| Bemisia tabaci is one of the most notorious agricultural pests in the world.A vicious circle has gradually formed between overuse of pesticides and an increase in pesticide resistance,damaging to the environment and human health.Beauveria sp.has long been a well-known fungus pathogenic to insects and an effective natural enemy of B.tabaci.On this basis,a Beauveria bassiana strain LY2 with rich secondary metabolites and significant inhibitory effect on B.tabaci was isolated and screened.The secondary metabolites of B.bassiana LY2 were isolated,purified and identified by a series of modern separation methods such as column chromatography,thin layer chromatography(TLC)and high-performance liquid chromatography(HPLC),as well as identification techniques such as nuclear magnetic resonance spectroscopy(NMR)and mass spectrometry(MS).The multi-directional activity of B.tabaci was determined by the tube membrane method and the leaf disc method,and the safety of the obtained bioactive compounds was evaluated with four kinds of crops.The main results of this thesis are as follows:1.Screening and identification of target strains:52 strains of entomopathogenic fungi were obtained by combining the trapping method,the tissue isolation method and Koch’s method.The 52 strains were fermented in SDY and Czapek-Dox liquid medium,and their crude extracts were obtained by macroporous adsorption resin.The results showed that strain LY2 not only had obvious effects on B-type whitefly(B.tabaci)adults,but also produced rich secondary metabolites.Therefore,strain LY2 was selected as the target strain.It grows slowly on PDA,the colony is powdery and loose.Conidiophores are bottle-shaped and solitary.Conidia are spherical to nearly spherical,colorless,transparent and thin-walled,2.41(1.53-3.26)μm ×2.62(1.56-3.11)μm.The blast results of its sequence in the NCBI database showed that the similarity between LY2 and Beauveria bassiana was the highest,reaching 100%.Phylogenetic analysis showed that LY2 was clustered in the same clade with other strains of B.bassiana.Combined with the results of morphological and molecular identification,the tested strain LY2 was identified as B.bassiana.2.Isolation,purification and identification of secondary metabolites of strain LY2:combined with various separation and purification methods,10 compounds were isolated and purified from the crude extract resulting from liquid fermentation extraction of strain LY2.Compounds 1-10 were identified as bassiatin(1),ergosterol peroxide(2),melithasterol B(3),(22E,24R)ergosta-8(14),22-diene-3β,5α,6β,7α-tetrol(4),6-dehydrocerevisterol(5),cerevisterol(6),methyl-1,4-dihydro-4-oxo-2-quinoline carboxylate(7),9-hydroxycerevisterol(8),cerebroside F(9)and cerebroside B(10).Compound 1 is a morpholinone,compounds 2-6 and compound 8 are sterols,compound 7 is a quinolinone,and compounds 9 and 10 are cerebrosides.Compound 9 is a new compound,compound 7 belongs to new natural product,and compounds 3-10 were firstly isolated from Beauveria spp..3.Bioactivity determination of compounds 1-10:compounds 1-10 were screened by the tube membrane method.The LC50 of compounds 1-2 and 4-7 via feeding assay were 11.42 μg/mL,22.06 μg/mL,13.83 μg/mL,5.65 μg/mL,22.29 μg/mL and 5.66 μg/mL respectively;the LC50 of compounds 1,3,5 and 7 via contact assay were 10.95 μg/mL,41.98 μg/mL,26.59 μg/mL and 19.05 μg/mL respectively;compounds 8-10 had no obvious activity.A series of biological activities of active compounds 1-7 were determined by the leaf disc method.Among them,the residual assay,antifeedant assay and selective/non-selective oviposition avoidance assay and selective adult avoidance activities of compounds 1 and 7 were very significant compared with the control group,the values of LC50/AFC50/EC50 were 9.05/5.86/2.02 μg/mL and 11.89/8.65/19.68 μg/mL respectively.In addition,compounds 1-7 have strong oviposition avoidance effects on Bemisia tabaci,the EC50 values of non-/selective were 5.94/3.22 μg/mL,6.13/6.13μg/mL,2.65/5.54μg/mL,2.81/2.00μg/mL,6.41/1.97μg/mL,1.61/1.95μg/mL and 16.88/1.98 μg/mL respectively.4.Crop safety evaluation of compounds 1-7:The safety evaluation assay was carried out at a concentration of 100 μg/mL for compounds 1-7,and the results showed that the plant height,leaf color and development degree of all treatment groups were not significantly different from those of the control group. |
