A New Record of Penicillium cainii from Soil in Korea.

Twenty Penicillium isolates were recovered during the investigation of fungal community in the soil samples collected from Wando (Jeonnam Province, Korea). Among them, one species was identified and described as P. cainii based on phylogentic analysis of internal transcribed spacer and β-tubulin (BT2) genes and morphological characteristics. This is a first report of P. cainii in Korea.

Highly diverse groups of fungi are presented in soil and have important and complex physiological and ecological functions in ecosystem [1]. Penicillium species are detected frequently by using dilution plate techniques in soil analyses [2]. Many Penicillium species have been found from different soil resources and varied with soil depth and nutrient conditions [3-6]. Identification of Penicillium based on phenotype is useful for species with recognized distinctions; however, it is difficult for closely related species with similar morphology. Molecular approaches have been popular in recent years for the identification, especially phylogenetic analysis using different genes [7, 8]. Twenty Penicillium isolates were recovered during the investigation of fungal community in the soil samples from Wando, (Jeonnam Province, Korea). Among them, one species, P. cainii was identified based on molecular and morphological characteristics. The species is a new record of Penicillium in Korea.

Soil samples were collected from different places in Wando, Jeonnam Province, Korea during the period of Aug. 2012. Each sample was taken from 10~15 cm depth, kept in sterile polyethylene tubes, and stored at 4℃ until examination. Soil fungi were obtained by soil dilution plate method [9]. Soil dilution (100 µg/L) using distilled water was spread on dichloran rose bengal chloramphenicol agar and incubated at 25℃ for 3~7 days. Individual colonies of filamentous fungi were picked up and pure cultures were transferred in potato dextrose agar (PDA; Difco, Detroit, MI, USA) slant tubes and deposited in the Culture Collection of Chungnam National University (CNU) Fungi Herbarium.

Genomic DNA was isolated from mycelia collected from PDA plate using the method with a modification of Park et al. [10]. For the amplification of internal transcribed spacer (ITS) region and β-tubulin (BT2) gene of the Penicillium speices (isolate CNU 114236), primers ITS5 and ITS4 [11], and primers Bt2a and Bt2b [12] were used respectively. The resulting sequences and relevant sequences available in the GenBank database were initially aligned with the CLUSTAL X program [13]. Sequences of the two genes were combined and completed with manual adjustment. The combined dataset was analyzed using RAxML software [14]. Maximum likelihood analysis was performed using the GTRGAT model of nucleotide substitution. The robustness of tree shown in Fig. 1 was evaluated by 1,000 bootstrap replications. ITS and BT2 gene sequences (accession Nos. KC424615 and KC424616, respectively) of the isolate CNU 114236 were identical to the type strain DAOM 239914 of P. cainii [15]. Meanwhile, in the phylogenetic tree, the isolate placed in a clade comprising reference isolates of P. cainii with 100% bootstrap values support (Fig. 1). The results indicated that the isolate CNU 114236 was P. cainii.

Fig. 1

Maximum likelihood tree for Penicillium cainii using RAxML based on a combined internal transcribed spacer and β-tubulin sequence data. Bootstrap values (> 70%) of the maximum likelihood analysis are presented at the nodes. The bar indicates the number of substitutions per site. The mark 'T' indicates type strain.

To confirm the molecular result, morphology of the isolate CNU 114236 was determined. Cultural features were observed on Czapek yeast extract agar (CYA; K2HPO4 1 g, Czapek concentrate 10 mg, yeast extract 5 g, sucrose 30 g, agar 15 g, distilled water 1 L), malt extract agar (MEA; Oxoid, Basingstoke, UK), and yeast extract sucrose agar (YES; yeast extract 20 g, sucrose 150 g, MgSO4·7H2O 0.5 g, CuSO4·5H2O 0.005 g, ZnSO4·7H2O 0.01 g, agar 20 g, distilled water 1 L). Three-point inoculated in 9 cm plastic Petri dishes plates using a dense conidial suspension were incubated in the dark at 25℃ for 7 days. Conidial morphology on MEA media was measured and compared with the previous description [15]. Morphology of the present isolate agreed with the previous description of P. cainii. Morphological structures of the isolate CNU 114236 are shown in Table 1 and Fig. 2.

Table 1

Comparison of cultural and morphological characteristics between the present isolate CNU 114236 and Penicillium cainii described previously

CYA, Czapek yeast extract agar; MEA, malt extract agar; YES, yeast extract sucrose agar.

a Sources of description [15].

Fig. 2

Penicillium cainii CNU 114236. Colonies grown on Czapek yeast extract agar (A, B), malt extract agar (C, D), and yeast extract sucrose agar (E, F) for 7 days at 25℃, conidiophores (G~I), conidia (J) (scale bars: G = 20 µm, H~J = 10 µm).

Penicillium cainii K. G. Rivera, Malloch & Seifert, 2011 (Fig. 2)

Colonies on CYA grown for 7 days at 25℃ (Fig. 2A and B)

37~40 mm in diameter, dense and velutinous, radially sulcae (8~16 sulcae) with several wrinkles, glaucous grey to pale olivaceous grey with 1~2mm white mycelia at the margins, golden yellow in the center covered with clear or pale yellow droplets of exudates, soluble pigment not produced, margin entire, reverse yellowish white to golden yellowish.

Colonies on MEA grown for 7 days at 25℃ (Fig. 2C and D)

36~38 mm in diameter, dense and velutinous, radially sulcate (7~11 sulcate), usually with some wrinkles, sclerotia not produced, grayish sky blue to pale greenish grey with white mycelia at the margins, olivaceous buffer in the center covered with yellowish exudates, soluble pigment not produced, margin entire, reverse pale luteous to orange.

Colonies on YES grown for 7 days at 25℃ (Fig. 2E and F)

39~42 mm in diameter, dense and velutinous, many irregularly radial sulcae, densely sulcae near the edges with many wrinkles, glaucous grey to olivaceous grey with 1~2 mm white mycelia at the margins, exudates absent, soluble pigment not produced, margin entire, reverse pale luteous to orange.

Conidiophores (Fig. 2G~I) were mostly monoverticillate on MEA, arising from felted hyphae or agar surface. Stipes were simple, septate, rough-walled, normally 35~85 × 2~3 (~3.3) µm, sometimes over 100 µm long, with vesicles 3.5~5.0 (~5.5) µm wide. Phialides were ampulliform, 7~10 (~12) × 1.5~2.5 µm with conspicuous neck 1~2.5 × 0.5~1 µm in size. Conidia (Fig. 2J) were born in chains, subglobose to globose, smooth-walled or finely roughened, 2~3 (~3.5) µm in diameter.

Culture examined

CNU 114236, isolated from soil samples, Wando, Jeonnam Province, Korea.

Note

Rivera and Seifert [15] designated the species in the Penicillium sclerotiorum complex. They also demonstrated that the species was easily recognized by short and rough-stiped conidiophores combined with the association of host plants in the family Juglandaceae. However, the present isolate was collected from soil and produced yellowish exudates in the center of colonies on MEA media. Phylogenetically, the species is closely related to Penicillium jacksonii but is distinguished by the conspicuously roughened conidiophores [15]. The fungus is only known from nuts of Juglans nigra and Carya ovata in Canada. It was firstly reported from soil in Korea.