| Partial replacement of chemical fertilizers by organic fertilizers can achieve the purpose of reducing chemical fertilizer application without reducing production.The impact of combined application of organic fertilizers on crop yield and nitrogen use efficiency is an important scientific issue that people are concerned about.Soil fertility is closely related to crop yield.Soil fertility is divided into physical fertility,chemical fertility and biological fertility.In the past,a large number of studies have studied the effects of different fertilization treatments on soil physical and chemical fertility by measuring soil physical and chemical properties.The effect of biological fertility remains to be further studied.Microorganisms are the most important living organisms in soil.The metabolic activities of soil microorganisms profoundly affect the formation and development of soil,material circulation and fertility evolution.Soil microorganisms are an important part of soil biological fertility.How organic fertilizer application affects soil microorganisms,especially nitrogen-fixing microorganisms that are closely related to soil biological fertility,which is rarely reported.Based on long-term field trials,this study investigated the effects of three different fertilization treatments: 1)no fertilization control treatment(CK),2)chemical fertilizer treatment(NPK),3)chemical fertilizer + 4500 kg/ha pig manure organic fertilizer treatment(NPKM)on soil chemical properties,microbial biomass carbon and nitrogen,crop yield,nitrogen utilization efficiency,soil bacterial and fungal microflora,and analyzed the effect of organic fertilizer on soil chemical and biological fertility,and NPK and NPKM are equal nutrient fertilization treatments in this study.Further through indoor cultivation,The effects of carbon and nitrogen sources and components of rice root exudates on soil nitrogenase activity were studied,and the effects of combined application of organic fertilizers on soil bacteria and nitrogen-fixing bacteria were studied by enrichment culture experiments combined with high-throughput sequencing and analysis techniques.The focus was on how to improve the effectiveness of carbon sources in organic fertilizers for nitrogen-fixing microorganisms through cooperation among the flora,and the following main results were obtained:1.Fertilization can increase crop yield and increase soil nutrient content.NPKM treatment had the highest crop yield,NPK and NPKM treatments had the highest soil total nitrogen and soil available nitrogen contents,and NPKM treatment had significantly higher soil organic carbon and soluble organic carbon contents than other treatments.The soil carbon-nitrogen ratio and nitrogen fertilizer use efficiency(the nitrogen fertilizer agronomic efficiency and nitrogen fertilizer partial productivity)of NPKM were significantly higher than those of NPK.NPK and NPKM treatments significantly increased soil microbial biomass carbon and nitrogen,and the soil microbial biomass carbon and nitrogen of NPKM treatment was significantly higher than other treatments.Therefore,the application of organic fertilizers can improve soil chemical and biological fertility and nitrogen use efficiency,and maintain stable and high yield of rice-wheat rotation system.2.Different fertilization treatments had significantly different effects on soil microflora.NPKM treatment significantly increased the Chao1 index of bacteria,and increased the Shannon index of fungi compared with NPK treatment.Fertilization treatments significantly affected the community structure,species composition and network structure of bacteria and fungi.Compared with NPK treatment,NPKM treatment enriched some nitrogen-fixing taxa.Fertilization had significantly different effects on soil microbial communities of bacteria and fungi,a comprehensive study of soil bacterial and fungal communities found that NPKM increased the complexity of soil microbial networks and the network parameters such as the average degree,the number of nodes,and the number of positive and negative correlation edges were improved.Crop yield was positively correlated with soil microbial richness,evenness and diversity.Therefore,soil microbial diversity and network complexity can be used as biological indicators of soil fertility.3.It was found that glucose could stimulate soil nitrogen fixation by the cultivation experiment,and rice root exudates such as L-lactic acid and diglycerol and other carbon sources could stimulate soil biological nitrogen fixation.Through carbon and nitrogen compound culture experiments,it was found that with the increase of nitrogen concentration,soil nitrogenase activity began to decline until it was completely inhibited,and increasing the concentration of glucose could delay the nitrogen concentration that was inhibited by nitrogen fixation.The proportional relationship between the effective carbon source and nitrogen source was the main factor limiting soil nitrogenase activity,the combined application of organic fertilizers significantly improved the nitrogen fixation capacity of the soil.The nitrogen fixation capacity of soil and nitrogenase activity of farmland soil were significantly positively correlated with crop yield.4.It was found that when the organic fertilizer was the only carbon source,the nitrogenase activity in the system was difficult to be detected by the subculture experiment.On the basis of the organic fertilizer as the carbon source,the easily available carbon source mannitol(MOF)was added that can significantly increase soil nitrogenase activity,which is significantly higher than that of the treatment with the same amount of mannitol as carbon source(MCK),indicating that in the presence of readily available carbon sources,the addition of organic fertilizers can improve the carbon source efficiency of nitrogen-fixing microorganisms.High-throughput sequencing data showed that MOF treatment decreased the evenness and Shannon diversity of nitrogen-fixing bacterial communities,but increased the Richness,Evenness and Shannon indices of bacterial communities.In subculture,MOF and MCK treatments directionally drove the development of nitrogen-fixing bacteria and bacterial community structures,forming their own stable flora.The species composition of MOF and MCK treatments differed significantly between the same generations,and the respective nitrogen-fixing and bacterial groups were enriched with the progress of subculture.The results of real-time fluorescence quantitative PCR showed that nitrogenase activity was not significantly correlated with nif H gene copy number,but was significantly positively correlated with 16 s rRNA gene copy number.PICRUSt-based functional prediction found that MOF treatment had stronger carbohydrate metabolism,which is embodied by a stronger citric acid cycle,fructose and mannose metabolism,galactose metabolism,glycolysis/gluconeogenesis,pentose and glucuronic acid interconversion,pyruvate metabolism and nitrogen metabolism in energy metabolism.The functional prediction based on FAPROTAX element cycle found that MOF treatment was significantly(P < 0.05)stronger than MCK in aliphatic non-methane hydrocarbon degradation,aromatic compound degradation,cellulose degradation,fermentation,hydrocarbon degradation,iron respiration,and xylan decomposition functions,and especially the cellulose degradation function,MOF treatment was significantly higher than MCK treatment in each generation of subculture treatment.Further analysis found that the relative abundance of Clostridium XIVa of the Lachnospiraceae family with cellulose degradation function,the relative abundance of Geobacter and Rhizomicrobium with iron respiration function,and the relative abundance of Melioribacter,which has the functions of cellulose degradation,xylan decomposition and iron respiration at the same time,was significantly higher in MOF treatment than in MCK treatment,and nitrogenase activity was significantly positively correlated with the relative abundance of these strains.The above results show that the availability of carbon sources has an important impact on the activity of nitrogenase,and the available carbon sources are the inexhaustible driving force for nitrogen-fixing bacteria to perform nitrogen-fixing.In subculture,MOF treatment enriched key auxiliary groups such as Clostridium XIVa of the Lachnospiraceae family,Clostridium III and Melioribacter,which could decompose carbon sources that were not easy to be directly used by nitrogen-fixing bacteria into carbon sources tha were easy to use,which solved the energy source problem of nitrogen-fixing bacteria and promoted the biological nitrogen-fixing level of the whole system,and then revealed the microbiological mechanism of combined application of organic fertilizer to promote nitrogen-fixing bacteria to exert high nitrogen-fixing ability.In this research,through field experiments,it was found that the application of organic fertilizers could significantly improve the yield of rice-wheat rotation system by improving soil chemical fertility and biological fertility,in which the diversity of soil bacteria and fungi and the complexity of the community co-occurrence network can be used as indicators to measure soil biological fertility.soil carbon source availability has an important impact on soil nitrogenase activity,high concentration of effective carbon source can effectively alleviate the inhibition of high concentration nitrogen source on nitrogenase activity.Carbon sources such as L-lactic acid and diglycerol in root exudates can stimulate soil biological nitrogen fixation,and the application of organic fertilizers significantly increases the abundance of microorganisms in the soil with carbon source degradation functions that are difficult to use by nitrogen-fixing bacteria such as cellulose and xylan,thereby promoting the conversion of these difficult-to-use carbon sources into carbon sources that nitrogen-fixing bacteria could easily use,thereby promoting soil biological nitrogen fixation.This study provides theoretical support for revealing that combined application of organic fertilizers can improve the efficient utilization of nitrogen in rice-wheat rotation systems. |
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