Spatial Differentiation Of Asymbiotic Diazotrophs And Nitrogen Fixation Activity In Alpine Meadows Of Qinghai-Tibet Plateau

Under the influence of extreme climate,the primary productivity of alpine meadows on the Qinghai-Tibet Plateau is limited by nitrogen availability.Biological nitrogen fixation has become the main source of nitrogen in the Qinghai-Tibet Plateau due to the lack of nitrogen fertilizer application.As compared to symbiotic nitrogen fixation,asymbiotic nitrogen fixation can fix nitrogen without relying on a symbiotic relationship with other organisms and is more widely distributed in space and time.Therefore,asymbiotic nitrogen fixation is the main source of nitrogen input to the alpine meadow ecosystem on the Qinghai-Tibet Plateau.However,there is currently limited research on the community structure and nitrogen fixation activity of asymbiotic diazotrophs,and their mutual relationships in the soil-plant system of alpine meadow on the Qinghai-Tibet Plateau.In this study,dominant individual plants and those from plant community were collected from five typical alpine meadow sites on the Qinghai-Tibet Plateau.The community structure of asymbiotic diazotrophs and nitrogen fixation activity of soil and plant at different sites and in different plants were determined by using high-throughput sequencing and acetylene reduction assay.The spatial differentiation of asymbiotic diazotrophs and nitrogen fixation activity in different parts of different dominant plant species in alpine meadow were compared by bioinformatics methods.The community of asymbiotic diazotrophs,the spatial difference of nitrogen fixation activity in soil-plant system,and the key driving factors of aymbiotic nitrogen fixation in soil-plant systems under different spatial geographical locations were studied.The results are of great significance for understanding the role of aymbiotic nitrogen fixation in maintaining grassland productivity and promoting sustainable development of alpine meadows on Qinghai-Tibet Plateau.The main conclusions are as follows:1.Spatial differences between soil and plant nitrogen fixation activities in alpine meadows of Qinghai-Tibet Plateau:（1）The plant nitrogen fixation activity of alpine meadows ranged from 0.618 to 18.466nmol C₂H₄g^-1h^-1,while the average value of soil nitrogen fixation activity ranged from 0.023 to 0.084 nmol C₂H₄g^-1h^-1.（2）Among the sampling sites,Haibei and Namucuo had significantly higher plant nitrogen fixation activity than other sites,while Naqu had the highest soil nitrogen fixation activity.（3）The nitrogen fixation activity of dicotyledonous plants,such as Ajania tenuifolia,Oxytropis ochrocephala,Potentilla anserina L.,Aster flaccidus and Leontopodium nanum,showed an absolute advantage over monocotyledonous plants.However,there was no significant difference in soil nitrogen fixation activity among plant species.2.Spatial differences in diversity and community structure of asymbiotic diazotrophs in soil-plant systems in alpine meadows on the Qinghai-Tibet Plateau:（1）The abundance of nif H gene and Shannon diversity of diazotrophs decreased gradually from bulk soil to rhizosphere and then to endosphere,with no significant differences observed among plant species.（2）Compared to plant species and different parts of plants,the spatial geographical position exerted the most significant influence on the community structure of asymbiotic diazotrophs.In five sampling sites,the influence of different plant parts on the community structure of diazotrophs was greater than that of plant species.（3）Anabaena was significantly enriched in bulk soil and rhizosphere at the Namucuo site,while Naqu site showed significant enrichment of Bradyrhizobium in endosphere.In addition,Cyanobacteria was significantly enriched in dicotyledons relative to monocotyledons in bulk soil,rhizosphere and endosphere.3.Key driving factors of asymbiotic nitrogen fixation potential in alpine meadows of Qinghai-Tibet Plateau:（1）Microbial life was the major contributor to the differences in plant nitrogen fixation activity,while soil physical and chemical properties were the major contributors to the differences in soil nitrogen fixation activity.（2）Plant nitrogen fixation activity was significantly correlated with the diversity and community structure of asymbiotic diazotrophs.The relative abundance of Anabaena in endosphere and rhizosphere was the key driving factor,and it was positively correlated with plant nitrogen fixation activity.（3）There was a significant positive correlation between soil nitrogen fixation activity and the copy number of nif H gene in bulk soil.TOC and NH₄⁺-N were the key driving factors which had a positive impact on soil nitrogen fixation activity.The present study revealed significant spatial differences in asymbiotic diazotrophs and nitrogen fixation activity in the soil-plant system of the alpine meadow on the Qinghai-Tibet Plateau.The nitrogen-fixing activity of dicotyledonous plants was found to be higher than that of monocotyledonous plants.The community structure of asymbiotic diazotrophs emerged as a key driver of plant nitrogen fixation activity,while TOC and NH₄⁺-N were identified as key drivers of soil nitrogen fixation activity.