Aspergillus fumigatus is the main cause of invasive aspergillosis (IA) with a high annual global incidence and mortality rate. Recent studies have indicated an increasing prevalence of azole-resistant A. fumigatus (ARAF) strains, with agricultural use of azole fungicides as a potential contributor. China has an extensive agricultural production system and uses a wide array of fungicides for crop production, including in modern growth facilities such as greenhouses. Soils in greenhouses are among the most intensively cultivated. However, little is known about the occurrence and distribution of ARAF in greenhouse soils. Here, we investigated genetic variation and triazole drug susceptibility in A. fumigatus from greenhouses around metropolitan Kunming in Yunnan, southwest China. Abundant allelic and genotypic variations were found among 233 A. fumigatus strains isolated from nine greenhouses in this region. Significantly, ∼80% of the strains were resistant to at least one medical triazole drug, with >30% showing cross-resistance to both itraconazole and voriconazole. Several previously reported mutations associated with triazole resistance in the triazole target gene cyp51A were also found in our strains, with a strong positive correlation between the frequency of mutations at the cyp51A promoter and that of voriconazole resistance. Phylogenetic analyses of cyp51A gene sequences showed evidence for multiple independent origins of azole-resistant genotypes of A. fumigatus in these greenhouses. Evidence for multiple origins of azole resistance and the widespread distributions of genetically very diverse triazole-resistant strains of A. fumigatus in greenhouses calls for significant attention from public health agencies.IMPORTANCE The origin and prevalence of azole-resistant Aspergillus fumigatus have been attracting increasing attention from biologists, clinicians, and public health agencies. Current evidence suggests agricultural fungicide use as a major cause. In southwest China, greenhouses are used to produce large amounts of fruits, flowers, and vegetables for consumers throughout China as well as those in other countries, primarily in southeast Asia. Here, we found a very high frequency (∼80%) of triazole-resistant A. fumigatus in our sample, the highest reported so far, with a significant proportion of these strains resistant to both tested agricultural fungicides and medical triazole drugs. In addition, we found novel allelic and genotypic diversities and evidence for multiple independent origins of azole-resistant genotypes of A. fumigatus in greenhouse populations in this region. Our study calls for a systematic evaluation of the effects of azole fungicide usage in greenhouses on human health.

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