Microbial Response Mechanism Of Variation In Soil Reactive Nitrogen Driven By Land-Use Changes In Paddy Ecosystem

Soil nitrogen status is closely related to agricultural production and ecological environment.The change in soil reactive nitrogen（Nr）pool is an important factor affecting soil fertility,environment and health quality.Microorganisms are the key factors of soil nitrogen transformation processes,and the succession of microbial communities and functions are closely related to Nr components and their environmental effects.Currently,soil Nr change and its related functional response of microbiomes upon agricultural land-use change driven by rapid urbanization are largely unknown.Taking farmland around Chengdu as the research object,soil profile samples（0～20,20～40,40～60,60～100 cm）were collected from 141 sites in 3 land use patterns（rice-wheat rotations,rice-vegetable rotations and afforest land）.Using spatial-substitution-time,microbial 16S rRNA sequencing,metagenomic sequencing,structural equation modeling and path analysis,the changes of soil Nr pool and soil acidity,the succession of microbial community structure,the characteristics of nitrogen transformation functional microorganisms and soil acidification functional microorganisms caused by nitrogen transformation were studied after the paddy use change caused by the rapid expansion of typical cities.The primary results are as the follows:（1）Paddy use change caused significant changes in soil Nr pool and soil acidity,especially in conversion from rice-wheat rotations（RW）to rice-vegetable rotations（RV）.After the RW-to-RV conversion,the average concentrations of nitrate nitrogen content in soil profile was increased 98～431%,and the deeper soil layer had a greater increase indicating that nitrate nitrogen shifted downward through the RV soil profiles.The concentrations of microbial biomass nitrogen and dissolved organic nitrogen in0～40 cm RV soils were decreased 41%～44%and 30%～36%than those in the RW respectively,indicating that the increase of soil Nr pool in the RV was mainly from inorganic nitrogen.Ammonium nitrogen concentrations were increased significantly only in 0～20 cm.Soil exchangeable cation pool showed similar changes as the soil Nr pool,in which the RV enriched soil exchangeable acid cations and decrease a large number of exchangeable base cations.After the RW-to-RV conversion,the Al³⁺and H⁺concentrations in 0～100 cm soils were 3～8.65 times and 1.79～2.87 times higher than those before the conversion respectively,while the Ca²⁺and Mg²⁺concentrations in0～40 cm soils decreased by 43%～83%and 52%～70%respectively,and the average base cation saturation decreased from 95.50%to 54.68%.This indicated that the soil inorganic nitrogen accumulation and base cations loss and acid cations enrichment were important related factors of soil acidification after the conversion to RV;this change was particularly significant in 0～60 cm（p<0.05）,in which The pH value decreased by1.31 units in 0～20 cm,and decreased by 0.70 and 0.65 in 20～40 cm and 40～60 cm respectively.After the RW-to-RV conversion,except for soil microbial biomass nitrogen and exchangeable Ca²⁺concentrations decreased significantly and Al³⁺concentrations increased significantly in 0～20 cm,the concentrations of Nr components and pH in other soil profiles with no significant change,which indicated that under the condition of rapid urbanization,the soil quality change after the RW-to-RV conversion was worthy of high attention.（2）Changes of inorganic and organic nitrogen components in soil Nr pool are the important factors leading to the difference of soil acidification intensity driven by paddy use change.Using slope failure test,we found that the pH threshold of soil acidification was 5.17,their corresponding threshold of inorganic nitrogen concentrations was 38.67mg kg^-1.The acidogenic intensity of inorganic nitrogen was also affected by soil organic Nr components,and the thresholds of microbial biomass nitrogen and dissolved organic nitrogen were 18.22 and 28.87 mg kg^-1 respectively.The path analysis of structural equation showed that:under the condition of inorganic nitrogen concentrations<38.67mg kg^-1,the high microbial growth rate significantly reduced the concentrations of dissolved organic nitrogen,and then consumed soil inorganic nitrogen and increased soil microbial biomass nitrogen.At this time,the soil pH value was not decreased significantly.When the inorganic nitrogen concentrations>38.67 mg kg^-1,the low microbial growth rate led to a significant decrease in microbial consumption for inorganic nitrogen,caused the decrease of microbial biomass nitrogen.At this time,a decrease of soil microbial biomass nitrogen（path coefficient 0.35^*）and an increase of inorganic nitrogen(path coefficient-0.85^***)led to a significant decrease in soil pH.After the RW-to-RV conversion,the changes of soil acidity caused by soil active nitrogen pool were particularly significant.Compared to the RW,the pH of 0-20 cm soil layer decreased by 0.5 and 1.5 in 10-year and 20-year RV respectively,the microbialβdiversity decreased by 17.43%and 43.18%respectively,and these decrease of microbialβdiversity caused by pH decline significantly increased soil acid actions.In addition,path analysis showed that the continuous decrease of soil pH in the long-term RV was significantly correlated with excessive nitrogen application and high irrigation.Therefore,the microbial diversity loss was an important factor of soil acidification upon long-term excessive nitrogen application in the RV soils.（3）After the RW-to-RV conversion,the change of soil Nr was significantly affected the diversity of soil microbial communities and its nitrogen transformation function,and its influence varied with the change in conversion years and soil depths.Compared with the RW,soil microbial communities had a lower community turnover and resistance in 20～40 cm,microbial communities in which were significantly more similar in composition and abundance across soil profiles（0～40 cm）,namely the homogenization of microbial communities in soil 0～40 cm.This homogenization was driven by increased concentrations of nitrate N and decreased soil pH and soluble organic N concentrations after the RW-to-RV conversion.At 0～20 cm,the RV decreased the abundance of microbial N-fixation genes（nifHDK,anfG and vnfHGDK）,whereas increased the abundance of microbial nitrification genes（amoCAB,pmoCAB and hao）.Moreover,the RV significantly increased the potential denitrification and N₂O emissions from soil 0～40 cm via decreasing the abundance of bacterial nosZ genes（encoding nitrous-oxide reductase）and increasing archaeal nir KS（nitrite reductase）genes and bacterial nor BC genes（nitric-oxide reductase）.Therefore,the long-term RV led to homogenization of soil microbial communities,and weakened microbial nitrogen fixation functions,strengthened microbial nitrification ability,especially enhanced microbial denitrification and N₂O emission potential.（4）After the RW-to-RV conversion,inorganic nitrogen enrichment significantly increased the diversity of acidification-associated microorganisms and their dehydrogenation function genes.Linear discriminant analysis and cross validation showed that 7 biomarkers were significantly enriched in the RV soils compared with the RW.After the RW-to-RV conversion,the increase in abundance of Rhodanobacter,Gemmatrosa,Sphingomonas,Candidatus＿Solibacter and Streptomyces were significantly correlated with the increase in H⁺,Al³⁺and NO₃^--N concentrations,which could be defined as acid-dependent biomarkers.The abundance of 2 biomarkers were significantly decreased in the RV soils compared with the RW,the decrease in abundance of Nitrospira and Candidatus＿Entotheonella were significantly correlated with the decrease in Ca²⁺,Mg²⁺and Na⁺concentrations,which could be defined as base-dependent biomarkers.Random forest classification prediction also showed that the prediction accuracy of the 9 biomarkers for soil acidification in human management systems（farm and afforest land）was more than 90%.Compared with the RW,the RV significantly increased the abundance of dehydrogenation genes in acid-dependent biomarkers.On the one hand,the abundance ofγ-aminobutyric acid（M00135,GABA）synthesis and assimilation nitrate reduction（M00531）genes were increased significantly in the RV,which improved microbial H⁺consumption potential and maintained the pH homeostasis of acid-dependent microorganisms;on the other hand,the abundance of respiratory chain（M00115,M00151,M00417）and pentose phosphate pathway dehydrogenation（M0004）genes were increased significantly,which improved the ability of microbial H⁺transport and efflux in acid-dependent microorganisms.We inferred that this is the potential microbial mechanism of soil acidification in the RV soils.