* Corresponding author: Tel: +262 35 76 34; Fax: +262 35 76 41; e-mail: luisetti@cirad.fr.
1 Laboratoire de Phytopathologie, Centre de coopération Internationale en Recherche Agronomique pour le Développement, 97448 Saint-Pierre Cedex, La Réunion, France.
2 Station de Pathologie Végétale, Institut National de la Recherche Agronomique, BP94, 84143 Montfavet Cedex, France.
3 Cooperative Research Centre for Tropical Plant Protection, Department of Microbiology and Parasitology, The University of Queensland, Brisbane Qld 4072, Australia.
running title: Genetic diversity within Ralstonia solanacearum
S u m m a r y
We determined partial hrpB and endoglucanase genes sequences for 30 strains o f Ralstonia solanacearum and one strain of the blood disease bacterium (BDB), a close relative o f Ralstonia solanacearum. Sequence comparisons showed high levels of variability within these two regions o f the genome involved in pathogenicity. Phylogenetic analysis based upon sequence comparisons o f these two regions revealed three major clusters comprising all Ralstonia solanacearum isolates, the BDB strain constituted a phylogenetically distinct entity. Cluster 1 and cluster 2 corresponded to the previously defined divisions 1 and 2 of Ralstonia solanacearum. Moreover, two subclusters could be identified within cluster 2. The last cluster, designated cluster 3 in this study, included biovar 1 and N2 strains originating from Africa. This recently described group o f strains was confirmed to be clearly different from the other strains suggesting a separate evolution from those o f both divisions 1 and 2.
Key words: Ralstonia solanacearum - bacterial wilt - hrpB gene sequences - endoglucanase gene sequences - genetic diversity - phylogeny - African strains.
In tro d u ctio n
Bacteria] wilt caused by Ralstonia solanacearum is one o f the most important bacterial plant diseases worldwide (Hayward, 1991). R. solanacearum is a heterogeneous species traditionally classified into five races on the basis of differences in host range (Buddenhagen et al., 1962; He et al., 1983; Pegg and Moffett, 1971) and six biovars on the basis o f biochemical properties (Hayward, 1964, 1991, 1994). Studies of DNA-DNA homology of R. solanacearum
strains have revealed that the relatedness between isolates of this species is often less than the 70% threshold level commonly expected within a species (Palleroni and Doudoroff, 1971; Roberts et al., 1990). Restriction fragment length polymorphism (RFLP) analysis (Cook et al., 1989; Cook and Sequeira, 1994) has shown that R. solanacearum is divided into two divisions: division 1 grouping strains belonging to biovars 3, 4 and 5 and division 2 comprising strains belonging to biovars 1, 2 and N2. These two divisions distinguish strains mainly originating in the Old and the New World respectively. Several other investigations have confirmed this dichotomy within
R. solanacearum-. PCR amplification with tRNA consensus primers (Seal et al., 1992), PCR-RFLP o f a polygalacturonase gene fragment (Gillings et al., 1993) and partial and complete sequencing o f the 16S rRNA gene (Li et al., 1993; Seal et al., 1993; Taghavi et al., 1996; Poussier et al., 2000). Taghavi et al. (1996) also revealed the existence of a subdivision within division 2 comprising isolates of R. solanacearum from Indonesia including the closely related organisms the blood disease bacterium (BDB) and Pseudomonas syzygii. Further sequencing of the 16S-23S rRNA gene intergenic spacer region, the polygalacturonase gene and the endoglucanase gene (Fegan et al., 1998) has supported the existence of the two divisions and the existence o f the group o f strains originating in Indonesia.
To accurately assess the phylogenetic variation amongst closely related bacterial populations, markers with greater resolving power than the commonly used rRNA genes are required. For instance, transposable elements, avirulence genes (Nelson et al., 1994; Adhikari et al., 1995), hrp genes (Leite et al., 1994) or polygalacturonase genes (Gillings et al., 1993) were successfully investigated. A recent PCR-RFLP analysis o f the hrp gene region, which is involved in hypersensitive reaction and pathogenicity of most plant pathogenic bacteria, showed th at certain African biovar 1 strains did not cluster with other biovar 1 isolates as expected, these isolates were more closely related to division 1 (biovars 3, 4 and 5) isolates rather than to division 2 isolates (Poussier et al., 1999). An extended PCR-RFLP analysis o f the hrp gene region complemented by amplified fragment length polymorphism (AFLP) and sequencing o f the 16S rRNA gene (Poussier et al., 2000) has provided further evidence for the existence o f this group o f strains. However, the relationship o f this group of strains to strains o f divisions 1 and 2 was not clear since the strains either fell close to R. solanacearum strains o f division 1 or division 2 depending on the method employed to measure diversity (Poussier et al., 2000). Sequencing o f the endoglucanase gene has already provided phylogenetically valuable information (Fegan et al., 1998) and the hrpB gene, the regulatory gene of the hrp gene region (Genin et al., 1992) has already revealed great variability (Poussier et al., 1999; Poussier et al., 2000). The sequence analysis o f the hrpB and endoglucanase genes in this study were undertaken to clarify the relationships between strains belonging to the “African” group of strains and other strains o f
R. solanacearum.
M a te ria ls a n d M eth o d s
B acterial stra in s: All R. solanacearum and blood disease bacterium (BDB) isolates used in this study are listed in Table 1 and were cultured as described previously (Hayward, 1964; Poussier et al., 1999).
DNA isolation: Genomic DNA was isolated and purified following three different methods: the hexadecyltrimethylammonium bromide method (Ausubel et al., 1991), the method of Boucher et al. (1987) or the method o f Marmur (1961).
S e q u e n c in g :
hrpB gene. PCR amplification o f the hrpB gene was performed with the forward primer RShrpBf
(5’-TGCCATGCTGGGAAACATCT-3’) and the reverse primer RShrpBr (5 ’-
Strain8 O ther designation6 Biovar or species G eographic origin Host Cluster/ Subclusterc hrpB gene sequence, G enBank accession no. Endoglucanase gene sequence, G enB ank accession no. M AFF 211266 JT690 1/4 Japan Lycopersicon esculentum 1 AF295603 AF295250 GMI 1000 JS753 3 G uyana Lycopersicon esculentum 1 AF295604 A F295 2 51 JT 523 3 Reunion Island Solanum tuberosum 1 AF295605 AF295252 NCPPB 3190 JS941 4 M alaysia Lycopersicon esculentum 1 AF295606 AF295253 UW 151 JS834 4 Australia Zingiber officinale 1 AF295607 AF295254 R 292 JT661 5 China Morus alba 1 AF295608 AF295255 UW 162 JT648 1 Peru Musa sp. cv. plantain 2 a AF295609 AF295256 UW 9 JT644 1 Costa Rica Heliconia sp. 2a AF295610 AF295257 JT 516 2 Reunion Island Solanum tuberosum 2 a A F 295611 AF295258 CFBP 3858 JS907 2 The Netherlands Solanum tuberosum 2 a AF295612 AF295259 UW 477 JT654 N2 Peru Solanum tuberosum 2a AF295613 AF295260 NCPPB 3987 JT677 N 2 Brazil Solanum tuberosum 2a AF295614 AF295261 CFBP 2047d JR659 1 United-States Lycopersicon esculentum 2b AF295615 AF295262 ICMP 7963 JS967 1 K enya Solanum tuberosum 2b AF295616 AF295263 CFBP 2972 JS734 1 Martinique Solanum tuberosum 2b AF295617 AF295264 CFBP 2957 JS717 1 Martinique Lycopersicon esculentum 2b AF295618 AF295265 CFBP 2958 JS728 1 Guadeloupe Lycopersicon esculentum 2b AF295619 AF295266 CFBP 712 JS770 1 Burkina Faso Solanum melongena 2b AF295620 AF295267 CFBP 715 JS779 1 Burkina Faso Lycopersicon esculentum 2b AF295621 AF295268 UW 469 JT652 1 Brazil Solanum tuberosum 2b AF295622 AF295269 CFBP 3059 JS904 1 Burkina Faso Solanum melongena 3 AF295623 AF295270 NCPPB 1018 JS950 1 Angola Solanum tuberosum 3 AF295624 AF295271 JT 525 1 Reunion Island Pelargonium asperum 3 AF295625 AF295272 JT 528 1 Reunion Island Solanum tuberosum 3 A F295626 AF295273 CFBP 734 JS767 1 M ad ag ascar Solanum tuberosum 3 AF295627 AF295274 NCPPB 283 JS946 1 Zim babwe Solanum pandura/orne 3 AF295628 AF295275 NCPPB 332 JS949 1 Zim babwe Solanum tuberosum 3 AF295629 AF295276 NCPPB 505 JS951 1 Zim babwe Symphytum sp. 3 AF295630 A F295277 NCPPB 342 JS952 1 Zim babwe Nicotiana tabacum 3 A F295631 AF295278 J 25 N2 K enya Solanum tuberosum 3 AF295632 AF295279 R 230 JT657 BDB Indonesia Musa sp. AF295633 AF295280 ‘ Abbreviations: CFBP, Collection Française de Bactéries Phytopathogènes, Angers, France; NCPPB, National Collection o f Plant Pathogenic B acteria, Harpenden, UK; ICMP, International Collection o f Microorganisms from Plants, Auckland, New Zealand; UW , D. Cook and L. Sequeira, Department o f Plant Pathology, University o f Wisconsin-Madison, USA; GM I, M. Arlat and P. Barberis, CNRS-INRA, Auzeville, Castanet-Tolosan Cedex, France; MAFF, Ministry o f Agriculture Forestry and Fisheries, National Institute o f Agrobiological Resources, Japan; R, Institute o f Arable Crops Research-R othamsted, Harpenden, UK. b Designation o f strains o f the laboratoire de phytopathologie, CIR AD-F LHOR, 97448 Saint-Pierre, La Réunion, France. # C lusters and subclusters as defined in Figures 1 and 2.
d T ype strain o f Ralstonia solanacearum.
Table 2. Number o f nucleotides specific to the BDB strain and clusters or subclusters o f
Ralstonia solanacearum isolates identified within hrpB and endoglucanase genes sequences. Clusters and subclusters as per Table
1 and Figures 1 and 2
Number of unique nucleotide positions for the genes indicated hrpB endoglucanase C lu ster 1 4 13 C luster 2 7 9 subcluster 2a 1 2 subcluster 2b 9 2 C lu ster 3 3 7 BDB 2 0 2 2
r
the basis o f the nucleotide sequence o f the hrp genes cluster o f the strain GMI 1000 o f R. solanacearum (accession no.: Z14056; EMBL/GenBank/DDBJ databases). Primers were selected with the aid of the Oligo 5.0 software package (National Biosciences, 1996) and synthesized by Genosys Biotechnologies, Cambridge, United Kingdom. The PCR mixture (50 ul, total volume) contained 0.7 U of Taq and Pwo DNA polymerases used with buffer 3 (Expand Long Template PCR System; Boehringer Mannheim, Meylan, France), 100 pM o f each dNTP (Boehringer Mannheim), 0.25 pM o f each primer and 50 ng o f template DNA. PCR amplifications were carried out in a GeneAmp PCR system 9600 thermocycler (Perkin-Elmer Corporation, Norwalk, USA) programmed for an initial dénaturation step of 95 °C for 5 min, followed by 10 cycles of95°C for 30 s, 64°C for 30 s, 68°C for 2 min. The final 20 cycles were the same as the first 10 except that an additional 20 s was added to the elongation step for each new cycle, and a final extension step at 68°C for 7 min.
PCR products were electrophoresed onto 1% agarose gels at 5V/cm and visualized with UV light after ethidium bromide staining. Amplification products were purified from agarose gel slice by using the QIAquick purification kit PCR (Qiagen S. A., Courtaboeuf, France) according to the manufacturer’s instructions. The sequences of the PCR products were determined using BigDye™ terminator chemistry by Cambridge Bioscience, Cambridge, United Kingdom. All hrpB gene sequences have been deposited in the GenBank data library under the accession numbers shown in Table 1.
Endoglucanase gene. A 750 bp region of the endoglucanase gene was amplified using the primer
pair Endo-F (5’-ATGCATGCCGCTGGTCGCCGC-3’) and Endo-R ( 5 ’-
GCGTTGCCCGGCACGAACACC-3’). The reaction mixture (100 pi, total volume) contained PCR buffer (67 mM Tris-HCi, pH 8.8; 16.6 mM (N H ^ S O ^ 0.45% (vol/vol) Triton X-100; 200 p g o f gelatin per ml), 1.5 mM MgCl2, 200 pM of each dNTP; 0.25 pM of each primer; 100 ng DNA or 2 pi o f a turbid bacterial suspension as template and 1.1U o f Tth Plus DNA polymerase (Biotech International Ltd., Perth, Australia). PCR was performed on a MJ Research PTC 100 thermocycler (MJ Research, Waltham, MA, USA) by using the following protocol: initial dénaturation at 96 °C for 5 min, followed by 30 cycles of 95°C for 1 min, 70°C for 1 min, 72°C for 2 min, with a final extension step of 72°C for 10 min. PCR products were electrophoresed onto 2% agarose gels at 5V/cm and visualized with UV light after ethidium bromide staining.
PCR products were purified using the Promega Wizard® PCR Preps DNA Purification System according to the manufacturer’s instructions, then sequenced using BigDye™ term inator chemistry (Applied Biosystems, Foster City, USA) according to the manufacturer’s instructions. Primers used for sequencing were Endo-F and Endo-R. Sequencing products were purified as recommended by the manufacturer and sequences determined at the Australian Genome Research Facility, The University o f Queensland, St. Lucia, Australia. All endoglucanase gene sequences have been deposited in the GenBank data library under the accession numbers shown in Table 1. Sequence data analysis: The sequences of the hrpB gene and the endoglucanase gene were analysed using the PHYLIP (Felsenstein, 1995) software package. Sequences were aligned with the aid o f the CLUSTAL W software package (Thompson et al., 1994). Evolutionary distances between sequences were computed by using the algorithm o f Jukes and Cantor (1969) o f the DNADIST program o f the PHYLIP package. Phylogenetic trees were constructed from genetic distance values by using the neighbor-joining method (Saitou and Nei, 1987) o f the NEIGHBOR program o f the PHYLIP package. Finally, the strength of tree topologies was tested by 100 bootstrap resamplings o f the data.
R e su lts
ItrpB gene sequences
The hrpB gene sequences o f 30 isolates o f R. solanacearum representing all biovars and one isolate of the BDB, a closely related bacterium, were determined and compared to the published sequence o f reference strain GMI 1000. A phylogenetic tree (Fig. 1) was generated by comparing 1049 nucleotides, representing approximately 75% of the whole hrpB gene sequence, omitting all ambiguous nucleotides. This tree revealed four phylogenetically distinct groups. High bootstrap values indicated that these groups were well supported and the tree was robust. All R. solanacearum isolates o f biovar 3, 4 and 5 formed a monophyletic cluster designated cluster 1. All R. solanacearum strains o f biovar 1 from the Americas, three biovar 1 strains from Africa (ICMP 7963, CFBP 712, CFBP 715), arfd all biovar 2 and N2 strains, with the exception o f
Cluster 1
^ MAFF 211266 JT 523 NCPPB 3190 , CFBP 734 / NCPPB 505 , NCPPB 283 NCPPB342 I NCPPB 33 \ JT 525 ' J 25 JT 528Cluster 3
CFB P712 UW 469 \ CFBP 2957 CFBP 715 CFBP 2972 CFBP 2958 ICMP 7963 / / ’•..CFBP 2 0 4 7 T t \ ... "" /\ S u b clu ster 2b / /
\ / V / R 230 0.01Fig. 1. Neighbor-joining tree show ing the phylogenetic relationships o f the BDB strain and clusters or subclusters of
Ralstonia solanacearum strains based on partial hrpB gene sequence comparisons. The numbers at the branch points are the percentages o f bootstrap replicates in which the clusters or subclusters were found.
isolate J 25, grouped together in a monophyletic cluster designated as cluster 2. Cluster 2 is further separable into two subclusters; one subcluster, 2a, was comprised o f biovar 2 and N2 strains isolated from potato and biovar 1 strains isolated from Musaceous hosts; the second subcluster, 2b, consisted o f biovar 1 strains not isolated from Musaceous plants. Other biovar 1 isolates o f R. solanacearum mainly originating from Southern Africa and a biovar N2 isolate from Kenya were gathered into another group, which has been termed cluster 3. The BDB strain fell outside o f the 3 major clusters.
Based on the genetic distance matrix, strains included in cluster 1 were almost identical, showing greater than 97.1% sequence similarity, whereas strains of cluster 2 were the most diverse in some cases sharing less than 94% sequence similarity. Strains belonging to cluster 3 showed greater than 96.8% sequence similarity. Clusters 1 and 3 shared an average sequence homology o f 94.3%, clusters 1 and 2 shared 89.2% similarity, and clusters 2 and 3 shared 89.5% similarity.
HrpB gene sequences displayed a high degree of polymorphism with approximately 20% of the nucleotide positions o f the hrpB sequences (206 o f 1049 nucleotide positions) exhibiting variation. Many nucleotide positions were useful in differentiating between the clusters and subclusters (Table 2). For instance, 4, 7 and 3 nucleotide positions were specific to clusters 1, 2 and 3, respectively (Table 2). The sequence o f the BDB strain had 20 unique nucleotide positions. Strains o f cluster 1 and the BDB strain shared one nucleotide position whereas 16 nucleotide positions were shared by cluster 2 strains and the BDB strain suggesting a close link between cluster 2 strains and the BDB (results not shown). Biovar 1 strains of sub-cluster 2a, originating from French West Indies, Burkina Faso and South America, shared 9 unique nucleotide positions. E n d o g l u c a n a s e g e n e s e q u e n c e s
The endoglucanase gene sequences were completed for the same strains for which the hrpB gene sequences were determined. The phylogenetic tree (Fig. 2) was obtained by comparing 694 nucleotides, representing approximately 45% o f the complete endoglucanase gene sequence, omitting all ambiguous nucleotides. The sequences belonging to the strains MAFF 211266, GMI 1000, JT 523, NCPPB 3190, UW 151 and R 292 were six bp shorter than those o f all other strains since they have a 6-base deletion at the beginning of the area sequenced. The tree was very similar to the hrpB gene sequence-based tree with the same isolates belonging to the same clusters and sub-clusters as observed in the hrpB gene tree. This clustering was also well supported by high bootstrap values.
The genetic distance matrix showed that isolates of cluster 1 shared the greatest sequence similarity (98.5%) and isolates of cluster 2 shared the least similarity (95%). Strains belonging to cluster 3 shared 96.8% sequence similarity or greater. Cluster 3 shared similar sequence similarities with both clusters 1 and 2 (91.6% sequence similarity with cluster 1 and 91.4% sequence similarity with cluster 2). Cluster 1 and cluster 2 shared only 88.5 % sequence similarity.
As for the hrpB gene sequences, endoglucanase gene sequencing revealed many polymorphisms with 22% o f the nucleotide positions in the endoglucanase gene sequence (154 o f 694 nucleotide positions) being variable. Many o f the polymorphic nucleotides were specific for clusters and subclusters seen in the tree (Table 2). Cluster 1, 2 and 3 were characterized by 13, 9 and 7 unique nucleotide positions, respectively and 22 nucleotide positions were unique for the BDB strain (Table 2). In contrast to hrpB-based results, the BDB shared the same number (7) o f nucleotide positions in common with clusters 1 and 2 (results not shown). Furthermore, only two nucleotide positions were unique to all strains o f subcluster 2b (Table 2).
D isc u ss io n
Partial sequences of the hrpB and endoglucanase genes were generated for one isolate o f the BDB and for 30 isolates o f R. solanacearum, including the type strain CFBP 2047, selected to take account of the known diversity o f the organism including host range, geographic distribution and phenotype.
Phylogenetic analysis o f both the endoglucanase and hrpB gene sequences showed that the R. solanacearum isolates examined grouped into three major clusters (clusters 1-2-3; Fig. 1 and 2), and the BDB isolate formed a single isolate cluster. Cluster 1 contains all isolates of biovars 3, 4 and 5 and is equivalent to division 1 as defined by Cook et al. (1989); cluster 2 contains isolates o f biovars 1, 2 and N2 from Africa, the Antilles, USA and Central and South America, and is equivalent to division 2 as defined by Cook et al. (1989); cluster 3 contains isolates of biovars 1