INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Oct. 1991, p. 535-542 Vol. 41, No. 4 OO20-7713/91/O40535-08$02.00/0 Copyright 0 1991, International Union of Microbiological Societies Differentiation of Xanthomonas campestris pv. Citri Strains by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis of Proteins, Fatty Acid Analysis, and DNA-DNA Hybridization L. VAUTERIN,l* P. YANG,l B. HOSTE,l M. VANCANNEYT,I E. L. CIVEROL0,2 J. SWINGS,l AND K. KERSTERSl Laboratoriurn voor Microbiologie en Microbiele Genetica, Rijksuniversiteit, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium,' and Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland 207052 A total of 61 strains, including members of all five currently described pathogenicity groups of Xanthomonas campestris pv. citri (groups A, B, C, D, and E) and representing a broad geographical diversity, were compared by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins, gas chromatographic analysis of fatty acid methyl esters, and DNA-DNA hybridization. We found that all of the pathogenicity groups were related to each other at levels of DNA binding of more than 60%, indicating that they all belong to one species. Our results do not confirm a previous reclassification of X. campestris pathogens isolated from citrus in two separate species (Gabriel et al., Int. J. Syst. Bacteriol. 39:14-22, 1989). Pathogenicity groups A and E could be clearly delineated by the three methods used, and group A was the most homogeneous group. The delineation of pathogenicity groups B, C, and D was not clear on the basis of the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins and gas chromatography of fatty acid methyl esters, although these groups constituted a third subgroup on the basis of the DNA homology results. Bacterial canker disease of citrus, which is caused by predominantly on Swingle citrumelo, a hybrid between Xanthomonas campestris pv. citri, is characterized by raised trifoliate orange and grapefruit. The second most important lesions on leaves, twigs, and fruits. Severe infestations can host is grapefruit. result in abscission of leaves, premature fruit drop, and Although it is known that the xanthomonads that are dieback (1, 21). On the basis of host range, serology, phage associated with the respective disease forms are different typing, and geographical distribution, four forms of citrus biological entities (14), the relationships among the different canker were recognized prior to 1984 (1, 3, 9, 21). The most groups of organisms are not yet fully understood. On the severe and damaging form, form A (the Asiatic form), is basis of the results of a comparison of restriction endonu- native to Asia, but is found in many citrus-growing regions clease fingerprints of genomic DNAs (13), group A and B all over the world. This form occurs on sweet orange (Citrus strains of X. campestris pv. citri were clearly distinguished sinensis), grapefruit (Citrus paradisi), lemon (Citrus limon), from each other; each of these groups constitutes a clonal Mexican lime (Citrus reticulata) and, to a lesser degree, on group. Group B and D strains could not be differentiated other rutaceous plants, such as trifoliate orange (Poncirus from each other. The causal agents of the disease in Florida trifoliata). Form B (cancrosis B) occurs primarily in Argen- have been referred to as members of X. campestris pv. citri tina, but it also occurs in Uruguay and Paraguay. The most group E (13) or, more tentatively, Florida citrus nursery susceptible hosts are Mexican lime and lemon. Form C strains (14). On the basis of the results of a RFLP analysis of (Mexican lime cankrosis) is found in Brazil; this form is genomic DNAs, the Florida nursery strains did not consti- restricted to Mexican lime. Form D of citrus canker (Mexi- tute a single homogeneous group (7, 12, 14). Only the can bacteriosis) occurs in Mexico on Mexican lime. aggressive group E strains were found to be clonally related In 1984, a new bacterial citrus disease which was thought (12). A RFLP analysis (7) revealed that group B, C, and D to be caused by X. campestris pv. citri was reported in strains of X. campestris pv. citri were clonally related to Florida citrus nurseries (19). This disease was referred to each other but highly distinct from group A strains (7). initially as the Florida nursery form of citrus canker (18) and However, the results of another RFLP comparison sug- most recently as citrus bacterial spot (10, 12, 14). On the gested that there is a significant relationship between group basis of distribution, host range, and symptom development A and groups B, C, and D (14). Gabriel et al. (8) proposed and later on the basis of restriction fragment length polymor- that strains belonging to the former taxon X. campestris pv. phism (RFLP) patterns, it became clear that the pathogens citri should be reclassified into Xanthomonas citri (group A), associated with the Florida nursery disease were substan- X. campestris pv. aurantifolii (groups B, C, and D), and X. tially different from those associated with citrus canker. The campestris pv. citrumelo (Florida nursery strains [group El). typical symptoms are flat water-soaked spots of various In a comment on the article by Gabriel et al., Young et al. sizes, sometimes surrounded by necrosis, that appear on (25) stated that the reclassification proposals made were leaves and twigs of infected trees. In contrast to citrus defective in terms of current taxonomy and plant pathology canker, fruits are usually not infected, and defoliation or standards. These authors proposed provisional retention of dieback does not occur (18). Extensive variations in aggres- the subdivisions as groups A, B, C, D, and E of X. siveness have been observed (11, 12). The disease occurs campestris pv. citri to designate the different pathogenicity groups, until further study established the overall relation- ships among the xanthomonads isolated from citrus. Below, * Corresponding author. we use this nomenclature. 535 536 VAUTERIN ET AL. INT.J. SYST.BACTERIOL. We describe here the results of a comparative study of 61 tions were calculated. We included all of the fatty acids for X. campestris strains that were isolated worldwide and which the product of the mean relative amount times the represented the causative agents of five currently described percentage of samples containing the fatty acid was more disease forms of citrus bacterial canker and bacterial leaf than 0.25%. spot. The strains were analyzed and compared numerically DNA-DNA hybridization. DNAs were extracted from 19 by determining sodium dodecyl sulfate (SDS)-polyacryl- representative strains. Each DNA was purified as described almide gel electrophoresis (PAGE) patterns of cellular pro- by Marmur (17). The degrees of DNA binding were deter- teins and gas chromatographic fingerprints of cellular fatty mined from the initial renaturation rates by using a spectro- acid methyl esters (FAMEs). In order to reveal the overall photometer (5). The renaturation rates were measured in 2 x genomic relationships among strains formerly classified as SSC (IX SSC is 0.15 M NaCl plus 0.015 M trisodium citrate, X. campestris pv. citri, it was necessary to compare com- pH 7.0) at the optimal renaturation temperature (80.8"C), plete genomes by using DNA-DNA hybridization (24, 25); a which was calculated from the G+C content (4). Each subset of representative strains was selected for DNA-DNA experiment was repeated at least once. hybridization. RESULTS MATERIALS AND METHODS SDS-PAGE protein patterns. The reproducibility of the Bacterial strains. A total of 61 X. campestris strains that electrophoresis technique which we used was checked by were isolated from citrus species were studied. These in- preparing 30 protein extracts in duplicate and running all of cluded 24 group A strains, 3 group B strains, 3 group C the extracts in at least two gels. In all cases, a level of strains, 1 group D strain, 18 group E strains, and 12 strains reproducibility (r)of more than 0.90 was obtained. obtained from culture collections, for which the group as- We observed considerable heterogeneity in SDS protein signments were not known. The pathogenicities and types of profiles among the X. campestris pv. citri strains. On the reactions of all of the strains except the culture collection dendrogram in Fig. 1, the lowest level of similarity (r) strains have been determined previously (2,6,8,11,14). The between groups of strains was about 0.73. Four SDS protein strains which we used are listed in Table 1. clusters were delineated (Fig. 1). Electropherograms of SDS-PAGE of whole-cell proteins. All of the strains were representative strains belonging to each cluster are shown analyzed by electrophoresis of whole-cell proteins. The in Fig. 2. Cluster 1 was composed of 19 group A strains methods used for cultivation of cells and preparation and and 10 culture collection strains. Cluster 2 contained the electrophoresis of SDS protein extracts have been described three group B strains which we studied, the only available elsewhere (23). Profiles were densitometrically recorded and group D strain, and, at a lower correlation level, four group istored as normalized records of 400 points on a PC-AT A strains. Cluster 3 consisted of the group C strains and two (computer.The level of similarity between pairs of traces was culture collection strains, strains ICMP 8432 and ICMP (calculatedby using the Pearson product-moment correlation 8435. Cluster 4 contained all of the group E strains which we (coefficient (r) (20). A cluster analysis was performed by studied. using the unweighted average algorithm. Strain XC222 (group A) had a clearly atypical protein Gas chromatographic analysis of FAMEs. Bacterial strains pattern (Fig. 2) and occurred separately on the dendrogram. were precultured for 3 days on nutrient agar slants (0.1% FAME analysis.