Interactions among Escovopsis, Antagonistic Microfungi Associated with the Fungus-Growing Ant Symbiosis

Fungi in the genus Escovopsis (Ascomycota: Hypocreales) are prevalent associates of the complex symbiosis between fungus-growing ants (Tribe Attini), the ants’ cultivated basidiomycete fungi and a consortium of both beneficial and harmful microbes found within the ants’ garden communities. Some Escovopsis spp. have been shown to attack the ants’ cultivated fungi, and co-infections by multiple Escovopsis spp. are common in gardens in nature. Yet, little is known about how Escovopsis strains impact each other. Since microbe–microbe interactions play a central role in microbial ecology and evolution, we conducted experiments to assay the types of interactions that govern Escovopsis–Escovopsis relationships. We isolated Escovopsis strains from the gardens of 12 attine ant genera representing basal (lower) and derived groups in the attine ant phylogeny. We conducted in vitro experiments to determine the outcome of both intraclonal and interclonal Escovopsis confrontations. When paired with self (intraclonal interactions), Escovopsis isolated from lower attine colonies exhibited antagonistic (inhibitory) responses, while strains isolated from derived attine colonies exhibited neutral or mutualistic interactions, leading to a clear phylogenetic pattern of interaction outcome. Interclonal interactions were more varied, exhibiting less phylogenetic signal. These results can serve as the basis for future studies on the costs and benefits of Escovopsis coinfection, and on the genetic and chemical mechanisms that regulate the compatibility and incompatibility observed here.

Melanin production and laccase mediated oxidative stress alleviation during fungal-fungal interaction among basidiomycete fungi

Fungal-fungal interaction often leads to the change in metabolite profile of both the interacting fungus which may have potential implication in industry or agriculture. In the present study, we performed two sets of fungal-fungal interaction—Trametes coccinea (F3) with Leiotrametes lactinea (F9) and T. coccinea (F3) with T. versicolor (F1) to understand the changes in the metabolite profile during the interaction process and how this process impacts the hyphal/mycelial morphology of the participating fungi. The metabolites produced during interaction of T. coccinea (F3) with L. lactinea (F9) and T. coccinea (F3) with T. versicolor (F1) was analysed through liquid chromatography coupled to mass spectroscopy (LC-MS). Most of the metabolites secreted or produced during interaction are associated with defensive response. Further, visualization with scanning electron microscopy revealed that interaction between the tested fungi led to the changes in the hyphal morphology. The bipartite fungal interaction resulted in the production of a dark brown colour pigment—melanin as confirmed by the LC-MS, FTIR and NMR analysis. Moreover, the fungal–fungal interaction also led to increase in the production of laccase, a group of multicopper oxidases involved in detoxification of toxic compounds. Further, increased activity of superoxide dismutase, an enzyme that catalyzes the dismutation of the superoxide anion to hydrogen peroxide was also recorded during fungal–fungal interaction. Quantitative real-time PCR revealed upregulation of lcc1 (encoding a laccase enzyme) and few other stress related genes of T. versicolor during its hyphal interaction with T. coccinea, suggesting a direct correlation between laccase production and melanin production.

Direct Ethanol Production from Xylan and Acorn Using the Starch-Fermenting Basidiomycete Fungus Phlebia acerina

During our search for ethanol-producing basidiomycete fungi for a wide range of substrates, we isolated Phlebia acerina, which is a white rot basidiomycete fungus. It favorably converted starch into ethanol with approximately 70% yield. Although the yield decreased as the starch concentration increased, growth and fermentation were observed even at 200 g/L of starch. P. acerina produced ethanol from glucose, galactose, mannose, xylose, cellobiose, and maltose with 93%, 91%, 86%, 72%, 92%, and 68% yields, respectively. Additionally, P. acerina, which secreted xylanase and xylosidase, was capable of assimilating xylan and directly converting it to ethanol with a yield of 63%. Furthermore, P. acerina produced ethanol directly from acorns, which are plant fruits containing starch and tannins, with a yield of 70%. Tannin delayed mycelia growth, thus prolonging ethanol production; however, this did not particularly affect the yield. These results were similar to those of fermentation in a medium with the same amounts of starch and tannin as the target crop acorn, thus suggesting that P. acerina could successfully produce environmentally friendly ethanol from starch-containing lignocellulosic biomass, unlike previously reported ethanol-producing basidiomycete fungi.

Host Susceptibility Modulates Escovopsis Pathogenic Potential in the Fungiculture of Higher Attine Ants

Health and disease emerge from intricate interactions between genotypes, phenotypes, and environmental features. The outcomes of such interactions are context-dependent, existing as a dynamic continuum ranging from benefits to damage. In host-microbial interactions, both the host and environmental conditions modulate the pathogenic potential of a microorganism. Microbial interactions are the core of the agricultural systems of ants in the subtribe Attina, which cultivate basidiomycete fungi for food. The fungiculture environment harbors a diverse microbial community, including fungi in the genus Escovopsis that has been studied as damage-causing agent. Here, we consider the ant colony as a host and investigate to what extent its health impacts the dynamics and outcomes of host-Escovopsis interactions. We found that different ant fungal cultivars vary in susceptibility to the same Escovopsis strains in plate-assays interactions. In subcolony-Escovopsis interactions, while healthy subcolonies gradually recover from infection with different concentrations of Escovopsis conidia, insecticide-treated subcolonies evidenced traits of infection and died within 7 days. The opportunistic nature of Escovopsis infections indicates that diseases in attine fungiculture are a consequence of host susceptibility, rather than the effect of a single microbial agent. By addressing the host susceptibility as a major modulator of Escovopsis pathogenesis, our findings expand the understanding of disease dynamics within attine colonies.

Cytoplasmic Mixing, Not Nuclear Coexistence, Can Explain Somatic Incompatibility in Basidiomycetes

Nonself recognition leading to somatic incompatibility (SI) is commonly used by mycologists to distinguish fungal individuals. Despite this, the process remains poorly understood in basidiomycetes as all current models of SI are based on genetic and molecular research in ascomycete fungi. Ascomycete fungi are mainly found in a monokaryotic stage, with a single type of haploid nuclei, and only briefly during mating do two genomes coexist in heterokaryotic cells. The sister phylum, Basidiomycota, differs in several relevant aspects. Basidiomycete fungi have an extended heterokaryotic stage, and SI is generally observed between heterokaryons instead of between homokaryons. Additionally, considerable nuclear migration occurs during a basidiomycete mating reaction, introducing a nucleus into a resident homokaryon with cytoplasmic mixing limited to the fused or neighboring cells. To accommodate these differences, we describe a basidiomycete model for nonself recognition using post-translational modification, based on a reader-writer system as found in other organisms. This post-translational modification combined with nuclear migration allows for the coexistence of two genomes in one individual while maintaining nonself recognition during all life stages. Somewhat surprisingly, this model predicts localized cell death during mating, which is consistent with previous observations but differs from the general assumptions of basidiomycete mating. This model will help guide future research into the mechanisms behind basidiomycete nonself recognition.

Transcriptomics reveals the mycoparasitic strategy of the mushroom Entoloma abortivum on species of the mushroom Armillaria

During mycoparasitism, a fungusーthe hostーis parasitized by another fungusーthe mycoparasite. The genetic underpinnings of these relationships have been best characterized in Ascomycete fungi. However, within Basidiomycete fungi, there are rare instances of mushroom-forming species parasitizing the reproductive structures, or sporocarps, of other mushroom-forming species. One of the most enigmatic of these occurs between Entoloma abortivum and species of Armillaria, where hyphae of E. abortivum are hypothesized to disrupt the development of Armillaria sporocarps, resulting in the formation of carpophoroids. However, it remains unknown whether carpophoroids are the direct result of a mycoparasitic relationship. To address the nature of this unique interaction, we analyzed gene expression of field-collected Armillaria and E. abortivum sporocarps and carpophoroids. Transcripts in the carpophoroids are primarily from E. abortivum, supporting the hypothesis that this species is parasitizing Armillaria. Most notably, we identified differentially expressed E. abortivum β-trefoil-type lectins in the carpophoroid, which we hypothesize bind to Armillaria cell wall galactomannoproteins, thereby mediating recognition between the mycoparasite and the host. The most significantly upregulated E. abortivum transcripts in the carpophoroid code for oxalate decarboxylasesーenzymes that degrade oxalic acid. Oxalic acid is a virulence factor in many plant pathogens, including Armillaria species, however, E. abortivum has evolved a sophisticated strategy to overcome this defense mechanism. The number of gene models and genes that code for carbohydrate-active enzymes in the E. abortivum transcriptome were reduced compared to other closely related species, perhaps as a result of the specialized nature of this interaction.

A comparative study on the laccase activity of four basidiomycete fungi with different lignocellulosic residues via solid-state fermentation

The laccase producing abilities of four Basidiomycete fungi species were compared using solid-state fermentation using four different lignocellulosic residues. The biosynthetic potential of the Basidiomycetes was highly dependent on the type of fungi. In general, the laccase secreting ability of Cerrena unicolor Han 849 was greater than Lenzites betulinus Han 851, Stropharia rugosoannulata Han 1321, and Auricularia heimuer Han 1333. The maximum laccase production of C. unicolor Han 849 was approximately 11.25, 122.26, and 15.27 times higher than L. betulinus Han 851, S. rugosoannulata Han 1321 and A. heimuer Han 1333, respectively. Different species of fungi had a preference in lignocellulosic residues. The presence of Firmiana platanifolia was conducive to secreting laccase via C. unicolor Han 849 during solid-state fermentation. A continuous and stable laccase production via C. unicolor Han 849 was an obvious advantage of solid-state fermentation with any of the four lignocellulosic residues used. The maximum laccase production of C. unicolor Han 849 using Firmiana platanifolia was approximately 2.12, 1.68, and 6.13 times higher than Populus beijingensis, Sorghum bicolor, and Oryza sativa, respectively. These findings will be helpful for developing new productivity strains in industrial applications and selecting suitable lignocellulosic residues for laccase production.

Resistance mechanisms of Cryptococcus spp. and plant compounds as tools to combat them

Cryptococcus is a genus of dimorphic basidiomycete fungi found in the form of yeasts and filaments. Cryptococcosis has as main etiological agents the species Cryptococcus neoformans and Cryptococcus gattii. This disease is considered a public health problem and has becoming more alarming because of the limitations of antimicrobials available to its treatment, in addition to an increase in reports of fungal resistance. In this sense, the present review sought to survey information on the resistance mechanisms of Cryptococcus spp. against the main drugs used in cryptococcosis therapy as well as on the antimicrobial activities of plants against these fungi. Studies have reported that several mechanisms may be involved in fungal resistance to drugs including drug inactivation by enzymes, expression of efflux pumps and others drug transporters, as well as changes in the drug target and/or implementation of alternative metabolic pathways. As an alternative to conventional antimicrobials, substances and molecules extracted from plants have demonstrated potential for controlling these pathogens. These phytochemicals can trigger the inhibition and/or death of Cryptococcus through morphological changes on fungi cells, inhibition of ergosterol synthesis, cell leakage, capsular decrease, interference in cell division, reduction of activity of several enzymes such as laccase and urease, inhibition of biofilm formation, among others. In this sense, plants are an important source of bioactive compounds with antimicrobial activity that can be studied in the search for new drugs that are increasingly effective, specific and less toxic in the control of cryptococcosis.

Protein Engineering Approaches to Enhance Fungal Laccase Production in S. cerevisiae

Laccases secreted by saprotrophic basidiomycete fungi are versatile biocatalysts able to oxidize a wide range of aromatic compounds using oxygen as the sole requirement. Saccharomyces cerevisiae is a preferred host for engineering fungal laccases. To assist the difficult secretion of active enzymes by yeast, the native signal peptide is usually replaced by the preproleader of S. cerevisiae alfa mating factor (MFα1). However, in most cases, only basal enzyme levels are obtained. During directed evolution in S. cerevisiae of laccases fused to the α-factor preproleader, we demonstrated that mutations accumulated in the signal peptide notably raised enzyme secretion. Here we describe different protein engineering approaches carried out to enhance the laccase activity detected in the liquid extracts of S. cerevisiae cultures. We demonstrate the improved secretion of native and engineered laccases by using the fittest mutated α-factor preproleader obtained through successive laccase evolution campaigns in our lab. Special attention is also paid to the role of protein N-glycosylation in laccase production and properties, and to the introduction of conserved amino acids through consensus design enabling the expression of certain laccases otherwise not produced by the yeast. Finally, we revise the contribution of mutations accumulated in laccase coding sequence (CDS) during previous directed evolution campaigns that facilitate enzyme production.