Study On The Tolerance Mechanism Of Ectomycorrhizal Fungi To As(?) And Its Effect With Pinus Thunbergii To Remediate As Contaminated Soil

The arsenic(As)is an important environmental pollutant.In recent years,the combined plant-microbe remediation has gradually become a research hotspot in terms of heavy metal contaminated soil remediation technology.Ectomycorrhizal fungi has many functions,such as promoting the growth of host plants,regulating the absorption of nutrients by plants,and improving the stress resistance of plants.This study screened for arbuscular mycorrhizal fungi with higher As(?)tolerance,explored the physiological and molecular mechanisms to As(?)tolerance,and at the same time,we studied the effect of ectomycorrhizal fungi on the remediation of As contaminated soil by Pinus thunbergii Parl.The main research results are as follows:1.By comparing the growth status of 15 strains of ectomycorrhizal fungi on different concentrations of As(?)solid medium containing,it was found that 1000 mg·L-1 As(?)had inhibited the growth of most fungi,the growth inhibition rates of Hebeloma cylindrosporum(Hc),Laccaria laccata(L1),and Hebeloma crustuliniforme(He)were 10.1%,19.8%and 26.2%,respectively.However,the growth of Hebeloma vinosophyllum(Hv)was promoted 162.6%under the treatment of 1000 mg·L-1As(?).In order to better compare the differences in the tolerance of different strains to As,according to the growth curve and homology analysis results of the fungi,two ectomycorrhizal fungi(Hv and Hc)of the Hebeloma genus were selected as As(?)resistant strains and to explore the mechanism of tolerance.2.In order to explore the physiological mechanism of tolerance of Hv and He strains to As(?),liquid culture experiments were used to further study the changes of biomass?pH and Eh of Hv and Hc under different concentrations of As(?)stress.The results showed that the half inhibitory concentration of As(?)on the growth of both strains was 300 mg·L-1.We founded that Hv was more sensitive to As(?)stress compared with Hc by using scanning electron microscopy to observe the morphology of fungal mycelium under As(?)stress.In the presence or absence of As stress,the pH of the medium increased and the Eh value decreased with time.By analyzing the morphology of As(?)in the medium after the strains were cultured at different times,it was founded that after 100 mg·L-1As(?)treatment for 10 days,As in the medium of Hc and Hv strains almost all existed in the form of As(?),but In the ten days after the growth of the Hc,34.9%As(?)was oxidized to As(?)again,but there was no such oxidation phenomenon in the Hv medium.After adding high concentration of phosphate,the reduction process of As(?)by two strains was inhibited,and the reduction process could not occur in anaerobic environment.It indicated that the two fungi conform to the characteristics of arsenic-resistant microorganisms(ARMs).Further study of the oxidation process of As in the two strains of culture found that Hc lost its ability to reduce As(?)in the late growth stage,and the increase of pH in the culture medium will promote the oxidation reaction;The oxidation process did not occur in the Hv'medium because the Hv had the ability to reduce As(?)within 20 days of culture.Moreover,the reduction rate was higher than the rate at which As(?)was oxidized due to the increase of pH.The subcellular distribution of As in the mycelium of the two strains under As(?)treatment showed that the enrichment ability of both fungi to As was weak,so the reduction of As(?)by the two strains is their arsenic tolerance The main physiological mechanism.3.To find related genes involved in the regulation of As(?)tolerance in Hv and He strains,Hebeloma cylindrosporum h7 was used as a reference strain,and the BGISEQ-500 platform was used to perform transcriptome sequencing on He and Hv hyphae under As treatment and control conditions.The results showed that there were 1114 genes that were significantly differentially expressed in Hc treated with As(?),of which 555 genes were up-regulated and 559 genes were down-regulated;377 genes in Hv under As(?)treatment occurred Significantly differential expression,of which 197 genes were up-regulated and 180 genes were down-regulated;79 genes were genes that were significantly differentially expressed in both Hc and Hv strains.Significance enrichment analysis of GO function showed that genes of He strain were mainly involved in cellular process,membrane and catalytic activity;genes of Hv strain were mainly involved in catalytic activity,membrane and cellular process.KEGG pathway annotation results show that the metabolic pathways related to heavy metal tolerance and detoxification processes involved in He and Hv strains include glutathione metabolism pathway(ko00480),antioxidant system pathway(ko04146),ABC transport system(ko02010).Among the significantly differentially expressed genes,four genes highly related to arsenic stress were found in Hc,three of which were genes of arsenite transporter(ACR3)and one was arsenite methyltransferase gene(AS3MT);A gene of arsenite transporter(ACR3)was found in the Hv strain,but the genes related to As(?)reduction and transport have not been found yet,the reason is for further study.4.The investigation of the soil at six sites near the tailings reservoir of the abandoned arsenic mine in Heshan Village,Shimen County,Changde City,Hunan Province found that the excess As rate in soil reached 100%,and The highest was 30.8 times higher than the limit.In view of the soil with severe arsenic contamination in this area,We inoculated the root of Pinus thunbergii Parl with two strains of two As(?)tolerant strains Hv and Hc and the sensitive strain Tylopilus felleus(Tf),an infection rate higher than 70%and non-mycorrhizal seedlings(control)is a research material to explore its remediation potential for arsenic-contaminated soil.The results of the study showed that:compared with non-mycorrhizal seedlings,inoculation with EMF significantly improved the survival rate of black pine seedlings and promoted the growth of black pine,while mycorrhizal plants showed a stronger adaptability to arsenic-contaminated soil.Compared with non-mycorrhizal seedlings,the dry weight of Hv and Hc mycorrhizal seedlings increased by 1.9 and 2.1 times,respectively.At the same time,the As content of Hv and Hc mycorrhizal seedlings was significantly higher than that of Tf mycorrhizal and non-mycorrhizal seedlings.In addition,the analysis of the effective content of As in the soil before and after remediation found that planting mycorrhizal seedlings significantly reduced the content of As in water-soluble,surface-adsorbed,and Fe/Al combined states in the soil,and alleviated the toxic effects of As on plants.