| In traditional agricultural,due to over-reliance on,chemical pesticides for plant disease control,cause the resistance of plant pathogens become increasingly prominent.Meanwhile,residual pesticide contaminated soil and crops,and caused more negative effects on human health and the environment.However,the biocontrol agents conform to the needs of green sustainable development of Agriculture,because of non-toxic,environmentally friendly,safety and high efficiency,exploiting and exploring of high-quality bacterial resources is great significance in plant disease protection.In this study,the preserved beneficial bacteria in the laboratory were used,and six pathogenic fungi including Botrytis cinerea and Fusarium oxysporum were regarded as target pathogens.The strain19573-3 was found to have broad-spectrum antibacterial properties by dual culture and mycelial growth rate.For clarifying the taxonomic status of strain 19573-3,Biolog GEN III Omnilog Microbial ID system,phylogenetic analysis based on 16S r RNA and gyr A gene sequence,and analysis of ANI value were adopted,the strain 19573-3 was identified as Bacillus velezensis.Detection of potential antimicrobial peptides in strain 19573-3 by using specific primers,showed that iturin A,Fengycin and Bacilysin could be tested.Based on selective identification culture experiment and UPLC-MS,that strain 19573-3 has the ability to produce protease,cellulase and siderophore.In addition,it can also produce salicylic acid,cytokinins(ip and ip R)and growth hormone and other bio-promoting substances.To further mine the genetic information and its genes function of B.velezensis 19573-3,the genome was sequenced by Pacific Biosciences(Pac Bio)single-molecule real-time sequencing technology(SMRT).The whole genome of B.velezensis 19573-3,possesses a single circular chromosome of 3,990,203 bp with a 46.56%G+C content,including 86 t RNA,27 r RNA,and 8 s RNA.The bacterial genome contained 4146 genes that occupied 90.03%of the genome.And strain 19573-3was accurately determined as a member of the B.velezensis,using the phylogenomic analysis of core genes.Thirteen biosynthetic gene clusters(BGCs)encoding secondary metabolites with biocontrol functions were predicted,including kijanimicin,bacilysin,difficidin,bacillaene and so on through anti SMASH.In order to analyze the antibacterial mechanism of B.velezensis 19573-3 and clarify the components of the antibacterial substance,a crude extract of B.velezensis 19573-3 with antibacterial activity was obtained through the combination of ethyl acetate extraction and rotary evaporation.The components of the crude extract were analyzed by HPLC,LC-QTOF-MS and it was preliminarily determined that the bacteriostatic components of the crude extract of B.velezensis 19573-3 were mainly Fengycin,C15 iturin A[M+H~+]or C14 Bacillomycin F[M+H~+],C16 iturin A[M+H~+]or C15Bacillomycin F[M+H~+]and C14 Surfactin[M+H~+],C15 Surfactin[M+H~+],C14 Surfactin[M+Na~+],C15 Surfactin[M+Na~+],which were consistent with the results of anti SMASH.With pot experiment,the disease prevention and growth promotion effect of B.velezensis 19573-3on tomato was verified.Compared with the non-inoculated control treatment(CK),the tomato plants treated with fermentation broth of B.velezensis 19573-3,that 31.41%increase in the plant height by,23.32%in stem thickness,83.35%in fresh weight,and 59.84%in dry weight.The diameter of the Botrytis cinerea spots on the tomato plant leaves after the fermentation broth treatment was significantly smaller than that of the control group in detached leaves method,that means B.velezensis 19573-3 has significant biocontrol functions.In addition,the tomato leaves treated with the fermentation broth of B.velezensis 19573-3 were associated with the expression of catalase(CAT),superoxide dismutase(SOD),and phenylalanine ammonia lyase(PAL),which showed a trend of first increasing and then decreasing.And the expression of PR2b gene that related to induced systemic resistance reached maximum at 12 h,while the expression of TGA1a gene expression was higher at 72 h,which indicating that B.velezensis19573-3 can induce plant production systems resistance. |
