Genomic Insights into a Multispecies Bacterial Pathogen Complex Driving Bacterial Blotch in White Button Mushrooms.

Bacterial blotch remains a major constraint to global white button mushroom (Agaricus bisporus) production, yet its etiological complexity has been underestimated. Through a genome-resolved, polyphasic approach applied to symptomatic mushrooms from two U.S. farms, we uncovered an unexpectedly diverse complex of Pseudomonas species driving blotch disease. Beyond classical pathogens (P. tolaasii, P. gingeri, P. agarici, and P. "reactans", P. yamanorum, Pseudomonas sp. NC02), our analyses revealed a striking prevalence of P. azotoformans, a species not previously associated with mushroom pathology, alongside P. pergaminensis, P. monsensis, P. tensinigenes, P. simiae, Pseudomonas sp. Irchel 3A7, Pseudomonas sp. REP124 and two putatively novel lineages. Comparative genomics demonstrated pronounced heterogeneity in accessory genome content, with P. azotoformans exhibiting exceptional genomic plasticity indicative of broad ecological adaptability. Secondary metabolite profiling and white-line assays further delineated species-specific chemotaxonomic signatures, underscoring the multifactorial nature of virulence. Collectively, this study provides the most comprehensive genomic and phenotypic characterization of blotch-associated Pseudomonas to date, overturning the long-held paradigm of a single dominant pathogen. By establishing that bacterial blotch is a regionally structured, multispecies disease complex, our findings redefine its epidemiology and lay the foundation for improved diagnostics, surveillance, and integrated management strategies in mushroom production systems. SIGNIFICANCE STATEMENTBacterial blotch disease has long been attributed to a narrow set of Pseudomonas species, shaping decades of diagnostic and management practices in mushroom production. Our study overturns this paradigm by revealing that blotch is caused by a highly diverse, regionally structured pathogen complex Pseudomonas species, including multiple species never before linked to mushrooms. Using a genome-resolved, polyphasic framework, we demonstrated that accessory genome expansion and secondary metabolite diversity collectively drive pathogenic variability. The unexpected dominance of Pseudomonas azotoformans, a species previously known only from plant and environmental contexts, signals an emerging threat with broad ecological implications. These findings redefine the epidemiology of blotch disease, establish a new foundation for genomic surveillance, and highlight the urgent need for integrated management strategies that account for multispecies pathogen dynamics. By bridging microbial ecology, comparative genomics, and applied plant pathology, this work provides actionable insights for improving food security and sustainability in one of the worlds most economically important specialty crops.