Discovery of a Sexual Cycle in Aspergillus lentulus,a Close Relative of A. fumigatus

Aspergillus lentulus was described in 2005 as a new species within the A. fumigatus sensu lato complex. It is an opportunistic human pathogen causing invasive aspergillosis with high mortality rates, and it has been isolated from clinical and environmental sources. The species is morphologically nearly identical to A. fumigatus sensu stricto, and this similarity has resulted in their frequent misidentification. Comparative studies show that A. lentulus has some distinguishing growth features and decreased in vitro susceptibility to several antifungal agents, including amphotericin B and caspofungin. Similar to the once-presumed-asexual A. fumigatus, it has only been known to reproduce mitotically. However, we now show that A. lentulus has a heterothallic sexual breeding system. A PCR-based mating-type diagnostic detected isolates of either the MAT1-1 or MAT1-2 genotype, and examination of 26 worldwide clinical and environmental isolates revealed similar ratios of the two mating types (38% versus 62%, respectively). MAT1-1 and MAT1-2 idiomorph regions were analyzed, revealing the presence of characteristic alpha and high-mobility-group (HMG) domain genes, together with other more unusual features such as a MAT1-2-4 gene. We then demonstrated that A. lentulus possesses a functional sexual cycle with mature cleistothecia, containing heat-resistant ascospores, being produced after 3 weeks of incubation. Recombination was confirmed using molecular markers. However, isolates of A. lentulus failed to cross with highly fertile strains of A. fumigatus, demonstrating reproductive isolation between these sibling species. The discovery of the A. lentulus sexual stage has significant implications for the management of drug resistance and control of invasive aspergillosis associated with this emerging fungal pathogen.     

Double-Stranded RNA Mycovirus Infection of Aspergillus fumigatus Is Not Dependent on the Genetic Make-Up of the Host

Aspergillus fumigatus is a fungus that causes opportunistic infections in immunocompromised patients, with high morbidity and mortality. In its turn, A. fumigatus can become infected with mycoviruses. Most mycoviruses have a dsRNA genome and can cause fungal hypovirulence. For that reason, mycoviruses could theoretically be used as therapeutic tools to combat fungal infections. We determined if a certain genetic make-up of A. fumigatus was associated with the presence of mycoviruses in 86 clinical A. fumigatus isolates. Mycovirus screening was performed by isolating dsRNA from mycelial cultures using a Trizol/Chloroform method. The genetic relatedness of dsRNA infected A. fumigatus was determined by cell surface protein (CSP) typing and determination of the mating type. Sixteen (18.6%) of the 86 clinical A. fumigatus isolates contained dsRNA. The A. fumigatus collection could be divided into 11 different CSP types. DsRNA infected A. fumigatus isolates had similar CSP types as non-infected isolates. In both cases, the CSP types t01, t02, t03 and t04 were the most prevalent and the distribution comparable to the CSP types observed in other Dutch collections. Mating types MAT1-1 and MAT1-2 were evenly distributed among all A. fumigatus strains, regardless of CSP type. No difference was observed in mycovirus infections between MAT1-1 and MAT1-2 isolates. DsRNA mycovirus infections in A. fumigatus are not related to either CSP or mating type and therefore represent an interesting future therapeutic tool to combat fungal infections.     

Evaluation of the potential for sexual reproduction in field populations of Cercospora beticola from USA

Cercospora leaf spot, caused by the hemibiotrophic fungal pathogen Cercospora beticola, is the most economically damaging foliar disease of sugarbeet worldwide. Although most C. beticola populations display characteristics reminiscent of sexual recombination, no teleomorph has been described. To assess whether populations in northern United States have characteristics consistent with sexual reproduction, 1024 isolates collected over a 3-y period were analyzed for frequency and distribution of mating type genes. After clone correction, an approximately equal distribution of mating types was found for each sampling year. Mating type frequency was also assessed in individual lesions. Lesions always consisted of isolates with a single mating type and microsatellite haplotype, but both mating types and up to five microsatellite haplotypes could be found on an individual leaf. The MAT1-1-1 and MAT1-2-1 genes were sequenced from 28 MAT1-1 and 28 MAT1-2 isolates, respectively. Three MAT1-1-1 nucleotide haplotypes were identified that encoded a single amino acid sequence. For MAT1-2-1, five nucleotide haplotypes were identified that encoded four protein variants. MAT1-1-1 and MAT1-2-1 gene expression analyses were conducted on plants inoculated with either or both mating types. MAT1-1-1 expression remained low, but MAT1-2-1 spiked during late stages of colonization. A segment of the MAT1-2-1 coding sequence was also found in MAT1-1 isolates. Taken together, these results suggest that C. beticola has the potential for sexual reproduction.     

Sexual Reproduction as the Cause of Heat Resistance in the Food Spoilage Fungus Byssochlamys spectabilis (Anamorph Paecilomyces variotii)

Paecilomyces variotii is a common cosmopolitan species that is able to spoil various food- and feedstuffs and is frequently encountered in heat-treated products. However, isolates from heat-treated products rarely form ascospores. In this study we examined by using molecular techniques and mating tests whether this species can undergo a sexual cycle and form ascospores. The population structure of this species was examined by analyzing the nuclear ribosomal internal transcribed spacer 1 (ITS1) and ITS2 and the 5.8S rRNA gene, as well as partial β-tubulin, actin, and calmodulin gene sequences. Phylogenetic analyses revealed that P. variotii is a highly variable species. Partition homogeneity tests revealed that P. variotii has a recombining population structure. In addition to sequence analyses, mating experiments indicated that P. variotii is able to form ascomata and ascospores in culture in a heterothallic manner. The distribution of MAT1-1 and MAT1-2 genes showed a 1:1 ratio in the progeny of the mating experiments. From the sequence analyses and mating data we conclude that P. variotii is the anamorph of Talaromyces spectabilis and that it has a biallelic heterothallic mating system. Since Paecilomyces sensu stricto anamorphs group within Byssochlamys, a new combination Byssochlamys spectabilis is proposed.     

Presence and expression of the mating type locus in Paracoccidioides brasiliensis isolates

Paracoccidioides brasiliensis has been classified in the phylum Ascomycota, order Onygenales, family Ajellomycetaceae, even in the absence of a known sexual cycle or mating system. The objective of this work was to determine the presence of the mating type locus in 71 P. brasiliensis isolates from various sources. A PCR assay using specific primers for the MAT 1 gene was developed and applied for the detection of such genes. Two heterothallic groups (MAT1-1 or MAT1-2) were recognized and, in some isolates, gene expression was confirmed indicating the existence of a basal gene expression. The distribution of two mating type loci in the studied population suggested that sexual reproduction might occur in P. brasiliensis. This finding points towards the possibility of applying a more precise definition of the concept of biological species to P. brasiliensis. Further studies should be conducted to confirm the sexual capacity of this fungus and its implications among phylogenetic species and geographical distribution.     

Heterothallism revealed in the root rot fungi Berkeleyomyces basicola and B. rouxiae

Berkeleyomyces basicola and Berkeleyomyces rouxiae, two sister species previously treated collectively as Thielaviopsis basicola, reside in the Ceratocystidaceae (Microascales, Ascomycota). Both species are important root pathogens of many important agricultural crops and ornamental plants. Although T. basicola has been known for more than 150y, a sexual state has never been found and it has been assumed to be an asexual pathogen. The aim of this study was to determine the mating strategy of the two Berkeleyomyces species. Investigation of the genome sequences of two B. basicola isolates allowed for the complete characterization of the MATlocus, revealing that it has a typical heterothallic mating system with the MAT1-1andMAT1-2 idiomorphs occurring in different isolates. PCR amplification using mating type primers developed in this study, showed that the MAT1-1-1andMAT1-2-1 genes were also present in different isolates of B. rouxiae. Pairing of isolates representing the two mating types of both species,using a variety of techniques failed to produce sexual structures. Although we have found no direct evidence that they reproduce sexually, these fungi are clearly heterothallic with both mating types occurring in some countries suggesting that a cryptic sexual cycle could exist for them.     

Sexualidad y patogenicidad en Aspergillus fumigatus: ¿existe alguna relación?

BackgroundAspergillus fumigatus, like many other fungal species of clinical relevance, has been traditionally regarded as an asexual organism. However, in last few years several pieces of evidence question this such assumption, suggesting that the sexual state of A. fumigatus may still be undiscovered. These investigations have finally led to the recent discovery of a teleomorph stage of A. fumigatus, which has been named Neosartorya fumigata.AimsTo review the most important findings on A. fumigatus sexuality and discuss the possible implications of such findings on its pathogenicity.MethodsA bibliographic search was performed to find the main works that study the sexuality of fungal pathogens and, especially, of A. fumigatus. Moreover, data from our recent investigations in this field were also introduced to the discussion.ResultsThe existence of a teleomorph for A. fumigatus could have significant clinical repercussions, as sexual reproduction might produce offspring with increased virulence and/or resistance to antifungal agents. In this sense, the results of our investigations suggest the existence of an association between the MAT1-1 mating type and the invasiveness of A. fumigatus isolates.ConclusionsThe study of the sexual reproduction of the fungal pathogens and its possible relationship with virulence will continue to be a topic of interest during the next years, not only because of its basic interest, but also for the possible clinical repercussions.     

Characterization of the mating-type genes in Leptographium procerum and Leptographium profanum

Leptographium procerum and the closely related species Leptographium profanum, are ascomycetes associated with root-infesting beetles on pines and hardwood trees, respectively. Both species occur in North America where they are apparently native. L. procerum has also been found in Europe, China New Zealand, and South Africa where it has most probably been introduced. As is true for many other Leptographium species, sexual states have never been observed in L. procerum or L. profanum. The objectives of this study were to clone and characterize the mating type loci of these fungi, and to develop markers to determine the mating types of individual isolates. To achieve this, a partial sequence of MAT1-2-1 was amplified using degenerate primers targeting the high mobility group (HMG) sequence. A complete MAT1-2 idiomorph of L. profanum was subsequently obtained by screening a genomic library using the HMG sequence as a probe. Long range PCR was used to amplify the complete MAT1-1 idiomorph of L. profanum and both the MAT1-1 and MAT1-2 idiomorphs of L. procerum. Characterization of the MAT idiomorphs suggests that the MAT genes are fully functional and that individuals of both these species are self-sterile in nature with a heterothallic mating system. Mating type markers were developed and tested on a population of L. procerum isolates from the USA, the assumed center of origin for this species. The results suggest that cryptic sexual reproduction is occurring or has recently taken place within this population.     

Frequency and Genetic Diversity of the MAT1 Locus of Histoplasma capsulatum Isolates in Mexico and Brazil

The MAT1-1 and MAT1-2 idiomorphs associated with the MAT1 locus of Histoplasma capsulatum were identified by PCR. A total of 28 fungal isolates, 6 isolates from human clinical samples and 22 isolates from environmental (infected bat and contaminated soil) samples, were studied. Among the 14 isolates from Mexico, 71.4% (95% confidence interval [95% CI], 48.3% to 94.5%) were of the MAT1-2 genotype, whereas 100% of the isolates from Brazil were of the MAT1-1 genotype. Each MAT1 idiomorphic region was sequenced and aligned, using the sequences of the G-217B (+ mating type) and G-186AR (− mating type) strains as references. BLASTn analyses of the MAT1-1 and MAT1-2 sequences studied correlated with their respective + and − mating type genotypes. Trees were generated by the maximum likelihood (ML) method to search for similarity among isolates of each MAT1 idiomorph. All MAT1-1 isolates originated from Brazilian bats formed a well-defined group; three isolates from Mexico, the G-217B strain, and a subgroup encompassing all soil-derived isolates and two clinical isolates from Brazil formed a second group; last, one isolate (EH-696P) from a migratory bat captured in Mexico formed a third group of the MAT1-1 genotype. The MAT1-2 idiomorph formed two groups, one of which included two H. capsulatum isolates from infected bats that were closely related to the G-186AR strain. The other group was formed by two human isolates and six isolates from infected bats. Concatenated ML trees, with internal transcribed spacer 1 (ITS1) -5.8S-ITS2 and MAT1-1 or MAT1-2 sequences, support the relatedness of MAT1-1 or MAT1-2 isolates. H. capsulatum mating types were associated with the geographical origin of the isolates, and all isolates from Brazil correlated with their environmental sources.     

Genetic Analysis Using an Isogenic Mating Pair of Aspergillus fumigatus Identifies Azole Resistance Genes and Lack of MAT Locus’s Role in Virulence

Invasive aspergillosis (IA) due to Aspergillus fumigatus is a major cause of mortality in immunocompromised patients. The discovery of highly fertile strains of A. fumigatus opened the possibility to merge classical and contemporary genetics to address key questions about this pathogen. The merger involves sexual recombination, selection of desired traits, and genomics to identify any associated loci. We constructed a highly fertile isogenic pair of A. fumigatus strains with opposite mating types and used them to investigate whether mating type is associated with virulence and to find the genetic loci involved in azole resistance. The pair was made isogenic by 9 successive backcross cycles of the foundational strain AFB62 (MAT1-1) with a highly fertile (MAT1-2) progeny. Genome sequencing showed that the F₉ MAT1-2 progeny was essentially identical to the AFB62. The survival curves of animals infected with either strain in three different animal models showed no significant difference, suggesting that virulence in A. fumigatus was not associated with mating type. We then employed a relatively inexpensive, yet highly powerful strategy to identify genomic loci associated with azole resistance. We used traditional in vitro drug selection accompanied by classical sexual crosses of azole-sensitive with resistant isogenic strains. The offspring were plated under varying drug concentrations and pools of resulting colonies were analyzed by whole genome sequencing. We found that variants in 5 genes contributed to azole resistance, including mutations in erg11A (cyp51A), as well as multi-drug transporters, erg25, and in HMG-CoA reductase. The results demonstrated that with minimal investment into the sequencing of three pools from a cross of interest, the variation(s) that contribute any phenotype can be identified with nucleotide resolution. This approach can be applied to multiple areas of interest in A. fumigatus or other heterothallic pathogens, especially for virulence associated traits.     

Sexuality Generates Diversity in the Aflatoxin Gene Cluster: Evidence on a Global Scale

Aflatoxins are produced by Aspergillus flavus and A. parasiticus in oil-rich seed and grain crops and are a serious problem in agriculture, with aflatoxin B₁ being the most carcinogenic natural compound known. Sexual reproduction in these species occurs between individuals belonging to different vegetative compatibility groups (VCGs). We examined natural genetic variation in 758 isolates of A. flavus, A. parasiticus and A. minisclerotigenes sampled from single peanut fields in the United States (Georgia), Africa (Benin), Argentina (Córdoba), Australia (Queensland) and India (Karnataka). Analysis of DNA sequence variation across multiple intergenic regions in the aflatoxin gene clusters of A. flavus, A. parasiticus and A. minisclerotigenes revealed significant linkage disequilibrium (LD) organized into distinct blocks that are conserved across different localities, suggesting that genetic recombination is nonrandom and a global occurrence. To assess the contributions of asexual and sexual reproduction to fixation and maintenance of toxin chemotype diversity in populations from each locality/species, we tested the null hypothesis of an equal number of MAT1-1 and MAT1-2 mating-type individuals, which is indicative of a sexually recombining population. All samples were clone-corrected using multi-locus sequence typing which associates closely with VCG. For both A. flavus and A. parasiticus, when the proportions of MAT1-1 and MAT1-2 were significantly different, there was more extensive LD in the aflatoxin cluster and populations were fixed for specific toxin chemotype classes, either the non-aflatoxigenic class in A. flavus or the B₁-dominant and G₁-dominant classes in A. parasiticus. A mating type ratio close to 1∶1 in A. flavus, A. parasiticus and A. minisclerotigenes was associated with higher recombination rates in the aflatoxin cluster and less pronounced chemotype differences in populations. This work shows that the reproductive nature of the population (more sexual versus more asexual) is predictive of aflatoxin chemotype diversity in these agriculturally important fungi.     

Asexual Propagation of a Virulent Clone Complex in a Human and Feline Outbreak of Sporotrichosis

Sporotrichosis is one of the most frequent subcutaneous fungal infections in humans and animals caused by members of the plant-associated, dimorphic genus Sporothrix. Three of the four medically important Sporothrix species found in Brazil have been considered asexual as no sexual stage has ever been reported in Sporothrix schenckii, Sporothrix brasiliensis, or Sporothrix globosa. We have identified the mating type (MAT) loci in the S. schenckii (strain 1099-18/ATCC MYA-4821) and S. brasiliensis (strain 5110/ATCC MYA-4823) genomes by using comparative genomic approaches to determine the mating type ratio in these pathogen populations. Our analysis revealed the presence of a MAT1-1 locus in S. schenckii while a MAT1-2 locus was found in S. brasiliensis representing genomic synteny to other Sordariomycetes. Furthermore, the components of the mitogen-activated protein kinase (MAPK)-pheromone pathway, pheromone processing enzymes, and meiotic regulators have also been identified in the two pathogens, suggesting the potential for sexual reproduction. The ratio of MAT1-1 to MAT1-2 was not significantly different from 1:1 for all three Sporothrix species, but the population of S. brasiliensis in the outbreaks originated from a single mating type. We also explored the population genetic structure of these pathogens using sequence data of two loci to improve our knowledge of the pattern of geographic distribution, genetic variation, and virulence phenotypes. Population genetics data showed significant population differentiation and clonality with a low level of haplotype diversity in S. brasiliensis isolates from different regions of sporotrichosis outbreaks in Brazil. In contrast, S. schenckii isolates demonstrated a high degree of genetic variability without significant geographic differentiation, indicating the presence of recombination. This study demonstrated that two species causing the same disease have contrasting reproductive strategies and genetic variability patterns.     

Natural populations of the entomopathogenic fungus Beauveria bassiana in Chinese forest ecosystems are diverse and reveal equal frequencies of mating types within phylogenetic species

Beauveria bassiana is an important entomopathogenic fungus with widespread application in the biological control of harmful insect pests. This species is widely distributed as an anamorph while only two teleomorph specimens have been found in eastern China. However, little is known about the ecological conditions for sexual reproduction in natural populations of B. bassiana. Here, we collected 488 isolates of Chinese B. bassiana sensu stricto from five sites, in which teleomorph or anamorph occurred, and used molecular phylogenetic and haplotype information to determine phylogenetic diversity, mating types, and sexual reproductive potential in these populations. Molecular identification based on denaturing gradient gel electrophoresis (DGGE) and combined data of the nuclear intergenic region Bloc and translation elongation factor-1a (TEF) assemblage resolved five B. bassiana s.s phylogenetic species labeled according to their geographic origin: Europe/N. Africa 1, Asia 3, Asia 4, AFNEO_1, and N. America 2. In Guniujiang and Manshuihe collection sites, teleomorph isolates RCEF 0771 and RCEF 0382 were both identified as Europe/N. Africa 1 phylogenetic species. In addition, more than half of the isolates in five representative sites belonged to Europe/N. Africa 1. However, the teleomorph of B. bassiana s.s. was not detected in Kuankuoshui while isolates within Europe/N. Africa 1 were present at this site, and isolates belonging to Europe/N. Africa 1 were not found in either Jingyuetan or Dinghushan collection sites. Distribution of MAT1 and MAT2 mating type idiomorphs in Europe/N. Africa 1 were 51:69, 37:24, and 15:15 in Guniujiang, Manshuihe, and Kuankuoshui, respectively. The presence of teleomorph and roughly equal frequencies of opposite mating types indicate regular sexual reproduction in B. bassiana natural populations. The data offer a better understanding of the ecological conditions of sexual reproduction in natural populations of B. bassiana. These results also yield insights into the potential for sexual reproduction in other supposedly ‘asexual’ fungi.     

Recombination between Clonal Lineages of the Asexual Fungus Verticillium dahliae Detected by Genotyping by Sequencing

Most asexual species of fungi have either lost sexuality recently, or they experience recombination by cryptic sexual reproduction. Verticillium dahliae is a plant-pathogenic, ascomycete fungus with no known sexual stage, even though related genera have well-described sexual reproduction. V. dahliae reproduces mitotically and its population structure is highly clonal. However, previously described discrepancies in phylogenetic relationships among clonal lineages may be explained more parsimoniously by recombination than mutation; therefore, we looked for evidence of recombination within and between clonal lineages. Genotyping by sequencing was performed on 141 V. dahliae isolates from diverse geographic and host origins, resulting in 26,748 single-nucleotide polymorphisms (SNPs). We found a strongly clonal population structure with the same lineages as described previously by vegetative compatibility groups (VCGs) and molecular markers. We detected 443 recombination events, evenly distributed throughout the genome. Most recombination events detected were between clonal lineages, with relatively few recombinant haplotypes detected within lineages. The only three isolates with mating type MAT1-1 had recombinant SNP haplotypes; all other isolates had mating type MAT1-2. We found homologs of eight meiosis-specific genes in the V. dahliae genome, all with conserved or partially conserved protein domains. The extent of recombination and molecular signs of sex in (mating-type and meiosis-specific genes) suggest that V. dahliae clonal lineages arose by recombination, even though the current population structure is markedly clonal. Moreover, the detection of new lineages may be evidence that sexual reproduction has occurred recently and may potentially occur under some circumstances. We speculate that the current clonal population structure, despite the sexual origin of lineages, has arisen, in part, as a consequence of agriculture and selection for adaptation to agricultural cropping systems.     

Isolation of the MAT1-1 mating type idiomorph and evidence for selfing in the Chinese medicinal fungus Ophiocordyceps sinensis

Ophiocordyceps sinensis is one of the most valued medicinal fungi in China. Research on the mating system and sexual development is vitally important to this endangered species. Previous efforts devoted to investigate the mating type (MAT) locus of O. sinensis, however, resulted in an incomplete understanding. In this study, the MAT1-1 locus of O. sinensis was investigated. The conserved α-box and HMG-box regions of the MAT1-1-1 and MAT1-1-3 genes, respectively, and a conserved region of the DNA lyase gene were successfully amplified using degenerate PCR. A combination of TAIL-PCR and long-range PCR were used to connect these genes and obtain the sequence of the MAT1-1 locus. Screening of 22 single spore isolates by PCR demonstrated that both the MAT1-1-1 and MAT1-2-1 genes cooccurred within the same isolate. Additionally, both MAT1-1-1 and MAT1-2-1 are expressed in vegetative mycelia, providing evidence that O. sinensis is likely capable of selfing. DAPI (4,6-diamidino-2-phenylindole) staining of ascospores and hyphae showed that a majority of hyphal compartments are binucleate, suggesting that O. sinensis may be pseudohomothallic. Analyses of sequence diversity showed lower levels of genetic diversity in MAT1-1-1 compared to MAT1-2-1, indicating the possibility that different selective pressures act on the two MAT idiomorphs. The MAT1-1-1 sequences of O. sinensis and Tolypocladium inflatum cluster as a monophyletic group consistent with phylogenetic classification of Ophiocordycipitaceae. Comparison of the structure of the MAT1-1 locus across hypocrealean taxa showed that O. sinensis contains all three mating type genes (MAT1-1-1, MAT1-1-2, and MAT1-1-3) and supported previous observations that of the four families in Hypocreales, MAT1-1-3 has undergone a lineage specific loss only in some members of the Cordycipitaceae.     

Characterization and population analysis of the mating-type genes in Aspergillus flavus and Aspergillus parasiticus

We characterize the mating-type genes in Aspergillus flavus,Aspergillus parasiticus and Petromyces alliaceus. A single MAT1-1 or MAT1-2 gene was detected in the genomes of A. flavus and A. parasiticus, which is consistent with a potential heterothallic organization of MAT genes in these species. In contrast, the only known, functionally homothallic species in Aspergillus section Flavi, P. alliaceus, has tightly linked (<2kb) MAT1-1 and MAT1-2 genes, typical of other self-fertile homothallic euascomycetes. This is the first example of linked MAT genes within a homothallic species of Aspergillus. We tested the null hypothesis of no significant difference in the frequency of MAT1-1 and MAT1-2 in A. flavus and A. parasiticus sampled from a single peanut field in Georgia. For each species, mating-type frequencies were determined for the total population samples and for samples that were clone-corrected based on vegetative compatibility groups (VCGs) and aflatoxin gene cluster haplotypes. There was no significant difference in the frequency of the two mating types for A. flavus and A. parasiticus in either VCG or haplotype clone-corrected samples. The existence of both mating-type genes in equal proportions in A. flavus and A. parasiticus populations, coupled with their expression at the mRNA level and the high amino acid sequence identity of MAT1-1 (77%) and MAT1-2 (83%) with corresponding homologs in P. alliaceus, indicates the potential functionality of these genes and the possible existence of a sexual state in these agriculturally important species.     

Revisiting phylogenetic diversity and cryptic species of <Emphasis Type="Italic">Cenococcum geophilum</Emphasis> sensu lato

The fungus Cenococcum geophilum Fr. (Dothideomycetes, Ascomycota) is one of the most common ectomycorrhizal fungi in boreal to temperate regions. A series of molecular studies has demonstrated that C. geophilum is monophyletic but a heterogeneous species or a species complex. Here, we revisit the phylogenetic diversity of C. geophilum sensu lato from a regional to intercontinental scale by using new data from Florida (USA) along with existing data in GenBank from Japan, Europe, and North America. The combination of internal transcribed spacer (ITS) ribosomal DNA and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene resolved six well-supported lineages (87–100 % bootstrap values) that are closely related to each other and a seventh lineage that is phylogenetically distinct. A multi-locus analysis (small subunit (SSU), large subunit (LSU), translational elongation factor (TEF), and the largest and second-largest subunits of RNA polymerase II (RPB1 and RPB2)) revealed that the divergent lineage is the sister group to all other known Cenococcum isolates. Isolates of the divergent lineage grow fast on nutrient media and do not form ectomycorrhizas on seedlings of several pine and oak species. Our results indicate that C. geophilum sensu lato includes more phylogenetically distinct cryptic species than have previously been reported. Furthermore, the divergent lineage appears to be a non-mycorrhizal sister group. We discuss the phylogenetic diversity of C. geophilum sensu lato and argue in favor of species recognition based on phylogenetic and ecological information in addition to morphological characteristics. A new genus and species (Pseudocenococcum floridanum gen. et sp. nov.) is proposed to accommodate a divergent and putatively non-mycorrhizal lineage.     

The Molecular Identification and Antifungal Susceptibilities of Aspergillus Species Causing Otomycosis in Tochigi,Japan

Aspergillus species are the most common pathogenic fungi involved in otomycosis, an infection of the outer ear canal. In this study, we examined the incidence of Aspergillus infections and the antifungal susceptibilities of 30 Aspergillus species isolates from patients with otomycosis who visited Saiseikai Utsunomiya Hospital between August 2013 and July 2016. Based on the morphological test results, the strains were identified as Aspergillus niger sensu lato (20 strains), A. terreus sensu lato (7 strains), and A. fumigatus sensu lato (3 strains). In contrast, the molecular identifications based on analyzing the isolates’ partial β-tubulin gene sequences revealed them to be A. niger sensu stricto (12 strains), A. tubingensis (8 strains), A. terreus sensu stricto (7 strains), and A. fumigatus sensu stricto (3 strains). The antifungal susceptibility test results indicated that strains of A. tubingensis and A. niger sensu stricto displayed lower susceptibilities to ravuconazole, compared with the other isolates. The Aspergillus strains from this study showed low minimum inhibitory concentrations toward the azole-based drugs efinaconazole, lanoconazole, and luliconazole. Therefore, these topical therapeutic agents may be effective for the treatment of otomycosis.

     

Chickpea Ascochyta Blight: Disease Status and Pathogen Mating Type Distribution in Syria

Chickpea fields were surveyed in nine major chickpea‐growing provinces of Syria in 2008 and 2009 to determine the prevalence and severity of Ascochyta blight, and the distribution of Didymella rabiei mating types (MATs) in the country. A total of 133 Ascochyta rabiei isolates were assayed for mating type, including isolates from older collections that date back to 1982. Multiplex MAT‐specific PCR with three primers was used for MAT analysis. Out of the 133 tested isolates, 64% were MAT1‐1 and 36% were MAT1‐2. Both MATs were found in six provinces but MAT1‐1 alone was found in three provinces. Chi‐squared analysis was used to test for a 1 : 1 ratio of MAT frequencies in all samples. The MAT ratios in the six provinces were not significantly different from 1 : 1, suggesting that there is random mating of the pathogen population under natural conditions. The presence of the two MATs is expected to play a role in the evolution of novel virulence genes that could threaten currently resistant chickpea varieties. Overall analysis of the 133 isolates showed a significant deviation from the 1 : 1 ratio with almost twice as many MAT1‐1 isolates than MAT1‐2 isolates, which indicates a competitive advantage associated with MAT1‐1 in Syria. However, the overall picture of an unequal frequency in MATs indicates that there may be limited sexual recombination occurring in the Syrian population.