Keywords: Ralstonia solanacearum, classification, phylogeny, phylotype, sequevar. Abstract. Ralstonia solanacearum strains are highly variable and adaptable ...
Recent Developments in the Phylogeny and Classification of Ralstonia solanacearum P. Prior INRA, Unité de Pathologie Végétale Domaine St Maurice, BP 94 F-84140, Montfavet France
M. Fegan CRCTPP School of Molecular and Microbial Sciences The University of Queensland Australia
Keywords: Ralstonia solanacearum, classification, phylogeny, phylotype, sequevar Abstract Ralstonia solanacearum strains are highly variable and adaptable as attested by worldwide distribution and a large and expanding host range. R. solanacearum (Rs) and its close relatives, the blood disease bacterium and R. syzygii, constitute a species complex, a diverse group of related isolates that represent more than one species. Biovar (Bv) typing and race assessment are methods commonly used for assessing the diversity of Rs strains. However, recent genetic evidence has indicated that these phenotypically-based schemes are not sufficient to encompass the diversity of strains represented in the species Rs. Here we present a classification system based upon phylogenetic analysis of sequence data generated from the 16S23S internal transcribed spacer (ITS) region, the endoglucanase gene and the mutS gene. From the phylogenetic analysis of the sequence data the Rs species complex can be subdivided into four monophyletic cluster of strains, termed phylotypes. Strains within each phylotype broadly originate from the same geographic location. Phylotype I includes all strains belonging to Bv 3, 4, and 5 and strains are isolated primarily from Asia. Phylotype II includes strains belonging to Bv 1, 2 and 2T isolated primarily from America. The Rs race 3 potato pathogen, which has a worldwide distribution, and the race 2 banana pathogen are both members of phylotype II. Phylotype III contains strains primarily isolated from Africa and surrounding islands. Strains in this group belong to Bv 1 and 2T. Phylotype IV contains strains isolated primarily from Indonesia belonging to Bv 1, 2 and 2T. Each phylotype is composed of a number of groups of strains with a highly conserved sequence termed sequevars. Some sequevars contain strains which are pathogenic on the same hosts or strains of common geographic origin. The phylotyping scheme is highly discriminatory, flexible, additive, and should allow better prediction of the properties of strains. This in turn will aid in the successful control of the many bacterial wilts caused by Rs, including bacterial wilt of tomato. INTRODUCTION The genus Ralstonia is within the β-subdivision of the Proteobacteria and includes five species, R. picketii, R. insidiosa, R. mannitolilytica, R. syzygii and R. solanacearum. R. solanacearum (Yabuuchi et al., 1995), a soilborne vascular pathogen of worldwide distribution, causes bacterial wilt of an unusually broad host range of plants (more than 200 species) from highly diverse botanical families including monocots and dicots (Hayward, 1964). This wide geographic distribution, large host range and the exceptional capacity of this organism to adapt to many different environments is mirrored in the astonishing phenotypic and genetic diversity at the strain level. Driving forces for evolution of R. solanacearum strains need to be considered with respect to the unusual genomic plasticity of the organism (Boucher and Genin, 2004), the ability of this organism for natural transformation (Bertolla et al., 1999) and the secretive and subtle life of this plant pathogen as a xylem inhabitant (Schell, 1996, 2000). The large diversity of strains which belong to the R. solanacearum species complex results in the need to accurately classify R. solanacearum at the infra-specific level. Studies to characterize the genetic diversity of strains of R. solanacearum are needed to allow identification of infraProc. 1st IS on Tomato Diseases Eds. M.T. Momol, P. Ji and J.B. Jones Acta Hort 695, ISHS 2005
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subspecific groups of strains that have common biological properties, evolutionary relationships or geographic origins. Such understanding may result in improving breeding strategies for obtaining durable resistance to bacterial wilt in many different plant species affected by this organism. It is well known that the subtle host-pathogen-environment interactions may lead to a breakdown of the genetic gain for resistance resulting from breeding programmes. One reason for this breakdown may be the lack of information on the different strains of the organism present in different regions of the world, and different regions within a country. A better knowledge of the genetic diversity of R. solanacearum is needed to identify groups of R. solanacearum strains that are associated with pathogenicity for certain hosts, to rapidly stop or recognize quarantine organisms, and to develop targeted diagnostic tests. The race and biovar classification systems are the international standard adopted to type R. solanacearum strains. The races of R. solanacearum should more correctly be termed pathovars as they are named based upon the pathotype of the strain as the system is based on strain host range (Buddhenhagen et al., 1962), not susceptibility of host cultivars. Biovar typing is based on the metabolic profile of a given strain, especially the ability to metabolise disaccharides and hexosealcohol (Hayward, 1964). More recently, molecular based methods have resulted in a fine tuning of the understanding of the diversity of R. solanacearum strains. Cook et al. (1989) and Cook and Sequeira (1994) reported 46 multilocus genotypes (MLG) following RFLP analysis, these MLG groups clustered into two major branches: Division I made up of strains from race 1; biovars 3, 4 and 5 originating from the Old World (Asiaticum); and Division II including strains of race 1 and 2; and biovar 2 (race 3) originating from the New World (Americanum). Additional evidence for revising the classification of R. solanacearum was provided by genetic fingerprinting analysis using AFLP and PCRRFLP on the HRP gene cluster (Poussier et al., 2000a) and nucleotide sequence analysis of 16S rRNA gene (Li et al., 1993; Taghavi et al., 1996; Poussier et al., 2000a), the non coding but transcribed intergenic spacer (ITS) region between the 16S and 23S rRNA genes, and partial sequencing of the endoglucanase gene and hrpB gene (Fegan et al., 1998; Poussier et al., 2000b). From the genetic fingerprinting and sequence analyses two other genetic groups of strains were identified which also correlate with geographical origin of the strains: a group of biovar 1 and 2 strains from Africa, and a group of strains from Indonesia. The group of strains from Indonesia, the most diverse genetic group, includes R. solanacearum strains belonging to biovars 1, 2 and 2T (isolated primarily from solanaceous plant species and cloves), but also two closely related organisms; R. syzigii, a pathogen from cloves, and the agent of blood disease of banana (Taghavi et al., 1996). Thus, genetic diversity within the R. solanacearum species complex can be split into at least four phylogenetically different groups and two related species (Fig. 1). At the 3rd International Bacterial Wilt Symposium (Allen et al., 2004), Fegan and Prior (2004) made a proposal for a new and hierarchical classification scheme to describe intra-specific variations among strains of R. solanacearum. The overriding reason for this proposal was that the phenotypic (metabolic and pathogenic) measures of diversity are not consistent with genetic groupings: R. solanacearum strains belonging to biovars 1 and 2T show a high degree of genetic variation as they are present in three out of four genetic groups; biovar 2 is present in two of the four genetic groups and are not always equivalent to race 3; and strains of biovars 3 and 4 overlap in their phenotype and RFLP groups. In addition, based on the race classification scheme, most strains fall into race 1, a large and highly heterogeneous group of strains of R. solanacearum. Strains which do not belong to race 2 or 3 tend to be placed here, by default. However, races 2 and 3, which contain strains with a narrower host range, exhibit much less genetic diversity. As stated by Buddenhagen (1986), “to be meaningful biologically, taxonomy of plant pathogenic bacteria must be correlated with pathogenic potential or geographic origin”. Consequently, it should be expected from a classification scheme that the genetic group to which a strain belongs should reflect its biological and ecological properties. Here we build upon the reference work based upon phylogenetic analysis of the endoglucanase gene and the classification scheme for genotyping R. solanacearum 128
published by Fegan and Prior (2004). In this paper, additional phylogenetic evidence is presented to reinforce the phylotyping scheme, based on original sequence information from the gene mutS, which encodes for a DNA mismatch repair protein. MATERIALS AND METHODS Table 1 presents some characteristics of the 86 strains used in this phylogenetic analysis of the mutS partial gene. Most strains were shared by the Franco-Australian consortium for phylogenetic analysis based on ITS 16S-23S, hrpB and endoglucanase partial gene (Poussier et al., 2000b; Fegan and Prior, 2004; Prior and Fegan, 2004). In this table, phylotype and sequevar designation are from Fegan and Prior (2004). PCR amplification of a 758 bp fragment of the terminal portion of the mutS gene was performed using a MJ Research PTC200 thermocycler. Reactions were carried out according to the protocol provided by A. Mercier (pers. comm.); in a total volume of 25 µl containing 1 x PCR buffer (Promega) 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.8 µM DMSO, 1.25 U of Taq Polymerase (Promega), 9 pmoles of the primers mutS-RsF.1570 (5'-ACA GCG CCT TGA GCC GGT ACA-3') and mutS-RsR.1926 (5'-GCT GAT CAC CGG CCC GAA CAT-3'). Reactions were heated to 96°C for 5 min and then cycled through 35 cycles of 94°C for 60s, 66°C for 60s and 72°C for 90s, followed by a final extension period of 5 min at 72°C. Samples (5 µl) of reaction mixtures were examined by electrophoresis through 2% agarose gels and bands were revealed by staining in 0.5 µg mL-1 ethidium bromide. PCR products were purified and sequencing reaction performed on both strands by MWG-Biotech sequencing services (Ebersberg, Deutschland). The sequences of the mutS gene were analysed by using the ARB Software Environment (Ludwig et al., 2004). Sequences were manually aligned using the ARB sequence editor. Evolutionary distances between sequences were computed by using the algorithm of JUKES and CANTOR (1969) and phylogenetic trees were constructed from genetic distance data by using the neighbor-joining method (Saitou and Nei, 1987) with 5000 bootstrap resamplings of the data to test the tree topologies. RESULTS The partial mutS gene sequences (583 to 694 nucleotides) of 82 strains of R. solanacearum representing the known geographical, phenotypic and pathotype diversity were determined and aligned. This included strains already used to infer phylogenetic trees based on the ITS region and partial hrpB and endoglucanase (Fig. 2) gene sequences (Poussier et al., 2000b; Fegan and Prior, 2004). Two strains of the clove pathogen R. syzygii (R24 and R28) and two strains of the blood disease bacterium (JT656 and JT657) were included in this study as they fall within the R. solanacearum species complex (Taghavi et al., 1998; Prior and Fegan, 2004). A phylogenetic tree with robust bootstrap values was generated which shows five distinct genetic groups (Fig. 3A). The first group contains a single strain, ACH0732, which was isolated from tomato in the North of Australia. All strains representing biovars 3, 4 and 5 cluster together in a second group equivalent to phylotype I described by Fegan and Prior (2004). Strains equivalent to Phylotype II (Fegan and Prior, 2004) resolved into two distinct and robust clusters named Phylotype II broad host range (bhr) and Phylotype II narrow host range (nhr). This clustering is mirrored in the clustering of R. solanacearum strains in phylotype II observed in the phylogenetic tree based upon the endoglucanase gene (Fig. 2). Phylotype II-bhr contains race 1/biovar 1 strains from the new world primarily from solanaceous hosts from America. This group sub-clustered in two branches: one branch consisted of strains equivalent to sequevar 5 and 6 (from tomato and banana respectively) (Fegan and Prior, 2004), strain CIP301 (MLG3), strains equivalent to sequevar 7 (Fegan and Prior, 2004) and CIP239 and a Brazilian strain (MLG38) pathogenic to potato (Fig. 3B). Phylotype II-nhr included biovar 1 strains pathogenic to Musa sp. (Sequevar 3, MLG 24; sequevar 4, MLG 25; Fegan and Prior, 2003) and strains pathogenic to potato (brown rot) which have adapted to low temperature (race 3, MLG 26, 27 and 34). The third group of strains is equivalent to Phylotype III (Fegan and Prior, 2004) is made of strains of biovar 129
1 and biovar N2 from South Africa, Madagascar and Reunion Is. Finally, the fourth group of strains is equivalent to phylotype IV (Fegan and Prior, 2004) and includes strains of R. solanacearum primarily isolated from Indonesia, and close relatives R. syzygii and the agent of the blood disease of banana. DISCUSSION Phylogenetic trees inferred from partial mutS gene sequence analysis were fully consistent with the previous reports of trees based on phylogenetic analysis of sequence data from other genomic regions: the hrpB gene (Poussier et al., 2000b), the ITS region (Fegan et al., 1998) and the endoglucanase gene (Fegan et al., 1998; Poussier et al., 2000b). This work re-enforces the picture which has emerged indicating that the genetic and phenotypic diversity within the R. solanacearum species complex is comprised of four broad groups related to geographical origin of strains. These were defined by Fegan and Prior (2004) as phylotypes: “monophyletic cluster of strains revealed by phylogenetic analysis of sequence data”. Strain ACH0732 is unique in the collection as it is the only isolate which varies in its position depending on the area of the genome analysed: based on the endoglucanase gene sequence analysis it belongs to Phylotype I (Poussier et al., 2000b), whereas based on the ITS (Fegan et al., 1998) and mutS sequences this strain appears to stand alone with a branching depth equivalent to that of a phylotype. The only other differences between the phylogenetic trees produced here and in other reports are the relative relationships of each phylotype to other phylotypes and the genetic diversity observed within each phylotype. Based on the ITS (Fegan et al., 1998; Fegan and Prior, unpublished) and the mutS gene sequence analysis phylotype I, III and IV are phylogenetically distant from ACH0732 and phylotype II (America). The diversity observed within each phylotype is the smallest in the tree based upon ITS sequence data and the largest in the endoglucanase tree (Poussier et al., 2000b; Prior and Fegan, 2004). This ITS region sequence similarity between members within each phylotype has been exploited to designed a multiplex PCR to diagnose the phylotype to which a strain belongs (Fegan and Prior, 2004). From phylogenetic analysis of the endoglucanase and the hrpB genes (Poussier et al., 2000b) each phylotype was equally distant to all other phylotypes. However, the relative position of phylotype I (Asia) varies, being closer to phylotype IV or to phylotype III, in the endoglucanase gene based tree and the hrpB based tree respectively. Within each phylotype the composition and number of sequevars (“a sequence variant defined as a group of strains with a highly conserved sequence within the area sequenced”; Fegan and Prior, 2004) in each phylotype was the same. Strains within phylotype I exhibit the least genetic diversity of all the phylotypes regardless of the area sequenced. It is anticipated that strains already sequenced within phylotypes III and IV are representative of the diversity within these groups but to fully appreciate the full diversity within these phylotypes more strains will need to be sequenced. Importantly, little is known about the pathotype of strains falling in phylotype III, and the full pathotype diversity of strains in phylotype IV still remains to be unravelled. Most spectacular is the clear-cut phylogenetic separation within phylotype II strains of R. solanacearum. Genetic diversity within this phylotype is as large as is the phenotypic and ecological diversity of strains which fall in this phylotype. Phylogenetic analysis based on the endoglucanase gene and the mutS gene sequences identified two sub-clusters with high bootstrap support (Fig. 3A). These two sub-clusters have been called phylotype II-bhr and -nhr for broad and narrow host range, respectively. The former group (phylotype II-bhr) is made of R. solanacearum strains which are pathogenic to both solanaceous hosts, including tomato, and Musa spp. Phylotype II-nhr can be resolved into R. solanacearum race 3 strains pathogenic to potato grown in the highlands, and R. solanacearum race 2 moko disease causing strains. It is remarkable that strains of R. solanacearum races 2 and 3 both with narrow host range fell in this subcluster (Phylotype II-nhr) within phylotype II, regardless the genome region analysed (hrpB, 130
endoglucanase and mutS). Such phylogenetic evidence suggests a close evolutionary relationship of these strains which may help in the exploration of genetic determinants for host specificity. Phylotyping strains of R. solanacearum provides fast and reliable information. However, not all laboratories can access sequencing facilities. Hence PCR-based molecular diagnostic protocols to identify the phylotype to which a strain belongs (Fegan and Prior, 2004), and to identify the race 2 strains in sequevar 3, 4 and 6 (Prior and Fegan, 2004) have been developed. The phylotyping scheme has many advantages compared to the race and biovar classification schemes: (i) it is additive; (ii) it is highly discriminatory and can distinguish among a large number of genotypes and (iii) it is based on genetic variations which accumulate at a low rate and this provides a long term global epidemiology. In addition, the phylotype/sequevar classification has proven to be independent of the area sequenced, which indicates that the scheme mirrors the evolutionary histories of the organism. The phylotyping scheme is consistent with RFLP classification scheme of Cook et al. (1989) and Cook and Sequeira (1994). Unfortunately the RFLP probes developed by Cook et al. (1989) are no longer available which makes this RFLP typing scheme obsolete. The phylotyping scheme has already proven extremely useful in identifying the emergence of a group of biovar 1 strains (ANT307 and ANT1121) pathogenic to anthurium in Martinique. These strains were rapidly identified to belong to phylotype II and to a group of strains that cause moko disease, and while these strains were not pathogenic to banana (NPB) they were able to cause disease in Heliconia and plantain. Like moko disease causing strains these strains are strongly suspected to be insect transmitted (see Wicker et al., this symposium). However, unlike moko disease causing strains these strains cause disease in many unusual hosts including cucurbits (see Wicker et al., this symposium). A further example of the predictive value of the phylotyping scheme is the identification of a biovar 1 strain, CIP 418, isolated from groundnut in Indonesia which was surprisingly related to moko disease causing strains within phylotype II. Following pathogenicity tests on Musa ‘Cavendish’, this strain was proved to be highly pathogenic for banana (P. Prior, unpublished results). Identifying the infra-specific genetic variability in R. solanacearum species complex by using the phylotyping scheme accurately identifies the evolutionary relationships between strains and the scheme relates well to phenotypic and ecological traits of R. solanacearum strains. From Fig. 2, it can be clearly seen that tomato (in particular) and solanaceous hosts (in general) may wilt due to strains belonging to all phylotypes. This may explain the difficulty in breeding durable resistance for bacterial wilt in tomato. In the future, it is anticipated that by using the phylotyping scheme breeders will be able to develop durable resistance to bacterial wilt of tomato. ACKNOWLEDGEMENTS We thank A. Mercier and F. Bertolla (UMR CNRS 5557, Université ClaudeBernard Lyon 1) for providing sequence information and PCR primers for the mutS gene. Thanks to Caroline Guilbaud for technical support in this study. Literature Cited Allen, C., Prior, P. and Hayward, A.C. 2004. Bacterial wilt: the disease and the Ralstonia solanacearum species complex. APS Press. Bertolla, F., Frostegard, A., Brito, B., Nesme, X. and Simonet, P. 1999. During infection of its host, the plant pathogen Ralstonia solanacearum naturally develops a state of competence and exchanges genetic material. Mol. Plant-Microbe Interact. 12:467-472. Boucher, C. and Genin, S. 2004. The Ralstonia solanacearum-plant interaction. p.92-112. In: N.J. Talbot (ed.), Plant-pathogen interactions, Blackwell Publishing, Oxford. Buddenhagen, I., Sequeira, L. and Kelman, A. 1962. Designation of races in Pseudomonas solanacearum. Phytopathology 52:726. Cook, D. and Sequeira, L. 1994. Strain differentiation of Pseudomonas solanacearum by 131
molecular genetic methods. p.77-93. In: A.C. Hayward and G.L. Hartman (eds.), Bacterial wilt: the disease and its causative agent, Pseudomonas solanacearum, CAB International, Wallingford, UK. Cook, D., Barlow, E. and Sequeira, L. 1989. Genetic diversity of Pseudomonas solanacearum: detection of restriction fragment polymorphisms with DNA probes that specify virulence and hypersensitive response. Mol. Plant-Microbe Interact. 2:113121. Fegan, M., Taghavi, M., Sly, L.I and Hayward, A.C. 1998. Phylogeny, diversity and molecular diagnostics of Ralstonia solanacearum. p.19-33. In: P. Prior, C. Allen and J. Elphinstone (eds.), Bacterial wilt disease: Molecular and ecological aspects, Springer. Fegan, M. and Prior, P. 2004. How complex is the Ralstonia solanacearum species complex. In: C. Allen, P. Prior and A.C. Hayward (eds.), Bacterial wilt: the disease and the Ralstonia solanacearum species complex, APS Press (in press). Gillings, M.R. and Fahy, P. 1994. Genomic fingerprinting: towards a unified view of the Pseudomonas solanacearum species complex. p.95-112. In: A.C. Hayward and G.L. Hartman (eds.), Bacterial wilt: the disease and its causative agent, Pseudomonas solanacearum, CAB International, Wallingford, UK. Hayward, A.C. 1964. Characteristics of Pseudomonas solanacearum. J. Appl. Bacteriol. 27:265-277. Hayward, A.C. 1994. The hosts of Pseudomonas solanacearum. p.9-25. In: A.C. Hayward and G.L. Hartman (eds.), Bacterial wilt: the disease and its causative agent, Pseudomonas solanacearum, CAB International, Wallingford, UK. Jukes, T.H. and Cantor, C.R. 1969. Evolution of protein molecules. p.21-132. In: H.N. Munro (eds.), Mammalian protein metabolism, New York, Academic Press. Li, X., Dorsch, M., Del Dot, T., Sly, L.I., Stackebrandt, E. and Hayward, A.C. 1993. Phylogenetic studies of the rRNA group II pseudomonads based on 16S rRNA gene sequences. J. Appl. Bacteriol. 74:324-329. Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H., Yadhukumar, Buchner, A., Lai, T., Steppi, S., Jobb, G., Förster, W., Brettske, I., Gerber, S., Ginhart, A.W., Gross, O., Grumann, S., Hermann, S., Jost, R., König, A., Liss, T., Lüßmann, R., May, M., Nonhoff, B., Reichel, B., Strehlow, R., Stamatakis, A., Stuckmann, N., Vilbig, A., Lenke, M., Ludwig, T., Bode, A. and Schleifer, K.H. 2004. ARB: a software environment for sequence data. Nucl. Acids Res. 32(4):1363-1371. Poussier, S., Trigalet-Demery, D., Vandewalle, P., Goffinet, B., Luisetti, J. and Trigalet, A. 2000a. Genetic diversity of Ralstonia solanacearum as assessed by PCR-RFLP of the hrp gene region, AFLP and 16S rRNA sequence analysis, and identification of an African subdivision. Microbiology 146:1679-1692. Poussier, S., Prior, P., Luisetti, J., Hayward, A.C. and Fegan, M. 2000b. Partial sequencing of the hrpB and endoglucanase genes confirms and expands the known diversity within the Ralstonia solanacearum species complex. Syst. Appl. Microbiol. 23:479-486. Prior, P. and Fegan, M. 2004. Diversity and molecular detection of Ralstonia solanacearum race 2 strains. In: C. Allen, P. Prior and A.C. Hayward (eds.), Bacterial wilt: the disease and the Ralstonia solanacearum species complex, APS Press (in press). Saitou, N. and Nei, M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425. Schell, M.A. 2000. Control of virulence and pathogenicity genes of Ralstonia solanacearum by an elaborate sensory network. Annu. Rev. Phytopathol. 38:263-292. Schell, M.A. 1996. To be or not to be: how Pseudomonas solanacearum decides whether or not to express virulence genes. Eur. J. Plant Pathol. 102:459-469. Taghavi, M., Hayward, A.C., Sly, L.I. and Fegan, M. 1996. Analysis of the phylogenetic relationships of strains of Burkholderia solanacearum, Pseudomonas syzygii, and the Blood Disease Bacterium of banana based on 16S rRNA gene sequences. Int. J. Syst. 132
Bacteriol. 46:10-15. Yabuuchi, E., Kosako, Y., Yano, I., Hotta, H. and Nishiuchi, Y. 1995. Transfer of two Burkholderia and an Alcaligenes species to Ralstonia Gen. Nov.: proposal for Ralstonia pickettii, Ralstonia solanacearum and Ralstonia eutropha. Microbiol. Immunol. 39:897-904. Tables Table 1. Ralstonia solanacearum, R. syzigii and the BDB strains used in this study. Strain1 GMI 1000 JT 523 UW393 UW397 UW399 PSS240 PSS194
Biovar Other or designation species JS753 3 3 ND ND ND 3 3
Geographic origin
Host
Guyana Reunion Is. Rep. South Africa Rep. South Africa Rep. South Africa Taiwan Taiwan
Lycopersicon esculentum Solanum tuberosum Lycopersicon esculentum Solanum melongena Lycopersicon esculentum Lycopersicon esculentum Lycopersicon esculentum
GA4
3 3 3 3
Australia Malaysia Philippines Guadeloupe (FWI)
Strelitzia reginae Lycopersicon esculentum Solanum tuberosum Solanum melongena
ACH92
4
Australia
Zingiber officinale
MAFF211266 CFBP765
JT690 JS771
4 4
Japan Japan
Lycopersicon esculentum Nicotiana tabacum
R292 R288
CIP277 JT659
5 5
China China
Morus alba Morus alba
CIP117 CFBP3858 JT516 JT510 JS529 UW394 UW477
UW453 JS907
Nigeria Netherlands Reunion Is. Reunion Is. Reunion Is. Rep. South Africa Peru
Solanum tuberosum Solanum tuberosum Solanum tuberosum Solanum tuberosum Pelargonium asperum Solanum tuberosum Solanum tuberosum
CIP351 NCPPB3190 CIP365 CFBP2968 UW151
ACH1023 JS941
JT654
2 2 2 2 2 ND N2
CIP10
R569
N2
Peru
Solanum tuberosum
NCPPB3987 CIP430
JT677
N2 1
Brazil Peru
Solanum tuberosum Solanum tuberosum
UW469
1
Brazil
Solanum tuberosum
UW 9
JT644
1
Costa Rica
Heliconia sp.
CFBP 1409 CFBP 1482 CIP 418 MOLK 2 UW 11 CFBP1183
JS775 JS730 MOH6
1 1 1 1 1 1
Honduras Panama Indonesia Philippines Costa Rica Costa Rica
Musa sp. Musa sp. Arachis hypogaea Musa sp. Heliconia sp. Heliconia sp.
1
Peru
Musa sp. plantain
JT648
1
Peru
Musa sp. plantain
UW156
1
Peru
Musa sp. plantain
1 1
Peru Peru
Musa sp. plantain Musa sp. plantain
CIP239
S167 JS793
UW129 UW162 CIP20 UW163 UW131
Phylotype, Sequevar, MLG2 I, 13-18 I, 13-18 I, 13-18 I, 13-18 I, 13-18 I, 13-18 I, 13-18 I, 13-18, MLG10a I, 13-18 I, 13-18 I, 13-18 I, 13-18, MLG22 I, 13-18 I, 13-18 I, 12, MLG19 I, 12 II-nhr, 1-2, MLG34 II-nhr, 1-2 II-nh, 1-2 II-nhr, 1-2 II-nhr, 1-2 II-nhr, 1-2 II-nhr, 1-2 II-nhr, 1-2, MLG29 II-nhr, 1-2 II-nhr, 1-2 II-nhr, 1-2, MLG38 II-nhr, 3, MLG24 II-nhr, 3 II-nhr, 3 II-nhr, 3 II-nhr, 3 II-nhr, 3 II-nhr, 3 II-nhr, 4, MLG25 II-nhr, 4, MLG25 II-nhr, 4, MLG25 II-nhr, 4 II-nhr, 4
GenBank3 AY756804 AY756803 AY756781 AY756819 AY756820 AY756741 AY756790 AY756802 AY756738 AY756787 AY756800 AY756764 AY756791 AY756740 AY756801 AY756797 AY756796 AY756748 AY756783 AY756739 AY756778 AY756767 AY756821 AY756789 AY756785 AY756759 AY756808 AY756744 AY756751 AY756805 AY756809 AY756813 AY756780 AY756749 AY756782 AY756795 AY756756 AY756779 AY756793
133
Table 1. continued. II-nhr, 4, AY756784 MLG25 II-nhr, 4, UW175 1 Colombia Musa sp. plantain AY756768 MLG25 R368 1 Colombia Musa sp. plantain II-nhr, 4 AY756818 II-nhr, 4, UW70 CIP70 1 Colombia Musa sp. plantain AY756794 MLG25 UW170 1 Colombia Musa sp. plantain II-nhr, 4 AY756737 UW166 CIP27 1 Costa Rica Musa sp. plantain II-nhr, 4 AY756761 ANT1121 1 Martinique (FWI) Anthurium andreanum II-nhr, 4 AY756769 ANT307 1 Martinique (FWI) Anthurium andreanum II-nhr, 4 AY756742 CFBP 2972 JS734 1 Martinique Solanum tuberosum II-bhr, 5 AY756807 CFBP 2957 JS717 1 Martinique Lycopersicon esculentum II-bhr, 5 AY756755 CFBP 2958 JS728 1 Guadeloupe Lycopersicon esculentum II-bhr, 5 AY756806 CFBP 712 JS770 1 Burkina Faso Solanum melongena II-bhr, 5 AY756773 CFBP 715 JS779 1 Burkina Faso Lycopersicon esculentum II-bhr, 5 AY756762 JQ1143 1 Reunion Is. Solanum tuberosum II-bhr, 5 AY756771 CFBP702 1 Burkina Faso Lycopersicon esculentum II-bhr, 5 AY756770 CFBP3256 GT1 1 Guadeloupe Lycopersicon esculentum II-bhr, 5 AY756798 CFBP3057 JS912 1 Burkina Faso Lycopersicon esculentum II-bhr, 5 AY756765 II-bhr, 5, CIP120 R563 1 Peru Solanum tuberosum AY756774 MLG04 II-bhr, 5, CIP 301 R311 1 Peru Solanum tuberosum AY756745 MLG03 A3907 1 Hawaii USA Heliconia sp. II-bhr, 6 AY756757 A3909 1 Hawaii USA Heliconia sp. II-bhr, 6 AY756753 A3911 1 Hawaii USA Heliconia sp. II-bhr, 6 AY756747 II-bhr, 6, UW181 JT649 1 Venezuela Musa sp. plantain AY756754 MLG28 II-bhr, 6, Musa sp. AY756758 UW21 R371 1 Honduras MLG28 ICMP 7963 JS967 1 Kenya Solanum tuberosum II-bhr, 7 AY756776 II-bhr, 7, UW134 S221 1 Kenya Solanum tuberosum AY756792 MLG01 II-bhr, 7, K60 UW25 1 USA Lycopersicon esculentum AY756799 MLG01 JT 525 1 Reunion Is. Pelargonium asperum III, 19-23 AY756786 NCPPB1029 1 Reunion Is. Pelargonium capitatum III, 19-23 AY756736 CFBP 734 JS767 1 Madagascar Solanum tuberosum III, 19-23 AY756746 NCPPB 283 JS946 1 Zimbabwe Solanum panduraforme III, 19-23 AY756814 NCPPB 332 JS949 1 Zimbabwe Solanum tuberosum III, 19-23 AY756760 NCPPB 505 JS951 1 Zimbabwe Symphytum sp. III, 19-23 AY756816 NCPPB 342 JS952 1 Zimbabwe Nicotiana tabacum III, 19-23 AY756815 UW34 K179 1 N. Rhodesia Nicotiana tabacum III, 19-23 AY756763 NCPPB 1018 JS950 1 Angola Solanum tuberosum III, 19-23 AY756772 CFBP 3059 JS904 1 Burkina Faso Solanum melongena III, 19-23 AY756766 J 25 N2 Kenya Solanum tuberosum III, 19-23 AY756810 CIP358 N2 Cameroon Solanum tuberosum III, 19-23 AY756750 MAFF301558 JS934 N2 Japan Solanum tuberosum IV, 8 AY756812 R24 R. syzygii NA Indonesia Syzygium aromaticum IV, 9 AY756775 R28 R. syzygii NA Indonesia Syzygium aromaticum IV, 9 AY756777 PSI07 2 Indonesia Lycopersicon esculentum IV, 10 AY756752 PSI36 3 Indonesia Lycopersicon esculentum IV, 10 AY756817 JT656 BDB NA Indonesia Musa sp. IV, 10 AY756811 JT657 BDB NA Indonesia Musa sp. IV, 10 AY756788 ACH0732 UW433 2 Australia Lycopersicon esculentum NA, 11 AY756743 1 CFBP, Collection Française de Bactéries Phytopathogènes, Angers, France; NCPPB, National Collection of Plant Pathogenic Bacteria, Harpenden, UK; ICMP, International Collection of Microorganisms from Plants, Auckland, New Zealand; UW, D. Cook and L. Sequeira, Department of Plant Pathology, University of Wisconsin-Madison, USA; GMI, M. Arlat and P. Barberis, CNRS-INRA, Auzeville, Castanet-Tolosan Cedex, France; MAFF, Ministry of Agriculture Forestry and Fisheries, National Institute of Agrobiological Resources, Japan; R, Institute of Arable Crops Research-Rothamsted, Harpenden, UK; JS, JT, JR, JQ, Laboratoire de Phytopathologie, CIRAD-FLHOR, 97448 Saint-Pierre, La Réunion, France. NA: not applicable. ND, not done. BDB, blood disease of banana. 2 from this work and Fegan and Prior (2004); 3 mutS gene partial sequence GenBank accession number. UW160
134
R282
1
Peru
Musa sp. plantain
Figures
Species Subspecies Biovars RFLP group
3 8 9 10 12 13 14
Clones Races
Ralstonia solanacearum Division Division Group Group I II III IV Asia America Africa Indonesia 4 5 2T 1 2 2T 1 2T 1 2 R B 15 21 29 30 1 2 24 26 19 11 17 23 31 32 3 4 25 27 20 16 22 33 5 6 28 34 1
4
5
1
2
3
Fig. 1. Schematic diagram resuming actual schemes and how they are related for classifying the intra-specific variability within the R. solanacearum species. Two additional genetic groups (III and IV) were unravelled by Taghavi et al. (1998) and Poussier et al. (2000 a, b). R: Ralstonia syzygii. B: blood disease of banana (Pseudomonas celebense). Modified from Gillings and Fahy, 1994.
"American" Bv 1 (7) "Antillean" Bv1 (5) & MLG 28
Phylotype I (Asia)
(6)
MLG 25 SFR/A
(4)
MLG 24 H/B
(3)
GMI 1000
(12-18)
0.01
Biovar 2/2T (1-2) Rs BDB
(8-11) Rs, P.syzygii
(19-23)
Phylotype III (Africa)
Phylotype II (America)
Phylotype IV (Indonesia)
Fig. 2. Phylogenetic tree based on partial endoglucanase gene sequence analysis which shows phylogenetic distance between phylotypes and relationship with sequevar in brackets. R. solanacearum strains pathogenic to tomato, in extenso to solanaceous, are symbolized with tomatoes. Strains with narrow host range are symbolized with banana or potato. The bar indicates 1 nucleotide change per 100 nucleotide positions. Adapted from Prior and Fegan (2004).
135
A 64 98
99
65
30 83
0.01
62
53 79
B
64 CIP 239
26 98
75
0.01
Fig. 3. Tree based on partial mutS gene sequences analysis showing phylogenetic distances between phylotypes. As reported from the ITS tree (Fegan and Prior, 2004), strain ACH732 did not fit with any of the four phylotypes (A). Phylotype II splitted in two major group with ecological divergence based on narrow or broad host range. Phylogenic distances between sequevar of the phylotype II are detailed in the sub-treeing B. Numbers on the branch of the phylogenic tree are bootstrap value when different of 100%. The bar indicates 1 nucleotide change per 100 nucleotide positions.
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