Integrative taxonomy of Metarhizium anisopliae species complex, based on phylogenomics combined with morphometrics, metabolomics, and virulence data.

Metarhizium anisopliae (Clavicipitaceae, Hypocreales) is a globally distributed entomopathogenic fungus, which has been largely studied and used in agriculture for its potent entomopathogenicity. Since its taxonomic establishment as a member of Metarhizium, many closely related taxa have been described with highly similar morphology (cryptic species). A holotype specimen of M. anisopliae is not extant, and the ex-neotype strain (CBS 130.71) does not form a monophyletic clade with other strains, up to now, recognized as M. anisopliae sensu stricto. In this study, we have conducted an integrative taxonomic treatment of M. anisopliae sensu lato by including the ex-neotype strain of M. anisopliae, other unknown strains from our collections identified as M. anisopliae s. lat., as well as other known species that have been previously delimited as closely related but distinct to M. anisopliae. By including whole-genome sequencing, morphometric analysis, LC-MS based metabolomics, and virulence assays, we have demonstrated that M. anisopliae s. str. should also include M. lepidiotae (synonym), and that M. anisopliae s. str. differentiates from the other species of the complex by its metabolome and less severe entomopathogenicity. New taxa, namely M. hybridum, M. neoanisopliae and M. parapingshaense spp. nov., are proposed. The novel taxa proposed here have strong phylogenomics support, corroborated by fine-scale differences in the length/width of conidia/phialides, while the metabolomics and virulence data still largely overlap. We have also demonstrated via population genomics data the existence of local clonal lineages, particularly the one corresponding to the persistence of a biocontrol candidate strain that has been used in the field application for three years. This study showcases the utility of combining various data sources for accurate delimitation of species within an important group of fungal biocontrol agents against pest insects.

Bioactive Bioxanthracene and Cyclodepsipeptides from the Entomopathogenic Fungus Blackwellomyces roseostromatus BCC56290.

In the course of our ongoing research targeting the identification of potential biocontrol agents from entomopathogenic fungi (EPF), we explored a solid-state rice fungal extract of Blackwellomyces roseostromatus BCC56290 derived from infected lepidopteran larvae. Chemical and biological prospections afforded four unprecedentedly reported natural products differentiated into a dimeric naphthopyran bioxanthracene ES-242 derivative (1) and three cyclodepsipeptides (2-4) in addition to two known cyclodepsipeptides, cardinalisamides B (5) and C (6). Chemical structures of the isolated compounds were elucidated through comprehensive 1D/2D NMR and HR-ESI-MS data together with comparisons to the reported literature. The absolute configuration of the isolated cyclodepsipeptides was determined using Marfey's method. All isolated compounds were assessed for their antimicrobial, cytotoxic, and nematicidal activities with some compounds revealing significant activities.

Bioprospection of Tenellins Produced by the Entomopathogenic Fungus Beauveria neobassiana.

Fungi are known as prolific producers of bioactive secondary metabolites with applications across various fields, including infectious diseases, as well as in biological control. However, some of the well-known species are still underexplored. Our current study evaluated the production of secondary metabolites by the entomopathogenic fungus Beauveria neobassiana from Thailand. The fermentation of this fungus in a liquid medium, followed by preparative high-performance liquid chromatography (HPLC) purification, resulted in the isolation of a new tenellin congener, namely pretenellin C (1), together with five known derivatives (2-6). Their chemical structures were elucidated by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy in combination with high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). We evaluated the antimicrobial and cytotoxic activities from all isolated compounds, as well as their inhibitory properties on biofilm formation by Staphylococcus aureus. Generally, tenellins displayed varying antibiofilm and cytotoxic effects, allowing us to propose preliminary structure-activity relationships (SARs). Among the tested compounds, prototenellin D (2) exhibited the most prominent antibiofilm activity, while its 2-pyridone congener, tenellin (4), demonstrated potent cytotoxic activity against all tested cell lines. Given the fact that the biological activities of the tenellins have so far been neglected in the past, our study could provide a good starting point to establish more concise structure-activity relationships in the near future.

Targeted sequencing analysis pipeline for species identification of human pathogenic fungi using long-read nanopore sequencing.

Among molecular-based techniques for fungal identification, Sanger sequencing of the primary universal fungal DNA barcode, the internal transcribed spacer (ITS) region (ITS1, 5.8S, ITS2), is commonly used in clinical routine laboratories due to its simplicity, universality, efficacy, and affordability for fungal species identification. However, Sanger sequencing fails to identify mixed ITS sequences in the case of mixed infections. To overcome this limitation, different high-throughput sequencing technologies have been explored. The nanopore-based technology is now one of the most promising long-read sequencing technologies on the market as it has the potential to sequence the full-length ITS region in a single read. In this study, we established a workflow for species identification using the sequences of the entire ITS region generated by nanopore sequencing of both pure yeast isolates and mocked mixed species reads generated with different scenarios. The species used in this study included Candida albicans (n = 2), Candida tropicalis (n = 1), Nakaseomyces glabratus (formerly Candida glabrata) (n = 1), Trichosporon asahii (n = 2), Pichia kudriavzevii (formerly Candida krusei) (n = 1), and Cryptococcus neoformans (n = 1). Comparing various methods to generate the consensus sequence for fungal species identification, the results from this study indicate that read clustering using a modified version of the NanoCLUST pipeline is more sensitive than Canu or VSEARCH, as it classified species accurately with a lower abundance cluster of reads (3% abundance compared to 10% with VSEARCH). The modified NanoCLUST also reduced the number of classified clusters compared to VSEARCH, making the subsequent BLAST+ analysis faster. Subsampling of the datasets, which reduces the size of the datasets by approximately tenfold, did not significantly affect the identification results in terms of the identified species name, percent identity, query coverage, percentage of reads in the classified cluster, and the number of clusters. The ability of the method to distinguish mixed species within sub-populations of large datasets has the potential to aid computer analysis by reducing the required processing power. The herein presented new sequence analysis pipeline will facilitate better interpretation of fungal sequence data for species identification.

Is Hyperdermium Congeneric with Ascopolyporus? Phylogenetic Relationships of Ascopolyporus spp. (Cordycipitaceae, Hypocreales) and a New Genus Neohyperdermium on Scale Insects in Thailand.

During surveys of insect pathogenic fungi (IPF) in Thailand, fungi associated with scale insects and plants were found to represent five new species of the genus Ascopolyporus in Cordycipitaceae. Their macroscopic features resembled both Hyperdermium and Ascopolyporus. Morphological comparisons with the type and known Ascopolyporus and Hyperdermium species and phylogenetic evidence from a multigene dataset support the appointment of a new species of Ascopolyporus. Moreover, the data also revealed that the type species of Hyperdermium, H. caulium, is nested within Ascopolyporus, suggesting that Hyperdermium is congeneric with Ascopolyporus. The specimens investigated here differ from other Ascopolyporus species by phenotypic characters including size and color of stromata. Phylogenetic analyses of combined LSU, TEF1, RPB1 and RPB2 sequences strongly support the notion that these strains are distinct from known species of Ascopolyporus, and are proposed as Ascopolyporus albus, A. galloides, A. griseoperitheciatus, A. khaoyaiensis and A. purpuratus. Neohyperdermium gen. nov. is introduced for other species originally assigned to Hyperdermium and Cordyceps occurring on scale insects and host plants as epiphytes, accommodating two new combinations of Hyperdermium pulvinatum and Cordyceps piperis.

Mushroom poisoning in Thailand between 2003 and 2017.

The frequent occurrence of mushroom poisoning cases in the wet season in Thailand has long been recognized but has never been quantitatively analyzed. This study aims to analyze mushroom poisoning cases in Thailand between 2003 and 2017 and focused on their association with the rainfall. The results revealed 22,571 cases and 106 deaths in this period. Cases were higher for females than males, adults than children, and agriculturists than people in other occupations. Cases were higher in the northeastern and northern regions than in the central, south, and east regions. There are strong effects of seasonality on mushroom poisoning cases. Over the 15-y period, 17,337 cases occurred in the wet season (May-Sep) compared with 5,234 cases in the dry season (Oct-Apr). The number of monthly poisoning cases were high in men, mature adults, agriculturists, people living in the rural areas and people living in the Northeastern and Northern provinces. Moreover, a strong positive correlation was shown between the number of monthly poisoning cases and the amount of monthly rainfall.

Phallus chiangmaiensis sp. nov. and a Record of P. merulinus in Thailand.

During the rainy season in Thailand, specimens of Phallus chiangmaiensis sp. nov. and P. merulinus were collected from Chiang Mai and Samut Sakhon Provinces, respectively. Molecular phylogenetic analyses based on sequences of the nuclear ribosomal large subunit (LSU), nuclear ribosomal 5.8S gene including the internal transcribed spacer regions 1 and 2 (ITS), and the protein-coding gene atp6 (mitochondrial adenosine triphosphate [ATP] synthase subunit 6) support the placement of the new species within Phallus. Phallus chiangmaiensis has a well-developed white indusium and campanulated caps with reticulate surfaces. It differs morphologically from the related species, as supported by the phylogenetic data. Phallus merulinus is reported here as a species that was re-encountered in Thailand. The descriptions of the species are accompanied by illustrations of macro- and micro- morphological features, and a discussion of the related taxa is presented.

Using High-Throughput Amplicon Sequencing to Evaluate Intragenomic Variation and Accuracy in Species Identification of Cordyceps Species.

While recent sequencing technologies (third generation sequencing) can successfully sequence all copies of nuclear ribosomal DNA (rDNA) markers present within a genome and offer insights into the intragenomic variation of these markers, high intragenomic variation can be a source of confusion for high-throughput species identification using such technologies. High-throughput (HT) amplicon sequencing via PacBio SEQUEL I was used to evaluate the intragenomic variation of the ITS region and D1-D2 LSU domains in nine Cordyceps species, and the accuracy of such technology to identify these species based on molecular phylogenies was also assessed. PacBio sequences within strains showed variable level of intragenomic variation among the studied Cordyceps species with C. blackwelliae showing greater variation than the others. Some variants from a mix of species clustered together outside their respective species of origin, indicative of intragenomic variation that escaped concerted evolution shared between species. Proper selection of consensus sequences from HT amplicon sequencing is a challenge for interpretation of correct species identification. PacBio consensus sequences with the highest number of reads represent the major variants within a genome and gave the best results in terms of species identification.

In Vitro Antibacterial and Anti-Inflammatory Effects of Novel Insect Fungus Polycephalomyces phaothaiensis Extract and Its Constituents against Propionibacterium acnes.

Propionibacterium acnes plays an important role in the pathophysiology of acne vulgaris, the most common chronic inflammatory skin disease of the pilosebaceous unit. This study was conducted to investigate whether the entomopathogenic fungus Polycephalomyces phaothaiensis components have antibacterial and anti-inflammatory effects against P. acnes that may serve for acne treatment. A chemical study by spectroscopic analysis resulted in the identification of seven known compounds. The anti-P. acnes potency of extracts and test compounds was determined by both agar diffusion and broth dilution methods. The ethyl acetate extract from culture broth along with cordytropolone (1) and stipitalide (2) exhibited strong anti- P. acnes activity while (+)-piliformic acid (3) showed mild inhibitory activity. The anti-inflammatory effect of ethyl acetate extract and 1-3 was then examined by the quantification of pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α on heat-killed P. acnes induced cytokine production by THP-1 cells. The result demonstrated that the extract and its constituents (1-3) showed a potent significant effect by inhibiting the P. acnes-induced pro-inflammatory cytokines production in THP-1. Our results suggest for the first time that P. phaothaiensis and its constituents (1 and 2) hold therapeutic value for further studies as a new alternative treatment for acne.

Population genomics revealed cryptic species within host-specific zombie-ant fungi (Ophiocordyceps unilateralis).

The identification and delimitation of species boundaries are essential for understanding speciation and adaptation processes and for the management of biodiversity as well as development for applications. Ophiocordyceps unilateralis sensu lato is a complex of fungal pathogens parasitizing Formicine ants, inducing zombie behaviors in their hosts. Previous taxonomic works with limited numbers of samples and markers led to the "one ant-one fungus" paradigm, resulting in the use of ant species as a proxy for fungal identification. Here, a population genomics study with sampling on three ant species across Thailand supported the existence of host-specific species in O. unilateralis s.l. with no footprints of long term introgression despite occasional host shifts and first-generation hybrids. We further detected genetic clusters within the previously delimited fungal species, with each little footprints of recombination, suggesting high levels of inbreeding. The clusters within each of O. camponoti-leonardi and O. camponoti-saundersi were supported by differentiation throughout the genome, suggesting they may constitute further cryptic species parasitizing the same host, challenging the one ant-one fungus paradigm. These genetic clusters had different geographical ranges, supporting different biogeographic influences between the north/center and the south of Thailand, reinforcing the scenario in which Thailand endured compartmentation during the latest Pleistocene glacial cycles.

Pigmentosins from Gibellula sp. as antibiofilm agents and a new glycosylated asperfuran from Cordyceps javanica.

In the course of our exploration of the Thai invertebrate-pathogenic fungi for biologically active metabolites, pigmentosin A (1) and a new bis(naphtho-α-pyrone) derivative, pigmentosin B (2), were isolated from the spider-associated fungus Gibellula sp. Furthermore, a new glycosylated asperfuran 3, together with one new (6) and two known (4 and 5) cyclodepsipeptides, was isolated from Cordyceps javanica. The pigmentosins 1 and 2 showed to be active against biofilm formation of Staphylococcus aureus DSM1104. The lack of toxicity toward the studied microorganism and cell lines of pigmentosin B (2), as well as the antimicrobial effect of pigmentosin A (1), made them good candidates for further development for use in combination therapy of infections involving biofilm-forming S. aureus. The structure elucidation and determination of the absolute configuration were accomplished using a combination of spectroscopy, including 1D and 2D NMR, HRMS, Mosher ester analysis, and comparison of calculated/experimental ECD spectra. A chemotaxonomic investigation of the secondary metabolite profiles using analytical HPLC coupled with diode array detection and mass spectrometry (HPLC-DAD-MS) revealed that the production of pigmentosin B (2) was apparently specific for Gibellula sp., while the glycoasperfuran 3 was specific for C. javanica.

New nematicidal and antimicrobial secondary metabolites from a new species in the new genus, Pseudobambusicola thailandica.

During the course of a study on the functional biodiversity of the mycobiota inhabiting rainforests in Thailand, a fungal strain was isolated from a plant sample and shown to represent an undescribed species, as inferred from a combination of morphological and molecular phylogenetic methods. Molecular phylogenetic analyses, based on four DNA loci, revealed a phylogenetic tree with the newly generated sequences clustering in a separate branch, together with members of the Sulcatisporaceae (Pleosporales, Ascomycota). The Thai specimen morphologically resembled Neobambusicola strelitziae in having pycnidial conidiomata with phialidic conidiogenous cells that produce both fusoid-ellipsoid macroconidia and subcylindrical microconidia. However, the new fungus, for which the name Pseudobambusicola thailandica is proposed, differs from N. strelitziae in having conidiomata with well-defined necks, the presence of globose to subglobose thick-walled cells adjacent to conidiomata and the production of chlamydospores in culture. When cultures of P. thailandica, growing on water agar, were confronted with Caenorhabditis elegans nematodes, worms approaching the fungal mycelia were killed. This observation gave rise to a study of its secondary metabolites and six novel and two known compounds were isolated from submerged cultures of P. thailandica. The structures of metabolites 1-6, for which the trivial names thailanones A-F are proposed, were elucidated using a combination of spectral methods, including extensive 1 and 2D NMR analysis and high resolution mass spectrometry. Compounds 4 and 8 showed strong nematicidal and weak antifungal activity, whereas all other tested compounds showed moderate to weak nematicidal activity but no significant effects in the serial dilution assay against various fungi and bacteria. Compounds 1 and 8 also inhibited growth of the pathogenic basidiomycete Phellinus tremulae in a plate diffusion assay.

Akanthopyrones A-D, α-Pyrones Bearing a 4-O-Methyl-ß-d-glucopyranose Moiety from the Spider-Associated Ascomycete Akanthomyces novoguineensis.

Hypocrealean fungi have proved to be prolific bioactive metabolite producers; they have caught the attention of mycologists throughout the world. However, only a few studies on the insect and spider parasitic genus Akanthomyces have so far been carried out. In this study, we report the isolation, structural elucidation and biological activities of four unprecedented glycosylated α-pyrone derivatives, akanthopyrones A-D (1-4), from a culture of Akanthomyces novoguineensis collected in Thailand. The chemical structures of the akanthopyrones were determined by extensive 1D- and 2D-NMR, and HRMS spectroscopic analysis. Their absolute configurations were determined. Akanthopyrone A (1) exhibited weak antimicrobial activity against Bacillus subtilis DSM10 and cytotoxicity against the HeLa cell line KB-3-1, while akanthopyrone D (4) showed weak activity against Candida tenuis MUCL 29892.

Five Unprecedented Secondary Metabolites from the Spider Parasitic Fungus Akanthomyces novoguineensis.

Five new compounds including the glycosylated ß-naphthol (1, akanthol), a glycosylated pyrazine (2, akanthozine), and three amide derivatives including a hydroxamic acid derivative (3-5) were isolated from the spider-associated fungus Akanthomyces novoguineensis (Cordycipitaceae, Ascomycota). Their structures were elucidated by using high resolution mass spectrometry (HRMS) and NMR spectroscopy. In this study, the antimicrobial, cytotoxic, anti-biofilm, and nematicidal activities of the new compounds were evaluated. The distribution pattern of secondary metabolites in the species was also revealed in which more isolates of A. novoguineensis were encountered and their secondary metabolite profiles were examined using analytical HPLC with diode array and mass spectrometric detection (HPLC-DAD/MS). Remarkably, all isolated compounds are specifically produced by A. novoguineensis.

Insights from the genome of Ophiocordyceps polyrhachis-furcata to pathogenicity and host specificity in insect fungi.

BACKGROUND: Ophiocordyceps unilateralis is an outstanding insect fungus for its biology to manipulate host ants' behavior and for its extreme host-specificity. Through the sequencing and annotation of Ophiocordyceps polyrhachis-furcata, a species in the O. unilateralis species complex specific to the ant Polyrhachis furcata, comparative analyses on genes involved in pathogenicity and virulence between this fungus and other fungi were undertaken in order to gain insights into its biology and the emergence of host specificity. RESULTS: O. polyrhachis-furcata possesses various genes implicated in pathogenicity and virulence common with other fungi. Overall, this fungus possesses protein-coding genes similar to those found on other insect fungi with available genomic resources (Beauveria bassiana, Metarhizium robertsii (formerly classified as M. anisopliae s.l.), Metarhizium acridum, Cordyceps militaris, Ophiocordyceps sinensis). Comparative analyses in regard of the host ranges of insect fungi showed a tendency toward contractions of various gene families for narrow host-range species, including cuticle-degrading genes (proteases, carbohydrate esterases) and some families of pathogen-host interaction (PHI) genes. For many families of genes, O. polyrhachis-furcata had the least number of genes found; some genes commonly found in other insect fungi are even absent (e.g. Class 1 hydrophobin). However, there are expansions of genes involved in 1) the production of bacterial-like toxins in O. polyrhachis-furcata, compared with other entomopathogenic fungi, and 2) retrotransposable elements. CONCLUSIONS: The gain and loss of gene families helps us understand how fungal pathogenicity in insect hosts evolved. The loss of various genes involved throughout the pathogenesis for O. unilateralis would result in a reduced capacity to exploit larger ranges of hosts and therefore in the different level of host specificity, while the expansions of other gene families suggest an adaptation to particular environments with unexpected strategies like oral toxicity, through the production of bacterial-like toxins, or sophisticated mechanisms underlying pathogenicity through retrotransposons.

New species of Ophiocordyceps unilateralis, an ubiquitous pathogen of ants from Thailand.

Ophiocordyceps unilateralis is an ubiquitous pathogen of ants with hidden phylogenetic diversity associated with host specificity. In this study, we describe two new species to this species complex: Ophiocordyceps septa and Ophiocordyceps rami. Both were found on unidentified ants of the genus Camponotus (C. sp.1 and C. sp2 respectively). Ophiocordyceps septa is very similar to Ophiocordyceps polyrhachis-furcata, Ophiocordyceps camponoti-leonardi, and Ophiocordyceps camponoti-saundersi (found respectively on the ants Polyrhachis furcata, Camponotus leonardi, and Camponotus saundersi) but differs in the size, the shape and the septation of the ascospores, while O. rami is clearly identifiable with macro-morphological features including multiple stromata similar to Ophiocordyceps halabalaensis on Camponotus gigas. A thorough morphological examination was also provided for O. polyrhachis-furcata, O. camponoti-leonardi, and O. camponoti saundersi, showing that the first was apparently distinguishable from the others by the absence of septation of the ascospores. A combined molecular phylogeny also supports O. septa and O. rami as distinct new species.