Effects And Mechanism Of Nitrogen Addition And Subshrub Encroachment On Soil Microbial Community In A Grassland Of The Loess Plateau

Grassland is the largest terrestrial ecosystem in China,which plays an important role in climate regulation,windbreak and sand fixation,soil and water conservation,carbon fixation and oxygen release,and biodiversity protection,and is of strategic significance to ecological and food security.However,due to global climate change and human unreasonable use,most grassland in China is facing degradation,especially in the fragile ecological environment of the Loess Plateau region,which is related closely to the ecological protection and high-quality development of grassland and animal husbandry in the Yellow River Basin.Anthropogenic nitrogen deposition is a global environmental problem that can alter community compositions and functions,and consequently,the ecosystem services.However,the effect of nitrogen deposition on plants,soil and microorganisms is highly regional and closely related to ecosystem types,soil types and climate factors.In addition,subshrub/shrub encrochment is a typical phenomenon in arid and semi-arid grassland in recent years,which has changed the structure and function of biological community.However,it is not clear how the encrochment of Artemisia sacroorum（subshrub）in grassland of the Loess Plateau affects the grassland vegetation-soil characteristics and microbial community.In this study,we take the grassland ecosystem of Yunwu Mountain in Ningxia on the Loess Plateau as the research object,by arranging four nitrogen addition levels and three subshrub encrochment intensities（light,moderate and heavy subshrub encrochment）,we research the effects of nitrogen addition,subshrub encrochment and their interaction on grassland vegetation-soil characteristics and microbial community（bacterial and fungal communities）diversity and composition,and analyze the driving factors affecting of the diversity and composition of soil microbial community.At the same time,the effects of nitrogen addition and subshrub encrochment on microbial co-occurrence network and the assembly process of soil microbial community were analyzed.The main results are as follows:（1）Nitrogen addition,subshrub encrochment and their interaction had significant effects on vegetation-soil characteristics.Nitrogen addition had significant positive effects on the community weighted mean of leaf nitrogen content(LN_CWM),leaf nitrogen phosphorus ratio,and soil alkali hydrolyzable nitrogen（AN）,but had significant negative effects on the species evenness index（J）,community weighted mean of leaf carbon content(LC_CWM)and soil p H.Plant community species richness index（s）,diversity index（H）,dominance index（D）,functional richness index（FRic）,functional evenness index（FEve）,functional dispersion index（FDis）,community weighted mean of specific leaf area(SLA_CWM),community weighted mean of leaf dry matter content(LDMC_CWM),community weighted mean of leaf phosphorus content(LP_CWM),aboveground biomass（AB）,Belowground biomass（BB）,soil water content（SWC）,organic carbon（OC）,total nitrogen（TN）,total phosphorus（TP）,and available phosphorus（AP）had no significant response to nitrogen addition.Moderate and heavy subshrub encrochment significantly increased the LC_CWM,LN_CWM,LP_CWM,leaf nitrogen phosphorus ratio,AB,SWC,OC,TN,AN,TP and AP,but decreased the D,LDMC_CWM,J and soil p H.Heavy subshrub encrochment reduced S,J and FDis.Nitrogen addition had no significant effect on plant community composition,but subshrub encrochment changed plant community composition.The interaction of nitrogen addition and subshrub encrochment only had significant effects on LC_CWM,leaf nitrogen phosphorus ratio and soil p H.（2）Subshrub encrochment significantly changed the soil microbial communityα-diversity and composition,nitrogen addition and the interaction between nitrogen addition and subshrub encrochment on soil microbial communityα-diversity and composition had no significant effect.The dominant phyla of soil bacterial community are Proteobacteria,Acidobacteria,Actinobacteria and Gemmatimonadetes,and the dominant phyla of soil fungal community are Ascomycota,Mortierellomycota,Basidiomycota and Chytridiomycota.Two factor analysis of variance showed that the effects of nitrogen addition and the interaction between nitrogen addition and subshrub encrochment on soil microbial communityα-diversity and relative abundance of microbial dominant phyla had no significant effect.LEf Se,principal coordinate analysis（PCo A）,permutational multivariate analysis of variance（PERMANOVA）and multi responser permutation procedure（MRPP）showed that nitrogen addition had little effect on the composition of soil microbial community.In contrast to nitrogen addition,the subshrub encrochment significantly affected microbial communityα-diversity and composition.Two factor analysis of variance showed thatα-diversity of soil bacterial community was the highest in heavy subshrub encrochment grassland,but the fungal communityα-diversity has no significant change in the three subshrub encrochment grasslands.PCo A,PERMANOVA and MRPP showed that the composition of soil bacterial and fungal communities was different in light,moderate and heavy subshrub encrochment grassland.Partial least squares structural equation model showed that subshrub encrochment affected soil microbial community indirectly by directly affecting vegetation characteristics and soil properties.The determinants of bacterial communityα-diversity are vegetation structure(SLA_CWM and plant community composition)and soil properties（OC,AN,TP,AP and SWC）.The composition of bacterial community is mainly regulated by plant aboveground biomass and soil properties,and the main driving factor of fungal community composition is vegetation structure.（3）Nitrogen addition and subshrub encrochment significantly changed the structure and complexity of soil microbial co-occurrence network.We built the overall co-occurrence network of soil bacterial and fungal communities based on all samples（36）,and also built the specific co-occurrence networks of nitrogen addition and subshrub encroachment,respectively.The results showed that nitrogen addition reduced the network complexity of soil bacterial and fungal communities.Soil bacterial and fungal communities had the highest network complexity in moderate and heavy subshrub encrochment grassland,respectively.All bacterial specific co-occurrence networks are simple networks,and fungal specific co-occurrence networks have certain complexity.We used Kolmogorov-Smirnov test to evaluate the overall distribution pattern of node attributes（degree,betweenness,closeness and transitivity）of specific co-occurrence networks with nitrogen addition and subshrub encrochment,the results showed that there were significant differences in the overall distribution of node attributes of microbial specific co-occurrence network,that is,nitrogen addition and subshrub encrochment changed the structure of microbial network.Spearman correlation analysis showed that the topological properties of soil bacterial network（nodes number,edges number,degree,average path length and betweenness）were significantly related to many environmental factors(SLA_CWM,LDMC_CWM,LC_CWM,LN_CWM,LP_CWM,FRic,FEve,H,D,J,AB,OC,TN,AN,TP,AP,p H and SWC),LN_CWM and AN have the higest correlation coefficients.The topological properties of soil fungal network are only significantly related to AN and FEve.（4）The assembly process of soil microbial community is mainly stochastic process.The migration rate of soil microbial community reached the peak in heavy subshrub encrochment grassland,which indicating that the soil microbial community has higher diffusion ability and is more conducive to soil microbial communityα-diversity increase.Spearman correlation analysis and Mantel test showed that the microbial communityα-diversity and composition have little correlation with environmental factors（vegetation characteristics and soil properties）.Variance partitioning analysis and hierarchical partitioning further showed that the explanatory variance of vegetation characteristics and soil properties on the difference of microbial community composition was low（<20%）,and the deterministic process played a secondary role in the construction of soil microbial community.Neutral community model（NCM）,PER-SIMPER and dispersal-niche continuum index showed that the formation of soil microbial community was mainly regulated by stochastic process.In addition,the effects of stochastic process on different taxa（geuns to phylum）of soil microbial community are consistent,which indicating that the taxa in the same phylogenetic lineage have similar responses to stochastic process.NMDS shows that the microbial taxa of neutral and non neutral partitions formed based on NCM have different compositions.The richness and abundance of microbial taxa in neutral partition were significantly higher than those in non neutral partition.The migration rate of microbial taxa is the main reason for the formation of neutral and non neutral partitions.In this study,we analyzed the effects of nitrogen addition,subshrub encrochment and their interaction on grassland vegetation-soil characteristics and microbial community,and supposed to the following conclusions:（1）Compared with nitrogen addition and the interaction between nitrogen addition and subshrub encrochment,subshrub encrochment has significant effects on grassland vegetation-soil characteristics and microbial community（α-diversity and composition）.（2）Subshrub encrochment indirectly affects soil microbial community by affecting vegetation-soil characteristics.（3）Nitrogen addition and subshrub encrochment change the structure and complexity of microbial co-occurrence network.AN can be used as a predictor of the topological properties of microbial co-occurrence network.（4）The assembly process of soil microbial community in grassland of Loess Plateau is mainly stochastic process.