中国农业科学 ›› 2013, Vol. 46 ›› Issue (9): 1818-1826.doi: 10.3864/j.issn.0578-1752.2013.09.009

• 土壤肥料·节水灌溉·农业生态环境 • 上一篇    下一篇

稻田土壤nirS型反硝化细菌群落对氮肥水平的响应

 宋亚娜, 吴明基, 林艳   

  1. 福建省农业科学院生物技术研究所/福建省农业遗传工程重点实验室,福州 350003
  • 收稿日期:2012-12-12 出版日期:2013-05-01 发布日期:2013-01-15
  • 通讯作者: 宋亚娜,Tel:0591-87863144;E-mail:syana@sina.com
  • 作者简介:宋亚娜,Tel:0591-87863144;E-mail:syana@sina.com
  • 基金资助:

    国家自然科学基金项目(40801097)、福建省自然科学基金项目(2012J01107)

Response of the Denitrifying Bacterial nirS Gene Community to Nitrogen Fertilizer in Paddy Field

 SONG  Ya-Na, WU  Ming-Ji, LIN  Yan   

  1. Institute of Biological Technology, Fujian Academy of Agricultural Sciences/Fujian Province Key Laboratory of Genetic Engineering for Agriculture, Fuzhou 350003
  • Received:2012-12-12 Online:2013-05-01 Published:2013-01-15

摘要: 【目的】基于水稻田间定位试验,利用nirS功能基因研究不同氮肥水平对稻田土壤反硝化细菌群落多样性的影响。【方法】运用PCR-DGGE(聚合酶链反应-变性梯度凝胶电泳)结合DNA克隆测序和荧光定量PCR(Real-time PCR)技术对反硝化细菌nirS基因进行检测,分析田间定位试验第4年不同氮肥水平下稻田nirS型反硝化细菌群落结构和丰度的变化。【结果】依据DGGE图谱计算的群落多样性指数显示,与不施肥对照处理(CK)比较,施用氮肥处理(N1:75 kg N•hm-2,N2:150 kg N•hm-2和N3:225 kg N•hm-2)可促进稻田土壤nirS型反硝化细菌群落多样性指数提高,尤其在水稻生长的齐穗期和成熟期后者均显著高于前者(P<0.05)。但群落多样性指数在N1、N2 和N3处理间的差异主要表现在水稻分蘖期和齐穗期的表层土壤中,N3可显著高于N1(P<0.05)。冗余分析结果显示水稻生育时期对稻田土壤nirS型反硝化细菌群落结构的影响较大,表层和根层土壤的群落结构都与生育时期存在显著相关性(P=0.002,0.002);而不同氮肥水平对群落结构的显著性影响仅表现在稻田表层土壤中(P=0.002)。荧光定量PCR结果显示氮肥水平提高可促进稻田土壤nirS型反硝化细菌丰度增加,在水稻分蘖期和齐穗期内表层和根层土壤的nirS基因拷贝数均存在CK<N1<N2<N3的趋势,且以齐穗期时在表层土壤中的差异最大,不同处理间的差异都达到显著水平(P<0.05)。同时,本研究获得稻田反硝化细菌nirS基因片段序列8条登录GenBank(登录号:JX997923、JX997924、JX997926—JX997931)。此外,本试验中N1、N2和N3处理比CK处理分别增产59%、92%和107%,表层和根层土壤中NO3--N含量也随氮肥用量提高而增加。【结论】氮肥用量增加促进了稻田土壤nirS型反硝化细菌丰度及群落多样性指数的提高,尤其在稻田表层土壤中。氮肥水平提高还可改变稻田表层土壤nirS型反硝化细菌的群落结构。综合分析表明稻田表层土壤的nirS型反硝化细菌群落对氮肥水平提高的响应程度更明显。

关键词: 氮肥水平 , 反硝化细菌 , nirS 基因 , 群落结构 , 群落丰度

Abstract: 【Objective】The aim of this study was to investigate the effect of the nitrogen input amount on diversity of denitrifying bacterial community in paddy soil by nirS gene based on field location test of rice. 【Method】 Composition and abundance of denitrifying bacterial community in paddy soil were studied with the aid of denaturing gradient gel electrophoresis, cloning sequencing and real-time PCR by assay nirS gene in the forth year of a field experiment.【Result】Shannon-Wiener index of denitrifying bacterial nirS gene community in paddy field was calculated according to the image of DGGE. In the three treatments of nitrogen fertilizer (N1: 75 kg N•hm-2, N2: 150 kg N•hm-2 and N3: 225 kg N•hm-2), the Shannon-Wiener index was higher than that in the treatment of CK (without fertilizer), and the differences reached a significant level at heading and maturate stages of rice (P<0.05). Whereas, among treatments of N1, N2 and N3 only Shannon-Wiener index of N3 was markedly higher than that of N1 in surface soil (P<0.05) at tillering and heading stages of rice. Redundancy discriminate analysis (RDA) showed that the denitrifying bacterial nirS gene community structure in surface soil or in root zone soil all markedly correlated to growth stages of rice (P=0.002, 0.002), while there was a significantly correalation (P=0.002) between different levels of nitrogen fertilizer treatments and denitrifying bacterial nirS gene community structure only in surface soil. Real-time PCR analysis of nirS genes copies manifested that the increase in nitrogen input amount enhanced the abundance of denitrifying bacteria. At heading or maturate stages of rice, there was a upward trend of CK<N1<N2<N3 in nirS genes copies in surface soil or in root zone soil, especially in surface soil at heading stage of rice (P<0.05). At the same time, 8 DNA sequences of nirS gene cloning from DGGE band were got and logged in GenBank (Accession Number: JX997923, JX997924, JX997926-JX997931). In addition, the yield of rice in treatments of N1, N2 or N3 was 59%, 92%, or 107% more than that of CK. NO3--N in surface soil or in root zone soil increased with the increase of nitrogen input amount.【Conclusion】The findings demonstrated that the increase in nitrogen input amount promoted the enhancement in abundance and Shannon-Wiener index of denitrifying bacterial nirS gene community in paddy field, especially in surface soil. Changes in community structure of denitrifying bacterial nirS gene among different levels of nitrogen fertilizer treatments also took on in the surface soil. So there is an obvious response of denitrifying bacterial nirS gene community to nitrogen input amount in surface soil of the paddy field.

Key words: nitrogen fertilizer levels , denitrifying bacteria , nirS gene , community structure , community abundance