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作物学报 ›› 2015, Vol. 41 ›› Issue (05): 698-707.doi: 10.3724/SP.J.1006.2015.00698

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

绿肥还田对双季稻根际土壤产甲烷古菌群落结构的影响

赵晓萌1,**,刘婧娜2,**,易丽霞3,朱波3,代红翠2,胡跃高2,曾昭海2,*   

  1. 1,北京农学院生物科学与工程学院，北京102206；2,中国农业大学农学与生物技术学院，北京100193；3长江大学农学院，湖北荆州434025
  • 收稿日期:2014-10-16 修回日期:2015-04-02 出版日期:2015-05-12 网络出版日期:2014-04-01
  • 通讯作者: 曾昭海, E-mail: zengzhaohai@cau.edu.cn, Tel: +86-10-62733847
  • 基金资助:

    本研究由国家自然科学基金项目(31171509, 30671222)和国家“十二五”循环农业科技工程(2012BAD14B03)资助。

Community Structure of Methanogens from Double-rice Rhizosphere Soil as Affected by Green Manure Incorporation

ZHAO Xiao-Meng1,**,LIU Jing-Na2,**,YI Li-Xia3,ZHU Bo3,DAI Hong-Cui2,HU Yue-Gao2,ZENG Zhao-Hai2,*   

  1. 1 College of Biological Science and Engineering, Beijing University of Agriculture, Beijing 102206, China; 2China Agricultural University, Beijing 100193, China; 3 School of Agronomy, Yangtze University, Jingzhou 434025, China
  • Received:2014-10-16 Revised:2015-04-02 Published:2015-05-12 Published online:2014-04-01
  • Contact: 曾昭海, E-mail: zengzhaohai@cau.edu.cn, Tel: +86-10-62733847

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摘要：

利用PCR-DGGE技术及克隆文库构建方法研究尿素、紫云英鲜草翻压还田、黑麦草鲜草翻压还田和不施氮肥4种处理对双季稻不同生育时期(早稻季：分蘖期，拔节期，成熟期；晚稻季：分蘖期，扬花期，成熟期)稻田根际土壤中产甲烷群落结构的影响。结果表明，双季稻不同取样时期和各处理中产甲烷古菌群落结构稳定且相似，早稻季和晚稻季的优势群落均为甲烷微菌目(Methanomicrobiales)、Rice Cluster I (RC-I)、甲烷鬃菌科(Methanosaetaceae)、甲烷杆菌属(Methanobacterium)。但早稻季产甲烷古菌群落的Shannon-Weiner指数(H)和丰富度指数(R)整体低于晚稻季。紫云英和黑麦草鲜草翻压还田处理较尿素处理更为明显地提高了双季稻(一年)稻田根际土壤中产甲烷古菌群落的Shannon-Weiner指数和丰富度指数，但均暂未对产甲烷群落结构产生决定性影响。

关键词: 产甲烷古菌, 群落结构双季稻, 紫云英, 黑麦草

Abstract:

 With the potential to prevent nitrogen loss through nitrate leaching and resulted non-point source pollution, green manure incorporation is widely regarded as an important soil management practice. However, massive input of organic matters from green manure supplies sufficient substrate for methanogens and may change the methanogenic archaeal community structure. To investigate the variation of methanogenic archaeal community structure as influenced by green manure incorporation, we designed four treatments of urea application, Chinese milk vetch (Astragalus sinicus L.) incorporation, ryegrass (Lolium multiflorum Lam.) incorporation and control (without nitrogen fertilizer). Rhizosphere soil at different double-rice growing periods (tillering stage, jointing stage and mature stage for early rice; tillering stage, flowering stage and mature stage for late rice) was collected. Polymerase chain reaction-denaturing gradient gel electrophoresis technology (PCR-DGGE) and clone library method were employed to analyze methanogenic archaeal community structure. Methanogenic archaeal 16S rRNA gene was amplified using the 1106F/1378R primer set. A GC-clamp was added to the forward primers, 1106F, to enable denaturing gradient gel electrophoresis analyses. Selected Electrophoresis bands were cloned with pEASY-T3 vectors and sequenced. Relative band intensity and positions were used for principal component analysis (PCA). Methanotrophic bacterial community diversity was evaluated by the Richness index (R) and Shannon-Weiner index (H) from DGGE band data. The obtained sequences were analyzed with DNAMAN software and manually aligned with the sequences from GenBank database using the BLAST search program on NCBI (National Center for Biotechnology Information) web site. The phylogenetic trees were constructed by 1000-fold bootstrap analysis using a neighbor-joining method with Mega 4.0. Results indicated that methanogenic community structure was relative stable with high diversity and had small variations among sampling stages and treatments. Dominant populations were Methanomicrobiales, RC-I, Methanosaetaceae and Methanobacterium over the entire double-rice season. Methanohalophilus mahii only showed in early-rice season and fewer methanogens affiliated to Crenarchaeota in late-rice season. Methanogenic community structure in mature stages of early-rice and late-rice showed slightly low similarity with that in other growth stages of double-rice, which may caused by the decrease of organic matter content in soil. Higher Shannon-Weiner index (H) and Richness index (R) were observed in late-rice season by comparison with those in early-rice season. This may due to the improved activity of soil microbe and methanogens caused by increased average temperature in late-rice season. Additionally, three different fertilizer treatments increased Shannon-Weiner index and Richness index of methanogenic community structure by comparison with control, especially treatments with green manure incorporation. Increasing soil organic matter and ammonium nitrogen content, development of soil reducing conditions may contribute to this result. Whereas they didn’t have a crucial impact on methanogenic community structure in this study

Key words: Methanogens, Community structure, Double-rice, Chinese milk vetch, Ryegrass

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