Melanin is a complex natural pigment that imparts a variety of colors to the fruiting bodies of edible fungi, influencing both their nutritional quality and commercial value. Stropharia rugosoannulata is an emerging type of edible fungus that has been widely cultivated in recent years. It can be categorized into red and yellow varieties based on cap color, while its pigment characteristics remain unclear. In this study, the melanins from the two varieties were obtained using an alkaline extraction and acid precipitation method, followed by comprehensive characterization of their chemical properties and ultrastructural features. Both melanins displayed distinct absorption maxima at approximately 211 nm. The melanin extracted from the red variety consisted of 55.63% carbon (C), 7.40% hydrogen (H), 30.23% oxygen (O), 5.99% nitrogen (N), and 0.64% sulfur (S), whereas the yellow variety comprised 52.22% C, 6.74% H, 29.70% O, 5.91% N, and 0.99% S. Both types of melanin included eumelanin and phaeomelanin forms, with eumelanin being the predominant type. Variations in the quantities and relative proportions of eumelanin and phaeomelanin contributed to the observed color differences in the mushroom caps. Ultrastructural micrographs revealed the melanins were primarily localized in the cell wall, consistent with findings in other fungal species. These findings contribute valuable insights into fundamental knowledge and potential applications of mushroom pigments.

Ilyonectria is a common soil-inhabiting fungal genus that comprises numerous plant phytopathogenic species capable of infecting a wide array of crops, medicinal herbs, and horticultural plants. However, the lack of a reliable and efficient genetic transformation method has severely hindered the elucidation of the pathogenic mechanisms of Ilyonectria pathogens. In this study, we established an efficient protoplast-mediated genetic transformation method for two dominant Panax root rot pathogens, I. robusta and I. vredehoekensis. Key parameters governing high-quality protoplast preparation, including mycelium culture time, enzyme composition, osmotic stabilizer type, digestion speed, and digestion time, were systematically optimized. Subsequently, orthogonal experiments were conducted to optimize the PEG-CaCl₂-mediated transformation conditions and to screen regeneration media for protoplasts. The optimal enzymatic system is composed of 20 mg/mL driselase and 10 mg/mL lysing enzyme, with 0.7 M NaCl as the osmotic stabilizer. Under these conditions, high-viability and high-quality protoplasts were obtained from I. vredehoekensis after 3 h of digestion at 150 rpm, and from I. robusta after 2 h of digestion at 100 rpm, yielding 5.52 × 10⁷ CFU/mL and 5.75 × 10⁷ CFU/mL protoplasts, respectively. Efficient transformation was achieved using a mannitol-prepared STC buffer mediated by 40% PEG4000. PCR and fluorescence microscopy verified positive transformants. Additionally, pathogenicity assays showed no significant differences in virulence between the transformed and wild-type strains, suggesting that the transformation procedure did not alter virulence. To the best of our knowledge, this is the first study to successfully establish genetic transformation methods for I. robusta and I. vredehoekensis, providing an essential technical platform for functional gene analysis, pathogenicity studies, and host–pathogen interaction research. In addition, the optimized transformation strategy may serve as a valuable reference for studies on other Ilyonectria species.

The Chinese black truffle (Tuber sinense) is an economically vital ectomycorrhizal fungus threatened by unsustainable harvesting. Cultivating truffles using mycorrhizal seedlings is essential for sustainable production, yet the rhizosphere microbiome dynamics remain unclear. This study explored microbial community succession in the rhizosphere of Chinese chestnut (Castanea mollissima) seedlings inoculated with T. sinense over 8 months. High-throughput sequencing and soil physicochemical analysis were conducted at 1, 3, and 8 months post-inoculation. Significant changes in soil properties, such as decreased pH and increased total nitrogen (TN), total potassium (TK), available phosphorus (AP), and calcium (Ca), influenced microbial assembly. Tuber relative abundance rose from 0.02% in non-inoculated samples to 8.81% at 8 months. Inoculation altered microbial structures, enriching fungal genera like Tuber, Staphylotrichum, and Sphaerosporella. Network analysis showed 79.23% positive bacterial-fungal interactions, crucial for rhizosphere stability. Tuber correlated positively with Staphylotrichum and Spizellomyces, indicating potential synergies in mycorrhizal development and nutrient cycling. Tuber also showed significant positive correlations with TN, TK, AP, and Ca, highlighting its preference for nutrient-enriched conditions. This study provides the first comprehensive profile of microbial succession during the mycorrhizal development of T. sinense on chestnut, offering a scientific basis for optimizing truffle seedling production and supporting sustainable cultivation.

Stropharia rugosoannulata is efficiently cultivated using agricultural and forestry wastes, such as sawdust and straw, and has gained popularity in China for its rich nutrition and excellent taste. However, traditional cultivation methods, which lack nitrogen supplementation, often result in low yields and poor fruiting body quality. This study evaluated the effects of protein-rich supplements (PRSs)—feather meal (FM), soybean meal (SM), and their mixture (FS)—on substrate properties, yield, and nutritional quality. PRS significantly increased substrate total nitrogen (TN), reduced the C/N ratio, and improved fruiting body yield, with FM achieving the highest yield (6.80 kg·m⁻²). Moreover, FM and FS significantly enhanced the contents of crude fat, crude fiber, crude protein, crude polysaccharides, total amino acids (TAAs), essential amino acids (EAAs), and umami amino acids (p < 0.05). Additionally, FS significantly enhanced antioxidant activity and 1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capacity of crude polysaccharides (p < 0.05). These findings demonstrate the potential of PRS to optimize S. rugosoannulata cultivation, offering a cost-effective strategy to improve commercial production.