| Rice blast is a disastrous fungal disease on rice,which poses an serious threat to global food security.At present,control of this disease mainly rely on rice resistant varieties and chemical fungicides.However,the breeding cycle of resistant varieties is long,the resistance to rice blast is easy to lose,and chemical fungicides have potential harms to the environment and food safety.Therefore,it is quite important to develop microbial resources with the ability of biological control of rice blast and regulation of rice growth.In this study,Bacillus subtilis JN005,which has a high inhibitory effect on Magnaporthe oryzae,was used to evaluate the ability to control rice blast and improve rice growth.The mechanism of control disease and growth promotion of the strain was analyzed by whole genome sequencing and related experiments.Finally,antagonistic active substances of the strain were studied.The main results are shown as follows:Rice leaves inoculated with B.subtilis JN005 strain resulted in increased activities of defense-related enzymes compared to the water and the M.oryzae-inoculated treatments.In vitro inoculated rice leaves with 1×10~7cfu/mL strain JN005 suspension compared to sterile water treatment exhibited lower disease incidence in the curative and preventive groups by 76%and 79%,respectively.The2-year field experiment showed that after spraying with 1×10~7cfu/mL bacterial suspension,efficacy rates on controlling rice blast on plants were 50%～60%at seedling and maturity stages,and the rice production yield increased by 41%and 50%in 2 years respectively than that used no the controlling rice blast fungicide.Compared with the control group,after 1×10~7cfu/mL bacterial suspension treatment,rice seeds had 16%germination energy,14%germination rate,15%germination index and 270%vigor index.The plant height,root length,stem circumference,aboveground fresh weight and underground fresh weight of the treated rice plants were increased by 46%,127%,54%,130%and 90%,respectively.In addition,the chlorophyll concentration of rice leaves and root activity index of rice seedlings were increased.The whole genome of B.subtilis JN005 was sequenced using Illumina Hiseq Ⅱ platform and PacBio RS Ⅲ platform,and uploaded to NCBI(accession number PRJNA717641).A ring chromosome of strain JN005 was acquired,with the length of4,167,853 bp and G+C content of 43.86%.No plasmids were found,but 4294 coding DNA sequences,85 tRNA genes,30 rRNA genes and 92 sRNA genes were predicted.Furthermore,42 tandem repeat sequences,4 genomic islands,2 prophages and 7CRISPR-Cas were contained.Genes related to colonization,growth promotion,response to oxidative stress,and synthetic secondary metabolites were found in strain JN005.The genome of strain JN005 contained a variety of extracellular enzyme genes such as Subtilisin E、Bacillopeptidase F and neutral protease.Genome analysis identified gene clusters encoding secondary metabolites synthesis of Bacillaene,Fengycin,Bacillibactin,Subtilosin A,Bacilysin,Surfactin and chalcone synthase.Compared with B.subtilis 168 and XF-1,the genome of strain JN005 had characteristic genes,such as arsenate reductase,tunicamycin resistance protein,nebramycin synthase and siderophore biosynthesis protein.B.subtilis JN005 secreted protease,cellulase,amylase,β-1,3-glucanase,siderophore and a small amount of indoleacetic acid.The strain was able to dissolve phosphorus and potassium,and form biofilm,but does not produce ACC deaminase and fix nitrogen.The volatile substances produced by strain JN005 had no antifungal effect on M.oryzae,but fermentation supernatant had activity against M.oryzae.The fermentation supernatant interfered melanin synthesis of M.oryzae,and destroyed the cell wall and cell membrane of M.oryzae,resulting in the leakage of alkaline phosphatase,soluble protein,soluble sugar and macromolecular substances.Both protein substances and lipopeptide substances extracted from B.subtilis JN005 had inhibitory effects on M.oryzae,but lipopeptide substances were significantly more effective than protein substances.Both compounds had thermal stability,as well as acid and alkali resistance.Both compounds kept antifungal activity even they were irradiated for 12 h under ultraviolet light or were saved at 4℃for 6 months,and they also had low sensitivity to papain,trypsin,pepsin,alkaline protease and protease K.The lipopeptide substances produced by strain JN005 had a low concentration of anionic surfactant and had the ability of expelling oil.Both protein substances and lipopeptide substances of strain JN005 caused hyphae of M.oryzae to distort and hindered conidia to germinate.Surfactin and Fengycin derivatives of strain JN005 were identified by the UPLC-QTOP-MS analysis.By analyzing protein extracts of strain JN005 through shotgun method quantitative proteomics technology,the serine protease,bacilysin,amylase and other substances were detected,mainly serine protease. |
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