Journal of Fungi

12 pages, 3929 KiB

Open AccessArticle

Establishment of a Mutant Library for Infection Cushion Development and Identification of a Key Regulatory Gene in Botrytis cinerea

by Maoyao Tang, Kexin Wang, Pan Zhang, Jie Hou, Xiaoqian Yu, Hongfu Wang, Yangyizhou Wang and Guihua Li

J. Fungi 2025, 11(1), 16; https://doi.org/10.3390/jof11010016 (registering DOI) - 29 Dec 2024

Abstract

Botrytis cinerea, the grey mould fungus affecting over 1400 plant species, employs infection cushion (IC), a branched and claw-like structure formed by mycelia, as a critical strategy to breach host surface barriers. However, the molecular mechanisms underlying IC formation remain largely unexplored. [...] Read more.

Botrytis cinerea, the grey mould fungus affecting over 1400 plant species, employs infection cushion (IC), a branched and claw-like structure formed by mycelia, as a critical strategy to breach host surface barriers. However, the molecular mechanisms underlying IC formation remain largely unexplored. In this study, we utilized a forward genetics approach to establish a large T-DNA tagged population of B. cinerea, which contained 14,000 transformants. Through phenotype screening, we identified 161 mutants with defects in IC development. Detailed analyses revealed that these mutants exhibited various degrees of impairment in IC formation, ranging from complete failure to form ICs to a reduction in the number and maturity of ICs. Further genetic analysis of one of the mutants led to the identification of EXO70, a gene encoding a component of the exocyst complex, as a key regulatory factor in IC development. Mutants with deletion of EXO70 failed to form ICs, confirming its crucial role in the process. The mutant library reported here provides a rich resource for further large-scale identification of genes involved in IC development. Our findings provide valuable insights into the genetic and molecular basis of IC formation and offer new targets for controlling B. cinerea pathogenicity. Full article

(This article belongs to the Special Issue Biodiversity, Systematics, and Evolution of Plant Pathogenic Fungi)

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Figure 1
Phenotypes of several representative infection cushion (IC) development-deficient mutants of B. cinerea. (a) Mutants that do not form ICs or whose ICs cannot mature. Mycelial plugs of the tested strains were inoculated on glass slides to induce IC development and observed at 40 h post inoculation (HPI). Bar = 50 μm. (b) Mutants with a significantly reduced number of ICs. Conidial suspensions (105 conidia/mL in 1/2 PDB) of the tested strains were inoculated on glass slides to induce IC development and observed at 40 HPI. Bar = 100 μm. WT, the wild-type strain B05.10. Other strains are selected typical T-DNA-tagged mutants. Full article ">Figure 2
EXO70 is essential for IC development of B. cinerea. (a) Strategy for generation of EXO70 knockout strain Δexo70 via gene replacement approach. WT, the wild-type strain B05.10; pEXO-KO, EXO70 knockout vector. HPH, the hygromycin resistance gene. Blue lines and green lines indicate genomic sequences and EXO70 homologous flanks, respectively. (b) Molecular identifications of knockout mutants Δexo70 (KO1 and KO2) and the complemented strain Δexo70-C (KO1-C). PCR amplifications were used for detecting HPH upstream recombination (up-rec) with primers NU/Hb-RC, downstream recombination (down-rec) with primers Ha-RC/ND, and EXO70 loss with primers EI-F/EI-R, respectively. Relative EXO70 expression levels in the indicated strains were determined by quantitative reverse transcription PCR (qRT-PCR). (c) Colony of tested strains cultured on PDA. (d) Pathogenicity assays with mycelial plugs inoculated on detached green-bean leaves at 48 HPI. (e) IC development on glass slides at 40 HPI. Bar = 100 μm. (f) Quantification of the colony sizes at 72 h shown in (c). (g) Quantification of the lesion sizes shown in (d). (h) Quantification of the IC numbers shown in (e). ND, not detected. Data represent means ± standard deviations (SD) from at least three independent experiments. **, ***, significance at p < 0.01, 0.001, respectively. Full article ">

20 pages, 13390 KiB

Open AccessArticle

Genome Sequencing Providing Molecular Evidence of Tetrapolar Mating System and Heterothallic Life Cycle for Edible and Medicinal Mushroom Polyporus umbellatus Fr

by Shoujian Li, Youyan Liu, Liu Liu, Bing Li and Shunxing Guo

J. Fungi 2025, 11(1), 15; https://doi.org/10.3390/jof11010015 (registering DOI) - 28 Dec 2024

Abstract

Polyporus umbellatus is a species whose sclerotia have been extensively employed in traditional Chinese medicine, which has diuretic, antitumor, anticancer, and immune system enhancement properties. However, prolonged asexual reproduction has resulted in significant homogenization and degeneration of seed sclerotia. In contrast, sexual reproduction [...] Read more.

Polyporus umbellatus is a species whose sclerotia have been extensively employed in traditional Chinese medicine, which has diuretic, antitumor, anticancer, and immune system enhancement properties. However, prolonged asexual reproduction has resulted in significant homogenization and degeneration of seed sclerotia. In contrast, sexual reproduction has emerged as an effective strategy to address these challenges, with a distinct mating system serving as the foundation for the implementation of sexual breeding. This study presents the first sequencing and assembly of the genome of P. umbellatus, thereby providing an opportunity to investigate the mating system at the genomic level. Based on the annotated mating-type loci within the genome, monokaryotic offspring exhibiting different mating-types were identified. Through the integration of traditional mating tests, the tetrapolar mating system of P. umbellatus was distinctly elucidated. The resequencing of monokaryotic strains with four different mating-types, along with comparative analyses of mating-type loci, revealed the HD1 and HD2 (HD, homeodomain) genes determined the mating A types, and the PR4, PR5, and PR6 (PR, pheromone receptor) genes determined the mating B types. Meanwhile, this study offers a successful case study in the molecular investigation of mating systems. Additionally, the number of sterigma and basidiospores on each basidium was examined using scanning electron microscopy, while the nuclei of basidiospores and basidia at various developmental stages were analyzed through DAPI staining. This research clarifies the heterothallic life cycle of P. umbellatus. The findings of this study are expected to facilitate advancements in genetic research, breeding development, strain improvement, and the industry of P. umbellatus. Full article

(This article belongs to the Special Issue Molecular Biology of Mushroom)

28 pages, 1784 KiB

Open AccessArticle

Omics-Based Comparison of Fungal Virulence Genes, Biosynthetic Gene Clusters, and Small Molecules in Penicillium expansum and Penicillium chrysogenum

by Holly P. Bartholomew, Christopher Gottschalk, Bret Cooper, Michael R. Bukowski, Ronghui Yang, Verneta L. Gaskins, Dianiris Luciano-Rosario, Jorge M. Fonseca and Wayne M. Jurick II

J. Fungi 2025, 11(1), 14; https://doi.org/10.3390/jof11010014 (registering DOI) - 28 Dec 2024

Abstract

Penicillium expansum is a ubiquitous pathogenic fungus that causes blue mold decay of apple fruit postharvest, and another member of the genus, Penicillium chrysogenum, is a well-studied saprophyte valued for antibiotic and small molecule production. While these two fungi have been investigated [...] Read more.

Penicillium expansum is a ubiquitous pathogenic fungus that causes blue mold decay of apple fruit postharvest, and another member of the genus, Penicillium chrysogenum, is a well-studied saprophyte valued for antibiotic and small molecule production. While these two fungi have been investigated individually, a recent discovery revealed that P. chrysogenum can block P. expansum-mediated decay of apple fruit. To shed light on this observation, we conducted a comparative genomic, transcriptomic, and metabolomic study of two P. chrysogenum (404 and 413) and two P. expansum (Pe21 and R19) isolates. Global transcriptional and metabolomic outputs were disparate between the species, nearly identical for P. chrysogenum isolates, and different between P. expansum isolates. Further, the two P. chrysogenum genomes revealed secondary metabolite gene clusters that varied widely from P. expansum. This included the absence of an intact patulin gene cluster in P. chrysogenum, which corroborates the metabolomic data regarding its inability to produce patulin. Additionally, a core subset of P. expansum virulence gene homologues were identified in P. chrysogenum and were similarly transcriptionally regulated in vitro. Molecules with varying biological activities, and phytohormone-like compounds were detected for the first time in P. expansum while antibiotics like penicillin G and other biologically active molecules were discovered in P. chrysogenum culture supernatants. Our findings provide a solid omics-based foundation of small molecule production in these two fungal species with implications in postharvest context and expand the current knowledge of the Penicillium-derived chemical repertoire for broader fundamental and practical applications. Full article

(This article belongs to the Special Issue Plant Pathogens and Mycotoxins)

21 pages, 7825 KiB

Open AccessArticle

Effects of Drying Treatments on the Physicochemical Characteristics and Antioxidant Properties of the Edible Wild Mushroom Cyttaria espinosae Lloyd (Digüeñe Mushroom)

by Marcelo Villalobos-Pezos, Ociel Muñoz Fariña, Kong Shun Ah-Hen, María-Fernanda Garrido Figueroa, Olga García Figueroa, Alexandra González Esparza, Luisbel González Pérez de Medina and José Miguel Bastías Montes

J. Fungi 2025, 11(1), 13; https://doi.org/10.3390/jof11010013 (registering DOI) - 28 Dec 2024

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Abstract

The wild mushroom Cyttaria espinosae, also known as digüeñe, was a parasitic ascomycete of Nothofagus trees endemic to southern Chile. This species of wild mushroom was of great nutritional importance, especially for the Mapuche indigenous communities, and this edible mushroom is highly [...] Read more.

The wild mushroom Cyttaria espinosae, also known as digüeñe, was a parasitic ascomycete of Nothofagus trees endemic to southern Chile. This species of wild mushroom was of great nutritional importance, especially for the Mapuche indigenous communities, and this edible mushroom is highly sought after. Edible wild mushrooms, rich in bioactive compounds, are a potential source of health components. In the case of C. espinosae, research on its bioactive compounds is still lacking and, due to its perishability, on the effect of preservation treatments on these bioactive compounds. This study evaluates different drying treatments, such as freeze-drying, hot-air drying, and microwave–vacuum drying. The rehydration capacity, color, and microstructural properties using scanning electron microscopy (SEM) were evaluated on dried mushrooms, and total phenolic content, antioxidant activity determined by DPPH and ORAC assays, and levels of ergothioneine were investigated in both fresh and dried extracts of C. espinosae. The results showed that freeze-drying and microwave–vacuum drying could be advisable treatments for the digüeñe mushroom, due to better outcomes in rehydration rate, color, and structural properties observed through SEM images. Total phenolic content and antioxidant activity showed superior results for mushroom extracts dried by microwave–vacuum compared to the other drying treatments. Full article

(This article belongs to the Special Issue Fungal Wonders: Investigating the Diverse Biological Activities of Fungal-Derived Compounds)

15 pages, 4712 KiB

Open AccessArticle

A Protein with Unknown Function, Ps495620, Is Critical for the Sporulation and Oospore Production of Phytophthora sojae

by Xiaoran Du, Yan Zeng, Yiying Li, Qin Peng, Jianqiang Miao and Xili Liu

J. Fungi 2025, 11(1), 12; https://doi.org/10.3390/jof11010012 - 27 Dec 2024

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Abstract

While the rapid rise in bioinformatics has facilitated the identification of the domains and functions of many proteins, some still have no domain annotation or largely uncharacterized functions. However, the biological roles of unknown proteins were not clear in oomycetes. An analysis of [...] Read more.

While the rapid rise in bioinformatics has facilitated the identification of the domains and functions of many proteins, some still have no domain annotation or largely uncharacterized functions. However, the biological roles of unknown proteins were not clear in oomycetes. An analysis of the Phytophthora sojae genome database identified the protein Ps495620, which has no domain annotations and functional predictions in Phytophthora. This study used a CRISPR/Cas9-mediated gene replacement system to knock out Ps495620 to elucidate its function. The Ps495620-knockout mutants exhibited significantly increased oospore production and decreased sporangium formation compared to the wild-type strain P6497. Transcriptomics showed that it is a key regulator of nitrogen, pyruvate, ascorbate, and adorate metabolism in P. sojae. Our findings indicate that Ps495620 is critical in regulating sporangium formation and oospore production in P. sojae. Full article

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19 pages, 29438 KiB

Open AccessArticle

Description of the New Species Laccaria albifolia (Hydnangiaceae, Basidiomycota) and a Reassessment of Laccaria affinis Based on Morphological and Phylogenetic Analyses

by Francesco Dovana, Roberto Para, Gabriel Moreno, Edoardo Scali, Matteo Garbelotto, Bernardo Ernesto Lechner and Luigi Forte

J. Fungi 2025, 11(1), 11; https://doi.org/10.3390/jof11010011 - 27 Dec 2024

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Abstract

Laccaria is a diverse and widespread genus of ectomycorrhizal fungi that form symbiotic associations with various trees and shrubs, playing a significant role in forest ecosystems. Approximately 85 Laccaria species are formally recognised, but recent studies indicate this number may be an underestimation, [...] Read more.

Laccaria is a diverse and widespread genus of ectomycorrhizal fungi that form symbiotic associations with various trees and shrubs, playing a significant role in forest ecosystems. Approximately 85 Laccaria species are formally recognised, but recent studies indicate this number may be an underestimation, highlighting the need for further taxonomic studies to improve our understanding of species boundaries. This manuscript focuses on Laccaria affinis, originally described by Singer in 1967 as Laccaria laccata var. affinis, and details a comprehensive study of its morphological and molecular characteristics, including the examination of its holotype and recent collections from Italy and the United Kingdom. Our findings reveal significant micromorphological traits that enhance the original description. Phylogenetic analyses indicate that L. affinis occupies a distinct clade within Northern Hemisphere Laccaria species, although minimal genetic differences challenge its independence from L. macrocystidiata. Consequently, we propose that these two taxa be considered synonymous. This study not only contributes to the understanding of Laccaria diversity but also proposes the formal designation of an epitype for L. affinis, thereby providing a foundation for future research on this ecologically significant genus. Furthermore, a new species named Laccaria albifolia belonging to the “/Laccaria bicolor complex clade” is described here on the base of six collections from Italy and Spain. Full article

(This article belongs to the Special Issue Taxonomy, Systematics and Evolution of Forestry Fungi, 2nd Edition)

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2 pages, 150 KiB

Open AccessCorrection

Correction: Roles of Three FgPel Genes in the Development and Pathogenicity Regulation of Fusarium graminearum. J. Fungi 2024, 10, 666

by Lu Cai, Xiao Xu, Ye Dong, Yingying Jin, Younes M. Rashad, Dongfang Ma and Aiguo Gu

J. Fungi 2025, 11(1), 10; https://doi.org/10.3390/jof11010010 - 27 Dec 2024

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Abstract

In the original article [...] Full article

17 pages, 2947 KiB

Open AccessArticle

Light Regulates Secreted Metabolite Production and Antagonistic Activity in Trichoderma

by Edgardo Ulises Esquivel-Naranjo, Hector Mancilla-Diaz, Rudi Marquez-Mazlin, Hossein Alizadeh, Diwakar Kandula, John Hampton and Artemio Mendoza-Mendoza

J. Fungi 2025, 11(1), 9; https://doi.org/10.3390/jof11010009 - 26 Dec 2024

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Abstract

Secondary metabolism is one of the main mechanisms Trichoderma uses to explore and colonize new niches, and 6-pentyl-α-pyrone (6-PP) is an important secondary metabolite in this process. This work focused on standardizing a method to investigate the production of 6-PP. Ethanol and ethyl [...] Read more.

Secondary metabolism is one of the main mechanisms Trichoderma uses to explore and colonize new niches, and 6-pentyl-α-pyrone (6-PP) is an important secondary metabolite in this process. This work focused on standardizing a method to investigate the production of 6-PP. Ethanol and ethyl acetate were both effective solvents for quantifying 6-PP in solution and had limited solubility in potato–dextrose–broth media. The 6-PP extraction using ethyl acetate provided a rapid and efficient process to recover this metabolite. The 6-PP was readily produced during the development of Trichoderma atroviride growing in the dark, but light suppressed its production. The 6-PP was purified, and its spectrum by nuclear magnetic resonance and mass spectroscopy was identical to that of commercial 6-PP. Light also induced or suppressed other unidentified metabolites in several other species of Trichoderma. The antagonistic activity of T. atroviride was influenced by light, as suppression of plant pathogens was greater in the dark. The secreted metabolite production on potato–dextrose–agar was differentially regulated by light, indicating that Trichoderma produced several metabolites with antagonistic activity against plant pathogens. Light has an important influence on the secondary metabolism and antagonistic activity of Trichoderma, and this trait is of key relevance for selecting antagonistic Trichoderma strains for plant protection. Full article

(This article belongs to the Section Fungal Cell Biology, Metabolism and Physiology)

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The absorbance of 6-pentyl-α-pyrone in different solvents. (A) Absorbance spectrum of 6-PP (100 mg/L) dissolved in n-hexane, ethanol, ethyl acetate, and PDB. Absorbance was scanned using a UV-1600PC VWR Spectrophotometer for 200 nm to 500 nm wavelengths. (B,C) Linear dynamic range. Absorbance was measured at 320 nm using a gradient of concentrations of 6-PP dissolved in ethanol (B) and ethyl acetate (C). Full article ">Figure 2
Effect of mixing time and media concentration on extraction of 6-pentyl-α-pyrone. (A) Mixing time for recovery of 6-PP (100 mg/L) from the PDB media. The 6-PP dissolved in PDB was mixed with the solvent ethyl acetate, vortexed for the time indicated, and the supernatant recovered by centrifuging. (B) Media:Solvent (M:S) ratio for recovery of 6-PP (20 mg/L). Volume relation was 1M:1S (X1), 2M:1S (X2), 3M:1S (X3), and 4M:1S (X4). Mixtures were vortexed for 30 s and centrifuged for 3 min at 3000 rpm. The 6-PP was quantified using the equation in <a href="#jof-11-00009-f001" class="html-fig">Figure 1</a>C. Full article ">Figure 3
Effect of incubation in the dark or light on T. atroviride IMI206040 growth and secretion of organic compounds. (A) Dry weight of mycelium after growth in PDB for 7 days at 27 °C. (B) Absorbance following ethyl acetate extraction from the T. atroviride IMI206040 cultures. (C,D) Compounds extracted from cultures of T. atroviride IMI206040 growing in light or dark for 7 days. The TLCs were exposed to short-wave UV (C, 254 nm) or long-wave UV (D, 350 nm) to detect the compounds produced in light (LC) or dark (DC). Full article ">Figure 4
A 6-pentyl-α-pyrone purified from T. atroviride IMI206040. (A) The Nuclear magnetic resonance of 6-PP purified. (B) Stacked NMR spectra of commercially available 6-PP and the 6-PP-like compound isolated from a submerged culture grown in the dark. Full article ">Figure 5
Effect of incubation in the light on growth of Trichoderma species/ strains and metabolite production. (A) Dry weight of mycelium of the Trichoderma strains after growing in PDB for 3 days at 27 °C under light or dark. (B,C), Metabolite pattern of different strains of Trichoderma growing for 3 days at 27 °C in light (L) and dark (D). (B) The TLCs exposed to short-wave UV (254 nm) (left) or long-wave UV (350 nm) (right) to detect the compounds produced in light (LC) or dark (DC). The strains used were T. atroviride IMI206040, Trichoderma sp. atroviride B LU132, LU584, and LU633, T. hamatum LU592, T. asperellum LU697, T. gamsii LU755, and T. viridescens LU1369. The bars have the (+/−) standard deviation of data generated from three replicates, and different letters over the bars indicate significant differences between dark and light. Full article ">Figure 6
Effect of growth in the dark and light on antagonistic activity of T. atroviride IMI206040 as assessed by plant pathogen growth. (A) Antagonistic activity on PDA plates. (B) Colony growth: colony diameter was measured using ImageJ, and the average diameter of the control was taken as 100 % growth. The vertical bars on each bar are (+/−) standard deviation of data generated from three replicates, and different letters over the bars indicate significant differences between dark and light. Full article ">Figure 7
Effect of light on antagonistic activity of several species of Trichoderma against four plant pathogens. Antagonistic activity was assayed by growing Trichoderma strains on PDA plates covered with a cellophane sheet for 48 h in the dark or light at 27 ° C. After removing the Trichoderma colony, the plant pathogens were inoculated, and as a control, fresh PDA was inoculated. Each treatment had three replicates (<a href="#app1-jof-11-00009" class="html-app">Table S1</a>). The heat map matrix shows the average inhibition percentage of two treatments: dark and light. Full article ">Figure 8
Metabolites secreted by Trichoderma spp. grown on PDA in the dark or light. PDA plates holding Trichoderma growth for 60 h in the dark or light were treated to isolate the metabolites produced by Trichoderma strains and analyzed by TLC. The compounds from three cultures were detected using short-wave UV. As a control, metabolite extraction was performed using fresh PDA, and as a reference, 5 µg 6-PP was used. Full article ">

21 pages, 20591 KiB

Open AccessArticle

New Species of Diaporthales (Ascomycota) from Diseased Leaves in Fujian Province, China

by Xiayu Guan, Taichang Mu, Nemat O. Keyhani, Junya Shang, Yuchen Mao, Jiao Yang, Minhai Zheng, Lixia Yang, Huili Pu, Yongsheng Lin, Mengjia Zhu, Huajun Lv, Zhiang Heng, Huiling Liang, Longfei Fan, Xiaoli Ma, Haixia Ma, Zhenxing Qiu and Junzhi Qiu

J. Fungi 2025, 11(1), 8; https://doi.org/10.3390/jof11010008 - 26 Dec 2024

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Abstract

Fungal biota represents important constituents of phyllosphere microorganisms. It is taxonomically highly diverse and influences plant physiology, metabolism and health. Members of the order Diaporthales are distributed worldwide and include devastating plant pathogens as well as endophytes and saprophytes. However, many phyllosphere Diaporthales [...] Read more.

Fungal biota represents important constituents of phyllosphere microorganisms. It is taxonomically highly diverse and influences plant physiology, metabolism and health. Members of the order Diaporthales are distributed worldwide and include devastating plant pathogens as well as endophytes and saprophytes. However, many phyllosphere Diaporthales species remain uncharacterized, with studies examining their diversity needed. Here, we report on the identification of several diaporthalean taxa samples collected from diseased leaves of Cinnamomum camphora (Lauraceae), Castanopsis fordii (Fagaceae) and Schima superba (Theaceae) in Fujian province, China. Based on morphological features coupled to multigene phylogenetic analyses of the internal transcribed spacer (ITS) region, the large subunit of nuclear ribosomal RNA (LSU), the partial beta-tubulin (tub2), histone H3 (his3), DNA-directed RNA polymerase II subunit (rpb2), translation elongation factor 1-α (tef1) and calmodulin (cal) genes, three new species of Diaporthales are introduced, namely, Diaporthe wuyishanensis, Gnomoniopsis wuyishanensis and Paratubakia schimae. This study contributes to our understanding on the biodiversity of diaporthalean fungi that are inhabitants of the phyllosphere of trees native to Asia. Full article

(This article belongs to the Special Issue Diversity of Microscopic Fungi)

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Figure 1
Consensus tree of Diaporthe virgiliae species complex inferred from Bayesian inference analyses based on the combined ITS, cal, his3, tef1 and tub2 sequence dataset, with Diaporthe shennongjiaensis (CNUCC 201905) as the outgroup. The Maximum likelihood (ML) bootstrap support values and Bayesian posterior probabilities (BPPs) above 80% and 0.90 are shown at the nodes. Strains marked with “T” are ex-type, ex-epitype and ex-neotype. The isolates from this study are indicated in red. Full article ">Figure 2
Consensus tree of Gnomoniopsis inferred from Bayesian inference analyses based on the combined ITS, tef1 and tub2 sequence dataset, with Melanconis stilbostoma (CBS 109778) as the outgroup. The Maximum likelihood (ML) bootstrap support values and Bayesian posterior probabilities (BPPs) above 80% and 0.90 were shown at the nodes. Strains marked with “T” are ex-type, ex-epitype and ex-neotype. The isolates from this study are indicated in red. Full article ">Figure 3
Consensus tree of Tubakiaceae inferred from Bayesian inference analyses based on the combined ITS, LSU, rpb2, tef1 and tub2 sequence dataset, with Greeneria uvicola (FI12007) as the outgroup. The Maximum likelihood (ML) bootstrap support values and Bayesian posterior probabilities (BPPs) above 80% and 0.90 were shown at the nodes. Strains marked with “T” are ex-type, ex-epitype and ex-neotype. The isolates from this study are indicated in red. Full article ">Figure 4
Diaporthe wuyishanensis (HMAS 352949). (a) Diseased leaves of Cinnamomum camphora; (b,c) surface and reverse sides of colony after 7 days on PDA (d,e) and 14 days; (f,g) conidiomata; (h) conidiogenous cells and conidia; and (i,j) alpha conidia. Scale bars: (h–j) 10 µm. Full article ">Figure 5
Gnomoniopsis wuyishanensis (HMAS 353149). (a) Diseased leaves of Castanopsis fordii; (b) surface and reverse sides of colony after 7 days on PDA (c) and 14 days; (d,e) conidiomata; (f–k) conidiogenous cells and conidia; and (l,m) conidia. Scale bars: (f–m) 10 µm. Full article ">Figure 6
Paratubakia schimae (HMAS 353150). (a) Diseased leaves of Schima superba; (b) surface and reverse sides of colony after 7 days on PDA (c) and 14 days; (d,e) conidiomata; (f–j) conidiogenous cells and conidia; and (k,l) conidia. Scale bars: (f–l) 10 µm. Full article ">

16 pages, 3754 KiB

Open AccessArticle

Molecular Mechanism During Mycelium Subculture Degeneration of Volvariella volvacea

by Lidan Feng, Lujuan Wang, Yuanxi Lei, Jie Li and Fengyun Zhao

J. Fungi 2025, 11(1), 7; https://doi.org/10.3390/jof11010007 - 25 Dec 2024

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Abstract

Periodic mycelial subculture is a method commonly used for the storage of edible mushrooms, but excessive subculturing can lead to the degeneration of strains. In this study, the Volvariella volvacea strain V971(M0) was successively subcultured on PDA medium every 4 days, and one [...] Read more.

Periodic mycelial subculture is a method commonly used for the storage of edible mushrooms, but excessive subculturing can lead to the degeneration of strains. In this study, the Volvariella volvacea strain V971(M0) was successively subcultured on PDA medium every 4 days, and one generation of strains was preserved every 4 months. Thus, five generations of subcultured strains (M1–M5) were obtained after 20 months of mycelial subculturing, their production traits were determined, and transcriptomic analysis was performed using RNA-seq; the differentially expressed genes were verified via RT-qPCR. The results showed that as the number of subcultures increased, the diameter of the mycelium and biological efficiency gradually decreased; in addition, the time in which the primordium formed increased and the production cycle was lengthened, while strains M4 and M5 lacked the ability to produce fruiting bodies. There were 245 differentially expressed genes between the M1–M5 and M0 strains, while the highest number of differentially expressed genes was between M3 and M0, at 1439; the smallest number of differentially expressed genes was between M2 and M0, at 959. GO enrichment analysis showed that the differentially expressed genes were mainly enriched in metabolic processes, organelle components, and catalytic activities. KEGG enrichment analysis showed that the differentially expressed genes were mainly enriched in metabolic pathways. The further annotation of differentially expressed genes showed that 39, 24, and 24 differentially expressed genes were related to substrate degradation, amino acid synthesis and metabolism, and reactive oxygen species metabolism, respectively. The downregulation of the related differentially expressed genes would lead to the excessive accumulation of reactive oxygen species, inhibit nutrient absorption and energy acquisition, and lead to the degradation of V. volvacea. These findings could form a theoretical basis for the degeneration mechanism of V. volvacea, and also provide a basis for the molecular function study of the genes related to strain degradation. Full article

(This article belongs to the Special Issue Edible and Medicinal Macrofungi, 3rd Edition)

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16 pages, 1313 KiB

Open AccessArticle

Development of Aspergillus oryzae BCC7051 as a Robust Cell Factory Towards the Transcriptional Regulation of Protease-Encoding Genes for Industrial Applications

by Sarocha Panchanawaporn, Chanikul Chutrakul, Sukanya Jeennor, Jutamas Anantayanon and Kobkul Laoteng

J. Fungi 2025, 11(1), 6; https://doi.org/10.3390/jof11010006 - 25 Dec 2024

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Abstract

Enzyme-mediated protein degradation is a major concern in industrial fungal strain improvement, making low-proteolytic strains preferable for enhanced protein production. Here, we improved food-grade Aspergillus oryzae BCC7051 by manipulating the transcriptional regulation of protease-encoding genes. Genome mining of the transcription factor AoprtR and [...] Read more.

Enzyme-mediated protein degradation is a major concern in industrial fungal strain improvement, making low-proteolytic strains preferable for enhanced protein production. Here, we improved food-grade Aspergillus oryzae BCC7051 by manipulating the transcriptional regulation of protease-encoding genes. Genome mining of the transcription factor AoprtR and computational analysis confirmed its deduced amino acid sequence sharing evolutionary conservation across Aspergillus and Penicillium spp. The AoPrtR protein, which is classified into the Zn(II)2-Cys6-type transcription factor family, manipulates both intra- and extracellular proteolytic enzymes. Our transcriptional analysis indicated that the regulation of several protease-encoding genes was AoPrtR-dependent, with AoPrtR acting as a potent activator for extracellular acid-protease-encoding genes and a likely repressor for intracellular non-acid-protease-encoding genes. An indirect regulatory mechanism independent of PrtR may enhance proteolysis. Moreover, AoPrtR disruption increased extracellular esterase production by 2.55-fold, emphasizing its role in protein secretion. Our findings highlight the complexity of AoPrtR-mediated regulation by A. oryzae. Manipulation of regulatory processes through AoPrtR prevents secreted protein degradation and enhances the quantity of extracellular proteins, suggesting the low-proteolytic variant as a promising platform for the production of these proteins. This modified strain has biotechnological potential for further refinement and sustainable production of bio-based products in the food, feed, and nutraceutical industries. Full article

(This article belongs to the Special Issue Current Trends in Mycological Research in Southeast Asia)

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Growth characteristics of the AoprtR-deficient A. oryzae strain. (A) Colonial growth and protease secretion of the AoprtR-deficient (ΔpyrG,ΔprtR::pyrG) and parental (ΔpyrG::pyrG) strains grown on skim milk agar (left panel) and PDA (right panel) media. (B) Mycelial growth and residual glucose profiles of the AoprtR-deficient strain (solid line) and the parental strain (dotted line) in SM broth at 30 °C and 200 rpm. Symbols indicate dry cell weight (circles) and residual glucose concentration (triangles). The data are represented as the mean value with standard error (mean ± SE). Full article ">Figure 2
Proteolytic activities of the AoprtR-deficient A. oryzae strain. (A) Intracellular and (B) extracellular proteolytic activities of the AoprtR-deficient (ΔpyrG,ΔprtR::pyrG as black bar) and parental (ΔpyrG::pyrG as gray bar) strains after 3 days of culture in SM broth at 30 °C and 200 rpm. Asterisks above the bars indicate statistically significant differences in proteolytic activity between strains (p < 0.05). (C) The time course of extracellular proteolytic activities in the AoprtR-deficient (black line) and parental (gray line) strains was measured at different culture times. The left, middle, and right panels represent acidic, neutral, and alkaline proteolytic activities, respectively. The data are represented as the mean value with standard error (mean ± SE). Full article ">Figure 3
Proteolytic activities of AoprtR-overexpressing A. oryzae. (A) Intracellular and (B) extracellular proteolytic activities of AoprtR-overexpressed (ΔpyrG::OEprtR::pyrG as black bars) and parental (ΔpyrG::pyrG as gray bars) strains after 3 days of cultivation. Asterisks above the bars denote statistically significant differences in proteolytic activity between strains (p < 0.05). The data are represented as the mean value with standard error (mean ± SE). Full article ">Figure 4
Effects of AoprtR disruption on the transcript levels of protease-encoding genes. RT-qPCR analysis of protease-encoding genes in AoprtR-deficient (ΔpyrG,ΔprtR::pyrG as black bar) and parental (ΔpyrG::pyrG as gray bar) strains. The relative gene expression was calculated as a fold change by normalizing the transcript level of the AoprtR-deficient strain to that of the parental strain, which was adjusted to one. Asterisks above the bars indicate statistically significant differences in the transcript levels of each gene among the strains (p < 0.05). The data are represented as the mean value with standard error (mean ± SE). Full article ">Figure 5
Extracellular esterase production by the AoprtR-deficient A. oryzae strain. The esterase secretion profiles of the AoprtR-deficient (ΔpyrG,ΔprtR::pyrG as black line) and parental (ΔpyrG::pyrG as gray line) strains grown for 96 h. The data are represented as the mean value with standard error (mean ± SE). Full article ">

12 pages, 2715 KiB

Open AccessArticle

Utility of Cand PCR in the Diagnosis of Vulvovaginal Candidiasis in Pregnant Women

by Eduardo García-Salazar, Paola Betancourt-Cisneros, Xóchitl Ramírez-Magaña, Hugo Díaz-Huerta, Erick Martínez-Herrera and María Guadalupe Frías-De-León

J. Fungi 2025, 11(1), 5; https://doi.org/10.3390/jof11010005 - 25 Dec 2024

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Abstract

Vulvovaginal candidiasis (VVC) can lead to multiple complications when it occurs during pregnancy, so it is necessary to diagnose it promptly for effective treatment. Traditional methods for identifying Candida spp. are often too time-consuming and have limited specificity and sensitivity. In this work, [...] Read more.

Vulvovaginal candidiasis (VVC) can lead to multiple complications when it occurs during pregnancy, so it is necessary to diagnose it promptly for effective treatment. Traditional methods for identifying Candida spp. are often too time-consuming and have limited specificity and sensitivity. In this work, we evaluated the diagnostic utility of an endpoint PCR assay (Cand PCR) in vaginal swab specimens. Using a cotton swab, 108 vaginal swab samples were taken from pregnant women who consented to participate in the study. The samples were inoculated in Sabouraud agar plates (the gold standard) and subsequently used to extract DNA directly from the exudate. The yeasts isolated from the Sabouraud agar were identified in CHROMagar™ Candida. DNA extracted from vaginal swabs was amplified by Cand PCR. Based on the results of the Cand PCR and the gold standard, sensitivity (S), specificity (E), positive predictive values (PPVs), and negative predictive values (NPVs) were determined. Cand PCR presented an S = 65%, E = 100%, PPV = 100% and NPV = 91%. Cand PCR showed low sensitivity for detecting Candida spp. directly from vaginal swabs, but it was useful for identifying the etiologic agent and reducing the time to obtain the result, which is usually at least 48 h. Full article

(This article belongs to the Special Issue Diagnosis of Human Pathogenic Fungi)

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Frequency of Candida species isolated from vaginal swabs from pregnant women. Full article ">Figure 2
Electrophoretic analysis of DNA samples extracted directly from vaginal swab specimens from pregnant women. Electrophoresis was conducted in 1.0% agarose gel stained with Midori Green in a 0.5× TBE buffer. Electrophoresis was run at 70 V for two hours. Full article ">Figure 3
Amplification of DNA samples obtained directly from vaginal swab samples from pregnant women. Electrophoresis was performed in 1.5% agarose gel in a 0.5× TBE buffer stained with Midori Green. M: 100 bp molecular size marker. C−: negative control, C+: positive control (DNA of C. albicans ATCC® 18804™). Full article ">Figure 4
Amplification of DNA samples obtained directly from vaginal swab samples from pregnant women. Electrophoresis was performed in 1.5% agarose gel in a 0.5× TBE buffer stained with Midori Green. M: 100 bp molecular size marker. C−: negative control, C+: positive control (DNA of C. albicans ATCC® 18804™). Full article ">Figure 5
Amplification of DNA samples obtained directly from vaginal swab samples from pregnant women. Electrophoresis was performed in 1.5% agarose gel in a 0.5× TBE buffer stained with Midori Green. M: 100 bp molecular size marker. C−: negative control, C+: positive control (DNA of C. albicans ATCC® 18804™). Full article ">Figure 6
Frequency of Candida species identified by Cand PCR in vaginal swab samples from pregnant women. Full article ">

14 pages, 679 KiB

Open AccessReview

Biocontrol of Mycotoxigenic Fungi by Actinobacteria

by Louise Maud, Nathalie Barakat, Julie Bornot, Selma P. Snini and Florence Mathieu

J. Fungi 2025, 11(1), 4; https://doi.org/10.3390/jof11010004 - 24 Dec 2024

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Abstract

Actinobacteria are well known for their production of metabolites of interest. They have been previously studied to identify new antibiotics in medical research and for their ability to stimulate plant growth in agronomic research. Actinobacteria represents a real source of potential biocontrol agents [...] Read more.

Actinobacteria are well known for their production of metabolites of interest. They have been previously studied to identify new antibiotics in medical research and for their ability to stimulate plant growth in agronomic research. Actinobacteria represents a real source of potential biocontrol agents (BCAs) today. With the aim of reducing the use of phytosanitary products by 50% with the different Ecophyto plans, a possible application is the fight against mycotoxin-producing fungi in food matrices and crops using BCAs. To deal with this problem, the use of actinobacteria, notably belonging to the Streptomyces genus, or their specialized metabolites seems to be a solution. In this review, we focused on the impact of actinobacteria or their metabolites on the development of mycotoxigenic fungi and mycotoxin production on the one hand, and on the other hand on their ability to detoxify food matrices contaminated by mycotoxins. Full article

(This article belongs to the Special Issue Mycotoxin Contamination and Control in Food)

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14 pages, 2611 KiB

Open AccessArticle

Assessment of the Chemical Diversity and Functional Properties of Secondary Metabolites from the Marine Fungus Asteromyces cruciatus

by María Paz González-Troncoso, Catalina Landeta-Salgado, Javiera Munizaga, Ruth Hornedo-Ortega, María del Carmen García-Parrilla and María Elena Lienqueo

J. Fungi 2025, 11(1), 3; https://doi.org/10.3390/jof11010003 - 24 Dec 2024

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Abstract

Natural compounds derived from microorganisms, especially those with antioxidant and anticancer properties, are gaining attention for their potential applications in biomedical, cosmetic, and food industries. Marine fungi, such as Asteromyces cruciatus, are particularly promising due to their ability to produce bioactive metabolites [...] Read more.

Natural compounds derived from microorganisms, especially those with antioxidant and anticancer properties, are gaining attention for their potential applications in biomedical, cosmetic, and food industries. Marine fungi, such as Asteromyces cruciatus, are particularly promising due to their ability to produce bioactive metabolites through the degradation of marine algal polysaccharides. This study investigates the metabolic diversity of A. cruciatus grown on different carbon sources: glucose, Durvillaea spp., and Macrocystis pyrifera. Crude extracts of fungal biomass were analyzed for total phenolic content (TPC), antioxidant capacity (TAC), toxicity, and phenolic compound identification using ultra-high-performance liquid chromatography coupled with high-resolution electrospray ionization mass spectrometry (UHPLC-MS/MS). The analysis revealed the presence of anthraquinone compounds, including emodin (0.36 ± 0.08 mg/g DW biomass) and citrereosein in glucose medium and citrereosein and endocrocin in M. pyrifera medium. No such compounds were detected in Durvillaea spp. medium. The glucose-grown extract exhibited the highest TPC (3.09 ± 0.04 mg GAE/g DW) and TAC (39.70 ± 1.0 µmol TEq/g biomass). Additionally, no detrimental effects were observed on a neuronal cell line. These findings highlight the influence of carbon sources on the production of bioactive metabolites and their functional properties, providing valuable insights into the biotechnological potential of A. cruciatus. Full article

(This article belongs to the Special Issue The Gift of Marine Fungi: Abundant Secondary Metabolites)

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Chemical structure depiction of the anthraquinone compounds emodin, citreorosein, and endocrocin from the PubChem Compound Summary [<a href="#B29-jof-11-00003" class="html-bibr">29</a>,<a href="#B30-jof-11-00003" class="html-bibr">30</a>,<a href="#B31-jof-11-00003" class="html-bibr">31</a>]. Full article ">Figure 2
Growth of A. cruciatus on different culture media. (a) Ergosterol concentration’s (b) pH variation after 2, 3, 4, 5, and 6 days of fungal culture on glucose (20 g/L), Durvillaea spp. (30 g/L), or M. pyrifera (30 g/L) as carbon sources (n = 3). Full article ">Figure 3
Metabolomic data of A. cruciatus extracts grown with different carbon sources based on HPLC-MS/MS analysis. (a) Heatmap of metabolites in the crude extracts. Rows represent metabolites, and columns represent different extracts. White and blue boxes represent higher and lower metabolite abundance, respectively. The red box highlights anthraquinone pigments (citreorosein, emodin, and endocrocin). (b) Principal Component Analysis (PCA) of metabolite profiles of crude extracts. Score plots show the differential metabolites along Principal Components 1 and 2 (n = 2). Full article ">Figure 4
Characterization of crude extracts from A. cruciatus grown on glucose medium. Color change of the extract at different pH levels. Full article ">Figure 5
Cytotoxicity evaluation of crude extracts on neuronal cell lines. Cell viability percentages of PC12 cells relative to untreated control cells (control -). Results are expressed as the mean ± standard deviation. DMSO 0.1% served as the negative control. ns: not significant (n = 2). Full article ">

16 pages, 1191 KiB

Open AccessArticle

Gigaspora roseae and Coriolopsis rigida Fungi Improve Performance of Quillaja saponaria Plants Grown in Sandy Substrate with Added Sewage Sludge

by Guillermo Pereira, Diyanira Castillo-Novales, Cristian Salazar, Cristian Atala and Cesar Arriagada-Escamilla

J. Fungi 2025, 11(1), 2; https://doi.org/10.3390/jof11010002 - 24 Dec 2024

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Abstract

The use of living organisms to treat human by-products, such as residual sludge, has gained interest in the last years. Fungi have been used for bioremediation and improving plant performance in contaminated soils. We investigated the impact of the mycorrhizal fungus (MF) Gigaspora [...] Read more.

The use of living organisms to treat human by-products, such as residual sludge, has gained interest in the last years. Fungi have been used for bioremediation and improving plant performance in contaminated soils. We investigated the impact of the mycorrhizal fungus (MF) Gigaspora roseae and the saprophytic fungus (SF) Coriolopsis rigida on the survival and growth of Quillaja saponaria seedlings cultivated in a sandy substrate supplemented with residual sludge. Q. saponaria is a sclerophyllous tree endemic to Chile, known for its high content of saponins. We inoculated plants with the MF, the SF, and a combination of both (MF + SF). Following inoculation, varying doses of liquid residual sludge equivalent to 0, 75, and 100% of the substrate’s field capacity were applied. After 11 months, we found a positive influence of the utilized microorganisms on the growth of Q. saponaria. Particularly, inoculation with the SF resulted in higher plant growth, mycorrhizal colonization percentage, and higher enzymatic activity, especially after the application of the sludge. This increase was more evident with higher doses of the applied sludge. These results highlight the potential of combined microorganism and residual sludge application as a sustainable strategy for enhancing plant growth and reducing waste. Full article

(This article belongs to the Special Issue Fungi Activity on Remediation of Polluted Environments, 2nd Edition)

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16 pages, 3183 KiB

Open AccessArticle

Comparative Analysis of Freeze-Dried Pleurotus ostreatus Mushroom Powders on Probiotic and Harmful Bacteria and Its Bioactive Compounds

by Gréta Törős, Áron Béni, Ferenc Peles, Gabriella Gulyás and József Prokisch

J. Fungi 2025, 11(1), 1; https://doi.org/10.3390/jof11010001 - 24 Dec 2024

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Abstract

Pleurotus ostreatus (oyster mushroom) holds excellent promise worldwide, bringing several opportunities and augmenting the tool sets used in the biotechnology field, the food industry, and medicine. Our study explores the antimicrobial and probiotic growth stimulation benefits of freeze-dried P. ostreatus powders (OMP-TF, oyster [...] Read more.

Pleurotus ostreatus (oyster mushroom) holds excellent promise worldwide, bringing several opportunities and augmenting the tool sets used in the biotechnology field, the food industry, and medicine. Our study explores the antimicrobial and probiotic growth stimulation benefits of freeze-dried P. ostreatus powders (OMP-TF, oyster mushroom powder from the total fresh sample; OMP-CSR, oyster mushroom powder from the cooked solid residue; OMP-CL, oyster mushroom powder from the cooked liquid), focusing on their bioactive compounds and associated activities. Our research examined polysaccharide fractions—specifically total glucans and α- and β-glucans—alongside secondary metabolites, including polyphenols and flavonoids, from freeze-dried mushroom powders. Additionally, carbon nanodots (CNDs) were also characterized. The growth inhibition was tested against Escherichia coli and Staphylococcus epidermidis, while the capacity for stimulating probiotic growth was evaluated using Lactobacillus plantarum and Lactobacillus casei. Evidence indicates that OMP-CL and OMP-CSR exhibit significant antimicrobial properties against S. epidermidis Gram-positive bacteria. OMP-CL notably promoted the growth of L. casei. OMP-CL, containing the most significant number of CNDs, has shown to be a valuable source for gut microbiota modulation, with its antimicrobial and probiotic-stimulating efficacy. However, further in vitro and in vivo studies should be performed to explore CNDs and their behavior in different biological systems. Full article

(This article belongs to the Special Issue Fungal Wonders: Investigating the Diverse Biological Activities of Fungal-Derived Compounds)

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J. Fungi, Volume 11, Issue 1 (January 2025) – 16 articles

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