Screening,Identification,and Biocontrol Mechanism Of PVY Antagonistic Bacteria

Tobacco potato virus Y disease is a systemic infectious disease caused by potato virus Y（PVY）,which is one of the main diseases restricting the production of flue-cured tobacco in China.In order to explore the resources of biocontrol strains for the prevention and control of tobacco potato virus Y disease,this study used tobacco PVY as the target,screened excellent biocontrol bacteria from the rhizosphere soil of healthy tobacco,and used morphological,physiological,biochemical and molecular biological identification to clarify its taxonomic status.The pot control effect on tobacco potato virus Y disease and the growth-promoting effect on tobacco plants of antagonistic bacteria were verified.At the same time,the broad-spectrum antibacterial activity and functional metabolites of the strain were determined to explore the systemic resistance of tobacco to PVY induced by the strain,and the fermentation of the strain was optimized by response surface method（RSM）.The results are as follows:1.A total of 216 strains were isolated from 30 soil samples,and 4 strains with better PVY passivation effect were screened from 216 strains,among which ZF-10 had the best antagonistic effect.The strain ZF-10 was identified as Bacillus velezensis by morphological characteristics,physiological and biochemical indexes,16S r DNA and gyr B molecular identification.2.B.velezensis ZF-10 fermentation broth treatment of tobacco re-inoculated with PVY virus significantly reduced the incidence and disease index.The incidence rate was 55.57%and the disease index was 15.23.The relative control effect on tobacco potato virus Y disease was 61.85%,which was slightly lower than that of 8%ningnanmycin and 5.6%cytosintetidemycin+moroxydine hydrochloride wettable powder,but there was no significant difference with 8%Ningnanmycin 1200 times solution treatment,indicating that strain ZF-10 had better control effect on tobacco potato virus Y disease.The soaking of fermentation broth of strain ZF-10 could promote the germination of tobacco seeds.The pot root irrigation experiment showed that the agronomic traits of tobacco were significantly improved.The strain ZF-10 had broad-spectrum antibacterial activity and had good inhibitory effects on 8 plant pathogenic fungi such as Thielaviopsis basicola,Alternaria alternata and Alternaria solani.Through special medium detection,it was found that strain ZF-10 also had the ability to produce ammonia,cellulase,protease,siderophore and phosphate solubilization,and no potassium solubilization ability was found.3.B.velezensis ZF-10 could cause the accumulation of H₂O₂and O^2-in tobacco,enhance the activities of defense enzymes such as superoxide dismutase（SOD）,peroxidase（POD）,catalase（CAT）,polyphenol oxidase（PPO）and phenylalanine ammonia-lyase（PAL）in tobacco leaves,and reduce the content of MDA,indicating that strain ZF-10 could activate the response of tobacco defense system,weaken the membrane lipid peroxidation caused by PVY invasion,and improve tobacco resistance to cope with PVY invasion.4.The results of single factor test showed that the optimum carbon source,nitrogen source and inorganic salt for strain ZF-10 fermentation were soluble starch,yeast extract powder and magnesium sulfate,respectively.The optimum temperature,inoculation amount,liquid volume,initial pH,and fermentation time were 32℃,1%,250 mL shake flask liquid volume 60 mL,7.0 and 36 hours,respectively.Three significant factors were determined by Plackett-Burman test as liquid volume,magnesium sulfate and yeast extract.The response surface analysis obtained the optimal response value when the corresponding yeast extract content was 2.023%,the magnesium sulfate content was 0.384%,and 250 mL shake flask liquid volume 38.6 mL.The predicted absorbance value of the strain was 3.814,and the absorbance value of the strain obtained by the verification test was 3.809.The optimized fermentation biomass reached 3.61×10⁹CFU/mL,which was 14.6 times higher than that before optimization.In summary,B.velezensis ZF-10 has broad application prospects in the prevention and control of tobacco potato virus Y disease.