Journal of Fungi

An efficient Agrobacterium-mediated genetic transformation based on the plant binary vector pPZP-RCS2 was carried out for the multiple heterologous protein production in filamentous fungus Thermothelomyces thermophilus F-859 (formerly Myceliophthora thermophila F-859). The engineered fungus Th. thermophilus was able to produce plant storage proteins of Zea mays (α-zein Z19) and Amaranthus hypochondriacus (albumin A1) to enrich fungal biomass by valuable nutritional proteins and improved amino acid content. The mRNA levels of z19 and a1 genes were significantly dependent on their driving promoters: the promoter of tryptophan synthase (PtrpC) was more efficient to express a1, while the promoter of translation elongation factor (Ptef) provided much higher levels of z19 transcript abundance. In general, the total recombinant proteins and amino acid contents were higher in the Ptef-containing clones. This work describes a new strategy to improve mycoprotein nutritive value by overexpression of plant storage proteins. Full article

Four yeast strains, representing one genus and two novel anamorphic yeast species, were isolated from pineapple leaves collected in Thailand. Analysis of the sequences of the D1/D2 domains of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) regions showed the two strains (DMKU-PAL186 and DMKU-PAL178) were closely related to the type strains of the Protomyces and Taphrina species, but with high nucleotide divergence. Two strains (DMKU-PAL39 and DMKU-PAL18) were found to be closely related to the type strains of Goffeauzyma iberica, but with eight nucleotide substitutions in the D1/D2 domains and 26 nucleotide substitutions in the ITS regions. In phylogenetic analyses, the strains DMKU-PAL186 and DMKU-PAL178 formed a well-separated lineage from Protomyces and Taphrina genera, confirming that they represented a distinct genus, while the strains DMKU-PAL39 and DMKU-PAL18 represented a species in the genus Goffeauzyma, which was phylogenetically distinct from other recognized species of the genus. Based on molecular analyses and phenotypic characteristics, the names Savitreella gen. nov. (Taphrinomycetes, Ascomycota) and Savitreella phatthalungensis sp. nov. are proposed to accommodate the strains DMKU-PAL186 and DMKU-PAL178, and the name Goffeauzyma siamensis sp. nov. (Tremellomycetes, Basidiomycota) is proposed to accommodate the strains DMKU-PAL39 and DMKU-PAL18. Full article

The antifungal and antiaflatoxinogenic activities of the essential oils (EOs) from the leaves of Cymbopogon schoenanthus, Cymbopogon citratus, Cymbopogon nardus, and their pair combinations were investigated. Antifungal susceptibility and the efficacy of paired combinations of EOs were assessed using agar microdilution and checkerboard methods, respectively. Identification and quantification of chemical components of the EOs were carried out by gas chromatography-mass spectrometry and gas chromatography-flame ionization detector (GC-MS and GC-FID), respectively. Aflatoxins were separated and identified by High-Performance Liquid Chromatography (HPLC) and then quantified by spectrofluorescence. The EO of C. nardus exhibited the highest inhibitory activity against Aspergillus flavus and Aspergillus parasiticus. The combination of C. citratus and C. nardus and that of C. nardus and C. schoenanthus exhibited a synergistic effect against Aspergillus flavus and Aspergillus, respectively. Both C. citratus and C. schoenanthus EOs totally inhibited the synthesis of aflatoxin B1 at 1 µL/mL. C. citratus blocked the production of aflatoxins B2 and G2 at 0.5 µL/mL. Both C. citratus and C. schoenanthus totally hampered the production of the aflatoxin G1 at 0.75 µL/mL. The combination of C. citratus and C. schoenanthus completely inhibited the production of the four aflatoxins. The study shows that the combinations can be used to improve their antifungal and antiaflatoxinogenic activities. Full article

Globally, candidemia displays geographical variety in terms of epidemiology and incidence. In that respect, a nationwide Greek study was conducted, reporting the epidemiology of Candida bloodstream infections and susceptibility of isolates to antifungal agents providing evidence for empirical treatment. All microbiologically confirmed candidemia cases in patients hospitalized in 28 Greek centres during the period 2009–2018 were recorded. The study evaluated the incidence of infection/100,000 inhabitants, species distribution, and antifungal susceptibilities of isolated strains. Overall, 6057 candidemic episodes occurred during the study period, with 3% of them being mixed candidemias. The average annual incidence was 5.56/100,000 inhabitants, with significant increase over the years (p = 0.0002). C. parapsilosis species complex (SC) was the predominant causative agent (41%), followed by C. albicans (37%), C. glabrata SC (10%), C. tropicalis (7%), C. krusei (1%), and other rare Candida spp. (4%). C. albicans rates decreased from 2009 to 2018 (48% to 31%) in parallel with a doubling incidence of C. parapsilosis SC rates (28% to 49%, p < 0.0001). Resistance to amphotericin B and flucytosine was not observed. Resistance to fluconazole was detected in 20% of C. parapsilosis SC isolates, with a 4% of them being pan-azole-resistant. A considerable rising rate of resistance to this agent was observed over the study period (p < 0.0001). Echinocandin resistance was found in 3% of C. glabrata SC isolates, with 70% of them being pan-echinocandin-resistant. Resistance rate to this agent was stable over the study period. This is the first multicentre nationwide study demonstrating an increasing incidence of candidemia in Greece with a species shift toward C. parapsilosis SC. Although the overall antifungal resistance rates remain relatively low, fluconazole-resistant C. parapsilosis SC raises concern. Full article

The in vitro interaction of amphotericin B in combination with colistin was evaluated against a total of 86 strains comprising of 47 Candida species (10 Candida albicans, 15 Candida auris, five Candida glabrata, three Candida kefyr, five Candida krusei, four Candida parapsilosis and five Candida tropicalis), 29 Aspergillus species (five Aspergillus flavus, 10 Aspergillus fumigatus, four Aspergillus nidulans, five Aspergillus niger, and five Aspergillus terreus), and 10 Rhizopus species (seven Rhizopus arrhizus, one Rhizopus delemar and two Rhizopus microsporus) strains. For the determination of the interaction, a microdilution checkerboard technique based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) reference method for antifungal susceptibility testing was used. Results of the checkerboard technique were evaluated by the fractional inhibitory concentration index (FICI) based on the Loewe additivity model for all isolates. Different inhibition endpoints were used to capture both the interaction at MIC and sub-MIC levels. Additionally, checkerboard technique results for Candida species were evaluated by response surface analysis based on the Bliss independence model. Against common Candida species, the combination was synergistic for 75% of the strains by FICI and for 66% of the strains by response surface analysis. For C. tropicalis, the interaction was antagonistic for three isolates by FICI, but antagonism was not confirmed by response surface analysis. Interestingly, synergistic and antagonistic FICIs were simultaneously present on checkboard microplates of all three strains. Against C. auris the combination was synergistic for 73% of the strains by response surface analysis and for 33% of the strains by FICI. This discrepancy could be related to the insensitivity of the FICI to detect weak interactions. Interaction for all other strains was indifferent. For Aspergillus and Rhizopus species combination exhibited only indifferent interactions against all tested strains. Full article

Although aquatic and parasitic fungi have been well known for more than 100 years, they have only recently received increased awareness due to their key roles in microbial food webs and biogeochemical cycles. There is growing evidence indicating that fungi inhabit a wide range of marine habitats, from the deep sea all the way to surface waters, and recent advances in molecular tools, in particular metagenome approaches, reveal that their diversity is much greater and their ecological roles more important than previously considered. Parasitism constitutes one of the most widespread ecological interactions in nature, occurring in almost all environments. Despite that, the diversity of fungal parasites, their ecological functions, and, in particular their interactions with other microorganisms remain largely speculative, unexplored and are often missing from current theoretical concepts in marine ecology and biogeochemistry. In this review, we summarize and discuss recent research avenues on parasitic fungi and their ecological potential in marine ecosystems, e.g., the fungal shunt, and emphasize the need for further research. Full article

Apple Valsa canker is one of the most serious diseases, having caused significant apple yield and economic loss in China. However, there is still no effective biological methods for controlling this disease. Our present study focused on the inhibitory activity and mechanisms of Trichoderma longibrachiatum (T6) fermentation on Valsa mali that causes apple Valsa canker (AVC). Our results showed that the T6 fermentation exhibited effective antifungal activity on the mycelial growth and conidia germination of V. mali, causing mycelium malformation and the hyphal disintegrating in comparison to the control. The activity of pathogenically related enzymes that are secreted from V. mali and the expression level of gene of V. mali were significantly inhibited and downregulated by treatment with T6 fermentation. In addition, the lesion area and number of pycnidia of V. mali formed on the branches were significantly reduced after treatment with the T6 fermentation through the pathogenicity test on the detached branches. Our results indicate that the possible mechanism of T6 fermentation against V. mali occurs through inhibiting its growth and reproduction, the pathogenic enzyme activity, and its related gene expression. Full article

The present study focused on the use of sewage sludge (SS) as a casing material amendment and the potential uptake of metal elements by the cultivated white button (Agaricus bisporus: MS-39) mushroom. Laboratory experiments were performed under controlled environmental conditions to grow A. bisporus on the composted wheat straw substrate for 50 days. Different treatments (0, 50, 100, 150, and 200 g/kg) of casing material were prepared by mixing garden and dried SS and applied on the mushroom substrate after proper sterilization. The results revealed that SS application was significant (p < 0.05) in accelerating mushroom yield with a biological efficiency of 65.02% for the mixing rate of 200 g/kg. Moreover, the maximum bioaccumulation of selected metal elements (Cu, Cr, Cd, Fe, Mn, and Zn) was observed using the same treatment. Additionally, the multiple regression models constructed for the uptake prediction of metal elements showed an acceptable coefficient of determination (R² > 0.9900), high model efficiency (ME > 0.98), and low root mean square error (RMSE < 0.410) values, respectively. The findings of this study represent sustainable use of SS for the formulation of mushroom casing material contributing toward synergistic agro-economy generation and waste management. Full article

Blue mold caused by Penicillium italicum is one of the most serious postharvest diseases of citrus fruit. The aim of this study was to investigate the inhibitory effect of a novel oligosaccharide ester, 6-O-β-L-mannopyranosyl-3-O-(2-methylbutanoyl)-4-O-(8-methyldecanoyl)-2-O-(4-methyl-hexanoyl) trehalose (MTE-1), against P. italicum. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM), along with transcriptome and proteome analysis also, were conducted to illuminate the underlying mechanism. Results showed that MTE-1 significantly inhibited P. italicum growth in vitro in a dose-dependent manner. Moreover, MTE-1 suppressed the disease development of citrus fruit inoculated with P. italicum. Furthermore, ultrastructure observation, as well as transcriptome and proteome analysis, indicated that MTE-1 treatment damaged the cell wall and plasma membrane in spores and mycelia of P. italicum. In addition, MTE-1 regulated genes or proteins involved in primary metabolism, cell-wall metabolism, and pathogenicity. These results demonstrate that MTE-1 inhibited P. italicum by damaging cell walls and membranes and disrupting normal cellular metabolism. These findings contribute to the understanding of the possible molecular action of MTE-1. Finally, MTE-1 also provides a new natural strategy for controlling diseases in postharvest fruit. Full article

Chronic pulmonary aspergillosis (CPA) is a fungal lung infection associated with high morbidity and mortality. Yet, it remains under-recognized worldwide, with few Australian clinical data available. This retrospective study aimed to investigate CPA at a major tertiary referral hospital in Sydney. We identified patients having International Classification of Diseases (ICD-10) codes for “aspergillosis” and/or positive respiratory microbiology samples for Aspergillus species from January 2012–December 2018 at Westmead Hospital. Eligible cases were classified using European Respiratory Society 2016 CPA guidelines. We diagnosed 28 CPA patients: median age 60 years (IQR: 57–66), with 17 (60.7%) being males. Most had chronic cavitary pulmonary aspergillosis phenotype (n = 17, 60.7%). Twenty-three patients had outcomes data returned. Nineteen (82.6%) received antifungal therapy (median duration: 10.5 months (IQR: 6.5–20.7)). Eight (34.7%) patients received <6 months of antifungals, including three (38%) deaths. Two (13%) patients receiving ≥6 months of antifungals died. Chronic obstructive pulmonary disease (COPD) (n = 9, 32.1%) was the leading predisposing factor for CPA in our cohort. This contrasts with the global picture, where prior tuberculosis generally predominates, but is similar to findings from other high-income countries. Nevertheless, further larger-scale studies are required to determine whether these results are generalizable to the wider Australian population. Full article

With the increasing world population, demand for industrialization has also increased to fulfill humans’ living standards. Fungi are considered a source of essential constituents to produce the biocatalytic enzymes, including amylases, proteases, lipases, and cellulases that contain broad-spectrum industrial and emerging applications. The present review discussed the origin, nature, mechanism of action, emerging aspects of genetic engineering for designing novel proteases, genome editing of fungal strains through CRISPR technology, present challenges and future recommendations of fungal proteases. The emerging evidence revealed that fungal proteases show a protective role to many environmental exposures and discovered that an imbalance of protease inhibitors and proteases in the epithelial barriers leads to the protection of chronic eosinophilic airway inflammation. Moreover, mitoproteases recently were found to execute intense proteolytic processes that are crucial for mitochondrial integrity and homeostasis function, including mitochondrial biogenesis, protein synthesis, and apoptosis. The emerging evidence revealed that CRISPR/Cas9 technology had been successfully developed in various filamentous fungi and higher fungi for editing of specific genes. In addition to medical importance, fungal proteases are extensively used in different industries such as foods to prepare butter, fruits, juices, and cheese, and to increase their shelf life. It is concluded that hydrolysis of proteins in industries is one of the most significant applications of fungal enzymes that led to massive usage of proteomics. Full article

Parabambusicolaceae is a well-studied family in Massarineae, Pleosporales, comprising nine genera and approximately 16 species. The family was introduced to accommodate saprobic bambusicola-like species in both freshwater and terrestrial environments that mostly occur on bamboos and grasses but are also found on different host substrates. In the present study, we surveyed and collected ascomycetes from bamboo and submerged grass across Yunnan Province, China. A biphasic approach based on morphological characteristics and multigene phylogeny demonstrated five new taxa in Parabambusicolaceae. A novel genus Scolecohyalosporium is introduced as a monotypic genus to accommodate S. submersum sp. nov., collected from dead culms of grass submerged in a freshwater stream. The genus is unique in forming filiform ascospores, which differ from other known genera in Parabambusicolaceae. Multigene phylogeny showed that the genus has a close relationship with Multiseptospora. Moreover, the novel monotypic genus Neomultiseptospora, isolated from bamboo, was introduced to accommodate N. yunnanensis sp. nov. Neomultiseptospora yunnanensis formed a separated branch basal to Scolecohyalosporium submersum and Multiseptospora thailandica with high support (100% ML, 1.00 PP). Furthermore, the newly introduced species, Parabambusicola hongheensis sp. nov. was also isolated from bamboo in terrestrial habitats. Parabambusicola hongheensis clustered with the other three described Parabambusicola species and has a close relationship with P. bambusina with significant support (88% ML, 1.00 PP). Parabambusicola hongheensis was reported as the fourth species in this genus. Detailed description, illustration, and updated phylogeny of Parabambusicolaceae were provided. Full article

Aspergillus niger strain CSR3 is an endophytic fungus that regulates plant endogenous hormones, secondary metabolites, and promotes plant growth during abiotic stress conditions. In this study, we sequenced the genome of A. niger (CSR3) and compared it with previously available A. niger strains. The final genome assembly was 35.8 Mb in size, consisting of 23 scaffolds with N50 scaffold length of 2.4 Mb. A total of 12,442 protein coding genes, 270 tRNA, and 57 rRNA were predicted in the CSR3 genome. We used comparative genomic analysis to provide insights into the genome’s evolution and to elucidate the adaptive genomic signatures for bioactive secondary metabolite biosynthesis, hormones biosynthesis, and plant growth promoting activities. We also analyzed the transposable elements (TEs), simple sequence repeats (SSRs), CAZymes families, genes involved in gibberellin biosynthesis, and secondary metabolite clusters in the CSR3 genome. A total of 21 secondary metabolite biosynthesis gene clusters were detected, with 18 essential enzymes involved in the mevalonate pathway (MVA). The repeat analysis revealed about 3431 SSR, 274 TEs, and 205 inverted repeats (IR). Further gene family analysis revealed that 124 gene families were gained, whereas 125 gene families were lost in CSR3 genome, compared to A. niger ASM151534V and A. niger ASM285V2 genomes. The results improve our understanding of the CSR3 genome and will assist in future investigations on the genetic basis of A. niger CSR3, including the identification of CSR3 phytostimulant properties. Full article

Entomopathogenic fungi play important roles in the control of populations of agricultural and disease vector pests in nature. The shortcomings of mycoinsecticides for pest management in the field cannot be completely overcome by improving single biocontrol properties of fungi. Therefore, enhancing the biocontrol potential of entomopathogenic fungi in multiple respects by genetic engineering is desirable. Transcription factors are usually involved in various important processes during fungal growth and pathogenesis via regulating a series of genes, and are important candidates for fungal improvement via genetic engineering. Herein, overexpression of MaSom1, a key transcription factor gene in the cAMP/PKA pathway, improves the biocontrol traits of Metarhizium acridum in multiple respects. When compared with WT, the MaSom1-overexpression strains exhibit enhanced tolerances to UV-B and heat shock, with increased mean 50% inhibition times by 66.9% and 155.2%, respectively. Advanced conidiation emerged accompanied by increased conidial yield up to 3.89 times after 3-day incubation for the MaSom1-overexpression strains compared to WT. Furthermore, when compared with WT, the virulence of the MaSom1-overexpression strains was also increased with the mean 50% lethality times reduced by 21.8% to 23.8%. Taken together, the MaSom1-overexpression improved the biocontrol potential of M. acridum in multiple respects. Our results provide insights into the application of key transcription factors for genetic engineering and offer a credible way to further improve the biocontrol potential of entomopathogenic fungi. Full article

We aimed to establish an etiology-based connection between the symptoms experienced by the occupants of a workplace and the presence in the building of toxic dampness microbiota. The occupants (5/6) underwent a medical examination and urine samples (2/6) were analyzed by LC-MS/MS for mycotoxins at two time-points. The magnitude of inhaled water was estimated. Building-derived bacteria and fungi were identified and assessed for toxicity. Separate cytotoxicity tests using human THP-1 macrophages were performed from the office’s indoor air water condensates. Office-derived indoor water samples (n = 4/4) were toxic to human THP-1 macrophages. Penicillium, Acremonium sensu lato, Aspergillus ochraceus group and Aspergillus section Aspergillus grew from the building material samples. These colonies were toxic in boar sperm tests (n = 11/32); four were toxic to BHK-21 cells. Mycophenolic acid, which is a potential immunosuppressant, was detected in the initial and follow-up urine samples of (2/2) office workers who did not take immunosuppressive drugs. Their urinary mycotoxin profiles differed from household and unrelated controls. Our study suggests that the presence of mycotoxins in indoor air is linked to the morbidity of the occupants. The cytotoxicity test of the indoor air condensate is a promising tool for risk assessment in moisture-damaged buildings. Full article

Acetic acid is a major inhibitory compound in several industrial bioprocesses, in particular in lignocellulosic yeast biorefineries. Cell envelope remodeling, involving cell wall and plasma membrane composition, structure and function, is among the mechanisms behind yeast adaptation and tolerance to stress. Pdr18 is a plasma membrane ABC transporter of the pleiotropic drug resistance family and a reported determinant of acetic acid tolerance mediating ergosterol transport. This study provides evidence for the impact of Pdr18 expression in yeast cell wall during adaptation to acetic acid stress. The time-course of acetic-acid-induced transcriptional activation of cell wall biosynthetic genes (FKS1, BGL2, CHS3, GAS1) and of increased cell wall stiffness and cell wall polysaccharide content in cells with the PDR18 deleted, compared to parental cells, is reported. Despite the robust and more intense adaptive response of the pdr18Δ population, the stress-induced increase of cell wall resistance to lyticase activity was below parental strain levels, and the duration of the period required for intracellular pH recovery from acidification and growth resumption was higher in the less tolerant pdr18Δ population. The ergosterol content, critical for plasma membrane stabilization, suffered a drastic reduction in the first hour of cultivation under acetic acid stress, especially in pdr18Δ cells. Results revealed a crosstalk between plasma membrane ergosterol content and cell wall biophysical properties, suggesting a coordinated response to counteract the deleterious effects of acetic acid. Full article

In addition to being key pathogens in plants, animals, and humans, fungi are also valuable resources in agriculture, food, medicine, industry, and the environment. The elimination of pathogenic fungi and the functional enhancement of beneficial fungi have been the major topics investigated by researchers. Non-thermal plasma (NTP) is a potential tool to inactivate pathogenic and food-spoiling fungi and functionally enhance beneficial fungi. In this review, we summarize and discuss research performed over the last decade on the use of NTP to treat both harmful and beneficial yeast- and filamentous-type fungi. NTP can efficiently inactivate fungal spores and eliminate fungal contaminants from seeds, fresh agricultural produce, food, and human skin. Studies have also demonstrated that NTP can improve the production of valuable enzymes and metabolites in fungi. Further studies are still needed to establish NTP as a method that can be used as an alternative to the conventional methods of fungal inactivation and activation. Full article

The boletoid genera Butyriboletus and Exsudoporus have recently been suggested by some researchers to constitute a single genus, and Exsudoporus was merged into Butyriboletus as a later synonym. However, no convincing arguments have yet provided significant evidence for this congeneric placement. In this study, we analyze material from Exsudoporus species and closely related taxa to assess taxonomic and phylogenetic boundaries between these genera and to clarify species delimitation within Exsudoporus. Outcomes from a multilocus phylogenetic analysis (ITS, nrLSU, tef1-α and rpb2) clearly resolve Exsudoporus as a monophyletic, homogenous and independent genus that is sister to Butyriboletus. An accurate morphological description, comprehensive sampling, type studies, line drawings and a historical overview on the nomenclatural issues of the type species E. permagnificus are provided. Furthermore, this species is documented for the first time from Israel in association with Quercus calliprinos. The previously described North American species Exsudoporus frostii and E. floridanus are molecularly confirmed as representatives of Exsudoporus, and E. floridanus is epitypified. The eastern Asian species Leccinum rubrum is assigned here to Exsudoporus based on molecular evidence, and a new combination is proposed. Sequence data from the original material of the Japanese Boletus kermesinus were generated, and its conspecificity with L. rubrum is inferred as formerly presumed based on morphology. Four additional cryptic species from North and Central America previously misdetermined as either B. frostii or B. floridanus are phylogenetically placed but remain undescribed due to the paucity of available material. Boletus weberi (syn. B. pseudofrostii) and Xerocomus cf. mcrobbii cluster outside of Exsudoporus and are herein assigned to the recently described genus Amoenoboletus. Biogeographic distribution patterns are elucidated, and a dichotomous key to all known species of Exsudoporus worldwide is presented. Full article