Different Fertilization Modes And Cultivation Years Affect Soil Phosphorus Availability And Carbon-nitrogen-phosphorus Coupling Transformations And Their Microbial Mechanisms

Phosphorus?P?is a critical nutrient for plant growth,but its availability is mostly limited,which reduces crop production in around 40%arable soil worldwide.Thus,mineral P is continuously applied to the soil to meet the demand of high crop yield and quality in agro-ecosystems.However,mineral P is easily fixed in soils rather than absorbed by plants,with the continuous accumulation of soil residual P?85%in soil?and low P use efficiency?10%-25%in the first cycle of crop use?.These not only reduce the eco-efficiency of mineral P fertilizer and cause non-renewable P resource scarcity,but also increase potential environmental risks.Soil microbes,the most important decomposers in the agroecosystem,largely drive the organic and inorganic P mobilization and the coupling processes with carbon?C?and nitrogen?N?,and thus increase soil P availability.Hence,more understanding of how soil microbial community potentially drive nutrient cycling and their coupling processes,is critical for contributing to high productivity,high nutrient utilization and sustainable agriculture.Although the effects of soil fertilization regimes and short-term changes in land use on soil nutrient cycling and microbial communities have been well studied,the responses of soil C-,N-,P-processes mediated by the interactions of soil microorganisms and the edaphic variables to the long-term sustainable management are not well understood.Hence,by applying soil enzymatic method,stable isotope tracing techniques,high throughput sequencing and high throughput quantitative PCR?HT-q PCR?analysis,we used long-term field experiments with different fertilization regimes and two soil chronosequence with different land use,to explore soil microbial community together with microbial metabolism and abiotic properties,on soil C-,N-,P-key processes and their driving factors.The major results of the investigation are as follows:1. We analyzed the oxygen isotope ratios in phosphate(?¹⁸O_P)of sequentially extracted inorganic P?P_i?pools?H₂O-P_i,Na HCO₃-P_i,Na OH-P_i,and HCl-P_i?in a long-term agricultural research field in Henan,China with different fertilization regimes and coupled with soil enzyme activity and P-cycling bacteria gene abundance studies.Results showed that after long-term P fertilization especially the N,P and potassium?K?fertilization,the?¹⁸O_P values of H₂O-P_i,Na HCO₃-P_i and Na OH-P_i pools approached to or achieved equilibrium,suggesting that the externally applied P was actively mobilized and cycled by soil microorganisms and speciated into different P pools.Results from the isotope mass balance calculation showed that the extracellular hydrolysis of organic P compounds generated lighter isotopes in Na HCO₃-P_iand Na OH-P_ithan the equilibration,and the new formed Ca-P was derived from P fertilizer but through multistep transformation reactions.The correlation and variation partitioning analysis?VPA?showed that the?¹⁸O_P values were significantly related with phosphatase activities and P functional gene abundances,indicating that the turnover of different soil P pools was regulated by the expression of P functional genes and phosphatase activities.Overall,the long-term P fertilization especially the balanced fertilization with NPK was found to be beneficial for the microbial transformations among different soil P pools,and then greatly increased soil P availability.2. We conducted a rice-rice-crop rotation to investigate the responses of pho D-and pqq C-harboring bacterial communities by high-throughput sequencing to the partial replacement of inorganic fertilizer by organic manure.The genes pho D and pqq C are responsible for regulating organic mineralization and inorganic P solubilizing,respectively.Results showed that in comparison with the conventional fertilization,the organic-inorganic fertilization with reduced P input significantly enhanced soil P bioavailability?H₂O-P_i,Na HCO₃-P_i and Na OH-P_i?and maintained high rice yield,and also largely increased the absolute abundance of pho D gene and the relative abundances of Bradyrhizobium and Methylobacterium in pho D-harboring bacterial community.Adonis test showed that the organic-inorganic fertilization notably reshaped the structure and composition of pho D-and pqq C-harboring bacterial communities,also formed the phosphate mobilizing bacterial community with higher network complexity and stability by network analysis.Moreover,the structural equation models?SEM?indicated that the phosphate mobilizing bacterial community was more important as an up-regulator of phosphatase expression than the gene abundance.Moreover,except for the supply of carbon,the increased p H was the most impact factor for the pho D-and pqq C-harboring bacterial communities,by promoting microbial P turnover and greatly increasing soil bioavailable P.Hence,our study demonstrated that the partial replacement of mineral fertilizer with organic manure could reshape the phosphate solubilizing and alkaline-phosphomonoesterase encoding bacterial communities towards more resilient and effective to the high P utilization and high productivity over intense cultivation,providing insights into the potential of soil microbes in the efficient management of agricultural.3. We explored the effects of the microbial community associated with its functional potentials on soil C,N and P turnover under the organic-inorganic fertilization by using high-throughput sequencing?including bacteria and fungi?and HT-q PCR of functional genes.The combination of mineral and organic fertilization greatly increased the diversity of bacterial community that dominated by copiotrophic bacteria,and enhanced microbial functional potentials and soil multifunctionality?characterized by the enzyme activities involved in soil C,N and P cycles?,but not significantly changed the fungal community.Particularly,positive relationships were observed among bacterial diversity,copiotrophic taxa abundance,functional gene abundance,and multiple enzyme activities.Random forest analysis and SEM further demonstrated that soil multifunctionality was directly affected by the edaphic variables,and also positively regulated by the microbial functional potentials via shifts of bacterial communities?i.e.,composition and diversity?combined with soil environments.Moreover,resource availability?SOC,NH₄⁺and available P?,soil p H and functional potentials are reliable predictors of multi soil functions,and these factors mainly driven the process-level microbial transformations with the fertilization strategy of the partial replacement of mineral fertilizer by organic manure.Thus,by increasing the resource availability with proper management practice,we could potentially take advantage of the positive effects of microbial functions to enhance soil multifunctionality and biodiversity.4. The microbial abundances?HT-q PCR?and community composition?bacteria and fungi?as well as their controlling soil properties were investigated in soil depth profiles along the 2000-year paddy and 700-year dryland soil chronosequence,to understand the microbial succession and the functions related to soil C-,N-,P-transformations along the long-term soil development with different land use.Results showed that across multiple time-scales ranging from decades to millennia,significant shifts in soil microbial communities with soil depths and chronosequence occur,indicating the occurrence of soil development and microbial succession since reclamation from tidal wetlands.With the prolonged soil cultivation,paddy soils significantly increased the relative abundance of bacterial groups including GCA004,env OPS12,Nitrospirales and Clostridiales,which could facilitate soil nutrient turnovers.Moreover,bacteria,fungi and microbial functions showed different patterns in relation to soil age and depth:bacterial community was more affected by soil age linked to changes in soil properties,whereas soil depth had stronger impacts on fungal community and microbial functions.Particularly,in contrast to dryland soils,the microbial community in paddy soil chronosequence was transited from the“rapid succession stage”to the“progressive succession stage”,forming the homogeneous and stable bacterial community with the prolonged cultivation.Additionally,RDA and VPA analysis indicated the mutual promotion and feedback relationship between soil stoichiometry and microbial community succession.Furthermore,with the prolonged paddy cultivation,the interactions between soil p H,C:N ratio and oligotrophic bacteria significantly increased the microbial potentials in topsoil C-,N-,and P-transformations,and thus promoted soil nutrients turnover and enhanced their availabilities.5. By collecting soils from the 2000-year-old paddy chronosequence at two different soil depths?0-20 cm and 20-50 cm?,we measured microbial C use efficiency?CUE?,growth and biomass turnover rate using a substrate-independent H₂¹⁸O labeling method,gross rate of organic N cycling to estimate microbial N use efficiency?NUE?,as well as microbial abundances,to explore how microbial physiology in paddy soil responds to soil cultivation year and how this affects soil organic C?SOC?content.Results showed a significant accumulation of SOC under the prolonged paddy cultivation occurred in topsoil,SOC content was increased by 65%and 125%in 50-and 2000-year,respectively,while C loss appeared in subsoil after 700 years of paddy cultivation.Vector analysis of soil enzyme stoichiometry indicated the significant N and relative C limitations in both depths of paddy soils,but the N limitation and C limitation decreased after 1000 years.For topsoil and subsoil,microbial CUE reached to the highest values in 1000-and 700-year,along with microbial growth which both increased 5.2-and 3.3-fold in 1000-year,respectively.We found the similar increasing trends between microbial CUE and soil C:P and N:P ratios,the reduction of N limitation and functional potentials including N-and P-cycling,C degradation,C-fixation?acs A gene?,microbial community homogenization and microbial biomass across soil chronosequence in topsoil.Moreover,the structural equation model revealed that with the prolonged paddy cultivation,the decline in soil p H had positive effects on microbial functional potentials and microbial biomass carbon.The enhanced functional potentials directly positively affected microbial growth,and thereby on microbial biomass carbon.Finally,the prolonged paddy cultivation increased SOC content via its direct positive effect and indirect positive influence on microbial biomass carbon.We conclude that the prolonged paddy cultivation captures the cumulative microbial anabolism on SOC sequestration in the plough layer,with the shifts in abiotic and biotic conditions towards increased nutrient availability and homogenous microbial community with higher functional potentials.