Mechanisms Of Mineral Weathering By ECM Fungi And Their Ecological Effects

Ectomycorrhizal(ECM)fungi,a group of special biotrophic fungi in the soil,form symbiotic associations with fine roots of woody plants,which greatly promote the uptake of water and mineral nutrients of host plants.This reciprocal association greatly accelerates soil mineral weathering,elemental biogeochemical cycle,and carbon allocation underground and accumulation in the soil.Although many studies have revealed that ECM fungi can weather mineral through biomechanical and biochemical mechanisms,the regulation mechanism at the molecular level in the mineral weathering by ECM fungi is still unclear.Especially for some ECM fungi with weak weathering ability,how do they obtain elements from minerals to meet the nutritional needs of themselves and host plants?Can these ECM fungi select specific accompanying bacterial groups to compensate for their deficiency?It is still in a‘black box'to date.In addition,oxalic acid excreted by ECM fungi is an important agent in mineral bioweathering,but it is easy to form insoluble compounds with metal ions in soils and cover the surface of minerals or ectomycorrhizas,which could impede the further weathering of minerals and the absorption of nutrients.In addition,it is also unknown that whether ECM fungi can select specific bacterial community to alleviate the adverse effects caused by oxalate.Based on the questions above,in this study,environmental adaptation and host specificity of ECM fungi,and bacterial communities in ectomycorrhizosphere were analyzed using high-throughput sequencing(HTS).We used methods of molecular biology,bioinformatics,mineralogy,etc.to explore the mechanism of molecular regulation of ECM fungi weathering minerals.The weathering-related genes were verified in the model fungus Aspergillus nidulans using molecular genetics method.Then,the hypothesis that ECM fungi select high-weathering potential bacteria to promote mineral weathering and nutrient uptake was determined using pot planting and in situ incubation experiments.Finally,the population and diversity of oxalate-degrading bacteria in ectomycorrhizosphere of different forest trees were analyzed using quantitative PCR and HTS as well as their characteristics of oxalate degrading and roles in carbon migration and transformation.The main results obtained in this study are as follows:1.Four ECM fungi were obtained using tissue separation,which are Suillus placidus LS07,Amanita pantherina LS08,Scleroderma bovista LS62 and Pisolithus orientalis LS65,respectively.The results of growth characteristics of these four fungi showed that the optimum growth temperature and p H differed among species,but they all can utilize multiple types of carbon sources,such as monosaccharides,disaccharides and oligosaccharides,and preferred to utilize ammonium nitrogen.2.The ECM fungal composition of ectomycorrhizas and bacterial diversity in the ectomycorrhizaosphere of Pinaceae Pinus massoniana and Fagaceae Quercus acutissima and Quercus serrata were determined using HTS,showing that plants can symbiose with large number of distinct ECM fungi,and a single plant can be colonized by 7–10 ECM fungal species.Compared to the non-ectomycorrhizosphere,ECM fungi prefer to shape similar bacterial community structures,but not to increase bacterial richness.The environmental adaptability of ECM fungi was analyzed by ITS amplicon sequencing.The results showed that these fungi have strong environmental adaptability,but environmental pollution can affect the community structure and composition characteristics.Among them,ascomycetes ECM fungi such as Tomentella,Trichophaea,and Wilcoxina have strong adaptability to stress.In addition,environmental pollution can significantly affect the formation of ECM fungal fruiting bodies as well.3.Combining RNA-seq with real-time quantitative PCR(RT-q PCR),the gene expression of A.pantherina in weathering potassium-containing mineral and apatite was investigated.The results showed that the fungus could respond to the lack of soluble potassium and phosphorus deficiency and up-regulate relevant metabolic processes.The results also demonstrated that there was no up-regulation of any organic acid biosynthesis pathways,reflecting the weak weathering capacity of A.pantherina LS08,especially its inability to utilize P in apatite.However,it can significantly up-regulate the expression of ion transporter genes,such as potassium ion transporter gene Ap HAK1,belonging to the HAK family.In addition,the results of semi-quantitative PCR and RT-q PCR also found that the carbonic anhydrase(CA)gene Ap CA2 of strain LS08 was closely related to mineral weathering,and the expression level was significantly up-regulated in the condition of potassium and/or phosphorus deficiency,while the expression level of Ap CA3 was even down-regulated in the condition of phosphorus deficiency,suggesting that they may have different function.The different function of CAs in A.nidulans were verified using gene knockout and overexpression techniques,showing that the two CA genes(can A and can B)have markedly distinct function,where can A not only participates in the weathering of silicate minerals,but also can be accompanied by the formation of carbonate mineral(calcite)while can B is essential for fungi to adapt to low CO₂ surroundings.4.The results of potting experiment using mycorrhized plants showed that ECM fungi with weak weathering ability can select mineral-weathering bacteria to promote the weathering of mineral(apatite),and have a significant enrichment effect on Sphingomonas,which relative abundance was as high as 10.53%,significantly higher than other bacterial genera.The results of in-growth mesh bag experiment showed that ECM fungi can select high-weathering potential bacteria to promote mineral dissolution.Meanwhile,ECM fungi can select different weathering bacteria species based on the type of minerals when obtaining structural elements in minerals.In the weathering of apatite and potassium feldspar,ECM fungi tend to enrich bacteria such as Pseudomonas and Sphingomonas,while in the case of weathering serpentine,they tend to enrich bacteria such as Bacillus and Paenibacillus.5.The results of HTS and quantitative PCR showed that oxalate-degrading bacteria communities not only differed from bulk soil,but also the number increased(2.60×10⁸–5.03×10⁸copies/g soil).A bacterium with highly oxalate degradation efficiency,Streptomyces sp.NJ10,was obtained by plate separation and was able to form carbonate precipitates in the degradation of calcium oxalate.Taken all together,ECM fungi can select various mineral-weathering bacteria to promote soil mineral weathering to compensate for their functional deficiencies in mineral weathering,enrich oxalotrophic bacteria to mitigate the adverse effects caused by oxalate and promote carbon fixation.This study reveals a new mechanism that ECM fungi can promote soil mineral weathering and improve plant nutrition,and further deepens the understanding of the ecological functioning of mineral-microbial interactions and provides a potential application of vegetation restoration and forest ecosystem reconstruction in wasteland,degraded forest and other special environments.