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. All of the strains were analyzed at least [wt/vol] Lab Lemco, 0.2% [wt/vol] yeast extract, 0.5% twice, and the results were compared by performing a [wtfvol] bacteriological peptone, 0.5% [wt/vol] NaCl, 2.0% cluster analysis (principal component analysis and un- [wt/vol] agar). The precultures were streaked onto plates weighted average grouping). Duplicates of the same strain containing 3.0% (wt/vol) Trypticase soy broth (BBL) sup- clustered closely at a level of about 2 Euclidian distances on plemented with 1.5% (wtfvol) Bacto Agar (Difco), and the a dendrogram that was produced by using the unweighted plates were incubated at 28°C for 48 h. A loopful of cells average grouping method (data not shown). from the overlap area of the second and third series of On the basis of their FAME profiles the strains which we streaks was harvested with a sterile plastic loop (diameter, investigated fell into three major types (Fig. 3). FAME type 4 mm) and inoculated into a small test tube (13 by 100 1consisted of 17 strains belonging to pathogenicity group A, mm) that was capped with a Teflon-lined screw cap. The 3 strains belonging to group B, and 9 culture collection procedure described by Stead (22) was used to prepare strains. FAME type 2 was composed of three strains belong- FAMEs. FAMEs were separated by gas-liquid chromatog- ing to group C and two culture collection strains, strains raphy with a model 5890A instrument (Hewlett-Packard Co., ICMP 8432 and ICMP 8435. FAME type 3 contained 16 Avondale, Pa.) by using a fused silica capillary column (25 m strains, all of which belonged to group E. by 0.2 mm) that was coated with methyl phenyl silicone A total of 24 fatty acids were found. The eight major fatty (Hewlett-Packard Co.). The computer-controlled parame- acids (fatty acids with mean percentages higher than 5.0% ters for the instrument were those described by Korn- for at least one FAME type) were 1l:O iso, 13:O is0 30H, Wendisch et al. (15). FAME fingerprints were identified by 15:O iso, 15:O anteiso, 16:l cis 9, 16:0,17:1 is0 F, and 17:O is0 using a Microbial Identification System software package (Table 2). There was a significant difference in fatty acids (MIS version no. 3.2) obtained from Microbial ID, Inc., 15:O iso, 16:l cis 9, and 17:l is0 F among the three FAME Newark, Del.) and a calibration mixture of known standards types. FAME type 2 strains contained the highest levels of (Hewlett-Packard). 15:O is0 (48.0%) and 17:l is0 F (14.7%) but the lowest level FAME profiles were compared by subjecting them to a of 16:l cis 9 (6.0%). In contrast, FAME type 3 strains principal component analysis and unweighted, arithmetic contained the lowest levels of 15:O is0 (25.7%) and 17:l is0 F average clustering. The software used for cluster analysis (5.4%) but the highest level of 16:l cis 9 (18.3%). FAME type was provided by Microbial ID, Inc. Within each FAME 1 strains had an intermediate profile, with 33.6% 15:O iso, group, the fatty acid mean percentages and standard devia- 8.0% 17:l is0 F, and 11.5% 16:l cis 9. VOL. 41. 1991 DIFFERENTIATION OF X. CAMPESTRZS PV. CITRI STRAINS 537

TABLE 1. Strains tested

Protein FAME DNA Code Source strain no.'' LMG no." Host' Groupb clusterC subgroup Place and year of isolation no. 1 NCPPB 405 680 Citrus sp. 1 1 New Zealand, 1956 2 NCPPB 408 68 1 C. reticulata 1 1 1 New Zealand, 1956 3 NCPPB 409d 682 C limon 1 1 1 New Zealand, 1956 4 NCPPB 410 683 C. sinensis 1 1 New Zealand, 1956 5 ICMP 28 8650 1 1 1 India 6 ICMP 31 8651 C. limon 1 1 New Zealand, 1958 7 ICMP 594 8652 Citrus sp. 1 1 Japan, 1962 8 ICMP 5809 8653 Citrus aurantifolia 1 AT Brazil, 1976 9 ICMP 7494 8654 Citrus latifolia 1 1 1 Brazil, 1981 10 ICMP 8436 8657 C. aurantifolia 1 1 1 Brazil, 1983 11 E. L. Civerolo XC59 9176 C. aurantifolia A 1 1 1 Brazil 12 E. L. Civerolo XC62 9177 C. reticulata A 1 AT Japan 13 E. L. Civerolo XC63 9178 C. reticulatu A 1 AT Japan 14 D. W. Gabriel 3210 9321 A 1 1 1 Florida 15 D. W. Gabriel 3213 9322 A 1 1 Florida 16 E. L. Civerolo XC124 9652 Tangerine A 1 1 Korea 17 E. L. Civerolo XC221 9653 Orange A 1 1 Brazil 18 E. L. Civerolo XC206 9655 C. aurantifolia A 2 AT Oman 19 E. L. Civerolo XC114 9656 Citrus sp. A 1 1 People's Republic of China 20 E. L. Civerolo XC223 9657 Persian lime A 1 1 Brazil 21 E. L. Civerolo XC102 9659 Citrus sp. A 1 1 Guam 22 E. L. Civerolo XC126 9660 Tangerine A 1 1 Korea 23 E. L. Civerolo XC112 9661 Citrus sp. A 1 1 People's Republic of China 24 E. L. Civerolo XC176 9662 C. auruntifolia A 2 1 Saudi Arabia 25 E. L. Civerolo XC224 9663 Lime A 1 1 Brazil 26 E. L. Civerolo XC227 9664 C. reticulata A 1 1 Brazil 27 E. L. Civerolo XClOO 9665 Citrus sp. A 2 AT 1 Pakistan 28 E. L. Civerolo XC113 9666 Citrus sp. A 1 1 People's Republic of China 29 E. L. Civerolo XC98 9667 C; aurantifolia A 1 1 Yemen 30 E. L. Civerolo XC104 9668 West Indian lime A 1 1 Australia 31 E. L. Civerolo XC220 9669 C. aurantifolia A 2 AT Fiji 32 E. L. Civerolo XC226 9670 Orange A 1 1 Brazil 33 E. L. Civerolo XC222 9671 Lime A AT AT 1 Brazil 34 E. L. Civerolo XC107 9672 Lime A 1 AT Australia 35 E. L. Civerolo XC64 9179 C. limon B 2 1 2 Argentina 36 E. L. Civerolo XC96 9183 C. limon B 2 1 2 37 E. L. Civerolo XC148 9185 C. limon B 2 1 Argentina 38 ICMP 8432 8655 C. aurantifolia 3 2 2 Brazil, 1981 39 ICMP 8435 8656 C. aurantifoliu 3 2 Brazil, 1982 40 E. L. Civerolo XC70 9181 C. aurantilolia C 3 2 2 Brazil 41 E. L. Civerolo XC171 9654 C. aurantifolia C 3 2 Brazil 42 E. L. Civerolo XC172 9658 C. aurantifolia C 3 2 2 Brazil 43 E. L. Civerolo XC90 9182 C. aurantifolia D 2 AT 2 Mexico 44 E. L. Civerolo F1 9160 P. trifoliata x C. sinensis E 4 AT 3 Florida 45 E. L. Civerolo F3 9161 C. paradisi E 4 3 Florida 46 E. L. Civerolo F6 9163 C. paradisi E 4 3 Florida 47 E. L. Civerolo F21 9165 C. paradisi E 4 3 Florida 48 E. L. Civerolo F56 9167 P. trifoliata x C. paradisi E 4 3 3 Florida 49 E. L. Civerolo F96 9168 E 4 3 Florida 50 E. L. Civerolo FlOO 9169 P. trifoliata x C. paradisi E 4 3 Florida 51 E. L. Civerolo F224 9170 Citrus sp. E 4 3 Florida 52 E. L. Civerolo F230 9171 C. paradisi E 4 3 Florida 53 E. L. Civerolo F231 9172 C. paradisi E 4 AT 3 Florida 54 E. L. Civerolo F257 9173 C. paradisi E 4 3 Florida 55 E. L. Civerolo F263 9174 C. sinensis E 4 3 Florida 56 E. L. Civerolo F294 9175 C. paradisi E 4 3 3 Florida 57 D. W. Gabriel 4600 9323 E 4 3 Florida 58 D. W. Gabriel 3401 9324 Citrus sp. E 4 3 Florida 59 D. W. Gabriel 0498 9326 E 4 3 Florida 60 D. W. Gabriel 6260 9327 E 4 3 Florida 61 D. W. Gabriel 3162 9331 E 4 3 Florida " NCPPB, National Collection of Plant Pathogenic Bacteria, Harpenden, England; ICMP, International Collection of Microorganisms from Plants, Auckland, New Zealand; LMG, Laboratorium voor Microbiologie Ghent Culture Collection, Ghent, Belgium. X. curnpestris pv. citri group based on pathogenicity. AT, atypical strain. Pathovar reference strain. 538 VAUTERIN ET AL. INT. J. SYST.BACTERIOL.

r 1.00 0.90 0.80 0.70 0.60 group D strain tested, strain XC90. Group D strain XC90 had 1 I I I I a fatty acid profile similar to that of FAME type 2 strains, but - XC102 with a lower level of 15:O is0 (43.4%). Group E strain F231 was related to FAME type 3 strains, but had a higher level of 15:O is0 (31.4%), whereas group E strain F1 had much higher levels of 15:O (13.2%) and 17:l B (7.6%). XC107 DNA-DNA hybridization. A total of 46 DNA hybridization XC113 XC114 experiments were performed with 19 strains that represented xc223 pathogenicity groups A, B, C, D, and E of X. campestris XC124 pv. citri. Figure 4 shows a matrix of mean values for DNA- XC59 xc63 DNA hybridization between pairs of strains. The average xc112 overall error of the method, as calculated from duplicates, XC98 L3%. Cluster 1 ICMP 28 was All of the values for homology between X. ICMP 5809 campestris pv. citri strains were about 60% or greater. A ICMP 594 clear division of the matrix could be made at the 80% ICMP 7494 ICMP 31 homology level, yielding the following three homogeneous NCPPB 408 DNA homology subgroups: subgroup 1, comprising group NCPPB 405 A isolates and all of the culture collection strains except strain ICMP 8432; subgroup 2, which included pathogenicity group B, C, and D isolates together with collection strain XC62 ICMP 8436 ICMP 8432; and subgroup 3, consisting of all of the group E NCPPB 409 strains tested. The culture collection strains, for which NCPPB410 3 pathogenicities are not known, are genotypically related to - 3213 group A, a result which is also consistent with the results - XC220 I XC176 ‘h of the protein electrophoresis analysis and the FAME anal- ysis. Cluster 2 XC96 XC90 - xc148 DISCUSSION - lCMP8432 ICMP 843!j On the basis of the results of SDS-PAGE of proteins and (Cluster3 XC70 XC172 a fatty acid analysis, pathogenicity group A of X. campestris - XC171 pv. citri is a clearly defined cluster. With both techniques, F56 however, a limited number of group A strains were found to F263 F294 be atypical. Interestingly, four of these strains were the same Fl in both analyses. However, total genomic DNA homology F6 FlOO measurements showed that all of the group A strains, F230 including those having atypical protein and fatty acid pro- F257 files, exhibited DNA homology values of more than 88%. Cluster 4 6260 F21 The homogeneity of group A is in agreement with the results F96 of comparisons of genomic DNA restriction fragment pat- 3162 4600 terns (13) and RFLP analyses (7, 14), in which group A F224 strains were found to be clonally related and to display very F3 little polymorphism. Nearly all of the culture collection F231 strains, which were received without any phytopathological L I assignments, belong to group A of X. carnpestris pv. citri. xcm 1 Strains ICMP 8432 and ICMP 8435, which were originally FIG. 1. Dendrogram based on average linkage cluster analysis of isolated from Citrus nurantifolii in Brazil, unambiguously SDS-PAGE protein patterns from 61 X. cumpestris pv. citri strains. belong to group C. The analysis of the protein electrophoresis data showed that group E is not as heterogeneous as might be expected on the basis of RFLP patterns. Except for the strain 3401 and Seven group A strains, one culture collection strain, two 0498 patterns, the group E protein patterns are rather group E strains, and the single available group D strain did uniform (see the electropherograms in Fig. 2). According to not belong to any of the FAME types (Fig. 3). Group A the results of the FAME analysis, the group E strains istrains XC63, XC107, XC100, and XC220 had fatty acid constitute a very uniform group (FAME type 3), which is ]profiles that were somewhat similar to the FAME type 1 even more homogeneous than group A (FAME type 1). This ;profile; strains XC63 and XC107 differed mainly in 15:O is0 finding contrasts with the data of Graham et al. (12), who Fatty acid contents (26.4 and 29.1%), and strains XClOO and found considerable heterogeneity among group E strains, XC220 differed mainly in 16:l cis 9 fatty acid contents (14.8 which could to be related to virulence. This apparent dis- ,and 15.7%). The other group A strains, strains XC222 and crepancy is rather surprising, since some of our strains were XC206, and culture collection strain ICMP 5809 had different the same as those used by Graham et al. However, we were profiles, with much higher levels of 1S:O (7.7 to 9.5%) and not able to compare fatty acid levels and standard devia- lower levels of 15:O is0 (26.9 to 19.5%). Group A strain XC62 tions, as quantitative data were not provided by Graham et (41.3% 15:O iso, 10.4% 16:l cis 9, and 8.1% 17:l is0 F) a1 . occupied a position between FAME type 1 and the single At the genotypic level also, the group E strains constitute VOL.41, 1991 DIFFERENTIATION OF X. CAMPESTRIS PV. CITRI STRAINS 539

FIG. 2. SDS-PAGE protein patterns of 11 representative X. campestris pv. citri strains belonging to the groups obtained by cluster analysis. a definite DNA homology group (DNA subgroup 3). Ge- citri originated from an endemic population that was char- nomic methods, such as RFLP analysis, have revealed acterized by a broad host range and had a latent ability to significant polymorphism among group E strains. Hartung attack certain citrus hosts (13). According to this idea, the and Civerolo (14) found that although group E as a whole sudden occurrence of the disease could be explained by the was not clonal, it consisted of three related clonal sub- increased cultivation of the susceptible host Swingle cit- groups. Lazo et al. (16) have related moderate genomic rumelo since 1984 (18). Adaptations to the new host might diversity to a broad host range of the pathogen. These data have taken place in several locations, which could explain support the idea that the group E strains of X. campestris pv. the existence of several clonal subgroups.

14 - 8

44 12- 33 34 18

10-

8 FAME 08 *- V a i2 z 6- 3

43 4- FAME type 2 2- 040 39

ol I I I I 0 10 20 30 4'0

PRINCIPAL COMPONENT 1 FIG. 3. Principal component analysis of fatty acid prohles of 61 X. carnpestris pv. citri strains. The numbers are the strain code numbers given in Table 1. 540 VAUTERIN ET AL. INT. J. SYST.BACTERIOL.

TABLE 2. Fatty acid patterns of X. campesfris pv. citri FAME types

% of total fatty acids in:

~~~ FAME FAME FAME Fatty acid type 1 type 2 type 3 (n = 29)" (n = 5) (n = 16) Mean SD Mean SD Mean SD 110:o 0.9 0.2 l1:O is0 6.6 1.o 2.9 0.4 5 .O 0.7 IJnknown 11.7986 2.0 0.4 2.1 0.3 1.9 0.3 :11:0 is0 30H 3.2 0.6 2.1 0.3 2.4 0.4 :13:0 is0 0.9 0.2 12:O 30H 2.3 0.5 0.7 0.1 3.3 0.5 14:O is0 0.7 0.4 14:O 0.5 0.4 1.2 0.2 13:O is0 30H 8.1 1.1 6.9 0.6 5.3 0.7 15:O is0 33.6 2.2 48.0 0.8 25.7 1.5 15:O anteiso 9.1 1.1 4.6 0.4 8.1 1.0 15:l B" 0.4 0.2 15:O 0.7 0.4 1.2 0.2 FIG. 5. Schematic comparison of the groupings of X. campestris 16:O is0 3.6 1.3 1.3 0.3 2.3 0.5 pv. citri obtained by different methods in this study (a), the study of 16:l B' 1.5 0.5 Gabriel et al. (7) (b), and the study of Hartung and Civerolo (14) (c). 16:l cis 9 11.5 1.5 6.0 0.5 18.2 1.2 16:O 1.9 0.5 0.7 0.3 6.1 0.9 17:l is0 F' 8.0 1.7 14.7 1.6 5.4 0.6 17:O is0 5.9 1.3 5.8 0.4 8.3 0.8 Whereas the delineation of pathogenicity groups A and E 17:O anteiso 0.5 0.3 does not pose great problems, some discrepancies are found 17:l B" 0.5 0.5 0.3 0.2 1.1 0.2 in the delineation of groups B, C, and D. According to the 18:l cis 9 0.7 0.2 results of protein electrophoresis, group B and D strains Summed feature 2" 0.5 0.3 constitute one type, whereas according to the results of Summed feature 5" 0.5 0.3 FAME analysis, the group B strains are indistinguishable a n is the number of strains. from group A strains. According to the results of both The identity of the fatty acid which has an equivalent chain length of techniques, the group C strains constitute a distinct cluster. 11.798 is unknown. The double bond position represented by B or F is unknown. Thus, on the basis of protein and fatty acid profiles, there is Fatty acids 15:l is0 H, 15:l is0 I, and 13:O 30H could not be separated no clear indication that group B, C, and D strains form one from each other by gas chromatography by using the MIS system and together entity. In DNA hybridization experiments, however, these were considered summed feature 2. three pathogenicity groups are indistinguishable. This result " Fatty acids 17:l is0 I and 17:l anteiso B could not be separated from each other by gas chromatography by using the MIS system and together were is consistent with previous findings that were based on DNA considered summed feature 5. restriction patterns, in which the group B, C, and D strains were found to be clonally related (7, 13, 14). However, we are aware that the number of strains that belong to these groups and were available for study was too limited to arrive at valid conclusions.

NCPPB 409 NCPPB 408 ICMP 8436 ICMP 7494 ICMP 28 321 0 Yup xc59 XCl 00 xc222 XC64 XC96 XC70 XC172 ICMP 8432 XC90 F1 F23 1 F294 Ygroup F56 rr I-, j 83 I ild FIG. 4. Levels of DNA-DNA hybridization between representative strains of X. campestris pv. citri. VOL. 41, 1991 DIFFERENTIATION OF X. CAMPESTRZS PV. CITRI STRAINS 541

Figure 5 shows a schematic comparison of the results REFERENCES obtained by different methods in our study and other studies 1. Civerolo, E. L. 1984. Bacterial canker disease of citrus. J. Rio (7, 14). The phenotypic results (SDS-PAGE protein patterns Grande Val. Hortic. SOC.37:127-146. and FAME profiles) which we obtained confirmed previous 2. Civerolo, E. L. Unpublished data. findings that group A and group E can be clearly discrimi- 3. Civerolo, E. L., and F. Fan. 1982. Xanthomonas campestris pv. nated. However, the most important observation is the citri detection and identification by enzyme-linked immunoab- significant levels of relatedness among all of the pathogenic- sorbent assay. Plant Dis. 66:231-236. ity groups of X. campestris pv. citri, including group E, as 4. De Ley, J. 1970. Reexamination of the association between determined by DNA-DNA hybridization. All of the X. melting point, buoyant density, and chemical base composition campestris pv. citri strains which we studied exhibited DNA of deoxyribonucleic acid. J. Bacteriol. 101:738-754. 5. De Ley, J., H. Cattoir, and A. Reynaerts. 1970. The quantitative homology values of more than 60% and thus can be consid- measurement of DNA hybridization from renaturation rates. ered members of the same species. It has been established Eur. J. Biochem. 12:133-142. previously that of the different genomic methods available, 6. Egel, D. S., J. H. Graham, and T. D. Riley. 1991. Population DNA hybridization is the most reliable method for determin- dynamics of strains of Xanthornonas campestris differing in ing taxonomic relationships between and within species, aggressiveness on Swingle citrumelo and grapefruit. Phytopa- since this method essentially measures levels of total ge- thology 81:666-671. nomic homology. For taxonomic purposes, the results of 7. Gabriel, D. W., J. Hunter, M. Kingsley, J. Miller, and G. Lazo. 1988. Clonal population structure of Xanthornonas campestris other genomic methods, such as RFLP analysis, should be and genetic diversity among citrus canker strains. Mol. Plant interpreted cautiously, especially at species and higher lev- Microbe Interactions 159-65. els. This was clearly illustrated by the levels of relatedness 8. Gabriel, D. W., M. T. Kingsley, J. E. Hunter, and T. Gottwald. determined by RFLP analyses between pathogenicity group 1989. Reinstatement of Xanthomonas citri (ex Hasse) and A and groups B and C of X. campestris pv. citri. Gabriel et X. phaseofi (ex Smith) to species and reclassification of all al. (7) found almost no relationship between group A and X. campestris pv. citri strains. Int. J. Syst. Bacteriol. 39:14 groups B and C, whereas Hartung and Civerolo (14) ob- 22. 9. Goto, M., T. Takahashi, and M. Messina. 1980. A comparative served a significant level of relatedness, even though in these study of the strains of X. campestris pv. citri isolated from citrus studies the workers used a number of the same strains. A canker in Japan and cancrosis B in Argentina. Ann. Phyto- likely explanation for this discrepancy is that these workers pathol. SOC.Jpn. 46:329-338. used different probes and/or combinations of probes and 10. Gottwald, T. R., E. L. Civerolo, S. M. Garnsey, R. H. Brlansky, restriction enzymes. In studies in which RFLP analysis was J. H. Graham, and D. W. Gabriel. 1988. Dynamics and spatial used, no significant relationship was observed between distribution of Xanthomonas campestris pv. citri group E group A and group E; these findings were used by Gabriel et strains in simulated nursery and new grove situations. Plant Dis. al. (8) to argue that these two groups should be reclassified as 72:781-787. 11. Graham, J. H., and T. R. Gottwald. 1990. Variation in aggres- “Xanthomonas citri” and “X. campestris pv. citrumelo,” siveness of Xanthomonas campestris pv. citrumelo associated respectively. Our data do not support this reclassification, with citrus bacterial spot in Florida citrus nurseries. Phytopa- but clearly reveal that all of the pathogenic xanthomonads thology 80:190-196. isolated from citrus belong to one species. Young et al. (25) 12. Graham, J. H., J. S. Hartung, R. E. Stall, and A. R. Chase. 1990. have criticized the reclassification of X. campestris pv. Pathological, restriction-fragment length polymorphism, and citri by Gabriel et al.; we do not repeat their arguments fatty acid profile relationships between Xanthomonas campes- here. However, the proposal of Gabriel et al. to rename the tris from citrus and noncitrus hosts. Phytopathology 80:829- B, 836. group C, and D strains and the group E strains as two 13. Hartung, J. S., and E. L. Civerolo. 1987. Genomic fingerprints of separate pathovars (X. campestris pv. aurantifolii and X. Xanthomonas campestris pv. citri strains from Asia, South campestris pv. citrumelo, respectively) might be substan- America and Florida. Phytopathology 77:282-285. tially correct on the basis of phytopathological and taxo- 14. Hartung, J. S., and E. L. Civerolo. 1989. Restriction fragment nomic data. In light of the DNA homology relationships, a length polymorphisms distinguish Xanthomonas campestris more complex picture is appearing as all pathogenic xanth- strains isolated from Florida citrus nurseries from X. c. pv. citri. omonads isolated from citrus belong to DNA homology Phytopathology 79:793-799. group 1 as defined by Vauterin et al. (24), together with a 15. Korn-Wendisch, F., A. Kempf, E. Grund, R. M. Kroppenstedt, and H. J. Kutzner. 1989. Transfer of Faenia rectivirgufa Kurup number of pathovars isolated from leguminous and other and Agre 1983 to the genus Saccharopolyspora Lacey and hosts. The placement of all of the actual pathovars into the Goodfellow 1975, elevation of Saccharopofyspora hirsuta DNA homology groups should provide the framework for an subsp. taberi Labeda 1987 to species level, and emended improved classification of the genus Xanthomonas. Each of description of the genus Saccharopolyspora. Int. J. Syst. Bac- the DNA homology groups should be a new Xanthomonas teriol. 39:430441. species. As this work is still ongoing, it is premature to 16. Lazo, G. R., R. Roffey, and D. W. Gabriel. 1987. Restriction propose new Xanthomonas taxa on the basis of the presently fragment length pol ymorphisms distinguish pathovars of Xan- thomonas campestris. Int. J. Syst. Bacteriol. 37:214-221. available fragmentary data. 17. Marmur, J. 1961. A procedure for the isolation of deoxyribo- nucleic acid from microorganisms. J. Mol. Biol. 3:208-218. 18. Schoulties, C. L., E. L. Civerolo, J. W. Miller, R. E. Stall, C. J. Krass, S. R. Poe, and E. P. DuCharme. 1987. Citrus canker in ACKNOWLEDGMENTS Florida. Plant Dis. 71:388-395. 19. Schoulties, C. L., J. W. Miller, R. E. Stall, E. L. Civerolo, and L.V. is indebted to the National Fonds voor Wetenschappelijk M. Sasser. 1985. A new outbreak of citrus canker in Florida. Onderzoek. P.Y. acknowledges the Algemeen Bestuur van de Plant Dis. 69:361. Ontwikkelingssamenwerking. K.K. is indebted to the Fonds voor 20. Sneath, P. H. A., and R. R. Sokal. 1973. The principles and Geneeskundig Wetenschappelijk Onderzoek for personnel and re- practice of numerical classification. W. H. Freeman, San Fran- search grants. cisco. !i42 VAUTERIN ET AL. INT. J. SYST.BACTERIOL.

21. Stall, R. E., and C. P. Seymour. 1983. Canker, a threat to citrus E. L. Civerolo, A. C. Hayward, H. Maraite, R. E. Stall, A. K. in the Gulf-Coast states. Plant Dis. 67581-585. Vidaver, and J. F. Bradbury. 1990. Towards an improved 22. Stead, D. E. 1989. Grouping of Xanthornonas carnpestris patho- taxonomy of Xanthomonas. Int. J. Syst. Bacteriol. 40:312- vars of cereals and grasses by fatty acid profiling. Bull. OEPP 316. (Organ. Eur. Mediterr. Prot. Plant.)/EPPO (Eur. Mediterr. Plant Prot. Organ.) Bull. 195748. 2s. Young, J. M., J. F. Bradbury, L. Gardan, R. I. Gvozdyak, D, E. :23. Vauterin, L., J. Swings, and K. Kersters. 1991. Grouping of Stead, Y. Takikawa, and A. K. Vidaver. 1991. Comment on the Xanthomonas campestris pathovars by SDS-PAGE of proteins. reinstatement of Xanthornonas citri (ex Hasse 1915) and X. J. Gen. Microbiol. 137:1677-1687. phaseoli (ex Smith 1897) Gabriel et al. 1989: indication of the '24. Vauterin, L., J. Swings, K. Kersters, M. Gillis, T. W. Mew, need for minimal standards for the genus Xanthornonas. Int. J. M. N. Schroth, N, J. Palleroni, D. C. Hildebrand, D. E. Stead, Syst. Bacteriol. 41:172-177.