Rapid Identification Of Bacillus Subtilis And Its Lipopeptides Application In Biocontrol Of Plant Disease

Bacillus subtilis has significant advantages such as growing fast, producing abundantmetabolites and high food safety, etc. It is widely used in biocontrol of the plant disease.Therefore, it is significant to isolate and identify more Bacillus subtilis which can beapplied in agriculture.The B. subtilis strain B3, 38, 49, 85 were isolated from soil of the plant root andidentified using biochemical, physiological methods previously in our laboratory. In thisarticle, these strains were identified using 16S rDNA sequence analysis methods. It showedthat 16S rDNA sequences of the isolates were highly homologous to the B. subtilis. B3, 38,49,85 showed 99.4%,99.5%,93.8% and 99.4% similarity to B. subtilis 168 respectively. The16S rDNA sequence of the B3 had been deposited in the GenBank database, under theaccession number EF492885. The phylogenetic tree was constructed using DNAstarsoftware basing on their 16S rDNA sequence. From the phylogenetic tree, B3, 38, 49, 85and B. subtilis 168 got together on close genetic relationship.An innovative method was developed for rapid sensitive detection and efficientstructural characterization of compounds of the bacteria by matrix-assisted laser desorptionionization-time of flight (MALDI-TOF) mass spectrometry by using whole microbial cellsand crude culture filtrates as targets in combination with surface tension measurements. Inthis article, the isolates B.subtilis strain B2, B3, 43, 49,55, 72,85,90 were also identified byMALDI-TOF-MS technique, and standard strains were used in this experiment, B. subtilis168, ATCC6633, ATCC13915, ATCC21332 were present by American Bacillus GeneticStock Center, and A1/3, b213, C1 which were presented by Dr.Joahim Vater fromTechnical University of Berlin. The results showed that each strain usually had three to fourbands, among these characteristics bands, the bands with m /z 379,493,618, 714,843 and969 were regular and stable which could represent the secondary metabolites of the B.subtilis.The lipopeptide play very important role in biocontrol of the plant diseases. In this test,surfactin, bacillomycinD and fengycin which were belong to cyclic lipopeptide antibioticfamily were used for controlling the cucumber downy mildew and tobacco mosaic virusdisease, these lipopeptide was isolated and purified previously in our laboratory. The resultsshowed that 20μg / mL bacillomycinD and 5μg / mL fengycin could influence the conidiagermination of the Pyricularia grisea,which could induce the conidia formation thespherical swelling, 10μg / mL surfactin could induce the spherical spores getting smallerand forming root-like protuberances, when Fusarium graminearum was treated with thesame concentration lipopeptide the result showed that inhibition formation of the conidiawas not obvious to the Pyricularia grisea. The surfactin was also used for controlling thetobacco mosaic virus (TMV). The best effect was obtained when the surfactin was sprayedonto the tobacco one day before inoculation TMV. When tobacco was treated with 10 mg /L and 60 mg / L surfactin, the inhibition rate of TMV lesion was 50% and 95.65%respectively.After 15 days, the control plants were killed, but the plants treated withsurfactin were still alive, only part of the leaves appearance withered symptoms. Thebiocontrol effection of surfactin against cucumber downy mildew was similar with Mancozeb.Therefore, the lipopeptide compounds can be used as medicine for biocontrol of plantdisease.