| With the increasing frequency of human activities,a large amount of heavy metals had entered the soil environment.As a non-essential heavy metal element,Cd has a long half-life and is a persistent environmental pollutant that eventually threatens human health through the enrichment of the food chain.Plant-microbe combined remediation technology is considered as a safe and effective method to remediate heavy metal contaminated soil.As one of the main participants,understand its response mechanism to heavy metals is extremely important for us to apply this type of microorganisms to the remediation of heavy metal-contaminated soils.In order to study the accumulation and distribution characteristics of Cd by EMF,we collected more than 150 fruiting bodies and mycorrhizas from heavy metal contaminated areas and non-polluted areas,and purified 32 kinds of fungi.By rDNA-ITS sequencing analysis,15 kinds of EMF were isolated.After treatment with different concentrations of Cd,the Cd high-tolerance strain Hebeloma vinosophyllum(Hv)and the low-tolerance strain Lnccaria laccata(LI)were screened out.Under 0,0.2,2 mg L-1 Cd treatment,with the increase of Cd concentration,the biomass of L1 and Hv decreased gradually,but the decrease rate of LI biomass was significantly higher than that of Hv.In addition,the concentration of nutrients in L1 mycelia gradually decreased(P increased first and then decreased),while Hv gradually increased.With the increase of Cd concentration,the concentration Cd in the mycelium gradually increased.Under the same concentration of Cd treatment,the concentration of Cd in L1 mycelium was significantly higher than that of Hv mycelium.Cd in L1 and Hv mycelium was mainly distributed in the cell wall and soluble part.With the increase of Cd concentration,the main position of Cd distribution in LI mycelium changed from soluble part to cell wall,while in Hv the pattern was opposite.In the mycelia of both strains,the chemical forms of Cd was mainly water-extracted(Fw),sodium chloride-extracted(FNaCI)and acetic acid-extracted(FHAC).With the increase of Cd concentration,the ratio of FNacl-Cd in Ll mycelia increased,while that in FHAC-Cd decreased;the trend in Hv was opposite.The proportion of Fw-Cd of L1 and Hv mycelia increased with the increase of Cd concentration,and the Cd content and proportion of water extracts in L1 at the same concentration were significantly higher than that of Hv.In order to elucidate the molecular mechanism of EMF resistance to Cd,transcriptome sequencing and analysis were performed on L1 and Hv treated with 0,0.2,and 2 mg L-1 Cd.The differential expression analysis showed that under 0.2 mg L-1 Cd treatment,338 and 1242 genes were differentially expressed in L1 and Hv,respectively,accounting for 1.62%and 3.82%of their total genes,respectively.Under 2 mg L-1 Cd treatment,605 and 1582 genes were differentially expressed in L1 and Hv,respectively,accounting for 2.89%and 4.87%of the total genes respectively,indicating that the highly resistant strain Hv may respond to Cd stress through more pathways at the transcriptional level.Through GO enrichment analysis of differentially expressed genes,the results showed that the catalytic,metabolic and binding related functions were significantly enriched and may be involved in the Cd-tolerant process of EMF.Further analysis revealed that cytochrome P450 gene,MFS transporter family genes and cold shock protein gene were significantly differentially expressed in Hv,ABC transporter,vacuolar protein and MOP(multidrug/Oligosaccharidyl-lipid/Polysaccharide flippase)transporter family may actively participates in the efflux of Cd and its transport into vacuoles.These findings preliminarily revealed the molecular mechanism of EMF resistance to Cd,and provided a theoretical basis for the better application of EMF to plant-microbial joint restoration of heavy metal contaminated soil.In the root-bag test,external hyphae took the initiative into the Cu/Cd-contaminated bulk soil,absorb and transport Cu and Cd to the rhizosphere soils and further transport it to the shoots of the host plants.Inoculation with EMF also promoted the uptake of nutrients by host plants,thereby increasing their biomass and improving Cu/Cd tolerance compared with non-inoculated plants.Inoculation with EMF species with higher Cu or Cd tolerance generated more phytostabilization and phytoextraction of Cu or Cd by host plants.In a short-term exposure test,inoculation with EMF accelerated the absorption of Cu and Cd by P.thunbergii within 12 h of 3000 mg kg-1 Cu or 150 mg kg-1 Cd irrigation.Therefore,EMF do not act as a barrier inhibiting the absorption of heavy metals by host plants,but rather promote this absorption.Improving the plant's nutritional status and promoting growth,diluting heavy metal concentrations,thereby reducing the toxic effects of heavy metals on host plants.These indicated that EMF can simultaneously promote the effects of phytostablization and phytoextraction.In order to study the remediation effect of contaminated soils in the mining area by EMF,we chose two Cd,Pb,and Zn ore-contaminated soils.We planted non-mycorrhizal,LI and Hv mycorrhizal P.thunbergii in these two soils,by comparing plant growth,nutritional status,and accumulation of heavy metals.In addition,changes in the status of soil heavy metals,soil microbial richness,and soil enzyme activity before and after remediation were analyzed.The results showed that inoculation of EMF can improve the survival rate of P.thunbergii planted in the mining soil,promote the accumulation of Cd,Zn,and Pb by host plants and improve the status of plant nutrient.After planting mycorrhizal pine seedlings,it significantly increased the soil bacteria richness,and increased soil urease and acid phosphatase activity.These indicated that EMF can improve the effect of host plants on the remediation of heavy metal-contaminated soil,and improve the physicochemical properties of contaminated soil.Our results provide further theoretical support for the application of EMF to the in-situ remediation of Cd contaminated soils. |
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