Transformation Of Carbon And Nitrogen In Soil Aggregates As Affected By Long-term Fertilization In A Rice-wheat Rotation System

Organic fertilization has always been considered as an important practice for yield improvement of rice-wheat rotation system,which provides vital guarantee for sustainability of agroecosystem.As the key component of soil structure,soil aggregate plays the material basis for soil fertility and mediation for carbon?C?and nitrogen?N?turnover.Understanding the transformation features of C and N in soil aggregates is thus a basic precondition of reasonable fertilization.Based on an established long-term fertilizer experiment under rice-wheat rotation,we investigated the distribution of soil C and N,structural composition of soil humus,C,N-related enzyme activities,microbial community composition variation,soil microbial diversity that involved in ammonia oxidizing and cellulose decomposition processes across different soil particle-size fractions.In this research,biological techniques of solid-state cross-polarization and magic angle spinning ¹³C nuclear magnetic resonance(CP/MAS¹³C-NMR),phospholipid fatty acid?PLFA?,quantitative PCR?qPCR?,terminal restriction fragment length polymorphism?T-RFLP?and 454 pyrosequencing were explored to elucidate the transformation of C and N in soil aggregates in rice-wheat rotation under long-term fertilizations.The main findings were as follows:?1?Distribution of C and N across soil particle-size fractions under long-term fertilizations.Soil aggregate were separated as large macroaggregate?>63?m?,coarse sand?2000-200?m?,fine sand?200-63?m?,silt?63-2?m?and clay?2-0.1?m?fractions in this study.The proportions of different particle-size fractions were affected by long-term fertilizations with the least fine sand fraction obtained in both seasons.This yellow-brown paddy soil was dominated by silt and large macroaggregate in wheat season but by coarse sand and silt in rice season.Compared with inorganic fertilizer,extra organic fertilizer application significantly reduced large macroaggregate proportion but increased the percentage of coarse sand,resulting in soil structure improvement.The results also revealed the pronounced influence that particle-size fraction emphasized on soil organic carbon?SOC?and total N distribution,with the highest SOC,total N concentration and C:N ratio observed in the fine sand fraction.Except fine sand,SOC and C:N ratios were greater in larger fractions?>63?m?than silt and clay fractions?63-0.1?m?.Coarse sand and clay contained relatively higher total N content than the others in wheat and rice seasons,respectively.Generally,organic fertilizer application significantly enhanced SOC and total N,but decreased soil C:N ratios in both seasons.?2?Differences of extracellular enzyme activities across soil particle-size fractions under long-term fertilizations.The results indicated enzyme activities significantly varied with different particle-size fractions and fertilizer treatments,which were strongly correlated with SOC,total N and C:N ratios.The ten tested extracellular enzymes including phosphatase,sulfatase,?-glucosidase,?-cellobiosidase,N-acetyl-glucosaminidase,?-xylosidase,?-glucosidase,L-leucine-aminopeptidase,phenol oxidase and peroxidase were highest in fine sand fraction except phosphatase and sulfatase activities in wheat season.Furthermore,compared with inorganic fertilization,enzyme activities under organic and inorganic fertilizer combined treatments were significantly advanced except phosphatase,sulfatase,N-acetyl-glucosaminidase and?-xylosidase in wheat season and sulfatase,?-glucosidase and N-acetyl-glucosaminidase in rice season.?3?Characteristics of soil microbial community structure across soil particle-size fractions under long-term fertilizations.Both particle-size fraction and fertilizer treatments significantly affected soil microbial community structure.Total PLFAs,which were improved under organic fertilizer treatments,showed higher abundance in larger fraction?>63?m?than smaller fractions?63-0.1?m?in both seasons especially silt fraction.>200?m fraction held relatively lower gram-positive bacteria:gram-negative bacteria ratios and higher fungi:bacteria ratios in wheat season.It was demonstrated that total N,C:N ratio,phosphatase,sulfatase,N-acetyl-glucosaminidase and?-xylosidase activities were the major factors that impacted on soil microbial community in wheat season.Parameters SOC,total N,C:N ratio,?-glucosidase,sulfatase,?-glucosidase,?-cellobiosidase and phenol oxidase activities significantly affected soil microbial community in rice season.?4?Variations of ammonia-oxidizers across soil particle-size fractions under long-term fertilizations.We found fertilization and particle-size fractions had strong effects on potential nitrification activity and ammonia-oxidizers community,where 40%of the variations could be explained by C,N levels and involved extracellular enzyme activities.Compared with control,inorganic and organic fertilizer combined treatment significantly enhanced the potential nitrification activity of bulk soil and soil particle-size fractions,which also contained more ammonia-oxidizing bacteria?AOB?than inorganic fertilizer treatment.Generally,ammonia-oxidizing archaea?AOA?and AOB amoA gene copy numbers were higher in rice season than wheat season.AOA:AOB ratios with highest values in clay fraction were reduced under fertilizer treatment.Even though AOA population was hundreds of times greater than AOB,it seemed AOB responded more sensitively to ambient shifts.Pyrosequencing data revealed that the Thaumarchaeota Group I.1b and Nitrosospira Cluster 3a were the dominant clusters of AOA and AOB,respectively.?5?Responses of cellulolytic genes to soil particle-size fractions under long-term fertilizations.The calculated results revealed the remarkable effects of particle-size fraction and fertilization on the gene abundances of fungal glycosyl hydrolase cellobiohydrolase I?cbhI?and bacterial glycosyl hydrolase?GH48?that participated in cellulose decomposition.cbhI and GH48 which were most abundant in fine sand and poorest in clay fraction,showed general increases with organic fertilizer input.Based on the correlation analysis between cellulolytic gene copy numbers and soil humus structure,we observed larger fractions?>63?m?which showed lower humification degree with higher aromaticity,lower aliphaticity and alkyl:O-alkyl ratios of humic acid,always associated with higher abundance of cellulolytic microbes.However,smaller fraction?63-0.1?m?exhibited lower cbhI and GH48 gene abundances.In summary,long-term combined application of inorganic and organic fertilizers reduced the proportion of>2000?m fraction but increased that of coarse sand.Larger fractions?>63?m?generally contained higher C and N contents,enhanced C,N-involved enzyme activity,total PLFA,abundances of ammonia-oxidizing bacteria and cellulolytic genes than smaller fractions?63-0.1?m?.Compared with inorganic fertilization,organic amendments could enhance the above parameters which indicated the acceleration of C and N transformation in soil aggregate and improvement of soil fertility.