| As a kind of heavy metal,Chromium(Cr)is an important environmental pollutant.The main forms of Cr present are trivalent chromium(Cr(?))and hexavalent chromium(Cr(?))and the toxicity of Cr(?)is 100 times that of Cr(?).In recent years,the combination of plant-microbe remediation as a remediation technology for heavy metal contaminated soil has gradually become a hot spot.Ectomycorrhizal(ECM)fungi have the multi-faceted functions such as promoting plant growth,regulating nutrient absorptionin plants and improving the stress resistance of plants.This study screened out the ECM fungi with ability of Cr(?)tolerance and removal,explored the physiological and molecular mechanismsof Cr(?)tolerance and removal intarget strain,and investigated the potential of ECM symbiotic system which formed between ECM fungiand Pinus thunbergiion Cr(?)-contaminated soil remediation.The main results are as follows:1.The tolerance and the removal ability of five kinds of ECM fungi to Cr(VI),the growth parameters,Cr accumulation in mycelium under Cr(?)treatments were compared.The results showed that the tolerance index(TI)of Pisoli(hus(P.spl)significantly higher than that of other 4 strains under 10-50 mg/L Cr(?)treatments.P.sp1 showed the strongest ability of Cr accumulation and removal among five ECM fungi,expecially under high level of Cr(VI)treatment(50 mg/L),the Cr accumulation in P.spl significantly higher than that of other strains.We also found that the Cr(?)tolerance and accumulation ability of Cg and Hv were at moderate and poor levels respectively compared with P.sp1.2.The effects of pH,temperature,inoculum size,initial Cr(?)concentration,and metal ions on the Cr(?)removal by P.spl were studied in liquid culture medium.The results showed that with the decrease of pH,the removal rate of Cr(?)by P.sp1 was significantly increased;the optimum temperature for the Cr(?)removal by P.sp1 was 25?;too much or less inoculum size was not good for P.spl to Cr(?)removal;with the increasing of initial Cr(?)concentration,the removal of Cr(?)by P.spl significantly decreased;external added 0.2 mM of Cu2+ and Fe3+ have obvious promoting effect on the removal of Cr(?)byP.spl in different phases of Cr(?)treatment,while Ca2+?Zn2+?Mg2+and Co2+has no effect on this process,and Cd2+has an inhibitory effect on this process.3.In order to investigate the physiological mechanism of Cr(?)removal by P.spl,the total amount of protons,the species and quantity of low-molecular-weight organic acids(LMWOAs)produced by P.spl,and the pH of the culture system were analyzed.We also analyzed the variation rule among pH,organic acids and Cr(?)in the system.In addtion,the distribution of Cr in the culture system was analyzed by the subcellular distribution.To investigate whether protons and LMWOAs in the culture system have an effect on Cr(?)removal,the experimental results were verified by the external addition of proton pump inhibitor Na3VO4 and LMWOAs such as oxalic acid,malic acid,and citric acid.The results showed that P.spl strain accelerated the Cr(?)removal in the culture system by directly secreting H+ to promote Cr(?)reduction.The main physiological mechanism of Cr(?)removal by P.spl were extracellular reduction and cell wall adsorption,which can account for about 75%and 24%Cr(?)in the culture system respectively.4.To estimate the molecular mechanism of genes which associated with Cr(?)tolerance and reduction in P.spl,Pisolithus sp2(P.sp2)were selected as reference strain due to its higher homology and lower Cr(?)tolerance compared with P.spl.We used Illumina Solexa high-throughput sequencing technology to analyze the transcriptome of two strainsunderdifferent concentration of Cr(?)treatments.The results showed that atotal of 93642 assembled unique transcripts representing 47,3801 unigenes were obtained and 16103 unigenes were assigned into 50 GO functional classifications,and 6429 unigenes were assigned into 25 KOG functional categories.KEGG pathway analysis showed that 15481 unigenes were assigned into 239 pathways.In order to study the expression profile characteristics in response to Cr(?)stress in the mycelium of two Pisolithus spvarieties,the cDNA library was constructedfor mycelium samples from two Pisolithus spvarieties with different Cr(?)treatments and thensequenced with Illumina Solexa sequencing technology and mapped with de novotranscriptome of Pisolithus sp.The differential expression unigenes were then assigned to GO function and KEGG pathway annotation and enrichment analysis.In spl(0)vs spl(10)and sp2(0)vs sp2(10),72 and 756 differential expression unigenes were found respectively.GO functional enrichment analysis found that"response to integral to plasma membrane","high affinity secondayary active ammonium transmembrane transporter activity" and "response to peptidoglycan glycosyltrans tramsporterase" were significantly enriched in both Pisolithus sp mycelium under Cr(VI)treatment.Other differential expression unigenesincluded:(1)nitrate reductase;(2)L-sparaginase;(3)acohol dehydrogenase;(4)long-chain acyl-CoA synthetas;(5)aldehyde dehydrogenase;(6)posphoglycerate mutase.Under the 10 mg/L Cr(VI)treatment,2 nitrate reductase genes significantly up-regulatedand 1 L-asparaginase gene significantly down-regulated in P.sp1,which may play important role in Cr(VI)tolerance and removal.5.The previously screened 3 kinds of ECM fungi with different tolerance and accumulation ability to Cr(VI),Hv(sensitive),Cg(moderate tolerant)and P.sp1(tolerant)strain were used to inoculate with Japanese black Pine(Pinus thunbergii),and ECM or non-mycorrhizal(NM)seedlings were used as research objects by comparing the tolerance and remedation potential of 3 kinds of ECM seedlings to Cr(VI)in contaminated soil.The results showed that inoculation with Hv,Cg or P.sp1 significantly improved the growth and photosynthesis efficiency of P.thunbergii and alleviated the toxic of Cr(VI)to host plants;compared with NM and Cg,Hv ECM seedlings,inolulation with P.sp1 significantly improved Cr content per plant of P.thunbergii.In addition,the cultivation of P.sp1 ECM seedlings significantly reduced the percentage of exchangeable and organic-binded Cr in soil,alleviated the toxicity of Cr to host plant,which indicated that P.sp1 ECM seedlings are more suitable for the phytoremediation of Cr(VI)contaminated soil.6.In order to improve the remediation efficiency of ECM P.thunbergii,and consider the factors of cost and environment safety,the best biodegradable chelating agent-Cg fermentation liquor with obvious activation to Cr in contaminatedsoil were screened out from 20 kinds of fungal fermentation liquor and 3 kinds of commonly chemical chelating agents(EDTA,EDDS,NTA).At the same time,the effects of external added Cg fermentationliquor and inoculation with P.sp1 on the biomass,Cr content in shoots/roots,urease and acid phosphatase activity in soil were compared in pot and root-bag experiments to explore the effect of Cg fermentation liquoron P.sp1 ECM seedlings assisted remediation of Cr contaminated soil.The results of pot experiment showed that the Cg fermentation liquor significantly inhibited the growth of NM seedlings but did not significantly inhibited the growth of P.sp1 ECM seedlings,and signifncantly improving the enrichment of Cr in P.sp1 ECM seedlings.The results of root bag experiment showed that the low concentration of Cg fermentation liquor significantly increased the enrichment of Cr in P.sp1 ECM seedlings but high concentration of Cg fermentation liquor inhibited this process.In addition,compared with single P.sp1 ECM seedlings treatment,the combined treatments of Cg fermentation liquor and P.sp1 ECM seedlings significantly enhanced the activity of urease and acidic phosphatase in contaminated soil,improved soil and beneficial to survival of host plants. |
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