Studies On Genetic Diversity Of Plant Pathogenic Coryneform Bacteria And Ralstonia Solanacearum

Plant pathogenic coryneform bacteria and Ralstonia solanacearum are important plant pathogenic bacteria which constitute serious threats to crop production in many provinces of China. In order to study the classification and identification, phylogenesis, correlations between genetic type and phenotypic characteristic, geographic and host origins, the genetic diversity of plant pathogenic coryneform bacteria and R. solanacearum has been analyzed by using PCR-based genomic fingerprinting methods combined with biological programs.Twenty-four Clavibacter fangii strains collected from six cities of Jiangsu Province and twenty-one Curtobacterium flaccumfaciens pv. beticola strains collected from five cities of Inner Mongolia were analyzed by genotypic typing methods including BOX-PCR, ERIC-PCR, and ITS profiling (PCR ribotyping). The results revealed conspicuous heterogeneity among analyzed strains of Cl. fangii, and Cur. flaccumfaciens pv. beticola, and that geographic regions were important determinants of genetic diversity, but did not reveal clear and direct relationship between gene type and their geographical distribution in China, and suggested that ERIC- and BOX-PCR were highly discriminatory techniques for detecting genetic variation among strains of and identification of strains as well as classification studies.RAPD analysis was used for the taxonomy of plant pathogenic coryneform bacteria,especially for the classification of two new pathogens (Cur. flaccumfaciens pv. basellae pv. nov. and Cur. flaccumfaciens pv. beticola pv. nov.). 20 random primers were screened from 50 ones to detect polymorphism among the total strains used. 80.4% were polymorphic bands among the 225 ones produced. The results of pairwise similarity and UPGMA cluster analysis suggested that the two new pathovars of sugar beet (Beta vulgaris var. saccharifera) and malabar spinach (Basella rubra) were genetically close related with Cur. flacumfaciens, and the minimal similarity coefficient is 0.6511. According to the RAPD analysis and previous research, some newly made taxonomic changes of the plant pathogenic coryneform bacteria were discussed.Samples of diseased plants from 14 different hosts were collected in 15 provinces in China and the resulting unique collection comprising 319 R. solanacearum isolates was assessed for their phenotypic and genotypic diversity together with 30 other strains from 19 other countries. Of the 319 Chinese strains, 20 belonged to biovar 2,122 to biovar 3,162 to biovar 4, 3 to biovar 5 and 12 to biovar 3-1. The genomic DNA was extracted from all 319 strains and used as a template for PCR. ARDRA patterns of these 319 strains were identical. Restriction fragment length polymorphism (RFLP) analyses of fliC gene amplicons obtained with two primer systems gave two pattern types, A724-1/A400-1 and for all biovar 2/race 3 strains A724-2/A400-2. Thus fliC RFLP (AluI) provided an easy mean to distinguish biovar2/race 3 strains from other R. solanacearum strains. The genetic diversity was unraveled by BOX-PCR fingerprints to test for correlations with biovar, host plant, geographic origin. The genetic diversity assessed by BOX-PCR revealed a high diversity of Chinese R. solanacearum strains. The genetic diversity of R. solanacearum strains from China showed a stronger correlation with biovar and geographic distance than with host plant.Recently, hrpB-regulated genes homologous to the avrBs3 effector family of phytopathogenic Xanthomonas were discovered in R. solanacearum. Internal repeats predispose the gene to recombinational change which in Xanthomonas could result in modulation of the host specificity. In a diverse collection of 349 R. solanacearum strains, variants of the gene differing in the number and nucleotide sequence of the repeats could be detected in 309 strains of biovars 3,4 and 5, but not in any of the biovar 1 or 2 strains. Very similar strains, as evidenced by genomic fingerprints, could have different variants. A statistically significant association between the originating host plant of the strains and the size of the repeat region was found.