Journal of Fungi

Cordycepin is a key active component of Cordyceps militaris, but the molecular mechanism underlying temperature-regulated biosynthesis remains unclear. In this study, Cordyceps militaris strain KN-1 was used as experimental material, with low-temperature (15 °C), control (20 °C), and high-temperature (25 °C) treatments applied during the fruiting body stage. Transcriptomics, untargeted metabolomics, weighted gene co-expression network analysis (WGCNA), and Reverse Transcription quantitative PCR (RT-qPCR) validation were integrated to elucidate the molecular mechanism of temperature-mediated cordycepin biosynthesis. The results showed that 25 °C increased fruiting body cordycepin content by 84%, while 15 °C reduced it. Transcriptomic analysis identified differentially expressed genes (DEGs) enriched in transmembrane transport and fatty acid metabolism, and untargeted metabolomics revealed differential metabolites (DAMs) enriched in lipids and organic acids, indicating that temperature primarily affects Cordyceps militaris membrane function. WGCNA showed that the MEblue module was positively correlated with cordycepin (r = 0.93), with Major Facilitator Superfamily (MFS) members accounting for the highest proportion (47.1%) that may affect cordycepin transmembrane transport. Multi-omics analysis indicated that high temperature promotes cordycepin accumulation through the synergistic regulation of multiple pathways: upregulating genes in the pentose phosphate pathway, purine metabolism, and cordycepin biosynthetic gene cluster (Cns1–Cns3), increasing protective agent pentostatin content, downregulating cordycepin-degrading genes, and enhancing cordycepin transmembrane transport. This study clarifies the molecular mechanism of temperature-mediated cordycepin accumulation, providing a theoretical basis for improving cordycepin production via temperature regulation, optimizing Cordyceps militaris strain quality, and facilitating efficient industrial production.

Species of the ascomycetous genus Cladobotryum (Hypocreales, Hypocreaceae) are ecologically and economically important mycoparasites that cause cobweb disease in cultivated and wild mushrooms. Despite their significance as fungal pathogens and producers of bioactive metabolites, the taxonomy of Cladobotryum remains unresolved due to extensive morphological plasticity, complex teleomorph–anamorph connections, and the presence of cryptic species. This study employs an integrative approach combining micro- and macromorphological characterization, multi-locus phylogeny (ITS, rpb2, and tef-1a), and comparative genomics to clarify the taxonomic position of the Greek isolate Cladobotryum sp. ATHUM 6904, previously designated as an unclassified red-pigmented (URP) strain. Phylogenetic analyses demonstrated that URP strains form a distinct, well-supported clade closely related to C. tenue and C. rubrobrunnescens, yet genetically and morphologically distinct from both. Comparative genomic analyses of isolate ATHUM 6904 and the ex-type strains of C. tenue and C. rubrobrunnescens revealed pronounced divergence in transposable element content, mitochondrial genome architecture, gene order, orthologous gene composition, secondary metabolite biosynthetic potential, and overall genomic distance. Micro- and macromorphological comparisons further supported the differentiation of isolate ATHUM 6904 from both reference species. Based on the combined molecular, morphological, and genomic evidence, the Greek isolate ATHUM 6904 is described as a novel species, Cladobotryum rhodochroum sp. nov.

Although RNA sequencing (RNA-seq) enables rapid transcriptome profiling, functional annotation of fungal transcriptomes remains challenging. Existing tools prioritize broad taxonomic coverage, and reference genomes are scarce for non-model species. This study aimed to develop a fungal-specific functional annotation workflow to support rapid and accurate functional analyses downstream of RNA-seq, independent of reference genome availability. To evaluate the workflow, RNA-seq data from 57 samples of Lentinula edodes strain H600 (shiitake mushroom) were retrieved, along with full-length transcript sequencing (Iso-Seq) data and corresponding RNA-seq data from 20 samples of Phakopsora pachyrhizi (Asian soybean rust) from public databases. The workflow successfully annotated over 96% of protein-coding transcripts and demonstrated applicability to Iso-Seq data. Functional enrichment analyses revealed higher-resolution functional detection than existing annotation tools. Furthermore, integrating homology searches against fungal-specific databases with expression pattern-based annotations highlighted the workflow’s utility for target identification in genome editing and other applications. Overall, the results of this study highlight the potential of the developed workflow in facilitating the discovery of functionally important transcripts and their translation into biotechnological applications.