Fungal Genomics & Biology
Open Access

Action Mechanism, Benefits and Cultivation (ABC) of Cordyceps

Vijay Kumar Dalal^{* *}Correspondence: Vijay Kumar Dalal, Department of Botany, Dayalbagh Educational Institute, Agra, India, Tel: +91-562 280 1545, +91-9711453093, Email: ,

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Introduction

Cordyceps Fr. (L.) is a large and diverse genus of the family Cordycipitaceae (Hypocreales, Ascomycota) comprising more than 625 species according to Myco Bank [1]. They are parasitic fungi, mostly endoparasitoids of insects and other arthropods, while some of them are parasitic on other fungi [2]. Cordyceps genus includes many insect parasites and fungal parasites such as the tongue core club (Cordyceps ophioglossoides) which lives on deer truffles (Elaphomyces spp.) and plant parasites such as the grass kernel fungus (Epichloë typhina). The ergot fungus (Claviceps purpurea), Cordyceps militaris, Ophiocordyceps sinensis (O. sinensis, caterpillar fungus), Cordyceps nutans and Paecilomyces tenuipes are related species. Recently, several new Ophiocordyceps species parasitizing on a range of insect hosts have been identified, e.g., O. asiana and O. tessaratomidarum, O. flavida, O. mizoramensis, O.vespulae, O. bidoupensis, O. hydrangea, O. buquetii, O. laotii and O. puluongensis [3-9]. Recent changes in the nomenclature and modification in classifications of Cordyceps and related genera has been discussed by Shrestha, et al. [10].

Among these medicinal fungi, the most valued one in Chinese medicine is O. sinensis (formerly Cordyceps sinensis), O. sinensis mainly infects pupae but sometime larvae of insects. The healing powers of O. sinensis are documented in Chinese herbal books that are up to 2,000 years old. Another less explored Cordyceps species similar to O. sinensis, C. militaris and C. nutans is found in Japan, Taiwan and China. C. nutans is entomoparasitic ascomycete of hemipteran insects [11]. One more medicinal mushroom Paecilomyces is an anamorph of Cordyceps [12]. P. tenuipes had been used traditionally as an effective, nutritious health food and medicine in China [13,14].

There are many other Cordyceps species that grow on different kind of insects, infect their bodies and the ascomata emerges out from their body especially head to spread the spores. These species mostly infect only on host species thus widespread damage to different species is not possible by one fungus. It is a mechanism in nature that controls the unregulated growth of any species in check since the abundant species has higher chance of getting infected by a parasitic Cordyceps spore. Some of the Cordyceps species also show mind control behavior on their host e.g., Ophioglassus unilateralis after infecting host rainforest carpenter ant, Camponotus leonardi leads it to climb on a tree and bite the midrib on the lower side of a leaf to remain stuck in that position even after its death. This facilitates the dispersal of spores and infection of ants travelling on the ground.

O. sinensis (known as Dong-Chong Xia-Cao in Chinese means ‘worm in winter and grass in summer’) grows better in alpine areas (3000 to 5000 meters high) of Tibet, Chinese and Indian territory which are covered with snow in winter and become grassy in summer. It infects a range of host insects especially Hepialus/Thitarodes (Lepidoptera) [15,16]. Cattle dig and eat it up during summers in the grassy lands to become energetic. Overharvesting of O. sinensis has led to its categorization as endangered species in China (State Order of Chinese Government). O. sinensis fruiting body has been cultivated commercially recently (some patents filed by companies). Mycelial forms have also been cultivated in vitro [17,18].

Cordyceps species are not only recommended as an aphrodisiac (called Himalayan Viagra), but also for strengthening the lungs, kidneys and sperm production, against cough, cold and bleeding. Infected pupae and caterpillars are in great demand as medicines but are not so common in nature, thus the pupae of the silkworm (Bombyx mori) are artificially infected with the fungi to meet the demand. The fruiting bodies harvested in nature may contain high amounts of Arsenic and other heavy metals therefore their sale is stringently regulated by Chinese government since 2016 [19]. The fungus germinates in the living larva, kills and mummifies it. Sometimes, many species including O. sinensis simultaneously infect the insect body. A dark brown stalk-like fruiting body, ascus which is 1-8 cm long, club-shaped orange/red and cylindrical, emerges from the head of insect corpse and stands upright. The clubs are whitish to pale orange inside. Shape and size of ascomata vary widely based on the size of the host, number of stromata formed and on the substrate of mycelial growth [20]. The perithecia are present inside clubs which is covered with the stroma. Its spores are long, filiform, smooth, hyaline and often septate that disintegrate later into smaller 3-7 μm × 1-1.2 μm subspores.

C. militaris is considered as a cheaper substitute for the rare fungus O. sinensis and ranks top among the commercialized Cordyceps species, followed by O. sinensis [21]. C. militaris have higher content of cordycepin as compared to O. sinensis. The consumption of C. militaris have a more potent anti-tumor effect than O. sinensis at the same dose level [22] (Figure 1). Fruiting bodies obtained from cultured C. militaris are sold as health food product and drug material across South East Asia and China [23]. C. militaris is widely distributed in Europe, North and South America and Asia and have multiple medicinal properties [24-26]. Many bioactive compounds have been isolated from it such as cordycepin 3’-deoxyadenosine Monophosphate (3’-dAMP), Cordyceps polysaccharides, ergosterol, ergothioneine trehalose, mannitol etc., [27-29] (Figure 2). Its nutraceuticals are consumed either as culinary mushrooms or in several other forms, e.g., extracts, fermented powder and tinctures. C. militaris have antioxidant, antimicrobial, anti-inflammatory, hypolipidemic, hypoglycemic, antiviral, antimalarial, prosexual, neuroprotective and immuno-protective and anti-tumorigenic [30-32] (Figure 1). In diabetic rat models (induced with Streptozotocin (STZ)), C. militaris treatment improved the sexual parameters e.g. serum testosteron levels, copulatory behaviour, epididymal sperm count and mobility [33]. Aqueous extracts of C. militaris show hypoglycemic activity i.e. anti-Protein Tyrosine Phosphatase 1B (PTP 1B) activity due to the presence of four cerebrosides including cordycerebroside B and a disaccharide [34]. It is also used for stopping bleeding, reducing phlegm, hypnotizing, calming, benefiting liver and kidney, tonifying deficiency, relieving asthma and skin cosmetics [35-39].

Figure 1: Beneficial properties of Cordyceps.

Figure 2: Constituents of Cordyceps.

Literature Review

Active ingredients

Many components have been found from natural and cultured Cordyceps by Liquid Chromatography (LC), Gas Chromatography (GC), Nuclear Magnetic Resonance (NMR) spectrometry and Capillary Electrophoresis (CE) combined with Mass Spectrometry (MS) and UV-visible spectrophotometry such as cordycepin, adenosine, proteins, amino acids, carbohydrates, carboxylic acids, lipids, glycosides and minerals [40,41]. Among them, cordycepin is considered as the most valuable and economically important compound produced by this fungus (Figure 3). It is used to inhibit the growth of bacterial, fungal and viral pathogens including Clostridium paraputrificum and Clostridium perfringens, Bacillus subtilis, Candida, human poliovirus, tobacco mosaic virus and hepatitis C virus etc., [42-47]. In addition to this, cordycepin exhibits insecticidal effects. Cordycepin can induce cell death in pests, including Plutella xylostella, Trypanosoma brucei and Trypanosoma evansi [48-50]. Cordycepin induces programmed cell death in-vivo in P. xylostella. In T. evansi infected mice, a combination of cordycepin (2 mg/kg) and pentostatin (0.2 mg/kg) showed significant therapeutic potential and decreased toxicity. In addition, cordycepin also exhibited antifungal activity e.g. against different Candida isolates (Figure 3). Cordycepin has normally been detected by High-Performance Liquid Chromatography (HPLC), but a new method developed recently used Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS) to detect its concentration [51]. From another species, O. sinensis identified 8,541 putative antioxidant peptides using a high-throughput method [52].

Figure 3: Uses of Cordyceps.

Mechanism of action of active ingredients

Cordycepin is supposed to enter the cells via the human Equilibrative Nucleoside Transporters 1 (hENT1) and phosphorylated by Adenosine Kinase (AK) to 3’-deoxyadenosine Monophosphate (3’-dAMP). After being phosphorylated twice, 3’-dAMP is converted to the active 3’-deoxyadenosine Triphosphate (3’-dATP) by the concerted action of Adenosine Monophosphate Kinase (AMPK) and Nucleoside Diphosphate Kinase (NDPK) enzymes [53,54]. Cordycepin achieves its function by:

• Forming cordycepin triphosphate through phosphorylation which is erroneously recognized as ATP by target enzymes [55].

• Abnormal purine metabolism through erroneous reading of cordycepin as adenosine

• Cordycepin and/or cordycepin-triphosphate directly activate some protein kinases, e.g. AMPK probably due to an increase in the AMP/ATP ratio.

• Cordycepin and/or cordycepin-triphosphate inhibit targets kinases by replacing ATP binding with the highly structurally similar cordycepin-triphosphate binding and/or the activation of protein phosphatases [56,57].

• Cordycepin and/or cordycepin-triphosphate interrupt mRNA polyadenylation because of the erroneous recognition of cordycepin-triphosphate as ATP by poly A polymerase [58].

Cordycepin can specifically inhibit transcription efficiency and thus RNA elongation and also has the potential to act as a ligand. Recently diastereoisomers of ProTide, a modified form of cordycepin named NUC-7738, was synthesized artificially, which have up to 40 times greater potency for killing cancer cells than the cordycepin [59-61].

Total extracts with water or 50% ethyl alcohol and polysaccharides from C. militaris tend to promote type 1 immunity, whereas that with 70-80% ethyl alcohol promote type 2 immunity. Cordycepin also promotes type 2 immunity. Different immunomodulatory effects of C. militaris extracts obtained using different solvents might be due to the different polarities of the final products.

C. militaris is not only rich in proteins (39.37%; 1.8, 1.5 and 1.9 times of the protein content of pork, beef and mutton) and amino acids, but also in more than 30 trace elements required by human body. The content of Phosphorus (P), Zinc (Zn), Copper (Cu) and Iron (Fe) are respectively 1.8, 2.1, 8.8 and 3.5 times of that of O. sinensis. Additionally, the content of Selenium (Se) is equivalent to that of Astragalus. It contains an 18-kDa protein Cordyceps Militaris Protein-18 (CMP18), which induces apoptosis and might be toxic when the fungi are eaten fresh but gets destroyed upon cooking [62]. CMP18 induces apoptosis via mitochondria-dependent pathway.

Artificial cultivation and enhancement of active ingredients

Cost-effective, cheap, suitable and abundant resources such as by-products of the agricultural and agro-industrial activities are being explored for the development of commercial cultivation of C. militaris and other edible and medicinal mushrooms [63,64]. A lot of literature is available for media optimization and enhancing the active ingredients content.

A wide range of methodologies are used to produce fruiting bodies and mycelia of C. militaris viz. submerged static fermentation, solid-state fermentation, cultivation following larval or pupal infection and one-step non-static or repeated batch fermentation in liquid media [65-72]. There are many media on which C. militaris can be grown. One of the C. militaris culture medium comprises 40-50 g of glucose, 20-30 g of maltose, 5-15 g of beef extract, 5-15 g of peptone, 25-35 g of calcium nitrate, 0.2-0.6 g of magnesium sulfate, 0.2-2.0 g of dipotassium hydrogen phosphate, 0.2-0.6 g of calcium chloride, 30-60 g of silkworm chrysalis powder and 20-50 g of agar per 1000 ml of distilled water. 0.2-1.0 g of common salt per liter is also added (Chinese Patent #132542158). The content of three pharmaceutically important products of C. militaris viz. polysaccharides, cordecepin and adenosine varies with time in fruiting bodies. The use of corn cobs or cottonseed shells to wheat bran and rice at ratios of 8:1:1 (w/w/w) resulted in higher yields of fruit bodies compared to the conventional rice media, while corn cobs produced fruit bodies with the highest cordycepin content. Addition of vegetable oils such as rapeseed, olive, palm, peanut, corn, soybean and sunflower seeds in the static cultures of C. militaris promoted mycelium growth. Moreover, the addition of peanut oil significantly increased cordycepin content. Optimized media formulation for C. militaris contained 20% beef broth, 0.10% peptone, 2% glucose, 0.15% yeast extract, 0.20% KH₂PO₄ and 0.02% MgSO₄. Zhu, et al. [73], found that in C. militaris strain CM-H0810 which grows in the surrounding areas of Shanghai and Guangdong province, the polysaccharide content was highest at 45 d (3.46%), cordycepin at 60 d (3.57 μg/mg) and adenosine at 35 d (1.86 μg/mg). Liang, et al. [74], studied two C. militaris strains, “H” and “L” and obtained the highest yield and biological efficiency with Pearl Barley (PB) substrate for C. militaris H strain and with brown rice+peptone substrate for C. militaris strain L. Furthermore, they observed the highest cordycepin content on Wheat (W)+Monosodium Glutamate (MG) substrate, mannitol content on Plumule Rice (PR)+MG and adenosine content on PR+Yeast Extract (YE) substrates in the C. militaris H fruiting bodies. In C. militaris L, highest cordycepin was reported with W+MG substrate, mannitol with PB substrate and adenosine with PB+YE substrate. In another study, the concentration of bioactive compounds was compared between C. militaris and(i) Fruiting bodies on wheat; (ii) Fruiting bodies on pupae; (iii) Sclerotium (the pupae portion) and (iv) Sclerotium with fruiting bodies (stroma). The amounts of cordycepin, carotenoids and superoxide dismutase were found to be higher in the fruiting bodies on pupae than that in the fruiting bodies on wheat, whereas the amounts of adenosine, polysaccharides and mannitol were higher in the fruiting bodies on wheat than in the fruiting bodies on pupae. The adenosine, polysaccharide and mannitol contents in the sclerotium (the pupae portion) with fruiting bodies were significantly lower than those of the fruiting bodies on wheat.

Light and heat stress also influences cordycepin biosynthesis in C. militaris. It was suggested that during the late maturation stage of ascomata, heat and light stresses led to a significant increase in cordycepin biosynthesis without affecting biological efficiency and heat stress significantly promotes carotenoid production [75]. Moreover, it was observed that the optimal growth temperature for C. militaris is 20°C on agar medium, while growth at 25°C is compromised.

Mycelium growth of C. militaris also proved suitable for starch-processing-industry waste under solid-state and submerged culture conditions. High level production of adenosine was reported by Lim, et al. [76], using millet as solid substrate under dark for the first 7 days and harvested on day 40, which decreased till day 50. High cordycepin level was produced by using soybean solid substrate and keeping in the dark for the first 14 days and harvesting on 50^th day. In these conditions, Cordycepin increased from day 40 to day 50. Authors also reported a high level of D-mannitol using millet as solid substrate and keeping the culture in dark for first 7 days and harvesting on 50^th day. Tao, et al. [77], studied cultivation of six strains of C. militaris on different substrates e.g. rice, wheat and tussah (Antheraea pernyi) pupae and found that different strains grew better on different substrates e.g. strain CM3 showed best efficiency on rice and wheat (62.26% and 54.48% respectively), strain CM9 on tussah pupae (biological efficiency of 291.70%). Further they observed highest adenosine (2.62 mg g^-1) content in fruiting bodies of strain CM9 cultivated on tussah pupae and highest cordycepin (5.68 mg g^-1) in strain CM4 cultivated on wheat. A high amount of Cordycepin may be itself harmful for the C. militaris, therefore a feedback mechanism to produce pentostatin, an adenosine deaminase inhibitor exists in C. militaris [78]. Hypoxia adaptation by introduction of Vitreoscilla haemoglobin in media, improved the growth, biomass and crude polysaccharides’ content in C. militaris [79]. The efficient cordycepin production was explored using six edible insects as substrates. Highest yield of cordycepin was produced by the cultivation on Allomyrina dichotoma which was 34 times that on Bombyx mori pupae. Fat content of insects was found to be important for cordycepin production. A positive correlation was observed between oleic acid content and cordycepin production. The transcriptional levels of cns1 and cns2, genes involved in cordycepin. biosynthesis, were higher in cordyceps grown on A. dichotoma than on other five tested insects [80].

It is important that cultured fungus should replace the natural one to decrease the burden on natural resources. Wild, O. sinensis takes 1-2 years to complete its life cycle, while cultured, C. militaris takes only 4-6 weeks for fruiting in artificial culture. Artificial cultivation of O. sinensis was difficult but it has been achieved recently [81-84]. In vitro-cultured mycelia of O. sinensis showed only 5% genetic variability from natural counterparts and indicated almost similar D-mannitol content, supporting replacement of the natural fungus with the cultured one [85]. Tang, et al. [86], achieved the successful cultivation of O. sinensis fruiting body with high mycelial protein content of 2.11% by media optimization. Some companies (e.g. Aloha Medicinals, USA) have filed patents for O. sinensis cultivation methods. However, there are conflicting reports-whether these have optimized the process. Kaushik, et al. [87], reported higher amount of cordycepin with growth supplements: nucleosides hypoxanthine and adenosine; amino acids-glycine and glutamine; plant hormones-1-Naphthaleneacetic Acid (NAA) and 3-Indoleacetic Acid (IAA); vitamin-thiamine (B1) in O. sinensis.

Biosynthesis pathway of C. militaris contains four genes cns1, cns2, cns3 and cns4 (Figure 4). Genes cns1, cns2 and cns3 for cordycepin synthesis are present on adjacent loci. Functional verification of the cns1 and cns2, genes essential for cordycepin production was confirmed by generating Aspergillus nidulans knockout mutants and the heterologous gene expression in Saccharomyces cerevisiae and Metarhizium robertsii. Similarly, functional verification of the cns3 for pentostatin production was achieved through heterologous expression of cns3 in M. robertsii and C. bassiana. Cns1 and Cns2 protein interaction was also proved by yeast two-hybrid and co-localisation-studies. Agrobacterium tumefaciens mediated transformation protocol for O. sinensis was standardized by co-cultivation of fungal recipient strains [88]. Pathways in C. cicadae, C. kyushensis and alternative pathway in C. militaris have also been deciphered recently [89-92].

Figure 4: Cordycepin biosynthesis pathway.

Phenotypic degeneration during artificial cultivation

Phenotypic degeneration is a significant problem for mushroom growers. Usually fungi lose their virulence and their morphology is altered when they are repeatedly sub-cultured on artificial media. Morphological changes include colour and growth form modifications and reduced sporulation [93]. Various terms have been used to describe this phenomenon including phenotypic phenotypic degeneration, instability or deterioration, dual phenomenon, saltation and attenuation [94,95]. Particularly in C. militaris, degenerated cultures demonstrate longer growth cycle, slower mycelial growth, a lighter colour of hyphae (probably due to decrease in pigment content), reduced (or no) ability to produce fruit bodies, abnormal fruit body formation, decrease in the number of primordia, decrease in conidia production, decrease in secondary metabolites content, lower dehydrogenase activity and reduced cellulase and amylase activities, as well as lower extracellular and higher intracellular polysaccharides content [96-101]. It becomes more evident after fourth or fifth generations. Several factors that are either intrinsic/genetic or cultivation-related are involved in C. militaris degeneration. Developing suitable methodologies and interventions to avoid (or minimize) culture degeneration, improving ascomata and mycelia production and/or on enhancing cordycepin output by adopting various approaches and the use of both solid and liquid substrates are important research problems currently pursued in this field [102-111]. Beside this, use of breakthrough technologies such as genetic engineering can considerably improve C. militaris biomass content and various metabolites, including cordycepin [112-114]. A recent study standardized the Clustered Regularly Interspaced Short Palindromic Repeats-Cas9 (CRISPR-Cas9) gene editing technique for C. militaris [115].

He, et al. [116], determined that the mineral elements K+, Ca²⁺ and Zn²⁺ (at concentrations of 1.0 g L⁻¹, 0.02 g^L−1 and 250-375 μg^L−1 respectively) delayed the degeneration of different types of C. militaris and whereas Mn²⁺ and Mg²⁺ (at trace concentrations) promoted the degeneration. Degeneration of C. militaris was also affected by oxidative stress. Furthermore, homokaryosis promoted degeneration of C. militaris. The degenerated strains could be rejuvenated by cross-mating of their single ascospore isolates.

Molecular mechanisms of phenotypic degeneration

Molecular mechanisms that affect C. militaris culture degeneration are not much understood. Continuous culture induced Mating-Type (MAT) loci allele segregation is proposed to be one mechanism responsible for culture degeneration [117-120]. DNA methylation and gene mutation are also considered to be responsible for strain degeneration. A transcriptome-wide study during C. militaris sub culturing found that the genes involved in stress response such as the production of streptothricin acetyltransferase, glutathione S-transferase, gamma-glutamyl transpeptidase, alcohol dehydrogenase, the 30 kDa heat shock protein, Major Facilitator Superfamily (MFS) multidrug transporter and detoxification were up-regulated during strain degeneration. Similarly, expression of genes involved the metabolism of proteins, amino acids, carbohydrates, lipids, nucleic acids and nucleotides were significantly up-regulated e.g. mitochondrial co-chaperone GrpE, metalloprotease 1, trypsin-like serine protease, Suppressor of G2 allele of SKP1 (SGT1) and Cysteine and Histidine-Rich Domains (CHORD)-containing Proteins (CS), acetate transporter, formyl tetrahydrofolate deformylase, nucleoside triphosphate hydrolases and uracil phosphoribosyl transferase. On the basis of these findings, authors proposed that the strain degeneration mechanism in C. militaris was associated with genes involved in Deoxyribonucleic Acid (DNA) methylation, toxin biosynthesis, energy metabolism and chromosome remodeling.

Discussion

Transcriptomics, metabolics and lipidomics

Recent Omics studies have significantly improved our understanding of various aspects of Cordyceps fungi [121-125]. Transcriptomics of wild type and cultivated O. sinensis showed that the fatty acid metabolism was more active in wild O. sinensis compared to cultivated O. sinensis, evidenced by downregulation of genes encoding various enzymes involved in fatty acids metabolism e.g. acyl Coenzyme A (acyl- CoA) dehydrogenase, enoyl-CoA hydratase, 3-ketoacyl-CoA thiolase and acetyl-CoA acetyltransferase in cultivated O. sinensis. Liquid Chromatography Mass Spectroscopy (LC-MS)/MS-based metabolomics analysis in O. sinensis from Naqu (NCs) and Yushu (YCs), infected-stiff worm and their substituents i.e. artificially Cultivated Cordyceps species (CCs) and mycelia, identified 901 metabolites. The contents of adenosine and cordycepin were significantly higher in cultivated O. sinensis than in the natural types whereas the levels of mannitol and polysaccharides were lower. However, the contents in the stiff worms were not significantly different from those in natural ones. Mannitol was present maximally in natural types followed by cultivated types and stiff worm. An integrated metabolomics and transcriptomics analysis of O. sinensis suggested the involvement of Inosine 5'-Monophosphate Dehydrogenase (IMPDH), Adenyl Succinate Synthetase (ADSS), Adenosine Kinase (AK), Guanylate Kinase (GUK) and guanosine Monophosphate Synthetase (guaA) genes in the synthesis of purine nucleotides and nucleosides.

Transcript analysis of C. militaris cultivation on germinated soyabean seeds showed that the gene expression in the early stages of fungus development is important for cordycepin biosynthesis and isoflavone modification metabolic pathways. It further revealed that transcription profile changed after two weeks of incubation. Based on a transcriptomics study in C. militaris, Zhang, et al. [126], proposed that sclerotium showed increased oxidative stress and energy metabolism and mitogen-activated protein kinase signaling might induce the formation of fruiting body. Furthermore, n_os_milR16, n_os_milR21, n_os_milR34 and n_os_milR90 milRNAs could regulate the induction of fruiting body in it. In a lipidomics study, 435 lipids were detected from O. sinensis (Naqu, NCs and Yushu, YCs) and its substituents i.e. artificially CCs and mycelia from bailing capsules (BLs; fermented Cordyceps). There were less differences among bioactive lipids such as Sphingolipids (SLs), Glycolipids (GPs) and Fatty Acids (e.g., unsaturated Free Fatty Acids (FFA) and eicosanoid) between CCs and wild cordyceps while significant differences existed between BLs and wild Cordyceps. Majority of differentially expressed GPs and SLs were higher in BLs whereas most of differential Free Fatty Acids (FFAs) and eicosanoids were lower in BLs [127]. Li, et al. [128], carried out a transcriptomics study at six developmental stages i.e. Hyphae (HY), Sclerotium (ST), Primordium (PRm), Young Fruiting body (YF), Developed Fruiting body (DF) and Mature Fruiting body (MF) in O. sinensis, PR and MF stages grouped together, suggesting that primordium differentiation and sexual maturation had similar gene expression patterns. However, the ST and HY stages were far apart developmentally evidenced by more Diethylene Glycols (DEGs) between them, covering 47.5% of the genome. A recent study showed the cultivated O. sinensis contained more of amino acids and derivatives, carbohydrates and derivatives and phenolic acids than wild O. sinensis, but lesser contents of most nucleosides and nucleotides. There are other studies also that shed a light on the pathways and active ingredient contents in correlation with different treatments [129-131].

Conclusions

Cordyceps fungus has many species which have proven and potential therapeutic activities. O. sinensis is the most sought after of these, found only in the Himalayas. It is dug by the native people to sell in the high demand market. This process has created a threat to the survival of this valuable species. Therefore, alternative species C. militaris were discovered that could be easily cultivated on the artificial substrates. The active principles in natural and cultivated Cordyceps, responsible for various therapeutic potentials have been compared and being further increased with the help of elicitation. Media manipulations were performed to achieve high bioactive compounds such as cordycepin, mannitol, adenosine, polysaccharides, ergosterol and Superoxide Dismutase (SOD) in C. militaris. Recently cultivation of O. sinensis has also been achieved on artificial media but with good scope for improvement. The action mechanisms of bioactive compounds are intensively being investigated and newer mechanisms are being unfolded. Many companies have intensified their efforts for its commercial exploitation as drug candidate for several diseases for which comprehensive data need to be generated before getting regulatory approvals. Higher accumulation of cordycepin and other active ingredients and cheaper cultivation methods of Cordyceps spp., are the needs of the hour for its commercial exploitation and availability to the larger population.

Competing Interests

Author declares that there is no conflict of interest.

Author’s Contribution

Everything about the manuscript was planned and performed by VKD.

Acknowledgement/Funding

The funding for the work was provided by DEI minor project grant to VKD.

Data Availability

No data was generated to share.

References

1. MYCOBANK Database. MycoBank. 2023.

2. Nikoh N, Fukatsu T. Interkingdom host jumping underground: phylogenetic analysis of entomoparasitic fungi of the genus Cordyceps. Mol Biol Evol. 2000;17(4):629-638.

   [Crossref] [Google Scholar] [PubMed]

3. Khao-Ngam S, Mongkolsamrit S, Rungjindamai N, Noisripoom W, Pooissarakul W, Duangthisan J, et al. Ophiocordyceps asiana and Ophiocordyceps tessaratomidarum (Ophiocordycipitaceae, Hypocreales), two new species on stink bugs from Thailand. Mycol Prog. 2021;20:341-353.

   [Crossref] [Google Scholar]

4. Mongkolsamrit S, Noisripoom W, Pumiputikul S, Boonlarppradab C, Samson RA, Stadler M, et al. Ophiocordyceps flavida sp. nov. (Ophiocordycipitaceae), a new species from Thailand associated with Pseudogibellula formicarum (Cordycipitaceae), and their bioactive secondary metabolites. Mycol Prog. 2021;20:477-492.

   [Crossref] [Google Scholar]

5. Chawngthu Z, MC Vabeikhokhei JO, Zomuanpuii R, de Mandal S, Zothanzama J. A new species of Ophiocordyceps (Ophiocordycipitaceae) from Mizoram, India. Phytotaxa. 2021;500(1):11-20.

   [Crossref] [Google Scholar]

6. Long FY, Qin LW, Xiao YP, Hyde KD, Wang SX, Wen TC. Multigene phylogeny and morphology reveal a new species, Ophiocordyceps vespulae, from Jilin Province, China. Phytotaxa. 2021;478(1):33-48.

   [Crossref] [Google Scholar]

7. Zou W, Tang D, Xu Z, Huang O, Wang Y, Tran NL, et al.  Multigene phylogeny and morphology reveal Ophiocordycepshydrangea sp. nov. and Ophiocordycepsbidoupensis sp. nov. (Ophiocordycipitaceae). MycoKeys. 2022;92:109.

   [Crossref] [Google Scholar]

8. Mongkolsamrit S, Noisripoom W, Hasin S, Sinchu P, Jangsantear P, Luangsa-ard JJ. Multi-gene phylogeny and morphology of Ophiocordyceps laotii sp. nov. and a new record of O. buquetii (Ophiocordycipitaceae, Hypocreales) on ants from Thailand. Mycological Progress. 2023;22(1):5.

   [Crossref] [Google Scholar]

9. Xu ZH, Tran NL, Wang Y, Zhang GD, Dao VM, Nguyen TT, et al. Phylogeny and morphology of Ophiocordyceps puluongensis sp. nov.(Ophiocordycipitaceae, Hypocreales), a new fungal pathogen on termites from Vietnam. J Invertebr Pathol. 2022;192:107771.

   [Crossref] [Google Scholar] [PubMed]

10. Shrestha B, Sung GH, Sung JM. Current nomenclatural changes in Cordyceps sensu lato and its multidisciplinary impacts. Mycology. 2017;8(4):293-302.

    [Crossref] [Google Scholar] [PubMed]

11. Sasaki F, Miyamoto T, Yamamoto A, Tamai Y, Yajima T. Relationship between intraspecific variations and host insects of Ophiocordyceps nutans collected in Japan. Mycoscience. 2012;53(2):85-91.

    [Crossref] [Google Scholar]

12. Paterson RR. Cordyceps-a traditional Chinese medicine and another fungal therapeutic biofactory? Phytochemistry. 2008;69(7):1469-1495.

    [Crossref] [Google Scholar] [PubMed]

13. Hong IP, Nam SH, Sung GB, Chung IM, Hur H, Lee MW, et al. Chemical components of Paecilomyces tenuipes (Peck) samson. Mycobiology. 2007;35(4):215-218.

    [Crossref] [Google Scholar] [PubMed]

14. Nam KS, Jo YS, Kim YH, Hyun JW, Kim HW. Cytotoxic activities of acetoxyscirpenediol and ergosterol peroxide from Paecilomyces tenuipes. Life sciences. 2001;69(2):229-237.

    [Crossref] [Google Scholar] [PubMed]

15. Yan JK, Wang WQ, Wu JY. Recent advances in Cordyceps sinensis polysaccharides: Mycelial fermentation, isolation, structure, and bioactivities: A review. Journal of Functional Foods. 2014;6:33-47.

    [Crossref] [Google Scholar] [PubMed]

16. Cordyceps. Wikipedia, the free encyclopedia. 2023.

17. Wang L, Yan H, Zeng B, Hu Z. Research progress on cordycepin synthesis and methods for enhancement of cordycepin production in Cordyceps militaris. Bioengineering. 2022;9(2):69.

    [Crossref] [Google Scholar] [PubMed]

18. Olatunji OJ, Tang J, Tola A, Auberon F, Oluwaniyi O, Ouyang Z. The genus Cordyceps: An extensive review of its traditional uses, phytochemistry and pharmacology. Fitoterapia. 2018;129:293-316.

    [Crossref] [Google Scholar] [PubMed]

19. Anyu AT, Zhang WH, Xu QH. Cultivated cordyceps: a tale of two treasured mushrooms. 2021;4(4):221-227.

    [Crossref] [Google Scholar]

20. Cohen N, Cohen J, Asatiani MD, Varshney VK, Yu HT, Yang YC, et al. Chemical composition and nutritional and medicinal value of fruit bodies and submerged cultured mycelia of culinary-medicinal higherBasidiomycetes mushrooms. Int J Med Mushrooms. 2014;16(3):273-291.

    [Crossref] [Google Scholar] [PubMed]

21. Wang L, Zhang WM, Hu BA, Chen YQ, Qu LH. Genetic variation of Cordyceps militaris and its allies based on phylogenetic analysis of rDNA ITS sequence data. Fungal Divers. 2008;31:147-155.

    [Google Scholar]

22. Cui JD. Biotechnological production and applications of Cordyceps militaris, a valued traditional Chinese medicine. Crit Rev Biotechnol. 2015;35(4):475-484.

    [Crossref] [Google Scholar] [PubMed]

23. Reis FS, Barros L, Calhelha RC, Ćirić A, van Griensven LJ, Soković M, et al. The methanolic extract of Cordyceps militaris (L.) Link fruiting body shows antioxidant, antibacterial, antifungal and antihuman tumor cell lines properties. Food Chem Toxicol. 2013;62:91-98.

    [Crossref] [Google Scholar] [PubMed]

24. Frederiksen S, Malling H, Klenow H. Isolation of 3′-deoxyadenosine (cordycepin) from the liquid medium of Cordyceps militaris (L. ex Fr.) Link. Biochim Et Biophys Acta -Nucleic Acids Protein Synth. 1965;95(2):189-193.

    [Crossref] [Google Scholar] [PubMed ]

25. Tuli HS, Sandhu SS, Sharma AK. Pharmacological and therapeutic potential of Cordyceps with special reference to Cordycepin. 3 Biotech. 2014;4(1):1-2.

    [Crossref] [Google Scholar] [PubMed]

26. Zhang G, Yin Q, Han T, Zhao Y, Su J, Li M, et al. Purification and antioxidant effect of novel fungal polysaccharides from the stroma of Cordyceps kyushuensis. Ind. Crops Prod. 2015;69:485-491.

    [Crossref] [Google Scholar]

27. Kim HG, Shrestha B, Lim SY, Yoon DH, Chang WC, Shin DJ, et al. Cordycepin inhibits lipopolysaccharide-induced inflammation by the suppression of NF-κB through Akt and p38 inhibition in RAW 264.7 macrophage cells. Eur J Pharmacol. 2006;545(2-3):192-199.

    [Crossref] [Google Scholar] [PubMed]

28. Zhou X, Cai G, He YI, Tong G. Separation of cordycepin from Cordyceps militaris fermentation supernatant using preparative HPLC and evaluation of its antibacterial activity as an NAD+ -dependent DNA ligase inhibitor. Exp Ther Med. 2016;12(3):1812-1816.

    [Crossref] [Google Scholar] [PubMed]

29. Wu WC, Hsiao JR, Lian YY, Lin CY, Huang BM. Theapoptotic effect of cordycepin on human OEC-M1 oral cancer cell line. Cancer Chemother. Pharmacol. 2007;60:103-11.

    [Crossref] [Google Scholar] [PubMed]

30. van Nguyen T, Chumnanpuen P, Parunyakul K, Srisuksai K, Fungfuang W. A study of the aphrodisiac properties of Cordyceps militaris in streptozotocin-induced diabetic male rats. Veterinary World. 2021;14(2):537.

    [Crossref] [Google Scholar] [PubMed]

31. Sun J, Xu J, Wang S, Hou Z, Lu X, An L, et al. A new cerebroside from Cordyceps militaris with anti-PTP1B activity. Fitoterapia. 2019;138:104342.

    [Crossref] [Google Scholar] [PubMed]

32. Das G, Shin HS, Leyva-Gómez G, Prado-Audelo ML, Cortes H, Singh YD, et al. Cordyceps spp.: A review on its immune-stimulatory and other biological potentials. Front Pharmacol. 2021;11:602364.

    [Crossref] [Google Scholar] [PubMed]

33. Noh EM, Kim JS, Hur H, Park BH, Song EK, Han MK, et al. Cordycepin inhibits IL-1β-induced MMP-1 and MMP-3 expression in rheumatoid arthritis synovial fibroblasts. Rheumatology. 2009;48(1):45-48.

    [Crossref] [Google Scholar] [PubMed]

34. Taofiq O, González-Paramás AM, Martins A, Barreiro MF, Ferreira IC. Mushrooms extracts and compounds in cosmetics, cosmeceuticals and nutricosmetics—A review. Ind Crops Prod. 2016;90:38-48.

    [Crossref] [Google Scholar]

35. Zhang J, Wen C, Duan Y, Zhang H, Ma H. Advance in Cordyceps militaris (Linn) Link polysaccharides: Isolation, structure, and bioactivities: A review. Int J Biol Macromol. 2019;132:906-914.

    [Crossref] [Google Scholar] [PubMed]

36. Zhong X, Gu L, Xiong WT, Wang HZ, Lian DH, Zheng YM, et al. 1H NMR spectroscopy-based metabolic profiling of Ophiocordyceps sinensis and Cordyceps militaris in water-boiled and 50% ethanol-soaked extracts. J Pharm Biomed Anal. 2020;180:113038.

    [Crossref] [Google Scholar] [PubMed]

37. Lu Y, Zhi Y, Miyakawa T, Tanokura M. Metabolic profiling of natural and cultured Cordyceps by NMR spectroscopy. Scientific reports. 2019;9(1):7735.

    [Crossref] [Google Scholar] [PubMed]

38. Dong JZ, Wang SH, Ai XR, Yao L, Sun ZW, Lei C, et al. Composition and characterization of cordyxanthins from Cordyceps militaris fruit bodies. J Funct Foods. 2013;5(3):1450-1455.

    [Crossref] [Google Scholar]

39. Ahn YJ, Park SJ, Lee SG, Shin SC, Choi DH. Cordycepin: Selective Growth Inhibitor Derived from Liquid Culture of Cordyceps militaris against Clostridium spp. J Agric Food Chem. 2000;48(7):2744-2748.

    [Crossref] [Google Scholar] [PubMed]

40. Rottman, F, Guarino, AJ. The inhibition of purinebiosynthesisde novo in Bacillus subtilis by cordycepin. Biochim. Et Biophys Acta. 1964;80:640-647.

    [Crossref] [Google Scholar] [PubMed]

41. Sugar AM, McCaffrey RP. Antifungal activity of 3′-deoxyadenosine (cordycepin). Agents Chemother. 1998;42(6):1424-1427.

    [Crossref] [Google Scholar] [PubMed]

42. Nair CN, Panicali DL. Polyadenylate sequences of human rhinovirus and poliovirus RNA and cordycepin sensitivity of virus replication. J Virol. 1976;20(1):170-176.

    [Crossref] [Google Scholar] [PubMed]

43. Leinwand L, Ruddle FH. Stimulation of in vitro translation of messenger RNA by actinomycin D and cordycepin. Science. 1977;197(4301):381-383.

    [Crossref] [Google Scholar] [PubMed]

44. Luo G, Hamatake RK, Mathis DM, Racela J, Rigat KL, Lemm J, et al. De novo initiation of RNA synthesis by the RNA-dependent RNA polymerase (NS5B) of hepatitis C virus. Journal of virology. 2000;74(2):851-863.

    [Crossref] [Google Scholar] [PubMed]

45. Chen J, Li HF, Zhao G, Lin JM, He X. Matrix-assisted laser desorption ionization mass spectrometry based quantitative analysis of cordycepin from Cordyceps militaris. J Pharm Anal. 2021;11(4):499-504.

    [Crossref] [Google Scholar] [PubMed]

46. Tong X, Guo J. High throughput identification of the potential antioxidant peptides inOphiocordyceps sinensis. Molecules. 2022;27(2):438.

    [Crossref] [Google Scholar] [PubMed]

47. Kim JR, Yeon SH, Kim HS, Ahn YJ. Larvicidal activity against Plutella xylostella of cordycepin from the fruiting body of Cordyceps militaris. Pest Manag Sci. 2002;58(7):713-717.

    [Crossref] [Google Scholar] [PubMed]

48. Vodnala SK, Ferella M, Lundén-Miguel H, Betha E, van Reet N, Amin DN, et al. Preclinical assessment of the treatment of second-stage African trypanosomiasis with cordycepin and deoxycoformycin. PLoS Negl Trop Dis. 2009;3(8):e495.

    [Crossref] [Google Scholar] [PubMed]

49. Dalla Rosa L, da Silva AS, Gressler LT, Oliveira CB, Dambros MG, Miletti LC, et al. Cordycepin (3′-deoxyadenosine) pentostatin (deoxycoformycin) combination treatment of mice experimentally infected with Trypanosoma evansi. Parasitology. 2013;140(5):663-671.

    [Crossref] [Google Scholar] [PubMed]

50. Shelton J, Lu X, Hollenbaugh JA, Cho JH, Amblard F, Schinazi RF. Metabolism, biochemical actions, and chemical synthesis of anticancer nucleosides, nucleotides, and base analogs. Chem Rev. 2016;116(23):14379-1455.

    [Crossref] [Google Scholar] [PubMed]

51. Holbein S, Wengi A, Decourty L, Freimoser FM, Jacquier A, Dichtl B. Cordycepin interferes with 3′ end formation in yeast independently of its potential to terminate RNA chain elongation. RNA. 2009;15(5):837-849.

    [Crossref] [Google Scholar] [PubMed]

52. Holbein S, Freimoser FM, Werner TP, Wengi A, Dichtl B. Cordycepin-hypersensitive growth links elevated polyphosphate levels to inhibition of poly (A) polymerase in Saccharomyces cerevisiae. Nucleic Acids Res. 2008;36(2):353-363.

    [Crossref] [Google Scholar] [PubMed]

53. Wang Z, Wu X, Liang YN, Wang L, Song ZX, Liu JL, et al. Cordycepin induces apoptosis and inhibits proliferation of human lung cancer cell line H1975 via inhibiting the phosphorylation of EGFR. Molecules. 2016;21(10):1267.

    [Crossref] [Google Scholar] [PubMed]

54. Hueng DY, Hsieh CH, Cheng YC, Tsai WC, Chen Y. Cordycepin inhibits migration of human glioblastoma cells by affecting lysosomal degradation and protein phosphatase activation. J Nutr Biochem. 2017;41:109-116.

    [Crossref] [Google Scholar] [PubMed]

55. Levenson R, Kernen J, Housman D. Synchronization of MEL cell commitment with cordycepin. Cell. 1979;18(4):1073-1078.

    [Crossref] [Google Scholar] [PubMed]

56. Sakaguchi S, Fukuda T, Takano H, Ono K, Takio S. Photosynthetic electron transport differentially regulates the expression of superoxide dismutase genes in liverwort, Marchantia paleacea var. diptera. Plant Cell Physiol. 2004;45(3):318-324.

    [Crossref] [Google Scholar] [PubMed]

57. Cheutin T, O'Donohue MF, Beorchia A, Vandelaer M, Kaplan H, Deféver B, et al. Three-dimensional organization of active rRNA genes within the nucleolus. J Cell Sci. 2002;115(16):3297-3307.

    [Crossref] [Google Scholar] [PubMed]

58. Schwenzer H, de Zan E, Elshani M, van Stiphout R, Kudsy M, Morris J, et al. The novel nucleoside analogue ProTide NUC-7738 overcomes cancer resistance mechanisms in vitro and in a first-in-human phase I clinical trial. Clin Cancer Res. 2021;27(23):6500-6513.

    [Crossref] [Google Scholar] [PubMed]

59. Bai KC, Sheu F. A novel protein from edible fungi Cordyceps militaris that induces apoptosis. J Food Drug Anal. 2018;26(1):21-30.

    [Crossref] [Google Scholar] [PubMed]

60. Lee J, Cho K, Shin SG, Bae H, Koo T, Han G, et al. Nutrient recovery of starch processing waste to Cordyceps militaris: Solid state cultivation and submerged liquid cultivation. Appl Biochem Biotechnol. 2016;180:274-288.

    [Crossref] [Google Scholar] [PubMed]

61. Tang J, Qian Z, Wu H. Enhancing cordycepin production in liquid static cultivation of Cordyceps militaris by adding vegetable oils as the secondary carbon source. Bioresour Technol. 2018;268:60-67.

    [Crossref] [Google Scholar] [PubMed]

62. Lin Q, Long L, Wu L, Zhang F, Wu S, Zhang W, et al. Evaluation of different agricultural wastes for the production of fruiting bodies and bioactive compounds by medicinal mushroom Cordyceps militaris. J Sci Food Agric. 2017;97(10):3476-3480.

    [Crossref] [Google Scholar] [PubMed]

63. Kong BH, Yap CS, Razif MF, Ng ST, Tan CS, Fung SY. Antioxidant and cytotoxic effects and identification of Ophiocordyceps sinensis bioactive proteins using shotgun proteomic analysis. Food Technol Biotechnol. 2021;59(2):201-208.

    [Crossref] [Google Scholar] [PubMed]

64. Rao YK, Fang SH, Wu WS, Tzeng YM. Constituents isolated from Cordyceps militaris suppress enhanced inflammatory mediator's production and human cancer cell proliferation. J Ethnopharmacol. 2010;131(2):363-367.

    [Crossref] [Google Scholar] [PubMed]

65. Chiu CP, Liu SC, Tang CH, Chan Y, El-Shazly M, Lee CL, et al. Anti-inflammatory cerebrosides from cultivated Cordyceps militaris. J Agric Food Chem. 2016;64(7):1540-1548.

    [Crossref] [Google Scholar] [PubMed]

66. Guo M, Guo S, Huaijun Y, Bu N, Dong C. Comparison of major bioactive compounds of the caterpillar medicinal mushroom, Cordyceps militaris (Ascomycetes), fruiting bodies cultured on wheat substrate and pupae. Int J Med Mushrooms. 2016;18(4):327-336.

    [Crossref] [Google Scholar] [PubMed]

67. Shi K, Yang G, He L, Yang B, Li Q, Yi S. Purification, characterization, antioxidant, and antitumor activity of polysaccharides isolated from silkworm cordyceps. J Food Biochem. 2020;44(11):e13482.

    [Crossref] [Google Scholar] [PubMed]

68. Zeng Y, Han Z, Qiu P, Zhou Z, Tang Y, Zhao Y, et al. Salinity-induced anti-angiogenesis activities and structural changes of the polysaccharides from cultured Cordyceps Militaris. PLoS One. 2014;9(9):e103880.

    [Crossref] [Google Scholar] [PubMed]

69. Wang CC, Wu JY, Chang CY, Yu ST, Liu YC. Enhanced exopolysaccharide production by Cordyceps militaris using repeated batch cultivation. J Biosci Bioeng. 2019;127(4):499-505.

    [Crossref] [Google Scholar] [PubMed]

70. Zhu L, Zhang H, Liu Y, Zhang J, Gao X, Tang Q. Effect of culture time on the bioactive components in the fruit bodies of Caterpillar mushroom, Cordyceps militaris CM-H0810 (Ascomycetes). Int J Med Mushrooms. 2019;21(11):1107-1114.

    [Crossref] [Google Scholar] [PubMed]

71. Liang ZC, Liang CH, Wu CY. Various grain substrates for the production of fruiting bodies and bioactive compounds of the medicinal caterpillar mushroom, Cordyceps militaris (Ascomycetes). Int J Med Mushrooms. 2014;16(6):569-578.

    [Crossref] [Google Scholar] [PubMed]

72. Jiaojiao Z, Fen W, Kuanbo L, Qing L, Ying Y, Caihong D. Heat and light stresses affect metabolite production in the fruit body of the medicinal mushroom Cordyceps militaris. Appl Microbiol Biotechnol. 2018;102:4523-4533.

    [Crossref] [Google Scholar] [PubMed]

73. Lim L, Lee C, Chang E. Optimization of solid state culture conditions for the production of adenosine, cordycepin, and D-mannitol in fruiting bodies of medicinal caterpillar fungus Cordyceps militaris (L.: Fr.) Link (Ascomycetes). Int J Med Mushrooms. 2012;14(2);181-187.

    [Crossref] [Google Scholar] [PubMed]

74. Tao SX, Xue D, Lu ZH, Huang HL. Effects of substrates on the production of fruiting bodies and the bioactive components by different Cordyceps militaris strains (ascomycetes). Int J Med Mushrooms. 2020;22(1):55-63.

    [Crossref] [Google Scholar] [PubMed]

75. Xia Y, Luo F, Shang Y, Chen P, Lu Y, Wang C. Fungal cordycepin biosynthesis is coupled with the production of the safeguard molecule pentostatin. Cell Chem Biol. 2017;24(12):1479-1489.

    [Crossref] [Google Scholar] [PubMed]

76. Wang Y, Yang Z, Bao D, Li B, Yin X, Wu Y, et al. Improving hypoxia adaption causes distinct effects on growth and bioactive compounds synthesis in an entomopathogenic fungus Cordyceps militaris. Front Microbiol. 2021;12:698436.

    [Crossref] [Google Scholar] [PubMed]

77. Turk A, Abdelhamid MA, Yeon SW, Ryu SH, Lee S, Ko SM, et al. Cordyceps mushroom with increased cordycepin content by the cultivation on edible insects. Front Microbiol. 2022;13:1017576.

    [Crossref] [Google Scholar] [PubMed]

78. Yue K, Ye M, Lin X, Zhou Z. The artificial cultivation of medicinal caterpillar fungus, Ophiocordyceps sinensis (Ascomycetes): A review. Int J Med Mushrooms. 2013;15(5):425-434.

    [Crossref] [Google Scholar] [PubMed]

79. Liu G, Han R, Cao L. Artificial cultivation of the Chinese cordyceps from injected ghost moth larvae. Environ Entomol. 2019;48(5):1088-1094.

    [Crossref] [Google Scholar] [PubMed]

80. Li X, Liu Q, Li W, Li Q, Qian Z, Liu X, et al. A breakthrough in the artificial cultivation of Chinese cordyceps on a large-scale and its impact on science, the economy, and industry. Crit Rev Biotechnol. 2019;39(2):181-191.

    [Crossref] [Google Scholar] [PubMed]

81. Li X, Liu Q, Li W, Li Q, Qian Z, Liu X, et al. A breakthrough in the artificial cultivation of Chinese cordyceps on a large-scale and its impact on science, the economy, and industry. Crit Rev Biotechnol. 2019;39(2):181-191.

    [Crossref] [Google Scholar] [PubMed]

82. Singh R, Negi PS, Ahmed Z. Genetic variability assessment in medicinal caterpillar fungi Cordyceps spp.(Ascomycetes) in central Himalayas, India. Int J Med Mushrooms. 2009;11(2).

    [Crossref] [Google Scholar]

83. Tang CY, Wang J, Liu X, Chen JB, Liang J, Wang T, et al. Medium optimization for high mycelial soluble protein content of Ophiocordyceps sinensis using response surface methodology. Front Microbiol. 2022;13:1055055.

    [Crossref] [Google Scholar] [PubMed]

84. Kaushik V, Singh A, Arya A, Sindhu SC, Sindhu A, Singh A. Enhanced production of cordycepin in Ophiocordyceps sinensis using growth supplements under submerged conditions. Biotechnol Rep. 2020;28:e00557.

    [Crossref] [Google Scholar] [PubMed]

85. Xiong C, Xia Y, Zheng P, Wang C. Increasing oxidative stress tolerance and subculturing stability of Cordyceps militaris by overexpression of a glutathione peroxidase gene. Appl Microbiol Biotechno. 2013;97:2009-2015.

    [Crossref] [Google Scholar] [PubMed]

86. Chen A, Wang Y, Shao Y, Huang B. A novel technique for rejuvenation of degenerated caterpillar medicinal mushroom, Cordyceps militaris (Ascomycetes), a valued traditional Chinese medicine. Int J Med Mushrooms. 2017;19(1):87-91.

    [Crossref] [Google Scholar] [PubMed]

87. Sun SJ, Deng CH, Zhang LY, Hu KH. Molecular analysis and biochemical characteristics of degenerated strains of Cordyceps militaris. Arch Microbiol. 2017;199:939-944.

    [Crossref] [Google Scholar] [PubMed]

88. Yin J, Xin X, Weng Y, Gui Z. Transcriptome-wide analysis reveals the progress of Cordyceps militaris subculture degeneration. PLoS One. 2017;12(10):e0186279.

    [Crossref] [Google Scholar] [PubMed]

89. Wang H, Wei J, Lin N, Feng A, Chen M, Bao D. Distribution of mating-type genes in fruiting and non-fruiting forms of Cordyceps militaris. Acta Edulis Fung. 2010;17(4):1-4.

    [Google Scholar]

90. Xin X, Yin J, Zhang B, Li Z, Zhao S, Gui Z. Genome-wide analysis of DNA methylation in subcultured Cordyceps militaris. Arch Microbiol. 2019;201:369-375.

    [Crossref] [Google Scholar] [PubMed]

91. Meina L, Chunyan L, Bosen F, Qingwei L. Molecular analysis of degeneration of artificial flanted Cordyceps militaris. Mycosystema. 2003;22(2):277-282.

    [Google Scholar]

92. Raethong N, Wang H, Nielsen J, Vongsangnak W. Optimizing cultivation of Cordyceps militaris for fast growth and cordycepin overproduction using rational design of synthetic media. Comput Struct Biotechnol J. 2020;18:1-8.

    [Crossref] [Google Scholar] [PubMed]

93. Montesinos‐Matías R, Ordaz‐Hernández A, Angel‐Cuapio A, Colin‐Bonifacio Y, Garcia‐Garcia RE, Ángel‐Sahagún CA, et al. Principal component analysis of the biological characteristics of entomopathogenic fungi in nutrient‐limited and cuticle‐based media. J Basic Microbiol. 2021;61(2):147-156.

    [Crossref] [Google Scholar] [PubMed]

94. Wang LY, Liang X, Zhao J, Wang Y, Li SP. Dynamic Analysis of nucleosides and carbohydrates during developmental stages of Cordyceps militaris in silkworm (bombyxmori). J AOAC Int. 2019;102(3):741-747.

    [Crossref] [Google Scholar] [PubMed]

95. Li X, Wang J, Zhang H, Xiao L, Lei Z, Kaul SC, et al. Low dose of fluoride in the culture medium of Cordyceps militaris promotes its growth and enhances bioactives with antioxidant and anticancer properties. J Fungi. 2021;7(5):342.

    [Crossref] [Google Scholar] [PubMed]

96. Liu GQ, Qiu XH, Cao L, Han RC. Scratching stimuli of mycelia influence fruiting body production and ROS-scavenging gene expression of Cordyceps militaris. Mycobiology. 2018;46(4):382-387.

    [Crossref] [Google Scholar] [PubMed]

97. Lu Y, Xia Y, Luo F, Dong C, Wang C. Functional convergence and divergence of mating-type genes fulfilling in Cordyceps militaris. Fungal Genet Biol. 2016;88:35-43.

    [Crossref] [Google Scholar] [PubMed]

98. Chen BX, Wei T, Guo LQ, Lin JF. Transcriptome analysis reveals the flexibility of cordycepin network in Cordyceps militaris activated by L-alanine addition. Front Microbiol. 2020;11:524087.

    [Crossref] [Google Scholar] [PubMed]

99. Lou H, Ye Z, Yun F, Lin J, Guo L, Chen B, et al. Targeted gene deletion in Cordyceps militaris using the split-marker approach. Mol Biotechnol. 2018;60:380-385.

    [Crossref] [Google Scholar] [PubMed]

100. Lou HW, Ye ZW, Yu YH, Lin JF, Guo LQ, Chen BX, et al. The efficient genetic transformation of Cordyceps militaris by using mononuclear protoplasts. Sci Hortic. 2019;243:307-313.

     [Crossref] [Google Scholar]

101. Lou HW, Zhao Y, Chen BX, Yu YH, Tang HB, Ye ZW, et al. Cmfhp gene mediates fruiting body development and carotenoid production in Cordyceps militaris. Biomolecules. 2020;10(3):410.

     [Crossref] [Google Scholar] [PubMed]

102. Chen BX, Wei T. Efficient CRISPR-Cas9 gene disruption system in edible-medicinal mushroom Cordyceps militaris. Front microbiol. 2018;9:334783.

     [Crossref] [Google Scholar] [PubMed]

103. He L, Han C, Li P, Chen Y, Liu D, Geng L. Effect of mineral elements on colony types of Cordyceps militaris in subculturing. J Shenyang Agri Uni. 2009;40(6):672-677.

104. Grognet P, Bidard F, Kuchly C, Tong LC, Coppin E, Benkhali JA, et al. Maintaining two mating types: structure of the mating type locus and its role in heterokaryosis in Podospora anserina. Genetics. 2014;197(1):421-432.

     [Crossref] [Google Scholar] [PubMed]

105. Yin J, Xin XD, Weng YJ, Li SH, Jia JQ, Gui ZZ. Genotypic analysis of degenerative Cordyceps militaris cultured in the pupa of Bombyx mori. Entomol Res. 2018;48(3):137-144.

     [Crossref] [Google Scholar]

106. Yokoyama E, Yamagishi K, Hara A. Development of a PCR-based mating-type assay for Clavicipitaceae. FEMS Microbiol Lett. 2004;237(2):205-212.

     [Google Scholar] [PubMed]

107. Pradhan P, de J, Acharya K. Strategies in Artificial Cultivation of Two Entomopathogenic Fungi Cordyceps militaris and Ophiocordyceps sinensis. InApplied Mycol Agri Foods. 2024:315-345.

     [Google Scholar]

108. Zhang H, Yue P, Tong X, Gao T, Peng T, Guo J. Comparative analysis of fatty acid metabolism based on transcriptome sequencing of wild and cultivated Ophiocordyceps sinensis. PeerJ. 2021;9:e11681.

     [Crossref] [Google Scholar] [PubMed]

109. Guo S, Lin M, Xie D, Zhang W, Zhang M, Zhou L, et al. Comparative metabolic profiling of wild Cordyceps species and their substituents by liquid chromatography-tandem mass spectrometry. Front Pharmacol. 2022;13:1036589.

     [Crossref] [Google Scholar] [PubMed]

110. Zhou Y, Wang M, Zhang H, Huang Z, Ma J. Comparative study of the composition of cultivated, naturally grown Cordyceps sinensis, and stiff worms across different sampling years. PLoS One. 2019;14(12):e0225750.

     [Crossref] [Google Scholar] [PubMed]

111. Zhang J, Wang N, Chen W, Zhang W, Zhang H, Yu H, et al. Integrated metabolomics and transcriptomics reveal metabolites difference between wild and cultivated Ophiocordyceps sinensis. Food Res Int. 2023;163:112275.

     [Crossref] [Google Scholar] [PubMed]

112. Yoo CH, Sadat MA, Kim W, Park TS, Park DK, Choi J. Comprehensive transcriptomic analysis of Cordyceps militaris cultivated on germinated soybeans. Mycobiology. 2022;50(1):1-11.

     [Crossref] [Google Scholar] [PubMed]

113. Zhang H, Yue P, Tong X, Bai J, Yang J, Guo J. mRNA-seq and miRNA-seq profiling analyses reveal molecular mechanisms regulating induction of fruiting body in Ophiocordyceps sinensis. Sci Rep. 2021;11(1):12944.

     [Crossref] [Google Scholar] [PubMed]

114. Lin M, Guo S, Xie D, Li S, Hu H. Lipidomic profiling of wild Cordyceps and its substituents by liquid chromatography-electrospray ionization-tandem mass spectrometry. LWT. 2022;163:113497.

     [Crossref] [Google Scholar]

115. Li X, Wang F, Liu Q, Li Q, Qian Z, Zhang X, et al. Developmental transcriptomics of Chinese Cordyceps reveals gene regulatory network and expression profiles of sexual development-related genes. BMC genomics. 2019;20:1-8.

     [Crossref] [Google Scholar] [PubMed]

116. Wang YL, Wang ZX, Liu C, Wang SB, Huang B. Genome-wide analysis of DNA methylation in the sexual stage of the insect pathogenic fungus Cordyceps militaris. Fungal Biol. 2015;119(12):1246-1254.

     [Crossref] [Google Scholar] [PubMed]

117. Zheng ZL, Qiu XH, Han RC. Identification of the genes involved in the fruiting body production and cordycepin formation of Cordyceps militaris fungus. Mycobiology. 2015;43(1):37-42.

     [Crossref] [Google Scholar] [PubMed]

118. Wang L, Zhang WM, Hu BA, Chen YQ, Qu LH. Genetic variation of Cordyceps militaris and its allies based on phylogenetic analysis of rDNA ITS sequence data. Fungal Divers. 2008;31:147-155.

     [Google Scholar]

119. Wang J, Kan L, Nie S, Chen H, Cui SW, Phillips AO, et al. A comparison of chemical composition, bioactive components and antioxidant activity of natural and cultured Cordyceps sinensis. LWT. 2015;63(1):2-7.

     [Crossref] [Google Scholar]

120. Li W, Zhao S, Chen H, Yuan H, Wang T, Huang X. High-yielding cordycepin in Cordyceps militaris modified by low-energy ion beam. Sheng Wu Gong Cheng Xue Bao. 2009;25(11):1725-1731.

     [Google Scholar] [PubMed]

121. Qi W, Zhou X, Wang J, Zhang K, Zhou Y, Chen S, et al. Cordyceps sinensis polysaccharide inhibits colon cancer cells growth by inducing apoptosis and autophagy flux blockage via mTOR signaling. Carbohydr Polym. 2020;237:116113.

     [Crossref] [Google Scholar] [PubMed]

122. Liu G, Cao L, Rao Z, Qiu X, Han R. Identification of the genes involved in growth characters of medicinal fungus Ophiocordyceps sinensis based on Agrobacterium tumefaciens–mediated transformation. Appl Microbiol Biotechnol. 2020;104:2663-2674.

     [Crossref] [Google Scholar] [PubMed]

123. Liu T, Liu Z, Yao X, Huang Y, Qu Q, Shi X, et al. Identification of cordycepin biosynthesis-related genes through de novo transcriptome assembly and analysis in Cordyceps cicadae. R Soc Open Sci. 2018;5(12):181247.

     [Crossref] [Google Scholar] [PubMed]

124. Zhao X, Zhang G, Li C, Ling J. Cordycepin and pentostatin biosynthesis gene identified through transcriptome and proteomics analysis of Cordyceps kyushuensis Kob. Microbiol Res. 2019;218:12-21.

     [Crossref] [Google Scholar] [PubMed]

125. Wongsa B, Raethong N, Chumnanpuen P, Wong-Ekkabut J, Laoteng K, Vongsangnak W. Alternative metabolic routes in channeling xylose to cordycepin production of Cordyceps militaris identified by comparative transcriptome analysis. Genomics. 2020;112(1):629-636.

     [Crossref] [Google Scholar] [PubMed]

126. Zhang H, Yang J, Luo S, Liu L, Yang G, Gao B, et al. A novel complementary pathway of cordycepin biosynthesis in Cordyceps militaris. Int Microbiol. 2023:1-3.

     [Crossref] [Google Scholar] [PubMed]

127. Kawakami K. On the changes of characteristics of the silkworm muscardines through successive cultures. Bulletin Sericulture Experimental Station Japan. 1960;16:83-99.

     [Google Scholar]

128. Butt TM, Wang C, Shah FA, Hall R. Degeneration of entomogenous fungi. InAn ecological and societal approach to biological control. 2006:213-226.

     [Crossref] [Google Scholar]

129. Lou H, Lin J, Guo L, Wang X, Tian S, Liu C, et al. Advances in research on Cordyceps militaris degeneration. Appl Microbiol Biotechnol. 2019;103:7835-7841.

     [Crossref] [Google Scholar] [PubMed]

130. Lin Q, Qiu X, Zheng Z, Xie C, Xu Z, Han R. Characteristics of the degenerate strains of Cordyceps militaris. Mycosystema. 2010;29(5):670-677.

     [Google Scholar]

131. Ibrahim L, Jenkinson P. Effect of artificial culture media on germination, growth, virulence and surface properties of the entomopathogenic hyphomycete Metarhizium anisopliae. Mycol Res. 2002;106(6):705-715.

     [Crossref] [Google Scholar]