INTRODUCTION
The genus Acrophialophora is monophyletic and belongs to the family Chaetomiaceae [1]. Edward (1959) established Acrophialophora genus with A. nainiana Edward as the type [1]. The genus Taifanglania was considered synonymous to Acrophialophora, and accordingly, all Taifanglania species were transferred to genus Acrophialophora [2]. The species of Acrophialophora are saprophytic, thermotolerant, and have characteristics that may play an essential role in the degradation of cellulose [3-5]. A. ellipsoidea is characterized by solitary phialides tapering into thin necks and long chains of ellipsoidal to fusiform conidia. A. ellipsoidea is found most frequently in soil, and it is widely distributed in temperate and tropical regions. It is also commonly isolated as a decomposer of compost and other self-heating substrates [6, 7]. During a survey, the strain KNU-US-1802E was isolated and identified as A. ellipsoidea, which until now was an unreported fungal species in Korea. In this study, we describe morphological features and cultural characteristics of the A. ellipsoidea KNU-US-1802E strain and its phylogenetic relationship with allied species.MATERIALS AND METHODS
Soil sample collection and fungal isolation
In 2018, the soil sample was collected from a field in Uiseong, Korea (N 36°25'12.8", E 128°45'33.6") at a depth of 15 to 30 cm. The sample was transferred to polyethylene zipper bags after air drying, and then stored at 4℃ until use. KNU-US-1802E was isolated by a conventional dilution plating technique [8]. Briefly, 1 g of soil was suspended in 10 mL of sterile distilled water and gently vortexed. The suspension was serially diluted, and a defined volume spread on potato dextrose agar (PDA; Difco, Detroit, USA) plates.Morphological characterization
The isolate, KNU-US-1802E, was cultured on PDA media and incubated at 35°C. Colony characteristics such as color, shape, and size were identified and recorded, and the morphological description was made from colonies on PDA media after 6 days in culture [2]. The model BX-50 light microscope (Olympus, Tokyo, Japan) was used to observe the morphological structures of the isolate.Genomic DNA extraction, PCR amplification and sequencing
Using a HiGene Genomic DNA prep kit (Biofact, Daejeon, Korea) and following the instructions of the manufacturer, we extracted genomic DNA for molecular identification based on multiple genes. Polymerase chain reaction (PCR) amplification was performed to amplify two gene markers; ITS1F and ITS4 [9, 10] were used to amplify the internal transcribed spacer (ITS) regions, and Bt2a and Bt2b [11] were used to amplify a portion of the beta-tubulin (tub2) gene. Then, the amplified PCR products were purified with ExoSAP-IT (Thermo Fisher Scientific, Waltham, USA) and sequenced (Macrogen, Daejeon, Korea).Phylogenetic analyses
The DNA sequences obtained from our isolate were compared with reference sequences from the GenBank database of the National Center for Biotechnology Information (NCBI), using the basic local alignment search tool (BLAST). The isolates used to construct the phylogenetic tree are summarized in Table 1 with their strain and GenBank accession numbers. Based on Kimuras neighbor-joining (NJ) algorithm, the evolutionary distance matrices were generated [12]. Phylogenetic analyses were performed using the program MEGA 7 [13] with bootstrap values based on 1,000 replications. The analyzed sequences were deposited at the collection facility of NCBI GenBank, with accession numbers of LC485179 and LC485180 for the ITS region and partial of tub2 gene sequences, respectively.
RESULTS AND DISCUSSION
Morphology of the KNU-US-1802E isolate
Colony diameters were 42 mm at 6 days of culture at 35°C on PDA. Colonies on PDA were flat, light gray, dense, with entire margins; reverse dark gray to black, with white margins (Fig. 1). Conidia were hyaline, one-celled, ellipsoidal to fusiform, forming long chains of more than ten conidia with an average length × width of 5.0±0.3 × 2.9±0.2 μm (n=50). Although the isolate showed small differences in culture characteristics, most of morphological characteristics were like those previously reported for A. ellipsoidea CGMCC 3.15255 (Table 1). This result suggests that the fungal isolate KNU-US-1802E was closely related to A. ellipsoidea.
Molecular phylogeny of the KNU-US-1802E isolate
In studying the phylogenetic relationship between isolate KNU-US-1802E and the previously reported A. ellipsoidea, their ITS regions and a portion of their tub2 gene sequences were compared and analyzed. After the sequencing analysis, sequences of 507 bp and 488 bp were obtained from the ITS region and tub2 gene, respectively. BLAST search results indicated that the ITS region and the partial tub2 gene sequence showed 100% and 99% similarities, respectively, with A. ellipsoidea CGMCC 3.15255 collected from China. The concatenated sequences of the ITS region and tub2 gene were used to determine the molecular relationships between the sequences of A. ellipsoidea retrieved from GenBank (Table 2). A neighbor-joining method generated a phylogenetic tree showing that the strain KNU-US-1802E clustered in the same clade as other Acrophialophora strains, indicating that KNU-US-1802E is a strain of A. ellipsoidea (Fig. 2). Thus, fungal strain KNU-US-1802E was identified as A. ellipsoidea, and the fungal isolate KNU-US-1802E was deposited in the National Institute of Biological Resources (NBI RFGC000502237).Genus Acrophialophora has been known to be a monophyletic and belongs to the family Chaetomiaceae [1]. Some species of these fungi can produce highly active laccase and cellulose as well as useful thermostable enzymes [14], while other species have been predicted to be emerging opportunistic pathogens in humans, associated with keratitis pulmonary colonization and infection [15] and devastating cerebral infections requiring intensive antifungal therapy [16]. According to previous reports, A. ellipsoidea is thermotolerant, having ideal growth temperatures of 37~40℃ and maximum growth temperatures near 50℃, which are key to its acceptance into the Acrophialophora genus [6]. Therefore, the fungi can produce thermostable enzymes that help the food and paper industries and enhance agriculture, notwithstanding the fact that the species can cause infections in humans. Further studies are required to provide in-depth knowledge about this species. In this study, we report Acrophialophora ellipsoidea for the first time in Korea.
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Table 2. List of the sequences used in this study 
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T Type strain; bold letters: the isolates used in this study |
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Fig. 2. Phylogenetic tree constructed with neighbor-joining (NJ) method, based on the concatenated ITS and tub2 gene sequences, shows the phylogenetic position of Acrophialophora ellipsoidea KNU-US-1802E among members of the genus Acrophialophora. The strain isolated in this study is shown in boldface. Bootstrap values (based on 1,000 replications) are shown at the branch points. Chaetomium thermophilum var. coprophilum T ATCC 16451 was used as an outgroup. Bar means 0.02 substitutions per nucleotide position.
