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Genetics and Molecular Biology

Print version ISSN 1415-4757On-line version ISSN 1678-4685

Genet. Mol. Biol. vol.31 no.1 São Paulo  2008 



BOX-PCR-based identification of bacterial species belonging to Pseudomonas syringae - P. viridiflava group



Abi S.A. MarquesI; Anne MarchaisonII; Louis GardanII; Régine SamsonII

IEmbrapa Recursos Genéticos e Biotecnologia, Laboratório de Quarentena Vegetal,Unidade de Bacteriologia, Brasília, DF, Brazil
IIInstitut National de la Recherche Agronomique, Centre d'Angers, Station de Pathologie Végétale,Angers, France

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The phenotypic characteristics and genetic fingerprints of a collection of 120 bacterial strains, belonging to Pseudomonas syringae sensu lato group, P. viridiflava and reference bacteria were evaluated, with the aim of species identification. The numerical analysis of 119 nutritional characteristics did not show patterns that would help with identification. Regarding the genetic fingerprinting, the results of the present study supported the observation that BOX-PCR seems to be able to identify bacterial strains at species level. After numerical analyses of the bar-codes, all pathovars belonging to each one of the nine described genomospecies were clustered together at a distance of 0.72, and could be separated at genomic species level. Two P. syringae strains of unknown pathovars (CFBP 3650 and CFBP 3662) and the three P. syringae pv. actinidiae strains were grouped in two extra clusters and might eventually constitute two new species. This genomic species clustering was particularly evident for genomospecies 4, which gathered P. syringae pvs. atropurpurea, coronafaciens, garçae, oryzae, porri, striafaciens, and zizaniae at a noticeably low distance.

Key words: Pseudomonas syringae, phenotypic characters, genomospecies, BOX-PCR, bacterial identification.




Taxonomy of the large bacterial group Pseudomonas syringae (LOPAT I of Lelliott et al., 1966) and P. viridiflava is currently under revision, since nine genomospecies were described (Gardan et al., 1999). Many of the causal agents of plant bacterial diseases belong to this group of bacteria, which are either designated as species or as pathovars, i.e. infra-specific subdivision for strains specifically linked to a given host plant.

Identification of bacteria was traditionally performed by phenotypic descriptions, but this approach had some limits. Thus, not all genomospecies can be reliably distinguished by techniques other than quantitative DNA-DNA hybridization, which is not suitable for routine diagnosis. Alternative specific genomic fingerprints have been proposed as diagnostic tools (Versalovic et al., 1994; Rademaker and Bruijn, 1997) by means of amplification of interspersed repetitive DNA sequences present in bacterial genomes, referred to as rep-PCR (Rademaker and Bruijn, 1997) or by amplification of random sequences by arbitrary primers, RAPD (Williams et al., 1990). One of these methods appeared interesting for the delineation of species (Onfroy et al., 1999), subspecies (Louws et al., 1998) or pathovars (Louws et al., 1994) for instance. BOX-PCR, independent from the other rep-PCR techniques, has revealed the possibility of delineating P. syringae genomospecies (Marques et al., 2000), as well as for typing Aeromonas spp. strains (Tacao et al., 2005) and for identification of races and biovars of Ralstonia solanacearum (Galal et al., 2003). The technique has also been used to investigate bacterial inoculum sources (Greco et al., 2004), as a tool for unequivocal identification of strains belonging to a unique pathovar (El Tassa et al., 1999) or to define new species, as a part of a polyphasic approach (Catara et al., 2002).

At present, bacterial species discrimination is based on quantitative DNA-DNA hybridization, as recommended by Wayne et al. (1987). In view of the absence of diverse discriminating tests to distinguish the genomospecies assigned by Gardan et al. (1999), the objective of this study was to compare nutritional characteristics and genomic fingerprints of all the pathovars of the P. syringae - P. viridiflava group, thus checking the hypothesis that BOX-PCR could be correlated with those species discriminations and evaluating its potential for use as a taxonomic tool.


Material and Methods

Bacterial strains

Phytopathogenic fluorescent pseudomonads belonging to the Pseudomonas syringae group (Palleroni, 1984) were obtained from the "Collection Française des Bactéries Phytopathogènes" (CFBP, Angers, France), and comprised 120 strains (Table 1): strains of P. savastanoi pv. phaseolicola, including representatives of the nine races of the bacteria, isolated from a wide range of hosts and geographical origins (Taylor et al., 1996); representative strains of two other bacteria pathogenic to the bean (P. syringae pvs. tabaci and syringae); strains of the very closely related P. savastanoi pv. glycinea; strains of P. syringae pvs. syringae (CFBP 3388) and actinidiae because of their ability to produce phaseolotoxin (Tamura et al., 1989; Tourte and Manceau, 1995); strains which are type strains of species and pathovars included in the large group P. syringae-viridiflava and two strains 3650 and 3662 received as P. savastanoi pv. phaseolicola, but which differed considerably and are listed separately as unknown pathovars. In the following text, the ternary nomenclature will be designated by the abbreviation P. syr. syringae instead of P. syringae pv. syringae.

Nutritional characterization

Assimilation of 99 carbon sources (sugars, alcohols, amino acids and organic acids) was performed with the Biotype 100 system (BioMérieux, La Balme-les-Grottes, France). The strips were inoculated with Biotype Medium 1 as recommended by the manufacturer, and the results read visually at two, four and six days after incubation at 28 °C. In addition, 20 conventional biochemical tests were carried out: arginine dihydrolase, oxidase, gelatin, nitrate reduction, levan, fluorescence, hypersensitive reaction (HR) on tobacco, esculin, pectinolysis on calcium pectinate, Tween esterase, DNAse, polypectate hydrolysis at pH 5 and 8.3, and the utilization of sucrose, lactate, L(+)tartrate, D(-)tartrate, erythritol, mannitol, and sorbitol in ARJ medium (Gardan et al., 1999).

Bacterial cultures and genomic DNA preparation

Strains were grown at 26-28 °C on King's medium B for 24 h. From these cultures, cells were washed with sterile distilled water, and a suspension was prepared, which was adjusted to an O.D.560 of 0.2, corresponding to a bacterial cell suspension at 108 cfu mL-1. Aliquots of 500 mL in 2 mL cryotubes were stored at -20 °C. For utilization, after liquefying the suspension at room temperature, cells were lysed for 10 min in a boiling water bath, and the cryotubes kept on ice before use.

BOX primer and BOX-PCR reaction

The 22-mer BOXA1R oligonucleotide (Bioprobe Systems/Quantum, France) was used to generate BOX-PCR profiles (Versalovic et al., 1991; Martin et al., 1992). Amplification reactions were performed in volumes of 25 µL, containing 2 µM of the single BOX primer, 200 µM each of dATP, dCTP, dGTP and dTTP (Bioprobe Systems/Quantum, France), PCR reaction buffer (10 mM TrisHCl [pH 9.0], 50 mM KCl, 1.5 mM MgCl2, 0.1% TritonX100 and 0.2 mg mL-1 bovine serum albumin), 1.5 units of Taq DNA polymerase (Appligene-Oncor, France) and, as template DNA, 5 µL of a bacterial cell suspension at 108 cfu mL-1. Amplification was performed in an MJ Research, Inc. PTC-100 Thermal Cycler programmed for an initial denaturation step of 7 min at 95 °C, followed by 30 cycles of 1 min at 94 °C, 1 min at 53 °C and 8 min at 65 °C with a final elongation step of 15 min at 65 °C. PCR amplification products were detected by electrophoresis of 12 µL aliquots through 1.4% agarose gels in Tris-borate-EDTA (TBE) buffer (Sambrook and Russell, 2001), which were stained with ethidium bromide (EtBr 1.25 mg/L), visualized under UV light, and printed image through Bio-Print (Vilber Lourmat, France). DNA standards (1-kb DNA ladder Gibco BRL) were included in each electrophoresis gel. All of the amplifications were performed at least twice in separate assays, to ensure the reproducibility of the patterns, and only bands common to the replicate amplifications were scored. DNA fingerprints of strains were first compared for similarity by visual inspection of band patterns. They were considered identical when all scored bands in each pattern had the same apparent migration distance, even if a slightly different molecular weight was assigned to the same band over two or three different electrophoreses. Variations in intensity were not taken as differences.

Following the visual inspection, the patterns of all of the isolates were analyzed more rigorously using the Bio-Profil software (Vilber Lourmat, France). Band sizes were assigned by direct comparison to concurrently run DNA standards (1 kb). This information was used to construct a matrix table where each isolate was matched with a notation +/-, where (+) represents the identical presence and position of a band in the fingerprints to be compared.

Data analysis

Dendrograms were established using TAXONUM, software developed by G. Hunault and L. Gardan (Faculté des Sciences d'Angers, Angers, France). Cluster analysis was carried out using the unweighted pair-group method with averages (UPGMA) with the complement of Jaccard's similarity coefficient (Sneath and Sokal, 1973). Each fragment was considered as a separate marker in pairwise comparisons.

The BOX fragments as well as the biochemical tests characteristic of each cluster were identified by assessing the amount of information provided by each fragment or character, obtained by calculating the diagnostic ability coefficient (DAC) (Descamps and Véron, 1981).



Nutritional characteristics

All of the 120 bacterial strains (Table 1) fitted with the general characteristics of P. syringae sensu lato and P. viridiflava. They were obligate aerobes, presenting oxidative metabolism of glucose, and being positive for levan (except P. viridiflavaT, P. syr. ribicolapt and P. syr. primulaept) and tobacco hypersensitivity and negative for oxidase and arginine tests, produced fluorescent pigment on King's medium B (except for P. syr. actinidiae strains, P. sav. glycinea strain 3356, and P. sav. phaseolicola strains 3653 and 4706).

The numerical analysis of 119 nutritional characteristics (data not shown) evidenced two clusters. The first cluster contained P. sav. phaseolicola, P. sav. glycinea, P. syr. tabaci (two strains), P. syr. mori and P. syr. sesami, all belonging to genomospecies 2, and was distinguishable by two substrates only: sorbitol and meso-tartrate. The second cluster comprised all of the other strains evaluated, i.e. all nine genomospecies including other strains of P. syr. tabaci and other pathovars of genomospecies 2.

Comparing BOX fingerprints of 120 Pseudomonas strains

The amplification of genomic DNA of 88 Pseudomonas strains, followed by gel electrophoresis of resulting PCR products, showed 12 to 22 bands for the whole set of strains, and a total of 133 discrete bands were scored, ranging in size from 220 bp to 3.6 kb. From the first list of 89 strains, we were not able to amplify P. syr. theae DNA. The data matrix showing presence or absence of these 133 bands was analyzed by Jaccard coefficient and UPGMA, and a dendrogram displaying the distances between the 88 strains is shown in Figure 1.

At a distance of 0.72, all pathovars belonging to each one of the nine genomospecies described by Gardan et al. (1999) were clustered together. Cluster I included all of the strains of genomospecies 2. Into this group the great homogeneity of P. sav. phaseolicola strains obtained by BOX-PCR fingerprinting is illustrated in Figure 2, where only one different band is found and for only two strains. The eight remaining clusters, III, IV, V, VI, VII, VIII, IX and X corresponded to genomospecies 3, 6, 8, 5, 4, 7, 1 and 9, respectively (Gardan et al., 1999). Inside cluster X, three additional strains of P. syr. cannabina were evaluated and showed the same fingerprint as CFPB 2341 (data not shown). The two P. syringae strains of unknown pathovars (3650 and 3662) and the three P. syr. actinidiae strains were grouped in clusters II and XI, respectively.

The second step of analyses originated from the results shown in Figure 2: the homogeneity of a given pathovar. In order to confirm the utility of BOX-PCR to identify the genomospecies, a second analysis was performed. From the total of 41 strains of P. sav. phaseolicola, only the type strain was maintained representing the pathovar, based on its fingerprint homogeneity. This analysis was performed upon 60 pathovar-type strains and one more strain of P. syr. striafaciens. A total of 61 strains were included in this step of analysis.

The dendrogram displaying the distance relationships between the strains is shown in Figure 3. At a distance of 0.73 seven clusters were shown (I to VII), where five of them (I, II, III, IV and VII) corresponded strictly to genomospecies 3, 1, 6, 9 and 4. The remaining two clusters (V and VI) clustered together at the mentioned distance, the genomospecies 2 and 5 (cluster V) and 7 and 8 (cluster VI). Despite this fact, when analyzing the two groups at a distance of 0.65 the four genomospecies are separated in tight groups: V.a at 0.65 clustered together all the pathovars of genomospecies 2, V.b clustered all the pathovars of genomospecies 5, VI.a clustered all the pathovars of genomospecies 7 and VI.b all the pathovars of genomospecies 8. Figure 4 shows this similarity in fingerprints between isolates from the same genomospecies, but from different pathovars and the great homogeneity inside genomospecies 4, clustered at a distance of 0.35.



The results of the present study support the observation that BOX-PCR seems to be able to identify bacterial strains at species level.

The two clusters (II and XI, Figure 1) not included into the first description of the genomospecies (Gardan et al., 1999), might eventually constitute two new species.

The bands that most discriminated the ten clusters in the first study were selected by DAC analysis (Table 2).



The data obtained from the analyses of phenotypic and genetic diversity of a collection of P. syringae sensu lato group, P. viridiflava, and reference bacteria showed that pathovars belonging to the genomospecies designed after Gardan et al. (1999) could be separated at species level by BOX-PCR pattern.

Although bacteria are traditionally identified by phenotypic descriptions, it is not possible to obtain from nutritional studies a battery of discriminating substrates to the genomospecies as related by Gardan et al. (1999), and confirmed in this study.

A multiphasic approach has been proposed as being a reliable method of integrating different types of information, such as genotypic, phenotypic and phylogenetic data (Vandamme et al., 1996). Methods of fingerprinting based on the analysis of the total genome may constitute a valuable complement (Rademaker and Bruijn, 1997).

Amplification of Box primer by PCR from DNA of 88 strains belonging to 31 pathovars of P. syringae sensu lato group and P. viridiflava, led to the establishment of patterns that allowed the distinction of 11 BOX clusters, where nine of them cut the nine genomospecies described inside those pseudomonads (Gardan et al., 1999). When including 30 other strains representing the remaining species and pathovars for this first group, the genomospecies discrimination was confirmed, despite the necessity of cutting the dendrogram at different but close distances. Genomospecies 2 and 5 are very closely related, and the strain CFBP3229 of P. syr. tremae, originally included in genomospecies 5, could be clustered with genomospecies 2. A similar situation was found with genomospecies 7 and 8, whose strains could be separated at 0.65. In this study, P. syr. avellanae was clustered together with P. syr. theae, both corresponding to genomospecies 8. Using different molecular techniques, other authors found that the strains described by Gardan et al. (1999) as genomospecies 8 and 3 should be clustered together: Sarkar and Guttman (2004, utilizing multilocus sequencing typing, MLST), Inoue and Takikawa (2006, comparing the hrpZ and hrpA genes sequences). The present analysis, which used BOX-PCR fingerprinting, is capable of separating genomospecies 3 and 8. It also shows a remarkable homogeneity inside genomospecies 4, whose isolates are maintained together until a distance of 0.35.

Long after Louws et al. (1994), claimed BOX analysis could discriminate pathovars of P. syringae, it now appears that the authors were dealing with three different genomospecies: pv. morsprunorum (genomospecies 2), pv. syringae (genomospecies 1), and pv. tomato (genomospecies 3). According to Louws et al. (1995) BOX-PCR was able to distinguish strains A and B of X. campestris pv. vesicatoria, separated by DNA-DNA hybridization by Stall et al. (1994), and further named X. axonopodis pv. vesicatoria (ex A) and X. vesicatoria (ex B) (Vauterin et al., 1995).

Regarding the two extra groups obtained in this study (clusters II and XI, Figure 1), they are not included in the nine known genomic species of Gardan et al. (1999), and might constitute two new species. Cluster II is composed of two bacterial strains isolated from beans (CFBP 3650 and 3662), for which hybridization rate with type strain of P. sav. phaseolicola (CFPB 1390 pt) was only 50% and 51%, respectively (data not shown). In addition, they did not possess the phaseolotoxin gene, their esterase isozyme patterns were very distinct and they were aesculine positive (Marques et al., 2000). If there were no mistakes during the work, they might constitute another distinct bean pathogen belonging to the P. syringae group. Cluster XI of the dendrogram is composed of three strains of P. syr. actinidiae (CFPB 4909 the pathotype strain, 4911 and 5095). In order to define their inclusion as new genomospecies it would be necessary to provide quantitative DNA-DNA hybridization (Wayne et al., 1987).

Quantitative DNA-DNA hybridization still constitutes the reference for the description of a new species, but it is not adapted to a routine base identification of bacteria. It seems very important to propose alternative techniques, which could reproduce identical results, in order to identify bacterial species.

Since our data basis is constituted from BOX-PCR profiles of 31 or 61 pathovars of P. syringae - P. viridiflava, new pathovars will be easily assigned to a known genomic species. Furthermore, considering the hypothesis that it may be possible to verify that discriminating bands detected by BOX-PCR technique could amplify specific DNA fragments of each genomic species, a new route would be offered to help identify the P. syringae group of plant bacteria at species level.



We are grateful to Marion Le Saux for helping to update information and to Gláucia S.C. Buso for critical reading of the manuscript; to J.D. Taylor for kindly providing the great majority of P. savastanoi pv. phaseolicola strains; to Alain Huard and Wesley R. Souza for preparing the figures. This study was supported in part by Région Pays de la Loire (France). Abi S.A. Marques was supported by Embrapa (Empresa Brasileira de Pesquisa Agropecuária, Brazil).



Catara V, Sutra L, Morineau A, Achouak W, Christen R and Gardan L (2002) Phenotypic and genomic evidence for the revision of Pseudomonas corrugata and proposal of Pseudomonas mediterranea sp. nov. Int. J Syst Evolution Microbiol 52:1749-1758.        [ Links ]

Cross JE, Kennedy BW, Lambert JW and Cooper RL (1966) Pathogenic races of the bacterial blight pathogen of soybeans, Pseudomonas glycinea. Plant Dis Rep 50:557-560.        [ Links ]

Descamps P and Véron M (1981) Une méthode de choix des caractères d'identification basée sur le théorème de Bayes et la mesure de l'information. Ann Inst Pasteur/Microbiol 132B:157-170.        [ Links ]

El Tassa SOM, Moraes MG and Duarte V (1999) Identificação de Pseudomonas syringae pv. coronafaciens através de ERIC- e BOX-PCR. Fito Bras 24:503-508. (Abstract in English).        [ Links ]

Galal AA, Kehil YEI, el Daoudi YH, Shihata ZA and Ouf MF (2003) A comparative study on the identification of races and biovars of some Egyptian isolates of Ralstonia solanacearum. Egypt J Phytopathol 31:103-117.        [ Links ]

Gardan L, Shafik H, Belouin S, Brosch R, Grimont F and Grimont PAD (1999) DNA relatedness among the pathovars of Pseudomonas syringae and description of Pseudomonas tremae sp. nov. and Pseudomonas cannabina sp. nov. (ex. Sutic and Dowson 1959). Int J Syst Bacteriol 49:469-478.        [ Links ]

Greco S, Bella P, Tessitori M and Catara V (2004) Indagini sulla disseminazione in vivaio di Xanthomonas hortorum pv. pelargonii. Colture Protette 33:65-68. (Abstract in English).        [ Links ]

Inoue Y and Takikawa Y (2006) The hrpZ and hrpA genes are variable, and useful for grouping Pseudomonas syringae bacteria. J Gen Plant Pathol 72:26-33.        [ Links ]

Lelliott RA, Billing E and Hayward AC (1966) A determinative scheme for the fluorescent plant pathogenic pseudomonads. J Appl Bacteriol 29:470-489.        [ Links ]

Louws FJ, Fulbright DW, Stephens CT and DeBruijn FJ (1994) Specific genomic fingerprints of phytopathogenic Xanthomonas and Pseudomonas pathovars and strains generated with repetitive sequences and PCR. Appl Environ Microbiol 60:2286-2295.        [ Links ]

Louws FJ, Fulbright DW, Taylor SE and DeBruijn FJ (1995) Differentiation of genomic structure by rep-PCR fingerprinting to rapidly classify Xanthomonas campestris pv. vesicatoria. Phytopathology 85:528-536.        [ Links ]

Louws FJ, Bell J, Medina-Mora CM, Smart CD, Opgenorth D, Ishimaru CA, Hausbeck MK, Bruijn FJ and Fulbright DW (1998) rep-PCR-mediated genomic fingerprinting: a rapid and effective method to identify Clavibacter michiganensis. Phytopathology 88:862-868.        [ Links ]

Marques ASA, Corbière R, Gardan L, Tourte C, Manceau C, Taylor JD and Samson R (2000) Multiphasic approach for the identification of the different classification levels of Pseudomonas savastanoi pv. phaseolicola. Eur J Plant Pathol 106:715-734.        [ Links ]

Martin B, Humbert O, Camara M, Guenzi E, Walker J, Mitchell T, Andrew P, Prudhomme M, Alloing G and Hakenbeck R (1992) A highly conserved repeated DNA element located in the chromosome of Steptococcus pneumoniae. Nucleic Acids Res 20:3479-3483.        [ Links ]

Onfroy C, Tivoli B, Corbière R and Bouznad Z (1999) Cultural, molecular and pathogenic variability of Mycospharella pinodes and Phoma medicaginis var. pinodella isolates from dried pea (Pisum sativum) in France. Plant Pathol 48:218-229.        [ Links ]

Palleroni NJ (1984) Genus I. Pseudomonas Migula 1894. In: Krieg NR and Holt JG (eds) Bergey's Manual of Systematic Bacteriology. v. 1. Williams and Wilkins, Baltimore, pp 141-199.        [ Links ]

Rademaker JLW and DeBruijn FJ (1997) Characterization and classification of microbes by rep-PCR genomic fingerprinting and computer assisted pattern analysis. In: Gaetano-Anolles G (ed) DNA Markers: Protocols, Applications and Overviews. Willey & sons, New York, pp 151-171.        [ Links ]

Sambrook J and Russell DW (2001) Molecular Cloning: A Laboratory Manual. 3rd edition. Cold Spring Harbor Laboratory Press, New York, 999 pp.        [ Links ]

Sarkar SF and Guttman DS (2004) Evolution of the core genome of Pseudomonas syringae, a highly clonal, endemic plant pathogen. Appl Environ Microbiol 70:1999-2012.        [ Links ]

Sneath PHA and Sokal RP (1973) Numerical Taxonomy: The Principles and Practice of Numerical Classification. WH Freeman and Company, San Francisco, 573 pp.        [ Links ]

Stall RE, Beaulieu C, Egel D, Hodge NC, Leite RP, Minsavage GV, Bouzar H, Jones JB, Alvarez AM and Benedict AA (1994) Two genetically diverse groups of strains are included in Xanthomonas campestris pv. vesicatoria. Int J Syst Bacteriol 44:47-53.        [ Links ]

Tacao M, Alves A, Saavedra MJ and Correia A (2005) BOX-PCR is an adequate tool for typing Aeromonas spp. Antoine-van-Leeuwenhoek 88:173-179.        [ Links ]

Tamura K, Takikawa S, Tsuyumu S and Goto M (1989) Characterization of the toxin produced by Pseudomonas syringae pv. actinidiae, the causal bacterium of kiwifruit canker. Ann Phytopathol Soc Japan 55:512        [ Links ]

Taylor JD, Teverson DM, Allen DJ and Pastor-Corrales MA (1996) Identification and origin of races of Pseudomonas syringae pv. phaseolicola from Africa and other bean growing areas. Plant Pathol 45:469-478.        [ Links ]

Tourte C and Manceau C (1995) A strain of Pseudomonas syringae which does not belong to pathovar phaseolicola produces phaseolotoxin. Eur J Plant Pathol 101:483-490.        [ Links ]

Vandamme P, Pot B, Gillis M, de Vos P, Kersters K and Swings J (1996) Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol Rev 60:407-438.        [ Links ]

Vauterin L, Hoste B, Kersters K and Swings J (1995) Reclassification of Xanthomonas. Int J Syst Bacteriol 45:472-489.        [ Links ]

Versalovic J, Koeuth T and Lupski JR (1991) Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res 19:6823-6831.        [ Links ]

Versalovic J, Schneider M, de Bruijn FJ and Lupski JR (1994) Genomic fingerprinting of bacteria using repetitive sequence-based Polymerase Chain Reaction. Methods Mol Cell Biol 5:25-40.        [ Links ]

Wayne LG, Brenner DJ, Colwell RR, Grimont PAD, Kandler O, Krichevsky MI, Moore LH, Moore WEC, Murray RGE, Stackebrandt E, Starr MP and Trüpper HG (1987) International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37:463-464.        [ Links ]

Williams JGK, Kubelik AR, Livak KJ, Rafalski JA and Tingey SV (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res 18:6531-6535.        [ Links ]

Young JM, Saddler GS, Takikawa Y, DeBoer SH, Vauterin L, Gardan L, Gvozdyak RI and Stead DE (1996) Names of plant pathogenic bacteria 1864-1995. Rev Plant Pathol 75:721-762.        [ Links ]



Send correspondence to:
Abi S.A. Marques.
Embrapa Recursos Genéticos e Biotecnologia,
Parque Estação Biológica, Final Av. W5 Norte, Caixa Postal 02372,
70770-900 Brasília, DF, Brazil.

Received: June 28, 2007; Accepted: January 16, 2008.



Associate Editor: Luis Carlos de Souza Ferreira

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