Biocontrol Mechanisms,Identification Of Clonostachys Rosea Fermentation Products And Preparation Of Its Formulations

Long-term and excessive application of chemical pesticides has been caused high resistance of crop diseases and pests and environmental pollution.The Promotion and application of biopesticides are restricted for the few varieties of biological pesticides and their narrow fungicidal or insecticidal spectrum,slow control effect,and higher cost.The best solution to the above problems is to develop effective and low cost biopesticides.The fungus Clonostachys rosea is a widely distributed saprophyte in the soil and has been considered a potential biocontrol agent,since it has many advantages of strong sporulation ability,diverse mechanisms of action,wide parasitic,environmentally friendly.C.rosea has been applied abroad in the prevention and treatment of various crop diseases and it shows good control effects.Relevant researches on its parasitic and antagonistic effects on plant pathogens have been conducted in China.However,there are few reports on the identification of fermentation products,mechanisms of biocontrol and preparation of its formulations of C.rosea.In this paper,based on the identification of the fermentation products of C.rosea and study of its biocontrol mechanisms,C.rosea Wetteble Powder and Dry Flowable were developed with the optimization of its additives.Our research work not only provides the theoretical basis for its industrial production and commercial application,but also develops new biological control agents for the green prevention and control of plant diseases.The stability of C.rosea fermentation products and its effects on the protective enzymes of plant pathogens were studied,and the secondary metabolites from C rosea were analyzed and identified by LC-MS.Its biocontrol mechanisms were explored jointly by biological and chemical methods.Based on the optimization of the additives,C.rosea WP and DF were developed.The inhibitory rate of C.rosea fermentation products against Physalospora piricola,Alternaria solani.and Fusarium oxysporum were 50.68%,40.48%and 37.74%respectively.The C.rosea fermentation products were very sensitive to temperature and pH.The Ultrasonic had little effects on the activity of C.rosea fermentation products,but the UV affected the activity of C.rosea fermentation products.Hydrogen peroxide greatly decreased the activity of C.rosea fermentation products,however,the metal ions of Mg2+,Fe3+ K+and Na+had little effects on the activity of C.rosea fermentation products.Being treated with C.rosea fermentation products the activity of 5 protective enzymes and Lipid Peroxidation of MDA,GSH and conductivity of P.piricola were analyzed.MDA content of both treated group and control group was increased.C.rosea fermentation products inhibited all the activity of CAT,SOD,GSH,ACP and PAL.GSH content was decreased and the activity of AKP was increased by 3.97%?9.88%.The conductivity of the treated group was 4.90 times higher than that of the control group at 30h time point.The above results showed that C.rosea fermentation products inhibited the activity of protective enzymes in P.piricola,promoted the Lipid Peroxidation and decreased the activity of its detoxification enzymes.C.rosea fermentation products inhibited the activity of POD and SOD in Sclerotinia sclerotiorum;but they increased the activity of CAT.Being treated with C.rosea fermentation products GSH content in S.sclerotiorum was decreased with time,however,GSH content in the control group was increased with time.MDA content in S.sclerotiorum was increased with time and it was much higher than that of the control group.At each treatment time point,C.rosea fermentation products inhibited the activity CAT in A.solani.C.rosea fermentation products inhibited SOD activity in A.solani in the early stage and slightly increased its activity in the later stage.Except for 6-hour time point,the activity of POD in A.solani of treated group was significantly higher than that of the control group;and the POD activity of treatment group was gradually increased by C.rosea fermentation products.The content of GSH and MDA in A.solani of treated group was higher than those of the control group at every treatment stage.The bioassay results showed that the C.rosea fermentation products had good fungicidal activity on four pathogens.The analysis of non-target metabolomics data showed that 42,128 metabolites were found in the pos mode and 27,917 metabolites were found in the neg mode.7 compounds were separated and purified from the secondary metabolites from C.rosea fermentation products,5 compounds of them were identified as p-Hydroxybenzoic acid,Caffeic acid,Bisorbicillinolide,Bisvertinoquinol and Sorbicillin by LC-MS,and the other two compounds were deducted as the new compounds by compound databank analysis.C.rosea Wettable Powder and Dry Flowable were developed with the screening and optimization of adjuvants.The spray drying process was used to develop C.rosea DF.The evaluation of C.rosea WP and DF was conducted in pot and field trials.The results showed that the control efficacy of C.rosea WP against S.sclerotiorum was from 60.52%to 85.74%.The control efficacy of C.rosea DF against S.sclerotiorum is more than 80%.This result indicated that C.rosea conidia powder and its fermentation products had good synthetic control effect.