Study On The Microbial Community Structure Of Fungus-comb And The Culture Of Purified Termitomyces In Vitro

Termitomyces spp.is a precious wild edible fungus,which is only growing on ant nests（ie"fungus-comb"）.The fungus-comb formed by interaction of termites,Termitomyces and the microbial of fungus-comb collaborative development.The mycelium of Termitomyces develops into fruit bodies under suitable conditions.This process shows that the Termitomyces spp.forms a complex symbiotic relationship with the microbes of fungus-comb,which means that the symbiotic microbes play a vital role in the growth of Termitomyces spp..This study analyzed the macrogenomics of the fungus-comb and its surrounding soil by the 16S rDNA high-throughput sequencing techniques,and to identify the bacterial community structure and its differences between fungus-comb and its surrounding soil,and to provide background support for related microecological studies;2)In order to clarify the main group of culturable microorganisms in the fungus-comb,the microorganisms in the fungus-comb are separated and purified;3)The Termitomyces spp.were isolated,purified and identified.Study the biological characteristics and functions of fungal and in order to obtain a large number of mycelium,we optimize the culture conditions of the fungus（especially the liquid culture conditions）,and lay a foundation for the domestication and utilization of the Termitomyces spp.The main results of this study are as follows.（1）Analysis of microbial diversity of fungus-comb and near to the soil of fungus-comb:The 16S rDNA high-throughput sequencing technique was used to analyze the microbial community structure of fungus-comb and its surrounding soil.Theαdiversity index of microbial showed that the abundance and diversity of bacteria in the fungus-comb and comb-wall were lower than the environmental soil.In all samples,there were 11dominant gates with sequence abundance greater than 1%,accounting for approximately 97%of the subjects,including Proteobacteria,Actinobacteria,Acidobacteria,Chloroflexi,Firmicutes,Nitrospirae,Gemmatimonadetes,Latescibacteria,Bacteroidetes,Planctomycetes,Verrucomicrobia.Except for the Firmicutes,Actinobacteria,and Bacteroidetes,the total abundance of the other eight phylum are gradually decreased in the above order in the sample.Proteobacteria is the main dominant phylum of all samples,and its relative abundance in the fungus-comb and comb-wall is significantly higher than that in the environmental soil,and the relative abundance of fungus-comb and comb-wall is 65.19%and 70.11%,respectively.The relative abundances ofα-Proteobacteria andβ-Proteobacteria were more than 67%and 56%in the fungus-comb and comb-wall,respectively,which was significantly higher than that the environmental soil which the abundance of the two were 14.87%and6.94%).And the relative abundance of Bradyrhizobium that was inα-Proteobacteria was only high in fungus-comb which was 24.82%.The relative abundance of Burkholderia-Para Burkholderia that inβ-Proteobacteria was higher in fungus-comb and comb-wall than the environmental soil,and their relative abundance is 43.89%and 37.58%,respectively.Other dominant bacteria include Actinobacteria,Acidobacteria,Chloroflexi,and Firmicutes.Actinobacteria,Acidobacteria,Chloroflexi,and their relative abundance in environmental soils were not significantly different,and significantly higher than the fungus-comb and comb-wall.The relative abundances in environmental soils,the fungus-comb and comb-wall were 18.18±1.48%,11.44%,5.45%;17.27±1.15%,4.88%,3.66%;14.77±1.91%,8.14%,and 6.02%.Compared with other samples,the relative abundance of Firmicutes which in fungus-comb was the highest are 11.07%,and the relative abundance of Clostridia was the highest.There are 15 OTUs with high abundance in environmental soils,mostly Proteobacteria and Actinomycetes.There are 22 OTUs with high abundance in the wall of the cavity,and there are 15 OTUs in total.Among them,9 OTUs are Burkholderia-Para Burkholderia genus,and Actinomycetes have 4 OTUs.There are 8 OTUs with high abundance in the Bacillus,2OTUs from the green cloaca,3 OTUs from Proteobacteria,and OTU2881（Bradyrhizobium）and OTU2694（Burkholderia-Paraburkholderia）.There are 2 OTUs from the thick-walled bacteria,OTU2713（Bacillus）and OTU2884（Lachnospiraceae）.（2）Isolation and preliminary identification of microorganisms in the fungus-comb:The cultivable microorganisms in the fungus-comb were separated,and 383 strains of bacteria and actinomycetes were obtained,of which 339 were Gram-positive and 44 were Gram-negative;47 filamentous fungi were obtained.According to the morphological characteristics of the strain,there were 43 strains,including 17 strains of Trichoderma,6 strains of Aspergillus,3 strains of Xylaria and 5 strains of Penicillium.3 strains of Alternaria;3 strains of Fusarium;1 strain of Streptomyces;1 strain of Alpina;1 strain of Mucoraceae;1 strain of Truncatells;1 strain of Clodosporium;1 strain of Termitomyces,and 4 strains were not identified.（3）Characteristics of in vitro culture of Termitomyces spp.:In the solid culture condition,the morphological observation and physiological were developed and white,and the convex part of the bacterium had a transparent or yellow droplet,and the droplet contained a large number of elliptical spores.As time goes by,the liquid gradually solidifies,and the gelatinous part is mainly composed of ethnic elliptic conidia,and then develops into snow-white mature hyphae,which was slightness,little diaphragm,rod shaped at the top,finally the hyphae are yellow.The morphology of fungi in the pre-fermentation period（4d）under liquid culture conditions have burrs,and the hyphae are coarse and the cross-section is obvious.The hyphae which was fermented for 7 days became slender and the diaphragm was reduced.tight and elliptical,with few branches.At this time,the hyphae were slender and the cross-section was small.（4）Optimization of in vitro culture conditions of Termitomyces spp.:Under the condition of solid culture,the optimum value of the growth of the mycelial growth of the fungal was studied by single factor experiment.It was found that the optimal carbon source for the growth of the mycelium of G.faecalis was glucose,the nitrogen source was peptone,and the magnesium sulfate was 2.0 g/L.Potassium dihydrogen phosphate 2.5 g/L,p H 4.0,culture temperature 28°C.The previous three factors have a great influence.The combination of these experiments showed that the mycelium of G.faecalis grew best with glucose 20g/L,peptone 2.0g/L and magnesium sulfate 2.0g/L,which was 1.41mm.d^-1.Under the condition of liquid culture,the optimum value of the growth of the mycelial growth of the fungal was determined by single factor experiment.It was found that the optimum carbon source for the growth of the mycelium of the genus Mycelia was glucose,the nitrogen source was soybean meal,and the magnesium sulfate was 1.0g/L.Potassium dihydrogen phosphate 1.0 g/L,p H 6.0,culture temperature 28°C.The carbon source,magnesium sulfate and p H were greatly affected,and they were combined.The results showed that the mycelium of fungal broth was the highest in soybean meal,magnesium sulfate 1.5g/L and p H 6.0,which was 11.6g/L.