Response of denitrifying bacteria community structure and abundance to nitrogen in paddy fields
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摘要: 以氮肥田间定位试验为研究对象, 利用PCR-DGGE(聚合酶链反应-变性梯度凝胶电泳)和荧光定量PCR(real-time PCR)技术, 通过对反硝化细菌nirS基因的检测, 分析了定位试验第2年稻田反硝化细菌群落结构和丰度的变化。DGGE图谱及依据其条带位置和亮度数字化数值进行的主成分分析(PCA)结果均显示: 在氮肥定位试验第2年, 与不施肥对照(CK)比较, 在水稻各个生育期(分蘖期、齐穗期和成熟期)内, 施用氮肥[150 kg(N)·hm-2]的稻田根层土或表土中的反硝化细菌群落结构均无明显变化; 且稻田根层土或表土中的反硝化细菌群落结构在水稻各个生育期间也均无明显差异。荧光定量PCR结果显示, 在水稻生长发育过程中, 施用氮肥的稻田根层土或表土中的反硝化细菌nirS基因拷贝数始终显著(P<0.05)高于其对应的不施肥对照。此外, 无论施用氮肥与否, 根层土中的反硝化细菌nirS基因拷贝数在水稻成熟期时都会显著(P<0.05)降低; 但表土中的nirS基因拷贝数在水稻各生育期间无明显变化; 且水稻成熟期时施用氮肥和不施肥的稻田表土中nirS基因拷贝数都显著(P<0.05)高于根层土。同时, 与对照比较施用氮肥可促进水稻增产44%。研究表明, 短期定位试验中施用氮肥能够显著提高稻田土壤反硝化细菌的丰度, 但对其群落结构没有明显影响。Abstract: Denitrification is critical for nitrogen cycle in the ecosystem, where fixed nitrogen is released into the atmosphere as N2. Nitrite reductase, the product of nirS or nirK nitrite reductase genes, is the key enzyme of bacteria dissimilatory denitrification process. Denitrifying bacteria community composition varies with environmental factors such as temperature, moisture, pH, O2 and nutrient availability. There is obvious denitrification process in flooded paddy fields. Hence denitrifying bacteria community structure and abundance in paddy fields are used to investigate the response of denitrifying bacteria to nitrogen fertilizer application in paddy fields. The experiment was conducted in a second-year nitrogen fertilization field with the aid of denaturing gradient gel electrophoresis and real-time PCR assay copies of nirS gene. DGGE images of nirS gene in root-zone soil and surface soil showed rich abundance of denitrifying bacteria in paddy soils. DGGE band number in surface soil image was higher than that in root-zone soil. Principle components analysis (PCA) of nirS gene DGGE profile showed that denitrifying bacteria community structure in root-zone or surface soil of paddy fields with nitrogen fertilizer [N: 150 kg(N)·hm-2] was similar to that of paddy fields without fertilizer (CK) during rice growth stages of tillering, heading and maturity. Also no difference was noted in denitrifying bacteria community structure in root-zone soil or surface soil among different growth stages of rice. Denitrifying bacteria nirS gene copy abundance in root-zone or in surface soil with nitrogen fertilizer treatment was significantly (P < 0.05) higher than that of CK treatment during rice growth. In both nitrogen fertilizer and CK treatments, denitrifying bacteria nirS gene copies in root-zone soil markedly (P < 0.05) dropped at maturity stage of rice growth. There were, however, no differences in nirS gene copies in surface soil among the different rice growth stages. At maturity stage, nirS gene copies in surface soils of both nitrogen fertilizer and CK treatments were higher (P < 0.05) than those in root-zone soils. Furthermore, rice yield in nitrogen fertilizer treatment was 44% higher than that of CK. In conclusion, denitrifying bacteria abundance was not only variable but also actively responded to nitrogen fertilizer supply. On the other hand, denitrifying bacteria community structure was not only relatively stable but largely unresponsive to nitrogen fertilizer supply. The study demonstrated that nitrogen fertilizer enhanced denitrifying bacteria abundance, which was critical for nitrogen cycling in paddy field ecosystem.
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