Features Associated With Nitrogen Loss And Microbial Communities In Rice-frog Cultivation

Advances in intensive agriculture,such as the use of fertilizers,have increased rice production every year.A key part of the intensification is the use of fertilizer.However,excessive fertilizer application is a common problem in China’s rice crop industry.Particularly excessive amounts of nitrogen（N）fertilizer,coupled with a low utilization rate,results in its accumulation of N in the soil.The situation is exacerbated by runoff and leakage into water bodies from paddy fields,causing eutrophication and a series of environmental pollution problems such as red tide and water blooms,that seriously threaten the safety of drinking water and food production.It is,therefore,important to understand green production patterns and their interaction with ecological breeding and cultivation in rice paddy fields to improve China’s food security and environmental protection systems.In this paper,we evaluated N loss of ecologically cultivated rice frogs in the Qingpu Modern Agricultural Park in Shanghai.Specifically,we analyzed N leakage,runoff loss and soil microbial community structure（especially nitrifying bacteria）in the green rice-frog cultivation（GR）and the organic rice-frog cultivation（OR）,with conventional rice cultivation（CR）as a control,between 2016and 2017.With this,we aimed to understand the regulation of N loss and the associated microbial communities during rice-frog cultivation and provide a theoretical reference and technical support to guide the production of high-quality rice and reduce N loss.The main findings were as follows:（1）The highest total nitrogen（TN）runoff and leakage loss,12.94 kg·hm^-2,was recorded in CR,which was significantly higher than those from GR or OR,indicating that the latter significantly reduced N runoff and leakage loss relative to CR.Both GR and OR significantly reduced the leakage loss of ammonium nitrogen（NH₄⁺-N）and nitrate-nitrogen（NO₃^–-N）.Particularly,NH₄⁺-N represented the main form of inorganic N in the leakage water,accounting for 55.07%of the TN content,followed by dissolved organic nitrogen（DON）,which was about 37.99%.OR recorded a significantly higher rate of DON loss than CR or GR.In addition,GR required reduced levels of chemical fertilizers while ensuring optimum yield,while replacing chemical with 50%organic fertilizer led to reduced N loss.OR was found to minimize N loss and reduce non-point source pollution of paddy rice fields.（2）GR and OR significantly increased soil TN content by 16.84 and 16.53%,and also significantly reduced soil NO₃^–-N content by 12.93 and 32.67%,respectively relative to CR.Moreover,OR significantly reduced NH₄⁺-N content in the soil by24.69%over two years.Correlation analysis revealed a significant negative relationship between TN content and its leakage loss,and a significant positive association between NH₄⁺-N content with its leakage loss as well as that of NO₃^–-N.Furthermore,NO₃^–-N and NH₄⁺-N content in the soil were closely related to N loss in CR and OR,soil-available phosphorus（AP）,TN content in the soil,and soil organic carbon（SOC）.（3）GR and OR recorded significantly lower soil urease activity,of 43.39 and25.52%,respectively relative to CR.Similarly,GR and OR also recorded higher soil protease activity,by 14.22 and 33.62%respectively,as well as soil nitrate reductase activity,by 63.09 and 35.95%,respectively.Correlation analysis revealed a significant positive relationship between NH₄⁺-N and NO₃^–-N leakage with soil urease activity.Conversely,NH₄⁺-N and TN leakage had a significantly negative correlation with soil protease activity.In addition,OR and GR significantly increased soil microbial biomass carbon（SMBC）,by 31.89 and 42.65%,respectively relative to CR.NH₄⁺-N leakage showed a significantly negative relationship with SMBC,microbial biomass nitrogen（SMBN）and the SMBC/SMBN ratio,whereas an increase in microbial biomass mediated a significant reduction in NH₄⁺-N and NO₃^–-N leakage.Overall,the level of leakage had a significantly negative relationship with SMBC and the SMBC/SMBN ratio.（4）A significantly higher community richness of bacteria,archaea and fungi in the rhizospheric soil was recorded in the OR system,while that of soil fungal communities was significantly higher in GR mode,relative to CR.In addition,GR and OR had a significant effect on the structure and composition of soil microbial communities.The main driving factors for changes in soil bacterial,archaea,and fungal communities included SOC,as well as NH₄⁺-N and NO₃^–-N contents.Contents of NH₄⁺-N and NO₃^–-N in the soil as well as changes in the structure of soil microbial communities were also closely related to the N leakage loss.Overall,GR and OR recorded significantly higher enrichment of OTUs in soil ammonia-oxidizing archaea（AOA）,but lower enrichment of OTUs in ammonia-oxidizing bacteria（AOB）,relative to CR.Correlation analysis revealed a close relationship between N leakage loss with AOA and AOB abundance.（5）OR recorded significantly higher（by 690.30%）expression levels of AOA amo A gene,relative to CR.On the other hand,GR and OR had significantly lower levels of AOB amo A copies,by 96.16 and 94.55%,respectively,compared to the CR.AOB mainly drove nitrification in the rhizospheric soil of CR,while AOA mainly dominated nitration in the OR system.In addition,GR and OR affected community structure and composition of soil AOA and AOB,by significantly increasing the species richness indices（ACE index）of both microbes.Apart from this,GR significantly reduced the species diversity index（simpson index）of AOA,while OR significantly increased the AOB species diversity index（simpson index）.Moreover,OR significantly reduced the relative abundance of Betaproteobacteria and Nitrosospira,but significantly increased the relative abundance of the unclassified genus Nitrosomonadales and Nitrosotalea.Overall,GR and OR affected AOA and AOB communities by changing SOC and NH₄⁺-N contents,thereby reducing N leakage loss from rice paddy fields.In summary,both GR and OR significantly reduced N loss in paddy fields.Furthermore,nitrification in OR was mainly done by AOA,whereas AOB was the main microorganism in CR.The best approach for reducing NO₃^–-N leakage entailed controlling community abundance of AOA and AOB.Therefore,to ensure the production of high-quality rice,with reduced N loss,we recommend the use of GR.