ABSTRACT Denitrification could lead to N loss from agricultural soil resulting in a low rate of N availability by crops. We investigated the response of nirK and nirS denitrifiers in a paddy soil to the incorporation of green manure and reduced chemical fertilizer. Soil samples were collected from plots of a short-term fertilization experiment initiated in 2009. The treatments were no chemical fertilizer, no Chinese milk vetch (Astragalus sinicus L., CK), chemical fertilizer (NPK), Chinese milk vetch (MV) plus 80% chemical fertilizer (MF80), MV plus 60% chemical fertilizer (MF60) and MV plus 40% chemical fertilizer (MF40). Abundance and community composition of nirK and nirS denitrifiers were analyzed using quantitative PCR and Miseq sequencing. Reduced chemical fertilizer did not reduce content of total N and available N in soils amended with green manure. Abundances of nirK and nirS genes in different treatments were 1.04 ×108 to 4.89×108 and 1.22 ×107 to 7.04 ×107 copies g-1 soil, respectively, which were significantly higher in NPK treatment than those in soils with green manure. NirK abundance was positively correlated with the potential denitrifying activity (PDA) (r2 = 0.827, p < 0.01). Green manure combination with reduced chemical fertilizer significantly changed the community structure of nirK denitrifiers but not nirS denitrifiers relative to soils amended with chemical fertilizer and unfertilized soil. Shifts of community structure of nirK denitrifiers were closely associated with soil organic matter (r2 = 0.623, p = 0.003), available N (r2 = 0.507, p = 0.01), pH (r2 = 0.661, p = 0.006), and PDA (r2 = 0.633, p = 0.005). In conclusion, nirK-type denitrifying community was more sensitive to the incorporation of green manure and reduced chemical fertilizer and they played a more important role in the denitrification process in this study.

ABSTRACT Cold waterlogged paddy field is a type of low-yield paddy soil, and opening a drainage ditch has been suggested to improve waterlogged paddy soil and to increase the rice yield. However, little has been done on the role of a drainage ditch in ameliorating waterlogged soil. In the present study, rice (Oryza sativa L.) yield, soil physicochemical properties, microbial abundances and community structures in the surface soil of a cold waterlogged paddy field, which has three treatments with different distances from a drainage ditch including 5, 15 and 25 m were investigated. The results showed that opening a drainage ditch had a significant effect on the rice yield and soil physicochemical properties. Sampling sites near the drainage ditch such as 5 m-site had higher yield and soil nutrients, however, 25 m-site had lower values. Bacterial 16S rRNA gene did not exhibit a regular pattern but fungal abundance decreased sharply with increasing distance from the drainage ditch. The most abundant bacterial phyla across all soil samples were Proteobacteria, Acidobacteria, Planctomycetes and Chloroflexi. Abundant fungal phyla were Ascomycota, Zygomycota and Basidiomycota. Three treatments had similar microbial community compositions but with different relative abundances. The Bray-Curtis distance demonstrated that microbial community structures were divided into two major groups, samples of 5 m- and 15 m-site clustered into one group and samples of 25 m-site was another group. Moreover, redundancy analysis revealed that soil redox potential and total reducing substances were significant factors in shaping microbial community structures. The abundant aerobic bacteria were observed at the 5 m- and 15 m-site, in contrast, abundant anaerobic bacteria were observed at the 25 m-site. In conclusion, opening a drainage ditch in cold waterlogged paddy fields affects rice yield, soil nutrients, microbial abundances and community structures.

A field experiment established in 1980 was conducted to evaluate the effects of open drainage ditch applied for water removal on bacterial and fungal communities of cold waterlogged paddy soils in 2011. In this experiment, traditional plate counting and temperature gradient gel electrophoresis were employed to characterize the abundance and diversity of soil bacterial and fungal communities. Four different distances from the open drainage ditch, 5, 15, 25 and 75 m with different degrees of drainage were designed for this study. Maximum populations of culturable aerobic bacteria and fungi were at 15-m distance while minimum populations were at 75-m distance. Significant differences (p < 0.05) in fungal populations were observed at all distances from open drainage ditch. The highest diversity of the bacterial community was found at a distance of 25 m, while that of the fungal community was observed at a distance of 5 m. Sequencing of excised TGGE bands indicated that the dominant bacteria at 75-m distance belonged to anaerobic or microaerobic bacteria. Relationships between microbial characteristics and soil physicochemical properties indicated that soil pH and available nitrogen contents were key factors controlling the abundance of culturable aerobic bacteria and fungi, while soil water capacity also affected the diversity of fungal community. These findings can provide the references for better design and advanced management of the drainage ditches in cold waterlogged paddy soils.

Eight silver-staining protocols were applied to detect DNA in polyester-backed gels to select the optimal. Results showed important differences in staining quality and that four methods were well-suited for TGGE gels due to high sensitivity and low background, including the Bassam et al. methods, the manufacturer method and our improved method.