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Applied and Environmental Microbiology, October 1998, p. 3961-3965, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Relatedness of Strains of Xanthomonas fragariae by
Restriction Fragment Length Polymorphism, DNA-DNA Reassociation,
and Fatty Acid Analyses
P. D.
Roberts,1
N. C.
Hodge,2
H.
Bouzar,1
J. B.
Jones,1,2,*
R. E.
Stall,2
R. D.
Berger,2 and
A.
R.
Chase3
Gulf Coast Research and Education Center,
University of Florida, Bradenton, Florida
342031;
Plant Pathology Department,
University of Florida, Gainesville, Florida
326112; and
Apopka Research and
Education Center, University of Florida, Apopka, Florida
327033
Received 3 March 1997/Accepted 20 July 1998
 |
ABSTRACT |
The levels of relatedness of strains of Xanthomonas
fragariae collected over several years from locations in Canada
and the United States were compared by determining fatty acid methyl
ester profiles, restriction fragment length polymorphisms
(RFLP) based on pulsed-field gel electrophoresis (PFGE) analysis, and
DNA-DNA reassociation values. Based on qualitative and quantitative
differences in fatty acid profiles, the strains were divided into nine
groups and four groups by the MIDI "10% rule" and unweighted pair
analysis, respectively. Restriction analysis of genomic DNA by PFGE
with two endonucleases (XbaI and SpeI) revealed
four distinct profiles. When a third endonuclease (VspI)
was used, one group was divided into three subgroups. The profile of
the American Type Culture Collection type strain differed from the
profile of every other strain of X. fragariae.
Considerable diversity was observed within X. fragariae,
although the majority of the strains represented a clonal population.
The four groups based on fatty acid profiles were similar to the four
groups based on RFLP, but neither method related groups to the
geographic origins of the strains. The DNA-DNA reassociation values
were high for representative strains, providing evidence that all of
the strains belong to the same species.
| |
INTRODUCTION |
Xanthomonas fragariae
causes angular leaf spot disease on strawberries (Fragaria
species and Fragaria × ananassa Duch.),
which results in decreased yields (7, 18, 32). International movement of infected plants is blamed for the introduction of angular
leaf spot into Greece and New Zealand (5, 30). The disease
has recently become more important in fruit production fields in
Florida, but control measures are generally ineffective (25,
26). The variation within X. fragariae must be
determined in order to design effective control strategies.
Although strawberry cultivars exhibit different levels of resistance
to X. fragariae (14, 15, 20), no known
races of the pathogen have been identified. Thus, a screening program
to identify resistance genes must include representatives of the
major genetic variants in an attempt to identify genes for
resistance to all strains. Although the relationship of X. fragariae to other members of the genus Xanthomonas has been examined (4, 17, 39), no extensive analysis of strains belonging to this species from diverse locations has been performed previously.
Variability within bacterial populations has been examined by
biochemical and molecular biological techniques. Protein staining and
fatty acid analyses have identified differences at the metabolic level
in Xanthomonas species (1, 12, 37). The PCR and
restriction fragment length polymorphisms (RFLP) have been used to
identify genetic variability (3, 6, 11, 13, 16, 22, 23, 36-38). The hrp gene cluster primers developed by
Leite et al. (23) were used to amplify a region of the
X. fragariae genomic DNA from which primer sequences
specific for X. fragariae were selected
(33). Fifty strains of X. fragariae produced
identical restriction enzyme patterns following amplification of
a 448-bp fragment with the X. fragariae-specific
primers and restriction endonuclease digestion of the PCR product
(33). Primer sets derived from conserved repetitive
bacterial DNA sequences (repetitive extragenic palindromic
[REP], BOX, and enterobacterial repetitive intergeneric
consensus [ERIC]) generated genomic fingerprints that were used to
differentiate phytopathogenic Xanthomonas spp. and
Pseudomonas spp. (4, 24). A unique genomic
fingerprint was generated by REP-PCR and ERIC-PCR for reference strains
of X. fragariae which were used to identify field
strains of the pathogen (29). Three PCR methods identified
closely related genetic variants within a population of 25 strains of
X. fragariae (31). Random amplified
polymorphic DNA PCR and REP-PCR performed with REP and ERIC primers
identified several genotypes among 25 strains of X. fragariae; however, there was no correlation between genotype and
geographic origin.
Although other workers (29, 31) have determined genetic
variation within X. fragariae, this study was
undertaken to characterize the genetic and phenotypic diversity
of X. fragariae strains collected over several
years from diverse locations in the United States and Canada. Profiles
used to identify variation were generated by pulsed-field gel
electrophoresis (PFGE) and by gas-liquid chromatography of
cellular fatty acids. A DNA-DNA reassociation analysis of
representative strains belonging to RFLP-fatty acid methyl ester (FAME)
groups was performed to confirm the species affiliations of the strains in an attempt to determine if diversity resulted from infraspecific or
interspecific variation.
| |
MATERIALS AND METHODS |
Bacterial strains.
The strains of X. fragariae used in this study are listed in Table
1. These strains were previously tested
for pathogenicity on strawberry plants (33). The strains
were isolated from diseased strawberry plants from the major
strawberry-growing regions in the United States and Canada. The strains
were cultured on Wilbrink's medium (21) at 24°C, and
long-term storage was in 15% glycerol at 
70°C. Overnight cultures
used for PFGE plugs were prepared by inoculating single colonies into 5 ml of nutrient broth (Difco Laboratories, Detroit, Mich.) and shaking
the preparations for 16 to 20 h at 200 rpm at 24°C.
RFLP.
The method used to perform the restriction
endonuclease analysis was the method described by Egel et al.
(6) and Cooksey and Graham (3), with the
following modifications. Cells (1.5 ml of a 5 × 109-CFU/ml suspension) grown in nutrient broth were washed
in 1 ml of TE buffer (10 mM Tris, 1 mM EDTA; pH 8.0) and resuspended in 0.5 ml of TE buffer. An equal volume of a 1% Seakem Gold agarose solution (10 mM Tris [pH 8.0], 10 mM MgCl2, 0.1 mM EDTA
[pH 8.0], 1% [wt/vol] Seakem Gold agarose [FMC BioProducts,
Rockland, Maine]) in sterile filtered water was added to the washed
cells. Plugs containing DNA were made and stored as described by Egel
et al. (6). Sections of the plugs that were 4 by 8 and 4 by
4 mm were digested in 200 µl of restriction buffer (as recommended by
the manufacturer [Promega, Madison, Wis.]) and used in wells made with 10- and 20-well combs (Bio-Rad, Richmond, Calif.), respectively. Restriction enzymes were added in the following units: XbaI,
40 U; SpeI, 30 U; and VspI, (Promega), 30 U. The
pieces of plugs were placed into wells in a 1.2% Seakem GTG agarose
gel made with 0.5× TBE (45 mM Tris, 45 mM boric acid, 1 mM EDTA, pH
8.0), and the wells were sealed with the 1% Seakem Gold agarose
solution. The gel was placed in a CHEF DR II unit (Bio-Rad) containing
1.8 liters of 0.5× TBE and electrophoresed at 200 V (15 V/cm of gel). The pulse times for plugs digested with XbaI or
SpeI were 4 s for 1 h and then 8 s for
18 h. The pulse times for plugs digested with VspI were
4 s for 1 h and then 12 s for 17 h. Lambda DNA in
48.5-kb concatamers (FMC BioProducts) was used in the outside lanes.
The gels were stained in a solution containing 0.5 mg of ethidium
bromide per liter and were photographed with type 55 Polaroid film.
The positions of bands were assessed visually or by analysis with the
Gelmeas computer program (
3). Similarity values were
calculated as described by Egel et al. (
6) by using the
mathematical
equation proposed by Nei and Li (
28) based on
the proportion
of shared DNA fragments. The number of nucleotide
substitutions
per site was estimated by the iterative method of Nei
(
27) by
using the SAS program as described by Leite et al.
(
22). The
KITSCH program of the PHYLIP computer package
(
9) was used
to create a rooted phylogenetic tree by the
Fitch-Margoliash method
(
10). A strain of
Xanthomonas
campestris pv. vesicatoria was
included as the outgroup. The input
data was a distance matrix
of pairwise estimates of the number of
nucleotide substitutions
per site between strains for the combined
SpeI,
XbaI, and
VspI
digestion data
obtained as described above, and negative branching
was not allowed
(
22,
37).
Fatty acid composition.
Strains of X. fragariae were inoculated onto Trypticase soy broth agar and grown
for 48 h at 24°C. The X. fragariae strains produced insufficient growth with the standard MIDI protocol (growth for 24 h at 28°C). Cellular fatty acids were extracted and
derivatized to their FAME as described previously (35). FAME
were analyzed by using the MIDI (Newark, Del.) Microbial Identification
System, software version TSBA 3.50. A library of strains of
X. fragariae was created by using the MIDI Library
Generation System, software version 3.30. Qualitative and quantitative
differences in the fatty acid profiles were used to compute the
Euclidian distance for each strain relative to the other strains in the
population. Strains within 6 Euclidian distance units, the value
determined for subspecies according to the MIDI protocol
(34), were grouped in the same cluster.
DNA-DNA hybridization.
DNA-DNA relatedness studies were
performed in microplates by using the fluorometic assay of Ezaki et al.
(8), with minor modifications. High-molecular-weight DNA
were extracted from strains of X. fragariae that were
representatives of the RFLP groups identified in this study and the
type strains of Xanthomonas campestris, Xanthomonas
albilineans, Xanthomonas oryzae, Xanthomonas
axonopodis, and Xanthomonas graminis. DNA extraction
and purification were performed by using Marmur's procedure as
described by Johnson (19). The DNA was fragmented by three
passages through a French pressure cell at 16,000 lb/in2,
which resulted in DNA fragments that were ca. 0.5 kb long. The DNA was
heat denatured and either used to coat microdilution plates (MicroFluor
type B; Dynatech Laboratory, Alexandria, Va.) or biotinylated for use
as a probe. Each microtiter well was coated with 3 µg of fragmented,
denatured DNA. The probe contained 20 to 50 ng of DNA labeled with
Photoprobe biotin (Vector Laboratories, Burlingame, Calif.) by the
manufacturer's protocol. Hybridization was carried out at 52°C for
12 h. DNA reassociation ratios were determined fluorometrically
(model 7630 microplate fluorometer; Cambridge Technology, Inc.,
Watertown, Mass.) 1 h after binding of the beta-galactosidase avidin D (Vector Laboratories) and addition of the substrate
4-methylumbelliferyl-
-D-galactoside (Sigma Chemical Co.,
St. Louis, Mo.).
| |
RESULTS |
RFLP-PFGE analysis.
Restriction endonucleases XbaI
and SpeI generated DNA fragments that were 5 to 400 kb long
(Fig. 1). Typically, a strain profile contained 10 DNA fragments more than 100 kb long. Analysis of the
XbaI profiles of 50 strains resulted in four RFLP groups, designated groups A through D. The same groups were obtained when SpeI was used. Group A contained only the American Type
Culture Collection (ATCC) strain, ATCC 33239. Groups B, C, and D
contained 76, 16, and 6% of the strains, respectively. A third
endonuclease, VspI, separated the strains belonging to
groups A, C, and D. However, analysis of the B strains with
endonuclease VspI divided a representative subsample of the
strains into three subgroups, designated subgroups B1, B2, and B3.
Subgroups B1, B2, and B3 contained 9, 27, and 64% of the group B
strains, respectively. The phylogenetic tree (Fig.
2) derived from RFLP analysis of these
strains showed that the three group B subgroups exhibited very little
divergence.
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FIG. 1.
Agarose gel showing the RFLP of genomic DNA of strains
representing the four major RFLP groups of X. fragariae
after restriction digestion with endonucleases XbaI and
SpeI and separation by PFGE.
|
|
FAME analysis.
The 47 strains of X. fragariae
were clustered into nine subgroups based on the MIDI "10% rule"
(34) (Fig. 3, Table 1). The
majority of the strains were identified as members of six closely
related subgroups which could be visualized quantitatively on the basis
of the data for three major acids: 16:1 
7 cis, 15:0
anteiso, and 15:0 iso (Fig. 4). The most
abundant acid, 15:0 iso, accounted for 34 to 54% of the total FAME
profile. The ATCC type strain (ATCC 33239) and strains 1238, 1240, and
119 were qualitatively differentiated from the other strains by the
absence of palmitoleic acid (16:1 7 cis).
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FIG. 3.
X. fragariae groups as determined by the
MIDI 10% rule and distinguished by qualitative and quantitative
differences in three major acids. Symbols:  , groups 1 and 4 through 8;  , groups 2 and 9;  ,
group 3.
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FIG. 4.
Dendrogram based on the results of the cluster analysis
of fatty acid methylase profiles, showing the four clusters (clusters 1 through 4) of X. fragariae strains. The strains were
also divided into groups by using the MIDI 10% rule. A Euclidian
distance of 6 was the cutoff point for groups determined by the cluster
analysis.
|
|
Groups identified on the basis of the MIDI 10% rule and by RFLP had
similar compositions. The type strain (ATCC 33239) and
strain 119 comprised FAME groups 9. Strains belonging to RFLP
groups A and D also
formed groups distinct from other strains
on the basis of FAME analysis
data. The three strains in RFLP
group C examined in the FAME analysis
were uniform and clustered
in FAME group 5. Two strains in RFLP group B
also clustered in
FAME group 5.
The MIDI dendrogram unweighted pair group analysis, a cluster analysis
program, separated the strains into four clusters at
a Euclidian
distance of 12 U (Fig.
4). The four clusters, designated
FAME clusters
1 through 4, contained 16, 6, 4, and 74% of the
strains, respectively.
The ATCC type strain and strains 1238,
1240, and 119 formed cluster 2.
DNA-DNA homology.
Although the strains isolated from
strawberry plants produced diverse restriction patterns, the levels of
hybridization between DNA from the type strain of X. fragariae (ATCC 33239) and DNA from representative strains
belonging to the different RFLP groups were greater than 70% (Table
2). Furthermore, the hybridization values
obtained in the reciprocal hybridization experiments were also greater
than 70%. The levels of homology between DNA from the X. fragariae strains that were representatives of the RFLP groups and
DNA from other Xanthomonas species were always less than
40%. Therefore, the strains that were isolated from strawberry plants
and represented the different RFLP groups are closely related genetically but distantly related to the other Xanthomonas
species.
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TABLE 2.
Similarity values generated by DNA reassociation
experiments performed with X. fragariae strains
representing the different RFLP groups
|
|
| |
DISCUSSION |
Our FAME and RFLP-PFGE analyses of X. fragariae
strains revealed considerable diversity. Four groups or clusters
of X. fragariae strains were identified by
RFLP-PFGE and unweighted pair group analysis of FAME; four strains (the
ATCC type strain and strains 1238, 1240, and 119) formed a group
distinct from the other strains used in this study. However, the
majority of the strains represented a clonal population with some
variation, as determined by the two analyses. Approximately one-half of
the strains were members of RFLP group B despite having been isolated
over a 3-year period from plants from diverse geographic locations.
Genomic fingerprinting by REP-PCR of field isolates collected in
California over a 4-year period revealed that population was
homogeneous (29). This finding supports the conclusion
that there was a high degree of clonality in the more diverse
population (strains isolated from locations throughout the United
States and Canada) analyzed in this study. The analysis of 25 strains
of X. fragariae by three PCR methods identified a
maximum of nine groups of closely related genetic variants
(31). Random amplified polymorphic DNA PCR, REP-PCR, and
ERIC-PCR assays identified nine, four, and two genotypes, respectively, among 25 strains of X. fragariae
that did not correlate with geographic origin. The high degrees of
similarity among pathogenic strains of X. fragariae
observed in our more extensive survey support the conclusions of the
PCR-based study, although our data suggest that most strains belong to
one clonal group (group B). The predominance of one clonal group
containing strains diverse geographic regions might be due to extensive
transportation of infected plant material which distributed a clonal
population of the pathogen. None of the groups was correlated with
plant material from a particular region of the United States or Canada. Similarly, international movement of infested plants makes it difficult
to determine if endemic populations of the organism exist outside North
America due to distribution of the pathogen from the origin of
propagation.
Sasser (34) indicated that a Euclidean distance greater than
10 was an indication that distinct species may exist. The cluster analysis in this study revealed dendrogram distances greater than 10. Based on the cluster analysis of the FAME results in this study, there
was evidence that more than one species may exist. Further evidence
that unique genetic groups may exist was provided by the fact that
distinct RFLP-PFGE groups were identified. DNA-DNA hybridization was
useful in clarifying the extent of genetic variation present within
X. fragariae. As a result of the more than 70% homology between representative strains of the four groups, the strains
of X. fragariae should be considered members of the
same species (40). Although FAME and PFGE results indicated
that distinct species may exist within X. fragariae,
the DNA hybridization data indicated otherwise. A similar situation was
observed by Egel et al. (6), who examined the citrus
bacterial spot pathogen by the DNA reassociation method and found very
high (>88% homology) levels of similarity among strains, whereas
these strains were very diverse as determined by RFLP analysis. The
differences among X. fragariae strains determined by
the RFLP and FAME analyses were not apparent when the DNA reassociation
method was used; thus, the strain diversity was within a single
species.
The FAME and RFLP-PFGE methods used in this research identified four
strains, including the ATCC type strain, that were distinct from a
collection of 50 strains. As determined by RFLP analysis, group A
contained only the ATCC type strain from Minnesota, which was collected
more than 20 years ago; however, as determined by FAME cluster
analysis, one other strain (119, which was isolated from an infected
plant from Canada in 1993) grouped with the ATCC type strain. In the
RFLP-PFGE analysis, the 119 profile was the same as the profile
obtained for group D, which contained strains 1238 and 1240, which were
isolated from samples from California in 1990. Dendrogram analysis
placed these two groups close to each other. The FAME statistical
analysis revealed that these four strains lacked 16:1 7 cis acid, which distinguished them from the other
X. fragariae strains. It is interesting to speculate that perhaps strain 119 represents a "bridge" between RFLP groups A
and D because of its intergroup relationship; its RFLP profile is a
group D profile, but it is more closely related to group A as
determined by the FAME analysis.
| |
ACKNOWLEDGMENTS |
We acknowledge Gary Stark for development of computer software
used in this study. We also acknowledge Trish Strickler for technical
assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Plant Pathology
Department, University of Florida, FL 34203. Phone: (352) 392-7244. Fax: (352) 392-6532. E-mail:
jbjones{at}nersp.nervm.ufl.edu.
Florida Agriculture Experiment Station Journal Series paper
R-05657.
| |
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
