Introduction
Pseudeurotium bakeri is an ascomycetous fungus belonging to the Pseudeurotiaceae family. The general features of this fungal species include having hyaline or brown ascospores in a thin walled ascus [1]. Pseudeurotium bakeri has been reported as an endophytic fungus having a cosmopolitan distribution, and can survive in high concentrations of diesel fuel in soil [2]. The conidial phase of P. bakeri has been noted as sporothrix, beauvaria, or acremonium-like structures [3].
Soil fungi play vital ecological roles as decomposers, pathogens, and symbionts [4]. Fungi have a cosmopolitan distribution and grow in a vast area of habitats. Despite their ecological importance, little is known about their distribution and biodiversity. Therefore, we collected fungal strains to discover the diversity of fungi in the crop field soil from Suncheon-si, Jeollanam-do, Korea. Among the fungal isolates, Pseudeurotium bakeri, which has not been reported officially from Korea, was encountered. The purpose of this study was to describe the isolated fungal species of Pseudeurotium by morphologically comparing it with previously described Pseudeurotium spp.
Materials and Methods
Sampling of soil and fungal isolation
Soil samples were obtained from different areas of Suncheon-si, Jeollanam-do, Korea. Each soil sample was taken at a depth of 1~15 cm. All the collected soil samples were stored in plastic bags and stored at 4°C. The fungi were isolated by the conventional dilution method [5] and cultured on potato dextrose agar (PDA; Difco, Detroit, MI, USA) supplemented with 100 μg chloramphenicol (bacteriostatic agent) per liter of PDA for 5~7 days at 26°C. For further use, pure cultures of PDA slants were maintained at 4°C at the plant microbiology and biotechnology lab of Kangwon National University.
Morphological examination
Morphological features of the isolate were observed in PDA by performing one point inoculation in 9 cm petri plates. Inoculated plates were incubated in the dark at 26°C. The morphological characteristics were observed by using a differential contrast microscope. The color, size, and shape of the microstructures of the isolate were observed by using an HK3.1 CMOS digital camera (KOPTIC, Seoul, Korea) attached to an Olympus BX50F-3 microscope (Olympus, Tokyo, Japan). Scanning electronic microscopy images of the conidia and conidiophores were taken by a LEO Model 1450VP Variable Pressure (SEM; Carl Zeiss, Oberkochen, Germany).
DNA extraction, PCR amplification, sequencing and data analysis
Genomic DNA was extracted from fully grown mycelia on PDA media. DNA was extracted using a DNeasy Plant Mini Kit (Qiagen, Germantown, MD, USA) following the manufacturer’s instructions. For amplification of the internal transcribed spacer (ITS) gene region, primers ITS1 (5'-TCCGTAGGTGAACCTGCG-3') and ITS4 (5'-TCCT CCGCTTATTGATATGC-3') [6] were used. The amplified PCR products were sequenced using an ABI Prism 3730 DNA analyzer (Applied Biosystems, Foster City, CA, USA). The sequences were compared with reference ITS sequences deposited in GenBank at the National Center for Biotechnology Information (NCBI) using the basic local alignment search tool (BLAST) [7]. The nucleotide sequence obtained from the isolate KNU14-21 was deposited in GenBank and was assigned an accession number KP055601. The isolates used for the construction of the phylogenetic tree are shown in Table 2. The neighborjoining relationship was analyzed using MEGA 6.0 [8]. A neighbor-joining tree was constructed using the Kimura 2-parameter substitution model [9]. The branch robustness was tested by 1,000 replicates of bootstrap analysis.
Results and Discussion
Morphological features of the isolate KNU14-21 assessed by optical and scanning electron microscopes are shown in Fig. 1. The colony grew moderately, attaining a diameter of 25~32 mm. The isolate had a smooth colony surface. The colony was white in color at the backside and light yellow on the front side. The center of the isolate was slightly powdery (Fig. 1A, 1B). The conidiophores were produced from the aerial or substrate mycelium (Fig. 1F). Short side-branches of the conidiophores were observed (23~57 μm in length) (Fig. 1G). Conidia were subglobose or ellipsoid (Fig. 1E). The size of the conidia was 4.8~5.5 × 3.0~3.2 μm (Table 1). Conidia were present in a chain-like structure (Fig. 1H). The conidial head was loosely columnar. The terminal conidiogenous cells were 9~21 μm in size and lageniform. The morphological description of our study isolate reasonably fits with the description of P. bakeri [10]. Formation of conidia and conidiophores by the isolate is similar to that of the Teberdinia state of P. bakeri reported by Sogonov et al. [10].
To confirm the identification, molecular analysis was carried out. ITS gene region was used for determination of the phylogenetic relationship with the previously described Pseudeurotium species. Our results showed that the isolate is most closely related to P. bakeri (CBS878.71), with a bootstrap value of 99% (Fig. 2). Additional sequence similarity analysis of the ITS region using BLAST search also supported that KNU14-21 is P. bakeri (Table 3). Although this is a common fungal species with a cosmopolitan distribution, this is the first report of its incidence in Korea.
Since Pseudeurotium bakeri has been reported as afungal species resistant to biodiesel contaminated soil [2], this finding has important biotechnological implications. However, the main purpose of our present study was limited to identify and describe the isolate morphologically and molecularly. Further studies on its use and implications in the field of biotechnology are thus needed.
Fig. 1. Morphological characteristics of Pseudeurotium bakeri KNU14-21 grown for 7 days on potato dextrose agar. A, reverse view; B, obverse view; C, D, Simple microscopy picture of conidiophores; E, Simple microscopy picture of conidia; F, G, Scanning electronic microscopic pictures of conidiophores; H, Scanning electronic microscopic pictures of conidia.
Table 2. Sequences of Pseudeurotium and allied species used in this study with their GenBank accession numbers
Fig. 2. Neighbor-joining phylogenetic analysis of the partial 18S-ITS1-5.8S-ITS2-28S rDNA sequences of Pseudeurotium bakeri KNU14-21 obtained from crop field soil in Korea. The phylogenetic tree was constructed using the MEGA 6 program. The sequence obtained in the study is shown in boldface. The mark (T) indicates type strain. Numerical values (> 50) on branches are the bootstrap values as percentage of bootstrap replication from a 1,000 replicate analysis. The scale bar represents the number of substitutions per site.
