The Effects Of Organic Fertilizers And Inhibitors On Soil Nitrogen Transformation Functional Microbes, N₂O And NH₃ Emissions On Calcareous Soil

【Objective】The consecutive application of mineral nitrogen fertilizer has resulted in a series of negative effects in China,which has attracted more and more attention.Thus,it is critical to find methods which can reduce nitrogen loss and increase nitrogen use efficiency.From the perspective of nitrogen optimized management,the combination of chemical fertilizers and organic fertilizers or urease/nitrification inhibitors can achieve high efficient utilization of nitrogen nutrients in croplands.The nitrogen transformation is violent in the calcareous soil and easily leads to the loss of nitrogen,among them,N₂O emission and NH₃ volatilization are important nitrogen loss pathways.However,the effects of the combination of organic fertilizers and chemical fertilizer and urease inhibitor（UI）or nitrification inhibitor（NI）or double inhibitors（DIs）on the characteristics of nitrogen transformation are not clear,and those impacts on the NH₃ and N₂O emissions are unknown,moreover,the microbiological mechanism of nitrogen transformation is lack of data.Therefore,we studied the effects of chemical fertilizer combined with organic fertilizer as well as adding UI/NI on the community abundance,structure and diversity of nitrogen transformation microbes in calcareous soil,and simultaneously monitored the N₂O and NH₃emission characteristics.This information will aid in understanding the mechanism of organic fertilizers and inhibitors on the nitrification and denirification mircobes,and provide a theoretical basis for optimizing management of fertilization in agriculturally-productive calcareous soils.【Method】This study includes two parts:a 4-yr continuous fertilization experiment and indoor simulation culture text.According to the field location fertilization experiment,the effects of organic fertilizers combined with chemical fertilizer on the abundance of soil nitrification microorganisms（ammonia oxidizing bacteria（AOB）and ammonia oxidizing archaea（AOA））and denitrification functional genes（nirS/nirK/nosZ）were studied using the real-time PCR,the community structure and diversity of nitrogen transformation functional microbes were analyzed by the high throughput sequencing technology,and the N₂O emissions from drip-irrigated cotton fields were measured using static chamber-gas chromatography.In the incubation text,two culture temperatures（25 and 35°C）were set up,the impacts of the amendments of organic fertilizer combined with UI（N-（n-Butyl）thiophosphoric Triamide,NBPT）or NI（2-Chloro-6-trichloromethyl,CP）or DIs on the N transformation characteristics were studied,the abundance and diversity of nitrogen transformation functional microbes was determined by the same technology with the field experiment.Using the Picarro（G2508）machine to measure the fluxes of NH₃ and N₂O.The enzymatic method was used to study the activities of key N transformation related enzymes.【Result】1)The incorporation of organic fertilizer,chemical fertilizer and UI or DIs could delay urea hydrolysis to 22 days and 15 days,respectively,in 25 and 35℃incubated conditions.Addition of NI or DIs can maintain the high NH₄⁺concentrations for 69d and 56d in the soil at 25 and 35°C culture conditions,meanwhile,reduced the production of NO₃^-in soil.The application of organic fertilizer with NI or DIs can significantly reduce the microbial biomass,the apparent nitrification rate and potential nitrification rate（PNR）,whereas,increase soil NH₄⁺/NO₃^-and soil nitrification inhibition rate in the initial stage of cultivation.The high culture temperature decrease effective time of NBPT and CP.2)The application of organic fertilizer combined with UI can significantly reduce soil urease activity at the initial stage of culture（P<0.05）.Among them,the amendment of COM+NBPT and COM+NBPT+CP reduced urease activity by 47.6%and by 41.2%,respectively,compared to COM at 35°C incubation conditions.Nitrite reductase（NiR）and hydroxylamine reductase（HyR）activities in soil were significantly reduced by organic fertilizer mixed with NI or DIs,whilst nitrate reductase（NaR）was significantly increased（P<0.05）.For example,in the 35°C culture experiment,COM+CP and COM+NBPT+CP treatments reduced NiR activity by 25.6 and by 28.8%,respectively,compared with COM treatment,and HyR activity was decreased by 8.36 and by 26.1%,respectively,but NaR activity was increased by 74.8and 63.8%,respectively.The enzyme activities were much higher in the 35°C incubation conditions than those in the 25°C incubation conditions.Soil factors,NH₄⁺,pH^-were the main physiochemical properties that lead to the change of soil enzyme activities at the initial and middle time of incubation.3)The 4-yr consecutive application of chemical and organic fertilizers(3000,6000kg·hm^-2 manure and bio-fertilizer)had no significant effect on the AOA community（P>0.05）,however,organic fertilizers combined with chemical fertilizer significantly increased the AOB community richness andα-diversity.In the incubation experiment,organic fertilizer combined with CP or NBPT+CP significantly reduced the AOB community richness but had no significant effect on AOA community.qPCR results showed that organic fertilizer combined with chemical fertilizers significantly increased the abundance of AOB genes compared with CK treatment and only chemical fertilizers（CF）treatment（P<0.05）,in both field and incubation experiments.The mixed application of organic fertilizer,chemical fertilizer and NI or DIs significantly reduced the AOB gene copy numbers,which was reduced by 76.5 and by 85.8%in contrast to the COM treatment,respectively.However,organic fertilizer mixed with UI or NI had no significant effects on AOA gene abundance（P>0.05）.PCA analysis showed that the AOA community structure did not significantly separate among chemical fertilizer treatment,chemical fertilizer mixed with cow manure or bio-fertilizer treatments,whilst the AOB community structure in the treatments of chemical fertilizer mixed with different ratios of cow manure or bio-fertilizer significantly separated with that in the CF or CK treatment.Similarly,the AOB community structure in the amendments of the organic fertilizer combined with the CP or NBPT+CP was significantly different from in that of the CF or organic fertilizer treatment（COM）（heat map analysis,Fig.5-6B）.Correlation analysis showed that soil potential nitrification rate and soil AOB gene copy number had a significant correlation at the P<0.001 level in the field test and a significant correlation at the P<0.05（P=0.0179）in the incubation experiment.These indicate that continuous supply of chemical and organic fertilizers or the combined application of chemical,organic fertilizer and urease/nitrification inhibitors has a significant effect on the abundance,structure and functional diversity of AOB,but the AOA community did not change significantly under these fertilization amendments.Thus,we suggest that AOB instead of AOA may predominante the nitrification process on calcareous soil.4)The application of chemical fertilizers for 4 consecutive years increased the the richness and diversity of nirK,while the combination of chemical fertilizer and organic fertilizers increased the Simpson and Shannon indices of nirS and nosZ more significantly compared with CF（P<0.05）.In the culture experiment,organic fertilizer combined with NI（COM+CP）or DIs（COM+NBPT+CP）significantly reduced the Shannon index and Simpson index of nirS and nirK compared with COM.qPCR analysis showed that CF treatment improved the nirK gene copy numbers by 36.5 and 20.9%compared with CBF2and COM2 treatments,respectively,in the field,while the gene copy numbers of nirS and nosZ treated with COM2 and CBF2 increased by an average of 35.8 and 31.7%in contrast to CF treatment.The application of NBPT or CP or both of them significantly reduced the nirK gene copy numbers（P<0.05）.However,only COM+NBPT+CP can significantly reduce the abundances of nirS and nosZ by 40.9 and 20.0%,respectively,related to COM treatment.The denitrifier community structure in COM2 treated amendment was similar with that in CBF2,which were distinctly different from that in CK and CF treatments.In the incubation experiment,a similar structure of denitrifying bacteria between COM+CP and COM+NBPT+CP treatments was found,and there was a significant difference of nirS/nirK structure between COM treatment and COM+NI/DIs treatments.Correlation analysis showed that nitrite reductase activity（NiR）was significantly correlated with nirS gene abundance but had no correlation with the abundance of nirK genes in the field,while it was significantly correlated with nirK and nirS at the level of P<0.001 and P<0.05,respectively,in the incubation experiment.（5）The application of organic fertilizer with NI or DIs can significantly reduced the flux peak of N₂O during the culture period,and the COM+DIs was the best treatment.COM+CP or COM+DIs treatment significantly reduced cumulative N₂O emissions by 66.2 and 76.8%（35°C）compared to COM treatment.The cumulative N₂O emission under the conditions of 35°C was higher than that under the conditions of25°C in the fertilizer added treatments,but the addition of CP did not increase the cumulative N₂O emission with temperature increasing.The application of UI significantly reduced the flux of NH₃ and the cumulative emissions of NH₃,however,the application of NI or DIs significantly increases the flux of NH₃ and the cumulative emission of NH₃（P<0.05）.The peak of NH₃ emissions in the COM+NBPT+CP treatment decreased by 24.2 and 57.5%at 25 and 35°C conditions,compared with COM+CP treatment,respectively.In the consideration of loss of two kinds of gaseous nitrogen,the amount of nitrogen lost is highest with the treatment of organic fertilizer mixed with ony CP,account for 9.62%and 15.7%of the nitrogen application rate,respectively,at 25 and 35℃.The combination of COM with DIs can significantly reduce the sum of the two gas losses compared with COM plus CP alone,with a decrease of 38.7%at 25°C incubation condition.（6）The key factor that caused the changes of AOB community structure were pH after continuous fertilization in the field.NO₃^-,NH₄⁺and NH₄⁺were the key factors that lead to the changes of nirK,nirS and nosZ community structure.Under culture conditions,NO₃^-,SOC and pH were the significant factors affecting the gene abundance of nitrifiers and denitrifiers.Soil factors NO₃^-,pH and NH₄⁺contributed more to N₂O emissions in both field and indoor culture experiments,AOB and nirS were key microbial factors that affect N₂O emissions.The NH₃ emissions from soil were mainly influenced by pH and NH₄⁺.【Conclusion】1)The incorporation of organic fertilizer,chemical fertilizer and NBPT can significantly delay the hydrolysis of urea and reduced the urease activity,thus,significantly increase the inhibition rate of urease.Organic fertilizer mixed with CP or DIs significantly prolonged NH₄⁺residence time in the soil,on the contrary,reduced soil nitrification potential and soil microbial biomass at the initial time of cultivation.NiR and HyR activity was significantly decreased when COM combined with NI or DIs,but NaR activity was significantly increased.2)The 4 years consecutive application of organic fertilizers and combined with NI or DIs increased the community richness and functional diversity as well as the abundance of AOB genes,and changed the structure of the community.However,there was no significant effect on the community of AOA.The combination of organic fertilizers with NI or DIs significantly reduced the AOB community diversity and gene abundance,and changed its community structure,but did not have a significant effect on AOA community,indicating that AOB is more sensitive to organic fertilization and NI application.Ammonia-oxidizing microorganisms involved in nitrification on calcareous soil may be AOB but not AOA.3)Consecutive 4-year application of chemical fertilizer and organic fertilizers increased the functional diversity and gene abundance of nirS and nosZ,while the nirK community was more sensitive to chemical fertilizer alone.The application of organic fertilizer with NI or DIs significantly reduced the functional diversity and gene abundance of the community of nirK,and changed its community composition.Organic fertilizer mixed with UI or NI had weaker effects on the nirS and nosZ structure composition and species richness than that on the nirK community.4)The application of organic fertilizer with chemical fertilizer reduced N₂O emissions compared with chemical fertilizers alone treatment,but had no significant effect on the NH₃ emission.The application of COM combined with NBPT significantly reduced NH₃ volatilization.The application of organic fertilizer with NI or DIs significantly reduced the N₂O flux and accumulated emissions,but significantly increased the volatilization loss of NH₃.The combination of organic fertilizers and DIs reduced more of nitrogen loss compared to COM mixed with only CP.5)High cultivation temperature significantly increased soil enzyme activities and promoted the loss of N₂O and NH₃.NBPT and CP still have significant effects on the blocking urea hydrolysis and decreasing nitrification process with the increase of culture temperature from 25 to 35℃（P<0.05）,but their efficiency were markly shorten in the soil.6)The key soil factor affecting the structure of AOB in the soil with consective organic amendments were pH.NO₃^-was key factor influencing the change of soil nitrifier and denitrifier abundance and N₂O emissions.The top two most important microbial factors affecting N₂O emissions were AOB and nirS gene abundance.The soil pH and NH₄⁺were the critical factors impacting NH₃ volatilization on calcareous soil.