Study On The Micro-environment From Sclerotia To Habitat Soil And Emulationally Ecological Cultivation Of Isaria Cicadae

In this paper,high-throughput sequencing technique was used to detect the microbial community composition of sclerotia,external mycelial layer covering ossified cicada,habitat soil(including soil adhering to surface of ossified cicada,soil 1 cm around ossified cicada,soil 2 cm around ossified cicada,soil 5 cm around ossified cicada)and without Isaria cicadae growing environment soil.Comparing the community composition of Isaria cicadae from inside to outside,detecting the core microbiome and ecological function in sclerotia,external mycelial layer covering ossified cicada and soil adhering to surface of ossified cicada.The correlation between soil physical and chemical indexes and core microbial community in Isaria cicadae was analyzed.In addition,the emulationally ecological cultivation of Isaria cicadae has been developed,and its cultivated effect was verified in Masson pine forest and tea plantation.The cultivated experiments were proceeded with using tussah pupa to replace the natural host cicada nymph of Isaria cicadae and imitating the growth process in its natural environment.The emulationally ecological cultivation technology was confirmed by screening of high-quality strains,inoculation method,inoculation concentration,soil-covering time and soil-covering thickness.The main conclusions in this paper are as follows:(1)The diversity and richness of microbial community in habitat soil were significantly higher than those in sclerotia and external mycelial layer covering ossified cicada.43,58 and 260 known fungal genera and 341,291 and 308 known bacterial genera were detected in sclerotia,external mycelial layer covering ossified cicada and soil adhering to surface of ossified cicada,respectively.The microbial community structure of sclerotia and external mycelial layer covering ossified cicada was similar,in which there were a few colonized fungi and bacteria.In sclerotia,the dominant fungal genus was Isaria,and dominant bacterial genera were Pseudomonas and Serratia.In external mycelial layer covering ossified cicada,the dominant fungal genus was Isaria,dominant bacterial genus was Pseudomonas,and Sphingobacterium,Ensifer,Aminobacter also had a highly relative abundance.Fungus of Isaria,Mortierella,Beauveria,Cryptococcus,Penicillium and bacteria of Bradyrhizobium,unknown genera of Acidobacteria and Xanthobacteria had a highly relative abundance in soil adhering to surface of ossified cicada.Pseudomonas and Serratia in sclerotia might originate from cicada,which could be involved in the infection process of Isaria cicadae.(2)10,5 and 20 core fungi OTU were detected in sclerotia,external mycelial layer covering ossified cicada and soil adhering to surface of ossified cicada,respectively.11 ecologically functional groups were annotated,including ectomycorrhizal fungi,undefined saprophytic fungi,animal pathogens,fungal parasite,litter saprotroph,dung saprotroph,endophyte,plant pathogens,arbuscular mycorrhizal fungi,plant saprophytic fungi and wood saprotroph.26,12,56 core bacteria OTU were detected in sclerotia,external mycelial layer covering ossified cicada and soil adhering to surface of ossified cicada,respectively.Function of bacterial community in sclerotia was mainly related to pathogen,nitrogen cycle,chemoheterotrophy,inorganic and organic compound degradation.Function of bacterial community in external mycelial layer covering ossified cicada was mainly related to nitrogen cycle,chemoheterotrophy,inorganic and organic compound degradation.Function of bacterial community in soil adhering to surface of ossified cicada was mainly related to carbon cycle,nitrogen cycle,chemoheterotrophy,inorganic and organic compound degradation.It is speculated that the nitrogen cycle in vivo of Isaria cicadae and carbon cycle in vitro are related to the exchange of nutrient elements.(3)The main soil factors affecting the microbial community of Isaria cicadae were total nitrogen(TN),total phosphorus(TP)and total potassium(TK).Isaria in sclerotia,external mycelial layer covering ossified cicada and soil adhering to surface of ossified cicada was negatively correlated with soil TK and positively correlated with TP and total organic carbon(TOC).(4)The tussah pupae were inoculated with 5×10~7 conidia/m L spore suspension of Isaria cicadae.After 3 days of culture,the ossified pupae were cultured with soil covering 1 cm thick for 35-45 d,and mature Isaria cicadae would be harvested.The whole process is the emulationally ecological cultivation technology obtained from this research.The main active substances(such as polysaccharide and adenosine,cordycepin and mannitol)of Isaria cicadae fruit body obtained by this cultivation method were similar to those of wild,and their amino acid content was significantly higher than that of wild.In addition,the cultivation effect was verified in Masson pine forest and tea plantation.It was found that the rate of growing fruit body reached 82±12%and 89±5%respectively,indicating that the cultivation method could be cultivated in the natural environment.The emulationally ecological cultivation from this research can provide support for the study of the infection mechanism of Isaria cicadae and the transformation of nutrient elements between Isaria cicadae and soil.