The Korean Journal of Mycology
www.kjmycology.or.kr
RESEARCH ARTICLE
Penicillifer diparietisporus: a New Record
from Field Soil in Korea
Kallol Das1, Chang-Gi Back2, Seung-Yeol Lee1, Hee-Young Jung1*
School of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National University,
1
Daegu 41566, Korea
Horticultural and Herbal Crop Environment Division, National Institute of Horticultural and Herbal
2
Science, Wanju 55365, Korea
*Corresponding author: heeyoung@knu.ac.kr
ABSTRACT
A fungus was isolated from field soil collected from Daegu, Korea. The colony of the isolated
fungus showed short, branched, and light to dark yellow pigments with hyaline, yellowish
red to orange brown aerial mycelia. In addition, the fungus produced solitary to aggregated
perithecia, ovoid to pyriform, short neck, and asci as well as biseriately arranged ascospores.
Phylogenetic analysis using the internal transcribed spacer region and translation elongation
factor 1-α sequences and morphological characteristics identified the isolated fungus as
Penicillifer diparietisporus , which belongs to the family Nectriaceae. To our knowledge, this is
the first report of Penicillifer diparietisporus in Korea.
Keywords: Nectriaceae, Penicillifer diparietisporus , Soil fungi
INTRODUCTION
Approximately 2,700 fungal species from 240 genera are currently recognized in eight families
OPEN ACCESS
pISSN : 0253-651X
eISSN : 2383-5249
Kor. J. Mycol. 2018 September, 46(3): 227-233
https://doi.org/10.4489/KJM.20180029
Received: August 11, 2018
Revised: August 18, 2018
Accepted: August 18, 2018
© 2018 THE KOREAN SOCIETY OF MYCOLOGY.
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distributed under the terms of
theCreativeCommonsAttributionNon-Commercial
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in the order Hypocreales [1, 2]. The species produce light- to bright-colored, ostiolate, perithecial
ascomata containing unitunicate asci with hyaline ascospores. The asexual morphs found in
nature are, most frequently, moniliaceous and phialidic [3-7]. The fungal species are globally
found in various environments; they are of great importance in agriculture and medicine and
extensively exploited for industrial and commercial applications [5]. Moreover, several species
have been reported as important opportunistic human pathogens [8-10], whereas others produce
mycotoxins that are of medical concern [5].
The purpose of this study was to screen for unreported fungal species in field soil in Korea.
This study provides information for further studies on the use of such organisms in agriculture
and medicine as well as for industrial applications. On the basis of morphological and molecular
characteristics, the strain KNU16-010, identified as Penicillifer diparietisporus, was reported
for the first time in Korea.
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Das et al.
MATERIALS AND METHODS
Collection of soil samples
In 2016, samples were collected from field soil during the screening of fungal species in Daegu,
Korea (35°54′11.3″N, 128°36′56.1″E). A sterile trowel and spatula were used to collect the soil
samples from a depth of 15~30 cm. The collected soil samples were placed in polythene zipper
bags and transferred to the laboratory. Then, the soil samples were prepared for serial dilutions,
and 1 g of soil was diluted with 10 mL of sterile distilled water. The serial dilutions were
performed until a concentration of 10-3 was achieved, and, then, 100 µL of each sample was
spread on potato dextrose agar (PDA; Difco, Detroit, MI, USA) plates and incubated for 2~3
days at 25°C. Single germinating fungal colonies were transferred to fresh PDA plates and
incubated under the same conditions. The strain KNU16-10 was selected for further
morphological and molecular phylogenetic analyses.
Morphological characterization
To study the cultural and morphological characteristics, PDA, oatmeal agar (OA; Difco), and
malt extract agar (MEA; Difco) were used; the incubation period was 10 days at 25°C. Then,
cultural characteristics such as colony color, fungal growth, and texture were observed and
recorded, and the morphological characteristics were observed under a light microscope (BX50; Olympus, Tokyo, Japan).
Genomic DNA extraction, PCR amplification, and sequencing
Genomic DNA was extracted from 7-day-old colonies of the strain KNU16-010 grown on
PDA by using the HiGene Genomic DNA Prep kit (BIOFACT, Daejeon, Korea), according to
the manufacturer’s instructions. Partial gene sequences were amplified by the internal
transcribed spacer (ITS) region, ITS1/ITS4, and translation elongation factor (TEF) 1-α gene,
EF1-728F/EF2, as described previously [11-13]. Then, the amplified PCR products were
purified with EXOSAP-IT (Thermo Fisher Scientific, Waltham, MA, USA) and sequenced by
Solgent (Daejeon, Korea). The similarities of the sequences were analyzed using BLAST of
NCBI. The sequences obtained from KNU16-010 were deposited in NCBI GenBank (accession
numbers LC387549 and LC387601 for the ITS region and TEF gene, respectively).
Phylogenetic analysis
The consensus sequences were compared with other sequences in the NCBI database by using
BLAST to determine the percentage of shared sequence identity with other sequences of
fungal species. The alignments were performed using MEGA 7.0 [14] with 1,000 bootstrap
replicates, and the evolutionary distance matrices were generated based on Kimura’s neighborjoining algorithm model [15].
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First report ofPenicillifer diparietisporus isolated from Korean soil.
RESULTS AND DISCUSSION
Morphology of the strain KNU16-010
To observe the morphological characteristics, the strain KNU16-010 was cultured on PDA,
OA, and MEA at 25°C for 10 days, and the diameter of the colonies was 28.7~29.7 mm,
32.0~32.7 mm, and 31.9~32.5 mm, respectively (Fig. 1). The morphology of the strain KNU16-
010 was compared with previous descriptions of Penicillifer diparietisporus [16] and
Pseudonectria diparietospora [17] (Table 1). Pseudonectria diparietospora produced reddish
brown colonies on PDA [17]. Moreover, Neocosmospora arxii (basionym: Pseudonectria
diparietispora) colonies are composed of hyaline or sometimes yellow-pigmented, branched,
septate, smooth-walled, hyphae that are 2.5~5 μm wide, and they produce semi-immersed to
immersed perithecia, scattered or aggregated in small groups, yellowish orange to yellowish
brown, broadly ovoid to somewhat pyriform, 220~340 × 160~270 μm in diameter, almost
glabrous, conical shaped with short neck [18]. However, our strain formed orange brown to
yellowish red colonies in all the media, and PDA was used to observe the mycological
characteristics (Fig. 1). The fungal colonies showed wavy mycelia at the edges and rough wartlike structures. They also formed septate, irregular, and short hyphae, and the average width of
the hyphae was 2.42 μm. They produced submerged perithecia, solitary to aggregated,
yellowish orange to yellowish brown, ovoid to pyriform, smooth, conical shaped with short
neck (Fig. 2A, 2C). Asci clavate, 70.9~81.1 × 16.3~19.7 μm, thin wall, and 8-spored (Fig. 2D,
2E). Ascospores were oval, septate, guttulate, 2-celled, distichous, double wall, biseriate,
tubular, hyaline and pale green, smooth and blunt at both ends with a diameter (n = 30) of
17.9~30.7 × 6.7~10.9 μm and no conidia (Fig. 2F, 2G). The ascospores were distichous,
elliptical, continuous, hyaline, with thick, double wall, smooth 18~24 × 12~14 μm and no
Fig. 1. Cultural characteristics of KNU16-010. A, B, Front and reverse sides of the colony on potato
dextrose agar; C, D, Front and reverse sides of the oatmeal agar; E, F, Front and reverse sides of the malt
extract agar.
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Fig. 2. Morphological characteristics of KNU16-010. A, B, Mature perithecia on potato dextrose agar;
C, Perithecium; D, E, Asci; F, G, Ascospores (scale bars: A, B = 500 μm, C = 50 μm, D~G = 10 μm).
Table 1. Morphological characteristics of the strain KNU16-010 with reference to Penicillifer diparietisporus
a
Characteristics
Colony
b
Color
c
Penicillifer diparietisporus
Pseudonectria diparietospora
Reddish-brown color on PDA and
Yellowish to orange and dark brown in Reddish white edge, changing to
reverse on PDA. Light yellow in center, greenish yellow on OA and yellowish colonies composed of tangled masses
white edge and brown color in reverse brown to brown, light brownish orange of conidial chains.
to dark brown on PDA. Dark brown
on MEA.
color in reverse both media.
KNU16-010
Size (Diam.)
PDA: 28.7~29.7 mm; OA: 32.0~32.7
mm and MEA: 31.9~32.5 mm in 10
days at 25°C.
PDA: 22mm; OA:30 mm in 10 days at N/A
25°C; MEA: N/A
Shape
Edge margin irregular, wavy and
inadequate aerial mycelium.
Edges submerged into irregular margin Lightly floccose or plane and appressed
and scant of aerial mycelium.
and thin.
Asci
Size (Diam.) and
Shape
Clavate, 70.9~81.1 × 16.3~19.7 μm,
thin wall and 8-spored.
Clavate, 60~85 × 12~25 μm,
unitunicate and 8-spored.
Broadly clavate to spindle shaped with
very thin wall, short stalked, 60~80 ×
19~22 μm and 8-spored.
Ascosspores
Size (Diam.)
17.9~30.7 × 6.7~10.9 μm.
21~25 × 12~15 μm.
18~24 × 12~14 μm.
Shape and Color
Oval, septate, distichous, double wall,
tubular, hyaline to pale green, smooth
and blunt in both ends.
Biseriate, thick walled, broadly
ellipsoidal, surrounded by a thin
walled sheath collapsing at maturity
and sometimes giving the ascospores
roughened appearance.
Distichous, elliptical, continuous,
hyaline, with thick, double wall,
smooth.
Superficial, transparent wall, globoseSolitary to densely aggregated,
Submerged, solitary to aggregated,
yellowish orange to yellowish brown, superficial, globose, ovoid to pyriform, conoid with very short neck, smooth,
yellowish-red, 400~500 × 300~400
270~300 × 240~270 μm diameter,
ovoid to pyriform,smooth, conical
μm.
brown, red-orange to orange.
shaped and short neck.
PDA, potato dextrose agar; MEA, malt extract agar; OA, oatmeal agar; Diam., diameter; N/A, not available in previous references.
a
Fungal strain studied in this paper.
b
Sources of the descriptions [16].
c
Sources of the descriptions [17].
Perithecia
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First report ofPenicillifer diparietisporus isolated from Korean soil.
conidia [17]. Thus, these morphological characteristics of the strain KNU16-010 were very
similar to those of Penicillifer diparietisporus.
Notes: Currently, the genus Viridispora is composed of four species, Viridispora diparietispora
(= Penicillifer furcatus), Viridispora alata (= Penicillifer bipapillatus), Viridispora fragariae (=
Penicillifer fragariae), and Viridispora penicilliferi (= Penicillifer macrosporus), each with its
own Penicillifer asexual morphs [7, 16, 19, 20]. Lombard et al. [21] reported that the epithet
Pseudonectria diparietispora (1957) pre-dates that of Penicillifer furcatus (1991), and the
basionyms are provided below.
Penicillifer diparietisporus (J.H. Miller, Giddens & A.A. Foster) Rossman, L. Lombard &
Crous, comb. nov.
Basionym: Pseudonectria diparietispora J.H. Miller, Giddens & A.A. Foster, Mycologia
49:793. 1957.
≡ Neocosmospora diparietispora (J.H. Miller, Giddens & A.A. Foster) Rossman,
Samuels & Lowen, Mycologia 85:699. 1993.
≡ Viridispora diparietispora (J.H. Miller, Giddens & A.A. Foster) Samuels &
Rossman, Stud. Mycol. 42:167. 1999.
= Neocosmospora arxii Udagawa, Horie & P. Cannon, Sydowia 41:353. 1989.
= Neocosmospora endophytica Polishook, Bills & Rossman, Mycologia 83:798. 1991.
= Penicillifer furcatus Polishook, Bills & Rossman, Mycologia 83:798. 1991.
Molecular phylogeny of the strain KNU16-010
Genetic sequences of the ITS regions and TEF1-α were analyzed to determine the evolutionary
relationships (Fig. 3) with the strains obtained from GenBank (Table 2). After analyzing the
nucleotide sequences, 523 bp and 438 bp sequences were obtained from the ITS regions and
TEF1-α, respectively. The BLAST results showed 100% similarity with Penicillifer
diparietisporus (CBS 376.59) and 99% with Penicillifer diparietisporus (KM231861) in the
ITS regions and TEF1-α, respectively. The combined BLAST results suggested that the strain
KNU16-010 showed 100% similarity with the strain Viridispora diparietispora (= Penicillifer
diparietisporus) (CBS 102797) and Penicillifer diparietisporus (CBS 376.59, NR154310). The
phylogenetic tree was constructed on the basis of the ITS regions and TEF1-α to the strain
Penicillifer diparietisporus [21]. The strain KNU16-010 was closely clustered with Penicillifer
diparietisporus (CBS 376.59), with a 100% bootstrap value (Fig. 3). The phylogenetic results
support the fact that the strain KNU16-010 is Penicillifer diparietisporus.
Members of the family Nectriaceae are pleomorphic fungi that display both asexual and sexual
morphs during their life cycles [22]. In this study, we used molecular analysis, which provides
a considerable challenge to conventional fungal systematics. Thus, more genomic studies of
members of the family Nectriaceae are urgently required. To our knowledge, this is the first
report of Penicillifer diparietisporus isolated from field soil in Korea.
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Das et al.
Fig. 3. Neighbor-joining phylogenetic tree based on the combined internal transcribed spacer sequences
and translation elongation factor 1-α. Pseudonectria buxi CBS 324.56 was used as an outgroup. The
strain isolated in this study is indicated in bold, and the bootstrap values are based on 1,000 replications.
Bar, 0.02 substitutions per nucleotide position.
Table 2. GenBank accession numbers used in this study for the phylogenetic analyses
Species
Strains
Penicillifer bipapillatus
CBS 420.88
Penicillifer diparietisporus
CBS 376.59
Penicillifer macrosporus
CBS 423.88
Penicillifer pulcher
CBS 560.67
Penicillifer diparietisporus
KNU16-010
CBS 324.56
Pseudonectria buxi
ITS, internal transcribed spacer; TEF, translation elongation factor.
GenBankaccession numbers
ITS
TEF
KM231740
KM231860
NR 154310
KM231861
KM231739
KM231859
KM231742
KM231862
LC387549
LC387601
KM231778
KM231909
ACKNOWLEDGEMENTS
This research was supported by a grant from the National Institute of Biological Resources
(NIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea for the
project on survey and discovery of indigenous fungal species.
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