The Physiological Mechanism Of Pisolithus Sp.1 In Response To Hexavalent Chromium Stress And The Remediation Effect Of Ectomycorrhizal Pinus Thunbergii On Chromium Contaminated Soil

With the development of industrialization and urbanization,chromium（Cr）pollution in the environment has increased.Cr mainly exists in two valence states in the natural environment:hexavalent chromium（Cr（VI））and trivalent chromium（Cr（III））,of which the toxicity of Cr（VI）is 100 times that of Cr（III）.In recent years,the technology of plant-microbe joint remediation of heavy metal contaminated soil has broad application prospects due to its environmental friendliness and low cost.Ectomycorrhizal fungi（EMF）are widely present in nature and can form Ectomycorrhiza（ECM）with most woody plants,which can help promote plant growth and enhance the stress tolerance of the host plant.In the early stage,our laboratory screened a Cr（VI）-resistant ectomycorrhizal fungi Pisolithus sp.1（Sp1）,which can remove Cr（VI）by extracellular reduction.At the same time,the expression of two nitrate reductase genes（nid D,nii A）in Sp1 was significantly up-regulated under Cr（VI）stress.Therefore,this paper will explore the regulation mechanism of nitrate reductase on the extracellular reduction of Cr（VI）by Sp1,the regulation mechanism of nitric oxide on Sp1 alleviating Cr（VI）-induced oxidative stress,and the remediation potential of Sp1-Pinus thunbergii symbionts on Cr contaminated soil was studied.Through liquid culture experiments,taking Pisolithus sp.1 as the research object,the regulation mechanism of nitrate reductase（NR）on the extracellular reduction of Cr（VI）in Sp1 was explored.The results showed that Cr（VI）stress significantly increased the NR activity in Sp1,and the NR activity reached the highest level after 10 mg/L Cr（VI）treatment for 48 h.The addition of NR inhibitor（Tungstate）has no significant effect on the total Cr content in the liquid Kottke mediums,but it can significantly reduce the Cr（VI）reduction rate.Under Cr（VI）stress,the H⁺efflux rate of Sp1 increased significantly,and NR inhibitor treatment could significantly reduce the H⁺efflux rate.Adding proton pump inhibitor（Na₃VO₄）can significantly inhibit the Cr（VI）reduction process.However,Sp1 can not reduce Cr（VI）in the liquid Kottke mediums under anaerobic conditions,and it can not grow normally.Therefore,NR reduces Cr（VI）to Cr（III）by regulating the extracellular secretion of H⁺by Sp1,thereby reducing the toxicity of Cr（VI）.In addition,the reduction process is aerobic reduction.Through liquid culture experiments,the physiological mechanism of nitric oxide（NO）regulating Sp1 alleviating Cr（VI）-induced oxidative stress was explored.The results showed that Cr（VI）stress significantly increased the endogenous NO content in Sp1,and the NO content reached the highest level after 10 mg/L Cr（VI）treatment for 48 h.Adding NR inhibitor treatment under Cr（VI）stress can significantly reduce the NO content in Sp1,while the NOS inhibitor（L-NAME）has no significant effect on the NO content in Sp1.Adding NO scavengers（c PTIO）to remove endogenous NO has no significant effect on the accumulation of Cr in Sp1,but it aggravates the accumulation of ROS,membrane lipid peroxidation and decreased metabolic activity in Sp1 induced by Cr（VI）stress.Under Cr（VI）stress,the activity of CAT in Sp1 increased significantly,and its activity decreased significantly after removing NO.At the same time,GSH content,GR and GSNOR activities in Sp1 were significantly increased under Cr（VI）stress,while GSH content and enzyme activities related to its synthesis and recycling were significantly decreased after NO was eliminated.However,NO has no significant effect on ASA content,MDHAR and DHAR activity.Therefore,NO does not directly affect the accumulation of Cr,but can alleviate the oxidative stress caused by Cr（VI）by selectively increasing the activity of CAT in Sp1 and promoting the synthesis and recycling of GSH.In order to explore the potential of Sp1 to assist phytoremediation of Cr contaminated soil,another Cr（VI）sensitive bacterium,Pisolithus sp.2,was selected,including two mycorrhizal P.thunbergii and non-mycorrhiza P.thunbergii.They were planted in Cr contaminated soil.Four months later,the plant growth,nutrient elements and Cr content were measured,and the soil physical and chemical properties,DTPA-Cr and Cr（VI）content and changes in enzyme activity were analyzed.The results show that the inoculation of ectomycorrhizal fungi can promote the absorption of nutrient elements（N,P,Mg）of P.thunbergii,thereby increasing the biomass of P.thunbergii and the survival rate in Cr contaminated soil.Moreover,compared with the non-mycorrhizal P.thunbergii,the mycorrhizal P.thunbergii significantly enhances the ability to accumulate Cr,and increases the soil available phosphorus content and the activities of urease,acid phosphatase,and alkaline phosphatase,and reduces the soil p H,DTPA-Cr and Cr（VI）content.At the same time,mycorrhizal P.thunbergii can significantly increase the relative abundance of beneficial microorganisms in Cr contaminated soil.Among them,the P.thunbergii inoculated with Sp1 has higher biomass and stronger nutrient absorption capacity,and has a better effect on improving the soil ecological environment.Therefore,using Cr（VI）tolerant ectomycorrhizal fungi to assist P.thunbergii to remediation Cr contaminated soil has great practical value and application prospects.