Sang-Kuk Han

Cultural Characteristics and Fruiting Body Production in Cordyceps bassiana

Abstract Single ascospore isolates of Cordyceps bassiana were observed for their colony pigmentation on Sabouraud Dextrose agar plus Yeast Extract (SDAY) plates and were inoculated in a brown rice medium for production of fruiting bodies. Colony pigmentation did not show any relationship with perithecial stromata formation. The isolates were also grown on opposite sides of SDAY agar plates and were observed for vegetative compatibility. Neither vegetative compatibility nor perithecial stromata could be found to be related to each other. It was concluded that fertile fruiting body production was independent of colony pigmentation and vegetative compatibility. Synnemata formation was found to be more common than perithecial stromata formation. This might be due to its highly conidiogenous anamorphic stage, i.e., Beauveria bassiana.

Optimum Conditions for Artificial Fruiting Body Formation of Cordyceps cardinalis

Abstract Stromatal fruiting bodies of Cordyceps cardinalis were successfully produced in cereals. Brown rice, German millet and standard millet produced the longest-length of stromata, followed by Chinese pearl barley, Indian millet, black rice and standard barley. Oatmeal produced the shortest-length of fruiting bodies. Supplementation of pupa and larva to the grains resulted in a slightly enhanced production of fruiting bodies; pupa showing better production than larva. 50~60 g of brown rice and 10~20 g of pupa mixed with 50~60 mL of water in 1,000 mL polypropylene (PP) bottle was found to be optimum for fruiting body production. Liquid inoculation of 15~20 mL per PP bottle produced best fruiting bodies. The optimal temperature for the formation of fruiting bodies was 25°C, under conditions of continuous light. Few fruiting bodies were produced under the condition of complete darkness, and the fresh weight was considerable low, compared to that of light condition.

Fruiting Body Formation of Cordyceps militaris from Multi-Ascospore Isolates and Their Single Ascospore Progeny Strains.

Interest in commercial cultivation and product development of Cordyceps species has shown a recent increase. Due to its biochemical and pharmacological effects, Cordyceps militaris, commonly known as orange caterpillar fungus, is being investigated with great interest. Cultivation of C. militaris has been practiced on a large scale in order to fulfill a demand for scientific investigation and product development. Isolates of C. militaris can be easily established from both spores and tissue. For isolation of spores, ascospores released from mature stromata are trapped in sterile medium. Multi-ascospore isolates, as well as combinations of single ascospore strains, are used for production of fruiting bodies. Progeny ascospore strains can be isolated from artificial fruiting bodies, thus, the cycle of fruiting body production can be continued for a long period of time. In this study, we examined fruiting body production from multi-ascospore isolates and their progeny strains for three generations. F1 progeny strains generally produced a larger number of fruiting bodies, compared with their mother multi-ascospore isolates; however, F2 and F3 progeny strains produced fewer fruiting bodies. Optimum preservation conditions could help to increase the vitality of the progeny strains. In order to retain the fruiting ability of the strains, further testing of various methods of preservation and different methods for isolation should be performed.

Successful Development of Cordyceps bassiana Stromata from Beauveria bassiana

Abstract A specimen of Beauveria bassiana was collected from Yang-yang of Gangwon province, Korea in October 2006. Conidial isolates were prepared from the specimen by the dilution method and inoculated in brown rice medium for fruiting body production. After nearly two months incubation for perithecial stromata developed from single isolates as well as from their combinations. They were determined as Cordyceps bassiana by observing the stromatal characters and their conidial structures. This is the first report of the development of C. bassiana from B. bassiana cultures.

Distribution and in vitro Fruiting of Cordyceps militaris in Korea

Cordyceps militaris specimens were continuously collected by Entomopathogenic Fungal Culture Collection (FFCC), Kangwon National University from different mountains, national parks and recreation parks of Korea from 1986 to 2002, mainly from late May to October of each year. Dry specimens of C. militaris along with their isolates have been preserved in EFCC. Fruiting of C. militaris was induced from single aseospore isolates as well as their combinations in brown rice medium. Fruiting experiments showed that combinations of single aseospore isolates produced fertile fruiting bodies, but single isolates could not produce any fruiting bodies. It was shown that two isolates of the opposite mating types were required to produce fertile stromata. However, combinations of the same mating type isolates produced no fruiting body, showing that C. militaris is a bipolar, heterothallie fungus.

Effect of Preservation Periods and Subcultures on Fruiting Body Formation of Cordyceps militaris In Vitro

Effects of various preservation periods and subcultures on fruiting body formation of Cordyceps militaris were investigated using EFCC C-10995 single ascospore strains. Fruiting body formation by original strains was profuse when preserved at 4°C for 5~6 months. Fruiting from subcultures was stable till second to sixth subcultures, after which it decreased sharply. The more the colony color of subcultures changed, the less the fruiting bodies formed. Liquid inoculum preparation of single ascospore strains in the same or separate broths did not affect fruiting body formation. Similarly, two strains C-10995–3 and C-10995–6 in different numbers during liquid inoculum preparation produced similar fruiting bodies.

Mushroom Flora of Ulleung-gun and a Newly Recorded Bovista Species in the Republic of Korea

Abstract We conducted five times surveys, in June, September and October in 2012; June and September 2013, to catalog the mushroom flora in Ulleung-gun, Republic of Korea. More than 400 specimens were collected, and 317 of the specimens were successfully sequenced using the ribosomal DNA internal transcribed spacer barcode marker. We also surveyed the morphological characteristics of the sequenced specimens. The specimens were classified into 2 phyla, 7 classes, 21 orders, 59 families, 122 genera, and 221 species, and were deposited in the herbarium of Korea National Arboretum. Among the collected species, 72% were saprophytic, 25% were symbiotic, and 3% were parasitic. The most common order was Agaricales (189 specimens, 132 species), followed by Polyporales (47 specimens, 27 species), Russulales (31 specimens, 22 species), Boletales (10 specimens, 7 species), and so on. Herein, we also reported the first Bovista species in Korea, which was collected from Dokdo, the far-eastern island of Korea.

Taxonomic Study of Amanita Subgenus Lepidella and Three Unrecorded Amanita Species in Korea

Abstract Amanita Pers. is a well-known monophyletic mushroom genus with a broad distribution. However, the diversity of Korean Amanita species has been underestimated, and most taxonomic studies conducted in Korea have only investigated their morphological characteristics. This approach is frequently insufficient for correct identification in fungal classification; therefore, we constructed a phylogeny of Amanita subgen. Lepidella in order to understand the phylogenetic placements of 16 Amanita specimens collected in Korea in 2012. The phylogeny constructed using the sequence data of the internal transcribed spacers and the partial large subunit of ribosomal RNA identified nine Amanita species (A. citrina, A. excelsa var. spissa, A. flavipes, A. fritillaria, A. oberwinklerana, A. pallidorosea, A. rubescens, A. subjunquillea, and A. volvata); of these, A. fritillaria, A. oberwinklerana, and A. pallidorosea are new to Korea.

Growth and Cultural Characteristics of Cordyceps cardinalis Collected from Korea

Cordyceps cardinalis was reported in Japan and the USA in 2004, and its fruiting bodies have recently been cultured in Korea. Herbarium specimens preserved at the Cordyceps Research Institute, Mushtech, Korea were revised and identified as C. cardinalis, based on morphological characters and conidial structures. Most of the C. cardinalis specimens were collected from Mt. Halla in Jeju-do. The effects of various nutritional sources and environmental conditions such as temperature and pH on mycelial growth of C. cardinalis were studied. Oatmeal agar, Martins peptone dextrose agar, and Schizophyllum (mushroom) genetics complete medium plus yeast extract resulted in the best mycelial growth. Among carbon sources, cereals, and nitrogen sources, maltose, oatmeal, and peptone resulted in the best mycelial growth respectively. Mineral salts helped to increase growth rate but only resulted in thin mycelial density, similar to water agar. A temperature of 25°C and a pH of 7 resulted in the highest mycelial growth. Based on these results, a Cordyceps cardinalis composite medium (CCM) was formulated with 1% maltose, 2% oatmeal, 1% peptone, and 2% agar. Use of the CCM resulted in slightly better mycelial growth than that of other commonly used agar media. Only organic nitrogen sources imparted a reddish pigmentation to the agar media, but this character diminished after several subcultures. A 7 day culture duration resulted in the best mycelial growth.

Morphological Characteristics of Conidiogenesis in Cordyceps militaris

Conidial development of Cordyceps militaris was observed from germinating aseospores and vegetative hyphae through light and scanning electron microscopy (SEM). Aseospores were discharged from fresh specimens of C. militaris in sterile water as well as Sabouraud Dextrose agar plus Yeast Extract (SDAY) plates. We observed aseospore germination and conidial formation periodically. Under submerged condition in sterile water, most part-spores germinated unidireetionally and eonidia were developed directly from the tips of germinating hyphae of part-spores within 36 h after aseospore discharge, showing mieroeyelie eonidiation. First-formed eonidia were cylindrical or elavate followed by globose and ellipsoidal ones. Germination of aseospores and conidial development were observed on SDAY agar by SEM. Slimy heads of eonidia on variously arranged phialides, from solitary to whorl, developed 5 days after aseospore discharge. Besides, two distinct types of eonidia, elongated pyriform or cylindrical and globose, were observed in the same slimy heads by SEM. Conidia were shown to be uninucleate with 4,6-diamidino-2-phenylindole staining. Conidiogenous cells were more slender than vegetative hyphae, having attenuated tips. Mieroeyelie eonidiation, undifferentiated conidiogenous hyphae (phialides), polymorphic eonidia and solitary, opposite to whorled type of phialidie arrangement are reported here as the characteristic features of asexual stage of C. militaris, which can be distinguished from other Cordyceps species.