Variations in the N2 Fixation and CH4 Oxidation Activities of Type I Methanotrophs in the Rice Roots in Saline-Alkali Paddy Field Under Nitrogen Fertilization

The root-associated methanotrophs contribute to N₂ fixation and CH₄ oxidation in paddy fields under N-limited conditions. However, the impact of nitrogen inputs on N₂ fixation and CH₄ oxidation by methanotrophs is largely unknown, especially in saline-alkali paddy fields with higher nitrogen application. This study explored the impact of nitrogen fertilization on N₂ fixation and CH₄ oxidation by root-associated active diazotrophic and methanotrophic communities in a saline-alkali paddy field using ¹⁵N-N₂ and ¹³C-CH₄ isotope feeding experiments along with RNA-based sequencing. The ¹⁵N and ¹³C isotope feeding experiments showed that the CH₄ oxidation-dependent nitrogen fixation rate of methanotrophs (¹⁵N and ¹³C) in the roots of two rice cultivars was significantly higher than the CH₄ oxidation-independent nitrogen fixation rate of heterotrophic diazotrophs (only ¹⁵N) under nitrogen fertilization (SN) in a saline-alkali environment (P < 0.05). For Kongyu131 rice, the CH₄ oxidation-dependent nitrogen fixation rate ranged from 1.17 to 4.15 μmol/h/g, while the CH₄ oxidation-independent nitrogen fixation rate was determined to be 1.10 to 3.17 μmol/h/g. In J3 rice, these rates were 7.30 to 9.22 μmol/h/g and 5.76 to 4.85 μmol/h/g, respectively (P < 0.05). Moreover, both N₂ fixation and CH₄ oxidation rates of methanotrophs in the roots of salt-alkali tolerant J3 cultivar (9.22 μmol/h/g for N₂ fixation; 0.09 μmol/h/g for CH₄ oxidation) were significantly higher than those in the roots of the common rice cultivar Kongyu131 (4.15 μmol/h/g for N₂ fixation; 0.03 μmol/h/g for CH₄ oxidation) under nitrogen fertilization (P < 0.01). Thus, methanotrophs associated with J3 rice roots demonstrated improved N₂ fixation and CH₄ oxidation activities under saline-alkali stress in the presence of nitrogen fertilizer. Even heterotrophic diazotrophs in J3 rice roots showed enhanced N₂ fixation with (SN) or without (LN) nitrogen inputs. The RNA-based amplicon sequencing showed that nitrogen fertilizer had a greater influence on diazotrophic and methanotrophic communities than the differences between rice cultivars. Further, active Methylomonas (type I methanotrophs) dominated the root-associated diazotrophic (9.8-20.9%) and methanotrophic (46.8-80.3%) communities. Within these, Methylomonas methanica (13.3 vs. 3.8%) and Methylomonas paludis (8.8 vs. 27.4%) were determined as the common genera in the diazotrophic and methanotrophic communities, respectively, with both proportions undergoing significant shifts under SN conditions. Whereas the LN condition led to high CH₄ oxidation activity and a relatively high abundance of Methylocystis (26.0%) in the roots of Kongyu131 rice, which sharply decreased under the SN condition (0.3%). The findings revealed that CH₄ oxidation-dependent N₂ fixation and CH₄ oxidation activities of root-associated type I methanotrophs were significantly affected under nitrogen fertilization, with a more pronounced effect in the salt-alkali tolerant J3 rice cultivar compared to Kongyu131. This study highlights the potential of aerobic diazotrophic methanotrophs in enhancing symbiotic diversity and environmental adaptability while contributing to CH₄ emission reduction and bioavailable nitrogen accumulation in saline-alkali paddy fields.