| Pinus massoniana Lamb.or masson's pine,is a coniferous evergreen tree from pinaceae.It is used as the main timber and important tree species for afforestation in southern China.Due to its well-developed root system and strong stress resistance,it is easy to form ectomycorrhizal that further promote plant growth,heavy metal absorption,and improve the efficiency of plant remediation of heavy metal pollution through various ways,which has also become the pioneer tree species for phytoremediation in heavy metal contaminated mining areas.In order to understand the potential and remediation mechanism of P.massoniana and Suillus luteus symbiotic system in heavy metal contaminated soil,the symbiotic system of P.massoniana and S.luteus were selected as research objects,the morphological and structural changes,physiological and biochemical characteristics and molecular level of individual growth were investigated to reveal the response rule and regulatory mechanism of P.massoniana and its symbiotic system to heavy metal stress.The main results were as follows:1.The biomass,morphological structure,dynamic absorption and enrichment abilities,antioxidant ability and the secretion of organic acid under Pb and Zn stress were investigated by environmental scanning electron microscopy,X ray diffractometer and atomic absorption spectrometry.The results showed that S.luteus showed strong resistance to Pb and Zn,and more tolerance to Zn than that of Pb.When the concentrations of Pb were 50,200 and 500 mg/L,the biomass of mycelium were 41.46%,51.22%and 75.61%lower than that of control,respectively,while the biomass was 25%-60%lower than that of control under the strees of Zn.The mass fractions of Pb and Zn of S.luteus were 4.6%and 1.9%,respectively,using by X ray spectrometer(EDS)element scanning system.The enrichment of S.luteus under different Pb concentrations(50,200,500 mg/L)were 35.56 ?g/g,165.88 ?g/g and 188.57 ?g/g,respectively;and Zn were 312.2 ?g/g,343.2 ?g/g and 425.56 ?g/g,respectively.Meanwhile,mycelium had strong antioxidant and free radical scavenging abilities.The total antioxidant capacity(ABTS)of S.luteus showed a significant negative correlation with the concentration Pb(R=-0.74).The Zn treatment at different concentrations obviously promoted the SOD,CAT and free radical scavenging capacity,moreover,the scavenging capacity of Zn stress was better than that of Pb.The pH values of Pb and Zn treatments were significantly lower than those of control,which caused by tartaric acid and oxalic acid.2.A symbiotic system of P.massoniana-S.luteus was constructed.The seeding germination,growth,physiological and biochemical characteristics of seedlings and heavy metal morphology in rhizosphere soil of P.massoniana were determinated.The result showed that:1)the mycelium of S.luteus could alleviate the toxicity of heavy metals and promote the germination of P.massoniana seeds.When the concentration of Pb reached 800 mg/kg,the seed was ungerminaed,while the germination rate of the seed was maintain 36.67%with S.luteus inoculation.A low concentration of Zn could increase the germination rate of P.massoniana seedings,when the concentration of Zn reached 400 mg/kg,the germination rate of P.massoniana seedings were 86.67%,while the germination rate were about 60%without ectomycorrhizal fungi inoculation;2)inoculation of ectomycorrhizal fungi could promote the growth and root development of P.massoniana seedlings.Compared with that of non-inoculated ectomycorrhizal fungi seedlings,the plant height of mycorrhizal seedlings increased by 10.27%,19.68%,4.67%and 12.43%,respectively,when the Pb stress concentration were 0,50,200 and 500mg/kg;while increased by 16.86%,17.31%,8.57%and 7.07%under different Zn concentrations.The ground diameter and root-crown ratio of the seedlings with ectomycorrhizal fungi were significantly higher than those of the seedlings without ectomycorrhizal fungi.Meanwhile,the root development and root activity were promoted,for example,under different concentrations Zn stress,the lateral root number of the seedlings inoculated with ectomycorrhizal fungi increased by 5.70%-107.1%,and the root activity increased by 23.74%-163.62%compared with that of seedlings without ectomycorrhizal fungi;3)the contents of free proline,soluble sugar,soluble protein,POD activity,SOD activity,chlorophyll content and malondialdehyde increased with ectomycorrhizal fungi inoculation.Correlation analysis showed that there was a significant negative correlation between malondialdehyde and seedling height,root infection rate,carotenoid,root activity and POD,and the correlation coefficients were-0.709,-0.784,-0.802,-0.699 and-0.734,respectively;4)chemical speciation and potential mobility of heavy metals in rhizosphere soil of P.massoniana inoculated with or without ectomycorrhizal fungi(S.luteus)was changed.The content of acid-soluble Pb in rhizosphere soil of P.massoniana seedlings inoculated with S.luteus increased significantly(p<0.05);the content of acid-soluble Zn increased significantly,while decreased the reducible Zn significantly inoculated with S.luteus;the major chemical speciation of heavy metals in rhizosphere soil were acid soluble and residue speciation under the combined stress of Pb and Zn.3.The roots and needles of P.massoniana seedlings inoculated with or without ectomycorrhizal were sequenced by RNA-Seq transcriptome sequencing under different heavy metal stress.Approximately 606,276,306bp high quality clean reads were generated,these clean reads were trimmed and assembled into 309,172 non-redundant putative transcripts.Of these Unigenes,28,414 differentially expressed Unigenes(DEGs)were screened out Among them,there were 11,023 expressed Unigenes up-regulated,and 17,3 91 expressed Unigenes down-regulated in roots and needles of P.massoniana.The results of GO functional annotation showed that the DEGs of needles of P.massoniana was mainly involved in the biological construction process,and its molecular function was mainly related to ion binding and catalytic reaction.Moreover,the root system of P.massoniana was involved in the reception and conduction of defense response,stress signal,and enzyme activity.KEGG pathway showed that genes manipulation of ZIP,YSL,NRAMP and TGA could be identified as the main factor that related to the heavy metal transmembrane transportation;genes such as PCS,MTP,BIP3 and ABC transporter family protein genes involved in regulating heavy metal efflux;and genes such as PROT1,PERK 13 and HSP83 and genes related to antioxidant enzymes(SOD,POD and CAT)and osmotic protective substances in P.massoniana were established.The results of RT-qPCR verification showed that it was feasible to use RNA-Seq technology to sequence the transcriptome and screen the different genes of P.massoniana under heavy metal stress.4.The Illumina Miseq high-throughput sequencing platform was further utilized,and soil microbial community structure was determined by 16s rRNA and 18s rRNA,also the physicochemical properties of rhizosphere soil,the accumulation and transportation of heavy metals in plants were determinated to explore the tolerance mechanism and accumulating potential of symbiotic system and its adaptability to soil environment in lead-zinc tailing area.The results showed that:1)the soil total carbon/total nitrogen,moisture,available nitrogen,phosphorus,and potassium were significantly higher inoculated with ectomycorrhizal fungi,while the pH,bulk density and heavy metals showed opposite trend when compared with control(p<0.05).The heavy metals accumulating in plants' roots with ectomycorrhizal fungi were greater than that without ectomycorrhizal fungi.Conversely,they were lower in shoots with ectomycorrhizal fungi;2)the bacterial community were affiliated with 23 bacterial phyla,70 classes,115 orders,201 families,and 363 genera.Constrained Principal Coordinate Analysis and redundancy analysis demonstrated that bacterial communities structure in the soil inoculated with or without ectomycorrhizal fungi and bulk soil were distinguishable,but no difference between rhizosphere and non-rhizosphere.The LEfSe analysis showed Acidobacteria,Actinobacteria,and Proteobacteria were the dominant phyla that contributed to these difference;3)the rhizospheric fungi belong to 6 phylum,25 classes,65 orders,115 families and 150 genera and the dominant phyla were Chytridiomycota(50.49%),Ascomycota(38.54%),and Basidiomycota(9.02%).The relative abundance of Suillus,Paraglomus,Agaricus,and Tulasnella were the highest with ECM fungi inoculation.RDA analysis showed that the community structure significantly changed with ECM fungi inoculation,which was significantly related to soil water content,carbon nitrogen ratio,bulk density,available potassium,and soil enzymes.All together,this study demonstrated S.luteus had strong tolerance,absorption and accumulation properties under the stress of heavy metals of Pb and Zn,promoted growth,heavy metal transportation,physiological and biochemical resistance in P.massoniana,and potentially improved symbiotic system for remediation of contaminated soil in mining area.The research provided theoretical and technical guidance for the practical application of plant-mycorrhizal symbiotic system in remediation of heavy metal contaminated mining area. |
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