| Nomuraea rileyi is a well-known environmentally friendly fugal pathogen thrivingin a range of obligate hosts, particularly the noctuid pests (such as Spodopterafrugiperda, Spodoptera exigua, Helicoverpa armigera and Heliothis assulta)and can beused as a mycoinsecticide. However, the lack of reliable cost-effective protocols formass production of entomopathogenic fungus conidia and the low toxicity and storageof liquidspores limit its commercialization and application. Therefore, it’s necessary tofind another active agent for commercial production of the fungal pathogen. Themicrosclerotia (MS)are pseudoparenchymatous aggregation of hyphae, can be producedby many phytopathogenic fungi for persistence in the soil and decaying plant material.The stability of dried microsclerotia preparations and their potential to produce infectiveconidia when conditions are suitable for growth make this propagule a promising fungalform for use as a mycoinsecticide. The MS formation was successfully induced inN.rileyi.However, the molecular mechanism of MS development and the scale-upfermentation ofN. rileyi MS productionhave not been done.Based on the successfully induced of N. rileyi microsclerotia formation in liquidamended medium (AM) culture, we investigated the molecular mechanism of MSdevelopment, the optimization of MS formation, andthe optimization of submergedferemeation of MS production. The results suggest that the microsclerotia can be usedas a biocontrol agent of mycopesticide of N. rileyi andpromote the commercial scale-upand cost-effective of fermentation of this fungal pathogen.The main results were as follows:1) Optimization of culture medium for MS production by N. rileyi.To improve MS production, the culture medium was varied. Three quantitativevariables, carbon, nitrogen, and basal salt sources, were investigated using the AM. Anorthogonal experiment [L9(3)3] design was applied to optimize the culture medium.Maximum MS production (21.9×104MS/ml) was obtained using the optimized culturemedium (32.0g/l of glucose,2.0g/l of ammonium citrate, and0.15g/l of ferroussulfate). The influence of the medium constituents on MS yields decreased as follows:ferrous sulfate> glucose> ammonium chloride. Thus, treatment with ferrous sulfatewas noted to be the most important determinant of the MS yields. 2) Analysis of MS viability for use as a mycoinsecticide.The MS, collected from the optimized culture medium, exhibited virulence andgreater thermotolerance than conidia, and maintained86.4%germinability after1-yearroom temperature storage. These results suggested that the MS can be used as amycoinsecticide.3) Comparative transcriptome analysis of MS development.To study the molecular mechanism of microsclerotia development, comparetranscriptomes were analyzed using next-generation sequencing technology to find thegenes involved in microsclerotia development. A total of4.69Gb of clean nucleotidescomprising32,061sequences was obtained, and20,919sequences were annotated(about65%). Among the annotated sequences, only5,928were annotated with34geneontology (GO) functional categories, and12,778sequences were mapped to165pathways by searching against the Kyoto Encyclopedia of Genes and Genomes pathway(KEGG) database. Furthermore, we assessed the transcriptomic differences betweencultures grown in minimal and amended medium. In total,4,808sequences were foundto be differentially expressed;719differentially expressed unigenes were assigned to25GO classes and1,888differentially expressed unigenes were assigned to161KEGGpathways, including25enrichment pathways. Subsequently, we examined theup-regulation or uniquely expressed genes following amended medium treatment,which were also expressed on the enrichment pathway, and found that most of themparticipated in mediating oxidative stress homeostasis. To elucidate the role of oxidativestress in microsclerotia development, we analyzed the diversification of unigenes usingquantitative reverse transcription-PCR (RT-qPCR).Our findings suggest that oxidativestress occurs during microsclerotia development, along with a broad metabolic activitychange. Our data provide the most comprehensive sequence resource available for thestudy of the developmentof N. rileyimicrosclerotia.4) Identification of the functional gene of regulating the MS development.Transmembrane proteins, Sho1p and Sln1p, act as sensing growth stress andregulating cell signaling to respond. We first investigated the changing cultureconditions during N. rileyi MS development and these effects on MS differentiation. Onthe other hand, the sho1and sln1genes from N. rileyi were cloned and shown to encode306and1,118amino acid proteins, respectively. The transcription levels of sho1andsln1were up-regulated in response to the changing culture conditions. To determine thefunctions of sho1and sln1,gene-silencing mutants (sholRM, sln1RM, and shol&sln1RM, respectively) were generated using RNA silencing technology. The mutants weresensitive to osmotic pressure and oxidative stress and growth rates, conidial yields andvirulence were reduced. Furthermore, the mutants caused a noticeable decrease in MSyields. In conclusion,the results obtained suggested that sho1and sln1appear to sharesome essential functions in N. rileyi,including hyphal growth, conidiation, virulence,and sensing growth stress of regularly changed culture medium during N. rileyiMSdevelopment.5) Statistical optimization of process variables of N. rileyi MS.We first used two-level fraction design to confirm the factors, including shakerspeed, inoculum density, initial pH, and temperature, affecting N. rileyi MS production.Three factors were found to be important. Then, a23full factorial central compositedesign (CCD) and response surface methodology (RSM) were applied to determine theoptimal level of each variable. A second-order polynomial was determined and shakerspeed and inoculum density were found to be the uppermost factors.6) Development of mass scale production of N. rileyi MS.To develop an optimized process for the submerged fermentation, the variousinoculums, the different culture times of mycelium and conidium inoculum, and thevarious inoculum sizes, affecting N. rileyi MS yields and biomass, were investigated.The results showed that the N. rileyi MS yields were1.0×104MS/mL with inoculums,cultured in24-48hour and the mycelia could be used as the effective inoculums for onestep fermentation. Furthermore, the N. rileyi MS yields of fermentation in30litersbioreactor were optimized (150rpm in the previous three days and200rpm in thefollowing days; agitation rate of800L/h sterile air) and up to20-30%. The results willprovide scientific data for N. rileyi MS fermentation in large scale. |
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