Gene editing using CRISPR/Cas9 is an innovative tool for developing new mushroom strains, offering a promising alternative to traditional breeding methods that are time-consuming and labor-intensive. However, plasmid-based gene editing presents several challenges, including the need for selecting appropriate promoters for Cas9 expression, optimizing codons for the Cas9 gene, the unintended insertion of fragmented plasmid DNA into genomic DNA (gDNA), and regulatory concerns related to genetically modified organisms (GMOs). To address these issues, we utilized a Ribonucleoprotein (RNP) complex consisting of Cas9 and gRNA for gene editing to modify the A mating-type gene of Lentinula edodes. To overcome the challenges posed by the large size of the Cas9 protein, which limits its penetration through the protoplast membrane, and the susceptibility of sgRNA to degradation, we developed a nanoparticle complex using calcium phosphate and polyacrylic acid. This approach significantly improved gene editing efficiency. Consequently, we successfully edited the mating-controlling genes hd1 and hd2 in L. edodes and examined the effects of their disruption on mating. Disruption of the hd1 gene, which is known to influence mycelial growth, did not significantly affect growth or mating. In contrast, editing the hd2 gene disrupted mating with compatible partners, highlighting its critical role in the mating process. The RNP-based transformation technology presented here offers significant advancement over traditional plasmid-based methods, enhancing the efficiency of targeted gene modification while avoiding the insertion of foreign genetic material, thereby mitigating GMO-related regulatory concerns.

Digital phenotyping is increasingly recognized as a critical tool for quantitative analysis of fungal morphology, particularly in controlled indoor cultivation systems where large numbers of fruiting bodies must be assessed consistently and non-destructively. While YOLOv8-based deep learning approaches have been previously applied to phenotypic analysis of edible mushrooms, the applicability of newer YOLO architectures to fungal phenotyping remains unexplored. In this study, we present a controlled-environment digital phenotyping framework for indoor mushroom cultivation and evaluate the feasibility of YOLOv11 for phenotypic analysis through a direct comparison with YOLOv8. Using bottle-cultivated Pleurotus ostreatus and Flammulina velutipes as representative edible basidiomycetes, we conducted a systematic comparison of YOLOv8-seg and YOLOv11-seg under identical datasets, preprocessing pipelines, and hyperparameter configurations. The results demonstrate that YOLOv11 achieves segmentation performance comparable to YOLOv8 across all evaluated metrics (ΔmAP50–95 < 0.01), while substantially reducing computational complexity, including fewer trainable parameters, lower FLOPs, and decreased gradient load. Validation against caliper-based physical measurements revealed moderate, trait-dependent agreement, whereas inter-model consistency between YOLOv8 and YOLOv11 was consistently high across diverse morphological and segmentation scenarios. These findings indicate that architectural refinements in YOLOv11 primarily enhance computational efficiency and training behavior without altering phenotypic interpretation. Collectively, this study provides the first validation of YOLOv11 for mushroom phenotyping and highlights its potential as an efficient analytical tool for future high-throughput fungal morphology studies.

Digital phenotyping is increasingly recognized as an essential tool for the quantitative analysis of fungal morphology, particularly in controlled indoor cultivation systems where large numbers of fruiting bodies must be assessed consistently and non-destructively. While YOLOv8-based deep learning approaches have previously been applied in phenotypic analyses of edible mushrooms, the applicability of newer YOLO architectures to fungal phenotyping remains largely unexplored. In this study, we present a controlled-environment digital phenotyping framework for indoor mushroom cultivation and conduct a systematic benchmarking evaluation of YOLOv11 for phenotypic segmentation in comparison with YOLOv8. Using bottle-cultivated Pleurotus ostreatus and Flammulina velutipes as representative edible basidiomycetes, we performed a controlled comparison of YOLOv8-seg and YOLOv11-seg using identical datasets, preprocessing pipelines, and hyperparameter configurations. The results demonstrate that YOLOv11 achieves segmentation performance comparable to that of YOLOv8 across all evaluated metrics (ΔmAP_50–95 < 0.01) while substantially reducing computational complexity, including fewer trainable parameters, lower FLOPs, and decreased gradient load. Validation against caliper-based physical measurements revealed moderate, trait-dependent agreement, whereas inter-model consistency between YOLOv8 and YOLOv11 remained consistently high across diverse morphological and segmentation scenarios. These findings suggest that recent developments in object detection architectures can improve computational efficiency without compromising phenotypic measurement fidelity. More broadly, this study highlights the importance of periodically evaluating emerging detection architectures within biological phenotyping pipelines to ensure scalable, sustainable, and high-throughput fungal phenotyping under controlled-environment cultivation systems.

This study aimed to develop and evaluate new hybrid strains of Pleurotus cystidiosus with enhanced functional and physiological characteristics. Hybridization between the parental strains KMCC01257 and KMCC05164 yielded four promising hybrid lines (PA-054, PA-104, PA-122, and PA-132), which were selected based on superior mycelial growth and yield performance. Morphological traits and productivity were evaluated across three developmental stages: primordium formation (C1), fruiting body development (C2), and maturation (C3). As cultivation progressed, the number of fruiting bodies decreased, whereas total yield per cultivation bag increased, indicating that late-stage management plays a critical role in maximizing productivity. Chemical analyses revealed that water extracts contained higher levels of polysaccharides, ergothioneine, and total phenolics than EtOAc extracts. Among the tested strains, PA-132 exhibited the highest phenolic content and strongest antioxidant activity, while PA-104 showed greater polysaccharide and ergothioneine accumulation than the parental strains. Antioxidant activity increased over developmental stages and was consistently higher in water extracts, whereas α-glucosidase inhibitory activity was detected primarily in EtOAc extracts with minimal variation among strains or stages. Overall, the results demonstrate that hybridization effectively enhanced the biosynthesis of bioactive metabolites and the functional properties of P. cystidiosus. The selected hybrid strains, particularly PA-132 and PA-054, represent promising candidates for the development of high-value functional mushroom cultivars and nutraceutical applications.

Tricholoma matsutake is a valuable edible mushroom in Eastern Asia. Due to the challenges of artificial cultivation, T. matsutake cultivation has relied on foraging from pine forests. Changes in global climate variables, such as temperature and precipitation, could affect the amount of foraged T. matsutake. In this study, we investigated the correlation between the amount of foraged T. matsutake and climatic factors (average monthly temperature and precipitation) in Yangyang-gun using the augmented Dickey–Fuller test and the ordinary least squares method. Among the assessed correlations between the foraged T. matsutake and different climatic factors, the average temperature in August was significantly correlated with the amount of foraged T. matsutake, increasing by 1.5 tons when the temperature in August increased by 1 unit. Overall, this study identified a potentially strong positive correlation between the average temperature in August and amount of foraged T. matsutake.

Mushrooms have long been economically and nutritionally important crops, and recent advances in digital agriculture have increased interest in automating phenotypic evaluation. Due to the limitation of traditional phenotype assessment, various artificial intelligence (AI) models including YOLOv8 have been introduced to evaluate mushroom phenotypes non-destructively and efficiently. However, unlike previous models, few studies of mushroom phenotype assessment with YOLOv11 were published. In this study, using Pleurotus ostreatus and Flammulina velutipes, comparison of mushroom phenotype analysis between YOLOv8 and YOLOv11 was processed. All images were captured under controlled conditions and conducted to be preprocessed for the model evaluation. The results demonstrated that YOLOv11 achieved segmentation accuracy comparable to YOLOv8 (ΔmAP50–95 < 0.01) while substantially improving computational efficiency with a reduction of approximately 15–20%. In validation with the physical measurements of mushroom phenotype, both models showed biologically meaningful and moderate correlations across phenotypic traits (r ≈ 0.2–0.44; R² ≈ 0.72–0.83), confirming that YOLO-derived measurements captured essential dimensional variation. Inter-model comparisons revealed strong consistency (r ≥ 0.94, R² ≥ 0.96, MAE ≤ 0.40), indicating that YOLOv11 maintained the predictive reliability of YOLOv8 while operating with superior computational efficiency. This study establishes YOLOv11 as a robust foundation for AI-assisted digital breeding and automated quality monitoring systems in fungal research and precision agriculture.