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      A pmoA-based study reveals dominance of yet uncultured Type I methanotrophs in rhizospheres of an organically fertilized rice field in India

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          Abstract

          Rice fields are one of the important sources of anthropogenic methane. Methanotrophs can oxidize up to 30 % of the produced methane and thus have a pivotal environmental role in methane mitigation. India occupies the largest region under rice cultivation; however, most of the studies done on methanotrophic communities have focused on the Northern region. We studied methanotrophic community of a flooded, organically fertilized rice field using pmoA clone library approach. Organic rice fields impose a more serious threat as they produce more methane. pmoA gene is the main functional gene which is primarily used for taxonomical analysis of methanotrophs. Our results showed that the pmoA clone libraries from two growth stages of rice were dominated by pmoA sequences which were very distant from cultivated Type Ia methanotrophic genera (80–82 % nucleotide similarity) indicative of the presence of a putatively novel genus. We designated this group of clones as ‘rice field clones’ as this also includes many pmoA sequences originating from other rice fields. Thus, our current knowledge of methanotroph diversity from Indian rice fields has been expanded revealing that a substantial portion of methanotrophic diversity is unexplored.

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          The online version of this article (doi:10.1007/s13205-016-0453-3) contains supplementary material, which is available to authorized users.

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          The global methane cycle: recent advances in understanding the microbial processes involved.

          The global budget of atmospheric CH4 , which is on the order of 500-600 Tg CH4 per year, is mainly the result of environmental microbial processes, such as archaeal methanogenesis in wetlands, rice fields, ruminant and termite digestive systems and of microbial methane oxidation under anoxic and oxic conditions. This review highlights recent progress in the research of anaerobic CH4 oxidation, of CH4 production in the plant rhizosphere, of CH4 serving as substrate for the aquatic trophic food chain and the discovery of novel aerobic methanotrophs. It also emphasizes progress and deficiencies in our knowledge of microbial utilization of low atmospheric CH4 concentrations in soil, CH4 production in the plant canopy, intestinal methanogenesis and CH4 production in pelagic water. © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd.
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            Molecular ecology techniques for the study of aerobic methanotrophs.

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              Quantitative detection of methanotrophs in soil by novel pmoA-targeted real-time PCR assays.

              Methane oxidation in soils is mostly accomplished by methanotrophic bacteria. Little is known about the abundance of methanotrophs in soils, since quantification by cultivation and microscopic techniques is cumbersome. Comparison of 16S ribosomal DNA and pmoA (alpha subunit of the particulate methane monooxygenase) phylogenetic trees showed good correlation and revealed five distinct groups of methanotrophs within the alpha and gamma subclasses of Proteobacteria: the Methylococcus group, the Methylobacter/Methylosarcina group, the Methylosinus group, the Methylocapsa group, and the forest clones group (a cluster of pmoA sequences retrieved from forest soils). We developed quantitative real-time PCR assays with SybrGreen for each of these five groups and for all methanotrophic bacteria by targeting the pmoA gene. Detection limits were between 10(1) and 10(2) target molecules per reaction for all assays. Real-time PCR analysis of soil samples spiked with cells of Methylococcus capsulatus, Methylomicrobium album, and Methylosinus trichosporium recovered almost all the added bacteria. Only the Methylosinus-specific assay recovered only 20% of added cells, possibly due to a lower lysis efficiency of type II methanotrophs. Analysis of the methanotrophic community structure in a flooded rice field soil showed (5.0 +/- 1.4) x 10(6) pmoA molecules g(-1) for all methanotrophs. The Methylosinus group was predominant (2.7 x 10(6) +/- 1.1 x 10(6) target molecules g(-1)). In addition, bacteria of the Methylobacter/Methylosarcina group were abundant (2.0 x 10(6) +/- 0.9 x 10(6) target molecules g of soil(-1)). On the other hand, pmoA affiliated with the forest clones and the Methylocapsa group was below the detection limit of 1.9 x 10(4) target molecules g of soil(-1). Our results showed that pmoA-targeted real-time PCR allowed fast and sensitive quantification of the five major groups of methanotrophs in soil. This approach will thus be useful for quantitative analysis of the community structure of methanotrophs in nature.
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                Author and article information

                Contributors
                +91-20-25325120 , monalirahalkar@aripune.org
                Journal
                3 Biotech
                3 Biotech
                3 Biotech
                Springer Berlin Heidelberg (Berlin/Heidelberg )
                2190-5738
                16 June 2016
                16 June 2016
                December 2016
                : 6
                : 2
                : 135
                Affiliations
                [ ]MACS Agharkar Research Institute, G.G. Agarkar Road, Pune, Maharashtra 411004 India
                [ ]Microbial Culture Collection, NCCS, Sai-Trinity Building, Pashan, Pune, Maharashtra 411021 India
                Article
                453
                10.1007/s13205-016-0453-3
                4910840
                28330207
                9c53c004-276e-4aa8-b7b2-e16f30332ec9
                © The Author(s) 2016

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

                History
                : 23 March 2016
                : 3 June 2016
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100001407, Department of Biotechnology , Ministry of Science and Technology;
                Award ID: BT/BioCARe/06/840/2012
                Award Recipient :
                Categories
                Short Reports
                Custom metadata
                © The Author(s) 2016

                methanotrophs,rice fields,molecular approach,clone libraries,pmoa: gene for particulate methane monooxygenase subunit

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