Multi-Omics Analysis Of Russula Griseocarnosa And Its Interaction With Mycosphere Microorganisms

Russula griseocarnosa is an important edible and medicinal ectomycorrhizal fungi,which can form mycorrhizal symbiosis with Castanopsis,Quercus and other Fagaceae plants,and it cannot be cultivated artificially at present.In this study,the fresh fruiting bodies and mycosphere soil of Russula griseocarnosa were collected in the field.The whole genome sequencing analysis of R.griseocarnosa was conducted to understand the gene composition and potential function of R.griseocarnosa.Transcriptome sequencing and metabolome analysis were performed on different tissues of R.griseocarnosa to understand the differential expression of metabolites and related genes,revealing their related nutrient metabolism and symbiotic mechanism.We also carried out microbial(fungi and bacteria)diversity analysis on the mycosphere soil and bulk soil of R.griseocarnosa,combined with the relevant soil physicochemical properties,to find out beneficial mycorrhizal helper bacteria(MHB)and growth factors of R.griseocarnosa.Metagenomic sequencing analysis was performed on mycosphere and bulk soil of the typical sites to find out the growth-associated microbes and corresponding functions,and explore the cooperative symbiotic mechanism of R.griseocarnosa and mycosphere microorganisms.The objective of this study aims to understand the potential function,nutritional and medicinal values of R.griseocarnosa and its symbiotic mechanism,and provide a theoretical basis for the sustainable use of R.griseocarnosa.The results are as follows:(1)The assembly process of the whole genome sequencing of R.griseocarnosa resulted in471 scaffolds with a total genome size of 64.81 Mb,a GC content of 49.41%,and a total of16128 protein-coding genes.There are a large number of genes related to nutrient metabolism,medicinal function in the genome of R.griseocarnosa genome,as well as carbohydrate enzyme(CAZys)genes related to symbiotic degradation plants,fungi and bacterial cell wall enzymes.Based on the whole genome sequences of R.griseocarnosa and Macrofungi published genome,phylogenetic analysis showed that the R.griseocarnosa was closedly related to Heterobasidion irregulare and Stereum hirsutum.The mitochondrial genome size of R.griseocarnosa was60995 bp,encoding 53 genes,including 14 protein-coding genes.(2)A total of 17,911 genes were obtained from the transcriptome sequencing and 709 metabolites were detected from different tissues(ectomycorrhizal,stipe,lamellae and pileus).Among them,some genes and metabolites were related to the formation of the mycorrhizal symbiosis of R.griseocarnosa and its host.It was speculated that gene04979 and gene07798,D(+)-Glucose,N-Acetyl-D-glucosamine and D-(+)-Mannose might be related to cell wall degradation and synthesis of oligosaccharides during mycorrhizal symbiosis,which can be recognized by the hosts and activate general symbiotic signaling pathway.It was also speculated that gene01852,N-hexosyl-p-coumaroyl serotonin and N-Feruloyl tryptamine might be related to auxin synthesis.Auxin can inhibit the growth of host roots and induce the generation of host lateral roots.(3)The diversity of bacteria and fungi in mycosphere soil of Russula griseocarnosa is lower than that in bulk soil.The mycosphere soil fungi,such as Penicillium,Tolypocladium,Tomentella and Chaunopycnis,and bacteria,such as Burkholderia-Paraburkholderia,Mycobacterium,Roseiarcus,Candidatus＿Xiphinematobacter,Sorangium,Acidobacterium and Singulisphaera,were significantly higher than that in bulk soil.It is indicated that these bacteria might be mycorrhizal helper bacteria which might help the formation of the symbiotic system of R.griseocarnosa and its host.Russula griseocarnosa prefers acidic soils with a p H of 3.99-4.55.The content of available nitrogen(AN),available phosphorus(AP),available potassium(AK)and the organic carbon(SOC)in mycosphere soil is significantly higher than that in bulk soil(p<0.05).AP and elevation have a significant effect on the fungal community structure of mycosphere soil of R.griseocarnosa,while soil p H,SOC,AN,AP,and AK have significant effects on the community structure of soil bacteria,especially p H and AN.The KEGG functional annotation of soil bacteria using PICRUSt showed that the main functions bacteria are two-component system,bacterial chemotaxis,bacterial secretion system,tyrosine metabolism,unsaturated fatty acid biosynthesis,ascorbic acid and uronic acid metabolism,cofactors and vitamin metabolism,and the activity of these metabolic pathways in mycosphere soil are significantly higher than that in bulk soil(p<0.05).(4)The metagenome sequencing results showed that bacteria dominated in mycosphere soil,and fungi and archaea were relatively few.The microorganisms in mycosphere soil of R.griseocarnosa are significantly higher than the bulk soil in terms of energy conversion,signal transduction,material transport metabolism,and degradation of plant and fungal cell walls.It also indicated that the mycosphere microbes might help the formation of the symbiotic system of R.griseocarnosa and its host.Soil p H and AK have significant effects on the KEGG metabolic pathway,and soil SOC and AN have significant effects on CAZys.