INTRODUCTION
The genus Albifimbria L. Lombard & Crous 2016 belongs to the family Stachybotryaceae of Ascomycotic fungi. These fungi are characterized by verrucose setae and conidia bearing a funnel-shaped mucoidal appendage [1]. These fungi are distributed in the soil, leaves, fibers, fruit, and air [2]. To date, Albifimbria consists of four species, i.e. A. lateralis, A. terrestris, A. verrucaria, and A. viridis, of which A. terrestris has been previously reported in Korea [3]. Albifimbria verrucaria was previously classified under the genus Myrothecium [2]. M. verrucaria is associated with mycotoxicoses of livestock and humans [4-7]. This fungus is also associated with dieback and leafspot of various plant hosts [1, 8] resulting in exploitation of bioherbicides [9-11]. Deconica coprophila (Strophariaceae, Agaricales, Basidomycota), commonly known as coprophilous and small mushroom, is distributed worldwide [12] and most commonly in tropical regions [13]. It is also known as Psilocybe coprophila [12]. A fungal diversity survey was carried out in 2017 in Sancheong and Yangpyeong, South Korea, through which two fungal species were assigned to the genera Albifimbria and Deconica. The objective of the present study is to morphologically and molecularly characterize these two unrecorded fungal species A( lbifimbria verrucaria and Deconica coprophila) in Korea.
MATERIALS AND METHODS
Soil sampling and isolation of fungi
Soil sample collection was carried out in 2017 at different crop field locations at Gangwon-do province, Korea. In the present study, morphologically distinct fungal isolates, KNU17-1 and KNU17-199, were isolated from different crop field sites, 35.161771oN, 127.561884oE and 37.320693oN, 127.412046oE, respectively. Soil samples were collected from 15 cm below the surface, avoiding crop debris. Each soil sample was air dried and stored in a sterile polythene bag at 4℃. Collected soil was sieved through an autoclaved brass sieve of 2 mm aperture size. A conventional soil dilution technique [14] was followed to isolate the fungi from the soil. One g of fine soil was mixed with 9 mL of sterile distilled water and serial dilutions were made ranging from 10-1 to 10-5. A 0.1 mL aliquot from each dilution was transferred into potato dextrose agar (PDA; Difco Laboratories, Detroit, MI, USA) supplemented with 100 μgL-1 chloramphenicol (a bacteriostatic agent) in a Petri dish of size 90 mm and incubated at 25℃ for 7-10 days. Thereafter, individual colonies of varied morphologies were transferred onto PDA plates. Pure isolates were maintained in PDA slant and stored in 20% glycerol at -80℃. KNU17-1 and KNU17-199 were deposited at the culture collection of the National Institute of Biological Resource (NIBR, Incheon, Korea) and received NIBR numbers of NIBRFGC000499998 and NIBRFG0000501887, respectively.
Morphological Characterization
For detailed morphological studies, the fungal isolates KNU17-1 and KNU17-199 were cultured on five different agar media, namely potato dextrose agar (PDA), oatmeal agar (OMA), malt extract agar (MEA), Czapek yeast extract agar (CYEA), and yeast extract sucrose agar (YESA). The isolates were inoculated at three points on Petri plates containing each medium and incubated at 26℃ for 7 days under dark conditions. The fungal structures were examined using a light microscope (Olympus BX50F-3; Olympus Co., Tokyo, Japan). Photomicrographs were taken using an HK 3.1 CMOS digital camera (KOPTIC Korea Optics, Seoul, Korea) attached to an Olympus BX50F microscope. The micromorphology of the fungal isolates was examined using a scanning electron microscope (LEO Model 1450VP Variable Pressure Scanning Electron Microscope; Carl Zeiss, Cambridge, MA, USA).
Genomic DNA extraction, PCR, sequencing
For molecular identification, genomic DNA of the study isolates was extracted using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). The internal transcribed spacer (ITS) region was amplified with the primer pairs ITS1 and ITS4 [15]. Polymerase chain reaction(PCR) amplification mixtures (total volume, 20μL) for each gene were prepared in a 50 μL reaction comprising fungal DNA template, primers for each gene, Tag DNA polymerase, dNTPs, buffer, and a tracking dye. The amplification program included the following conditions: an initial denaturation step of 2 min at 95℃ followed by 35 cycles of denaturation for 30 s at 95℃, annealing for 1 min at 72℃, and a final extension at 72℃ for 10 min [16]. PCR amplification products were purified with MicroconTM filters (Millipore, Burlingtong, MA, USA). The amplified PCR products were sequenced using an ABI Prism 3730 DNA analyzer (Applied Biosystem, Foster City, CA, USA).
Phylogenetic analysis
The fungal sequences obtained from the GenBank database (Table 1) were aligned using Clustal W [17]. All sequences were analyzed using the Basic Local Alignment Search Tool (BLAST) program (NCBI, Bethesda, MD, USA). Phylogenetic trees were constructed by neighbor-joining method using Kimuras two parameter model [18] implemented in MEGA7 software [19]. The statistical confidence of tree topology was evaluated based on bootstrap analysis of 1,000 replicates. The ITS nrDNA sequences of the isolates KNU17-1 and KNU17-99 have been deposited in GenBank and have received accession numbers MH231755 and MH231771, respectively.
RESULTS AND DISCUSSION
Morphological characteristics of isolate KNU17-1
Colony of KNU17-1 in PDA attained a diameter of 60-65 mm in 7 days at 26℃. The front of the colony was white, whereas the back was light yellow (Fig. 1A, 1F). The shape of the colony was irregular, with moderate to dense sporulation. Elevation was flat. Margin was undulate. In OMA, the colony grew fast attaining a diameter of 70-75 mm. The front of the colony was cottony white and the back was rosy buff (Fig. 1B, 1G). The shape of the colony was irregular with moderate to dense sporulation. Margin was entire. Elevation was flat. In CYEA, the colony attained a diameter of 55-60 mm in 7 days at 26℃. The front of the colony was white and the back was light yellow (Fig. 1C, 1G). Sporulation was moderate. Margin was entire. Surface was smooth. Elevation was flat. In YESA, the colony attained a diameter of 65-70 mm in 7 days at 26℃. The front of the colony was white and the back was light yellow (Fig. 1D, 1I). Sporulation was moderate to dense. Margin was entire. Surface was smooth. Elevation was flat. In MEA, the colony attained a diameter of 60-65 mm in 7 days at 26℃. The front of the colony was white and the back was light yellow (Fig. 1F, 1J). Sporulation was moderate to dense. Margin was entire. Surface was smooth. Elevation was flat.
Fig. 1. Morphological characteristics of Deconica coprophila (KNU17-1) grown for 7 days at 26oC on potato dextrose agar (PDA), oatmeal agar (OMA), Czapek yeast extract agar (CYEA), yeast extract sucrose agar (YESA), and malt extract agar (MEA). (A-E) Observe colony from left to right. (F-J) Reverse colony from left to right grown on PDA, OMA, CYEA, YESA, and MEA. (K) Hyphae with anastomose (L) Intercalary chlamydospore (M, N) Hyphae with short branches.
Micromorphology of the isolate KNU17-1
Aerial mycelium: hyphae (1.5-5 μm) were branched septate with clamp connection, with short branches, presence of anastomose. Hyphae abundant; chlamydospores present, Figure 1 (K-N). The morphological characteristics of the colonies were compared with previous descriptions of Deconica coprophila (Table 2) [20].
|
Table 2. Morphological comparison of the present study isolate KNU17-1 with previously reported Deconica coprophila isolate 
|
|
|
aSilva et al. 2016 [20]. N/A: Not available |
|
Morphological characteristics of isolate KNU17-199
Colony of KNU17-199 in PDA attained a diameter of 40-45 mm in 14 days at 26℃. The front of the colony was white, whereas the back was reverse rosy buff (Fig. 2A, 2F). Shape was circular. Moderate to dense sporulation. Elevation was flat. Margin was entire. In OMA, the colony attained a diameter of 50-55 mm in 14 days at 26℃. The front of the colony was rosy buff and the back was light brown. Shape was irregular. Elevation was raised. Margin was irregular (Fig. 2B, 2G). In CYEA, the colony attained a diameter of 42-47 mm in 14 days at 26℃. The front side of the colony was white, whereas the back was dark brown in the center and light yellow on the edges. The elevation was flat and margin was filiform (Fig. 2C, 2H). In YESA, the colony grew 35-40 mm in diameter in 7 days at 26℃. Front side of the colony was white while back was rosy buff in color. Shape was circular, margin was entire and flat elevation (Fig 2D, 2I). In MEA, colony attained diameter of 45-50 mm. Front side of the colony was white in color while back side was creamy light brown in color. Shape was irregular, elevation was flat and margin was entire (Fig 2E, 2J).
Fig. 2. Morphological characteristics of Albifimbria verrucaria (KNU17-199) grown for 7 days at 25oC on potato dextrose agar (PDA), oatmeal agar (OMA), Czapek yeast extract agar (CYEA), yeast extract sucrose agar (YESA), and malt extract agar (MEA). (A-E) Observe colony from left to right. (F-J) Reverse colony from left to right grown on PDA, OMA, CYEA, YESA, and MEA. (K) Phialides. (L) Conidia on hyphae. (M, N) Spores.
Micromorphology of the isolate KNU17-199
Hyphae were rarely branched, phialides were 3.6 in number and were cylindrical in shape and pointed towards the ends (Fig. 2K, 2L). Conidia were fusiform and pointed towards one end, 7-8 x 2.5-3.1 μm (Fig. 2M, 2N). The morphological characteristics observed for strain KNU17-199 were very similar to the previously described characteristics of Albifimbria verrucaria (Table 3) [2].
|
Table 3. Morphological comparison of the present study isolate KNU17-199 with the previously reported Albifimbria verrucaria isolate. 
|
|
|
aLombard et al. 2016 [2]. N/A: Not available |
|
Phylogenetic analysis of study isolates
Molecular phylogenetic analysis was carried out by comparing the ITS sequences of the study isolates with those of the reference strains in GenBank using the BLAST program. The phylogenetic tree evaluation based on a bootstrap analysis of 1,000 replicates revealed that the ITS region of strain KNU17-1 shared 100% identity with that of the strain JX235960 of Psilocybe coprophila. A concatenated dataset of ITS gene sequences was used to determine the molecular relationship between the present Korean isolate and P. coprophila retrieved from GenBank. A neighbor joining tree showed that KNU17-1 strain clustered in the same clade as a P. coprophila strain, indicating that KNU17-1 is most closely related to P. coprophila (Fig. 3). P. coprophila is a synonym of Deconica coprophila, which was transferred to the genus Psilocybe by Paul Kummer in 1871 [21]. Similarly, the phylogenetic relationship from ITS sequence analysis indicated that KNU17-199 was grouped with the type strain A. verrucaria NR153550, KU845895, and KU845896 in a highly supported clade (98%) in our phylogenetic tree (Fig. 4). A. verrucaria was previously well known as Myrothecium verrucaria [2]. To our knowledge, this is the first report of A. verrucaria and D. coprophila in Korea and its identity is supported by the morphological and molecular characterization presented here.
Fig. 3. Neighbor-joining phylogenetic analysis of the partial 18S-ITS1-5.8S-ITS2-partial 28S rDNA sequences for Psilocybe coprophila KNU17-1, obtained from field soil in Korea. Species examined in this study are in bold. Bootstrap scores of >60 are presented at the nodes. The scale bar represents the number of substitutions per sites.
Fig. 4. Neighbor-joining phylogenetic analysis of the partial 18S-ITS1-5.8S-ITS2-partial 28S rDNA sequences for Albifimbria verrucaria KNU17-199, obtained from field soil in Korea. Species examined in this study are in bold. Bootstrap scores of >60 are presented at the nodes. The scale bar represents the number of substitutions per sites.
ACKNOWLEDGEMENTS
This research was sponsored by the Ministry of Environment (MOE) of the Republic of Korea from the National Institute of Biological Resources (NIBR) grant for the survey and discovery of indigenous fungal species of Korea. This study was also supported by the Research Grant from the University Industry Cooperation Foundation of Kangwon National University.
