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Applied and Environmental Microbiology, February 2000, p. 850-854, Vol. 66, No. 2
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Pathovars of Pseudomonas syringae
Causing Bacterial Brown Spot and Halo Blight in Phaseolus
vulgaris L. Are Distinguishable by Ribotyping
Ana J.
González,1,*
Elena
Landeras,2 and
M.
Carmen
Mendoza3
Servicio Regional de Investigación y
Desarrollo Agrario, Consejería de Agricultura,
Ganadería y Pesca, 33300-Villaviciosa,1
Laboratorio de Sanidad Vegetal, Consejería de
Agricultura, Ganadería y Pesca,
33071-Oviedo,2 and Departamento de
Biología Funcional, Area Microbiología, Facultad de
Medicina, Universidad de Oviedo,
33006-Oviedo,3 Principado de Asturias, Spain
Received 26 July 1999/Accepted 19 November 1999
 |
ABSTRACT |
Ribotyping was evaluated as a method to differentiate between
Pseudomonas syringae pv. phaseolicola and pv. syringae
strains causing bacterial brown spot and halo blight diseases in
Phaseolus vulgaris L. Ribotyping, with restriction enzymes
BglI and SalI and using the Escherichia
coli rrnB operon as the probe, differentiated 11 and 14 ribotypes, respectively, and a combination of data from both procedures
yielded 19 combined ribotypes. Cluster analysis of the combined
ribotypes differentiated the pathovars phaseolicola and syringae, as
well as different clonal lineages within these pathovars. The potential
of ribotyping to screen for correlations between lineages and factors
such as geographical region and/or bean varieties is also reported.
| |
TEXT |
Phytopathogenic bacteria of
the Pseudomonas syringae group cause diseases in all
major groups of higher plants and can be divided into more than 50 pathovars. The term pathovar is used to refer to strains with similar
features that are differentiated at the subspecies level on the basis
of differences in plant host range and types of symptoms, and
additionally by biochemical profiles (7, 12, 16). Such a
phenotypically based classification is of practical interest, but it
does not reveal the genetic relatedness within or between pathovars
(1, 3). In this work, a two-way ribotyping procedure was
evaluated as a method to distinguish between P. syringae pv.
syringae and pv. phaseolicola organisms isolated from Phaseolus
vulgaris L., which are the causal agents of bacterial brown spot
and halo blight, respectively.
Isolation procedure and phenotypic characterization of P. syringae strains.
The P. syringae isolation
procedure used has been described elsewhere (6, 10, 14). The
samples of bean plants analyzed were collected at different times from
different bean fields in a geographical area of about 100,000 km2 in the north of Spain, over the period 1991 to 1997. This area includes two different climatic regions: the Cantabrian coast area, wet and temperate (WT), and the northern inland meseta, dry and
continental (DC). P. syringae organisms were isolated from
19.0 and 40.9% of sets of infected plant material (leaves and/or pods
showing either typical or suspected symptoms of bacterial brown spot
and halo blight diseases) collected in the WT and DC regions,
respectively, and from 26.1% of damaged seed sets, each supplied by a
different farmer in the WT region. Of the isolated organisms, only the
61 strains compiled in Table 1 were
characterized in this work.
The initial strain identification was based on the Hugh-Leifsson
reaction and the LOPAT scheme (production of levane, oxidase,
pectinases, and arginine dihydrolase, as well as tobacco
hypersensitivity)
(
7). Pathovar confirmation included tests
for esculin hydrolysis
and acid production from mannitol, erythritol,
and myoinositol
(
6,
10) as well as an enzyme-linked
immunosorbent assay-double-antibody
sandwich indirect procedure using
an antiserum specific for flagellar
antigens of pathovar phaseolicola
strain CFBP 1390. These tests
led us to assign the
P. syringae strains to two well-defined pathovars;
phaseolicola (46 strains) and syringae (15 strains). It is noticeable
that in our series
a total correlation between negative esculin
hydrolysis and pathovar
phaseolicola and between positive esculin
hydrolysis and pathovar
syringae was revealed; besides, this test
is rapid, cheap, and easily
performed. The assignment of the pathovar
phaseolicola strains to
defined races according to the scheme
published in reference
15 was carried out in the Servicio de
Investigación, Desarrollo y Tecnología Agraria,
Valladolid,
Spain. By this procedure, 32 strains were differentiated
into
races 2, 5, 6, 7, and 9, whereas 10 other strains could not be
assigned to a defined race and were labeled as nonidentified (NI).
Findings of epidemiological interest were that in leaves and/or
pods
from the two climatic regions both pathovars were found but
that
pathovar phaseolicola was much more frequent in the DC region.
Conversely, from seeds (all collected from a single autochthonous
bean
variety named Granja Asturiana within the WT region) only
pathovar
syringae organisms were
identified.
Two-way ribotyping of P. syringae group strains.
Isolation of genomic DNA and ribotyping were carried out as reported in
reference 9. Ribotyping was performed with the enzymes BglI and SalI (Stratagene, La Jolla,
Calif.), and hybridization was performed with an rrnB operon
from Escherichia coli and use of the nonradioactive DNA
labeling and detection kit of Boehringer GmbH (Mannheim, Germany). The
polymorphic restriction sites along the ribosomal DNA regions were
inferred from the presence or absence of bands in the overall ribotypes
from each enzyme. In addition to the 61 strains cited above, three
other strains of P. syringae were analyzed and used as
outgroup strains: pathovar phaseolicola strain ATCC 19304 (Colección Española de Cultivos Tipo; CECT 321); pathovar
tomato strain ATCC 10862 (CECT 126); and one unspecified-pathovar strain, Boelema P2 (CECT 94), collected from Pisum sativum.
With BglI, 13 different B ribotypes were found in the series
(Fig. 1A), two of which (B1 and B2) were
represented by pathovar phaseolicola and nine of which (B21 to B29)
were represented by pathovar syringae organisms. Pathovar phaseolicola
strain ATCC 19304 generated the B1 ribotype, while the
unspecified-pathovar strain Boelema 2 and pathovar tomato strain ATCC
10862 generated ribotypes not represented among the Spanish strains.
The B ribotypes included between four and eight fragments that appeared
in the region below 5.2 kb. Only one fragment of about 1.4 kb was
common to all of them, and 20 polymorphic restriction sites were
revealed. The number and size of the fragments indicate the presence of
one or more BglI sites within the five rrn
operons of P. syringae strains. With SalI, 15 different S ribotypes were found in the series (Fig. 1B), six of these
(S1 to S6) being represented by pathovar phaseolicola and eight (S21 to
S28) being represented by pathovar syringae. Pathovar phaseolicola
strain ATCC 19304 fell into the S6 ribotype, the unspecified-pathovar
strain Boelema P2 generated an S ribotype not represented among the
Spanish strains, and pathovar tomato strain ATCC 10862 could not be
identified by this procedure (successive SalI digestions
failed). The S ribotypes included between three and five fragments that
appeared in the region between 14.2 and 6.6 kb. None of the fragments
were common to all S ribotypes, but a fragment of about 7 kb appeared
in the S ribotypes represented by pathovar phaseolicola strains and
another fragment of about 12 kb appeared in the S ribotypes from
pathovar syringae strains. These data suggest that the strains have no
SalI cutting points within rrn operons and that
each band usually carries only one rnn operon
(2). However, in S ribotypes with fewer than five bands some
fragments could carry two neighbor rrn operons or, alternatively, could correspond to two different DNA fragments of very
similar sizes.
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FIG. 1.
Ribotypes from P. syringae strains causing
disease in the common bean. Ribotypes were generated by BglI
(A) and SalI (B). Lane T, ribotype from pathovar tomato
strain ATCC 10862; lane UP, ribotype from the unspecified-pathovar
Boelema P2 strain; Phas. and Phaseolicola, ribotypes from pathovar
phaseolicola strains; Syringae, ribotypes from pathovar syringae
strains. Values to the left of the gels indicate sizes, in kilobases.
Data for phytopathogenic strains representing each ribotype are
compiled in Table 1.
|
|
The performance of two-way ribotyping was evaluated according to
several criteria (
13). With the enzymes used, all the
P. syringae group strains, except pathovar tomato strain
ATCC 10862,
which failed with
SalI, could be assigned to
well-differentiated
ribotypes. Reproducibility and ease of
interpretation of the band
profiles were very good. The profiles from
each enzyme showed
a certain degree of similarity with one another and
were pathovar
specific, thus revealing the relatedness of
P. syringae organisms.
The discriminatory power (not including the
three reference strains)
was tested by considering the number of
ribotypes generated using
each enzyme and by the calculation of the
discrimination index
(DI) (
3,
5). The number of ribotypes
(and DIs) obtained
was lower with
BglI than with
SalI: 11 and 14 ribotypes (DI =
0.48 and 0.84),
respectively. By combining profiles from both
enzymes, 19 combined
ribotypes (DI = 0.86) were generated, and
the two pathovars were
subdivided: pathovar phaseolicola into
7 (DI = 0.73) and pathovar
syringae into 12 (DI = 0.96) combined
ribotypes. Within pathovar
phaseolicola, when ribotypes were combined
with races, a further
differentiation was revealed, 15 subtypes
(DI = 0.93). For these
calculations, only the 42 strains tested
for races could be used, and
the NI strains were considered a
single and different type. Ribotyping
presents other advantages
such as accessibility (requiring basic DNA
analysis equipment,
as well as commercial materials and reagents) and
flexibility
(it can be used for different bacterial species). On the
other
hand, it cannot be overlooked that ribotyping is a laborious
procedure,
requiring multiple
steps.
It must also be noted that
BglI ribotyping can be a useful
tool to identify pathovar phaseolicola organisms, on the basis
of the
specific band profiles generated. However, it is not a
good tool to
differentiate pathovar phaseolicola organisms, because
in the series
studied all except two strains generated a single
band profile. A
correlation between ribotypes and races was not
revealed, with the
following exceptions. The two race 2 strains
generated the B2 ribotype,
which was not represented by any other
strain of the series. The five
strains from the Granja Asturiana
bean variety were assigned to race 5, B1-S3 combined ribotype
and race NI, B1-S6 combined ribotype, which are
feature combinations
not registered among the strains from the
remaining bean varieties.
However, strains of different races fell into
a single combined
ribotype (races 5, 7, and NI into B1-S1 and races 6 and 7 into
B1-S3), while strains assigned to NI generated a single B
ribotype
(B1) but four different S ribotypes (S1, S3, S5, and
S6).
For the phylogenetic analysis, data from ribotypes were used. A
combined numerical analysis of the different banding profiles
revealed
by each enzyme was performed with a software package
as previously
described (
9). A high heterogeneity of ribosomal
DNA regions
of the strains under study (similarity between 22
and 93%) was
registered, and the similarity dendrogram showed
that different
groupings could be observed by varying levels of
similarity (Fig.
2). At a low level (
S = 0.46), the strains were
distributed into two clusters labeled A
(including only pathovar
phaseolicola strains) and B (including all
pathovar syringae strains
as well as the Boelema P2 strain). These
groupings support previous
studies assigning strains of the pathovars
phaseolicola and syringae
to different genospecies (
1,
3,
4,
8,
11). At a
higher level (
S = 0.66), both clusters
were differentiated into
subclusters and branches. Organisms falling
into each one of these
groupings could be considered members of the
same clonal lineage.
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FIG. 2.
Dendrogram obtained from cluster analysis of
BglI and SalI ribotypes of P. syringae
group strains. Each branch represents a combined ribotype. A and B and
A1, B1, and B2 are the clusters and
subclusters revealed at similarity coefficients of 0.43 and 0.68, respectively. The pathovar, race, and climatic area where the strains
falling into each branch were collected are indicated. Other features
of the strains and clusters are shown in Table 1 and/or described in
the text.
|
|
The above-mentioned data have also been applied to further our
knowledge in the contemporary molecular epidemiology of
P. syringae causing bacteriosis in the common bean in two Spanish
climatic regions which are very important bean production areas.
Some
findings to emphasize are the following. (i) Ribotyping procedures
are
useful tools for distinguishing individual phytopathogenic
strains of
P. syringae, allowing the assignment of organisms with
defined ribotypes to specific pathovars and the grouping of strains
with identical ribotypes into clones. (ii) Different pathogen
clones
have been selected, but only some pathovar phaseolicola
clones appeared
with a wide geographic spread. (iii) The cluster
analysis grouped
clones into subclusters which were considered
clonal lineages, enabling
us to screen relationships between lineages
with some bean varieties
and/or climatic regions. Two lineages
(pathovar phaseolicola subcluster
A
1 and pathovar syringae subcluster
B
1)
presented a wide range of hosts and were widespread, while
a third
lineage (pathovar syringae subcluster B
2) showed a specific
host range and could be considered endemic in the WT region. Organisms
of this last lineage were collected only from the Granja Asturiana
bean
variety (which is large, with a highly appreciated sensory
quality). It
is also noteworthy that the only two pathovar phaseolicola
race 2 strains appeared as members of a fourth lineage. These
findings support
ribotyping as a useful tool for specific epidemiological
studies such
as establishing the endemic types versus those introduced
for the first
time in a country or geographical area and ascertaining
the pathogenic
strains carried by bean seeds for sowing or in
seeds from other
countries with different endemic
P. syringae lineages. This
last point could be particularly useful to protect
the production of
specific autochthonous dry bean varieties of
high market value, such as
the Spanish Granja
Asturiana.
| |
ACKNOWLEDGMENTS |
We thank M. Altwegg for the pKK3535 plasmid, the source of the
rrn probe; F. Uruburu of CECT for the P. syringae
reference strains; C. Jordá of the Universidad Politécnica
de Valencia for the specific antiserum of pathovar phaseolicola strain
CFBP 1390; C. Asensio of the Servicio de Investigación,
Desarrollo y Tecnología Agraria, Valladolid, for the
identification of pathovar phaseolicola races and samples of infected
plants from the DC region; and R. Marquinez of the Instituto de
Semillas y Plantas de Vivero, Vitoria, for the País Vasco
strains. We are also indebted to M. R. Rodicio for her critical
revision of the manuscript.
This work has been supported by a grant from the Instituto Nacional de
Investigación Agraria y Alimentaria, Ministerio de Agricultura,
Pesca y Alimentación, Spain (reference SC-94-051).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: SERIDA, Apartado
13, 33300 Villaviciosa, Asturias, Spain. Phone: 34-985890066. Fax: 34-985891854. E-mail: anagf{at}princast.es.
| |
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Applied and Environmental Microbiology, February 2000, p. 850-854, Vol. 66, No. 2
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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