Isolation,Identification And Bioactivity Of Entomogenous Fungi In Soils Of Hunan,Hubei,Henan And Hebei Provinces,China

Entomogenous fungi are important resources of microbial pesticide.Isolation,identification and collection of entomogenous fungi can enrich the sources of insecticidal active substances of microbial pesticides.The soil contains abundant fungal resources,which is a resource library for excavating entomogenous fungi.This paper is a part of the project of isolation and identification of soil entomogenous fungi in China.The soils of Hunan,Hubei,Henan,Hebei provinces were collected to isolate and identify the species of fungi in soil.There are few reports on the entomogenous fungi resources in these four provinces,and the four provinces have complex and diverse topography,large latitude spans,and diverse climate types.They are located in the Yellow River and Yangtze River basins,and their agricultural production is developed.In this study,soil samples from the above areas will be collected,and the entomogenous fungi will be isolated by selective medium.The morphological and molecular biology methods will be used to identify the strains and analyze the diversity of entomogenous fungi.Then selected representative strains to determine its biological activity against Phyllostachys striolata.The main results are as follows:(1)Total 245 soil samples were collected from four provinces,and 910 strains of fungi were isolated from 220 soil samples.The effective separation rate was 89.8%.Among them,233 strains of fungi were isolated from 59 soil samples in Hunan,with an isolation rate of91.5%;256 strains were isolated from 63 soil samples in Hubei,with an isolation rate of85.7%;181 strains were isolated from 70 soil samples in Henan,with an isolation rate of88.6%;240 strains were isolated from 53 soil samples in Hebei,with an isolation rate of94.3%.The average number of isolates per soil sample was 3.9 in Hunan,4.1 in Hubei,2.6in Henan,and 4.5 in Hebei.According to the vegetation type,290 fungi were isolated from arbor soil samples,the separation rate was 95.5%;307 from farmland soil samples,the separation rate was 88.2%;35 from bamboo forest soil samples,the separation rate was81.8%;278 from weed soil samples,the separation rate was 88%;the average number of isolates per soil sample was 4.4 from arbor,3.3 from farmland,3.2 from bamboo forest and3.7 from weeds.(2)According to morphological characteristics and ITS sequence analysis,429 strains were identified,belonging to 44 genera and 103 species.Among which there were 7 species and 12 species belonging to the known entomogenous fungi,including Purpureocillium lilacinum 110 strains,Metarhizium anisopliae 48 strains,Metarhizium robertsii 1 strain,M.marquandii 32 strains,Isaria fumosorosea 3 strains,I.farinosa 1 strain,I.javanica 1 strain,Beauveria bassiana 10 strains,Paecilomyces carneus 19 strains,P.tenuis 2 strains,Clonostachys rosea 4 strains,Pochonia chlamydosporia 5 strains.The average number of isolates per soil sample was 1.9 in Hunan,1.8 in Hubei,1.3 in Henan,and 2.1 in Hebei.The average number of isolates per soil sample was 1.8 from arbor,1.6 from farmland,1.9 from weeds and 1.4 from bamboo forests.(3)Under laboratory conditions,the biological activities of 96 non-entomogenous fungi strains and 12 entomogenous fungi against the adults of Phyllostachys striolata were determined by immersion method.Among non-entomogenous fungi,only 9 soil fungi,Aspergillus auricomus Aa H4601,Talaromyces verruculosus Tv H24S01,Fusarium solani Fs H5001,Lecanicillium saksenae Ls H29S05,A.granulosus Ag H3104,A.candidus Ac H3103,Monascus purpureus Mp H57S01,A.pseudoviridinutans Ap H3102 and A.oryzae Ao H28S02 had lower toxicity when the treatment concentration is 1×10~8spores/m L,corrected mortality rates were 45.24%,31.58%,28.07%,26.32%,26.19%,23.81%,22.81%,21.43%and 21.05%,respectively.Among the entomogenous fungi,I.fumosorosea If H6102and M.anisopliae Ma B15B01 had good activity when the treatment concentration was 1×10~8spores/m L,the corrected mortality rates were 67.89%and 23.56%.(4)Different photoperiods had effects on the growth and pathogenicity of I.fumosorosea If H6102.Under the whole light condition,the spores were the fastest and the spore activity was the strongest;under the whole dark condition,the mycelial growth of the colonies was the fastest and the spore activity was the weakest;under the light and dark alternate,sporulation and mycelial growth were in the middle,but its pathogenicity was the strongest.With the increase of I.fumosorosea bacterial liquid concentration,the lethality rate of Phyllotreta striolata was also higher and higher.(5)Scanning electron microscopy(SEM)showed that conidia of I.fumosorosea If H6102 were mainly adhered to intersegmental membranes and the dense parts of stiff hair on Phyllotreta striolata,such as antennae,foot joints,chest and pereiopoda.After 12 hours of treatment,conidia began to germinate and grow dental tube and appressorium;24 hours later,a large number of appressorium formed,and the strain began to invade the surface of onychophora;48 hours later,mycelia began to penetrate from the thinnest internode membrane on the surface of the body,and new conidia stalks and conidia grew;48 hours to72 hours,mycelia continued to grow and gradually covered the surface of Phyllotreta striolata.