Soil N₂O Emission Feature And Its Microbial Mechanism In Tea Orchard

N2O is one of the green-house gases. It not only participates in photochemical reaction and destruction of stratospheric ozone, but also has negative effect on biosphere. Soil is the major N2O source and contributes70%of N2O emission to atomosphere. Tea orchard soil is highly acid and widely distributed in the southern hilly area of China. Soil acidification and N2O emission were enhanced due to heavy nitrogen fertilization application in tea orchard. The agricultural managements, e.g. liming, fertilizer application in tea orchard can affect the changes in microbial community composition and the rate of N2O emission. However, the mechanism between soil microbial community and high N2O emission rate in tea orchard remains unclear. In this study, a series of tea orchard soils, adjacent wasteland and forest derived from the same parent material were collected from Meijiawu tea orchard, located in West Lake district of Hangzhou, Zhejiang Province in Southeast of China. A series of traditional and molecular methods (quantitative PCR, clone library and terminal restriction fragment length polymorphism (TRFLP)) were carried out to study the effect of fertilization treatments on N2O emission and its microbiological mechanism.The main results are as follows:1With the succeeding development of tea orchard ecosystems, the primary chemical and microbial properties in tea orchard soils, i.e., soil acidity, organic carbon, total N, inorganic N, available P showed a significantly increasing trend with increasing ages. The higher nitrification potential and denitrification enzyme activity were observed in the50-year-old (0.99mg/kg-d) and100-year-old tea orchard soil (0.79mg/kg-d). The application of nitrogen fertilizers and nitrification inhibitors showed that the highest amount of N2O emission was observed in KNO3treatments (0.87mg/kg) which was7times higher than that in (NHO2SO4alone. Effect of KNO3with DCD or DMPP on N2O emission was not significant whereas (NH4)2SO4with DCD or DMPP could significantly inhibit N2O emission. Quantitative PCR of nosZ gene, a key gene encoded N2O reduction enzyme, suggested that nosZ gene abundance in KNO3-associated treatments was the lowest. The application of different nitrogen fertilizers (KNO3,(NH4)2SO4, Urea, NH4NO3, control) showed that N2O emission in KNO3treatment was744.8ng/g, that was16times higher than control. The nitrification potential in Urea or (NH4)2SO4treatments was significantly higher than that in KNO3treatment, but N2O emission was lower. Soil pH (3.71) was adjusted to5.11,6.19and7.41after liming. With (NH4)2SO4addition, soil nitrification potential in soil with pH7.41was increased by88%and N2O emission was7.3times higher than that in pH3.71treatment. Combined (NH4)2SO4with C2H2, soil nitrification potential and N2O emission decreased by91%and93%, respectively in soils with pH7.41. In soils with pH3.71, though soil nitrification potential was decreased by79%, N2O emission showed no significant difference with (NH4)SO4alone treatment. The above results suggested that nitrification is the dominant source of N2O emission in the neutral soil, but denitrification dominated in N2O emission in highly acidic soil. The inhibition of nosZ by NO3" addition may result in high N2O emission at low pH.2The15N tracer method showed dentirification is the dominant source of15N-N2O during3days or7days aerobic incubation, accounting for71.1%and78.9%of total15N-N2O, respectively. The contribution of autotrophic nitrification to15N-N2O was smaller than denitrification, accounting for23.7%and17.8%, respectively. Using C2H2inhibition method, the contribution of autotrophic nitrification was19.4%(3days) and11.1%(7days), respectively and denitrification accounted for80.6%(3days) and88.9%(7days), respectively.3In the nitrate amendment experiment, the highest N2O emission (2.0mg/kg,21days) was produced at1000mg/kg NO3-in soils at pH3.71, ranging from21.1-65.2times higher than the rest three pH soils.10Pa C2H2was used to inhibit N2O reduction and the result suggested that the reduced N2O accounted for89%-99%of total N2O production in soils with pH3.71-6.19, which is significantly higher than that in soils with pH7.41. We provide strong evidence that the impaired reduction of N2O under acidic conditions is not likely to be responsible for the high N2O emission in this acidic soil. Using TRFLP, cloning and sequencing, the acid-tolerant denitrifiers were found in clone library3.71-5.11, which were also found in the acidic soils in the previous studies. The detected denitrifiers may have a high activity in the acidic soils and the large amounts of N2O emission was mainly related with the adaptation of acid-tolerant denitrifying populations.4Using fungal and bacterial inbitors, cycloheximide and streptomycin, fungal denitrification accounted for70%of N2O emission in tea soil and therefore fungal denitrification in tea soil plays an important role in N2O emission. Then, two acid-tolerant fungi, were isolated in soils with pH3.8using rose-bengal medium. According to the morphological characteristics and the phylogenetic analysis based on18S rDNA sequence, the two strains were identified to be penicillium and hypocrea, respectively. With the inoculation of the two strains into the liquid medium, the N2O emission from penicillium was328mg/kg, accounting for16.4%of added NO3-N, but the hypocrea showed no significant N2O emission with control. The representative penicillium was also inoculated into soils with y-sterilization. The result suggested that on day3, N2O emission rate was significantly higher than that in the nonsterile soils with NO3-N addition. The above results suggested N2O emission in the tested100-year-old tea soil is closely related with fungal denitrification.