Response Of Medicago Sativa To Copper And Zinc Stress After Co-inoculation With S.meliloti And A.tumefaciens

Bioremediation of heavy metal-contaminated soils must overcome problems such as high concentrations of residual heavy metals,compound pollution of multiple heavy metals,and lack of nutrient elements?such as N,P?that are common in heavy metal-contaminated areas.Inthisstudy,theimportantphysiologicalprocessofS.meliloti CCNWSX0020-Medicago lupulina symbiotic nitrogen fixation was combined with the remediation under copper and zinc stress by using alfalfa as material.The mechanism of plant copper and zinc tolerance by co-inoculation of rhizobium S.meliloti CCNWSX0020and probiotic bacteria A.tumefaciens CCNWGS0286 were studied through the plant antioxidant enzyme system,lipid peroxidation level,heavy metal extraction,chlorophyll content and symbiotic nitrogen fixation ability,and further explore the practical application potential of the Medicago lupulina-co-inoculation system in heavy metal pollution remediation.The results of the study were as follows:1)Under different concentration gradient Cu and Zn stress,double inoculation could significantly promote the growth and development of alfalfa,increase the biomass,promote the enrichment of Cu,Zn in alfalfa,improve the tolerance of plants to heavy metals and form effective symbionts.The results showed that when the concentration of Cu²⁺and Zn²⁺reached 400 mg.kg^-1,the plant height,root length,aboveground biomass,root biomass and nodule number of alfalfa co-inoculated with S.meliloti and A.tumefaciens showed a significant increase.Compared with single inoculated S.meliloti or A.tumefaciens,the total uptake of Zn by double inoculated plants increased 89.22%?28.36%and the total absorption of Cu increased significantly by 154.11%and 306.01%,respectively.It further enriches the theoretical basis of plant tolerance to high heavy metal and multiple heavy metal stress;it also provides the necessary theoretical basis for agricultural production in areas with severe environmental pollution.2)The tested strains S.meliloti CCNWSX0020 and A.tumefaciens CCNWGS0286were co-inoculated under 400 mg.kg^-11 Cu²⁺and Zn²⁺double stress,which were higher than that of the non-inoculated strain,with higher root length,dry weight on the ground and dry weight under the ground,increased 32.56%and 14.06%,34.35%,34.35%and 31.21%respectively.Under any heavy metal stress,the enrichment coefficient of root and the total uptake of Cu,Zn in co-inoculated plants were significantly higher than those in other treatments.And the double inoculated plants were cultured at 400 mg.kg^-11 Cu²⁺or Zn²⁺stress,the transfer coefficient was significantly lower than that of non-inoculated treatment by 80.24%and 52.60%?P<0.05?.Under all stress conditions,the transport coefficients of heavy metals in double inoculation plants were far less than 1,indicating that heavy metals were mainly accumulated in the roots of plants,and only a small amount of Cu,Zn was transported to the aboveground part.The chlorophyll content of double inoculated plants increased by 93.39%and 147.89%under the 400 mg.kg^-11 Cu²⁺stress and Cu²⁺,Zn²⁺dual stress,respectively,compared with the un-inoculated plants.The results showed that the double inoculated alfalfa could tolerate higher concentrations of heavy metal stress,which also showed the potential and safety of the application of Medicago lupulina-co-inoculation system in plant immobilization technology.3)Through the analysis of antioxidant enzyme activity system of alfalfa,the results showed that in different stages of symbiotic system development,high concentration of Cu,Zn stress resulted in a large amount of MDA accumulation in plants,and the highest accumulation amount was found in non-inoculated plants.Under different heavy metal stress conditions,the MDA content of roots and shoots of the co-inoculated plants was significantly lower than that of non-inoculated plants after 8 days of inoculation,and had different degrees decreased compared with the single inoculated plants.Under the same heavy metal concentration,the SOD,CAT,POD and APX of double inoculated plants were higher than that of single inoculation treatment.The antioxidant enzyme activity system of alfalfa was seriously damaged by the combined pollution of Cu and Zn,which was lower than that of single Cu stress plant antioxidant enzyme.4)Under 200 mg.kg^-11 Cu²⁺treatment,the nodule number of different inoculation treatments had no significant change,but the nitrogenase activity of double inoculated plants were 15.55%higher than that of single inoculated with S.meliloti.The nodulation number and nitrogenase activity of S.meliloti and A.tumefaciens co-inoculated plants were significantly higher than that of single inoculation plants under 400 mg.kg^-1Cu²⁺and Cu²⁺,Zn²⁺dual stress.As a result,the total amount of N in the aboveground or underground parts of the double inoculated plants was the highest.It can be seen from the paraffin sections of the rhizobia that the number of bacterial cells in the rhizobia decreases with the increase of the concentration of heavy metals in the matrix.Under the same stress condition,the number of infected cells in the rhizobia formed by double inoculated plants was significantly higher than that of single inoculated plants.The synergistic effect of the two can promote the successful invasion of host plants by rhizobia in Cu-Zn contaminated substrates form an effective symbiotic system thereby could promote the growth,nodulation and nitrogen fixation of leguminous plants,and at the same time slow down the stress on the plants.