Eco-agricultural systems aim to reduce the use of chemical fertilizers in order to improve sustainable production and maintain a healthy ecosystem. The aim of this study was to explore the effects of rice-frog farming on the bacterial community and N-cycling microbes in paddy rhizosphere soil. This experiment involved three rice cultivation patterns: Conventionally cultivated rice (CR), green rice-frog farming (GR), and organic rice-frog farming (OR). The rice yield, paddy soil enzyme activities, physicochemical variables and bacterial and N-cycling bacterial abundances were quantitatively analyzed. Rice-frog cultivations significantly increased soil protease, nitrate and reductase activity. Additionally, the nirS gene copy number and the relative abundance of denitrifying bacteria also increased, however urease activity and the relative abundance of nitrifying bacteria significantly decreased. The bacterial community richness and diversity of OR soil was significantly higher than that of the GR or CR soil. Nitrogen use efficiency (NUE) of GR was highest. The N-cycling bacterial community was positively correlated with the total carbon (TC), total nitrogren (TN) and carbon to nitrogen (C:N) ratio. The present work strengthens our current understanding of the soil bacterial community structure and its functions under rice-frog farming. The present work also provides certain theoretical support for the selection of rational rice cultivation patterns.

Integrated rice-frog farming (IRFF), as a mode of ecological farming, is fundamental in realizing sustainable development in agriculture. Yet its production of greenhouse gas (GHG) emissions remains unclear. Here, a randomized plot field experiment was performed to study the GHG emissions for various farming systems during the rice growing season. The farming systems included: conventional farming (CF), green integrated rice-frog farming (GIRF), and organic integrated rice-frog farming (OIRF). Results indicate that the cumulative methane (CH₄) emissions from the whole growth period were divergent for the three farming systems, with OIRF having the highest value and CF having the lowest. For nitrous oxide (N₂O) emissions, the order is reversed. IRFF significantly increased the dissolved oxygen (DO), soil redox potential (Eh), total organic carbon (TOC) content, and soil C:N ratio, which is closely related to GHG emissions in rice fields. Additionally, the average emissions of carbon dioxide (CO₂) from soils during rice growing seasons ranged from 2312.27 to 2589.62 kg ha⁻¹ and showed no significant difference in the three treatments. Rice yield in the GIRF and OIRF were lower (2.0% and 16.7%) than the control. The CH₄ emissions contributed to 83.0–96.8% of global warming potential (GWP). Compared to CF, the treatment of GIRF and OIRF increased the GWP by 41.3% and 98.2% during the whole growing period of rice, respectively. IRFF significantly increased greenhouse gas intensity (GHGI, 0.79 kg CO₂-eq ha⁻¹ grain yield), by 91.1% over the control. Compared to the OIRF, GIRF decreased the GHGI by approximately 39.4% (0.59 kg CO₂-eq ha⁻¹ grain yield), which was 44.2% higher than that of the control. The results of structural equation model showed that the contribution of fertilization to CH₄ emissions in paddy fields was much greater than that of frog activity. Moreover, frog activity could decrease GWP by reducing CH₄ emissions from rice fields. And while GIRF showed a slight increase in GHG emissions, it could still be considered as a good strategy for providing an environmentally-friendly option in maintaining crop yield in paddy fields.

Identification of the carbon (C) sources of methane (CH₄) and methanogenic community structures after organic fertilization may provide a better understanding of the mechanism that regulate CH₄ emissions from paddy soils. Based on our previous field study, a pot experiment with isotopic ¹³C labelling was designed in this study. The objective was to investigate the main C sources for CH₄ emissions and the key environmental factor with the application of organic fertilizer in paddies. Results indicated that 28.6%, 64.5%, 0.4%, and 6.5% of ¹³C was respectively distributed in CO₂, the plants, soil, and CH₄ at the rice tillering stage. In total, organically fertilized paddy soil emitted 3.51 kg·CH₄ ha⁻¹ vs. 2.00 kg·CH₄ ha⁻¹ for the no fertilizer treatment. Maximum CH₄ fluxes from organically fertilized (0.46 mg·m⁻²·h⁻¹) and non-fertilized (0.16 mg·m⁻²·h⁻¹) soils occurred on day 30 (tillering stage). The total percentage of CH₄ emissions derived from rice photosynthesis C was 49%, organic fertilizer C < 0.34%, and native soil C > 51%. Therefore, the increased CH₄ emissions from paddy soil after organic fertilization were mainly derived from native soil and photosynthesis. The 16S rRNA sequencing showed Methanosarcina (64%) was the dominant methanogen in paddy soil. Organic fertilization increased the relative abundance of Methanosarcina, especially in rhizosphere. Additionally, Methanosarcina sp. 795 and Methanosarcina sp. 1H1 co-occurred with Methanobrevibacter sp. AbM23, Methanoculleus sp. 25XMc2, Methanosaeta sp. HA, and Methanobacterium sp. MB1. The increased CH₄ fluxes and labile methanogenic community structure in organically fertilized rice soil were primarily due to the increased soil C, nitrogen, potassium, phosphate, and acetate. These results highlight the contributions of native soil- and photosynthesis-derived C in paddy soil CH₄ emissions, and provide basis for more complex investigations of the pathways involved in ecosystem CH₄ processes.

Rice–frog co-culture is an organic farming practice known to effectively reduce nitrogen (N) loss from paddy fields. Ammonia-oxidizing archaea (AOA) and bacteria (AOB) mediate the rate-limiting step of nitrification; however, their relative contributions to this process remain unclear in certain soil types. This study aimed to evaluate the effects of rice–frog co-culture on N leaching and the associated AOA and AOB communities. A two-year field experiment was conducted in Shanghai, comparing three cultivation patterns: conventional rice (CR), green rice–frog (GR) co-cultivation, and organic rice–frog co-cultivation (OR). Nitrogen leaching was monitored, soil properties were analyzed, and the abundance and community composition of AOA and AOB were assessed. Nitrogen leaching losses were significantly lower in GR and OR than in CR, by 14.82% and 28.45%, respectively. Ammonium (NH₄⁺) was the predominant form of leached N, accounting for 58.10%, 56.20%, and 49.02% of the total N in CR, GR, and OR, respectively. Rice–frog co-culture significantly increased amoA gene copy numbers of AOA but suppressed those of AOB. The community structures of both AOA and AOB were strongly influenced by soil properties, including NH₄⁺, soil organic carbon (SOC), total nitrogen (TN), and pH. Specifically, the AOA community was primarily shaped by NH₄⁺ and SOC, whereas the AOB community was mainly driven by SOC content. These findings indicate that integrated rice–frog co-cultivation systems mitigate nitrogen leaching losses, modify soil physicochemical properties, and shift the abundance and community composition of ammonia-oxidizing archaea and bacteria.