Fungal Endophytes: Discovering What Lies within Some of Canada's Oldest and Most Resilient Grapevines.

Plant diseases and pests reduce crop yields, accounting for global crop losses of 30% to 50%. In conventional agricultural production systems, these losses are typically controlled by applying chemical pesticides. However, public pressure is mounting to curtail agrochemical use. In this context, employing beneficial endophytic microorganisms is an increasingly attractive alternative to the use of conventional chemical pesticides in agriculture. A multitude of fungal endophytes are naturally present in plants, producing enzymes, small peptides, and secondary metabolites due to their bioactivity, which can protect hosts from pathogens, pests, and abiotic stresses. The use of beneficial endophytic microorganisms in agriculture is an increasingly attractive alternative to conventional pesticides. The aim of this study was to characterize fungal endophytes isolated from apparently healthy, feral wine grapes in eastern Canada that have grown without agrochemical inputs for decades. Host plants ranged from unknown seedlings to long-lost cultivars not widely propagated since the 1800s. HPLC-MS was used to identify unique endophyte-derived chemical compounds in the host plants, while dual-culture competition assays showed a range in endophytes' ability to suppress the mycelial growth of Botrytis, which is typically controlled in viticulture with pesticides. Twelve of the most promising fungal endophytes isolated were identified using multilocus sequencing and morphology, while DNA barcoding was employed to identify some of their host vines. These fungal endophyte isolates, which consisted of both known and putative novel strains, belonged to seven genera in six families and five orders of Ascomycota. Exploring the fungal endophytes in these specimens may yield clues to the vines' survival and lead to the discovery of novel biocontrol agents.

Genome sequences of three genetic lineages of the fungus Nothophaeocryptopus gaeumannii, the causal agent of Swiss needle cast on Douglas-fir trees.

Here, we present the nearly complete genome sequences of the three main genetic lineages of Nothophaeocryptopus gaeumannii, an endophytic ascomycete fungus responsible for Swiss needle cast, a foliar disease that is emerging as a significant threat to the Douglas-fir tree in its natural distribution range.

Non-targeted Screening of Natural Products from 288 Fungal Endophytes from Canadian Fruit Crops.

Many diverse species of fungi naturally occur as endophytes in plants. The majority of these fungi produce secondary metabolites of diverse structures and biological activities. Culture extracts from 288 fungi isolated from surface-sterilized blueberries, cranberries, raspberries, and grapes were analyzed by LC-HRMS/MS. Global Natural Products Social (GNPS) Molecular Networking modeling was used to investigate the secondary metabolites in the extracts. This technique increased the speed and simplicity of dereplicating the extracts, targeting new compounds that are structurally related. In total, 60 known compounds were dereplicated from this collection and seven new compounds were identified. These previously unknown compounds are targets for purification, characterization, and bioactivity testing in future studies. The fungal endophytes characterized in this study are potential candidates for providing bio-protection to the host plant with a reduced reliance on chemical pesticides.

Apothecial Ancestry, Evolution, and Re-Evolution in Thelebolales (Leotiomycetes, Fungi).

Closed cleistothecia-like ascomata have repeatedly evolved in non-related perithecioid and apothecioid lineages of lichenized and non-lichenized Ascomycota. The evolution of a closed, darkly pigmented ascoma that protects asci and ascospores is conceived as either an adaptation to harsh environmental conditions or a specialized dispersal strategy. Species with closed ascomata have mostly lost sterile hymenial elements (paraphyses) and the capacity to actively discharge ascospores. The class Leotiomycetes, one of the most speciose classes of Ascomycota, is mainly apothecioid, paraphysate, and possesses active ascospore discharge. Lineages with closed ascomata, and their morphological variants, have evolved independently in several families, such as Erysiphaceae, Myxotrichaceae, Rutstroemiaceae, etc. Thelebolales is a distinctive order in the Leotiomycetes class. It has two widespread families (Thelebolaceae, Pseudeurotiaceae) with mostly closed ascomata, evanescent asci, and thus passively dispersed ascospores. Within the order, closed ascomata dominate and a great diversity of peridia have evolved as adaptations to different dispersal strategies. The type genus, Thelebolus, is an exceptional case of ascomatal evolution within the order. Its species are the most diverse in functional traits, encompassing species with closed ascomata and evanescent asci, and species with open ascomata, active ascospore discharge, and paraphyses. Open ascomata were previously suggested as the ancestral state in the genus, these ascomata depend on mammals and birds as dispersal agents. In this scheme, species with closed ascomata, a lack of paraphyses, and passive ascospore discharge exhibit derived traits that evolved in adaptation to cold ecosystems. Here, we used morphological and phylogenetic methods, as well as the reconstruction of ancestral traits for ascomatal type, asci dehiscence, the presence or absence of paraphyses, and ascospore features to explore evolution within Thelebolales. We demonstrate the apothecial ancestry in Thelebolales and propose a new hypothesis about the evolution of the open ascomata in Thelebolus, involving a process of re-evolution where the active dispersal of ascospores appears independently twice within the order. We propose a new family, Holwayaceae, within Thelebolales, that retains the phenotypic features exhibited by species of Thelebolus, i.e., pigmented capitate paraphyses and active asci discharge with an opening limitation ring.

Fungal Endophytes and Their Role in Agricultural Plant Protection against Pests and Pathogens.

Virtually all examined plant species harbour fungal endophytes which asymptomatically infect or colonize living plant tissues, including leaves, branches, stems and roots. Endophyte-host interactions are complex and span the mutualist-pathogen continuum. Notably, mutualist endophytes can confer increased fitness to their host plants compared with uncolonized plants, which has attracted interest in their potential application in integrated plant health management strategies. In this review, we report on the many benefits that fungal endophytes provide to agricultural plants against common non-insect pests such as fungi, bacteria, nematodes, viruses, and mites. We report endophytic modes of action against the aforementioned pests and describe why this broad group of fungi is vitally important to current and future agricultural practices. We also list an extensive number of plant-friendly endophytes and detail where they are most commonly found or applied in different studies. This review acts as a general resource for understanding endophytes as they relate to potential large-scale agricultural applications.

Metabolomic-guided discovery of cyclic nonribosomal peptides from Xylaria ellisii sp. nov., a leaf and stem endophyte of Vaccinium angustifolium.

Fungal endophytes are sources of novel bioactive compounds but relatively few agriculturally important fruiting plants harboring endophytes have been carefully studied. Previously, we identified a griseofulvin-producing Xylaria species isolated from Vaccinium angustifolium, V. corymbosum, and Pinus strobus. Morphological and genomic analysis determined that it was a new species, described here as Xylaria ellisii. Untargeted high-resolution LC-MS metabolomic analysis of the extracted filtrates and mycelium from 15 blueberry isolates of this endophyte revealed differences in their metabolite profiles. Toxicity screening of the extracts showed that bioactivity was not linked to production of griseofulvin, indicating this species was making additional bioactive compounds. Multivariate statistical analysis of LC-MS data was used to identify key outlier features in the spectra. This allowed potentially new compounds to be targeted for isolation and characterization. This approach resulted in the discovery of eight new proline-containing cyclic nonribosomal peptides, which we have given the trivial names ellisiiamides A-H. Three of these peptides were purified and their structures elucidated by one and two-dimensional nuclear magnetic resonance spectroscopy (1D and 2D NMR) and high-resolution tandem mass spectrometry (HRMS/MS) analysis. The remaining five new compounds were identified and annotated by high-resolution mass spectrometry. Ellisiiamide A demonstrated Gram-negative activity against Escherichia coli BW25113, which is the first reported for this scaffold. Additionally, several known natural products including griseofulvin, dechlorogriseofulvin, epoxy/cytochalasin D, zygosporin E, hirsutatin A, cyclic pentapeptides #1-2 and xylariotide A were also characterized from this species.

Placement of Triblidiaceae in Rhytismatales and comments on unique ascospore morphologies in Leotiomycetes (Fungi, Ascomycota).

Triblidiaceae is a family of uncommonly encountered, non-lichenized discomycetes. A recent classification circumscribed the family to include Triblidium (4 spp. and 1 subsp.), Huangshania (2 spp.) and Pseudographis (2 spp. and 1 var.). The apothecia of these fungi are persistent and drought-tolerant; they possess stromatic, highly melanized covering layers that open and close with fluctuations of humidity. Triblidialean fungi occur primarily on the bark of Quercus, Pinaceae and Ericaceae, presumably as saprobes. Though the type species of Huangshania is from China, these fungi are mostly known from collections originating from Western Hemisphere temperate and boreal forests. The higher-rank classification of triblidialean fungi has been in flux due in part to an overemphasis on ascospore morphology. Muriform ascospores are observed in species of Triblidium and in Pseudographiselatina. An intense, dark blue/purple ascospore wall reaction in iodine-based reagents is observed in species of Pseudographis. These morphologies have led, in part, to these genera being shuffled among unrelated taxa in Hysteriaceae (Dothideomycetes, Hysteriales) and Graphidaceae (Lecanoromycetes, Ostropales). Triblidiaceae has been placed within the monofamilial order Triblidiales (affinity Lecanoromycetes). Here, we demonstrate with a three-gene phylogenetic approach that triblidialean fungi are related to taxa in Rhytismatales (Leotiomycetes). We synonymize Triblidiales under Rhytismatales and emend Triblidiaceae to include Triblidium and Huangshania, with Pseudographis placed within Rhytismataceae. A history of Triblidiaceae is provided along with a description of the emended family. We discuss how the inclusion of triblidialean fungi in Rhytismatales brings some rarely observed or even unique ascospore morphologies to the order and to Leotiomycetes.

Diagnostic fragmentation filtering for the discovery of new chaetoglobosins and cytochalasins.

RATIONALE: Microbial natural products are often biosynthesized as classes of structurally related compounds that have similar tandem mass spectrometry (MS/MS) fragmentation patterns. Mining MS/MS datasets for precursor ions that share diagnostic or common features enables entire chemical classes to be identified, including novel derivatives that have previously been unreported. Analytical data analysis tools that can facilitate a class-targeted approach to rapidly dereplicate known compounds and identify structural variants within complex matrices would be useful for the discovery of new natural products. METHODS: A diagnostic fragmentation filtering (DFF) module was developed for MZmine to enable the efficient screening of MS/MS datasets for class-specific product ions(s) and/or neutral loss(es). This approach was applied to series of the structurally related chaetoglobosin and cytochalasin classes of compounds. These were identified from the culture filtrates of three fungal genera: Chaetomium globosum, a putative new species of Penicillium (called here P. cf. discolor: closely related to P. discolor), and Xylaria sp. Extracts were subjected to LC/MS/MS analysis under positive electrospray ionization and operating in a data-dependent acquisition mode, performed using a Thermo Q-Exactive mass spectrometer. All MS/MS datasets were processed using the DFF module and screened for diagnostic product ions at m/z 130.0648 and 185.0704 for chaetoglobosins, and m/z 120.0808 and 146.0598 for cytochalasins. RESULTS: Extracts of C. globosum and P. cf. discolor strains revealed different mixtures of chaetoglobosins, whereas the Xylaria sp. produced only cytochalasins; none of the strains studied produced both classes of compounds. The dominant chaetoglobosins produced by both C. globosum and P. cf. discolor were chaetoglobosins A, C, and F. Tetrahydrochaetoglobosin A was identified from P. cf. discolor extracts and is reported here for the first time as a natural product. The major cytochalasins produced by the Xylaria sp. were cytochalasin D and epoxy cytochalasin D. A larger unknown "cytochalasin-like" molecule with the molecular formula C38 H47 NO10 was detected from Xylaria sp. culture filtrate extracts and is a current target for isolation and structural characterization. CONCLUSIONS: DFF is an effective LC/MS data analysis approach for rapidly identifying entire classes of compounds from complex mixtures. DFF has proved useful in the identification of new natural products and allowing for their partial characterization without the need for isolation.

New 1,3-benzodioxin-4-ones from Synnemapestaloides ericacearum sp. nov., a biosynthetic link to remarkable compounds within the Xylariales.

Surveys of foliar endophytes from the Acadian forest region over the past three decades have identified numerous phylogenetically diverse fungi producing natural products toxic to forest pests and diseases. The life histories of some conifer endophytes can be restricted to plant foliage or may include saprotrophic phases on other plants tissues or even alternate hosts. Considering the potentially broad host preferences of conifer endophytes we explored fungi isolated from understory species and their metabolites as part of an ongoing investigation of fungal biodiversity from the Acadian forest. We report a hitherto unidentified Xylariomycetidae species isolated from symptomatic Labrador tea (Rhododendron groenlandicum) leaves and mountain laurel (Kalmia latifolia) collected in coastal southern New Brunswick, Canada. Morphological and phylogenetic evidence demonstrated the unknown species was a novel Synnemapestaloides (Sporocadaceae) species, described here as Syn. ericacearum. A preliminary screening assay indicated that the culture filtrate extract of the new species was potently antifungal towards the biotrophic pathogen Microbotryum violaceum, warranting an investigation of its natural products. Two natural products possessing a rare 1,3-benzodioxin-4-one scaffold, synnemadoxins A-B (1-2), and their postulated precursor, synnemadiacid A (3), were characterized as new structures and assessed for antimicrobial activity. All isolated compounds elicited in vitro inhibitory antifungal activity towards M. violaceum at 2.3 µg mL-1 and moderate antibiotic activity. Further, the characterization of synnemadoxins A-B provided a perspective on the biosynthesis of some related 1,3-benzodioxin-4-ones produced by other fungi within the Xylariales.

Natural Products of Picea Endophytes from the Acadian Forest.

Endophytes of healthy needles were collected from Picea rubens (red spruce) and P. mariana (black spruce) in a survey of southeastern New Brunswick, Canada. Four endophyte strains were selected for further investigation based on the production of biologically active extracts from culture filtrates during screening as well as phylogenetic relationship to species known to produce natural products or taxonomic novelty. A novel endophyte within the family Rhytismataceae produced two new dihydropyrones (1 and 2) as major metabolites together with phthalides (3 and 4), isocoumarins (5 and 6), and tyrosol (7). Lachnum cf. pygmaeum synthesized a new chlorinated para-quinone, chloromycorrhizinone A (8), and the nematicidal compounds (1'Z)-dechloromycorrhizin A (9), mycorrhizin A (10), and chloromycorrhizin A (11). A new isocoumarin (12) and four related structures (13-16) were isolated from an undescribed taxon in the Mycosphaerellaceae. The known antifungal metabolites cryptosporiopsin (17), 5-hydroxycryptosporiopsin (18), (+)-cryptosporiopsinol (19), and mellein (20) were produced by Pezicula sporulosa. Phylogenetically diverse conifer endophytes from the Acadian forest continue to be a productive source of new biologically active natural products.

Production of antifungal and antiinsectan metabolites by the Picea endophyte Diaporthe maritima sp. nov.

The genus Diaporthe comprises close to 800 species, with around 2000 names attributed to it and its asexual morphs previously recognized in Phomopsis. Diaporthe species are common plant associates, including saprotrophs, pathogens, and endophytes affiliated with a diverse range of hosts worldwide. In this study, an unknown Diaporthe sp. was frequently isolated as an endophyte from healthy Picea mariana and Picea rubens needles in the Acadian forest of Eastern Canada. Morphological observations and the application of the genealogical concordance phylogenetic species recognition concept using four unlinked loci (internal transcribed spacer (ITS), DNA-lyase (Apn2), translation elongation factor 1-α (EF1-α), and beta-tubulin (TUB)) support the distinctiveness of this species, described here as Diaporthe maritima. Crude liquid culture extracts from this new species showed potent antifungal activity towards the biotrophic pathogen Microbotryum violaceum in a screening assay necessitating an investigation of its natural products. Three dihydropyrones, phomopsolides A (1), B (2), and C (3), and a stable alpha-pyrone (4), were characterized by mass spectrometry and spectroscopic techniques. All isolated metabolites individually demonstrated in vitro antifungal and antibiotic activity towards Bacillus subtilis. The Acadian forest has proven to be a rich source of biodiversity that has the potential to deliver environmentally sustainable pest management tools.

Sexual and asexual states of some endophytic Phialocephala species of Picea.

Unidentified DNA sequences in isolation-based or culture-free studies of conifer endophytes are a persistent problem that requires a field approach to resolve. An investigation of foliar endophytes of Picea glauca, P. mariana, P. rubens and Pinus strobus in eastern Canada, using a combined field, morphological, cultural and DNA sequencing approach, resulted in the frequent isolation of Phialocephala spp. and the first verified discovery of their mollisia-like sexual states in the field. Phialocephala scopiformis and Ph. piceae were the most frequent species isolated as endophytes from healthy conifer needles. Corresponding Mollisia or mollisioid sexual states for Ph. scopiformis, Ph. piceae and several undescribed species in a clade containing Ph. dimorphospora were collected in the sampling area and characterized by analysis of the nuc internal transcribed spacer rDNA (ITS) and gene for the largest subunit of RNA polymerase II (RPB1) loci. Four novel species and one new combination in a clade containing Ph. dimorphospora, the type of Phialocephala, are presented, accompanied by descriptions of apothecia and previously undocumented synanamorphs. An epitype culture and corresponding reference sequences for Phialocephala dimorphospora are proposed. The resulting ITS barcodes linked with robust taxonomic species concepts are an important resource for future research on forest ecosystems and endophytes.

Xerotolerant fungi in house dust: taxonomy of Spiromastix, Pseudospiromastix and Sigleria gen. nov. in Spiromastigaceae (Onygenales, Eurotiomycetes).

During a global investigation of fungi in house dust, we isolated six novel arthroconidial fungi. Phylogenies from combined analysis of nuc rDNA 18S, 28S and internal transcribed spacers sequences demonstrated that these fungi and two species preserved in culture collections represent undescribed species of Spiromastigaceae, Onygenales. Seven of the eight species lacked sexual states and only characters of asexual states and growth rates on different media could be used to characterize them. The eighth species produced ascomata only on water agar. We introduce six new species and one new combination in Spiromastix and validate the recently proposed family Spiromastigaceae, genus Pseudospiromastix and combination Ps. tentaculata. The new genus Sigleria is proposed for two new species that differ from Spiromastix by conidiophore branching patterns, slower growth and a limited ability to utilize nitrate as a sole N source. A key to the three genera of Spiromastigaceae, Spiromastix, Pseudospiromastix and Sigleria, is provided. Phylogenetic analyses support the placement of Spiromastigaceae within Onygenales.

A century later: rediscovery, culturing and phylogenetic analysis of Diploöspora rosea, a rare onygenalean hyphomycete.

Nearly 100 years after its first discovery, Diploöspora rosea was detected on biologically damaged parchment paper in Rome, Italy and isolated from house dust collected in Micronesia. The isolation of this culture permitted morphological study of colony characters, conidium and conidiophore development, and phylogenetic investigations using sequences of nuc 18S rDNA, internal transcribed spacers, and 28S rDNA. The results indicate that D. rosea is an onygenalean fungus, of uncertain taxonomic position, basal or sister to the Gymnoascaceae. Based on observations of the parchments using SEM-Energy Dispersive Spectroscopy, we speculate that the fungus occurs in archival and domestic environments subject to periodic wetting. Its ability to grow on all low water activity media used in the study, including malt extract agar amended with 60% sucrose, confirms its xerophilic nature.

Application of the phylogenetic species concept to Wallemia sebi from house dust and indoor air revealed by multi-locus genealogical concordance.

A worldwide survey of Wallemia occurring in house dust and indoor air was conducted. The isolated strains were identified as W. sebi and W. muriae. Previous studies suggested that the W. sebi phylogenetic clade contained cryptic species but conclusive evidence was lacking because only the internal transcribed spacer (ITS) marker was analyzed. The ITS and four protein-coding genes (MCM7, RPB1, RPB2, and TSR1) were sequenced for 85 isolates. Based on an initial neighbor joining analysis of the concatenated genes, W. muriae remained monophyletic but four clades were found in W. sebi, which we designated as W. sebi clades 1, 2, 3, and 4. We hypothesized that these clades represent distinct phylogenetic species within the Wallemia sebi species complex (WSSC). We then conducted multiple phylogenetic analyses and demonstrated genealogical concordance, which supports the existence of four phylogenetic species within the WSSC. Geographically, W. muriae was only found in Europe, W. sebi clade 3 was only found in Canada, W. sebi clade 4 was found in subtropical regions, while W. sebi clade 1 and 2 were found worldwide. Haplotype analysis showed that W. sebi clades 1 and 2 had multiple haplotypes while W. sebi clades 3 and 4 had one haplotype and may have been under sampled. We describe W. sebi clades 2, 3, and 4 as new species in a companion study.

Rasamsonia pulvericola sp. nov., isolated from house dust.

In the course of a global survey of the indoor mycobiota, we sampled and analysed settled dust from 87 buildings from 14 countries, using both a modified dilution-to-extinction method and 454-pyrosequencing. Rasamsonia is a recently established genus including thermotolerant or thermophilic species, five of which have been isolated from humans, including the emerging pathogen R. argillacea. A new species, R. pulvericola, was recovered from one residence in Songkhla, Thailand, and is morphologically characterised and compared phylogenetically with other members of the genus. Rasamsonia pulvericola forms a clade with R. brevistipitata and shares morphological characters such as usually biverticillate and never terverticillate conidiophores, and subglobose to ellipsoidal conidia. It has a lower maximum growth temperature and is the first mesophilic species added to the genus. The ITS sequence of R. pulvericola was not detected in the 454-pyrosequencing data for Thailand or other countries, but a similar ITS sequence was detected in Micronesia, probably representing another undescribed Rasamsonia species.

The effects of glyphosate on the in vitro linear growth of selected microfungi from a boreal forest soil.

Glyphosate-based herbicides are used extensively in forestry and agriculture to control broadleaf plant competition. A review of the literature offers conflicting results regarding the impact of glyphosate on fungal growth. This study investigated the effects of 7 glyphosate concentrations (1, 2, 5, 10, 50, 100, and 1000 microg*mL-1) of Roundup (35.6% glyphosate) on the number of colony-forming units (CFUs) of soilborne microfungi from a boreal forest soil sample and on the in vitro linear growth of 20 selected species of microfungi representative of this boreal forest soil. Concentrations of glyphosate at 50 microg*mL-1 and higher significantly decreased the number of CFUs observed. At glyphosate concentrations equal to 5 microg*mL-1, 13 fungal species exhibited colony diameters less than 50% than that of their respective controls. Several species showed an inhibition of pigmentation and sporulation when subjected to glyphosate concentrations of 1 microg*mL-1. Differential sensitivity was observed among species at the various concentrations, suggesting the possibility of a shift towards tolerant species of fungi when they are exposed to glyphosate.