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X. arboricola pv. juglandis



 
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İletiTarih: 16 Mar Pts, 2015 8:20 pm    İleti konusu: X. arboricola pv. juglandis Alıntıyla Cevap Gönder

Journal of Plant Pathology (2012), 94 (1, Supplement), S1.23-S1.27 Edizioni ETS Pisa, 2012 S1.23
CHARACTERIZATION OF XANTHOMONAS ARBORICOLA pv. JUGLANDIS
ISOLATED FROM WALNUTS IN LITHUANIA
D. Burokiene1 and J. Pulawska2
1Institute of Botany at the Nature Research Centre, Laboratory of Phytopathogenic Microorganisms,
Zaliuju Ezeru Str. 49, 08406 Vilnius, Lithuania
2Research Institute of Horticulture, Pomology Division, ul. Pomologiczna 18, 96-100 Skierniewice, Poland
SUMMARY
This study records the presence of bacterial walnut
blight, caused by Xanthomonas arboricola pv. juglandis
(Xaj), on walnuts (Juglans spp.) in Lithuania and confirms
that its geographic distribution has been underestimated.
The pathogen caused the most serious symptoms
on J. cinerea and J. mandshurica trees. DNA typing
methods (rep-PCR and MLST) used for characterization
of Xaj isolates disclosed a high similarity among
them. Strains from Lithuania, however, showed a still
not fully understood difference from Xaj strains from
Poland, a neighbouring country, that needs further investigation.
Key words: Juglans spp., bacterial walnut blight, rep-
PCR, MLST, diagnosis.
Lithuania is the northernmost country in Europe
where walnuts are successfully overwintering, fruits
ripen and can be harvested. Walnuts are grown mainly
individually in private and botanical gardens and parks
and in a limited number of nurseries (Navasaitis, 2004).
Thus, walnuts have no economic importance in Lithuania,
although the interest for the crop is growing and
new species and varieties are introduced. At the moment,
five walnut species and a few interspecific hybrids
are grown in the country and the nuts harvested.
Bacterial walnut blight caused by Xanthomonas arboricola
pv. juglandis (Xaj) is one of the most serious and economically
important diseases of walnut, occurring in all
major walnuts-growing areas of the world (CABI-EPPO,
2001). There are no effective control methods, thus bacterial
walnut blight may cause considerable yield losses
(up to 70%) under favourable conditions for the disease
(Gironde et al., 2009; Lang and Evans, 2010; Mulrean
and Schroth, 1981). Till now, walnut bacterial diseases
have not been investigated in Lithuania.
In the years 2008-2009, different disorders were observed
on leaves and nuts of Juglans spp. trees. Leaves
showed necrotic leaf veins and irregularly shaped, small,
dark brown spots, gradually increasing in size (Fig. 1).
Characteristic sunken brown to black spots with a
chlorotic margin were found on fruits and exudates
were observed under humid environmental conditions
(Vasinauskiene et al., 2007). These symptoms corresponded
to those of walnut blight caused by Xanthomonas
arboricola pv. juglandis (Xaj) as described in
literature (Mulrean and Schroth, 1982) and bacteria,
forming yellow colonies on semi-selective brilliant cresyl
blue starch agar were consistently isolated from them.
The aim of this study was to identify and to characterize
genetically the causal agent of bacterial walnut
blight in Lithuania using genetic diversity analysis of the
pathogen with nucleic acid-based techniques (rep-PCR
and MLST).
Five walnut species, Juglans cinerea L., J. mandshurica
Maxim., J. regia L., J. nigra L., J. ailantifolia Carr. and two
hybrids, J. x bixbyi Rehd. (J. cinerea x J. ailantifolia) and J.
x quadrangulata (Carr.) Rehd. (J. cinerea x J. regia) were
inspected. Thirty plant samples showing typical blight
symptoms were collected and analysed. A total of 59
Xanthomonas-like isolates were recovered from sample
homogenized with mortar and pestle and streaked on
brilliant cresyl blue starch agar (Mulrean and Schroth,
1981). When DNA extracts from all isolates were submitted
to PCR using the Xanthomonas genus-specific
primers X1 and X2 (Maes, 1993), 8 isolates yielded the
Xanthomonas-specific PCR amplification product of ca.
480 bp. Five strains out of 8 were identified as Xaj after
PCR with the pathovar-specific primers XajF and XajR
(Gironde et al., 2009), showing the expected DNA product
of 216 bp.
The five Xaj-specific PCR-positive isolates were ultimately
identified through pathogenicity, biochemical
and physiological tests. Strain Xaj LMG 746 and data
from the literature served as reference (Klement et al.,
1990; Lelliott and Stead, 1987; Schaad et al., 2001). Xajlike
isolates grew on potato dextrose agar, yeast extractglucose-
calcium carbonate and yeast extract-dextrosecalcium
carbonate agar as slimy yellow colonies (Lelliott
and Stead, 1987). All isolates were Gram-negative with
oxidative metabolism of glucose only, did not produce
oxidase and catalase, grew at 37°C, hydrolysed starch
Corresponding author: D. Burokiene
Fax: +370.5.2729950
E-mail: daiva.burokiene@botanika.lt
005_COST(Burokiene)_S23_COL 14-06-2012 18:01 Pagina 23
and esculin, induced hypersensitive reactions on tomato
leaves (Schaad et al., 2001; Lelliott and Stead, 1987),
caused necrosis on immature walnut fruits when tested
according to Aletà et al. (2001) and grew on succinate
quinate medium (Lee et al., 1992). All these features
were comparable to those of the reference strain LMG
746. Thus, based on disease symptoms and the characteristics
they were tested for, the five isolates in question
were identified as X. arboricola pv. juglandis, and denoted
NRCIB X1, NRCIB X2, NRCIB X3, NRCIB X4 and
NRCIB X5.
To determine their genetic diversity multilocus sequence
typing (MLST) and repetitive sequence-based
polymerase chain reaction (rep-PCR) were conducted
on total DNA prepared according to Aljanabi and Martinez
(1997). As reference, 4 known Xaj strains from
Poland (RIPF GH1, RIPF X04, RIPF X06, RIPF X07)
and 3 strains from other European culture collections,
LMG 746 (UK), CFBP 7179 (France), I-391 (Portugal)
were used.
In our MLST study partial sequences of three genes
were investigated, i.e. gyrB (DNA gyrase subunit B),
fyuA (tonB-dependent receptor) and rpoD (RNA polymerase
sigma factor). The fragment of gyrB gene was
amplified according to Parkinson et al. (2007). PCR
products from fyuA and rpoD genes were obtained using
the primers described by Young et al. (2008). Sequences
of Xaj strains from Lithuania were deposited in
GenBank with accession numbers HE610436 to
HE610450. Concatenated sequences of sequenced loci
with a total length of 1,939 bp were submitted to phylogenetical
analysis using the MEGA 5 software package
S1.24 X. arboricola pv. juglandis in Lithuania Journal of Plant Pathology (2012), 94 (1, Supplement), S1.23-S1.27
Fig. 1. Symptoms of bacterial blight on walnut shoots (A), leaves (B) and fruit (C) observed in Lithuania in 2009.
Fig. 2. Maximum likelihood tree showing the phylogenetic relationship between strains Xaj strains from
Lithuania, Poland and other reference strains based on concatenated sequences of genes fyuA, gyrB and
rpoD. Bootstrap values (expressed as percentages of 1000 replications) are given at the nodes. Bar – estimated
nucleotide substitutions per site is 0.01.
005_COST(Burokiene)_S23_COL 14-06-2012 18:01 Pagina 24
with the Maximum likelihood method and the Tamura-
Nei model (Tamura et al., 2011). Bootstrap analysis with
500 replicate data sets was performed to assess the validity
of the clusters found.
All Xaj strains were grouped in one monophyletic
cluster the similarity of their concatenated sequences
ranging from 98.2 to 100%. The Lithuanian strains had
identical sequences for all tested genes. There was a difference
with sequences of strains from other countries,
but the level of similarity was still high, e.g. for Polish
strains it was 98.3-100%, 97.3% and 100% in the case
of genes fyuA, gyrB and rpoD, respectively (Fig. 2).
For rep-PCR, carried out according to Louws et al.
(1994) and Versalovic et al. (1991, 1994), the primers
targeting the conserved repetitive sequences REP, ERIC
and BOX were used. Rep-PCR-amplified bands were
scored as 1 (present) or 0 (absent) for all strains. Only
reproducible, unambiguous, and clear-cut genomic fingerprints
were taken into account. Reproducibility of
the rep-PCR results was tested by repeating the reactions
at least twice under the same conditions. No
changes in DNA fingerprints were observed in any of
the replicated experiments. Cluster analysis was performed
using Free Tree software (Hampl et al., 2001)
and dendrograms were constructed from Jaccard’s similarity
coefficient data by UPGMA.
Rep-PCR was suitable for the molecular characterization
of Xaj strains (Fig. 3). Fingerprints obtained in our
assays disclosed a substantial genetic diversity among the
few Xaj strains studied, coming from two different geographical
areas (Lithuania and Poland). Fingerprint profiles
generated with BOX, REP or ERIC primers, were
different for Lithuanian and Polish strains of Xaj and two
clusters were observed. REP PCR primers were the best
for discrimination of Xaj (Fig. 4). No differences were
Journal of Plant Pathology (2012), 94 (1, Supplement), S1.23-S1.27 Burokiene and Pulawska S1.25
Fig. 3. Dendrogram of relationships between Xanthomonas arboricola pv. juglandis strains analysed
by rep-PCR using primers BOX, ERIC and REP. The results per strain were clustered by an unweighted
average pair group method (UPGMA) using Jaccard’s coefficient.
Fig. 4. Characteristic rep-PCR fingerprint patterns of Xaj using
REP PCR primers.
M
LMG 746
CFBP 7179
I-391
RIPF X04
NRCIB X1
NRCIB X2
NRCIB X3
NRCIB X4
NRCIB X5
RIPF GH1
RIPF X06
RIPF X07
M
005_COST(Burokiene)_S23_COL 14-06-2012 18:01 Pagina 25
observed among the 5 Lithuanian strains, even though
they were isolated from different Juglans species, i.e.
Juglans cinerea (NRCIB X1 and NRCIB X2) and J. mandshurica
(NRCIB X3, NRCIB X4 and NRCIB X5).
In the present study, Xaj was isolated from J. cinerea
and J. mandshurica in Lithuania but not from other
Juglans species that showed symptoms resembling those
of walnut blight but were infected by Melanconium oblongum
Berk. and Gnomonia leptostyla (Fr.) Ces. et De
Not., as shown by the successful recovery of these fungi.
In our hands, both MLST and rep-PCR proved useful
for the characterisation of bacterial isolates at the pathovar
level and for discriminating strains within the pathovar.
The genetic diversity between Xaj strains had already
been studied using AFLP, rep-PCR, MLST (Loreti et al.,
2001; Marcelletti et al., 2010; Rademaker et al., 2005) and
some differences between populations were found. Some
relation between genetic characteristics geographical origin
and virulence of the pathogen was found in one study
(Scortichini et al., 2001). Marcelletti et al. (2010) after
analysis of four housekeeping genes of 45 Xaj strains did
not support the notion that the genetic diversity of the
strains is associated with geographical isolation, in contrast
with our finding that Lithuanian Xaj isolates could
be discriminated from some Polish ones. However, to
what extent the observed difference is to be attributed to
geographical separation remains to be elucidated with
further studies using more strains.
Our study shows that in Lithuania, as in many other
European countries, walnut trees are vulnerable to walnut
blight caused by Xaj. Since walnut blight have not
been investigated in Lithuania so far, the present investigation
is epidemiologically important and provides
new information on the spread of this disease and
pathogen in Europe. A better understanding of the
structure and dynamics of pathogenic bacteria populations
may stimulate the development of more efficient
control measures and of more advanced and specific diagnostic
protocols.
ACKNOWLEDGEMENTS
This work was conducted within the framework of
COST Action 873 and was supported by the Science
Council of Lithuania and the Polish Ministry of Science
and Higher Education Grant 118/N-COST/2008/0.
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S1.26 X. arboricola pv. juglandis in Lithuania Journal of Plant Pathology (2012), 94 (1, Supplement), S1.23-S1.27
005_COST(Burokiene)_S23_COL 14-06-2012 18:01 Pagina 26
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Journal of Plant Pathology (2012), 94 (1, Supplement), S1.23-S1.27 Burokiene and Pulawska S1.27
005_COST(Burokiene)_S23_COL 14-06-2012 18:01 Pagina 27
005_COST(Burokiene)_S23_COL 14-06-2012 18:01 Pagina 28
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Tarih: 16 Mar Pts, 2015 8:20 pm    İleti konusu: X. arboricola pv. juglandis

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İletiTarih: 16 Mar Pts, 2015 8:23 pm    İleti konusu: Re: X. arboricola pv. juglandis Alıntıyla Cevap Gönder

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