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

1.Structured Abstract1.1 AbstractSoybean red crown rot, caused by the soil-borne fungus Calonectria ilicicola, causes substantial yield losses, but the response of the root-associated bacterial microbiome remains poorly understood. Here, we combined 16S rRNA gene sequencing, shotgun metagenomics, and single-cell genomics to characterize bacterial communities in soybean root-associated soils. 16S rRNA gene sequencing showed that diseased plants had rhizosphere and, more strikingly, rhizoplane microbiomes distinct from those of healthy plants, often with increased Enterobacterales. Shotgun metagenomics further revealed enrichment of genes associated with antibiotic resistance, particularly cationic antimicrobial peptide resistance, in diseased rhizoplane samples. Single-cell genomics recovered seven nonredundant Enterobacterales genomes and showed that plant pathogenicity-related genes were broadly distributed across these lineages. In contrast, dlt genes, which are associated with cationic antimicrobial peptide resistance, were detected only in the Enterobacterales lineages enriched in diseased rhizoplane soils. These results support a model in which soybean red crown rot is accompanied by microbiome restructuring and opportunistic enrichment of specific Enterobacterales lineages carrying putative cationic antimicrobial peptide resistance genes. More broadly, this study highlights the value of strain-resolved single-cell genomics for linking disease-associated community shifts to specific bacterial traits. 1.2 ImportanceUnderstanding crop disease requires resolving not only the primary pathogen but also the root-associated bacteria that respond to infection. Here, we used 16S rRNA gene sequencing, shotgun metagenomics, and single-cell genomics to examine the soybean rhizoplane microbiome under red crown rot. Diseased plants showed reproducible shifts in bacterial composition, including frequent enrichment of Enterobacterales and antimicrobial resistance-related functions. Strain-resolved genomes further revealed that the Enterobacterales lineages enriched in diseased rhizoplane soils specifically carried putative dlt-mediated resistance to cationic antimicrobial peptides, whereas general pathogenicity-related genes were broadly shared. These findings suggest that host defense-associated selection, rather than pathogenicity genes alone, may help shape disease-associated root microbiomes. This study demonstrates how single-cell genomics can uncover strain-level traits hidden within bulk community data and thereby clarify plant-pathogen-microbiome interactions.