Virulence And Pathology Of Three Strains Of Beauveria Bassiana Against Mythimna Separata

Mythimna separata（Walker）was listed as one of the major pests in the agricultural development of China as early as the 1950s.The larvae of armyworm are widely distributed in the world,they feed on more than 300 plant types in 14 families and seriously do harm to crops.Beauveria bassiana is the first entomogenous fungus to be discovered.It is pathogenic to many agricultural and forestry pests.However,armyworm is mainly controlled by chemical pesticides at present.It is easy to produce more serious drug resistance and cause environmental pollution,thus it is dangerous to human and animal health.Therefore,it is urgent to use B.bassiana for biological control of armyworm.In this study,entomopathogenic fungi were isolated from soil using Tenebrio molitor larvae as bait and three strains of B.bassiana were obtained by morphological observation and molecular identification.The pathogenesis of Z-G2-5,a highly virulent strain,to the larva of M.separata was studied.The main results were as follows:（1）The results of strains identification:Through the construction of phylogenetic tree,it was found that the three strains isolated and selected from the soil were in the smallest branch with the closest genetic relationship and the highest homology with several B.bassiana strains.Combined with morphological characteristics,the above three strains were identified as B.bassiana,and named Z-G2-5,M-G3 and M-G4 in turn.（2）Virulence of three strains of Beauveria bassiana:The three strains of B.bassiana can infect the larvae of M.separata effectively.Their virulence to the 3rd instar larvae was different.At the concentration of 5×10⁸spores/m L,the cumulative corrected mortality of strain Z-G2-5 reaches the maximum at the 6th day,which is 96.77%.The LT₅₀value was3.02 d,and its LC₅₀value was 4.95×10⁶spores/m L.But the cumulative mortality of M-G3and M-G4 strains were only 66.67%and 71.11%.The LT₅₀values were 4.89 d and 3.97 d and the LC₅₀values were 1.18×10⁸spores/m L and 1.13×10⁷spores/m L,respectively.Strain Z-G2-5 had stronger pathogenicity and faster lethal rate to armyworm larvae than strain M-G3 and M-G4.Commercialized B.bassiana preparation had high pathogenicity to armyworm larvae only at a high concentration（10⁹spores/m L）,and the highest lethality rate reached 75%.At low concentration（10⁸spores/m L or less）,the lethality rate was less than20%,indicating that the strain Z-G2-5 is more virulent to armyworm larvae than the biological preparation of B.bassiana.under the same conditions.（3）Histopathology of the Mythimna separata infected by strain Z-G2-5:By scanning electron microscopy and tissue sections,results showed that conidia of strain Z-G2-5 could attach to most areas of the integument of armyworm larvae,and were mainly distributed in the parts containing hair such as the spiracle,vestigial bristle and appendages.Spores or hyphae can enter the body of the armyworm directly through the spiracle or cuticle,resulting in the death of the armyworm.At the early stage of invasion（1 d after infection）,conidia began to germinate.Some of the conidia at the integument had a faster germination rate and could generate hyphae attaching to the surface,while some of the hyphae generated appressoria.2 d after infection,the hyphae began to grow quickly and could penetrate the epidermis entering the subcutaneous tissue.Some of the hyphae extended to the inside of the spiracle and then proliferated rapidly in the cavity,resulting in melanism in spiracle.Some of the hyphae invaded through the spiracle and the roots of vestigial bristle.After 3 days of infection,the hyphae grew wildly and interlaced with each other to form a network structure covering the surface of the insect body.The conidia entered the coelomic cavity through the curticle of midgut and germinated a large number of hyphae,which continue to proliferate and invade the surrounding organs such as fat body,muscle,digestive tract and curticle,making them decompose and broken.6 d after infection,lots of hyphae invaded the head,malpighian tube and hindgut of armyworm,and a large area of melanism appeared.Finally,the whole coelomic cavity,including the curticle of the body,was surrounded by hyphae,and lots of conidia were produced.（4）Molecular pathology of the Mythimna separata infected by strain Z-G2-5:Transcriptome sequencing results showed that a total of 2930 differentially expressed genes（DEGs）were obtained on the first day of infection,among which 894 genes were down-regulated and 2036 genes were up-regulated.There were 250 down-regulated genes and 1288 up-regulated genes for 3 d infection.According to Unigenes annotation,73 and 91immune-related genes which may affect the virulence of fungi were excavated after 1 d and3 d infection,and they were classified into four categories,including signal recognition genes,signal modulation and amplification genes,signal transduction genes and immune effector.The signal recognition genes includeβ-1,3-glucan-binding protein（β-GBP）,peptidoglycan-recognition protein（PGRP）and C-type lectin（CTL）.Signal modulation and amplification genes include serine protease（SP）and serine protease inhibitor（Serpin）.Signal transduction genes include spaetzle protein,toll protein,glutathione S-transferase（GST）,nuclear factor NF-kappa-B and transcription inhibitor cactus.Immune effectors include lysozyme and antibacterial peptides.The q RT-PCR results showed that:The relative expression levels of the five DEGs Attacin2、SP7、Cecropin C、β-GBP、Serpin were all up regulated after1 d and 3 d infection of strain Z-G2-5,which were basically consistent with the results of transcriptome sequencing.These genes are significantly expressed after fungal infection,which are closely related to the virulence of fungi to insects,and can be used as target genes for detecting the virulence of fungi.Compared with the commercial B.biologics,the relative expression levels of Attacin2、SP7、Cecropin C、β-GBP、Serpin in group infected by the strain Z-G2-5 were more significant.The above results indicated that the B.bassiana strain Z-G2-5 had strong pathogenicity to M.separata,Combined with histopathological and molecular pathology data,the results showed that the B.bassiana strain Z-G2-5 was more pathogenetic than the commercial insecticide,and it can be used as a candidate strain with potential application value for biological control of M.separata.