Comparative analysis in diagnosis by real-time polymerase chain reaction versus direct microscopy, culture, and histology in fungal infections of the nails, scalp, and skin.

BACKGROUND: The diagnosis of superficial fungal infections is the subject of intensive research in many countries around the world. The diagnostic methods used are diverse, including both conventional and innovative techniques. METHODS: This study evaluates the sensitivity, specificity, and efficacy of the real-time polymerase chain reaction (PCR) methodology and compares them with those of the conventional methods - direct microscopic, cultural, and histological examinations of materials from hair, skin, and nails - in order to demonstrate the benefits and significance of real-time PCR for the diagnosis of dermatophytic infections. RESULTS: The values obtained for the sensitivity, specificity, and efficacy of direct microscopic, cultural, histological, and real-time PCR studies are as follows: 63.71, 88.89, and 72.96% (P < 0.001); 58.06, 100, and 73.47% (P < 0.001); 85.96, 100, and 90.70% (P < 0.001); 88.52, 100, and 92.63% (P < 0.001). CONCLUSION: The use of real-time PCR in the diagnosis of dermatophytic infections is a relatively new approach in mycology and is subject to testing and experience from its use. The results are promising, but the method has not yet established itself as a new gold standard in the diagnosis of superficial fungal infections caused by dermatophytes, though its application would be very useful in identifying isolates without conidiogenesis or absence of growth.

Microbiological culture combined with PCR for the diagnosis of onychomycosis: Descriptive analysis of 121 patients.

BACKGROUND: Onychomycosis is the most common nail pathology, involving various pathogens such as dermatophytes, moulds and yeasts. OBJECTIVE: The objective of this study was to observe the prevalence of onychomycosis, analyse the most appropriate diagnostic test, and assess the distribution of pathogens based on age, sex, quarter of the year, duration of symptoms and previous treatment. METHODS: Retrospectively, mycological culture and PCR data and results were collected from 121 patients. RESULTS: Of the 121 samples, 57% (69/121) tested positive when both microbiological study techniques were combined. The prevalence of onychomycosis was higher when PCR was performed (52.1%) compared to microbiological culture (33.1%). Among the 81 samples negative by microbiological culture, 31 were positive by PCR. Similarly, of the 58 samples negative by PCR, eight were positive by microbiological culture. Diagnostic accuracy data (with 95% confidence intervals) for PCR, using microbiological culture as the gold standard, were as follows: sensitivity of 0.8, specificity of 0.62, positive predictive value of 0.51 and negative predictive value of 0.86. The most frequently identified pathogen was Trichophyton rubrum, and the hallux nail plate was the most commonly affected location. However, no statistically significant associations were found between sex, age, quarter of the year and affected area with culture and PCR results. CONCLUSION: Combining microbiological culture and PCR can increase the detection rate of onychomycosis and help avoid false-negative results.

Improved RAPD Method for Candida parapsilosis Fingerprinting.

Recently, methods based on the analysis of arbitrarily amplified target sites of genome microorganisms have been extensively applied in microbiological studies, and especially in epidemiological studies. The range of their application is limited by problems with discrimination and reproducibility resulting from a lack of standardized and reliable methods of optimization. The aim of this study was to obtain optimal parameters of the Random Amplified Polymorphic DNA (RAPD) reaction by using an orthogonal array as per the Taguchi and Wu protocol, modified by Cobb and Clark for Candida parapsilosis isolates. High Simpson's index values and low Dice coefficients obtained in this study indicated a high level of interspecies DNA polymorphism between C. parapsilosis strains, and the optimized RAPD method proved useful in the microbiological and epidemiological study.

Mucormycosis-causing fungi in humans: a meta-analysis establishing the phylogenetic relationships using internal transcribed spacer (ITS) sequences.

Introduction. Mucormycosis is a severe angio-invasive fungal infection caused by mucormycetes, a group of fungi that are ubiquitous in the environment. The incidence of mucormycosis has been surging rapidly due to the global corona virus disease 2019 (COVID-19) pandemic.Gap Statement. The complete picture of the causative fungi associated with mucormycosis and their phylogenetic relationships are not well defined.Aim. This meta-analysis aimed to collate all confirmed fungal pathogens that cause mucormycosis, and assess their taxonomic relationships.Methodology. All types of articles in the PubMed database that report fungi as a cause of mucormycosis were reviewed. We summarized the fungal morphological characteristic up to the genus level. The internal transcribed spacer (ITS) nucleotide sequences of these fungi were retrieved from the National Center for Biotechnology Information (NCBI) and UNITE databases whenever available, and multiple sequence analysis was conducted using Clustal W. The phylogenetic tree was constructed using mega version 7.Results. Forty-seven fungal species were identified as pathogens causing mucormycosis in humans. Thirty-two fungal species were phylogenetically grouped into three clades, and it was evident that the ITS sequences have well-conserved regions in all clades, especially from the 400th to 500th base pairs.Conclusion. The findings of this work contribute to the descriptive data for fungi that cause mucormycosis, emphasizing the need for robust phylogenetic approaches when identifying clinical isolates from infected patients.

Time and cost-efficient identification of Trichophyton indotineae.

BACKGROUND: During the past 5 years, an outbreak of recalcitrant dermatophytosis due to a novel Trichophyton species generally resistant to terbinafine, T. indotineae, has spread out from South Asia to many countries around the World. These isolates cannot be reliably differentiated from other Trichophyton spp. on the basis of morphological traits and the current laboratory diagnostics relies on sequencing of ribosomal DNA ITS region. OBJECTIVES: In this study, we aimed to introduce two inexpensive and rapid PCR-based assays for differentiation between T. indotineae and other dermatophytes. METHODS: The first introduced assay is based on PCR-restriction fragment length polymorphism (PCR-RFLP) analysis, involving the amplification of TOP2 sequences and the digestion of PCR products by Cfr13I restriction enzyme. The second assay is proposed as conventional endpoint species-specific PCR amplification of the C120-287 intergenic locus. To validate the assays, a total of 191 Trichophyton spp. and 2 Microsporum canis strains with known ITS region sequences were used. From the T. mentagrophytes / T. interdigitale species complex (TMTISC), strains with 18 different ITS genotypes were tested. The sample of TMTISC isolates included 41 T. indotineae strains. RESULTS: TOP2 PCR-RFLP and T. indotineae-specific PCR were positive with testing on DNA of all 41 T. indotineae isolates and two strains of T. mentagrophytes belonging to ITS Types XIII and XVI, but negative with other species and other TMTISC ITS genotypes (n = 152). Therefore, the specificity of both new assays was 99%. CONCLUSION: The two developed diagnostic assays provide accurate and cost-effective means of identifying cultured T. indotineae isolates.

Pan-cancer analyses reveal cancer-type-specific fungal ecologies and bacteriome interactions.

Cancer-microbe associations have been explored for centuries, but cancer-associated fungi have rarely been examined. Here, we comprehensively characterize the cancer mycobiome within 17,401 patient tissue, blood, and plasma samples across 35 cancer types in four independent cohorts. We report fungal DNA and cells at low abundances across many major human cancers, with differences in community compositions that differ among cancer types, even when accounting for technical background. Fungal histological staining of tissue microarrays supported intratumoral presence and frequent spatial association with cancer cells and macrophages. Comparing intratumoral fungal communities with matched bacteriomes and immunomes revealed co-occurring bi-domain ecologies, often with permissive, rather than competitive, microenvironments and distinct immune responses. Clinically focused assessments suggested prognostic and diagnostic capacities of the tissue and plasma mycobiomes, even in stage I cancers, and synergistic predictive performance with bacteriomes.

Evaluation of different DNA extraction methods based on steel-bullet beating for molecular diagnosis of onychomycosis.

BACKGROUND: Considering increased trends toward molecular methods for detection/identification of fungi causing onychomycosis, the aim of this study is comparison three DNA extraction methods based on steel-bullet beating to extract DNA from nail. METHODS: Ex -vivo onychomycosis model was developed using bovine hoof with Candida albicans and Aspergillus flavus. For two models, total DNA was extracted using the three different methods. In method 1, the extraction and purification were performed by steel-bullet beating and phenol chloroform protocol, respectively. In method 2, a freezing step were applied before beating. The purification step in method 3 was carried out using a commercial kit, although DNA extraction was done similarly to method 1 in that approach. To evaluate the efficacy of each method, the extracted genomic DNA was amplified with Polymerase Chain Reaction (PCR) using Internal Transcribed Spacer (ITS) regions. Moreover, 50 nail samples were evaluated for onychomycosis using direct microscopy examination as well as PCR in order to evaluate the diagnostic efficiency of the optimal DNA extraction method. RESULTS: Regarding the desirable quality of the extracted DNA, cost effectiveness, and simplicity, method 1 could be used to extract DNA effectively. Additionally, the obtained data showed that PCR had a higher detection rate of fungal agents in the nail samples than direct microscopic examination. CONCLUSIONS: This study demonstrated that the mechanical disruption of the cell wall by steel-bullet beating is a useful and practical method to improve the quantity and quality of fungal DNA thorough the extraction process.

Toxigenicity of F. graminearum Residing on Host Plants Alternative to Wheat as Influenced by Environmental Conditions.

Fusarium graminearum is an important pathogen that causes Fusarium head blight (FHB) in several cereal crops worldwide. The potential of this pathogen to contaminate cereals with trichothecene mycotoxins presents a health risk for both humans and animals. This study aimed to evaluate the potential of different trichothecene genotypes of F. graminearum isolated from an alternative host plant to produce mycotoxins under different spring wheat grain incubation conditions. Fourteen F. graminearum strains were isolated from seven alternative host plants and identified as 3-acetyl-deoxynivalenol (3-ADON) and 15-acetyl-deoxynivalenol (15-ADON) genotypes. These strains were cultivated on spring wheat grains at 25 °C and 29 °C for 5 weeks. The mycotoxins produced were analysed with a high-performance liquid chromatograph (HPLC) coupled to a Thermo Scientific TSQ Quantiva MS/MS detector. The obtained results showed that the F. graminearum strains from alternative host plants could produce nivalenol (NIV), deoxynivalenol (DON), fusarenon-X (FUS-X), 3-ADON, deoxynivalenol-3-ß-d-glucoside (D3G), 15-ADON, and zearalenone (ZEA). F. graminearum strains produced DON and ZEA under both temperatures, with the mean concentrations varying from 363 to 112,379 µg kg-1 and from 1452 to 44,816 µg kg-1, respectively. Our results indicated the possible role of dicotyledonous plants, including weeds, as a reservoir of inoculum sources of F. graminearum-induced Fusarium head blight, associated with the risk of mycotoxin contamination in spring wheat.

Genotypes analysis of Candida albicans species complex from healthy individual saliva in Ahvaz, Iran.

Although Candida albicans is a part of the mycoflora of healthy individuals, it is causing mild to severe forms of candidiasis in patients with underlying diseases. The recent molecular investigations were classified three genotypes, A, B and C for C. albicans. The aim of the present study was to detect different genotypes of C. albicans complex species in a normal population in Iran. Saliva was randomly collected from a normal population, homogenized and cultured on CHROMagar Candida. Classical and molecular methods were used for the detection of isolates. Candida 25S rDNA gene was amplified using the primer pairs of CA-INT-L and CA-INT-R for ABC genotyping of C. albicans. Candida albicans complex was recovered from 103/194 (53·1%) normal papulation. Genotype A with a frequency of 41·7% was the most common isolate, followed by genotype C (34%), genotype B (20·4%) and genotype D (3·9%). Genotyping of C. albicans species complex from healthy individuals showed the presence of three A, B and C genotypes of C. albicans and one D genotype of C. dubliniensis.

Optimization of a Quantitative PCR Methodology for Detection of Aspergillus spp. and Rhizopus arrhizus.

INTRODUCTION: Multiplex quantitative polymerase chain reaction (qPCR) methods for the detection of Aspergillus spp. based only on SYBR Green and melting curve analysis of PCR products are difficult to develop because most targets are located within ITS regions. The aim of this study was to adapt our previously developed methodology based on a multiplex PCR assay coupled with GeneScan analysis to provide a qPCR method. METHODS: A SYBR Green-based real-time PCR assay was optimized to detect A. fumigatus, A. flavus, A. niger, A. terreus, and R. arrhizus in a multiplex assay and applied to cultured fungi and spiked plasma. RESULTS: Different melting temperatures allowed identification of all five pathogens and discrimination between them, even in samples with low amounts of fungal gDNA (from 1.3 to 33.0 pg/µL), which has been reported previously as problematic. No false-positive results were obtained for non-target species, including bacteria and human DNA. This method allowed detection of fungal pathogens in human plasma spiked with fungal DNA and in coinfections of A. niger/R. arrhizus. DISCUSSION: This work provides evidence for the use of a qPCR multiplex method based on SYBR Green and melting curve analysis of PCR products for the detection of A. fumigatus, A. flavus, A. niger, A. terreus, and R. arrhizus. The proposed method is simpler and less expensive than available kits based on fluorescent probes and can be used for aiding diagnosis of the most relevant invasive filamentous fungi, particularly in low-income health care institutions.

DNA SEQUENCING OF NOVEL YEAST ISOLATED FROM BLOODSTREAM INFECTIONS IN AL-NAJAF PROVINCE.

OBJECTIVE: The aim: To identify of fungal isolates using PCR techniques with universal primer (ITS1-ITS4 and ITS3-ITS4). A total of 533 blood samples from cancer patients, renal failure patients and patients who underwent cardiac catheterization have been included in this study. PATIENTS AND METHODS: Materials and methods: Devices and tools were used to preceding the study as shown in table (I), while biological and chemical materials are given in table (II). RESULTS: Results: Three groups, 44 isolates of Candida spp were isolated: 35(79.6%) isolates from cancer patients, 6(13.6%) isolates from patients with renal failure, and 3 (6.8%) isolates from patients with cardiac catheterization. These yeasts were diagnosed by conventional methods and by CHROMagar Candida medium, as well as by molecular methods to detect the regions of ITS2, ITS1, and the isolates were diagnosed as belonging to the yeast Candida spp. These isolates were also diagnosed using DNA sequencing detection technology and 12 new strains were recorded for the first time In the name of the researcher by the Japan Gene Bank. CONCLUSION: Conclusions: It was concluded that there was high susceptibility of the polymerase chain reaction technique based on ITS 1, ITS2 primers in diagnosing the types of yeasts isolated from the bloodstream with high accuracy and speed compared to other traditional methods. Therefore, the DNA sequencing method is considered one of the best rapid standard methods for the diagnosis of fungi.

A Quantitative PCR Assay for Detection and Quantification of Fusarium sambucinum.

Fusarium sambucinum is an ascomycete that has been isolated from a broad range of plant hosts, including hop (Humulus lupulus L.), where it acts as a causal agent of Fusarium canker, a disease that can impact cone quality and yield in severe cases. Current diagnostic methods rely on isolation of the fungus from plant tissue, a time- and resource-intensive process with limited sensitivity, complicated by the potential presence of other Fusarium spp. that have been reported on hop. Our objective was to develop a rapid and sensitive diagnostic tool to detect and quantify F. sambucinum in plant tissues. Using a modified random amplified polymorphic DNA PCR assay, we identified a F. sambucinum-specific marker that serves as the target in a TaqMan (hydrolysis) probe quantitative PCR (qPCR) assay that can be used to detect F. sambucinum DNA in a background of plant DNA. When used to screen 52 isolates of F. sambucinum and isolates representing 13 other Fusarium spp., the assay was robust in detecting F. sambucinum while discriminating between F. sambucinum and closely related Fusarium spp., including F. venenatum. Furthermore, this assay reliably detects as little as 1 pg of F. sambucinum DNA in a background of total DNA from plant tissue. Within-sample comparisons of this qPCR assay with traditional cultural isolation methods demonstrated the greater sensitivity of the qPCR-based method for detection of F. sambucinum. When used to screen 220 asymptomatic stem samples, the qPCR assay detected F. sambucinum in 100 samples (45.5%); by comparison, F. sambucinum was detected in only 3 samples (1.4%) by culturing methods. Moreover, quantification of F. sambucinum DNA was possible for 60 of these samples, indicating the utility of the qPCR assay for early detection. This assay should be useful in diagnostic and epidemiological applications to detect and quantify F. sambucinum from multiple hosts and environmental samples.

Detection of multiple mycetoma pathogens using fungal metabarcoding analysis of soil DNA in an endemic area of Sudan.

Mycetoma is a tropical disease caused by several fungi and bacteria present in the soil. Fungal mycetoma and eumycetoma are especially challenging to treat; therefore, prevention, early diagnosis, and early treatment are important, but it is also necessary to understand the geographic distribution of these pathogenic fungi. In this study, we used DNA metabarcoding methodology to identify fungal species from soil samples. Soil sampling was implemented at seven villages in an endemic area of Sennar State in Sudan in 2019, and ten sampling sites were selected in each village according to land-use conditions. In total, 70 soil samples were collected from ground surfaces, and DNA in the soil was extracted with a combined method of alkaline DNA extraction and a commercial soil DNA extraction kit. The region for universal primers was selected to be the ribosomal internal transcribed spacer one region for metabarcoding. After the second PCR for DNA library preparation, the amplicon-based DNA analysis was performed using next-generation sequencing with two sets of universal primers. A total of twelve mycetoma-causative fungal species were identified, including the prime agent, Madurella mycetomatis, and additional pathogens, Falciformispora senegalensis and Falciformispora tompkinsii, in 53 soil samples. This study demonstrated that soil DNA metabarcoding can elucidate the presence of multiple mycetoma-causative fungi, which may contribute to accurate diagnosis for patient treatment and geographical mapping.

Prevalence of Fusarium fungi and Deoxynivalenol Levels in Winter Wheat Grain in Different Climatic Regions of Poland.

Fusarium head blight (FHB) caused by fungi of the genus Fusarium is one of the most dangerous crop diseases, which has a wide geographic distribution and causes severe economic losses in the production of major cereal species. The infection leads to the accumulation of mycotoxins in grains, which compromises its suitability for human and animal consumption. The study demonstrated that grain samples from warmer regions of Poland, including Sulejów and Tomaszów Boleslawicki (results differed across years of the study), were colonized mainly by F. graminearum and were most highly contaminated with deoxynivalenol (DON). Samples from Northeastern Poland, i.e., Ruska Wies, which is located in a cooler region, were characterized by a predominance of Fusarium species typical of the cold climate, i.e., Fusarium poae and Penicillium verrucosum. A Spearman's rank correlation analysis revealed that the severity of grain infection with F. avenaceum/F. tricinctum was affected by the mean daily temperature and high humidity in May, and the corresponding values of the correlation coefficient were determined at R = 0.54 and R = 0.50. Competitive interactions were observed between the F. avenaceum/F. tricinctum genotype and DON-producing F. culmorum and F. graminearum, because the severity of grain infections caused by these pathogens was bound by a negative correlation.

Host Genotype and Weather Effects on Fusarium Head Blight Severity and Mycotoxin Load in Spring Barley.

Epidemiology of Fusarium Head Blight (FHB) of spring barley is relatively little understood. In a five-year study, we assessed quantitative resistance to FHB in an assortment of 17 spring barley genotypes in the field in southern Germany. To this end, we used soil and spray inoculation of plants with F. culmorum and F. avenaceum. This increased disease pressure and provoked genotypic differentiation. To normalize effects of variable weather conditions across consecutive seasons, we used a disease ranking of the genotypes based on quantification of fungal DNA contents and multiple Fusarium toxins in harvested grain. Together, this allowed for assessment of stable quantitative FHB resistance of barley in several genotypes. Fungal DNA contents were positively associated with species-specific Fusarium toxins in single years and over several years in plots with soil inoculation. In those plots, plant height limited FHB; however, this was not observed after spray inoculation. A multiple linear regression model of recorded weather parameter and fungal DNA contents over five years identified time periods during the reproductive phase of barley, in which weather strongly influenced fungal colonization measured in mature barley grain. Environmental conditions before heading and late after anthesis showed strongest associations with F. culmorum DNA in all genotypes, whereas for F. avenaceum, this was less consistent where we observed weather-dependent associations, depending on the genotype. Based on this study, we discuss aspects of practical resistance breeding in barley relevant to improve quantitative resistance to FHB and associated mycotoxin contaminations.

Biodiversity and biocatalyst activity of culturable hydrocarbonoclastic fungi isolated from Marac-Moruga mud volcano in South Trinidad.

Mud volcanoes (MVs) are visible signs of oil and gas reserves present deep beneath land and sea. The Marac MV in Trinidad is the only MV associated with natural hydrocarbon seeps. Petrogenic polyaromatic hydrocarbons (PAHs) in its sediments must undergo biogeochemical cycles of detoxification as they can enter the water table and aquifers threatening ecosystems and biota. Recurrent hydrocarbon seep activity of MVs consolidates the growth of hydrocarbonoclastic fungal communities. Fungi possess advantageous metabolic and ecophysiological features for remediation but are underexplored compared to bacteria. Additionally, indigenous fungi are more efficient at PAH detoxification than commercial/foreign counterparts and remediation strategies remain site-specific. Few studies have focused on hydrocarbonoclastic fungal incidence and potential in MVs, an aspect that has not been explored in Trinidad. This study determined the unique biodiversity of culturable fungi from the Marac MV capable of metabolizing PAHs in vitro and investigated their extracellular peroxidase activity to utilize different substrates ergo their extracellular oxidoreductase activity (> 50% of the strains decolourized of methylene blue dye). Dothideomycetes and Eurotiomycetes (89% combined incidence) were predominantly isolated. ITS rDNA sequence cluster analysis confirmed strain identities. 18 indigenous hydrocarbonoclastic strains not previously reported in the literature and some of which were biosurfactant-producing, were identified. Intra-strain variability was apparent for PAH utilization, oil-tolerance and hydroxylase substrate specificity. Comparatively high levels of extracellular protein were detected for strains that demonstrated low substrate specificity. Halotolerant strains were also recovered which indicated marine-mixed substrata of the MV as a result of deep sea conduits. This work highlighted novel MV fungal strains as potential bioremediators and biocatalysts with a broad industrial applications.

Bioclimatic and anthropogenic variables shape the occurrence of Batrachochytrium dendrobatidis over a large latitudinal gradient.

Amphibian chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), has caused the greatest known loss of biodiversity due to an infectious disease. We used Bd infection data from quantitative real-time PCR (qPCR) assays of amphibian skin swabs collected across Chile during 2008-2018 to model Bd occurrence with the aim to determine bioclimatic and anthropogenic variables associated with Bd infection. Also, we used Bd presence/absence records to identify geographical Bd high-risk areas and compare Bd prevalence and infection loads between amphibian families, ecoregions, and host ecology. Data comprised 4155 Bd-specific qPCR assays from 162 locations across a latitudinal gradient of 3700 km (18º to 51ºS). Results showed a significant clustering of Bd associated with urban centres and anthropogenically highly disturbed ecosystems in central-south Chile. Both Bd prevalence and Bd infection loads were higher in aquatic than terrestrial amphibian species. Our model indicated positive associations of Bd prevalence with altitude, temperature, precipitation and human-modified landscapes. Also, we found that macroscale drivers, such as land use change and climate, shape the occurrence of Bd at the landscape level. Our study provides with new evidence that can improve the effectiveness of strategies to mitigate biodiversity loss due to amphibian chytridiomycosis.

Solving a long-standing enigma: Myrmicinosporidium durum belongs to Blastocladiomycota, a phylum of primarily aquatic fungi.

Myrmicinosporidium durumHölldobler (1933) is a widely distributed fungal endoparasite of ants. However, little is known about its biology, ecology, or evolutionary history. Our study investigated the phylogenetics of this entomopathogenic fungus using a molecular approach. Samples of M. durum were obtained from infected Solenopsis fugax workers collected in Warsaw (Poland). Analyses of rDNA markers revealed that M. durum belongs to a phylum of primarily aquatic fungi, Blastocladiomycota. It is currently the only species from this group known to parasitise hymenopterans. Our findings have clarified this fungus' taxonomy and suggest future directions for research into its biology, ecology, and infection dynamics.

High Genetic Diversity in Predominantly Clonal Populations of the Powdery Mildew Fungus Podosphaera leucotricha from U.S. Apple Orchards.

Apple powdery mildew (APM), caused by Podosphaera leucotricha, is a constant threat to apple production worldwide. Very little is known about the biology and population structure of this pathogen in the United States and other growing regions, which affects APM management. A total of 253 P. leucotricha isolates, sampled from 10 apple orchards in Washington, New York, and Virginia, were genetically characterized with novel single sequence repeat and mating type markers. Eighty-three multilocus genotypes (MLGs) were identified, most of which were unique to a given orchard. Each isolate carried either a MAT1-1 or a MAT1-2 idiomorph at the mating type locus, indicating that P. leucotricha is heterothallic. Virulence tests on detached apple leaves showed that the 10 most frequent P. leucotricha MLGs were avirulent on a line containing a major resistance gene. Analysis of molecular variance showed significant differentiation (P < 0.001) among populations, a result supported by principal coordinate analysis revealing three genetic groups, each represented by nonoverlapping MLGs from Washington, New York, and Virginia. A Bayesian cluster analysis showed genetic heterogeneity between Washington populations, and a relative migration analysis indicated substantial gene flow among neighboring orchards. Random mating tests indicated that APM epidemics during the active cycle were dominated by clonal reproduction. However, the presence of sexual structures in orchards, the likelihood that five repeated MLGs resulted from sexual reproduction, and high genotypic diversity observed in some populations suggest that sexual spores play some role in APM epidemics. IMPORTANCE Understanding the population biology and epidemiology of plant pathogens is essential to develop effective strategies for controlling plant diseases. Herein, we gathered insights into the population biology of P. leucotricha populations from conventional and organic apple orchards in the United States. We showed genetic heterogeneity between P. leucotricha populations in Washington and structure between populations from different U.S. regions, suggesting that short-distance spore dispersal plays an important role in the disease's epidemiology. We presented evidence that P. leucotricha is heterothallic and that populations likely result from a mixed (i.e., sexual and asexual) reproductive system, revealing that the sexual stage contributes to apple powdery mildew epidemics. We showed that the major resistance gene Pl-1 is valuable for apple breeding because virulent isolates have most likely not emerged yet in U.S. commercial orchards. These results will be important to achieve sustainability of disease management strategies and maintenance of plant health in apple orchards.

Molecular identification and antifungal susceptibility profiles of Candida dubliniensis and Candida africana isolated from vulvovaginal candidiasis: A single-centre experience in Iran.

BACKGROUND: Vulvovaginal candidiasis (VVC) is a common and debilitating long-term illness affecting million women worldwide. This disease is caused mainly by Candida albicans and a lesser extent by other species, including the two phylogenetically closely related pathogens Candida africana and Candida dubliniensis. OBJECTIVES: In this study, we report detailed molecular epidemiological data about the occurrence of these two pathogenic yeasts in Iranian patients affected by VVC, or its chronic recurrent form (RVVC), and provide, for the first time, data on the antifungal activity of two new drugs, efinaconazole (EFN) and luliconazole (LUL). METHODS: A total of 133 vaginal yeast isolates, presumptively identified as C albicans by phenotypic and restriction analysis of rDNA, were further analysed by using a specific molecular method targeting the HWP1 gene. All C africana and C dubliniensis isolates were also tested for their in vitro susceptibility to a panel of modern and classical antifungal drugs. RESULTS AND CONCLUSIONS: Based on the molecular results, among 133 germ-tube positive isolates, we identify 119 C albicans (89.47%), 11 C africana (8.27%) and 3 C dubliniensis (2.26%) isolates. C africana and C dubliniensis showed low MIC values for most of the antifungal drugs tested, especially for EFN and LUL, which exhibited a remarkable antifungal activity. High MIC values were observed only for nystatin and terbinafine. Although C albicans remains the most common Candida species recovered from Iranian VVC/RVVC patients, our data show that its prevalence may be slightly overestimated due to the presence of difficult-to-identify closely related yeast, especially C africana.