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.

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.

Flammulina Velutipes (F. velutipes) is widely planted all over the world and is rich in nutrients, which is of great benefit to the human body. However, the research on the aroma of F. velutipes is relatively rare, which limits the application of F. velutipes in deep processing, resulting in a single product and edible method of F. velutipes. The purpose of this study was to find out the aroma compounds contributing to the sensory properties of F. velutipes to promote the application of different varieties of F. velutipes in deep processing. Aromas of 7 species of F. velutipes were described and evaluated by sensory evaluation experiment. The volatile compounds in seven kinds of F. velutipes were detected by headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (GC-MS). A total of 74 volatile compounds were found, including 23 alcohols, 5 aldehydes, 2 phenols, 1 acid, 16 esters, 7 ketones, 1 ether, 13 hydrocarbons, 1 sulfide, 1 acyl compound, and 4 heterocyclic compounds. It was also found that the sensory evaluation results of sample F, C, and E had a high correlation with the content of compound, and the correlation between sample B and sample A was also high. A lexicon for describing aroma attributes of F. velutipes was developed and they could be grouped into categories, such as fruity (apple-like, banana-like, cucumber-like, citrus-like and berry-like), alcoholic (whisky-like, fermented fruit-like), milky (creamy-like), floral (hyacinth-like, phoenix-like, iris-like and mint-like), sulfurous (onion-like), and musty (mud-like). This research will provide a theoretical basis for the future study of F. velutipes aroma and the development and application of F. velutipes products.

Recognized for its dual nutritional and therapeutic value, the fungus Hericium erinaceus is increasingly acknowledged as a rich resource of naturally derived α-glucosidase inhibitors. In this study, eight compounds were isolated from H. erinaceus, including three novel compounds designated as erinacerins X−Z (1, 2, and 6). Their absolute configurations were definitively elucidated using a combination of NMR, HR-MS, and ECD calculations. Furthermore, an integrated screening strategy combining molecular docking, molecular dynamics (MD) simulations, and surface plasmon resonance (SPR) analysis identified two isoindolin-1-ones (2 and 3) as potent naturally derived α-glucosidase inhibitors. Notably, in vitro testing established compounds 2 and 3 as robust α-glucosidase inhibitors, affording IC₅₀ values of 17.80 ± 1.03 μM and 19.50 ± 1.33 μM, respectively. MD simulations revealed that electrostatic interactions and van der Waals forces are the primary drivers of this intermolecular association. These findings were further corroborated by SPR analysis, which quantified their high-affinity binding kinetics to the enzyme. Overall, this combined approach establishes a solid foundation for the discovery and development of natural α-glucosidase inhibitors from H. erinaceus.