Isolation And Identification Of Endophytic Fungi From Rhododendron And Their Activities Against Pests Of Traditional Chinese Medicinal Materials

At present,the problems of diseases and pests in the planting and storage of Chinese medicinal materials have greatly reduced or lost their medicinal and commercial value.Currently,highly toxic chemical agents are used to prevent and control the pests in the planting and storage of Chinese medicinal materials.Plant endophytic fungi are not only rich in species,but also have a variety of compound structures,and may metabolize and produce novel structural compounds.It is an important source of new natural active products as well as a valuable resource of new pollution-free pesticides.In the preliminary screening of this study,it was found that Rhododendron plants had obvious biological activities against Ditylenchus destructor and Tribolium castaneum.Therefore,in order to obtain active strains,four species of Rhododendrons were selected for the separation and purification of endophytic fungi,and the control effects of their metabolites against T.castaneum and D.destructor were studied.In addition,the research on chemical constituents of the metabolites and the material basis of pest control are also carried out,so as to conduct basic research for the development of ecological planting and green storage of Chinese medicinal materials.The research contents and experimental results are as follows:1.Isolation of plant endophytic fungi:22 strains of endophytic fungi were isolated from different tissues of R.thymifolium,R.capitatum,R.anthopogonoide and R.przewalskii by means of tissue cutting method and streak plate method.The result is as follow:5 strains were obtained from R.thymifolium,7 strains from R.capitatum,3strains from R.anthopogonoide and 7 strains from R.przewalskii.Among them,13 and9 strains of endophytic fungi were isolated from branches and leaves,accounting for59.09%and 40.91%,respectively.2.Activity screening of strains:The crude extracts of 22 endophytic fungi and their secondary metabolites were prepared by PDB medium liquid fermentation technology and methanol reflux extraction.A total of 44 crude extracts were obtained.The contact and antifeedant activities of 44 crude extracts against T.castaneum and their nematicidal activities against D.destructor were determined.The results showed that the crude extract of mycelium of LS-Y-1 had the best contact activity against T.castaneum(LD₅₀=69.31μg/adult),while the crude extract of fungal fluid of LS-Y-1 had obvious antifeedant activity against T.castaneum（FDI=84.28%）and nematicidal activities against D.destructor(LC₅₀=0.06 mg/m L).In addition,the crude extract of strain of TH-Z-1 had obvious nematicidal activity against D.destructor(LC₅₀=0.07mg/m L).The crude extract of fungal fluid and mycelium of LS-Y-4 had obvious antifeedant activity against T.castaneum（FDI=73.12%）and nematicidal activity against D.destructor(LC₅₀=0.18 mg/m L),respectively.Based on the above results,LS-Y-1,LS-Y-4 and TH-Z-1 were found to have obvious biological activities.3.Identification of active strains:Three strains with high activity were identified by means of molecular biology and morphological analysis,and the attribution of the target strains were determined.Strains LS-Y-1 and LS-Y-4 have a high similarity with Alternaria sp.Alternaria sp.belongs to the Moniliales,It is common in plant endophytic fungi.The strain TH-Z-1 has a high similarity with Trametes sp.4.Separation and identification of chemical constituents:After preliminary activity screening,the chemical composition of LS-Y-1 strain with good insect-resistant activity and comprehensive effect was studied.The chemical constituents of secondary metabolites from endophytic fungi of LS-Y-1 were isolated and purified by TLC,slica column chromatography and gel column chromatography.Six monomer compounds were obtained.Finally,five compounds were identified,which were 2,4-Dimethyldodecan-1-ol（1）,mannitol（2）,4-O-β-D-galactopyranosyl-erythritol（3）,alternariol 9-O-methyl ether（4）,allantoin（5）.Among them,the compound2,4-Dimethyldodecan-1-ol was first isolated from fungi of Alternaria sp..5.Activity determination of isolated compounds:The activity tests of the five compounds were carried out,and the results showed that alternariol 9-O-methyl ether indicated the best biological activity,showing obvious contact activity not only against the adults(LD₅₀=38.26μg/adult)and larvae(LD₅₀=51.89μg/larvae)of T.castaneum,but also against D.destructor(LC₅₀=0.56 mg/m L).Secondly,2,4-dimethyldodecan-1-ol showed obvious toxic activity against T.castaneum adult(LD₅₀=43.43μg/adult)D.destructor(LC₅₀=1.64 mg/m L).On this basis,mannitol and alternariol-9-O-methyl ether were mixed in different proportions,and it was found that when the volume ratio was 6:2,the contact activity against the adult of T.castaneum was significantly enhanced,showing a synergistic effect.In summary,22 strains of endophytic fungi of Rhododendron were isolated and purified in this paper.One strain of endophytic fungus was obtained by screening for its insect-resistant activity.The structure of five compounds in the endophytic fungus was obtained by isolation,and the insect-resistant activities of these compounds against pests in the planting and storage of Chinese medicinal materials were investigated.The results show that plant endophytic fungi have a certain value in the green control of Chinese herbal medicine pests.