Three new records of penicillium species isolated from insect specimens in Korea.

Three Penicillium species have been isolated from insect specimens in Korea; Penicillium sp., P. steckii, and P. polonicum. Penicillium sp. (KNU12-3-2) was isolated from Lixus imperessiventris, while P. polonicum (KNU12-1-8) and Penicillium steckii (KNU12-2-9) were isolated from Muljarus japonicas and Meloe proscarabaeus, respectively. The identification was based on the morphological characteristics of the fungi and in internal transcribed spacer analysis. This is the first report on the isolation of these three species of Penicillium from insects in Korea.

Penicillium species are usually regarded as soil fungi [1], but many species inhabit well-defined habitats other than soil. They function as decomposers of dead materials and are especially important postharvest, where they spoil food commodities [2-5]. Some reports have shown that Penicillium species can be isolated from insects and their body parts [6]. In this study, we report three species of Penicillium isolated from three different insect species in Korea.

Collection of insects and fungal isolation

Insect samples were collected from preserved specimens of the Insect Museum at Kangwon National University in Chuncheon (Gangwon Province, Korea). Collected insects were placed in a laboratory clean box for isolation of fungi. Collected insects were surface sterilized in a 2% sodium hypochlorite solution for 3 min, rinsed with plenty of sterile distilled water, then dried using filter paper. Surface sterilized cadavers were plated onto potato dextrose agar (PDA) containing 0.25 mg/mL chloramphenicol to inhibit bacterial growth and incubated at 25℃. Hyphae of the fungi growing and sporulating on cadavers (and on PDA medium) were cut, transferred to fresh PDA plates and incubated at 25℃.

ITS sequencing analysis

Fungal genomic DNA samples were extracted using InstaGene Matrix (Bio-Rad Laboratories, Hercules, CA, USA). The primers ITS1 primer (5'-TCCGTAGGTGAACCTGCGG-3') and ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3') and ITS4 primer (5'-TCCTCCGCTTATTGATATGC-3') were used for the PCR. The PCR reaction was performed with 20 ng of genomic DNA as the template in a 30 µL reaction mixture by using EF-Taq (SolGent, Daejeon, Korea) as follows: activation of Taq polymerase at 95℃ for 2 min, 35 cycles of 95℃ for 1 min, 55℃, and 72℃ for 1 min each were performed, finishing with a 10-min step at 72℃. The amplification products were purified with a multiscreen filter plate (Millipore Corp., Bedford, MA, USA). Sequencing reactions were performed using the PRISM BigDye Terminator v3.1 Cycle Sequencing Kit. The DNA samples containing the extension products were added to Hi-Di formamide (Applied Biosystems, Foster City, CA, USA). The mixture was incubated at 95℃ for 5 min, followed by 5 min on ice, and then analyzed by the ABI Prism 3730XL DNA analyzer (Applied Biosystems, Foster City, CA, USA). The sequences were compared using the NCBI BLAST program (http://www.ncbi.nlm.nih.gov/Blast) for identification of the isolates. Sequences used to calculate phylogeny were first determined using BLAST results from databases [7] and a phylogenetic tree was subsequently prepared by the neighbor-joining method [8].

Morphological characteristics and identification

Penicillium polonicum

Conidiophores are observed as two-stage branched (terverticillate) with all elements adpressed and stipes rough-walled. Conidia were smooth, globose to subglobose, and 3~4 µm in diameter. Morphologically, conidia and conidiophores of our strain were similar to those of Penicillium polonicum (Fig. 1). When the internal transcribed spacer (ITS) sequences of the strain were compared with related species retrieved from GenBank, sequence analysis by BLAST indicated that KNU12-3-2 was highly related to P. polonicum, with a 99% sequence similarity (Table 1, Fig. 2).

Fig. 1

Morphological characteristics of Penicillium polonicum isolated from Muljarus japonicas. A, Morphology of the host insect (M. japonicas); B, Colony on potato dextrose agar after 7 days of incubation; C, Mycelia of P. polonicum; D, Spores of P. polonicum; E, Conidia of P. polonicum (scale bars: C~E = 10 µm).

Table 1

ITS sequence analysis for the identification of Penicillium species

Fig. 2

Phylogenetic tree (using internal transcribed spacer sequences) showing the closest known relatives of newly reported Penicillium species in Korea. Numbers above the branches indicate bootstrap values of distance.

Penicillium steckii

Conidia observed were broadly ellipsoidal. Conidiophores from surface hyphae, symmetrically biverticillate, stipes smooth, and width 2.2~3.0 µm; metulae in whorls of 3~6, 13~18 × 2.5~3.3 µm; ampulliform phialides, 7.0~10 × 2.2~3.0 µm; conidia smooth-walled, broadly ellipsoidal, and slightly fusiform in some strains, 2.3~3.1 × 2.0~2.6 µm (Fig. 3). When the ITS sequences of the strain were compared with related species retrieved from GenBank, sequence analysis by BLAST indicated that KNU12-1-8 was highly related to P. steckii with 99% sequence similarity (Table 1, Fig. 2).

Fig. 3

Morphological characteristics of Penicillium steckii isolated from Meloe proscarabaeus. A, Morphology of the host insect (M. proscarabaeus); B, Morphology of P. steckii on potato dextrose agar; C, Spores of P. steckii under a microscope; D, Mycelia of P. steckii (scale bars: D, E = 10 µm).

Penicillium sp.

The mycelium typically consists of a highly branched network of multinucleate, septate, and usually colorless hyphae. Many-branched conidiophores sprout on the mycelia, bearing individually constricted conidiospores. The conidiospores are the main dispersal route of the fungi, and are often greenish in color. Conidia are globose, ellipsoidal, cylindrical or fusiform, hyaline or greenish, and smooth or rough-walled (Fig. 4). When ITS sequences of the strain were compared with related species retrieved from GenBank, sequence analysis by BLAST indicated that KNU12-2-9 was highly related to Penicillium sp. with 100% sequence similarity (Table 1, Fig. 2).

Fig. 4

Morphological characteristics of Penicillium sp. isolated from Lixus imperessiventris. A, Morphology of host insect (L. imperessiventris); B, Morphology of Penicillium sp. on potato dextrose agar; C, D, Mycelia and spores of Penicillium sp. under a microscope (scale bars: D, E = 10 µm).