The Functional Mechanism Of Bacillus Subtilis GXHZ16 Against Phytophthora Nicotiana

Tobacco black shank（caused by the fungus Phytophthora nicotianae）is a soil-borne disease with wide distribution and serious damage in tobacco production.Chemical control is the main control method at present,but long-term single application of chemical pesticides is easy to cause resistance of Phytophthora nicotianae.In the context of promoting green low-carbon agricultural transformation,it is of great significance to screen efficient antagonistic strains for biological control of Phytophthora nicotianae.In this study,Phytophthora nicotiana was used as the target,and then biocontrol bacteria of Phytophthora nicotiana were isolated,screened and identified from rhizosphere soil of healthy tobacco plants.Pot experiment was conducted to study the effect of biocontrol bacteria on tobacco black shank disease.On this basis,the possible inhibition mechanism of biocontrol bacteria against Phytophthora nicotiana was further explored by means of the study of antibacterial lipopeptides,scanning electron microscopy and high throughput transcriptomic sequencing（RNA-Seq）.The main research results are as follows:1.By confrontation culture method,47 strains of bacteria with different morphology were isolated from the root soil samples of healthy tobacco plants and 33 strains of biocontrol bacteria preserved in the laboratory were screened to obtain 4 strains with inhibitory effect on Phytophthora nicotiana.The bacterium GXHZ16 with the best inhibition effect was selected for further study.The biocontrol bacterium GXHZ16 was identified as Bacillus subtilis by observing its colony morphology,Gram staining results,physiological and biochemical test results and molecular biological identification.2.Antibacterial spectrum analysis of Bacillus subtilis GXHZ16,the results showed thatGXHZ16 had antagonistic effects on 10 species of pathogens,including Alternaria alternata,Rhizoctonia solani and Cochliobolus heterostrophus.Among them,the inhibition rate of mycelium growth of Alternaria alternata was the highest,reaching 84.66±0.61%.3.In the pot experiment of Bacillus subtilis GXHZ16 fermentation broth（1×10⁸ cfu/m L）,the relative pot control effect of GXHZ16 fermentation broth on tobacco black shank disease was 51.49±2.69%.4.The bacteriostatic activity of Bacillus subtilis GXHZ16 against Phytophthora nicotiana was analyzed.The results showed that antimicrobial lipopeptides crude extract of GXHZ16 inhibited Phytophthora nicotiana with EC₅₀value of 2.9432mg/m L and EC₉₀value of 5.6810mg/m L.And PCR detection showed that GXHZ16 contained a variety of functional genes for synthesis of antimicrobial lipopeptides,such as pps B,ynd J,srf AA,srf AB,fen D,yng G.In particular,the results of scanning electron microscopy showed that under the stress of GXHZ16,the mycelia of Phytophthora nicotiana showed distortion,contraction and other malformation.5.Transcriptome sequencing（RNA-Seq）technology was used to further analyze the differential expression profile of Bacillus subtilis GXHZ16.A comparative analysis of the mixed samples of GXHZ16 and Phytophthora nicotiana cultured for 0 and 36 h showed that 1607 genes of GXHZ16 were differently expressed.Moreover,GO enrichment analysis demonstrated that differentially expressed genes（DEGs）were significantly enriched in the cellular anatomical entity of the cell component category.KEGG enrichment analysis showed that DEGs was significantly enriched in Microbial metabolism in diverse environments.Other major metabolic pathways include Flagellar assembly and Starch and sucrose metabolism.Most of the DEGs enriched in Flagellar assembly metabolic pathways and Bacterial chemotaxis metabolic pathways were up-regulated,suggesting that GXHZ16 may achieve inhibition against Phytophthora nicotiana through cell movement and outward diffusion growth.In addition,due to the active expression of genes related to the synthesis of plipastatin,surfactin and fengycin in DEGs,it is speculated that GXHZ16 may inhibit the growth of Phytophthora nicotiana mycelia by producing surfactin,fengycin and plipastatin during the growth process.In conclusion,the results of this study not only provided a highly effective biocontrol strain for the biological control of tobacco black shank disease,but also provided a basis for further elucidate the biocontrol mechanism of Bacillus against plant pathogenic fungi（including Phytophthora nicotiana）,and also had a certain significance for expanding people’s understanding of Bacillus biocontrol of pathogenic fungi.