Induction Culture Microsclerotia Of Nomuraea Rileyi And Cloning And Expression Analysis Of An Hydrophobin Gene Nrhyd In Nomuraea Rileyi

Entomopathogenic fungi play an important role in biological control of insects.The development of research on basis and application is very rapid due to its advantageof pest control and maintenance of species diversity. Nomuraea rileyi is anentomopathogenic fungi capable of infecting several lepidopterous pests and is able tocause desease in natural environment. Aerial conidia is active ingredients of fungalbiocontrol agents because fungal infect insect by it. Because of the specificrequirements of N. rileyi, attempts to develop cost-effective, rapid multiplicationprotocols have met with limited success. The research was done on the two sides: one isto clone genes involved in sporulation, the other is to develop new formulation to takethe place of aerial conidia. The results are as follows:①The experimental results show the new isolated strain is Nomuraea rileyi. Wenamed it for SDNr02.②The full-length cDNA of a hydrophobin gene Nrhyd gene was cloned from theinsect pathogenic fungus Nomuraea rileyi using SMART RACE RT-PCR. The Basicphysicochemical properties and protein structure of deduced amino acid was analyzedusing SMART and Expasy software and phylogenetic tree was made by softwareClustalx and MEGA. Sequence analysis showed that the cDNA contained an openreading frame of339bp,132bp5’ noncoding region,262bp3’ noncoding region and a29bp signal added poly (A). Analysis of the structure and function domain of Nrhyd andprediction of Signal peptide indicated5’ region was integrated. Sequence analysis showsthat Nrhyd encoding a polypeptide with111amino acids. The precursor protein has amolecular mass of10.6and a calculated pI of6.19. It contains an Functional Domainsof HYDRO（43－106amino acid residues）, Prediction of Signal peptide indicated theprotein contain18amino acid residues. Phylogenetic tree shows that Nrhyd ofNomuraea rileyi had highest genetic relationship with Metarhizium acridum CQMa102,Secondly Metarhizium anisopliae.③The expression of Nrhyd gene by Nomuraea rileyi under different cultureconditions was analyzed using Realtime-PCR. The RT-qPCR results showed that theexpression of Nrhyd gene was consistent with the Dynamic spore yield. It wassuppressed under liquid culture condition. While under solid agar culture condition, theexpression level of Nrhyd gene increased along with the formation of conidia and reached the highest level8d post inoculation, and then decreased gradually.④Two strains of Nomuraea rileyi formed MS in shake flask cultures using mediawith varying carbon concentrations and carbon-to-nitrogen (C:N) ratios. Under theconditions of this study, the two strains both produced MS, compact hyphal aggregatesthat became pigmented with culture age in addition to more mycelia.⑤The forming of MS was affected by carbon concentrations andcarbon-to-nitrogen ratios. While two strains formed desiccation tolerant MS, liquidmedium of low carbon concentrations produced more MS than high carbonconcentrations medium. CQNr01and SDNr02produced1.3×104MS/mL，1.2×104MS/mL respectively in poor medium with C:N ratios of4:1,While they produced0.8×102MS/mL，0.78×102MS/mL in rich medium with C:N ratios of20:1. The two strainsboth produced higher biomass concentrations in rich medium than they did in poormedium with the same C:N ratios. In rich medium with C:N ratios of20:1, CQNr01andSDNr02produced biomass concentrations37mg/mL，38mg/mL respectively. In poormedium with C:N ratios of20:1, they produced biomass concentrations30mg/mL,29mg/mL respectively.⑥Microsclerotial granules of N. rileyi containing diatomaceous earth survivedafter air-drying (to <5%moisture) with no significant loss in viability. Rehydration andincubation of air-dried MS granules on water agar plates resulted in hyphal germination2days later and sporogenic germination to produce high concentrations of conidia8days later. Bioassays using air-dried MS preparations resulted in significant infectionand mortality in larvae of the Spodoptera litura. The mortality was more than80%twodays after inoculation.Conclusion: Nrhyd gene might play an important role in the formation of conidia in N.rileyi. The expression level of Nrhyd gene is affected by the environmental conditions.We succesfully induced Nomuraea rileyi to produce microsclerotia. The forming of MSis affected by carbon concentrations and carbon-to-nitrogen ratios. Bioassays usingair-dried MS preparations resulted in significant infection and mortality in larvae of theSpodoptera litura.