Microbiological Mechanism Of Soil Carbon And Nitrogen Transformation Under Long-Term Straw Return In A Double-Cropped Rice Paddy

As the main grain producing area in China,the southern double-cropping rice system plays an important role in ensuring China’s food security.In recent decades,straw chopping and returning in situ has become an important way to increase soil carbon and fertilize and improve the quality of cultivated land.The results of previous study showed that there were seasonal differences in the content of soil organic carbon and nitrogen in double-cropping paddy field by returning straw to field.However,under the condition of exogenous carbon and nitrogen input mediated by different ways of straw returning for a long time,the characteristics of soil carbon and nitrogen transformation and the driving mechanism of soil microorganisms in double-cropping paddy field are not clear.Based on the existing long-term positioning experiment of double-cropping rice field,under the same amount of N,P and K nutrient input,this project was divided into four treatments:no chemical fertilizers application with straw removal（CK）,single application of chemical fertilizer（F）,chemical fertilizers application with straw removal（SBR）,and straw return with reducing chemical fertilizers（SR）.Long-term field experiment,pot experiment of ¹³C and ¹⁵N isotope double labeling and soil flooding culture were used to clarify the effects of different straw returning methods on carbon and nitrogen fixation and transformation of double cropping rice plants and soil,the transport and distribution characteristics of photosynthetic carbon and exogenous nitrogen in rice-soil system,and the transformation and supply characteristics of soil carbon and nitrogen.Combined with enzymology,soil microbial 16S r RNA and metagenomic next-generation sequencing technology,the key functional microbial mechanism of soil carbon and nitrogen transformation in double-cropping rice field under long-term different straw returning methods was revealed.The main results are as follows:1.Effect of long-term straw returning on yield,nutrient absorption and accumulation of double cropping rice.Under the same nutrient conditions,long-term straw returning to the field has a significant cumulative yield increase effect on double-cropping rice,and the increase of SR yield is significantly higher than that of SBR,especially in early rice season.Compared with F treatment for 13 years,the cumulative yield increase rates of SBR and SR for early rice reached 3.53%and 5.16%respectively,and the yield increase rates for late rice season reached 2.41%and 3.65%,respectively.The reason of increasing yield lies in the increase of effective panicle number and grain number per panicle.SR significantly improved the dry matter quality of stem and sheath and the total dry matter accumulation of plants at the heading stage of early rice,which was beneficial to the accumulation of dry matter and total dry matter in plant organs at various growth stages of late rice.At the same time,it also significantly increased the total root surface area（increase by 34.6%-61.3%）,total root length,total root volume and root tip number,as well as root activity and active absorption area.In addition,SR is beneficial to improve the carbon fixation of early and late rice plants,nitrogen（N）absorption in the late growth stage（heading stage and maturity stage）,and improve nutrient utilization efficiency.2.Effects of long-term straw returning on soil physical and chemical properties and carbon and nitrogen fixation in double cropping rice fields.Long-term total straw returning significantly improved soil physical properties,increased soil moisture content,total porosity and capillary porosity,and significantly reduced soil bulk density.Compared with F,SR can significantly improve the content of macroaggregates in 0-15 cm soil layers and the stability of soil structure,while SBR can significantly reduce the content of macroaggregates in the cultivated layer（0-15 cm）.SR significantly increased the total organic carbon content in 0-30 cm soil layer and the total nitrogen content in deep soil（10-30 cm）in double cropping rice field,which was related to the increase of ammonium nitrogen and nitrate nitrogen.In addition,SBR significantly increased the content of available potassium in different growth stages and soil layers of rice,while SR only significantly increased the content of slowly available potassium.Except for early rice maturity,SR significantly increased soil total organic carbon and soil total nitrogen content in early and late rice maturity,mainly due to the increase of soil alkali-hydrolyzable nitrogen,soluble organic carbon and nitrogen content.In addition,SR is beneficial to improve soil enzyme activity,and then accelerate soil carbon and nitrogen turnover in double-cropping rice fields.3.Transport and distribution characteristics of photosynthetic carbon and exogenous nitrogen in rice-soil system under long-term straw returning.The results of double labeling of ¹³C and ¹⁵N isotopes in potted plants showed that the photosynthetic carbon(¹³C)was mainly distributed in aboveground plants,accounting for76.8%-79.0%,followed by underground plants.Compared with F treatment,SBR and SR significantly reduced the transport and distribution of photosynthetic carbon to the underground part of plants.Compared with SBR,SR significantly increased the proportion of photosynthetic carbon distribution in leaves and stems,but it was not conducive to the storage of rice ears and soil.In addition,about 61.76%-73.43%of ¹⁵N was accumulated in panicle grains,followed by stem and leaf organs.However,SR significantly increased the total accumulation and distribution rate of ¹⁵N in panicle,promoted the absorption of fertilizer nitrogen and other nitrogen sources by double cropping rice,and was beneficial to improve nitrogen utilization rate.4.Characteristics of carbon and nitrogen transformation in soil under long-term straw returning.Through waterlogged incubation experiment,it was found that the nitrogen mineralization amount of SR was higher than that of SBR and F treatment,especially in the late stage of soil mineralization culture（42-49 days）,but there was no significant difference in nitrogen mineralization rate.In addition,SR significantly increased the accumulation of soil nitrogen mineralization and nitrogen mineralization potential,indicating that returning all straw to the field was beneficial to improve the nitrogen supply capacity of paddy soil.However,SBR significantly reduced the proportion of soil mineralizable nitrogen,resulting in relatively insufficient nitrogen mineralization potential in the later stage of soil.In addition,SR significantly reduced the cumulative amount of soil carbon mineralization（CO₂-C）,but increased the cumulative release of CH₄and N₂O,which enhanced the stability of soil organic carbon in paddy fields to some extent.5.Effects of long-term straw returning on soil microbial diversity in double cropping rice fields.SR significantly increased the richness of soil bacterial community at the young panicle differentiation stage and heading stage of double-cropping rice,and increased the unique OTUs number in the soil at the young panicle differentiation stage and early rice maturity stage.At the same time,SR mainly increased the species abundance of Proteobacteria,Acidoptera and Bacteroides in the young panicle differentiation period,as well as Firmicutes and Cyanophyta in each main growth period;However,SBR only increased the species richness of Actinomycetes at heading stage,but decreased the species richness of Chlorocurvata and Acinetobacter.LEf Se analysis showed that the species enrichment degree of different straw returning treatments varied with growth period and season.In addition,the changes of soil microbial diversity and species richness during the growth period of double cropping rice mainly affect soil p H value,total organic carbon,soluble organic carbon,total nitrogen and available potassium content.6.Effect of long-term straw returning on the structure and function of rhizosphere soil microbial community in double cropping rice.The results of rhizosphere soil metagenome sequencing showed that the microbial community of early and late rice was mainly bacteria,accounting for 98.23%-99.11%.SR significantly increased the relative abundance of Archaea of early rice and Eukaryote of late rice,while F and SBR only significantly increased the relative abundance of late rice eukaryote.At the phylum level,SR was beneficial to improve the relative abundance of Bacteroidea,Archaea and Archaea in early rice soil,and Microfungi in late rice,especially Proteobacteria in double-cropping rice field.However,SBR only significantly increased the relative abundance of Phomophila and Archaea in late rice.At the species level,the unique species number of SR is obviously more than that of F and SBR,which indicates that the soil microorganisms with total straw returning to the field have higher species diversity.At the same time,the dominant species of rhizosphere soil microorganisms such as Proteobacteria,Bacteroides,Euryarchaeota,Bathyarchaeota and Verrucomicrobia affect the changes of p H value,total organic carbon,carbon-nitrogen ratio,available potassium and soil enzyme activities in paddy soil.The function of rhizosphere soil microbial community was analyzed.The results showed that metabolic functional proteins accounted for the largest proportion in all treatments,and there were still a large number of unknown functional proteins.SR significantly increased the transport of inorganic ions and the relative abundance of functional proteins such as metabolism class（P）,early rice cell movement class（N）,post-translational modification of late rice,protein transformation,chaperone class（O）and extracellular structural class（W）of eukaryotic cells.Under the condition of total straw returning to the field,7 kinds of glycoside hydrolases and 2 kinds of carbohydrate binding modules were significantly enriched in paddy soil in the morning and evening.At the same time,SR also increased the gene abundance of enzymes involved in carbon metabolism pathway（glycolytic pathway,pentose phosphate pathway,tricarboxylic acid cycle and methane metabolism）and nitrogen-fixing enzyme,glutamate synthase and nitrite reductase,especially nitrogen metabolism pathway.Moreover,SR increased the relative abundance of carbon metabolism（carbohydrate metabolism,carbon fixation and methane metabolism）and nitrogen metabolism-related functional genes such as glt B,GDH2,ncd2,npd,nas A,nif H and nif D,especially nitrogen metabolism functional genes.In addition,the abundance of metabolic pathways and functional genes involved in carbon and nitrogen cycle is significantly correlated with total organic carbon,C/N ratio,available potassium,sucrase and cellulase activities in rhizosphere soil.In general,long-term different straw returning methods affect soil microbial species composition and community structure in double-cropping paddy fields,resulting in changes in carbon and nitrogen metabolic pathways and functional gene abundance,thus driving carbon and nitrogen turnover and stability in the soil system.Among them,the total amount of straw returning is more beneficial to increase the diversity and richness of soil microbial community,to the abundance and function of enzyme genes involved in carbon and nitrogen metabolism pathway,to improve soil quality,to enhance the carbon fixation and nitrogen supply capacity of paddy soil,and to better promote soil carbon sequestration and fertilizer cultivation and double cropping rice yield.The results can provide a theoretical basis for the scientific utilization of straw resources and the improvement of cultivated land quality in double-cropping rice system in South China.