| Aflatoxin is a kind of carcinogenic and mutagenic substance produced by Aspergillus,which does great harm to the biosafety and human health.Peanut is vulnerable to aflatoxin contamination due to its characteristics of above-ground flowering and underground fruit,and the pod is in direct contact with the soil.The prevention and control of aflatoxin contamination in peanut is very important,and it’s urgent to find a safe and effective prevention and control method.Much attention has been paid to biocontrol due to its low cost,effectiveness and no secondary pollution to the environment.The use of non-aflatoxic Aspergillus flavus to reduce aflatoxin contamination in grains through competitive inhibition has been certified to be an effective method.In this study,a combination of morphological identification and PCR-RFLP technique was used to isolate and screen non-aflatoxic Aspergillus flavus from the soils in main growing areas of peanut in China.Immobilized microorganism technique was used to prepare immobilized microbeads biocontrol agents with temperature sensitivity,the carbon based double layer microbeads biocontrol agents were prepared based on that.The moisture absorption characteristics of carbon based microbeads biocontrol agents and releasing mechanism in the soil was analyzed.The field experiment and pot experiment were used to evaluate the biocontrol agents in the prevention and control effect on peanut aflatoxin contamination.The main work is as follows:（1）The non-aflatoxic Aspergillus flavus was screened as biocontrol agents and its characteristics were evaluated.By using morphological identification combined with PCR-RFLP,non-toxigenic Aspergus was screened out from the soil in Henan province,which is the main peanut producing area in China.The screened non-aflatoxic Aspergillus flavus H4-5 had a strong antagonistic and inhibitory effect on CD3-1,the toadstogenic strain with the strongest AFB1 production capacity,and the inhibitory rate of AFB1 production on soybean culture medium and peanut seed could reach 67.52%and 59.76%,respectively.In normal application concentration range,the pesticides used in peanut cultivation have no inhibitory effect on the application of non-aflatoxic Aspergillus flavus as biocontrol agents.（2）The temperature-responsive intelligent biocontrol agents were prepared.The microbeads biocontrol agents was prepared by immobilized microorganism technology.Starch and sodium alginate were used as the substrate,and the suspension of non-aflatoxic Aspergillus flavus was used as the source of biocontrol agents.The optimal preparation process of the encapsulated biocontrol agents was determined by single factor and orthogonal experiment,which was 1.5%w/v of sodium alginate,10%w/v of starch,2%w/v of Ca Cl2,10%of spore suspensions,and the curing time was 1 h.On this basis,the temperature sensitive gel was introduced to prepare the temperature sensitive beads,the sensitive temperature was in the range of 29～30°C,which met the temperature requirement of the initial flowering of peanut in the critical period when the biocontrol agent was applied.The release of biocontrol agents in the temperature responsive microbeads was continuous,controllable and had good temperature sensitivity.The temperature-responsive intelligent biocontrol agents could meet the requirements of slow release effect in soil after early application,so as to weaken the influence of adverse conditions on biocontrol fungus in the environment.However,when the temperature reaches the temperature of initial flowering of peanut（30.5～33.2℃）,it can be suddenly released and expanded into the environment in large quantities,so as to better meet the practical application effect.（3）Carbon-based bilayer immobilized beads with bacteriostatic effect were prepared.Carbon-based bilayer immobilized beads were prepared based on temperature-responsive biocontrol agents.The addition of biochar to the immobilized beads has a sustained-release effect similar to that of kaolin.The addition of biochar can significantly improve the embedding rate of bacteria in the microspheres and improve the water retention performance of the soil.The carbon-based beads coated with chitosan could inhibit Bacillus subtilis and Aspergillus niger growth in a certain time.The carbon-based bilayer immobilized beads had good temperature response and controlled release properties,and their release characteristics were similar to those of temperature responsive beads.（4）The hygroscopic properties and release mechanism in soil of carbon-based biocontrol agents were analyzed.The hygroscopicity of biochar and carbon-based biocontrol agents was better than that of soil,and the application of them can increase the equilibrium moisture content of soil and improve soil water retention.In the range of 15℃～35℃,the modified Henderson model can better evaluate the relationship between the equilibrium moisture content of biochar and soil and the temperature and humidity,and more accurately predict the equilibrium water content of biochar and soil.The equilibrium moisture content model of carbon-based biocontrol agents was related to temperature.The model of relative humidity,water content and temperature can be used to evaluate the water activity of carbon-based biocontrol agents and provide reference for the application of biocontrol microorganisms.The release mechanism of three-layer diffusion was consistent with the release process of biocontrol agents in soil,which could predict the release characteristics of microbial agents in soil.It has a certain theoretical significance for the development of biocontrol agents and the study of controlled release characteristics.（5）The application effect of non-toxic Aspergillus flavus in preventing peanut aflatoxin contamination was studied.The biological control method of aflatoxin contamination in peanut by non-aflatoxic Aspergillus flavus through competition inhibition has been proved to be effective in main growing areas of peanut in China.The ratio of non-aflatoxic Aspergillus flavus in soil can be changed by the application of biocontrol agents.The early flowering period of peanut was the best period for the application of biocontrol agents,and the application of biocontrol agents with rice as carrier in the early flowering period could increase the proportion of non-aflatoxic Aspergillus flavus in the soil to 92.41%.When evaluating the amount of biocontrol agents,1 kg/mu of biocontrol agents with rice as carrier in the early flowering period could reduce the content of AFB1 in peanuts by 75.66%under the stress of high temperature and drought weather conditions.The bacteriostatic property of the double-layer microspheres had an inhibitory effect on the mixed bacteria in the soil within a certain time range,which reduced the interference on the biocontrol fungus.The application of carbon-based double-layer immobilized beads agents in the soil during sowing period has a good effect on the prevention and control of aflatoxin contamination in peanut and can provide convenience for the application in agriculture.The research in this paper can provide data guidance for the biocontrol of peanut aflatoxin pollution in China,and provide theoretical and technical support for the development and application of biological pesticides for the control of peanut aflatoxin contamination,which is of great significance for the realization of sustainable agricultural development. |
