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Diversity and Genetic Relatedness within Genera Xanthomonas and Stenotrophomonas Using Restriction Endonuclease Site Differences of PCR-amplified 16S rRNA Gene
System. Appl. Microbiol. 18, 127-135 (1995) © Gustav Fischer Verlag, Swugart . Jena . New York
Diversity and Genetic Relatedness within Genera Xanthomonas and Stenotrophomonas Using Restriction Endonuclease Site Differences of PeR-amplified 165 rRNA Gene XA VIER NESME J ,2* , MARIO VANEECHOlfITE 3 , STEPHANIE ORSO J , BART HOSTE 4 , and JEAN SWINGS 4 I
3 4
Laboratoire de Microbiologie du Sol, Centre National de la Recherche Scientifique, URA 1977, Universite Lyon 1, F-69622 Villeurbanne cedex, France Institut National de la Recherche Agronomique (INRA) Laboratorium voor Bacteriologie en Virologie, Universitair Ziekenhuis, Universiteit Gent, B-9000 Ghent, Belgium Laboratorium voor Microbiologie, Universiteit Gent, B-9000 Ghent, Belgium Received November 30, 1994
Summary Restriction maps of PCR-amplified 16s rRNA genes (rrs for ribosomal rRNA small subunit) were prepared and compared for 43 Xanthomonas and Stenotrophomonas strains belonging to representative DNA-DNA similarity groups. Nineteen restriction fragment length polymorphisms (RFLPs) were distinguished. forming two main clusters: one contained all Xanthomonas species, the other contained strains of Stenotrophomonas spp. (formerly named X. maltophilia). The genera Xanthomonas and Stenotrophomonas appeared to form a coherent group excluding Xylella fastidiosa. The rrs RFLP permitted to differentiate X. fragariae, X. oryzae, X. axonopodis, X. populi pv. populi from most pathovars of X. campestris. The latter species had very closely related patterns, contrary to X. albilineans, X. campestris pv. hyacinthi and pv. graminis, and a X. campestris strain (isolated from Saccharum officinarum, Guadeloupe) whic formed a more distiner subcluster. All genospecies of Stenotrophomonas had different rrs patterns, and appeared to form a less coherent taxon. Relationships between the percentage of DNADNA hybridization and the rate of nucleotide substitution in rrs confirmed the clear delineation of the two genera.
Key words: Xanthomonas - Stenotrophomonas - PCR-RFLP of 16S rDNA - rrs - Genetic distance Phylogeny - DNA:DNA hybridization groups
Introduction Up to recently, the genus Xanthomonas has been defined as containing both species that cause diseases of diverse plants (X. campestris, X. albilineans, X. axonopodis, X. 0ryzae, X. fragariae, X. populi), and X. maltophilia which is associated to clinical and to plant material, but which is not a plant pathogen (Bradbury, 1984; Swings et aI., 1993). Over 140 known plant pathogens presently have the status of pathovars of Xanthomonas campestris. They are characterized by their high host specificity but they cannot be differentiated readily from other species, subspecies or pathovars using current diagnostic methods • To whom requests should be addressed.
(Dye et aI., 1980; Vauterin et aI., 1990, 1993). The transfer of Pseudomonas maltophilia to the genus Xallthomonas has been proposed by Swings et al. (1983) on the basis of comparative biochemical and molecular biological data; but recently Pal/eroni and Bradbury (1993) have proposed to transfer X. maltophilia to a separate genus Stenotrophomonas. The classification of the genus Xanthomonas is therefore in constant evolution. To a large extent, this is due to the introduction of a number of new andlor more discriminating techniques. Taxonomic techniques based on DNA-DNA similarity have received much attention since they do provide a direct access to the genomic information and therefore to the
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genealogical relationships upon which taxa should be defined. Twenty-one DNA similarity groups have been delineated within genus Xanthomonas (Vauterin et aI., 1993). This scheme comprises 5 DNA similarity groups corresponding to the species X. albilineans, X. frageriae, x. oryzae, X. popllii and the former species X. maltophilia together with 16 new genomic species. It provides no information about the relatedness of the 21 genomic species within Xanthomonas. The DNA similarity-based classifi-
cation does not allow to determine whether a plant pathogen e.g. X. populi is more related to a non-pathogen like X. maltophilia, than to a plant pathogen like X. campestris or even to the related genus Xylella. Using TI oligonucleotide catalogs, Woese et al. (1984) and Wells et al. (1987) showed the close relatedness of X. campestris to X. maltophilia, and various xanthomonads to Xyle/la fastidiosa. At present, sequence analyses of the 165 ribosomal RNA (rRNA) or of the 165 rRNA gene
Table I. DNA-DNA similarity groups and rrs RFLP groups of the Xanthomonas (X.) and Stellotrophonmas (5.) strains used in this study Strain no' LMG LMG LMG LMG LMG LMG CFBP LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG UvlG CFBP CFBP CFBP LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG a
b < d
568 7455 847
T
736T2
673 12141 2112 911Tl 689 688 8660 8658 947 8662 8047 5403 8680 852 5047 708 538Tl 8672 5743 5753 974 2787 1923 2205 471 494 742 726 8678 11114 10883 10857 11108 11087 10882 10853 10996 11002 958
T T T T
T
T
Name
Genospecies b
rrs RFLP'
X. X. X. X. X. X. X. X. X. X. X. X.
XII VIII XIII VI XIV XV
A A A A A
campestris. pv. campestris c. pv. phaseoli c pv. pisi c. pv. holcicola c. pv. cassavae A c. pv. populi populi pv. salicis c. pv. vesicatoria A c. pv. corylina c. pv. corylina c. pv. corylina c. pv. corylina X. c. (Bromus carinatus) X. c. pv. CIIcurbitae X. c. pv. juglandis X. c. pv. poinsettiicola C. X. c. pv. prulli X. c. pv. pruni X. oryzae pv. oryzae X. fragariae X. axollopodis X. c. pv. melonis X. populi pv. populi X. populi pv. populi X. populi pv. populi X. populi pv. populi X. populi pv. populi X. populi pv. populi X. c. (Saccharum officillarum) X. albilinealls X. c. pv. hyacinthi X. c. pv. graminis X. c. pv. poinsettiicola B s. maltophilia S. maltophilia S. maltophilia S. maltophilia S. maltophilia s. maltophilia S. maltophilia s. maltophilia S. maltophilia s. maltophilia
A
nd
A
XVI XV XV XV XV
A A A A A A A A A
V
XX XV XV XV XV VII
A
B
Ao Af C C D
II
VIII XXI IV IV
D D D D D
IV
nd nd nd
XVII
E
I X
F F G H L L I
IX
XIX M3 M3 M8 M5 M2
III III III III III
J (MIh)d (MI?) (MIa) (MIg) (MIb)
N K M
o P
Q
T, type strain; ., pathovar reference strain; LMG, Laboratorium voor Microbiologie Gent Culrure Collection, Gent, Belgium; CFBP, Collection Fran"aise de Bacteries Phytopathogenes, Angers, France. DNA-DNA similivity groups (Vauterin et aI., 1993); nd, not determined. rrs, gene encoding the ribosomal RNA small subunit or I6S rRNA; rrs RFLP groups are defined in Table 2. DNA-DNA subgroups of genospecies III (see text).
rrs RFLP Phylogeny within Genus Xanthomonas
(called rrs ribosomal for rRNA small subunit) is the method of choice to determine phylogenetic relationships between organisms (Woese, 1987). A rapid and versatile method based on restriction endonuclease site differences of PCR-amplified rrs has alternatively be used by several authors (Gurtler et al., 1991; Navarro et al., 1992; Ponsonnet and Nesme, 1994; Vaneechoutte et al., 1992, 1993). The PCR-RFLP study of rrs is simpler and faster than sequencing and suited for use in almost any laboratory. If sufficient enzymes are used and the position of the recognition sites are known, one will obtain a pretty good picture of relatedness. In the present paper, we report on the use of 14 restriction enzymes to show the diversity of PCR-amplified rrs of 43 strains of Xanthomonas and Stenotrophomonas belonging to 23 DNA similarity groups and evaluate their genetic relatedness, and to determine the taxonomic resolution of the rrs PCR-RFLP method. Materials and Methods Microorganisms and DNA isolation. According to Palleroni and Bradbury (1993), we will use the name Stenotrophomonas to indicate the strains formerly classified as X. ma/tophi/ia. The strains investigated by the PCR-RFLP method belong ra 19 genomic species of Xanthomonas and Stenotrophomonas as previously characterized by biochemical features and DNA-DNA hybridization studies (Vauterin et aI., 1993), and to 4 new groups found among Stenotrophomonas (Table 1). Genomic DNA was isolated by the protocol of Marmur (1961). DNA amplification. For DNA amplification, the proracol of MlllIis and Faloona (1987) was followed, with some modifications. The reaction mixtures of 50 J.lI contain 100 ng of genomic DNA (in 10 mM Tris-HCI, pH 8.3, 50 mM KCl, 1.5 mM MgCl2J 0.01 % wlv gelatin), the four dNTPs (at 20 J.IM), primers (at 0.1 ~) and 2.5 units of Taq polymerase (Gibco BRL, Cergy-Ponraise, France). PCR reactions were performed under a thin layer of paraffin oil in a dry-block thermal cycler (PHC-3, Techne Inc, Princeton, NJ, USA) using the following conditions: initial denaturation for 3 min at 95°C, followed by 35 cycles of denaturation (1 min at 95 0q, annealing (1 min at 55°C) and extension (2 min at 72 0q, and a final extension (3 min at 72 0C). The mixture was then stored at - 20°C. Reaction efficiency was estimated by electrophoresing 3 f.ll of the amplification product on 0.8% (w/v) horizontal agarose gels. The prokaryote specific primers used for PCR - FGPS6 (5'-GGA GAG TTA GAT err GGC TCA G-3') and FGPSI509' (5'-AAG GAG GGG ATC CAG CCG CA-3') were designed from the conserved regions of the 16S, and allowed amplification of 99.5% of the gene (Ponsonnet and Nesme, 1994). Restriction enzyme analysis. Three to 5 J.lI of PCR products were digested in a 15 f.ll final volume, for 1 hour. The endonuc1eases were used as specified by the manufacturers: AluI, CfoI, RsaI (Gibco BRL), Ndell (Appligene, Strasbourg, F), HpaII, TaqI, Sau96I, (Boehringer-Mannheim, FRG) and SerFi (Ozyme, Montigny Ie Bretonneux, F), BsoFI, BfaI, Bas]I, Tsp509I, MseI, BstUI, (New England Biolab). The restriction fragments were separated by horizontal electrophoresis in TBE buffer (89 mM Tris borate, 89 mM boric acid, 2 mM EDTA, pH 8.0) with a 3% (w/v) Nusieve (FMC Bioproducts, Rockland, ME, USA) aparose gel containing 1 f.lg. ml· t ethidium bromide. Gels were run at 4 VI em for 2 hours, and immediately photographed with llford FP4 films with a 302 nm UV source. The technique permitted the 9
System. Appl. MICroblol. Vol. 18/1
129
dear visualization as well as the estimation of the length of DNA fragments longer than 70 bp, and only permitted the visualization of poorly resolved fragments berween 40 bp ra 70 bp. Data analysis. The restriction maps were determined from RFLPs by comparison with those inferred from the published rrs sequences of the type strain of S. maltophilia LMG 958 (ATCC 13637, Genbank M59158) and Xylella fastidiosa (ATCC 35880, Genbank M26601). In this study, restriction and nudeotidic sites were numbered according to their position in the rrs sequence of S. ma/tophilia LMG 958 after corrections of some sequence un· certainties in the (41-300) region. Restriction site differences were analyzed by the Wagner parsimony method using the MIX sofware of the Phylip package (Felsenstein 1993). The numbers of nucleotide substitution per nucleotidic site was estimated by d = (-lnS)/r, where S = 2 mx/mx + mr (Nei and Li, 1979; Nei et aI., 1985). Here, fix and mv are the numbers of restriction sites for DNA sequences x and y', respectively, mxy the number of restriction sites shared by the rwo sequences, and r the number of nucleorides in the recognition sequence of the restriction enzymes (r = 4 in all eases in the present study). The distance matrix (d values) was used to construct dendrograms using methods of Fitch and Margo/iash (1967), UPGMA (Sokal and Sneath, 1963), Neighbour-Joining (Saitou and Nei, 1987) and Maximum-Likelihood (Felsenstein, 1992).
Results and Discussion Stringency conditions of PCR allowed the amplification of a single band of the same 1545 bp expected size for all tested strains (data not shown). Digestion of PCR products with fourteen endonucleases provided generally unambiguous patterns from which positions of restriction sites could be inferred from the published sequences of strain LMG 958 or Xylella fastidiosa. Ultimately, 19 different rrs patterns were distinguished (Table 2). Complex patterns were obtained with strains LMG 11002 and LMG 10853 indicating the occurrence of variable sites (459 AluI and 472 HpaII) which were not found on all rrs alleles. Such restriction site differences between alleles within the same genome have already been detected in bacteria (Mevarech et al., 1989; Gurtler et aI., 1991). Further data analysis has been done considering sites variable within strains as effectively present. The hundred trees obtained by the Wagner parsimony method were all equally parsimonious (71 steps for 55 polymorphic sites) and had identical topology (Fig. 1). Slight differences in branch length were only due to site states at upper node of sites 471 SerF I and 1269 BastUI (not shown). The parsimony method provided thus an unambiguous tree of pattern relationships. Genetic distances between the various rrs patterns were also calculated (Table 3). The trees obtained by the distance-based methods, Fitch-Margoliash, Neighbour-Joining, UPGMA, Maximum-Likelihood, are very similar to the parsimonious tree (data not shown). All methods showed two main clusters corresponding to Xanthomonas and to Stenatrophomonas strains, respectively. The most striking difference results in the branching position of pattern L (i.e. DNA:DNA group M3), which appeared either as an outgroup with the parsimony and UPGMA methods, or more related to the Stenotrophomonas cluster with Fitch-Mar-
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X. Nesme et al.
Table 2. Restriction maps of the rrs gene of the various rrs RFLP groups of Xanthamanas. Stenotrophamanas and X)'/e/la fastidiosa. Bacterial rrs RFLP groupsh
Site'
A/ul 83 248
(AGCT)
395 459 651 735 862
1070 (181+195) 1279
efal 220
(GCGC)
380
1109
1464
Hpall
201 472 505
(CCGG)
(33+450)
(173+473)
1165
Ndell 17
(GATe)
206 283
390
(.17+260)
707 (315+338) 1264 Rsal
487
893 1235 1397 Sau971
205
(GTAC)
201 471
614
625
D
E
F
G
H
+c
+
+ +
+
+ + +
+
+ + +
+
+
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+
+
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+
+ + +
+
K
L
M
N
o
p
Q
Xf
+
+ + +
+ + +
+
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+
+
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+
+ + +
+ +
+/-
+ + + +
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+1-
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+ + +
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+ + +
+
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+ + + +
nk
+
+ + + + +
+
+
+
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+/- +
+
+
+
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+ + +
+
+
+ +
+ +
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+
(CCNGG)
+ +
J
+
(GGNCq
932 1210
20
C
+
213 267 333 743
1390 SerFl
B
+
307
1360 1535
Ao
+
1176 1267
1306 1388
Af
+ +
290
1446
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+ + +
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nk
rrs RFLP Phylogeny within Genus Xanthomonas
131
Table 2. Continued. Bacterial rrs RFLP groupsb
Site'
681 740
798
Af
Ao
B
C
D
E
F
G
H
+ +
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+
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(GCNGC) +
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64
(195-400)
859 964
+
+
(50+110)
+122 353 401
(100+70) •
523/526 733 881 1053 1259 1370 1526 BfaI (CTAG) 250 473 653 1348 MseJ (TIAA)
+
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Tsp5091
561 631
677
843 921
961
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1093 1153 1449 BstUI (CGCG) 219/221 403 528
+ +
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Xf
+
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873 959
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+
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0
+
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+ +
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M
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+
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L
+ +
+
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+
K
+
+ +
+
621
+ + +
+
J
+ + + + +
+
465 595
459
+
+ +
1221 1387/1388 TaqI (TCGA)
869
+
+ +
987
973 1269 1454
+
+
+
885
1002 1041 1325 BsoFl 40 71
A
+
+
+
+ + + +
+ +
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+
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+
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+
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+
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+ +
+
+ +
+
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(AATI)
+
+
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+ + nk
+ + + + +
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+ + + +
132
X. Nesme et al.
Tahle 2. Continued. Bacterial
TTS
F
G
H
+
+
+ +
+ + + +
+ + +
+
Site" Af
A
Bs,ljl
C
D
E
+
188 201 27] 527
+ + +
+
+
+ + + + +
+
+
+
+
+
+
+
+ + +
+
+
6U 625
722 740 "'97 885 (I07+2J2) 1221 1387 1416 I·PO
b
B
(CCNNGG)
90
.•
Ao
+
+
+
+
+ +
+
+ +
+ + +
+
+
+ + + + + + + + + + + +
+
+
+
+ + +
+
+ +
+ +
+ +
+ +
+
+ +
+
+
+
+ + +
+ + +
+ +
+ +
+
+ +
+
+
+
+
+ +
+
+
+
+
+
+
RFLP groups!> K
L
M
N
o
P
Q
Xf
+ +
+ +
+ + + + +
+ + + +
+
+ + +
+ +
+ +
+ +
+ +
+
+
+ + + +
+
+ +
+
+
+ + +
+ + +
+ +
+
+ + +
+ +
+
+
+
+ +
+
+ +
+ + + +
+ +
+ +
+ +
+ + +
+ + + +
+
+
+
+ + +
+ + +
+
+ +
nk
+
+ + + + +
+ +
+ +
+
+
+ +
+
+ + +
+ +
+ +
+
+
+
+
+
+ +
+ + + +
+ + + +
nk
site numh~ring is that of the 16S rRNA of Stellotrophol/loll
V. VI. VIII. XII. XIII XIV. XV. XVI. XX VII srte changes
o ,
2
L......L....J
XV
Xanrhomonas
VIII. XXI IV XVII
I. X IX
(i
XIX M5 M8
M?
l'\ M
IIIIMPI
K
o P
XI
IIIIM1., IlIlMlgl
Q L
srenorrophomonas
IIllMlhl
III (Mlb)
M3
I
Xy/eJ/a /asr,d,osa
Fig. 1. Dendrogram of genetic relatedness of Xanthomollas and Stenotrophomonas species based on RFLPs of the rrs gene. The tree was constructed hv using the Wagner parsi~onr method with Tahle 2 data. The scale indicates the number of restriction site changes between two consecutive nodes. Letters correspond to rrs patterns in Tahle 2 and roman characters indicate DNA-DNA similaritv groups or sub-groups in Tabl~ 1.
rrs RFLP Phylogeny within Genus Xanthomonas
133
Table 3. Genetic distances between the different rrs RFLP groups of Xanthomonas, Stenotrophomonas and Xylella fastidiosa. "s RFLP groups·
A
Af
A
941-
Af
13
1 95 40
Ao B
C D E F
G H I
J
K
L
M N 0
P Q
Xf
27 13 13 13
27
27 27
69 55 82 69 68 82 40 27 109 122 94 108 194 207 225 238 180 194 165 178 196 209 209 222 194 207 390 403
Ao
B
2 I 3 2 96 3 40 93 40 27 40 27 82 63 95 83 \09 96 53 40 135 123 121 \09 220 209 252 241 207 196 191 180 222 211 236 225 220 209 416 408
C
D
E
1 2 3 2 95 27 69 83 96 40 122 108 207 238 194 178 209 222 207 403
2 3 2 2 93 69 83 96 40 123 \09 209 241 196 180 211 225 209 376
I
4 5 6 5 5 5 90 42 56 82 169 153 213 230 214 198 230 244 213 464
F
G
5 6 7 6 6 6 3
91 14 95 182 167 256 274 257 241 274 288 271 413
6 5 8 7 7 7 4 1
H
2 3 4 3 3 3 6 7
8 9 10 9 9 9 12 13
92 14 8 109 96 8 196 108 94 180 121 40 269 191 81 288 222 196 271 178 68 255 135 54 288 194 82 301 207 95 284 191 109 426 416 453
7 8 9 8 8 8
II
12
13
K
L
M
N
14
16 17 18 17 17 17 16 19 20 16 14
13
12
IS
16 15 15 15 15 18 19 14 6 9 94 196
9 3 95 122 209 109 13 94 54 123 54 137 40 122 54 435 485
15
14 92 182 196 140 211 196 491
14 15 14 14 14 15 18 19 13
5 8 1 13 93 40 41 27 40 472
13
14 13
13 13
14 17 18 10 4 7 4 14 3 94 54 68 67 469
0
p
Q
Xf
14 IS 14 15 16 15 17 16 16 15 16 15 15 16 15 15 16 15 16 17 IS 19 20 19 20 21 20 14 15 14 7 6 8 \0 9 9 4 4 3 10 15 14 3 2 3 4 5 5 92 1 2 14 93 1 27 13 94 491 505 485
27 28 29 28 28 26 31 28 29 29 31 30 33 33 32 32 33 34 33 93
• rrs RFLP groups are described in Tables 1 and 2: A ro H Xanthom01!aS spp., I ro Q Stenotrophomas spp. b Numbers on and above the diagonal are the rota I number of restriction sites and the number of differing restriction sites, respective-
ly, for fourteen 4-base enzymes. Numbers below the diagonal are the substitutions per nucleotide site (x 10-4) estimated from restriction site homologies according ro Nei et al. (1985).
goliash and Neighbour-Joining methods. All these data confirmed the robustness of the resulting relatedness inference. The present study showed that the variability and the resolution of the rrs PCR-RFLP method depends upon the taxonomic level considered. In the gamma 3 subclass of the Proteobacteria, Xanthomonas, Stenotrophomonas, and Xyllela shared 69 restriction sites. This is much more than the number shared with any other genus of the same subclass (not shown). This therefore confirms the close relatedness of Xylella and Xanthomonas (sensu lato) already shown by Wells et al. (1987). However, 8 restriction sites were found almost exclusively in Xanthomonas and Stenotrophomonas spp. and 10 exclusively in Xylella, allowing the clear differentiation of the latter genus. No rrs pattern was present simultaneously in Xanthomonas and Stenotrophomonas. Moreover, sites Sau961 (213), SerF I (471), Alul (735), Taql (1041) and HpaII (1267) are present more particularly in Stenotrophomonas strains, while BsoFI (40), BsoFI (401) and BsaJI (located bet\veen 885 and 1221) sites are found exclusively in Xanthomonas spp. This confirms the clear difference bet\veen the twO clusters already shown by polyamine patterns (Yang et ai., 1993), supporting the divergence of Xallthomonas and Stellotrophomonas. In the genus Xanthomonas, the rrs RFLP allowed to differentiate X. fragariae, X. oryzae, X. axonopodis, X. populi pv. populi from most pathovars of X. campestris. Difference with the main pattern A were due to single site
differences (Table 2). The respective polymorphic sites were BsaJI (188), BsoFI (two sites located between 71 and 353), Ndell (located between 390 and 707), Cfol (220) for X. fragariae (rrs pattern Af), X. oryzae (Ao), X. axonopodis (C), X. populi pv. populi (D), respectively. Strains belonging to different DNA similarity groups had the same rrs pattern: pattern A was found in strains belonging to DNA similarity groups V, VI, VIII, XII, XIII, XIV, XV, XVI, XX; pattern C in groups VIII and XXI; pattern F in groups I and X. In two instances, strains belonging to the same DNA similarity group were found to have different rrs patterns (group XV with patterns A and B; group VIII which patterns A and C). In these cases, patterns differed only by a single restriction site (sites Alul [248) and NdeII [unlocated), respectively). In all likelihood, there is thus a core of closely related genospecies corresponding to patterns A, Af, Ao, B, C, D, H consisting of most pathovars of X. campestris plus X. axonopodis, X. fragariae, X. oryzae and X. populi. Patterns E, F and G consist of strains belonging to X. albilineans, to a group originally received as X. albilineans (isolated from Saccharum officinarum, Guadeloupe) but later identified as a particular DNA-DNA group of X. campestris (Vauterin et ai., 1990, 1993), to X. c. pv. hyacinthi and to X. c. pv. graminis. The relatively large distances found by rrs RFLP between X. albilineans and the core of other plant pathogenic xanthomonads confirm other results (Berthier et ai., 1993), but the exact positions ov X. c. pv. hyacinthi, X. c. pv. graminis require additional analysis.
134
X. Nesme et al.
From our results, X. populi undoubtedly belongs to the core of plant pathogenic xanthomonads, confirming recent studies based on chemical and biochemical features (Vauterin et al., 1993). Although physiological races of X. populi (Nesme et al., 1994) could not be differentiated by the method, the two pathovars populi and salicis described by De Kam (1977, 1981) were distinguished by RFLPs (Fig. 1). These results showed that it is pertinent to look for differences between rrs sequences in order to differentiate pathovars which can be relatively far removed, but not to differentiate races which may be very closely related since race differences are often due to one single gene. In the genus Stenotrophomonas, rrs pattern N corresponds to DNA-DNA group M2, L to M3, J to M5, I to M8 (Table 1). Different patterns M, K, 0, P and Q were observed within strains belonging to the same DNA-DNA hybridization group III (Table 1). The rrs differences between K and Q amounted 0.0054 nucleotide substitution per nucleotidic site (4/94 restriction sites) (Table 3). Due to the 50% D (degree of binding) hybridization threshold commonly used to determine xanthomonads groups, the group III consisted however of rather distantly related strains like LMG 958 and LMG 10853 which hybridized only 41 % D together (Hoste et al., unpublished results). By using a threshold of 70% D according to the criterion of Wayne et al. (1987), group III could be readily split in more homogeneous sub-groups such as MIa, MIb, MIg, M Ih, M I? (Hoste et al., unpublished data), corresponding to rrs pattern 0, Q, P, K, M, respectively (Fig. 1). Groups obtained by DNA-DNA hybridization as well as rrs RFLP confirmed the discrimination obtained by other methods: strain LMG 10882 (DNA group M1h and rrs pattern K) can be distinguished by fatty acid and Biolog patterns, strain LMG 10996 (MIa and 0) by fatty acids, and LMG 10857 (M8 and I) by Biolog pattern IV although other tested strains were classified in Biolog patterns I or II (unpublished data). Although, the resolution of the PCR-RFLP method in the rrs gene is situated at the level of clusters of genospecies in Xanthomonas, in Stenotrophomonas this is at the level of the DNA-DNA hybridization subgroups. The relationship between DNA-DNA similarity and genetic distance estimated from rrs appeared different in the two genera. As indicated in Fig. 2, distances higher than 0.003 nucleotide substitution per nucleotidic site were found to occur between strains hybridizing less than 50% D within Xanthomonas (X:X), and less than 60% D within Stenotrophomonas (5:5). Considering the rrs as a molecular clock (Ochman and Wilson, 1987), hybridization results suggest thus that for the same period of time (indicated by an identical rrs distance), whole genomic differences (obtained from DNA-DNA hybridization studies) are greater between Xanthomonas spp. than between Stenotrophomonas spp. This is perhaps an adaptative response to the highly variable environment of plant pathogens. In addition, the large rrs distances (d > 0.019) found between Xanthomonas and Stenotrophomonas strains (X:S in Fig.2) were always related to low DNA:DNA similarity « 30% D), confirming the delineation of the two genera. Using this double criterion for genus delineation, the large
Rate of nucleotide substitution in rrs (d) 0.03
A
A
~
A
A~.
A
~~ 0.02
AJ
X:X
i
3:S
+
o 0 00 0 0 (]) ODO + OOO~ 0 + 00 ~OO+OO+ +
0.01
o
&A
o
+ S:S a x:s
O
o aDO[]) o
20
am ++
00 0
40
60
0
000 80
0
100
DNA/DNA hybridization (%) Fig. 2. Relationships between genetic distances estimated by the rate of nucleotide substitution in rrs RFLP and the percentage of DNA-DNA hybridization within Xanthomonas spp., Stenotrophomonas spp., and between Xanthomonas and Stenotrophomonas spp. Dots indicate strain combinations: X:X, Xanthomonas: Xanthomonas; 5:5, Stenotrophomonas: Stenotrophomonas; x:s Xanthomonas: Stenotrophomonas; 3:5, Stenotrophomonas genospecies M3: Stenotrophomonas. d values are calculated from rrs RFLP in Table 3. % of DNA:DNA hybridization are from Vauterin et al. (1993).
rrs distances and the low DNA:DNA similarity calculated between M3 strains and other Stenotrophomonas strains (3:5 in Fig. 2, d > 0.02, < 20% D), strongly suggested that M3 strains differed readily from other Stenotrophomonas spp., and may thus belong to another genus. Xanthomonas and Stenotrophomonas are composed of two species complexes, which can be clearly separated and further differentiated by rrs RFLPs. This may be retained as an additional argument to differentiate the two taxa and to rename X. maltophilia to Stenotrophomonas maltophilia as proposed by Pal/eroni and Bradbury (1993). However, the relatedness tree obtained requires additional information, especially entire rrs sequences, to clearly establish the phylogeny of the Xanthomonadaceae family. Acknowledgments. This work was supported by EEe interdisciplinary Research for Poplar Improvement contract N° AiRlCT92-0349 for XN; SO received a fellowship from the French Ministere de la Recherche et de la Technologie; jS was supported by the National Fund of Scientific Research, Belgium; MV and BH were supported by the Fund of Medical Scientific Research, Belgium. We thank J. Haurat and M. A. Poirier for technical assistance.
rrs RFLP Phylogeny within Genus Xanthomonas
References Berthier, Y., Verdier, V., Guesdon, j. L., Chevrier, D., Denis, j. B., Decoux, G., Lemattre, M.: Characterization of Xanthomonas campestris pathovars by rRNA gene restriction patterns. Appl. Environ. Microbiol. 59, 851-859 (1993) Bradbury, j. F.: Xmrthomonas Dowson 1939, pp. 199-210. In: Bergey's Manual of Systematic Bacteriology (N. R. Krieg, j. G. Holt, eds.), Baltimore, Williams and Wilkins 1984 De Kam, M.: A bacterial disease of Salix dasyclada, caused by a Xanthomonas species and its relation to Aplanobacter populi. Eur. J. For. Path. 7, 257-262 (1977) De Kam, M.: The identification of the two subspecies of Xanthomonas populi in vitro. Eur. J. For. Path. 11,25-29 (1981) Dye, D. W., Bradbury, j. F., Goto, M., Hayward, A. c., Lelliot, R. A., Schroth, M. N.: International standards for naming pathovars of phytopathogenic bacteria and list of pathovar names and pathotype strains. Rev. Plant Pathol. 59, 153-168 (1980) Felsenstein, j.: Phylogenesis from restriction sites: a maximumlikelihood approach. Evolution 46, 159-173 (1992) Felsenstein, j.: Phylip (Phylogeny Inference Package) version 3.5 c. Distributed by the author. Department of Genetics, University of Washington, Seattle, USA (1993) Fitch, F. W., Margoliash, E.: Construction of phylogenetic trees. Science 155, 279-284 (1967) Gurtler, V., Wilson, V. A., Mayall, B. c.: Classification of medically important clostridia using restriction endonuclease site differences of PCR-amplified 16S rDNA. J. Gen. Microbiol. 137,2673-2679 (1991) Marmur, j.: A procedure for the isolation of DNA from microorganisms. J. Mol. BioI. 3, 208-218 (1961) Mevarech, M., Hirsch- Twizer, 5., Goldman,S., Yakobson, E., Eisenberg, H., Dennis, P. P.: Isolation and characterization of the rRNA gene clusters of Halobacterium marismortui.]. Bacteriol. 171, 3479-3485 (1989) Mullis, K. B., Faloona, F. A.: Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction. Methods Enzymol. 155, 335-350 (1987) Navarro, E., Simonet, P., Normand, P., Bardin, R.: Characterization of natural populations of Nitrobacter spp. using PCRI RFLP analysis of the ribosomal intergenic spacer. Arch. MicrobioI. 157, 107-115 (1992) Nei, M., Li, W. H.: Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. 79,5269-5273 (1979) Nei, M., Stephens, j. c., Saitou, N.: Methods for computing the standard errors of branching points in an evolutionary tree and their application to molecular data from human and apes. Mol. BioI. Evol. 2, 66-85 (1985) Nesme, X., Steenackers, M., Steenackers, V., Picard, c., Menard, M., Ride,S., Ride, M.: Differntial host-pathogen interactions among clones of poplar and strains of Xanthomonas populi pv. populi. Phytopathol. 84, 101-107 (1994) Ochman, H., Wilson, A. c.: Evolution in bacteria: evidence for a universal substitution rate in cellular genomes. J. Mol. Evol. 26, 74-86 (1987)
135
Palleroni, N. j., Bradbury, j. F.: Stenotrophomonas, a new bacterial genus for Xanthomonas maltophilia (Hugh 1980) Swings et al. 1983. Int. J. Syst. Bacteriol. 43, 606-609 (1993) Ponsonnet, C, Nesme, X.: Identification of Agrobacterium strains by PCR-RFLP analysis of pTi and chromosomal regions. Arch. Microbiol. 161,300-309 (1994) Saitou, N., Nei, M.: The Neighbour-Joining method: a new method for reconstructing phylogenic tres. Mol. BioI. Evol. 4, 406-425 (1987) Sokal, R. R., Sneath, P. H. A.: The construction of a taxonomic system, pp. 169-210. In: Principles of numerical taxonomy, chap. 7. Freeman, San Francisco, U.S.A. (1963) Swings, j., Vauterin, L., Kersters, K.: The bacterium Xanthomonas, pp. 121-156. In: X,mthomonas U. G. Swings, E. L. Civerolo, eds.), London, Chapman and Hall 1993 Swings, j., De Vos, P., Van Den Moorter, M., De Ley,).: Transfer of Pseudomonas maltophilia Hugh 1981 to the genus Xanthomonas as Xanthomonas maltophilia (Hugh 1981) comb. nov. Int.]. Syst. Bacteriol. 33, 409-413 (1983) Vaneechoutte, M., De Beenhouwer, H., Claeys, G., Verschraegen, G., De Rouck, A., Paepe, N., Elaichollni, A., Portaels, F.: Identification of Mycobacterium species by using amplified ribosomal DNA restriction analysis. J. Clin. Microbiol. 31, 2061-2065 (1993) Vaneechoutte, M., Rossau, R., De Vas, F., Gillis, M., janssens, D., Paepe, N., De Rouck, A., Fiers, T., Claeys, G., Kersters, K.: Rapid identification of bacteria of the Comamonadaceae with amplified ribosomal DNA-restriction analysis (ARDRA). FEMS Microbiol. Lett. 93, 227-234 (1992) Vauterin, L., Swings, j., Kersters, K., Gillis, M., Mew, T. W., Schroth, M. N., Pa/leroni, N. j., Hildebrand, D. c., Stead, D. E., Civerolo, E. L., Hayward, A. C, Maraite, H., Stall, R. E., Vidaver, A. K., Bradbury, J. F.: Toward an improved taxonomy of Xanthomonas. Int. J. Syst. Bacteriol. 40, 312-316 (1990) Vallterin, L., Hoste, B., Yang, P., Alvarez, A., Kersters, K., Swings, J. : Taxonomy of the genus Xanthomonas, pp. 157-192. In: Xanthomonas U. G. Swings, E. L. Civerolo, eds.), London, Chapman and Hall 1993 Wayne, L. G., Brenner, D. j., Colwell, R. R., Grimont, P. A. D., Kandler, 0., Krichevsky, M. i., Moore, L. H., Murray, R. G. E., Stackebrandt, E., Starr, M. P., Truper, H. G.: Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int. J. SYSt. Bacteriol. 37, 463 (1987) Wells,). M., Raju, B. c., Hung, H. Y., Weisburg, W. G., Nandelco-Paul, L., Brenner, D. ).: Xylella fastidiosa gen. nov., sp. nov.: Gram-negative, xylem-limited, fastidious plant bacteria related to Xanthomonas spp. Int. J. Syst. Bacteriol. 37, 136-143 (1987) Woese, C. R.: Bacterial evolution. Microbial. Rev. 51, 221-271 (1987) Woes .., C. R., Blanz, P., Hahn, eM.: What isn't a pseudomonad: the importance of nomenclature in bacterial classification. Syst. Appl. Microbiol. 5,179-195 (1984) Yang, P., Vauterin, L., Vancanneyt, M., Swings, j., Kersters, K.: Application of farry acid methyl esters for the taxonomic analysis of the genus Xanthomonas. Syst. Appl. Microbiol. 16, 47-71 (1993)
Xavier Nesme, Laboraroire de Microbiologie du Sol, Centre National de la Recherche Scientifique, URA 1977, Universite Lyon 1, F-69622 Villeurbanne cedex, France
Diversity and Genetic Relatedness within Genera Xanthomonas and Stenotrophomonas Using Restriction Endonuclease Site Differences of PeR-amplified 165 rRNA Gene XA VIER NESME J ,2* , MARIO VANEECHOlfITE 3 , STEPHANIE ORSO J , BART HOSTE 4 , and JEAN SWINGS 4 I
3 4
Laboratoire de Microbiologie du Sol, Centre National de la Recherche Scientifique, URA 1977, Universite Lyon 1, F-69622 Villeurbanne cedex, France Institut National de la Recherche Agronomique (INRA) Laboratorium voor Bacteriologie en Virologie, Universitair Ziekenhuis, Universiteit Gent, B-9000 Ghent, Belgium Laboratorium voor Microbiologie, Universiteit Gent, B-9000 Ghent, Belgium Received November 30, 1994
Summary Restriction maps of PCR-amplified 16s rRNA genes (rrs for ribosomal rRNA small subunit) were prepared and compared for 43 Xanthomonas and Stenotrophomonas strains belonging to representative DNA-DNA similarity groups. Nineteen restriction fragment length polymorphisms (RFLPs) were distinguished. forming two main clusters: one contained all Xanthomonas species, the other contained strains of Stenotrophomonas spp. (formerly named X. maltophilia). The genera Xanthomonas and Stenotrophomonas appeared to form a coherent group excluding Xylella fastidiosa. The rrs RFLP permitted to differentiate X. fragariae, X. oryzae, X. axonopodis, X. populi pv. populi from most pathovars of X. campestris. The latter species had very closely related patterns, contrary to X. albilineans, X. campestris pv. hyacinthi and pv. graminis, and a X. campestris strain (isolated from Saccharum officinarum, Guadeloupe) whic formed a more distiner subcluster. All genospecies of Stenotrophomonas had different rrs patterns, and appeared to form a less coherent taxon. Relationships between the percentage of DNADNA hybridization and the rate of nucleotide substitution in rrs confirmed the clear delineation of the two genera.
Key words: Xanthomonas - Stenotrophomonas - PCR-RFLP of 16S rDNA - rrs - Genetic distance Phylogeny - DNA:DNA hybridization groups
Introduction Up to recently, the genus Xanthomonas has been defined as containing both species that cause diseases of diverse plants (X. campestris, X. albilineans, X. axonopodis, X. 0ryzae, X. fragariae, X. populi), and X. maltophilia which is associated to clinical and to plant material, but which is not a plant pathogen (Bradbury, 1984; Swings et aI., 1993). Over 140 known plant pathogens presently have the status of pathovars of Xanthomonas campestris. They are characterized by their high host specificity but they cannot be differentiated readily from other species, subspecies or pathovars using current diagnostic methods • To whom requests should be addressed.
(Dye et aI., 1980; Vauterin et aI., 1990, 1993). The transfer of Pseudomonas maltophilia to the genus Xallthomonas has been proposed by Swings et al. (1983) on the basis of comparative biochemical and molecular biological data; but recently Pal/eroni and Bradbury (1993) have proposed to transfer X. maltophilia to a separate genus Stenotrophomonas. The classification of the genus Xanthomonas is therefore in constant evolution. To a large extent, this is due to the introduction of a number of new andlor more discriminating techniques. Taxonomic techniques based on DNA-DNA similarity have received much attention since they do provide a direct access to the genomic information and therefore to the
128
X. Nesme et al.
genealogical relationships upon which taxa should be defined. Twenty-one DNA similarity groups have been delineated within genus Xanthomonas (Vauterin et aI., 1993). This scheme comprises 5 DNA similarity groups corresponding to the species X. albilineans, X. frageriae, x. oryzae, X. popllii and the former species X. maltophilia together with 16 new genomic species. It provides no information about the relatedness of the 21 genomic species within Xanthomonas. The DNA similarity-based classifi-
cation does not allow to determine whether a plant pathogen e.g. X. populi is more related to a non-pathogen like X. maltophilia, than to a plant pathogen like X. campestris or even to the related genus Xylella. Using TI oligonucleotide catalogs, Woese et al. (1984) and Wells et al. (1987) showed the close relatedness of X. campestris to X. maltophilia, and various xanthomonads to Xyle/la fastidiosa. At present, sequence analyses of the 165 ribosomal RNA (rRNA) or of the 165 rRNA gene
Table I. DNA-DNA similarity groups and rrs RFLP groups of the Xanthomonas (X.) and Stellotrophonmas (5.) strains used in this study Strain no' LMG LMG LMG LMG LMG LMG CFBP LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG UvlG CFBP CFBP CFBP LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG LMG a
b < d
568 7455 847
T
736T2
673 12141 2112 911Tl 689 688 8660 8658 947 8662 8047 5403 8680 852 5047 708 538Tl 8672 5743 5753 974 2787 1923 2205 471 494 742 726 8678 11114 10883 10857 11108 11087 10882 10853 10996 11002 958
T T T T
T
T
Name
Genospecies b
rrs RFLP'
X. X. X. X. X. X. X. X. X. X. X. X.
XII VIII XIII VI XIV XV
A A A A A
campestris. pv. campestris c. pv. phaseoli c pv. pisi c. pv. holcicola c. pv. cassavae A c. pv. populi populi pv. salicis c. pv. vesicatoria A c. pv. corylina c. pv. corylina c. pv. corylina c. pv. corylina X. c. (Bromus carinatus) X. c. pv. CIIcurbitae X. c. pv. juglandis X. c. pv. poinsettiicola C. X. c. pv. prulli X. c. pv. pruni X. oryzae pv. oryzae X. fragariae X. axollopodis X. c. pv. melonis X. populi pv. populi X. populi pv. populi X. populi pv. populi X. populi pv. populi X. populi pv. populi X. populi pv. populi X. c. (Saccharum officillarum) X. albilinealls X. c. pv. hyacinthi X. c. pv. graminis X. c. pv. poinsettiicola B s. maltophilia S. maltophilia S. maltophilia S. maltophilia S. maltophilia s. maltophilia S. maltophilia s. maltophilia S. maltophilia s. maltophilia
A
nd
A
XVI XV XV XV XV
A A A A A A A A A
V
XX XV XV XV XV VII
A
B
Ao Af C C D
II
VIII XXI IV IV
D D D D D
IV
nd nd nd
XVII
E
I X
F F G H L L I
IX
XIX M3 M3 M8 M5 M2
III III III III III
J (MIh)d (MI?) (MIa) (MIg) (MIb)
N K M
o P
Q
T, type strain; ., pathovar reference strain; LMG, Laboratorium voor Microbiologie Gent Culrure Collection, Gent, Belgium; CFBP, Collection Fran"aise de Bacteries Phytopathogenes, Angers, France. DNA-DNA similivity groups (Vauterin et aI., 1993); nd, not determined. rrs, gene encoding the ribosomal RNA small subunit or I6S rRNA; rrs RFLP groups are defined in Table 2. DNA-DNA subgroups of genospecies III (see text).
rrs RFLP Phylogeny within Genus Xanthomonas
(called rrs ribosomal for rRNA small subunit) is the method of choice to determine phylogenetic relationships between organisms (Woese, 1987). A rapid and versatile method based on restriction endonuclease site differences of PCR-amplified rrs has alternatively be used by several authors (Gurtler et al., 1991; Navarro et al., 1992; Ponsonnet and Nesme, 1994; Vaneechoutte et al., 1992, 1993). The PCR-RFLP study of rrs is simpler and faster than sequencing and suited for use in almost any laboratory. If sufficient enzymes are used and the position of the recognition sites are known, one will obtain a pretty good picture of relatedness. In the present paper, we report on the use of 14 restriction enzymes to show the diversity of PCR-amplified rrs of 43 strains of Xanthomonas and Stenotrophomonas belonging to 23 DNA similarity groups and evaluate their genetic relatedness, and to determine the taxonomic resolution of the rrs PCR-RFLP method. Materials and Methods Microorganisms and DNA isolation. According to Palleroni and Bradbury (1993), we will use the name Stenotrophomonas to indicate the strains formerly classified as X. ma/tophi/ia. The strains investigated by the PCR-RFLP method belong ra 19 genomic species of Xanthomonas and Stenotrophomonas as previously characterized by biochemical features and DNA-DNA hybridization studies (Vauterin et aI., 1993), and to 4 new groups found among Stenotrophomonas (Table 1). Genomic DNA was isolated by the protocol of Marmur (1961). DNA amplification. For DNA amplification, the proracol of MlllIis and Faloona (1987) was followed, with some modifications. The reaction mixtures of 50 J.lI contain 100 ng of genomic DNA (in 10 mM Tris-HCI, pH 8.3, 50 mM KCl, 1.5 mM MgCl2J 0.01 % wlv gelatin), the four dNTPs (at 20 J.IM), primers (at 0.1 ~) and 2.5 units of Taq polymerase (Gibco BRL, Cergy-Ponraise, France). PCR reactions were performed under a thin layer of paraffin oil in a dry-block thermal cycler (PHC-3, Techne Inc, Princeton, NJ, USA) using the following conditions: initial denaturation for 3 min at 95°C, followed by 35 cycles of denaturation (1 min at 95 0q, annealing (1 min at 55°C) and extension (2 min at 72 0q, and a final extension (3 min at 72 0C). The mixture was then stored at - 20°C. Reaction efficiency was estimated by electrophoresing 3 f.ll of the amplification product on 0.8% (w/v) horizontal agarose gels. The prokaryote specific primers used for PCR - FGPS6 (5'-GGA GAG TTA GAT err GGC TCA G-3') and FGPSI509' (5'-AAG GAG GGG ATC CAG CCG CA-3') were designed from the conserved regions of the 16S, and allowed amplification of 99.5% of the gene (Ponsonnet and Nesme, 1994). Restriction enzyme analysis. Three to 5 J.lI of PCR products were digested in a 15 f.ll final volume, for 1 hour. The endonuc1eases were used as specified by the manufacturers: AluI, CfoI, RsaI (Gibco BRL), Ndell (Appligene, Strasbourg, F), HpaII, TaqI, Sau96I, (Boehringer-Mannheim, FRG) and SerFi (Ozyme, Montigny Ie Bretonneux, F), BsoFI, BfaI, Bas]I, Tsp509I, MseI, BstUI, (New England Biolab). The restriction fragments were separated by horizontal electrophoresis in TBE buffer (89 mM Tris borate, 89 mM boric acid, 2 mM EDTA, pH 8.0) with a 3% (w/v) Nusieve (FMC Bioproducts, Rockland, ME, USA) aparose gel containing 1 f.lg. ml· t ethidium bromide. Gels were run at 4 VI em for 2 hours, and immediately photographed with llford FP4 films with a 302 nm UV source. The technique permitted the 9
System. Appl. MICroblol. Vol. 18/1
129
dear visualization as well as the estimation of the length of DNA fragments longer than 70 bp, and only permitted the visualization of poorly resolved fragments berween 40 bp ra 70 bp. Data analysis. The restriction maps were determined from RFLPs by comparison with those inferred from the published rrs sequences of the type strain of S. maltophilia LMG 958 (ATCC 13637, Genbank M59158) and Xylella fastidiosa (ATCC 35880, Genbank M26601). In this study, restriction and nudeotidic sites were numbered according to their position in the rrs sequence of S. ma/tophilia LMG 958 after corrections of some sequence un· certainties in the (41-300) region. Restriction site differences were analyzed by the Wagner parsimony method using the MIX sofware of the Phylip package (Felsenstein 1993). The numbers of nucleotide substitution per nucleotidic site was estimated by d = (-lnS)/r, where S = 2 mx/mx + mr (Nei and Li, 1979; Nei et aI., 1985). Here, fix and mv are the numbers of restriction sites for DNA sequences x and y', respectively, mxy the number of restriction sites shared by the rwo sequences, and r the number of nucleorides in the recognition sequence of the restriction enzymes (r = 4 in all eases in the present study). The distance matrix (d values) was used to construct dendrograms using methods of Fitch and Margo/iash (1967), UPGMA (Sokal and Sneath, 1963), Neighbour-Joining (Saitou and Nei, 1987) and Maximum-Likelihood (Felsenstein, 1992).
Results and Discussion Stringency conditions of PCR allowed the amplification of a single band of the same 1545 bp expected size for all tested strains (data not shown). Digestion of PCR products with fourteen endonucleases provided generally unambiguous patterns from which positions of restriction sites could be inferred from the published sequences of strain LMG 958 or Xylella fastidiosa. Ultimately, 19 different rrs patterns were distinguished (Table 2). Complex patterns were obtained with strains LMG 11002 and LMG 10853 indicating the occurrence of variable sites (459 AluI and 472 HpaII) which were not found on all rrs alleles. Such restriction site differences between alleles within the same genome have already been detected in bacteria (Mevarech et al., 1989; Gurtler et aI., 1991). Further data analysis has been done considering sites variable within strains as effectively present. The hundred trees obtained by the Wagner parsimony method were all equally parsimonious (71 steps for 55 polymorphic sites) and had identical topology (Fig. 1). Slight differences in branch length were only due to site states at upper node of sites 471 SerF I and 1269 BastUI (not shown). The parsimony method provided thus an unambiguous tree of pattern relationships. Genetic distances between the various rrs patterns were also calculated (Table 3). The trees obtained by the distance-based methods, Fitch-Margoliash, Neighbour-Joining, UPGMA, Maximum-Likelihood, are very similar to the parsimonious tree (data not shown). All methods showed two main clusters corresponding to Xanthomonas and to Stenatrophomonas strains, respectively. The most striking difference results in the branching position of pattern L (i.e. DNA:DNA group M3), which appeared either as an outgroup with the parsimony and UPGMA methods, or more related to the Stenotrophomonas cluster with Fitch-Mar-
130
X. Nesme et al.
Table 2. Restriction maps of the rrs gene of the various rrs RFLP groups of Xanthamanas. Stenotrophamanas and X)'/e/la fastidiosa. Bacterial rrs RFLP groupsh
Site'
A/ul 83 248
(AGCT)
395 459 651 735 862
1070 (181+195) 1279
efal 220
(GCGC)
380
1109
1464
Hpall
201 472 505
(CCGG)
(33+450)
(173+473)
1165
Ndell 17
(GATe)
206 283
390
(.17+260)
707 (315+338) 1264 Rsal
487
893 1235 1397 Sau971
205
(GTAC)
201 471
614
625
D
E
F
G
H
+c
+
+ +
+
+ + +
+
+ + +
+
+
+ +
+
+
+ +
+
+
+
+ + +
+
K
L
M
N
o
p
Q
Xf
+
+ + +
+ + +
+
+ + +
+
+
+ +
+
+ + +
+ +
+/-
+ + + +
+ +
+
+1-
+
+
+ +
+ +
+
+
+
+
+
+
+ +
+ + +
+ + + +
+ + + +
+ + + +
+ + +
+/- + + +
+
+
+
+
+ +
+
+
+
+ +
+ + + + +
+ +
+ + + +
+
+
+
+
+
+
+
+ +
+ +
+ +
+ +
+
+ +
+ +
+ +
+ +
+
+
+
+
+
+
+
+
+
+
+
+
+
+ +
+ +
+ + + +
+
+
+ +
+ + +
+ + + +
+
+
+
+ +
+ + + +
+ + +
+ + + +
+ + + +
+ +
+ +
+ +
+
+
+
+ +
+
+
+
+
+ +
+ +
+ + +
+
+ + +
+ +
+
+
+ +
+ + + +
+ +
+ +
+ +
+
+ +
+ +
+
+
+
+
+
+
+
+
+
+ + + +
+
+
+
+
+
+
+
+
+
+
+ + +
+ +
+ +
+ +
+
+
+
+
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ + +
+ +
+ +
+ + +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+ + + + +
+
+
+ +
+
+
+ +
+
+ +
+
+
+ +
+
+
+ +
+
+
+ + +
+
+ + + + +
+
+
+
+
+ +
+
+ +
+ +
+
+ +
+ +
+ +
+
+
+
+
+
+ +
+ +
+ +
+
+ +
+ +
+
+ +
+
+
+
+
+
+ +
+ +
+ + +
+
+
+
+ + + +
+
+
+ +
+
+
+
+
+
+
+ + +
+ +
+
+
+
+
+
+
+
+
+
+ +
+
+
+
+
+
+
+
+
+
+
+
+ +
+ +
+ +
+
+
+ +
+ +
+
+
+
+
+
+
+
+
+
+
+
+ +
+
+
+
+ + + +
+ + + +
+ + + +
+
+ +
+
+
+
+
+ +
+ +
+
+
+ + +
+
+
+
+
+
+
+
+
+
+
+
+
+ +
+ + +
+ + +
+ +
+ + +
+
+
+
+ + + +
nk
+
+ + + + +
+
+
+
+
+
+
+ + +
+ +
+
+
+
+
+
+ + + + +
+
+
+ + + + +
+ +
+ + +
+ + + + + + + +
+
+
+
+
+
+
+
+ +
+
+ +
+ +
+ +
+ +
+ + +
+ +
+
+
+
+
+
+
+ +
+ +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+ +
+ +
+
+
+
+
+
+
+
+ +
+
+ + +
+ + +
+
+
+
+ +
+ +
+
+
+
+
+ +
+ + + + +
+ + + +
+
+
+
+/- +
+
+
+
+
+
+
+
+
+
+
+
+ + +
+
+
+ +
+ +
+
+
(CCNGG)
+ +
J
+
(GGNCq
932 1210
20
C
+
213 267 333 743
1390 SerFl
B
+
307
1360 1535
Ao
+
1176 1267
1306 1388
Af
+ +
290
1446
A
+ + +
+
+
+
+ + + +
+
+
+
+ +
+
+
+
+ +
+
+
+
+ +
+ + +
+ +
+
+
+ + +
+ + + +
+
+ + + +
+
+ +
+ + + + +
+
+
+ +
+
+
+
+
+ +
+
+ +
+ +
+
+
+
+
+
+
+
+
+
+
+ + +
+ +
+
+ +
nk
rrs RFLP Phylogeny within Genus Xanthomonas
131
Table 2. Continued. Bacterial rrs RFLP groupsb
Site'
681 740
798
Af
Ao
B
C
D
E
F
G
H
+ +
+ +
+
+ +
+
+
+
+ + +
+ + +
+ +
+ +
+ + + + + +
+
+ + + +
+ + + + + + +
+
+
+
+ + +
+ + + + + + +
+
+
+
+
+
+
+ + +
+
+ +
+
+
+
+
+
+
+
+
+ +
+
+ + +
+ + +
+
+
+ +
+ + +
+
+
+ + +
+
+
+ +
+ + +
+
+
+
+
+
+
+
+
+
+
+ +
(GCNGC) +
+
+ + + + + +
+
+
+
+
+ +
+ +
+ +
+ +
+
+ +
+ +
+ +
+ +
+
+
+ + + +
+
+ +
+ +
+ + + +
+
+ + + + + + +
+ +
+
+
+
+
+
+
+ +
+ +
64
(195-400)
859 964
+
+
(50+110)
+122 353 401
(100+70) •
523/526 733 881 1053 1259 1370 1526 BfaI (CTAG) 250 473 653 1348 MseJ (TIAA)
+
+
+
+
+ +
+ + +
+
+
+ +
+
+ +
+
+
+ +
+
+
+
+
+
+
+
+ + + +
+
+
+ +
+
+
+
+ + +
+ + + + + +
+ +
+ +
+
+
+ +
+
+
+ + +
+ + + + + +
+
+
+
+
Tsp5091
561 631
677
843 921
961
+ + +
+ +
+
+
+
+ + +
+ + +
+
+
+
+
+ + +
+
+ + +
+
+ +
+ + +
+ + +
+
+ + +
+ +
+
+ +
+
+
+
+
+
+
+ +
+ +
+
+
+ +
+ + +
+
+
+
+
+ + + + + +
+ + + + +
+ + +
+ +
+ +
+
+
+ + + + + +
+
+
+
+
+
+
+ +
+
+ + +
+
+
+
+ +
+ + +
+
+
+ + +
+
+
+ +
+
+
+
+
+ + + +
+ +
+
+
+ + + +
+
+
+ + +
+
+
+
+ +
+
+ + +
+
+
+
+
+ +
+
+
+
+
+
+
+
+ + +
+
+ + + +
+
+
+
+
+
+ + + + +
+
+
+
+
+
+ +
+ + + +
+
+
+
+ +
+
+
+
+ + + +
+
+ +
+
1093 1153 1449 BstUI (CGCG) 219/221 403 528
+ +
+
+
+ + +
+
+ +
+
+
+ +
+
+ + + +
+ + + + + + +
+
+ +
+ + +
+
+
+ +
+ + +
+ + + + +
+
+
+ +
+ + +
+
+
+ + +
Xf
+
+
873 959
Q
+
+
+
P
+
+
+ +
0
+
+
+ +
N
+
+
+ +
M
+
+
+ +
+
+
L
+ +
+
+
+
K
+
+ +
+
621
+ + +
+
J
+ + + + +
+
465 595
459
+
+ +
1221 1387/1388 TaqI (TCGA)
869
+
+ +
987
973 1269 1454
+
+
+
885
1002 1041 1325 BsoFl 40 71
A
+
+
+
+ + + +
+ +
+
+
+ +
+ + +
+
+
+ +
+
+
+
+ + +
+
+
+
+ +
+
+
+
+
+
+
+
+
+
+
+
+
+
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+
+ +
+
+
+
+
+
+ + +
+ +
+ + +
+
+
+
+
+ + +
+
+ +
+ + +
+ +
+
+
+
+
+
+
+
+
+
+
+ +
+ +
+
+ + + + +
+ + + + +
+
+
+ + +
+
+
+
+
+
+ + + +
+ + + +
+ + + +
+ +
+
+ + + +
+ +
+
+ +
+
+ +
+
+
+
+
+ + + +
+
+ + +
+
+
+
+
+ +
+
+
+ +
+
+ +
+
+
+
(AATI)
+
+
+ + + +
+ + +
+ + +
+
+ + + +
+ + + + + +
+ +
+
+
+
+ + +
+ +
+
+ +
+ +
+
+
+
+ + + + + +
+ + + + +
+ + + +
+ + + +
+ +
+
+
+
+ +
+
+ + + +
+ +
+ +
+
+ +
+ +
+ + nk
+ + + + +
+
+ + +
+ +
+ + +
+
+ + + +
+ + + +
132
X. Nesme et al.
Tahle 2. Continued. Bacterial
TTS
F
G
H
+
+
+ +
+ + + +
+ + +
+
Site" Af
A
Bs,ljl
C
D
E
+
188 201 27] 527
+ + +
+
+
+ + + + +
+
+
+
+
+
+
+
+ + +
+
+
6U 625
722 740 "'97 885 (I07+2J2) 1221 1387 1416 I·PO
b
B
(CCNNGG)
90
.•
Ao
+
+
+
+
+ +
+
+ +
+ + +
+
+
+ + + + + + + + + + + +
+
+
+
+ + +
+
+ +
+ +
+ +
+ +
+
+ +
+
+
+
+ + +
+ + +
+ +
+ +
+
+ +
+
+
+
+
+ +
+
+
+
+
+
+
RFLP groups!> K
L
M
N
o
P
Q
Xf
+ +
+ +
+ + + + +
+ + + +
+
+ + +
+ +
+ +
+ +
+ +
+
+
+ + + +
+
+ +
+
+
+ + +
+ + +
+ +
+
+ + +
+ +
+
+
+
+ +
+
+ +
+ + + +
+ +
+ +
+ +
+ + +
+ + + +
+
+
+
+ + +
+ + +
+
+ +
nk
+
+ + + + +
+ +
+ +
+
+
+ +
+
+ + +
+ +
+ +
+
+
+
+
+
+ +
+ + + +
+ + + +
nk
site numh~ring is that of the 16S rRNA of Stellotrophol/loll
V. VI. VIII. XII. XIII XIV. XV. XVI. XX VII srte changes
o ,
2
L......L....J
XV
Xanrhomonas
VIII. XXI IV XVII
I. X IX
(i
XIX M5 M8
M?
l'\ M
IIIIMPI
K
o P
XI
IIIIM1., IlIlMlgl
Q L
srenorrophomonas
IIllMlhl
III (Mlb)
M3
I
Xy/eJ/a /asr,d,osa
Fig. 1. Dendrogram of genetic relatedness of Xanthomollas and Stenotrophomonas species based on RFLPs of the rrs gene. The tree was constructed hv using the Wagner parsi~onr method with Tahle 2 data. The scale indicates the number of restriction site changes between two consecutive nodes. Letters correspond to rrs patterns in Tahle 2 and roman characters indicate DNA-DNA similaritv groups or sub-groups in Tabl~ 1.
rrs RFLP Phylogeny within Genus Xanthomonas
133
Table 3. Genetic distances between the different rrs RFLP groups of Xanthomonas, Stenotrophomonas and Xylella fastidiosa. "s RFLP groups·
A
Af
A
941-
Af
13
1 95 40
Ao B
C D E F
G H I
J
K
L
M N 0
P Q
Xf
27 13 13 13
27
27 27
69 55 82 69 68 82 40 27 109 122 94 108 194 207 225 238 180 194 165 178 196 209 209 222 194 207 390 403
Ao
B
2 I 3 2 96 3 40 93 40 27 40 27 82 63 95 83 \09 96 53 40 135 123 121 \09 220 209 252 241 207 196 191 180 222 211 236 225 220 209 416 408
C
D
E
1 2 3 2 95 27 69 83 96 40 122 108 207 238 194 178 209 222 207 403
2 3 2 2 93 69 83 96 40 123 \09 209 241 196 180 211 225 209 376
I
4 5 6 5 5 5 90 42 56 82 169 153 213 230 214 198 230 244 213 464
F
G
5 6 7 6 6 6 3
91 14 95 182 167 256 274 257 241 274 288 271 413
6 5 8 7 7 7 4 1
H
2 3 4 3 3 3 6 7
8 9 10 9 9 9 12 13
92 14 8 109 96 8 196 108 94 180 121 40 269 191 81 288 222 196 271 178 68 255 135 54 288 194 82 301 207 95 284 191 109 426 416 453
7 8 9 8 8 8
II
12
13
K
L
M
N
14
16 17 18 17 17 17 16 19 20 16 14
13
12
IS
16 15 15 15 15 18 19 14 6 9 94 196
9 3 95 122 209 109 13 94 54 123 54 137 40 122 54 435 485
15
14 92 182 196 140 211 196 491
14 15 14 14 14 15 18 19 13
5 8 1 13 93 40 41 27 40 472
13
14 13
13 13
14 17 18 10 4 7 4 14 3 94 54 68 67 469
0
p
Q
Xf
14 IS 14 15 16 15 17 16 16 15 16 15 15 16 15 15 16 15 16 17 IS 19 20 19 20 21 20 14 15 14 7 6 8 \0 9 9 4 4 3 10 15 14 3 2 3 4 5 5 92 1 2 14 93 1 27 13 94 491 505 485
27 28 29 28 28 26 31 28 29 29 31 30 33 33 32 32 33 34 33 93
• rrs RFLP groups are described in Tables 1 and 2: A ro H Xanthom01!aS spp., I ro Q Stenotrophomas spp. b Numbers on and above the diagonal are the rota I number of restriction sites and the number of differing restriction sites, respective-
ly, for fourteen 4-base enzymes. Numbers below the diagonal are the substitutions per nucleotide site (x 10-4) estimated from restriction site homologies according ro Nei et al. (1985).
goliash and Neighbour-Joining methods. All these data confirmed the robustness of the resulting relatedness inference. The present study showed that the variability and the resolution of the rrs PCR-RFLP method depends upon the taxonomic level considered. In the gamma 3 subclass of the Proteobacteria, Xanthomonas, Stenotrophomonas, and Xyllela shared 69 restriction sites. This is much more than the number shared with any other genus of the same subclass (not shown). This therefore confirms the close relatedness of Xylella and Xanthomonas (sensu lato) already shown by Wells et al. (1987). However, 8 restriction sites were found almost exclusively in Xanthomonas and Stenotrophomonas spp. and 10 exclusively in Xylella, allowing the clear differentiation of the latter genus. No rrs pattern was present simultaneously in Xanthomonas and Stenotrophomonas. Moreover, sites Sau961 (213), SerF I (471), Alul (735), Taql (1041) and HpaII (1267) are present more particularly in Stenotrophomonas strains, while BsoFI (40), BsoFI (401) and BsaJI (located bet\veen 885 and 1221) sites are found exclusively in Xanthomonas spp. This confirms the clear difference bet\veen the twO clusters already shown by polyamine patterns (Yang et ai., 1993), supporting the divergence of Xallthomonas and Stellotrophomonas. In the genus Xanthomonas, the rrs RFLP allowed to differentiate X. fragariae, X. oryzae, X. axonopodis, X. populi pv. populi from most pathovars of X. campestris. Difference with the main pattern A were due to single site
differences (Table 2). The respective polymorphic sites were BsaJI (188), BsoFI (two sites located between 71 and 353), Ndell (located between 390 and 707), Cfol (220) for X. fragariae (rrs pattern Af), X. oryzae (Ao), X. axonopodis (C), X. populi pv. populi (D), respectively. Strains belonging to different DNA similarity groups had the same rrs pattern: pattern A was found in strains belonging to DNA similarity groups V, VI, VIII, XII, XIII, XIV, XV, XVI, XX; pattern C in groups VIII and XXI; pattern F in groups I and X. In two instances, strains belonging to the same DNA similarity group were found to have different rrs patterns (group XV with patterns A and B; group VIII which patterns A and C). In these cases, patterns differed only by a single restriction site (sites Alul [248) and NdeII [unlocated), respectively). In all likelihood, there is thus a core of closely related genospecies corresponding to patterns A, Af, Ao, B, C, D, H consisting of most pathovars of X. campestris plus X. axonopodis, X. fragariae, X. oryzae and X. populi. Patterns E, F and G consist of strains belonging to X. albilineans, to a group originally received as X. albilineans (isolated from Saccharum officinarum, Guadeloupe) but later identified as a particular DNA-DNA group of X. campestris (Vauterin et ai., 1990, 1993), to X. c. pv. hyacinthi and to X. c. pv. graminis. The relatively large distances found by rrs RFLP between X. albilineans and the core of other plant pathogenic xanthomonads confirm other results (Berthier et ai., 1993), but the exact positions ov X. c. pv. hyacinthi, X. c. pv. graminis require additional analysis.
134
X. Nesme et al.
From our results, X. populi undoubtedly belongs to the core of plant pathogenic xanthomonads, confirming recent studies based on chemical and biochemical features (Vauterin et al., 1993). Although physiological races of X. populi (Nesme et al., 1994) could not be differentiated by the method, the two pathovars populi and salicis described by De Kam (1977, 1981) were distinguished by RFLPs (Fig. 1). These results showed that it is pertinent to look for differences between rrs sequences in order to differentiate pathovars which can be relatively far removed, but not to differentiate races which may be very closely related since race differences are often due to one single gene. In the genus Stenotrophomonas, rrs pattern N corresponds to DNA-DNA group M2, L to M3, J to M5, I to M8 (Table 1). Different patterns M, K, 0, P and Q were observed within strains belonging to the same DNA-DNA hybridization group III (Table 1). The rrs differences between K and Q amounted 0.0054 nucleotide substitution per nucleotidic site (4/94 restriction sites) (Table 3). Due to the 50% D (degree of binding) hybridization threshold commonly used to determine xanthomonads groups, the group III consisted however of rather distantly related strains like LMG 958 and LMG 10853 which hybridized only 41 % D together (Hoste et al., unpublished results). By using a threshold of 70% D according to the criterion of Wayne et al. (1987), group III could be readily split in more homogeneous sub-groups such as MIa, MIb, MIg, M Ih, M I? (Hoste et al., unpublished data), corresponding to rrs pattern 0, Q, P, K, M, respectively (Fig. 1). Groups obtained by DNA-DNA hybridization as well as rrs RFLP confirmed the discrimination obtained by other methods: strain LMG 10882 (DNA group M1h and rrs pattern K) can be distinguished by fatty acid and Biolog patterns, strain LMG 10996 (MIa and 0) by fatty acids, and LMG 10857 (M8 and I) by Biolog pattern IV although other tested strains were classified in Biolog patterns I or II (unpublished data). Although, the resolution of the PCR-RFLP method in the rrs gene is situated at the level of clusters of genospecies in Xanthomonas, in Stenotrophomonas this is at the level of the DNA-DNA hybridization subgroups. The relationship between DNA-DNA similarity and genetic distance estimated from rrs appeared different in the two genera. As indicated in Fig. 2, distances higher than 0.003 nucleotide substitution per nucleotidic site were found to occur between strains hybridizing less than 50% D within Xanthomonas (X:X), and less than 60% D within Stenotrophomonas (5:5). Considering the rrs as a molecular clock (Ochman and Wilson, 1987), hybridization results suggest thus that for the same period of time (indicated by an identical rrs distance), whole genomic differences (obtained from DNA-DNA hybridization studies) are greater between Xanthomonas spp. than between Stenotrophomonas spp. This is perhaps an adaptative response to the highly variable environment of plant pathogens. In addition, the large rrs distances (d > 0.019) found between Xanthomonas and Stenotrophomonas strains (X:S in Fig.2) were always related to low DNA:DNA similarity « 30% D), confirming the delineation of the two genera. Using this double criterion for genus delineation, the large
Rate of nucleotide substitution in rrs (d) 0.03
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DNA/DNA hybridization (%) Fig. 2. Relationships between genetic distances estimated by the rate of nucleotide substitution in rrs RFLP and the percentage of DNA-DNA hybridization within Xanthomonas spp., Stenotrophomonas spp., and between Xanthomonas and Stenotrophomonas spp. Dots indicate strain combinations: X:X, Xanthomonas: Xanthomonas; 5:5, Stenotrophomonas: Stenotrophomonas; x:s Xanthomonas: Stenotrophomonas; 3:5, Stenotrophomonas genospecies M3: Stenotrophomonas. d values are calculated from rrs RFLP in Table 3. % of DNA:DNA hybridization are from Vauterin et al. (1993).
rrs distances and the low DNA:DNA similarity calculated between M3 strains and other Stenotrophomonas strains (3:5 in Fig. 2, d > 0.02, < 20% D), strongly suggested that M3 strains differed readily from other Stenotrophomonas spp., and may thus belong to another genus. Xanthomonas and Stenotrophomonas are composed of two species complexes, which can be clearly separated and further differentiated by rrs RFLPs. This may be retained as an additional argument to differentiate the two taxa and to rename X. maltophilia to Stenotrophomonas maltophilia as proposed by Pal/eroni and Bradbury (1993). However, the relatedness tree obtained requires additional information, especially entire rrs sequences, to clearly establish the phylogeny of the Xanthomonadaceae family. Acknowledgments. This work was supported by EEe interdisciplinary Research for Poplar Improvement contract N° AiRlCT92-0349 for XN; SO received a fellowship from the French Ministere de la Recherche et de la Technologie; jS was supported by the National Fund of Scientific Research, Belgium; MV and BH were supported by the Fund of Medical Scientific Research, Belgium. We thank J. Haurat and M. A. Poirier for technical assistance.
rrs RFLP Phylogeny within Genus Xanthomonas
References Berthier, Y., Verdier, V., Guesdon, j. L., Chevrier, D., Denis, j. B., Decoux, G., Lemattre, M.: Characterization of Xanthomonas campestris pathovars by rRNA gene restriction patterns. Appl. Environ. Microbiol. 59, 851-859 (1993) Bradbury, j. F.: Xmrthomonas Dowson 1939, pp. 199-210. In: Bergey's Manual of Systematic Bacteriology (N. R. Krieg, j. G. Holt, eds.), Baltimore, Williams and Wilkins 1984 De Kam, M.: A bacterial disease of Salix dasyclada, caused by a Xanthomonas species and its relation to Aplanobacter populi. Eur. J. For. Path. 7, 257-262 (1977) De Kam, M.: The identification of the two subspecies of Xanthomonas populi in vitro. Eur. J. For. Path. 11,25-29 (1981) Dye, D. W., Bradbury, j. F., Goto, M., Hayward, A. c., Lelliot, R. A., Schroth, M. N.: International standards for naming pathovars of phytopathogenic bacteria and list of pathovar names and pathotype strains. Rev. Plant Pathol. 59, 153-168 (1980) Felsenstein, j.: Phylogenesis from restriction sites: a maximumlikelihood approach. Evolution 46, 159-173 (1992) Felsenstein, j.: Phylip (Phylogeny Inference Package) version 3.5 c. Distributed by the author. Department of Genetics, University of Washington, Seattle, USA (1993) Fitch, F. W., Margoliash, E.: Construction of phylogenetic trees. Science 155, 279-284 (1967) Gurtler, V., Wilson, V. A., Mayall, B. c.: Classification of medically important clostridia using restriction endonuclease site differences of PCR-amplified 16S rDNA. J. Gen. Microbiol. 137,2673-2679 (1991) Marmur, j.: A procedure for the isolation of DNA from microorganisms. J. Mol. BioI. 3, 208-218 (1961) Mevarech, M., Hirsch- Twizer, 5., Goldman,S., Yakobson, E., Eisenberg, H., Dennis, P. P.: Isolation and characterization of the rRNA gene clusters of Halobacterium marismortui.]. Bacteriol. 171, 3479-3485 (1989) Mullis, K. B., Faloona, F. A.: Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction. Methods Enzymol. 155, 335-350 (1987) Navarro, E., Simonet, P., Normand, P., Bardin, R.: Characterization of natural populations of Nitrobacter spp. using PCRI RFLP analysis of the ribosomal intergenic spacer. Arch. MicrobioI. 157, 107-115 (1992) Nei, M., Li, W. H.: Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. 79,5269-5273 (1979) Nei, M., Stephens, j. c., Saitou, N.: Methods for computing the standard errors of branching points in an evolutionary tree and their application to molecular data from human and apes. Mol. BioI. Evol. 2, 66-85 (1985) Nesme, X., Steenackers, M., Steenackers, V., Picard, c., Menard, M., Ride,S., Ride, M.: Differntial host-pathogen interactions among clones of poplar and strains of Xanthomonas populi pv. populi. Phytopathol. 84, 101-107 (1994) Ochman, H., Wilson, A. c.: Evolution in bacteria: evidence for a universal substitution rate in cellular genomes. J. Mol. Evol. 26, 74-86 (1987)
135
Palleroni, N. j., Bradbury, j. F.: Stenotrophomonas, a new bacterial genus for Xanthomonas maltophilia (Hugh 1980) Swings et al. 1983. Int. J. Syst. Bacteriol. 43, 606-609 (1993) Ponsonnet, C, Nesme, X.: Identification of Agrobacterium strains by PCR-RFLP analysis of pTi and chromosomal regions. Arch. Microbiol. 161,300-309 (1994) Saitou, N., Nei, M.: The Neighbour-Joining method: a new method for reconstructing phylogenic tres. Mol. BioI. Evol. 4, 406-425 (1987) Sokal, R. R., Sneath, P. H. A.: The construction of a taxonomic system, pp. 169-210. In: Principles of numerical taxonomy, chap. 7. Freeman, San Francisco, U.S.A. (1963) Swings, j., Vauterin, L., Kersters, K.: The bacterium Xanthomonas, pp. 121-156. In: X,mthomonas U. G. Swings, E. L. Civerolo, eds.), London, Chapman and Hall 1993 Swings, j., De Vos, P., Van Den Moorter, M., De Ley,).: Transfer of Pseudomonas maltophilia Hugh 1981 to the genus Xanthomonas as Xanthomonas maltophilia (Hugh 1981) comb. nov. Int.]. Syst. Bacteriol. 33, 409-413 (1983) Vaneechoutte, M., De Beenhouwer, H., Claeys, G., Verschraegen, G., De Rouck, A., Paepe, N., Elaichollni, A., Portaels, F.: Identification of Mycobacterium species by using amplified ribosomal DNA restriction analysis. J. Clin. Microbiol. 31, 2061-2065 (1993) Vaneechoutte, M., Rossau, R., De Vas, F., Gillis, M., janssens, D., Paepe, N., De Rouck, A., Fiers, T., Claeys, G., Kersters, K.: Rapid identification of bacteria of the Comamonadaceae with amplified ribosomal DNA-restriction analysis (ARDRA). FEMS Microbiol. Lett. 93, 227-234 (1992) Vauterin, L., Swings, j., Kersters, K., Gillis, M., Mew, T. W., Schroth, M. N., Pa/leroni, N. j., Hildebrand, D. c., Stead, D. E., Civerolo, E. L., Hayward, A. C, Maraite, H., Stall, R. E., Vidaver, A. K., Bradbury, J. F.: Toward an improved taxonomy of Xanthomonas. Int. J. Syst. Bacteriol. 40, 312-316 (1990) Vallterin, L., Hoste, B., Yang, P., Alvarez, A., Kersters, K., Swings, J. : Taxonomy of the genus Xanthomonas, pp. 157-192. In: Xanthomonas U. G. Swings, E. L. Civerolo, eds.), London, Chapman and Hall 1993 Wayne, L. G., Brenner, D. j., Colwell, R. R., Grimont, P. A. D., Kandler, 0., Krichevsky, M. i., Moore, L. H., Murray, R. G. E., Stackebrandt, E., Starr, M. P., Truper, H. G.: Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int. J. SYSt. Bacteriol. 37, 463 (1987) Wells,). M., Raju, B. c., Hung, H. Y., Weisburg, W. G., Nandelco-Paul, L., Brenner, D. ).: Xylella fastidiosa gen. nov., sp. nov.: Gram-negative, xylem-limited, fastidious plant bacteria related to Xanthomonas spp. Int. J. Syst. Bacteriol. 37, 136-143 (1987) Woese, C. R.: Bacterial evolution. Microbial. Rev. 51, 221-271 (1987) Woes .., C. R., Blanz, P., Hahn, eM.: What isn't a pseudomonad: the importance of nomenclature in bacterial classification. Syst. Appl. Microbiol. 5,179-195 (1984) Yang, P., Vauterin, L., Vancanneyt, M., Swings, j., Kersters, K.: Application of farry acid methyl esters for the taxonomic analysis of the genus Xanthomonas. Syst. Appl. Microbiol. 16, 47-71 (1993)
Xavier Nesme, Laboraroire de Microbiologie du Sol, Centre National de la Recherche Scientifique, URA 1977, Universite Lyon 1, F-69622 Villeurbanne cedex, France