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Boeremia was established to accommodate phoma-resembling fungi. Its species occur in terrestrial ecosystems as endophytes, saprobes and pathogens, except one species reported from a marine ecosystem. Boeremia species are characterized by hyaline, thin-walled, and aseptate (occasionally 1(-2)-septate) conidia that are variable in shape, and hyaline, straight or slightly curved, thick-walled, and 1-septate ascospores that are usually constricted at the septum. In the past, host associations were used to delimit Boeremia species. However, since Boeremia taxa have overlapping morphological characters and are cryptic, it renders taxonomic identification arduous. Therefore, the use of other approaches including multi-gene phylogenetic analyses are imperative. Recommended DNA markers for species delineation are the internal transcribed spacer (ITS, nuclear rDNA consisting of ITS1-5.8S-ITS2) and large subunit (28S, D1-D2 domains of nuclear 28S rDNA) loci, and the genes for actin (ACT1), beta-tubulin (TBB1), RNA polymerase 2 (RPB2) and translation elongation factor 1α (TEF1). Here, we applied morphological and molecular phylogenetic analyses to establish a new taxon (B. albae), and a new host and geographical record for B. maritima associated with leaf spots of Morus alba (Moraceae) in northern Thailand. By providing sequence data for three additional gene regions, our phylogenetic analyses impart a stable phylogenetic placement of the ex-type strain of B. maritima, as illustrated. This is the first study that reports Boeremia species from M. alba, and B. maritima from a terrestrial habitat., Competing Interests: Declaration of competing interest On behalf of all co-authors, I confirm that the submitted research is original and has not been published, nor is it currently under consideration for publication elsewhere. We have no conflicts of interest to disclose., (Copyright © 2024 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Eucalyptus spp. in plantations are negatively affected by canker and wilt diseases caused by several species of Ceratocystis, particularly those in the Latin American Clade (LAC). Ceratocystis eucalypticola and Ceratocystis manginecans are of particular concern where disease epidemics are reported globally, with recent outbreaks emerging in South African and Indonesian Eucalyptus plantations. Consequently, a rapid screening protocol is required for these pathogens. In this study, a high-resolution melting curve analysis (HRMA) was developed to detect C. eucalypticola and C. manginecans that bypasses time-consuming isolation and post-PCR procedures. Primers targeting a 172 bp region of the cerato-platanin (CP) gene were designed. Using these primers, the accuracy of HRMA to detect and distinguish between these two LAC species was assessed using pure fungal DNA, and DNA extracted directly from Eucalyptus samples naturally infected with C. eucalypticola. The assay accurately detected the presence of C. eucalypticola and C. manginecans and quantifies their DNA, both from cultures, and directly from wood samples. HRMA further differentiated these two species from all other tested LAC individuals. This assay was also able to detect the presence of all the tested LAC species and distinguish seven of these, including C. fimbriata, to species level. Ceratocystis polyconidia was the only non-LAC off-target species detected. Based on these results, the developed assay can be used to rapidly identify C. eucalypticola and C. manginecans directly from infected plant material or fungal cultures, with the potential to also screen for several other LAC species., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The cajuzinho do cerrado (Anacardium humile-Anacardiaceae), a shrub species native to Brazil, is harvested for multiple uses in food and medicine. Members of a harvesting community, near the municipality of Bonito de Minas, Minas Gerais state, Brazil reported characteristic symptoms of shoot blight and dieback reducing pseudofruit and seed production by this plant. This study aimed to identify the etiological agent of this disease. Two fungal isolates were obtained from symptomatic leaf samples and morphologically and molecularly characterized. The fungus was identified, based on morphological analyses, as a probable new species of Pseudoplagiostoma. Phylogenetic analyses based on a combination of DNA sequence data (nuc rDNA ITS1-5.8S-ITS2 region, tef1-α and tub2), confirmed this hypothesis. The isolates obtained were allocated to a distinct, well-supported clade (IB = 0.99, ML = 100%), placed as a unique lineage here proposed as a new species named Pseudoplagiostoma humilis. The pathogenicity test confirmed that this new species was the causal agent of shoot blight and dieback on A. humile. This is the fourteenth Pseudoplagiostoma species reported in the world and the third in Brazil., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Detection and quantification of pathogen propagules in the air or other environmental samples is facilitated by culture-independent assays. We developed a quantitative PCR assay for the hop powdery mildew fungus, Podosphaera macularis , for detection of the organism from air samples. The assay uses primers and a TaqMan probe designed to target species-specific sequences in the 28S large subunit of the nuclear ribosomal DNA. Analytical sensitivity was not affected by the presence of an exogenous internal control or potential PCR inhibitors associated with DNA extracted from soil. The level of quantification of the assay was between 200 and 350 conidia when DNA was extracted from a fixed number of conidia. The assay amplified all isolates of P. macularis tested and had minimal cross-reactivity with other Podosphaera species when assayed with biologically relevant quantities of DNA. Standard curves generated independently in two other laboratories indicated that assay sensitivity was qualitatively similar and reproducible. All laboratories successfully detected eight unknown isolates of P. macularis and correctly discriminated Pseudoperonospora humuli and a water control. The usefulness of the assay for air sampling for late-season inoculum of P. macularis was demonstrated in field studies in 2019 and 2020. In both years, airborne populations of P. macularis in hop yards were detected consistently and increased during bloom and cone development., Competing Interests: The author(s) declare no conflict of interest.

Eucalyptus spp. are undoubtedly one of the most favored plantation trees globally. Accurately identifying Eucalyptus pathogens is therefore crucial for timely disease prevention and control. Recently, symptoms of a leaf blight disease were observed on Eucalyptus trees in plantations at Jhajjar and Karnal in the state of Haryana, northern India. Asexual morphs resembling the features of the Botryosphaeriaceae were consistently isolated from the symptomatic leaves. Morphological features coupled with DNA sequence analysis confirmed a novel species, which is described and illustrated here as Botryosphaeria eucalypti sp. nov. Conidia of the new taxon are longer and wider than those of its phylogenetic neighbors. A distinct phylogenetic position for the new taxon was established through combined analysis of the internal transcribed spacer (ITS), partial translation elongation factor-1α (tef1) and partial β-tubulin (tub2) regions. Recombination analysis provided additional support for the new species hypothesis. The pathogenicity of the novel species was proved on Eucalyptus leaves, and Koch's postulates were fulfilled. The discovery of new Botryosphaeria species is important because it will help in understanding the species diversity, host range, possible threats and disease control in the long run., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The genetic variety and habitats of Camptophora species, generally known as black yeast, have not been clarified. In this study, we re-evaluated Camptophora based on morphological observations and phylogenetic analyses. Because prior investigations on Camptophora only included a few strains/specimens, 24 Camptophora-related strains were newly obtained from 13 leaf samples of various plant species to redefine the genetic and species concepts of Camptophora. Their molecular phylogenetic relationships were examined using small subunit nuclear ribosomal DNA (nSSU, 18S rDNA), the internal transcribed spacer (ITS) rDNA operon, the large subunit nuclear ribosomal DNA (LSU, 28S rDNA), β-tubulin, the second largest subunit of RNA polymerase II (rpb2), and mitochondrial small subunit DNA (mtSSU). Single- and multi-locus analyses using nSSU-ITS-LSU-rpb2-mtSSU revealed a robust phylogenetic relationship among Camptophora species within Chaetothyriaceae. Camptophora species could be distinguished from other chaetothyriaceous genera by their snake-shaped conidia with microcyclic conidiation and loosely interwoven mycelial masses. Based on the results of phylogenetic analyses, two undescribed lineages were recognized, and Ca. schimae was excluded from the genus. ITS sequence comparison with environmental DNA sequences revealed that the distribution of the genus is restricted to the Asia-Pacific region. Camptophora has been isolated or detected from abrupt sources, and this was attributed to its microcycle. The mechanisms driving genetic diversity within species are discussed with respect to their phyllosphere habitats., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: Lichens, traditionally considered as a simple partnership primarily between mycobiont and photobiont, are, in reality, complex holobionts comprised of a multitude of microorganisms. Lichen mycobiome represents fungal community residing within lichen thalli. While it is acknowledged that factors like the host lichen species and environmental conditions influence the structure of the lichen mycobiome, the existing research remains insufficient. To investigate which factor, host genus or location, has a greater impact on the lichen mycobiome, we conducted a comparative analysis of mycobiomes within Parmelia and Peltigera collected from both Turkey and South Korea, using high-throughput sequencing based on internal transcribed spacer region amplification., Results: Overall, the lichen mycobiome was dominated by Capnodiales (Dothideomycetes), regardless of host or location. At the order level, the taxonomic composition was not significantly different according to lichen genus host or geographical distance. Hierarchical clustering of the top 100 abundant ASVs did not clearly indicate whether the lichen mycobiome was more influenced by host genus or location. Analyses of community similarity and partitioning variables revealed that the structure of the lichen mycobiome is more significantly influenced by location than by host genus. When analyzing the core mycobiome by host genus, the Peltigera mycobiome contained more ASV members than the Parmelia mycobiome. These two core mycobiomes also share common fungal strains, including basidiomycete yeast. Additionally, we used chi-squared tests to identify host genus-specialists and location-specialists., Conclusions: By comparing lichen mycobiomes of the same genera across different countries, our study advances our comprehension of these microbial communities. Our study elucidates that, although host species play a contributory role, geographic distance exerts a more pronounced impact on the structure of lichen mycobiome. We have made foundational contributions to understanding the lichen mycobiome occupying ecologically crucial niches. We anticipate that broader global-scale investigations into the fungal community structures will provide more detailed insights into fungal residents within lichens., (© 2024. The Author(s).)

Most species of Dothiora are known from the dead parts of various host plants as saprobic fungi in terrestrial habitats occurring in tropical and temperate regions. In the present study, samples of Dothiora were collected from dead twigs and branches of Capparis spinosa , Rhaponticum repens , and an unknown angiosperm plant from the Tashkent and Jizzakh regions of Uzbekistan. Multi-gene phylogenetic analyses based on a combined ITS, LSU, SSU, TEF1 , and TUB2 sequence data revealed their taxonomic positions within the Dothideaceae. Three new species of Dothiora , namely, Dothiora capparis , Dothiora rhapontici , and Dothiora uzbekistanica were proposed by molecular and morphological data. Likewise, the phylogenetic relationship and morphology of Dothiora are discussed. In addition, we provide a list of accepted Dothiora species, including host information, distribution, morphology descriptions, and availability of sequence data, to enhance the current knowledge of the diversity within Dothiora ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author SH declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Senwanna, Hongsanan, Khuna, Kumla, Yarasheva, Gafforov, Abdurazakov and Suwannarach.)

Many fungi require specific growth conditions before they can be identified. Direct environmental DNA sequencing is advantageous, although for some taxa, specific primers need to be used for successful amplification of molecular markers. The internal transcribed spacer region is the preferred DNA barcode for fungi. However, inter- and intra-specific distances in ITS sequences highly vary among some fungal groups; consequently, it is not a solely reliable tool for species delineation. Ampelomyces, mycoparasites of the fungal phytopathogen order Erysiphales, can have ITS genetic differences up to 15%; this may lead to misidentification with other closely related unknown fungi. Indeed, Ampelomyces were initially misidentified as other pycnidial mycoparasites, but subsequent research showed that they differ in pycnidia morphology and culture characteristics. We investigated whether the ITS2 nucleotide content and secondary structure was different between Ampelomyces ITS2 sequences and those unrelated to this genus. To this end, we retrieved all ITS sequences referred to as Ampelomyces from the GenBank database. This analysis revealed that fungal ITS environmental DNA sequences are still being deposited in the database under the name Ampelomyces, but they do not belong to this genus. We also detected variations in the conserved hybridization model of the ITS2 proximal 5.8S and 28S stem from two Ampelomyces strains. Moreover, we suggested for the first time that pseudogenes form in the ITS region of this mycoparasite. A phylogenetic analysis based on ITS2 sequences-structures grouped the environmental sequences of putative Ampelomyces into a different clade from the Ampelomyces-containing clades. Indeed, when conducting ITS2 analysis, resolution of genetic distances between Ampelomyces and those putative Ampelomyces improved. Each clade represented a distinct consensus ITS2 S2, which suggested that different pre-ribosomal RNA (pre-rRNA) processes occur across different lineages. This study recommends the use of ITS2 S2s as an important tool to analyse environmental sequencing and unveiling the underlying evolutionary processes., Competing Interests: No authors have competing interests.

Human infections by pleosporalean fungi (class Dothideomycetes, phylum Ascomycota) are rarely reported. Because their identification is challenging using morphological characterization, several phylogenetic markers must be sequenced for an accurate identification and taxonomical placement of the isolates. Three isolates of clinical origin were phenotypically characterized, but due to the absence of relevant morphological traits, D1-D2 domains of the 28S nrRNA gene (LSU), the internal transcribed spacer region (ITS) of the nrRNA, and fragments of the RNA polymerase II subunit 2 (rpb2) and translation elongation factor 1-alpha (tef1) genes were sequenced to allow a phylogenetic analysis that would solve their phylogenetic placement. That analysis revealed that these isolates did not match any previously known pleosporalean genera, and they are proposed here as the new fungal genus, Gambiomyces. Unfortunately, the isolates remained sterile, which, consequently, made the morphological description of the reproductive structures impossible. Future studies should try to understand the behaviour of this fungus in nature as well as its characteristics as an opportunistic fungal pathogen. Molecular identification is becoming an essential tool for proper identification of Dothideomycetes of clinical origin., Lay Abstract: We describe a new pleosporalen pathogenic fungus, Gambiomyces profunda, found in superficial to deep samples from a human patient. Because all strains remained sterile, the fungus was finally identified following a phylogenetic analysis by using four different molecular markers., (© The Author(s) 2020. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Three species of Xenosporium producing ellipsoidal or ovoid conidia were collected from decaying culms of Miscanthus floridulus submerged in freshwater streams of Alishan area, Chiayi County, Taiwan. Xenosporium formosiforme and X. parvulum are described and illustrated as new species. Xenosporium africanum is described in this paper as a new record for Taiwan. The phylogenetic relationships of Xenosporium species and related taxa were sought by comparing the sequences of their internal transcribed spacer (ITS) barcode, 28S rDNA, and RNA polymerase II second largest subunit ( RPB2 ) gene segments. Phylogenetic analysis of these Xenosporium species supports their placement in the Tubeufiaceae. A synopsis of Xenosporium species producing ellipsoidal or ovoid conidia is given. The criteria used to differentiate species are discussed.

Dictyochaeta (Chaetosphaeriaceae) is a phialidic dematiaceous hyphomycete with teleomorphs classified in Chaetosphaeria . It is associated with significant variability of asexual morphological traits, which led to its broad delimitation. In the present study, six loci: nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS barcode), nuc 18S rDNA (18S), nuc 28S rDNA (28S), DNA-directed RNA polymerase II second largest subunit gene ( RPB2 ), translation elongation factor 1-α ( TEF1-α ), and β-tubulin ( TUB2 ), along with comparative morphological and cultivation studies, are used to reevaluate the concept of Dictyochaeta and establish species boundaries. Based on revised species, morphological characteristics of conidia (shape, septation, absence or presence of setulae), collarettes (shape), and setae (presence or absence) and an extension of the conidiogenous cell proved to be important at the generic level. The dual DNA barcoding using ITS and TEF1-α , together with TUB2 , facilitated accurate identification of Dictyochaeta species. Thirteen species are accepted, of which seven are characterized in this study; an identification key is provided. It was revealed that D. fuegiana , the type species, is a complex of three distinct species including D. querna and the newly described D. stratosa . Besides, a new species, D. detriticola , and two new combinations, D. callimorpha and D. montana , are proposed. An epitype of D. montana is selected. Dictyochaeta includes saprobes on decaying wood, bark, woody fruits, and fallen leaves. Dictyochaeta is shown to be distantly related to the morphologically similar Codinaea , which is resolved as paraphyletic. Chaetosphaeria talbotii with a Dictyochaeta anamorph represents a novel lineage in the Chaetosphaeriaceae; it is segregated from Dictyochaeta , and a new genus Achrochaeta is proposed. Multigene phylogenetic analysis revealed that D. cylindrospora belongs to the Vermiculariopsiellales, and a new genus Tubulicolla is introduced.

Plant disease is one of the most important causes of crop losses worldwide. The effective control of plant disease is related to food security. Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum leads to serious yield losses in rapeseed (Brassica napus) production. Hypovirulent strain DT-8 of S. sclerotiorum, infected with Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), has the potential to control SSR. In this study, we found rapeseed bio-priming with strain DT-8 could significantly decrease the disease severity of SSR and increase yield in the field. After bio-priming, strain DT-8 could be detected on the aerial part of the rapeseed plant. By 16S rRNA gene and internal transcribed spacer (ITS) sequencing technique, the microbiome on different parts of the SSR lesion on bioprimed and non-bioprimed rapeseed stem was determined. The results indicated that SSR and bio-priming treatment could influence the structure and composition of fungal and bacterial communities. Bio-priming treatment could reduce the total abundance of possible plant pathogens and enhance the connectivity and robustness of the interaction network at the genus level. This might be one of the mechanisms that rapeseed bioprimed with strain DT-8 had excellent tolerance on SSR. It might be another possible mechanism of biocontrol and will provide a theoretical guide for agricultural practical production.

Truffles in the genus Tuber are hypogeus fungi that have a worldwide distribution. Despite this, knowledge about their diversity in the Middle East is very limited. In recent years, large quantities of truffles have been imported from Iran for being sold in Italy. While analyzing certain commercial batches of T. aestivum from Iran, we found some ascomata that resembled T. excavatum but had macro- and micromorphological features that were distinct from this species. They were subglobose, or depressed to slightly irregular, with a conspicuous basal cavity, grayish brown, brown, or pinkish gray, with a minutely papillose peridium. The gleba was pinkish gray in youth, brown at maturity, marbled with cream branched veins. Ascospores were broadly ellipsoid, with an irregular reticulum and distinctive long crests along the longitudinal axis, up to 9 µm high. Analysis of internal transcribed spacer (ITS) and large subunit (LSU) rDNA sequences showed that these specimens form a monphyletic and well-supported taxon within the Excavatum clade. Morphological and molecular analyses supported the proposal of the new species T. iranicum .

Morels are precious and rare edible mushroom, which contain 61 species. It is difficult to identify the species of morels based on morphological characteristics. In recent years, morels cultivation technology has made great progress, Morchella sextelata has been cultivated in China widely. In this study, a PCR-based approach was developed for identification of Morchella sextelata, based on a 190-bp segment of internal transcribed spacer (ITS). The species-specific primer, MS1F/MS1R, was designed based on the alignment of sequence obtained from the 20 representative cultivated body fruit of Morchella sextelata as well as 10 sequence data available in Genbank. Verifying the species-specific primer pair demonstrated generation of an amplicon from extracted DNA of Morchella sextelata. The primer pair was also applied and tested for five other species of morels and eight common mushrooms from other genus. This novel PCR assay allowed a rapid and effective quality evaluation of Morchella sextelata.

Exploring the secondary structure information of nuclear ribosomal internal transcribed spacer 2 (ITS2) has been a promising approach in species delimitation. However, Compensatory base changes (CBC) concept employed in this approach turns futile when CBC is absent. This prompted us to investigate the utility of insertion/deletion (INDELs) and substitutions in fungal delineation at species level. Upon this rationale, 116 strains representing 97 species, belonging to 6 genera (Colletotrichum, Boeremia, Leptosphaeria, Peyronellaea, Plenodomus and Stagonosporopsis) of Ascomycota were retrieved from Q-bank for molecular morphometric analysis. CBC, INDELs and substitutions between the species of their respective genus were recorded. Most species combinations lacked CBC. Among the substitution events, transitions were predominant. INDELs were less frequent than the substitutions. These evolutionary events were mapped upon the helices to discern species specific variation sites. In 68 species unique variation sites were recognised. The remaining 29 species shared absolute similarity with distinctly named species. The variation sites catalogued in them overlapped with other distinct species and resulted in the blurring of species boundaries. Species specific variation sites recognized in this study are the preliminary results and they could be discerned with absolute confidence when larger datasets encompassing all described species of genera were investigated. They could be of potential use in barcoding fungi at species level. This study also concludes that the ITS2 molecular morphometric analysis is an efficient third dimensional study of the fungal species delimitation. This may help to avoid the false positives in species delimitations and to alleviate the challenges in molecular characterization., (Copyright © 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Desert truffles (mycorrhizal hypogeous Ascomycota) are found in arid and semi-arid areas of the globe and have great ecological and economic importance. Terfezia is undoubtedly the most diversified of all desert truffle genera, but its taxonomy is far from resolved. Specifically, the large number of newly described species plus the high intraspecific morphological variability observed within some Terfezia lineages as rendered the use of molecular techniques mandatory for specimen's discrimination. But the subsequent increasing amount of sequence data produced also a huge number of undescribed taxa that required determination. We compiled and used the public available ITS data on Terfezia spp. on the custom-curated UNITE database to reconstruct the genus phylogeny. We found at least 17 distinct lineages within the genus and successfully resolved some of the more pressing taxonomic issues, namely the T. leptoderma/olbiensis complex and some misapplied synonymy. Based on this resolved phylogeny, and motivated by the recent new described species, we proposed an identification key to Terfezia genus highlighting the importance of morphological and ecological characterization., (Copyright © 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Fungal spoilage remains a significant issue in dairy product quality, especially for cultured dairy products such as yogurt formulated without preservatives such as potassium sorbate. Fungal contamination can occur throughout the processing continuum, from the dairy farm environment to the finished product processing environment. As molecular characterization of fungal isolates is used more frequently, we obtained fungal isolates obtained in 2 yogurt processing facilities as part of routine fungal testing of raw materials (e.g., fruit preparations, added ingredients), in-process product samples, environmental samples (e.g., air plates, equipment surfaces such as valves, face plates, air nozzles), and finished product samples, to determine whether internal transcribed spacer (ITS) barcoding data would be helpful to support source tracking of fungal contamination issues. Internal transcribed spacer PCR amplification and sequencing allowed us to classify the 852 isolates from these 2 facilities into 200 unique ITS allelic types (AT), representing the phyla Ascomycota (743 isolates), Basidiomycota (97 isolates), and Mucoromycota (12 isolates). Thirty ITS AT were isolated from both facilities; 62 and 108 ITS AT were isolated from only facility A or only facility B, respectively. Nine ITS AT were each represented by more than 20 isolates; these AT comprised 53% of the 852 isolates. The considerable diversity of fungal isolates even within a single facility illustrates the challenge associated with controlling fungal contamination of dairy products. The ITS barcoding technique, however, did show promise for facilitating the source tracking of fungal contamination, particularly for ITS AT over-represented in a given facility. For example, we found evidence for equipment-specific reservoirs for 2 AT (14 and 219) in facility B. Our data suggest that despite its limited discriminatory power, ITS sequencing can provide initial information that can help trace fungal contamination along the processing continuum. However, development and implementation of discriminatory subtyping methods will be needed to further improve the ability to identify sources of fungal contamination in dairy facilities. Developing and implementing sampling plans that comprehensively capture yeast and mold diversity in a given processing facility remain a considerable challenge., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Neoscytalidium (or N.) dimidiatum and N. novaehollandiae are two aggressive plant pathogenic species that affect several agricultural crops. Early detection and identification of these fungi are of critical importance to bring about the effective minimization to the threat they pose to the infected plants. Herein, two species of Neoscytalidium were rapidly discriminated by utilizing the rRNA internal transcribed (ITS4-5.8S-ITS5) PCR primers. A total of 100 isolates of Neoscytalidium species, which were isolated from Iraqi canker-infected fig trees, were included in this study. Two discrete electrophoretic PCR bands were observed in Neoscytalidium isolates-A-variants were about 546 bp, while B-variants were about 993 bp in length. The comprehensive phylogenetic analysis of both DNA variants revealed that A-variants resided between N. novaehollandiae and N. hyalinum, while B-variants were closely related to N. dimidiatum. Furthermore, the highly specific re-constructed tree of both electrophoretic variants demonstrated that B-variants share a high similarity with N. novaehollandiae. Additionally, the secondary structures for both variants were predicted computationally to reveal the structural patterns that each variant follows. In conclusion, a small rRNA locus comprising 22 nucleotides that differs in the two variants is potentially responsible for this species-specific classification. The main divergence in the amplified loci led to the classification of these fungal variants into two main species, namely N. dimidiatum and N. novaehollandiae, demonstrating that the amplification by ITS4-ITS5 rRNA fragment is a beneficial strategy that can be employed for the assessment of Neoscytalidium diversity in the natural ecosystems.

Previous studies for enumerating fungal communities on pine needles relied entirely on phenotypic diversity (microscopy) or identification based on DNA sequence data from those taxa recovered via cultural studies. To bypass limitations of the culturing methods and provide a more realistic diversity estimate, we employed and assessed a PCR-DGGE based method coupled with rDNA phylogenetic sequence analyses to characterize fungal taxa associated with pine needles. Fresh (living) and decayed needles from three hosts of the Pinaceae (Keteleeria fortunei, Pinus elliottii and P. massoniana) were examined. Morphological studies reveal that the most abundant species associated with decayed needles were Cladosporium cladosporioides and an unidentified Trichoderma species followed by Gliocephalotrichum sp., Gliocladium sp., Lophodermium pinastri, Paecilomyces varioti, Phaeostalagmus cyclosporus and a Phoma sp, which are commonly occurring fungi. Community genomic data from freshly collected and decayed pine needles recovered 40 operational taxonomic units, which appear to be mostly undetected members of the natural fungal consortium. Sequence analyses revealed a number of phylotypes or "species" that were not recovered using traditional morphological and cultural approaches previously used. Phylogenetic data from partial 18S rDNA sequence data reveal that most phylotypes represent potential novel phylogenetic fungal lineages with affinities to the Dothideomycetes, Leotiomycetes, Lecanoromycetes and Sordariomycetes and were not identical to previously known endophytes or saprobes. Although the major ecological roles of these phylotypes in pine needles are still enigmatic, this study provides new insights in hidden fungal diversity that mycologists are possibly ignoring given the discrepancies associated with available methods. To what extent do previously recovered identified species (either as saprobes or endophytes) from morphological or culturing studies act as pioneer decomposers or constitute an integral part of endophytic community warrants further investigation.

Determination and assessment of airborne fungal particles is complex and results of different sampling and analytical strategies are hard to compare due to limitations of each of the techniques. Here, an indoor mold detection system based on quantitative polymerase chain reaction (qPCR) is described and validated for its reliability and stability to identify airborne fungal particles collected. Data obtained from testing the system with fungal DNA, spore suspensions and bioaerosols indicated a need for spiking and normalization of measurements due to material loss and assay specific bias. Considering the loss of material during sample processing, detection limits defined for suspensions of Tritirachium oryzae spores were roughly 18 spores per sample. Detection of fungal spore mixtures nebulized under controlled conditions in a bioaerosol chamber showed generally 2-3 times higher normalized values measured with the molecular system compared to cultivation. Data obtained from a mold infested indoor sampling site and its corresponding outdoor reference measurement showed good correlations between qPCR and high-throughput sequencing (rho = 0.83, p < 0.01), if Cladosporium species were excluded. Taking necessary data normalization into account, the described qPCR detection system shows great potential to complement commonly used culture based approaches with the aim to improve the precision of indoor mold assessments. In contrast to already available qPCR assays that detect certain molds on a species level, this system covers a broad range of relevant fungal communities, serving as a promising alternative to high-throughput sequencing to identify indoor molds.

The taxonomy of the fungi that produce human infections and that develop asexual fruiting bodies in culture has become very complex. Recent molecular studies have produced dramatic changes in their classification. Currently, the coelomycetes traditionally included in Sphaeropsidales and Melanconiales are in fact distributed across at least three different classes of the Phylum Ascomycota. Approximately 1000 genera and 7000 species have been grouped in the classes Dothideomycetes, Leotiomycetes and Sordariomycetes and their proper identification can only be made by analysing their DNA sequences and comparing them with those corresponding to type strains available in the adequate databases. To facilitate this task for scientists and clinicians involved in the study of these complex, and every day more numerous taxa, we have updated the knowledge about the taxonomy of the commonest coelomycetes of clinical interest with the aim of improving their identification and antifungal treatment., (© 2018 Blackwell Verlag GmbH.)

Botryosphaeria dieback, caused by species of Botryosphaeriaceae, is an important grapevine trunk disease in Australia. Inocula produced by the pathogens are primarily dispersed by rain splash and wind and infect pruning wounds leading to cankers, dieback, and eventually death of vines. The objective of this study was to develop molecular tools to detect and quantify Botryosphaeriaceae inocula from the environment. These tools are essential for investigating spore dispersal patterns of Botryosphaeriaceae pathogens in Australian vineyards. DNA extraction protocols were evaluated and one modified protocol was found suitable for extracting Botryosphaeriaceae DNA from artificially and naturally inoculated Burkard volumetric spore sampler tapes. Multispecies primers and a hydrolysis probe for quantitative PCR (qPCR) were further developed to detect and quantify Botryosphaeriaceae inocula from environmental samples. Specificity tests showed that the multispecies primers were able to amplify the DNA of 10 Botryosphaeriaceae species (58 isolates) found in Australia while none of the 27 nontarget fungal species (90 isolates) tested were amplified. The qPCR assay was suitable for amplifying purified DNA, synthetic DNA fragments (gBlocks), and mixed DNA from spore trap tapes. The qPCR method developed in this study was shown to be rapid and sensitive in detecting Botryosphaeriaceae inocula from the environment using spore traps.

Fonsecaea pedrosoi, a melanized fungal pathogen that causes Chromoblastomycosis, a human disease with a worldwide distribution. Biolistic is a widely used technique for direct delivery of genetic material into intact cells by particles bombardment. Another well-established transformation method is Agrobacterium-mediated transformation (ATMT), which involves the transfer of a T-DNA from the bacterium to the target cells. In F. pedrosoi there are no reports of established protocols for genetic transformation, which require optimization of physical and biological parameters. In this work, intact conidia of F. pedrosoi were particle bombarded and subjected to ATMT. In addition, we proposed hygromycin B, nourseothricin and neomycin as dominant selective markers for F. pedrosoi and vectors were constructed. We tested two parameters for biolistic: the distance of the particles to the target cells and time of cells recovery in nonselective medium. The biolistic efficiency was 37 transformants/μg of pFpHYG, and 45 transformants/μg of pAN7.1. Transformants expressing GFP were successfully obtained by biolistic. A co-culture ratio of 10: 1 (bacterium: conidia) and co-incubation time of 72 h yielded the largest number of transformants after ATMT. Southern blot analysis showed the number of foreign DNA insertion into the genome is dependent upon the plasmid used to generate the mutants. This work describes for the first time two efficient methods for genetic modification of Fonsecaea and these results open new avenues to better understand the biology and pathogenicity of the main causal agent of this neglected disease., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

The dispersion of pathogenic microorganisms consists of the transport of pathogens from their source to inoculate a new host. Agricultural and economic importance of the Soybean root rot justifies studying this disease, especially the role of insects as dispersers. The aim of this study was to evaluate the role of the ladybird beetle, Cycloneda sanguinea Linnaeus (Coleoptera: Coccinellidae) in the dispersion of pathogens that cause Soybean root rot. Three pathogen species, Macrophomina phaseolina (Tassi) (Sphaeropsidales: Botryosphaeriaceae), Fusarium incarnatum-equiseti species complex (FIESC), and F. commune (Skovgaard) O'Donnell & Nirenberg were isolated from the midgut of ladybird beetles and cultured. Macrophomina phaseolina was identified by morphology while for the other two species, DNA was sequenced. The DNA extracted was amplified in the Internal Transcriber Spacer (ITS) region, sequenced and compared to voucher sequences deposited in the GenBank. Sequences of nucleotide ITS1-5.8 S were identified in the regions of rDNA-ITS4 ribosomal DNA. This is the first report of Macrophomina phaseolina (Tassi) (Sphaeropsidales: Botryosphaeriaceae), Fusarium incarnatum-equiseti species complex (FIESC), and F. commune (Skovgaard) O'Donnell & Nirenberg, being dispersed by C. sanguinea in Brazilian soybean fields.

Background: Chromoblastomycosis (CBM) is a chronic fungal disease. In China, the principle etiologic agent was a group of dematiaceous fungi, including Fonsecaea monophora, Fonsecaea nubica, and Cladophialophora carrionii. Although the Fonsecaea species have similar morphology, their pathogenicity is quite different. This study aims to establish a new solution for early identification of Fonsecaea species because of their distinctive potential infection risk., Methods: Five reference strains and 35 clinical isolates from patients with CBM, preserved in our laboratory, were used in this study. The universal primer ITS1 and ITS2 were chosen to amplify the highly conserved regions of rDNA. High-resolution melting (HRM) analysis was performed using the LIGHTCYCLER ® 96 System. All the amplicons were verified by direct sequencing and the sequence were aligned with those in GenBank by BLAST analysis., Results: We successfully differentiated the five strains according to their different Tm values and curve shapes. The 35 clinical isolates from patients were identified as 24 strains for F. monophora and 11 strains for F. nubica, which is consistent with the DNA sequencing results., Conclusion: It is the first time to use HRM analysis for identification of Fonsecaea species. Since the CBM etiologic agent in South China is mainly F. monophora and F. nubica, this strategy is sufficient to be applied in the clinical examination with high accuracy, speed, and throughput., (© 2017 Wiley Periodicals, Inc.)

White-nose syndrome, first diagnosed in North America in 2006, causes mass deaths among bats in North America. We found the causative fungus, Pseudogymnoascus destructans, in a 1918 sample collected in Europe, where bats have now adapted to the fungus. These results are consistent with a Eurasian origin of the pathogen.

Taxa in the genus Melanelia (Parmeliaceae, Ascomycota) belong to a group of saxicolous lichens with brown to black foliose thalli, which have recently undergone extensive changes in circumscription. Taxa belonging to Parmeliaceae are prolific producers of bioactive compounds, which have also been traditionally used for chemotaxonomic purposes. However, the chemical diversity of the genus Melanelia and the use of chemical data for species discrimination in this genus are largely unexplored. In addition, identification based on morphological characters is challenging due to few taxonomically informative characters. Molecular identification methods, such as DNA barcoding, have rarely been applied to this genus. This study aimed to identify the Melanelia species from Iceland using DNA barcoding approach, and to explore their chemical diversity using chemical profiling. Chemometric tools were used to see if lichen metabolite profiles determined by LC-MS could be used for the identification of Icelandic Melanelia species. Barcoding using the fungal nuclear ribosomal internal transcribed spacer region (nrITS) successfully identified three Melalenlia species occurring in Iceland, together with Montanelia disjuncta (Basionym: Melanelia disjuncta). All species formed monophyletic clades in the neighbor-joining nrITS gene tree. However, high intraspecific genetic distance of M. stygia suggests the potential of unrecognized species lineages. Principal component analysis (PCA) of metabolite data gave a holistic overview showing that M. hepatizon and M. disjuncta were distinct from the rest, without the power to separate M. agnata and M. stygia due to their chemical similarity. Orthogonal partial least-squares to latent structures-discriminate analysis (OPLS-DA), however, successfully distinguished M. agnata and M. stygia by identifying statistically significant metabolites, which lead to class differentiation. This work has demonstrated the potential of DNA barcoding, chemical profiling and chemometrics in identification of Melanelia species.

The current literature reveals that the intrathalline coexistence of multiple microalgal taxa in lichens is more common than previously thought, and additional complexity is supported by the coexistence of bacteria and basidiomycete yeasts in lichen thalli. This replaces the old paradigm that lichen symbiosis occurs between a fungus and a single photobiont. The lichen Ramalina farinacea has proven to be a suitable model to study the multiplicity of microalgae in lichen thalli due to the constant coexistence of Trebouxia sp. TR9 and T. jamesii in long-distance populations. To date, studies involving phycobiont diversity within entire thalli are based on Sanger sequencing, but this method seems to underestimate the diversity. Here, we aim to analyze both the microalgal diversity and its community structure in a single thallus of the lichen R. farinacea by applying a 454 pyrosequencing approach coupled with a careful ad hoc-performed protocol for lichen sample processing prior to DNA extraction. To ascertain the reliability of the pyrosequencing results and the applied bioinformatics pipeline results, the thalli were divided into three sections (apical, middle and basal zones), and a mock community sample was used. The developed methodology allowed 40448 filtered algal reads to be obtained from a single lichen thallus, which encompassed 31 OTUs representative of different microalgae genera. In addition to corroborating the coexistence of the two Trebouxia sp. TR9 and T. jamesii taxa in the same thallus, this study showed a much higher microalgal diversity associated with the lichen. Along the thallus ramifications, we also detected variations in phycobiont distribution that might correlate with different microenvironmental conditions. These results highlight R. farinacea as a suitable material for studying microalgal diversity and further strengthen the concept of lichens as multispecies microecosystems. Future analyses will be relevant to ecophysiological and evolutionary studies to understand the roles of the multiple photobionts in lichen symbioses.

Citrus greasy spot (CGS) is a disease of citrus with worldwide distribution and recent surveys have revealed a high level of incidence and severity of symptoms of the disease in Sicily, southern Italy. Although Mycosphaerel la citri (anamorph Zasmidium citri-griseum) and other related species are generally considered as causal agents, the etiology of CGS is still unclear. Here, we report the use of an amplicon metagenomic approach to investigate the fungal communities on citrus leaves symptomatic or asymptomatic for CGS from an orchard in Sicily showing typical CGS symptoms. A total of 35,537 high-quality chimeric free reads were obtained and assigned to 176 operational taxonomic units (OTUs), clustered at 99 % similarity threshold. Data revealed a dominating presence of the phylum Ascomycota (92.6 %) over other fungal phyla. No significant difference was observed between symptomatic and asymptomatic leaves according to both alpha and beta diversity analyses. The family Mycosphaerellaceae was the most abundant and was represented by the genera Ramularia, Mycosphaerella, and Septoria with 44.8, 2.4, and 1.7 % of the total detected sequences, respectively. However, none of the species currently reported as causal agents of CGS was detected in the present study. The most abundant sequence type (ST) was associated to Ramularia brunnea, a species originally described to cause leaf spot in a perennial herbaceous plant of the family Asteraceae. Results exclude that CGS symptoms observed in Sicily are caused by Z. citri-griseum and, moreover, they indicate that a considerable part of the fungal diversity in citrus leaves is still unknown.

Lasiodiplodia species (Botryosphaeriaceae, Ascomycota) infect a wide range of typically woody plants on which they are associated with many different disease symptoms. In this study, we determined the identity of Lasiodiplodia isolates obtained from baobab (Adansonia species) trees in Africa and reviewed the molecular markers used to describe Lasiodiplodia species. Publicly available and newly produced sequence data for some of the type strains of Lasiodiplodia species showed incongruence amongst phylogenies of five nuclear loci. We conclude that several of the previously described Lasiodiplodia species are hybrids of other species. Isolates from baobab trees in Africa included nine species of Lasiodiplodia and two hybrid species. Inoculation trials with the most common Lasiodiplodia species collected from these trees produced significant lesions on young baobab trees. There was also variation in aggressiveness amongst isolates from the same species. The apparently widespread tendency of Lasiodiplodia species to hybridise demands that phylogenies from multiple loci (more than two and preferably four or more) are compared for congruence prior to new species being described. This will avoid hybrids being incorrectly described as new taxa, as has clearly occurred in the past., (Copyright © 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Fonsecaea pedrosoi sensu lato is a major causative agent of dematiaceous fungal infection in Japan. Recent sequence analysis of the internal transcribed spacer (ITS) regions of the ribosomal RNA gene has shown that this species can be separated into three species: F. pedrosoi sensu stricto, F. monophora and F. nubica. The cell walls of dematiaceous fungi including the genus Fonsecaea contain melanin, which is important for their virulence. Polyketide synthase (PKS1) is an enzyme required for melanin synthesis. This study analyzed the phylogeny of strains of F. pedrosoi sensu lato isolated in Japan by sequencing the PKS1 gene and ITS regions and identifying molecular polymorphism. Sixty strains morphologically identified as F. pedrosoi isolated worldwide, including 37 strains isolated in Japan, were analyzed. ITS regions of the ribosomal RNA gene and part of the PKS1 gene region were amplified, yielding sequences of approximately 600 and 450 bp, respectively. Polymerase chain reaction products were sequenced, and cluster analysis was performed. The proposed phylogenetic tree based on PKS1 sequences closely matched that based on the ITS regions. Sequencing of both regions showed that the isolates from Japan belonged to the clade of F. monophora. Molecular variations of these Japanese strains were evaluated by assessing both ITS and PKS1 sequences. The 37 isolates could be divided into at least seven molecular subtypes. The combination of these two molecular markers provides a most robust method for intraspecies subtyping and further epidemiological study of F. monophora., (© 2016 Japanese Dermatological Association.)

The fungal pathogen, Ceratocystis manginecans, has caused serious canker and wilt disease on Mangifera indica (mango), on legume tree species in Oman and Pakistan and on Acacia spp. in Indonesia. A Ceratocystis species, with similar morphology to C. manginecans, has recently been reported in Vietnam, causing severe disease of Acacia trees. Previous population genetic studies on isolates from M. indica in Oman and Pakistan have shown that the pathogen represents a single clonal haplotype, indicative of an introduced pathogen. The aim of this study was to investigate the genetic diversity and population structure of 160 C. manginecans isolates, from four host-associated populations from Oman, Pakistan, Indonesia and Vietnam. This was done by applying a combination of 14 previously developed microsatellite markers and a new set, designed in this study from two different C. manginecans genomes. Sequence data confirmed that the isolates in Vietnam are the same species as those in Indonesia and were thus identified as C. manginecans. Unlike the populations in Oman and Pakistan, relatively high levels of genetic variation were found for the isolates from Indonesia and Vietnam. The Vietnam population was significantly differentiated from the other populations and isolates from this area had the highest level of genetic diversity thus far encountered for the pathogen., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Unlabelled: Traditional culture methods for identifying the plant fungal pathogens Sclerotinia sclerotiorum (Lib.) de Bary and Botrytis cinerea Pers.:Fr. are slow and laborious. The goal of this study was to develop a multiplex real-time PCR (qPCR) assay to detect and quantify DNA from S. sclerotiorum and B. cinerea. A primer set (SsIGS&#95;5) for S. sclerotiorum was designed that targeted the intergenic spacer (IGS) regions of the ribosomal DNA. Addition of a probe to the assay increased its specificity: when the primer/probe set was tested against 21 fungal species (35 strains), amplification was detected from all S. sclerotiorum strains and no other species. For qPCR, the SsIGS&#95;5 primer and probe set exhibited a linear range from 7·0 ng to 0·07 pg target DNA (R(2)  = 0·99). SsIGS&#95;5 was then multiplexed with a previously published primer/probe set for B. cinerea to develop a high-throughput method for the detection and quantification of DNA from both pathogens. When multiplexed, the sensitivity and specificity of both assays were not different from individual qPCR reactions. The multiplex assay is currently being used to detect and quantify S. sclerotiorum and B. cinerea DNA from aerosol samples collected in commercial seed alfalfa fields., Significance and Impact of the Study: A primer and probe set for the quantification of Sclerotinia sclerotiorum DNA in a PCR assay was developed. The probe-based nature of this assay signifies an improvement over previous assays for this species by allowing multiplex reactions while maintaining high sensitivity. The primer/probe set was used in a multiplex real-time PCR assay for the quantification of S. sclerotiorum and Botrytis cinerea DNA, enabling rapid analysis of environmental samples. In crops susceptible to both pathogens, this multiplex assay can be used to quickly quantify the presence of each pathogen., (© 2016 Her Majesty the Queen in Right of Canada © 2016 The Society for Applied Microbiology. Reproduced with the permission of the Office of the Agriculture and Agri-Food Canada.)

A reliable identification method was developed for three closely related Curvularia species, which are frequently isolated from human keratomycoses. Since the traditionally used morphological method and the increasingly used internal transcribed spacer (ITS)-based molecular method proved to be insufficient to discern C. australiensis, C. hawaiiensis and C. spicifera, other molecular targets, such as β-tubulin, translation elongation factor 1-α and the nuclear ribosomal intergenic spacer (IGS), were tested. Among them, the use of the highly divergent IGS sequence is suggested and the species-specific discriminating characters were determined in appropriate reference strains. It was also concluded that C. hawaiiensis and C. spicifera can be predominantly isolated from eye infections among the three species. The in vitro antifungal susceptibility of 10 currently used antifungal agents against 32 Curvularia isolates was also investigated. MICs were determined in each case. Isolates of C. spicifera proved to be less susceptible to the tested antifungals than those of C. hawaiiensis, which underline the importance of the correct identification of these species., (© 2015 Blackwell Verlag GmbH.)

The objective of this study was to develop a rapid method to isolate DNA from Venturia inaequalis spores for use in diagnostic DNA mutation analysis. Chelex-100 resin was evaluated and compared with a well established DNA exctraction method, utilizing CTAB in order to have a robust comparison. In this research we demonstrated that Chelex-100 efficiently makes extraction of the DNA from V. inaequalis spores available for direct use in molecular analyses. Also, the quantity and quality of extracted DNA were shown to be adequate for PCR analysis. Comparatively, the quality of DNA samples isolated using Chelex method was better than those extracted using CTAB. In conclusion, the Chelex method is recommended for PCR experiments considering its simplicity and cost-effectiveness., (Copyright © 2015. Published by Elsevier B.V.)

White mold is a common bean disease caused by the fungus Sclerotinia sclerotiorum, resulting in economic losses in Brazil and worldwide. Lack of knowledge about the population structure of the pathogen makes it difficult to control the disease. The aim of this study was to characterize strains of S. sclerotiorum obtained from ex-perimental and commercial common bean fields in Brazil. We analyzed 50 strains of S. sclerotiorum collected at several locations in the state of Minas Gerais. The strains were characterized according to their ability and time for developing apothecia. Morphological and physiological analyses such as the mycelial growth index, colony color, the time re-quired to form the first sclerotia on media, the number of sclerotia per plate, average sclerotium size, and sclerotium shape were performed. We determined the mycelial compatibility, conducted molecular analy-sis of microsatellites, and evaluated the aggressiveness of 28 strains. Most strains had the ability to form apothecia. A small group of strains showed mycelial compatibility, and the strains showed different aggres-siveness levels. Overall, the population studied here demonstrated wide variability based on the morphological, physiological, and molecular traits analyzed. The average size and shape of sclerotia presented a cor-relation of 0.617, whereas the times required to form sclerotia and the number of sclerotia per plate showed a correlation of -0.455. The char-acterization of the pathogen population described herein will provide an important tool for promoting the development of bean cultivars re-sistant to white mold.

Fungal communities in Norway spruce (Picea abies) logs in two forests in Sweden were investigated by 454-sequence analyses and by examining the ecological roles of the detected taxa. We also investigated the relationship between fruit bodies and mycelia in wood and whether community assembly was affected by how the dead wood was formed. Fungal communities were highly variable in terms of phylogenetic composition and ecological roles: 1910 fungal operational taxonomic units (OTUs) were detected; 21% were identified to species level. In total, 58% of the OTUs were ascomycetes and 31% basidiomycetes. Of the 231 337 reads, 38% were ascomycetes and 60% basidiomycetes. Ecological roles were assigned to 35% of the OTUs, accounting for 62% of the reads. Wood-decaying fungi were the most common group; however, other saprotrophic, mycorrhizal, lichenized, parasitic and endophytic fungi were also common. Fungal communities in logs formed by stem breakage were different to those in logs originating from butt breakage or uprooting. DNA of specific species was detected in logs many years after the last recorded fungal fruiting. Combining taxonomic identification with knowledge of ecological roles may provide valuable insights into properties of fungal communities; however, precise ecological information about many fungal species is still lacking., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Black yeast-like fungi and relatives as agents of cerebral phaeohyphomycosis are often encountered in human fatal brain abscesses and lead to almost 100% mortality despite the application of antifungal and surgical therapy. We report to our knowledge the first case of brain infection due to Rhinocladiella mackenziei in a 54-year-old immunocompetent male in Iran where R. mackenziei has not been reported previously. The initial diagnosis was brain fungal infection because of pigmented, irregular, branched, septated hyphae based on histopathological staining. The patient was treated with intravenous amphotericin B deoxycholate (0.5mg/kg/day) combined with oral itraconazole (200mg twice daily), nevertheless, his neurological function deteriorated rapidly and ultimately the patient died due to respiratory failure later two weeks. R. mackenziei was identified based on the sequencing of internal transcribed spacer (ITS rDNA region) (KJ140287). Therefore, considerable attention for this life-threatening infection is highly recommended., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Chalara fraxinea is the causal agent of ash dieback, a disease affecting Fraxinus excelsior and F. angustifolia across Europe. Loop-mediated isothermal amplification (LAMP) is a rapid, DNA-based method which can be used for specific detection of plant pathogens in infected material. The combination of a rapid LAMP assay for C. fraxinea with a simple sample preparation method in a user-friendly kit format raises the potential for testing to be carried out away from conventional laboratory facilities, to expedite measure to manage this damaging disease.

A major challenge in obtaining a full molecular description of evolutionary adaptation is to characterize how transcription factor (TF) DNA-binding specificity can change. To identify mechanisms of TF diversification, we performed detailed comparisons of yeast C2H2 ZF proteins with identical canonical recognition residues that are expected to bind the same DNA sequences. Unexpectedly, we found that ZF proteins can adapt to recognize new binding sites in a modular fashion whereby binding to common core sites remains unaffected. We identified two distinct mechanisms, conserved across multiple Ascomycota species, by which this molecular adaptation occurred. Our results suggest a route for TF evolution that alleviates negative pleiotropic effects by modularly gaining new binding sites. These findings expand our current understanding of ZF DNA binding and provide evidence for paralogous ZFs utilizing alternate modes of DNA binding to recognize unique sets of noncanonical binding sites., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The causal agent of Septoria tritici blotch, Mycosphaerella graminicola, is currently the most frequently reported pathogen on wheat crops worldwide. The populations of this fungus were previously studied in Tunisia using microsatellite markers, but no information is available on the mitochondrial (mt) genetic structure of the fungus in this country. The objective of this study was thus to examine the genetic diversity and population structure of M. graminicola in Tunisia using mtDNA markers. A set 105 monoconidial isolates of the pathogen were sampled in 2012 from four geographical locations in Tunisia (Bizerte, Béja, Kef and Jendouba) and assessed for diversity and polymorphism of three mtDNA sequences (Mg1: 388 bp, Mg2: 570 bp, Mg3: 929 bp) using PCR-RFLP-SSCP. Mg2 and Mg3 were the more polymorphic sequences with 12 and 9 observed alleles, respectively, while Mg1 was the less polymorphic one with only two alleles. Data analyses revealed a moderate mt gene diversity and a low mt haplotype diversity within the population. Further analyses showed a low population differentiation (G(ST) = 0.16) and a moderate gene flow (Nm = 2.61) within the population. Both UPGMA and Bayesian clustering methods confirmed this lack of mt genetic structuration. Our study allowed us to characterize for the first time mt genetic structure of M. graminicola in Tunisia and to report a moderate mt gene diversity, a low mt genotype diversity and a lack of mt genetic structure of the parasite in this country.

Grapevines are sensitive to a wide range of fungal pathogens, including agents such as Phaeomoniella chlamydospora and Phaeoacremonium aleophilum that cause tracheomycosis. In the present study, a procedure for DNA extraction from grapevine woody tissue is first evaluated and shown to be suitable for quantitative analysis. Next, a multiplex real-time PCR method targeting the β-tubulin gene of the pathogens and the actin gene of plant material is developed and its quantitative capability is verified. This protocol was evaluated in inoculated grapevine-wood samples and in young vines from a nursery and was found to be reliable and highly specific. Results obtained from inoculated cuttings show that the fungal colonization process must be considered regardless of the wood phenotype. An analysis of samples of young vines from the nursery shows that a high rate of contamination occurs at the basis of plants and that this contamination is associated with low quantitative values. This finding provides evidence that in vine nurseries, these fungi may be efficient soil-borne pathogens.

Some tropical plant species possess hollow structures (domatia) occupied by ants that protect the plant and in some cases also provide it with nutrients. Most plant-ants tend patches of chaetothyrialean fungi within domatia. In a few systems it has been shown that the ants manure the fungal patches and use them as a food source, indicating agricultural practices. However, the identity of these fungi has been investigated only in a few samples. To examine the specificity and constancy of ant-plant-fungus interactions we characterised the content of fungal patches in an extensive sampling of three ant-plant symbioses (Petalomyrmex phylax/Leonardoxa africana subsp. africana, Aphomomyrmex afer/Leonardoxa africana subsp. letouzeyi and Tetraponera aethiops/Barteria fistulosa) by sequencing the Internal Transcribed Spacers of ribosomal DNA. For each system the content of fungal patches was constant over individuals and populations. Each symbiosis was associated with a specific, dominant, primary fungal taxon, and to a lesser extent, with one or two specific secondary taxa, all of the order Chaetothyriales. A single fungal patch sometimes contained both a primary and a secondary taxon. In one system, two founding queens were found with the primary fungal taxon only, one that was shown in a previous study to be consumed preferentially. Because the different ant-plant symbioses studied have evolved independently, the high specificity and constancy we observed in the composition of the fungal patches have evolved repeatedly. Specificity and constancy also characterize other cases of agriculture by insects.

Botryosphaeria dothidea is a widespread and economically important pathogen on various fruit trees, and it often causes die-back and canker on limbs and fruit rot. In characterizing intraspecies genetic variation within this fungus, group I introns, rich in rDNA of fungi, may provide a productive region for exploration. In this research, we analysed complete small subunit (SSU) ribosomal DNA (rDNA) sequences of 37 B. dothidea strains, and found four insertions, designated Bdo.S943, Bdo.S1199-A, Bdo.S1199-B and Bdo.S1506, at three positions. Sequence analysis and structure prediction revealed that both Bdo.S943 and Bdo.S1506 belonged to subgroup IC1 of group I introns, whereas Bdo.S1199-A and Bdo.S1199-B corresponded to group IE introns. Moreover, Bdo.S1199-A was found to host an open reading frame (ORF) for encoding the homing endonuclease (HE), whereas Bdo.S1199-B, an evolutionary descendant of Bdo.S1199-A, included a degenerate HE. The above four introns were novel, and were the first group I introns observed and characterized in this species. Differential distribution of these introns revealed that all strains could be separated into four genotypes. Genotype III (no intron) and genotype IV (Bdo.S1199-B) were each found in only one strain, whereas genotype I (Bdo.S1199-A) and genotype II (Bdo.S943 and Bdo.S1506) occurred in 95% of the strains. There is a correlation between B. dothidea genotypes and hosts or geographic locations. Thus, these newly discovered group I introns can help to advance understanding of genetic differentiation within B. dothidea.

The systematics of the ascomycete genera Apiorhynchostoma, Endoxyla and Pseudovalsaria are reevaluated based on the comparison of cultural characteristics, teleomorph morphology and DNA sequence data. Analyses of sequences of the internal transcribed spacer (ITS) of the ribosomal DNA operon and the large subunit (LSU) of the nuclear ribosomal DNA gene resolve Boliniales as a robustly supported lineage comprising Apiorhynchostoma, Camarops, Camaropella, Cornipulvina, Endoxyla and Pseudovalsaria. Within Boliniales, species of Endoxyla form a strongly supported lineage. Apiorhynchostoma curreyi and Pseudovalsaria ferruginea group with Cornipulvina ellipsoides. Species of Camarops are paraphyletic and comprise two clades, one of which includes Camaropella. Boliniaceae is emended, Endoxyla mallochii is described as new and Apiorhynchostoma trabicola is considered a synonym of Apiorhynchostoma altipetum. We also propose the combinations Endoxyla occulta, Endoxylina luteobasis and Jobellisia peckii. Keys to genera included in the Boliniaceae and to species of Apiocamarops, Apiorhynchostoma and Endoxyla are provided.

Four new species of Hymenoscyphus (H. brevicellulus, H. hyaloexcipulus, H. microcaudatus, and H. subsymmetricus) and a new Chinese record (H. subpallescens) are described. These five species share common characteristics: small apothecia (<3 mm in diameter); hymenium whitish, pale yellow, to yellow in color; ectal excipulum of textura prismatica; asci arising from simple septa; ascospores scutuloid and guttulate; saprophytic nutrition; and leaf habitats, except for H. subsymmetricus, which grows on herbaceous stems. Phylogenetic analyses of internal transcribed spacer nuclear ribosomal DNA sequences, the universal DNA barcode for fungi, for 16 species in the genus indicated that these taxa were closely related to H. microserotinus, in accordance with their morphological features, but represented independent species. The distinguishing features of each new species from its relatives are discussed, and their phylogenetic relationships explored.

Premise of the Study: In spite of the recent advances in generic and species circumscriptions and in recognizing species diversity in lichen-forming fungi, the timing of speciation and the factors that promote diversification in lichens remain largely unexplored. We used brown parmelioids as a model to assess the timing of divergence and explore the impact of geological and climatic events on lineage divergence and diversification in lichenized fungi. Additionally, to clarify the phylogenetic position of the species currently placed in Melanelia disjuncta group, we evaluated the taxonomic status and phylogenetic relationships within Parmeliaceae. •, Methods: Phylogenetic relationships and divergence time estimates were inferred from a four-loci data set. Alternative hypotheses were tested using Shimodaira-Hasegawa and expected likelihood weights tests. •, Key Results: The M. disjuncta group forms a strongly supported, monophyletic lineage independent from Melanelia s.s. The M. disjuncta clade arose ca. 23.1 million years ago (Ma). Our results suggest that most of the lineages within the clade diversified during the Miocene (17.6 to 11.2 Ma). The split of other brown parmelioids, such as Emodomelanelia-Melanelixia occurred ca. 41.70 Ma, and the radiation of Melanelixia began during the Eocene-Oligocene transition (ca. 33.75 Ma). •, Conclusions: Montanelia is described here as a new genus to accommodate species of the Melanelia disjuncta group. Further, the study indicates that the current species delimitation within the newly described genus requires revision. We provide evidence of lineage divergence of Montanelia at the Oligocene-Miocene boundary. Our results indicate that the diversification during Miocene would have happened during major mountain uplifts.

Bupleurum gibraltarium is an evergreen shrub endemic to southern Spain and northern Algeria and Morocco. We have collected and cultured an undescribed Kabatiella species that is consistently associated with the flower rachises and leaves of B. gibraltarium in the province of Granada. The fungus forms melanized acervuli on overwintered flower rachises. It also can be isolated from yeast-like conidial masses that emerge from senescing leaves and from surface-disinfected healthy leaves. Like other Kabatiella species, the fungus forms blastic falcate to lunate conidia simultaneously from the apex of conidiogenous cells in acervuli. In culture, melanized single-septate conidia form blastically from the vegetative hyphae that accumulate in yeast-like masses. These conidia germinate by budding to form secondary yeast-like cells or directly as hyphae. In culture, the fungus behaves like, and could be confused with, Aureobasidium and Hormonema species. We describe the growth of this species in agar media and its phylogenetic position based on the analyses of nuclear ribosomal RNA gene sequences. This new species is a sister species of the morphologically similar clover pathogen, K. caulivora.

New collections of T. gennadii, T. oligospermum and T. asa (Tuberaceae, Pezizales) from the Mediterranean basin were compared on the basis of morphology and DNA sequences. Phylogenetic inference based on the internal transcribed spacer region (ITS) and the 28S nuclear large ribosomal subunit (nrLSU) indicates that T. gennadii includes at least two lineages with characteristic morphological traits. T. gennadii s. str. has small subglobose ascomata with conspicuous glebal locules that form a continuous labyrinth when mature and has a prosenchymatic peridium. T. lacunosum has medium to large, deeply lobed ascomata with small to very small isolated locules when mature and has a pseudoparenchymatic peridium. Two major clades were identified within the T. oligospermum group. On the basis of morphological, ecological and biogeographical data, these are identified with the taxa T. oligospermum (= T. asa) and T. sphaerospermum. Tuber oligospermum forms small to medium globose or subglobose, whitish ascomata, with globose to broadly ellipsoidal ascospores and is found in alkaline, sandy soils. Tuber sphaerospermum forms medium to large, irregularly globose or deeply lobed, yellowish ascomata, with regularly globose ascospores and is found in acidic soils. Finally, a new truffle species with a prosenchymatic peridium and globose to ellipsoid reticulate spores, collected in acidic argillaceous soils of Spain under Cistus ladanifer is proposed here as the new species T. cistophilum.