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.

Flammulina filiformis (F. filiformis) is called the ‘benefiting intelligence’ mushroom. There is a notable difference between a yellow cultivar (with a robust aroma) and a white mutant cultivar (with a high yield) of F. filiformis. A thorough analysis of aroma differences is essential to improve the aroma of high-yield strains. This study employed a combination of gas chromatography–mass spectrometry–olfactometry (GC-MS-O) and aroma extract dilution analysis (AEDA) to analyze the variations in aroma compounds. Then, the contribution of the odorants was determined using flavor dilution (FD) factors and odor activity values (OAVs). Aroma omission and recombination experiments were used to identify the key odorants. A total of 16 key aroma compounds were characterized in F. filiformis, along with four eight-carbon volatiles (3-octanone, 3-octanol, octanal, and 1-octen-3-ol). Finally, the dominant aroma characteristic was “sweet” for the yellow strain, while it was “green” for the white strain. More research is required to investigate the enzymes and corresponding genes that regulate the synthesis of aroma compounds in F. filiformis for future breeding programs.

Pleurotus citrinopileatus Singer (PCS) has attracted increasing attention as a raw material for medicine and food. Its quality is greatly affected by the accumulation of metabolites, which varies with the applied drying methods. In this study, we utilize an approach based on ultra-high-performance liquid chromatography/Q Exactive mass spectrometry (UHPLC-QE-MS) to reveal the metabolic profiles of PCS from three different drying methods (natural air-drying, NAD; hot-air-drying, HAD; vacuum freeze-drying, VFD). The results showed that lipids, amino acids and their derivatives were all important secondary metabolites produced during NAD, HAD and VFD treatments, with the key differential metabolites of PCS during drying including fifteen lipids and seven amino acids. Meanwhile, VFD was the best way for long-term preservation of dried PCS. Hot-drying methods, especially HAD, can improve the medicinal component of PCS. Furthermore, KEGG enrichment analysis highlighted 16 pathways and indicated that amino acid metabolism might be the key metabolite pathway for the PCS drying process. Our study elucidates the relationship between drying methods and metabolites or metabolic pathways of PCS to determine the mechanisms affecting the quality of PCS, and finally provides reference values for further development and application in functional food and medications.

Flammulina filiformis (F. filiformis) is one of the four major edible types of fungus in the world and has been cultivated in China since 800 CE (Anno Domini). Some of the most essential criteria for evaluating the quality of F. filiformis are the types and contents of volatile components present. A focused study on screened the terpene synthase genes involved in the aroma of offspring and compared key terpenoids between parents and offspring, which is helpful for the development and application of F. filiformis. Firstly, the volatile aroma components of parent and offspring F. filiformis were extracted using two pretreatment procedures, and then were semi-quantified by an internal standard. Forty-eight, fifty-eight, and forty-eight volatile compounds were identified in parents and offspring of three different strains, and 15, 22, and 12 aroma compounds (OAVs ≥ 1) were further screened out via calculating their odor activity values (OAVs). Terpenoids, in particular linalool and eucalyptol, which contribute more to the aroma, result in the unique green and grassy aroma of the offspring. At last, the F. filiformis genome was resequenced and the coordinates of genes related to terpenoid synthase were determined. The results showed that Scaffolds, including scaffold3.t874 and scaffold9.t157 were connected to terpenoid synthesis of offspring (No. 61523). The variant genes g269 and g61 were related to terpenoid synthase sequences. This study provides a theoretical foundation for the cultivation of more diverse and unique varieties of F. filiformis.

In this study, the differences in gene expression of Stropharia rugosoannulata at different treatment times under high temperature and drought stress were analyzed by transcriptomics. Here, a total of 74,571 transcripts and 16,233 unigenes were identified, with an average assembly length of 3002 bp. A total of 10,248 differentially expressed genes (DEGs) were identified. DEG analysis indicated that the numbers of DEGs under high-temperature stress for 1 d, 2 d, and 3 d were 798, 851, and 1484, respectively. These DEGs were involved in 96 GO functional categories and 69 KEGG metabolic pathways. Meanwhile, the numbers of DEGs under drought stress for 3 d, 6 d, and 9 d were 421, 1072, and 2880, respectively. These DEGs were involved in 108 GO functional categories and 78 KEGG metabolic pathways. Further analysis of the metabolic pathway (ko04011) commonly enriched by DEGs identified 15 candidate genes responding to high-temperature or drought stress. Eight candidate genes were randomly selected for qRT-PCR verification, and the qRT-PCR results were basically consistent with the transcriptome datasets. These findings provide critical candidate genes for understanding the molecular regulation mechanism of S. rugosoannulata in response to high temperature and drought stress and have important reference value for its stress resistance breeding.

Light is a crucial environmental regulator for Tricholoma giganteum (T. giganteum). This study investigated the effects of light quality and photoperiod on its growth, physiology, and nutritional composition. During the mycelial stage, blue light (BL) exposure for 5 d promoted the highest growth rate (0.74 mm d⁻¹, 45% higher than dark control, p < 0.05). Red light (RL) enhanced antioxidant capacity, elevating superoxide dismutase (SOD) activity to 240.20 U·mL⁻¹ (after 5 d) and DPPH radical-scavenging activity to 276.11% (after 3 d). Ultraviolet (UV) suppressed polyphenol oxidase (PPO) activity. BL also increased mycelial polysaccharide content (6.45 g·100 g⁻¹). In the fruiting stage, green light (GL) improved agronomic traits and first-grade yield (3.75 kg), while also promoting the accumulation of glutamate (4.39 g·100 g⁻¹), a key umami compound. Further photoperiod optimization revealed that 4 h of daily GL exposure shortened the fruiting cycle, achieved the highest biological efficiency (98.4%), and maximized both polysaccharide (38.17 g·100 g⁻¹) and glutamate contents (5.70 g·100 g⁻¹). These results recommend a two-stage lighting protocol: BL for mycelial growth and a 4 h daily GL for fruiting, providing a scientific basis for the industrial cultivation of T. giganteum.