Against the backdrop of global food security and circular agriculture development, edible fungi, as a high-protein food source, have both ecological and economic value in their production model using agricultural and forestry wastes. Based on the “Cycle Production of Plants, Animals, and Fungi” theory, this paper systematically reviews the research progress of alternative substrates for edible fungi. First, alternative substrates are categorized into plant-derived, animal-derived, and microbial-derived types according to their sources. The physicochemical properties, application status, and bottlenecks of each type are analyzed, such as difficult lignin degradation in plant-derived substrates, pollutant risks in animal-derived substrates, and lack of unified application standards for microbial-derived substrates. Second, the mechanisms of key influencing factors including substrate nutritional content, pH and moisture content are elaborated. Furthermore, the paper points out current industrial challenges such as regional resource heterogeneity, difficult control of pretreatment parameters, pollutant residues, and poor batch stability, and summarizes targeted optimization strategies, including regional substrate formulations, precise pretreatment technologies, nutritional regulation, and circular utilization models. Finally, future directions are prospected from four aspects: localized resource utilization, technological innovation, circular model upgrading, and standardized governance, providing theoretical support for the large-scale and sustainable development of the edible fungi industry and contributing to agricultural waste resource utilization and the achievement of “dual carbon” goals.

The sampling technique is commonly used in research investigations to more accurately estimate data with greater precision, at a lower cost and in less time. In plant DUS (distinctness, uniformity, and stability) testing, many quantitative characteristic data usually need to be obtained through individual measurements. However, there is currently no scientific method for determining the appropriate sampling size. The minimum number of testing samples for DUS testing was calculated based on the theory of sample size in descriptive studies and was validated through simple random sampling. The results show that the quantitative characteristics for the edible mushroom Shiitake (Lentinula edodes) in DUS testing were uniform. The calculated results show that 10 fruiting bodies for a single measurement were sufficient. Furthermore, the outcomes of the random sampling revealed that the mean of 10 samples did not significantly differ from the mean of all data. When the sample size exceeded 10, Cohen’s kappa statistic suggested that the conclusion of distinctness was very close to the near-perfect agreement. Reducing the number of samples did not affect the uniform assessment. This study suggests that the theory of sample size in descriptive studies could be applied to calculate the minimum sample size in DUS testing, and for Shiitake DUS testing, measuring 10 fruiting bodies was sufficient.

Grifola frondosa is a commercially important edible mushroom, yet the molecular mechanisms underlying fruiting body development in this species remain poorly understood. In this study, five sample types were collected from the same cultivation bag bearing mature fruiting bodies to represent different stages of a developmental continuum: spawn (S), white mycelial film (WM), gray mycelial mat (GM), fruiting body base (B), and pileus (P). Transcriptomic, biochemical, and morphological analyses were integrated to investigate the tissue-specific characteristics and potential roles of these tissues during fruiting body formation. The results revealed that S exhibited enhanced cellulase expression and activity, consistent with its proposed role in carbon supply for the developing fruiting body. WM showed upregulation of genes involved in H₂O₂ production and ATP synthesis, together with hydrophobin-related genes, consistent with active energy metabolism and a possible role in surface protection in this light-exposed layer. GM displayed distinct morphological features, including thicker hyphae with rounded tips and elevated expression of lectin, transcription-related, and signal transduction genes, consistent with its possible role as the site of primordium initiation. Both B and P specifically upregulated meiosis-related genes, suggesting their involvement in spore production. Collectively, these findings provide a comprehensive molecular resource for understanding tissue-specific genetic programs governing fruiting body formation in G. frondosa, while also highlighting candidate genes and testable hypotheses for future functional validation.