牛场肥水灌溉对土壤nirK、nirS型反硝化微生物群落结构的影响

以徐水县梁家营长期定位施肥试验田为研究对象,利用末端限制性片段长度多态性(T-RFLP)分析和克隆文库构建,研究了5种施肥处理(清水灌溉CK、无机肥灌溉CF、牛场肥水不同浓度、不同次数灌溉T4、T5和T11)下土壤中nirK、nirS型反硝化细菌群落多样性及其群落结构的演变。结果表明,不同施肥处理下nirK、nirS型反硝化细菌群落多样性无显著差异,但群落结构却有明显变化:nirK型反硝化细菌群落结构既受施肥种类又受施肥量影响,优势种群尤其对施肥种类和施肥量响应显著;nirS型反硝化细菌则主要受施肥种类影响,施肥量影响微弱。牛场肥水处理和无机肥处理分别促进和抑制不同的nirS型反硝化细菌,群落主成分受无机肥促进、牛场肥水抑制。系统发育分析结果表明,土壤中nirK型反硝化细菌主要与假单胞菌属(Pseudomonas)、产碱杆菌属(Alcaligenes)和根瘤菌属(Rhizobium)的反硝化细菌具有较近的亲缘关系;nirS型反硝化细菌主要与劳尔氏菌(Ralstonia)和红长命菌属(Rubrivivax)有较近的亲缘关系。试验土壤中反硝化微生物多与目前已报道的好氧反硝化细菌亲缘关系较近,这可能与微生物分析取自表层土有关。

Effects of cattle fertilizer on community structure of nirK- and nirS- type denitrifying bacteria in soil

Field experiments of winter wheat-summer maize rotation were conducted in Xushui, Hebei Province, North China Plain irrigation area, to explore the effects of dairy effluent irrigation on diversity and community structure of nirK- and nirS- type denitrifying bacteria through terminal restriction fragment length polymorphism (T-RFLP) analysis and gene sequence analysis. Five treatments were designed, including no fertilizers treatment (CK), conventional fertilization (CF) and dairy effluent irrigations with 3 fertilizer levels (T4, T5 and T11). The results showed that no significantly differences were noted in diversity indexes of nirK- and nirS- denitrifying bacteria in all treatments. And T-RFLP analysis of nirK and nirS genes addressed significant differences in community composition: both fertilizer type and amount affected the relative abundance of nirK- T-RFs significantly, especially for dominant T-RFs, and therefore affected nirK- community composition; organic and inorganic fertilizer treatments increased or decreased different nirS- T-RFs'' relative abundance, resulting in significant differences in nirS- community composition. Phylogenetic analysis of nirK and nirS gene indicated that the nirK- type denitrifiers were mainly composed of Pseudomonas, Alcaligeneand and Rhizobium, nirS- type denitrifiers were mainly composed of Ralstonia and Rubrivivax. Most of these identified denitrifying bacteria were belong to families of aerobic denitrifying bacteria. A possible reason was that samples for biological analysis were taken from topsoil.