CRISPR/Cas9 ribonucleoprotein (RNP)-mediated gene editing has recently been applied to Ganoderma lucidum as a promising tool for functional genomics and strain improvement. However, the multinuclear nature of this basidiomycete can result in genetic mosaicism, raising concerns about the long-term stability of edited strains. In this study, we report the occurrence of revertant phenotypes in CRISPR/Cas9-edited transformants of G. lucidum. Although the edited colonies initially exhibited the expected phenotype, repeated subculturing led to the reappearance of wild-type phenotypes. PCR and sequencing analyses revealed the coexistence of edited and non-edited nuclei, and the progressive loss of edited genotypes over successive generations. These findings demonstrate that multinuclearity is a key factor contributing to the instability of CRISPR/Cas9-based edits in G. lucidum. This brief report provides the first direct documentation of revertant occurrence in edited G. lucidum strains and underscores the need for rigorous selection strategies and novel approaches to secure stable homokaryotic transformants in mushroom genetic engineering.

This study reports the development of a new pure white Flammulina velutipes variety, ‘Seoldan’. The breeding process involved a primary cross between a brown strain (KMCC02267) collected by the Rural Development Administration and a white cultivar ‘Baekwoon’ (KMCC05472). From this cross, a white line ‘Fv-23-553’ was selected and subsequently crossed with a superior pure white resource (KMCC05474), resulting in the novel cultivar Seoldan. Mycelial growth tests showed that both varieties grew best at 25°C; however, Seoldan exhibited faster mycelial growth than the control variety under four temperature conditions except at 20°C. In media tests, Seoldan also demonstrated superior growth on three media types, with the exception of YM medium. In sawdust bottle cultivation, The time from scratching to the first fruiting was 2 days shorter in Seoldan than in the control variety, contributing to an overall reduction in cultivation time. The most distinct morphological trait of Seoldan is its pure white cap color. The average yield was 218.8 ± 10.6 g per bottle, comparable to that of the control. Morphologically, Seoldan produced smaller pileus and thinner stipes than the control. Furthermore, somatic incompatibility tests confirmed that Seoldan is genetically distinct from the control variety. These results indicate that Seoldan is a promising pure white variety with stable productivity, improved cultivation efficiency, and clear genetic differentiation from existing varieties.

Tricholoma matsutake, one of the most famous edible mushroom in Asia, has been cultivated in red pine forest. Because of its difficulty in artificial cultivation, T. matsutake cultivation has relied on foraging in the forest. Under certain environmental conditions, T. matsutake form the Shiro, the condensed mycelium, and develop into fruiting bodies. Among the certain environmental conditions, fungal communities play a major role in the mushroom development. Therefore, fungal community investigations for Bonghwa and Yangyang have been conducted on soil with fairy rings in the past, soil with existing fairy rings, and soil with presumptive fairy rings developing in the future. From the six soil samples, total 163 genera of fungi were detected and species diversity and species abundance of each sample were also analyzed. In result, the species diversity and species abundance of the fairy rings in Yangyang were lower than those in Bonghwa. In comparison with the fairy rings in Yangyang and Bonghwa, the dominance of the genus Tricholoma was higher and that of the genus Motilella was lower in Yangyang. Through the continuous study, establishment of optimal environmental conditions to promote the T. matsutake cultivation is highly expected.

Despite their historical use, studies on the genetic functions of mushrooms and varietal improvement via biomolecular techniques are limited compared to other organisms. Recent advancements in CRISPR/Cas9 have enabled precise genetic modifications in mushrooms, with RNP-based systems offering high editing efficiency without foreign gene insertion. In this study, we optimized gene-editing conditions for Ganoderma lucidum (Yongji 2) by utilizing RNP/nanoparticle complexes to enhance efficiency. The optimal conditions included a 0.2 M sorbitol buffer (pH 7.0) and a protoplast-to-complex ratio of 10:1. Among eight gRNAs designed for the catA gene, three were identified with high activity, and PEG-mediated transformation resulted in successful gene edits, primarily involving 1 bp deletions. The editing efficiency reached 7–8%, demonstrating that nanoparticle-supported RNP systems are effective for marker-free gene editing in mushrooms. These findings highlight a promising approach for advancing genetic research and varietal improvement in G. lucidum and other mushroom species.

In order to develop a stable production and delicious unique cultivar of beech mushrooms, we generated hybrids using wild resources collected domestically, and we developed excellent strains by identifying the mycelial cultivability of the hybrid strains. The developed strains were cultivated according to the type of spawn and incubation time, and strains with excellent yield and shape were first selected, and second strains with less bitterness and excellent taste were selected through quantitative descriptive analysis. This was verified in farms, and ‘HM6-6’, which had a good reputation in the field, was directly developed cultivars as ‘Maruking’. In addition, the yield was high overall in the 90-day culture of the growth substrate inoculated with solid spawn, and the ‘Maruking’ cultivar, was also best in the 90-day culture. The yield was high overall in the 75-day culture of the liquid spawn, but ‘Maruking’ showed excellent yield when cultured for 70 days.

The Grifola frondosa cultivar KMCC03118 was used to isolate monokaryotic strains via spore separation, resulting in the successful crossbreeding of strains KMCC03118-11 and KMCC03118-23, which produced F1 hybrids. These F1 hybrids were then further crossed with various monokaryotic strains to generate F2 progeny. In evaluating the effects of different medium compositions on fruit body development, it was found that a substrate consisting of wheat bran and dried sawdust, with a carbon-to-nitrogen (C/N) ratio of 66-68, provided the most favorable conditions for mycelial growth. Among the strains tested, KMCC03137 and GF-18-50 demonstrated superior characteristics, including a larger fruit body diameter, thicker pileus, and greater stipe thickness, with the highest productivity observed at 143.6 ± 13.3 g and 144.7 ± 15.2 g, respectively. Furthermore, the color of the caps (L: 29.7 ± 7.1, a: 2.6 ± 0.7, b: 8.2 ± 1.8) remained consistent, indicating stable high-quality production. Based on these results, the optimal substrate composition for enhancing both the quality and productivity of the fruit bodies was determined to be 42% Quercus sp. sawdust, 42% Quercus sp. fermented sawdust, 6% wheat bran, and 10% dried tofu residue. This study provides a crucial foundation for the commercial cultivation and breeding improvement of Grifola frondosa, offering valuable insights into its genetic enhancement, and providing essential data for future research aimed at increasing the species' genetic diversity and productivity.

In this study, Pleurotus ostreatus were grown in bottles at temperatures set to 15°C, 20°C, and 25°C inside the cultivation room. Changes in temperature, CO2 concentration, and humidity inside the bottles were measured, and growth characteristics according to the temperature conditions were evaluated. CO2 concentration increased overall as the temperature increased and was particularly stable at 20°C, suggesting that 20°C is the optimal condition for the physiological respiration of P. ostreatus . While humidity was relatively constant at 15°C, it decreased over time at 20°C and was maintained at a stable level at 25°C, suggesting that water retention capacity may occur at high temperatures. As a result of the growth investigation, the yield per bottle and individual weight were the highest at 20°C, confirming that 20°C is the most suitable temperature condition for the growth of oyster mushrooms. At 25°C, the yield per bottle was maintained but the individual weight decreased and the color tended to change. These results suggest that the interaction between CO2 concentration, humidity, and temperature has a significant effect on the growth and quality of oyster mushrooms, and that it is effective to control the cultivation room temperature to 20°C for optimal growth.

Oyster mushroom is one of the most widely cultivated and consumed mushrooms in Korea, and mechanization and automation of cultivation systems have enabled mass production. Many cultivars have been developed to replace the old ones such as ‘Suhan‘ and ‘Chunchuneutari 2 ho,‘ which have been cultivated for over 20 years. Among these, ‘Soltari‘ was developed in 2015. Although it has excellent quality, its cultivation is challenging and the productivity is somewhat lower. To address these issues, the Mushroom Division at the National Institute of Horticultural and Herbal Science selected the genetic resource KMCC05165 and attempted hybridization between monokaryons from KMCC05165 and ‘Soltari(KMCC04940)’. Through repeated cultivation tests and evaluation of fruiting body characteristics, the superior strain ‘Po-2019-smj22’ was selected and finally named ‘Otari‘. The optimal mycelial growth temperature of ‘Otari’ was between 25 and 30°C and optimal fruiting body growth temperature was between 13 and 18°C. Mycelial growth on PDA medium was best at 25°C, and at the same temperature, mycelial growth was similar across four media: PDA, MEA, MCM, and YM. In 1,100 mL bottle cultivation, the yield was approximately 174 g, which is about 5% higher than the control cultivar ‘Soltari‘, and the number of valid individuals was also higher at about 25. The diameter and height of the pileus were 29.8 mm and 17.6 mm, respectively, slightly smaller than ‘Soltari‘, and the stipe was thin and long with a thickness of 12.2 mm. Additionally, the pileus’ lightness index (L index) was 30.7, indicating a darker brown color compared to 'Soltari.' With excellent mycelial growth, ease of cultivation, and high yield, the new cultivar ‘Otari‘ is expected to be widely adopted by domestic oyster mushroom farms.

Recently, active research in Korea and worldwide has begun to focus on gene function and cultivar development using gene editing tools. This research, in addition to studies on edible mushroom, aims to enhance the physical and biochemical characteristics of mushrooms for applications in materials and substance production. For these studies, efficient isolation of protoplasts from the target mushroom is critical. However, several commercial cell wall-lysing enzyme cocktails, including Novozyme234, Glucanex, and Lysing enzymes, have recently been discontinued. In this study, we aimed to identify alternative enzyme systems to replace the discontinued cell wall-lysing enzymes for stable isolation of protoplasts from Ganoderma lucidum. To select an optimal osmotic buffer, enzyme function in 0.6 and 1.2 M Sorbitol, Sucrose, Mannitol, and KCl was assessed. The effect of reaction time was also evaluated. Protoplast isolation efficiency of each alternative enzyme was tested using lysing enzymes from Trichoderma harzianum, Chimax-N, and Yatalase, either individually or in combination. This matrix of studies identified enzymes and optimal conditions that could replace the discontinued lysing enzymes.

Plastics are widely used in industries in human society and because of their structural stability, degradation is a serious global issue. To estimate the degradation of plastic, 31 edible mushrooms were cultured with the selected plastic films (polyethylene [PE], polystyrene [PS], and poly(ethylene terephthalate) [PET]) for 3 months at 25 °C. Measuring the weight of the films showed that four species of mushrooms, namely Porostereum spadiceum, Ganoderma lucidum, Coprinellus micaceus, and Pleurotus ostreatus, exhibited the highest degrees of plastic degradation. In addition, the mushrooms and fungi that exhibited the most significant plastic degradation were cross-cultured to promote this degradation. As a result, cross-cultivation of G. lucidum and Aspergillus niger showed a weight loss of 2.49% for the PET film. For the PS film, Aspergillus nidulans showed a weight loss of 4.06%. Cross-cultivation of A. nidulans and C. micaceus, which showed a weight loss of 2.95%, was noted as an alternative for PS biodegradation, but is harmful to humans. These bio-degradation effects of edible mushroom will contribute to the development of alternatives for eco-friendly plastic degradation.

As a member of ectomycorrhizal fungi, Tricholoma matsutake has a symbiotic relationship with its host, Pinus densiflora. To cultivate T. matsutake artificially, the co-cultivation of T. matsutake mycelia and bacteria from shiro was introduced. In this study, bacteria were isolated from soil samples in Bonghwa-gun, and seven bacterial isolates (B22_7_B05, B22_7_B06, B22_7_B07, B22_7_B08, B22_7_B10, B22_7_B13, and B22_7_B14) promoted the growth of T. matsutake mycelia (147.48, 232.11, 266.72, 211.43, 175.17, 154.62, and 177.92%, respectively). Sequencing of the 16S rRNA region of the isolated bacteria was performed. B22_7_B05 and B22_7_B10 were identified as Bacillus toyonensis, B22_7_B06 and B22_7_B08 as Paenibacillus taichungensis, B22_7_B07 and B22_7_B14 as P. gorilla, and B22_7_B13 as P. odorifer. These bacterial isolates were associated with the shiro community and are expected to contribute to the cultivation of T. matsutake.

Tricholoma matsutake is a traditional favorite food in East Asia, cultivated in fairy rings called “shiro,” which are found near Pinus densiflora. For effective artificial cultivation of Tri. matsutake, microorganisms from symbiotic fairy rings are co-cultivated. In this study, one bacterial isolate (Y22_B35) and two fungal isolates (Y22_F64 and Y22_F68) displayed growth-promoting effects on Tri. matsutake mycelium (158.47, 125.00, and 122.26% enhanced growth, respectively). For identification, 16S rRNA or ITS regions from the microorganisms¡¯ genomes were sequenced. Other sequences, including BenA, CaM, and RPB2 were sequenced in the fungal isolates. The bacterial isolate Y22_B35 was identified as Bacillus cereus. Y22_F64 and Y22_F68 were identified as Umbelopsis nana and Aspergillus parvulus, respectively. To identify the effects of the dominant microorganisms on Tri. Matsutake cultivation, metagenomic analyses were performed. Discovery of these Tri. matsutake mycelium growth-promoting microorganisms and metagenomics analyses are expected to contribute to our understanding of Tri. matsutake fruiting body growth and construction of biomimicry.