서론
MetaCordyceps chlamydosporia is an entomophagous fungus belonging to the Clavicipitaceae family of the phylum Ascomycota and the order Hypocreales. MetaCordyceps chlamydosporia was previously named Cordyceps chlamydosporia[1]. The genus MetaCordyceps, which was erected from the genus Cordyceps along with other genera, OphioCordyceps and ElaphoCordyceps, contains more than 400 species attacking a range of insects and fungal hosts [2,3]. MetaCordyceps includes species with solitary or several simple and branched stromata, fleshy or tough stipe, partially or completely immersed perithecia in stromata, and cylindrical ascospores [3].
MetaCordyceps species are particularly abundant and diverse in humid temperate and tropical forests of Asia.However, a wide range of entomopathogenic fungi are still hidden in the island and mountain regions of Korea.Ulleung-do, one of Korea’s many islands, is located 150 kmeast of the Korean Peninsula. The island is volcanic in origin and has high variation in geographical and ecological indices, including temperature, altitude, biodiversity,soil physicochemical properties, etc. [4]. These factors contribute to a rich fungal diversity. Thus, examination of the fungal diversity of this solitary area so far away from the main land of Korea is important. The aim of this study was to isolate soil fungi from Ulleung-do, Korea, and confirm the first record of fungal species in Korea.The current study compares a previously unknown fungal species, M. chlamydosporia KNU14-22, with previously described MetaCordyceps species with respect to morphological and phylogenetic characteristics.
Soil sample collection and isolation of fungi
Soil samples were collected in 2014 from the forest of Nari Basin, Ulleungdo Island, Korea (37o 31'06.67'' N, 120o 51'54.27'' E). The samples were taken from a depth of 0~15cm, air dried, and stored in plastic bags at 4oC until used.The fungus was isolated using a conventional dilution method[5] and grown on potato dextrose agar (PDA) for 5~7 days at 25oC. The isolate was preserved at 20oC on PDA slants for further studies.
Morphological characterization
The isolate KNU14-22was cultured on PDA at 25oC for 14 days and colony characteristics including color, shape, and size were recorded. Photomicrographs were taken with an HK 3.1 CMOS digital camera (KOPTIC, Seoul, Korea) attached to an Olym-pus BX50F-3 microscope (Olympus, Tokyo, Japan) and a scanning electron microscope (LEO Model 1450VP; Carl Zeiss, Oberkochen, Germany). The morphological characteristics of the identified species are summarized in Table1. The colony attained a diameter of 80~90 mm. The colony was white to pale white in color, floccose mycelium,margin entire, and lacking exudate (Fig. 1A, 1B). Phialides were usually scanty and produced on prostrate aerial hyphae, solitary, sometimes in whorls of 2~3 numbers,tapered towards the tip and 15~25 μm in diameter. Conidia were ellipsoidal, smooth walled, one-celled, and 2.5~3.5 μm in size. Dichlomydospores were abundant, produced in the aerial mycelium, and stalked (Fig. 1C~1F).
Genomic DNA extraction, sequencing, and data analysis
The genomic DNA of the isolate was extracted using the DNeasy Plant Mini Kit (QIAGEN, Germantown, MD,USA) following the manufacturer's instructions. The internal transcribed spacer (ITS) region of rDNA was amplified using the ITS1 and ITS4 primers [6]. The purified DNA was sequenced directly using the Macrogen Sequencing Service (Macrogen, Seoul, Korea). The obtained 579-bp sequence was deposited in GenBank at the National Center for Biotechnology Information under accession no. KP055595. In a GenBank BLAST search the ITS sequenceshowed 99% similarity with M. chlamydosporia (AB 709846). For phylogenetic analyses, all available ITS rDNA sequences of reference species belonging to the MetaCordyceps were retrieved from GenBank. M. owariensis (JN049894) was used as an outgroup taxon. All retrieved sequences were aligned using the Multalin program. The phylogenetic tree was constructed using neighbor-joining method with Kimura 2-parameter model in MEGA6 software[7]. Bootstrap analysis was performed with 1,000replications to determine the support for each clade. The ITS sequence of KNU14-22 matched with M. chlamydosporia(AB709846) with 99% similarity (576/576 bp) and clustered together in a clade with a 99% bootstrap value(Fig. 2).
Fig. 2. Neighbor-joining tree of the partial 18S, ITS1-5.8S-ITS2 and partial 28S rDNA sequence of MetaCordyceps chlamydosporia KNU14-22. The sequence was compared with the reference sequences in NCBI GenBank. The sequences obtained in the present study are shown in boldface. Numerical values (>50) above/below branches are the percentage of 1,000 bootstrap replicates that support the branch. MetaCordyceps owariensis was used as an outgroup. The scale bar represents the number of substitutions per site.
This fungal species is a new record for Korea and previous research results have proved that M. chlamydosporia is pathogenic to nematodes and insects. Thus, further investigations on the biocontrol aspect of the current isolate would be worthwhile.
