Study On The Insecticidal Activity Of The Fermentation Products Of Endophytic Fungi From Stephania Dielsiana Y.C.wu And Citrus Against Solenopsis Invicta

Red fire ants（Solenopsis invicta Buren）are one of the most dangerous invasive organisms in the world,having invaded more than 20 countries.They could cause serious harm to human health,public safety,agricultural and forestry production and ecological environment,etc.How to control S.invicta safely and effectively is an urgent problem to be solved.It is one of the improved strategies to find the control agent of red fire ant from natural products.Endophytic fungi,an important resource for searching for natural active substances,can produce natural compounds with significant activity and rich structure types.In order to explore the secondary metabolite activity of secondary metabolites of plant endophytic fungi against S.invicta,and to provide a new idea for the control of S.invicta,we conducted the study on the isolation and identification of plant endophytic fungi,as well as the insecticidal activity of their fermentation products against S.invicta.In this study,firstly,we collected healthy plant samples of Chinses traditional medicinal plant Stephania dielsiana Y.C.Wu,and the important characteristic commercial crop citrus from different regions of Guangxi.Then,endophytic fungi were isolated by the tissue culture method,and identified by morphology combined with molecular identification technology.The representative strains were selected for fermentation culture,and the crude extraction were prepared.In order to explore the toxicity potential of the secondary metabolites of endophytic fungi and the possible mechanism of action to S.invicta,the contact toxicity and feeding activity of the crude extracts to S.invicta were determined.The main results are as follows.1.A total of 143 endophytic fungi were isolated from the healthy plant tissues by a tissue culture method,including 72 endophytic fungi of citrus and 71 endophytic fungi of Stephania dielsiana.By morphological observation and ITS sequence analysis,30 citrus endophytic fungi were identified,belonging to 14 genera and 13 families;38 endophytic fungi of Stephania dielsiana were identified,belonging to 19 genera in 12 families.2.35 endophytic fungi were selected to culture in rice solid medium.The fermentation was extracted with ethyl acetate.A total of 35 ethyl acetate crude extracts of endophytic fungi were obtained by evaporation under vacuum.3.The insecticidal activities of the ethyl acetate extracts of 35 endophytic fungi against S.invicta were determined by a drug film method.It was found that the crude extracts from different plants displayed contact toxicity to some extent.Among them,the crude extracts of Stdif9,Stdif6,J-12,STJ-J-3-6 and WZMG-Y-24 showed the most significant contact activity against S.invicta.When the concentration was 250 mg/m L,the cumulative corrected mortality of24 h of S.invicta was over 80%.4.The contact virulence of the ethyl acetate extracts of the above 5 strains Stdif9,Stdif6,J-12,STJ-J-3-6 and WZMG-Y-24 against S.invicta were further determined.The results indicated that J-12,Stdif9 and Stdif6 had the most significant toxicity to S.invicta,with LC₅₀values of 24 h at 21.372 8 mg/m L,38.495 5 mg/m L and 37.055 2 mg/m L,respectively.The 3endophytic fungi Stdif6,Stdif9 and J-12 from Stephania dielsiana were more virulent to S.invicta than the citrus endophytic fungi WZMG-Y-24 and STJ-J-3-6.5.The liquid feeding activity of the ethyl acetate extracts of Stdif6,Stdif9 and J-12 to S.invicta was determined in vitro by a“water tube”method.The results showed that the 3endophytic fungi had good liquid feeding toxicity and delayed toxicity to S.invicta.The mortality of S.invicta treated with different concentrations of poison baits was less than 30%within 24 h.Moreover,in the tested time（within 15 d）,the insecticidal activity of different agents with the same concentration to S.invicta was different,and the insecticidal activity increased with the increase of treatment concentration of different concentrations of the same agent to S.invicta.When the concentration was 1.000 mg/m L,the cumulative mortality of S.invicta,treated with Stdif6,reached 100%.The mortality rates of S.invicta,treated with Stdif9and J-12,were not more than 80%.When the concentration was 0.100 mg/m L,the cumulative mortality of S.invicta,treated with Stdif6,more than 80%.The mortality rates of S.invicta,treated with Stdif9 and J-12,were not more than 70%.When the concentration was 0.010mg/m L,the mortality rate of S.invicta treated with Stdif6 and J-12 was more than 50%.When the concentration was 0.001 mg/m L,while the mortality rate of 3 endophytic fungi was more than50%.The above results indicated that Stdif6 had the most significant toxicity to S.invicta by liquid feeding.6.Baits were used to investigate the toxicity of crude extracts of Stdif6,Stdif9 and J-12 on S.invicta.The results showed that the baits of 3 endophytic fungi had good feeding toxicity and delayed toxicity to S.invicta.The mortality rates of S.invicta increased with the increase of bait concentration.The mortality of S.invicta treated with different concentrations of poison baits was less than 5%within 24 h.Moreover,in the test time（within 15 d）,when the concentration of poison baits was 50.000 mg/g,the cumulative mortality of S.invicta treated with 3 endophytic fungi baits was close to 100%,and the cumulative mortality of S.invicta was more than 90%at the concentration of 25.000 mg/g.While the cumulative mortality of S.invicta reached 90%,treated with J-12 bait with the concentration at 12.500 mg/g.The mortality rates of S.invicta,treated with Stdif6 and Stdif9,were not more than 60%.When the concentration was 6.250 mg/g,the cumulative mortality of S.invicta treated with Stdif6,Stdif9 and J-12 bait for 16 days was more than 40%,70%and 80%,respectively.When the concentration was 3.125 mg/g,the cumulative mortality of S.invicta treated with Stdif6,Stdif9 and J-12 bait was more than 30%,20%and 80%,respectively.The results showed that Stdif6,Stdif9 and J-12 showed strong feeding toxicity to S.invicta,and J-12 showed obvious feeding toxicity to S.invicta when the concentration was 3.125 mg/g.In this study,it was found that the plant endophytic fungi Stdif9,Stdif6,J-12,STJ-3-6,WZMG-Y-24 displayed remarkable insecticidal activity against S.invicta,especially the 3endophytic fungi Stdif9,Stdif6,J-12 isolated from Stephania dielsiana.The results laid a foundation for further study on the toxicity mechanism of the endophytic fungi of the Stephania dielsiana to S.invicta,and provided a basis for their application as a natural control agent for S.invicta.