Characterization and Biological Activities of Melanin from the Medicinal Fungi Ophiocordyceps sinensis.

Melanin is a complex natural pigment that is widely present in fungi. The mushroom Ophiocordyceps sinensis has a variety of pharmacological effects. The active substances of O. sinensis have been extensively studied, but few studies have focused on the O. sinensis melanin. In this study, the production of melanin was increased by adding light or oxidative stress, namely, reactive oxygen species (ROS) or reactive nitrogen species (RNS), during liquid fermentation. Subsequently, the structure of the purified melanin was characterized using elemental analysis, ultraviolet-visible absorption spectrum, Fourier transform infrared (FTIR), electron paramagnetic resonance (EPR), and pyrolysis gas chromatography and mass spectrometry (Py-GCMS). Studies have shown that O. sinensis melanin is composed of C (50.59), H (6.18), O (33.90), N (8.19), and S (1.20), with maximum absorbance at 237 nm and typical melanin structures such as benzene, indole, and pyrrole. Additionally, the various biological activities of O. sinensis melanin have been discovered; it can chelate heavy metals and shows a strong ultraviolet-blocking ability. Moreover, O. sinensis melanin can reduce the levels of intracellular reactive oxygen species and counteract the oxidative damage of H2O2 to cells. These results can help us to develop applications of O. sinensis melanin in radiation resistance, heavy metal pollution remediation, and antioxidant use.

Stable reference gene selection for Ophiocordyceps sinensis gene expression studies under different developmental stages and light-induced conditions.

The molecular mechanism of Chinese cordyceps formation has received a substantial amount of attention because of its usage as traditional Chinese medicine. The formation process of Chinese cordyceps includes two parts: asexual proliferation (Ophiocordyceps sinensis proliferates in the hemolymph of Thitarodes armoricanus larvae) and sexual development (formation and development of fruiting bodies). Therefore, validation of reference genes under different development stages and experimental conditions is crucial for RT-qPCR analysis. However, there is no report on stable reference genes at the development stage of O. sinensis fruiting body. In this study, 10 candidate reference genes, Actin, Cox5, Tef1, Ubi, 18s, Gpd, Rpb1, Try, Tub1 and Tub2, were selected and calculated their expression stability using four methods: geNorm, NormFinder, BestKeeper, and Comparative △Ct. After comprehensive analysis of the results of these four methods with RefFinder, we determined that the most stable reference genes during asexual reproduction of O. sinensis were Tef1 and Tub1, while the most stable reference genes during fruiting body development were Tyr and Cox5, and the most stable reference genes under light-induced conditions were Tyr and Tef1. Our study provides a guidance for reference genes selections at different proliferation processes with light stress of O. sinensis, and represents a foundation for studying the molecular mechanism of Chinese cordyceps formation.

Detection of the pathogenic fungus Cordyceps farinosa in the Thitarodes armoricanus soil-rearing environment based on nucleic acid targets.

Cordyceps farinosa, an entomopathogenic fungus, infects and leads to high mortality of Thitarodes armoricanus larvae, which die soon after the infection of C. farinose, usually before the colonization of Ophiocordyceps sinensis owing to competitive inhibition and fruiting body formation. Therefore, monitoring C. farinosa in the O. sinensis cultivation environment is critical for minimizing the C. farinosa infection-induced losses. In this study, we initially designed a PCR primer pair (Tar-1F/Tar-1R) through open reading frame prediction and homology comparison of the C. farinosa genome sequence. This primer pair can detect both C. farinosa and Samsoniella hepiali. To further distinguish, primers (ITS5-172/ITS4-95) were then designed to selectively amplify the large ribosomal subunit sequences in the C. farinosa genome. All these primers were applied in combination for detection of C. farinosa in soil samples. The sensitivity reached a detection limit of 1 × 106 spores/g soil. In addition, these primers can detect the presence of C. farinosa in dead T. armoricanus larval samples. This newly established rapid detection method provides important information for C. farinosa control during O. sinensis cultivation.

Study of Pathogenesis Using Fluorescent Strain of Cordyceps farinosa Revealed Infection of Thitarodes armoricanus Larvae via Digestive Tract.

Cordyceps farinosa is often utilized as a biocontrol agent because of its wide host range, strong lethality, and safety for mammals. Artificial rearing of Thitarodes armoricanus larvae is a prerequisite for the artificial cultivation of Chinese cordyceps, and C. farinosa is the most lethal pathogenic fungus during the rearing process. However, the infection process of C. farinosa is still unclear. In this study, we cloned the promoter of the C. farinosa glyceraldehyde 3-phosphate dehydrogenase gene, constructed the EGFP expression cassette, and integrated it into the C. farinosa genome via Agrobacterium transformation. We obtained a fluorescent strain for better observation of the infection process. Using two different inoculation methods of the fluorescent strain, we observed the traditional infection process through the body surface as well as through the digestive tract via feeding. Both infection modes can lead to larval death and mummification. Our findings demonstrated that during the artificial rearing of T. armoricanus, preventing C. farinosa pollution should be an important part of the disinfection of the rearing environment.