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Acta Agron Sin ›› 2015, Vol. 41 ›› Issue (05): 698-707.doi: 10.3724/SP.J.1006.2015.00698

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2015-05-12 Published:2014-04-01
  • Contact: 曾昭海, E-mail: zengzhaohai@cau.edu.cn, Tel: +86-10-62733847 E-mail:zgq2091010@163.com

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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