Effects Of Long-term Fertilization On Carbon And Nitrogen Cycle And Microbial Regulation In Black Soil

Carbon（C）and nitrogen（N）cycle play an important role in maintaining soil quality and crop growth,and directly affect the stability and productivity of agricultural ecosystem.Fertilization application has a significant effect on the C and N cycle.However,there have been few reports on the mechanism of microbial regulation of the C and N cycle,especially on the effect of microorganisms in the roots,and the interaction between plants,soil and microorganisms needs further investigation.Therefore,based on the long-term positioning experiment of Jilin Agricultural University,combined with situ experiment,culture experiment and pot experiment,this paper systematically analyzed the effects of long-term fertilization on the conversion of C and N in maize rhizosphere soil by using multi-omics techniques of high-throughput sequencing（16s and ITs）and metagenomic sequencing,as well as gas phase and liquid phase mass spectrometry.Microbial community diversity and structural composition in rhizosphere soil and root endophytic and regulation of functional genes related to the C and N cycle;effects of plant-soil-microbial interactions on the C and N cycle and the effect of straw addition on the C and N nutrient content and greenhouse gas emissions.This study provides insight into the regulation of the C and N cycle by microorganisms under long-term fertilization and provides a theoretical basis for soil nutrient balance and sustainable development of farmland.The results showed that:1.Long-term fertilization affects soil C and N nutrient content and greenhouse gas emissionN fertilization significantly reduced organic carbon（SOC）content,which was 2.78 g/kg lower than CK treatment.The addition of straw increased the content of various forms of C in the soil,especially the straw combined with fertilizers promoted the accumulation of soil organic carbon,increased the dissolution rate and oxidation rate of soil organic carbon.Moreover,the addition of straw also improved the farmland management index（CMPI）,and the CPMI index under the treatment of NPKS was the highest.Long-term fertilization significantly increased soil total nitrogen（TN）content,and the highest TN content was 1.56 g/kg under NPKS treatment.The application of chemical fertilizer increased the percentage of ammonium nitrogen（NH₄⁺-N）and nitrate nitrogen（NO₃^--N）in TN.Ammonia nitrogen was the main organic nitrogen in maize rhizosphere soil,and it increased significantly under N treatment.The addition of straw promoted the formation of acid hydrolyzed organic nitrogen,especially the content of acid hydrolyzed total nitrogen（ATN）under NPKS treatment reached 243.39 mg/kg.The addition of straw promoted the emission of CO₂,and the application of fertilizer promoted the emission of N₂O.In addition,long-term fertilization can indirectly affect greenhouse gas emissions by affecting soil physicochemical properties.In particular,CO₂ is significantly correlated with soil SOC,water-soluble carbon（DOC）,easily oxidized carbon（ROC）,TN,NO₃^--N,ATN,acid-insoluble nitrogen（AIN）,available phosphorus（AP）,available potassium（AK）and total potassium（TK）.2.Long-term fertilization regulates C and N cycle by influencing soil microorganismsLong-term fertilization did not alter the microbial composition and dominant community of the maize rhizosphere soil.The microorganisms in rhizosphere soil were predominantly bacteria,accounting for 98.24%of the microbiome.Proteobacteria were dominant bacteria,ascomycetes were dominant fungi,and Bradyrhizobium and Rhizophagus were the highest relative abundance of bacteria and fungi at the genus level.However,it had significant effects on soil microbial diversity and species abundance.N fertilizer was the main factor affecting soil microbial communities.The application of N fertilizer reduced soil microbial diversity and thus soil SOC,TN and organic nitrogen contents,promoted the generation of NH₄⁺-N and NO₃^--N,and also reduced CO₂ emissions.Fertilization can regulate the conversion of soil carbon and nitrogen conversion by affecting the abundance of soil participating in C and N functional genes.The application of N fertilizer had a significant effect on the functional genes related to C cycle.Maize rhizosphere soil solidifies primarily through the reductive tricarboxylic acid cycle（r TCA cycle）,accounting for23.48～23.97%of the total C fixation pathway.Application of chemical fertilizer increased the abundance of C4-dicarboxylic acid cycle and crassulacean acid metabolism（CAM pathway）,and dicarboxylate-hydroxybutyrate（DC/4-HB cycle）.CH₄ is primiarily produced by the acetate substrate metabolic pathway,which accounted for 68.08～72.94%of methane metabolic pathway.Fertilizer application reduced the abundance of methanogenic genes（mtt B and mtt C）and CH₄oxidation genes（pmo A and pmo B）.The functional genes related to C degradation were mainly involved in the degradation of hemicellulose,cellulose and starch.The abundance of functional genes related to carbon degradation was higher in the straw treatment than in the manure treatment.The application of N fertilizer inhibited N degradation,nitrification and dissimilatory nitrate reduction,and promoted denitrification and assimilatory nitrate reduction.In addition,the application of N fertilizer also reduced the abundance of nif A gene in soil microbial nitrogen fixation.These results indicated that long-term application of N fertilizer had the highest effect on the C and N cycle in maize rhizosphere soil.In addition,functional genes related to soil C and N cycle were significantly correlated with soil physical and chemical properties,SOC（C:r²=0.79,N r²=0.83）and NO₃^--N（C:r²=0.64,N r²=0.68）were the main contributing factors of soil C and N cycle.3.Fertilization regulates C and N cycle by influencing microorganisms in maize root endophyticLong-term fertilization changed the dominant bacterial community of endophytic bacteria in the maize root.In PKS treatment,Cyanobacteria was the dominant bacteria,accounting for95.98%of the total bacteria,and Proteobacteria was the dominant bacteria in the other treatments.The correlation between endophytic microorganisms and soil nutrient content was lower than that of rhizosphere soil microorganisms.The C transformation of soil morphology was only related to Bacteroides.TN and hydrolyzed nitrogen were significantly correlated with Proteobacteria and Cyanobacteria,but not with other microorganisms.In addition,the correlation between root microorganisms and greenhouse gases was low,with no significant correlation between endophytic bacteria and N₂O and CH₄ emissions and no significant correlation between endophytic fungi and CO₂ and CH₄ emissions.Compared with rhizosphere soil,the carbon sequestration pathway in rhizosphere was also dominated by reducing citric acid cycle,but the abundance of carbon sequestration pathway in Calvin cycle was higher than that in soil.Fertilization had a significant effect on the abundance of functional genes involved in C and N cycle,but had no significant effect on CH₄ metabolism.Long-term fertilization can enhance N fixation in roots and reduce denitrification and nitrate dissimilation reduction.The effect of microorganisms in roots on greenhouse gas emissions was less,and the proportion of N-fixation in the N cycle was increased.PKS treatment was the main factor affecting the N cycle in the root.The root fixation C pathway was dominated by r TCA,accounting for 18.31～28.86%of the total metabolic pathways,and increased significantly under PKS treatment,followed by Calvin cycle and DC/4HB cycle.Long-term fertilization increased the abundance of cbbs genes in the Calvin cycle,and fertilizer combined with straw treatment significantly promoted CO oxidation.S treatment increased the abundance of the functional genes xyl F and xyl H related to hemicellulose decomposition.However,long-term fertilization had no significant effect on CH₄metabolism.Moreover,TP（r²=0.55）has the highest contribution rate to the C cycle.Long-term fertilization could enhance N fixation in roots and reduced denitrification and nitrate dissimilation reduction.The effect of microorganisms in roots on greenhouse gas emissions was less,and the proportion of N fixation in the N cycle was increased.PKS treatment had the highest influence on the C and N cycle in the root.Compared to maize rhizosphere soil,rhizosphere microorganisms had a smaller impact on greenhouse gas emissions.The proportion of N fixation in N cycle was increased.Moreover,fertilization had a higher effect on microbial diversity and C and N cycle function genes.The rate at which soil physical and chemical properties contribute to C and N cycle was decreased,and the key factors also was changed.4.Fertilization regulates C and N cycle by influencing plant-soil-microbial interactions.Fertilization had significant effects on soil enzyme activity and root exudates.Protease,amylase,β-glucase and nitrification enzyme were beneficial to SOC accumulation,DOC and ROC,TN and organic nitrogen content increasing.Peroxidase and anti-digestive enzymes accelerated the loss of soil C and N nutrients.Sucrose,tyrosine,and citric acid are the substances with the highest content of sugar,amino acids,and organic acids in root exudates,respectively.There was a significant interaction between most substances in maize root exudates and soil SOC,active carbon,TN and organic nitrogen.There is a positive interaction between soil environmental factors and soil enzyme activity,as well as between microorganisms inside and outside the roots,and a negative interaction with root exudates.For soil C cycle,the contribution rate of each component to soil C cycle was as follows:microorganisms>root exudates>soil environment>soil enzyme activity.The contribution rate to soil N cycle was microbial>soil environment>root exudates>soil enzyme activity.The contribution rate of C cycle in root was root exudates>soil environment>endophytic fungi>soil enzyme activity.The contribution rate of root N cycle was root exudates>endophytic bacteria>soil environment>soil enzyme activity.5.Straw addition had significant effect on soil C and N cycleThe addition of straw increased the content of soil C and N nutrients,the maximum value was reached under S100 treatment,and compared with CK treatment,SOC,ROC,TN,and NO₃^--N in S100 treatment increased 6.26 g/kg,0.46 g/kg,0.24 g/kg,and 18.48 mg/kg,respectively.But with the increase of straw addition,the percentage of active carbon in organic carbon and the percentage of NO₃^--N and acid hydrolyzed nitrogen in TN increased first and then decreased,and reached the highest level when the straw addition level was 50%.The addition of straw also promoted the increase of CO₂ and CH₄ emissions.The emission of N₂O decreased significantly when the straw dosage was more than 50%.