Intensive rice cropping drives shifts in abundance, activity, and assembly of root-associated methanotrophic community

Abstract

Rice is a staple crop relevant to present and future human feeding. However, these agroecosystems significantly contribute to greenhouse gas methane emissions. In Uruguay, a traditional low-intensity, reduced-tillage rice system alternates annual rice crops with pastures for livestock. We hypothesize that rice crop intensification impacts aerobic methanotrophic communities associated with rice roots, which are crucial in mitigating methane emissions. The pmoA gene abundance, methane oxidation potential (MOP), and methanotrophic community composition by 16S rRNA gene Illumina MiSeq (V4 region) allowed us to determine the dynamics of these communities in bulk and rhizospheric soils from continuous rice (CR) and rice-pastures (RP) rotations throughout the crop cycle. Results showed that rice crop intensification significantly affected MOP and pmoA abundance in both compartments. The tillering stage showed the greatest pmoA abundance and MOP. Rhizospheric methanotrophic communities from the CR and RP systems at flowering differed greatly. While Methylocystis dominated rhizospheric CR soil, Methylocella predominated in those from RP rotation. Active rhizospheric methanotrophic communities at flowering detected by 13CH4 DNA-SIP were dominated by distinct Methylocystis-affiliated ASVs in both cropping systems. However, other active genera were differentially enriched in the two contrasting cropping systems. These results suggest aerobic methanotrophs could be a microbial guild sensitive to crop intensification.

Keywords: Methylocella; Stable Isotope Probing; pmoA gene; aerobic methane oxidation; crop rotations; intensification; rice rhizosphere.

Figure 1.

Dynamics of pmoA gene abundance in bulk (A) and rhizospheric (B) soils throughout the crop season. For each compartment, different letters indicate significant statistical differences (n = 3) in log₁₀  pmoA copies g of soil dw⁻¹ according to ANOVA and Tukey’s Test performed for bulk (P-value= 3.62×10⁻⁵) or rhizospheric soils (P-value= 1.95×10⁻⁷). CR: Continuous Rice. RP: Rice- Pastures rotation.

Figure 2.

Dynamics of MOP in bulk (A) and rhizospheric (B) soils throughout the crop season. According to ANOVA and Tukey’s test performed, a significant crop stage * crop rotation interaction was found for bulk (P-value= 0.00147) and rhizospheric soils (P-value= 0.00129). Different letters indicate statistically significant differences (n = 3). CR: Continuous Rice. RP: Rice-Pastures rotation.

Figure 3.

¹³CH₄ DNA-SIP experiment for rhizospheric soils from continuous rice (CR) and rice-pastures (RP) rotations at the flowering stage. (A) Methanotrophic enrichment, as pmoA relative abundance in each fraction, obtained across the different buoyant densities in CsCl gradient fractionation. (B) Bacterial community composition at the Class level (16S rRNA gene amplicon-sequencing Illumina MiSeq; relative abundances >5%) for heavy (H) and light (L) fractions obtained from ¹³CH₄ and control (¹²CH₄) slurry incubations. (C) Abundance of methanotrophic genera detected in heavy (H) fractions from ¹³CH₄ and control (¹²CH₄) slurry incubations.

Figure 4.

Active rhizospheric bacterial communities from continuous rice (CR) and rice-pastures (RP) rhizospheric soils at the flowering stage detected in heavy (H) fractions by ¹³CH₄ DNA-SIP and 16S rRNA gene amplicon-sequencing. Dominant active genera (relative abundance >5%) are shown.

Figure 5.

Total and active methanotrophic communities from continuous rice (CR) and rice-pastures (RP) rhizospheric soils at the flowering stage. Relative abundance heatmap of dominant methanotrophic genera (relative abundances >5%; mean values n=2) inhabiting original rhizospheric soils (Rh) and detected by ¹³CH₄ DNA-SIP in heavy fractions (H).

Figure 6.

Differential abundance analysis for active rhizospheric methanotrophic genera detected in heavy fractions of ¹³CH₄ slurry incubations for CR and rice-pastures (RP) at the flowering stage. Methanotrophic genera showing significant differential abundances (α=0.05) are shown at an ASV level. The log2 fold change shown was calculated by comparing CR with RP.