Seasonal Variation Of Free-Living Nitrogen Fixation And Net Nitrogen Mineralization In Alpine Meadow Of Qinghai-Tibet Plateau

Biological nitrogen fixation is the process by which diazotrophs reduce N₂to ammonium through nitrogenases.This process includes both symbiotic and free-living nitrogen fixation.The latter is widely distributed in alpine meadow ecosystems.Net nitrogen mineralization refers to the process by which microorganisms convert complex organic nitrogen compounds into inorganic nitrogen and release it into soil.Biological nitrogen and net nitrogen mineralization are important nitrogen sources for plants and play a critical role in alleviating nitrogen limitation of primary productivity in alpine meadows.However,there has been a lack of systematic research on free-living nitrogen fixation and soil net nitrogen mineralization of alpine meadows.To address this gap,this study investigated nitrogenase activity,soil net nitrogen mineralization,and associated microbial communities by using different plant phenophases（green-up,reproduction,and wilting）,different plant species（Elymus nutans,Poa annua,Kobresia humilis,Carex moorcroftii,moss and mixed community）and different niches within the soil-plant system as materials,in order to reveal the phenological distribution characteristics and key drivers of free-living nitrogen fixation of the soil-plant system and soil net nitrogen mineralization in alpine meadows of Qinghai-Tibet Plateau.Our results could provide a theoretical basis for evaluating the nitrogen supply capacity of alpine meadows and grassland management.The key results are as follows:（1）Nitrogenase activity was influenced by plant phenology,plant species,and different niches within the soil-plant system.In the soil-plant system,nitrogenase activity was found to be highest in leaves,followed by roots and soil.Soil and root nitrogenase activity generally increased as phenological periods progressed.Significant seasonal changes in leaf nitrogenase activity were observed only in mixed plant communities.In situ nitrogenase activities of mosses,Carex moorcroftii,and mixed plant communities showed significant seasonal changes with a pattern of initial decline followed by an increase.Mosses were the primary contributors to nitrogen fixation in alpine meadows,exhibiting greater leaf and in situ nitrogenase activities compared to other plant species at all phenological stages.Soil ammonium nitrogen,available soil potassium,soil respiration,and the ratio of total soil organic carbon to total nitrogen were important environmental factors influencing soil,root,leaf,and in situ nitrogenase activities,respectively.However,environmental factors had a low degree of explanation for changes in nitrogenase activity.The diazotrophic community in alpine meadows was dominated by the genus Bradyrhizobium,its relative abundance showed significant seasonal changes in various niches of the soil-plant system（bulk soil,rhizosphere soil,and root endosphere）,but the shifting rules were different.The patterns of seasonal variation in alpha diversity in diazotrophic communities were different in different niches.Diazotrophic community in rhizosphere soil was most affected by phenology,but seasonal variation in the diazotroph community in bulk soil was not significant.Compared with different plant species and niches,variation in diazotrophic community structure was least explained by plant phenology.Phenological period had a stronger influence on diazotroph community structure in rhizosphere soil compared to bulk soil and root endosphere,and diazotrophic community structure was strongly influenced by phenological period in the rhizosphere soil of Carex moorcroftii,rather than in that of other plant species.In addition,plants also had a selective effect on diazotrophic community,resulting in the enrichment of Bradyrhizobium in the roots of individual plants.Cyanophyceae was enriched in the rhizosphere soil during the green-up period,and was most abundant in the rhizosphere soil of Carex moorcroftii.Alpha diversity was highest in bulk soil,followed by rhizosphere soil and lowest in root endosphere.Nitrate nitrogen in the soil was an important environmental driver of alpha diversity and community structure of diazotrophs in alpine meadows.The characteristics of diazotrophic community were correlated with the activity of azotobacter in the corresponding parts only in the specific parts in the specific period,and the characteristics of diazotrophic community in rhizosphere soil were strongly correlated with root nitrogenase activity in the green-up stage.（2）Soil net nitrogen mineralization potential was primarily composed of net nitrification rate.The overall trend of seasonal variation in net nitrification rate and net potential nitrogen mineralization showed a reduction throughout the plant growing season,but seasonal variation in the net ammonification rate of soil under different plant community type was different.Plant species can regulate soil inorganic nitrogen content by influencing soil net nitrogen mineralization.At each phenological stage,soil under the Cyperaceae（Kobresia humilis and Carex moorcroftii）had significantly lower rate of net nitrification,net nitrogen mineralization potential,and inorganic nitrogen content compared to other plant species.Soil net nitrification rate and net nitrogen mineralization potential were positively correlated with most environmental factors（such as nitrate nitrogen,ammonium nitrogen,total nitrogen,etc.）,but negatively correlated with p H.Environmental factors related to net ammonification rate were mostly negatively correlated,but as the period of phenology progressed,the negative correlation changed to a positive one.Nitrate nitrogen is a key environmental factor for net nitrification rate and net nitrogen mineralization potential,while monthly average temperature and monthly average rainfall were key environmental factors for net ammonification rate.Bacteroidia was the most abundant class of bacterial communities in the bulk soil of alpine meadows,and its relative abundance decreased significantly over time.The alpha diversity and community structure of bacterial community were affected by plant phenology.In the fungal community,the relative abundance of dominant classes,alpha diversity and community structure were not sensitive to the changes of plant phenology.Bacteria that can synthesize arginase,dehydrogenase,N-acetylglucaminidase,protease and urease,and saprophytic fungi were selected as potential functional groups of soil nitrogen mineralization in the functional prediction results.The seasonal variation of the relative abundances of bacteria that can synthesize arginase and dehydrogenase showed an increasing trend,while the seasonal variation of the relative abundances of bacteria that can synthesize N-acetylglucosaminidase showed a decreasing trend.There was no significant seasonal variation in the relative abundance of Saprophytic fungi.There was a strong correlation between the net nitrogen mineralization and the relative abundance of potential functional groups in the reproduce stage.The relative abundance of bacteria that can synthesize urease was significantly positively correlated with the net ammonia rate,net nitrification rate and net nitrogen mineralization potential.（3）Nitrogen mineralization is related to free-living nitrogen fixation.Net nitrogen mineralization promoted alpha diversity of diazotrophic communitiy in bulk soil by increasing nitrate nitrogen content,and inhibited nitrogenase activity by increasing ammonium nitrogen content in soil.