Optimizing conditions of mycelial inoculum immobilized in Ca-alginate beads: a case study in ectomycorrhizal fungus Astraeus odoratus.

Ectomycorrhizal inoculum has emerged as a critical tool for forest restoration, especially under challenging climate change conditions. The inoculation of selective ectomycorrhizal fungi can enhance seedling survival and subsequent growth in the field. This study optimized the liquid media for mycelial growth of Astraeus odoratus strain K1 and the sodium alginate solution composition for enhanced mycelial viability after entrapment. Using Modified Melin-Norkrans as the optimal media for mycelial cultivation and 2% sodium alginate supplemented with Czapek medium, 0.25% activated charcoal, 5% sucrose, and 5% sorbitol in the alginate solution yielded the highest viability of A. odoratus mycelia. Preservation in distilled water and 10% glycerol at 25 °C for 60 days proved to be the most effective storage condition for the alginate beads. Both fresh and preserved alginate beads were tested for colonizing on Hopea odorata Roxb. seedlings, showing successful colonization and ectomycorrhizal root formation, with over 49% colonization. This study fills a crucial gap in biotechnology and ectomycorrhizal inoculum, paving the way for more effective and sustainable forest restoration practices.

Comparative transcriptomics reveal different mechanisms for hyphal growth across four plant-associated dimorphic fungi.

Fungal dimorphism is a phenomenon by which a fungus can grow both as a yeast form and a hyphal form. It is frequently related to pathogenicity as different growth forms are more suitable for different functions during a life cycle. Among dimorphic plant pathogens, the corn smut fungus Ustilago maydis serves as a model organism to understand fungal dimorphism and its effect on pathogenicity. However, there is a lack of data on whether mechanisms elucidated from model species are broadly applicable to other fungi. In this study, two non-model plant-associated species in the smut fungus subphylum (Ustilaginomycotina), Tilletiopsis washingtonensis and Meira miltonrushii, were selected to compare dimorphic mechanisms in these to those in U. maydis. We sequenced transcriptomic profiles during both yeast and hyphal growth in these two species using Tween40, a lipid mimic, as a trigger for hyphal growth. We then compared our data with previously published data from U. maydis and a fourth but unrelated dimorphic phytopathogen, Ophiostoma novo-ulmi. Comparative transcriptomics was performed to identify common genes upregulated during hyphal growth in all four dimorphic species. Intriguingly, T. washingtonensis shares the least similarities of transcriptomic alteration (hyphal growth versus yeast growth) with the others, although it is closely related to M. miltonrushii and U. maydis. This suggests that phylogenetic relatedness is not correlated with transcriptomic similarity under the same biological phenomenon. Among commonly expressed genes in the four species, genes in cell energy production and conversion, amino acid transport and metabolism and cytoskeleton are significantly enriched. Considering dimorphism genes characterized in U. maydis, as well as hyphal tip-associated genes from the literature, we found only genes encoding the cell end marker Tea4/TeaC and the kinesin motor protein Kin3 concordantly expressed in all four species. This suggests a divergence in species-specific mechanisms for dimorphic transition and hyphal growth.

Model Choice, Missing Data, and Taxon Sampling Impact Phylogenomic Inference of Deep Basidiomycota Relationships.

Resolving deep divergences in the tree of life is challenging even for analyses of genome-scale phylogenetic data sets. Relationships between Basidiomycota subphyla, the rusts and allies (Pucciniomycotina), smuts and allies (Ustilaginomycotina), and mushroom-forming fungi and allies (Agaricomycotina) were found particularly recalcitrant both to traditional multigene and genome-scale phylogenetics. Here, we address basal Basidiomycota relationships using concatenated and gene tree-based analyses of various phylogenomic data sets to examine the contribution of several potential sources of bias. We evaluate the contribution of biological causes (hard polytomy, incomplete lineage sorting) versus unmodeled evolutionary processes and factors that exacerbate their effects (e.g., fast-evolving sites and long-branch taxa) to inferences of basal Basidiomycota relationships. Bayesian Markov Chain Monte Carlo and likelihood mapping analyses reject the hard polytomy with confidence. In concatenated analyses, fast-evolving sites and oversimplified models of amino acid substitution favored the grouping of smuts with mushroom-forming fungi, often leading to maximal bootstrap support in both concatenation and coalescent analyses. On the contrary, the most conserved data subsets grouped rusts and allies with mushroom-forming fungi, although this relationship proved labile, sensitive to model choice, to different data subsets and to missing data. Excluding putative long-branch taxa, genes with high proportions of missing data and/or with strong signal failed to reveal a consistent trend toward one or the other topology, suggesting that additional sources of conflict are at play. While concatenated analyses yielded strong but conflicting support, individual gene trees mostly provided poor support for any resolution of rusts, smuts, and mushroom-forming fungi, suggesting that the true Basidiomycota tree might be in a part of tree space that is difficult to access using both concatenation and gene tree-based approaches. Inference-based assessments of absolute model fit strongly reject best-fit models for the vast majority of genes, indicating a poor fit of even the most commonly used models. While this is consistent with previous assessments of site-homogenous models of amino acid evolution, this does not appear to be the sole source of confounding signal. Our analyses suggest that topologies uniting smuts with mushroom-forming fungi can arise as a result of inappropriate modeling of amino acid sites that might be prone to systematic bias. We speculate that improved models of sequence evolution could shed more light on basal splits in the Basidiomycota, which, for now, remain unresolved despite the use of whole genome data.

Broad Genomic Sampling Reveals a Smut Pathogenic Ancestry of the Fungal Clade Ustilaginomycotina.

Ustilaginomycotina is home to a broad array of fungi including important plant pathogens collectively called smut fungi. Smuts are biotrophs that produce characteristic perennating propagules called teliospores, one of which, Ustilago maydis, is a model genetic organism. Broad exploration of smut biology has been hampered by limited phylogenetic resolution of Ustilaginiomycotina as well as an overall lack of genomic data for members of this subphylum. In this study, we sequenced eight Ustilaginomycotina genomes from previously unrepresented lineages, deciphered ordinal-level phylogenetic relationships for the subphylum, and performed comparative analyses. Unlike other Basidiomycota subphyla, all sampled Ustilaginomycotina genomes are relatively small and compact. Ancestral state reconstruction analyses indicate that teliospore formation was present at the origin of the subphylum. Divergence time estimation dates the divergence of most extant smut fungi after that of grasses (Poaceae). However, we found limited conservation of well-characterized genes related to smut pathogenesis from U. maydis, indicating dissimilar pathogenic mechanisms exist across other smut lineages. The genomes of Malasseziomycetes are highly diverged from the other sampled Ustilaginomycotina, likely due to their unique history as mammal-associated lipophilic yeasts. Despite extensive genomic data, the phylogenetic placement of this class remains ambiguous. Although the sampled Ustilaginomycotina members lack many core enzymes for plant cell wall decomposition and starch catabolism, we identified several novel carbohydrate active enzymes potentially related to pectin breakdown. Finally, ∼50% of Ustilaginomycotina species-specific genes are present in previously undersampled and rare lineages, highlighting the importance of exploring fungal diversity as a resource for novel gene discovery.

The Suhomyces clade: from single isolate to multiple species to disintegrating sex loci.

Candida tanzawaensis clade members are now placed in Suhomyces. The group was virtually unknown until the early 2000s. Here, we review progress made on Suhomyces over the last two decades and provide data from reports of new members of the group from distant localities worldwide, their habitats and a new study of mating loci that helps explain earlier failed compatibility tests. Phylogenetic studies indicate early diverging members are mostly associated with plants, but later diverging species are usually fungus-feeding insect associates. The genome of S. tanzawaensis was known to have a heterothallic mating allele arrangement with a single MAT α idiomorph. For this review, we generate sequence data and compare the MAT gene arrangement of 30 strains from nine Suhomyces species. These varied from MAT α loci containing mating genes α1 and α2, hypothetical MAT a loci without detectable mating genes a1 and a2 to truncated, possibly completely dissociated MAT loci with intraspecific variation. The absence of a second MAT in a genome locus precludes the possibility of mating type switching. Sympatric speciation likely occurred after MAT locus deterioration began in isolated habitats. Although asexual reproduction may be an effective short-term strategy, theory predicts it will not endure over the extreme long term.

Rare or rarely detected? Ceraceosorus guamensis sp. nov.: a second described species of Ceraceosorales and the potential for underdetection of rare lineages with common sampling techniques.

Ceraceosorales is a monotypic order in Ustilaginomycotina. Its namesake, Ceraceosorus bombacis, was described as a phytopathogen of Bombax ceiba in India. In this study, we describe Ceraceosorus guamensis sp. nov., collected on the South Pacific island of Guam, which appears to represent the second isolation of any member of this order in over 40 years. Ceraceosorus species are monokaryotic and filamentous in culture, producing conidia on potato dextrose agar. However, both species behave yeast-like when cultured on corn meal agar. The internal transcribed spacer (ITS) region (spanning the ITS1-5.8S-ITS2) in both species of Ceraceosorus is highly heterogeneous containing multiple disparate copies that can vary intragenomically by up to 3.5 %. Moreover, this region could not be amplified using the fungal ITS primers most frequently used for culture-independent methods of assessing fungal biodiversity. This fact, combined with the extremely slow growth rates on commonly employed media, may indicate that members of this lineage are potentially underdetected by current sampling methods.

Systems biology-guided understanding of white-rot fungi for biotechnological applications: A review.

Plant-derived biomass is the most abundant biogenic carbon source on Earth. Despite this, only a small clade of organisms known as white-rot fungi (WRF) can efficiently break down both the polysaccharide and lignin components of plant cell walls. This unique ability imparts a key role for WRF in global carbon cycling and highlights their potential utilization in diverse biotechnological applications. To date, research on WRF has primarily focused on their extracellular 'digestive enzymes' whereas knowledge of their intracellular metabolism remains underexplored. Systems biology is a powerful approach to elucidate biological processes in numerous organisms, including WRF. Thus, here we review systems biology methods applied to WRF to date, highlight observations related to their intracellular metabolism, and conduct comparative extracellular proteomic analyses to establish further correlations between WRF species, enzymes, and cultivation conditions. Lastly, we discuss biotechnological opportunities of WRF as well as challenges and future research directions.

Roles of Alternative Sigma Factors in Invasion and Growth Characteristics of Listeria monocytogenes 10403S Into Human Epithelial Colorectal Adenocarcinoma Caco-2 Cell.

Listeria monocytogenes is a Gram-positive facultative intracellular bacterium with a broad host range. With its housekeeping sigma factor and four alternative ones (namely SigB, SigC, SigH, and SigL), L. monocytogenes can express genes in response to changing environments. However, the roles of these sigma factors in intracellular survival are still unclear. The objectives of this study were to characterize the role of each alternative σ factor on L. monocytogenes invasion and growth inside human epithelial colorectal adenocarcinoma Caco-2 cells. We used L. monocytogenes 10403S wild type and its 15 alternative sigma factor deletion mutants at a multiplicity of infection of 100 and 1 in invasion and intracellular growth assays in the Caco-2 cells, respectively. At 1.5, 2, 4, 6, 8, 10, and 12 h post-infection, Caco-2 cells were lysed, and intracellular L. monocytogenes were enumerated on brain-heart infusion agar. Colony-forming and growth rates were compared among strains. The results from phenotypic characterization confirmed that (i) SigB is the key factor for L. monocytogenes invasion and (ii) having only SigA (ΔsigBCHL strain) is sufficient to invade and multiply in the host cell at similar levels as the wild type. Our previous study suggested the negative role of SigL in bile stress response. In this study, we have shown that additional deletion of the rpoN (or sigL) gene to ΔsigB, ΔsigC, or ΔsigH could restore the impaired invasion efficiencies of the single mutant, suggesting the absence of SigL could enhance host invasion. Therefore, we further investigated the role of SigL during extracellular and intracellular life cycles. Using RNA sequencing, we identified 118 and 16 SigL-dependent genes during the extracellular and intracellular life cycles, respectively. The sigL gene itself was induced by fivefolds prior to the invasion, and 5.3 folds during Caco-2 infection, further suggesting the role of SigL in intracellular growth.

Draft Genome Sequence of the Ectomycorrhizal Fungus Astraeus odoratus from Northern Thailand.

We report the draft genome sequence of Astraeus odoratus, an edible ectomycorrhizal fungus from northern Thailand. The assembled genome has a size of 45.1 Mb and 13,403 annotated protein-coding genes. This reference genome will provide a better understanding of the biology of mushroom-forming ectomycorrhizal fungi in the family Diplocystidiaceae.

A Multiomic Approach to Understand How Pleurotus eryngii Transforms Non-Woody Lignocellulosic Material.

Pleurotus eryngii is a grassland-inhabiting fungus of biotechnological interest due to its ability to colonize non-woody lignocellulosic material. Genomic, transcriptomic, exoproteomic, and metabolomic analyses were combined to explain the enzymatic aspects underlaying wheat-straw transformation. Up-regulated and constitutive glycoside-hydrolases, polysaccharide-lyases, and carbohydrate-esterases active on polysaccharides, laccases active on lignin, and a surprisingly high amount of constitutive/inducible aryl-alcohol oxidases (AAOs) constituted the suite of extracellular enzymes at early fungal growth. Higher enzyme diversity and abundance characterized the longer-term growth, with an array of oxidoreductases involved in depolymerization of both cellulose and lignin, which were often up-regulated since initial growth. These oxidative enzymes included lytic polysaccharide monooxygenases (LPMOs) acting on crystalline polysaccharides, cellobiose dehydrogenase involved in LPMO activation, and ligninolytic peroxidases (mainly manganese-oxidizing peroxidases), together with highly abundant H2O2-producing AAOs. Interestingly, some of the most relevant enzymes acting on polysaccharides were appended to a cellulose-binding module. This is potentially related to the non-woody habitat of P. eryngii (in contrast to the wood habitat of many basidiomycetes). Additionally, insights into the intracellular catabolism of aromatic compounds, which is a neglected area of study in lignin degradation by basidiomycetes, were also provided. The multiomic approach reveals that although non-woody decay does not result in dramatic modifications, as revealed by detailed 2D-NMR and other analyses, it implies activation of the complete set of hydrolytic and oxidative enzymes characterizing lignocellulose-decaying basidiomycetes.

Investigating the Smuts: Common Cues, Signaling Pathways, and the Role of MAT in Dimorphic Switching and Pathogenesis.

The corn smut fungus Ustilago maydis serves as a model species for studying fungal dimorphism and its role in phytopathogenic development. The pathogen has two growth phases: a saprobic yeast phase and a pathogenic filamentous phase. Dimorphic transition of U. maydis involves complex processes of signal perception, mating, and cellular reprogramming. Recent advances in improvement of reference genomes, high-throughput sequencing and molecular genetics studies have been expanding research in this field. However, the biology of other non-model species is frequently overlooked. This leads to uncertainty regarding how much of what is known in U. maydis is applicable to other dimorphic fungi. In this review, we will discuss dimorphic fungi in the aspects of physiology, reproductive biology, genomics, and molecular genetics. We also perform comparative analyses between U. maydis and other fungi in Ustilaginomycotina, the subphylum to which U. maydis belongs. We find that lipid/hydrophobicity is a potential common cue for dimorphic transition in plant-associated dimorphic fungi. However, genomic profiles alone are not adequate to explain dimorphism across different fungi.

A new species of Cintractiella (Ustilaginales) from the volcanic island of Kosrae, Caroline Islands, Micronesia.

Cintractiella is an unusual genus of smut fungi containing two described species that produce sori as adventitious gall-like spikelets on members of tribe Hypolytreae (subfam. Mapanioideae, Cyperaceae). In September 200, during a botanical expedition on the volcanic island of Kosrae located in the eastern Caroline Islands and within the Federated States of Micronesia, a specimen of Mapaniapacifica was collected displaying Cintractiella-like sori in adventitious spikelets on the host leaves. Sori were hypophyllous, occurring in groups of spikelets composed of olivaceous-brown scale-like leaves, 1-1.5 mm wide and up to 6 mm long. Microscopic comparison with the protologue and drawings of the type material of C.lamii show several differences in teliospore and sori characters between it and the newly collected material on Mapania. To our knowledge, this represents only the second known collection of any member of Cintractiella on vegetative organs of Hypolytreae and a third species for this genus and the only known smut species infecting Mapania, herein described as Cintractiellakosraensis sp. nov.

Dose-dependent cytotoxic effects of menthol on human malignant melanoma A-375 cells: correlation with TRPM8 transcript expression.

BACKGROUND: Transient receptor potential melastatin 8 (TRPM8), a principle membrane receptor involved in calcium ion influx and cell signal transduction, has been found to be up-regulated in some cancer types, including melanomas. Efficiency of menthol, an agonist of TRPM8, in killing melanoma cancer cells has been reported previously, but the mechanisms remain unclear. We here determined whether in vitro cytotoxic effects of menthol on A-375 human malignant melanoma cells might be related to TRPM8 transcript expression. MATERIALS AND METHODS: The PrestoBlue® cell viability assay was used to assess the in vitro cytotoxic effect of menthol after 24h of treatment. RT-PCR was used to quantify TRPM8 transcript expression levels in normal and menthol- treated cells. Cell morphology was observed under inverted phase contrast light microscopy. RESULTS: TRPM8 transcript expression was found at low levels in A-375 cells and down-regulated in a potentially dose-dependent manner by menthol. Menthol exerted in vitro cytotoxic effects on A-375 cells with an IC50 value of 11.8 µM, which was at least as effective as 5-fluorouracil (IC50=120 µM), a commonly applied chemotherapeutic drug. Menthol showed no dose-dependent cytotoxicity on HeLa cells, a TRPM8 non-expressing cell line. CONCLUSIONS: The cytotoxic effects on A-375 cells caused by menthol might be related to reduction of the TRPM8 transcript level. This suggests that menthol might activate TRPM8 to increase cytosolic Ca2+ levels, which leads to cytosolic Ca2+ imbalance and triggers cell death.