Phylogeny of the genus Pseudomonas: intrageneric structure reconstructed from the nucleotide sequences of gyrB and rpoD genes

Satoshi Yamamotoa,1, Hiroaki Kasai1, Dawn L. Arnold2, Robert W. Jackson2, Alan Vivian2 and Shigeaki Harayama1

Marine Biotechnology Institute, Kamaishi Laboratories, Kamaishi City, Iwate 026, Japan1
Centre for Research in Plant Science, Faculty of Applied Sciences, University of the West of England, Coldharbour Lane, Bristol BS16 1QY, UK2

Author for correspondence: Satoshi Yamamoto. Tel: +81 43 203 0226. Fax: +81 43 203 0216. e-mail: yamamotost{at}nichirei.co.jp


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Phylogenetic analysis of the genus Pseudomonas was conducted by using the combined gyrB and rpoD nucleotide sequences of 31 validly described species of Pseudomonas (a total of 125 strains). Pseudomonas strains diverged into two major clusters designated intrageneric cluster I (IGC I) and intrageneric cluster II (IGC II). IGC I was further split into two subclusters, the P. aeruginosa complex’, which included P. aeruginosa, P. alcaligenes, P. citronellolis, P. mendocina, P. oleovorans and P. pseudoalcaligenes, and the ‘P. stutzeri complex’, which included P. balearica and P. stutzeri. IGC II was further split into three subclusters that were designated the ‘P. putida complex’, the ‘P. syringae complex’ and the ‘P. fluorescens complex’. The ‘P. putida complex’ included P. putida and P. fulva. The ‘P. syringae complex’ was the cluster of phytopathogens including P. amygdali, P. caricapapayae, P. cichorii, P. ficuserectae, P. viridiflava and the pathovars of P. savastanoi and P. syringae. The ‘P. fluorescens complex’ was further divided into two subpopulations, the ‘P. fluorescens lineage’ and the ‘P. chlororaphis lineage’. The ‘P. fluorescens lineage’ contained P. fluorescens biotypes A, B and C, P. azotoformans, P. marginalis pathovars, P. mucidolens, P. synxantha and P. tolaasii, while the ‘P. chlororaphis lineage’ included P. chlororaphis, P. agarici, P. asplenii, P. corrugata, P. fluorescens biotypes B and G and P. putida biovar B. The strains of P. fluorescens biotypes formed a polyphyletic group within the ‘P. fluorescens complex’.

Keywords: gyrB, rpoD, Pseudomonas, phylogeny, PCR

Abbreviations: IGC, intrageneric cluster; NJ, neighbour-joining; UPGMA, unweighted pair group method with arithmetic averages

The GenBank accession numbers for the sequences determined in this work are: gyrB, D37926, D37297, D86005D86019 and AB039381AB039492; rpoD, D86020D86036 and AB039493AB039624.

a Present address: Nichirei Corporation, Research & Development Center, 9 Shinminato, Mihama-Ku, Chiba-Shi, Chiba 261-8545, Japan.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Pseudomonads are ubiquitous bacteria in nature. They possess variable metabolic abilities that enable them to utilize a wide range of organic compounds, and occupy an important ecological position in the carbon cycle. They are also important as pathogens of animals and plants. Therefore, the ecology of pseudomonads in the biosphere has been a matter of interest. An essential prerequisite for a detailed investigation of the roles and evolution of pseudomonads is an accurate system of classification and identification. However, the classification of Pseudomonas strains is not fully established due to the lack of an accurate taxonomic system.

The classificatory criteria for the genus Pseudomonas have been revised along with progress in bacterial taxonomy. Today, DNA–DNA hybridization is the recommended standard for the delineation of a bacterial species (Wayne et al., 1987 ), but it has the drawback that it is not effective in the estimation of genetic distances between distantly related species. As a complement to DNA–DNA hybridization, sequence analysis of 16S rRNA or its gene (16S rDNA) is frequently used (Laguerre et al., 1994 ; Moore et al., 1996 ; Bennasar et al., 1998 ). However, the degree of resolution obtained with 16S rRNA sequence analysis is not sufficiently discriminatory to permit resolution of intrageneric relationships because of the extremely slow rate of evolution of 16S rRNA. Due to the gap between the valid genetic ranges of the two methods, a detailed intrageneric structure of the genus Pseudomonas remains to be resolved.

In this article, we discuss the intrageneric structure of the genus Pseudomonas on the basis of the nucleotide sequences of their genes for DNA gyrase B subunit (gyrB) and {sigma}70 factor (rpoD). DNA gyrase is the enzyme responsible for introducing negative supercoils into bacterial chromosomes and plays a crucial role in the replication of chromosomes (Watt & Hickson, 1994 ). The {sigma}70 factor, on the other hand, is one of the sigma factors that confer promoter-specific transcription initiation on RNA polymerase (Lonetto et al., 1992 ). Both proteins are ubiquitous in bacteria and essential for their cell growth. We previously reported that these protein-encoding genes evolved much faster than rDNAs and provided higher resolution than the use of 16S rRNA sequences (Yamamoto & Harayama, 1998 ). We further demonstrated that the phylogenetic clustering of Acinetobacter strains based on gyrB sequence analysis is almost equivalent to the genomic species delineated by DNA–DNA hybridization (Yamamoto et al., 1999 ). These results suggest that a phylogenetic analysis using the gyrB and rpoD sequences may fill the resolution gap between 16S rRNA sequence analysis and DNA–DNA hybridization studies.

Molecular phylogeny deduced from a single locus may be unreliable due to the stochastic nature of base substitutions or to rare horizontal gene transfer events. Consequently, we decided to use a combination of the gyrB and rpoD genes to establish the phylogenetic relationships of strains in the genus Pseudomonas. This strategy allowed, first, the comparison of the gyrB-based phylogenetic tree with the rpoD-based one to confirm the consistency of the analyses, and second, the improvement of the reliability of the phylogenetic tree.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and DNA preparation.
Thirty-one validly described Pseudomonas species, comprising a total of 125 strains, were examined in this study (Table 1). Each bacterial sample was grown aerobically in nutrient broth at 30 °C. Chromosomal DNAs from Pseudomonas strains used as the PCR template were prepared by the methods described by Brenner et al. (1982) or by using the Puregene DNA Isolation kit (Gentra Systems) according to the supplier’s instructions.


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Table 1. List of Pseudomonas strains analysed

 
PCR amplification and sequencing of gyrB and rpoD.
PCR amplification of gyrB and rpoD genes was done following the method described previously (Yamamoto & Harayama, 1995 , 1998 ; Yamamoto et al., 1999 ). PCR amplification was performed with a Progene thermal cycler (Techne) by using PCR buffer (Perkin-Elmer) containing each of the deoxynucleoside triphosphates at a concentration of 200 µM, each of the primers at a concentration of 1 µM, 1 µg template DNA and 2·5 U AmpliTaq Gold DNA polymerase (Perkin-Elmer) in a total volume of 100 µl. A total of 35 cycles of amplification was performed, with the template DNA denaturation at 94 °C for 1 min, the primer annealing for 30 s and the primer extension at 72 °C for 2 min. The annealing temperature ranged from 58 to 63 °C depending on the G+C content of the template DNA. Higher annealing temperatures were used for higher G+C contents. Amplified products were electrophoresed on 0·8% low-melting-temperature agarose gels (SeaPlaque GTG; FMC Bioproducts), and purified by using QIAquick (Qiagen) following the manufacturer’s instructions. The nucleotide sequences of gyrB and rpoD genes were determined directly from the PCR fragments. PCR primers and sequencing primers used in this study are summarized in Table 2. Sequencing was carried out using an ABI PRISM BigDye Terminator Cycle Sequencing kit and a 377 DNA sequencer (Perkin-Elmer) according to the manufacturer’s instructions.


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Table 2. PCR primers and sequencing primers used in this study

 
Data analysis.
GyrB and RpoD sequences translated from gyrB and rpoD sequences, respectively, were aligned using the CLUSTAL W computer program (Thompson et al., 1994 ). The gyrB and rpoD sequences were aligned manually according to the alignments of the GyrB and RpoD sequences, respectively. Phylogenetic trees were constructed with the PHYLIP computer program package (Felsenstein, 1989 ), using the neighbour-joining (NJ) method (Saitou & Nei, 1987 ) with genetic distances computed using Kimura’s two-parameter model (Kimura, 1980 ). Phylogenetic trees were reconstructed not only from the data sets of gyrB (888–891 bp) and rpoD (798–816 bp), but also from the combined nucleotide sequences of these two genes (1686–1707 bp), assuming that the analysis using longer sequences would result in a better resolution and reliability. In the latter analysis, gyrB and rpoD sequences from the same strain were combined in series and treated as a single nucleotide sequence. The nucleotide sequences of the gyrB and rpoD genes from Esherichia coli K-12 (ECGYRB and ECORPSPRO) were used as the outgroup for phylogenetic tree reconstruction.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Reconstruction of phylogenetic tree
As the first step in the analysis, NJ trees from the gyrB and rpoD nucleotide sequences were reconstructed from the individual data sets. The basic topologies of these two NJ trees were similar to each other but slightly different in detail (data not shown). The most conspicuous difference between them was the branching order of the clusters that included the ‘P. aeruginosa lineage’ strains and those that included the ‘P. stutzeri lineage’ strains. In the gyrB NJ tree, these two clusters branched off after they had diverged from the remaining mass of Pseudomonas, whilst in the rpoD NJ tree, the ‘P. stutzeri lineage’ cluster branched off first, followed by the ‘P. aeruginosa lineage’, which diverged from the rest. Theoretically, the influence of stochastic drift on the rate of evolution cannot be eliminated from the molecular phylogeny. Hence, these minor discrepancies may have their origin in such drift. If this were the case, the use of longer sequences, for example the combined gyrB and rpoD sequences, in the analysis would give a more accurate estimate of the phylogeny.

We thus conducted the phylogenetic analysis by using the combined nucleotide sequences of the gyrB and rpoD genes. A NJ tree reconstructed from the combined gyrB and rpoD nucleotide sequences (gyrB-rpoD tree) is shown in Fig. 1. The topology of trees reconstructed from the combined sequences of gyrB and rpoD by the unweighted pair group method with arithmetic averages (UPGMA) (Sokal & Michener, 1958 ) and the maximum-parsimony method (Fitch, 1971 ) were almost identical to that of the NJ tree. One notable difference between them was the position of P. straminea IAM 1598T. In the NJ and maximum-parsimony trees, P. straminea IAM 1598T was excluded from both of the clusters of the ‘P. aeruginosa lineage’ and the ‘P. stutzeri lineage’, whereas in the UPGMA tree, P. straminea IAM 1598T was included in the cluster of the ‘P. aeruginosa lineage’. The phenotypic characteristics of P. straminea, such as its considerably lower maximum growth temperature (no growth at 37 °C) in comparison with that of the ‘P. aeruginosa lineage’ and the ‘P. stutzeri lineage’ (can grow at 41 °C), may account for its divergence as in the NJ tree and maximum-parsimony tree. Consequently, we chose the combined gyrB and rpoD NJ tree as the basis of Pseudomonas phylogeny in this study.



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Fig. 1. Phylogenetic tree of 125 Pseudomonas strains that have been assigned to 31 validly described species based on the nucleotide sequences of the gyrB and rpoD genes. The tree was reconstructed by using the NJ method, using the genetic distances computed by using the Kimura’s two-parameter model (Kimura, 1980 ). The scale bar indicates a genetic distance of 0·1. The number shown next to each node indicates the percentage bootstrap values of 1000 replicates that exceeded 50% (except for the important node). Sequences from E. coli K-12 were treated as the outgroup. The topological characteristics of the phylogenetic trees produced by the NJ method, the maximum-parsimony method and UPGMA were almost identical (data not shown).

 
Outline of the intrageneric structure of the genus Pseudomonas
The Pseudomonas strains examined, with the exception of two strains (P. stanieri ATCC 27130T and P. iners IAM 1419T), formed a monophyletic group that corresponds to the rRNA group I of Palleroni (1984) . The intrageneric relationships of the authentic Pseudomonas species observed in the combined gyrB and rpoD NJ tree are summarized in Fig. 2. Within the genus Pseudomonas, two major intrageneric clusters were recognized. The first intrageneric cluster includes P. aeruginosa, P. alcaligenes, P. balearica, P. citronellolis, P. mendocina, P. oleovorans, P. pseudoalcaligenes, P. straminea and P. stutzeri. The second intrageneric cluster includes P. agarici, P. amygdali, P. asplenii, P. azotoformans, P. caricapapayae, P. chlororaphis (including ex-P. aureofaciens strains), P. cichorii, P. corrugata, P. ficuserectae, P. fluorescens (biotypes A, B, C and G), P. fulva, P. marginalis pathovars, P. mucidolens, P. putida (biovars A and B), P. savastanoi pathovars, P. synxantha, P. syringae pathovars, P. taetrolens and P. tolaasii. These two intrageneric divisions seem consistent with the ‘P. aeruginosa intrageneric cluster’ and the ‘P. fluorescens intrageneric cluster’ that have been designated using 16S rRNA gene sequence analysis (Moore et al., 1996 ). However, phylogenetic relationships within each of the ‘intrageneric clusters’ in the combined gyrB and rpoD NJ tree were eminently different from that of the 16S rRNA gene sequence trees (Moore et al., 1996 ; Verhille et al., 1999 ). Consequently, in this article, we propose to designate the first and second major clusters as ‘intrageneric cluster I’ (IGC I) and ‘intrageneric cluster II’ (IGC II).



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Fig. 2. Schematic dendrogram summarizing the intrageneric structure of the genus Pseudomonas. The number shown next to each node indicates the percentage bootstrap value of 1000 replicates (only those for the important nodes are indicated).

 
IGC I
IGC I includes the type species of the genus Pseudomonas, P. aeruginosa. Many of the constituents of this cluster were isolated from clinical specimens. The general characteristics of IGC I species are: a single polar flagellum; a higher range of G+C contents (60·6–66·3 mol%) than IGC II (59·0–63·6 mol%); growth at 41 °C, except for P. straminea (no growth at 37 °C); non-production of fluorescent pigments, except for P. aeruginosa and P. straminea (Palleroni, 1984 ). No consensus profile of carbon source utilization was recognized.

IGC I was further split into at least two subclusters, the ‘P. aeruginosa complex’, which included P. aeruginosa, P. alcaligenes, P. citronellolis, P. mendocina, P. oleovorans and P. pseudoalcaligenes, and the ‘P. stutzeri complex’, which included P. balearica and P. stutzeri. P. aeruginosa strains were monophyletic within the ‘P. aeruginosa complex’. The type strains of P. oleovorans and P. pseudoalcaligenes, IFO 13583T and IFO 14167T, respectively, were genetically almost identical. As we discussed previously, the phylogenetic position of P. straminea in IGC I was uncertain as its bootstrap probability was 46%. P. straminea possibly represents an independent third subcluster.

IGC II
The general characteristics of IGC II are: more than one polar flagellum; a lower range of G+C contents (59·0–63·6 mol%) than IGC I (60·6–66·3 mol%); inability to grow at 41 °C and ability of some strains to grow at 4 °C; production of fluorescent pigments by most strains (Palleroni, 1984 ). IGC II is more species-rich than IGC I despite the comparable extent of genetic diversity of the two clusters. Within IGC II, at least three distinct monophyletic groups were recognized. We designated these subclusters the P. putida complex’, the ‘P. syringae complex’ and the ‘P. fluorescens complex’.

The ‘P. putida complex’ included P. putida and P. fulva. All of the P. putida strains characterized as biovar A belonged in this group; however, all of the P. putida biovar B strains were included in the ‘P. fluorescens complex’. Strains of P. putida biovar B have been distinguished from biovar A mainly by their ability to grow on L-tryptophan and L-kynurenine. Collectively, the phenotypes of the P. putida biovar B strains resemble more closely those of P. fluorescens strains than those of biovar A. For example, P. putida biovar B strains can grow at 4 °C and can utilize a broader range of sugars than biovar A strains (Stanier et al., 1966 ). These physiological differences support the phylogenetic separation of biovar B strains from the clade of P. putida including the type strain and biovar A strains.

The ‘P. syringae complex’ was the clade of phytopathogenic Pseudomonas, representing a huge ‘pathogenicity island’. The ‘P. syringae complex’ included P. amygdali, P. caricapapayae, P. cichorii, P. ficuserectae, P. viridiflava, P. savastanoi pathovars (pv. savastanoi, pv. phaseolicola and pv. glycinea) and P. syringae pathovars (pv. antirrhini, pv. coriandricola, pv. coronafaciens, pv. lachrymans, pv. maculicola, pv. morsprunorum, pv. pisi and pv. syringae).

The ‘P. fluorescens complex’ was the most species-rich subcluster and at least 16 validly described species belonged in this clade. The majority of the constituents of the ‘P. fluorescens complex’ were fluorescent, with the exception of P. corrugata. Many are saprophytic and/or psychrophilic (growth at 4 °C), and are frequently associated with food spoilage, especially at chilling temperatures (1–10 °C). Some members are also important as pathogens of plants and fungi. This cluster was further divided into two subpopulations, the P. fluorescens lineage’ and the ‘P. chlororaphis lineage’. The ‘P. fluorescens lineage’ contained P. fluorescens biotypes A, B and C, P. azotoformans, P. marginalis pathovars, P. mucidolens, P. synxantha and P. tolaasii, whilst the ‘P. chlororaphis lineage’ included P. chlororaphis, P. agarici, P. asplenii, P. corrugata, P. fluorescens biotypes B and G and P. putida biovar B. The strains of P. fluorescens biotypes, especially those of biotype B, were positioned either in the ‘P. fluorescens lineage’ or in the ‘P. chlororaphis lineage’. It is clear that a reclassification of the P. fluorescens strains is required. On the other hand, P. agarici, P. asplenii, P. chlororaphis, P. corrugata and P. tolaasii formed tight monophyletic branches and can be considered as solid species.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Phylogenetic relationships of pseudomonads resolved by using gyrB and rpoD sequences were eminently different from those resolved by using 16S rRNA sequences (Moore et al., 1996 ; Verhille et al., 1999 ). These discrepancies appear to have their origins in the eccentric evolutionary process of 16S rRNA genes. The secondary structures of 16S rRNA determined by the complementary sequences in the small helices are functionally important since 16S rRNA provides a scaffold for the assembly of ribosomal proteins into the small subunit and interacts with mRNA. The majority of base substitutions in 16S rRNAs between closely related organisms are located in these helices, called variable regions, and generally these substitutions are compensatory, i.e. they maintain the base pairing within the helices (Hancock et al., 1988 ; Rousset et al., 1991 ; Dixon & Hillis, 1993 ). We observed that the genetic distances in the variable regions of 16S rRNAs correlated poorly with the synonymous distances in the gyrB and rpoD genes (Yamamoto & Harayama, 1998 ). This observation suggests that the base substitutions in these helices might not be accumulated by successive point mutations, but might be caused by single-event mutations introducing multiple substitutions. Thus, the genetic distances calculated from the whole 16S rRNA sequences could be erroneous because the numbers of base substitutions outside the variable regions are much fewer. Frequently, the results of the 16S-rRNA-based analysis did not correlate with the DNA–DNA reassociation values determined by DNA hybridization experiments that have been used as the criterion for bacterial species definition (Fox et al., 1992 ; Stackebrandt & Goebel, 1994 ). In contrast, results of gyrB sequence analysis correlated very well with DNA reassociation values (Yamamoto et al., 1999 ). Obviously, the use of appropriate protein genes like gyrB is more suitable for the delineation of intrageneric relationships because they evolved mainly by synonymous substitutions (the ‘molecular clock’).

The extent of genetic diversity among the Pseudomonas species was quite variable. The ‘P. stutzeri complex’ and the ‘P. putida complex’ consisted of single species, P. stutzeri and P. putida, respectively. In contrast, the ‘P. fluorescens complex’, which is genetically less divergent than either the ‘P. stutzeri complex’ or the ‘P. putida complex’, contained no less than 16 species.

Three validly described, but as yet unconfirmed, Pseudomonas species (Kersters et al., 1996 ), P. azotoformans, P. fulva and P. straminea, were newly confirmed as genuine members of the genus Pseudomonas in this study. Conversely, type strains of two valid Pseudomonas species, P. stanieri ATCC 27130T and P. iners IAM 1419T, were excluded from the genus.

Some of the phenotypic traits of the strains in the genus Pseudomonas did not reflect their phylogenetic relationships. The fluorescent strains were polyphyletic and belonged to both IGC I and IGC II. Within IGC II, non-fluorescent pseudomonads formed several independent clusters (e.g. P. corrugata in the ‘P. fluorescens complex’ and P. amygdali in the ‘P. syringae complex’). We presume that the common ancestor of the genus Pseudomonas was originally fluorescent and that non-fluorescent species have appeared by losing their ability to produce a fluorescent pigment.

Phytopathogenic Pseudomonas species were also distributed throughout the genus Pseudomonas, not only in the ‘P. syringae complex’. The polyphyletic relationship of phytopathogenic pseudomonads suggests that pathogens in separate clades have independently acquired their phytopathogenicity, presumably by the intergeneric transmission of virulence factors. It is likely that the common ancestor of the ‘P. syringae complex’ had acquired a set of factors that determine pathogenicity to plants, after the divergence of the ‘P. syringae complex’ and the ‘P. fluorescens complex’. More recently, such genes would probably be horizontally acquired via plasmid transfer and involve virulence and/or avirulence genes such as avrPpiB described in P. syringae pv. pisi race 3 (Cournoyer et al., 1995 ) or the pathogenicity island in P. savastanoi pv. phaseolicola (Jackson et al., 1999 ). If so, virulence/avirulence genes would be expected to evolve along with the evolutionary course of the ‘P. fluorescens complex’. However, correlation was not necessarily observed between the phylogenetic relationships of phytopathogens and their host range. For example, strains of P. viridiflava (pathogenic on bean) were scattered at various phylogenetic positions in the ‘P. syringae complex’, whilst those of P. caricapapayae (pathogenic on pawpaw) formed a tight cluster. Polyphyly of host range may be due to the horizontal transfer of virulence and/or avirulence genes.

Within the ‘P. syringae complex’, P. cichorii strains formed an independent monophyletic cluster with two strains of P. syringae pv. syringae. P. cichorii strains are oxidase-positive, as are the majority of pseudomonads, whilst exceptionally, the P. syringae strains were oxidase-negative. This phenotypic difference corroborates the hypothesis that the ‘P. cichorii lineage’ branched at an early stage after the acquisition of phytopathogenicity by the common ancestor, and subsequently the ancestor of the P. syringae lineage’ lost the ability to produce oxidase. The detached P. syringae cluster in the ‘P. cichorii lineage’ might also have lost the ability to produce oxidase by an independent event.

As stated above, reclassification of P. fluorescens biotype strains, the major constituents of the ‘P. fluorescens complex’, is required since they were polyphyletic. Thus, a DNA–DNA hybridization study accompanied by a phenotypic analysis should be carried out to propose new classificatory criteria for the ‘P. fluorescens complex’. Experiments with an optimal combination of strains for DNA–DNA hybridization should be designed, guided by data from the gyrB and rpoD sequence analysis rather than that from 16S rDNA analysis (Yamamoto & Harayama, 1998 ). The close relationship between P. savastanoi pathovars was clearly demonstrated in the gyrB and rpoD sequence analyses presented here. The new species P. savastanoi was created to include the P. syringae pathovars glycinea, phaseolicola and savastanoi, on the basis of their DNA relatedness obtained by DNA–DNA hybridization experiments (Gardan et al., 1992 ).

Because of the high ratios of evolution in the gyrB and/or rpoD nucleotide sequences, it was very easy to design PCR primers or probes having specificity for clusters of species, subspecies or higher classes such as the ‘lineages’ and ‘complexes’ designated in this paper (data not shown). On the other hand, the design of such primers or probes based on the variable regions of 16S rRNA presents a problem because sequence similarity among the variable regions does not always guarantee a close phylogenetic relationship (Yamamoto & Harayama, 1998 ). In conclusion, classification, identification and detection systems for pseudomonads based on gyrB and/or rpoD sequences can be very useful in microbial ecology and other fields of bacteriology.


   ACKNOWLEDGEMENTS
 
We are grateful to Ms Atsuko Katsuta, Ms Syouko Komukai, Ms Yuuka Takahashi and Ms Ikuko Hiramatsu for technical assistance. We are also grateful to Dr Elena A. Kiprianova for providing us with bacterial strains. This work was performed as a part of The Industrial Science and Technology Frontier Program supported by New Energy and Industrial Technology Development.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 4 April 2000; revised 17 July 2000; accepted 21 July 2000.