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A multigene phylogeny was constructed, including a significant number of representative species of the main lineages in the Xylariaceae and four DNA loci the internal transcribed spacer region (ITS), the large subunit (LSU) of the nuclear rDNA, the second largest subunit of the RNA polymerase II (RPB2), and beta-tubulin (TUB2). Specimens were selected based on more than a decade of intensive morphological and chemotaxonomic work, and cautious taxon sampling was performed to cover the major lineages of the Xylariaceae; however, with emphasis on hypoxyloid species. The comprehensive phylogenetic analysis revealed a clear-cut segregation of the Xylariaceae into several major clades, which was well in accordance with previously established morphological and chemotaxonomic concepts. One of these clades contained Annulohypoxylon, Hypoxylon, Daldinia, and other related genera that have stromatal pigments and a nodulisporium-like anamorph. They are accommodated in the family Hypoxylaceae, which is resurrected and emended. Representatives of genera with a nodulisporium-like anamorph and bipartite stromata, lacking stromatal pigments (i.e. Biscogniauxia, Camillea, and Obolarina) appeared in a clade basal to the xylarioid taxa. As they clustered with Graphostroma platystomum, they are accommodated in the Graphostromataceae. The new genus Jackrogersella with J. multiformis as type species is segregated from Annulohypoxylon. The genus Pyrenopolyporus is resurrected for Hypoxylon polyporus and allied species. The genus Daldinia and its allies Entonaema, Rhopalostroma, Ruwenzoria, and Thamnomyces appeared in two separate subclades, which may warrant further splitting of Daldinia in the future, and even Hypoxylon was divided in several clades. However, more species of these genera need to be studied before a conclusive taxonomic rearrangement can be envisaged. Epitypes were designated for several important species in which living cultures and molecular data are available, in order to stabilise the taxonomy of the Xylariales.

Fusarium wilt, caused by Fusarium oxysporum f. sp. cucumerinum J. H. Owen, results in considerable yield losses for cucumber plants. A bio-organic fertilizer (BIO), which was a combination of manure composts with antagonistic microorganisms, and an organic fertilizer (OF) were evaluated for their efficiencies in controlling Fusarium wilt. Application of the BIO suppressed the disease incidence by 83% and reduced yield losses threefold compared with the application of OF. Analysis of microbial communities in rhizosphere soils by high-throughput pyrosequencing showed that more complex community structures were present in BIO than in OF treated soils. The dominant taxonomic phyla found in both samples were Proteobacteria, Firmicutes, Actinobacteria and Acidobacteria among bacteria and Ascomycota among fungi. Abundance of beneficial bacteria or fungi, such as Trichoderma , Hypoxylon , Tritirachium , Paenibacillus , Bacillus , Haliangium and Streptomyces , increased compared to the OF treatment, whereas the soil-borne pathogen, Fusarium , was markedly decreased. Overall, the results of this study demonstrate that the application of the BIO was a useful and effective approach to suppress Fusarium wilt and that the high-throughput 454 pyrosequencing was a suitable method for the characterization of microbial communities of rhizosphere soil of cucumber.

Two new diterpenoids, hypoxyterpoids A (1) and B (2), and four new isocoumarin derivatives, hypoxymarins A–D (4–7), together, with seven known metabolites (3 and 8–13) were obtained from the crude extract of the mangrove-derived fungus Hypoxylon sp. The structures of the new compounds were elucidated on the basis of 1- and 2-dimensional (1D/2D) nuclear magnetic resonance (NMR) spectroscopic and mass spectrometric analysis. The absolute configurations of compounds 1, 2, 4, 5, and 7 were determined by comparison of experimental and calculated electronic circular dichroism (ECD) spectra, and the absolute configurations of C-4′ in 6 and C-9 in 7 were determined by [Rh2(OCOCF3)4]-induced ECD spectra. Compound 1 showed moderate α-glucosidase inhibitory activities with IC50 values of 741.5 ± 2.83 μM. Compounds 6 and 11 exhibited DPPH scavenging activities with IC50 values of 15.36 ± 0.24 and 3.69 ± 0.07 μM, respectively.

Infections involving biofilms are difficult to treat due to increased resistances against antibiotics and the immune system. Hence, there is an urgent demand for novel drugs against biofilm infections. During our search for novel biofilm inhibitors from fungi, we isolated linoleic acid from the ascomycete Hypoxylon fragiforme which showed biofilm inhibition of several bacteria at sub-MIC concentrations. Many fatty acids possess antimicrobial activities, but their minimum inhibitory concentrations (MIC) are high and reports on biofilm interferences are scarce. We demonstrated that not only linoleic acid but several unsaturated long-chain fatty acids inhibited biofilms at sub-MIC concentrations. The antibiofilm activity exerted by long-chain fatty acids was mainly against Gram-positive bacteria, especially against Staphylococcus aureus. Micrographs of treated S. aureus biofilms revealed a reduction in the extracellular polymeric substances, pointing to a possible mode of action of fatty acids on S. aureus biofilms. The fatty acids had a strong species specificity. Poly-unsaturated fatty acids had higher activities than saturated ones, but no obvious rule could be found for the optimal length and desaturation for maximal activity. As free fatty acids are non-toxic and ubiquitous in food, they may offer a novel tool, especially in combination with antibiotics, for the control of biofilm infections.

Members of the fungal genus Hypoxylon of the family Xylariaceae are known to produce secondary metabolites with significant chemical diversity. There are more than 200 species in the genus, including the filamentous fungus Hypoxylon fendleri. To the best of our knowledge, there have been no reports of mycoviruses in H. fendleri. In this study, a novel mycovirus, designated “Hypoxylon fendleri mitovirus 1” (HfMV1), was isolated from this fungus. The genome of HfMV1 is 2850 nt in length with a G + C content of 36% and contains a large open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp). BLASTp analysis revealed that the RdRp domain of HfMV1 had 28.30–50.90% sequence identity to those of members of the genus Duamitovirus and had the highest identity (50.90%) to Fusarium graminearum mitovirus 2-2 (FgMV2-2). Phylogenetic analysis further indicated that HfMV1 is a member of the genus Duamitovirus of the family Mitoviridae. This is the first report of a mycovirus in H. fendleri.

During a survey of xylarialean Sordariomycetes in North-western Argentina, several collections representing a new species morphologically resembling the pantropical Hypoxylon monticulosum was encountered. They were studied by a combination of morphological and chemotaxonomic studies along with a multi-locus phylogeny of Hypoxylon and related genera of the Hypoxylaceae based on a combined ITS, LSU, TUB2, and RBP2 dataset. The phylogenetic tree revealed that DNA sequences of the new fungus clustered together with Hypoxylon monticulosum and Hypoxylon submonticulosum in a separate clade. In addition, all representatives of this clade produced the antifungal polyketides of the sporothriolide type in standardized submerged cultures. Furthermore, a comparison of the morphology of this group of species formerly placed in Hypoxylon , including H. austrobahiense and H. rubigineoareolatum , also revealed certain morphological similarities. They are therefore accommodated in the new genus Hypomontagnella , with Hypomontagnella monticulosa as type species, and the new Argentine fungus is described as Hypomontagnella barbarensis.

Members of the genus Hypoxylon (Ascomycota) are pleomorphic fungi mostly forming conspicuous teleomorphs, consisting of perithecia embedded into stromal tissue, and their morphology has traditionally served for species delineation. However, analysis in tandem with other phenotypic characters, such as chemical and genetic traits, proved to be a more stable predictor of interspecies and intergeneric relationships. During 2014 and 2015, a set of species identified as Hypoxylon were described from the Neotropics, exclusively relying on morphological traits. The secondary metabolite profiles of their stromata were analysed by HPLC/DAD-ESI-MS, corroborating their classification within Xylariales. Additionally, molecular data for ex-type strains of H. dussii and H. sofaiense were incorporated into an inferred molecular phylogeny of the Hypoxylaceae and allies. Furthermore, a freshly collected specimen from North Carolina was selected as epitype of Sphaeria perforata Schweinitz (syn. Hypoxylon perforatum), as its morphological/chemotaxonomic characters matched those of the holotype. Our findings demonstrate that the secondary metabolism of Hypoxylon closely correlates with both morphological features and molecular data, serving as a complement for species identification.

Nodulisporic acids (NAs) are indole diterpene fungal metabolites exhibiting potent systemic efficacy against blood-feeding arthropods, e.g., bedbugs, fleas and ticks, via binding to arthropod specific glutamate-gated chloride channels. Intensive medicinal chemistry efforts employing a nodulisporic acid A template have led to the development of N-tert-butyl nodulisporamide as a product candidate for a once monthly treatment of fleas and ticks on companion animals. The source of the NAs is a monophyletic lineage of asexual endophytic fungal strains that is widely distributed in the tropics, tentatively identified as a Nodulisporium species and hypothesized to be the asexual state of a Hypoxylon species. Inferences from GenBank sequences indicated that multiple researchers have encountered similar Nodulisporium endophytes in tropical plants and in air samples. Ascomata-derived cultures from a wood-inhabiting fungus, from Martinique and closely resembling Hypoxylon investiens, belonged to the same monophyletic clade as the NAs-producing endophytes. The hypothesis that the Martinique Hypoxylon collections were the sexual state of the NAs-producing endophytes was tested by mass spectrometric analysis of NAs, multi-gene phylogenetic analysis, and phenotypic comparisons of the conidial states. We established that the Martinique Hypoxylon strains produced an ample spectrum of NAs and were conspecific with the pantropical Nodulisporium endophytes, yet were distinct from H. investiens. A new species, H. pulicicidum, is proposed to accommodate this widespread organism. Knowledge of the life cycle of H. pulicicidum will facilitate an understanding of the role of insecticidal compounds produced by the fungus, the significance of its infections in living plants and how it colonizes dead wood. The case of H. pulicicidum exemplifies how life cycle studies can consolidate disparate observations of a fungal organism, whether from environmental sequences, vegetative mycelia or field specimens, resulting in holistic species concepts critical to the assessment of the dimensions of fungal diversity.

Two new species and a new combination of Hypoxylon from Texas were identified and described based on morphological, multigene phylogenetic (ITS [nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2], 28S [5′ 1200 bp of nuc 28S rDNA], RPB2 [partial second largest subunit of the DNA-directed RNA polymerase II], TUB2 [partial β-tubulin]), and chemotaxonomic data. Hypoxylon olivaceopigmentum is characterized by its pulvinate to glomerate stromata, olivaceous KOH-extractable pigments, equilateral ascospores, and indehiscent perispore. Hypoxylon texense can be distinguished from morphologically similar species by its rust to dark brick KOH-extractable pigments and the high-performance liquid chromatography (HPLC) profile of its stromatal secondary metabolites. Hypoxylon hinnuleum is proposed as the sexual morph of Nodulisporium hinnuleum, featuring dark vinaceous glomerate stromata with dark brick KOH-extractable pigments composed of cohaerin-type azaphilones and smooth equilateral ascospores with indehiscent perispore. Based on these diagnostic characters, H. hinnuleum forms a complex with H. croceum and H. minicroceum. More than 50 ITS sequences with high identity originating from North American and East Asian environmental isolates formed a well-supported clade with the type of N. hinnuleum, demonstrating the widespread distribution of the species complex. In addition, updated descriptions and comprehensive illustrations with detailed information on the diagnostic features of H. fendleri and H. perforatum are provided. The multilocus phylogenetic reconstruction of Hypoxylon supported the status of the new species and broadened the knowledge about intergeneric relationships.

During a mycological survey of the Democratic Republic of the Congo, a fungal specimen that morphologically resembled the American species Hypoxylon papillatum was encountered. A polyphasic approach including morphological and chemotaxonomic together with a multigene phylogenetic study (ITS, LSU, tub2 , and rpb2 ) of Hypoxylon spp. and representatives of related genera revealed that this strain represents a new species of the Hypoxylaceae. However, the multi-locus phylogenetic inference indicated that the new fungus clustered with H. papillatum in a separate clade from the other species of Hypoxylon . Studies by ultrahigh performance liquid chromatography coupled to diode array detection and ion mobility tandem mass spectrometry (UHPLC-DAD-IM-MS/MS) were carried out on the stromatal extracts. In particular, the MS/MS spectra of the major stromatal metabolites of these species indicated the production of hitherto unreported azaphilone pigments with a similar core scaffold to the cohaerin-type metabolites, which are exclusively found in the Hypoxylaceae. Based on these results, the new genus Parahypoxylon is introduced herein. Aside from P. papillatum , the genus also includes P. ruwenzoriense sp. nov. , which clustered together with the type species within a basal clade of the Hypoxylaceae together with its sister genus Durotheca .