Azole resistance mechanisms and population structure of the human pathogen Aspergillus fumigatus on retail plant products.

Aspergillus fumigatus is a ubiquitous saprotroph and human-pathogenic fungus that is life-threatening to the immunocompromised. Triazole-resistant A. fumigatus was found in patients without prior treatment with azoles, leading researchers to conclude that resistance had developed in agricultural environments where azoles are used against plant pathogens. Previous studies have documented azole-resistant A. fumigatus across agricultural environments, but few have looked at retail plant products. Our objectives were to determine if azole-resistant A. fumigatus is prevalent in retail plant products produced in the United States (U.S.), as well as to identify the resistance mechanism(s) and population genetic structure of these isolates. Five hundred twenty-five isolates were collected from retail plant products and screened for azole resistance. Twenty-four isolates collected from compost, soil, flower bulbs, and raw peanuts were pan-azole resistant. These isolates had the TR34/L98H, TR46/Y121F/T289A, G448S, and H147Y cyp51A alleles, all known to underly pan-azole resistance, as well as WT alleles, suggesting that non-cyp51A mechanisms contribute to pan-azole resistance in these isolates. Minimum spanning networks showed two lineages containing isolates with TR alleles or the F46Y/M172V/E427K allele, and discriminant analysis of principle components identified three primary clusters. This is consistent with previous studies detecting three clades of A. fumigatus and identifying pan-azole-resistant isolates with TR alleles in a single clade. We found pan-azole resistance in U.S. retail plant products, particularly compost and flower bulbs, which indicates a risk of exposure to these products for susceptible populations and that highly resistant isolates are likely distributed worldwide on these products.IMPORTANCEAspergillus fumigatus has recently been designated as a critical fungal pathogen by the World Health Organization. It is most deadly to people with compromised immune systems, and with the emergence of antifungal resistance to multiple azole drugs, this disease carries a nearly 100% fatality rate without treatment or if isolates are resistant to the drugs used to treat the disease. It is important to determine the relatedness and origins of resistant A. fumigatus isolates in the environment, including plant-based retail products, so that factors promoting the development and propagation of resistant isolates can be identified.

Key Challenges in Plant Pathology in the Next Decade.

Plant diseases significantly impact food security and food safety. It was estimated that food production needs to increase by 50% to feed the projected 9.3 billion people by 2050. Yet, plant pathogens and pests are documented to cause up to 40% yield losses in major crops, including maize, rice, and wheat, resulting in annual worldwide economic losses of approximately US$220 billion. Yield losses due to plant diseases and pests are estimated to be 21.5% (10.1 to 28.1%) in wheat, 30.3% (24.6 to 40.9%) in rice, and 22.6% (19.5 to 41.4%) in maize. In March 2023, The American Phytopathological Society (APS) conducted a survey to identify and rank key challenges in plant pathology in the next decade. Phytopathology subsequently invited papers that address those key challenges in plant pathology, and these were published as a special issue. The key challenges identified include climate change effect on the disease triangle and outbreaks, plant disease resistance mechanisms and its applications, and specific diseases including those caused by Candidatus Liberibacter spp. and Xylella fastidiosa. Additionally, disease detection, natural and man-made disasters, and plant disease control strategies were explored in issue articles. Finally, aspects of open access and how to publish articles to maximize the Findability, Accessibility, Interoperability, and Reuse of digital assets in plant pathology were described. Only by identifying the challenges and tracking progress in developing solutions for them will we be able to resolve the issues in plant pathology and ultimately ensure plant health, food security, and food safety.

Azole-resistant Aspergillus fumigatus in the environment: Identifying key reservoirs and hotspots of antifungal resistance.

Aspergillus fumigatus is an opportunistic human pathogen that causes aspergillosis, a spectrum of environmentally acquired respiratory illnesses. It has a cosmopolitan distribution and exists in the environment as a saprotroph on decaying plant matter. Azoles, which target Cyp51A in the ergosterol synthesis pathway, are the primary class of drugs used to treat aspergillosis. Azoles are also used to combat plant pathogenic fungi. Recently, an increasing number of azole-naive patients have presented with pan-azole-resistant strains of A. fumigatus. The TR34/L98H and TR46/Y121F/T289A alleles in the cyp51A gene are the most common ones conferring pan-azole resistance. There is evidence that these mutations arose in agricultural settings; therefore, numerous studies have been conducted to identify azole resistance in environmental A. fumigatus and to determine where resistance is developing in the environment. Here, we summarize the global occurrence of azole-resistant A. fumigatus in the environment based on available literature. Additionally, we have created an interactive world map showing where resistant isolates have been detected and include information on the specific alleles identified, environmental settings, and azole fungicide use. Azole-resistant A. fumigatus has been found on every continent, except for Antarctica, with the highest number of reports from Europe. Developed environments, specifically hospitals and gardens, were the most common settings where azole-resistant A. fumigatus was detected, followed by soils sampled from agricultural settings. The TR34/L98H resistance allele was the most common in all regions except South America where the TR46/Y121F/T289A allele was the most common. A major consideration in interpreting this survey of the literature is sampling bias; regions and environments that have been extensively sampled are more likely to show greater azole resistance even though resistance could be more prevalent in areas that are under-sampled or not sampled at all. Increased surveillance to pinpoint reservoirs, as well as antifungal stewardship, is needed to preserve this class of antifungals for crop protection and human health.

Population Genomic- and Phylogenomic-Enabled Advances to Increase Insight Into Pathogen Biology and Epidemiology.

Population genetics has been a key discipline in phytopathology for many years. The recent rise in cost-effective, high-throughput DNA sequencing technologies, allows sequencing of dozens, if not hundreds of specimens, turning population genetics into population genomics and opening up new, exciting opportunities as described in this Focus Issue. Without the limitations of genetic markers and the availability of whole or near whole-genome data, population genomics can give new insights into the biology, evolution and adaptation, and dissemination patterns of plant-associated microbes.

Assessing Fitness Costs and Phenotypic Instability of Fentin Hydroxide and Tebuconazole Resistance in Venturia effusa.

Sensitivity monitoring of Venturia effusa, cause of pecan scab, has revealed insensitivity to fentin hydroxide and tebuconazole, but recent research indicates that the insensitivity to fentin hydroxide is not stable. A study was undertaken to determine if a fitness cost may be responsible for this instability. In this study, experiments were conducted to evaluate fitness components and phenotypic stability of insensitivity of V. effusa to fentin hydroxide and tebuconazole. Conidial production, conidial germination, microcolony growth, sensitivity to osmotic stress, and sensitivity to oxidative stress in the absence of fungicide were compared for isolates with differing sensitivities to both fungicides. Percent conidial germination decreased linearly with increasing fentin hydroxide insensitivity, and microcolony growth on 1.0 mM H2O2 decreased linearly with increasing tebuconazole insensitivity. Stability of resistance was assessed on concentrations of 1.0, 3.0, and 10 µg/ml of both fungicides prior to and after five transfers on non-fungicide-amended medium. Tebuconazole insensitivity was stable after transfers, but fentin hydroxide insensitivity on 1.0 and 3.0 µg/ml decreased significantly after transfers, indicating instability. Here we provide evidence that in V. effusa tebuconazole insensitivity is stable and fentin hydroxide insensitivity is not. These results suggest that fentin-hydroxide-resistant V. effusa isolates have reduced conidial viability compared with sensitive isolates, which may allow the population to regain sensitivity in the absence of this frequently used fungicide.

Fusarium wilt of cotton may commonly result from the interaction of Fusarium oxysporum f. sp. vasinfectum with Belonolaimus longicaudatus.

The interaction between Fusarium oxysporum f. sp. vasinfectum (Fov) and Meloidogyne incognita (root-knot nematode) resulting in Fusarium wilt (FW) of cotton is well-known. Although Belonolaimus longicaudatus (sting nematode) can also interact with Fov and cause FW, it has long been believed that virtually all of the FW in Georgia is caused by the interaction of Fov with M. incognita. In recent years, FW has been reported more frequently in Georgia, which suggests that something affecting the disease complex may have changed. In 2015 and 2016, a survey of 27 Georgia cotton fields in 10 counties was conducted. At least 10 soil and stem samples per field were collected from individual plants showing symptoms of FW to quantify plant-parasitic nematode levels and identify Fov races. Fov race 1 was identified in all samples in 2015, but one sample also had the LA110 genotype and another sample also had the LA108 genotype. In 2016, all Fov races and genotypes found in 2015 were present, however, MDS-12 and LA127/140 also were found. Meloidogyne incognita was present in 18% of fields in 2015 and 40% in 2016, whereas B. longicaudatus was present in all fields in 2015 and 75% of fields in 2016. Regardless of whether they occurred separately or together, M. incognita and B. longicaudatus were present, respectively, in 18% and 55% of individual samples in 2015 and 40% and 51% in 2016. However, M. incognita without B. longicaudatus was found in 7% of samples in 2015 and 34% in 2016, whereas B. longicaudatus without M. incognita was found in 45% of samples in 2015 and 44% in 2016. We conclude that Fov race 1 continues to be the dominant race in Georgia and many instances of FW in Georgia may be due to Fov interacting with B. longicaudatus and not M. incognita as previously believed.The interaction between Fusarium oxysporum f. sp. vasinfectum (Fov) and Meloidogyne incognita (root-knot nematode) resulting in Fusarium wilt (FW) of cotton is well-known. Although Belonolaimus longicaudatus (sting nematode) can also interact with Fov and cause FW, it has long been believed that virtually all of the FW in Georgia is caused by the interaction of Fov with M. incognita. In recent years, FW has been reported more frequently in Georgia, which suggests that something affecting the disease complex may have changed. In 2015 and 2016, a survey of 27 Georgia cotton fields in 10 counties was conducted. At least 10 soil and stem samples per field were collected from individual plants showing symptoms of FW to quantify plant-parasitic nematode levels and identify Fov races. Fov race 1 was identified in all samples in 2015, but one sample also had the LA110 genotype and another sample also had the LA108 genotype. In 2016, all Fov races and genotypes found in 2015 were present, however, MDS­12 and LA127/140 also were found. Meloidogyne incognita was present in 18% of fields in 2015 and 40% in 2016, whereas B. longicaudatus was present in all fields in 2015 and 75% of fields in 2016. Regardless of whether they occurred separately or together, M. incognita and B. longicaudatus were present, respectively, in 18% and 55% of individual samples in 2015 and 40% and 51% in 2016. However, M. incognita without B. longicaudatus was found in 7% of samples in 2015 and 34% in 2016, whereas B. longicaudatus without M. incognita was found in 45% of samples in 2015 and 44% in 2016. We conclude that Fov race 1 continues to be the dominant race in Georgia and many instances of FW in Georgia may be due to Fov interacting with B. longicaudatus and not M. incognita as previously believed.

Phylogenetic Diversity and Host Specialization of Corynespora cassiicola Responsible for Emerging Target Spot Disease of Cotton and Other Crops in the Southeastern United States.

Corynespora cassiicola is a ubiquitous fungus causing emerging plant diseases worldwide, including target spot of cotton, soybean, and tomato, which have rapidly increased in incidence and severity throughout the southeastern United States. The objectives of this study were to understand the causes for the emerging target spot epidemics in the United States by comparing phylogenetic relationships of isolates from cotton, tomato, soybean, and other crop plants and ornamental hosts, and through the determination of the host range of isolates from emerging populations. Fifty-three isolates were sampled from plants in the southeastern United States and 1,380 nucleotides from four nuclear loci were sequenced. Additionally, sequences of the same loci from 23 isolates representing each of the distinct lineages of C. cassiicola described from previous studies were included. Isolates clustered based on host of origin, regardless of the geographic location of sampling. There was no genetic diversity detected among isolates from cotton, which were genetically distinct from isolates from other host species. Furthermore, pathogenicity and virulence assays of 40 isolates from various hosts onto cotton, soybean, tomato, and cucumber showed that isolates from cotton were more aggressive to cotton than those from other hosts. Soybean and tomato were most susceptible to isolates that originated from the same host, providing evidence of host specialization. These results suggest that emerging target spot epidemics in the United States are caused by either the introduction of host-specific isolates or the evolution of more aggressive strains on each host.

Spatial Genetic Structure and Population Dynamics of Gummy Stem Blight Fungi Within and Among Watermelon Fields in the Southeastern United States.

The epidemiology of gummy stem blight (GSB) of cucurbits, particularly the sources of inoculum for epidemics, and the regional population genetic structure of the causal fungi Stagonosporopsis cucurbitacearum (syn. Didymella bryoniae), S. citrulli, and S. caricae are not well understood. Our goal was to better understand the population structure and fine-scale spatial genetic structure of Stagonosporopsis spp. in the southeastern United States. Overall, 528 isolates collected from nine fields in 2012, 2013, and 2014 were genotyped with 16 microsatellite markers. In 2013, S. caricae was first detected in the southeastern United States; however, S. citrulli remained the dominant species, representing 96.4% of the isolates. Principal coordinates analysis, discriminant analysis of principle components, and analysis of molecular variance indicated that most populations of S. citrulli were genotypically diverse, yet dominated by widely distributed clones that contributed to regional population structure. Spatial genetic structure resulting from aggregation of clonal genotypes at distances of less than 10 meters was detected within two of three fields in which isolate location was recorded. Studies on the epidemiological and fitness differences between S. citrulli and S. caricae and of prevalent and widespread clones will provide insight into the population structure and species dynamics observed in GSB epidemics.

Differences in Sensitivity to a Triazole Fungicide Among Stagonosporopsis Species Causing Gummy Stem Blight of Cucurbits.

Gummy stem blight (GSB) is a destructive disease of cucurbits caused by three closely related Stagonosporopsis species. In the southeastern United States, GSB management relies heavily on triazole fungicides. Our objectives were to determine if resistance to triazoles has developed in populations of GSB fungi in the southeastern United States, and if so, to investigate the molecular basis of resistance. A tebuconazole sensitivity assay was conducted on 303 Stagonosporopsis citrulli and 19 S. caricae isolates collected from the southeastern United States in 2013 and 2014, as well as three S. citrulli, three S. cucurbitacearum, and six S. caricae isolates from other regions or years. Tebuconazole resistance was detected for all 19 S. caricae isolates from the southeastern United States and one S. caricae isolate from Brazil. All S. citrulli and S. cucurbitacearum isolates were sensitive to tebuconazole. For resistant and sensitive isolates of S. caricae, coding and promoter regions of the target gene Cyp51 were sequenced and expression levels of Cyp51 and ScAtrG (an ATP-binding cassette transporter) were measured. Tebuconazole resistance was not associated with mutations within Cyp51, multiple copies of Cyp51, changes in the promoter region, or increased expression of Cyp51 or ScAtrG. Tebuconazole resistance may explain the increase in frequency of S. caricae isolates recovered from GSB-infected cucurbits in Georgia.

Genetic Diversity and Population Structure of Cucurbit Gummy Stem Blight Fungi Based on Microsatellite Markers.

Combining population genetics with epidemiology provides insight into the population biology of pathogens, which could lead to improved management of plant diseases. Gummy stem blight, caused by three closely related species of Stagonosporopsis-Stagonosporopsis cucurbitacearum (syn. Didymella bryoniae), S. citrulli, and S. caricae-is a devastating disease of cucurbits worldwide. Sources of inoculum for epidemics, mechanisms of dispersal, and the mating system of these species are not well understood. To improve our knowledge of gummy stem blight epidemiology, we developed 18 polymorphic microsatellite markers by combining microsatellite motif enrichment with next-generation sequencing. When tested on 46 isolates from diverse cucurbit hosts and regions, the markers were robust for the dominant and widely distributed S. citrulli. Within this species, we found no population structure based on broad-scale geographic region or host of origin. Using the microsatellites, a rapid polymerase chain reaction-based method was developed to distinguish the three morphologically similar species causing gummy stem blight. To better understand dispersal, reproduction, and fine-scale genetic diversity of S. citrulli within and among watermelon fields, 155 isolates from two field populations in Georgia, United States were genotyped with the 18 microsatellite loci. Although dominant and widespread clones were detected, we found relatively high genotypic diversity and recombinant genotypes consistent with outcrossing. Significant population genetic structure between the two field populations demonstrated that there is regional geographic structure and limited dispersal among fields. This study provides insight into the fine-scale genetic diversity and reproductive biology of the gummy stem blight pathogen S. citrulli in the field.

Exobasidium maculosum, a new species causing leaf and fruit spots on blueberry in the southeastern USA and its relationship with other Exobasidium spp. parasitic to blueberry and cranberry.

Exobasidium leaf and fruit spot of blueberry (Vaccinium section Cyanococcus) is an emerging disease that has rapidly increased in prevalence throughout the southeastern USA. To determine whether this disease is caused by a new species of Exobasidium, we studied the morphology and phylogenetic relationship of the causal fungus compared with other members of the genus, including the type species E. vaccinii and other species that parasitize blueberry and cranberry (V. macrocarpon). Both scanning electron microscopy and light microscopy were used for morphological characterization. For phylogenetic analyses, we sequenced the large subunit of the rDNA (LSU) from 10 isolates collected from leaf or fruit spots of rabbiteye blueberry (V. virgatum), highbush blueberry (V. corymbosum) and southern highbush blueberry (Vaccinium interspecific hybrid) from Georgia and North Carolina and six isolates from leaf spots of lowbush blueberry (V. angustifolium) from Maine and Nova Scotia, Canada. LSU was sequenced from isolates causing red leaf disease of lowbush blueberry and red leaf spot (E. rostrupii) and red shoot (E. perenne) of cranberry. In addition, LSU sequences from GenBank, including sequences with high similarity to the emerging parasite and from Exobasidium spp. parasitizing other Vaccinium spp. and related hosts, were obtained. All sequences were aligned and subjected to phylogenetic analyses. Results indicated that the emerging parasite in the southeastern USA differs morphologically and phylogenetically from other described species and is described herein as Exobasidium maculosum. Within the southeastern USA, clustering based on host species, host tissue type (leaf or fruit) or geographic region was not detected; however, leaf spot isolates from lowbush blueberry were genetically different and likely represent a unique species.

Temperature regulates the initiation of chasmothecia in powdery mildew of strawberry.

The formation of chasmothecia by the strawberry powdery mildew pathogen (Podosphaera aphanis) is widespread but often sporadic throughout the range of strawberry cultivation. In some production regions, notably in warmer climates, chasmothecia are reportedly rare. We confirmed that the pathogen is heterothallic, and that initiation of chasmothecia is not only dependent upon the presence of isolates of both mating types but also largely suppressed at temperatures >13°C. Compared with incubation at a constant temperature of 25°C, progressively more chasmothecia were initiated when temperatures were decreased to 13°C for progressively longer times. At lower temperatures, production of chasmothecia was associated with a decline in but not total cessation of conidial formation, and pairings of compatible isolates sporulated abundantly at 25°C. We developed mating-type markers specific to P. aphanis and used these to confirm the presence of both mating types in populations that had not yet initiated chasmothecia. The geographic discontinuity of chasmothecia production and the sporadic and seemingly unpredictable appearance of chasmothecia in P. aphanis are possibly due to the combined influence of heterothallism and suppression of chasmothecia formation by high temperatures.

Detection of azole-resistant Aspergillus fumigatus in the environment from air, plant debris, compost, and soil.

Aspergillus fumigatus is a ubiquitous fungus, a saprophyte of plants, and an opportunistic pathogen of humans. Azole fungicides are used in agriculture to control plant pathogens, and azoles are also used as a first line of treatment for aspergillosis. The continued exposure of A. fumigatus to azoles in the environment has likely led to azole resistance in the clinic where infections result in high levels of mortality. Pan-azole resistance in environmental isolates is most often associated with tandem-repeat (TR) mutations containing 34 or 46 nucleotides in the cyp51A gene. Because the rapid detection of resistance is important for public health, PCR-based techniques have been developed to detect TR mutations in clinical samples. We are interested in identifying agricultural environments conducive to resistance development, but environmental surveillance of resistance has focused on labor-intensive isolation of the fungus followed by screening for resistance. Our goal was to develop assays for the rapid detection of pan-azole-resistant A. fumigatus directly from air, plants, compost, and soil samples. To accomplish this, we optimized DNA extractions for air filters, soil, compost, and plant debris and standardized two nested-PCR assays targeting the TR mutations. Sensitivity and specificity of the assays were tested using A. fumigatus DNA from wild type and TR-based resistant isolates and with soil and air filters spiked with conidia of the same isolates. The nested-PCR assays were sensitive to 5 fg and specific to A. fumigatus without cross-reaction with DNA from other soil microorganisms. Environmental samples from agricultural settings in Georgia, USA were sampled and tested. The TR46 allele was recovered from 30% of samples, including air, soil and plant debris samples from compost, hibiscus and hemp. These assays allow rapid surveillance of resistant isolates directly from environmental samples improving our identification of hotspots of azole-resistant A. fumigatus.

Fine mapping of fw3.2 controlling fruit weight in tomato.

Tomato (Solanum lycopersicum) is an important crop in the Solanaceae family. One of the key traits selected during domestication is fruit mass which is controlled by many quantitative trait loci. The fruit weight locus fw3.2 is one of the major loci responsible for fruit mass in tomato. Identification of the underlying gene will improve our understanding of the molecular mechanism of fruit development while also providing insights into genes that were selected during domestication. We fine mapped fw3.2 to a 51.4-kb interval corresponding to a region comprising seven candidate genes. Gene action showed that the allele from cultivated tomato was additive to dominant in giving rise to an enlarged fruit. Fruit shape analysis indicated that fw3.2 primarily played a role in controlling fruit weight, with a minor effect on fruit shape. Gene expression and nucleotide diversity were investigated and the likelihood of the genes control fruit mass is discussed.

Linkage disequilibrium and spatial aggregation of genotypes in sexually reproducing populations of Erysiphe necator.

Random mating and recombination in heterothallic ascomycetes should result in high genotypic diversity, 1:1 mating-type ratios, and random associations of alleles, or linkage equilibrium, at different loci. To test for random mating in populations of the grape powdery mildew fungus Erysiphe necator, we sampled isolates from vineyards of Vitis vinifera in Burdett, NY (NY09) and Winchester, VA (VA09) at the end of the epidemic in fall 2009. We also sampled isolates from the same Winchester, VA vineyard in spring 2010 at the onset of the next epidemic. Isolates were genotyped for mating type and 11 microsatellite markers. In the spring sample, which originated from ascospore infections, nearly every isolate had a unique genotype. In contrast, fall populations were less diverse. In all, 9 of 45 total genotypes in VA09 were represented by two or more isolates; 3 of 40 total genotypes in NY09 were represented by two or more isolates, with 1 genotype represented by 20 isolates. After clone correction, mating-type ratios in the three populations did not deviate from 1:1. However, even with clone correction, we detected significant linkage disequilibrium (LD) in all populations. Mantel tests detected positive correlations between genetic and physical distances within vineyards. Spatial autocorrelation showed aggregations up to 42 and 3 m in VA09 and NY09, respectively. Spatial autocorrelation most likely results from short dispersal distances. Overall, these results suggest that spatial genetic aggregation and clonal genotypes that arise during the asexual phase of the epidemic contribute to persistent LD even though populations undergo sexual reproduction annually.

Comparative genomics of host-specialized populations of Corynespora cassiicola causing target spot epidemics in the southeastern United States.

Numerous plant-pathogenic fungi secrete necrotrophic effectors (syn. host-selective toxins) that are important determinants of pathogenicity and virulence in species that have a necrotrophic lifestyle. Corynespora cassiicola is a necrotrophic fungus causing emerging target spot epidemics in the southeastern United States (US). Previous studies revealed that populations of C. cassiicola from cotton, soybean, and tomato are clonal, host specialized and genetically distinct. Additionally, cassiicolin - the necrotrophic effector identified in some C. cassiicola isolates - is an important toxin for virulence on rubber. It is encoded by seven Cas gene variants. Our goal was to conduct comparative genomic analyses to identify variation among putative necrotrophic effector genes and to determine if lack of one of the mating-types explained clonal populations in C. cassiicola causing outbreaks in the southeastern US and the apparent absence of sexual reproduction worldwide. A total of 12 C. cassiicola genomes, with four each from isolates from tomato, soybean, and cotton, were sequenced using an Illumina Next Seq platform. Each genome was assembled de novo, compared with the reference genome from rubber, and searched for known Cas, and other gene clusters with homologs of secondary metabolites. Cas2 and/or Cas6 were present in isolates from soybean in the southeastern US, whereas Cas1 and Cas2 were present in isolates from cotton in the southeastern US. In addition, several toxin genes, including the T-toxin biosynthetic genes were present in all C. cassiicola from cotton, soybean, and tomato. The mating-type locus was identified in all of the sequenced genomes, with the MAT1-1 idiomorph present in all cotton isolates and the rubber isolate, whereas the MAT1-2 idiomorph was present in all soybean isolates. We developed a PCR-based marker for mating-type in C. cassiicola. Both mating types were present in isolates from tomato. Thus, C. cassiicola has both mating-types necessary for sexual reproduction, but the absence of both mating-types within soybean and cotton populations could explain clonality in these populations. Variation in necrotrophic effectors may underlie host specialization and disease emergence of target spot on cotton, soybean, and tomato in the southeastern US.

Analysis of Cyp51 protein sequences shows 4 major Cyp51 gene family groups across fungi.

Azole drugs target fungal sterol biosynthesis and are used to treat millions of human fungal infections each year. Resistance to azole drugs has emerged in multiple fungal pathogens including Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum, and Aspergillus fumigatus. The most well-studied resistance mechanism in A. fumigatus arises from missense mutations in the coding sequence combined with a tandem repeat in the promoter of cyp51A, which encodes a cytochrome P450 enzyme in the fungal sterol biosynthesis pathway. Filamentous members of Ascomycota such as A. fumigatus have either 1 or 2 of 3 Cyp51 paralogs (Cyp51A, Cyp51B, and Cyp51C). Most previous research in A. fumigatus has focused on Cyp51A due to its role in azole resistance. We used the A. fumigatus Cyp51A protein sequence as the query in database searches to identify Cyp51 proteins across fungi. We found 435 Cyp51 proteins in 295 species spanning from early-diverging fungi (Blastocladiomycota, Chytridiomycota, Zoopagomycota, and Mucormycota) to late-diverging fungi (Ascomycota and Basidiomycota). We found these sequences formed 4 major Cyp51 groups: Cyp51, Cyp51A, Cyp51B, and Cyp51C. Surprisingly, we found all filamentous Ascomycota had a Cyp51B paralog, while only 50% had a Cyp51A paralog. We created maximum likelihood trees to investigate the evolution of Cyp51 in fungi. Our results suggest Cyp51 is present in all fungi with 3 paralogs emerging in Pezizomycotina, including Cyp51C which appears to have diverged from the progenitor of the Cyp51A and Cyp51B groups.

Evidence for the agricultural origin of resistance to multiple antimicrobials in Aspergillus fumigatus, a fungal pathogen of humans.

Pathogen resistance to clinical antimicrobial agents is an urgent problem. The fungus Aspergillus fumigatus causes 300,000 life-threatening infections in susceptible humans annually. Azoles, which are widely used in both clinical and agricultural settings, are currently the most effective treatment, but resistance to clinical azoles is emerging worldwide. Here, we report the isolation and analysis of azole-sensitive and azole-resistant A. fumigatus from agricultural environments in the southeastern United States (USA) and show that the USA pan-azole-resistant isolates form a clade with pan-azole-resistant isolates from the United Kingdom, the Netherlands, and India. We show that several pan-azole-resistant isolates from agricultural settings in the USA and India also carry alleles with mutations conferring resistance to agricultural fungicides from the benzimidazole (MBC) and quinone outside inhibitor (QoI) classes. We further show that pan-azole-resistant A. fumigatus isolates from patients in clinical settings in the USA, India, and the Netherlands also carry alleles conferring resistance to MBC and QoI agricultural fungicides. The presence of markers for resistance to agricultural-use fungicides in clinical A. fumigatus isolates is strong evidence for an agricultural origin of pan-azole resistance in patients. The presence of multiple fungicide-resistance alleles in agricultural and clinical isolates further suggests that the unique genetics of the pan-azole-resistant clade enables the evolution and/or persistence of antimicrobial resistance mutations leading to the establishment of multifungicide-resistant isolates.

Identification and structure of the mating-type locus and development of PCR-based markers for mating type in powdery mildew fungi.

In ascomycetes, mating compatibility is regulated by the mating-type locus, MAT1. The objectives of this study were to identify and sequence genes at the MAT1 locus in the grape powdery mildew fungus, Erysiphe necator, to develop a PCR-based marker for determining mating type in E. necator, and to develop degenerate primers for amplification by PCR of conserved regions of mating-type idiomorphs in other powdery mildew fungi. We identified MAT1-2-1 of the MAT1-2 idiomorph in E. necator based on the homologous sequence in the genome of Blumeria graminis f. sp. hordei and we found MAT1-1-1 and MAT1-1-3 of the MAT1-1 idiomorph from transcriptome sequences of E. necator. We developed and applied a reliable PCR-based multiplex marker to confirm that genotype correlated with mating phenotype, which was determined by pairing with mating-type tester isolates. Additionally, we used the marker to genotype populations of E. necator from different Vitis spp. from throughout the USA. We found both mating types were present in all populations and mating-type ratios did not deviate from 1:1. The mating-type genes in E. necator are similar to those of other Leotiomycetes; however, the structure of the MAT1 locus in E. necator, like the MAT1-2 idiomorph of B. graminis, is markedly different from other ascomycetes in that it is greatly expanded and may contain a large amount of repetitive DNA. As a result, we were unable to amplify and sequence either idiomorph in its entirety. We designed degenerate primers that amplify conserved regions of MAT1-1 and MAT1-2 in E. necator, Podosphaera xanthii, Microsphaera syringae, and B. graminis, representing the major clades of the Erysiphales. These degenerate primers or sequences obtained in this study from these species can be used to identify and sequence MAT1 genes or design mating-type markers in other powdery mildew fungi as well.