Background: Pleurotus ostreatus is a popular edible mushroom in East Asian markets. Research on the responses of P. ostreatus under different carbon dioxide concentrations is limited. Methods: Label-free LC-MS/MS quantitative proteomics analysis technique was adopted to obtain the protein expression profiles of P. ostreatus fruiting body pileus collected under different carbon dioxide concentrations. The Pearson correlation coefficient analysis and principal component analysis were performed to reveal the correlation among samples. The differentially expressed proteins (DEPs) were organized. Gene ontology analysis was performed to divide the DEPs into different metabolic processes and pathways. Results: The expansion of stipes was inhibited in the high CO₂ group compared with that in the low CO₂ group. There were 415 DEPs (131 up- and 284 down-regulated) in P. ostreatus PH11 treated with 1% CO₂ concentration compared with P. ostreatus under atmospheric conditions. Proteins related to hydrolase activity, including several amidohydrolases and cell wall synthesis proteins, were highly expressed under high CO₂ concentration. Most of the kinases and elongation factors were significantly down-regulated under high CO₂ concentration. The results suggest that the metabolic regulation and development processes were inhibited under high CO₂ concentrations. In addition, the sexual differentiation process protein Isp4 was inhibited under high CO₂ concentrations, indicating that the sexual reproductive process was also inhibited under high CO₂ concentrations, which is inconsistent with the small fruiting body pileus under high CO₂ concentrations. Conclusions: This research reports the proteome analysis of commercially relevant edible fungi P. ostreatus under different carbon dioxide concentrations. This study deepens our understanding of the mechanism for CO₂-induced morphological change in the P. ostreatus fruiting body, which will facilitate the artificial cultivation of edible mushrooms.

Pleurotus giganteus is a commercially cultivated high-temperature mushroom. Investigating the molecular mechanism of fruiting body development will help us to better understand the regulation of substrates and energy in this process. However, little information has been reported on the development and nutrients of the P. giganteus fruiting body. In the present study, P. giganteus is cultivated in a climate chamber, and comparative transcriptome, proteome, and nutritional analysis of P. giganteus fruiting bodies were performed. Our results revealed that Cytochrome P450 monooxygenases and hydrophobin proteins play important roles during the differentiation in the elongation stage. Later, carbon metabolism dominate the fruiting body metabolism and genes related to the carbohydrate metabolic process, glycolytic process, and gluconeogenesis were up-regulated in the mature fruiting bodies. The up-regulation of carbohydrate substrates utilization CAZymes genes and inconsistent protein expression in pileus indicated a reverse transportation of mRNA from the fruiting body to vegetative mycelia. In addition, protein concentration in the pileus is higher than that in the stem, while the stem is the major nitrogen metabolic and amino acid synthetic location. The integrated transcriptomic, proteomic, and nutritional analysis indicated a two-way transportation of substrates and mRNAs in P. giganteus. Stem synthesizes amino acids and transported them to pileus with reducing sugars, while pileus induces the expression of substrate degradation mRNA according to the needs of growth and development and transports them in the other direction.

CRISPR/Cas9 systems were established in some edible fungi based on in vivo expressed Cas9 and guide RNA. Compared with those systems, the in vitro assembled Cas9 and sgRNA ribonucleoprotein complexes (RNPs) have more advantages, but only a few examples were reported, and the editing efficiency is relatively low. In this study, we developed and optimized a CRISPR/Cas9 genome-editing method based on in vitro assembled ribonucleoprotein complexes in the mushroom Flammulina filiformis. The surfactant Triton X-100 played a critical role in the optimal method, and the targeting efficiency of the genomic editing reached 100% on a selective medium containing 5-FOA. This study is the first to use an RNP complex delivery to establish a CRISPR/Cas9 genome-editing system in F. filiformis. Moreover, compared with other methods, this method avoids the use of any foreign DNA, thus saving time and labor when it comes to plasmid construction.

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.

Agronomic traits are key components in variety protection, cultivar development, and the formulation of DUS (distinct, uniform, and stable) test guidelines. P. giganteus is an increasingly popular and commercially promising edible macrofungi. In this study, both mycelial performance and fruiting body characters of 15 Pleurotus giganteus strains were investigated. The temperature gradient culture test indicated that, although most of the strains achieved optimal mycelial growth between 24 and 28 °C, a statistical difference in mycelial growth rates and temperature adaptability among strains were found, supporting that this trait has the potential to be adopted as an indicator in distinguishing strains. In the fruiting performance tests, the coefficient of variation (CV) of tested traits ranged from 5.30% (pileus diameter) to 18.70% (individual mushroom weight). The mushroom yields ranged from 103.37 g/bag (strain No. 15) to 275.76 g/bag (strain No. 9). The large divergence observed in individual mushroom weight tested strains, ranging from 40.88 g to 78.39 g (with median between 37.69 and 79.395 g), make it highly selective and a potential indicator in variety development. Strain No. 9 had the advantages of forming larger, heavier fruiting bodies and a more obvious funnel shape, which also exhibited the highest biological efficiency (15.61%). The results suggested some morphological traits showed high variety difference, such as pileus diameter (55.75 mm to 66.48 mm), stipe length (92.59 mm to 177.51 mm), stipe diameter (16.14 mm to 23.52 mm), and pileus thickness (13.38 mm to 19.75 mm). In the cluster analysis, the tested strains were grouped into four clusters based on agronomic traits: cluster Ⅰ comprised six strains (No. 6, No. 11, No. 8, No. 1, No. 14, and No. 9) with high mushroom yield; cluster Ⅱ included four strains (No. 3, No. 10, No. 7, and No. 4) with large pileus diameter and short stipe; cluster ⅡI consisted of four strains (No. 5, No. 12, No. 13, and No. 15) with relatively lower yields; and cluster Ⅳ included only strain No. 2 which was low in yield, individual mushroom weight, and biological efficiency, accompanied by smaller pileus size and shorter stipe. The results of the correlation analysis indicated three traits, including individual mushroom weight, stipe length, and pileus weight, were positively associated with high yield. This study suggested P. giganteus germplasm resources are of high abundance and their agronomic diversity is useful in distinguishing and developing different varieties. The findings of this work provide knowledge on the agronomic traits and cultivation performance of various P. giganteus strains, laying a foundation for the development of its DUS test guidelines and variety protection, as well as providing reference for the breeding and phenotype selection of high-quality cultivars.

Light quality is a pivotal environmental signal governing the morphogenesis and metabolic programming of edible fungi. This study evaluated the effects of eight light qualities—red (R), green (G), blue (B), red-green (RG), red-blue (RB), green-blue (GB), red-green-blue (RGB), and dark control (CK)—on the agronomic traits of Lentinula edodes. Among all treatments, blue light (B) emerged as the most effective regulator, yielding the highest productivity per log (228.12 g), and significantly enhancing pileus diameter (45.17 mm) and stipe thickness (29.45 mm). To elucidate the underlying molecular mechanisms, integrated transcriptomic and metabolomic analyses were performed on primordia and mature fruiting bodies. Transcriptomic profiling identified 4280 differentially expressed genes (DEGs) under blue light, which were significantly enriched in energy metabolism and structural development pathways. Metabolomic analysis revealed 45 differentially expressed metabolites (DEMs), highlighting a 7.64-fold upregulation of 9(S)-HPODE and a marked downregulation of L-arginine at the harvest stage under blue light. Multi-omics integration demonstrated that blue light orchestrates a strategic metabolic shift: it activates arginine and proline metabolism during the primordial stage, maintains linoleic acid metabolism throughout development, and triggers alanine, aspartate, and glutamate metabolism during the transition to maturity. These pathways facilitate the conversion of amino acids into energy precursors and enhance cell membrane fluidity through unsaturated fatty acid synthesis to support rapid growth. Conversely, red light treatment triggered stress-related MAPK signaling, delayed primordium formation, and redirected resources toward stipe elongation via phenylalanine accumulation, resulting in significantly lower yields. In conclusion, this study confirms that blue light is the optimal condition for L. edodes cultivation, providing a robust molecular foundation for precision light-regulation strategies to maximize yield and quality in commercial production.