Paul S S, Deb S M, Dey A, Somvanshi S P S, Singh D, Rathore R, Stiverson J
ICAR-Central Institute for Research on Buffaloes, Sirsa Road, Hisar 125001, Haryana, India.
ICAR-Central Institute for Research on Buffaloes, Sirsa Road, Hisar 125001, Haryana, India.
Anaerobe. 2015 Oct;35(Pt B):3-10. doi: 10.1016/j.anaerobe.2015.06.002. Epub 2015 Jun 21.
The molecular diversity of rumen methanogens was investigated using 16S rDNA gene library prepared from the rumen contents of Nili-Ravi buffaloes. Microbial genomic DNA was isolated from four adult male fistulated buffaloes and PCR conditions were set up using specific primers. Amplified product was cloned into a suitable vector, and the inserts of positive clones were sequenced. A total of 142 clones were examined, and the analysis revealed 46 species level (0.01 distance) operational taxonomic units (OTUs). Twenty six OTUs comprising 89 clones (63% of the total clones) were taxonomically assigned to Methanobacterium genus and the majority of them had highest percent identity with Methanobacterium flexile among cultured methanogens. Five OTUs comprising 27 clones (19% of total clones) were taxonomically assigned to Methanomicrobium genus and these clones showed highest sequence identity with Methanomicrobium mobile. Only two OTUs comprising 6 clones (4% of total clones) were assigned to Methanobrevibacter genus. A total of 17 clones belonging to 10 species level OTUs showed highest percent identity (ranging from 85 to 95%) with Methanomassilicoccus luminyensis and were taxonomically classified as Methanomassiliicocaceae. Out of the 142 rDNA clones, 112 clones, which constitute 79% of the total clones representing 42 OTUs, had less than 98.5% sequence identity with any of the cultured strains of methanogens and represent novel species of methanogens. This study has revealed the largest assortment of hydrogenotrophic methanogen phylotypes ever identified from the rumen of Nili-Ravi buffaloes. The study indicates that Methanobacterium is the most dominant methanogen in the rumen of Nili-Ravi buffalo. This is also the first report on the presence of methanogens phylogenetically close to M. luminyensis, an H2 dependent methylotrophic methanogen, in the rumen of buffaloes at such a high level of abundance.
利用从尼里-拉菲水牛瘤胃内容物制备的16S rDNA基因文库，对瘤胃产甲烷菌的分子多样性进行了研究。从四只成年雄性瘘管水牛中分离出微生物基因组DNA，并使用特异性引物设置PCR条件。将扩增产物克隆到合适的载体中，并对阳性克隆的插入片段进行测序。共检测了142个克隆，分析显示有46个物种水平（距离为0.01）的操作分类单元（OTU）。26个OTU（包含89个克隆，占总克隆数的63%）在分类学上被归为甲烷杆菌属，其中大多数与培养的产甲烷菌中最具柔韧性的甲烷杆菌具有最高的序列相似性百分比。5个OTU（包含27个克隆，占总克隆数的19%）在分类学上被归为甲烷微菌属，这些克隆与运动甲烷微菌具有最高的序列相似性。只有2个OTU（包含6个克隆，占总克隆数的4%）被归为甲烷短杆菌属。共有17个属于10个物种水平OTU的克隆与卢米尼产甲烷球菌具有最高的序列相似性百分比（范围为85%至95%），并在分类学上被归类为甲烷粒菌科。在142个rDNA克隆中，有112个克隆（占总克隆数的79%，代表42个OTU）与任何培养的产甲烷菌菌株的序列相似性均低于98.5%，代表了产甲烷菌的新物种。这项研究揭示了从尼里-拉菲水牛瘤胃中鉴定出的最大种类的氢营养型产甲烷菌系统发育型。该研究表明，甲烷杆菌是尼里-拉菲水牛瘤胃中最主要的产甲烷菌。这也是关于在水牛瘤胃中如此高丰度水平下存在与卢米尼产甲烷球菌系统发育关系密切的产甲烷菌的首次报道。
