DIEFFENBACHIA MACULATA

Transcription

Research Institute of Pomology and Floriculture, Skierniewice, Poland
PECTOLYTIC BACTERIA ASSOCIATED WITH SOFT ROT
OF DIEFFENBACHIA (DIEFFENBACHIA MACULATA)
A. Mikiciński, J. Puławska, P. Sobiczewski and L.B. Orlikowski
Abstract
On dieffenbachia potted plants growing in a greenhouse in central Poland,
beige water soaked as well as necrotic lesions located at the base of shoots and on
the leaves near the main vein were observed. Bacteria producing either circular,
convex, flat, white-grey or orange colonies were isolated from the border of diseased and apparently healthy tissue. Three isolates showed pectolytic activity, but
none of them induced HR on tobacco leaves. Pathogenicity tests indicated that isolates DLO1.1 and 2DLO2.3 caused symptoms on 37.5% of tested dieffenbachia
leaf petiole segments whereas DLO2.2 on 18.7% of such segments. Bacteria of the
same colony morphology were re-isolated from diseased tissue of artificially inoculated leaves. On the basis of phenotypic characterization and phylogenetic analysis
of 16S rRNA gene sequence, isolate DLO1.1 was found to be closely related to Bacillus pumilus, 2DLO2.3 to Flavobacterium defluvii and F. johnsoniae group and DLO2.2
to Chryseobacterium vrystaatense.
Key words: dieffenbachia, pectolytic bacteria, Bacillus pumilus, Chryseobacterium vrystaatense, Flavobacterium spp.
Introduction
Dieffenbachia (Dieffenbachia maculata), plant of the Araceae family, originates
from the tropical regions of Brazil (Chmiel 2000). Some species are cultivated in
Poland as potted ornamental plants. Bacterial leaf spot caused by bacterium
Xanthomonas axonopodis pv. dieffenbachiae was the first detected bacterial disease of
dieffenbachia (McCulloch and Pirone 1939). This disease was found regularly on
many other species of ornamental plants belonging to the family of Araceae, but
only occasionally on dieffenbachia (EPPO... 2004, Janse 2005). The most dangerous disease, however, is caused by soft rot bacterium Pectobacterium carotovorum
Phytopathologia 58: 21–32
© The Polish Phytopathological Society, Poznań 2010
ISSN 2081-1756
22
subsp. carotovorum, which can cause crop losses in greenhouses ranging up to 40%
(Cetinkaya-Yildiz et al. 2004). The bacterium causing the disease was isolated
mainly from the dieffenbachia leaves with soft rot symptoms. Dickeya dieffenbachiae
can also be the causal agent of soft rot of this plant (Janse 2005). This species was
discriminated in genus Dickeya. On the basis of polyphasic taxonomic study, genus
Dickeya contains all former strains of Erwinia chrysanthemi and Brenneria paradisiaca
(Samson et al. 2005).
In 2009, dieffenbachia plants grown in greenhouses in central Poland showed
various symptoms. The necrotic or watersoaked spots, located at the base or at different levels in the shoots and leaves near the midrib were observed. The aim of
this study was to investigate the etiology of these lesions.
Material and methods
Isolation of bacteria
From the leaves of D. maculata cv. ‘Camilla’ with disease symptoms (Phot. 1),
pieces of tissue from the edge of the spots were aseptically cut out and crushed in
sterile water. The resulting macerate was transferred with bacterial loop on nutrient agar medium with 5% sucrose (NAS) and streaked to obtain single colonies.
After 48 h of incubation at 24°C, some representative colonies were passaged on a
new NAS medium and used in further studies.
Test on potato tubers
Potato tubers were disinfected with
50% ethanol, cut up into slices of about
5 mm thick, and then placed on moist
filter paper in sterile Petri dishes. Approximately two loops of each bacterial
isolate grown on NAS medium were
uniformly spread onto the surface of the
slices. Development of rot on the slices
was examined 24–48 h after incubation
at 25°C (Mikiciński et al. 2010). Four
slices were inoculated with each isolate.
The experiment was repeated three
times.
Pectolytic activity on CVP medium
Phot. 1. The leaves of Dieffenbachia maculata cv.
‘Camilla’ with disease symptoms
(photo by A. Mikiciński)
Tests for the development of liquefied pits were observed on CVP (crystal
violet pectate) medium as described by
Pectolytic bacteria associated with soft rot of dieffenbachia...
23
Cuppels and Kelman (1974). The water-bacterial suspension of each isolate was
uniformly distributed with a glass rod on the surface of medium. Observations of
pits created around colonies were done within five days of incubation at 27°C.
Hypersensitive response on tobacco leaves
Leaves of tobacco cv. ‘Samsun’ were infiltrated with aqueous suspension
(107 cfu per 1 ml) of the tested bacterial strains according to Klement (1963). The
results were observed after 24 and 48 h.
Phenotypic characters of pectolytic bacteria
All pectolytic isolates were characterized using physiological and biochemical
tests which were selected according to keys of Buchanan and Gibbons (1974),
Bradbury (1988), Holt et al. (1994) and Schaad et al. (2001). The following properties of bacteria were determined: Gram reaction – staining, KOH reaction (Schaad
et al. 2001, Suslow et al. 1982), presence of L-alanine aminopeptydase (Merck,
Bacident®), spore production (Schaad et al. 2001), cell morphology (Holt et al.
1994), fluorescent pigment production (King et al. 1954), presence of oxidase,
catalase (Kovacs 1956, Bradbury 1988), oxidative/fermentative test (O/F) (Hugh
and Leifson 1953), levan production from sucrose (Lelliott and Stead 1987), hydrolysis of: gelatin, starch and aesculin (Fahy and Persley 1983), nitrate reduction,
presence of arginine dihydrolase, acid production from D-glucose, lactose,
D-mannitol, D-sorbitol, D-raffinose and D-xylose (Lelliott and Stead 1987).
For comparison, the reference strains of 853 (Pectobacterium carotovorum subsp.
carotovorum) and RIPF X02 (Xanthomonas axonopodis pv. dieffenbachiae) from own
collection and LMG 8337 (Chryseobacterium indologenes) (MCC-NIES Ghent, Belgium), were used. All tests were performed at least twice for each isolate.
Determination of the 16S rRNA gene sequences of pectolytic bacteria
All isolates were identified by sequence analysis of the PCR products of the 800
bp-long fragment of the 5’ end of 16S rRNA gene. Amplified DNA fragments with
fD1 and 800r primers (Weisburg et al. 1991, Drancourt et al. 1997) were directly
sequenced. Obtained sequences were compared to those available in the NCBI
GeneBank (http://www.ncbi.nlm.nih.gov) using BLAST N analysis to find the
closest relatives. Based on BLAST N analysis and classification of analysed isolates
to the genus level, 16S rRNA gene sequences of reference strains, of each species
within identified genera, were used for construction of phylogenetic trees. The genetic distances between the sequences were estimated by using the Kimura
two-parameter correction method (Kimura 1980). A phylogenetic tree was then
constructed by the neighbor-joining method (Saitou and Nei 1987). Inclusion of
16S rRNA gene sequences of isolates DLO2.2 and 2DLO2.3 to the analysis allowed
for recognition of their closest relatives.
24
Pathogenicity test on detached dieffenbachia leaves (Test I)
Young, 10 cm long leaves of D. maculata cv. ‘Camilla’ with 1 cm long segments
of petiole, were cut out from the plant with a sterile scalpel. The petiole base was
dipped in suspensions of the tested bacteria (109 cfu per 1 ml) for 5 min. Afterwards the leaves were placed on moist filter paper in Petri dishes and incubated at
26°C. For each isolate tested, 16 leaves were used. After seven days, the lesions
similar to those occurring on naturally infected plants were observed. These lesions were evaluated using the scale: (+ + +) – necrosis of half the length of the
midrib of the leaf, (+ +) – necrosis covering up to 1 cm of the petiole, (+) – necrosis of petiole smaller than 4 mm, ( ) – no necrosis. Each isolate was tested on
seven leaves. To fulfill the Koch’s postulates, bacteria were re-isolated from the
border between diseased and healthy tissue on NAS medium and their physiological characteristic was determined.
Pathogenicity test on dieffenbachia leaf petiole (Test II)
Leaf petioles of D. maculata cv. ‘Camilla’ were disinfected with 50% ethanol,
washed in sterile distilled water and cut in 5 cm long segments. The base of each
segment was dipped up to 1 cm into a 109 cfu per 1 ml suspension of the isolate
tested in a sterile test tube. The top of each test tube was covered with aluminum
foil. Pathogenicity of each isolate was tested on 16 leaf segments, which were incubated for seven days at 26°C. To fulfill the Koch’s postulates, bacteria were re-isolated from symptomatic tissue and plated on NAS medium.
Results and discussion
Bacteria of different colony morphology were isolated from leaves of dieffenbachia with rot symptoms. On NAS medium, in the case of DLO1.1, the bacteria
formed circular, flat colonies, which were white-gray, and in the case of DLO2.2,
slightly convex and orange. Isolate 2DLO2.3 grew as flat, orange colonies. Morphological and biochemical characteristics of the tested isolates varied from the
reference strains: 853 (P. carotovorum subsp. carotovorum), LMG 8337 (Ch. indologenes) and RIPF X02 (X. axonopodis pv. dieffenbachiae).
Common features of all tested bacteria, both obtained from dieffenbachia and
reference strains, were the lack of ability to synthesize fluorescent dye, the presence of catalase, levan production and lack of ability to produce acid from D-sorbitol. The obtained isolates macerated potato tissue and showed pectolytic activity
on CVP medium (Table 1, Phots. 2 and 3), as did the reference strains 853 and
RIPF X02. But, none of the tested isolates elicited a hypersensitivity reaction on tobacco leaves, even 48 h after infiltration. Only the reference strain 853 demonstrated this ability 24 h after the injection of bacteria to leaf mesophyll (Table 1).
Phenotypic characteristic of strains isolated from dieffenbachia
Test
Gray-white colony
25
Table 1
Orange colony
DLO1.1
853*
2DLO2.3
staining
+
–
–
–
–
–
L-alanine aminopeptydase
+
–
–
–
–
–
Gram reaction:
KOH reaction
+
–
–
DLO2.2 LMG 8337* RIPF X02*
–
–
–
Spore production
+
–
–
–
–
–
Cell morphology
rod
rod
rod
rod
rod
rod
Fluorescent pigment
–
–
–
–
–
–
Oxidase
–
–
+
+
+
–
Catalase
+
+
+
+
+
+
O/F
O/F
O
O
O
O
–
–
–
–
–
–
gelatin
+
–
+
+
+
+
aesculin
–
+
–/+
+
–/+
+
Nitrate reduction
+
–
–
–
–
–
Arginine dihydrolase
–
–
–/+
–
–
–
D-glucose
+
+
+
–/+
–/+
D-mannitol
+
+
–
–
–
D-raffinose
–
+
+
+
+
+
+
Oxidative/fermentative test (O/F)
Levan
Hydrolysis of:
starch
Acid production from:
lactose
D-sorbitol
D-xylose
Pectolytic activity on CVP
–
–
–
+
–
+
–
+
–
–
–
+
–
–
–
–
+
+
–
–
–
+
–/+
–
–
–
–/+
–
+
–
–/+
*Reference strains: 853 – Pectobacterium carotovorum subsp. carotovorum, LMG 8337 – Chryseobacterium indologenes), RIPF X02 – Xanthomonas axonopodis pv. dieffenbachiae.
Based on the analysis of phenotypic traits and sequence comparison of 16S
rRNA gene fragment, isolate DLO1.1 was classified to the species Bacillus pumilus.
This species is known as a biocontrol agent in the protection against fungal pathogens (Swadling and Jeffries 1998), as well as a factor inducing resistance in tomato
26
Phots. 2 and 3. Pectolytic activity on CVP medium (pits created around colonies)
of DLO2.2 and DLO1.1 isolates, respectively (photo by A. Mikiciński)
against cucumber mosaic cucumovirus (Zehnder et al. 2000). Among strains belonging to B. pumilus there are also those showing the ability to produce pectolytic
enzymes (Hallarsela et al. 1991). These enzymes cause soft rot of potato tubers
mainly during storage (Bradbury 1986). Isolates of B. pumilus with pectolytic activity, were also obtained from fruits of peach and apple trees with symptoms of
brown blotch (Saleh et al. 1997). For the first time, it was demonstrated that the
bacterium B. pumilus is able to induce soft rot on dieffenbachia.
Isolate DLO1.1 degradated the surface layers of potato tissue just 24 h after inoculation. Full degradation of slice was observed after three days. In the pathogenicity test (Test I) on the detached dieffenbachia leaves, symptoms of soft rot were
observed on three out of seven inoculated with this isolate leaves. In one case, the
necrosis covered the entire leaf petiole segment, in two others only part of it (Table
2). In test on leaf petiole segments (Test II, Table 2) seven days after being placed
in the test tubes with a suspension of the tested isolate 37.5% petiole segments
showed soft rot symptoms (Table 2). From the margin of rot on the petioles, bacteria were re-isolated and their morphology was identical to those that were used for
inoculation. In contrast, isolate 853 (P. carotovorum subsp. carotovorum) induced
very strong and rapidly spreading rot on almost all segments already after 48 h
from their deeping into inoculum. Strain RIPF X02 showed much lower virulence
but LMG 8337 was not able to cause any symptoms.
In the test on potato tubers, isolates 2DLO2.3 and DLO2.2 degraded tissue after 24 h, but the rot did not include the entire thickness of the slice. A similar tissue maceration was observed in case of strain RIPF X02 (X. axonopodis pv.
dieffenbachiae). As in the case of isolate DLO1.1, total tissue maceration was observed after three days. In the test on detached dieffenbachia leaves (Test I, Table
2), DLO2.2 caused soft rot on all seven leaf petioles (Table 2, Phot. 4 B). Isolate
2DLO2.3 caused rot on five of seven infected leaf petioles (Table 2, Phot. 4 C). In
test on leaf petiole segments (Test II, Table 2) symptoms of soft rot were visible on
Table 2
Pathogenicity of tested isolates
Isolate
Maceration
of potato
on tobacco tuber tissue
HR
Negative
control (water)
–
–
DLO1.1
2DLO2.3
–
DLO2.2
–
–
Test on detached
leaves* (Test I)
Test on leaf petioles** (Test II)
number of leaves
number of leaf
percentage
with disease
petioles with
of necrosis
symptoms
symptoms after:
in relation to
(in brackets – scale
entire length of
2 days 7 days petiole after 7 days
of infection***)
0
0
0
0.0
+
3 (1++, 2+)***
0
6
74.9
+
7 (7+)
0
3
+
5 (1+++, 4+)
0
6
853
+
+
7 (7+++)
15
16
RIPF X02
–
+
nt
0
3
LMG 8337
–
27
–
0
0
0
84.4
97.1
100 .0
0.
35.4
(+) – positive reaction, (–) – negative reaction, nt – not tested.
*Each isolate/strain was used for inoculation of 7 leaves.
**Each isolate/strain was used for inoculation of 16 leaf petioles ranging in length from 38.1 to 58.8
mm.
***(+++) – necrosis for half the length of the midrib and the petiole, (++) – only 1 cm long necrosis
on petiole, (+) – up to 4 mm necrosis on petiole.
37.5% inoculated of such segments, in the case of both isolates. However, isolate
2DLO2.3 caused soft rot on 84.4% of the length of all affected petioles (Table 2,
Phot. 5). Isolate DLO2.2 showed the lowest virulence – soft rot developed on only
18.7% of inoculated petioles, but it encompassed almost all of their length. In all
tests, from the border of healthy and diseased tissue, bacteria were re-isolated and
their morphology was identical to those used for inoculation.
Phot. 4. Pathogenicity test on detached dieffenbachia leaves: A – negative control, B and C – after
inoculation with isolates DLO2.2 and 2DLO2.3, respectively (photo by A. Mikiciński)
28
On the basis of phenotypic analysis
(Table 1) and comparison of sequences
of the 16S rRNA gene fragment, isolate
2DLO2.3 was classified to the genus
Flavobacterium while DLO2.2 to the genus Chryseobacterium. Based on phylogenetical analysis of 16S rRNA gene of
all available in GenBank species of the
Chryseobacterium genus, isolate DLO2.2
was found as most similar to Ch. vrystaatense while 2DLO2.3 as most similar both to Flavobacterium defluvii or
to F. johnsoniae. The genus Chryseobacterium was delineated from Flavobacterium by using the DNA-DNA
hybridization technique (Vandame et
Phot. 5. Pathogenicity test on dieffenbachia
al. 1994), and consists of 49 different
leaf petioles: left – inoculation of isolate
2DLO2.3, right – negative control
species, according to “List of Proka(photo by A. Mikiciński)
ryotic Names with Standing in Nomenclature” (http://www.bacterio.cict.fr).
Chryseobacterium vrystaatense was isolated from raw chicken the first time and it
is closely related to Ch. indologenes, Ch. joostei and Ch. gleum (De Beer et al. 2005).
These bacteria are widespread in the environment. They were found in water, soil
and also in food products such as milk, meat or fish (Jooste and Hugo 1999). Some
isolates of this species originating from plants, have the ability to limit the growth
of plant pathogenic fungi. Moline et al. (1999) attempted to control strawberry
against Botrytis cinerea with phyllosphere isolate of Ch. indologenes from this plant.
Our previous studies showed that Ch. indologenes having pectolytic activity is also
associated with soft rot of Zantedeschia spp. (Mikiciński et al. 2010). The only information about the Flavobacterium isolate with pectolytic activity concerns F. pectinovorum. This isolate is able to degrade the tori, bordered pit membranes and the
crossfield pits in Sitaka spruce (Fogarty and Owen 1973).
The results of pathogenicity tests on both the detached leaves and segments of
leaf petioles are remarkable. Pathogenicity of the tested isolates were variable. In
the case of isolate DLO2.2 all parts of petioles indicate disease symptoms, but not
very intensive. The remaining isolates did not infect all the leaves and petioles, but
on some of them, the disease severity was very high (Table 2). The observed differences could be associated with individual variation of the plant material used in the
study – each leaf and petiole came from a different plant. The ability to penetrate
deeply into plant tissue and fast spread of the rot may also be associated with the
ability of some bacteria to grown and produce pectolytic enzymes in anaerobic conditions, which is a case for facultative anaerobes belonging to the genus Pectobacterium spp. Łojkowska and Kelman (1994) showed that E. carotovora subsp.
carotovora in conditions of limited amount of oxygen can even increase the ability
to macerate potato tissue. It is also important that the tested bacteria are not typi-
29
cal pathogens of dieffenbachia but rather occasional ones and occurred on this
plant accidentally. In addition, the study of phenotypic traits of these bacteria
showed that they do not have the ability to use of a wide spectrum of carbon
sources, especially glucose (isolate DLO2.2, Table 1). This inability is a feature
that will undoubtedly influence the size of the bacterial population after penetrating into the tissues and its further spread. It is also proved that transcription of
particular pel genes coding pectolytic enzymes can depend on bacteria growth
phase and on many environmental factors like temperature, high osmolarity, nitrogen starvation or catabolite repression (Hugouvieux-Cotte-Pattat et al. 1992).
In study of strain E. chrysanthemi 3937 aimed on the role of pel and pem genes in
pathogenesis of soft rot on various plant species (Saintpaulia ionatha, Pisum sativum,
Solanus tuberosum and Cichorium intybus) it was proved that deletion of those genes
decreased virulence of this strain on all plants tested (Beaulieu et al. 1993). It was
also found that disease severity caused by this strain on various species can be related to one or more genes depending on plant inoculated. During pathogenesis pel
genes transcribe enzymes which can differ with activity depending on plant growth
phase as well as degree of disease development (Jafra et al. 1999). Thus, it is possible that our isolates studied possess very narrow spectrum of genes coding
pectolytic enzymes or that none of them are expressed in dieffenbachia tissues.
It is worth noting that among the bacteria obtained during this work from infected dieffenbachia plants, no Pectobacterium carotovorum subsp. carotovorum,
Dickeya dieffenbachiae or Xanthomonas axonopodis pv. dieffenbachiae, commonly known
as pathogens of this plant, were found.
Conclusions
1. The study showed for the first time that Bacillus pumilus, Chryseobacterium vrystaatense, Flavobacterium defluvii or F. johnsoniae can cause soft rot on dieffenbachia.
2. The virulence of the bacteria tested was estimated as low to medium.
3. From diseased tissues of dieffenbachia leaves and petioles Pectobacterium carotovorum and Dickeya dieffenbachiae (typical causal agents of soft rot) were
not isolated.
Streszczenie
BAKTERIE PEKTYNOLITYCZNE TOWARZYSZĄCE
MIĘKKIEJ ZGNILIŹNIE DIFENBACHII (DIEFFENBACHIA MACULATA)
Na roślinach difenbachii pochodzących ze szklarni w centralnej Polsce występowały objawy chorobowe w postaci uwodnionych, beżowych i nekrotycznych plam,
zlokalizowanych u podstawy pędów oraz w pobliżu głównego nerwu liści. W prób-
30
kach pobranych z pogranicza chorych i pozornie zdrowych tkanek wykryto bakterie,
tworzące na pożywce (agar odżywczy z sacharozą) lekko wypukłe lub płaskie, szarobiałe lub pomarańczowe, okrągłe kolonie. Trzy spośród pozyskanych izolatów wykazały aktywność pektynolityczną, ale żaden z nich nie wywoływał reakcji nadwrażliwości na liściach tytoniu. Na podstawie testu patogeniczności stwierdzono, że
izolaty DLO1.1 i 2DLO2.3 powodowały objawy chorobowe na 37,5% zainokulowanych fragmentów ogonków liściowych difenbachii, a DLO2.2 – na 18,7% takich fragmentów. W celu spełnienia postulatów Kocha z chorych tkanek reizolowano bakterie o morfologii podobnej do tych, których użyto do inokulacji. Analiza cech
fenotypowych i analiza filogenetyczna przeprowadzona z wykorzystaniem sekwencji
genu 16S rRNA pozwoliła na identyfikację izolatu DLO1.1 jako Bacillus pumilus
i stwierdzenie, że izolat 2DLO2.3 jest najbliżej spokrewniony z Flavobacterium defluvii
lub F. johnsoniae, a DLO2.2 – z Chryseobacterium vrystaatense.
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32
Authors’ address:
Artur Mikiciński M.Sc., Dr. Joanna Puławska, Prof. Dr. hab. Piotr
Sobiczewski, Prof. Dr. hab. Leszek B. Orlikowski, Research Institute of
Pomology and Floriculture, ul. Pomologiczna 18, 96-100 Skierniewice, Poland,
e-mail: Artur.Mikicinski@insad.pl
Accepted for publication: 7.11.2010

DIEFFENBACHIA MACULATA

Transcription

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