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[BBC02-03] Expanding the eco-collection of methane-oxidizing bacteria from rice roots
Keywords:methane, methane-oxidizing bacteria, methanotroph, rice paddy field, rice cultivation
Rice (Oryza sativa L.) is one of the most important staple food crops in the world. However, flooded rice paddy fields emit methane (CH4), a strong greenhouse gas. Therefore, the reduction of CH4 emission from the paddy fields is an important issue for sustainable agriculture. Aerobic methane-oxidizing bacteria (MOB) utilize CH4 as sole carbon and energy sources and they are classified to Methylococcaceae (Type I), Methylocystaceae (Type IIa), Beijerinckiaceae (Type IIb), and Methylacidiphilaceae (Type III) phylogenetically, in which Type Ia and Ib, subgroups of Type I, and IIa MOB are major groups inhabiting the paddy fields. The CH4 emission is the net balance of the production by anaerobic methanogenic archaea in the reduced soil layer and the oxidation by MOB at the oxic microsites. Rice root is one of the most important compartments where both of CH4 and O2 pass through. However, only a few MOB strains have been isolated from rice roots and no strain has been identified at species levels (1–3). In this research, we aimed to isolate and characterize MOB strains from rice roots to further expand the eco-collection of MOB. Functional and ecological analysis of MOB in the paddy fields will be helpful to reduce CH4 emission from the paddy fields and to understand the roles of MOB in the ecosystem.
The root samples collected from three varieties of rice plants grown in a paddy field without nitrogen fertilizer application for the long term were incubated in the nitrate mineral salt (NMS) liquid medium with about 14% CH4 gas for the enrichment of MOB. The cultures were subcultured on NMS agar plates, and then formed colonies were inoculated into a new NMS liquid medium to check the CH4 oxidation activity. The cultures showing the activity were further purified by colony isolation and/or extinction dilution methods. The purity of isolates (no contamination of heterotrophic bacteria) was checked by inoculating them into nutrient-rich media. Genomic DNA was extracted from the purified strains, and sequences of 16S rRNA and particulate methane monooxygenase (pmoA) genes were determined to infer the phylogeny of the strains. Presence of nitrogenase (nifH) gene was assessed by PCR. Genome sequences of some strains were analyzed by PacBio Sequel II. The values of average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) were calculated between the strains and phylogenetically related MOB strains.
Twelve MOB strains were obtained, of which two and ten strains belonged to Type Ia and Type IIa, respectively, according to phylogenetic analysis of 16S rRNA gene and pmoA genes. Two Type Ia MOB strains were closely related to Methylomonas koyamae Fw12E-YT with the ANI and dDDH values of 86% and 31%, respectively, indicating that these strains represent novel Type Ia MOB species. Among the ten strains belonging to Type IIa MOB, three strains were related to Methylocystis echinoides IMET10491T. The ANI and dDDH values between two of the strains and M. echinoides IMET10491T were 85% and 28%, respectively, indicating that these strains represent novel Type IIa MOB species. The rest seven strains were related to Methylosinus sporium 5T. All the isolated strains possessed nifH genes, suggesting a potential of nitrogen-fixing ability. These results revealed that unexplored MOB members including novel species inhabited rice roots. The present study contributed significantly to expanding the eco-collection of MOB from the paddy field ecosystem. Further characterization and genome analysis of the MOB strains from rice roots will be conducted to clarify their functions and notable genetic features.
(1) K. Takeda et al. (2008) Soil Sci. Plant Nutr., 54: 876–885.
(2) R. Shinoda et al. (2019) Soil Biol. Biochem., 132: 40–46.
(3) S. Asakawa (2021) Soil Sci. Plant Nutr., 67: 520–526
The root samples collected from three varieties of rice plants grown in a paddy field without nitrogen fertilizer application for the long term were incubated in the nitrate mineral salt (NMS) liquid medium with about 14% CH4 gas for the enrichment of MOB. The cultures were subcultured on NMS agar plates, and then formed colonies were inoculated into a new NMS liquid medium to check the CH4 oxidation activity. The cultures showing the activity were further purified by colony isolation and/or extinction dilution methods. The purity of isolates (no contamination of heterotrophic bacteria) was checked by inoculating them into nutrient-rich media. Genomic DNA was extracted from the purified strains, and sequences of 16S rRNA and particulate methane monooxygenase (pmoA) genes were determined to infer the phylogeny of the strains. Presence of nitrogenase (nifH) gene was assessed by PCR. Genome sequences of some strains were analyzed by PacBio Sequel II. The values of average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) were calculated between the strains and phylogenetically related MOB strains.
Twelve MOB strains were obtained, of which two and ten strains belonged to Type Ia and Type IIa, respectively, according to phylogenetic analysis of 16S rRNA gene and pmoA genes. Two Type Ia MOB strains were closely related to Methylomonas koyamae Fw12E-YT with the ANI and dDDH values of 86% and 31%, respectively, indicating that these strains represent novel Type Ia MOB species. Among the ten strains belonging to Type IIa MOB, three strains were related to Methylocystis echinoides IMET10491T. The ANI and dDDH values between two of the strains and M. echinoides IMET10491T were 85% and 28%, respectively, indicating that these strains represent novel Type IIa MOB species. The rest seven strains were related to Methylosinus sporium 5T. All the isolated strains possessed nifH genes, suggesting a potential of nitrogen-fixing ability. These results revealed that unexplored MOB members including novel species inhabited rice roots. The present study contributed significantly to expanding the eco-collection of MOB from the paddy field ecosystem. Further characterization and genome analysis of the MOB strains from rice roots will be conducted to clarify their functions and notable genetic features.
(1) K. Takeda et al. (2008) Soil Sci. Plant Nutr., 54: 876–885.
(2) R. Shinoda et al. (2019) Soil Biol. Biochem., 132: 40–46.
(3) S. Asakawa (2021) Soil Sci. Plant Nutr., 67: 520–526