서 론
Oidiodendron flavum Svilv., Lent. Bakto. belongs to genus Oidiodendron, a cosmopolitian genus whose mem- bers can usually be found in a wide range of habitats, including soils, different cellulose substrates (litter, wood pulp, bark, mosses, paper), and occasionally from lichens or from air [1]. Some Oidiodendron species have been reported as ericoid mycorrhizal fungi [2]. Plants in the Ericaceae have a distinctive ericoid mycorrhizal associa- tion, which plays important roles in plant growth, nutri- ent uptake, and soil mineralization [5]. The ability of some ericoid mycorrhizal fungi (Oidiodendron maius) to dissolve Zn oxide has been reported [6]. In addition, ericoid mycorrhizal fungus, Oidiodendron cf. truncatum have medical value as novel antifungal agent producers used in treatment of life-threatening fungal infections in immunocompromised hosts such as human immunodefi- ciency virus (HIV) infected persons and cancer patients [7]. Among the species of Oidiodendron, O. flavum is a thermophillic fungus with the capacity for production of thrombolytic agents utilized in treatment of thrombosis [8,9]. The fibrinolytic enzyme obtained from thermophi- llus fungus O. flavum, exhibits a profound fibrinolytic activity and also exhibits relatively high pH and tempera- ture stabilities [10]. Tahany et al., [8] also reported that a O. flavum released maximum amounts of either ammo- nia, peptides, or total soluble nitrogen. Thus, there has been considerable recent interst of mycologists in working with O. flavum.
During the studies of fungal diversity in agricultural soils in Korea, a species of Oidiodendron was discoverd that was not previously reported in Korea. Based on morphological and molecular characteristics, this species was identified as O. flavum.
Collection of soil samples and fungal isolation. Soil samples were collected from different locatoins in Taebaek city, Korea in 2013. Soil from (0-15) depth, air dried and stored in plastic bags at 4ºC until used. The fungi were isolated by conventional dilution and supplemented with 100 μ g chloramphenicol per mL potato dextrose agar (PDA; Difco Laboratories, Detroit, USA) and grown for 7 d at 28ºC until the growth of colonies was observed.
ITS sequencing analysis. Genomic DNA of the strain was extracted using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instruc- tions. The ITS regions, including the 5.8S were amplified with the primers ITS and ITS4 [11] The amplified PCR product was purified using a QIA quick PCR purification Kit (Qiagen, Valencia, CA, USA) following the manufac- turer's recommendations. The PCR product was sequen- ced using an ABI Prism 3730 DNA analyzer (Applied Biosystems, Foster city, CA, USA). The sequence was compared with reference ITS1-ITS4 rDNA sequences in GenBank using BLAST analysis (http://www.ncbi.nlm. nihgob/blast). The sequences of closely related strains were aligned using the MultAlin program. The DNA sequences were analyzed for phylogenetic relationship using Molecular Evolutionary Genetics Analysis (MEGA 5) software [12] The sequence of present isolate, KNU 13-6, was compared with the sequences in GenBank using Basic Local Alignment Search Tool (BLAST). Neighbor-Joining tree was constructed using Kimmura 2-parameter substitution model bootstrap analysis was performed with 1,000 replications in order to determine the support for each clade. ITS regions of the KNU13-6 were 100% identical to the culture collection of O. flavum (accession no. KJ921607) [13] (Fig. 1). Phylogenetic tree of the ITS regions of the isolate (KNU13-6) was identical to O. flavum with 97% bootstrap value support (Fig. 1). The results strongly suggest that the isolate is O. flavum. Consequently, the nucleotide sequence of the isolate re- ported here has been registerd in the NCBI GenBank (Accession no. KJ921607).
Fig. 1. Neighbor-joining phylogenetic analysis of Oidiodendron flavum KNU13-6 partial 18S-ITS1-5.8S-ITS2-28S rDNA region sequence obtained from crop field soil in Korea. The sequence obtained in the study is shown in boldface. Numerical values (>50) on branches are the bootstrap values as percentage of bootstrap replication from 1,000 replicate analysis. Leotiomycetes sp. (JF273533) was used as the outgroup
Morphological characteristics and identification. Mor- phological features were observed on potato dextrose agar (PDA) by doing three point inoculations in 9 cm petri plates which were incubated in the dark at 28ºC for 7 days. The morphological characteristics were identified with the aid of differential interference contrast micro- scopy. Photomicrographs were taken with a Kodak14n digital camera attached to the microscope. Slide material was mounted in water and sometimes with aniline blue staining. Colonies on PDA were slow growing, pale brown, cream, attaining 20~30 mm after growing for 10 days at 28ºC. Conidiophores were hyaline, branched alternately, oppositely or rarely verticillately in the medium (Fig. 2). Conidiophores arising from the mycelial substrate, 60-90 μ m tall from base to branching site. Conidia 1.8-3.8 μ m in diameter. Conidia arthrospores, hyaline, globose or irregular, occasionally with a fragment of conidiophore, one celled readily detached (Fig. 2). Conidia were hya- line, irregular, 2-3.8 × 2.5-3.5 μ m in diameter. Morpho- logical characteristics of the isolate agreed with the des- cription of O. flavum [9,15]. Based on the phylogenetic analysis and morphological characteristics of strain KNU 13-6 was O. flavum.
Fig. 2. Morphological characterization of Oidiodendron flavum KNU13-6 observed using a compound microscope and scanning electron microscope (SEM). A, Colony in front; B, Colony in reverse; C, Conidiophores (Compound miscroscope image; bar = 10 µm). D and E, Conidiophores and conidia (SEM micrograph; bar = 2 and 10 µm), and F, Conidia (SEM micrograph; bar = 20 µm).
In conclusion, we identified and described Oidiodendron flavum KNU13-6 as an unrecorded species in Korea. The species of Oidiodendron have the ability to produce phy- tohormones, solubilize insoluble phosphate and convert complex organic substances to simple forms and fibrino- lytic enzymes. Thus, in the future, further inverstigation in this respect would be worthwhile.
