Nitrification In Greenhouse Vegetable Soils And Corresponding Microbial Mechanisms

The application of nitrogen (N) fertilizer is an effective agronomic practice for high yield of vegetables, and it subsequently leads to a series of transformation processes of nitrogen in soil. Nitrification, one important process of N cycles driven by microbes, not only affects N utilization efficiency directly, but also relates to the ecological environment problems caused by nitrate leaching and greenhouse gas emissions. Greenhouse cultivation is one of the important practices of vegetable production, and it is very important for the vegetable supply. Annual N fertilizer inputs and water irrigation are significantly greater in the greenhouse vegetable systems than in other cropping systems, and these intensive production practices accelerate nitrification, induce significant shifts of microbial community composition and activity. Previous studies on soil nitrification and ammonia-oxidizing microbial community diversity in farmland ecosystem have been well documented. However, studies about intensive production system--greenhouse vegetable soils were rarely reported, the role that ammonia oxidizers played in N cycling of vegetable soil remains unclear. Incubation studies about the nitrification of greenhouse vegetable soils were performed using chemical testing and microbial molecular ecology technique (real-time PCR, terminal restriction fragment length polymorphism (T-RFLP) and clone library). This study aimed at evaluating soil nitrification characteristics and corresponding microbial mechanisms of different cultivation patterns and different soil types, exploring the interaction effects of biochar and nitrogen, nitrification inhibitor and soil moisture, the comparative effects of different nitrification inhibitors on soil nitrification and ammonia oxidizers. The main results were listed as follows:(1) Soil samples under open field and greenhouse vegetable cultivation patterns were collected from Huajiachi campus, Zhejiang University (HJC) and Xinglong, Hangzhou City (XL) to evaluate the characteristics of soil nitrification, as well as abundance and community structure diversity of ammonia oxidizers. It was shown that nitrification potentials and AOB amoA gene copies were significantly greater in greenhouse soil than those in open field soil, but no significant difference in AOA abundance was observed. The nitrification potential had significantly positive correlations with AOB abundance and NH4+-N content, but was negatively related to soil pH (r=-0.98*). T-RFLP analysis showed that AOA T-RFs 63 bp and 299 bp, belonging to group 1.1b, was the dominant AOA species in vegetable soils. Nitrosomonas cluster 8-like and Nitrosospira were the predominant AOB species in open field soil, however, Nitrosospira was dominant in greenhouse soil. Redundancy analysis (RDA) showed that environmental factors had no significant effect on the diversity of the AOA community structure, but the effects of soil pH, EC, C/N ratio, NO3--N and NH4+-N contents on AOB community structure were significant. The results indicated that AOB was the main driver of vegetable soil nitrification, and soil pH and NH4+-N content were main factors that affected the differences of nitrification, AOB population and the shift of AOB community structure between open field and greenhouse vegetable soils.(2) Five soil types, including black soil of Harbin (HEB), fluvo aquic soil of Henan (HN), fluvo aquic soil of Hangzhou (HZ), red soil of Jinhua (JH) and paddy soil of Jiaxing (JX) were collected from greenhouse vegetable soils to evaluate different characteristics of soil nitrification, and abundance and community structure diversity of ammonia oxidizers among the soil types. The results showed that soil nitrification potential of HN was the highest, and that of JH and JX was the lowest. AOA populations in JH and JX soils were the highest, while AOB population in HN was the highest. Soil nitrification potential had significantly positive correlations with AOB abundance (r=0.93*) and soil pH (r=0.88*), while no relationship between AOA and nitrification potential was observed. T-RFLP analysis revealed that AOA T-RFs 579 bp,340 bp and 283 bp were unique in HZ, JH and JX soils, respectively. The relative abundance of AOB T-RFs 60 bp and 156 bp, belonging to Nitrosospira, was the highest in HN and HZ soil, respectively, and AOB community diversity was the greatest in JH and JX soils. RDA analysis demonstrated that effects of soil pH, organic matter and C/N ratio on differences of microbial community diversity among soil types were significant. The results indicated that soil pH was the major factor of the abundance and community structure of AOB, which directly affecting nitrification of different soil types.(3) An incubation study was conducted to study the interaction effects of biochar and nitrogen fertilizer (urea and (NH4)2SO4) on nitrification and ammonia-oxidizing community diversity in Hangzhou greenhouse vegetable soil. The results showed that combined application of fertilizer and biochar significantly increased soil net nitrification rate and AOB population, and AOB abundance was significantly related with soil net nitrification rate (r=0.72**), but the biochar and nitrogen had no effects on AOA amoA gene copies. The AOB community composition was different among treatments, and relative abundance of T-RF 60 bp, belonging to Nitrosospira, increased in fertilizer and biochar combined treatments. Further analysis showed that AOB community diversity dramatically decreased. However, biochar and nitrogen fertilizers had no effects on AOA community composition and diversity. Therefore, the increase of AOB population and the shifts of its community structure in greenhouse vegetable soil were the main microbial mechanisms of soil nitrification stimulated by the combined application of biochar and nitrogen fertilizer.(4) An incubation study was demonstrated to compare the impacts of nitrification inhibitors (NIs) 3,4-dimethylpyrazole phosphate (DMPP) and dicyandiamide (DCD) on soil nitrification, AOA and AOB abundance as well as their community structure in a vegetable soil collected form Hangzhou greenhouse. Our results showed that urea application significantly increased the net nitrification rates, but was significantly inhibited by both NIs, and the inhibitory effect of DMPP was significantly greater than that of DCD. AOB growth was more greatly inhibited by DMPP than by DCD, and the net nitrification rate was significantly related to AOB abundance, but not to AOA abundance. Application of urea and NIs did not change the diversity of the AOA community, with the T-RFs remaining in proportions that were similar to control soils, while the community structure of AOB exhibited obvious shifts within all different treatments compare to the control. Phylogenetic analysis showed that all AOA sequences fell within group 1.1a and group 1.1b, and the AOB community consisted of Nitrosospira cluster 3, cluster 0 and unidentified species. These results suggested that DMPP exhibited a stronger inhibitory effect on nitrification than DCD by inhibiting AOB rather than AOA.(5) An incubation study about the interaction effects of moisture and nitrification inhibitor DMPP on ammonia oxidizers was performed. The greenhouse vegetable soils, which were collected from Hangzhou, China, were maintained 65% water holding capacity (WHC, CW), or four cycles of 65% WHC followed by a 3-day dry down to 15% WHC, and then rewetted to and kept at 65% WHC for 4 days (DW). The results showed that compared with CW, DW sharply reduced soil nitrification, especially under the soil drying condition. The inhibitory effect of DMPP on soil net nitrification rate was markedly higher in DW than in CW condition. Under CW condition, AOB population was significantly higher than in DW condition, and the inhibitory effect of DMPP was greater under DW condition. Soil net nitrification rate had a significantly positive relationship with AOB (r=0.73**), but not with AOA. T-RFLP analysis showed that the relative abundance of AOB T-RF 60 bp increased, and community diversity decreased under DW condition, especially when soil was drying. Moreover, under DW condition, AOB community diversity in urea+ DMPP treatment was significantly lower than in urea treatment, but there was no significant difference under CW condition. Phylogenetic analysis showed that all AOA sequences belonged to group 1.1b, and AOB sequences belonged to Nitrosospira, Nitrosomonas and uncultured Nitrosomonas-like genus. These results indicated that under DW condition, DMPP exhibited greater inhibitory effect, especially when soil was drying, and the inhibitory effect of DMPP on soil nitrification was demonstrated via its inhibition on AOB population and shift of community composition in the greenhouse vegetable soil.