Study On The Mechanism Of Arbuscular Mycorrhizal Fungi On The Adaption Of Elsholtzia Splendens To Copper Stress

Arbuscular mycorrhizal fungi(AMF),which engage a mutualistic symbiosis with the roots of most plant species,have been well studied for their ability to alleviate heavy metal stress in plant,including copper(Cu).Elsholtzia splendens,a widely distributed copper-tolerant plant,is usually found in the copper mining areas of southern China and is colonized by symbiotic AMF.In order to explore the mechanism of AMF on the adaptation of E.splendens to copper stress,we conducted a greenhouse experiment to compare the effects of AMF inoculation and copper addition on the photosynthetic pigment contents,anti-oxidation enzymatic activities,antioxidant contents and the lipid peroxidation of E.splendens and the chemial and physical properties and enzyme activities,structure and functional diversity of microbial communities of soil at different stage,including vegetative stage and flowering stage.The plants were grown on a mixture of sterilized soil and sand inoculated or not inoculated with AMF,addition or not addition with copper(0 mg kg-1 and 1000 mg·kg-1),with four treatments in total:control(CK),copper stress(Cu),arbuscular mycorrhizal fungi inoculation(AMF),copper stress and AMF inoculation(Cu+AMF).The results were shown in the followings.1.Compared with the CK,the chlorophyll a(Chl a)and chlorophyll b(Chl b)contents,chlorophyll(a+b)contents[Chl(a+b)]and carotenoid content(Car)under Cu stress significantly decreased.And the superoxide dismutase(SOD),peroxidase(POD),ascorbate peroxidase(APX),catalase(CAT),glutathione(GSH),ascorbic acid(AsA),non-protein thiol(NPT)and phytochaletins(PCs)contents in the leaves and roots of E.splendens under Cu stress significantly decreased except the AsA contents of leaves at vegetative stage and the GSH contents of roots at flowering stage,while the membrane relative permeability(MRP)and malondialdehyde(MDA)contents significantly increased.At the vegetative stage and flowering stage,inoculation with AMF significantly increased the Chl a,Chl b and Chl(a+b)contents in E.splendens leaves under copper stress.At the vegetative stage,inoculation with AMF significantly increased the SOD,POD,CAT,APX activities in leaves and AsA contents in roots,while decreased the roots' MRP,MDA contents.At the flowering stage,inoculation with AMF significantly increased the SOD,POD,APX activities in leaves and APX activities in roots,but the roots' MDA contents significantly decreased.In conclusion,AMF inoculations could effectively alleviate the damage of Cu stress on E.splendens by increasing the photosynthetic pigments contents and the antioxidant capacities and decreasing the lipid peroxidation of cell membranes and the relative conductivity,which might be important for Cu stress adaptation and the improved productivity of E.splendens.2.At August(vegetative stage)and October(flowering stage),the colonization of AMF to the roots of E.splendens treated with Cu and Cu+AMF were high which indicated that AMF could colonize E.splendens no matter the existing of Cu Copper content in different organs of E.splendens randed in the orders:root>stem>leaf.Under copper stress,AMF inoculation significantly decreased the copper content of E.splendens,especially for the above-ground part.The copper contents in the root,stem and leaf of E.splendens at vegetative stage weredifferent life history stages,56.2%,50.9%,and 41.9%,respectively,while those at flowering stage were 53.5%,47.6%and 35.0%,respectively.These results suggested that AMF could enhance the heavy metal tolerance ability of E.splendens and alleviating the damage of copper stress by inhibiting the transfer of Cu from the root to the above-ground part.3.Cu stress decreased significantly pH value of soil,while increased significantly the contents of soil total nitrogen,available phosphorus,available potassium and organic matter while the inoculation of AMF significantly increased them at the growth stages.The inoculation of AMF significantly decreased the contents of available phosphorus and organic matter in the rhizoshperic soil of E.splendens.Compared with CK,the soil acid phosphase,urease,sucrase,catalase activities and glomalin content of E.splendens under copper stress significantly decreased,while the inoculation of AMF significantly increased them,expect for acid phosphase activities at the October.There were significant positive correlations among acid phosphase,urease,sucrase and catalase activities of soil.There were significant negative correlations between soil enzyme activities and soil total nitrogen,available phosphorus,available potassium and organic matter at the vegetative stage and flowering stage,and significant positive correlations between soil enzyme activities and pH value of soil at the flowering stage.These results suggested that AMF-colonization could enhanced the activities of soil enzymes,promoted the uptake of soil mineral elements,such as N,P,K,which could relieve the damage of copper stress on the plant and enhanced the adaptability of E.splendens to copper stress.4.At the vegetative stage and flowering stage,the soil microbial biomass carbon(MBC),soil microbial biomass nitrogen(MBN),soil microbial biomass phosphorus(MBP)of E.splendens under copper stress were lower than in the CK.Inoculation with AMF significantly increased the MBC,MBP of soil under copper stress at the vegetative stage and and significantly increased MBN,MBP at the flowering stage,indicating an alleviation effects on the inhibition of copper on the soil microbial biomass of E.splendens.Compared with CK,the bacteria and actinomycetes number of soil under copper stress significantly decreased at the vegetative stage and flowering stage,while the fungal number of soil under copper stress were lower,but there were significant differences between them.Compared to only copper treatment,the fungal and bacteria number of soil inoculated with AMF significantly increased in the vegetative and flowering stage.The soil fungal number in vegetative stage and soil fungal,bacteria,actinomycetes number of flowering stage inoculated with the AMF were higher than in the CK.At both vegetative and flowering stages,the average well color development(AWCD)of soil microbes in four treatments randed in the orders:AMF>CK>Cu+AMF>Cu,indicating copper stress could decrease the soil microbial activity and the inoculation of AMF could increase the soil microbial activity.Base on the data of carbon utilization ability,copper stress decreased the Margalef index,Shannon diversity index and Pielou evenness index significantly while the inoculation of AMF significantly increased them in the vegetative stage.In the flowering stage,copper stress significantly decreased the Margalef index and Pielou evenness index while the inoculation of AMF significantly increased them.Soil DNA was extracted and the 16S and 18S rDNA genes were amplified using polymerase chaim reaction(PCR).Then,denaturing high performance liquid chromatography(DHPLC)was used to investigate the soil bacteria and fungal structural diversity by comparing their peak profiles.Under CK treatment,the soil bacteria community structure diversity were higher among different treatments at vegetative stage.However,no significant differences were found for the soil bacteria community structure with Cu,Cu+AMF and AMF treatments.The Shannon-weiner index,Margalef index,Simpson index and Richness index of AMF community under Cu+AMF and AMF treatment were higher than in the Cu and CK treatments,but did not markedly affect AMF community structure in the Cu and CK treatments.At flowering stage,no significant difference was found for the Shannon-weiner index,Margalef index,Simpson index and Richness index of soil bacteria community structure with different treatments.The Shannon-weiner index,Margalef index,Simpson index and Richness index of AMF community under Cu and CK treatments were lower than those in Cu+AMF and AMF treatments,and those in Cu+AMF treatment were lower than those in AMF treatment.These results suggested that AMF-colonization and Cu stress could change the soil microbial functional and structure diversity but inoculation with AMF could effectively contrubitue to AMF community,but did not markedly affect soil bacteria community structure.