Caihong Dong

In vitro evaluation of antioxidant activities of aqueous extracts from natural and cultured mycelia of Cordyceps sinensis

Abstract Cordyceps sinensis, one of the best known traditional Chinese medicines and health foods, has been highly valued for the treatment of a wide range of diseases and reported to have antioxidant properties. In the present study, the antioxidant activities of hot-water extracts from natural and cultured mycelia of C. sinensis were investigated and evaluated using six in vitro assays, including inhibition of linoleic acid peroxidation; scavenging abilities on DPPH, hydroxyl and superoxide anion radicals; the reducing power and the chelating ability on ferrous ions. Among these assays, the extracts showed the best effect on the inhibition of linoleic peroxidation with the lowest IC50 values and with an inhibition rate over 90% at concentration of 0.8–1.6mg/ml, more stable than that of α-tocopherol, a recognised natural antioxidant. The scavenging activities on superoxide anion and hydroxyl radicals of the two extracts were slightly lower than that of butylated hydroxytoluene. DPPH scavenging activities of both extracts reached over 80% inhibition at 4–8mg/ml. Both extracts showed moderate reducing power and ferrous ion chelating activity. The IC50 value of the extract from cultured mycelia in all the tests, except for linoleic acid peroxidation, was significantly lower than that of natural mycelia. There was no evident correlation between the antioxidant activity and the content of protein, polysaccharides and mannitol of extracts from C. sinensis; the antioxidant activity may be due to a combined effect of these or some other compounds. These results suggested that both the extracts from cultured and natural mycelia have direct and potent antioxidant activities and that the cultured mycelia of the fungus could be used for the antioxidant activity to reduce the human demands on the natural resources of the fungus, an endangered species.

Nutritional requirements of mycelial growth of Cordyceps sinensis in submerged culture

Aims:  The nutritional requirements for mycelial growth of Cordyceps sinensis in semi‐synthetic liquid media were investigated. The results provide a basis for further physiological study and industrial fermentation of the fungus.

A survey of the geographic distribution of Ophiocordyceps sinensis

Ophiocordyceps sinensis is one of the best known fungi in Traditional Chinese Medicine. Many efforts have been devoted to locating the production areas of this species resulting in various reports; however, its geographic distribution remains incompletely understood. Distribution of O. sinensis at the county level is clarified in this work based on both a literature search and fieldwork. More than 3600 publications related to O. sinensis were investigated, including scientific papers, books, and online information. Herbarium specimens of O. sinensis and field collections made by this research group during the years 2000–2010 were examined to verify the distribution sites. A total of 203 localities for O. sinensis have been found, of which 106 are considered as confirmed distribution sites, 65 as possible distribution sites, 29 as excluded distribution sites and three as suspicious distribution sites. The results show that O. sinensis is confined to the Tibetan Plateau and its surrounding regions, including Tibet, Gansu, Qinghai, Sichuan, and Yunnan provinces in China and in certain areas of the southern flank of the Himalayas, in the countries of Bhutan, India and Nepal, with 3,000 m as the lowest altitude for the distribution. The fungus is distributed from the southernmost site in Yulong Naxi Autonomous County in northwestern Yunnan Province to the northernmost site in the Qilian Mountains in Qilian County, Qinghai Province, and from the east edge of the Tibetan Plateau in Wudu County, Gansu Province to the westernmost site in Uttarakhand, India. The clarification of the geographic distribution of O. sinensis will lay the foundation for conservation and sustainable use of the species.

Isolation, characterization of melanin derived from Ophiocordyceps sinensis, an entomogenous fungus endemic to the Tibetan Plateau.

Melanins are pigments of high molecular weight formed by oxidative polymerization of phenolic or indolic compounds. In this present study, a black pigment was isolated from the fermentation broth of Ophiocordyceps sinensis, an entomogenous fungus which is endemic to the Tibetan Plateau by alkaline extraction, acid hydrolysis, and repeated precipitation. It was designed as melanin since the physical and chemical properties including its ultraviolet (UV) and infrared (IR) spectra of the black pigment conformed to the characteristic of melanin and similar to the commercial synthetic melanin. The antioxidant activity of melanin derived from O. sinensis was evaluated. They showed much stronger scavenging abilities on 1,1-diphenyl-2-picrylhydrazyl (DPPH•) and the chelating ability on ferrous ions than that of the water extract from the mycelia of O. sinensis, with IC(50) value 18.51 ± 0.85 μg/ml and 2.58 ± 0.26 μg/ml, separately. This is the first report of melanin from O. sinensis and will be helpful for the study on the physiology and the artificial cultivation of this fungus, an endangered species.

The blue-light receptor CmWC-1 mediates fruit body development and secondary metabolism in Cordyceps militaris

Light is an essential factor for pigment formation and fruit body development in Cordyceps militaris, a well-known edible and medicinal fungus. Cmwc-1, a homolog of the blue-light receptor gene white collar-1 (wc-1) in Neurospora crassa, was cloned from the C. militaris genome in our previous study. Here, Cmwc-1 gene inactivation results in thicker aerial hyphae, disordered fruit body development, a significant reduction in conidial formation, and carotenoid and cordycepin production. These characteristics were restored when the ΔCmwc-1 strains were hybridized with wild-type strains of the opposite mating type. A genome-wide expression analysis revealed that there were 1042 light-responsive genes in the wild-type strain and only 458 in the ΔCmwc-1 strain. Among five putative photoreceptors identified, Vivid, cryptochrome-1, and cyclobutane pyrimidine dimer photolyase are strongly induced by light in a Cmwc-1-dependent manner, while phytochrome and cryptochrome-2 were not induced. The transcription factors involved in the fungal light reaction were mainly of the Zn2Cys6 type. CmWC-1 regulates adenylosuccinate synthase, an important enzyme for adenosine de novo synthesis, which could explain the reduction in cordycepin production. Some G protein-coupled receptors that control fungal fruit body formation and the sexual cycle were regulated by CmWC-1, and the cAMP pathway involved in light signal transduction in N. crassa was not critical for the photoreaction in the fungus here. A transcriptional analysis indicated that steroid biosynthesis was more active in the ΔCmwc-1 strain, suggesting that CmWC-1 might switch the vegetative growth state to primordia differentiation by suppressing the expression of related genes.

Photo morphogenesis and photo response of the blue-light receptor gene Cmwc-1 in different strains of Cordyceps militaris.

Light is a necessary environmental factor for stroma formation and development of Cordyceps militaris, a well-known edible and medicinal fungus. In this study, photo morphogenesis and the blue-light receptor gene were studied using five representative strains of C. militaris. The results suggest that light was essential for colony pigmentation and could promote conidia production. Cmwc-1, the homologe of the blue-light photoreceptor of Neurospora crassa, was cloned from the genome of C. militaris by Hi-tail PCR. The protein CmWC-1 was characterized by the presence of the LOV and PAS domains and a GATA-type Znf domain. Genetic variation analysis of Cmwc-1 in different strains showed that 15-bp deletions occurred in three strains that resulted in 5-Gln deletions in the transcription activation domain. Phylogenetic analysis based on the Sordariomycetes WC-1-like proteins suggested that the sequence of WC-1 could be used as a candidate marker for phylogenetic analysis in fungi. Cmwc-1 mRNA was light inducible and the expression level increased significantly after irradiation in all tested strains. The sequence of CmWC-1 and the relative expressions responding to irradiation in degenerate and albino strains were similar as the cultivated one. This report will help to open the still-unexplored field of stroma development for this fungus.

Cordyceps industry in China

Cordyceps, as a general term, describes a group of ascomycetous fungi growing on arthropods and other related fungi. Some cordyceps have been used in traditional Chinese medicine for centuries and cordyceps-derived products are currently a big industry in China. A number of medicinal and health products have been developed and extensively commercialized from natural Chinese cordyceps, its anamorphic fungus (Hirsutella sinensis), and other fungi known as Chinese cordyceps. The lack of a defined classification system for medicinal cordyceps fungi is a source of confusion in the industry and the public, and even among pharmaceutical scientists. This review summarizes the cordyceps fungi currently used in the industry in China with a special reference to clarify Chinese cordyceps and associated fungi. Cordyceps militaris, Cordyceps guangdongensis and Isaria cicadae are well recognized and commercialized cordyceps fungi in China. Except the natural Chinese cordyceps and its anamorphic fungus, Paecilomyces hepiali, Mortierella hepiali, Cephalosporium sinensis and Clonostachys rosea isolated from natural Chinese cordyceps are classified as Chinese cordyceps–associated fungi. P. hepiali is a cordyceps fungus based on current phylogenetic analysis of Hypocreales, while M. hepiali is a fungus in the Zygomycetes and should only be treated as associated fungus of Chinese cordyceps. C. sinensis and C. rosea belong to the Hypocreales and their relationship to cordyceps fungi should be further studied. The exploitation of the resources of cordyceps fungi and their quality control in the industry should be major topics for future studies. Cooperation between the industry and the research community will enhance the whole cordyceps industry.

Reliable reference gene selection for Cordyceps militaris gene expression studies under different developmental stages and media

Cordyceps militaris is considered a model organism for the study of Cordyceps species, which are highly prized in traditional Chinese medicine. Gene expression analysis has become more popular and important in studies of this fungus. Reference gene validation under different experimental conditions is crucial for RT-qPCR analysis. In this study, eight candidate reference genes, actin, cox5, gpd, rpb1, tef1, try, tub, and ubi, were selected and their expression stability was evaluated in C. militaris samples using four algorithms, genorm, normfinder, bestkeeper, and the comparative ∆Ct method. Three sets of samples, five different developmental stages cultured in wheat medium and pupae, and all the samples pool were included. The results showed that rpb1 was the best reference gene during all developmental stages examined, while the most common reference genes, actin and tub, were not suitable internal controls. Cox5 also performed poorly and was less stable in our analysis. The ranks of ubi and gpd were inconsistent in different sample sets by different methods. Our results provide guidelines for reference gene selection at different developmental stages and also represent a foundation for more accurate and widespread use of RT-qPCR in C. militaris gene expression analysis.

Artificial cultivation of true morels: current state, issues and perspectives

Abstract Morels (Morchella, Ascomycota), which are some of the most highly prized edible and medicinal mushrooms, are of great economic and scientific value. Morel cultivation has been a research focus worldwide for more than 100 years, and the outdoor cultivation of morels has succeeded and expanded to a large scale in China in recent years. In this study, we review the progress in recent research regarding the life cycle and reproductive systems in the genus Morchella and the current state of outdoor cultivation. Sclerotia formation and conidia production are two important phases during the life cycle. The morel species cultivated commercially in America is M. rufobrunnea based on molecular phylogenetic analysis. The species currently cultivated in China are black morels, including M. importuna, M. sextalata and M. eximia. The field cultivation of morels expanded in the majority of the provinces in China with a yield of fresh morels of 0–7620 kg per ha. The key techniques include spawn production, land preparation and spawning, the addition of exogenous nutrition, fruiting management and harvesting. The application of exogenous nutrition is the most important breakthrough in the field of morel cultivation, but the mechanism remains unclear. It was estimated that the total amount of field cultivated fresh morels was ∼500 t in 2015–2016. We also discuss the potential issues remaining in the current literature and suggest directions for future studies.

Functional convergence and divergence of mating-type genes fulfilling in Cordyceps militaris.

Fungal sexual lives are considerably diversified in terms of the types of mating systems and mating-control gene functions. Sexual fruiting bodies of the ascomycete fungus Cordyceps militaris have been widely consumed as edible and medicinal mushrooms, whereas the regulation of fruiting-body development and sex in this fungus remain elusive. Herein, we performed the comprehensive functional analyses of mating-type (MAT) genes in C. militaris. Interspecies functional convergence was evident that MAT1-1 and MAT1-2-1 null mutants were sterile and lost the ability to produce stromata in outcrosses with the opposite mating-type partner. In contrast to other fungal species, functional divergence of MAT1-1-1 and MAT1-1-2 was also observed that ΔMAT1-1-1 produced barren stromata in outcrosses, whereas ΔMAT1-1-2 generated fruiting bodies morphologically similar to that of the parental strain but with sterile perithecia. The homothallic-like transformants MAT1-2::MAT1-1-1 (haploidic MAT1-2 isolate transformed with the MAT1-1-1 gene) produced sterile stromata, whereas the MAT1-1::MAT1-2-1 (haploidic MAT1-1 isolate transformed with the MAT1-2-1 gene) mutant was determined to be completely fruitless. The findings relating to the fully fertile gene-complementation mutants suggest that the genomic location is not essential for the MAT genes to fulfill their functions in C. militaris. Comparison of the production of bioactive constituents cordycepin and adenosine provides experimental support that the fungal sexual cycle is an energy consuming process. The results of the present study enrich our knowledge of both convergent and divergent controls of fungal sex.