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.

Polyporus umbellatus is a medicinal fungus primarily used for diuresis, with its sclerotium serving as the medicinal component. The growth and development of sclerotia are reliant on a symbiotic relationship with Armillaria. However, the impact of different Armillaria species on the yield and quality of sclerotia remains unclear. In this study, three Armillaria strains, A35, A541, and A19, were identified through TEF-1α sequence analysis and phylogenetic classification. These strains were classified into three distinct species: A35 as A. ostoyae, A541 as A. gallica, while the taxonomic status of A19 remains unresolved. After four years of co-cultivation with these Armillaria strains, three groups of P. umbellatus sclerotia were harvested and labeled as A35-P, A541-P, and A19-P, respectively. The yields of A35-P, A541-P, and A19-P exhibited significant variations, with A541-P achieving the highest yield (1221 ± 258 g·nest⁻¹), followed by A35-P (979 ± 201 g·nest⁻¹), and A19-P yielding the least (591 ± 54 g·nest⁻¹). HPLC revealed significant differences in the levels of polyporusterone A and polyporusterone B among the groups. The total polysaccharide content, determined via the phenol-sulfuric acid method, also varied significantly, with A541-P recording the highest content (0.897 ± 0.042%), followed by A19-P (0.686 ± 0.058%), and A35-P showing the lowest value (0.511 ± 0.083%). PCA based on these data indicated clear distinctions among A35-P, A541-P, and A19-P, with the three groups forming separate clusters. This study, for the first time, demonstrates the effects of three different Armillaria species on the yield and active compound content of P. umbellatus. These findings provide valuable insights for selecting high-quality Armillaria strains and offer guidance for the artificial cultivation of P. umbellatus.

Armillaria sp. 541, a genus of root-infecting fungi, forms a symbiosis with traditional Chinese medicine Gastrodia elata (Orchid) and Polyporus umbellatus via extensive networks of durable rhizomorphs. It is not clear the hallmarks of comparative transcriptome between the rhizomorphs and hyphae of Armillaria sp. 541. In the present study, transcriptomic analysis of Armillaria sp. 541 identified 475 differentially expressed genes (DEGs) between Armillaria rhizomorphs (AR) and hyphae (AH). Of them, 285 genes were upregulated and 190 were downregulated. Bioinformatics analyses and tests demonstrated DEGs involved in oxidoreductase activity and peptidoglycan binding were significantly enriched in this process when rhizomorph formed from hyphae. We accordingly obtained 14 gene-encoding proteins containing the LysM domain, and further consensus pattern and phylogenetic analysis indicated that their amino acid sequences were conserved and their biological functions may be peptidoglycan binding for recognition between the fungus and host. Among these genes, one, named Armillaria LysM domain recognition gene (aLDRG), was expressed significantly when rhizomorphs were differentiated from hyphae. It was located in the cortical cells of the rhizomorph by in situ hybridization. Furthermore, biolayer interferometry (BLI) assay demonstrated that aLDRG can bind specifically to chitin oligosaccharide of the fungal cell wall, including N,N′,N″-Triacetylchitotriose (CO3) and N,N′,N″,N′″,N″″-Pentaacetylchitopentaose (CO5). Therefore, we deduced that Armillaria sp. 541 expressed higher levels of LysM protein aLDRG for better binding of oligosaccharide after rhizomorphs were generated. This study provides functional genes for further studies on the interaction between Armillaria sp. 541 and its host.

Aims: Polyporus umbellatus sclerotium, known for its diuretic properties, relies on a symbiotic association with Armillaria for its growth and quality development. However, the impact of soil microorganisms on this symbiosis remains uncertain and warrants investigation. The primary objective of this research is to characterize the microorganisms capable of enhancing the symbiotic interaction between Armillaria gallica and Polyporus umbellatus sclerotia in the rhizosphere soil.Methods: Symbiotic cultivation experiments were conducted in woodland habitats with four groups: symbiotic group (Z0), control group (Z1), A. gallica-only group (Z2), and P. umbellatus-only group (Z3). Rhizosphere soil community profiling analysis was conducted using high-throughput sequencing of the bacterial 16S rRNA gene. Subsequently, bacterial strains were isolated, purified, and back-inoculated with A. gallica to assess their effects on this symbiotic relationship.Results: A total of 10,009 operational taxonomic units (OTUs) were identified, with the symbiotic group (Z0) showing higher bacterial richness and diversity (ACE, Chao1, Shannon indices) compared to Z2 and Z3. Dominant phyla such as Proteobacteria, Acidobacteriota, and Bacteroidota were notably more abundant in Z0. Notably, Rhodococcus sp. Z2-1 significantly promoted A. gallica rhizomorph growth (diameter increased by 112.2%, branches by 160.9%) and symbiosis establishment (100% contact rate in inoculated pots vs. 0–22.2% in controls).Conclusion: The symbiotic relationship between P. umbellatus and A. gallica shapes rhizosphere bacterial communities, with specific bacteria like Rhodococcus sp. enhancing fungal growth and symbiotic efficiency. This study presents the potential for developing a bio-bacterial fertilizer for cultivation of medicinal material.

The sclerotia of Wolfiporia hoelen are one of the most important traditional Chinese medicines and foods commonly used in China, Japan, Korea, and other Asian countries. To provide a high-quality reference genome and deepen our understanding of the genome of W. hoelen to elucidate various biological phenomena. In this study, we assembled three genomes of W. hoelen using a combination of Nanopore and Illumina sequencing strategies. The fifteen-chromosome genome L7 of W. hoelen was assembled with two-sided telomere and rDNA sequences for the first time. The chromosome count was subsequently confirmed through collinearity analysis, correcting the previous belief that W. hoelen had only fourteen chromosomes. Moreover, the aneuploid genome was discovered in W. hoelen for the first time through sequencing depth analysis of different chromosomes, and only some strains of W. hoelen exhibit aneuploid genomes. According to the genome analysis of homokaryotic offspring and protoplast-isolated strains, a potential variation in chromosome allocation patterns was revealed. Moreover, the gene function enrichment analysis of genes on reduplicated chromosomes demonstrated that aneuploidy in the genome may be the result of environmental adaptation for W. hoelen. The discovery of an aneuploid genome also provides new ideas for genetic improvement of W. hoelen.

Polyporus umbellatus is a valuable fungus with both dietary and medicinal applications. However, heterogeneous germplasm and chemical variability constrain its sustainable use. To elucidate the drivers of this variation, whole-genome resequencing and metabolic profiling were integrated. Genome-wide analysis of representative accessions revealed six distinct genetic clusters across China, identifying the Qinling–Daba Mountains as a putative center of diversity. Population analysis indicated severe genetic erosion with significant heterozygote deficits, likely driven by inbreeding and long-term clonal propagation. Multivariate analysis demonstrated that genetic lineage, rather than traditional commercial morphotypes (Zhushiling and Jishiling), is the primary determinant of metabolite accumulation. Specific lineages were identified as superior germplasm candidates: Group 2 consistently exhibited the highest genetic potential for accumulating steroids, whereas Group 4 attained the highest polysaccharide yield. Although the global genetic–chemical correlation was weak, implying environmental plasticity, the distinct clustering of superior lineages confirms that core accumulation patterns are genetically canalized. These findings advocate for shifting quality control from morphological grading to molecular-assisted selection. Ultimately, this framework provides an evidence-based foundation for urgent in situ conservation to restore genetic diversity and facilitates precision breeding of high-efficacy cultivars.