APPLIED AND ENvIRONMENTAL MICROBIOLOGY, Mar. 1993, p. 851-8590099-2240/93/03851-09$02.00/0Copyright © 1993, American Society for Microbiology

Characterization of Xanthomonas campestris Pathovars byrRNA Gene Restriction Patterns

YVETTE BERTHIER,1* VALERIE VERDIER,2 JEAN-LUC GUESDON,3 DANIELE CHEVRIER,3JEAN-BAPTISTE DENIS,' GUY DECOUX,' AND MONIQUE LEMATTRE'

Station de Pathologie Vegetale, Institut National de la Recherche Agronomique, Route de Saint Cyr, 78026Versailles, 1 and Laboratoire de Predeveloppement des Sondes, Institut Pasteur, 75724 Paris Cede-x 15, 3

France, and Laboratoire de Phythopatologie, ORSTOM, Brazzaville, Congo2

Received 30 September 1992/Accepted 11 November 1992

Genomic DNA of 191 strains of the family Pseudomonadaceae, including 187 strains of the genus

Xanthomonas, was cleaved by EcoRI endonuclease. After hybridization of Southern transfer blots with2-acetylamino-fluorene-labelled Escherichia coli 16+23S rRNA probe, 27 different patterns were obtained. Thestrains are clearly distinguishable at the genus, species, and pathovar levels. The variability of the rRNA gene

restriction patterns was determined for four pathovars ofXanthomonas campestris species. The 16 strains ofX.campestris pv. begoniae analyzed gave only one pattern. The variability of rRNA gene restriction patterns ofX. campestris pv. manihotis strains could be related to ecotypes. In contrast, the variability of patterns observedfor X. campestris pv. malvacearum was not correlated with pathogenicity or with the geographical origins ofthe strains. The highest degree of variability of DNA fingerprints was observed within X. campestris pv.

dieffenbachiae, which is pathogenic to several hosts of the Araceae family. In this case, variability was relatedto both host plant and pathogenicity.

Because of the economic importance of plant diseasescaused by xanthomonads, the genus Xanthomonas has beenthe subject of many taxonomic and determinative studies.The taxonomy of the genus Xanthomonas has been recentlyreviewed (42). In Bergey's Manual of Systematic Bacteriol-ogy, characteristics useful for differentiating the genera ofthe family Pseudomonadaceae are limited to the require-ment for growth factors and the production of xanthomona-dins (8). Biochemical characteristics were used to differen-tiate five species of the genus; however, Xanthomonasampelina was later reclassified as Xylophilus ampelinus (46).The species Xanthomonas campestris, the most complexspecies as described by Dye et al. (14), has been divided intomore than 125 pathovars. Accordingly, the term pathovar isused to refer to strains with similar characteristics that are

differentiated at the infraspecific level on the basis of patho-genicity to one or more host plants.Attempts to differentiate X. campestris pathovars by

methods other than pathogenicity have included serology (2,4, 30), phenotypic analysis and protein electrophoretic pat-terns (35, 41, 43), and fatty acid profiling (10, 18, 38).Although Xanthomonas pathovars are generally distinguish-able by these methods, some of them are heterogeneous.Molecular approaches are used increasingly in the taxon-

omy and epidemiology of Xanthomonas spp. DNA-DNAhybridization has demonstrated the heterogeneity of thegenus (23, 33). Restriction fragment length polymorphism(RFLP) analysis of plasmid DNAs (26) and genomic DNA,based on hybridization with different probes, have been usedto differentiate X. campestris pathovars (17, 22, 27, 28).By using 23S rRNA probes from Pseudomonas and Xan-

thomonas spp., De Vos and De Ley (13) distinguishedseparate rRNA branches in the family Pseudomonadaceaeand demonstrated that the genus Xanthomonas is distinct.Recently, the use of 16S rRNA genus-specific sequences for

* Corresponding author.

the identification of phytopathogenic bacteria was proposed(12). In the medical field, the use of rRNA gene restrictionpatterns as a taxonomic and epidemiologic tool has beendemonstrated (20, 34). Recently, Grimont et al. (19) de-scribed a test based on rRNA-rDNA hybridization in theabsence of radioactive material. This test involves the use of2-acetylamino-fluorene (AAF)-labelled 16+23S rRNA ofEscherichia coli as the probe and anti-AAF monoclonalantibody in an immunoenzymatic detection procedure. On thebasis of its value for the characterization of human patho-genic bacteria (5, 20, 34), ribotyping appeared to be a usefultaxonomic tool for phytopathogenic bacteria. The conservednature of rRNA genes allowed the use of a single probe tocharacterize phylogenetically distant bacteria. The use ofnonradioactive labelling of the probe overcomes the radio-active hazards and the instability of radiolabelled probes. Aprobe assay based on a label which provides signal amplifi-cation (e.g., enzymes) is likely to be more sensitive than anassay using a label which provides only a single signal permolecule (e.g., fluorochromes). Hapten-labelled probes aremore stable than enzyme-labelled probes (25), and an indi-rect method is more flexible since one given hapten-labelledprobe can be detected with various enzyme reactions (21).Moreover, non-radioactively-labelled probes give sharperbands than 32P-labelled probes in rRNA gene patterns (19).The purpose of this study was to characterize Xanthomo-

nas spp. by rRNA gene restriction patterns at the genus,species, and pathovar levels and to determine the relation-ship, if any, between the variability of four pathovars to thegeographical origins of the strains and their pathogenicity tohost plants.

MATERIALS AND METHODS

Bacterial strains. A total of 191 strains (Tables 1 and 2),including 4 strains of Pseudomonas solanacearum, 19strains from the different Xanthomonas species other than

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TABLE 1. Pseudomonas species, Xanthomonas species, and X. campestris pathovars studied

Species or pathovar Straina Host Location Serovar Pattern

Pseudomonas solanacearum ORST 1153 b Solanum melongena Congo 1

Xylophilus ampelinus

Xanthomonas fragariae

Xanthomonas axonopodis

Xanthomonas albilineans

ORST 1153 2cORST 1155 2a1000

CFBP 2098NCPPB 2220NCPPB 3026

CFBP 2157

NCPPB 2375NCPPB 457

G 7GP 5HV 5R 8USA 083 AKNA 003 aLKA 070 AG 55MDG 065 AMQE 58BF 60CIV 035 A2375 84

Solanum melongenaSolanum melongenaLycopersicon esculentum

Vits viniferaVitis viniferaV4tis vinifera

Fragaria sp.

Axonopus scopariusAxonopus scopariusSaccharum sp.Saccharum sp.Saccharum sp.Saccharum sp.Saccharum sp.Saccharum sp.Saccharum sp.Saccharum sp.Saccharum sp.Saccharum sp.Saccharum sp.Saccharum sp.Saccharum sp.

Xanthomonas campestris pv.campestrisvesicatoriacassavaevasculorumjuglandisjuglandisphaseoliphaseolicitriglycinesmangiferae indicaeincanaepelargoniioryzicolaoryzae

NCPPB 52810601HMB 29CFBP 1289CFBP 1023CFBP 1024ORST 1159CFBP 1816CFBP 1814ORST 1144 E5CFBP 1716CFBP 143810342CFBP 2286CFBP 1948

Brassica oleraceaLycopersicum esculentumManihot esculentaSaccharum sp.Juglans regiaJuglans regiaPhaseolus vulgarisPhaseolus vulgarisCitrus sp.Glycine maxMangifera indicaMatthiola incanaPelargonium zonaleOryza sativa

Oryza sativa

United KingdomUndeterminedZaireReunionFranceFranceCongoGreeceReunionCongoIndiaUnited StatesFranceMalaysiaCameroon

a Abbreviations for sources of strains: CFBP, Collection Francaise des Bacteries Phytopathogenes, INRA, Angers, France; NCPPB, National Collection ofPlant Pathogenic Bacteria, Harpenden, United Kingdom; ORST, ORSTOM, Brazzaville, Congo. Strains of X albilineans were received from P. Rott,IRAT-CIRAD, Guadeloupe, and P. Baudin, CIRAD, Montpellier, France.

X. campestris, and 168 strains from 17 pathovars of X.campestns, were studied.DNA extraction. Each strain was grown with shaking

overnight at 27°C in 5 ml of liquid medium. NYGB medium(3 g of yeast extract, 5 g of peptone, 20 g of glycerol [each perliter]) was used for X. campestris, while Wilbrink's medium(10 g of sucrose, 5 g of peptone, 0.5 g of K2HPO4, 0.25 g ofMgSO4- 7H20, 0.05 g of Na2SO3, and 15 g of agar [each per

liter; pH 7]) was used for Xanthomonas albilineans andXanthomonas axonopodis. Cells were harvested from 1.5 mlof a suspension of 109 CFU/ml by low-speed contrifugationat 1,200 x g for 2 min. The pellet was washed twice in 1 mlof 0.5 M NaCl and once in 1 ml of TE buffer (10 mMTris-HCl, 1 mM EDTA [pH 8]). The pellet was resuspendedin 500 ,ul of TE buffer and heated for 15 min at 70°C. Lysissolution (1.5 ,ul of proteinase K [10 mg/ml; Boehringer], 30 ,ulof Sarkosyl [10%], 30 ,ul of lysozyme [10 mg/ml; Sigma]) was

added to the suspension at room temperature. Samples werethen incubated at 50°C for 15 h (7). Deproteinization was

performed by sequential phenol and chloroform-isoamylalcohol (24:1) extraction. After ethanol precipitation, thepellet was dried and suspended in 150 ,ul of TE buffer.Samples were incubated for 1 h at 37°C with 1 ,ul of RNase(10 mg/ml) and stored at -20°C.

Gel electrophoresis of endonuclease-cleaved DNA andSouthern transfer. DNA samples (2 to 5 pRg) were cleavedovernight at 37°C by restriction endonucleases EcoRI,BamHI, and HindIII (Boehringer) with 5 U of endonucleasesper ,ug of DNA. Horizontal agarose gel electrophoresis ofDNA samples was done as described by Maniatis et al. (31)by using 0.8% (wt/vol) agarose gel in Tris-borate buffer.DNA standard Raoul I (Appligene) containing 22 fragmentswas included. Transfer of DNA to a BA 83 nitrocellulose

CongoCongoFrench Guiana

FranceGreeceItaly

United States

111

222

ColombiaColombia

3121

GuadeloupeGuadeloupeBurkina FasoReunionUnited StatesSt. KittsSri LankaGuadeloupeMadagascarMartiniqueBurkina FasoIvory CoastCameroon

3

44

5555555S55555

67891010101010101112151314

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RIBOTYPING OF XANTHOMONAS CAMPESTRIS PATHOVARS 853

TABLE 2. Pathovars of X. campestris studied more extensively

X. campestris pv. Straina Host Location Race Pattern

begoniae 10122, 10125, 1012, 10129, Begonia elatior France 1610149, 10150, 10126

423, 442, 509, 65710132, 1013510144

ORST 9, 10, 11, 13, 14, 15, 16,18, 19, 20, 21, 23, 24, 59,60, 62, 64, 65

ORST 39, 40, 41ORST 44, 45, 46, 49, 50, 51X77, X80, X96B, X182AORST 35, 36ORST 43ATCC 23380CIAT 1060, CIAT 1120CFBP 1856CIAT 1061CFBP 1854, 1855CIAT 1111

111704, 11705, 1170611710, 1171111731(S2)1171511716

11717, 11729(SEN 1)11725(HV 25), 11728(BKF 18),11742(BKF 31)

11727(BKF 26), 11744(BKF 5)11748, 11718, 11719, 1172011721(CMR 8), 11722(CMR87),11741(CMR)

11724(CMR3)ORST 571174711730, 11703(CFBP)11742(ZAM 2)11733(RCA 1)11734(RCA 2)1173711738117391174011735(C.R.2)11736(C.R.1)1172311702(CFBP 2035 A)1173211701(CFBP 2012)11726(HV 21)11745(Zam 1)

Begonia elatiorBegonia rexBegoniabambusiformeBegoniabambusifonne

Manihot esculenta

Manihot esculentaManihot esculentaManihot esculentaManihot esculentaManihot esculentaManihot esculentaManihot esculentaManihot esculentaManihot esculentaManihot esculentaManihot esculenta

Gossypium hirsutumGossypium hirsutumGossypium hirsutumGossypium hirsutumGossypiumbarbadenseGossypium hirsutumGossypium hirsutum

Gossypium hirsutumGossypium hirsutumGossypium hirsutum

Gossypium hirsutumGossypium hirsutumGossypium hirsutumGossypium hirsutumGossypium hirsutumGossypium hirsutumGossypium hirsutumGossypium hirsutumGossypium hirsutumGossypium hirsutumGossypium hirsutumGossypium hirsutumGossypium hirsutumGossypium hirsutumGossypium hirsutumGossypium hirsutumGossypium hirsutumGossypium hirsutumGossypium hirsutumAnthurium sp.

The NetherlandsFranceGuadeloupe

Ivory Coast

Congo

Central African RepublicZaireTogoBeninNigeriaUnited StatesColombiaBrazilVenezuelaBrazilUnited States

SoudanSoudanSoudanSenegalSenegal

SenegalBurkina Faso

Burkina FasoMadagascarCameroon

CameroonIvory CoastBeninMaliZambiaCentral African RepublicCentral African RepublicUnited StatesUnited StatesUnited StatesUnited StatesCosta RicaCosta RicaNicaraguaArgentinaLaosBurkina FasoBurkina FasoZambiaGuadeloupe

161616

16

17

1717171717171717181819

122016-18

2020202020

2020

18

20

18

20

2020

202020

202020202020202020202020202020

21212223

182023187

718

20

2018

11001, 11003, 11005, 11006,11008, 11010, 11011, 11016,11017, 11018, 11021, 11024,11025, 11028, 11029, 11030,11037, 11038, 11039, 11040,11041, 11042, 11043, 11044,11045, 11046

11019, 11023, 11026, 1102711014(X1VEN)15711015(X2HAW)11052(NCPPB 1833)

Anthunum sp.Anthurium sp.Anthurium sp.Anthurium sp.Anthurium sp.

MartiniqueVenezuelaPuerto RicoHawaiiBrazil

2323232323

Continued on following page

10137, 10147

manihotis

malvacearum

dieffenbachiae

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TABLE 2-Continued

X. campestris pv. Strain' Host Location Race Pattern

11022(X3HAW) Anthurium sp. Hawaii 2458, 63, 197 Anthurium sp. Puerto Rico 2411050(NCPPB 985), Dieffenbachia sp. United States 2511051(NCPPB 986)

11002, 11004, 11007, 11020 Anthunium sp. Guadeloupe 2511012 Anthurium sp. Guadeloupe 2611201, 11202, 11203, 11204, Philodendron sp. United States 27

11206, 11207, 11208, 11209

a Strains ofX. campestris pv. malvacearum were received from J. C. Follin, IRCI, Montpellier, France. Strains ofX campestris pv. dieffenbachiae were fromeither P. Prior, INRA, Guadeloupe, or our study. CIAT, Centro International de Agricultura Tropical, Cali, Colombia; ATCC, American Type Culture Collection.For other abbreviations for sources of strains, see Table 1, footnote a.

membrane (Schleicher & Schuell) was done as described bySouthern (37).

Probes. AAF-labelled 16+23S rRNA from E. coli (Euro-gentec, Liege, Belgium) was used to detect fragments in thegenomic DNA of bacteria. AAF-labelled pBR322 DNA(Eurogentec) hybridized with 21 DNA fragments of thestandard Raoul I set.

Hybridization. The transfer membranes were prehybrid-ized at 60°C for 1 h with shaking in a solution of 6 x SSC (1 xSSC is 0.15 M NaCl plus 0.015 M sodium citrate [pH 7]), 5 xDenhardt's solution (lx Denhardt's solution is 0.2 g ofpolyvinylpyrollidone 350, 0.2 g of Ficoll 400, 0.2 g of bovineserum albumin [BSA], and H20 to 1 liter), sodium dodecylsulfate (SDS) at 0.5%, and 100 ,ug of denatured shearedsalmon sperm DNA per ml. The membranes were hybridizedfor 18 h at 60°C with shaking in 6x SSC, 5 x Denhardt'ssolution, 100 ,g of denatured sheared salmon sperm DNAper ml, 100 ng of denatured AAF-labelled pBR322 DNA perml, and 500 ng of labelled denatured rRNA probe per ml.These nonstringent conditions were necessary to allow effi-cient hybridization because of the great phylogenetic diver-gence between Xanthomonas spp. and E. coli. The mem-branes were washed three times in 2x SSC-0.1% SDS andonce in 0.lx SSC at 52°C for 15 min with shaking.Immunodetection ofrRNA-rDNA duplexes. The hybridized

AAF-labelled rRNA was detected by using the anti-AAFmonoclonal antibody (K16-16) developed by Masse et al.(32) and available from Eurogentec. The membranes wereincubated for 1 h with purified anti-AAF antibody diluted to1 ,ug/ml in Tris-buffered saline (TBS)-Tween-BSA (0.01 MTris-HCl [pH 7.5], 0.15 M NaCl, 0.1% Tween, 1% BSA).The membranes were then washed three times for 10 mineach in TBS-Tween and incubated further for 1 h withalkaline phosphatase-labelled sheep anti-mouse immuno-globulin G diluted to 1 ,ug/ml in TBS-Tween-BSA. After anadditional washing, membranes were incubated for 10 min inthe dark in 100 mM Tris-HCl buffer (pH 9.5) containing 100mM NaCl, 20mM MgCl2, 0.3 mg of nitroblue tetrazolium perml, and 0.15 mg of 5-bromo-4-chloro-3-indolyl phosphate(BCIP) per ml. The enzymatic reaction was stopped bywashing the membranes in distilled water.

Pathogenicity tests. Strains ofX. campestris pv. begoniae,malvacearum, manihotis, and dieffenbachiae were tested forpathogenicity on their hosts, i.e., begonia, cotton, cassava,and Anthurium and Dieffenbachia spp., respectively. Ineach case, only homologous reactions were scored positivefor pathogenicity.The 16 strains of X. campestris pv. begoniae were tested

on the three Begonia species from which the strains wereisolated. Leaf parenchyma was infiltrated with 200 ,ul of

bacterial suspension at 102, 104, and 108 CFU/ml. Afterinoculation, plants were incubated in saturated humid con-ditions at 28°C with a day-length period of 12 h of light.Water-soaked lesions leading to chlorosis and necrosis werescored positive for pathogenicity.

Twenty-five strains of X. campestris pv. dieffenbachiaefrom different geographical origins and representative of thedifferent restriction patterns were tested for pathogenicity ontheir host plants, Anthunum andreanum, Dieffenbachiaamonea, and Philodendron spp., respectively. In prelimi-nary assays, the reactions on young leaves were demon-strated to be more significant than those on lower, olderleaves under our conditions. For each strain, a young leafwas infiltrated with 200 ,ul of bacterial suspension at 102, 104,or 108 CFUI/ml. Strains 11002, 11004, 11007, and 11020,isolated fromAnthurium sp., were inoculated on D. amonea.After inoculation, plants were kept under saturated humidityat 28°C with a day-length period of 12 h of light and thenmoved to a greenhouse at 25°C for 2 months. Pathogenicitywas scored positive as it was forX. campestns pv. begoniae.

Strains of X. campestris pv. malvacearum were tested forpathogenicity on the susceptible cotton cultivar Acala asdescribed by Follin et al. (16).

Strains of X. campestris pv. manihotis were tested oncassava plants as described by Verdier (45).For each host, negative inoculation controls were infil-

trated with water or with a heterologous X. campestrissuspension containing 102, 104, or 108 CFU/ml.

Statistical analysis. Statistical analysis was carried out withbinary data matrices in which rows correspond to a selectedsubset of strains and columns correspond to hybridizingbands. In fact, for each hybridizing band there are twocolumns, namely, a presence column (1 if the band is presentfor the given strain, 0 if not) and an absence column (0 if theband is present, 1 if not). The redundancy obtained bydoubling the columns is necessary to give all of the strainsthe same weight: the number of 1 is equal for every strain. Acorrespondence analysis (3, 24, 29) was done to calculatedistances between all pairs of strains on the most importantcomponents; the number of components was chosen toattain at least 50% of the inertia, and three of four werenecessary. From these distances, a hierarchical clusteringtree (9) was constructed by using the average linkagemethod. All computations were carried out with S language(1). When two strains have an identical pattern, their dis-tance is null and they are identically represented in the tree.In general, the more similar the patterns of two strains are,the smaller the genetic distance between them is and thegreater the probability of merging them in the first steps ofthe tree is.

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RIBOTYPING OF XANTHOMONAS CAMPESTRIS PATHOVARS 855

1 20 3,4 5 678 9 1011 12

-9.0

i-4.0

.. .. .@

FIG. 1. Southern blot of genomic DNA of X. campestris patho-

vars, cleaved by EcoRI and probed with AAF-labelled rRNA.

Lanes: 1, pathovar glycines (strain 1144 E5, pattern 10); 2, pathovar

begoniae (strain 10150, pattern 16); 3, pathovar malvacearum (strain

11711, pattern 20); 4, pathovar dieffenbachiae (strain 11206, pattern

27); 5, pathovar manihotis (strain ATCC 2380, pattern 17); 6,

pathovar cassavae (strain HMB 29, pattern 8); 7, pathovar juglandis

(strain CFBP 1024, pattern 10); 8, pathovar phaseoli (strain CFBP

1816, pattern 10); 9, pathovar mangiferae indicae (strain CFBP 1716,

pattern 11); 10, pathovar oryzicola (strain CFBP 2286, pattern 13);

11, pathovar oryzae (strain CFBP 1948, pattern 14); 12, molecular

weight standard set Raoul I.

RESULTS AND DISCUSSION

Polymorphism of rRNA gene restriction patterns. Several

restriction enzymes were tested. EcoRI was chosen to

cleave chromosomal DNA of the 191 strains studied in the

present work because it gave the best variability in the

patterns (data not shown). AAF-labelled 16+23S rRNA from

E. coli was chosen as a probe to analyze the Pseudomonas

and Xanthomonas strains listed in Tables 1 and 2. As

examples, typical patterns obtained with various strains are

shown in Fig. 1. The 27 different patterns, numbered 1 to 27,

are schematically represented in Fig. 2 and 3, and the pattern

number corresponding to each strain is given in Tables 1 and

2. The number of fragmnents containing rRNA genes in the

strains studied varied from four to seven.

Vairiability of rRNA gene restriction patterns at the genus,,

species, and pathovar levels. The strains listed in Table 1

were used to determine the variability of the patterns at the

genus, species, and pathovar levels.

The four strains of P. solanacearum that were analyzed

gave the same pattern, 1 (Fig. 2), with five fragmentsbetween 2.9 and 9.0 kb. Xanthomonas strains gave patterns

with hybridization fragments from 1.5 to 18.5 kb. The 1.5-kb

fragment was common to 24 of the 26 pattens and seemed

characteristic ofXanthomonas patterns. The two exceptionsobserved were in pattern 2 of Xylophilus ampelinus, where

the smallest hybridization fragmnent was 1.4 kb, and in

patter 14 of Xanthomonas oryzae pv. oryzae, where the

smallest fragment was 2 kb (Fig. 2). Strains ofXanthomonas

species other than X. campestris (Xylophilus ampelinus,Xanthomonas fragariae, X. axonopodis, and X. albilineans)

gave patterns 2 to 5. The 13 strains of X. albilineans from

different geographical origins and from the three serovars

described by Rott et al. (36) gave the same pattern (i.e.,

pattern 5). The pattern obtained after cleavage of DNA with

other endonucleases (BamHI and HindIII) did not differen-

tiate these strains. On the basis of 13 strains from different

geographic regions, X. albilineans seems to be a homoge-neous taxon. As expected, and as it was shown with a fewexamples, strains were easily differentiated by their restric-tion pattern at the genus and species levels.

Fifteen strains of 13 X. campestris pathovars (Table 1)gave 10 different patterns, i.e., patterns 6 to 15. (Fig. 2). Thetwo strains of X. campestris pv. juglandis and phaseoli andthe only strain of X. campestris pv. citri and glycines gavethe same pattern, pattern 10. But, unlike the results obtainedwith X. albilineans, strains could be differentiated aftercleavage of the DNA with BamHI instead of EcoRI.To improve the accuracy of the analysis, DNA fingerprints

of Pseudomonas and Xanthomonas spp. schematically rep-resented in Fig. 2 were subjected to similarity analysis. Theresults are presented in the dendrogram in Fig. 4. Unclus-tered patterns 11 and 12 were excluded from the dendro-gram. Pattern 1, obtained from four strains of P. solan-acearum, belongs to the same cluster as Pseudomonassyringae pisi (36a) and Pseudomonas aeruginosa (20). Themost distinct pattern was that of Xylophilus ampelinus. Byusing a 23S rRNA probe in hybridization with genomic DNAfrom Pseudomonas and Xanthomonas spp., De Vos and DeLey (13) concluded that the genus Xanthomonas, with theexception of Xanthomonas ampelina, was a separate group.Willems et al. (46) proposed to transfer Xanthomonas am-pelina to another genus which included only one species,Xylophilus ampelinus. Our results agree with these findings.The three other species described by Bradbury (8), namelyX. fragariae, X. albilineans, and X. axonopodis, weregrouped in a cluster different from that of the X. campestrisspecies. On the basis of analysis of only a few strains, ourresults are in agreement with the commonly accepted tax-onomy at the genus and species levels.

Patterns of strains from different X. campestris pathovars,including X. campestris pv. oryzicola (strain CFBP 2286;pattern 13), were clustered in the same group. In contrast,the rRNA gene restriction pattern of X. campestris pv.oryzae (strain CFBP 1948; pattern 14) appeared to be distinctfrom other X. campestris patterns. However, X. campestrispv. oryzae and oryzicola were recently regrouped at thespecies level (39) and were found to be closely related byother workers (44). X. campestris pv. pelargonii (pattern 15)was in an intermediate position between the cluster of otherX. campestris pathovars and the cluster of different Xan-thomonas species.

Variability of rRNA gene restriction patterns betweenstrains of various geographical origins. The infraspecificvariability of pathovars was studied for X. campestris pv.begoniae, manihotis, malvacearum, and dieffenbachiae. Thepatterns obtained are schematically presented in Fig. 3, andthe strains are listed in Table 2. A dendrogram is presentedin Fig. 5.The 16 strains ofX. campestris pv. begoniae from different

geographical origins and isolated from hosts belonging todifferent species of begonia gave the same pattern (i.e.,pattern 16). Furthermore, the strains were indistinguishablefollowing digestion of DNA with HindIlI and BamHI.The 42 strains of X. campestris pv. manihotis gave three

patterns. Thirty-eight strains, including all of the strainsfrom Africa, were clustered and gave the major pattern (i.e.,pattern 17). In contrast, strains isolated from South Americawere heterogeneous and gave three patterns. These resultssuggest that, in spite of the small number of strains fromSouth America analyzed, the most important variability ofthe pathogen is in the area of origin of the host plant. Thehomogeneity of the African isolates may be attributed to the

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100 {Kbp

10-----~ 9.0

- - - 4.0

-- -- 1.4

1 2 3 4 5 a 7 a 9 10 11 12 13 14 16 18 17 20 23 R

FIG. L. Patterns of Pseudomonas spp., Xanthomonas species, and X campestris pathovars. Patterns: P. solanacearum, 1; Xylophilusampelinus, 2; X. fraganae, 3; X. axonopodis, 4; X. albilineans, 5. Patterns ofX campestnis pathovars: campestris, 6; vesicatoria, 7; cassavae,8; vasculorum, 9; juglandis, phaseoli, citri, and glycines, 10; mangiferae indicae, 11; incanae, 12; oryzicola, 13; oryzae, 14; pelargonii, 15;begoniae, 16; manihotis, 17; malvacearum, 20; dieffenbachiae, 23.

fact that the introduced clones were not yet subjected topressure long enough to induce variability, as in the case ofSouth America. In fact, the cassava plant was quite recentlyintroduced in Africa, and cassava bacterial blight was firstdescribed in Congo by Boccas et al. (6).The 42 strains ofX. campestis pv. malvacearum analyzed

gave three different patterns. The major pattern (i.e., pattern

20) was detected in 39 pathogenic strains. It was not possibleto distinguish strains that were from different geographicalorigins or the different pathogenic races identified by Follinet al. (15, 16). The avirulent strain (11745) of race 18 fromZambia was characterized by a special pattern consisting ofseven hybridization fragments (pattern 22). Vauterin et al.

-18.5

Pseudomonas [

Xanthomonasspecies L

- --- -99.0

-4.0

- 1.4II - I~W 1

16 17 18 19 20 21 22 23 24 25 26 27 R

FIG. 3. rRNA gene restriction patterns of four X campestnspathovars. Patterns: pathovar begoniae, 16 (16 strains); pathovarmanihotis, 17 (38 strains), 18 (3 strains), and 19 (1 strain); pathovarmalvacearum, 20 (39 strains), 21 (2 strains), and 22 (1 strain);pathovar dieffenbachiae, 23 (34 strains), 24 (4 strains), 25 (6 strains),26 (1 strain), and 27 (8 strains).

X. campestrispathovars

P

1

67-

A14

2

98

1316201017

FIG. 4. Dendrogram derived from the analysis of the patterns ofPseudomonas and Xanthomonas spp. schematically presented inFig. 2. Abbreviations: A, P. aeruginosa; P, Pseudomonas syringaepv. pisi. Numbers refer to the pattern numbers given in Tables 1 and2. The lengths of the branches are proportional to the geneticdistances.

1

100 TKbp

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261922

2721

25

1823-

1617

FIG. 5. Dendrogram derived from the analysis of X. campestrispatterns schematically presented in Fig. 3.

(41), by analyzing SDS-polyacrylamide gel electrophoresisprotein patterns, concluded that X. campestris pv. mal-vacearum constituted a fairly homogeneous electrophoreticgroup, although this worldwide pathogen of cotton consistsof a number of pathological races. Our results are in agree-

ment with this conclusion.The highest degree of variability was observed within the

pathovar dieffenbachiae. The 53 strains analyzed were char-acterized by five different patterns (i.e., patterns 23 to 27),with no correlation to the geographical origin of these strainsbut with clear correlation to the host plant.The majority (34 of 43) ofX. campestris pv. dieffenbachiae

strains isolated fromAnthunium sp. gave pattern 23 and were

pathogenic on Anthurium sp. Three strongly aggressivestrains recently isolated from Puerto Rico as well as a strainfrom Hawaii gave pattern 24. Avirulent strain 11012 exhib-ited a completely different pattern (pattern 26). Strains 11050and 11051 isolated from Dieffenbachia amonea gave pattern25. Four strains presumably isolated from Anthurium sp.

also gave pattern 25 but were avirulent onAnthurium sp. andpathogenic on Dieffenbachia sp., suggesting the close rela-tionship with the two strains isolated from Dieffenbachia sp.

The eight strains isolated from Philodendron sp. and fromthe same geographical origin gave pattern 27.To improve the accuracy of the analysis, a computer

analysis of patterns schematically presented in Fig. 3 was

done by weighting each pattern by the corresponding num-

ber of strains. The results are presented in a dendrogram inFig. 5. The most distant patterns, i.e., patterns 26, 22, and19, were given by single avirulent strains of X. campestrispv. dieffenbachiae, malvacearum, and manihotis, respec-

tively. The four major patterns of the pathovars studied, i.e.,patterns 16, 17, 20, and 23, are clustered with the other X.campestris patterns described.The use of the probe for epidemiological investigations,

specifically with reference to geographical distribution, isnot generally reliable and is sometimes difficult to interpret.However, for the pathovar manihotis, we were able toestablish a relationship between the pattern and the geo-

graphical origin of the strains. Cook et al. (11) identifiedthree distinct RFLP groups of P. solanacearum race 2, eachof them being associated with an epidemic outbreak in one

geographical origin.

For the pathovar dieffenbachiae, it was not possible torelate patterns to geographical origins of the strains. Thedistribution of Araceae ornamentals and, particularly, An-thunium sp. is greatly governed by the commercial marketson the basis of demand. The results obtained in this studyindicate that there is greater variability among the pathovarswith a wide host range (i.e., dieffenbachiae) than amongthose with a narrow host range such as manihotis, mal-vacearum, and begoniae. Vauterin et al. (43) and Benedict etal. (2) observed that X. campestris pathovars with a narrowhost range like begoniae or pelargonii formed homogeneousgroups compared with pathovars with a wider host rangesuch as dieffenbachiae. By using monoclonal antibodies togroup 323 strains of X. campestris pv. dieffenbachiae iso-lated from different aroids, Lipp et al. (30) identified 12serogroups. Anthunium strains formed seven groups, four ofwhich contained exclusively Anthurium strains. By usingphysiological, pathological, and fatty acid analyses, Chase etal. (10) determined the heterogeneous nature of X. campes-tis pv. dieffenbachiae. Vauterin et al. (41), by using SDS-PAGE of proteins, also classified X. campestnis pv. dieffen-bachiae among the heterogeneous X. campestris pathovars.The results obtained by these techniques and those of rRNAgene restriction patterns are similar.On the basis of rRNA gene restriction patterns, strains

isolated from Anthunium sp. were clearly distinct frompatterns of strains isolated from Philodendron spp. Strainsisolated from Anthunium sp. and sharing the same patternwith strains isolated from Dieffenbachia spp. appeared,under our conditions, to be avirulent on Anthunium sp. andpathogenic on Dieffenbachia spp. This suggests that thesestrains belong to the epiphytic and not the pathogenic floraon Anthunum sp. Nevertheless, recent studies (10, 30) onthe pathogenicity of X. campestris pv. dieffenbachiae iso-lated from aroids demonstrated that these strains are gener-ally more virulent on their host of origin than on other plantsbut are not strictly host specific.

Finally, the results of this study showed that for thepathovars begoniae, malvacearum, manihotis, and dieffen-bachiae, major patterns correlated with pathogenicity on thehost plant can be used to characterize strains. Recently,atypical isolates of X. campestris pv. vasculorum fromReunion Island were easily distinguished from the typestrains by using the rRNA probe. Although patterns given bya few strains of Pseudomonas spp., Xanthomonas species,and different X. campestris pathovars appeared to be inagreement with the current taxonomy, a study including alarger number of strains of each taxon should be conductedto confirm this approach for the taxonomy of the genusXanthomonas.

ACKNOWLEDGMENTS

We are very grateful to Edwin L. Civerolo for the critical readingof the manuscript. We thank Sami Freigoun for encouraging discus-sions and help in the English translation. We are also grateful toPatrick Marchegay for the computer representation of the patterns.

This work was supported by the Institut National de la RechercheAgronomique and the Region Antilles.

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