Phialocephala lagerbergii: A New Record from Crop Field Soil in Korea

Sampling and isolation

Soil samples were collected from various locations in Samcheok city (37º 26' 87.04 N, 128º 90' 04.62 E), Gangwon-do, Korea. Fungal strains were isolated from the soil samples by the conventional dilution technique [8]. After dilution, the isolates were cultured on a potato dextrose agar (PDA; Difco, Detroit, MI, USA), supplemented with 100 μg chloramphenicol (bacteriostat/L PDA) for 5~7 days at 25°C, until the growth of fungal colony was observed. Then, a representative isolate (KNU14-11) was preserved at 20°C on PDA slants until further use.

Morphological characterization

Four different types of media were used for the morphological characterization of the study isolate (KNU14-11): potato dextrose agar (PDA), oatmeal agar (OA), yeast extract sucrose agar (YES), and malt extract agar (MEA). All the growth media were prepared according to Samson [9]. The isolate KNU14-11 was cultured on 9 cm petri dishes with three point inoculation and incubated in dark at 25°C for 7 days. Obverse and reverse colony colors, as well as the degree of speculation, were determined. An HK 3.1 CMOS digital camera (KOPTIC, Seoul, Korea), attached to an Olympus BX50F-3 microscope (Olympus, Tokyo, Japan), was used for capturing microscopic pictures of the fungal isolate. Scanning electron microscope (LEO Model 1450VP Variable Pressure Scanning Electron Microscope; Carl Zeiss, Oberkochen, Germany) was used for scanning and capturing the micro-morphological features of the fungal isolate.

DNA extraction, PCR amplification, sequencing, and data analysis

Genomic DNA was extracted from 1 week old colonies grown on PDA media, using a DNeasy Plant Mini Kit (Qiagen, Germantown, MD, USA) following the manufacturer’s instructions. The internal transcribed spacer region (ITS) was amplified using ITS1 (5'- TCCGTAGG TGAACCTGCG-3') and ITS4 (5'- TCCTCCGCTTATTG ATATGC-3') primers [10]. The amplified PCR products were sequenced using the Applied Biosystems 3730 DNA analyzer (Foster city, CA, USA). The sequences were compared with the reference ITS sequences, retrieved from GenBank, National Center for Biotechnology Information (NCBI), using the basic local alignment search tool (BLAST) software [11]. The annotated nucleotide sequence of KNU14-11 isolate was deposited in GenBank, with the accession number KP055600. All sequences were aligned using the Molecular Evolutionary Genetics Analysis (MEGA 6.0) software [12]. Phylogenetic tree was generated by the neighbor-joining method, utilizing the Kimura 2-parameter model with a bootstrap analysis of 1,000 replications for each clade.

Morphology of the KNU14-11 isolate

Colony morphology: Detailed morphological features of the fungal isolate KNU14-11 are shown in Fig. 1. The KNU14-11 isolate grown on PDA media attained a diameter of 9~12 mm within 7 days at 25°C. The dorsal side of the mycelium was white in color, whereas the ventral side was hairy green in the middle (Fig. 1A and 1E). Sporulation was moderately dense, and the conidia were seen in mass, having irregular form and smooth surface. The KNU14-11 isolate grown on OA media attained a diameter of 11~16 mm within 7 days at 25°C. The front and the rear sides of the mycelium were dark black with a circle (Fig. 1B and 1F). Sporulation was moderate, and the conidia were in mass, irregular form and rough surface. The KNU14-11 isolate grown on YES media attained a diameter of 8~10 mm within 7 days at 25°C. The front side of the mycelium was white on the margin and black in the center, and the rear was black in color (Fig. 1C and 1G). Sporulation was moderate to dense and the conidia were in mass, having irregular form and smooth surface. On MEA media, the KNU14-11 isolate attained a diameter of 14~17 mm within 7 days at 25°C. The front and rear sides of the mycelium were black in color (Fig. 1D and 1H).

Fig. 1. Morphological characteristics of Phialocephala lagerbergii KNU 14-11 isolate grown for 7 days on potato dextrose agar, oatmeal agar, yeast extract sucrose agar, and malt extract agar media at 25°C. A~D, obverse colony from left to right; E~H, reverse colony from left to right; I~K, light microscopic picture of conidiophores; L, light microscopic pictures of conidia; M, N, scanning electronic pictures of conidiophores; O, P, scanning electronic pictures of conidia.

Micromorphology: The conidiophores were macronematous, unbranched, erect, septate, smooth, and dark brown in color. Conidiophores were observed to be paler towards the apex. Conidiophores were 40~109 μm high and 3~4.8 μm wide (Fig 1I, 1M, and 1N). Hyphae were hyaline and smooth of about 1.5~2.5 μm wide. Hyphae were observed to be brown near the conidiophores. Conidia were catenate, non-septate, smooth, hyaline with rounded apices and truncated bases, tapering modestly to narrower bases, and 2~2.8 × 1~1.5 μm in size (Fig 1L, 1O, and 1P). Phialides were 2~4 in number and 4.8~24 × 1~7.2 μm in size (Fig 1J and 1K). Stipe comprised single to multiseptate and were 4.9~700 μm long. Comparisons of the morphological characteristics of KNU14-11 isolate with those of previously reported species of Phialocephala are described in Table 1.

Table 1. Morphological characteristics of Phialocephala lagerbergii KNU 14-11 isolated in this study

^aSource of description [13].

PDA, potato dextrose agar; OA, oatmeal agar; YES, yeast extract sucrose agar; MEA, malt extract agar.

Molecular phylogeny

The KNU14-11 isolate was sampled from the crop field soil of Samcheok city, Gangwon-do, Korea. The isolates used for constructing the phylogenetic tree are shown in Table 2, along with their respective GenBank accession numbers. The KNU14-11 isolate was most closely related to P. lagerbergii (CBS 266.336) and formed a monophyletic group, supported by a bootstrap value of 99% (Fig. 2). Phylogenetic analysis revealed that the isolate was P.lagerbergii.

Table 2. Sequences of Phialocephala lagerbergii and allied species used in this study, along with their GenBank accession numbers

The KNU14-11 isolate belonged to the Phialocephala genus, based on its characteristics (colony, conidia, and conidiophores) that were similar to those of the Phialocephala genus. In the KNU14-11 isolate, the micromorphological structures were similar to those described by Jacobs et al. [13]. Phialocephala species closely resembles the Sporendocladia fumosa in terms of conidial arrangement [14]. Moreover, the arrangement of phialides and branching patterns of conidiophores, as well as the structure of phialides of P. lagerbergii, as described by Day et al. [6], was found to be identical to that of the KNU14-11 isolate (Fig. 1I-1K). This confirmed that our fungal isolate KNU14-11 was P. lagerbergii. In addition, the ITS sequence of CBS 266.33 was in close proximity (99% similarity) with that of P. lagerbergii. Based on the ITS sequence comparison, the present isolate was found to be closely related with P. lagerbergii, which was also strongly supported by their morphological comparison. Produc-tion of a wide variety of extracellular enzymes such as polyphenyl oxidases, laccases, and proteases by the Phialocephala species reflects its importance in the field of biotechnology. Further studies on its biotechnological importance are worthwhile in the future.

Fig. 2. Phylogenetic analysis based on the neighbor-joining method using the partial 18S-ITS1-5.8S-ITS2-28S rDNA region of Phialocephala lagerbergii KNU 14-11 isolate obtained from the crop field soil in Korea. Numerical values (> 50) on branches are the bootstrap values with 1,000 replicates.