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. 1998 Oct;64(10):3961–3965. doi: 10.1128/aem.64.10.3961-3965.1998

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, A R Chase 3
PMCID: PMC106585  PMID: 9758826

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.

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.

TABLE 1.

Geographic sources, collection dates, and FAME and RFLP groups of strains used in this study

Strain(s) Geographic origina Year of isolation Sourceb FAME group RFLP group or subgroup
1238, 1240 California 1990 ARC 2 D
1241—1243 California 1990 ARC 8 B
1245 California 1990 ARC 4 B
1246 California 1990 ARC 8 NDc
1249 California 1990 ARC 4 ND
1250 California 1990 ARC 5 C
1290 California 1989 ARC ND C
1291 California 1989 ARC 1 B
1293 California 1989 ARC 7 B
1295 California 1989 ARC 8 ND
1296 California 1989 ARC 8 B3
1298 California 1989 ARC 8 B
1424 Florida 1992 ARC ND C
1425, 1428 Florida 1992 ARC 3 B2
1426, 1431 Florida 1992 ARC ND B
1427, 1429 Florida 1992 ARC 3 B
1514 California 1993 ARC ND C
1515 California 1993 ARC 6 B3
1516 North Carolina 1993 ARC ND B2
1517 North Carolina 1993 ARC 7 B3
1518, 1526 North Carolina 1993 ARC 8 B
1519 North Carolina 1993 ARC ND B
1520, 1523, 1524 California 1993 ARC 5 C
1525 California 1993 ARC ND C
1532 Florida 1993 ARC 3 ND
1533 Wisconsin 1993 ARC 5 B
122 Florida 1993 CAN 5 C
1534 Wisconsin 1993 ARC ND B3
100, 104, 108 Florida 1993 This study 1 B
101, 107 Florida 1993 This study 1 ND
103 Florida 1993 This study ND B
105, 114 Florida 1993 This study 3 B
106 Florida 1993 This study 3 ND
113 Florida 1993 This study 5 B3
115 Florida 1993 This study 4 B
116, 124 Canada 1993 This study ND B
117 Canada 1993 This study ND B3
119 Canada 1993 This study 9 D
125, 127 Canada 1993 This study 6 ND
126 Canada 1993 This study 4 ND
128 Quebec, Canada 1993 This study 4 B
129 Canada 1993 This study 6 B3
138, 153 Canada 1993 This study 6 B
146 Quebec, Canada 1993 This study 4 B1
ATCC 33239 Minnesota ATCC 9 A
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a

Geographic origin of plant material from which bacteria were isolated. 

b

ARC, A. R. Chase. 

c

ND, not determined. 

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-β-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.

FIG. 1.

FIG. 1

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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.

FIG. 2.

FIG. 2

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Relationship of X. fragariae groups and subgroups as determined by the RFLP analysis.

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 ω 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 ω 7 cis).

FIG. 3.

FIG. 3

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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.

FIG. 4.

FIG. 4

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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.

TABLE 2.

Similarity values generated by DNA reassociation experiments performed with X. fragariae strains representing the different RFLP groups

Strain RFLP group % Hybridization witha:
ATCC 33239 1425 122 1240 119
ATCC 33239 A 100 76 81 97 81
1425 B 133 100 113 128 119
122 C 112 83 100 109 99
1240 D 98 71 90 100 90
119 D 111 90 100 117 100
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a

Values are averages obtained with three samples and represent percentages of the homologous value, which was defined as 100%. Strain ATCC 33239 is the type strain of X. fragariae. 

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 ω 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

†

Florida Agriculture Experiment Station Journal Series paper R-05657.

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