Screening For Pathogenic Fungi Of Hyphantria Cunea And Its Immune Response To Beauveria Bassiana Infection

Hyphantria cunea(Drury)(Lepidoptera: Arctiidae)is a severe economic pest that originated in North America,which is second only to pine wood nematode in damage.At present,the control for H.cunea is mainly depended on chemical pesticide.Beauveria bassiana is one of the most widely studied insect pathogen fungi in the biological control.The study on the interaction between pathogenic fungi and it’s hosts is an important tissue for the biological control of pest insects.Based on the above background,this paper intends to carry out the following research on the H.cunea: Isolation and acquisition for pathogenic fungal strains of H.cunea by field collection,evaluate pathogenic fungi of biological properties and obtain highly pathogenic strains by bioassay.On this basis,the study focused on the changes of immune genes related to H.cunea after infection by pathogenic fungi,and verified and analyzed the changes of key immune genes.The main results are as follows:(1)Combined with morphological and molecular identification,three strains of Beauveria bassiana were obtained and stored separately,numbered RCEF6669,RCEF6670 and RCEF6671.After the comparison of biological properties and bioassay of three B.bassiana,it was found that the growth rate of strain RCEF6670 was 3.54mm/d,the average spore yield reached 1.70×108 spores/cm2,and the spore germination rate reached 36.64%after UV irradiation,which was the highest among the three strains.The lethal time(LT50)of H.cunea infected by RCEF6670 at 1×107 spores/mL was 2.93,which is the shortest among the 3 strains.Therefore,RCEF6670 was identified as a highly pathogenic strain of H.cunea.(2)After Tween-80 and B.bassiana RCEF6670 spore suspension treatment of fourth instar larvae of H.cunea,four cDNA libraries were constructed from tissue samples of control group and infection group at 6 h,48 h and 72 h.A total of 43,169 transcripts were obtained by Illumian high-throughput sequencing,and 33,141 unigenes were obtained by de-redundant processing.Cluster analysis of differential expressed genes showed that 1,036 genes were significantly up-regulated and 359 genes were significantly down-regulated after6 hours of B.bassiana infection,2,346 genes were significantly up-regulated and 468 genes were significantly down-regulated 48 hours after infection,and finally 3,205 genes were significantly up-regulated and 476 genes were significantly down-regulated 72 hours after infection.KEGG enrichment analysis of differential genes showed that genes related to energy metabolism and carbohydrate metabolism were significantly up-regulated after fungal infection,as were immune-related signaling molecules.(3)Based on sequence similarity search and homology analysis,226 immune-related genes were identified,including pattern recognition receptors,signal regulators,effector molecules and almost all members of Toll,IMD,JAK/STAT signaling pathway.It was found that PGRPs,β-GRPs,cSP and the whole Toll pathway were activated in large quantities induced by fungi.(4)Based on the level of gene expression,the expression profiles of Toll pathway and IMD pathway were constructed,and it was confirmed that Toll pathways played a major role after infection,and the expression changes of activated executive recognition functions(PGRP-S2,β GRP2 III,etc.),signal transduction(cSP6,Spz2,etc.)and signal regulated genes were up-regulated at first and then down-regulated after infection.However,the genes related to the activated effector factors(Diapasue1,Defensin,Lys1,Lys2,etc.)are continuously up-regulated after activation to deal with the infection of B.bassiana.Finally,the key immune genes after fungal infection were verified by qRT-PCR.The results showed that PGRP-S2,βGRP2,Toll4,Spz2,Defensin and PPO were significantly up-regulated after fungal infection.The results of this study laid a foundation for further elucidating the molecular mechanism of the interaction between host and pathogen.At the same time,our research will provide a scientific basis for the prevention and control of pathogenic fungi and the development of specific molecular targets.