Study Of The Inhibition Mechanism Of Bacillus Vallismortis TA-1 To Fusarium Graminearum And Its Genome Sequencing Analysis

Biological pesticides have the advantages of safe-use,low toxicity and less side effects,and no pollution to the environment.It is an inevitable trend to develop biological pesticides.Bacillus has a strong stress resistance and antifungal activity.Bacillus-based biocontrol agents play an important role in biological pesticides.In this study,based on the previous results,B.vallismortis TA-1 and its crude extract of lipopeptide was used to deterrmine the toxicity against five pathogenic fungi.The antifungal activity of crude lipopeptide extract on F.graminearum was determined by mycelial growth rate method.Then the antifungal activity mechanism of B.vallismortis TA-1 to F.graminearum was explained by the change of malondialdehyde content in F.graminearum mycelium.High performance liquid chromatography was used to further separate and purify the lipopeptides of B.vallismortis TA-1,identification of crude lipopeptide composition and structure.Finally,complete genome sequencing of B.vallismortis TA-1 was performed to further explains the reason of its biological control efficacy.The results of the study were summarized as follows:1.The antifungal effect of B.vallismortis TA-1 against five pathogenic fungi was conducted by the inhibition zone method.The results indicated that the inhibition rates of mycelium growth of F.graminearum,F.oxysporum,Phytophthora nicotianae,Rhizoctonia solani and Botrytis cinerea were 72.8%,70.6%,64.5%,62.3%and 59.7%,respectively.The crude extract of B.vallismortis TA-1 lipopeptide was extracted and its inhibitory effect on five pathogenic fungi was also determined.It was proved that the crude lipopeptide extract was the main inhibitory substance.Based on the assessments,the inhibition rate of F.graminearum was the highest,so it was selected for further study.It was found that there was a positive correlation between inhibition rate and lipopeptide dose.When concentration of lipopeptide was 200 mg·L^-1,the mycelial inhibition rate was 70.74%.2.The conductivity method was used to determine the effect of crude extract of B.vallismortis TA-1 lipopeptide on the membrane permeability of F.graminearum.The results showed that the crude lipopeptide extract could destroy the cell membrane of F.graminearum and cause leak of small molecules and ions in the mycelium.The conductivity of F.oxysporum mycelia cells increased continuously within 0-9 h after treatment with crude lipopeptide extracts,which was higher than that of the control.At 9 h,the conductivity of the treatment group of 1.00 mg·mL^-1 was about 2.3 times than that of the control group.3.The POD,PAL activities and MDA content of mycelium were measured by UV spectrophotometry for 0²4 h.The results showed that treated after24 h,the crude lipopeptide concentration at 1.00 mg·mL was 1.66 times than that of 0.25 mg·mL^-1,which was 3.52 times that of the control group.The PAL activity gradually increased and reached the highest value within 1.5 h,and the lipopeptides of 1.00 mg·ml^-1 The PAL activity of the crude extract was 357.87 U/g·h,which was 1.23 times than that of the control group;then the viability decreased slightly.The change trend of MDA content was basically similar as that of PAL,but reached the maximum value of 1.53μmol·g^-1 FW at 6 h,then the viability decreased,but the content of each concentration treatment was higher than the control.The results indicated that the crude lipopeptide extract could induce increase activitiesof the defense enzymes POD and PAL and MDA content in the F.graminearum mycelium to resist adverse environment.4.Complete genome sequencing of B.vallismortis TA-1 was performed using Illumine second generation sequencing and Pac-Bio three generation sequencing technology.The whole genome size was 3,929,778 bp and the GC content was 46.50%.The genome contained 3968 CDS,of which 3954（99.75%）had predictive functionality and functional annotation and parsing.By ANI analysis,it was found that the homology between strain TA-1and B.vallismortis NBIF-001 reached 99.95%,and the relationship was the closest,so it was finally identified as B.vallismortis.It was predicted that there were 8 antibiotic synthetic gene clusters in the TA-1 genome,and the antibiotics were closely related to the antifungal activity of Bacillus,which provided a genic explanation for its antifungal activity.