Effects Of Different Management Models On The Community Structure Of Free-living Nitrogen-fixing Microbes In The Alpine Meadow Soil On Tibet Plateau

Nitrogen,as one of the essential macronutrients for living organisms,can not only influence the healthy growth and reproduction of above-ground plants and limit terrestrial net primary production(NPP),but also change the soil nutrient gradient and microbe's community structure and its functions.The global nitrogen biogeochemical cycling processes are predominantly driven by microorganisms and it has become one of the hotspots for people to study the mechanism of nutrient cycles and the changes of environment and climate globally.Tibet Plateau is fundamental for sustainable development of animal husbandry and environmental protection because of its unique alpine meadow ecological environment and protective screen.However,alpine meadow have suffered from sever degradation driven by coupled adverse effects of climate change with improper and overload utilization,which is viewed as a serious threat to the balance of ecosystem and local economic development.Recently,numbers of reports have found the interacted relationship between nitrogen cycling and plants in Tibet Plateau's natural conditions and different management models,but little research focused on the effect of different management models on free-living nitrogen fixers in soil.To better understand how N-fertilization and grazing intensity impact free-living N-fixers,we investigated the long-term fertilization and grazing experiment at a study site of Alpine Meadow Ecosystem Research Station of Lanzhou University in Gannan Tibetan autonomous prefecture song county,using clone library analysis and quantitative polymerase chain reaction(qPCR)under different fertilizer inputs(0,30,60,90,120 g m-2year-1)and grazing intensity(prohibition-grazing,seasonal-grazing and annual-grazing).The objectives were to explore the relationship between soil physicochemical properties and free-living diazotroph's community structure and abundance,so that we can provide theoretical debate to help manage and recover alpine meadow to some expectations.The main results of this study are as follows:1.The effects of long-term fertilization on the free-living N-fixers' community structure and their abundance Clone library results based on nifH gene showed that all retrieved DNA sequences in 15 libraries were classified into 9 clusters.Among them,Proteobacteria(?-,?-,?-,?-)were the dominant in amount,followed by Cyanobacteria,Firmicutes,Actionbacteria,Archaeal and unidentified cluster.The amount of each cluster differed from fertilization treatments,and the main cluster in each treatment varies obviously.Overall,uncultured bacterium was the dominant cluster,and its relative proportion in all clusters ranged from 42.00%(F30)to 61.33%(F120).In the aspect of cultured bacteria,the four kinds of Proteobacteria distributed more widely and were higher in abundance than the other four clusters.From a diversity point of view,every Shannon-weaver index in all libraries stayed above 2.10.F60 fell into the lowest(2.14),but F120 got into the highest(2.84).It revealed that N-fixers showed a high and increasing diversity from F0 to F120 with no significance.The results estimated by qPCR indicated that the abundance of nifH genes decreased sharply,responsible for fertilization.The copy of nifgenes lay from 3.15×106 to 7.76×106copies g-1 dry soil,and it decreased significantly(p<0.05)with increased fertilizing amount.In the most fertilizer treatment F120,gene copies were 59.4%lower than the control F0.2.The effect of grazing intensity on the free-living N-fixers' community structureAll retrieved nifH sequences based on the phylogenetic tree could be classified into 5 clusters:Alphaproteobacteria,Betaproteobacteria,Cyanobacteria,Firmicutes and unidentified cluster.These clusters spread in all the grazing treatments with different proportion,and their amount varied within various grazing intensities to some extent.The amount of Alphaproteobacteria and unidentified cluster were increasing gradually,while that of Firmicutes was decreasing regularly.Overall,both Betaproteobacteria and Cyanobacteria had reduction signs in amount.3.Relationships between the community structure and abundance of N-fixers and soil physical and chemical factors in fertilizer treatmentMantel test explaining the effects of those soil characteristics on the bacteria community composition showed that the change of community structure was strong response to pH(p=0.009),NO3--N(p=0.007),available N(p=0.030)and AN/AP(p=0.028).Moreover,there was a marginal significant positive correlation between community structure and available P(p=0.076),whereas other soil characteristics analyzed had small impact on the structure change.Pearson's correlation coefficients between soil physicochemical characteristics and nifH gene copies indicated that NO3--N(p=0.030),available N(p=0.022),available P(p=0.015)and AN/AP(p<0.010)had a general or highly significant effect on the abundance of nifH gene.In addition,NO-3-N(r=-0.559),available N(r=-0.584)and available P(r=-0.615)affected significantly negatively,but AN/AP correlated highly significantly positively.4.Relationships between the community structure of N-fixers and soil physicochemical characteristics in grazing treatmentNMDS analysis revealed the difference of diazotrophic community structure was strongly related to soil physicochemical characteristics.Specifically,community change was remarkably causedby pH(r2=0.654,p=0.045)and available P(r2=0.764,p=0.017).In conclusion,long-term fertilization and grazing intensity were found to significantly altered free-living N-fixers' community structure and change the diversity,and fertilization had a dramatic effect on the abundance of N-fixers.Soil physicochemical characteristics can significantly affect N-fixers' community structure and their abundance.