| Aflatoxin is a class of secondary metabolites with carcinogenic,teratogenic and mutagenic effects produced by Aspergillus flavus,Aspergillus parasiticus and the like.Aspergillus flavus is widely distributed in nature and is a common pathogen of humans and animals and plants.During the growth of crops in the field,Aspergillus flavus can induce a variety of diseases.Although infestation does not necessarily lead to a significant reduction in crop production,aflatoxin contamination not only severely restricts the development of agricultural production,but also jeopardizes food safety and human health.The contamination of agricultural products with Aspergillus flavus and the production of toxins may occur before and after the crop is harvested.The traditional aflatoxin removal methods after harvesting include physical and chemical methods,including ammoniation,alkaline,high-temperature,oxidation,and adsorption.However,these methods have the disadvantages of unstable effects,large loss of nutrient components,and difficulty in scale production.Although the cultivation and planting of new varieties resistant to Aspergillus flavus is the fundamental method to solve aflatoxin contamination of agricultural products,the new varieties of crops resistant to Aspergillus flavus and high yield still need further breakthroughs and promotion.At present,the use of environment-friendly bio-control technology has become a hot topic among scientists around the world.Trichoderma harzianum is a type of Trichoderma spp.,an environment-friendly fungus with important biocontrol value.Due to its wide range of existence,strong adaptability,and broad-spectrum high-efficiency,it has been recognized by the world as a beneficial bacteria with great biological and biological potential.Trichoderma harzianum H-13L is a mutant strain with the characteristics of stress resistance,disease resistance and plant growth growth obtained by N~+injection screening in this experiment.In this study,H-13L and wild H.harzianum strain GIM3.442 were used to study the antagonism of Aspergillus flavus,based on various mechanisms such as Trichoderma competition,heavy parasitism,antibiotic,induced resistance and synergistic antagonism.In this study,Trichoderma spp.and Aspergillus flavus solids were used to investigate the antagonism of Aspergillus flavus and the induction of Trichoderma by Aspergillus flavus.To develop a vaccine-based trichoderma biocontrol agent against soil-borne aflatoxin.1.Antagonism of Aspergillus flavus against Trichoderma harzianum and Screening of Optimum Antagonistic Groups:According to Matarese et al.modified plate pairing experiment method,different concentrations of Trichoderma and Aspergillus flavus cakes were combined.Trichoderma and Aspergillus flavus cakes were placed in the center of the side of the PDA(φ9cm)plate for counterculture,while the Aspergillus flavus cake was placed on a blank PDA plate as a control,and photographs of each group were recorded daily for 24 hours.In the case of the same or similar antibacterial effect,the anti-fungus group consisting of low concentrations of Trichoderma and high concentrations of Aspergillus flavus was screened out,and the best antagonistic group,Trichoderma harzianum GIM3.442,was screened out by measuring the antibacterial rate and aflatoxin content.Trichoderma harzianum GIM3.442(10~2 cfu/m L)-Aspergillus flavus(10~5cfu/m L)and Trichoderma harzianum H-13L(10~2cfu/m L)-Aspergillus flavus(10~5cfu/m L)to the aphid culture group.The antibacterial and inhibitory rates of Trichoderma harzianum GIM3.442 against Aspergillus flavus were 52%and 96%,respectively.Trichoderma harzianum H-13L antibacterial rate and inhibitory rate on Aspergillus flavus were20%and 57%,respectively.2.Optimum antagonistic group screening in liquid mixed fermentation culture:Based on the results of the solid-to-silicone experiment,the mixed cultures of Trichoderma harzianum and Aspergillus flavus with 10~2:10~4,10~2:10~5,and 10~2:10~6 spores were designed.By mycelial morphology,aflatoxin content,chitinase activity,β-1,3-glucanase activity assay and scanning electron microscopy mycelium morphology.The best antagonistic group was selected as the mutant T.harzianum:Aspergillus flavus(10~2:10~5)mixed culture group.The mutant T.harzianum inhibited the production of Aspergillus flavus by 96%and the aflatoxin content was 0.06μg/kg.The enzyme activity andβ-1,3-glucanase activity were increased by 48%and 28%,respectively.Scanning electron microscope observation showed that the surface of mycelium of Aspergillus flavus was wrinkled,damaged and collapsed,and was obviously damaged.3.Mixed Fungus Determination and Fungus Safety Evaluation:Based on the strong antagonism of Trichoderma in the mixed liquid culture against Aspergillus flavus,and the good induction of Trichoderma by Aspergillus flavus.In this study,mutant Trichoderma harzianum:Aspergillus flavus(10~2:10~5)mixed culture broth was selected as the best mixed Fungus agent,and its safety was evaluated.The spore count of the haemocytometer was used to obtain a spore concentration of 1.56×10~8 cfu/m L.The bacterial agent and its dilution were plated and cultured for 14 days.No colony of Aspergillus flavus was observed,indicating the presence of Aspergillus flavus in the fungus.The spores cannot germinate.The spores in the fungicide are Trichoderma spores.The fungicide is safe and reliable.4.Evaluation of Biological Effects of Mixed Fungus:The Fungus and its dilutions were mixed with Aspergillus flavus spores and plated and cultured.No colony of Aspergillus flavus was observed in the Fungus solution and 10-fold dilution plates.There was a small amount of yellow in the 100-fold and 1000-fold dilutions of the Fungus agent.The growth of Aspergillus flavus colonies indicates that the fungicide contains secondary metabolites that can inhibit the spore germination and growth of Aspergillus flavus.The peanut filtrate was treated with the filtrate and its diluent to observe the effects of the filtrate on the seed vigor and seedling growth of peanut and corn.The experimental results showed that 10-fold,50-fold,and 100-fold dilutions of filtrates promoted the seed germination and hypocotyl growth of peanuts,and the 100-fold dilution had the best effect on the germination and hypocotyls growth of peanut seeds.The germination rate and germ length increased by 15%and 49%,respectively.The 10-fold filtrate dilution had the best effect on the growth of peanut seedlings.The stem height,main root length,lateral branch length,branch number,and fresh weight of the peanut seedlings increased by 84%,33%,39%,23%,and 33%,respectively.;The 10-fold dilution of the filtrate not only promotes the growth of peanut seedlings,but also increases the chlorophyll content,defense enzyme activity,and lipase and lipoxygenase activity of the peanut seedlings.The 100-fold filtrate diluent had the best effect on the seed vigor and seedling growth of maize.The germination rate,radicle and shoot length,and number of radicles increased by 9%,39%,46%,and 25%,respectively,compared with the control group.The plant height,root length,root number,fresh weight,and chlorophyll content of seedlings increased by 9%,30%,30%,20%,and 13%,respectively.It shows that the fungicide contains activators that can promote plant growth.5.Optimization of the mixed Fungus formulation side:In the first scenario,the addition of chitinase hydrolysates at different time intervals in the formulation side further optimizes and enriches the formulation.The results showed that the addition of chitinase hydrolysate at different time points all contributed to the reduction of the aflatoxin content in the mixed culture fluid,but the chitinase andβ-1,3-glucanase activity decreased.In the second scheme,chitosan hydrolysates were added for 2h and 5h in the fungicide and further fermented.The results showed that the addition of 5h chitin hydrolysate could increase the chitinase activity by 35%,and the activity of the bacteria was further optimized. |
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