High Throughput Identification of the Potential Antioxidant Peptides in Ophiocordyceps sinensis.

Ophiocordyceps sinensis, an ascomycete caterpillar fungus, has been used as a Traditional Chinese Medicine owing to its bioactive properties. However, until now the bio-active peptides have not been identified in this fungus. Here, the raw RNA sequences of three crucial growth stages of the artificially cultivated O. sinensis and the wild-grown mature fruit-body were aligned to the genome of O. sinensis. Both homology-based prediction and de novo-based prediction methods were used to identify 8541 putative antioxidant peptides (pAOPs). The expression profiles of the cultivated mature fruiting body were similar to those found in the wild specimens. The differential expression of 1008 pAOPs matched genes had the highest difference between ST and MF, suggesting that the pAOPs were primarily induced and play important roles in the process of the fruit-body maturation. Gene ontology analysis showed that most of pAOPs matched genes were enriched in terms of 'cell redox homeostasis', 'response to oxidative stresses', 'catalase activity', and ' integral component of cell membrane'. A total of 1655 pAOPs was identified in our protein-seqs, and some crucial pAOPs were selected, including catalase, peroxiredoxin, and SOD [Cu-Zn]. Our findings offer the first identification of the active peptide ingredients in O. sinensis, facilitating the discovery of anti-infectious bio-activity and the understanding of the roles of AOPs in fungal pathogenicity and the high-altitude adaptation in this medicinal fungus.

Biochemical Profiling and Cultivation of Medicinal Fungus Isaria cicadae (Ascomycetes) from India.

Isaria cicadae is an entomopathogenic fungus possessing several therapeutic properties and has a potential role in traditional Chinese medicine. The present study was designed to describe the taxonomic details of a new isolate of I. cicadae collected from the Northern Himalayas of India and to study its vegetative and reproductive growth responses under in vitro conditions. Proximate composition, biochemical profiling, and radical scavenging activities were studied to establish the bioactivity of the isolate. Micromorphological characteristics of conidia and conidiophore formation were studied using scanning electron microscopy. The optimum temperature and pH for mycelial growth was 25°C and 7.0, respectively. Pinhead initiation was observed at day 10 after inoculation, but the fully developed, branched, and coral to club-shaped fruiting bodies could be observed after 30 days of inoculation. Proximate analysis indicated that carbohydrates are the major constituents (50.2%) of the fruit bodies, along with a lower quantity of protein (4.46%), crude fat (6.4%), and crude fiber (1.55%). Vitamin D content of I. cicadae was 3,605.84 IU/g. Radical scavenging activity based on the DPPT (1,1-diphenyl-2-picrylhydrazyl) assay was 21.2%. ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid] and potassium ferricyanide reducing activity were quite high, at around 93% and 99.3%, respectively. The findings of this study provide insight into the biochemical constituents of I. cicadae and its cultivation practices for further exploitation of this mushroom at a larger scale.

Review on Research Progress and Prospects of Cicada Flower, Isaria cicadae (Ascomycetes).

Cicada flower, Isaria cicadae Miq., has been a traditional Chinese medicine for approximately 1600 years. Many works on its identification, bioactivities, and clinical use against some disorders have been published, but some inaccuracies and inconsistencies need to be further clarified. In combination with our > 20 years of research and application of cicada flower and examination of the literature and patents published in recent years, this article summarizes and reviews the life cycle and taxonomy, genome size and mating type, molecular systematic classification and cultivation, active ingredients, and pharmacological functions of I. cicadae.

Transcriptomic analysis of the orchestrated molecular mechanisms underlying fruiting body initiation in Chinese cordyceps.

Chinese cordyceps, the fruiting body of the Chinese caterpillar fungus (Ophiocordyceps sinensis, syn. Cordyceps sinensis), is among the most valuable traditional Chinese medicine fungi. Transcriptomic analysis of O. sinensis has revealed several aspects of its life cycle and ecological importance. However, the molecular mechanisms involved in fruiting body initiation remain unclear. The developmental transcriptomes were analyzed from three tissues at the fruiting body initiation stage, namely, the mycelium, sclerotium and primordium. Principal component analysis showed that in the three tissues, the gene expression patterns differed from each other. The functional analysis of differentially expressed genes showed that DNA synthesis and cell division were active in the primordium. In addition, the function of the mycelium was to absorb certain substances from the environment and the sclerotium was the metabolism center of O. sinensis. Genes participating in the mitogen-activated protein kinase (MAPK) signal pathway were involved in fruiting body initiation. Two environmental sensing genes, including a pheromone receptor gene (OSIN6252) and an amino acid sensing gene (OSIN6398), were highly expressed in the primordium, suggesting their important roles in initiation. These results provided insights into the orchestrated functions and gene profiles of different O. sinensis tissues at the key stage. These findings will aid in revealing the underlying mechanisms of fruiting body initiation, which will further benefit artificial cultivation.

Bioactive Metabolites and Potential Mycotoxins Produced by Cordyceps Fungi: A Review of Safety.

Ascomycete Cordyceps fungi such as C.militaris, C. cicadae, and C.guangdongensis have been mass produced on artificial media either as food supplements or health additives while the byproducts of culture substrates are largely used as animal feed. The safety concerns associated with the daily consumption of Cordyceps fungi or related products are still being debated. On the one hand, the known compounds from these fungi such as adenosine analogs cordycepin and pentostatin have demonstrated different beneficial or pharmaceutical activities but also dose-dependent cytotoxicities, neurological toxicities and or toxicological effects in humans and animals. On the other hand, the possibility of mycotoxin production by Cordyceps fungi has not been completely ruled out. In contrast to a few metabolites identified, an array of biosynthetic gene clusters (BGCs) are encoded in each genome of these fungi with the potential to produce a plethora of as yet unknown secondary metabolites. Conservation analysis of BGCs suggests that mycotoxin analogs of PR-toxin and trichothecenes might be produced by Cordyceps fungi. Future elucidation of the compounds produced by these functionally unknown BGCs, and in-depth assessments of metabolite bioactivity and chemical safety, will not only facilitate the safe use of Cordyceps fungi as human food or alternative medicine, but will also benefit the use of mass production byproducts as animal feed. To corroborate the long record of use as a traditional medicine, future efforts will also benefit the exploration of Cordyceps fungi for pharmaceutical purposes.

Effects of Substrates on the Production of Fruiting Bodies and the Bioactive Components by Different Cordyceps militaris Strains (Ascomycetes).

Cordyceps militaris is a type of food and medicinal species and is widely cultured in Asia. Substrate and strain are important factors for the production of fruiting bodies and bioactive components contents in fruiting bodies of C. militaris. This study aimed to select the excellent strains and suitable substrates by six strains of C. militaris cultivated on rice, wheat, and tussah (Antheraea pernyi) pupae. The results showed that the rice and wheat were suitable for fruiting body formation of strain CM3, with yields of 23.19 and 19.07 g per bottle, and biological efficiency of strain CM3 were 62.26% and 54.48%, respectively, which were significantly higher than other strains. Tussah pupae is suitable for fruiting body formation of strain CM9, with fruiting body length, yield, and biological efficiency of 5.57 cm, 6.80 g per each, and 291.70%, respectively, which were significantly higher than other strains. The content of adenosine in fruiting bodies of strain CM9 cultivated on tussah pupae was 2.62 mg g-1, followed by that of strain CM3 on rice of 2.51 mg g-1. The content of cordycepin in fruiting bodies of strain CM4 cultivated on wheat was 5.68 mg g-1, followed by that of strain CM9 on wheat of 5.41 mg g-1. To improve the product quality and the contents of bioactive components, C. militaris strains and substrates should both be considered, that is, different strains should be appropriate for different substrates.

Vegetative development and host immune interaction of Ophiocordyceps sinensis within the hemocoel of the ghost moth larva, Thitarodes xiaojinensis.

Ophiocordyceps sinensis is an entomopathogenic fungus that infects ghost moth larva, forming the most valuable and rare traditional Chinese medicine, Chinese cordyceps. Our knowledge of the basic morphology and developmental biology of Chinese cordyceps is limited. In this study, morphological and ultrastructural observations of O. sinensis development in the hemocoel of Thitarodes xiaojinensis were obtained by multiple light and electron microscopy techniques, and the host immune reaction activities were determined. Our results indicated that fungal cells in the host hemocoel underwent morphotype transformations from blastospores to prehyphae to hyphae in sequence. The fusiform yeast-like blastospores were the initial cell type present in the host hemocoel and remained for 5 months or more; the encapsulation reaction and phenoloxidase activity of T. xiaojinensis hemolymph were inhibited during this period. When larvae entered the last instar, the blastospores switched to prehyphae and expanded throughout the host tissues, and then hyphae germinated from the prehyphae and mycelia formed, which finally led to host death. Considering the distinct differences between blastospores and hyphae, we identified prehyphae, which play important roles in fungal expansion, hyphae germination, and fusion formation among filaments. Notably, the elongation of prehyphae was strongly presumed to occur through fission but without separation of the two sister cells, in contrast to blastospore budding. During the morphotype transformation, the amount and composition of lipid droplets changed greatly, suggesting their important roles in these events. Overall, we provide a morphological and ultrastructural characterization of O. sinensis vegetative development within the hemocoel of T. xiaojinensis, identify and name the prehypha fungal cell type in entomopathogenic fungi for the first time, and conclude that O. sinensis infection causes sustained immunosuppression in T. xiaojinensis.

Enhanced Cordycepin Production in Caterpillar Medicinal Mushroom, Cordyceps militaris (Ascomycetes), Mutated by a Multifunctional Plasma Mutagenesis System.

A multifunctional plasma mutation system (MPMS) method was used to create high cordycepin-yielding mutations from wild Cordyceps militaris, which yielded many viable mutants, many of which produced more cordycepin compared to the wild strain. One particular mutant strain (GYS60) produced 7.883 mg/mL, which is much higher than those reported to date and is more than 20 times higher than that of the wild strain, whereas the cordycepin production of another viable mutant (GYS80) was almost zero. The extraction and purification of cordycepin, using the fermentation broth of C. militaris GYS60, was also investigated. Cordycepin was extracted by using AB-8 macroporous resin and purified by using reversed-phase column chromatography. When the sample was adsorbed onto the macroporous resin, 20% ethanol was used as the desorption solvent yielding various fractions. The fractions containing cordycepin were loaded onto a reversed-phase chromatography column packed with octadecyl bonded silica as the stationary phase and ethanol (95%)/acetic acid solution (5%) at pH 6.0 as the mobile phase. The combination of this two-step extraction-purification process yielded cordycepin at 95% purity with a total recovery rate of 90%.

Carotenoid Production by Caterpillar Medicinal Mushrooms, Cordyceps militaris (Ascomycetes), under Different Culture Conditions.

Cordyceps militaris, a well-known edible and medicinal species, is a potential source of natural carotenoids. The optimized conditions for carotenoid production are important for high-quality cultivation since carotenoid contents are the key factors for the appearance of fruiting bodies. In this study, carotenoid production by C. militaris under different culture conditions was compared. It was found that two-stage culture and fruiting body cultivation were suitable for the carotenoid production. Light is one of the important factors for carotenoid accumulation in this fungus and carotenoid can be produced by continuous shaking under light irradiation. Blue light with intensity of 17.3-64.3 lux can promote carotenoid production under the three tested culture methods. The carotenoid content was 4410.42 ± 30.83 and 3747.92 ± 178.13 µg g-1 under blue light intensity of 64.3 lux for the velum under two-stage culture and fruiting body cultivation, respectively. Oat medium may be suitable for the cultivation of fruiting bodies due to its high carotenoid production compared with other grains such as rice, wheat, and millet. Oxygen vector and oxidative stress induced by hydrogen peroxide can promote carotenoid production significantly under the two-stage culture. The carotenoid extracted from C. militaris showed strong antioxidant activity with IC50 value of 0.219 ± 0.01 mg ml-1, even higher than that of BHT, a positive control. This study will benefit the high-quality cultivation of this fungus and confirm that the carotenoid from C. militaris has the potential to be developed as a health food.

Engineering Aspects in Production of Various Medicinal Mushrooms Biomass in Submerged Bioreactors.

Basidiomycetes of various species and their wide range of pharmaceuticaly interesting products in the past decades represents one of the most attractive groups of natural products in Asia and North America. Production of mushroom fruit bodies using farming technology is hardly covering the market. Development of comprehensive submerged technologies in stirred tank and air lift bioreactors are the most promising technologies for fast and large-amount cultivation of medicinal mushroom biomass and its pharmaceutically active products. Research in physiology, basic and applied studies in mushroom metabolism, process engineering aspects, and clinical studies in the past two decades represent a large cotribution to the development of this potential, which initiates the development of new drugs and some very attractive over-the-counter human and veterinary remedies. The current article is an overview of the most relevant engineering achievements in submerged cultivation of some medicinal mushrooms-Grifola frondosa, Trametes versicolor, Hericium erinaceus, and Cordyceps militaris-and some other species biomass production in bioreactors.

Comprehensive evaluation of wild Cordyceps cicadae from different geographical origins by TOPSIS method based on the macroscopic infrared spectroscopy (IR) fingerprint.

Cordyceps cicadae is an entomogenous fungus that has been used as a valuable traditional Chinese herbal tonic, however, it can be difficult to discern the false from the genuine. In this study, the macroscopic IR fingerprint methods containing Fourier transform infrared spectroscopy (FT-IR) and second derivative infrared spectroscopy (SD-IR) were used to elucidate wild C. cicadae. The TOPSIS (Technique for Order Preference by Similarity to Ideal Solution) method was used to comprehensively evaluate C. cicadae from different geographical origins based on the macroscopic infrared spectroscopy (IR) fingerprint. The FT-IR spectra of C. cicadae exhibited the major characteristics of the absorptive peaks of carbohydrates, lipids and nucleosides at the position of 3291, 2925, 2845, 1651, 1547, 1455, 1080 and 950 cm-1. The high resolution of SD-IR further amplified the difference and revealed the potentially characteristic IR absorption spectrum. TOPSIS evaluation showed that C. cicadae from Anhui possess the strongest intensity of absorption bands among all the samples. Notably, FT-IR combined with SD-IR can effectively reveal the overall chemical components without damaging medicinal materials, and TOPSIS methods can provide a novel scientific evidence for comprehensively assessing different origins of wild C. cicadae.

Effect of Culture Time on the Bioactive Components in the Fruit Bodies of Caterpillar Mushroom, Cordyceps militaris CM-H0810 (Ascomycetes).

Cordyceps militaris are widely cultivated in China for an important raw material for health foods. CM-H0810 is a C. militaris strain used in the production of C. militaris in Shanghai, the surrounding areas of Shanghai, and Guangdong province in China. We evaluated the effect of culture time on the bioactive components in the fruit bodies of C. militaris CM-H0810 to provide scientific references for production of C. militaris fruit bodies with good quality. The results showed that the polysaccharide contents increased gradually during 35-45 d, but it declined with the prolongation of culture time. The highest polysaccharide content was 3.46% at 45 d. With the prolongation of culture time the cordycepin content gradually increased; the highest cordycepin content was 3.57 µg/mg at 60 d, which increased 321% compared to that at 35 d. Contrary to cordycepin, the adenosine content declined gradually, with the highest content of 1.86 µg/mg at 35 d and the lowest content of 1.48 µg/mg at 60 d. Our study indicates that it is necessary to select suitable harvest times in view of different compounds that are desirable to obtain in high quantities.

Increasing of the Contain of Carotenoids in Caterpillar Mushroom, Cordyceps militaris (Ascomycetes) by Using the Fungal Elicitors Cultivation.

As a natural pigment, cordycepic carotenoids have many bioactive functions, such as antiinflammation, anticancer, and antioxidation. In addition, the good coloring of this hydrophilic pigment enables it to have wide application in the food industry. This study investigated five species of fungal elicitors, namely, Rhodotorula glutinis, Saccharomyces cerevisiae, Monascus ruber, Blakeslea trispora, and Flammulina velutipes, to evaluate their effects on carotenoid accumulation in Cordyceps militaris. Results showed that all fungal elicitors, except Rh. glutinis, have no positive effect on the biosynthesis of cordycepic carotenoids. The Rh. glutinis elicitor remarkably stimulated the accumulation of carotenoids with a 13.72% increase compared with the control. Subsequently, the entire Rh. glutinis elicitor (part NHK) was divided into three parts, namely, exopolysaccharide (EPS) (part E), mixture of EPS and protein (part PE), and other components (part O), to analyze their effects on carotenoid accumulations. Results showed that part O may be the effective component that remarkably stimulates the biosynthesis of carotenoids with a 26% increase compared with the control. This research demonstrated that Rh. glutinis elicitor can effectively increase the content of natural carotenoids in C. militaris, and provided an important reference for the development and utilization of carotenoid industrialization.

Cosmetic and Skincare Benefits of Cultivated Mycelia from the Chinese Caterpillar Mushroom, Ophiocordyceps sinensis (Ascomycetes).

Mushrooms are potential sources of novel natural cosmeceutical ingredients. This study was conducted to evaluate the cosmetic (skincare) benefits of the valuable medicinal species Ophiocordyceps sinensis (=Cordyceps sinensis). The mycelial extracts of 2 O. sinensis strains, Cs-HK1 and Cs-4, prepared sequentially with ethyl acetate, ethanol, and hot water were tested with in vitro assays for tyrosinase-, collagenase-, and elastase-inhibitory activity. The ethyl acetate extracts of both fungal strains showed potent antityrosinase and antielastase activity, with low half-maximal inhibitory concentrations (0.14-0.47 mg/mL) comparable to those of the respective reference compounds (arbutin and epigallocatechin gallate). All mycelial extracts exhibited moderate or significant anticollagenase activity; most extracts showed a significant photoprotective effect with a sun protection factor up to 25. The results from this study show the potential use of O. sinensis as a source of cosmetic ingredients for skincare applications.

Influence of Strain Preservation Methods on Fruiting Body Growth and Metabolite Production by the Medicinal Mushroom Cordyceps militaris (Ascomycetes).

Cordyceps militaris is a highly valued edible and medicinal fungus because of its production of various metabolites including adenosine, cordycepin, and N6-(2-hydroxyethyl)-adenosine. Fruiting bodies of this fungus have been used successfully in industrial production and widely as a substitute for Ophiocordyceps sinensis (syn. C. sinensis) in traditional Chinese medicine and health supplements. Strain degeneration occurs with high frequency during the subculturing and preservation of C. militaris strains, which leads to significant losses during industrial production. In this study, we evaluated the effects of different strain preservation methods on fruiting body growth and metabolite production. We found that strain degeneration affects not only fruiting body differentiation but also metabolite production, and suitable preservation methods can avoid degeneration. Preservation in sterile water has a similar effect as cryopreservation in liquid nitrogen at -196°C with regard to maintaining the characteristics of C. militaris strains for at least 1 year, and it is a practical and satisfactory method for preserving C. militaris strains that can be used in factories. Ultracold freezing at -80°C is not suitable for this fungus. Lyophilization, which causes C. militaris strains to retain their inherent characteristics and avoid degeneration, is suitable for long-term preservation (at least 4 years). This study provides practical preservation methods for C. militaris strains over the short and long term and will be helpful to achieve stable and superior-quality production of C. militaris fruiting bodies.

An Evaluation of Common Cordyceps (Ascomycetes) Species Found in Chinese Markets.

The common Cordyceps (more than 20 species) found in Chinese markets are evaluated and illustrated. We systematically introduce their taxonomies, allied species, hosts, distributions, yields, selling statuses, and market prices. We summarize and comment on the medicinal value and food safety of Cordyceps. The concept of "cordyceps" is explained, the "best" cordyceps is explored, and Chinese cordyceps culture is analyzed. We suggest using the word "cordyceps" instead of the Latin Cordyceps to report these important edible and medicinal mushrooms.

[Climate change impacts on yield of Cordyceps sinensis and research on yield prediction model of C. sinensis].

Cordyceps sinensis is a Chinese unique precious herbal material, its genuine producing areas covering Naqu, Changdu in Qinghai Tibet Plateau, Yushu in Qinghai province and other regions. In recent 10 years, C. sinensis resources is decreasing as a result of the blindly and excessively perennial dug. How to rationally protect, develop and utilize of the valuable resources of C. sinensis has been referred to an important field of research on C. sinensis. The ecological environment and climate change trend of Qinghai Tibet plateau happens prior to other regions, which means that the distribution and evolution of C. sinensis are more obvious and intense than those of the other populations. Based on RS (remote sensing)/GIS(geographic information system) technology, this paper utilized the relationship between the snowline elevation, the average temperature, precipitation and sunshine hours in harvest period (April and may) of C. sinensis and the actual production of C. sinensis to establish a weighted geometric mean model. The model's prediction accuracy can reach 82.16% at least in forecasting C. sinensis year yield in Naqu area in every early June. This study can provide basic datum and information for supporting the C. sinensis industry healthful, sustainable development.

Photoperiodic Responses and Characterization of the Cmvvd Gene Encoding a Blue Light Photoreceptor from the Medicinal Caterpillar Fungus Cordyceps militaris (Ascomycetes).

Light is a necessary environmental factor for production of conidia and pigment, formation of stroma, and development of Cordyceps militaris, a well-known edible and medicinal mushroom. In this study, an obvious rhythm loop was observed in certain strains of C. militaris under conditions of alternating 12-hour intervals of dark and light. A possibly related gene, Cmvvd, the homologue of the blue-light photoreceptor of Neurospora crassa, was cloned from the genome of C. militaris. The protein CmVVD is predicted to be 203 amino acids in length and is characterized by the presence of a light, oxygen, or voltage domain. Analysis of the CmVVD sensor domain (light, oxygen, or voltage) suggested that it is a blue-light receptor. Cysteine 108 is essential for the in vivo function of VIVID (VVD) in N. crassa photoadaptation. However, proline is in this position instead in all of the tested CmVVD proteins, suggesting that CmVVD may have a different function or may function in ways different from VVD in N. crassa. Genetic variation analysis of CmVVD in 6 representative strains indicated that 3 informative sites exist. Cmvvd messenger RNA was able to be induced by light, and the expression level increased over 10 times after irradiation and was maintained at high levels in the nascent fruiting body. The light-induced expression of Cmvvd was abolished in Cmwc-1 mutants, suggesting that the expression of Cmvvd is dependent on the photoreceptor CmWC-1 or on a functional CmWC-1/WC-2 complex. This article will help to open the still-unexplored field of circadian rhythms for this fungus.

Uncovering the Molecular Mechanism of Anti-Allergic Activity of Silkworm Pupa-Grown Cordyceps militaris Fruit Body.

Cordyceps militaris has been widely used as an herbal drug and tonic food in East Asia and has also been recently studied in the West because of its various pharmacological activities such as antitumoral, anti-inflammatory and immunomodulatory effects. In this study, we examined the molecular mechanism underlying the anti-allergic activity of ethanol extract prepared from silkworm pupa-cultivated Cordyceps militaris fruit bodies in activated mast cells. Our results showed that ethanol extract treatment significantly inhibited the release of [Formula: see text]-hexosaminidase (a degranulation marker) and mRNA levels of tumor necrosis factor-[Formula: see text] as well as interleukin-4 in RBL-2H3 cells. The cells were sensitized with 2,4-dinitrophenol specific IgE and then stimulated with human serum albumin conjugated with 2,4-dinitrophenol. Oral administration of 300[Formula: see text]mg/kg ethanol extract significantly ameliorated IgE-induced allergic reaction in mice with passive cutaneous anaphylaxis. Western immunoblotting results demonstrated that ethanol extract incubation significantly inhibited Syk/PI3K/MEKK4/JNK/c-jun biochemical cascade in activated RBL-2H3 cells, which activated the expression of various allergic cytokines. In addition, it suppressed Erk activation and PLC[Formula: see text] evocation, which would respectively evoke the synthesis of lipid mediators and Ca[Formula: see text] mobilization to induce degranulation in stimulated RBL-2H3 cells. A compound, identified as [Formula: see text]-sitostenone, was shown to inhibit [Formula: see text]-hexosaminidase secretion from activated mast cells. Our study demonstrated that ethanol extract contained the ingredients, which could inhibit immediate degranulation and de novo synthesis of allergic lipid mediators and cytokines in activated mast cells.

A Novel Technique for Rejuvenation of Degenerated Caterpillar Medicinal Mushroom, Cordyceps militaris (Ascomycetes), a Valued Traditional Chinese Medicine.

Cordyceps militaris has been used in traditional Chinese medicine for many years, but its frequent degeneration during continuous maintenance in culture can lead to substantial commercial losses. In this study, a degenerated strain of C. militaris was obtained by subculturing a wild-type strain through 10 successive subcultures. The relative abundance of the 2 mating types seems to be out of balance in the degenerated strain. By cross-mating 4 single-ascospore isolates (2 for MAT 1-1 and 2 for MAT 1-2) from the degenerated strain, we were able to restore fruiting body production to wild-type levels. The rejuvenated strain not only produced well-developed fruiting bodies but also accumulated more cordycepin and adenosine than either the original wild-type strain or the degenerated strain. These new characteristics remained stable after 4 successive transfers, which indicates that the method used to rejuvenate the degenerated strain in this study is an effective approach.