Phylogenetie Diversity Analysis Of Bacteria Associated With Radopholus Similis In Different Habitats

Radopholus similis is one of important plant pathogenic nematodes, which induced disease commonly known as root rot disease, blackhead disease or decline disease of banana. It is classified as a quarantine pest of China, while it has the great potential threat of tropical fruit crops and ornamental plants, such as banana and flamingo. Radopholus similis has an extensive host range and wide geographical distribution, almost throughout most of the world banana production area, especially tropical and subtropical regions. Radopholus similis is a migration of parasitic nematode, living inside the root tissues, and transferring outside the roots under the bad environmental condition. Radopholus similis can be classified two physiological races, i.e. Radopholus similis and Radopholus citorphilu. The former can infect banana only, and the latter can infect banana and citrus. Radopholus similis distributes in producing banana areas around the world, but Radopholus citorphilu only is reported in the United States. Through the host identification, the samples from Sanya, Haikou and Guangzhou habitats were identified as Radopholus similis.The multiple interactions are existed between Radopholus similis and bacteria. Those bacteria, living on polypide surface or internal tissues of nematode, are called associated bacteria. The study on diversity of associated bacteria can understand the effects of bacteria on Radopholus similis, and then explain the interaction mechanism of microorganisms-Radopholus similis-hosts.In this paper, the phylogenetic diversity analysis was applicated to study associated bacteria of Radopholus similis. With the diversity factor contrast, the differences of associated bacteria on Radopholus similis from three regionsl were compared, through the establishment of16S rRNA gene library, ARDRA analysis, construction of rarefation curve using DOTUR software, blast alignment of the library sequence, phylogenetic tree construction using MEGA software.With the ARDRA analysis, the sample libraries contained16OUT,15OUT and15OUT from Sanya, Haikou and Guangzhou, respectively. With the sequencing and DOTUR software analysising, the sample libraries contained14OTU,12OUT and13OTU from Sanya, Haikou and Guangzhou, respectively. The obtained OUT number had few difference between two methods. The rarefation curve richness and diversity parameters from two sets of data also had few difference. The results indicated that the cloning sequence screened with ARDRA could also represent the majority of bacterial diversity in the library.16S rDNA of three constructed libraries belonged to Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria of Proteobacteria. The samples from Sanya contained10genera, with5genera of Betaproteobacteria and sequence number accounting45%for the library,4genera of Alphaproteobacteria and sequence number accounting50%for the library, and1genus of Gammaproteobacteria and sequence number accounting5%for the library. Alphaproteobacteria was the dominant microflora and Rhizobium was the dominant bacteria:the samples from Haikou contained8genera, with5genera of Betaproteobacteria and sequence number accounting69%for the library,2genera of Alphaproteobacteria and sequence number accounting20%for the library, and1genus of Gammaproteobacteria and sequence number accounting11%for the library; the samples from Guangzhou contained5genera, with1genus of Betaproteobacteria and sequence number accounting90%for the library,3genera of Alphaproteobacteria and sequence number accounting5%for the library, and1genus of Gammaproteobacteria and sequence number accounting5%for the library. Betaproteobacteria and Burkholderia of the samples from Haikou and Guangzhou were the dominant bacteria, respectively.The similarity of sequence from Sanya and the database sequence were more than97%. the similarity was95%between sequence2-4b from Haikou and16S rDNA sequence of Martelella. the similarities were95%.95%and96%. beteween sequence3-4-1d, sequence3-4-4f and sequence3-12b from Guangzhou and16S rDNA sequence of Escherichia coli, Shigella flexneri and Sinorhizobium, respectively.The diversity degree of associated bacteria on Radopholus similis from three regions was low, there was no significant difference from each other. However, the diversity and evenness of associated bacteria from Sanya was the highest, declaring conducive symbiosis of Radopholus similis and bacteria in the hot and humid environment.The diversity analysis of associated bacteria with Radopholus similis from different habitats indicated that the system of Radopholus similis and associated bacteria was opened. Radopholus similis could get own favorable bacteria, such as Phytopathogen ralstonia, from the environment, in order to improve its ability to adapt to survive and invade the hosts.