Molecular Mechanism Of Volatile Compounds From Bacillus Subtilis S-16 Inhibiting The Growth And Development Of Sclerotinia Sclerotiorum Based On Multi-omics Analyses

Sclerotinia of sunflower,caused by Sclerotinia sclerotiorum,is one of the main diseases in sunflower production,which is widely prevalent around the world and has brought huge economic losses to the sunflower industry.Due to the lack of resistant varieties,the current prevention and treatment of Sclerotinia sclerotiorum are mainly by agricultural measures and chemical methods S.sclerotiorum is sclerotinia that overwinters in the soil and can survive in the soil for a long time.Moreover,there are many host plants of S.sclerotiorum,so the effect of agricultural measures and chemical control is not ideal,and the application of a large number of chemical fungicides causes serious water and soil pollution.Therefore,new prevention and control measures need to be found.Research and practice at home and abroad showed that the use of microbial fungicides is an effective and environmentally friendly measure to control soil-borne crop diseases,and has been widely recognized by society.Bacillus subtilis is an important resource for the development of microbial fungicides because it can produce a variety of lipopeptide antibiotics and Volatile Organic Compounds（VOCs）,and can form resistant and heat-resistant buds.Lipopeptides have good antifungal activity,while VOCs have a good biological fumigation effect,which plays an important role in regulating microbial community structure and reducing harmful biomass in soil.We early from sunflower a strain isolated from rhizosphere soil of high antagonism activity of hay B.subtilis S-16 strains,the strain peptide antibiotics not only produce a variety of lipopeptide,still can produce a kind of inhibition of sclerotium formation of VOCs,the material also can inhibit the growth of sunflower S.sclerotiorum and mycelial.In this study,the biocontrol potential of B.subtilis S-16 was analyzed through Genome-wide data analysis.Biocontrol substances related to B.subtilis S-16 were analyzed and identified by lipopeptide antibiotics,VOCs,growth-promoting substances,and enzyme-producing ability analysis.The inhibitory effect of dimethylbenzothiazole in volatile compounds on sunflower fungus was studied,and the synthesis gene This was cloned and its function was verified.The biocontrol mechanism of B.subtilis S-16was studied by analyzing the transcriptome and proteome of dimethylbenzothiazole against S.sclerotiorum.The main results of this study are as follows:1.Analysis and identification of biocontrol related substances of Bacillus subtilis S-1623 compounds,including ketones,alkanes,esters,alcohols,and acids,were isolated from the ethyl acetate extract of S-16 fermentation broth by chromatography.The antifungal activity of crude extracts of S-16 metabolism against S.sclerotiorum was studied by plate confrontation test.The results showed that the crude extracts of S-16 secondary metabolism had higher antifungal activity against S.sclerotiorum.The volatiles of S-16 was extracted by solid-phase microextraction.The antibacterial activity test showed that the volatiles of S-16 had a strong antagonistic effect on pathogenic fungi.GC-MS analysis showed that there was 35 volatiles in the volatiles.The lipopeptides produced by S-16 were extracted by acid precipitation,and the extracts showed high antibacterial activity.HPLC analysis showed that there were at least two types of lipopeptides in S-16,surfactin and Fengycin.S-16 can promote the growth of tomatoes.Biochemical tests showed that the strain had nitrogen fixation ability,dissolved inorganic phosphorus,and could produce IAA,of which the yield of IAA was 1.23μg/m L.2.Complete genome sequence analysis of Bacillus subtilis S-16The complete genome sequencing results of B.subtilis S-16 showed that the strain had a Circos with a size of 4.2M and contained 9 gene clusters related to bacteriostasis,including:These bacteriostatic substances include polyketone compound synthase（PKS）antibiotics sactipeptide-head＿to＿tail,NRPS pathway antibiotics,NRPs-Transat PKS-otherk,terpenes and other compounds.3.Inhibition effect of VOCs dimethylbenzothiazole on S.sclerotiorumS.sclerotiorum was sensitive to dimethylbenzothiazole（2-MBTH）,and its inhibition rate increased with the increase of 2-MBTH concentration.The EC₅₀ of 2-MBTH against the mycelia was 0.87μL/L.2-MBTH had certain effect on sclerotium germination of sunflower.With the increase of fumigation concentration,the number and weight of sclerotia decreased significantly.Furthermore,the physical properties of the mycelia of sunflower were significantly changed after 2-MBTH treatment,and the mycelia morphology and internal structure of the mycelia were damaged to varying degrees by transmission electron microscopy and scanning electron microscopy.4.Cloning and functional verification of thiazole biosynthesis related gene This.The volatile dimethylbenzothiazole synthesis gene（This）was obtained by genome-wide sequence analysis.By homologous recombination,This gene was located and mutated to obtain the mutantδThis.Compared with the wild-type strain,the mutant significantly reduced the production amount and antibacterial activity of dimethylbenzothiazole.5.Transcriptome study of S.sclerotiorum treated with 2-MBTHTranscriptome sequencing was used to analyze 2-MBTH-treated endophytes.The results showed that dimethylbenzothiazole treatment inhibited the expression of genes involved in the Oxidation-Reduction Reactions reaction,indicating that the Oxidation-Reduction Reactions reaction was closely related to sclerotia formation.KEGG pathway analysis of differentially expressed genes revealed that differentially expressed genes were significantly enriched in ribosomal pathway,ribosomal biogenesis pathway in eukaryotes,n-Glycan biosynthesis,glycine,serine,and threonine metabolism,and other pathways.6.Proteome study of s.sclerotinia treated with 2-MBTHOn the basis of transcriptomics,proteome sequencing was performed.Transcriptomic and proteome association analysis results showed that there were 15differentially expressed genes and differentially expressed proteins in the analysis group TMTT3 vs.TMTCK3＿CHULI3vs CK3,and the known pathway significantly enriched was aflatoxin biosynthesis pathway,while the rest were not annotated and needed further analysis and verification.9 differentially expressed genes and differentially expressed proteins were down-regulated and significantly enriched in ribosomal and metabolic pathways.