Genetic Diverstiy And Pathogenicity Variation Of Ralstonia Solanacearum

As a diverse species complex, Ralstonia solanacearum has developed an extremely broad host range throughout the world, including more than 450 host species representing 54 plant families. Biovar typing and race assessment are methods commonly used for assessing the diversity of R. solanacearum 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 R. solanacearum. Fegan and Prior (2005) proposed a new hierarchical classification scheme to distinguish the genetic diversity within the R. solanacearum species complex. The phylotyping scheme is highly discriminatory, flexible, additive, and should allow better prediction of the properties of strains.Although considerable research has been conducted on bacterial wilt disease in China, less work has been done on the genetic diversity of R. solanacearum species complex. The aim of this study was to use the phylotyping scheme to determine the genetic diversity of R. solanacearum strains from China. Based on the pathogenic properties and phylogenetic relationships among the R. solanacearum strains, suppression subtractive hybridization (SSH) and two-dimensional isoelectric focussing/sodium dodecyl-sulphate polyacrylamide gel electrophoresis (2-D IEF/SDS-PAGE) were then used to isolate host-specific gene. Such understanding allowed identification of biological properties, evolutionary relationships and pathogenicity variation of infra-subspecific groups of strains and result in improving breeding strategies, developing targeted diagnostic tests and studying pathogenesis of R. solanacearum.1. Genetic Diversity of Ralstonia solanacearum Strains from ChinaA survey of bacterial wilt in China collected 286 strains of Ralstonia solanacearum from 17 plant species in 13 Chinese provinces. A phylotype-specific multiplex-PCR showed that 198 isolates belonged to phylotype I (bv3, 4 and 5) and 88 to phylotype II (bv2 and bv1). A phylogenetic analysis examined the partial sequence of the egl and hrpB gene of all strains and the genetic diversity of 95 representatives were reported, demonstrating that Chinese strains are partitioned into phylotype I (Asia) and II (America). Phylotype I strains (historically typed bv3, 4 and 5), had considerable phylogenetic diversity, including 10 different sequevars: seven previously described sequevars 12 to 18 and three new sequevars: 34, 44 and 48.2. Pathogenicity Variation of Ralstonia solanacearum StrainsPo82, a potato strain from Mexico, was resolved into phylotype II sequevar 4, in a group with phylotype II/4NPB and banana Moko disease-causing strains. It was identified as Moko strains'sequevar 4 (MLG25) according to Mmx-PCR. AFLP fingerprinting showed that Po82 was more closely related to NPB than to moko disease-causing strains. However, SDS-PAGE of total cellular protein extracted from Po82 indicated that its protein pattern was more closely related to Moko disease-causing strains than to NPB. Additionally, pathogenicity test revealed that Po82 possess pathogenic traits of both NPB and Moko strains thereby proving that it was a pathogenicity variation strain.3. Differences between Ralstonia solanacearum strains Revealed by Suppression Subtractive HybridizationSSH approach was used to investigate the pathogenic determinants of the R. solanacearum strain. The genome of the R. solanacearum tester strain was subtracted from the genome of the R. solanacearum driver strain, resulting in the identification of subtracted fragments. The majority of the fragment sequences were homologous to Rhs proteins, transmembrane proteins, transcription regulator proteins, signal peptide proteins and hypothetical proteins. Sequence analysis indicated that many fragments in the mulberry strain were disrupted by insertion sequences (IS). A PCR diagnostic test for detection of Ralstonia solanacearum race 5-biovar 5 strains was developed.4. Mutant construction of hrpB gene and Analysis of Type III-Dependent EffectorsA comparative proteome analysis was initiated to investigate the Type III-dependent effectors of Ralstonia solanacearum strain M7 (race5). The mutant of hrpB and popP1 were constructed and subsequently used for pathegenicity and proteome analysis. 2-D IEF/SDS-PAGE allowed the separation of Type III-Dependent Effectors.In summury, an in-deep study of genetic diversity of R. solanacearum strains from China was determined by modern phylotype-sequevar phylogenetic analysis method, Po82, a potato strain was first reported could be pathogenic to banana. Construction of the subtracted DNA library revealed that IS elements may play a role in the evolution of mulberry Ralstonia solanacearum strains. These new findings may provide important clues when studying pathogenicity determinants in the R. solanacearum strains.