| Wheat take-all caused by Gaeumannomyces graminis var.tritici(Ggt)spread rapidly and was highly destructive,which resulted in serious wheat yield reduction in various wheat producing areas.Biological control of wheat take-all is safer,more durable,more environmental protection,and has broad development prospects.The study in the early stage of the laboratory screening for the erosion of wheat has significant control effect of Bacillus Z-14,on the basis of research application of the bacteria agent influence on wheat rhizosphere micro-ecology,identification of Bacillus Z-14 and type of fat peptide antibiotics may structure and testing of the total eclipse of the inhibition of wheat germs,the mechanism of action,through the determination of Bacillus Z-14 of genome-wide gene cluster and analyze the second born metabolites to explore prevention mechanism of the strain gene level,total eclipse of the late for its safe and effective prevention and control of wheat disease and other fungal diseases.In this study,the effects of Z-14 strain on wheat rhizosphere soil nutrients,pH,microbial number and enzyme activity were measured.The results showed that the treatment group inoculated with Z-14 strain three times was compared with the control group when wheat grew for 24 days.The available N,P and K contents increased by 21.00 mg/kg,2.71 mg/kg and 13.34 mg/kg,respectively.The number of bacteria and actinomycetes in soil increased by 1.90×108 cfu/g and 6.40×106 cfu/g,respectively,while the number of fungi decreased by 4.60×103 cfu/g.The activities of alkaline phosphatase and cellulase increased by 6.00%and 18.69%,respectively,but had no significant effect on the activities of urease and catalase.In order to prevent the effects of Bacillus Z-14 on the carbon source metabolism of soil microorganisms,the dynamic changes of carbon source metabolism in each treatment group were detected by Biolog-ECO plate.Soil microbial metabolic activity AWCD value,Simpson richness,Shannon-Winener and evenness Pielou index of microbial community in Z1 and Z2 treatment groups were significantly higher than those in CKO and CK1 group,and Z2 was higher than Z1 group.Principal component analysis showed that carbohydrate,amino acid,carboxylic acid and polymer were the major carbon sources contributing to the first and second principal components.In addition,the utilization of carbohydrate,amino acid,carboxylic acid and polymer in the rhizosphere soil of wheat inoculated with three secondary biocontrol Bacillus was significantly higher than that of the other three groups.The effect of inoculation with Bacillus Z-14 on soil microbial diversity was detected by high-throughput sequencing.Dilution curves and Shannon curves of soil samples showed that the test results were accurate and reliable.Through Alpha diversity analysis,the bacterial and fungal community richness index and diversity index(Chao1,Ace,Shannon,Simpson)of the soil samples all indicated that the sequencing results could represent the real situation of microorganisms in the samples.Compared with the untreated blank control group,the relative abundance of Morphella,Hemicellus,Aspergillus and Chaetobacteria in the soil inoculated with three secondary Bacillus Z-14 increased by 3.25%,0.84%,2.19%and 0.50%,respectively,and the relative abundance of Discobacterium in the soil significantly decreased by 17.87%.The relative abundance of Bacillus increased by 1.08%and 2.21%in the treatment groups inoculated with 1 and 3 times control the biocontrol Bacillus Z-14,respectively.The inoculation of Bacillus Z-14 increased the Alpha diversity index of fungi and bacteria in the soil,as well as the species richness and diversity.The Matrix-assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry(MALDI-TOF-MS)and Electrospray Ionization Tandem Mass Spectrometry(ESM)were used to support the matrix-assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry.The lipopeptide antibiotics were identified as Fengycin A(C14 Fengycin A,C15 Fengycin A,C16 Fengycin B,C16 Fengycin B)and Iturin A(C14 Iturin A and C15 Iturin A)in the supernatant of Bacillus Z-14 fermentation.The observation results by fluorescence microscope,scanning electron microscope and transmission electron microscope showed that both fengycin and iturin A could dry up,curl up or even break the mycelia by making the cell wall loose or disappear,the cell membrane deformed and degenerated,the organelle dissolved,and the cytoplasm contracted,thus inhibiting the growth of Ggt and leading to cell apoptosis.Fengycin inhibits the growth of Ggt mainly by destroying organelles and digesting cytoplasm,while iturin A inhibits the growth of pathogens mainly by destroying the cell wall of Ggt and causing mycelia to fracture,but both of them can induce apoptosis.In order to further study of Bacillus Z-14 strains at the genetic level prevention mechanism,combined with the second generation of Illumina platform and PacBio platform of the third generation sequencing technology could prevent Bacillus Z-14 for whole genome sequencing,according to the result of whole genome sequencing,identification of Bacillus Z-14 species,analyzes the mechanism of plant pathogens of antagonism and explore the potential of its biological control functions.Measured strain genome size is 4,135,736 bp,a total of 4 233 genes encoding,its genome data with GO,COG and KEGG database for comparative analysis,complete genome annotation function.based on 16 s rRNA and gyrA gene sequences to construct phylogenetic tree,and the result identified strain Z-14 as Bacillus amyloliquefaciens Z-14,By analyzing the genes related to biocontrol characteristics of Bacillus amyloliticus Z-14,the genomes of Z-14 were found to contain 53,40,13 and 7 genes related to locomotion and chemotaxis,biofilm formation,polysaccharide degradation and found that fat peptide antibiotic gene cluster of 10 and ketones,Among them,there were 6 antibiotic synthesis gene clusters(Macrolactin H,Bacillaene,Fengycin.Difficidin,Bacillibactin,Bacilysin)with 100%similarity,1 antibiotic synthesis gene cluster with 91%similarity(Surfactin),1 antibiotic synthesis gene cluster with only 22%similarity(Rhizocticin)and 2 unknown function gene clusters.And the specific gene composition of 8 secondary metabolite synthase gene clusters.It is concluded that Bacillus amyloliquefaciens Z-14 has strong antagonistic potential against plant pathogens and broad agricultural application prospects. |
