Effect Of Exogenous Application Of Pseudomonas Fluorescens On Nitrogen Assimilation And Flavonoids Metabolism In Ginkgo Biloba L.under Nitrogen Deposition And Cadmium Stress

With the rapid development of industrialization and urbanization worldwide,heavy metal pollution of urban soils has become increasingly serious and the emission and deposition of reactive nitrogen（Nr）in the urban environment has reached high levels.Urban forest plants perform a purifying function in urban ecosystems but are also subject to the dual stress of nitrogen deposition and cadmium pollution,which can have an impact on both nitrogen metabolism and resistance of plants.Pseudomonas fluorescens（Pf）is a ubiquitous plant growth-promoting rhizobacteria that enhances host plant stress tolerance and improves soil physicochemical properties.Exogenous addition of microbial fertilizers with Pseudomonas fluorescens as the active ingredient can improve the host plant’s resistance to heavy metal toxicity and promote plant growth and development under nitrogen deposition stress.Therefore,we selected Ginkgo biloba L.,an important greening tree species in Shenyang urban forest,as the research material,used soil culture to simulate soil Cd pollution and N deposition conditions,and applied Pseudomonas fluorescens suspension as microbial fertilizer to the roots of Ginkgo biloba L.We investigated the effects of N deposition,Cd stress and the combination of the two on the nitrogen assimilation and flavonoids metabolism of Ginkgo biloba L.,as well as the effect of exogenous application of Pf on the mitigation of Ginkgo biloba L.stress.The experiment consisted of eight treatments:CK、Cd(100mg·kg^-1)、N（NH₄NO₃）(10 g·m^-2·y^-1)、Cd+N、Pf(4×10⁸ CFU·m L^-1)、Cd+Pf、N+Pf、Cd+N+Pf,respectively.The results are as follows:（1）Compared with the control,Cd treatment significantly reduced Ginkgo biloba L.plant height and inhibited root growth,while N addition did not significantly affect Ginkgo biloba L.growth,and Ginkgo biloba L.plant height and root length were lower under Cd+N treatment than Cd treatment alone.Exogenous addition of Pf alleviated the inhibitory effect of Cd stress and Cd+N treatment on the growth of Ginkgo biloba L.to some extent.Cd+N treatment significantly increased the concentration of Cd in the root system,stems,and leaves of Ginkgo biloba L.compared to Cd treatment.Pf application significantly increased Cd concentrations in roots and leaves of Ginkgo biloba L.under Cd treatment,but significantly decreased Cd concentrations in roots,stems,and leaves of Ginkgo biloba L.under Cd+N treatment.（2）Compared with the control,Cd treatment significantly reduced the activities of nitrogen（N）assimilation-related enzymes（nitrate reductase（NR）,nitrite reductase（NIR）,glutamine synthetase（GS）,and glutamate synthetase（GOGAT））,resulting in a decrease in nitrate nitrogen（NO₃^–-N）and an increase in ammonium nitrogen（NH₄⁺-N）content in Ginkgo biloba L.leaves;in addition,Cd treatment decreased the soluble protein content but increased the soluble sugar content in Ginkgo biloba L.leaves.N addition significantly increased the activities of N assimilation-related enzymes,NO₃^–-N,NH₄⁺-N,total nitrogen,and soluble protein content,but significantly decreased soluble sugar content in Ginkgo biloba L.leaves.The activities of N assimilation-related enzymes,NO₃^–-N,and total N content in Ginkgo biloba L.leaves under Cd+N treatment were higher than those of Cd treatment alone,but significantly lower than those of control and N addition treatment,while NH₄⁺-N and soluble sugar content were significantly higher than those of other treatments.The application of Pf significantly increased the activities of nitrogen assimilation-related enzymes under Cd and Cd+N treatments,except that exogenous addition of Pf did not affect NIR activity under Cd+N treatment,and in addition,exogenous addition of Pf significantly increased NIR and NR activity under N addition treatment.Exogenous addition of Pf increased NO₃^–-N,total nitrogen,and soluble protein content in Ginkgo biloba L.leaves under all treatments,but decreased NH₄⁺-N content in all treatments.（3）Compared with the control,Cd treatment significantly increased the levels of reactive oxygen species（ROS,including O₂.^-and H₂O₂）in Ginkgo biloba L.leaves,as well as significantly increased phenylalanine ammonialyase（PAL）,cinnamic acid 4-hydroxylase（C4H）,4-coumarate Co A ligase（4CL）activities,and total flavonoids content.Compared with the control,N addition significantly increased ROS levels in Ginkgo biloba L.leaves,while significantly decreasing PAL,C4H,and 4CL activities as well as total flavonoids content.Cd+N treatment significantly increased ROS levels,as well as PAL,C4H,and 4CL activities and total flavonoids content.Exogenous addition of Pf significantly reduced ROS levels under Cd,N,and Cd+N treatments.In addition,exogenous addition of Pf significantly increased PAL,C4H,and 4CL activities and total flavonoids content under Cd stress,but significantly reduced C4H and 4CL activities and total flavonoids content under N addition and Cd+N treatment.In conclusion,the combined treatment of N deposition and Cd stress significantly increased the enrichment of Cd in the root system of Ginkgo biloba L.compared with Cd stress alone,while the growth of Ginkgo biloba L.showed a more severe inhibitory effect.However,the complex stress promoted the process of nitrogen assimilation in Ginkgo biloba L.leaves under Cd stress and improved the osmoregulatory ability of plants in response to oxidative stress.Exogenous addition of Pf promoted the uptake of Cd by plants,but significantly alleviated the oxidative damage suffered by plants and facilitated the nitrogen assimilation process in Ginkgo biloba L.Therefore,Pf can be used as an effective microbial strain as a way to alleviate the inhibitory effect of Cd contamination and nitrogen deposition environment on plant growth.