The genus Simplicillium Gams W. & Zare R. was separated from the former Verticillium sect. Prostrata to accommodate entomogenous and fungicolous verticillium-like anamorphic fungi that mainly produce simple, solitary phialides [1]. Currently, Simplicillium belongs to the family Cordycipitaceae and includes approximately ten species [2, 3]. Members of this genus have been isolated from the soil, marine sediment, diseased plant tissues, fungi, nematodes, mushrooms, and human nails worldwide [1-6]. Some species of the genus Simplicillium, such as S. lanosoniveum and S. lamellicola, are parasites of rusts, such as soybean rust, coffee rust, and rust of the evergreen woody shrub Elaeagnus latifolia, and can be used as biological control agents [1, 7, 8]. Additionally, S. lanosoniveum and S. lamellicola cause plant diseases. S. lanosoniveum is a causal agent of brown spot on Salvinia auriculata and Sa. molesta [9] and S. lamellicola causes gill mildew and brown spots on Agaricus bisporus [1]. Generally, most species in the genus Simplicillium, including five recently described taxa, i.e., S. aogashimaense, S. cylindrosporum, S. obclavatum, S. subtropicum, and S. sympodiophorum, are not pathogenic to plants [2].
While screening for unrecorded fungal species in field soil in Daegu, Korea, a fungal strain, KNU16-006, was isolated. A soil sample (1 g) was suspended in 10 mL of sterile distilled water, and the prepared suspension was vortexed, serially diluted, and spread onto potato dextrose agar (PDA; Difco, Detroit, MI, USA) plates. The plates were incubated at 25°C for 3 days. For purification, single colonies on the plates were transferred to new plates, followed by incubation on PDA at 25°C. One isolate, KNU16-006, was selected for further morphological and molecular phylogenetic analyses.
The isolate KNU16-006 was subjected to molecular identification based on the internal transcribed spacer (ITS) region. For this purpose, genomic DNA was extracted from the mycelia using the HiGene Genomic DNA Prep Kit (BIOFACT, Daejeon, Korea). The ITS region, including 5.8S, was amplified with the primers ITS1F and ITS4 [10]. PCR amplification was initiated with 2 min of denaturation at 95°C, followed by 35 cycles of denaturation at 95°C for 30 sec, annealing at 55°C for 45 sec, and elongation at 72°C for 1 min. Amplification was completed with a final extension at 72°C for 7 min. The amplified PCR products were purified using ExoSAP-IT and sequenced using an ABI 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA) to obtain partial ITS region sequences of KNU16-006 consisting of 616 bp. BLAST searches against the GenBank database revealed that the ITS sequence of KNU16-006 has high similarity (i.e., 99.3~ 99.5%) with the ITS sequences of various strains of Simplicillium cylindrosporum (Fig. 1). This relationship was also evident in a phylogenetic tree including the ITS sequences of related Simplicillium strains obtained from GenBank. Evolutionary distance matrices were obtained using the neighbor-joining algorithm with Kimura’s two-parameter model [11]. Tree topology was inferred using the neighbor-joining method in MEGA7 [12], and branch support was assessed by bootstrapping with 1,000 replicates. The isolate KNU16-006 clustered with seven S. cylindrosporum strains in phylogenetic trees constructed based on ITS region sequences, confirming their close relationship at the species level (Fig. 1). The nucleotide sequence of the ITS region of isolate KNU16-006 was deposited in GenBank (accession number LC228053).
The isolate KNU16-006 was cultured at 25°C, and colony characteristics, such as color, shape, and size, were recorded. After 11 days of incubation on a PDA plate, the colony was 5.6~5.7 cm in diameter. The isolate produced a white floccose aerial mycelium and a creamy, slightly brownish pigment at the bottom of the medium (Fig. 2A, 2B). To observe the hyphae and conidia, the conventional slide culture method was applied, with some modifications [13]. A clean PDA block was cut from a fresh PDA plate and placed on a sterilized slide glass in an empty petri dish. A small piece of gauze soaked with sterile water was also placed in the petri dish to provide humidity. The sides of the PDA block were inoculated with the fungus, and a sterilized cover glass was placed on the top of the block. The petri dish with the slide culture was sealed and incubated at 25°C for 14 days. When the fungus grew, the cover glass was carefully detached from the PDA block, placed on a new slide glass, and fixed with 85% lactic acid. The remaining culture on the slide glass was also observed by removing the PDA block and mounting with 85% lactic acid. The morphology of the isolate was examined under an Olympus BX50 light microscope (Tokyo, Japan). Phialides were mainly solitary, arose from aerial hyphae, tapered towards the apex, without basal septum, and their sizes were 16~37 × 1.1~1.9 µm (Fig. 2C, 2D). Conidia were produced in small globose or subglobose heads at the apex of the long phialides. They were mostly cylindrical, smooth-walled, and one-celled. Conidia were 2.9~4.8 µm in length and 1.1~1.9 µm in width (Fig. 2E, 2F). As shown in Table 1, these morphological characteristics of isolate KNU16-006 were consistent with those previously reported for S. cylindrosporum [2], strongly supporting the results of the phylogenetic analysis of KNU16-001 based on ITS sequences. The fungal strain isolated in this study was deposited in the National Institute of Biological Resources (NIBR, http://www.nibr.go.kr) for further study (sample no. NIBRFG0000499835).
This is the first report on S. cylindrosporum in Korea. Members of the genus Simplicillium produce various bioactive compounds, such as cytotoxic linear peptides produced by S. obclavatum EIODSF 020 [6], antibacterial mannosyl lipids produced by S. lamellicola BCP [14], and antifungal pyrrolidine alkaloids produced by S. lanosoniveum TAMA 173 [15]. Thus, further studies of S. cylindrosporum KNU16-006 as a potential biocontrol agent and producer of bioactive compounds are needed.
