Laboratoire de Phytopathologie, CIRAD-FLHOR, 97448 Saint-Pierre Cedex, La Réunion, France1
Laboratoire de Biologie Moléculaire des Relations Plantes-Microorganismes, INRA-CNRS, BP27, 31326 Castanet-Tolosan Cedex, France2
Unité de Biométrie et dIntelligence Artificielle, INRA, BP27, 31326 Castanet-Tolosan Cedex, France3
Author for correspondence: Stéphane Poussier. Tel: +33 262 35 76 30. Fax: +33 262 35 76 41. e-mail: poussier{at}cirad.fr
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ABSTRACT |
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Keywords: bacterial wilt, PCR-RFLP, hrp, AFLP, 16S rRNA
Abbreviations: AFLP, amplified fragment length polymorphism; BDB, blood disease bacterium; HCA, hierarchical cluster analysis; UPGMA, unweighted pair group method with arithmetic averages
The GenBank accession numbers for the sequences determined in this work are AF207891AF207897.
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INTRODUCTION |
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However, recent PCR-RFLP analysis of the hrp gene region (Poussier et al., 1999 ), including many African strains, which were rarely included in previous analyses of the genetic diversity of R. solanacearum, was not totally consistent with the above classification scheme since an African biovar 1 strains group belonged to the Asiaticum division instead of the Americanum division. Therefore, to clarify the relationships between these biovar 1 strains originating from the Southern part of Africa and other R. solanacearum isolates, three different approaches were compared and are presented in this paper. Firstly, 59 additional strains of R. solanacearum, including biovar N2 and 5 strains and new African strains, were analysed using PCR-RFLP. Two close relatives of R. solanacearum, Pseudomonas syzygii (causal agent of Sumatra disease of cloves) and the blood disease bacterium (BDB, causal agent of blood disease of bananas) (Eden-Green, 1994
; Seal et al., 1993
; Taghavi et al., 1996
) were also analysed, permitting the specificity of our PCR-RFLP test and the phylogenetic relationships between these three bacteria to be assessed. Secondly, we have used the very powerful DNA fingerprinting technique AFLP (Vos et al., 1995
), which allows very fine whole genome analysis. AFLP methodology has already been used to study the diversity of race 3 isolates of R. solanacearum (Van der Wolf et al., 1998
) but has never been used to analyse a worldwide collection of R. solanacearum strains. Finally, we have determined nearly complete 16S rRNA gene sequences for seven (five African, one American and one Japanese) biovar 1 strains of R. solanacearum, and compared these with 19 previously sequenced R. solanacearum 16S rRNA gene sequences (Taghavi et al., 1996
).
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METHODS |
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PCR-RFLP analysis.
DNA amplification conditions and digestion with eight restriction endonucleases of the five PCR products were as previously described (Poussier et al., 1999 ). Each PCR-RFLP was duplicated to assure its reproducibility. Two different Hierarchical Cluster Analysis (HCA) methods were used to analyse the PCR-RFLP band data collected. Using STATLAB version 2.0 (SLP Statistiques, Monterey, CA, USA), clustering was based on the Euclidean distance between strains (Ward, 1963
). The truncation level in the resulting dendrogram was thus determined to be that which provided the smallest number of clusters for which the variance within clusters was significantly (P=0·05) different from the variance between clusters. Using the PHYLIP software package (Felsenstein, 1995
), a distance matrix was firstly constructed with the Nei (1973)
genetic distance of the GENDIST program. A dendrogram was then constructed from genetic distance values by using the unweighted pair group method with arithmetic averages (UPGMA) (Sneath & Sokal, 1973
) contained in the NEIGHBOR program. Finally, the strength of the tree topology was assessed by the bootstrap method (Felsenstein, 1985
) of the SEQBOOT program.
AFLP analysis.
Ninety-six R. solanacearum strains, one BDB strain and one Ralstonia pickettii strain were analysed using AFLP as described by Janssen et al. (1996) and Vos et al. (1995)
with slight modifications. We used MspI and SacI to digest DNA instead of EcoRI and MseI.
Genomic DNA (200 ng per sample) was digested for 2 h at 37 °C in 50 µl (final volume) containing 5 U MspI, 5 U SacI (Amersham Pharmacia Biotech), 0·125 µl BSA (10 µg µl-1) and 2·5 µl 10xOne Phor All buffer (Amersham). Next, 50 pmol double-stranded MspI-adapter (5'-GACGATGAGTCCTGAA-3', 5'-CGTTCAGGACTCATC-3') (50 pmol µl-1), 5 pmol double-stranded SacI-adapter (5'-CTCGTAGACTGCGTACAAGCT-3', 5'-TGTACGCAGTCTAC-3') (5 pmol µl-1) (Genset), 1 µl ATP (10 mM), 1 U T4 DNA ligase (5 U µl-1) (Appligene) and 2·5 µl 10xOne Phor All buffer were added to the digested DNA and the ligation reactions were performed for 3 h at 20 °C. Digested (D) and ligated (L) DNA were diluted (D) eightfold and the resulting DLD DNA was then stored at 4 °C until used.
Selective amplifications were done with two primers (MspI-primer and SacI-primer) (Genset) complementary to the adapter sequences, and the MspI and SacI restriction sites respectively, with additional selective nucleotides at their 3' ends (cytosine for the SacI-primer and cytosine plus guanine for the MspI-primer). The SacI-primer was labelled with -33P [10 µCi (370 kBq) per DNA amplification; Nen Life Science Products] and T4 polynucleotide kinase (Gibco-BRL).
DNA amplifications were carried out in a 50 µl reaction mixture. DLD DNA (5 µl) was added to 45 µl mixture containing 5 µl 10x PCR buffer (Gibco-BRL), 1·5 µl labelled SacI-primer (50 µg µl-1), 2 µl unlabelled MspI-primer (30 µgµl-1), 1·5 µl MgCl2 (50 mM), 8 µl of each dNTP (1·25 mM) and 0·6 µl Taq polymerase (5 U µl-1; Gibco-BRL). Amplifications were performed with a thermocycler (Mastercycler gradient Eppendorf) by using the following protocol: 30 cycles consisting of denaturation at 94 °C for 30 s, annealing at 56 °C for 1 min and extension at 72 °C for 1 min.
Amplified fragments were separated by electrophoresis on 5% polyacrylamide gels and fingerprint patterns were visualized as described by Vos et al. (1995) . The reproducibility of AFLP was assessed by comparing the DNA fingerprinting obtained from duplicate assays of 14 strains. Duplicate DNA fingerprints were produced using two aliquots from two different DNA amplifications that were run in different gels. AFLP data analysis was performed as described above for PCR-RFLP.
16S rRNA sequencing.
Seven biovar 1 strains of R. solanacearum were used: CFBP 1036, CFBP 712, NCPPB 1018, CFBP 2146, CFBP 734, NCPPB 342 and MAFF 211266. 16S rRNA genes were amplified using the PCR as detailed by Taghavi et al. (1996) with slight modifications. PCR amplifications were carried out in a thermocycler (GeneAmp PCR system 9600; Perkin-Elmer) in a 50 µl (total volume) reaction mixture containing 10x buffer (200 mM Tris/HCl, 500 mM KCl, pH 8·4; Gibco-BRL), 1·5 mM MgCl2 (Gibco-BRL) 200 µM of each dNTP (Boehringer Mannheim), 0·25 µM primer 27f, 0·25 µM primer 1525r (Genosys Biotechnologies) (Taghavi et al., 1996
), 1 U Taq polymerase (Gibco-BRL) and 100 ng template DNA. The following PCR profile was used: initial denaturation at 96 °C for 2 min; 30 cycles consisting of 94 °C for 30 s, 60 °C for 1 min and 72 °C for 1 min; and final extension at 72 °C for 10 min.
PCR products were electrophoresed using 1% agarose gels at 5 V cm-1 for 1 h and visualized with UV light after ethidium bromide staining. Amplification products were purified from the agarose gel slice by using the QIAquick purification kit PCR (Qiagen) according to the manufacturers instructions.
PCR product sequences were determined by Cambridge Bioscience, Cambridge, UK. The GenBank accession numbers of the seven resulting 16S rRNA gene sequences are shown in Table 3. They were aligned using the gap proceed program of the Genetics Computer Group software package (Genetics Computer Group, 1999
) with the published 16S rRNA gene sequences of R. solanacearum, BDB and P. syzygii isolates studied by Taghavi et al. (1996)
. Data analysis was performed using the DNADIST (Jukes & Cantor, 1969
) and NEIGHBOR (UPGMA method) programs of the PHYLIP software package, and using the STATLAB software.
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RESULTS |
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Depending on the strain, DNA patterns contained 5080 different DNA bands (Fig. 2). Comparison of AFLP fingerprints revealed that 95% of the fragments were polymorphic. Three fragments appeared to be specific to R. solanacearum species. Moreover, five AFLP fragments were common to all R. solanacearum strains: three of them were shared with the BDB and R. pickettii strains and two of them were shared with only the BDB strains. One fragment was specific to the BDB isolate. Many of the DNA fragments were useful in distinguishing subgroups within R. solanacearum species. For instance, one fragment was found only in all of the biovar 1, 2 and N2 strains (except for the biovar N2 strain MAFF 301558). Eight fragments were produced by all of the biovar 3, 4 and 5 strains (with one, two or three exceptions depending on the fragment). No DNA band appeared to be specific to all biovar 1 strains; however one fragment was common to all R. solanacearum strains other than biovar 1 strains. African and Japanese biovar 1 isolates were characterized by three and two fragments, respectively. Six particular DNA bands were specific to biovar 2 and N2 isolates, except for strains JT510 and MAFF 301558. Strain JT510 (biovar 2) was unusual since it shared less than 60% of the AFLP fragments with other biovar 2 isolates. One fragment was common to all biovar N2 strains, except for strains JT510, JQ1056 and MAFF 301558. Seventeen AFLP fragments could discriminate between the two similar biovars 2 and N2. One fragment was specific for biovar 3 strains (two exceptions) and one only for African biovar 3 strains. No fragments discriminating biovar 4 or 5 isolates were obtained, but these isolates were distinguished from biovar 3 strains by 14 DNA bands.
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A phylogenetic tree was produced using the PHYLIP software package by comparing 1431 nucleotide positions, omitting all of the ambiguous nucleotides, and revealed two divisions, each of which was split into two subdivisions (Fig. 4). This tree was not completely consistent with that obtained by Taghavi et al. (1996)
since the subdivision 2b appeared to be more closely related to division 1 than to subdivision 2a. However, this result could be explained by the branch point separating division 1 and subdivision 2b, which was not as well supported (bootstrap value only 43%) as the branch point between subdivisions 2a and 2b (bootstrap value 59%) in the study of Taghavi et al. (1996)
. Moreover, the latter was supported by the dendrogram generated by the STATLAB software (result not shown). The four African biovar 1 strains originating from Angola, Madagascar, Reunion Island and Zimbabwe were included in a new subdivision, which was designated subdivision 2c. The branch point between subdivisions 2a and 2c was stable (bootstrap value 79%). The three other biovar 1 strains were distributed either into subdivision 2a (strains CFBP 712 and CFBP 1036), containing R. solanacearum biovar 1, 2 and N2 strains, or into division 1 (strain MAFF 211266), containing R. solanacearum biovars 3, 4 and 5 and a biovar 2 isolate.
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DISCUSSION |
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Clustering of the PCR-RFLP and AFLP profiles
Five PCR products were shown to be specific to R. solanacearum, P. syzygii and the BDB strains. This result is consistent with many other studies showing the close relationships between these three bacterial taxa (Eden-Green, 1994 ; Eden-Green & Sastraatmadja, 1990
; Roberts et al., 1990
; Seal et al., 1993
; Taghavi et al., 1996
). Nevertheless, P. syzygii as well as the BDB strains were distinguished from R. solanacearum strains since specific PCR-RFLP profiles, including specific restriction patterns, were obtained for these bacteria. To our knowledge, this is the first report of a simple and rapid method for discriminating R. solanacearum strains from P. syzygii and the BDB strains. New PCR-RFLP profiles were also obtained with new strains of R. solanacearum, particularly biovar N2 and 5 isolates. Biovar N2 strains generated five different subclusters demonstrating and confirming that biovar N2 is a genetically heterogeneous group of strains (Cook & Sequeira, 1994
; Gillings & Fahy, 1994
). Notably, the very similar biovars 2 and N2 (Hayward et al., 1990
) showed a polymorphism within the RS80-RS81 amplified fragment spanning the hrpB regulatory gene (Genin et al., 1992
). Since other groups of R. solanacearum strains are distinguishable by polymorphism in this gene (Poussier et al., 1999
), the hrpB gene may be considered as a target for further phylogeny purposes, and for relating pathogenicity gene function with genetic variability. The PCR-RFLP and AFLP profiles were distributed into clusters which agreed well with biovar and geographical origin classifications, confirming the results obtained in the previous investigation (Poussier et al., 1999
). In addition, this clustering was conserved using two different software packages which use different genetic distance methods, underlining its robustness. Biovar 1 and biovar 2 strains displayed the highest and the lowest diversity, respectively. Biovar 3 strains showed lower genetic diversity than biovar 1 strains. AFLP allowed very fine discrimination, close to the strain level, and reliable determination of genetic relationships between strains. This result is consistent with many previous investigations showing the usefulness of the AFLP procedure in strain identification, and for epidemiology and phylogeny purposes (Aarts et al., 1998
; Arias et al., 1997
; Blears et al., 1998
; Clerc et al., 1998
; Folkertsma et al., 1996
; Hermans et al., 1995
; Janssen et al., 1996
, 1997
; Keim et al., 1997
; Lin et al., 1996
; Restrepo et al., 1999
). The BDB and R. pickettii strains, which showed specific AFLP profiles, could be distinguished from R. solanacearum strains. However, the overall level of polymorphism between these bacterial taxa was low, confirming their close relationship.
AFLP reveals a high level of polymorphism
The AFLP analysis revealed great variability within R. solanacearum since 60 different AFLP fingerprints were observed for the 96 strains. Thus, with 60 AFLP fingerprints (95% of fragments were polymorphic), AFLP has a higher resolution level for intraspecific differentiation of R. solanacearum strains than PCR-RFLP (20 profiles for 178 strains tested) and RFLP (46 profiles for 164 strains tested) (Cook et al., 1989 , 1991
; Cook & Sequeira, 1994
). Several DNA fragments were common to all R. solanacearum species. Other DNA fragments could distinguish the divisions defined by Cook et al. (1989)
within R. solanacearum species or differentiate strains according to their biovar or geographical origin and are therefore useful for the development of diagnostic tools and epidemiological studies. Although AFLP clustering was approximately the same as PCR-RFLP clustering, the AFLP procedure was more efficient for assessing intraspecific diversity since it permitted a clearer separation between biovar 2 and N2 and also between biovars 3, 4 and 5. AFLP and PCR-RFLP confirmed that biovar 2 strains are the least genetically diverse of all biovars (Cook et al., 1989
; Cook & Sequeira, 1994
; Smith et al., 1995
; Van der Wolf et al., 1998
); nevertheless AFLP, in contrast to PCR-RFLP, demonstrated genetic diversity among biovar 2 strains. In contrast, PCR-RFLP permitted strains isolated from musaceous plants to be grouped into two main clusters whereas AFLP grouped them into only one cluster.
New or unexpected groups
Trees resulting from PCR-RFLP analysis of the hrp gene region, AFLP and 16S rRNA sequencing showed the separation of R. solanacearum into two major groups, confirming and extending the conclusions of previous investigations using DNADNA hybridization (Palleroni & Doudoroff, 1971 ), and more recently of RFLP analyses (Cook et al., 1989
, 1991
; Cook & Sequeira, 1994
), of PCR amplification with tRNA consensus (Seal et al., 1992
), of 16S rRNA sequencing (Li et al., 1993
; Taghavi et al., 1996
), and of sequencing of the 16S23S rRNA gene spacer region, the endoglucanase gene and the polygalacturonase gene (Fegan et al., 1998
). The first division, named Americanum by Cook et al. (1989)
, includes biovars 1, 2 and N2; and the second division named Asiaticum contains biovars 3, 4 and 5.
However, our analyses revealed that there are numerous exceptions. Indeed, our extended PCR-RFLP analysis showed that an African biovar 1 strains group was associated with the Asiaticum division rather than the Americanum division, and so supported the conclusion of the first PCR-RFLP analysis (Poussier et al., 1999 ). The conclusions of AFLP and 16S rRNA sequencing were different to those of PCR-RFLP and appeared to be in complete agreement with the classification scheme proposed by Cook et al. (1989)
. Indeed, biovar 1 strains originating from the Southern part of Africa (Angola, Madagascar, Reunion Island, Zimbabwe) appeared to be more closely related to American strains even though they constituted a clearly separable group from American biovar 1 strains, and these were thus included in subdivision 2c, a new subdivision compared to the work of Taghavi et al. (1996)
. The differentiation between American and African biovar 1 strains is more remarkable within the hrp gene region (PCR-RFLP analysis). To further clarify whether the discrimination between these two biovar 1 populations is clearer using regions of the genome involved in pathogenicity, other genes such as those encoding endoglucanase or polygalacturonase should be sequenced. The most probable explanation for the distinction between African and American biovar 1 isolates is separate evolution of the two populations due to geographical isolation. The two populations may have diverged under different natural selection pressures. The observation that other African isolates (three coming from Burkina Faso and one from Kenya) fell into clusters containing only American members may result from their introduction from the Americas through commercial exchanges.
The Japanese strains, MAFF 211266 and MAFF 211267, confirmed to be biovar 1 in our laboratory, were considered to be atypical. Both PCR-RFLP analysis and 16S rRNA sequence analysis indicated that these Japanese isolates appeared to be closely related to strains belonging to the Asiaticum division, confirming the findings of Tsuchiya & Horita (1998) . The peculiarity of these strains, which underlines the level of heterogeneity existing in the R. solanacearum species, may result from horizontal genetic transfers from biovar 3 or 4 members, which predominate in Japan. Moreover, another Japanese strain, MAFF 301558, was also unusual since PCR-RFLP and AFLP analyses showed that this biovar N2 strain was distantly related to all other biovar N2 strains and closely related to biovars 3, 4 and 5. This is not the first report of atypical isolates since Li & Hayward (1994)
and Taghavi et al. (1996)
also mentioned one atypical (ACH0732) biovar 2 strain. Furthermore, the two strains JT510 and JQ1056, which were identified as biovar 2 on several independent examinations, were unusual in AFLP grouping. It is possible that limited genomic rearrangements or genetic exchanges which do not modify the biovar typing occur. These considerations reinforce the need for a classification system that is based upon polymorphism between genes encoding pathogenicity functions. This would in turn permit more meaningful comparisons with specific phenotypic characteristics such as host specificity and survival in natural settings to be made.
Clearly the species R. solanacearum comprises two divisions, which may represent subspecies as suggested by Li et al. (1993) . However, the Americanum and Asiaticum designations of these divisions proposed by Cook et al. (1989)
in relation to the presumed geographical origin of strains could be reconsidered since our analyses reveal an African biovar 1 subdivision, which may have its own centre of genetic diversity, and thus likely evolutionary origin, in Africa.
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ACKNOWLEDGEMENTS |
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Received 2 December 1999;
revised 1 March 2000;
accepted 27 March 2000.