Morphological analyses

To obtain samples for microscopic examination and growth rate determination, isolates CNUFC-1YSRS2-4 and CNUFC-GSNPF3-1 were cultured on each of the 3 media: PDA, malt extract agar (MEA; 33.6 g MEA in 1 L of deionized water; Becton, Dickinson and Co.), and oatmeal agar (OA; 1.5% oatmeal and 1.5% agar in 1 L of deionized water; Junsei, Tokyo, Japan). The plates were incubated at 25 in the dark for 7 days. Samples were mounted in a drop of distilled water on a glass slide and were examined under an Olympus BX51 microscope with DIC optics (Olympus, Tokyo, Japan). Fine structures of the fungi were analyzed by scanning electron microscopy (Hitachi S4700 field emission scanning electron microscope; Hitachi, Tokyo, Japan). Samples were fixed in 2.5% paraformaldehyde-glutaraldehyde buffer with 0.05M phosphate (pH 7.2) (Junsei) for 2 hr and washed in cacodylate buffer (Junsei). Cellular membranes were preserved by fixing the samples in 1% osmium tetroxide (diluted in cacodylate buffer; Electron Microscopy Sciences, Hatfield, PA, USA) for 1 hr, by washing again in cacodylate buffer, dehydrating in a graded series of ethanol solutions (Emsure, Darmstadt, Germany) and isoamyl acetate (Junsei), and by drying in a fume hood. Finally, samples were covered with gold in a sputter coater and examined at Korea Basic Science Institute, Gwangju, Korea.

DNA extraction, PCR, and sequencing

Total genomic DNA was directly extracted from mycelia using the Solg Genomic DNA Prep Kit for fungi (SolGent, Daejeon, Korea). The internal transcribed spacers (ITS1 and ITS2) and the 5.8S gene were amplified with primers ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) by the method of Cano et al. [18]. D1/D2 domains of the 28S rRNA gene were amplified with primers NL1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 (5′-GGTCCGTGTTTCAAGACGG-3′) according to the method of Gilgado et al. [20]. The PCR amplification mixture (total volume 20 µL) contained 10 ng of a fungal DNA template, 5 pmol/µL each primer, and the Accupower PCR Premix (Taq DNA polymerase, dNTPs, buffer, and a tracking dye; Bioneer Corp., Daejeon, Korea). The PCR products were purified by means of the Accuprep PCR Purification Kit (Bioneer Corp.). DNA sequencing was performed on an ABI 3700 Automated DNA sequencer (Applied Biosystems Inc., Foster City, CA, USA).

Taxonomy of CNUFC-1YSRS2-4-1

Acremonium variecolor Giraldo, Guarro, Gené & Cano, Mycologia 104: 1463 (2012) (Table 2, Fig. 3).

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Fig. 3

Morphology of Acremonium variecolor CNUFC-1YSRS2-4-1. A–F, Colonies on potato dextrose agar (A, D), malt extract agar (B, E), or oatmeal agar (C, F) (A–C, obverse view; D–F, reverse view); G–I, Branched conidiophores, an acicular phialide, and oval conidia; J–L, A curved or straight phialide and globose to subglobose or ovoid conidia supported by a calyxlike structure (arrows); M, Conidia detached from slimy heads and conidia with an apiculate base (arrows) (scale bars: G, H = 20 µm, I = 10 µm, J = 2.5 µm, K, L = 1.5 µm, M = 5 µm).

Table 2

Morphological characteristics of CNUFC-1YSRS2-4 and of the reference species Acremonium variecolor when grown on the potato dextrose agar medium at 25

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^a From the description by Giraldo et al. [19].

Description: Colonies were initially white and then changed to brown, reaching 62 mm in diameter after 15 days in culture at 25 on PDA. The colony reverse was pale yellow and regularly zonate. Conidiophores were erect, mostly branched, bearing whorls of up to 5 phialides. Phialides were straight or acicular, measuring 1.0–2.0 µm wide by 18.0–95.0 µm long at the base. Conidia were subglobose or ovoid, measuring 2.25–4.35 µm wide × 2.54–5.08 µm long.

Taxonomy of CNUFC-GSNPF3-1-1

Acremonium persicinum (Nicot) W. Gams, Cephalosporium-artige Schimmelpilze: 75 (1971) (Table 3, Fig. 4).

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Fig. 4

Morphological characteristics of Acremonium persicinum CNUFC-GSNPF3-1-1. A–F, Colonies on potato dextrose agar (A, D), malt extract agar (B, E), or oatmeal agar (C, F) (A–C, obverse view; D–F, reverse view); G, A long and sharp phialide with conidia; H, I, Oval conidia arranged in slimy heads (arrows) on the phialide; J, Conidia; K, Conidia clustered in the slimy head (arrow); L, Arranged conidia with rough walls in the slimy head; M, Oval conidia with rough walls (scale bars: G, H = 20 µm, I, J = 10 µm, K, L = 2.5 µm, M = 2 µm).

Table 3

Morphological characteristics of CNUFC-GSNPF3-1 and of the reference species Acremonium persicinum on potato dextrose agar medium at 24–25

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^a From the descriptions by Perdomo et al. [11], Sukapure et al. [24], and Lo Piccolo et al. [25].

= Paecilomyces persicinus Nicot, Bulletin de la Société Mycologique de France 74: 222 (1958).

= Cephalosporium purpurascens Sukapure & Thirum, Mycologia 55: 563 (1963).

Description: Colonies were initially white and then changed to light pinkish with age, reaching 53 mm in diameter after 15 days in culture at 25 on PDA. The middle of the colony obverse was initially dark white and became pale and formed a crumpled shape as the colony grew toward the edge. On MEA and OA, the colony reverse was light yellow. Conidiophores were erect, simple, or branched. Phialides were lateral and acicular, measuring 1.5–2.5 µm wide × 18–43 µm long at the base. Conidia were oval, measuring 1.0–2.0 µm wide × 2.5–3.0 µm long.

This work was supported by the Project on Survey and Discovery of Indigenous Species of Korea funded by NIBR, and by the Project on Discovery of Fungi from Freshwater and Collection of Fungarium funded by NNIBR, the Ministry of Environment (MOE), and in part carried out with the support of the Cooperative Research Program for Agriculture Science & Technology Development (PJ012957), Rural Development Administration, Republic of Korea.