The Characteristics Of Microbial Transformation Of Nitrogen Under Substitution Of Mineral Fertilizers By Organic Amendments In A Paddy Ecosystem

Partial substitution of chemical fertilizer with organic amendments in double rice cropping systems is one of the key technologies utilized for ensuring food security and achieving green and sustainable agriculture in China.In this study,rice yield,microbial community composition,enzymes activity,microbial ammonia oxidation and biological nitrogen fixation processes as well as their interaction mechanism in bulk soil from a long-term?34 years?fertilizer field trial were investiguate by employing microplate fluorometric protocol,phospholipid fatty acid?PLFAs?,¹⁵N isotope tracing technology,quantitative PCR?q PCR?,high-throughput sequencing and co-occurrence network analysis.The main findings were as follows:?1?Characteristics of rice yield and soil nutrients to the partial substitution of chemical N with organic N.Compared with the mineral NPK fertilization regime,the replacement of 30%,50%and 70%mineral N with organic N increased the yield of early rice by 6.4%,4.5%and 8.3%,respectively and that of late rice by 8.0%,6.7%and 9.6%,respectively.The rice yield tended to increase as the organic N substitution percentage increased.Moreover,increasing the organic N substitution percentage could significantly?p?0.05?improve the contents of total carbon?TC?,total nitrogen?TN?,NH₄⁺-N,available phosphorus?AP?and pH value in both seasons.However,the soil C/N tended to decrease.?2?Responses of soil extracellular enzymes activity to the partial substitution of chemical N with organic N.The fertilization,season and their interaction effect significantly affected soil extracellular enzyme activity.In the early rice season,substitution of 30%-70%mineral N with organic N increased?-cellobiosidase?CBH?activity relative to that in the NPK treatment by 39.4%-251.0%;similarly, increases under the substitution treatments were observed for?-xylosidase?XYL,25.2%-48.8%?, ?-glucosidase?BG,55.1%-83.1%?,?-glucosidase?AG,44.5%-111.7%?,N-acetyl-glucosaminidase?NAG,109.6%-175.1%?,L-leucine aminopeptidase?LAM,58.4%-139.9%?and urease?URE,17.6%-56.4%?;while in the late rice season,compared with NPK treatment,substitution of 30%-70%mineral N with organic N increased CBH activity by 137.9%-723.1%,and increased the activities of XYL?56.9%-140.1%?,BG?82.7%-155.5%?,AG?1.51%-32.4%?,NAG?54.4%-75.7%?,LAM?49.0%-178.5%?and URE?19.6%-47.3%?.Moreover,soil enzyme activities increased as the percentage of organic N substitution increased.RDA analysis showed that soil extracellular enzyme activities were significantly affected by TC and NH₄⁺-N in the early rice season;While in the late rice season,NH₄⁺-N was the crucial factor markedly affecting soil enzyme activities,followed by NN,AP and pH.?3?Variations of soil microbial biomass and community composition to the partial substitution of chemical N with organic N.In the early rice season,substitution of 30%-70%mineral N with organic N,the microbial biomass carbon?MBC?and the microbial biomass nitrogen?MBN?were 1.53-1.79times and 1.21-2.45 times than that of NPK treatment,respectively;While in the late rice season,the MBC and MBN were 1.38-1.99 times and 1.19-1.48 times than that of NPK treatment,respectively.The interaction effect of fertilization and season was stronger than their individual effects.Compared with the mineral NPK fertilization regime,substitution of 30%-70%mineral N with organic N increased the content of total PLFAs by 59.0%to 83.4%in the early rice season and by 70.1%to 88.5%in the late rice season.However,the relative abundance of Fungi and the ratio of G⁺to G^-as well as Fungi to Bacteria were decreased.Furthermore,RDA analiysis showed that AP and pH were the major variables affecting microbial composition in the early rice season;While,in the late rice seasons,the main influencing factors were NH₄⁺-N,pH and TN,respectively.The relative abundances of identified PLFA biomarkers 16:1?5c,16:1?7c and 18:1?7c were markedly higher?p?0.05?in the organic alternative treated soils than the NPKtreated soils in both seasons,indicating that methane-oxidizing bacteria,ammonia-oxidizing bacteria and Pseudomonas spp.played important roles under the organic substitution regimes.?4?Changes of ammonia-oxidizers to the partial substitution of chemical N with organic N.The fertilization,season and their interaction effect significantly affected the potential nitrification rate?PNR?,abundance and community composition of ammonia-oxidizing archaea?AOA?and ammonia-oxidizing bacteria?AOB?.AOA abundance were significantly higher?p?0.05?in the late rice season than in the early rice season,while the AOB abundance exhibited the opposite trend.Moreover,PNR,AOA and AOB abundance increased as the percentage of organic N substitution increased,but the ratio of AOA to AOB tended to decrease.The AOB abundance was strongly correlated with the PNR and influenced the PNR more strongly than the AOA,indicating that AOB rather than AOA plays a major role in the ammonia oxidizing process in organic substitution management in double rice cropping systems.The high-throughput sequencing analysis revealed that the archaeal Nitrososphaera and Nitrosotalea clusters,as well as bacterial Nitrosospira and Nitrosomonas genera,were dominant in paddy soils.Overall,the pH and TN/AP were the key factors shaping the composition of the ammonia oxidizers.?5?Differences of N-fixer to the partial substitution of chemical N with organic N.The fertilization,season and their interaction effect significantly affected the potential nitrogen fixation rate?PNFR?and diazotrophic abundance and community structure,and the effect of seasonal shift on free-living diazotrophic community composition was greater than that of fertilization.As the percentage of organic N substitution increased,the diazotrophic abundance markedly decreased,however,the PNFR was not be affected.In addition,RDA analiysis revealed that pH and Fe²⁺/Mo were the major variables affecting diazotrophic microbial composition in the early rice season;While,in the late rice seasons,the main influencing factors were NH₄⁺-N and TN/AP,respectively.High-throughput sequencing and network analyses disclosed that Bradyrhizobium and Geobacter dominated in paddy soil and the microbe-microbe interactions.In addition,“collaborative”relationships diazotrophic taxa were greater than“competing”relationships.Models incorporating C/N and Fe²⁺/Mo were more effective at predicting the PNFR than those integrating free-living diazotrophic community composition,including the co-occurrence and keystone patterns.To sum up,compared with the mineral NPK fertilization regime,partial substitution of chemical N with organic N under the same inputs of N,P and K can increase the rice yield,alter the N cycle efficiency of the paddy field and improve soil biochemical properties.