Screening,Identification And Control Efficacy Of Antagonistic Strain ZY-11-2 Against Phytophtora Parasitica Var. Nicotianae

In recent years,in response to the call of green agriculture in China that we should avoid the problem of"3R"caused by mass usage of chemical pesticides and take advantages of continuous control,protection of the ecological environment and low toxicity to humans and animals,biological control methods play an increasingly important role in plant disease management.In particular,the screening of various antagonistic strains,the study of antimicrobial activity of metabolites and derivatives and the optimization of production process have become the current research hotspots.In order to discover the antagonistic strains against Phytophtora parasitica var.nicotianae,providing excellent resources for the prevention and treatment of plant diseases.30 soil samples were collected from different ecological environments in Qinling,and were subjected to dilution coating,streak culture,directional screening of Phytophtora parasitica var.nicotianae,re-screening of cylinder-plate method,and plate confrontation of a variety of pathogenic bacterium.After that,an antagonistic strain ZY-11 was obtained which has obvious antagonistic activity against Phytophtora parasitica var.nicotianae and has good broad-spectrum antibacterial ability.The bacteriostatic band width of the sterile fermentation filtrate against Phytophtora parasitica var.nicotianae was 12.62±0.37 mm.Subsequently,ZY-11 strain was identified by traditional morphological observation,physiological and biochemical characteristics determination combined with analysis of 16S rDNA gene sequence analysis.The results showed that the morphological,physiological and biochemical characteristics of ZY-11 strain were basically consistent with the characteristics of the Bacillus model strain.The ZY-11 strain was identified as Bacillus amyloliquefaciens by analysis of 16S rDNA sequence.The results show that ZY-11 strain has certain hemolytic properties,which may cause unknown damage to health of human and animal.Therefore,in order to improve the safety of the ZY-11 strain,it is subjected to physical?UV?and chemical?DES?mutagenesis.There were three weak hemolytic mutant strains which were induced by UV mutagenesis for 2 min,4 min and DES mutagenesis for20 min,named ZY-11-1,ZY-11-2 and ZY-11-3,respectively.The result showed that the antibacterial bandwidth of ZY-11-2 against Phytophtora parasitica var.nicotianae is up to11.59±0.51 mm.There was no significant difference in antibacterial bandwidth between ZY-11-2 strain and ZY-11strain,and the effect of ZY-11-2 strain was the best.And transferred 7 generations,its weak hemolytic property can be stably inherited.Then,using the single factor control method and multi-factor orthogonal analysis,the optimal ratio of each component of the liquid fermentation medium of ZY-11-2 strain was obtained as follows:corn flour 10.0 g/L,soybean meal 10.0 g/L,NaCl 2.5 g/L and K₂HPO₄ 3.5g/L;the optimum culture conditions were as follows:pH 7.0,32°C of culture temperature,6%-8%of inoculum size,30 mL/250 mL of liquid volume,180 r/min of rotation speed,36h of culture time.It was found that the OD₆₀₀00 value,the viable cell count and the control effect of the sterile filtrate of ZY-11-2 fermentation broth were better than those of the original fermentation broth after optimization of fermentation conditions,indicating that the optimization effect was obvious.Finally,the protective and therapeutic effects of ZY-11-2 fermentation broth?10⁷ cfu/mL?on tobacco black shank were about 74.19%and58.34%in experiments of greenhouse pot.The control effect of ZY-11-2 fermentation broth?10⁷ cfu/mL?on tobacco black shank disease was 47.31%and 41.03%respectively at 15and 30 days after the last application.The test results show that ZY-11-2 strain can effectively control the occurrence and spread of tobacco black shank,which has certain application potential.