| As an important natural resource insect,Hermetia illucens has been widely used as raw feeding material,decomposition of kitchen waste and feces,synthesis of antibacterial peptide.The intestinal microorganisms of Hermitia illucens have always been the focus of research,but the report of biocontrol bacteria from the intestinal microorganisms of Hermitia illucens larva are relatively few.In this paper,we focused on the functional microorganisms with antibacterial activity isolated from the intestinal tract of Hermitia illucens larvae feeding on four kinds of feeds,namely bean residue,pig manure,sludge and chicken manure,explore the feasibility of using the intestines of Hermetia illucens to multiply beneficial microorganisms and whether their feces could produce antibacterial fertilizer.The bacterial strain Y4-39 was isolated from the intestines of Hermitia illucens larva feeding on sludge,strain Y4-39 could significantly inhibit the growth of hyphae of the Phytophthora capsici and Sclerotium rolfsii Sacc.Based on morphological and 16S r DNA sequence identification,Y4-39 was identified as Bacillus velezensis.The bacteriostatic activity of Phytophthora capsici and Sclerotium rolfsii Sacc were selected as screening indexes respectively.We optimized the medium formula and fermentation conditions of Y4-39.The optimal medium of Y4-39 to Phytophthora capsici was 8.0g·L-1 soluble starch,7.0g·L-1 yeast powder,4.0g·L-1 tryptone and Mg SO4 7.0g·L-1.Two different inorganic salts were selected,which may be because Y4-39 fermentation broth produced different substances with antibacterial activities against Phytophthora capsici and Sclerotium rolfsii Sacc,leading to different antibacterial effects against Phytophthora capsici and Sclerotium rolfsii Sacc.The optimal medium of Y4-39 to Sclerotium rolfsii Sacc was6.0g·L-1 soluble starch,6.0g·L-1 yeast powder,4.0g·L-1 tryptone and Na Cl 7.0g·L-1.The optimum fermentation condition was set as,temperature at 30?,initial p H as 7.0,initial inoculating amount as 0.7%,and rotating speed as 180 r·min-1.The inhibition rates of Phytophthora capsici and Sclerotium rolfsii Sacc were 68%and 62%respectively.The anti-microbe substrate of fermentation broth was sensitive to acidic conditions(p H3?6)and ultraviolet light.The genome of Y4-39 contains only one circular chromosome with 3,891,759 bp in length,encoding 3,713 protein genes and containing 9 complete r RNA gene clusters.Twelve candidate secondary metabolites synthesis gene clusters were identified from the genome,and among these gene clusters,seven products were identified as known AMPs,such as surfactin,macrolactin H,bacillaene,fengycin,difficidin,bacillibactin and bacilysin.A comparative genomic analysis was carried out on 295 Bacillus velezensis strains with whole-genome sequences.The results showed that Bacillus velezensis could be divided into two groups,strain Y4-39 in Group I,and PGPR(plant-growth-promoting rhizobacteria)FZB42 in Group II.Strain Y4-39 was clustered with Bacillus velezensis SCDB 291 and Bacillus velezensis SCGB1,which were respectively from soybean fermentation products.The mean nucleotide consistency(ANI)of Y4-39 against stain SCDB 291 and SCGB1 was99.65%and 99.63%,respectively.RT-PCR was used to detect the expression levels of key genes in each gene cluster at different fermentation stages.The results showed that the gene clusters for lanthipeptide,fengycin,difficidin,terpenoids had a relative high gene expression levels,which preliminarily indicated that they could produce a variety of antibacterial substances in the broth. |
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