Genomic characterization of greenhouse-associated Aspergillus fumigatus from China reveals population structure, antifungal resistance, and relationships with global lineages

ABSTRACT Aspergillus fumigatus is a globally distributed saprophyte and a major opportunistic pathogen causing invasive aspergillosis. The emergence of azole-resistant A. fumigatus in agricultural environments poses a growing threat to human health, yet the genetic basis of resistance and the role of agricultural hotspots remain poorly understood. Here, we analyzed 226 greenhouse-derived isolates from Yunnan, China, within a global genomic framework. Although Yunnan isolates shared overall diversity patterns with worldwide populations, they exhibited pronounced population structure, with one well-supported clade (Clade B) enriched in greenhouse environments. To investigate cross-environment relationships, we further analyzed an expanded collection of isolates from greenhouse soils, air, agricultural markets, and clinical sources using multilocus genotyping. Identical or highly similar genotypes were detected across environments, including resistant strains carrying the TR₃₄/L98H allele, demonstrating that greenhouse and clinical isolates are genetically connected. Genome-wide association analyses identified candidate loci associated with antifungal resistance and stress tolerance beyond canonical cyp51A mutations, including genes involved in cell wall remodeling and mitochondrial dynamics. Together, these results reveal the genomic landscape of greenhouse-associated A. fumigatus in China and provide evidence for genetic connectivity between agricultural and clinical settings, underscoring the importance of surveillance at agricultural–clinical interfaces. IMPORTANCE Azole-resistant Aspergillus fumigatus is an increasing global health concern because resistant infections are difficult to treat and often fatal. Agricultural environments have been implicated as important sources of resistance, yet the mechanisms underlying the emergence, persistence, and transfer of resistance to human patients remain unclear. By integrating genomic and phenotypic analyses of greenhouse-derived isolates from Yunnan, China, we identified a distinct clade that is enriched in greenhouse environments and exhibits stable antifungal resistance. Resistant genotypes were repeatedly detected across greenhouses, local markets, and patients; notably, identical multilocus genotypes and cyp51A mutations were shared between market air samples and a patient with invasive aspergillosis, demonstrating genetic connectivity between agricultural and clinical settings. Beyond the well-known cyp51A mutations, genome-wide association analyses highlighted several candidate loci associated with antifungal resistance—including a dynamin-related GTPase and a strongly induced cell-wall peptidase—that may represent non-canonical resistance mechanisms. These findings provide insights into fungal adaptation and resistance evolution in agricultural contexts. They underscore the urgent need for integrated surveillance and stewardship across agricultural and medical sectors to mitigate the spread of antifungal resistance.