Effects Of Nitrification/Urease Inhibitors On Soil Flora And Nitrogen Transformation In Greenhouse Vegetable Fields

Soil microbial communities and nitrogen cycling are important components of biogeochemical cycle in greenhouse vegetable fields,in which ammonia-oxidizing microorganisms and denitrifying bacteria play a key role in driving nitrogen conversion.Greenhouse vegetable fields is a special agricultural system.Due to the closed environment,large fertilizer application and frequent irrigation,it is easy to lead to the leaching of nitrogen in soil,which leads to surface and underground water pollution,soil degradation and global warming and other environmental problems.In addition,the planting years of greenhouse vegetable fields also affect the health and stability of ecological environment.Therefore,it is of significant practical value to study the application of nitrification and urease inhibitors to soil nitrogen conversion and microbial community in greenhouse vegetable fields,which can reduce nitrogen loss and reduce greenhouse gas emissions.In this study,the soil of greenhouse vegetable fields in Shouguang City,Shandong Province was studied.PCR amplification and Illumina Miseq were used to analyze the changes of soil microbial community structure and diversity in different planting years（0,3,9 and 13）and soil layers（0～20cm and 20～40 cm）.The effects of nitrification inhibitors[2-chloro-6-（trichloromethyl）pyridine（CP）,dicyandiamide（DCD）]and urease inhibitors[N-（n-butyl）thiophosphoric triamide（NBPT）]on soil inorganic nitrogen（NH₄⁺-N,NO₂^--N and NO₃^--N）,greenhouse gases（N₂O,CH₄and CO₂）and soil enzyme activities（Urease,hydroxylamine reductase,nitrite reductase and nitrate reductase）in greenhouse vegetable fields were studied by indoor incubation experiments.And modern microbial techniques were used to explore the changes in community structure and abundance of ammonia-oxidizing archaea（AOA）,ammonia-oxidizing bacteria（AOB）,and denitrifying bacteria（nir K and nir S）.The main research results are as follows:（1）The content of environmental factors increased with the increase of planting years,and the content of NO₃^--N in the 0～20 cm soil layer was as high as 1290～1390mg/kg in year 13,showing a cumulative trend.The OTU numbers of bacteria and archaea communities gradually decreased,and the OTU numbers of 0～20 cm soil layer in year 13 were reduced by 30.03%and 52.24%compared with that in year 0,respectively.Shannon index and Simpson index showed that microbial diversity of soil in more than year 9 was greatly reduced.The dominant phyla in soil flora mainly included Firmicutes,Actinobacteria,Proteobacteria,Crenarchaeota and Euryarchaeota.Nitrosopumilus and Nitrososphaera in the AOA community,as well as Nitrolancea and Nitrospira in the nitrifying bacterial community,occupy a dominant position among ammonia-oxidizing microorganisms.With the increase of planting years,soil flora structure changed significantly,and soil biomass and diversity decreased significantly in years 9 and 13.It is suggested to improve microbial activity with reasonable fertilization,so as to provide the theoretical basis for sustainable utilization and high-quality production of greenhouse vegetable fields.（2）CP and DCD significantly inhibited NH₄⁺-N conversion,and NO₂^--N,and NO₃^--N accumulation,NBPT slowed down urea hydrolysis and NH₄⁺-N production,and the apparent nitrification rates of soil were in the following order:NBPT>DCD>DCD+NBPT>CP+NBPT>CP.The inhibitory effect of CP was better than that of DCD.Compared with urea treatment,the peak N₂O production rate of inhibitor treatment decreased by 73.30–99.30%,and the production rate of CH₄and CO₂decreased by more than 66.16%.The significant decrease in the production rate of greenhouse gases N₂O,CH₄and CO₂is closely related to the effect of inhibitors on nitrification and denitrification processes.（3）There was a significant difference in urease activity between the 7th and the35th day of incubation,and the inhibitory or promoting effect was weakened.On the one hand,it may be because the effect of nitrogen fertilizer and inhibitor was lost in the later stage of cultivation.On the other hand,there were differences in the enzyme activities of greenhouse vegetable fields with different planting years,especially on the35th day of incubation.Compared with other planting years,the enzyme activities of soil in year 0 were relatively reduced.The effects of different treatments of nitrogen fertilizer coupled inhibitors on nitrite reductase activity were basically consistent.In conclusion,the application of nitrogen fertilizer can promote the activities of urease,hydroxylamine reductase,nitrite reductase and nitrate reductase in soil.Nitrogen fertilizer coupled with CP and DCD can reduce the activities of urease and hydroxylamine reductase,but for nitrite reductase and nitrate reductase,NBPT can inhibit the activities of three enzymes except for hydroxylamine reductase.（4）Application of nitrogen fertilizer and inhibitors can stimulate the growth of microorganisms,and different flora have different degrees of response.CP and DCD decreased the abundance of AOA and AOB,respectively.NBPT also prevented the growth of ammonia-oxidizing microorganisms and nir S-type denitrifying bacteria,and urea and nitrification inhibitors were adverse to the growth of Ensifer and Sinorhizobium in the nir K community.Nitrification and urease inhibitors can effectively suppress the growth of ammonia-oxidizing microorganisms and denitrifying bacteria,slow down the rate of nitrification and denitrification,and thus reduce greenhouse gas emissions.This provides scientific basis for the rational use of inhibitors,improving nitrogen fertilizer utilization efficiency,and promoting sustainable development of soil environment health.