Genetic Diversity and Azole Resistance Among Natural Aspergillus fumigatus Populations in Yunnan, China.

The emergence and spread of azole resistance alleles in clinical and environmental isolates of Aspergillus fumigatus is a global human health concern and endangers the "One Health" approach in our fight against antifungal resistance (AFR) in this pathogen. A major challenge to combat AFR in A. fumigatus is the massive aerial dispersal ability of its asexual spores. Our recent fine-scale survey of greenhouse populations of A. fumigatus near Kunming, Yunnan, China, suggested that the use of azole fungicides for plant protection was likely a major driver of the high-frequency azole-resistant A. fumigatus (ARAF) in greenhouses. Here, we investigated the potential spread of those ARAF and the structure of geographic populations of A. fumigatus by analyzing 452 isolates from 19 geographic locations across Yunnan. We found lower frequencies of ARAF in these outdoor populations than those in greenhouses near Kunming, but there were abundant new alleles and new genotypes, including those associated with azole resistance, consistent with multiple independent origins of ARAF across Yunnan. Interestingly, among the four ecological niches, the sediments of a large lake near Kunming were found to have the highest frequency of ARAF (~ 43%). While most genetic variations were observed within the 19 local populations, statistically significant genetic differentiations were found between many subpopulations within Yunnan. Furthermore, similar to greenhouse populations, these outdoor populations of A. fumigatus in Yunnan were significantly different from those in other parts of the world. Our results call for increased attention to local and regional studies of this fungal pathogen to help develop targeted control strategies against ARAF.

Genetic structure and triazole resistance among Aspergillus fumigatus populations from remote and undeveloped regions in Eastern Himalaya.

Aspergillus fumigatus is a ubiquitous mold and a common human fungal pathogen. Recent molecular population genetic and epidemiological analyses have revealed evidence for long-distance gene flow and high genetic diversity within most local populations of A. fumigatus. However, little is known about the impact of regional landscape factors in shaping the population diversity patterns of this species. Here we sampled extensively and investigated the population structure of A. fumigatus from soils in the Three Parallel Rivers (TPR) region in Eastern Himalaya. This region is remote, undeveloped and sparsely populated, bordered by glaciated peaks more than 6,000 m above sea level, and contained three rivers separated by tall mountains over very short horizontal distances. A total of 358 A. fumigatus strains from 19 sites along the three rivers were isolated and analyzed at nine loci containing short tandem repeats. Our analyses revealed that mountain barriers, elevation differences, and drainage systems all contributed low but statistically significant genetic variations to the total A. fumigatus population in this region. We found abundant novel alleles and genotypes in the TPR population of A. fumigatus and significant genetic differentiation between this population and those from other parts of Yunnan and the globe. Surprisingly, despite limited human presence in this region, about 7% of the A. fumigatus isolates were resistant to at least one of the two medical triazoles commonly used for treating aspergillosis. Our results call for greater surveillance of this and other human fungal pathogens in the environment. IMPORTANCE The extreme habitat fragmentation and substantial environmental heterogeneity in the TPR region have long known to contribute to geographically shaped genetic structure and local adaptation in several plant and animal species. However, there have been limited studies of fungi in this region. Aspergillus fumigatus is a ubiquitous pathogen capable of long-distance dispersal and growth in diverse environments. In this study, using A. fumigatus as a model, we investigated how localized landscape features contribute to genetic variations in fungal populations. Our results revealed that elevation and drainage isolation rather than direct physical distances significantly impacted genetic exchange and diversity among the local A. fumigatus populations. Interestingly, within each local population, we found high allelic and genotypic diversities, and with evidence ~7% of all isolates being resistant to two medical triazoles, itraconazole and voriconazole. Given the high frequency of ARAF found in mostly natural soils of sparsely populated sites in the TPR region, close monitoring of their dynamics in nature and their effects on human health is needed.

Historical Differentiation and Recent Hybridization in Natural Populations of the Nematode-Trapping Fungus Arthrobotrys oligospora in China.

Maintaining the effects of nematode-trapping fungi (NTF) agents in order to control plant-parasitic nematodes (PPNs) in different ecological environments has been a major challenge in biological control applications. To achieve such an objective, it is important to understand how populations of the biocontrol agent NTF are geographically and ecologically structured. A previous study reported evidence for ecological adaptation in the model NTF species Arthrobotrys oligospora. However, their large-scale geographic structure, patterns of gene flow, their potential phenotypic diversification, and host specialization remain largely unknown. In this study, we developed a new panel of 20 polymorphic short tandem repeat (STR) markers and analyzed 239 isolates of A. oligospora from 19 geographic populations in China. In addition, DNA sequences at six nuclear gene loci and strain mating types (MAT) were obtained for these strains. Our analyses suggest historical divergence within the A. oligospora population in China. The genetically differentiated populations also showed phenotypic differences that may be related to their ecological adaptations. Interestingly, our analyses identified evidence for recent dispersion and hybridization among the historically subdivided geographic populations in nature. Together, our results indicate a changing population structure of A. oligospora in China and that care must be taken in selecting the appropriate strains as biocontrol agents that can effectively reproduce in agriculture soil while maintaining their nematode-trapping ability.

Extensive Genetic Diversity and Widespread Azole Resistance in Greenhouse Populations of Aspergillus fumigatus in Yunnan, China.

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.

Analyses of the Global Multilocus Genotypes of the Human Pathogenic Yeast Candida tropicalis.

Candida tropicalis is a globally distributed human pathogenic yeast, especially prevalent in tropical and sub-tropical regions. Over the last several decades, a large number of studies have been published on the genetic diversity and molecular epidemiology of C. tropicalis from different parts of the world. However, the global pattern of genetic variation remains largely unknown. Here we analyzed the published multilocus sequence data at six loci for 876 isolates from 16 countries representing five continents. Our results showed that 280 of the 2677 (10.5%) analyzed nucleotides were polymorphic, resulting in a mean of 82 (a range of 38-150) genotypes per locus and a total of 633 combined diploid sequence types (DSTs). Among these, 93 combined DSTs were shared by 336 strains, including 10 by strains from different continents. Analysis of Molecular Variance (AMOVA) showed that 89% of the observed genetic variations were found within regional and national populations while < 10% was due to among-country separations. Pairwise geographic population analyses showed overall low but statistically significant genetic differentiation between most geographic populations, with the Singaporean and Indian populations being the most distinct from other populations. However, the Mantel test showed no significant correlation between genetic distance and geographic distance among the geographic populations. Consistent with high genetic variation within and limited variations among geographic populations, results from STRUCTURE analyses showed that the 876 isolates could be grouped into 15 genetic clusters, with each cluster having a broad geographic distribution. Together, our results suggest frequent gene flows among certain regional, national, and continental populations of C. tropicalis, resulting in abundant regional and national genetic diversities of this important human fungal pathogen.

The complete mitochondrial genome of the nematode-trapping fungus Dactylellina haptotyla.

The complete mitochondrial genome of Dactylellina haptotyla was studied in this study. This mitogenome is a closed circular molecule of 146,101 bp in length with a GC content of 22.92%, including 14 protein-coding genes, 26 transfer RNA genes, 2 ribosomal RNA genes. Phylogenetic analyses based on sequences at the 14 concatenated mitochondrial protein-coding genes showed that D. haptotyla was closely related to Pyronema omphalodes.