绿肥还田对双季稻根际土壤产甲烷古菌群落结构的影响

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

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。

With the potential to prevent nitrogen loss through nitrate leaching and resulted non-point source pollution, green ma- nure 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 arehaeal 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 Lain.） 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. Poly- merase 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 posi- tions were used for principal component analysis （PCA）. Methanotrophic bacterial community diversity was evaluated by the Richness index （R） and Sharmon-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. Methanogeniccommunity 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 fertil- izer 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.