Diversity And Ecological Function Of Endophytic Bacteria In The Fruiting Body Of Boletus

Ectomycorrhizae is a highly evolved symbiont between soil fungi and plant roots,and host plants absorb mineral nutrients from outside the roots through symbiotic fungal mycelia,while fungi obtain photosynthetic carbon assimilates from host plants.While ectomycorrhizal fungi are mutually symbiotic with their hosts,they also cooperate with ectomycorrhization helper bacteria(MHB)in other surrounding ecological niches.MHB not only involve the formation and function of ectomycorrhiza,but also related to the growth of fungi and the formation of fruiting bodies.Previous studies have found that ectomycorrhiza fungi fruiting body endofungal bacteria population exist obvious difference,fruiting body surface and underground mycorrhizal hyphae can provide a special niche for bacteria,under the chemotaxis of fungi secretion,the soil bacteria existing in the mycelium surface biofilm formation,gradually into the fruit body internal with the host fungi endosymbiosis.In addition,ectomycorrhizal fungi hyphae expand the absorption area of plant root hair,and release organic acids and phosphatase to dissolve soil mineral nutrients,which is an effective nutrient absorption system.However,previous studies have shown that ectomycorrhizal fungi have the problems of slow growth rate and limited ability to decompose insoluble phosphate in natural soil.Therefore,the selected endophytic bacterial population may play an important biological role in the fruiting body of ectomycorrhizal fungi.However,the synergistic effects of secondary metabolites of endophytic bacteria on the growth of fungi themselves and the promotion of soil nutrient uptake by plants with fungi remain to be further studied.The study aims to characterize the endofungal bacterial microbiota in three different boletes,including Tylopilus felleus,Tylopilus areolatus,and Boletus queletii,respectively,and explore the bacterial microbiota difference of diversity,composition,and function among fruiting bodies of boletes and mycosphere soil by high-throughput sequencing approach.Different boletes,including Tylopilus felleus,Tylopilus areolatus,and Boletus queletii harbored distinct endofungal bacterial microbiota,which was also separated from their corresponding mycosphere soil.And they were collected on the 36 and 59 islands of Qiandao Lake,Zhejiang Province in 2018.The relative abundance of Firmicutes in the fruiting bodies was much greater than that in the mycosphere soil,and Firmicutes was the dominant phylum of endofungal bacteria in the fruiting bodies.In addition,at class and genus level,the relative abundance of bacteria was significantly different.The results showed that the host identity of ectomycorrhizal fungus bolete could affect endofungal bacterial microbiota.In order to further explore the biological function of endofungal bacteria in the bolete fruiting bodies,and under the condition of pure culture of endophytic bacteria produce gas volatiles and metabolites in Suillus.grevillei fungus hyphal growth,the influence of promote the growth of fungal hyphae,and improve the content of soluble sugar,protein and starch in mycelium.In addition,in the interaction between plants,ectomycorrhizal fungi and endofungal bacteria,endofungal bacteria also participate in the absorption of nutrients by plants and assist fungal hyphae to promote the absorption of nutrients by plants to the soil.A total of 31 isolates from bolete fruiting bodies were obtained and identified to be 4 genera.The dominant genera were Bacillus(14 strains),Pseudomonas(12 strains),and Burkholderia.Through the experiment of bacterial volatiles-fungus co-culture,the results showed that B11(Bacillus pumilus strain NFS-Cap-1)and B12(Bacillus sp.strain CRE2)could promote the growth of mycelia,and increase the biomass and area of mycelia.The results of mass spectrometric analysis showed that it might be related to the specific volatile compounds of ethyl palmitate and 2,4-tert-butyl phenol in strain B11 and tetradecycloheptosiloxane in strain B12.The volatiles of strain B11 increased the contents of glucose and starch in mycelium,while the volatiles of strain B12 increased the contents of glucose,fructose and starch in mycelium.Through the experiment of bacterial metabolite-fungus co-culture,the results showed that the metabolites of Bacillus subtilis strain PK5-6 and Pseudomonas sp.strain 102515 had beneficial influence on mycelium growth,and increased the biomass and area of mycelia.The metabolites of Bacillus subtilis strain PK5-6increased the contents of glucose,fructose,sucrose and protein in the mycelia of Suillus.grevillei.The metabolites of Pseudomonas sp.strain 102515 increased the contents of sucrose and protein in the mycelia of Suillus.grevillei.Endofungal bacteria in the fruiting bodies assisted host fungi to promote the absorption and utilization of nutrient elements,which have the ability of nitrogen-fixation,phosphate solubilization,producing IAA and membranes.The results of co-culture of endofungal bacteria and fungal to dissolve inorganic and organic phosphate showed that the fungus itself did not have the ability to dissolve inorganic phosphorus,while the endofungal bacteria had the strong ability to dissolve inorganic phosphorus by secreting acidic substances.The amount of dissolved phosphorus in the culture medium was 30.07-60.81 mg/L,and the synergistic effect between fungi and endofungal bacteria was not significant.The ability of fungi and endofungal bacteria to dissolve lecithin alone was weak.The content of phosphorus in the culture medium inoculated with endofungal bacteria and fungi alone was 2.37 mg/L and 1.19 mg/L,respectively,while the average content of phosphorus in the culture medium after co-culture of endofungal bacteria and fungi was 7.91 mg/L.