Genetic Diversity And Phylogeny Of Antagonistic Bacteria Isolated From Tobacco Rhizosphere

A total of 6652 isolates were obtained from the 55 different tobacco rhizosphere soil samples of Guizhou province in China. And 267 bacterial strains demonstrating antagonistic activity against Phytophthora nicotianae, and 31 bacteria have strongly activity; 178 bacteria strains demonstrating antagonistic activity against Ralstinia solanacearum, and 26 bacteria have strongly activity.The genetic diversity of the antagonistic bacteria was examined by BOXAIR-PCR, 16S-RFLP, 16S rRNA sequence analysis methods.BOXAIR-PCR revealed that 267 isolates which antagonistic against Phytophthora nicotianae were separated to 35 clusters/genotypic groups at a level of 91% similarity. There is a high genotypic diversity. Group 5 and 6 are larger than any other, while groups 21 and 12 are consisted of only a single isolate, respectively. The 178 isolates which antagonistc against Ralstinia solanacearum were separted to 41 clusters/genotypic groups at a level of 90.5% similarity. Group 12 and 13 are larger than any other, while groups 19 and 20 are consisted of only a single isolate, respectively. The environmental factors caused its genetic diversity of antagonistic bacteria.The dendrogram based on 16S-RFLP showed that the 267 isolates were separated to 25 clusters/genotypic groups at the level of 88.5% similarity. Two large groups are group 5 and 6. It includes17 small groups and 6 groups which consisted of only a single isolate, respectively. 178 isolates were separated to 20 clusters/genotypic groups at the level of 89% similarity. This includes 5 large groups (group 4, 9, 12, 15 and 16), 6 small groups and 9 groups which consisted of only a single isolate. Therefore, the results indicate that antagonistic bacteria are highly diverse at a phylogenetic level.According to the results of BOXAIR-PCR and 16S-RFLP, representative isolates of each group were selected, its 16S rRNA gene were sequenced. Ninety-three isolates which against Phytophthora nicotianae were clustered into 17 different genera and 33 different species at least. Bacillus, Brevibacillus, Streptomyces and Pseudomonas were the dominant genera. 79 isolates which against Ralstinia solanacearum clustered into 12 different genera, and 36 different species at leaset. Bacillus, Brevibacillus, Streptomyces and Pseudomonas were the dominant genera. It is first report that Bacillus altitudinis, Delftia tsuruhatensis, Delftia lacustris, Streptomyces roseolus, Burkholderia arboris, Lysobacter capsici, Sinorhizobium adhaerens, Stenotrophomonas maltophilia, Advenella incenata, Kocuria palustris, Agrobacterium tumefaciens and Myroides odoratimimus have antagonistic activity against Phytophthora nicotianae. Furthermore, the majorities (75%) of the isolates assayed for antagonistic activity were Gram-positives bacteria, and it is first report that Bacillus altitudinis, Bacillus silvestris, Bacillus horikoshii, Bacillus mycoides, Bacillus pseudomycoides, Brevibacillus formosus, Streptomyces tanashiensis, Streptomyces achromogenesnbrc, Streptomyces polychromogenes, Kocuria palustris, Acinetobacter lwoffii, Ensifer adhaerens, Kocuria palustris and Delftia tsuruhatensis have antagonistic activity against Ralstinia solanacearum. Furthermore, the majority (135 isolates) of the isolates assayed for antagonistic activity were Gram-positives bacteria.