TABLE 2-Continued. X. campestris pv. Strain'. Host. Location. Race. Pattern. 11022(X3HAW). Anthurium sp. Hawaii. 24. 58, 63, 197. Anthurium sp. Puerto Rico.
APPLIED AND ENvIRONMENTAL MICROBIOLOGY, Mar. 1993, p. 851-859
Vol. 59, No. 3
0099-2240/93/03851-09$02.00/0 Copyright © 1993, American Society for Microbiology
Characterization of Xanthomonas campestris Pathovars by rRNA Gene Restriction Patterns YVETTE BERTHIER,1* VALERIE VERDIER,2 JEAN-LUC GUESDON,3 DANIELE CHEVRIER,3 JEAN-BAPTISTE DENIS,' GUY DECOUX,' AND MONIQUE LEMATTRE' Station de Pathologie Vegetale, Institut National de la Recherche Agronomique, Route de Saint Cyr, 78026 Versailles, 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 with 2-acetylamino-fluorene-labelled Escherichia coli 16+23S rRNA probe, 27 different patterns were obtained. The strains are clearly distinguishable at the genus, species, and pathovar levels. The variability of the rRNA gene restriction patterns was determined for four pathovars of Xanthomonas campestris species. The 16 strains ofX. campestris pv. begoniae analyzed gave only one pattern. The variability of rRNA gene restriction patterns of X. campestris pv. manihotis strains could be related to ecotypes. In contrast, the variability of patterns observed for X. campestris pv. malvacearum was not correlated with pathogenicity or with the geographical origins of the 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 related to both host plant and pathogenicity. the identification of phytopathogenic bacteria was proposed (12). In the medical field, the use of rRNA gene restriction patterns as a taxonomic and epidemiologic tool has been demonstrated (20, 34). Recently, Grimont et al. (19) described a test based on rRNA-rDNA hybridization in the absence of radioactive material. This test involves the use of 2-acetylamino-fluorene (AAF)-labelled 16+23S rRNA of Escherichia coli as the probe and anti-AAF monoclonal antibody in an immunoenzymatic detection procedure. On the basis of its value for the characterization of human pathogenic bacteria (5, 20, 34), ribotyping appeared to be a useful taxonomic tool for phytopathogenic bacteria. The conserved nature of rRNA genes allowed the use of a single probe to characterize phylogenetically distant bacteria. The use of nonradioactive labelling of the probe overcomes the radioactive hazards and the instability of radiolabelled probes. A probe assay based on a label which provides signal amplification (e.g., enzymes) is likely to be more sensitive than an assay using a label which provides only a single signal per molecule (e.g., fluorochromes). Hapten-labelled probes are more stable than enzyme-labelled probes (25), and an indirect method is more flexible since one given hapten-labelled probe can be detected with various enzyme reactions (21). Moreover, non-radioactively-labelled probes give sharper bands than 32P-labelled probes in rRNA gene patterns (19). The purpose of this study was to characterize Xanthomonas spp. by rRNA gene restriction patterns at the genus, species, and pathovar levels and to determine the relationship, if any, between the variability of four pathovars to the geographical origins of the strains and their pathogenicity to host plants.
Because of the economic importance of plant diseases caused by xanthomonads, the genus Xanthomonas has been the subject of many taxonomic and determinative studies. The taxonomy of the genus Xanthomonas has been recently reviewed (42). In Bergey's Manual of Systematic Bacteriology, characteristics useful for differentiating the genera of the family Pseudomonadaceae are limited to the requirement for growth factors and the production of xanthomonadins (8). Biochemical characteristics were used to differentiate five species of the genus; however, Xanthomonas ampelina was later reclassified as Xylophilus ampelinus (46). The species Xanthomonas campestris, the most complex species as described by Dye et al. (14), has been divided into more than 125 pathovars. Accordingly, the term pathovar is used to refer to strains with similar characteristics that are differentiated at the infraspecific level on the basis of pathogenicity 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 patterns (35, 41, 43), and fatty acid profiling (10, 18, 38). Although Xanthomonas pathovars are generally distinguishable by these methods, some of them are heterogeneous. Molecular approaches are used increasingly in the taxonomy and epidemiology of Xanthomonas spp. DNA-DNA hybridization has demonstrated the heterogeneity of the genus (23, 33). Restriction fragment length polymorphism (RFLP) analysis of plasmid DNAs (26) and genomic DNA, based on hybridization with different probes, have been used to differentiate X. campestris pathovars (17, 22, 27, 28). By using 23S rRNA probes from Pseudomonas and Xanthomonas spp., De Vos and De Ley (13) distinguished separate rRNA branches in the family Pseudomonadaceae and demonstrated that the genus Xanthomonas is distinct. Recently, the use of 16S rRNA genus-specific sequences for *
MATERIALS AND METHODS
Bacterial strains. A total of 191 strains (Tables 1 and 2), including 4 strains of Pseudomonas solanacearum, 19 strains from the different Xanthomonas species other than
Corresponding author. 851
852
APPL. ENvIRON. MICROBIOL.
BERTHIER ET AL.
TABLE 1. Pseudomonas species, Xanthomonas species, and X. campestris pathovars studied Species or pathovar
Straina
Host
Location
Serovar
Pattern
1
Pseudomonas solanacearum
ORST 1153 b ORST 1153 2c ORST 1155 2a 1000
Solanum melongena Solanum melongena Solanum melongena Lycopersicon esculentum
Congo Congo Congo French Guiana
1 1 1
Xylophilus ampelinus
CFBP 2098 NCPPB 2220 NCPPB 3026
Vits vinifera Vitis vinifera
V4tis vinifera
France Greece Italy
2 2 2
Xanthomonas fragariae
CFBP 2157
Fragaria sp.
United States
3
Xanthomonas axonopodis
NCPPB 2375 NCPPB 457
Axonopus scoparius
Colombia Colombia
4 4
G7 GP 5 HV 5 R8 USA 083 A KNA 003 a LKA 070 A G 55 MDG 065 A MQE 58 BF 60 CIV 035 A 2375 84
Saccharum Saccharum Saccharum Saccharum Saccharum Saccharum Saccharum Saccharum Saccharum Saccharum Saccharum Saccharum Saccharum
NCPPB 528 10601 HMB 29 CFBP 1289 CFBP 1023 CFBP 1024 ORST 1159 CFBP 1816 CFBP 1814 ORST 1144 E5 CFBP 1716 CFBP 1438 10342 CFBP 2286 CFBP 1948
Brassica oleracea Lycopersicum esculentum Manihot esculenta Saccharum sp. Juglans regia Juglans regia Phaseolus vulgaris Phaseolus vulgaris
Xanthomonas albilineans
Xanthomonas campestris pv. campestris vesicatoria cassavae vasculorum juglandis juglandis phaseoli phaseoli citri glycines mangiferae indicae incanae
pelargonii oryzicola oryzae
Axonopus scoparius sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp.
Citrus sp.
Glycine max Mangifera indica Matthiola incana Pelargonium zonale Oryza
sativa
Oryza sativa
Guadeloupe Guadeloupe Burkina Faso Reunion United States St. Kitts Sri Lanka Guadeloupe Madagascar Martinique Burkina Faso Ivory Coast Cameroon
United Kingdom Undetermined Zaire Reunion France France Congo Greece Reunion Congo India United States France Malaysia Cameroon
3 1 2 1
5 5 5 5 5 5
5 S 5 5 5 5 5 6 7 8 9 10 10 10 10 10 10 11 12 15 13 14
a Abbreviations for sources of strains: CFBP, Collection Francaise des Bacteries Phytopathogenes, INRA, Angers, France; NCPPB, National Collection of Plant 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 per liter]) 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 of MgSO4- 7H20, 0.05 g of Na2SO3, and 15 g of agar [each per liter; pH 7]) was used for Xanthomonas albilineans and Xanthomonas axonopodis. Cells were harvested from 1.5 ml of a suspension of 109 CFU/ml by low-speed contrifugation at 1,200 x g for 2 min. The pellet was washed twice in 1 ml of 0.5 M NaCl and once in 1 ml of TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH 8]). The pellet was resuspended in 500 ,ul of TE buffer and heated for 15 min at 70°C. Lysis solution (1.5 ,ul of proteinase K [10 mg/ml; Boehringer], 30 ,ul of Sarkosyl [10%], 30 ,ul of lysozyme [10 mg/ml; Sigma]) was
added to the suspension at room temperature. Samples were then incubated at 50°C for 15 h (7). Deproteinization was performed by sequential phenol and chloroform-isoamyl alcohol (24:1) extraction. After ethanol precipitation, the pellet 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 and Southern transfer. DNA samples (2 to 5 pRg) were cleaved overnight at 37°C by restriction endonucleases EcoRI, BamHI, and HindIII (Boehringer) with 5 U of endonucleases per ,ug of DNA. Horizontal agarose gel electrophoresis of DNA 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 fragments was included. Transfer of DNA to a BA 83 nitrocellulose
RIBOTYPING OF XANTHOMONAS CAMPESTRIS PATHOVARS
VOL. 59, 1993
853
TABLE 2. Pathovars of X. campestris studied more extensively
Straina
X. campestris pv.
begoniae
10122, 10125, 1012, 10129, 10149, 10150, 10126 423, 442, 509, 657 10132, 10135 10144
Race
Location
Host
Pattern
Begonia elatior
France
16
Begonia elatior Begonia rex Begonia
The Netherlands France
Guadeloupe
16 16 16
bambusiforme
manihotis
malvacearum
10137, 10147
Begonia bambusifonne
Ivory Coast
16
ORST 9, 10, 11, 13, 14, 15, 16, 18, 19, 20, 21, 23, 24, 59, 60, 62, 64, 65 ORST 39, 40, 41 ORST 44, 45, 46, 49, 50, 51 X77, X80, X96B, X182A ORST 35, 36 ORST 43 ATCC 23380 CIAT 1060, CIAT 1120 CFBP 1856 CIAT 1061 CFBP 1854, 1855 CIAT 1111
Manihot esculenta
Congo
17
Manihot esculenta Manihot esculenta Manihot esculenta Manihot esculenta Manihot esculenta Manihot esculenta Manihot esculenta Manihot esculenta Manihot esculenta Manihot esculenta Manihot esculenta
Central African Republic Zaire Togo Benin Nigeria United States Colombia Brazil Venezuela Brazil United States
17 17 17 17 17 17 17 17 18 18 19
Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum Gossypium barbadense Gossypium hirsutum Gossypium hirsutum
Soudan Soudan Soudan Senegal Senegal
1 2 20 16-18
20 20 20 20 20
Senegal Burkina Faso
20 20
20 20
Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum
Burkina Faso Madagascar Cameroon
18
20 20 20
Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum Anthurium sp.
Cameroon Ivory Coast Benin Mali Zambia Central African Republic Central African Republic United States United States United States United States Costa Rica Costa Rica Nicaragua Argentina Laos Burkina Faso Burkina Faso Zambia Guadeloupe
Anthunum Anthurium Anthurium Anthurium Anthurium
Martinique Venezuela Puerto Rico Hawaii Brazil
111704, 11705, 11706 11710, 11711 11731(S2) 11715 11716 11717, 11729(SEN 1) 11725(HV 25), 11728(BKF 18), 11742(BKF 31) 11727(BKF 26), 11744(BKF 5) 11748, 11718, 11719, 11720 11721(CMR 8), 11722(CMR87), 11741(CMR) 11724(CMR3) ORST 57 11747 11730, 11703(CFBP) 11742(ZAM 2) 11733(RCA 1) 11734(RCA 2) 11737 11738 11739 11740 11735(C.R.2) 11736(C.R.1) 11723 11702(CFBP 2035 A) 11732 11701(CFBP 2012) 11726(HV 21) 11745(Zam 1)
dieffenbachiae
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, 11027 11014(X1VEN)
157 11015(X2HAW) 11052(NCPPB 1833)
sp. sp. sp. sp. sp.
20 18
20 18 20 2 3 18 7 7 18
20
20 18
20 20 20 20 20 20 20 20 20 20 20 20 20 20 20
21 21 22 23
23 23 23 23 23 Continued on following page
854
APPL. ENvIRON. MICROBIOL.
BERTHIER ET AL.
TABLE 2-Continued X. campestris pv.
Strain'
11022(X3HAW) 58, 63, 197 11050(NCPPB 985), 11051(NCPPB 986) 11002, 11004, 11007, 11020 11012 11201, 11202, 11203, 11204, 11206, 11207, 11208, 11209
Host
Anthurium sp. Anthurium sp.
Location
Race
Pattern
Hawaii
24 24
Dieffenbachia sp.
Puerto Rico United States
Anthunium sp. Anthurium sp. Philodendron sp.
Guadeloupe Guadeloupe United States
25 26 27
25
a Strains of X. campestris pv. malvacearum were received from J. C. Follin, IRCI, Montpellier, France. Strains of X campestris pv. dieffenbachiae were from either 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 by Southern (37). Probes. AAF-labelled 16+23S rRNA from E. coli (Eurogentec, Liege, Belgium) was used to detect fragments in the genomic DNA of bacteria. AAF-labelled pBR322 DNA (Eurogentec) hybridized with 21 DNA fragments of the standard Raoul I set. Hybridization. The transfer membranes were prehybridized at 60°C for 1 h with shaking in a solution of 6 x SSC (1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate [pH 7]), 5 x Denhardt's solution (lx Denhardt's solution is 0.2 g of polyvinylpyrollidone 350, 0.2 g of Ficoll 400, 0.2 g of bovine serum albumin [BSA], and H20 to 1 liter), sodium dodecyl sulfate (SDS) at 0.5%, and 100 ,ug of denatured sheared salmon sperm DNA per ml. The membranes were hybridized for 18 h at 60°C with shaking in 6x SSC, 5 x Denhardt's solution, 100 ,g of denatured sheared salmon sperm DNA per ml, 100 ng of denatured AAF-labelled pBR322 DNA per ml, and 500 ng of labelled denatured rRNA probe per ml. These nonstringent conditions were necessary to allow efficient hybridization because of the great phylogenetic divergence between Xanthomonas spp. and E. coli. The membranes were washed three times in 2x SSC-0.1% SDS and once in 0.lx SSC at 52°C for 15 min with shaking. Immunodetection of rRNA-rDNA duplexes. The hybridized AAF-labelled rRNA was detected by using the anti-AAF monoclonal antibody (K16-16) developed by Masse et al. (32) and available from Eurogentec. The membranes were incubated for 1 h with purified anti-AAF antibody diluted to 1 ,ug/ml in Tris-buffered saline (TBS)-Tween-BSA (0.01 M Tris-HCl [pH 7.5], 0.15 M NaCl, 0.1% Tween, 1% BSA). The membranes were then washed three times for 10 min each in TBS-Tween and incubated further for 1 h with alkaline phosphatase-labelled sheep anti-mouse immunoglobulin G diluted to 1 ,ug/ml in TBS-Tween-BSA. After an additional washing, membranes were incubated for 10 min in the dark in 100 mM Tris-HCl buffer (pH 9.5) containing 100 mM NaCl, 20 mM MgCl2, 0.3 mg of nitroblue tetrazolium per ml, and 0.15 mg of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) per ml. The enzymatic reaction was stopped by washing the membranes in distilled water. Pathogenicity tests. Strains of X. campestris pv. begoniae, malvacearum, manihotis, and dieffenbachiae were tested for pathogenicity on their hosts, i.e., begonia, cotton, cassava, and Anthurium and Dieffenbachia spp., respectively. In each case, only homologous reactions were scored positive for pathogenicity. The 16 strains of X. campestris pv. begoniae were tested on the three Begonia species from which the strains were isolated. Leaf parenchyma was infiltrated with 200 ,ul of
bacterial suspension at 102, 104, and 108 CFU/ml. After inoculation, plants were incubated in saturated humid conditions at 28°C with a day-length period of 12 h of light. Water-soaked lesions leading to chlorosis and necrosis were scored positive for pathogenicity. Twenty-five strains of X. campestris pv. dieffenbachiae from different geographical origins and representative of the different restriction patterns were tested for pathogenicity on their host plants, Anthunum andreanum, Dieffenbachia amonea, and Philodendron spp., respectively. In preliminary assays, the reactions on young leaves were demonstrated to be more significant than those on lower, older leaves under our conditions. For each strain, a young leaf was 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 humidity at 28°C with a day-length period of 12 h of light and then moved to a greenhouse at 25°C for 2 months. Pathogenicity was scored positive as it was for X. campestns pv. begoniae. Strains of X. campestris pv. malvacearum were tested for pathogenicity on the susceptible cotton cultivar Acala as described by Follin et al. (16). Strains of X. campestris pv. manihotis were tested on cassava plants as described by Verdier (45). For each host, negative inoculation controls were infiltrated with water or with a heterologous X. campestris suspension containing 102, 104, or 108 CFU/ml. Statistical analysis. Statistical analysis was carried out with binary data matrices in which rows correspond to a selected subset of strains and columns correspond to hybridizing bands. In fact, for each hybridizing band there are two columns, namely, a presence column (1 if the band is present for the given strain, 0 if not) and an absence column (0 if the band is present, 1 if not). The redundancy obtained by doubling the columns is necessary to give all of the strains the same weight: the number of 1 is equal for every strain. A correspondence analysis (3, 24, 29) was done to calculate distances between all pairs of strains on the most important components; the number of components was chosen to attain at least 50% of the inertia, and three of four were necessary. From these distances, a hierarchical clustering tree (9) was constructed by using the average linkage method. All computations were carried out with S language (1). When two strains have an identical pattern, their distance 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 the greater the probability of merging them in the first steps of the tree is.
RIBOTYPING OF XANTHOMONAS CAMPESTRIS PATHOVARS
VOL. 59, 1993
20 3,4
1
5 6 78
9 1011
12
-9.0 .@
..
..
i-4.0
genomic DNA of X. campestris pathoby 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. FIG.
1.
Southern blot of
cleaved
vars,
RESULTS AND DISCUSSION
Polymorphism of restriction
rRNA gene restriction patterns. Several
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 E.
coli
was
not
shown). AAF-labelled 16+23S rRNA from as a probe to analyze the Pseudomonas
chosen
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
Vairiability
species, and pathovar were
seven.
of rRNA gene restriction patterns at the genus,, levels. The strains listed in Table
variability pathovar levels.
used to determine the
genus,
species,
and
The four strains of P. solanacearum that the
gave
same
pattern,
1
(Fig. 2),
1
of the patterns at the
with
were
five
analyzed fragments
between 2.9 and 9.0 kb. Xanthomonas strains gave patterns
hybridization fragments fragment was common to 24
with
characteristic observed the
patter
of the 26 pattens and seemed
of Xanthomonas patterns. The two exceptions
were
smallest
from 1.5 to 18.5 kb. The 1.5-kb
in pattern
2 of
hybridization
Xylophilus ampelinus,
fragmnent
was
1.4
kb,
where and
in
14 of Xanthomonas oryzae pv. oryzae, where the
(Fig. 2). Strains of Xanthomonas campestris (Xylophilus ampelinus, Xanthomonas fragariae, X. axonopodis, and X. albilineans) smallest
fragment
species
other
was
2 kb
than X.
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
855
geographic regions, X. albilineans seems to be a homogeneous taxon. As expected, and as it was shown with a few examples, strains were easily differentiated by their restriction 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). The two strains of X. campestris pv. juglandis and phaseoli and the only strain of X. campestris pv. citri and glycines gave the same pattern, pattern 10. But, unlike the results obtained with X. albilineans, strains could be differentiated after cleavage of the DNA with BamHI instead of EcoRI. To improve the accuracy of the analysis, DNA fingerprints of Pseudomonas and Xanthomonas spp. schematically represented in Fig. 2 were subjected to similarity analysis. The results are presented in the dendrogram in Fig. 4. Unclustered patterns 11 and 12 were excluded from the dendrogram. Pattern 1, obtained from four strains of P. solanacearum, belongs to the same cluster as Pseudomonas syringae pisi (36a) and Pseudomonas aeruginosa (20). The most distinct pattern was that of Xylophilus ampelinus. By using a 23S rRNA probe in hybridization with genomic DNA from Pseudomonas and Xanthomonas spp., De Vos and De Ley (13) concluded that the genus Xanthomonas, with the exception of Xanthomonas ampelina, was a separate group. Willems et al. (46) proposed to transfer Xanthomonas ampelina to another genus which included only one species, Xylophilus ampelinus. Our results agree with these findings. The three other species described by Bradbury (8), namely X. fragariae, X. albilineans, and X. axonopodis, were grouped in a cluster different from that of the X. campestris species. On the basis of analysis of only a few strains, our results are in agreement with the commonly accepted taxonomy 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 distinct from other X. campestris patterns. However, X. campestris pv. oryzae and oryzicola were recently regrouped at the species level (39) and were found to be closely related by other workers (44). X. campestris pv. pelargonii (pattern 15) was in an intermediate position between the cluster of other X. campestris pathovars and the cluster of different Xanthomonas species. Variability of rRNA gene restriction patterns between strains of various geographical origins. The infraspecific variability of pathovars was studied for X. campestris pv. begoniae, manihotis, malvacearum, and dieffenbachiae. The patterns obtained are schematically presented in Fig. 3, and the strains are listed in Table 2. A dendrogram is presented in Fig. 5. The 16 strains ofX. campestris pv. begoniae from different geographical origins and isolated from hosts belonging to different species of begonia gave the same pattern (i.e., pattern 16). Furthermore, the strains were indistinguishable following 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 strains from Africa, were clustered and gave the major pattern (i.e., pattern 17). In contrast, strains isolated from South America were heterogeneous and gave three patterns. These results suggest that, in spite of the small number of strains from South America analyzed, the most important variability of the pathogen is in the area of origin of the host plant. The homogeneity of the African isolates may be attributed to the
856
APPL. ENVIRON. MICROBIOL.
BERTHIER ET AL. 100
{Kbp
10 ~
-
-
4.0
--
1.4
-
--
9.0
1 2
1
3
7
a
5
4
9
a
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; Xylophilus ampelinus, 2; X. fraganae, 3; X. axonopodis, 4; X. albilineans, 5. Patterns of X 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 to pressure long enough to induce variability, as in the case of South America. In fact, the cassava plant was quite recently introduced in Africa, and cassava bacterial blight was first described in Congo by Boccas et al. (6). The 42 strains of X. campestis pv. malvacearum analyzed gave three different patterns. The major pattern (i.e., pattern
20) was detected in 39 pathogenic strains. It was not possible to distinguish strains that were from different geographical origins or the different pathogenic races identified by Follin et al. (15, 16). The avirulent strain (11745) of race 18 from Zambia was characterized by a special pattern consisting of seven hybridization fragments (pattern 22). Vauterin et al. 2
100
14
TKbp
A
Pseudomonas [
P
1
-18.5
Xanthomonas species
L
6 -99.0
--
-
-4.0
1.4
-
I I
16
17
18
19
20
21
I~W
22
23
24
1
25
26
27
R
FIG. 3. rRNA gene restriction patterns of four X campestns pathovars. Patterns: pathovar begoniae, 16 (16 strains); pathovar manihotis, 17 (38 strains), 18 (3 strains), and 19 (1 strain); pathovar malvacearum, 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).
7-
9
X. campestris pathovars
8 13 16 20
10 17 FIG. 4. Dendrogram derived from the analysis of the patterns of Pseudomonas and Xanthomonas spp. schematically presented in Fig. 2. Abbreviations: A, P. aeruginosa; P, Pseudomonas syringae pv. pisi. Numbers refer to the pattern numbers given in Tables 1 and 2. The lengths of the branches are proportional to the genetic distances.
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RIBOTYPING OF XANTHOMONAS CAMPESTRIS PATHOVARS
26 19 22 27 21 25
18 2316 17
FIG. 5. Dendrogram derived from the analysis of X. campestris patterns schematically presented in Fig. 3.
(41), by analyzing SDS-polyacrylamide gel electrophoresis protein patterns, concluded that X. campestris pv. malconstituted a fairly homogeneous electrophoretic although this worldwide pathogen of cotton consists of a number of pathological races. Our results are in agree-
vacearum
group,
ment with this conclusion.
The highest degree of variability was observed within the pathovar dieffenbachiae. The 53 strains analyzed were characterized by five different patterns (i.e., patterns 23 to 27), with no correlation to the geographical origin of these strains but with clear correlation to the host plant. The majority (34 of 43) of X. campestris pv. dieffenbachiae strains isolated fromAnthunium sp. gave pattern 23 and were pathogenic on Anthurium sp. Three strongly aggressive strains recently isolated from Puerto Rico as well as a strain from Hawaii gave pattern 24. Avirulent strain 11012 exhibited a completely different pattern (pattern 26). Strains 11050 and 11051 isolated from Dieffenbachia amonea gave pattern 25. Four strains presumably isolated from Anthurium sp. also gave pattern 25 but were avirulent onAnthurium sp. and pathogenic on Dieffenbachia sp., suggesting the close relationship with the two strains isolated from Dieffenbachia sp. The eight strains isolated from Philodendron sp. and from the 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 number of strains. The results are presented in a dendrogram in Fig. 5. The most distant patterns, i.e., patterns 26, 22, and 19, were given by single avirulent strains of X. campestris pv. dieffenbachiae, malvacearum, and manihotis, respectively. 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, is not generally reliable and is sometimes difficult to interpret. However, for the pathovar manihotis, we were able to establish a relationship between the pattern and the geographical origin of the strains. Cook et al. (11) identified three distinct RFLP groups of P. solanacearum race 2, each of them being associated with an epidemic outbreak in one geographical origin.
857
For the pathovar dieffenbachiae, it was not possible to relate patterns to geographical origins of the strains. The distribution of Araceae ornamentals and, particularly, Anthunium sp. is greatly governed by the commercial markets on the basis of demand. The results obtained in this study indicate that there is greater variability among the pathovars with a wide host range (i.e., dieffenbachiae) than among those with a narrow host range such as manihotis, malvacearum, and begoniae. Vauterin et al. (43) and Benedict et al. (2) observed that X. campestris pathovars with a narrow host range like begoniae or pelargonii formed homogeneous groups compared with pathovars with a wider host range such as dieffenbachiae. By using monoclonal antibodies to group 323 strains of X. campestris pv. dieffenbachiae isolated from different aroids, Lipp et al. (30) identified 12 serogroups. Anthunium strains formed seven groups, four of which contained exclusively Anthurium strains. By using physiological, pathological, and fatty acid analyses, Chase et al. (10) determined the heterogeneous nature of X. campestis pv. dieffenbachiae. Vauterin et al. (41), by using SDSPAGE of proteins, also classified X. campestnis pv. dieffenbachiae among the heterogeneous X. campestris pathovars. The results obtained by these techniques and those of rRNA gene restriction patterns are similar. On the basis of rRNA gene restriction patterns, strains isolated from Anthunium sp. were clearly distinct from patterns of strains isolated from Philodendron spp. Strains isolated from Anthunium sp. and sharing the same pattern with strains isolated from Dieffenbachia spp. appeared, under our conditions, to be avirulent on Anthunium sp. and pathogenic on Dieffenbachia spp. This suggests that these strains belong to the epiphytic and not the pathogenic flora on Anthunum sp. Nevertheless, recent studies (10, 30) on the pathogenicity of X. campestris pv. dieffenbachiae isolated from aroids demonstrated that these strains are generally more virulent on their host of origin than on other plants but are not strictly host specific. Finally, the results of this study showed that for the pathovars begoniae, malvacearum, manihotis, and dieffenbachiae, major patterns correlated with pathogenicity on the host plant can be used to characterize strains. Recently, atypical isolates of X. campestris pv. vasculorum from Reunion Island were easily distinguished from the type strains by using the rRNA probe. Although patterns given by a few strains of Pseudomonas spp., Xanthomonas species, and different X. campestris pathovars appeared to be in agreement with the current taxonomy, a study including a larger number of strains of each taxon should be conducted to confirm this approach for the taxonomy of the genus Xanthomonas. ACKNOWLEDGMENTS
We are very grateful to Edwin L. Civerolo for the critical reading of the manuscript. We thank Sami Freigoun for encouraging discussions and help in the English translation. We are also grateful to Patrick Marchegay for the computer representation of the patterns. This work was supported by the Institut National de la Recherche Agronomique and the Region Antilles. REFERENCES 1. Becker, R. A., J. M. Chambers, and A. R. Wilks. 1988. The new S language, a programming environment for data analysis and
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