Growth Promotion Of Bacillus Subtilis Strains 8-32 And A Nalysis Of Indoleacetic Acid (IAA) Synthesis Pathway

Since the 13th Five-Year Plan,the Ministry of Agriculture and Rural Affairs has vigorously carried out the zero-growth action of fertilizer and pesticide use.Rhizosphere growth promoting bacteria(PGPR)is a powerful weapon to achieve this action.In this study,a strain of Bacillus subtilis 8-32 with strong fungal disease resistance and growth promotion effect was screened in the previous study,which had the ability to produce high inole acetic acid(IAA),and was an ideal PGPR strain.In this study,response surface analysis and other methods were used to optimize the composition and conditions of the fermentation medium for IAA production of the strain.Seed germination tests were used to preliminatively evaluate the growth promotion effect of the strain.Finally,whole-genome,targeted metabolome and gene knockout methods were used to explore the growth promotion mechanism of strain 8-32 and the metabolic pathway of IAA.The results are as follows:1.Single factor test,Plackett-Burman test,steepest climb test and Box-Behnken test were used to optimize the optimum fermentation medium composition and conditions for IAA production of strain 8-32.The results were 5%sucrose,0.03%NaCl,7.92%peptone,96 h fermentation time,56.17 mL/250 mL flask,initial pH 7.0.The content of IAA in fermentation broth increased from 8.48 mg/L before optimization to 65.06 mg/L after optimization.2.The root test of cucumber cotyledon,seed germination test and seedling pot experiment were conducted to verify the preliminary effect of strain 8-32 fermentation broth in crop planting.Strains 8-32 had obvious promotion effect on seed germination,cotyledon rooting and crop growth at seedling stage.3.Use whole genome sequencing and targeted metabolome methods to analyze the characteristics of 8-32 genomes,genes related to growth promotion,and the metabolic pathway of IAA production.The results showed that the total length of Bacillus subtilis 832 genome was 4 071 539 bp,the GC content was 43.81%,and there were 3 991 coding genes,including 86 tRNA,10 rRNA,10 sRNA,8 gene islands,5 CRISPR-Cas and 4 prophage.3 173 genes were annotated into the GO database,3 196 genes into the COGs database,and 2 287 genes into the KEGG database.In the carbon metabolic pathway,genes related to the synthesis of organic acid-related enzymes were predicted to participate in the dissolution of inorganic phosphorus.4.Two enzymes in the IAA metabolic pathway,monoamine oxidase(EC:1.4.3.4)and aldehyde dehydrogenase(EC:1.2.1.3),were annotated in the whole genome of strain 8-32,and their corresponding genes were yobN and yfmT,aldX,dhaS and ywdH respectively.Based on targeted metabolomics analysis of indole compounds,the main metabolic pathways for IAA synthesis in strains 8-32 were tryptamine(TAM)pathway,indole acetamide pathway(IAN)and indole pyruvate(IPyA)pathway.5.The yobN gene was successfully knocked out by homologous recombination method in strain 8-32.The IAA production of mutant was significantly decreased by 60.63%after yobN gene was knocked out.The tryptamine pathway was the main pathway of IAA production of Bacillus subtilis 8-32.In conclusion,the fermentation conditions of Bacillus subtilis 8-32 producing IAA were optimized in this paper,and its growth promoting effect and mechanism were studied,which provided a theoretical basis for the further development and utilization of strain 8-32.