Identification And Lipopeptide Analysis Of Biocontrol Bacillus Spp. Isolated From Tibet

The genus Bacillus is comprised of Gram-positive, spore-forming, and rod-shaped organisms with an aerobic or facultatively anaerobic metabolism, are ubiquitous in many environments. And their endospores are highly resistant to hostile physical and chemical conditions, such as high temperature, low temperature, arid and UV. The Bacillus spp., which is one of the most important plant growth promoting rhizobacteria(PGPR), can also produce various antibacterial substances or elicit induced systemic resistance of plant. And the lipopeptide compounds are the major antibiotics produced by Bacillus spp., play an very important role in the plant disease controlling, include three categories:iturin, surfactin and fengycin. In this article, the MALDI-TOF-MS was also used to analysis the kinds of lipopeptides produced by the Bacillus isolates.Qinghai-Tibet Plateau is the highest plateau in the world, and the average altitude is 4000 meters above sea level. This isolated region has an extremely hostile environment, such as low temperature, hypoxia and strong ultraviolet exposure, and it is likely that may produce abundant Bacillus spp. specific to this region. In this work,1388 isolates were obtained from thirty-five soil samples from rhizospheres of plants were collected from Nyingchi, Lhasa, Shannan and Xigaze in Tibet. Xanthomonas oryzae pv. oryzae and Sclerotinia sclerotinorium were chosen as the indicator bacteria and fungi, respectively, for antagonistic activity screening of bacteria, and a total of 311 isolates were found to be antagonistic (diameter of the inhibition zone≥5mm) to at least one of the two pathogens. 150 isolates were obtained after the genomic fingerprints screening based on results of the rep-PCR (repetitive extragenic palindromic PCR)The 150 Bacillus isolates were identified according to the classical classification system described by Gordon et al. (1973), and the isolates were clustered into seven branches. Then 31 Bacillus isolates were chosen for phylogenetic analysis. The phylogenetic trees were constructed using fatty acid methyl ester (FAME),16S rDNA partial sequences, gyrB gene partial sequences, and repetitive extragenic palindromic PCR (rep-PCR). The 31 isolates were identified to be most closely related to Bacillus pumilus (11 isolates), Bacillus cereus (7 isolates), Bacillus thuringiensis (3 isolates), Bacillus amyloliquefaciens (3 isolates), Bacillus atrophaeus (3 isolates), Bacillus axarquiensis (2 isolates), Bacillus subtilis (1 isolate) and Bacillus licheniformis (1 isolate).Then 11 representative Bacillus isolates were chosen for lipopeptides compounds analysis by the MALDI-TOF-MS detection. The results showed that strain LLCG23 can produce surfactin, bacillomycins D and fengycin; strain NMTD81 can produce surfactin, iturin and fengycin; strains YBWC18 and LNXM78 can produce surfactin and fengycin; strains NMTD54 and GBSW11 can produce bacillomycins L and fengycin; strain LSSC22 can produce iturin and fengycin; strains GBSW2 and GBSW19 can produce surfactin; strain DJFZ40 can produce bacillomycins D only; strain NMSW23 can not produce surfactin, iturin family(Mycosubtilins, iturin, bacillomycins D and bacillomycins L) or fengycin. Two strains YBWC43 and RJGP16 that have high antagonistic activity towards Sclerotinia sclerotinorium were used for lipopeptides compounds analysis. The result showed that strain YBWC43 produced bacillomycins D and fengycin, strain RJGP16 produced surfactin and fengycin. And biocontrol efficacy of strains YBWC43 and RJGP16 to rape selerotiniose on the detached leaves were 100% and 50.24%, respectively.