Isolation, Screening And Identification Of Bacillus Sp.Antagonistic To Meloidogyne Spp.

Root-knot nematodes(Meloidogyne spp.), which are a kind of a plant soil-borne pathogens nematodes, can cause serious economic losses each year worldwide. Among all the root-knot nematode species, Meloidogy incognita are the most harmful pathogens for agricultural industry, having widest host range in our country. At present, chemical nematicides the effective method of antagonisticing to root knot nematode. Whereas, its high production costs and heavy dangers to creature prompt us to seek a safer method. Then, based its features of security pollution-free, biological control is attracted more and more attention of academics throughout the world. Hainan Island is located in tropical northern margin with tropical monsoon climate, which is extremely special. Therefore, the unique diverse and complex ecosystems create rich microbial resources. Bacillus species, which are the common groups of soil bacteria, can produce kinds of active substances and metabolites. Because of these characteristics and its wide existence, Bacillus-like species become the focus of biocontrol development.In this study, Bacillus-like bacteria were isolated from two places, including Yongxing Island and Yinggeling Nature Reserve. And then Bacillus strains, which were potential new resources isolated from these two soil samples, were screened anti-nematode activity. After screening, the three strong activity strains were preliminary identified. The main results were as follows:In order to isolate the particular resources of Bacillus-like species in Hainan, the dilution soil suspensions with heat water bath were spread for the selective isolation of Bacillus-like species. After removing of the redundant strains,645isolates of spore-forming bacteria were separated, there were187strains from Yongxing Island,458strains were collected from Yinggeling Nature Reserve. Their phylogeny, and diversity were characterized by16S rRNA gene sequence analysis and16S rDNA PCR-RFLP.187isolates which isolating from the soil of Yongxing Island, were distributed in five genera, namely Bacillus, Lysinibacillus, Paenibacillus, Brevibacillus and Sporosarcina;458isolates from Yinggeling Nature Reserve soil were consist of Bacillus, Lysinibacillus, Paenibacillus, Ammoniphilus, Cohnella, Brevibacillus and Rummeliibacillus. Of these two groups, Bacillus sp. was both the dominant one. Among all the isolates, we received50strains of potential new resources. The results showed that there were affluent resources of culturable Bacillus from two soil samples.50Bacillus-like strains of potential new resources was screened the resistance to Meloidogy incognita by24-well plate fluid model. When fermentation supernatant was diluted10times,9strains were obtained with better activity, which the corrected mortality were all above50%. After two screening, DB14828, DB14586, DB14881were affirmed that have the highest activity, the corrected mortality were76%,73%,65%.Three active strains were identified, together with the recent mode strains, by the characteristics of training and morphological, physiological and biochemical, phylogenetic analysis based on16S rDNA sequences. The strain DB14881was centaily evaluated new specie of Paenibacillus sp., naming as Paenibacillns nematocida sp. nov.; The DNA-DNA hybridization value of DB14828and its nearest edge type strain Cohnella boryungensis BR-29T (98.1%) was59.2%, so DB14828was centaily identified new specie of Cohnella sp., naming as Cohnella hainanensis sp. nov.; and the DNA-DNA hybridization value of DB14586and its nearest edge type strain mmoniphilus oxalivorans RAOx-FST、Ammoniphilus oxalaticus RAOxlTwas54.6%,57.5%, DB14586was identified as new specie of Ammoniphilus sp., naming as Ammoniphilus yinggelingensis sp. nov..