Dandie C E, Miller M N, Burton D L, Zebarth B J, Trevors J T, Goyer C
Agriculture and Agri-Food Canada, Potato Research Centre, Fredericton, NB, Canada.
Appl Environ Microbiol. 2007 Jul;73(13):4250-8. doi: 10.1128/AEM.00081-07. Epub 2007 Apr 20.
The quantification of denitrifying bacteria is a component in the further understanding of denitrification processes in the environment. Real-time PCR primers were designed to target two segments of the denitrifier population (cnorB(P) [Pseudomonas mandelii and closely related strains] and cnorB(B) [Bosea, Bradyrhizobium, and Ensifer spp.]) in agricultural soils based on functional cnorB (nitric oxide reductase) gene sequences. Total population numbers were measured using 16S rRNA gene real-time PCR. Two soil microcosm experiments were conducted. Experiment 1 examined the response of the indigenous soil microbial population to the addition of 500 mg/kg glucose-C daily over 7 days in soil microcosms. Changes in the total population were correlated (r = 0.83) between 16S rRNA gene copy numbers and microbial biomass carbon estimates. Members of the cnorB(P) population of denitrifiers showed typical r-strategy by being able to increase their proportion in the total population from starting levels of <0.1% to around 2.4% after a daily addition of 500 mg/kg glucose-C. The cnorB(B) guild was not able to increase its relative percentage of the total population in response to the addition of glucose-C, instead increasing copy numbers only in proportion with the total population measured by 16S rRNA genes. Experiment 2 measured population dynamics in soil after the addition of various amounts of glucose-C (0 to 500 mg/kg) and incubation under denitrifying conditions. cnorB(P) populations increased proportionally with the amount of glucose-C added (from 0 to 500 mg/kg). In soil microcosms, denitrification rates, respiration, and cnorB(P) population densities increased significantly with increasing rates of glucose addition. cnorB(B) guild densities did not increase significantly under denitrifying conditions in response to increasing C additions.
反硝化细菌的定量分析是进一步了解环境中反硝化过程的一个组成部分。基于功能性cnorB（一氧化氮还原酶）基因序列，设计了实时荧光定量PCR引物，用于靶向农业土壤中反硝化细菌群体的两个片段（cnorB(P) [曼氏假单胞菌及密切相关菌株] 和cnorB(B) [博斯氏菌属、慢生根瘤菌属和剑菌属等]）。使用16S rRNA基因实时荧光定量PCR测量总菌数。进行了两个土壤微观试验。试验1研究了在土壤微观环境中，每天添加500 mg/kg葡萄糖碳，持续7天，本土土壤微生物群体的响应。16S rRNA基因拷贝数与微生物生物量碳估计值之间的总菌数变化具有相关性（r = 0.83）。反硝化细菌的cnorB(P)群体成员表现出典型的r策略，即每天添加500 mg/kg葡萄糖碳后，其在总菌数中的比例能够从起始水平的<0.1%增加到约2.4%。cnorB(B)群体无法响应葡萄糖碳的添加而增加其在总菌数中的相对百分比，而是仅与通过16S rRNA基因测量的总菌数成比例增加拷贝数。试验2测量了添加不同量葡萄糖碳（0至500 mg/kg）并在反硝化条件下培养后土壤中的菌群动态。cnorB(P)群体与添加的葡萄糖碳量（从0至500 mg/kg）成比例增加。在土壤微观环境中，反硝化速率、呼吸作用和cnorB(P)群体密度随着葡萄糖添加速率的增加而显著增加。在反硝化条件下，cnorB(B)群体密度并未因碳添加量的增加而显著增加。
