Screening And Potential Of Biocontrol Of Biocontrol Bacteria Brevibacillus Formosus

As an important resource of biocontrol microorganisms,bacteria are widely distributed in different habitats such as soil,air and ocean.In this study,the soil samples were collected from Tongbai Mountain Scenic Area in Nanyang,Henan Province and Boshan,Zibo City,Shandong Province.The antagonistic strains were screened by the confrontation culture method,and screening the biocontrol strain with Fusarium oxysporum f.sp.momordicae as the target bacteria,and studing the control effect on Fusarium wilt of Bitter gourd,and determining the colonization ability of biocontrol strain in Bitter gourd,and optimizing the fermentation conditions of biocontrol strain,and exploring the active products generated by biocontrol bacteria,and exploring the disease prevention mechanism of biocontrol strain by analysing of genome sequencing,and evaluating the biocontrol potential of the strain against Fusarium wilt of Bitter gourd.The results are as follows:1.The screening results showed that 281 strains were isolated from different soil samples,and a biocontrol strain BF19 against Fusarium wilt of Bitter gourd was screened out.The strain BF19 was identified as Brevibacillus formosus based on clarifying its morphological,physiological and biochemical characteristics and 16 S r RNA gene sequence.The strain BF19 could promote the growth of Bitter gourd in potted test,and its control effect against Fusarium wilt of Bitter gourd in pot and field was 66.67% and 49.90%,respectively.2 The strain BF19 was labeled by antibiotic labeling method,and its colonization ability was measured by the double-layer filter paper method in the dish.The results showed that the colonization density of strain BF19 in Bitter gourd root tip was 1.11×103 cfu·g-1.In pot experiment,the strain BF19 could colonize stably on the roots,stems and leaves of Bitter gourd,and maintaining the colonization density above 103 cfu·g-1 after 28 d of inoculation.3.The fermentation conditions of strain BF19 were optimized by single factor analysis and orthogonal test.The results showed that the optimal medium components of strain BF19 were peptone 3%(g/V),glycerol 2%(g/V),dipotassium hydrogen phosphate 0.05%(g/V)and magnesium sulfate heptahydrate 0.2%(g/V).The optimal culture conditions were 1% inoculum,50 m L medium in 250 m L flask,p H 7.0,rotation speed of 220 r/min,and fermentation time of 48 h.4.The crude extract of strain BF19 were extracted by low temperature ethanol precipitation method with 80% saturation,and the inhibitory rate of crude extract against F.oxysporum f.sp.Momordicae was 58.33%.The crude extract maintains significantly antagonistic activity at p H 3-7 and temperature between 20-60°C,and isn’t sensitive to ultraviolet rays,and high temperature and alkaline conditions will reduce the antagonistic activity of fermentation broth.The results of ultra-high-performance liquid chromatography-tandem mass spectrometry(LC-MS/MS)showed that there were Macrobrevin and Petrobactin in the crude extract of strain BF19.5.The second-generation Illumina NovaSeq and the third-generation Oxford Nanopore technology were used to complete the whole genome sequencing of the strain BF19.The results showed that the total genome length of strain BF19 was 6426658 bp,GC content was 47.23%,including 44 r RNAs,127 t RNAs and 102 nc RNAs,and 6017 genes were predicted for coding genes in the Whole genome.The coding genes were annotated in the functional databases of Nr,egg NOG,KEGG,GO,Swiss-Prot and CAZY,and it was speculated that the strain BF19 could synthesize chitinase,β-1,3-glucanase and cellulose synthase.Through functional sequence alignment and structural domain analysis of B.subtilis,it was found that the BF19 genome contains 58 colonization-related genes,17 plant growth-related genes and 15 genes related to induction of resistance.The secondary metabolic gene clusters of Petrobactin,Tyrocidine,Gramicidin and Macrobrevin were found in the Whole genome of strain BF19.