APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1997, p. 498–505 0099-2240/97/$04.0010 Copyright q 1997, American Society for Microbiology
Vol. 63, No. 2
Assessment of Genetic Diversity among Strains of Pseudomonas syringae by PCR-Restriction Fragment Length Polymorphism Analysis of rRNA Operons with Special Emphasis on P. syringae pv. tomato CHARLES MANCEAU1*
AND
ALAIN HORVAIS2
Station de Pathologie Ve´ge´tale, Institut National de la Recherche Agronomique, 49071 Beaucouze´ cedex,1 and Laboratoire de Ge´nie Ge´ne´tique, Plateforme de Biotechnologie, CHRU, 49033 Angers cedex,2 France Received 23 April 1996/Accepted 27 November 1996
Phylogenetic relationships among 77 bacterial strains belonging to Pseudomonas syringae and Pseudomonas viridiflava species were assessed by analysis of the PCR-restriction fragment length polymorphism (RFLP) patterns of three DNA fragments corresponding to rrs and rrl genes and the internal transcribed spacer, ITS1. No difference among all strains in rrs and rrl genes was observed with 14 restriction enzymes, which confirms the close relationships existing between these two species. The nucleotidic sequence of the internal transcripted spacer (ITS1) between rrs and rrl for the P. syringae pv. syringae strain CFBP1392 was determined. Restriction maps of the PCR-amplified ITS1 region were prepared and compared for all 77 strains. Seventeen RFLP patterns, forming three main clusters, were distinguished. One contained all strains of P. syringae pv. tomato and of other pathovars which had been previously described as closely related by either pathogenicity studies or biochemical analyses. This cluster was equally far from P. viridiflava and from other P. syringae pathovars. These other pathovars of P. syringae formed a less coherent taxon. PCR-amplified rrs (9, 14, 18, 30, 31) has been used for taxonomic and strain-typing purposes. In the present paper, we report on the use of 14 restriction enzymes to show the diversity of PCR-amplified rrs, rrl, and ITS1 of 76 strains of P. syringae belonging to 30 pathovars. The pathovar tomato of P. syringae was used as a model in our study because it has been well characterized by phenotypic and genotypic analyses (3, 4, 11) and pathogenicity studies (11, 34).
Phytopathogenic, fluorescent Pseudomonas strains were formerly named at the species level according to their pathogenicity on plants. Previously, all phytopathogenic, fluorescent, oxidase-negative pseudomonads were included in the species Pseudomonas syringae (2). A few years later, Young et al. (37, 38) introduced the concept of the pathovar. All fluorescent and cytochrome oxidase-negative Pseudomonas species, with the exception of Pseudomonas viridiflava, were considered members of a single species; i.e., P. syringae, which was subdivided into pathovars according to pathogenicity on plants. Although this subdivision is not covered by the International Code of Nomenclature of Bacteria (15), the term pathovar is commonly used among plant pathologists. Classifications of these bacteria were made with phenotypic characters (12, 16). These classifications have been widely used to identify pathovars of P. syringae. In the last decade, several numerical taxonomy studies were based on phenotypic characters, making use of nutritional and biochemical traits (12, 36) and fatty acid (28) and protein (32) profiles. DNA-DNA hybridization techniques have been the standard method to define genomic species among bacteria (33). Using DNA-DNA hybridization, Pecknold and Grogan (23) have shown that P. syringae is a heterogeneous species. More recently, nine genomic species among P. syringae were described (8, 26). At present, sequence analysis of ribosomal operons (rrn) is the method of choice to determine phylogenetic relationships among organisms (35). These operons contain three genes. One, rrs, codes for RNA of the small subunit (16S) of ribosomes; another, rrl, codes for RNA of the large subunit (23S) of ribosomes. The third codes for 5S RNA. Between rrs and rrl exists a noncoding DNA sequence called ITS1 (internal transcribed spacer). A versatile and rapid method based on restriction endonuclease site differences of
MATERIALS AND METHODS Bacterial strains. The strains used in this study are listed in Table 1. They include reference and type strains of 30 P. syringae pathovars, with special attention to P. syringae pv. tomato and closely related pathovars (pathovars maculicola, antirrhini, and apii) and a reference strain of P. viridiflava. All strains were stored as lyophilized stocks and were restreaked for colony uniformity on King’s medium B (13) before use. Plant inoculation. The pathogenicity of selected strains on tomato plantlets (cv. Montfavet 63-4, which is susceptible to both races of P. syringae pv. tomato), was assayed. Inoculations were done by spraying bacterial suspensions (5 3 108 CFU ml21) over the foliage of 1-month-old tomato plants. Inoculated plants were then incubated in a growth chamber (16 h of light at 248C and 8 h of darkness at 208C) under almost 100% relative humidity. Symptoms were recorded 7 days after inoculation. Three plants per strain were inoculated. Sample preparation for direct PCR from cell culture. Bacterial cells, which were grown on King’s B agar medium (13) for 24 h, were resuspended in sterile distilled water. The cell suspensions (approximately 107 cells ml21) were boiled for 10 min and were used for PCR assaying. PCR. Primers were designed from conserved sequences in 16S RNA (19) and 23S RNA (10). For 23S RNA, primers were chosen by comparing nucleotide sequences of Pseudomonas aeruginosa (29) and Escherichia coli (1). Primers A1 (59-GAG TTT GAT CAT GGC TCA G-39) and B6 (59-TTG CGG GAC TTA ACC CAA CAT-39) amplified nearly full-length 16S rRNA genes. Primers D21 (59-AGC CGT AGG GGA ACC TGC GG-39) and D22 (59-TGA CTG CCA AGG CAT CCA CC-39) amplified internal transcribed spacers (ITS1) between 16S rRNA and 23S rRNA genes. Primers N1 (59-GGT GGA TGC CTT GGC AGT CA-39) and N2 (59-AGA TGC TTT CAG CGG TTA TC-39) amplified nearly full-length 23S rRNA genes. Oligonucleotides were synthesized by Eurogentec SA, Seraing, Belgium. PCRs were carried out in a 100-ml reaction volume. A 10-ml volume of boiled bacterial cells was added to 90 ml of PCR mixture (10 mM Tris-HCl [pH 9] at 258C, 50 mM KCl, 1% Triton X-100, 3 mM MgCl2, 0.25 mM [each] dATP, dCTP, dGTP, and dTTP [Eurogentec SA], 20 pmol of each primer, and 2 U of Taq DNA polymerase [Eurotaq; Eurogentec SA]). PCR amplifications in a PTC-100 thermocycler (MJ Research, Inc., Watertown,
* Corresponding author. Phone: (33) 2.41.22.57.17. Fax: (33) 2.41.22.57.05. E-mail:
[email protected]. 498
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TABLE 1. RFLP groups of Pseudomonas strains used in this study Strain(s)a
P. syringae pv. tomato CFBP1318, CFBP1319, CFBP1320, CFBP1321, and CFBP1322 CFBP1323, CFBP1325, CFBP1326, CFBP1426, CFBP1427, CFBP2545, and CFBP2546 CFBP1636, CFBP1916, CFBP1917, CFBP1918, CFBP1919, and CFBP1920 DC3000 TSK10, TSK38, T1, and 832F CFBP1696 CFBP1698 CFBP1785 CFBP2212T JN7 JN16-1 and JN15-1 P. syringae pv. maculicola CFBP1657T CFBP1738 CFBP1740 ICMP2744 CFBP1637 JN8-5 and JN8-10 P. syringae pv. antirrhini CFBP1620T and CFBP1722 P. syringae pv. apii CFBP1726 P. syringae pv. berberidis CFBP1727T P. syringae pv. lachrymans CFBP2440 P. syringae pv. passiflorae CFBP2346T P. syringae pv. persicae CFBP1573T P. syringae CFBP1777 CFBP1678 CFBP1688 CFBP1776 CFBP2278 CFBP1649 P. syringae pv. philadelphi CFBP2398 P. syringae pv. delphini CFBP1730 CFBP1641 CFBP2215T P. syringae pv. garcae CFBP1634T CFBP1733 P. syringae pv. coronafaciens CFBP2216T P. syringae pv. oryzae CFBP3228 P. syringae pv. tagetis CFBP1694T P. syringae pv. porri CFBP1908T P. syringae pv. striafaciens CFBP1674T CFBP1686 P. syringae pv. atropurpurea CFBP2340T P. syringae pv. viburni CFBP1702T P. syringae pv. mori CFBP1642T
Host
Country of isolation
Source or referenceb
Pathogenicity on tomato
RFLP groupc
Lycopersicon esculentum
Switzerland
CFBP
1
A
Lycopersicon esculentum
France
CFBP
1
A
Lycopersicon esculentum
Canada
CFBP
1
A
Lycopersicon Lycopersicon Lycopersicon Lycopersicon Lycopersicon Lycopersicon Lycopersicon Lycopersicon
Canada Canada Denmark United States New Zealand United Kingdom Reunion Island Portugal
D. Cuppels G. Bonn CFBP CFBP CFBP CFBP This study L. Cruz
1 1 1 1 1 1 1 1
A A A A A A A A
Brassica oleracea Brassica oleracea Brassica oleracea Brassica nigra Raphanus sativus Raphanus sativus
New Zealand United Kingdom Zimbabwe Unknown United States France
CFBP CFBP CFBP ICMP CFBP This study
1 1 1 1 1 1
A A A A A A
Antirrhinum sp.
United Kingdom
CFBP
1
A
Apium graveolens
United States
CFBP
1
A
Berberis sp.
New Zealand
CFBP
2
A
Cucumis sativum
United States
CFBP
2
A
Passiflora edulis
New Zealand
CFBP
1
A
Prunus persica
France
CFBP
2
A
Euphorbia pulcherina Magnolia sp. Magnolia sp. Callistemon viminelis Apium graveolens Brassica oleracea
Unknown United Kingdom The Netherlands New Zealand France United Kingdom
CFBP CFBP CFBP CFBP CFBP CFBP
1 1 1 2 2 2
A A A B M E
Philadelphus coronarius
United Kingdom
CFBP
2
C
Delphinium sp. Delphinium elatum Delphinium sp.
Unknown United Kingdom New Zealand
CFBP CFBP CFBP
2 2 2
D D D
Coffea arabica Coffea arabica
Brazil Kenya
CFBP CFBP
2 2
F F
Avena sativa
United Kingdom
CFBP
F
CFBP
F
esculentum esculentum esculentum esculentum esculentum esculentum esculentum esculentum
Oryza sativa Tagetes erecta
Zimbabwe
CFBP
G
Allium porrum
France
CFBP
H
Unknown Avena sativa
Unknown Zimbabwe
CFBP CFBP
H H
Lolium multiflorum
Unknown
CFBP
H
Unknown
Unknown
CFBP
I
Morus alba
Hungary
CFBP
J
Continued on following page
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MANCEAU AND HORVAIS
APPL. ENVIRON. MICROBIOL. TABLE 1—Continued
Straina
P. syringae pv. savastanoi CFBP1670T P. syringae pv. mors-prunorum CFBP1647 P. syringae pv. glycinea CFBP2214T P. syringae pv. aptata CFBP1617T P. syringae pv. syringae CFBP1392Td P. syringae pv. tabaci CFBP2106T P. syringae pv. pisi CFBP2105T P. syringae pv. phaseolicola CFBP1390T P. syringae pv. atrofaciens CFBP2213T P. syringae pv. papulans CFBP1754T P. viridiflava CFBP2107T
Host
Country of isolation
Source or referenceb
Pathogenicity on tomato
RFLP groupc
Olea europa
Yugoslavia
CFBP
J
Prunus avium
Italy
CFBP
K
Glycine max
New Zealand
CFBP
L
Beta vulgaris
United States
CFBP
M
Syringa vulgaris
United Kingdom
CFBP
Nicotiana tabacum
Hungary
CFBP
M
Pisum sativum
New Zealand
CFBP
N
Phaseolus vulgaris
Canada
CFBP
N
Triticum aestivum
New Zealand
CFBP
O
Malus sylvestris
Canada
CFBP
P
Phaseolus vulgaris
Switzerland
CFBP
Q
2
M
a
T, type strains. CFBP, Collection Franc¸aise de Bacte´ries Phythopathoge `nes, Angers, France; ICMP, International Collection of Microorganisms, Auckland, New Zealand. c RFLP groups are defined in footnote a in Table 2. d Species reference strain. b
Mass.) with the following steps were performed: an initial denaturation step at 948C (928C for A1-B6) for 2 min; 36 cycles of 948C (928C) for 1 min, 608C (528C) for 1 min, and 728C for 1 min; and a final extension step of 728C for 2 min. Amplified DNA fragments were examined by horizontal electrophoresis in 3% agarose gels (1% SeaKem GTG, 2% Nuseive GTG; FMC Bioproducts, Rockland, Maine) in TAE buffer (17) with 5-ml aliquots of PCR products. Gels were stained with ethidium bromide and were photographed under UV light (312 nm). Direct DNA sequencing. DNA fragments obtained after PCR were purified from primers and deoxynucleoside triphosphates with Centricon-100 columns (Grace S.a.r.l., Amicon Division, Epernon, France) as described by the manufacturer. A second set of PCRs was performed with the purified DNA fragments under the conditions described previously, except that PCR mixtures contained either 1 and 50 pmol of D21 and D22 primers, respectively, or the inverted concentrations (50 pmol for D21 and 1 pmol for D22). These asymmetric amplifications resulted in synthesis of single-stranded DNAs which were purified from primers and deoxynucleoside triphosphates by using Centricon-100 columns as mentioned above and then used as templates in sequencing reactions by the dideoxy chain termination method (25) with T7 DNA polymerase (Sequenase 2.0 Sequencing kit; U.S. Biochemical, Cleveland, Ohio) and a-35S-dATP. The primers used in the sequencing reactions were those used at low concentration in the respective asymmetric PCRs performed to obtain the DNA templates. Restriction fragment analysis. The following enzymes were used: AluI, Bsh1236I, EcoRI, HaeIII, HhaI, MspI, PstI, RsaI, and TaqaI (Eurogentec S.A.); BfaI, MseI, and Tsp509I (New England Biolabs, Ozyme, Montigny le Bretonneux, France); and Sau3AI and Sau96I (GIBCO-BRL, Life Technologies, Eragny, France). PCR products (95 ml) were ethanol precipitated and resuspended in 100 ml of sterile distilled water; 10 ml was used for each digestion. Digestions were performed as described by the supplier, but with an excess of enzyme (5 U per reaction mixture). Restricted DNA was analyzed by horizontal electrophoresis in 3% agarose gel as described above. The technique allowed clear visualization, as well as estimation of the length of DNA fragments longer than 70 bp, and visualization of poorly resolved fragments between 40 and 70 bp. Data analysis. The restriction maps were determined from restriction fragment length polymorphisms (RFLPs) by comparison with those inferred from the sequence of P. syringae pv. syringae CFBP1392. In this study, restriction and nucleotidic sites were numbered starting from the first nucleotide identified in strain CFBP1392’s sequence. Restriction site differences were analyzed by the Wagner parsimony method with the mix software of the PHYLIP package (6). The numbers of nucleotide substitutions per site were estimated by the formula d 5 (2lnS) 3 r21, where S 5 2mxy (mx 1 my)21 (20, 21). Here, mx and my are the numbers of restriction sites for DNA sequences x and y, respectively; mxy is the number of restriction sites shared by two sequences; and r is the number of nucleotides in the recognition sequence of the restriction enzymes (r 5 4 in all cases). The distance matrix (d
values) was used to construct dendrograms by the method described by Fitch and Margoliash (7) and the UPGMA (27), neighbor-joining (24), and maximumlikelihood (5) methods. Nucleotide sequence accession number. The Genbank accession number for the sequence reported in this paper is U40717.
RESULTS Restriction analysis of amplified 16S rDNA and 23S rDNA. Stringent PCR conditions allowed amplification of a single DNA fragment with the same size of 1,550 bp and of one with the expected size of about 2,900 bp for 16S rDNA and 23S rDNA, respectively, for all 31 Pseudomonas strains tested (data not shown). Digestion of PCR products with nine restriction enzymes (AluI, EcoRI, HaeIII, HhaI, MspI, PstI, RsaI, TaqaI, and Sau3AI) did not show any polymorphism in patterns inside both genes (data not shown). Nucleotide sequence of the 16S-23S rDNA spacer region (ITS1) of the P. syringae pv. syringae strain CFBP1392. The 558-bp nucleotide sequence which corresponds to almost all of the intergenic ITS1 sequence is given in Fig. 1. Eight restriction sites for AluI; four restriction sites for Sau3AI and Tsp509I; three restriction sites for TaqaI; two restriction sites for MspI, HhaI, MseI, and BfaI; and one restriction site for Sau96I and HaeIII were located in the sequence. Their occurrences and locations were verified by analysis of the electrophoretic patterns of digested amplified DNA fragments. Sequences which code for alanine-tRNA and isoleucine-tRNA were located from nucleotides 126 to 192 and from nucleotides 234 to 305, respectively. Parts of the ITS1 regions of the P. syringae pv. tomato strain CFBP2212 and P. viridiflava CFBP2107 were sequenced (data not shown). These sequences were used to locate restriction sites which were not present in strain CFBP1392. Restriction analysis of amplified ITS1 fragments. D21 and D22 primers allowed amplification of the 16S-23S rDNA spacer region for all tested strains. Although most strains gave
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parsimonious. Genetic distances between the various RFLP groups were also calculated (Table 3). The trees obtained by the distance-based methods (Fitch-Margoliash, UPGMA, maximum-likelihood, and neighbor-joining methods) are very similar to each other. The tree obtained by neighbor-joining analysis is shown (Fig. 3). In all of these trees, four groups of genotypes were always observed: the first group consisted of genotypes A, B, C, and D; the second group contained genotypes G, H, I, J, K, and L; the third one contained genotypes F, M, N, O, and P; and the fourth contained the Q genotype. Only the position of E changed between different trees. E was branched upstream of the third and the fourth groups by neighbor-joining and Fitch-Margoliash analyses but was branched with the group containing the F, M, N, O, and P genotypes by UPGMA and maximum-likelihood analyses. All of these data confirmed the robustness of the resulting relatedness inference. Pathogenicity tests. Comparative pathogenicity studies of selected strains on tomato plantlets were performed. Tomato leaves inoculated with all P. syringae pv. tomato strains developed typical symptoms of bacterial speck: small dark-brown to black spots which were surrounded by a distinct, yellow halo. Inoculation of all strains of P. syringae pv. maculicola, P. syringae pv. antirrhini, P. syringae pv. apii, and P. syringae pv. passiflorae and three P. syringae strains with undetermined pathovars, CFBP1777, CFBP1678, and CFBP1688, provoked the same type of symptoms as P. syringae pv. tomato strains did
FIG. 1. Nucleotidic sequence of internal transcribed spacer ITS1 of the P. syringae pv. syringae strain CFBP1392 and locations of restriction sites of restriction enzymes used in the RFLP study. Two tRNA genes are located on the sequence, i.e., tRNAIle and tRNAAla between nucleotides 126 and 193 and nucleotides 235 and 305, respectively.
a single band after electrophoresis on gels (about 550 bp), some of them gave two very closely spaced bands, which indicated that several operons occur in the genomes of these bacteria (data not shown). Digestion of PCR products with 14 endonucleases provided unambiguous patterns, from which positions of restriction sites could be inferred from the sequence of strain CFBP1392. As examples, gels of amplified ITS1 fragments from several pathovars of P. syringae digested with MspI, HaeIII, and TaqaI are shown in Fig. 2. Seventeen different patterns were distinguished (Table 2). Complex patterns which indicated the occurrence of at least two types of operons in the same bacterial genome were sometimes found. Groups A, C, E, H, I, J, K, L, M, and O had only one type of operon according to the obtained data. Groups B, D, F, G, N, P, and Q had band patterns indicating that some restriction sites were present on one type of operon and absent on the other. The data analysis was done with two operons in each strain being considered either identical or different. Genetic relationships calculated from RFLPs of ITS1 fragments. The restriction site differences for the 17 genotypes were analyzed by using genetic distance and parsimony analyses to estimate the level of divergence between PCR-amplified ITS1 fragments from different P. syringae pathovars. A P. viridiflava strain was included as an outlying group. Fifteen (of 75) trees obtained by the Wagner parsimony method were equally
FIG. 2. Gel electrophoresis of PCR-amplified ITS1 fragments of P. syringae strains digested with MspI (A), HaeIII (B), and TaqaI (C). Lanes: 1, 100-bp molecular size ladder (the smallest band is 100 bp, and the 600-bp band is doubled); 2, P. syringae pv. tomato CFBP1322; 3, P. syringae pv. tomato CFBP1320; 4, P. syringae pv. antirrhini CFBP1722; 5, P. syringae pv. tomato CFBP2546; 6, P. syringae pv. atrofaciens CFBP2213; 7, P. syringae pv. morsprunorum CFBP1647; 8, P. syringae pv. coronofaciens CFBP2216; 9, P. syringae pv. mori CFBP1642; 10, P. syringae pv. porri CFBP1908; 11, P. syringae pv. atropurpurea CFBP2340; 12, P. syringae pv. papulans CFBP1754; 13, P. syringae pv. glycinea CFBP2214; and 14, P. syringae pv. phaseolicola CFBP1390.
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TABLE 2. Restriction maps of the ITS1 DNA fragments of various RFLP groups of P. syringae and P. viridiflava strains Bacterial ITS1 RFLP groupsb
Sitea A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
AluI (AGCT) 13a 13b 74a 74b 88a 88b 95a 95b 116a 116b 135a 135b 243/248a 243/248b
1 1 1 1 2 2 2 2 2 2 1 1 1 1
1 1 1 1 2 2 2 2 2 2 1 1 1 1
1 1 1 1 2 2 2 2 2 2 1 1 1 1
1 1 1 1 2 2 2 2 2 2 1 1 1 1
1 1 1 1 2 2 2 2 2 2 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 2 2 2 2 2 2 2 2 1 1 1 1
Bsh1236I (CGCG) 250a 250b
1 1
1 1
1 1
2 1
2 2
2 2
2 2
2 2
2 2
2 2
2 2
2 2
2 2
2 2
2 2
2 2
2 1
HaeIII (GGCC) 237a 237b
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
2 1
HhaI (GCGC) 100a 100b 130a 130b 152a 152b 226a 226b 259a 259b
2 2 2 2 1 1 1 1 1 1
2 2 2 2 1 1 1 1 1 1
2 2 2 2 1 1 1 1 1 1
1 1 2 2 2 2 1 1 1 1
1 1 2 2 1 1 2 2 1 1
1 1 2 2 1 1 2 2 1 1
1 1 2 2 1 1 2 2 1 1
1 1 2 2 1 1 2 2 1 1
1 1 2 2 2 2 2 2 1 1
1 1 2 2 1 1 2 2 1 1
1 1 2 2 1 1 2 2 1 1
2 2 1 1 2 2 2 2 1 1
2 2 2 2 1 1 2 2 1 1
2 2 2 2 1 1 2 2 1 1
2 2 2 2 1 1 2 2 1 1
1 1 2 2 1 1 2 2 1 1
2 2 2 2 1 1 2 2 1 1
MspI (CCGG) 235a 235b 443a 443b 470a 470b
1 1 1 1 1 1
1 1 2 1 1 1
2 1 1 1 1 1
2 1 1 1 1 1
2 1 1 1 1 1
2 2 1 1 1 1
2 1 2 2 1 1
2 2 1 1 1 1
2 2 1 1 1 1
2 2 1 1 1 1
2 2 1 1 1 1
2 2 1 1 1 1
2 2 1 1 1 1
2 2 1 1 1 1
2 2 1 1 1 1
2 2 1 1 1 1
2 2 2 2 2 2
RsaI (GTAC) 231a 231b
1 1
1 1
1 1
1 1
2 2
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
Sau3A (GATC) 290a 290b 352/361a 352/361b 398a 398b
1 1 2 2 1 1
1 1 2 2 1 1
1 1 2 2 1 1
1 1 2 2 1 1
1 1 2 2 1 1
1 1 2 2 1 1
1 1 2 2 1 1
1 1 2 2 1 1
1 1 2 2 1 1
1 1 1 1 1 1
1 1 1 1 1 1
1 1 1 1 1 1
1 1 1 1 1 1
1 1 1 1 1 1
1 1 1 1 1 1
1 1 1 1 1 1
1 1 2 2 1 1
TaqI (TCGA) 2a 2b 181a 181b 190a 190b 240a 240b 292a 292b
1 1 1 1 2 2 2 2 1 1
1 1 1 1 2 2 2 2 1 1
1 1 1 1 2 2 2 2 1 1
1 1 2 2 1 1 2 2 1 1
1 1 1 1 2 2 1 1 1 1
1 1 1 1 2 2 1 1 1 1
1 1 2 2 1 1 2 2 1 1
1 1 2 2 1 1 2 2 1 1
1 1 2 2 1 1 2 2 1 1
1 1 2 2 1 1 2 2 1 1
1 1 2 2 1 1 2 2 1 1
1 1 2 2 1 1 2 2 1 1
1 1 1 1 2 2 1 1 1 1
1 1 1 1 2 2 1 1 1 1
1 1 1 1 2 2 1 1 1 1
1 1 1 1 2 2 1 1 1 1
1 1 1 1 2 2 2 2 1 1
Continued on following page
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TABLE 2—Continued Bacterial ITS1 RFLP groupsb
Sitea A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
Sau96I (GGNCC) 236a 236b
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
MseI (TTAA) 77a 77b 408a 408b
2 2 1 1
2 2 1 1
2 2 1 1
2 2 1 1
1 2 1 1
1 2 1 1
2 2 1 1
2 2 1 1
2 2 1 1
1 1 1 1
2 2 1 1
2 2 1 1
1 2 1 1
1 1 1 1
1 2 1 1
1 1 1 1
2 2 1 1
Tsp509I (AATT) 37a 37b 121a 121b 201a 201b 432a 432b
1 1 2 2 2 2 1 1
1 1 2 2 2 2 1 1
1 1 2 2 2 2 1 1
1 1 2 2 2 2 1 1
1 1 1 2 1 2 1 1
1 1 1 2 1 2 1 1
1 1 2 2 2 2 1 1
1 1 2 2 2 2 1 1
1 1 2 2 2 2 1 1
1 1 2 2 2 2 1 1
1 1 2 2 2 2 1 1
1 1 2 2 2 2 1 1
1 1 1 2 1 2 1 1
1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1
BfaI (CTAG) 103a 103b 385a 385b
2 2 1 1
2 2 1 1
2 2 1 1
2 2 1 1
1 2 1 1
1 2 1 1
2 2 1 1
2 2 1 1
2 2 1 1
2 2 1 1
2 2 1 1
2 2 1 1
1 2 1 1
1 1 1 1
1 2 1 1
1 1 1 1
2 2 1 1
a The site numbering is that on the ITS1 DNA of P. syringae pv. syringae CFBP1392 (pattern M). When the restriction site was not present in CFBP1392, site positions were obtained from partial sequence data of either P. syringae pv. tomato CFBP2212 (pattern A) or P. viridiflava CFBP2107 (pattern Q). Sites separated by shills were indistinguishable by this method. a and b, the two types of operon identified in P. syringae strains. b ITS1 RFLP groups are listed in Table 1. 1, presence of the site; 2, absence of the site.
(Table 1). Half of the strains of P. syringae pv. delphini (CFBP1624 and CFBP1641) caused weak reactions on tomato leaves. Black spots were surrounded by a typical yellow halo, but they were rare and were not detected on all inoculated plants. The inoculation of the other strains of P. syringae pv. delphini tested (CFBP1730 and CFBP2215) did not cause any of the symptoms on tomato plants as did other P. syringae strains tested (Table 1).
DISCUSSION In this study, we showed that PCR-RFLP analysis of the rrn operon is a reliable technique to evaluate genetic relatedness on a large number of strains; resolution of the PCR-RFLP methods depended on the part of rrn operon which was analyzed. The data obtained by RFLP analyses of the rrs and rrl genes showed that P. syringae and P. viridiflava belong to the
TABLE 3. Genetic distances between the different RFLP groups of P. syringae and P. viridiflavaa RFLP group
A B C D E F G H I J K L M N O P Q a
Distance from RFLP group A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
0.000
0.011 0.000
0.011 0.022 0.000
0.111 0.124 0.101 0.000
0.161 0.174 0.152 0.209 0.000
0.200 0.212 0.192 0.245 0.109 0.000
0.204 0.196 0.196 0.165 0.234 0.149 0.000
0.191 0.204 0.183 0.152 0.221 0.118 0.053 0.000
0.217 0.231 0.209 0.133 0.247 0.140 0.075 0.043 0.000
0.224 0.237 0.216 0.188 0.232 0.132 0.091 0.060 0.082 0.000
0.208 0.221 0.200 0.170 0.237 0.135 0.072 0.041 0.062 0.039 0.000
0.234 0.247 0.226 0.174 0.284 0.176 0.116 0.083 0.064 0.080 0.061 0.000
0.200 0.212 0.192 0.265 0.129 0.037 0.168 0.137 0.160 0.113 0.115 0.137 0.000
0.231 0.243 0.223 0.294 0.162 0.071 0.200 0.170 0.192 0.127 0.148 0.170 0.036 0.000
0.216 0.228 0.208 0.280 0.146 0.055 0.184 0.154 0.176 0.130 0.132 0.154 0.018 0.018 0.000
0.245 0.257 0.238 0.288 0.159 0.070 0.196 0.167 0.189 0.125 0.145 0.185 0.053 0.017 0.034 0.000
0.190 0.181 0.181 0.268 0.224 0.239 0.271 0.279 0.310 0.311 0.295 0.326 0.239 0.229 0.213 0.245 0.000
RFLP groups are described in Table 2. A to P, P. syringae; Q, P. viridiflava.
504
MANCEAU AND HORVAIS
FIG. 3. Dendrogram of genetic relatedness of P. syringae and P. viridiflava strains based on RFLPs of the ITS1 regions. The tree was constructed by the neighbor-joining method with data from Table 2. The scale indicates the rate of differences. Letters correspond to RFLP patterns described in Table 2.
same genomic group. Data from partial sequencing of rrs confirmed the homogeneity between the rrs genes of P. syringae and P. viridiflava (data not shown). However, a large variability was detected by this method in the internal transcribed spacer ITS1. Although the length of this DNA fragment is much shorter than the lengths of rrs and rrl (550 versus 1,600 and 2,300 bp), resolution of the PCR-RFLP in the ITS1 is situated at the level of clusters of pathovars and genospecies in P. syringae. The 17 RFLP patterns were distributed into three clusters. P. syringae pv. tomato strains and related pathovars were grouped in the first cluster, which contained four RFLP genotypes. Genotype A contained all 30 strains of P. syringae pv. tomato tested, all strains belonging to pathovars maculicola, antirrhini, apii, berberidis, lachrymans, passiflorae, and persicae, and strains isolated from various host plants (Euphorbia pulcherina and Magnolia sp.) whose pathovar was not determined because a disease caused by P. syringae on these plants has never been described and because pathogenicity on reference plants has not been tested. Genotype B, which identified a strain of P. syringae isolated from Callistemon viminelis, and genotype C, which identified strain CFBP2398 of P. syringae pv. philadelphi, differed from genotype A by only one restriction site. Genotype A was cut by MspI at three sites in both types of operon when genotype B and genotype C contained two types of operon. One was cut at three sites, and the other was cut at only two sites (positions 443 and 235, respectively). Genotype D contained all strains of P. syringae pv. delphinii. They were different from those of genotype A in 6 of 70 restriction sites studied (Table 2). All of these strains are, therefore, genetically very closely related. Our data obtained from the analysis of rrn operons, which are considered a molecular clock (22) describing the phylogenetic relationships between individuals, confirm the propensity to separate pathovars closely related to P. syringae pv. tomato from other pathovars of P. syringae, on the basis of the results of pathogenicity and biochemical tests (34) or of RFLP analyses with randomly obtained probes (3, 4, 11). In addition, strains belonging to the tomato group were genetically far removed from the other strains tested. Genotype A was different from genotype Q, which contained the P. viridiflava strain in 16 of 70 restriction sites. It also differed from any other genotypic groups by more than 20 restriction sites. These other groups gathered all other pathovars of P. syringae. Thus, P. syringae pv. tomato and related pathovars are
APPL. ENVIRON. MICROBIOL.
genetically closer to P. viridiflava than to other pathovars of P. syringae. These other pathovars are distributed into two subgroups which were different from each other by 15 restriction sites, on average. These results are partly in accordance with those obtained by the DNA-DNA hybridization technique to describe genomic species within P. syringae (8, 26). All pathovars belonging to the genomic species I described by Gardan et al. (8) and Shafik (26) fell in to grouped patterns M, N, O, and P. P. viridiflava, which belongs to genomic species IX, is described in pattern Q in our study. Furthermore, all strains belonging to grouped patterns A, B, C, and D belong to the same genomic species (III). Besides, strains belonging to genomic species IV were distributed in pattern H and pattern F, which were not grouped in our study. In addition, strains belonging to genospecies II were distributed in patterns L, J, and K, which were loosely grouped; furthermore, two strains, P. syringae pv. tabaci CFBP2106 and P. syringae pv. phaseolicola CFBP1392, showed genotypic patterns M and N, respectively. These two patterns were not closely related to patterns I, J, and K, and, furthermore, they contained a strain belonging to genomic species I described by Gardan et al. (8). This indicates that these bacterial species are probably less homogeneous than genospecies III (tomato). We conclude that PCR-RFLP analysis of ITS1 has a resolution level lower than that of genomic species defined by DNA-DNA hybridization, which can describe superspecies taxons, and that it is possible to evaluate genetic relatedness between pathovars by this approach. In the case of P. syringae pv. tomato and related pathovars, this approach allowed us to show that they formed a homogeneous group which is as phylogenetically distinct from the other P. syringae pathovars as is the species P. viridiflava. ACKNOWLEDGMENTS We thank Chrystelle Blanloeil and Olivier Chauveau for technical assistance and Xavier Nesme (INRA-CNRS, Lyon, France) for help in data analysis. This research was partly supported by a grant (Qualite´ des semences et valeur ajoute´e) from the Re ´gion des Pays de la Loire, France. REFERENCES 1. Brosius, J., T. J. Dull, and H. F. Noller. 1980. Complete nucleotide sequence of a 23S ribosomal RNA gene from Escherichia coli. Proc. Natl. Acad. Sci. USA 77:201–204. 2. Buchanan, R. E., and N. E. Gibbons (ed.). 1974. Bergey’s manual of determinative bacteriology, 8th ed., p. 1246. Williams and Wilkins, Baltimore, Md. 3. Denny, T. P. 1988. Phenotypic diversity in Pseudomonas syringae pv. tomato. J. Gen. Microbiol. 134:1939–1948. 4. Denny, T. P., M. N. Gilmour, and R. K. Selander. 1988. Genetic diversity and relationships of two pathovars of Pseudomonas syringae. J. Gen. Microbiol. 134:1949–1960. 5. Felsenstein, J. 1992. Phylogenies from restriction sites: a maximum-likelihood approach. Evolution 46:159–173. 6. Felsenstein, J. 1993. Phylip (phylogeny inference package) version 3.5c. Distributed by the author. Department of Genetics, University of Washington, Seattle, Wash. 7. Fitch, F. W., and E. Margoliash. 1967. Construction of phylogenetic trees. Science 155:279–284. 8. Gardan, L., H. L. Shafik, and P. A. D. Grimont. 1995. DNA relatedness among pathovars of P. syringae and related bacteria, p. 42. In Abstracts of the 5th International Conference on Pseudomonas syringae Pathovars and Related Pathogens. Biologische Bundesanstalt fu ¨r Land und Forstwirtschaft, Braunschweig, Germany. 9. Gurtler, V., V. A. Wilson, and B. C. Mayall. 1991. Classification of medically important clostridia using restriction endonuclease site differences of PCRamplified 16S rDNA. J. Gen. Microbiol. 137:2673–2679. 10. Gutell, R. R., M. N. Schnare, and M. W. Gray. 1990. A compilation of large subunit (23S-like) ribosomal RNA sequences presented in a secondary structure format. Nucleic Acids Res. 18:2319–2324. 11. Hendson, M., D. C. Hildebrand, and M. N. Schroth. 1992. Relatedness of Pseudomonas syringae pv. tomato, Pseudomonas syringae pv. maculicola and
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