Biosci. Biotechnol. Biochem., 76 (8), 1580–1583, 2012 Note Distribution of Methanotrophs in the Phyllosphere y Hiroyuki IGUCHI, Izuru SATO, Maiko SAKAKIBARA, Hiroya YURIMOTO, and Yasuyoshi SAKAI Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan Received April 10, 2012; Accepted May 17, 2012; Online Publication, August 7, 2012 [doi:10.1271/bbb.120281] Plants have been reported to emit methane as well as In this study, we employed the enrichment culture methanol originating in their cell-wall constituents. We method with methane to screen methanotrophs living on investigated methanotrophs in the phyllosphere by the plants. Various plant tissues were sampled in Japan (in enrichment culture method with methane as sole carbon Kyoto, Shiga, and Hyogo Prefectures) in the spring and source. We enriched methanotrophs from the leaves, summer of 2006 and 2007. Prior to use, root samples flowers, bark, and roots of various plants. Analysis of were washed twice with sterilized water. Samples were the pmoA and mxaF genes retrieved from the enrich- placed in a vial (volume 25 mL) containing 5 mL of ment cultures revealed that methanotrophs closely nitrate mineral salt (NMS) medium.13) The vials were related to the genera Methylomonas, Methylosinus, sealed with a butyl rubber cap, and then 5 mL of and Methylocystis inhabit not only the rhizosphere but methane was added as sole carbon source to achieve a also the phyllosphere, together with methanol-utilizing 20:80 methane-to-air ratio at 1.25 atm of pressure. The bacteria. vials were incubated at 28 C with shaking. Subculture was done by transferring 1% (50 mL) of the culture to Key words: methanotroph; methylotroph; methane; fresh medium. After repeated subculturing more than 5 methanol; plant times, turbidity indicating methanotrophic growth was observed in many of the plant samples: 14% of leaves Plants emit a large variety of volatile organic (herbaceous plants, 21/127; woody plants, 18/159), compounds, including methane and methanol.1) Methane 4.0% of flowers (2/50), 9.1% of wood bark (2/22), and emission from plants is known to originate from 59% of roots (16/27). Some of the enrichment cultures methanogenic archaea under anaerobic conditions, with were subjected to sequence analysis. subsequent transport to the atmosphere through aeren- Genomic DNA was extracted from the methane- chyma.2) However, recent studies indicate that some enrichment cultures by a method using SDS and plants if not all produce methane aerobically from the proteinase K, as described previously.14) Genes of the methyl ester group of pectin,3–5) which is also a source molecular marker were amplified from the genomic of methanol.6) Methanol is thought to be produced by DNA by PCR with Ex Taq polymerase (Takara Bio, almost all plant species.6) Our previous study showed Shiga, Japan) and primer sets targeting the following that the methanol concentration on growing Arabidopsis genes: A189-mb661 for pmoA encoding the particulate leaves oscillates through a daily cycle within a range of methane monooxygenage,15,16) and mxaF1003f-1561r 4–64 mM.7) for mxaF encoding the methanol dehydrogenase.17) Methylotrophs are aerobic bacteria with the ability to The PCR products were cloned into pGEM-T Easy utilize C1-compounds such as methane, methanol, and (Promega, Madison, WI) by TA cloning, and plasmid methylamine as sole source of carbon and energy. They DNAs from at least five clones were sequenced using a are widespread in nature, and play an important role in BigDye Terminator v3.1 Cycle Sequencing Kit and an the global carbon cycle.8) Methylobacterium species, ABI PRISM 377 DNA sequencer (Applied Biosystems, which are known as pink-pigmented facultative methyl- Foster City, CA). Table 1 summarizes the methano- otrophs (PPFMs), are the major bacterial inhabitant of trophs and methanol-utilizers (non-methanotrophic the phyllosphere.9,10) Their methanol-utilizing ability has methylotrophs) identified in the plant materials based been found to play an important role in the colonization on the sequences of pmoA and mxaF. of leaves,7,11) indicating that plant methanol is an The pmoA gene sequences were found to be closely important carbon source for these organisms on leaves. related to the genera Methylomonas, Methylosinus, and Methanotrophs are a subset of methylotrophs that Methylocystis (Table 1) (accession nos. AB683078 to utilize methane, and are often found in environments AB683115). The sequence identities of the deduced near where methane is produced, such as wetlands, bogs, amino acid sequences of the pmoA genes to the closest rice paddies, and landfills.12) Almost no methanotrophs sequences based on BLAST analysis were 97–100% for can utilize carbon substrates such as sugars, except for type I methanotrophs ( -Proteobacteria) and 93–99% C1-compounds.12) Hence, methanotrophs probably uti- for type II methanotrophs ( -Proteobacteria). A phylo- lize methane and/or methanol in natural environments, genetic tree based on the PmoA sequences indicated that but little is known about the life of methanotrophs in the the methanotrophs obtained from the leaves, roots, phyllosphere. flowers, and bark clustered in the clades Methylomonas, y To whom correspondence should be addressed. Tel: +81-75-753-6385; Fax: +81-75-753-6454; E-mail: [email protected] Methanotrophs in the Phyllosphere 1581 Table 1. Methanotrophs and Methanol-Utilizers Retrieved from the Methane-Enrichment Cultures Origin Sample name Methanotrophy Methanol-utilizery Leaf (Acorus calamus var. angustatus) OH8 Methylocystis sp. Methylobacterium sp. Leaf (Acorus calamus var. angustatus) SH14 Methylomonas sp. Methylobacterium sp. Methylosinus sp. Leaf (Calystegia soldanella) B14 Methylocystis sp. Hyphomicrobium sp. Leaf (Eichhornia crassipes) SH31 Methylocystis sp. Methylophilus sp. Methylosinus sp. Leaf (Ilex sp.) K4 Methylomonas sp. Not tested Leaf (Keteleeria davidiana)SM24Methylomonas sp. Methylobacterium sp. Methylophilus sp. Leaf (Magnolia sp.) NK3 Methylosinus sp. Not tested Leaf (Phragmites australis)B4Methylosinus sp. Hyphomicrobium sp. Leaf (Picea abies)SM45Methylomonas sp. Not tested Methylocystis sp. Leaf (Pinus parviflora)SM39Methylomonas sp. Not tested Methylocystis sp. Leaf (Poaceae sp.) SH20 Methylosinus sp. Hyphomicrobium sp. Methylocystis sp. Leaf (Pyrus calleryansa)SM14Methylomonas sp. Methylobacterium sp. Methylophilus sp. Leaf (Quercus phillyraeoides)SM41Methylomonas sp. Methylobacterium sp. Methylophilus sp. Leaf (Quercus phillyraeoides) SH19 Methylocystis sp. Not tested Leaf (Viola X wittrockiana)SM26Methylomonas sp. Methylobacterium sp. Methylocystis sp. Methylophilus sp. Methylosinus sp. Leaf (Viola X wittrockiana) SH17 Methylocystis sp. Not tested Methylosinus sp. Flower (Phragmites australis) B13 Methylomonas sp. Not tested Bark (Quercus sp.) KF31 Methylosinus sp. Not tested Root (Acorus calamus var. angustatus)B2 Methylomonas sp. Methylovorus sp. Root (Acorus calamus var. angustatus) OH7 Methylocystis sp. Not tested Root (Oryza sativa)R1Methylomonas sp. Methylovorus sp. Root (Oryza sativa)R4Methylomonas sp. Hyphomicrobium sp. Methylocystis sp. Root (Oryza sativa)R2Methylomicrobium sp. Not tested Methylocystis sp. Root (Oryza sativa)R3Methylocystis sp. Not tested Root (Oryza sativa)R5Methylomonas sp. Not tested Methylocystis sp. Root (Oryza sativa)R6Methylomonas sp. Not tested Root (Oryza sativa)R7Methylomonas sp. Not tested Root (Oryza sativa)R8Methylomonas sp. Not tested Root (Oryza sativa)R9Methylomonas sp. Not tested Root (Phragmites australis)B3Methylosinus sp. Methylovorus sp. yBLAST (tblastx) analysis was used to search for the nearest relative sequence of the pmoA and the mxaF gene for methanotroph and methanol- utilizer, respectively. Methylosinus, and Methylocystis (Fig. 1). They were not agar, which was cultivated in a desiccator supplied with affiliated with the Upland Soil Cluster (USC , USC / methane. The 16S rRNA gene of the methanotrophic RA14, and JR3), the members of which are thought to isolates was PCR-amplified with 27f-1492r primers21) represent methanotrophs responsible for atmospheric and sequenced. Based on the 16S rRNA gene sequences, methane consumption.18,19) The Rice Paddy Cluster they were found to belong to the genus Methylomonas, (RPC-1 and RPC-2) has been reported to be composed Methylosinus,orMethylocystis (Table 2). Their abilities of sequences of rice fields from various geographical to utilize methanol were examined to determine whether regions,20) but no such sequences were detected in this methanotrophs can utilize the methanol on plant study. In addition to methanotrophs, sequencing of the surfaces. All the methanotrophic isolates tested grew mxaF gene identified methanol-utilizing bacteria that on methanol, but availability differed. Strains B4S and were closely related to the genera Methylobacterium, B2Z showed a longer lag growth phase on 2.5 mM Methylophilus, Methylovorus, and Hyphomicrobium methanol and no growth on 25 mM methanol (Table 2). (Table 1) (accession nos. AB683121 to AB683140). High concentrations of methanol are assumed to be These results indicate that methanotrophs inhabit diverse ordinarily present on plant surfaces,7) and hence further tissues and species of plants, and cohabit with methanol- study is needed to characterize the in situ growth of utilizing bacteria. methanotrophs on plants, including the carbon substrates Methanotrophs were also isolated from methane- utilized. enrichment cultures