Study On The Mechanism Of ECM Fungi Promoting The Utilization Of Organic Phosphorus On Castanea Henryi Seedlings

Organic phosphorus is an important part of soil phosphorus pool,among which the organic phosphorus in acidic red soil of Hunan province was 165.3±78.46mg·kg-1,accounting for about 23.0%of total soil phosphorus.The efficient utilization of organic phosphorus in phosphorus pools is a key measure to relieve the pressure of low phosphorus(inorganic phosphate)in the acidic red soil regions of southern China.Ectomycorrhizal(ECM)fungi are a class of soil microorganisms associated with organic phosphorus mineralization,which could mineralize organic phosphorus in soil into inorganic phosphorus for plant uptake.However,under low phosphorus conditions,how much organic phosphorus could be mineralized by ECM fungi?what is the mechanism of ECM fungi promoting the utilization of organic phosphorus by host plants?Firstly,the screening of ECM strains that can efficiently decompose organic phosphorus was carried out.Secondly,under lowphosphorus conditions,the 1-year-old Castanea henryi seedlings of ’Huali 4’family were used as test materials,which inoculated a high-efficiency organic phosphorus-solubilizing ECM strain and applied with organic phosphorus(calcium phytate,C6H6Ca6O24P6).Four treatments were including inoculated and applied with organic phosphorus(+ECM+P),inoculated and without applied with organic phosphorus(+ECM-P),without inoculated and applied with organic phosphorus(-ECM+P),without inoculated and without applied with organic phosphorus(-ECM-P).The growth and physiological characteristics of C.henryi seedlings,root tip and rhizosphere soil enzyme activities,and the composition of organic phosphorus were measured.The amount of organic phosphorus mineralization and mineralization rate were calculated.The correlation between each index was analyzed.We wanted to explain the promotion of ECM fungi to C.henryi seedlings about the physiological effects of organic phosphorus utilization.Thirdly,through RNA-Seq technology and bioinformatics analysis of C.henryi roots,the differentially expressed genes related to the utilization of organic phosphorus.We revealed preliminarily the mechanism of ECM fungi promoting organic phosphorus utilization.The main findings are as follows:1.We obtained a strain with high efficiency for organic phosphorus-solubilizing fungi(LY-8),and its optimum growth organic phosphorus concentration was 100 mg P/kg.Five ECM strains were obtained by the isolation,purification and ITS molecular identification of the fruiting bodies of large ECM fungi under the chestnut forest.These strains were Pisolithus orientalis(LY-8)and Scleroderma citrinum(LY-9 and LY-20-2),Scleroderma sp.(LY-17-2 and MB),respectively.These five ECM strains and the Cenococcum geophilum(Cg)gifted by Professor Huang Jian of Northwest A&F University,a total of six ECM strains were screened for high efficiency for organic phosphorussolubilizing fungi.We found that the colony diameter diameter(3.50 cm)and dry weight(0.1800 g)and acid phosphatase activity(0.047 U·ml-1),alkaline phosphatase activity(0.17 U·ml-1)and available phosphorus content(4.46 mg)in the LY-8 strain,respectively.It was significantly higher than that of other strains,and the pH and organic phosphorus content(0.24 mg·L-1)were significantly lower than those of other strains.When the concentration of organic phosphorus was 100 mg P/kg,the colony diameter(3.88 cm),dry weight(0.4731 g),acid phosphatase activity(0.16 U·ml-1)and alkaline phosphatase activity(0.06 U·ml-1)in the fermentation broth of LY-8 strain,respectively.These were significantly higher than other concentrations of organic phosphorus,and its pH of 5.87 was significantly lower than other concentrations of organic phosphorus.2.We clarified the physiological effects of ECM fungi in promoting the utilization of organic phosphorus and growth by C.henryi seedlings,and we found that the potential of ECM fungi to mineralize soil organic phosphorus increased with the time.We observed that the surface of the inoculated roots has yellow bacterial coats and epitaxial mycelium,and Hartig net structure was found inside,which confirmed that the ECM symbionts were formed between the C.henryi seedlings and LY-8 strain.Under the two organophosphorus levels,the mycorrhizal infection rates of +ECM+P and+ECM-P treatments were up to 73.64%and 68.57%,respectively.Inoculation with ECM fungi improved the photosynthetic capacity of C.henryi seedlings,especially the response of net photosynthetic rate to photosynthetically active radiation,carbon dioxide concentration and diurnal variation in+ECM+P treatment.Inoculation with ECM fungi significantly increased the plant height,ground diameter,biomass and the contents of N,P and K in roots,stems and leaves of C.henryi seedlings.In September and November,the+ECM+P treatment significantly increased the seedling height by 9.85%and 29.31%,and the root dry weight by 57.14%and 67.35%,and the root P content by 21.15%and 355.81%compared to the-ECMP treatment(control),respectively.Inoculation with ECM fungi significantly increased the activities of acid phosphatase,alkaline phosphatase and phytase in root tips and rhizosphere soil.In September and November,the+ECM+P treatment significantly increased the acid phosphatase activities of root tips by 110%and 333.3%,and the acid phosphatase in the rhizosphere soil by 37.20%and 88.79%compared to the control,respectively.Inoculation of ECM fungi significantly increased the available phosphorus content and changed the organic phosphorus composition of the rhizosphere soil.In September and November,the+ECM+P treatment significantly increased the content of available phosphorus by 177.03%and 242.29%,and the content of organic phosphorus increased by 59.14%and 326.20%,and the content of active organic phosphorus by 43.75%and 64.13%compared to the control,respectively.Inoculation of ECM fungi significantly increased the mineralization and mineralization rate of organic phosphorus.In September and November,the mineralization of organic phosphorus in the+ECM+P treatment reached 48.67 mg·kg-1 and 56.57 mg·kg-1,and the mineralization rate of organic phosphorus reached 17.46%and 20.30%,respectively.3.We screened out 22 differentially expressed genes related to acid phosphatase and 10 differentially expressed genes related to phosphorus transporter.The RNA-Seq sequencing was performed on 12 samples by Illumina sequencing technology platform,which obtained a total of 1249,490,156 Clean Data.We found that 1398 genes were upregulated and 848 genes were down-regulated in +ECM-P and-ECM-P treatments,and 2767 genes were up-regulated and 2903 genes were down-regulated in+ECM+P and-ECM+P treatments,respectively.We found that 22 genes were differentially expressed associated with acid phosphatase.Among them,there are 7 genes related to acid phosphatase synthesis and 12 genes related to purple acid phosphatase.The expression levels of these genes were higher in+ECM+P and+ECM-P treatments than in-ECM+P and-ECM-P treatments.We found that 10 genes were differentially expressed associated with phosphorus transporters.Among them,the expression levels of 5 genes in+ECM+P and+ECM-P treatments were higher than in-ECM+P and-ECM-P treatments,and 3 genes had higher expression levels in+ECM+P treatment compared with+ECM-P,-ECM+P and-ECM-P treatments,and 2 genes had higher expression levels in+ECM-P treatment than in+ECM+P,-ECM+P andECM-P treatments,respectively.