Studies On Antifungal And Insect-resistant Activities And Fermentation Technology Of Epichlo(?) Grass Endophytes

Microbial pesticides play an irreplaceable role in chemical pesticides,which resources are abundant.However,there are few kinds of resources that have been exploited and utilized,and it is difficult to meet the needs of the development of pesticide industry.The grass-endophyte symbiosis is considered a case of ‘defensive mutualism',which produces secondary metabolites not only significantly improve the resistance of host grasses to biotic and abiotic stresses,but also lead to livestock narcosis.Therefore,it has the potential to develop into microbial pesticides.In this study,the fungal strains of Epichlo? grasses were observed as the research object.The fermentation broth was obtained by shaking flask fermentation,mainly including the seeds of Lolium perenne were soaked,determination of alkaloid content,screening of antifungal and insect-resistant strains,and optimizing fermentation conditions of strains by orthogonal design to improve the metabolic yield of active substances of strains,in order to lay a foundation for large-scale production alkaloids of Epichlo?grasses strains and development new microbial pesticide fungicides,and to provide theoretical basis for further field experiments.The main results are as follows:1.By testing mildew rate and germination index of perennial ryegrass seeds soaked with fermentation broth of four fungal endophyte strains of Epichlo?gansuensis,E.sinensis,E.bromicola(Hb)and E.bromicola(Ed)were isolated from Achnatherum inebrians,Festuca sinensis,Hordeum brevisubulatum and Elymus dahuricus through the germination test by top paper,it was found that the seed soaking with E.gansuensis fermentation broth reduced the mildew rate of perennial ryegrass seeds and significantly(P<0.05)promoted the germination,plumule and radicle growth of ryegrass.Under the Achnatherum inebrians Epichlo? fermentation broth seed soaking,the mildew rate was significantly(P<0.05)lower than those of E.sinensis and E.bromicola(Hb),while the germination index,vigor index,water content of plumule,plumule length and radicle length were larger than those of other treatments.2.By determining alkaloid content in fermentation broth of eight fungal endophyte strains of Epichlo? gansuensis(SN-Sunan,Gansu of China;JZ-Jianzha,Qinghai of China),E.sinensis(XH-Xiahe,Gansu of China;PA-Pingan,Qinghai of China),E.bromicola(Hb)(YZ-Yuzhong,Gansu of China;AKT-Aletai,Xinjiang of China)and E.bromicola(Ed)(LZ-Lize,Gansu of China;HY-Haiyan,Qinghai of China)were isolated from Achnatherum inebrians,Festuca sinensis,Hordeum brevisubulatum and Elymus dahuricus though high performance liquid chromatography(HPLC),it was found that the contents of ergonovine,ergine and lolitrem B were less than 1 mg/L,while the contents of peramine were 10.72-44.10mg/L.Among the eight fungal strains,the contents of ergine and lolitrem B in JZ and ergonovine in SN were the highest,and the content of peramine in PA was the highest and significantly(P<0.05)higher than those of other seven strains.3.By determining laboratory bioassay of five different pathogenic fungi(Alternaria alternata,Drechslera avenae,Bipolaris sorokiniana,Bipolaris zeae and Fusarium acuminatum)though dual-culture and spore germination,it was found that the fungal strain JZ significantly inhibited(P<0.05)the colony growth of B.sorokiniana and F.acuminatum than LZ and ALT.The fungal strain ALT significantly(P<0.05)inhibited the growth of D.avenae and B.zeae than SN,LZ,HY and YZ.The fungal strain JZ significantly(P<0.05)inhibited the spore germination of D.avenae,A.alternata,B.zeae and F.acuminatum than HY and YZ.The fungal strains SN and PA significantly(P<0.05)inhibited the spore germination of F.acuminatum than HY,XH and YZ.4.By investigation the effect of eight fungal strains fermentation broth of Epichlo? on Rhopalosiphum padi though feeding test and touching test,it was found that the insect resistance of 8 fungal strains fermentation broth of Epichlo? was broad-spectrum,but the insect resistant activity of different strains of fermentation broth was different.With the increase of dilution ratio of eight fungal strains fermentation broth,the insect resistance activity decreased.The result of feeding test showed that the insect resistance of PA strain fermentation broth under the original broth was significantly(P<0.05)higher than diluted 5 times.The result of touching test showed that the insect resistance of SN strain fermentation broth under the original broth was significantly(P<0.05)higher than diluted 5 times.However,there was no significant(P>0.05)effectiveness on insect resistance when fermentation broth diluted 10 times and 15 times in feeding test and touching test.The results offeeding test and touching test showed that the insect resistance of PA was significantly(P<0.05)higher than those of other seven strains.5.The fermentation conditions were optimized by single factor and orthogonal experiments,it was found that the contents of ergonovine,ergine,peramine,lolitrem B and mycelial biomass in JZ and PA fermentation broth before and after optimization were increased by 2.3,3.3,1.5,3.8,0.9 times and 3.3,1.7,1.2,2.5,0.7 times,respectively.