Two +ssRNA mycoviruses cohabiting the fungal cultivar of leafcutter ants.

Leafcutter ants are dominant herbivores in the Neotropics and rely on a fungus (Leucoagaricus gongylophorus) to transform freshly gathered leaves into a source of nourishment rather than consuming the vegetation directly. Here we report two virus-like particles that were isolated from L. gongylophorus and observed using transmission electron microscopy. RNA sequencing identified two +ssRNA mycovirus strains, Leucoagaricus gongylophorus tymo-like virus 1 (LgTlV1) and Leucoagaricus gongylophorus magoulivirus 1 (LgMV1). Genome annotation of LgTlV1 (7401 nt) showed conserved domains for methyltransferase, endopeptidase, viral RNA helicase, and RNA-dependent RNA polymerase (RdRp). The smaller genome of LgMV1 (2636 nt) contains one open reading frame encoding an RdRp. While we hypothesize these mycoviruses function as symbionts in leafcutter farming systems, further study will be needed to test whether they are mutualists, commensals, or parasites.

Sequencing, assembly, and genomic annotation of Leucoagaricus gongylophorus LEU18496, a dikarya mutualistic species.

The basidiomycete fungus Leucoagaricus gongylophorus is able to grow in the fungus garden of leaf-cutter ants. This mutualistic interaction has driven the evolutionary adaptation of L. gongylophorus, shaping its metabolism to produce enzymes adept at lignocellulosic biomass degradation. In this study, we undertook the comprehensive sequencing, assembly, and functional annotation of the genome of L. gongylophorus strain LEU18496, mutualistic fungus of the Atta mexicana. Our genomic analyses revealed a distinctive bimodal nature to the genome: a predominant region characterized by AT enrichment and low genetic density, alongside a smaller region exhibiting higher GC content and higher genetic density. The presence of transposable elements (TEs) within the AT-enriched region suggests genomic compartmentalization, facilitating differential evolutionary rates. With a gene count of 6748, the assembled genome of L. gongylophorus LEU18496 surpasses previous reports for this fungal species. Inspection of genes associated with central metabolism unveiled a remarkable abundance of carbohydrate-active enzymes (CAZymes) and fungal oxidative lignin enzymes (FOLymes), underscoring their pivotal roles in the life cycle of this fungus.

Integrated transcriptome and metabolism unravel critical roles of carbon metabolism and oxidoreductase in mushroom with Korshinsk peashrub substrates.

Edible fungi cultivation serves as an efficient biological approach to transforming agroforestry byproducts, particularly Korshinsk peashrub (KP) branches into valuable mushroom (Lentinus edodes) products. Despite the widespread use of KP, the molecular mechanisms underlying its regulation of mushroom development remain largely unknown. In this study, we conducted a combined analysis of transcriptome and metabolism of mushroom fruiting bodies cultivated on KP substrates compared to those on apple wood sawdust (AWS) substrate. Our aim was to identify key metabolic pathways and genes that respond to the effects of KP substrates on mushrooms. The results revealed that KP induced at least a 1.5-fold increase in protein and fat content relative to AWS, with 15% increase in polysaccharide and total sugar content in mushroom fruiting bodies. There are 1196 differentially expressed genes (DEGs) between mushrooms treated with KP relative to AWS. Bioinformatic analysis show significant enrichments in amino acid metabolic process, oxidase activity, malic enzyme activity and carbon metabolism among the 698 up-regulated DEGs induced by KP against AWS. Additionally, pathways associated with organic acid transport and methane metabolism were significantly enriched among the 498 down-regulated DEGs. Metabolomic analysis identified 439 differentially abundant metabolites (DAMs) in mushrooms treated with KP compared to AWS. Consistent with the transcriptome data, KEGG analysis on metabolomic dataset suggested significant enrichments in carbon metabolism, alanine, aspartate and glutamate metabolism among the up-regulated DAMs by KP. In particular, some DAMs were enhanced by 1.5-fold, including D-glutamine, L-glutamate, glucose and pyruvate in mushroom samples treated with KP relative to AWS. Targeted metabolomic analysis confirmed the contents of DAMs related to glutamate metabolism and energy metabolism. In conclusion, our findings suggest that reprogrammed carbon metabolism and oxidoreductase pathways act critical roles in the enhanced response of mushroom to KP substrates.

Mycovorax composti gen. nov., sp. nov., a member of the family Chitinophagaceae isolated from button mushroom compost.

Two Gram-stain-negative, aerobic, rod-shaped, orange-coloured bacterial strains, designated strain C216T and strain M2295, were isolated from mature mushroom compost from composting facilities in Victoria and South Australia, Australia, respectively. External structures such as flagella or pili were not observed on the cells under scanning electron microscopy. Optimal growth was found to occur at 45 °C, at pH 7.25 and in the absence of NaCl on Emerson's 350 YpSs medium. The genome sequence of strain C216T was 3 342 126 bp long with a G+C content of 40.5 mol%. Functional analysis of the genome of strain C216T revealed genes encoding chitinolytic and hemi-cellulolytic functions, with 166 predicted genes associated with carbohydrate metabolism (8.9% of the predicted genes). These functions are important for survival in the mushroom compost environment, which is rich in hemicelluloses. No antibiotic resistance genes were found in the genome sequence. The major fatty acids of strain C216T were iso-C15 : 0 (56.7%), iso-C17 : 0 3-OH (15.6%), C16 : 1 ω7c/iso-C15 : 0 2-OH (7.3%) and iso-C15 : 1 G (6.1%). The only respiratory quinone was MK-7. The major polar lipid of strain C216T was phosphatidylethanolamine, but three unidentified phospholipids, four unidentified aminophospholipids/aminolipids and one unidentified glycolipid were also detected. Phylogenetic analysis based on proteins encoded by the core genome (bac120, 120 conserved bacterial genes) showed that strain C216T forms a distinct lineage in the family Chitinophagaceae and that the closest identified relative is Niabella soli (69.69% ANI). These data demonstrate that strain C216T represents a novel genus and novel species within the family Chitinophagaceae, for which we propose the name Mycovorax composti. The type strain is C216T (=DSM 114558T=LMG 32998T).

Transcriptome analysis of the medicinal mushroom Sanghuangporus vaninii in response to white light stress.

Light is a vital environmental factor that promotes the growth and development of edible fungi mycelium. Under white light, the mycelium color of Sanghuangporus vaninii shifts during its growth stages. To investigate the impact of visible light on mycelial morphogenesis, a comparative transcriptomic analysis was conducted. This analysis revealed the molecular processes that underpin mycelial growth and development in S. vaninii when cultured in both darkness and light conditions. From the analysis, 13,643 genes were aligned using Illumina raw reads. Of these, 596 genes exhibited significant expression changes under white light exposure. Specifically, 226 genes were upregulated and 370 downregulated, spanning 55 different metabolic pathways. We further classified differentially expressed genes (DEGs), these genes play roles in photomorphogenesis, signal transduction, carbohydrate metabolism, and melanin production, among other processes. Some are also implicated in cell cycle regulation and the differential expression of respiratory functions. The validation of the differentially expressed transcripts using qRT-PCR showed complete agreement with RNA-Seq data for 9 transcripts. Meanwhile, the light had an inhibitory effect on the bioactive components in S. vaninii. These findings offer valuable insights into the transcriptional shifts and molecular mechanisms driving the color change in S. vaninii under light exposure, providing a basis for further research into mechanisms of light-response regulation.

Unroughing the cat's tongue mushrooms: Four new species of Pseudohydnum from Brazil based on morphological and molecular phylogenetic evidence.

Pseudohydnum, commonly known as cat's tongue mushrooms, is a monophyletic assemblage within Auriculariales, which encompasses species with gelatinous basidiomata, spathulate, flabellate, or shell-shaped pileus, hydnoid hymenophore, globose to ellipsoidal basidiospores, and longitudinally cruciate-septate basidia. According to the available literature, 16 species have been described in Pseudohydnum, mostly represented in temperate-boreal forests of the Northern Hemisphere. However, the limited morphological, molecular, and ecological information, especially from the Southern Hemisphere ecosystems, does not presently allow a reliable assessment of its taxonomic boundaries nor provide a complete picture of the species diversity in the genus. In an ongoing effort to examine specimens collected in dense and mixed ombrophilous forest fragments (Atlantic Rainforest domain) from Southeastern and Southern Brazil, additional taxa assigned to Pseudohydnum were identified. Four new species are recognized based mostly on characters of the pileus surface, stipe, hymenium, and basidiospores. Molecular phylogenetic analyses based on nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS barcode), partial nuc rDNA 28S, and partial RNA polymerase II largest subunit (RPB1) sequences supported the description of these new taxa. Here, we propose Pseudohydnum brasiliense, P. brunneovelutinum, P. cupulisnymphae, and P. viridimontanum as new species. Morphological descriptions, line drawings, habitat photos, and comparisons with closely related taxa are provided. A dichotomous key for identification of currently known Southern Hemisphere Pseudohydnum species is presented.

High rate of gene family evolution in proximity to the origin of ectomycorrhizal symbiosis in Inocybaceae.

The genomes of ectomycorrhizal (ECM) fungi have a reduced number of genes encoding Carbohydrate-Active EnZymes (CAZymes), expansions in transposable elements (TEs) and small secreted proteins (SSPs) compared with saprotrophs. Fewer genes for specific peptidases and lipases in ECM fungi are also reported. It is unclear whether these changes occur at the shift to the ECM habit or are more gradual throughout the evolution of ECM lineages. We generated a genomic dataset of 20 species in the ECM lineage Inocybaceae and compared them with six saprotrophic species. Inocybaceae genomes have fewer CAZymes, peptidases, lipases, secondary metabolite clusters and SSPs and higher TE content than their saprotrophic relatives. There was an increase in the rate of gene family evolution along the branch with the transition to the ECM lifestyle. This branch had very high rate of evolution in CAZymes and had the largest number of contractions. Other significant changes along this branch included expansions in transporters, transposons-related genes and communication genes such as fungal kinases. There is a high concentration of changes in proximity to the transition to the ECM lifestyle, which correspond to the identified key changes for the gain of this lifestyle.

RNA-Seq analysis of the fruiting bodies and mycelia of angel-wing mushroom Pleurocybella porrigens that cause acute encephalopathy.

OBJECTIVE: In 2004, after consuming angel-wing mushrooms, Pleurocybella porrigens, 59 incidents of food poisoning were reported in Japan. Consequently, 17 individuals died of acute encephalopathy. In 2023, we proved that a lectin, pleurocybelline, and pleurocybellaziridine from this mushroom caused damage to the brains of mice. Although we reported genomic and transcriptomic data of P. porrigens in 2013, the assembly quality of the transcriptomic data was inadequate for accurate functional annotation. Thus, we obtained detailed transcriptomic data on the fruiting bodies and mycelia of this mushroom using Illumina NovaSeq 6000. RESULTS: De novo assembly data indicated that the N50 lengths for the fruiting bodies and mycelia were improved compared with those previously reported. The differential expression analysis between the fruiting bodies and the mycelia revealed that 1,937 and 1,555 genes were significantly up-regulated in the fruiting bodies and the mycelia, respectively. The biological functions of P. porrigens transcripts, including PA biosynthetic pathways, were investigated using BLAST search, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes pathway analysis. The obtained results revealed L-valine, a predicted precursor of PA, is biosynthesized in the fruiting bodies and mycelia. Furthermore, real-time RT-PCR was performed to evaluate the accuracy of the results of differential expression analysis.

Genome editing tools based improved applications in macrofungi.

Macrofungi commonly referred to as Mushrooms are distributed worldwide and well known for their nutritional, medicinal, and organoleptic properties. Strain improvement in mushrooms is lagging due to paucity of efficient genome modification techniques. Thus, for advanced developments in research and commercial or economical viability and benefit, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) emerged as an efficient genome editing tool. The higher efficiency and precision of the desired genetic modification(s) are the most valuable attributes of this recent technology. The present review comprehensively summarizes various conventional methods utilized for strain improvement in mushrooms including hybridization, protoplast fusion, and di-mon mating. Furthermore, the problems associated with these techniques have been discussed besides providing the potential recluses. The significance of CRISPR/Cas9 strategies employed for improvement in various mushroom genera has been deliberated, as these strategies will paves the way forward for obtaining improved strain and effective cultivation methods for enhancing the yield and quality of the fruit bodies.

Runs of homozygosity reveal contrasting histories of inbreeding across global lineages of the edible porcini mushroom, Boletus edulis.

Inbreeding, the mating of individuals that are related through common ancestry, is of central importance in evolutionary and conservation biology due to its impacts on individual fitness and population dynamics. However, while advanced genomic approaches have revolutionised the study of inbreeding in animals, genomic studies of inbreeding are rare in plants and lacking in fungi. We investigated global patterns of inbreeding in the prized edible porcini mushroom Boletus edulis using 225 whole genomes from seven lineages distributed across the northern hemisphere. Genomic inbreeding was quantified using runs of homozygosity (ROHs). We found appreciable variation both among and within lineages, with some individuals having over 20% of their genomes in ROHs. Much of this variation could be explained by a combination of elevation and latitude, and to a lesser extent by predicted habitat suitability during the last glacial maximum. In line with this, the majority of ROHs were short, reflecting ancient common ancestry dating back approximately 200-1700 generations ago, while longer ROHs indicative of recent common ancestry (less than approximately 50 generations ago) were infrequent. Our study reveals the inbreeding legacy of major climatic events in a widely distributed forest mutualist, aligning with prevailing theories and empirical studies of the impacts of historical glaciation events on the dominant forest tree species of the northern hemisphere.

Advancements in genetic studies of mushrooms: a comprehensive review.

Genetic studies in mushrooms, driven by innovations such as CRISPR-Cas9 genome editing and RNA interference, transform our understanding of these enigmatic fungi and their multifaceted roles in agriculture, medicine, and conservation. This comprehensive review explores the rationale and significance of genetic research in mushrooms, delving into the ethical, regulatory, and ecological dimensions of this field. CRISPR-Cas9 emerges as a game-changing technology, enabling precise genome editing, targeted gene knockouts, and pathway manipulation. RNA interference complements these efforts by downregulating genes for improved crop yield and enhanced pest and disease resistance. Genetic studies also contribute to the conservation of rare species and developing more robust mushroom strains, fostering sustainable cultivation practices. Moreover, they unlock the potential for discovering novel medicinal compounds, offering new horizons in pharmaceuticals and nutraceuticals. As emerging technologies and ethical considerations shape the future of mushroom research, these studies promise to revolutionize our relationship with these fungi, paving the way for a more sustainable and innovative world.

Paramixta manurensis gen. nov., sp. nov., a novel member of the family Erwiniaceae producing indole-3-acetic acid isolated from mushroom compost.

There are numerous species in the Erwiniaceae family that are important for agricultural and clinical purposes. Here we described the Erwiniaceae bacterium PD-1 isolated from mushroom (Pleurotus eryngii) compost. Comparative genomic and phylogenetic analyses showed that the strain PD-1 was assigned to a new genus and species, Paramixta manurensis gen. nov., sp. nov. in the family Erwiniaceae. From the average amino acid index, we identified the five AroBEKAC proteins in the shikimate pathway as a minimal set of molecular markers to reconstruct the phylogenetic tree of the Erwiniaceae species. The strain PD-1 containing annotated genes for ubiquinone and menaquinone produced a higher level of ubiquinone (Q8) than demethylmenaquinone (DMK8) and menaquinone (MK8) in anaerobic condition compared to aerobic condition, as similarly did the reference strains from the genera Mixta and Erwinia. Results from fatty acid methyl ester and numerical analyses of strain PD-1 showed a similarity to species of the genera Mixta and Winslowiella. This study revealed that the strain's ability to utilize polyols, such as glycerol, erythritol, and D-arabitol, distinguished the strain PD-1 from the nearest relative and other type strains. The analyzed genetic markers and biochemical properties of the strain PD-1 suggest its potential role in the process of mushroom compost through the degradation of carbohydrates and polysaccharides derived from fungi and plants. Additionally, it can produce a high concentration of indole-3-acetic acid as a plant growth-promoting agent.

Characterization of linoleate dioxygenases in basidiomycetes and the functional role of CcLdo1 in regulating fruiting body development in Coprinopsis cinerea.

Coprinopsis cinerea, a model fungus, is utilized for investigating the developmental mechanisms of basidiomycetes. The development of basidiomycetes is a highly organized process that requires coordination among genetic, environmental, and physiological factors. Oxylipins, a class of widely distributed signaling molecules, play crucial roles in fungal biology. Among oxylipins, the sexual pheromone-inducing factors (psi factors) have been identified as key regulators of the balance between asexual and sexual spore development in Ascomycetes. Linoleate dioxygenases are enzymes involved in the biosynthesis of psi factors, yet their specific physiological functions in basidiomycete development remain unclear. In this study, linoleate dioxygenases in basidiomycetes were identified and characterized. Phylogenetic analysis revealed that linoleate dioxygenases from Basidiomycota formed a distinct clade, with linoleate dioxygenases from Agaricomycetes segregating into three groups and those from Ustilaginomycetes forming a separate group. Both basidiomycete and ascomycete linoleate dioxygenases shared two characteristic domains: the N-terminal of linoleate dioxygenase domain and the C-terminal of cytochrome P450 domain. While the linoleate dioxygenase domains exhibited similarity between basidiomycetes and ascomycetes, the cytochrome P450 domains displayed high diversity in key sites. Furthermore, the gene encoding the linoleate dioxygenase Ccldo1 in C. cinerea was knocked out, resulting in a significant increase in fruiting body formation without affecting asexual conidia production. This observation suggests that secondary metabolites synthesized by CcLdo1 negatively regulate the sexual reproduction process in C. cinerea while not influencing the asexual reproductive process. This study represents the first identification of a gene involved in secondary metabolite synthesis that regulates basidiocarp development in a basidiomycete.

High phenotypic and genotypic plasticity among strains of the mushroom-forming fungus Schizophyllum commune.

Schizophyllum commune is a mushroom-forming fungus notable for its distinctive fruiting bodies with split gills. It is used as a model organism to study mushroom development, lignocellulose degradation and mating type loci. It is a hypervariable species with considerable genetic and phenotypic diversity between the strains. In this study, we systematically phenotyped 16 dikaryotic strains for aspects of mushroom development and 18 monokaryotic strains for lignocellulose degradation. There was considerable heterogeneity among the strains regarding these phenotypes. The majority of the strains developed mushrooms with varying morphologies, although some strains only grew vegetatively under the tested conditions. Growth on various carbon sources showed strain-specific profiles. The genomes of seven monokaryotic strains were sequenced and analyzed together with six previously published genome sequences. Moreover, the related species Schizophyllum fasciatum was sequenced. Although there was considerable genetic variation between the genome assemblies, the genes related to mushroom formation and lignocellulose degradation were well conserved. These sequenced genomes, in combination with the high phenotypic diversity, will provide a solid basis for functional genomics analyses of the strains of S. commune.

A description of two novel Psilocybe species from southern Africa and some notes on African traditional hallucinogenic mushroom use.

Two new Psilocybe species (Hymenogastraceae), P. ingeli and P. maluti, are described from southern Africa. Morphology and phylogeny were used to separate the two novel fungi from their closest relatives in the genus. Psilocybe ingeli was found fruiting on bovine manure-enriched grasslands in the Kwa-Zulu Natal Province of South Africa and differs from its closest relative P. keralensis and others in the internal transcribed spacer ITS1-5.8S-ITS2, partial 28S nuc rDNA, and translation elongation factor 1-alpha regions, distribution, and having larger basidiospores. Similarly, P. maluti was collected from the Free State Province of South Africa and observed in the Kingdom of Lesotho, growing on bovine manure. A secotioid pileus, geographic distribution, and differences in the same DNA regions distinguish P. maluti from its closest relative P. chuxiongensis. Furthermore, the spore dispersal and traditional, spiritualistic use of P. maluti are discussed here.

New Cortinariaceae species associated with Dicymbe, Aldina, and Pakaraimaea in Guyana.

Species of the ectomycorrhizal (ECM) family Cortinariaceae (Agaricales, Agaricomycetes, Basidiomycota) have long been considered impoverished or absent from lowland tropical rainforests. Several decades of collecting in forests dominated by ECM trees in South America's Guiana Shield is countering this view, with discovery of numerous Cortinariaceae species. To date, ~12 morphospecies of this family have been found in the central Pakaraima Mountains of Guyana. Here, we describe three of these as new species of Cortinarius and two as new species of Phlegmacium from forests dominated by the ECM tree genera Dicymbe (Fabaceae subfam. Detarioideae), Aldina (Fabaceae subfam. Papilionoideae), and Pakaraimaea (Cistaceae). Macromorphological, micromorphological, habitat, and DNA sequence data are provided for each new species.

Complete mitochondrial genomes of edible mushrooms: features, evolution, and phylogeny.

Edible mushrooms are an important food source with high nutritional and medicinal value. They are a useful source for studying phylogenetic evolution and species divergence. The exploration of the evolutionary relationships among these species conventionally involves analyzing sequence variations within their complete mitochondrial genomes, which range from 31,854 bp (Cordyceps militaris) to 197,486 bp (Grifolia frondosa). The study of the complete mitochondrial genomes of edible mushrooms has emerged as a critical field of research, providing important insights into fungal genetic makeup, evolution, and phylogenetic relationships. This review explores the mitochondrial genome structures of various edible mushroom species, highlighting their unique features and evolutionary adaptations. By analyzing these genomes, robust phylogenetic frameworks are constructed to elucidate mushrooms lineage relationships. Furthermore, the exploration of different variations of mitochondrial DNA presents novel opportunities for enhancing mushroom cultivation biotechnology and medicinal applications. The mitochondrial genomic features are essential for improving agricultural practices and ensuring food security through improved crop productivity, disease resistance, and nutritional qualities. The current knowledge about the mitochondrial genomes of edible mushrooms is summarized in this review, emphasising their significance in both scientific research and practical applications in bioinformatics and medicine.

Genome-wide characterization and metabolite profiling of Cyathus olla: insights into the biosynthesis of medicinal compounds.

Cyathus olla, belonging to the genus Cyathus within the order Agaricales, is renowned for its bird's nest-like fruiting bodies and has been utilized in folk medicine. However, its genome remains poorly understood. To investigate genomic diversity within the genus Cyathus and elucidate biosynthetic pathways for medicinal compounds, we generated a high-quality genome assembly of C. olla with fourteen chromosomes. The comparative genome analysis revealed variations in both genomes and specific functional genes within the genus Cyathus. Phylogenomic and gene family variation analyses provided insights into evolutionary divergence, as well as genome expansion and contraction in individual Cyathus species and 36 typical Basidiomycota. Furthermore, analysis of LTR-RT and Ka/Ks revealed apparent whole-genome duplication (WGD) events its genome. Through genome mining and metabolite profiling, we identified the biosynthetic gene cluster (BGC) for cyathane diterpenes from C. olla. Furthermore, we predicted 32 BGCs, containing 41 core genes, involved in other bioactive metabolites. These findings represent a valuable genomic resource that will enhance our understanding of Cyathus species genetic diversity. The genome analysis of C. olla provides insights into the biosynthesis of medicinal compounds and establishes a fundamental basis for future investigations into the genetic basis of chemodiversity in this significant medicinal fungus.

Extreme overall mushroom genome expansion in Mycena s.s. irrespective of plant hosts or substrate specializations.

Mycena s.s. is a ubiquitous mushroom genus whose members degrade multiple dead plant substrates and opportunistically invade living plant roots. Having sequenced the nuclear genomes of 24 Mycena species, we find them to defy the expected patterns for fungi based on both their traditionally perceived saprotrophic ecology and substrate specializations. Mycena displayed massive genome expansions overall affecting all gene families, driven by novel gene family emergence, gene duplications, enlarged secretomes encoding polysaccharide degradation enzymes, transposable element (TE) proliferation, and horizontal gene transfers. Mainly due to TE proliferation, Arctic Mycena species display genomes of up to 502 Mbp (2-8× the temperate Mycena), the largest among mushroom-forming Agaricomycetes, indicating a possible evolutionary convergence to genomic expansions sometimes seen in Arctic plants. Overall, Mycena show highly unusual, varied mosaic-like genomic structures adaptable to multiple lifestyles, providing genomic illustration for the growing realization that fungal niche adaptations can be far more fluid than traditionally believed.

Identification and fungicides sensitivity evaluation of the causal agent of cobweb disease on Lyophyllum decastes in China.

BACKGROUND: Cobweb disease is a fungal disease that commonly affects the cultivation and production of edible mushrooms, leading to serious yield and economic losses. It is considered a major fungal disease in the realm of edible mushrooms. The symptoms of cobweb disease were found during the cultivation of Lyophyllum decastes. This study aimed to identify the causative pathogen of cobweb disease and evaluate effective fungicides, providing valuable insights for field control and management of L. decastes cobweb disease. RESULTS: The causal agent of cobweb disease was isolated from samples infected and identified as Cladobotryum mycophilum based on morphological and cultural characteristics, as well as multi-locus phylogeny analysis (ITS, RPB1, RPB2, and TEF1-α). Pathogenicity tests further confirmed C. mycophilum as the responsible pathogen for this condition. Among the selected fungicides, Prochloraz-manganese chloride complex, Trifloxystrobin, tebuconazole, and Difenoconazole exhibited significant inhibitory effects on the pathogen's mycelium, with EC50 values of 0.076 µg/mL, 0.173 µg/mL, and 0.364 µg/mL, respectively. These fungicides can serve as references for future field control of cobweb disease in L. decastes. CONCLUSION: This study is the first report of C. mycophilum as the causing agent of cobweb disease in L. decastes in China. Notably, Prochloraz-manganese chloride complex demonstrated the strongest inhibitory efficacy against C. mycophilum.