The Adaptation Mechanism Of Leymus Chinensis To Alkali Stress Based On Root Exudates Associated Regulation Of Microbial Nitrogen Transformation In Rhizosphere

Soil salinization caused by factitial and natural factors is not only a global problem that human beings have to face,but also is one of the most important stress factors to impact ecosystem structure,process and functions in terrestrial ecosystems.Saline-alkali stress includes salt stress and alkali stress,and the damage of alkali stress to plants is greater than that of salt stress.High pH is the most obvious characteristic of alkali stress,and easy to infect plant roots,invalidate soil nutrients and interfere with plant metabolic activities,which could affect plant growth and survival.N（N）is one of the most important of essential nutrients among the3 mineral elements.Ecosystem provides various forms of N for plant utilization through the process of N cycle.Nitrification is the key rate-limiting step of N cycling.Soil pH as the main regulating factor,low pH inhibits soil nitrification,while high pH is beneficial to soil nitrification to oxidize ammonium N（NH₄⁺-N）to nitrate N（NO₃^--N）.NO₃^--N is easy to be lost by leaching or denitrification,and high pH will volatilize NH₄⁺-N in the form of ammonia（NH₃）.Therefore,high soil pH is not conducive to soil N existence.Previous studies have shown that many dominant species of successional top plants have biological nitrification inhibition（BNI）,which is considered to be an ecological driving force developed by top ecosystems to adapt to low N environment.Leymus chinensis（Trin.）Tzvel.is not only a constructive species of dominant plant community in meadow steppe and typical steppe in Northern China,but also a kind of high-quality forage grass,which has the characteristics of cold tolerance,drought tolerance,saline-alkali tolerance,strong ecological adaptability and wide ecological range.L.chinensis is widely distributed in saline-alkalized meadow steppe in Northeast China,and it often forms a single dominant community.Although there have been a lot of studies on the mechanism of alkaline tolerance of L.chinensis,the characteristics of root exudates and their roles in the processes of alkaline adaptation of L.chinensis are not clear yet.In this study,we explored the evidence of L.chinensis’BNI from field experiments and controlled laboratory experiments,and then focused on the effects of different forms of N on L.chinensis growth and adaptation to alkali stress,aiming to explore the feedback effect of root exudates on its living environment under different forms of N,and analyze the ecological functions of different forms of N in the process of L.chinensis growth and adaptation to alkali stress.In addition,we also explored the rhizosphere effects of L.chinensis on the transformation process of soil microbial N under the condition of soil culture.Finally,our study revealed that L.chinensis controlled soil N supply patterns using biological nitrification inhibitors（BNIs）in root exudates to inhibit soil nitrification,and enhanced dissimilatory nitrate reduction to ammonium（DNRA）through rhizosphere effect,so as to optimize its own growth and adapt to alkali stress.The main results of this study are as following:（1）Taking the northeast saline-alkali meadow steppe as the research object,L.chinensis,Puccinellia tenuiflora and Suaeda salsa of vegetation as different succession stages were selected to determine the soil nutrient,soil pH,electrical conductivity,soil nitrification intensity,the absolute abundance of soil nitrification genes amo A-AOA and amo A-AOB,and the carbon utilization function of soil microorganisms.To explore the effects of vegetation succession on soil N nutrition patterns,soil microorganisms and the relationship between soil microorganisms and soil N transformation in saline-alkali meadow steppe.The results showed that the content of soil nutrients increased,and the proportion of NH₄⁺-N in soil inorganic N also increased,while soil pH and electrical conductivity decreased with the positive advance of vegetation succession.As a succession top plant community,L.chinensis community has the highest soil nutrient content,and the conditions of pH and electrical conductivity are more suitable for plant growth and development.Meanwhile,the nitrification intensity of L.chinensis community soil was the weakest,but the absolute abundance of nitrification functional gene amo A was much higher than that of Puccinellia tenuiflora and Suaeda salsa community soil.On the contrary,the nitrification intensity of P.tenuiflora and S.salsa community soil with inferior absolute abundance of amo A gene was significantly stronger than that of L.chinensis community soil,which indicated that the soil nitrification of L.chinensis community soil is inhibited by some substances.In addition,the absolute abundance of amo A-AOA in different vegetation soils of northeast saline-alkali meadow steppe was much higher than that of amo A-AOB,indicating that AOA is the dominant microorganism of soil nitrification in the experimental ecosystem.（2）The root exudates of L.chinensis were collected and its effect on soil nitrification was studied using self-made root exudates collection device.The results showed that the root exudates of L.chinensis had significant inhibitory effects on soil nitrification.However,due to the high pH in saline-alkali soil,NH₄⁺-N is easy to volatilize in the form of NH₃,which is not conducive to the preservation of NH₄⁺-N.It contradicts the theoretical mechanism that plant BNI takes advantage of the low mobility of NH₄⁺-N,which prolong the absorption and utilization of N nutrients,and increase of the soil N use efficiency（NUE）due to decrease NO₃^--N leaching or denitrification loss.Therefore,we proposed a new mechanism of L.chinensis adapting to alkali stress.L.chinensis has BNI-capacity,mainly because it needs to ensure the supply of NH₄⁺-N and make use of the metabolic process of NH₄⁺-N to adapt to alkali stress.Firstly,the H⁺produced by NH₄⁺-N metabolism and excreted outside of the roots can not only reduce the rhizosphere pH and improve the availability of rhizosphere nutrients,but also provide proton driving force for plants to absorb anion nutrients such as nitrate（NO₃^-）,sulfate(SO₄^2-)and phosphate(PO₄^3-),which is beneficial to L.chinensis to improve the absorption and utilization efficiency of these nutrients.Secondly,NH₄⁺-N metabolism increased the probability of L.chinensis synthesizing phytosiderophores（PS）,which is beneficial to L.chinensis absorbing and utilizing iron（Fe）nutrition in soil.In the processes of L.chinensis using this pathway to adapt to alkali stress,NH₄⁺-N mainly regulated the growth and adapted to alkali stress,while NO₃^--N mainly played a role in nutrient supply.（3）The soil culture method was used to study the effects of L.chinensis on the properties of soil under different N treatments.Our results showed that NH₄⁺-N in non-rhizosphere soil was oxidized into NO₃^--N due to the lack of BNIs of L.chinensis,when L.chinensis absorbed water,NO₃^--N was easily diffused from non-rhizosphere to rhizosphere soil with soil water runoff because of its strong mobility,and was absorbed and utilized by L.chinensis,so that the content of total inorganic N in non-rhizosphere soil was significantly lower.L.chinensis secreted BNIs and inhibited nitrification in rhizosphere soil,so the content of NH₄⁺-N in rhizosphere soil was significantly higher than that in non-rhizosphere.Meanwhile,it effectively reduced the pH of rhizosphere and created better environmental conditions for L.chinensis’s growth and development when it used NH₄⁺-N.The content of NH₄⁺-N in rhizosphere soil was significantly higher than that in non-rhizosphere soil when treated with NO₃^--N.We also found that the relative abundance of nitrifying bacteria and denitrifying bacteria in L.chinensis rhizosphere soil was significantly lower than that in non-rhizosphere soil,which provided strong evidence for BNI of L.chinensis.These results indicated that the root exudates of L.chinensis may inhibit soil denitrification.The absolute abundance of nitrate reduction bacteria and DNRA bacteria in rhizosphere soil of L.chinensis was significantly higher than that in non-rhizosphere soil,indicating that L.chinensis has the ability to strengthen soil DNRA process by using its rhizosphere effect,it effectively solved the problem of insufficient NH₄⁺-N in saline-alkali soil,and then affected the expression of BNI ability of L.chinensis.As a dominant plant species of climax plant community in saline-alkalized grassland,L.chinensis has evolved a series of adaptive strategies after long-term natural selection to cope with saline-alkali stress.L.chinensis can not only inhibit soil nitrification by secreting BNIs from roots,but also strengthen soil DNRA process through rhizosphere effect.From both sink and source aspects,L.chinensis ensures the stable supply of NH₄⁺-N to itself in saline-alkali environment,and further use the metabolism of NH₄⁺-N to improve the pH and nutrient status of its living environment.The important finding in our study that the alkali stress adaptation strategy of L.chinensis based on root exudates not only has important biological and ecological significance for restoration of degraded ecosystem,adaptive management of saline-alkalized meadow grassland ecosystem,but also plays an important reference role in enhancing the alkali tolerance and yield of crops in agricultural production.Our results can also provide new theoretical support for the restoration and reconstruction of saline-alkali grassland ecosystem and agricultural production.