Study On The Mechanism Of NIR Inoculation To Alleviate Cadmium Toxicity Of Medicago Sativa L.

Cadmium（Cd）pollution seriously endangers the environment and human health.Cd in soil is easily absorbed and accumulated by plants and affects plant growth,cell division and metabolic activities.Piriformospora indica（NIR）is a plant root endophytic fungus,which can not only promote the absorption of soil nutrients by the host plant,improve the phosphorus,nitrogen and mineral nutrient levels of the host plant,but also effectively improve the plant’s tolerance to biotic and abiotic stress factors.In this study,alfalfa（Medicago sativa L.）was used as the research object,and NIR was used as the test fungus.The methods of seed germination test and pot experiment were used to analyze the growth,physiological characteristics and heavy metal resistance of alfalfa inoculated with NIR under Cd stress.The expression changes of related genes were used to explore the response of alfalfa to Cd stress and the mechanism of NIR inoculation alleviating stress injury.The main conclusions obtained are as follows:1.Compared with the control group,when the concentration of cadmium chloride solution increased to 2mg/L,the germination potential,germination rate and radicle length of seeds were 64.3%,50.9%and 56.6%lower than those of the control group respectively（p<0.05）.It shows that 2mg/L cadmium chloride solution has a significant inhibitory effect on seed germination and seedling growth,and when it reaches 3mg/L,the effect is too great,the germination potential and germination rate of seeds are too low,the radicle hardly grows,and the follow-up cannot be carried out.Therefore,2 mg/L cadmium chloride solution was selected as the stress concentration for subsequent experiments.2.Under Cd stress,the aboveground and underground biomass（fresh weight）of alfalfa decreased by 45.3%and 58.2%compared with the control group,respectively（p<0.05）,indicating that Cd had a serious impact on the growth of alfalfa.Inoculation with NIR alleviated the inhibitory effect of Cd on the growth of alfalfa,and the biomass of aboveground and underground in the NIR+Cd treatment group was 44.5%and 34.2%higher than that in the Cd treatment group,respectively（p<0.05）.At the same time,compared with the Cd treatment group,the transport coefficient in the NIR+Cd treatment group decreased by 82.1%（p<0.05）,indicating that after inoculation with NIR,most of the Cd absorbed from the soil was fixed in the root system,thus reducing the transport of Cd to the aboveground part and alleviating the injury of Cd to the functional parts of the aboveground part of alfalfa.In addition,the total soil Cd content was not significantly different between Cd and NIR+Cd treatments,indicating that inoculation with NIR did not assist alfalfa to take up more Cd from soil.3.Under Cd stress,the geometric parameters and spatial distribution characteristics of alfalfa roots were significantly inhibited,while inoculation with NIR alleviated the inhibitory effect of Cd on alfalfa root growth.In addition,based on the principal component analysis（PCA）of root functional traits,it was found that the PC1 axis and the PC2 axis accounted for58.0%and 26.5%of the variance explained rates,respectively,with a total of 84.5%,which can define the presence of root economic spectrum（RES）in alfalfa.The PC1 axis and PC2axis represent the conservative and acquisition strategies of the root system for resource acquisition and allocation under different environmental conditions,respectively.Compared with the control group,the root growth of alfalfa under Cd treatment is limited.Invest in relatively inexpensive root construction for the basic metabolic activities needed to maintain survival for a quick return,the acquisition strategy is adopted.The NIR+Cd treatment is located on the conservative side of the PC1 axis,and long-term survival can be achieved by investing in more expensive root system construction,such as increasing root tissue density and average root diameter.The difference distribution of Cd and NIR+Cd treatments in PCA indicated that inoculation with NIR changed the trade-off strategy of resource acquisition and allocation in alfalfa roots under Cd stress,from acquisition strategy to conservative strategy.4.Compared with the control group,alfalfa chlorophyll content,Photo,Cond,φPSII,ETR,etc.were significantly decreased under Cd stress,and O₂^·-,MDA and REL were significantly increased（p<0.05）,indicating that alfalfa photosynthesis was inhibited,intracellular substances are leaked out,and the cells are seriously damaged.However,inoculation of NIR relieved the inhibition of Cd on photosynthesis of alfalfa and the oxidative stress on the body.On the one hand,NIR maintained the intracellular redox homeostasis by increasing the activity of antioxidant enzymes（SOD,CAT）,and decreased O₂^·-,MDA and REL by 43.6%,44.3%and58.7%,respectively,compared with the addition of Cd（p<0.05）.On the other hand,NIR alleviates the inhibitory effect of Cd stress on photosynthesis by adjusting stomatal opening,promoting chlorophyll synthesis,enhancing linear electron transfer rate and light utilization efficiency.5.Compared with the control group,the addition of Cd treatment increased the transcription levels of CDPK2,CBL2 and Cd-AS 8 in alfalfa by 11.4%,26.8%and 35.2%,respectively（p<0.05）,indicating that the above signal transduction after Cd entered the cells genes are activated.This in turn triggers downstream signal transmission and promotes the up-regulation of the transcription levels of heavy metal transport-related genes Zn maybe,Fe²⁺transporter,Mg²⁺transporter and Ca uptake-pro.At the same time,alfalfa alleviated the toxicity of Cd by enhancing the expression of Cd resistance genes（Cd-res-pro-4,HM-res-pro-4）and antioxidant enzyme genes（SOD,POD15）.In addition,NIR inoculation significantly decreased the transcription levels of some signal transduction-related genes,heavy metal transport-related genes and Cd resistance genes in alfalfa under Cd stress,while the transcription levels of antioxidant enzyme genes（SOD,CAT4）were significantly increased,indicating that Cd content in the ground part of alfalfa decreased after inoculation with NIR,and the toxic effect of Cd on alfalfa could be alleviated only by enhancing the transcription level of antioxidant enzyme genes.In summary,NIR inoculation can alleviate the growth and physiological inhibition of alfalfa caused by Cd to a certain extent.It can alleviate the toxicity of Cd by changing the balancing strategy of alfalfa root system on resource acquisition and distribution under Cd stress,regulating the relative distribution of Cd in the body,the expression of Cd resistance and transport-related genes,and improving the activity of antioxidant enzymes.The results of this study will provide theoretical basis for NIR inoculation to alleviate the toxic effect of Cd on alfalfa.