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.

Curvularia trifolii is an important pathogenic fungus that causes leaf spot disease in strawberry and other crops. Increased resistance in pathogenic fungi against chemical fungicides necessitates the search for biological alternatives to control plant fungal diseases. The present study aimed to perform transcriptome and metabolome analysis of C. trifolii fungi. We evaluated the potential of an alkaloid, namely alpha (α)-solanine, to inhibit the growth of Curvularia under in vitro conditions. Furthermore, transcriptomic and metabolomic analysis of treated C. trifolii was performed to identify the differential genes and metabolites. Results revealed that treatment with α-solanine resulted in the poor growth and development of fungal spores. The transcriptome analysis revealed that 1413 genes were differentially expressed (DEGs), among which 340 unigenes were up-regulated, 100 unigenes were down-regulated, and the rest were unaffected in treated samples. Gene ontology analysis revealed that the majority of the genes were related to oxidative stress in the fungus. Additionally, using ultra-high performance liquid chromatography-tandem mass spectrometry, we identified 455 metabolites, among which the majority of metabolites were related to lipid biosynthesis. The high number of genes related to lipid biosynthesis and reactive oxygen species revealed that α-solanine causes oxidative stress in Curvularia, leading to growth inhibition, and can be potentially used as an alternative to chemical fungicides.

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.

To reduce the environmental impact of plastic packaging in the edible fungi supply chain, this study developed an edible natural chitosan composite film loaded with chlorogenic acid–chitosan oligosaccharide nanoparticles (CGA/COS NPs). The effects of CGA/COS NPs as additives on the structure and overall performances of chitosan-based films were systematically studied, and the application effect of nanoparticles/chitosan (NPs/CS) composite films in the preservation of Pleurotus geesteranus was explored. The results showed that the NPs had good compatibility with the film matrix, filled the voids of the chitosan matrix, enhanced the comprehensive performance of the film, and significantly improved the antioxidant activity of the film (DPPH free radical scavenging activity increased from 16.95% to 76.47%). Among all the films, the 5%NPs/CS composite film performed the best, not only having stronger barrier properties against moisture, oxygen, and ultraviolet rays, but also having the best thermal stability and mechanical properties, which can effectively extend the shelf life of Pleurotus geesteranus. This study developed a high-performance edible composite film, which provides a new path of great value for solving the preservation problem of perishable agricultural products such as Pleurotus geesteranus and promoting the innovative development of the green food packaging industry.

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.