Identification Of The Entomopathogenic Fungus Cordyceps Javanica BE01 Isolated From Hyphantria Cunea And Study On Its Pathogenic Mechanism

Hyphantria cunea is a worldwide pest.Since it was introduced into China in 1979,it has caused serious harm to China’s forestry and agriculture.Numerous studies have shown that entomopathogenic fungi play an important role in pest control.At present,the most studied entomopathogenic fungi are mainly Beauveria bassiana and Metarhizium anisopliae.In addition,fungi of the genus Cordyceps have great potential in pest control.Cordyceps belongs to Ascomycota,Pezizomycotina,Sordariomycetes,Hypocrellales and Cordycetaceae.The fungi of genus Cordyceps are a globally distributed group that can control more than 40 species of insects from 8 orders.The most widely used ones in this genus are Cordyceps farinosa and Cordyceps fumosorosea.This paper screened and identified a pathogenic fungus from the mycelium present on the surfaces of the cadavers,and studied its effect and pathogenic mechanism.The main research results are as follows:1.Screening and identification and biological characterization of pathogenic bacteria of H.cunea.Nine strains of fungi screened from the the surfaces of the cadavers were respectively formulated into a conidia suspension with a concentration of 1×10 ⁷ conidia/ml,and sprayed on the body surface of the fourth instar larvae of H.cunea.Only one fungus caused the death of the larvae of H.cunea（mortality:66.67%）and was marked BE01.Through morphological and molecular identification,the strain was identified as Cordyceps javanica.After biological characteristics determination,in SDAY medium,C.javanica BE01 grew the fastest at 25℃,and the conidia yield was the highest at 30℃.The germination rate was the highest in the germination solution containing2%glucose,2%glucose and 1%tryptone,2%sucrose and 1%tryptone.The optimum conidia germination temperature was 25℃～30℃,and the optimum conidia germination p H was at p H 5.0.The optimum relative humidity for conidia germination was 100%2.Determination of the virulence of the pathogenic fungus of the H.cunea,C.javanica BE01.By spraying the BE01 conidia solution with a concentration of 1×10 ⁴～1×10 ⁸ conidia/ml onto the body surface of the 3^rd,4^th and 5^th instar larvae,the virulence of BE01 were analyzed.After 15 days of treatment,under the treatment of BE01 with a concentration of 1×10 ⁸ conidia/ml,the mortality rates of the 3^rd,4^th and 5^th instar larvae were all over 73%,and the LT ₅₀ was lower than 4.9 d.The study found that conidia concentration had a significant effect on larval mortality and LT₅₀ of H.cunea,but the instar had no significant effect.However,the effect of conidia concentration on mortality was not significant at 10 d to 14 d after treatment at lower conidia concentrations（1×10⁴to 1×10⁶ conidia/m L）,when instar was the key to mortality of H.cunea larvae.In addition,instar was also able to significantly affect mortality at the early stage（1-5 d）of treatment with higher conidia concentrations（1×10⁷to 1×10⁸ conidia/m L）.3.Genome sequencing of BE01.In this study,the genome of C.javanica BE01 was assembled based on Pac Bio and Illumina sequencing data.The genome size of C.javanica BE01was finally determined to be 35.90 Mb,Contig of N50 and Scaffold of N50 were 3,203 Kb and 4,774Kb in BE01,respectively.And the assembled BE01 had good continuity.A total of 10,540 protein-coding genes were annotated in BE01,of which 10,197（96.74%）were functionally annotated.In addition,repetitive sequences account for 3.5%of the entire genome of BE01（1,267 Kb）.In conclusion,this study obtained a relatively complete and high-quality BE01 genome.4.Screening of BE01 potentially pathogenic-related genes.First,10 strains of family Cordycipitaceae（9 strains of Cordyceps,1 strain of Beauveria）including BE01 and one strain M.anisopliae of family Clavicipitaceae were performed for comparative genomic analysis.Through gene family clustering analysis,11 species were clustered into 10815 gene families,and there were5201 gene families in common.Second,based on the CAFésoftware,the families found to be expanded by BE01 were mainly annotated into the functional categories of"Posttranslational modification,protein turnover,chaperones",and the families with the most genes annotated were"pathogenicity-associated subtilisin-like proteases"and"protease S 8 tripeptidyl peptidase I".These two types of proteases can play a role in the infection process of entomopathogenic fungi.This suggests that BE01 may have enhanced its function in infecting hosts during evolution.Last,Go enrichment analysis of the core genes in the subtilisin-like and tripeptidyl peptidase I families with more expansion genes showed that the core genes of these two families were enriched in the serine hydrolase activity term.The proteins of the two families may play a role in degrading the insect body wall when BE01 infects the host,thereby helping BE01 to infect the host.In this study,CJPRB（Pr1 A）,which is the core gene of the subtilisin-like protease family and reported to have the highest activity,and CJPRB1（Pr1 H）,which was recently found to play a role in fungal infection,tripeptidyl peptidase I CJCLN2-1 acquired by BE01 during evolution were selected in the evolutionary process of BE01.The RT-q PCR results of different stages of BE01-infected H.cunea indicated that CJPRB,CJPRB1 and CJCLN2-1 may continue to play a role in the infection process of BE01.5.Construct a polyethylene glycol（PEG）-mediated genetic transformation system of BE01and to study the functions of CJPRB,CJPRB1 and CJCLN2-1.Choose 800μg/m L G418 as the concentration of screening transformants.Mixed enzymes（driselase、lysing enzyme and chitinase）enzymatically hydrolyzed BE01 mycelium for 3 h at 25°C and 60 rpm to obtain sufficient protoplasts for subsequent transformation at a concentration of 1.31×10⁷ protoplasts/ml.In addition,studies have shown that the molecular weight and concentration of PEG,the number of plasmids and the recovery time all affect the transformation efficiency.Using 20%PEG2000 to transfer 2μg of plasmid into 1000 protoplasts,and recover for 6 h,the transformation efficiency was 12.33±1.42transformants/μg plasmid.The overexpression strain was obtained by RT-q PCR verification.The phenotype verification of 3 overexpression strains and 1 GFP plasmid transformed strain showed that the overexpression of 3 genes and the plasmid itself did not affect the growth,sporulation and conidia germination of BE01.Afterwards,bioassays of 5 strains（including wild type）were carried out,and the results showed that there was no significant difference in the mortality of these 5 strains after infecting the larvae of H.cunea larvae for 15 days.However,the LT₅₀of CJPRB,CJPRB1 and CJCLN2-1 overexpression strains were 1.32-fold,2.21-fold and 2.14-fold shorter than those of the wild type,respectively.This suggests that these 3 genes may have accelerated BE01 infestation of the H.cunea.6.Expression of recombinant CJPRB protein by Pichia pastoris and protein bioassay.The results showed that the highest concentration of CJPRB pure protein was 1166μg/m L after 4 days of induction with 1%methanol（v/v）,and the enzyme activity was 1 80.2 U/m L.After 3d induction,1021μg/ml of GFP pure protein can be obtained.CJPRB protein impregnated,fed and injected with H.cunea was able to elicit responses of protective enzymes（SOD,POD,CAT）and the enzymatic activity of polyphenol oxidase PPO was higher than the control throughout the observation period.Validation by RT-q PCR showed that CJPRB could induce immune responses in insects.The expression patterns of immune-related genes were consistent with the process of insect clearance of pathogens after CJPRB infiltration.In addition,CJPRB protein feeding to H.cunea induced the expression of antimicrobial peptide CEA and chitin deacetylase（CDA1 and CDA2）in midgut cells.Injection of CJPRB protein into the hemocoel of H.cunea was able to trigger a more extensive immune response in the insects than other methods,as evidenced by the upregulation of the expression of most immune defense-related genes for most of the post-injection period.7.Screening and identification of CJPRB protein interacting with H.cunea.Recombinant pure proteins CJPRB and GFP were injected into the hemocoel,and total proteins were extracted at different time periods after injection.The protein captured by the His-beads was detected by Western blot,the gel strips of the detected CJPRB protein and GFP protein were identified by mass spectrometry.After removing the common in GFP and some muscle heavy and light chain proteins,as well as the lack of complete CDS region and the proteins that cannot be amplified,11 CJPRB potential interacting proteins were identified.But the final yeast two-hybrid experiment showed that none of these 11 proteins could interact with CJPRB in vitro.Based on the above results,a strain of C.javanica BE01 was screened and identified in this study.Through genome sequencing and comparative genomic analysis,potential pathogenic related proteins CJPRB,CJPRB 1 and CJCLN2-1 were obtained as the follow-up research objects.A genetic transformation system was constructed to overexpress three candidate genes which were found to accelerate the rate of infestation of H.cunea by C.javanica BE01.Finally,the bioassay experiment of pure CJPRB protein found that CJPRB can play a role in insect epidermis,intestine and hemocoel,and induce immune response in insects.