Single-cell genomics reveals opportunistic Enterobacterales carrying putative cationic antimicrobial peptide resistance genes in red crown rot-affected soybean rhizoplanes

The productivity of soybean, a globally important agricultural crop, is strongly influenced by soil- and root-associated biotic interactions, including those that shape disease outcomes. Soybean red crown rot, caused by the soil-borne fungus Calonectria ilicicola , leads to substantial yield losses worldwide. While the primary pathogen has been well characterized, disease incidence and severity can vary markedly even within a single field, suggesting that factors beyond the primary pathogen, including the root-associated bacterial microbiome, may influence disease development. However, how infection reshapes these bacterial communities remains poorly understood. We combined 16S ribosomal RNA gene sequencing, shotgun metagenomics, and strain-resolved single-cell genomics to characterize bacterial communities in soybean root-associated soils from healthy and diseased plants. Amplicon sequencing revealed that diseased plants harbored rhizosphere and, more strikingly, rhizoplane microbiomes that were compositionally distinct from those of healthy plants, frequently marked by enrichment of the bacterial order Enterobacterales . Shotgun metagenomics further revealed that diseased rhizoplane samples were enriched in genes associated with antibiotic resistance, particularly resistance to cationic antimicrobial peptides. To link these community-level shifts to specific bacterial lineages, we applied single-cell genomics, recovering seven nonredundant Enterobacterales genomes. While genes related to plant pathogenicity were broadly distributed across these genomes, dlt genes — which confer putative resistance to cationic antimicrobial peptides — were detected exclusively in the Enterobacterales lineages that were enriched in diseased rhizoplane soils. Our results demonstrate that soybean red crown rot is associated with restructuring of the rhizoplane microbiome and the selective enrichment of specific Enterobacterales lineages harboring putative cationic antimicrobial peptide resistance genes, although the magnitude of enrichment varied among individual plants. The selective enrichment of lineages carrying dlt genes, rather than lineages defined solely by pathogenicity-related genes, suggests that host defense-associated pressures may be a key driver shaping disease-associated root microbiomes. More broadly, this study demonstrates the value of strain-resolved single-cell genomics for uncovering bacterial traits that remain hidden in bulk community analyses, and highlights its potential for clarifying the complex interactions among plants, pathogens, and the root microbiome.