Population structure of Hymenoscyphus pseudoalbidus and its genetic relationship to Hymenoscyphus albidus

The ascomycete fungus Hymenoscyphus pseudoalbidus (anamorph Chalara fraxinea) is responsible for ash dieback currently expanding over large parts of Europe. Our objective was to investigate the genetic structure of H. pseudoalbidus and to examine its relationship to the species H. albidus, known as a saprotroph. The study comprised 181 isolates of H. pseudoalbidus collected within the diseased area, 17 H. albidus isolates from six apothecia, collected outside the diseased area in Norway, and nine apothecia of H. pseudoalbidus collected in Sweden. By analysis of microsatellite markers developed for this study, combined with AP-PCR using the M13 primer, we demonstrated sexual heterothally in H. pseudoalbidus, detected high gene flow and low geographic structure of the H. pseudoalbidus population and found indications of a founder effect. Also, substantial genetic differences were detected between the two species of fungi; only four of seven microsatellite markers developed for H. pseudoalbidus were amplified for H. albidus, and no alleles were shared among the species. Furthermore, AP-PCR banding patterns were distinctly different for the two species. We conclude that even though the two fungi have a similar habitat and are morphologically virtually identical, they do not share a recent common ancestor.     

The invasive ash dieback pathogen Hymenoscyphus pseudoalbidus exerts maximal infection pressure prior to the onset of host leaf senescence

Shoot dieback disease of European ash caused by the ascomycete Hymenoscyphus pseudoalbidus threatens ash on a continental scale. A spore sampler placed in a diseased ash forest in Southern Norway, coupled with microscopy and DNA-based fungal species-specific real-time PCR assays, was employed to profile diurnal and within-season variation in infection pressure by ascospores of H. pseudoalbidus and the potentially co-existing non-pathogenic Hymenoscyphus albidus. Hymenoscyphus pseudoalbidus was found to be predominant in the stand. Massive simultaneous liberation, by active discharge of pathogen ascospores in the morning, peaked in mid-Jul. to mid-Aug. Accumulation of pathogen DNA on leaflets of current-year leaves reached a high level plateau phase before appearance of autumn coloration, suggesting that pathogen establishment in leaves is terminated before the onset of leaf senescence.     

Assessment ofGibberella fujikuroi mating type by PCR

Mating type (MAT)-specific fragments of the two idiomorphs ofGibberella fujikuroi (anamorph,Fusarium moniliforme) were obtained by PCR amplification using primers to conserved regions ofMAT homologs from other fungal species and used to assign mating type by molecular criteria rather than the arbitrary historical designation. Mating type—strains of mating populations A-E and a mating type+strain of mating population F carry an α-box motif and should therefore be designatedMAT-1. Mating type+strains of mating populations A-E and a mating type—strain of mating population F carry an HMG-box motif and should be designatedMAT-2. Thus, assessment of mating type ofG. fujikurol strains can be easily achieved usingMAT-specific primers.     

Genetic Variability of Hymenoscyphus pseudoalbidus on Ash Leaf Rachises in Leaf Litter of Forest Stands in Poland

Two hundred and thirty cultures of Hymenoscyphus pseudoalbidus were obtained from ascospores created in apothecia on the previous years' ash leaf rachises in the stand floor. Fruiting bodies of the pathogen were collected in four regions of Poland differing by geographical location, the altitude above sea level and climatic conditions. Isolates were identified based on the sequences of ribosomal DNA (ITS1‐5.8S‐ITS2) and the calmodulin gene. Only the presence of H. pseudoalbidus was identified in the decaying ash stands in Poland; morphologically similar, saprotrophic species of H. albidus was absent. Intrapopulation and interpopulation genetic variability of isolates was determined based on 84 RAMS markers obtained using four primers. Genetic variability of the fungus populations, measured by the Dice coefficient of genetic similarity and the Shannon coefficient of genetic diversity, decreased along with a decrease in the location of isolate collection area above sea level. A significant dependency was shown between intrapopulation genetic variability of isolates and altitude of regions above sea level. The Mantel test excluded existence of dependence between geographical and genetic distance among populations (r = ?0.038, P = 0.55). A significant correlation was found between the genetic distances of individuals within populations and locations above sea level. Based on PCA and geographical location of populations, it was shown that populations create four distinct groups. amova showed that a majority of total genetic variability (65.80%) constitutes intrapopulation variability. Variability between populations was high (28.7%), and individual regions had a smallest influence (5.5%) on the level of total variability.     

Population structure of the invasive forest pathogen Hymenoscyphus pseudoalbidus

Understanding the genetic diversity and structure of invasive pathogens in source and in introduced areas is crucial to the revelation of hidden biological features of an organism, to the reconstruction of the course of invasions and to the establishment of effective control measures. Hymenoscyphus pseudoalbidus (anamorph: Chalara fraxinea) is an invasive and highly destructive fungal pathogen found on common ash Fraxinus excelsior in Europe and is native to East Asia. To gain insights into its dispersal mechanisms and history of invasion, we used microsatellite markers and characterized the genetic structure and diversity of H. pseudoalbidus populations at three spatial levels: (i) between Europe and Japan, (ii) in Europe and (iii) at the epidemic's front in Switzerland. Phylogenetic and network analysis demonstrated that individuals from both regions are conspecific. However, populations from Japan harboured a higher genetic diversity and were genetically differentiated from European ones. No evident population structure was found among the 1208 European strains using Bayesian and multivariate clustering analysis. Only the distribution of genetic diversity in space, pairwise population differentiation (G_ST) and the spatial analysis of principal components revealed a faint geographical pattern around Europe. A significant allele deficiency in most European populations pointed to a recent genetic bottleneck, whereas no pattern of isolation by distance was found. Our data suggest that H. pseudoalbidus was introduced just once by at least two individuals. The potential source region of H. pseudoalbidus is vast, and further investigations are required for a more accurate localization of the source population.     

Virulence of Hymenoscyphus albidus and H. fraxineus on Fraxinus excelsior and F. pennsylvanica

European ash (Fraxinus excelsior) is currently battling an onslaught of ash dieback, a disease emerging in the greater part of its native area, brought about by the introduction of the ascomycete Hymenoscyphus fraxineus (= Hymenoscyphus pseudoalbidus). The closely-related fungus Hymenoscyphus albidus, which is indigenous to Europe, is non-pathogenic when in contact with F. excelsior, but could pose a potential risk to exotic Fraxinus species. The North American green ash (Fraxinus pennsylvanica) is planted widely throughout Europe and regenerates naturally within this environment but little is known about the susceptibility of this species to ash dieback. We performed wound inoculations with both fungi (nine strains of H. fraxineus and three strains of H. albidus) on rachises and stems of F. excelsior and F. pennsylvanica under field conditions in Southern Poland. Necrosis formation was evaluated after two months on the rachises and after 12 months on the stems. After inoculation of H. albidus, only small lesions (of up to 1.3 cm in length) developed on the F. excelsior and F. pennsylvanica rachises, but with no significant distinction from the controls. Hymenoscyphus albidus did not cause necrotic lesions on the stems of either Fraxinus species. In contrast, H. fraxineus induced necroses on all inoculated rachises of both ash species with mean lengths of 8.4 cm (F. excelsior) and 1.9 cm (F. pennsylvanica). Necroses also developed on all of the inoculated F. excelsior stems (mean length 18.0 cm), whereas on F. pennsylvanica such lesions only occurred on about 5% of the stems (mean length 1.9 cm). The differences between strains were negligible. No necroses were observed on the control plants. Reisolations of H. albidus were only successful in around 8–11% of the cases, while H. fraxineus was reisolated from 50–70% of the inoculated organs showing necrotic lesions. None of the Hymenoscyphus species were isolated from the control plants. Our data confirm H. fraxineus’ high virulence with regards to F. excelsior and demonstrate a low virulence in relation to F. pennsylvanica under field conditions in Poland. Hymenoscyphus albidus did not express any perceivable pathogenicity on both host species.     

Development of a multiplex PCR assay to discriminate native Hymenoscyphus albidus and introduced Hymenoscyphus fraxineus in Britain and assess their distribution

Two Hymenoscyphus species are associated with ash (Fraxinus) in Europe; the recently arrived ash dieback pathogen Hymenoscyphus fraxineus and apparently native Hymenoscyphus albidus. However, in parts of Europe invasive H. fraxineus appears to be replacing H. albidus which may, therefore, face extinction. We designed primers based on calmodulin sequences for use in singleplex and multiplex PCR assays to detect and discriminate between these two morphologically very similar species. The multiplex assay was tested against 50 isolates and 26 apothecial field samples of H. albidus/H. fraxineus from Europe and Asia, plus isolates of Hymenoscyphus fructigenus, Hymenoscyphus koreanus and Hymenoscyphus occultus. Use of the assay confirmed both H. albidus and H. fraxineus were present across southern Britain between 2014 and 2015, with H. albidus detected at eight geographically distinct sites. The sensitivity, specificity and potential applications of the multiplex assay are discussed.     

The life cycle of Hymenoscyphus fraxineus on Manchurian ash,Fraxinus mandshurica,in Japan

Hymenoscyphus fraxineus causes a lethal disease known as “ash dieback” in the common ash, Fraxinus excelsior, in Europe. It is hypothesized that the fungus originated from East Asia. This fungus is found on the leaf litter of the Manchurian ash, Fraxinus mandshurica, in Japan and is reported to produce apothecia on pseudosclerotial plates formed mainly on decomposing rachises. However, dieback disease has not been reported in Japan, and little is known about the life cycle of H. fraxineus. This study was conducted to explore the behavior and life cycle of this fungus. It was revealed that, after infection by ascospores, H. fraxineus endophytically inhabits the living leaves of F. mandshurica. On fallen leaves, the fungus behaves saprophytically, producing apothecia on pseudosclerotial plates formed mainly on the decomposing rachises. Analysis by real-time quantitative polymerase chain reaction (qPCR) revealed that the amount of H. fraxineus DNA sharply increased in rachises, while such sharp increase of DNA was not found in leaflets.     

PCR-Based Identification of MAT-1 and MAT-2 in the Gibberella fujikuroi Species Complex

All sexually fertile strains in the Gibberella fujikuroi species complex are heterothallic, with individual mating types conferred by the broadly conserved ascomycete idiomorphs MAT-1 and MAT-2. We sequenced both alleles from all eight mating populations, developed a multiplex PCR technique to distinguish these idiomorphs, and tested it with representative strains from all eight biological species and 22 additional species or phylogenetic lineages from this species complex. In most cases, either an ~800-bp fragment from MAT-2 or an ~200-bp fragment from MAT-1 is amplified. The amplified fragments cosegregate with mating type, as defined by sexual cross-fertility, in a cross of Fusarium moniliforme (Fusarium verticillioides). Neither of the primer pairs amplify fragments from Fusarium species such as Fusarium graminearum, Fusarium pseudograminearum, and Fusarium culmorum, which have, or are expected to have, Gibberella sexual stages but are thought to be relatively distant from the species in the G. fujikuroi species complex. Our results suggest that MAT allele sequences are useful indicators of phylogenetic relatedness in these and other Fusarium species.     

Mating-type-specific and nonspecific PAK kinases play shared and divergent roles in Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle involving fusion of haploid MATα and MATa cells. Virulence has been linked to the mating type, and MATα cells are more virulent than congenic MATa cells. To study the link between the mating type and virulence, we functionally analyzed three genes encoding homologs of the p21-activated protein kinase family: STE20α, STE20a, and PAK1. In contrast to the STE20 genes that were previously shown to be in the mating-type locus, the PAK1 gene is unlinked to the mating type. The STE20α, STE20a, and PAK1 genes were disrupted in serotype A and D strains of C. neoformans, revealing central but distinct roles in mating, differentiation, cytokinesis, and virulence. ste20α pak1 and ste20a pak1 double mutants were synthetically lethal, indicating that these related kinases share an essential function. In summary, our studies identify an association between the STE20α gene, the MATα locus, and virulence in a serotype A clinical isolate and provide evidence that PAK kinases function in a MAP kinase signaling cascade controlling the mating, differentiation, and virulence of this fungal pathogen.     

Identification of the Mating-Type (MAT) Locus That Controls Sexual Reproduction of Blastomyces dermatitidis

Blastomyces dermatitidis is a dimorphic fungal pathogen that primarily causes blastomycosis in the midwestern and northern United States and Canada. While the genes controlling sexual development have been known for a long time, the genes controlling sexual reproduction of B. dermatitidis (teleomorph, Ajellomyces dermatitidis) are unknown. We identified the mating-type (MAT) locus in the B. dermatitidis genome by comparative genomic approaches. The B. dermatitidis MAT locus resembles those of other dimorphic fungi, containing either an alpha-box (MAT1-1) or an HMG domain (MAT1-2) gene linked to the APN2, SLA2, and COX13 genes. However, in some strains of B. dermatitidis, the MAT locus harbors transposable elements (TEs) that make it unusually large compared to the MAT locus of other dimorphic fungi. Based on the MAT locus sequences of B. dermatitidis, we designed specific primers for PCR determination of the mating type. Two B. dermatitidis isolates of opposite mating types were cocultured on mating medium. Immature sexual structures were observed starting at 3 weeks of coculture, with coiled-hyphae-containing cleistothecia developing over the next 3 to 6 weeks. Genetic recombination was detected in potential progeny by mating-type determination, PCR-restriction fragment length polymorphism (PCR-RFLP), and random amplification of polymorphic DNA (RAPD) analyses, suggesting that a meiotic sexual cycle might have been completed. The F1 progeny were sexually fertile when tested with strains of the opposite mating type. Our studies provide a model for the evolution of the MAT locus in the dimorphic and closely related fungi and open the door to classic genetic analysis and studies on the possible roles of mating and mating type in infection and virulence.     

The earliest samples of <Emphasis Type="Italic">Hymenoscyphus albidus</Emphasis> vs. <Emphasis Type="Italic">H. fraxineus</Emphasis> in Estonian mycological herbaria

In 2006, the alien and pathogenic Hymenoscyphus fraxineus was differentiated by molecular methods from the long recognized saprotrophic H. albidus . Today, H. fraxineus seems to have replaced H. albidus in several countries, but the exact year of arrival of H. fraxineus in northern Europe is still debated. Investigations of herbaria specimens might help to ascertain it. Before the epidemic, H. albidus was not of significant interest to mycologists in the Baltic area and was not eagerly sampled and deposited. Nevertheless, the TAAM herbarium in Estonia holds 13 putative H. albidus specimens that were collected 1966–2006. Using newly developed species-specific PCR primers to differentiate between H. albidus and H. fraxineus, all available herbarium samples from TAAM were identified. In addition, the primers were also tested on pure cultures and ascocarps of H. fraxineus and H. albidus, symptomatic petioles of F. excelsior, pure cultures of 10 non-target fungal species, and 10 different soil samples. With the exception of the oldest specimen in TAAM from 1966 (collected in Lithuania), all herbarium samples were identified as H. fraxineus. The first record of H. fraxineus in Estonia dates back to 1997 and apparently represents the oldest record of the species in Europe. In spite of this, symptoms based on reliable observations were not detected until 2003 in Estonia. This temporal shift is difficult to explain. Possible reasons may represent (i) overlooking of symptoms during the first years; (ii) typical lag phase at the start of an epidemic; (iii) an abrupt shift towards higher virulence of H. fraxineus; or (iv) other environmental (climatic) factors. Closer investigations are needed to disentangle these possible reasons.     

Evidence of sexual recombination among Cryptococcus neoformans serotype A isolates in sub-Saharan Africa

The most common cause of fungal meningitis in humans, Cryptococcus neoformans serotype A, is a basidiomycetous yeast with a bipolar mating system. However, the vast majority (>99.9%) of C. neoformans serotype A isolates possess only one of the two mating type alleles (MATα). Isolates with the other allele (MATa) were recently discovered and proven to mate in the laboratory. It has been a mystery whether and where C. neoformans strains undergo sexual reproduction. Here, we applied population genetic approaches to demonstrate that a population of C. neoformans serotype A clinical isolates from Botswana contains an unprecedented proportion of fertile MATa isolates and exhibits evidence of both clonal expansion and recombination within two partially genetically isolated subgroups. Our findings provide evidence for sexual recombination among some populations of C. neoformans serotype A from sub-Saharan Africa, which may have a direct impact on their evolution.     

αADα Hybrids of Cryptococcus neoformans: Evidence of Same-Sex Mating in Nature and Hybrid Fitness

Cryptococcus neoformans is a ubiquitous human fungal pathogen that causes meningoencephalitis in predominantly immunocompromised hosts. The fungus is typically haploid, and sexual reproduction involves two individuals with opposite mating types/sexes, α and a. However, the overwhelming predominance of mating type (MAT) α over a in C. neoformans populations limits α–a mating in nature. Recently it was discovered that C. neoformans can undergo same-sex mating under laboratory conditions, especially between α isolates. Whether same-sex mating occurs in nature and contributes to the current population structure was unknown. In this study, natural αADα hybrids that arose by fusion between two α cells of different serotypes (A and D) were identified and characterized, providing definitive evidence that same-sex mating occurs naturally. A novel truncated allele of the mating-type-specific cell identity determinant SXI1α was also identified as a genetic factor likely involved in this process. In addition, laboratory-constructed αADα strains exhibited hybrid vigor both in vitro and in vivo, providing a plausible explanation for their relative abundance in nature despite the fact that AD hybrids are inefficient in meiosis/sporulation and are trapped in the diploid state. These findings provide insights on the origins, genetic mechanisms, and fitness impact of unisexual hybridization in the Cryptococcus population.     

Deletion and Complementation of the Mating Type (MAT) Locus of the Wheat Head Blight Pathogen Gibberella zeae

Gibberella zeae, a self-fertile, haploid filamentous ascomycete, causes serious epidemics of wheat (Triticum aestivum) head blight worldwide and contaminates grain with trichothecene mycotoxins. Anecdotal evidence dating back to the late 19th century indicates that G. zeae ascospores (sexual spores) are a more important inoculum source than are macroconidia (asexual spores), although the fungus can produce both during wheat head blight epidemics. To develop fungal strains to test this hypothesis, the entire mating type (MAT1) locus was deleted from a self-fertile (MAT1-1/MAT1-2), virulent, trichothecene-producing wild-type strain of G. zeae. The resulting MAT deletion (mat1-1/mat1-2) strains were unable to produce perithecia or ascospores and appeared to be unable to mate with the fertile strain from which they were derived. Complementation of a MAT deletion strain by transformation with a copy of the entire MAT locus resulted in recovery of production of perithecia and ascospores. MAT deletion strains and MAT-complemented strains retained the ability to produce macroconidia that could cause head blight, as assessed by direct injection into wheat heads in greenhouse tests. Availability of MAT-null and MAT-complemented strains provides a means to determine the importance of ascospores in the biology of G. zeae and perhaps to identify novel approaches to control wheat head blight.     

Control of cell type in yeast by the mating type locus: The α1-α2 hypothesis

We have extended the genetic analysis of four mutants carrying defective MATα alleles in order to determine how the mating type locus controls yeast cell types: a, a, and $\text{a}\text{α}$. First, we have mapped the defect in the mutant VC73 to the mating type locus by diploid and tetraploid segregation analysis. Second, we have determined that the mutations in these strains define two complementation groups, MATα1 and MATα2. The MATα1 gene is proposed to be a positive regulator of α mating functions. The MATα2 gene product is proposed to have two roles, as a negative regulator of a-specific mating functions and as a regulator of $\text{a}\text{α}$ cell functions (required for sporulation, for inhibition of mating and other processes). This view of MATα leads to the prediction that matα1^?matα2^? mutants should have the mating ability of an a cell and that matα1^?matα2^?/MATα strains should mate as α and be unable to sporulate. Such double mutants have been constructed and behave as predicted. We therefore propose that a-specific mating functions in MATa cells are constitutively expressed due to the absence of the MATα2 gene product and that α-specific mating functions are not expressed due to the absence of the MATα1 gene product.     

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.