected grain mineral compositon. Root
fungal colonisation frequencies of wheat (34%), common (43%) and tartary buckwheat (27%) were low.
Significant differences between the treatments were observed in arbuscular density, density of
microsclerotia and density of coils of colonised root fragments in wheat. A non-metric multi-dimensional
scaling (Oksanen et al. 2010) analysis confirmed dissimilarity of the grain mineral composition of the
three species studied. In addition, the mineral composition of wheat shows larger scattering for B
treatment, suggesting differences in grain mineral composition, with concentration of K and Zn being the
most affected.
Anderson M.J. (2001). Austral Ecology 26, 32-46.
Bray J.R., Curtis J.T. (1957). Ecological Monographs 27, 325-349.
Trouvelot A, Kough J., Gianinazzi-Pearson V. (1986). In: Gianinazzi-Pearson V, Gianinazzi S (eds) Physiological and genetical
aspects of mycorrhizae. Institut National de la Recherche Agronomique, Paris: 217–221 pp.
Oksanen J., Blanchet F.G., Kindt R., Legendre P., Minchin P.R., O'Hara R.B., Simpson G.L., Solymos P., Stevens M.H.H., Wagner H.
(2012). Vegan: Community Ecology Package. R package version 2.0-5. http://CRAN.R-project.org/package=vegan
Vogel-Mikuš K, Pongrac P, Kump P, Nečemer M, Regvar M, (2006). Environmental Pollution 139, 362-371.
51
4
P-3
Leaf spectral signatures differ among plant species growing along water/land
gradients
Katja Klančnik, Alenka Gaberščik*
University of Ljubljana, Biotechnical Faculty, Department of Biology, Večna pot 111, SI-1000 Ljubljana, Slovenia
* corresponding author (alenka.gaberscik@bf.uni-lj.si)
Intermittent water regime in Lake Cerknica creates a variety of habitats that support different ecological
groups of plants with different optical properties. This study aimed to point out the differences in
reflectance spectra of leaves in different plant groups thriving in habitats along water/land gradient as
well as biochemical and morphological traits of leaves that explain the most of the variability of
reflectance spectra. We examined biochemical and structural properties of leaves of different plant
groups and reflectance spectra between 280 and 887 nm. Our results showed that in submerged leaves of
Potamogeton lucens, P. perfoliatus, P. crispus, Sagittaria sagittifolia, Nuphar lutea and Ranunculus lingua
the most of spectra variance was explained by mesophyll thickness (25%) and leaf biochemical
properties (21%). In addition reflectance spectra of submerged leaves might be additionally affected by
epiphyton. The analysis of natant leaves of species Nuphar lutea, Polygonum amphibium and Sagittaria
sagittifolia revealed the importance of leaf morphological traits namely upper cuticle thickness (43%),
epidermis thickness (12%) and spongious tissue thickness (16%) for leaf reflectance. In emergent dicots
Sagittaria sagittifolia, Ranunculus lingua, Polygonum amphibium, Senecio paludosus and Gratiola officinalis
chlorophylls and UV absorbing substances explained 14% and 9% of reflectance spectra, respectively. In
leaves of grasses (Molinia caerulea, Phalaris arundinacea, Phragmites australis, Deschampsia cespitosa)
and sedge (Carex elata) the most of the spectra variability was explained by Si and Ca encrustations,
mainly prickle hairs (16%) and upper cuticle thickness (19%). Our results revealed that detailed
information is needed for proper interpretation of data collected on aquatic and wetland plants by
remote-sensing and other techniques that base on reflectance spectra.
52
4
P-4
2 B Interakcije rastlin z okoljem
in drugimi organizmi
53
Distribution and diversity of arbuscular mycorrhizal fungi in grapevines from
production vineyards along the eastern Adriatic coast
1
2
2
Matevž Likar *, Katarina Hančević , Tomislav Radić , Marjana Regvar
1
1 Department
of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia
for Adriatic Crops and Karst Reclamation, Put Duilova 11, 21000 Split, Croatia
* corresponding author (matevz.likar@bf.uni-lj.si)
2 Institute
Background
Arbuscular mycorrhizal fungi (AMF) are among the most important beneficial fungal root endophytes,
and are known to colonise the roots of the majority of land plants, including grapevines. Similar to other
plant species with low root densities or with relatively coarse fine roots, grapevines appear to be reliant
on AMF colonisation for normal growth and development, which is especially important in organic
vineyards that follow low-input practices, as these support higher AMF spore abundance and diversity.
Key results and discussion
The comparison of the AMF communities on the roots of grapevines from East Adriatic Karst region
showed that the fungal communities associated with grapevine roots seem to be relatively stable, with
inter-vineyard variability comparable to intra-vineyard variability. Some of the changes in the fungal
communities were attributed to environmental factors (eg. plant-available P) and geographical location of
the vineyard. The majority of the AMF belonged to various representatives of genus Glomus, with some
species that are less frequently observed in studies on AMF (eg. Glomus sinuosum and Glomus indicum).
Screening of vineyard weeds belonging to 11 plant families and showed varying level of fungal
colonization and seemed to promote different sets of dominant AMF, potentially providing a wider
spectrum of AMF for colonizing grapevine roots. This indicates the value of encouraging host plant
diversity in vineyards. In deed herbaceous weed Tanacetum cinerariifolium was shown to improve
colonization of grapevine plants in a pot culture experiment.
In the next stage of our studies, we were starting to focus on the other aspects of “terroir” (vineyard
management) and its consequences on the fungal endophyte communities. AMF spore abundance greatly
increased in organic vineyards, as has been seen for different agricultural systems, including vineyards. In
contrast, vineyard management had no effect on the overall diversity of AMF colonizing grapevine roots.
Our results confirm that the production vineyard harbor well formed AMF communities that are greatly
affected by the vineyard management. Change towards more sustainable vineyard practices improves the
abundance and diversity of AMF that can result in positive effects on the mineral nutrition of grapevines
due to modified nutrient availability related to changes in the abiotic and biotic characteristics of the soil.
54
2B
IL-1
Testing of Verticillium albo-atrum virulence on different plant hosts and
development of a transformation system for Verticillium albo-atrum gene knock-out
Marko Flajšman, Branka Javornik
*
University of Ljubljana, Biotechnical Faculty, Agronomy Department, Jamnikarjeva 101, Ljubljana SI-1000, Slovenia
* corresponding author (branka.javornik@bf.uni-lj.si)
Verticillium albo-atrum is a soil-borne plant pathogen that is responsible for Verticillium wilt diseases and
is a very important phytopathogen in Slovenia, infecting hop (Humulus lupulus). Hop is the primary host
of Verticillium albo-atrum and two different V. albo-atrum hop isolates specific for Slovenia are known:
PG1, which induces a mild form of hop wilt, and PG2, which kills the plants.
We successfully established a protocol for generating knock-outs of V. albo-atrum, which comprises two
methods. The first is the creation of knock-out plasmids by the USER Friendly cloning technique
(Frandsen et al., 2008.). The second method is transformation of the fungal pathogen by Agrobacterium
tumefaciens-mediated transformation (ATMT) (Knight et al., 2009). We generated knock-outs of some
candidate genes and wanted to test knock-out virulence. Although hop is the primary host of V. alboatrum, it is not very appropriate as a model plant for testing pathogen virulence because it is perennial
and needs to undergo a dormancy phase. We therefore tested some other host plants. We used PG1 and
PG2 of V. albo-atrum to infect various plants: tobacco (Nicotiana benthamiana), tomato (Solanum
lycopersicum), eggplant (Solanum melongena), camelina (Camelina sativa) and Arabidopsis thaliana.
To date, tobacco, tomato, eggplant and camelina have not proved to be suitable host plants because the
mild pathotype PG1 caused the same symptoms as the lethal pathotype PG2. Arabidopsis thaliana is still
being tested.
Frandsen R. J. N., Andersson J. A., Kristensen M. B., Giese H. (2008). BMC Molecular Biology 9, 70-81.
Knight C.J., Bailey A.M., Foster G.D. (2009). Journal of Plant Pathology 91, 745–750.
55
2B
SL-1
Transcriptional analysis of differential gene expression in hop following infection
with Verticillium albo-atrum
Vasja Progar *, Nejc Jakše, Nataša Štajner, Sebastjan Radišek, Anže Švara, Branka Javornik
Biotehniška fakulteta, Univerza v Ljubljani, Jamnikarjeva 101, 1000 Ljubljana
* corresponding author (vasja.progar@bf.uni-lj.si)
Hop is a perennial dioecious climbing plant that is primarily grown to be used as an essential ingredient
of beer and, recently, also for its pharmaceutical value. The production of hop is challenged by Verticillium
albo-atrum, a soil-borne phytopathogenic fungus that causes one of the most devastating hop diseases Verticillium wilt. The disease is not specific to hops and affects dicotyledonous plants in general; several
host-Verticillium interactions have been studied by different research groups, on hosts ranging from hop
and tomato to olive, cotton and a variety of others (Cregeen et al. 2014).
The severity of the disease in hops varies, which is attributable to different degrees of fungus
pathogenicity, as well as to the resistance to the disease exhibited by certain hop cultivars. The
mechanisms of hop resistance to Verticillium wilt are not yet fully understood; however, a QTL for it has
recently been identified (Jakše et al. 2013) and segregation pattern suggested a model of two dominant
complementary genes being responsible for the resistance.
In order to elucidate the underlying mechanism of pathogenesis and host resistance to Verticillium wilt
further, we conducted an extensive colonisation experiment by inoculating susceptible hop cultivar, Celia,
and resistant cultivar, Wye Target, with a highly aggressive strain of V. albo-atrum. RNA samples were
taken at multiple time-points from inoculated and control plants and were sequenced on an Illumina
next-generation sequencing platform. The acquired data enabled us to perform transcriptional analysis of
gene expression patterns in relation to infection of hop with V. albo-atrum. The results of this analysis will
be presented and discussed.
Jakše J., Čerenak A., Radišek S., Šatović Z. , Luthar Z., Javornik B. (2013). Theor Appl Genet 126 (6): 1431-43.
Cregeen S., Radišek S., Mandelc S., Turk, B., Štajner N., Jakše J., Javornik B. (2014). Plant Mol Biol Rep (in press)
56
2B
SL-2
Viroid-derived small RNA (vd-sRNA) target identification in hop
Tine Pokorn1*, Sebastjan Radišek2, Branka Javornik1, Nataša Štajner1, Jernej Jakše1
1 University
of Ljubljana, Biotechnical Faculty, Department of Agronomy, Jamnikarjeva 101, 1000 Ljubljana
Institute for Hop Growing and the Brewing, Cesta Žalskega tabora 2, 3310 Žalec
* corresponding author (tine.pokorn@bf.uni-lj.si)
2 Slovenian
Hop (Humulus lupulus L.) is a dioecious climbing plant, the female plants of which are of economic importance.
The major hop growing area in Slovenia is the Savinja valley. As with other crops, commercial hop production
is threatened by the occurrence of various plant pathogens, such as fungi, bacteria, pythoplasmas, viruses and
viroids.
Much of the research focused on plant-pathogen interactions studies the mechanisms of pathogenesis of
bacteria, viruses or fungi, while interactions between viroids, tiny RNA plant pathogens, and plants are still
poorly understood. Viroids are naked, circular RNA molecules with strong secondary structures, which are not
transcribed into proteins. Hop plants are hosts to four different viroids – hop stunt viroid (HSVd), hop latent
viroid (HLVd), citrus bark cracking viroid (CBCVd) and apple fruit crinkle viroid (AFCVd). Recent studies have
revealed that viroid-plant interactions involve viroid derived (vd-sRNA) small RNA molecules, which are
abundantly present in infected plants. In order to investigate the role of vd-sRNA in interaction with hop
plants, a de-novo reconstruction of the hop transcriptome was done and a bioinformatics search for possible
targets for vd-sRNAs was undertaken. Expression analysis of selected targets involved in known pathways of
defence response was further undertaken in viroid free and viroid infected hop plants using RT-qPCR.
Various hop tissues were collected from viroid free (healthy) and infected plans throughout the growing
season. RNA was then isolated and the quality and quantity of RNA was determined by spectrophotometry,
formaldehyde gel electrophoresis and Bioanalyzer runs. Bulked RNA from healthy plants was subjected to
Illumina sequencing using a 100 bp paired–end module. A total of 348 M sequences was received, yielding
roughly 33 Gb of cleaned data. De-novo assembly was performed using a de-novo algorithm of CLC Genomics
Server using a bubble size of 50 and word size 24. The total length of the assembled transcriptome was 74 Mb,
represented by 140,443 scaffolds with N50 length of 984 bp. RNA molecules from three different viroids
(HLVd, CBCVd and HSVd) were in silico cut to all possible 21 bp, 22 bp and 24 bp mers, which were searched
against the transcriptome using two different small RNA-target identification tools. Both tools revealed 12,221
possible targets for the action of vd-sRNA, while 980 targets were common. All predicted targets were
annotated using the BLAST2GO tool. For expression analysis, 37 targets were selected for RT-qPCR analysis,
with an emphasis on genes involved in hormonal pathways of plant response (etilen, gibberellins, jasmonates
and salicylic acid) and selected pathogenesis related (PR) genes. The analysis was carried out on viroid-free
(BVV), HLVd infected and CBCVd infected hop plants. Primers were designed with Primer Express 3.0 software
and an RT-qPCR experiment was performed on ABI 7500 FAST real-time PCR, with Roche FastStart Universal
SYBR Green Master (Rox). Data were analysed by qBASE+ software (Biogazelle). Both groups of viroid infected
plants showed strong up-regulation of PR genes, while the vd-sRNA target showed either up- or down
regulation.
57
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SL-3
What can "plant defence molecules" tell us about population structure of plant
virus?
1,2
1
1
1
3
Denis Kutnjak *, Matevž Rupar , Ion Gutierrez-Aguirre , Polona Kogovšek , Tomaž Curk , Jan
Kreuze4, Maja Ravnikar1
1 Department of Biotechnology and Systems Biology, National Institute of Biology, Ljubljana, Slovenia
2 Jožef Stefan International Postgraduate School, Jamova 39, 1000 Ljubljana, Slovenia
3 Faculty of Computer and Information Science, University of Ljubljana, Ljubljana, Slovenia
4 International Potato Center (CIP), Lima, Peru
* corresponding author (denis.kutnjak@nib.si)
RNA virus populations are one of the fastest evolving biological systems. They exist as a cloud of diverse
sequences (quasispecies), which can have several biological implications [1]. Using next generation
sequencing (NGS), variability of viral populations have been investigated in-depth for few important
human and animal viruses, but little research has been conducted on plant viruses. The focus of our
research is Potato virus Y (PVY), a single stranded (ss)-RNA virus. Sequences corresponding to plant
infecting ss-RNA viruses, constitute at least three distinct, but interconnected pools: (I) ss-RNA molecules
packed in viral particles, (II) double stranded (ds)-RNA molecules formed during replication and (III)
virus derived small interfering RNAs (vsiRNAs) [2]. The latter are a result of the well conserved plant
defence mechanism called RNA interference (RNAi), in which ds viral RNAs are cut in 21-24 long
fragments. We hypothesized that sequence diversity between these pools is similar but could slightly
differ due to the errors or genetic bottlenecks introduced during viral cycle. Illumina deep sequencing of
two different pools of viral sequences was employed: vsiRNAs [3] and ss-RNA isolated from purified viral
particles [4]. The data was analysed to search for variants present in each of the two pools. The results
show that both pools reflect highly similar mutational landscape. This indicates sequence-independent
targeting of RNAi mechanism towards invading PVY sequences in plants. Nevertheless, some differences
were observed between the two sequence pools - small RNAs showing higher level of variation. Further,
small RNAs did not allow to search for recombination events, which were commonly detected in viral
particles pool. Observed inter-host differences in viral diversity will enable better understanding of smallscale viral evolution processes.
[1] Domingo E., Esteban Domingo, Sheldon J., Peraleset C. (2012). Microbiol. Mol. Biol. Rev, 76, 159-216.
[2] Llave, C. (2010). Trends Plant Sci. 15, 701–707.
[3] Kreuze J. F., Perez A., Untiveros M., Quispe D., Fuentes S., Barker I., Simon, R. (2009). Virology 388, 1–7.
[4] Rupar M., Ravnikar M., Tušek-Žnidarič M., Kramberger P., Glais L., Gutiérrez-Aguirre I. (2013). J. Chromatogr. A 1272, 33–40.
58
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SL-4
Morphology and gene expression in lesions in compatible and incompatible potato –
potato virus Y interaction
1
1
1
1
Maruša Pompe-Novak *, Špela Baebler , Magda Tušek-Žnidarič , Neža Turnšek , Karolina
Morgiewicz2, Jacek Hennig2, Kristina Gruden1
1 National
Institute of Biology, Department of Biotechnology and Systems Biology, Ljubljana, Slovenia
of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland
* corresponding author (marusa.pompe.novak@nib.si)
2 Institute
Potato virus Y (PVY) is of extreme economic importance as it is responsible for yearly losses in
production of crops, and thus the subjects of investigation in many research groups all over the world.
The tuber necrotic strain of Potato virus Y (PVYNTN) causes potato tuber necrotic ringspot disease
(PTNRD), growth inhibition, faster senescence and leaf drop, chlorotic ringspots and/or spot necrosis on
inoculated leaves, and crinkles and mosaics on systemic infected leaves in sensitive potato (Solanum
tuberosum L.) cultivars. Viruses from PVYN-Wi group can also cause severe symptoms on potato. Symptom
development and their severity depend on the isolate of PVY, potato cultivar, environmental conditions
and other factors. Besides, virus-infected leaf tissues comprise a heterogeneous mixture of cells at
different stages of infection and spatial and temporal relationships between sites of virus accumulation
and the accompanying host responses are not well defined.
In our studies, we analysed lesion development in PVY-inoculated leaves of two genotypes: a resistant cv.
Rywal and a susceptible NahG-Rywal, compromised in salicylic acid signalling, after the infection with
two isolates of Potato virus Y, PVYNTN and PVYN-Wi. Spatio temporal analyses were preformed on the
macroscopic, microscopic and molecular (gene expression) levels.
Baebler Š., Krečič-Stres H., Rotter A., Kogovšek P., Cankar K., Kok E.J., Gruden K., Kovač M., Žel J., Pompe-Novak M., Ravnikar M.
(2009). PVYNTN Molecular plant pathology 10, 263-275.
Baebler Š., Stare K., Kovač M., Blejec A., Prezelj N., Stare T., Kogovšek P., Pompe-Novak M., Rosahl S., Ravnikar M., Gruden K. 2011.
(2011). PloS one 6, 1-12.
Baebler Š., Witek K., Petek M., Stare K., Tušek-Žnidarič M., Pompe Novak M., Renaut J., Szajko K., Strzelczyk-Żyta D., Marczewski
W., Morgiewicz K., Gruden K., Hennig J. (2014). Journal of Experimental Botany, 65, 1095-1109.
Kogovšek P., Pompe-Novak M., Baebler Š., Rotter A., Gow L., Gruden K., Foster G.D., Boonham N., Ravnikar M. (2010). Plant
Pathology 59, 1121-1132.
Kogovšek P., Kladnik A., Mlakar J., Tušek Žnidarič M., Dermastia M., Ravnikar M., Pompe-Novak M. (2011). Phytopathology 101,
1292-1300.
Rupar M., Kogovšek P., Pompe Novak M., Gutierrez-Aguirre I., Delaunay A., Jacquot E., Ravnikar M. (2013). Journal of virological
methods 189, 93-100.
59
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SL-5
Potato Virus Y infection hinders potato defence response and renders plants more
vulnerable to Colorado potato beetle attack
1
1
1
1
2
Marko Petek *, Ana Rotter , Polona Kogovšek , Špela Baebler , Axel Mithöfer , Kristina Gruden
1
1 National
Institute of Biology, Department of Biotechnology and Systems Biology, Ljubljana, Slovenia
Planck Institute for Chemical Ecology, Department of Bioorganic Chemistry, Jena, Germany
* corresponding author (marko.petek@nib.si)
2Max
In the field, plants are challenged by more than one biotic stressor at the same time. In the present study,
the molecular interactions between potato (Solanum tuberosum L.), Colorado potato beetle (Leptinotarsa
decemlineata Say; CPB), and Potato virus YNTN (PVYNTN) were investigated through analyses of gene
expression in the potato leaves and the gut of the CPB larvae, and of the release of potato volatile
compounds. CPB larval growth was enhanced when reared on secondary PVYNTN-infected plants, which
was associated with decreased accumulation of transcripts associated with the antinutritional properties
of potato. In PVYNTN-infected plants, the ethylene signalling pathway induction and induction of auxin
response transcription factors was attenuated, while no differences were observed in jasmonic acid (JA)
signalling pathway. Similarly to rearing on virus-infected plants, CPB larvae gained more weight when
reared on plants silenced in the JA receptor gene (coi1). Although herbivore induced defence mechanism
is regulated predominately by JA, response in coi1-silenced plants only partly corresponds to the one
observed in PVYNTN-infected plants, confirming the role of other plant hormones in modulating response.
The release of β-barbatene and benzyl alcohol was different in healthy and PVYNTN-infected plants before
CPB larvae infestation, implicating the importance of PVYNTN infection in plant-to-plant communication.
This was reflected in gene expression profiles of neighbouring plants showing different degree of defence
response. The present study thus contributes to our understanding of plant responses in agroecosystems.
60
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SL-6
Fluorescently labelled Potato virus Y (PVY): in vivo tracking and measuring the rate of
spread of PVY in the host plants
1
2
2
1
3
Matevž Rupar *, Florence Faurez , Michel Tribodet , Ion Gutiérrez-Aguirre , Agnes Delaunay , Glais
Laurent2, David Dobnik1, Kristina Gruden1, Emmanuel Jacquot3, Maja Ravnikar1
National Institute of Biology, Večna pot 111, 1000 Ljubljana, Slovenia
INRA, UMR1349 IGEPP, F-35653 Le Rheu, France
3 INRA-CIRAD-Montpellier SupAgro, UMR-BGPI TA A-54/K, Campus International de Baillarguet, 34398 Montpellier
Cedex 5, France
* corresponding author (matevz.rupar@nib.si)
1
2
Potato virus Y (PVY) is the 5th most important plant virus (Scholthof at al., 2011) infecting economically
important Solanaceous crops such as tobacco, potato and pepper. Until now, the studies of movement and
localisation of PVY in plants were limited to the detection of viral RNA or proteins ex vivo (Kogovšek et
al., 2011). In this study, PVY N605 isolate was tagged by a green fluorescent protein (GFP) using infective
clones. Thorough characterisation in Nicotiana tabacum (Nt) cv. Xanthii indicated that the PVY N605 GFP
construct is biologically comparable to the non-labelled wild type PVY N605. The GFP labelled PVY caused
comparable symptoms as the non-labelled wild type PVY at the same time post inoculation. Stability of
the PVY N605 GFP was estimated with a combination of RT-qPCR assays and fluorescence microscopy.
The GFP tagged PVY was stable at least through three plant-to-plant passages and for 4 months in the
inoculated plant. The GFP signal was detected before any symptoms were observed and the intensity of
the fluorescence signal correlated with PVY RNA concentration. The PVY N605 GFP enabled in vivo
tracking of the viral cell-to-cell and long distance movement in inoculated and systemically infected
leaves. Moreover, the fluorescence tool allowed the estimation of the cell-to-cell movement rate of the
PVY in Nt cv. Xanthii (7.1 +/- 1.5 cells/hour). In addition to N. tabacum, PVY N605 GFP is also able to
infect Nicotiana benthamiana, different potato (Solanum tuberosum) cultivars and selected wild potato
relatives. The PVY N605 GFP is therefore a powerful tool for future studies of PVY-plant host interactions
such as functional analysis of both viral and plant genes involved in viral movement.
Scholthof K. G., Adkins S., Czosnek H., Palukaitis P., Jacquot E., Hohn T., Hohn B., Saunders K., Candresse T., Ahlquist P., Hemenway
C., Foster G. D. (2011). Mol. Plant Pathol. (12), 938–954
Kogovšek P., Kladnik A., Mlakar J., Tušek-Žnidarič M., Dermastia M., Ravnikar M., Pompe-Novak M. (2011). Phytopathology 101
(11), 1292–1300
61
2B
SL-7
Pathogenic Agrobacterium tumefaciens antagonises
Meloidogyne ethiopica reproduction on tomato roots
root-knot
nematode
Janja Lamovšek*, Saša Širca, Irena Mavrič Pleško, Barbara Gerič Stare, Gregor Urek
Agricultural Institute of Slovenia, Hacquetova ulica 17, SI-1000, Ljubljana, Slovenia
* corresponding author (Janja.lamovsek@kis.si)
Root-knot nematodes (Meloidogyne spp.) and pathogenic soil bacteria Agrobacterium tumefaciens are
economically important plant pests that interfere with water and nutrient flow through the host plant.
The nematodes induce development of root galls by manipulating host defence responses. Similarly, A.
tumefaciens causes crown gall disease and subdues host immune response to stimulate tumour growth.
Both organisms share similar host range and their interaction is often described as synergistic (Süle et al.,
1995; Elwakil et al., 2003). On the other hand, Fakhouri et al. (1996) described interaction between M.
javanica and A. tumefaciens on tomato roots as antagonistic. Is the interaction between Meloidogyne spp.
and A. tumefaciens truly synergistic? To answer this question, we conducted a study of interaction
between Slovenian population of M. ethiopica (Širca et al., 2004) and Slovenian strain of pathogenic A.
tumefaciens on roots of susceptible tomato hybrid. The inoculum concentration was low enough that
neither pest by itself caused severe damage to the host. The reproduction of M. ethiopica was not affected
when bacteria were co-inoculated with the nematodes. Similar effect was observed when bacteria were
added one week after the nematodes. However, the reproduction of M. ethiopica was significantly lower
(two-fold, p<0.001) when nematodes were added to the roots previously colonized by A. tumefaciens.
Additionally, a non-pathogenic A. tumefaciens strain exerted similar reduction in nematode reproduction
when applied before the nematodes. Even though pathogenic agrobacteria act as a biocontrol agent and
reduce the nematode population after their 1st reproductive cycle, the agrobacterial effect is shorttermed. After the 2nd cycle we observed a five-time increase in the number of nematode eggs. The
synergistic effect observed by other authors might express in later cycles. We observed a stimulating
effect on the nematode reproduction in the 2nd cycle when bacteria were added to the roots already
infested by the nematodes. Further, we conducted a relative gene expression study (qPCR) targeting six
genes involved in either salicylic or jasmonic acid plant defence response to discern which pathway is
stimulated/inhibited in parts of tomato roots not in a direct contact with both studied organisms at 0, 4
and 12 dpi.
Elwakil M. A., Mohamed Z. A., El-Sherif A. G. (2003). Pakistan Journal of Plant Pathology, 2 (1): 1-9.
Fakhouri W. D., Khlaif H. and Abu-Gharbeih W. I. (1996). Pakistan Journal of Nematology, 14 (1), 49-54.
Süle S., Lehoczky J., Jenser G., Nagy P., Burr T. J. (1995). Journal of Phytopathology, 143: 169-171.
Širca S., Urek G. and Karssen G. (2004). Plant disease, 88, 680.
62
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SL-8
Shot gun proteomics as a tool to explore potato-PVY interaction
Katja Stare1*, Neža Turnšek1, Tjaša Stare1, Stefanie Wienkoop2, Kristina Gruden1
1 National
Institute of Biology, Department of Biotechnology and Systems Biology, Ljubljana, Slovenia
University of Vienna, Department of Molecular Systems Biology, Vienna, Austria
* corresponding author (katja.stare@nib.si)
2
Plants are in their environment exposed to different pathogens thus they have to develop defense
mechanisms to survive. Potato, which is very important food crop, has to fight against its pathogen potato
virus Y (PVY). Understanding of this plant-pathogen interaction enables selection of efficient strategies
for plant protection. We are studding this interaction by using systems biology approaches and one of
them is label-free shot gun proteomics. It enables identification and quantification of proteins in tissue.
First, three different protocols for protein extraction were tested. Extraction with TriZol was optimal thus
selected for subsequent analyses. Proteins were digested with Lys-C and Trypsin and further desalted
with solid phase extraction cartridges (SPEC). Peptides were subjected to LC MS/MS analysis which was
performed in University of Vienna using OrbiTrap mass spectrometer (Thermo Scientific). Relative
quantification of peptides was determined by spectral counting. Proteins were identified by searching
the database combining all known potato genes (prepared on National Institute of Biology (Ljubljana)
and Jožef Stefan Institute (Ljubljana)).
We investigated differences 4 days after infection with virus PVY on cultivar Desiree using nontransformed Desiree and two different transformants (defective in salicylic acid and jasmonic acid
signaling pathway). Approx. 300 proteins were identified. Some proteins were differentially expressed
after infection: mostly they are included in photosynthesis, others have role in calcium signaling, ATPases transport, biotic stress, amino acid metabolism (synthesis and degradation), protein degradation,
tetrapyrrole synthesis, etc.
Some of proteins were found interesting on transcriptional level but not identified with shot gun
proteomics. To observe some of those proteins we have in plan to use another approach like MRM
(multiple reaction monitoring) technique and detection with antibodies.
63
2B
P-1
Primary metabolites increase in potato plants after infection with PVY
Polona Kogovšek1,2*, Maruša Pompe Novak2, Nina Prezelj2, Marko Petek2, Lena Fragner3, Wolfram
Weckwerth3, Kristina Gruden2
1 University
of Ljubljana, Biotechnical Faculty, Department of Biology, Večna pot 111, 1000 Ljubljana
Institute of Biology, Department of Systems Biology and Biotechnology, Večna pot 111, 1000 Ljubljana
3 University of Vienna, Faculty of Life Sciences, Department of Molecular Systems Biology, Althanstrasse 14, 1090
Vienna, Austria
* corresponding author (polona.kogovsek@nib.si)
2 National
Response of potato plants (Solanum tuberosum) cv. Igor to infection with Potato virus Y (PVY) has already
been studied on morphological, biochemical and transcriptom levels. Expression of genes related to
defence response, photosynthesis, sugar metabolism and signalling was different in infected plants as
compared to healthy controles [Pompe-Novak et al., 2006; Baebler et al., 2009, Kogovšek et al., 2010].
Salicylic acid was shown to have an important role in the dynamics of the response of potato to infection
[Baebler et al., 2011]. To upgrade and deepen the understanding of the potato-PVY interaction, the potato
metabolome was analysed in the present study.
Changes of metabolome in potato plants infected with differently aggressive PVY isolates (PVYNTN and
PVYN) were analysed using GC-MS at different time points after infection. After infection with both
isolates, the amount of metabolites connected to primary metabolism, e.g. sugars, amino acids and Krebs
cycle intermediates, increased from 1st to 6th day after infection. Potato plants infected with PVYNTN and
expressing severe symptoms accumulated higher concentration of metabolites then plants inoculated
with milder PVYN isolate, which were not showing any symptoms. The metabolomic results were in
accordance with the results obtained on the transcriptomic level by analysis of the expression of genes
for key enzymes in selected metabolic pathways by qPCR. Data from metabolomic and transcriptomic
analysis were integrated and presented in the MapMan tool to visualise the changes in primary
metabolism during the development of the disease symptoms.
Baebler Š., Krečič-Stres H., Rotter A., Kogovšek P., Cankar K., Kok E.J., Gruden K., Kovač M., Žel J., Pompe-Novak M., Ravnikar M.
(2009). Molecular Plant Pathology 10 (2), 263-275.
Baebler Š., Stare K., Kovač M., Blejec A., Prezelj N., Stare T., Kogovšek P., Pompe-Novak M., Rosahl S., Ravnikar M., Gruden K.
(2011) Plos One 6 (12), 1-12.
Kogovšek P., Pompe-Novak M., Baebler Š., Rotter A., Gow L., Gruden K., Foster G.D., Boonham N., Ravnikar M. (2010) Plant
Pathology 59 (6), 1121-1132.
Pompe-Novak M., Gruden K., Baebler Š., Krečič-Stres H., Kovač M., Jongsma M., Ravnikar M. (2006) Physiological and Molecular
Plant Pathology 67, 237-247.
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Transcriptional regulatory network in potato – potato virus Y interaction signalling
Tjaša Lukan*, Anna Coll, Ana Lazar, Špela Baebler, Kristina Gruden
Department of Plant Physiology and Biotechnology, National Institute of Biology, Večna pot 111, 1000 Ljubljana,
Slovenia
* corresponding author (tjasa.lukan@nib.si)
Potato virus YNTN (PVYNTN) is a strain belonging to the necrotic group of potato virus Y (PVY) that causes a
potato tuber ring necrotic ringspot disease in potato, which significantly decreases the quality and quantity of
the yield. Potato (Solanum tuberosum L.) is the fourth-most-important food crop in the world after corn, rice
and wheat, with a yield of 365 million tonnes in 2012, and a continuous increase in the world production of
tubers (http://faostat.fao.org/). The decreasing availability of land for area expansion means that yields will
have to be improved.
Plant hormones are crucial signalling molecules that coordinate all aspects of plant growth, development,
reproduction and defence. Three hormones are especially important for plant immune response. The SA, JA
and ET signalling pathways represent the backbone of the defence signalling network, with other hormonal
signalling pathways feeding into it. The importance of salicylic acid (SA), jasmonic acid (JA) and ethylene (ET)
as dominant primary signals in local and systemic induced defence signalling has been well documented.
However, the way these signal molecules function in a complex network of interacting pathways is less well
understood and the majority of research has been done on model plant species and very little on potato. Our
aim is to upgrade our current understanding of the roles of SA, JA and ET in the plant’s immune system and
crosstalk between defence hormone signalling pathways, with a focus on promoter analysis of carefully chosen
genes from defence signalling network.
In order to understand transcriptional network of signalling components in potato following PVY infection we
decided to analyse the promoters of some genes that are crucial components in plant defence signalling
pathways. The carefully chosen genes are ACC oxidase (ACO) which regulates ethylene synthesis and it's
transcription factor ethylene response factor (ERF) from ethylene signalling pathway, potato cysteine protease
inhibitor (PCPI) which is the latest product in jasmonic acid pathway and transcription factor MYC2 that
regulates PCPI expression, whereas patogenesis-related protein (PR1) and its transcription factor TGA have
been chosen from salicylic acid pathway.
Promoters obtained from different potato cultivars were sequenced, compared to the available model genome
sequence and analysed with TRANSFAC and PlantCARE. The results showed that promoter sequences of the
same gene differ between cultivars. Furthermore, each gene can have different promoter sequences within the
same cultivar. Using TRANSFAC and PlantCARE we managed to characterized transcription factor binding sites
and established how transcription factor binding sites vary between promoters of the same gene within each
cultivar.
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Interactions of endophytic fungi from seeds of common buckwheat (Fagopyrum
esculentum Moench)
Eva Kovačec, Matevž Likar, Ivan Kreft, Marjana Regvar*
Biotechnical Faculty, University of Ljubljana, Večna pot 111, SI-1001 Ljubljana, Slovenia
* corresponding author (marjana.regvar@bf.uni-lj.si)
Common buckwheat is known by high contents of bioactive substances from the fibers, vitamins and
flavonoids with strong antioxidant activities, which make it functional food (Kreft et al., 2006). Studies on
buckwheat plants are mostly focused on phylogeny, genetics, production, its secondary metabolites and
its nutrition value, only few had touched endophytes and mycological scope. Endophytic fungi have their
own ecological niche and role in agricultural and medicinal plants, but are often overlooked (Ganley and
Newcombe, 2006). In this study common buckwheat seeds mycoflora was monitored over time and the
presence of various fungi was demonstrated.
After surface sterilization of buckwheat seeds with 30 % H2O2, seeds were placed on petri-dishes
containing 2 % PDA and hyphae growing from seeds were subcultured till monocultural colony. Fungal
isolates were identified by isolation of fungal genomic DNA followed by PCR reactions using ITS1F-ITS4
primer pair. The most frequently isolated species were pathogenic fungi (genera Botrytis sp., Alternaria
sp., Fusarium sp.) and saprophytic fungi (genera Aureobasidium sp., Epicoccum sp.). The frequency and
diversity of fungi in seeds after one year were significantly reduced. Effects of different fungal isolates on
seed germination were tested by putting sterilized buckwheat seeds on edge of fungi growing on PDA
plates. Moreover, compatibility and potential antagonistic effects between fungal isolates were tested by
dual culture assay and double-dish method for determination of possible antifungal volatile organic
compounds.
Kreft I., Fabjan N. and Yasumoto K. (2006). Food chemistry 98, 508–512.
Ganley R. J. and Newcombe G. (2006). Mycological research 110, 318-327.
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Black, grey and green alder and their roots symbionts
Melita Hrenko1*, Hojka Kraigher1, Tine Grebenc1, Robert Brus2, Franc Batič3
Slovenian Forestry Institute, Večna pot 2, 1000 Ljubljana, Slovenija
University of Ljubljana, Biotechnical Faculty, Department of Forestry and Renewable Forest Resources, Večna pot
83, 1000 Ljubljana
3 University of Ljubljana, Biotechnical Faculty, Department of Agriculture, Jamnikarjeva 101, 1000 Ljubljana
* corresponding author (Melita.hrenko@gozdis.si)
1
2
Three alder species are native to Slovenia: black (Alnus glutinosa (L.) Gaertn.), grey (Alnus incana (L.)
Moench.) and green alder (Alnus alnobetula (Ehrh.) Hartig(= Alnus viridis (Chaix) DC.). The three species
differ according to their morphological characteristics and growing sites, yet all share their roots with
mycorrhizal (ecto- and arbuscular) and actinorhizal symbionts (Kraigher et al 2011). Alders and their
symbionts are used as model systems for studies of belowground complexity (Arnebrant e tal 1993),
biogeography, co-evolution and specificity of the macro- and microbionts in symbiosis. At a global scale
there are about 35 species of alders, which form ectomycorrhiza with about 142 primarily narrowly
specialized ectomycorrhizal fungi. The biogeography of ectomycorrhizal fungi was found to be consistent
with ancient host migration patterns, indicating codispersal of hosts and their mycobionts (Po~lme et al
2013). In Slovenia a screening of actinorhizal symbiosis was done on the three species (Grebenc et al 1999), and
ectomycorrhiza of black and green alder was screened by Hrenko et al and is presented here.
Arnebrant K, Ek H, Finlay RD, Söderström B. 1993. New Phytol., 124: 231–242.
Grebenc T, Rupnik M, Hacin J, Janc M, Kraigher H. 1999. Phyton (Horn), 39 (3): 207–211.
Kraigher H, Brus R, Batič F. 2011. Tehnične smernice za ohranjanje in rabo genskih virov : črna, siva in zelena jelša = Alnus
glutinosa, Alnus incana, Alnus viridis : Slovenija. Gozdarski vestnik, 69 (10): [475-484].
Po~lme S, Bahram M, Yamanaka T, Nara K, Dai YC, Grebenc T, Kraigher H, Toivonen M, Wang P-H, Matsuda Y, Naadel T, Kennedy
PG, Ko~ljalg U, Tedersoo L. 2013. New Phytologist 198: 1239–1249.
Acknowledgements: The presentation was financed by the research programme P4-0107 and the projects L4-4318 and V4-1140
through SRA & MAE, and prepared for the occasion of the retirement of Prof. Dr. Franc Batič.
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Mycobioindication of stress and disturbance in forest ecosystems
Hojka Kraigher1*, Samar Al Sayegh Petkovšek2, Franc Batič3
Slovenian Forestry Institute, Večna pot 2, 1000 Ljubljana, Slovenija
ERICo Istitute for Ecological Research, Koroška 58, 3320 Velenje
3 University of Ljubljana, Biotechnical Faculty, Department of Agriculture, Jamnikarjeva 101, 1000 Ljubljana
* corresponding author (Hojka.kraigher@gozdis.si)
1
2
Mycorrhiza is the main spatial and temporal linkage between different constituents in a forest ecosystem.
The functional compatibility and stress tolerance of ectomycorrhizal types is species specific, and
therefore the information on the ectomycorrhizal community structure adds to the understanding of
processes in forest ecosystems and can also be applied as tools for bioindication of stress in forest soils.
Mycorrhizal fungi and types of ectomycorrhiza have been used as in situ indicators, passive and active
monitors and ex situ testers of stress due to pollution, green house gasses, drought or soil compaction in a
series of studies in Slovenian forests; a number of reviews were published and shall be presented
(Kraigher et al 1996; Kraigher et al 2007; Cudlin et al 2007; Kraigher and Al Sayegh Petkovšek 2011,
Kraigher et al 2013), contributing to a historical overview of the method and its application in Slovenia.
Kraigher H, Batič F, Agerer R. 1996. Phyton (Horn), 36 (3): 115-120.
Kraigher H, Al Sayegh-Petkovšek S, Grebenc T, Simončič P. 2007. Environ. monit. assess., 128 (1): 31-45.
Cudlin P, Grebenc T, Kraigher H et al. 2007. Plant Biosyst. (Firenze, Testo stamp.), 141 (3): 406-425.
Kraigher H, Al Sayegh-Petkovšek S. 2011. In: Rai M, Varma A. (Eds.). Diversity and biotechnology of ectomycorrhizae, (Soil
biology, ISSN 1613-3382, vol. 25). Heidelberg; New York: Springer, pp 301-322.
Kraigher H, Bajc M, Grebenc T. 2013. In: Matyssek R. (Ed.). Climate change, air pollution and global challenges : understanding
and perspectives from forest research, (Developments in environmental science, ISSN 1474-8177, 13). Amsterdam [etc.]:
Elsevier, pp 151-177.
Acknowledgements: The presentation was financed by the research programme P4-0107 and the project L4-4318 through SRA &
MAE, and prepared for the occasion of the retirement of Prof. Dr. Franc Batič.
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Potential of thyme essential oil as antifungal preservative for storage of wheat seeds
Sabina Anžlovar, Tjaša Pršin, Matevž Likar, Jasna Dolenc Koce*
Department of Biology, Biotehnical Faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia
* corresponding author (jasna.dolenc.koce@bf.uni-lj.si)
The aim of this study was to determine the antifungal activity and potential use of thyme (Thymus
vulgaris) essential oil as seed disinfectant against fungi grown on wheat (Triticum aestivum) seeds. The
wheat seeds were exposed to the thyme essential oil vapors or were directly soaked in the essential oil.
The assessment of fungus infection and seed germination was performed by direct plating method
(Sumalan et al 2013). Fungal isolates from wheat seedlings were molecularly identified. The inhibitory
effect of thyme essential oil on the mycelial radial growth was tested by agar dilution method (Zabka et al
2009). The thyme essential oil showed inhibitory effect on fungal contamination of wheat seeds. Both,
direct and indirect application of the thyme essential oil significantly reduced fungal contamination of
wheat seeds. However, direct application was much more significant than indirect application. The
fungitoxicity of thyme essential oil was recorded for a level of 0.01% for all tested fungi: Alternaria,
Fusarium, Aspergillus and Epiccocum. Our results show that thyme essential oil has the potential to be
used as a seed disinfectant and as a natural fungicide for management of plant fungal disease.
Sumalan R.M., Alexa E., Poiana M.A. (2013). Chemistry Central Journal (7), 32-44.
Zabka M., Pavela R., Slezakova L. (2009). Industiral Crops and Products (30), 250-253.
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5 Dodiplomska sekcija
70
Searching for answers in Epipactis taxa in Slovenia
Anja Ivanuš1*, Metka Šiško1, Andreja Urbanek Krajnc1, Zlata Luthar2, Vilma Šuštar1, Matej Lipovšek1*
1 Faculty
of Agriculture and Life Sciences, University of Maribor, Slovenia
Biotechnical Faculty, University of Ljubljana, Slovenia
* corresponding authors (anja.ivanus@um.si; matej.lipovsek@guest.arnes.si)
2
The genus Epipactis (Orchidaceae) represent a taxonomically problematic group, involving high variation
associated with genetic structure, phenotypic characteristics, growth and development which causes
difficulties in identification and taxonomic description of the species. Based on morphological
characteristics and molecular evaluation the description of lower taxonomic units is in progress. In
Slovenia, the Epipactis have not yet been included in systematic molecular evaluation. The aim of this
research was to define morphological features as well as to evaluate the genetic structure of the nine
Goričko populations studied on location and in the lab. Significant variations among some taxa were
determined for the ovary length, labellum (hipochil, epichil) length, petal length and sepal length and
width. In contrast, epichil width and petal width were found to be quite uniform. The population that
exhibited the highest plant growth was found in Lucova, whereas the lowest plants were found in
Martinje.
Flow cytometry determination of genome size of all studied individuals revealed three different
homogeneous subsets by using Tukey’s test. The lowest average genome size was 25.57 pg/2C nucleus,
whereas the highest average genome size was 27.32 pg/2C nucleus. The genetic variation among
randomly sampled individuals was determined by AFLP method using nine different combinations of
primers. The AFLP approach was found to be very useful in discrimination of closely related genotypes.
Based on AFLPs, three discrete clusters were determined. The most outlying cluster C 1 included
individuals from the populations Gornji Slaveči, Matjaševci, Trdkova and Neradnovci. The second outlying
cluster C 3 included individuals from the populations Kuzma, Lucova, Matjaševci and Čepinci. Some
sampled individuals were genetically more closely related than others. The cluster C 2 with populations
Martinje, Čepinci, Lucova, Boreča, was divided in three sub clusters. The individuals from Čepinci and
Lucova proved to be genetically the most distinct group. The investigation indicates that there were
obvious genetic differences among individuals. The combination of morphological and molecular markers
should be the most reliable way to solve taxonomic status of the Epipactis at present.
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Microsatellite markers in determination of the raspberry variety 'Glen Ample'
Vesna Novak*, Metka Šiško, Andrej Šušek, Anton Ivančič
Faculty of Agriculture and Life Sciences, University of Maribor, Slovenia
* corresponding author (vesna.novak82@gmail.com)
Raspberries (Rubus idaeus L.) are important commercial fruit plants, widespread throughout temperate
regions. They are used for many purposes such as food, as medicinal plants as well as in pharmaceutical
and cosmetic industry. The demands for raspberries in the market are constantly increasing. There are
many traditional and new improved varieties which are characterized by higher yields and are more
resistant to pests and diseases. Their fruits are larger and more aromatic, with higher content of
antioxidant molecules. Each variety is a result of systematic breeding and presents intellectual property
protected by law. For the protection of intellectual property rights, it is very important to have an
efficient method which enables fast and accurate determination of each variety and clear differentiation
from other varieties. Genetic variability can be evaluated in two ways: on the phenotypic level with
morphological descriptors and on the molecular level with molecular markers. Particularly DNA markers
can provide information about the relationships between different clones or strains that are difficult or
impossible to distinguish morphologically. The molecular markers have many advantages and, if properly
chosen, they are more accurate than morphological descriptors. The aim of our investigation was to
identify raspberry varieties by using molecular markers. The incorporation of new methodologies into
plant material certification schemes will accelerate and optimize the identification process by allowing
fingerprinting of each genotype at any stage of the development and independently of environmental
factors that may influence the phenotype. SSR molecular markers were used for the assessment of genetic
diversity and genetic relationship in order to identify the variety. Nineteen microsatellite primers used in
previous studies were selected for molecular evaluation of the genetic diversity. For a successful analysis,
the optimization of primers is essential due to different laboratory working conditions. The optimal
annealing temperature for each primer pair was determined by the PCR gradient. Based on the results of
the gradient optimization for all nineteen primer sets, a single common annealing temperature was
selected. The ‘Glen Ample’ variety was chosen to develop a protocol for identification. From the
completed data (allele sizes) obtained in 19 optimized SSR primers, the most specific microsatellite loci
for each of the seven selected cultivars was chosen. For identification of the ‘Glen Ample’ cultivar, seven
selected microsatellite loci were used. With the selected microsatellite loci a fingerprint for the variety
was obtained. The fingerprint is variety specific and can be used for its identification.
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How does taste accumulate during growth? - The case of sugars and acids
Nadja Sadar1*, Pol Tijskens2, Tatjana Unuk1, Andreja Urbanek Krajnc1
University of Maribor, Faculty of Agriculture and Life Sciences, Slovenia
Wageningen University, Horticulture and Product Physiology, the Netherlands
*corresponding author (sadar.nadja@gmail.com)
1
2
Apple production is in a state of hyper competition, so to stay in business the producers need to provide
the consumers with a consistent supply of high quality fruit. Two of the most important quality attributes
of apple fruit are skin colour and taste. There is no detailed knowledge on the mechanisms and kinetics
of quality development during growth or on the distribution of these quality components within fruit, as
the majority of studies employ destructive measuring techniques for obtaining the data, which are than
analysed with empirical statistics that ignore the presence of biological variation and thus completely
mask the spatio-temporal physiological mechanisms. To address some of these issues, a study was
conducted to determine the behaviour of colour (a*) and taste-defining components: malic acid, citric
acid, fumaric acid, shikimic acid, fructose, sucrose, and glucose during apple fruit development. For the
purpose of in-vivo monitoring of taste components, a novel biopsy sampling technique, was used. In the
present study the spatial and temporal distribution of individual quality components in cv. ‘Gala’ and cv.
‘Pinova’ apple fruit during on-tree development were monitored. Additionally, standard quality indices
and diameter were determined. Apart from the usual empirical statistics, the data were analysed with
either a logistic or exponential process oriented model. The information on the kinetic parameters and
variation, presented in the data, was extracted with indexed non-linear regression. The results on
temporal dynamics of quality components reveal a sigmoidal increase of a*, an exponential increase of
diameter and sugars towards an asymptote, and an exponential decrease of organic acids over time.
Taste, i.e. total sugars to total acids balance is strongly (R2 > 0.8) correlated to malic acid and diameter.
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Seasonal sap-flow patterns in pubescent oak (Quercus pubescens Willd.) growing in
abandoned karst grasslands
1
1,2
2
1
Martina Lavrič , Klemen Eler , Mitja Ferlan , Dominik Vodnik *
University of Ljubljana, Biotechnical Faculty, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia
Slovenian Forestry Institute, Večna pot 2, SI-1000 Ljubljana, Slovenia
* corresponding author (dominik.vodnik@bf.uni-lj.si)
1
2
The carbon sink strength of abandoned, woody plants invaded dry calcareous grasslands in Slovenian
sub-Mediterranean region relies on water availability. Eddy covariance measurements revealed
significant decrease of ecosystem carbon uptake in years when assimilation was limited by prolonged
summer droughts (Ferlan et al, submitted). At diurnal scale we observed a decrease in the C-uptake after
the midday. Since our parallel measurements indicate no enhancement of soil respiration under such
conditions (Eler et al., 2013) we conclude that this change of net ecosystem exchange (NEE) can be linked
to the stomatal limitation of photosynthesis. Furthermore, on the basis of overlapping seasonal NEE
patterns and phenology of woody plant species which dominate the area, it can be expected that their
photosynthetic activity and tolerance to environmental constraints such as drought are the key
determinants of seasonal NEE patterns. Since the pubescent oak (Quercus pubescens) foliage represents a
vast majority of the green canopy in the studied area we presume that its photosynthetic characteristics
are directly reflected at the ecosystem level.
In order to get a deeper insight into the water balance regulation in pubescent oak, and to assess the
contribution of this species to ecosystem carbon and water balance, sap-flow measurements (heat ratio
method; Burgess, 2001) were performed in 2014 in parallel to long-term eddy covariance and meteo
measurements. In our contribution seasonal sap-flow patterns will be discussed with respect to: i)
species phenology and productivity (followed by LAI measurements), ii) environmental conditions
(precipitation, soil water, air humidity, temperature, ...) and iii) water vapour and carbon fluxes measured
on the ecosystem level (evapotranspiration, NEE).
Burgess S. O., Adams M. A., Turner N. C., Beverly C. R., Ong C. K., Khan A. A. H., Bleby T. M. 2001. Tree Physiology, 21: 589-598.
Eler K., Plestenjak G., Ferlan M., Čater M., Simončič P., Vodnik D., 2013. European Journal of Soil Science 64, 210–218.
Ferlan M., Eler K., Simončič P., Batič F., Vodnik D., (2014). Agriculture, Ecosystems and Environment, submitted.
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Response of Berula erecta to presence of cadmium
Klavdija Plohl, Špela Mechora, Jana Ambrožič Dolinšek*
Faculty of Natural Sciences and Mathematics, University of Maribor, Koroška cesta 160, 2000 Maribor, Slovenia
*corresponding author (Jana.Ambrozic@um.si)
The world copes with increased environmental pollution due to the development of industry and spread
of cities. In the present study, we wanted to examine the effect of two different concentrations of
cadmium chloride (1 mg/L and 2 mg/L CdCl2) on Berula erecta and its morphological, physiological and
biochemical alterations after exposure. Plants were cultivated in vitro on MS medium for three weeks in
growth chambers and later on exposed to CdCl2 for three weeks. Plants exposed to 1 mg/L and 2 mg/L of
CdCl2 showed a decline in growth and consequently lower average fresh weight than controls. Later on,
chlorosis occurred on CdCl2 treated plants. The photochemical efficiency of control and CdCl2 exposed
plants did not differ significantly. There was no difference in measured parameters between 1 and 2
mg/L CdCl2 treatments.
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Effect of various selenite concentrations on Berula erecta
Metka Sotler1, Jana Ambrožič Dolinšek1, Andreja Urbanek Krajnc2, Špela Mechora1*
1 Faculty
of Natural Sciences and Mathematics, University of Maribor, Koroška cesta 160, 2000 Maribor, Slovenia
of Agriculture and Biosystematic Science, Pivola 10, 2311 Hoče, Slovenia
*corresponding author (spela.mechora@gmail.com)
2 Faculty
Selenium (Se) is a naturally occurring trace element which is toxic at high concentrations, but it is also an
essential element for many organisms. The effect of Se(IV) treatment on physiological and biochemical
characteristics was studied on Berula erecta under controlled in vitro condition. Plants were treated with
different aqueous solution of Na selenite: 0.01, 0.1, 1, 10 and 50 mg Se(IV)/L for three weeks. High Se
concentrations (10 and 50 mg Se(IV)/L) inhibited growth of Berula erecta when compared to controls. All
Se(IV) treatments increased the number of stolones, buds, roots and root length. Photochemical efficiency
was negatively affected by the addition of 1 mg Se(IV)/L in the first two weeks, and by the addition of 10
and 50 mg Se(IV)/L in all three weeks. Se(IV) treatment decreased the amount of chlorophyll a regardless
Se(IV) concentration, while the amount of chlorophyll b decreased in plants treated with higher Se(IV)
concentrations. The concentration of total carotenoids increased in 0.01 mg/L Se(IV) treatment, while the
amount of anthocyanins was significantly lowered with higher Se(IV) application. We can conclude that
higher Se(IV) concentrations negatively affected the process of photosynthesis, formation of chlorophylls
and anthocyanins.
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Response of Apium repens to various selenite concentrations
Anže Žerdoner Čalasan1, Jana Ambrožič Dolinšek1, Andreja Urbanek Krajnc2, Špela Mechora1*
1 Faculty
of Natural Sciences and Mathematics, University of Maribor, Koroška cesta 160, 2000 Maribor, Slovenia
of Agriculture and Biosystematic Science, Pivola 10, 2311 Hoče, Slovenia
*corresponding author (spela.mechora@gmail.com)
2 Faculty
Selenium is an essential trace element for many organisms, but its essentiality for plants is still debatable,
although it can exert positive effects on plants. In the present research, morphological, physiological and
biochemical responses to selenite (Se(IV)) treatment were studied on perennial and semi-aquatic plant,
Apium repens (Jacq.) Lag. Plants were cultivated indoors in a growth chamber and were treated after a
two-week long incubation period with five different solutions of Se(IV) (10 μg/L, 100 μg/L, 1 mg/L, 10
mg/L and 50 mg/L). Three weeks after treatment the photochemical efficiency of photosystem II
decreased significantly in plants treated with higher Se(IV) concentrations (1 mg/L, 10 mg/L and 50
mg/L) in comparing to control plants. Lower Se(IV) concentrations (10 μg/L, 100 μg/L and 1 mg/L)
promoted growth when compared to controls, while plants exposed to higher Se(IV) concentrations (10
mg/L and 50 mg/L) exhibited stunted growth. Similarly, the average number of stolones, buds, roots and
the average length of roots were considerably lower in plants exposed to higher Se(IV) treatments. After a
two-week treatment period, chloroses were observed on plants treated with higher Se(IV) solutions (10
mg/L and 50 mg/L). The amount of chlorophyll a increased in Se(IV) treated plants within the first two
weeks after treatment when compared to the controls. In contrast, the amount of chlorophyll b increased
in Se(IV) treated plants in the second week. When applying higher Se(IV) concentrations significantly
higher concentrations of total carotenoids were measured in all three weeks after treatment, while the
amount of total anthocyanins increased in weeks 2 and 3, gradually with higher Se(IV) treatments. The
concentrations of total glutathione and total cysteine increased in plants treated with lower Se(IV)
concentrations. Higher Se(IV) concentrations caused a significant degradation of both total glutathione
and cysteine, accompanied by significantly more oxidised glutathione pool. We can conclude that higher
Se(IV) concentrations negatively affected morphological and physiological characteristics of Apium
repens.
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Germination rate of stinkwort (Dittrichia graveolens (L.) Greuter) and false
yellowhead (Dittrichia viscosa (L.) Greuter) in relation to salinity and the impact of
their extracts on germination of selected plant species
Mateja Grašič *, Sabina Anžlovar, Simona Strgulc Krajšek
Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia
* corresponding author (mgrasic@gmail.com)
Stinkwort (Dittrichia graveolens) is a plant species belonging to the family Asteraceae. It originates in the
Mediterranean region. It was first detected in Slovenia in 2008, where it has been spreading rapidly along
the motorway ever since. As a result, it has been characterized as an invasive alien species in Slovenia.
False yellowhead (Dittrichia viscosa) is an indigenous species in Slovenia. However, it only thrives in the
Slovene Istria region by the seaside. These facts suggest it is very likely that both species grow mainly on
sites with higher level of salinity. In addition, these two species (especially false yellowhead) are known
to produce secondary metabolites with allelopathic activity that inhibit germination of some other plant
species. Our hypotheses were that stinkwort and false yellowhead only thrive on sites with higher level of
salinity and that aqueous extracts from both species effectively inhibit germination of some other plant
species. We did germination tests in agar plates to determine germination rate of stinkwort and false
yellowhead in three different concentrations of NaCl and distilled water (control). We also did
germination tests to assess the effect of aqueous extracts from both species. However, in this second test,
we planted wheat, common ragweed and salad seeds on filter paper. We used 2 different concentrations
of aqueous exctact from each species and distilled water as control. Germination rate of stinkwort and
false yellowhead was very low (under 50% in most cases). There were many infections with fungi and
bacteria that could have lowered germination rate. Results have shown that stinkwort as well as false
yellowhead germinate under all salinity conditions. However, germination rate of both species was higher
in control and lower concentrations of NaCl. We can conclude that stinkwort and false yellowhead are
very tolerant regarding salinity. Also, their aqueous exctacts have proven to be effective against
germination of other species, especially the highest concentrations of aqueous extracts from both species.
Scientists have already pointed out that aqueous extract from false yellowhead could be used as a natural
phytoherbicide. Our results have proven that aqueous extract from stinkwort is just as effective as the
one from false yellowhead, so it could be used for the same purpose.
78
5
P-8
Allelopathic effects of aquaeous leaf extracts of Fallopia japonica and Fallopia x
bohemica on growth of tested plant species Raphanus sativus and Zea mays
Katarina Šoln, Jasna Dolenc Koce*
University of Ljubljana, Biotechnical Faculty, Dept. of Biology, Večna pot 111, SI-1000 Ljubljana, Slovenia
* corresponding author (jasna.dolenc.koce@bf.uni-lj.si)
Fallopia japonica and Fallopia × bohemica are two very invasive taxa from the group Fallopia sect.
Reynoutria that cause serious problems in Europe and North America. Their most efficient way of spread
is vegetative reproduction with rhizomes and underground stolons. Other modes of invasiveness are
possible. In this study, the biological activity of leaf aquatic extracts was tested to determine possible
allelopathic effects of F. japonica and F. x bohemica.
F. japonica and F. x bohemica mature leaves were collected in the field, lyophilized, homogenized,
resuspended in distilled water and extracted to prepare aquaeous extracts with concentrations 0.5 and 5
% (w/v). After filltration, the extracts were used to water seeds of Raphanus sativus and Zea mays for a
short time (both for 7 days) to evaluate their effects on germination and for a longer period of time (37
and 44 days, respecitively) to evaluate changes in growth and function of exposed plants (morphological
measurements of plant roots and shoots, stress related biochemical parameters: the content of
photosynthetic pigments, activity of antioxidant enzymes catalase- CAT and guaiacol peroxidase– G-POD,
lipid peroxidation).
Extracts of both Fallopia species had no significant effect on the germination of R. sativus and Z. mays
seeds. After 7 days of exposure to extracts, the growth of seedling roots was inhibited up to 20%. Shoot
growth was less affected. The content of photosynthetic pigments mainly decreased in exposed plants
except in case of 0.5% extracts of F. japonica. The reduction of pigments is correlated with chlorophyll a
and b content. Lipid peroxidation increased in roots and shoots due to extract exposure, but other
biochemical parameters were less sensitive. The activity of CAT and G-POD mainly decreased and the
effect was not related to the extract concentration
Similar results were obtained when R. sativus and Z. mays plants were exposed to Fallopia extracts for a
longer period of time (37 and 44 days). The only difference was shown in lipid peroxidation in leaves
which increased only in Z. mays plants.
Eventhough the germination of treated seeds was not affected, the following growth of the seedlings was
more or less inhibited. The most affected were R. sativus seedlings watered for 7 days with 5% extract of
F. japonica. The most sensitive stress related biomarker was proved to be lipid peroxidation.
79
5
P-9
Antifungal and antibacterial activity of goldenrod (Solidago spp.) essential oils
Maja Jankovec, Jerneja Ambrožič Avguštin, Sabina Anžlovar, Jasna Dolenc Koce*
Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia
* corresponding author (jasna.dolenc.koce@bf.uni-lj.si)
Recent studies suggest antifungal and antibacterial activity of organic extracts from three goldenrod
species (Solidago spp.) growing in Slovenia (Anžlovar and Dolenc Koce, 2014). Since essential oils
indicate even stronger activity than organic extracts we focused our study on different essential oils
extracted from leaves and blossoms of all three goldenrod species grown in Slovenia (Morel et al., 2006).
Our main interest was to determine the differences in antifungal activity of essential oils extracted from
leaves and blossoms and also the differences among all three species, native European goldenrod (S.
virgaurea) and two non-native species; Canadian goldenrod (S. canadensis) and giant goldenrod (S.
gigantea). We also compared antibacterial activity of two S.canadensis essential oils of different origin.
Antifungal activity was tested with agar dilution method against five fungal species which may occur on
seeds and cause spoilage - Alternaria alternata, Alternaria infectoria, Epicoccum nigrum, Aspergillus flavus
and Fusarium poae. Antibacterial activity was tested with adopted agar plate diffusion assay against Gram
positive strains among which some are multiresistant and thus harmful for human health - Bacillus
subtilis, Listeria monocytogenes, Staphylococcus pseudintermedius, Paenibacillus and Staphylococcus
aureus. Essential oils extracted from leaves and blossoms of all three Solidago species showed antifungal
activity against all selected fungi. Antibacterial activity was present in some bacterial strains with evident
differences among S. canadensis essential oils of different origin. However, this might indicate a possible
use of Solidago spp. essential oils as a natural food preservatives as suggested previously (Willis and
Saidman, 2013).
Anžlovar S., Dolenc Koce J. (2014). Phyton (Horn, Austria) (54), 135-147.
Morel A.F., Dias G.O., Porto C., Simionatto E., Stuker C.Z., Dalcol I.I. (2006). Fitoterapia (77), 453-455.
Willis D., Saidman S. (2013). Botanical essential oils as natural food preservatives. University of Pittsburgh, Swanson School of
Engineering, Conference Session B4, Paper #3059.
80
5
P-10
The bark war against the fungal terror: Antioxidant defences of Norway spruce
against low density Ceratocystis polonica inoculation
1
2
2,3
1
2
1
Mateja Felicijan *, Metka Novak , Neja Zupanec , Janez Gorenšek , Nada Kraševec , Mario Lešnik ,
Leopold M.M. Tijskens4, Radovan Komel2,3, Andreja Urbanek Krajnc1
1 Faculty
of Agriculture and Life Sciences, University of Maribor, Slovenia
National Institute of Chemistry, Ljubljana, Slovenia
3 Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Slovenia
4 Wageningen University, Horticulture and Product Physiology, The Netherlands
* corresponding author (felicijan.mateja@gmail.com)
2
The European spruce bark beetle (Ips typographus L.) and the associated pathogenic blue stain fungus
Ceratocystis polonica (Siem.) C. Moreau are among the most devastating pests regarding Norway spruce.
The basic defence strategy of the host tree, constitutive defence, involves the preformed resin ducts,
concentric layers of the polyphenolic parenchyma (PP) cells, sclerenchyma cells and calcium oxalate
crystals. Inducible defences are activated after an initial attack and include the de-novo formation of
traumatic resin ducts and proliferation of PP cells, which are associated with qualitative and quantitative
changes in terpenoids and phenolics. The broad range of defence mechanisms includes also the action of
antioxidants to counteract the reactive oxygen species (ROS). The ascorbate-glutathione-cycle is
considered to be the main pathway for ROS removal, and both ascorbate and glutathione are recognised
as the heart of the redox hub within the cell. In this experiment, the temporal courses of individual
phenolics, thiols and ascorbate were studied in vivo in Norway spruce phloem over a five-month-period
after inoculation with C. polonica at low density. The earliest stage of pathogen infection is characterised
by rapid increase of antioxidant compounds. Catechin and total cysteine, however, decreased three days
after inoculation. An initial reaction was followed by increased cysteine and glutathione concentrations,
increased glutathione reductase activity, and a more reduced redox state of those molecules. Similarly,
total phenolics, catechin and taxifolin concentrations increased continuously in response to low density C.
polonica inoculation, whereas the astringin and isorhapontin contents diminished within the infected
bark from June onwards. These events were accompanied by a significant degradation of the total
ascorbate, which reached a new steady state in September. Changes in the phenolic contents reflect an
increased pressure on the ascorbate-glutathione system as the system is needed for reducing the
phenoxyl radicals formed during the pathogen defence. Based on these results, bark phenolic metabolism
has been recognised to integrate ascorbate-glutathione system as a redox hub in Norway spruce defence
against C. polonica infection.
81
5
P-11
INDEKS AVTORJEV
A
E
Al Sayegh Petkovšek, 17, 68
Alberti, 23
Ambrožič Avguštin, 80
Ambrožič Dolinšek, 75, 76, 77
Ambrožič-Dolinšek, 37
Anžlovar, 69, 78, 80
Egge-Jacobsen, 32
Eichert, 36
Eisenberg, 43
Elena, 4
Eler, 23, 50, 74
F
B
Baebler, 5, 8, 9, 59, 60, 64, 65
Bakran-Petricioli, 25
Batič, 30, 48, 67, 68
Bohanec, 38, 42
Brus, 67
Faurez, 61
Felicijan, 81
Ferlan, 23, 74
Flajšman, 55
Fragner, 64
Frank, 41
C
G
Chersicola, 47
Ciringer, 37
Coll, 8, 9, 65
Curk, 58
Cvrtila, 25
Gaberščik, 22, 52
García Martínez, 3
Gerič Stare, 62
Germ, 21
Golob, 21
Gorenšek, 81
Goreta Ban, 26
Gozukirmizi, 6, 28
Grašič, 78
Grebenc, 67
Gričar, 49, 50
Gruden, 7, 8, 9, 13, 59, 60, 61, 63, 64, 65
Gutierrez-Aguirre, 58
Gutiérrez-Aguirre, 61
Č
Čerenak, 20
D
Dandekar, 2
Delaunay, 61
Demšar, 39
Dermastia, 47
Dobnik, 7, 8, 9, 12, 39, 61
Dolenc Koce, 25, 69, 79, 80
Dumbrell, 19
H
Hančević, 54
Helgason, 19
Hennig, 59
Hong, 44
Hrenko, 67
82
Lavrič, 74
Lazar, 8, 9, 65
Lee, 44
Lenarčič, 12
Lers, 47
Lešnik, 81
Likar, 51, 54, 66, 69
Likić, 24
Lipovšek, 71
Lukan, 13, 65
Luthar, 41, 42, 71
I
Ivančič, 40, 72
Ivanuš, 71
J
Jacquot, 61
Jagodic, 50
Jakše, 56, 57
Jamnik, 7
Jankovec, 80
Janžekovic, 40
Javornik, 20, 55, 56, 57
Jeran, 18, 30, 48
Jevremović, 31
M
Maček, 19
Maltar, 25
Mandelc, 20
Marakli, 6, 28
Maras, 16, 27
Mavrič Pleško, 62
Mazej, 18
Mechora, 75, 76, 77
Meglič, 11, 16, 27
Miljković, 13
Mithöfer, 60
Morgiewicz, 59
Morisset, 12
Mrak, 48, 49
Müller, 15
Murovec, 38
K
Kaligarič, 37
Kartal-Alacam, 6
Katanović, 25
Kidrič, 16, 27
Kim, 44
Kladnik, 38, 47
Klančnik, 52
Kogovšek, 58, 60, 64
Kokalj, 35
Komel, 81
Kopušar, 17
Košmrlj, 38
Kovačec, 66
Kraigher, 49, 67, 68
Kraševec, 81
Kreft, I.21, 66
Kreft S. 35
Kreuze, 58
Krumpestar, 38
Kryštufek, 17
Kump, 51
Kutnjak, 58
N
Na, 44, 76
Naseem, 2
Nečemer, 51
Nerić, 29
Nikolić, 30
Novak, 72, 81
O
Oberländer, 43
Orožen, 51
L
Lamovšek, 62
Laurent, 61
83
Skudnik, 18, 30
Smiljanec, 25
Sober, 40
Sotler, 76
Stabentheiner, 43
Stajič, 38
Stare K., 8, 63
Stare T., 13, 63
Stibilj, 21
Strgulc Krajšek, 78
Subotić, 31
Suomalainen, 10
P
Pantović, 30
Peressotti, 23
Pérez Alonso, 3
Perica, 26
Petek, 60, 64
Petricioli, 25
Petrić, 31
Pevalek-Kozlina, 31
Pfanz, 46
Pipan, 11
Plohl, 75
Pokorn, 57
Pollmann, 3
Pompe Novak, 64
Pompe-Novak, 59
Pongrac, 51
Povše, 20
Prezelj, 64
Progar, 56
Pršin, 69
Š
Šefc, 50
Šibanc, 19
Širca, 62
Šiško, 71, 72
Šlejkovec, 48
Šoln, 79
Štajner, 56, 57
Štebih, 39
Šušek, 72
Šuštar, 71
Šuštar Vozlič, 27
Šuštar-Vozlič, 16, 32
Švara, 56
R
Radić, 54
Radić Brkanac, 31
Radišek, 56, 57
Rajčić, 29
Ramšak, 13
Ravnikar, 12, 58, 61
Razinger, 16, 27
Regvar, 51, 54, 66
Rotter, 60
Rudolf Pilih, 42
Rupar, 22, 58, 61
Rusak, 24
Rutar, 11
T
Tijskens, 34, 73, 81
Tkalec, 25
Tome, 17
Trajković, 50
Tribodet, 61
Trošt Sedej, 22
Turnšek, 59, 63
Tušek-Žnidarič, 59
S
U
Sabovljević, 29, 30
Sadar, 73
Schouten, 34
Simčič, 34
Simončič, 23
Unuk, 34, 73
Urbanek Krajnc, 40, 71, 73, 76, 77, 81
Urek, 62
84
V
Vidaković-Cifrek, 25
Vleeshouwers, 7
Vodnik, 16, 20, 23, 27, 74
Vogel-Mikuš, 51
Vouk, 11
Vujčić, 31
Vujičić, 29, 30
Vuletin Selak, 26
W
Weckwerth, 64
Wienkoop, 63
Willfurth, 43
Winkler-Hermaden, 43
Y
Yilmaz, 6
Z
Zadražnik, 16, 32
Zechmann, 15
Zimmermann, 43
Zupanec, 81
Zupanič, 25
Zupin, 16, 27
Ž
Žel, 7, 9, 39
Železnik, 49
Žerdoner Čalasan, 77
85
SEZNAM UDELEŽENCEV
Al Sayegh Petkovšek Samar
ERICo Velenje, Inštitut za ekološke raziskave d.o.o.
Koroška 58, SI-3320 Velenje, Slovenia
samar.petkovsek@erico.si
Ambrožič-Dolinšek Jana
University of Maribor, PEF, Department of Elementary Teacher Education, FNM, Department of Biology
Koroška cesta 160, SI-2000 Maribor, Slovenia
jana.ambrozic@um.si
Anžlovar Sabina
Univerza v Ljubljani, Biotehniška fakulteta, Odd. za biologijo
Večna pot 111, SI-1000 Ljubljana, Slovenia
sabina.anzlovar@bf.uni-lj.si
Baebler Špela
NIB
Večna pot 111, SI-1000 Ljubljana, Slovenia
spela.baebler@nib.si
Bančič Jon
Univerza v Ljubljani, Biotehniška fakulteta, Odd. za biologijo
Večna pot 111, SI-1000 Ljubljana, Slovenia
jon.bancic@gmail.com
Batič Franc
Biotehniška fakulteta, Odelek za agronomijo
Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia
franc.batic@bf.uni-lj.si
Bohanec Borut
Univerza v Ljubljani, Biotehniška fakulteta
Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia
borut.bohanec@bf.uni-lj.si
86
Chersicola Marko
Nacionalni Inštitut za Biologijo
Večna pot 111, SI-1000 Ljubljana, Slovenia
marko.chersicola@nib.si
Coll Rius Anna
National Institute of Biology
Vecna pot 111, SI-1000 Ljubljana, Slovenia
anna.coll@nib.si
Čepin Timotej
Univerza v Ljubljani, Biotehniška fakulteta, Odd. za biologijo
Večna pot 111, SI-1000 Ljubljana, Slovenia
tcepin@gmail.com
Čerenak Andreja
Inštitut za hmeljarstvo in pivovarstvo Slovenije
Žalskega tabora 2, SI-3310 Žalec, Slovenia
andreja.cerenak@ihps.si
Dobnik David
Nacionalni inštitut za biologijo
Večna pot 111, SI-1000 Ljubljana, Slovenia
david.dobnik@nib.si
Dolenc Koce Jasna
Univerza v Ljubljani, Biotehniška fakulteta, Odd. za biologijo
Večna pot 111, SI-1000 Ljubljana, Slovenia
jasna.dolenc.koce@bf.uni-lj.si
Eichert Diane
ELETTRA - Sincrotrone Trieste, S.S. 14 Km. 163.5 in Area Science Park
1419 Basovizza, TS, Italy
diane.eichert@elettra.eu
Elena Santiago
IBMCP (CSIC-UPV) Campus UPV CPI 8E
46022 Valencia, Spain
sfelena@ibmcp.upv.es
87
Eler Klemen
Univerza v Ljubljani, Biotehniška fakulteta
Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia
klemen.eler@bf.uni-lj.si
Felicijan Mateja
Fakulteta za kmetijstvo in biosistemske vede UM
Pivola 10, SI-2311 Hoce, Slovenia
felicijan.mateja@gmail.com
Flajšman Marko
Biotehniška falkulteta
Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia
marko.flajsman@bf.uni-lj.si
Golob Aleksandra
UL, Biotehniška fakulteta
Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia
aleksandra.golob@bf.uni-lj.si
Gozukirmizi Nermin
Istanbul University, Molecular Biology and Genetics Department
34134 Istanbul, Turkey
nermin@istanbul.edu.tr
Grašič Mateja
Univerza v Ljubljani, Biotehniška fakulteta, Odd. za biologijo
Večna pot 111, SI-1000 Ljubljana, Slovenia
mgrasic@gmail.com
Grebenc Tine
Gozdarski inštitut Slovenije
Večna pot 2, SI-1000 Ljubljana, Slovenia
Tine.grebenc@gozdis.si
Gruden Kristina
NIB
Vecna pot 111, SI-1000 ljubljana, Slovenia
kristina.gruden@nib.si
88
Hrenko Melita
Gozdarski inštitut Slovenije
Večna pot 2, SI-1000 Ljubljana, Slovenia
melita.hrenko@gozdis.si
Ivanuš Anja
Fakulteta za kmetijstvo in biosistemske vede UM
Pivola 10, SI-2311 Hoče, Slovenia
anja.ivanus@gmail.com
Jankovec Maja
Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za biologijo
Večna pot 111, SI-1000 Ljubljana, Slovenia
maja.jankovec@gmail.com
Jeran Zvonka
Institut "Jožef Stefan"
Jamova 39, SI-1000, Ljubljana, Slovenia
zvonka.jeran@ijs.si
Kladnik Aleš
Univerza v Ljubljani, Biotehniška fakulteta, Odd. za biologijo
Večna pot 111, SI-1000 Ljubljana, Slovenia
ales.kladnik@bf.uni-lj.si
Klančnik Katja
Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za biologijo
Večna pot 111, SI-1000 Ljubljana, Slovenia
katja.klancnik@bf.uni-lj.si
Kogovšek Polona
Nacionalni inštitut za biologijo
Večna pot 111, SI-1000 Ljubljana, Slovenia
polona.kogovsek@nib.si
Kokalj Meta
Fakulteta za farmacijo, Univerza v Ljubljana
Aškerčeva cesta 7, SI-1000 Ljubljana, Slovenia
meta.kokalj@ffa.uni-lj.si
89
Kovačec Eva
Univerza v Ljubljani, Biotehniška fakulteta, Odd. za biologijo
Večna pot 111, SI-1000 Ljubljana, Slovenia
eva.kovacec@bf.uni-lj.si
Kraigher Hojka
Gozdarski inštitut Slovenije
Večna pot 2, SI-1000 Ljubljana, Slovenia
hojka.kraigher@gozdis.si
Kraševec Nada
National Institute of Chemistry
Hajdrihova 19, SI-1000 Ljubljana, Slovenia
nada.krasevec@ki.si
Kutnjak Denis
Nacionalni Inštitut za biologijo
Večna pot 111, SI-1000 Ljubljana, Slovenia
denis.kutnjak@nib.si
Lamovšek Janja
Kmetijski inštitut Slovenije
Hacquetova ulica 17, SI-1000 Ljubljana, Slovenia
janja.lamovsek@kis.si
Lavrič Martina
Univerza v Ljubljani, Biotehniška fakulteta
Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia
martina.lavric@gmail.com
Lazar Ana
Nacionalni inštitut za biologijo
Večna pot 111, SI-1000 Ljubljana, Slovenia
ana.lazar@nib.si
Likar Matevž
Univerza v Ljubljani, Biotehniška fakulteta, Odd. za biologijo
Večna pot 111, SI-1000 Ljubljana, Slovenia
matevz.likar@bf.uni-lj.si
90
Likić Saša
Faculty of Science, University of Zagreb
Rooseveltov trg 6, 10000 Zagreb, Croatia
sasa.likic@biol.pmf.hr
Lukan Tjaša
NIB
Večna pot 111, SI-1000 Ljubljana, Slovenia
tjasa.lukan@nib.si
Luthar Zlata
Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za agronomijo
Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia
zlata.luthar@bf.uni-lj.si
Maček Irena
Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za agronomijo
Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia
irena.macek@bf.uni-lj.si
Marakli Sevgi
Istanbul University, Molecular Biology and Genetics Department
34134 Istanbul, Turkey
sevgi.marakli@istanbul.edu.tr
Marton Kristina
Univerza v Ljubljani, Biotehniška fakulteta
Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia
kristina.marton@bf.uni-lj.si
Mrak Tanja
Gozdarski inštitut Slovenije
Večna pot 2, SI-1000 Ljubljana, Slovenia
tanja.mrak@gozdis.si
Müller Maria
Institute of Plant Sciences
Schubertstrasse 51, 8010 Graz, Austria
maria.mueller@uni-graz.at
91
Murovec Jana
Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za agronomijo
Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia
jana.murovec@bf.uni-lj.si
Na Chae Sun
Korea University
Anam-ro, Seongbuk-gu 136-701, Seoul, Korea, South
chaesun.na@uibk.ac.at
Naseem Muhammad
University of Wuerzburg Germany, Functional Genomics and Systems Biology Group,Department of Bioinformatics,
Biocentrum
97074 Wuerzburg, Germany
muhammad@biozentrum.uni-wuerzburg.de
Novak Vesna
Fakulteta za kmetijstvo in biosistemske vede UM
Pivola 10, SI-2311 Hoce, Slovenia
vesna.novak82@gmail.com
Orožen Lea
Biotehniška fakulteta, Oddelek za biologijo
Večna pot 111, SI-1000 Ljubljana, Slovenia
lea.orozen@gmail.com
Petek Marko
Nacionalni inštitut za biologijo
Večna pot 111, SI-1000 Ljubljana, Slovenia
marko.petek@nib.si
Pfanz Hardy
Institute of Applied Botany, University of Duisburg-Essen
45117 Essen, Germany
hardy.pfanz@uni-due.de
Pipan Barbara
Kmetijski inštitut Slovenije
Hacquetova ulica 17, SI-1000 Ljubljana, Slovenia
barbara.pipan@kis.si
92
Plohl Klavdija
UM, Fakulteta za naravoslovje in matematiko
Koroška cesta 160, SI-2000 Maribor, Slovenia
plohl.klavdija@gmail.com
Pokorn Tine
Biotehniška fakulteta
Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia
tine.pokorn@bf.uni-lj.si
Pollmann Stephan
Centro de Biotecnología y Genómica de Plantas (UPM – INIA), Campus de Montegancedo, 28223 Pozuelo de Alarcón
(Madrid), Autovía M-40 km 38, Spain
stephan.pollmann@upm.es
Pompe Novak Maruša
Nacionalni inštitut za biologijo
Večna pot 111, SI-1000 Ljubljana, Slovenia
marusa.pompe.novak@nib.si
Povše Rozalija
Inštitut za hmeljarstvo in pivovarstvo Slovenije
Cesta Žalskega tabora 2, SI-3310 Žalec, Slovenia
zala.povse@ihps.si
Progar Vasja
Biotehniška fakulteta, Univerza v Ljubljani
Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia
vasja.progar@bf.uni-lj.si
Ramšak Živa
Nacionalni inštitut za biologijo
Večna pot 111, SI-1000 Ljubljana, Slovenia
ziva.ramsak@nib.si
Ravnikar Maja
Nacionalni inštitut za biologijo
Večna pot 111, SI-1000 Ljubljana, Slovenia
maja.ravnikar@nib.si
93
Regvar Marjana
Univerza v Ljubljani, Biotehniška fakulteta, Odd. za biologijo
Večna pot 111, SI 1000 Ljubljana, Slovenia
marjana.regvar@bf.uni-lj.si
Rudolf Pilih Katarina
Univerza v Ljubljani, Biotehniška fakulteta
Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia
katarina.rudolfpilih@bf.uni-lj.si
Rupar Matevž
Nacionalni inštitut za biologijo, Oddelek za biotehnologijo in sistemsko biologijo
Večna pot 111, SI-1000 Ljubljana, Slovenia
matevz.rupar@nib.si
Sabovljević Aneta
Faculty of Biology, University of Belgrade
Takovska 43, 11000 Belgrade, Serbia
aneta@bio.bg.ac.rs
Sadar Nadja
Fakulteta za kmetijstvo in biosistemske vede UM
Pivola 10, SI-2311 Hoce, Slovenia
sadar.nadja@gmail.com
Sotler Metka
Fakulteta za naravoslovje in matematiko
Koroška cesta 160, SI-2000 Maribor, Slovenia
metka.sotler@gmail.com
Stabentheiner Edith
University Graz
Schubertstrasse 51, 8010 Graz, Austria
edith.stabentheiner@uni-graz.at
Stančič Brina
Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za biologijo
Večna pot 111, SI-1000 Ljubljana, Slovenia
brina.stancic@gmail.com
94
Stare Katja
Nacionalni inštitut za biologijo
Večna pot 111, SI-1000 Ljubljana, Slovenia
katja.stare@nib.si
Šircelj Helena
Biotehniška fakulteta, Univerza v Ljubljani
Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia
helena.sircelj@bf.uni-lj.si
Ida Šmid
Nacionalni inštitut za biologijo
Večna pot 111, SI-1000 Ljubljana, Slovenia
Ida.smid@nib.si
Šoln Katarina
Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za biologijo
Večna pot 111, SI-1000 Ljubljana, Slovenia
katarina.soln@gmail.com
Štebih Dejan
Nacionalni inštitut za biologijo
Večna pot 111, SI-1000 Ljubljana, Slovenia
dejan.stebih@nib.si
Šuštar Vozlič Jelka
Kmetijski inštitut Slovenije
Hacquetova ulica 17, SI-1000 Ljubljana, Slovenia
jelka.vozlic@kis.si
Tijskens Pol
Wageningen University
Droevendaalse steeg 1, Wageningen, Netherlands
pol.tijskens@wur.nl
Trošt Sedej Tadeja
Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za biologijo
Večna pot 111, SI-1000 Ljubljana, Slovenia
tadeja.trostsedej@bf.uni-lj.si
95
Urbanek Krajnc Andreja
Faculty of Agriculture and Life Sciences
Pivola 10, SI-2311 Hoce, Slovenia
andreja.urbanek@um.si
Vidaković-Cifrek Željka
University of Zagreb, Faculty of Science, Department of Biology
Rooseveltov trg 6, HR-10000 Zagreb, Croatia
zcifrek@biol.pmf.hr
Vodnik Dominik
Univerza v Ljubljani, Biotehniška fakulteta
Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia
dominik.vodnik@bf.uni-lj.si
Vujčić Valerija
Faculty of Science
Rooseveltov trg 6, 10000 Zagreb, Croatia
vvujcic@biol.pmf.hr
Vuletin Selak Gabriela
Institute for Adriatic Crops
Put Duilova 11, 21000 Split, Croatia
gabriela@krs.hr
Zadražnik Tanja
Kmetijski inštitut Slovenije
Hacquetova ulica 17, SI-1000 Ljubljana, Slovenia
tanja.zadraznik@kis.si
Zupin Mateja
Kmetijski inštitut Slovenije
Hacquetova ulica 17, SI-1000 Ljubljana, Slovenia
mateja.zupin@kis.si
Žerdoner Čalasan Anže
Fakulteta za naravoslovje in matematiko
Koroška cesta 160, SI-2000 Maribor, Slovenia
anze.z.c@gmail.com
96
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