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.     

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.     

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.     

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.     

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.     

A PCR-based method for mating type determination inCochliobolus heterostrophus

A rapid and accurate method based on multiplex PCR using three different primers was developed for determining mating type inCochliobolus heterostrophus. The primers of MAT113 and MAT123 were uniquely derived fromMAT1-1 andMAT1-2, respectively, and the primer MATcon5 is conserved in the flanking regions of the idiomorphs. The amplification product was estimated to be 702 bp (MAT1-1) or 547 bp (MAT1-2). Crossing experiments confirmed the accuracy of this method, which requires less time than the conventional determination of mating type.     

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.     

Phylogenetic Analyses on the Diversity of Aspergillus fumigatus Sensu Lato Based on Five Orthologous Loci

One hundred isolates of Aspergillus fumigatus sensu lato mainly from China, as well as from Australia, France, India, Indonesia, Ireland, UK, and USA were analyzed to infer their sequence types (STs) and population diversity based on partial calmodulin, calcineurin regulatory subunit B, beta-tubulin, cytochrome C and calcineurin catalytic subunit A genes as well as their mating types, using ClonalFrame, Structure and MEGA software. Our results inferred 48 STs and showed that most of the STs or lineages evolved independently and without clear population structure among them. Whereas one lineage was recognized that could be a true population and in which one clade might diverge into another distinct lineage, namely, a cryptic species, A. neoellipticus. In addition, we found that mutation, parasexual, and sexual recombination could, respectively, play specific roles in the evolution of these fungi. Our results also showed that MAT1-1/MAT1-2 mating type ratios of A. fumigatus sensu lato was biased to nearly 1:1.4 (20/28) when clone-corrected, but when not clone-corrected, the ratio of MAT1-1/MAT1-2 was so biased as near 1:2 (35/65), which might mean that isolates with MAT1-2 are in the process of losing sexual ability preceding those with MAT1-1.     

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.     

Mating type gene analyses in the genus Diplodia: From cryptic sex to cryptic species

Cryptic species are common in Diplodia, a genus that includes some well-known and economically important plant pathogens. Thus, species delimitation has been based on the phylogenetic species recognition approach using multigene genealogies. We assessed the potential of mating type (MAT) genes sequences as phylogenetic markers for species delimitation in the genus Diplodia. A PCR-based mating type diagnostic assay was developed that allowed amplification and sequencing of the MAT1-1-1 and MAT1-2-1 genes, and determination of the mating strategies used by different species. All species tested were shown to be heterothallic. Phylogenetic analyses were performed on both MAT genes and also, for comparative purposes, on concatenated sequences of the ribosomal internal transcribed spacer (ITS), translation elongation factor 1-alpha (tef1-α) and beta-tubulin (tub2). Individual phylogenies based on MAT genes clearly differentiated all species analysed and agree with the results obtained with the commonly used multilocus phylogenetic analysis approach. However, MAT genes genealogies were superior to multigene genealogies in resolving closely related cryptic species. The phylogenetic informativeness of each locus was evaluated revealing that MAT genes were the most informative loci followed by tef1-α. Hence, MAT genes can be successfully used to establish species boundaries in the genus Diplodia.     

Structure and function of the mating-type locus in the homothallic ascomycete, Didymella zeae-maydis

Homothallic Didymella zeae-maydis undergoes sexual reproduction by selfing. Sequence analysis of the mating type (MAT) locus from this fungus revealed that MAT carries both MAT1-1-1 and MAT1-2-1 genes found in heterothallic Dothideomycetes, separated by ～1.0 kb of noncoding DNA. To understand the mechanistic basis of homothallism in D. zeae-maydis, each of the MAT genes was deleted and the effects on selfing and on ability to cross in a heterothallic manner were determined. The strain carrying an intact MAT1-1-1 but defective MAT1-2-1 gene (MAT1-1-1;ΔMAT1-2-1) was self-sterile, however strains carrying an intact MAT1-2-1 but defective MAT1-1-1 gene (ΔMAT1-1-1;MAT1-2-1), when selfed, showed delayed production of a few ascospores. Attempts to cross the two MAT deletion strains yielded fewer ΔMAT1-1-1;MAT1-2-1 than MAT1-1-1;ΔMAT1-2-1 progeny and very few ascospores overall compared to WT selfs. This study demonstrates that, as in the other homothallic Dothideomycetes, both MAT genes are required for full fertility, but that, in contrast to other cases, the presence of a single MAT1-2-1 gene can induce homothallism, albeit inefficiently, in D. zeae-maydis.     

Improvement of fruiting body production in Cordyceps militaris by molecular assessment

Cordyceps militaris is a heterothallic ascomycetous fungus that has been cultivated as a medicinal mushroom. This study was conducted to improve fruiting body production by PCR assessment. Based on single-ascospore isolates selected from wild and cultivated populations, the conserved sequences of α-BOX in MAT1-1 and HMG-BOX in MAT1-2 were used as markers for the detection of mating types by PCR. PCR results indicated that the ratio of mating types is consistent with a theoretical ratio of 1:1 (MAT1-1:MAT1-2) in wild (66:70) and cultivated (71:60) populations. Cross-mating between the opposite mating types produced over fivefold more well-developed fruiting bodies than self- or cross-mating between strains within the same mating type. This study may serve as a valuable reference for artificial culturing of C. militaris and other edible and medicinal mushrooms and may be useful to develop an efficient process for the selection, domestication, and management of strains for industrial-scale production.     

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.     

Effects of truffle inoculation on a nursery culture substrate environment and seedling of Carya illinoinensis

We inoculated Tuber aestivum and Tuber sinoaestivum on Carya illinoinensis to explore the effects of inoculation on host plant growth, enzyme activities, the physicochemical properties of rhizosphere soil, the denitrifying bacterial community in the rhizosphere, and the distribution of mating type genes in the rhizosphere. We found that the Tuber spp. inoculation increased the height of the host plant and that the stem circumference of the host was greater two months after inoculation. Six months after inoculation, the peroxidase activity of the seedlings inoculated with T. sinoaestivum was higher than that of the control. At four and six months after inoculation, the superoxidase dismutase activities of the seedlings inoculated with T. aestivum were higher than those of the seedlings inoculated with T. sinoaestivum. Six months after inoculation, nitrate nitrogen content was lowest in the control and highest in the T. sinoaestivum treatment. Among the nirS-type denitrifying bacteria community, the relative abundances of Proteobacteria were high. T. aestivum and T. sinoaestivum inoculation did not affect the diversity of denitrifying bacteria. The mating type genes MAT1-1-1 and MAT1-2-1 were detected in the rhizosphere of C. illinoinensis inoculated with T. sinoaestivum and T. aestivum, and MAT1-1-1 dominated over MAT1-2-1.     

Sexual reproduction and recombination in the aflatoxin-producing fungus Aspergillus parasiticus

The fungal phylum Ascomycota comprises a large proportion of species with no known sexual stage, despite high genetic variability in field populations. One such asexual species, Aspergillus parasiticus, is a potent producer of carcinogenic and hepatotoxic aflatoxins, polyketide-derived secondary metabolites that contaminate a wide variety of agricultural crops. In this study, individuals of A. parasiticus from a population showing an evolutionary history of recombination were examined for sexual reproduction. Crosses between strains with opposite mating-type genes MAT1-1 and MAT1-2 resulted in the development of ascospore-bearing ascocarps embedded within stromata. Sexually compatible strains belonged to different vegetative compatibility groups. Recombination through the independent assortment of chromosomes 3 and 6 was detected using loci for mating type, aflatoxin gene cluster, and a protein-encoding gene. Our discovery of the sexual stage in A. parasiticus has important implications for current biological control strategies using nontoxigenic strains to reduce aflatoxin contamination in crops.