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The Korean Journal of Mycology www.kjmycology.or.kr
Received: 10 February, 2017
Revised: 21 February, 2017
Accepted: 21 February, 2017
Ⓒ The Korean Society of Mycology
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Kor. J. Mycol. 2017 March, 45(1): 63-67
https://doi.org/10.4489/KJM.20170007
pISSN : 0253-651X
eISSN : 2383-5249
OPEN ACCESS
The Korean Journal of Mycology Vol. 45, No. 1, 2017 ∙63
RESEARCH NOTE
Neocosmospora rubicola, an Unrecorded
Endophytic Fungus Isolated from Roots of
Glycyrrhiza uralensis in Korea
Jin-Hee Kim1, Dong-Yeo Kim1, Hyeok Park1, Jae Hee Cho2, Ahn-Heum Eom1*
1Department of Biology Education, Korea National University of Education, Cheongju 28173, Korea
2Department of Earth Science Education, Korea National University of Education, Cheongju 28173,
Korea
*Corresponding author: eomah@knue.ac.kr
Abstract
Through a survey of symbiotic endophytic fungi, we isolated an endophyte fungal strain from
the roots of Glycyrrhiza uralensis in Korea. The isolated fungal strain was identified using its
morphological characteristics and through phylogenetic analysis of the internal transcribed
spacer, the large subunit rDNA region, and the translation elongation factor region. The strain
was identified as Neocosmospora rubicola. This species has not been previously reported in
Korea. In this study, we report its isolation from the roots of Glycyrrhiza uralensis in Korea,
followed by the characterization and identification of the strain.
Keywords: Endophyte, Glycyrrhiza uralensis, Licorice, Neocosmospora rubicola
Licorice (Glycyrrhiza uralensis Fisch.) is a perennial legume and it is known to be an
important medicinal plant that contains a variety of physiologically active substances[1].
Licorice root has been widely used as a pharmaceutical agent because of its anti- inflammatory
and hepatoprotective effects. Some key components of licorice root are glycyrrhizin,
liquiritin, licoricidin, glycyrrhetinic acid, and saponin[1,2]. Importantly, licochalcone A, a
phenolic component of licorice root, is known for its anti-proliferative and anti-inflammatory
properties[3]. In Korea, consumption of licorice is largely dependent on imports, as the
domestic production of licorice could not satisfy its demand for consumption; moreover, few
studies have been conducted in this regard.
Endophytic fungi refer to the fungi living inside of the tissues of plants, such as roots,
leaves, and stems, without obvious disease symptoms[4]. They are often closely related to
pathogens but have limited pathogenic effects; moreover, endophytes may help protect the
host plant against pathogens[5]. They are transmissible from the host to other plants[6].
Endophytic fungi stimulate seed germination[7] but also provide resistance to pathogens
that may be harmful to the host plants[8]. The study of endophytic fungi is essential since
they form symbiotic associations with various plants, ranging from terrestrial herbaceous
Jin-Hee Kim, Dong-Yeo Kim, Hyeok Park, Jae Hee Cho, Ahn-Heum Eom
64 ∙The Korean Journal of Mycology Vol. 45, No. 1, 2017
and woody plants to aquatic plants, providing beneficial help to the hosts[9]. Because
licorice is an important medicinal plant, it is essential to study the symbiotic presence of
endophytic fungi within licorice roots. In this study, we report an unrecorded species of
fungi identified while isolating endophytic fungi from licorice roots collected in Korea.
Licorice roots cultivated in Jecheon, Korea (N 37° 18΄, E 128° 22΄) were collected and
transported to the laboratory within 48 hours. Only roots without apparent disease
symptoms were selected. The roots were rinsed thoroughly in running tap water and their
surfaces were sterilized with 70% ethanol for 1 min, and 3% NaClO for 1 min and 30 sec.
Then, they were washed three times with sterile water and treated with streptomycin and
chloramphenicol dissolved in sterilized water at a concentration of 100 µm/mL for 10 min.
After removing the remaining moisture from the roots with sterilized filter paper, they were
cut into pieces of 0.5 cm in length. Four root pieces were placed on water agar (WA)
medium and cultured in a dark place at 25°C. The hyphae growing out from the root
fragments were transferred to potato dextrose agar (PDA) medium. The isolate was stored
in 20% glycerol at －80°C at the Mycology Laboratory of Korea National University of
Education (Strain 15C026), Cheongju, Korea and deposited as a glycerol stock at the
Culture Collection of National Institute of Biological Resources (NIBR), Incheon, Korea
(accession number NIBRFG0000499910).
The isolates were cultured on two different media, potato dextrose agar (PDA) and malt
extract agar (MEA). Their characteristics were determined after growth at 25°C for 7 days.
For the study of conidia, the strain mycelium was cultured using a slide culture method and
examined under an optical microscope (AXIO Imager A1; Carl Zeiss, Oberkochen, Germany).
Genomic DNA was isolated from the fungal mycelium using the DNeasy Plant mini kit
(Qiagen, Germantown, MD, USA). A PCR was performed using the following fungal
specific primers: 1) ITS1F and ITS4 to amplify the internal transcribed spacer (ITS) region
[10]; 2) LR0R and LR16 to amplify the large subunit (LSU) region of rDNA[11]; and 3)
EF-526F and EF-1567R to amplify translation elongation factor region (tef1)[12]. The PCR
products were electrophoresed on a 1.5% agarose gel for 20 minutes and then sequenced by
the company SolGent (Daejeon, Korea). The obtained sequences were deposited in GenBank
(accession numbers KY554751) and compared with the pool sequences available in
GenBank using BLAST. Phylogenetic analysis was conducted using the neighbor-joining
method in MEGA 6[13].
Neocosmospora rubicola L. Lombard & Crous, Studies in Mycology 80: 227
(2015) (Fig. 1, Table 1)
Colony grown on PDA at 25°C for 7 days was 35~40 mm in diameter. Its color was white
on both sides. The colony shape was uniformly round and raised at the margin. The surface
of the colony was flat and smooth and there was no exudate. Dense mycelial growth was
observed. Colony grown on MEA at 25°C for 7 days was 20~25 in diameter. It was white
color on both sides, and its shape was uniformly round at the margin. Dense mycelial
growth was observed, with aerial growth at the margins. Ascomatal state and chlamy-
diospores were not observed. The conidiophore was fibrous, simple, and with no branches.
Neocosmospora rubicola, an Unrecorded Endophytic Fungus Isolated from Roots of Glycyrrhiza uralensis in Korea
The Korean Journal of Mycology Vol. 45, No. 1, 2017 ∙65
Fibrous conidia were 6~10 µm long and they had a cylindrical shape with a pointed end
either with or without septum. Macroconidium was not found and microconidia were 0~or
1-septum, fusiform, or elliptical in shape, and 8~22 × 3~6 µm in size.
Specimen examined. Korea, Jecheon, N 37° 18΄, E 128° 22΄, August 14, 2015, isolated
from roots of Glycyrrhiza uralensis, J. Kim, 15C026 (NIBRFG0000499910, GenBank
accession number: KY554751).
Comparison of the ITS seguence of the Korean strain with the sequences of GenBank
using BLAST showed that it shares a 99% similarity to that of Neocosmospora rubicola
KM231800. The LSU region showed a 98% similarity to that of KM231667, and the EF1
region showed a 98% similarity to that of KM231928. A phylogenetic tree showed that N.
rubicola 15C026 isolated from G. uralensis in this study was closely related to CBS320.73
and CBS101018 (Fig. 2).
Neocosmospora rubicola was recently described as a new species by Lombard et al.[14]
after the taxonomic re-evaluation of the large family Nectriaceae (Hypocreales, Pezizomycotina,
EF
A
D
C
B
Fig. 1. Colony of Neocosmospora rubicola 15C026 grown on potato dextrose agar (PDA) (A,
front; B, reverse) and malt extract agar (MEA) (C, front; D, reverse) for 7 days at 25°C,
macrocondium grown on MEA (C) and PDA (D) at the same condition. Microconidia with
1-septum (E) and 0-septum (F) (scale bars: E, F = 10 µm).
Table 1. Morphological characteristics of Neocosmospora rubicola
Characteristics Neocosmospora rubicola 15C026 Neocosmospora rubicolaa
Colony PDA, 25°C, 7 days;
white, reverse concolorous
35~40 mm in diam, arranged in concentric rings, richly
sporulating on the aerial mycelium
PDA, 24°C, 7 days;
abundant white to pale luteous aerial mycelium, reverse
concolorous, 35~40 mm in diam, arranged in concentric
rings, richly sporulating on the aerial mycelium
Conidiophores Simple conidiophores, 20~30 µm long, 3~7 µm at the base,
hyaline, aseptate or septate
13~129 µm long, 3~7 µm at the base, hyaline,
aseptate or septate
Microconidia 0~1-septum, 8~22 × 3~6 µm, ellipsoidal, fusiform or
obovoid, straight to slightly curved,
apex acutely rounded
0~1(~2)-septate, 11~22 × 4~5 µm, ellipsoidal, fusiform or
obovoid, straight to slightly curved, apex acutely rounded,
base sometime flattened
PDA, potato dextrose agar.
aOriginal description by Lombard et al.[14]
Jin-Hee Kim, Dong-Yeo Kim, Hyeok Park, Jae Hee Cho, Ahn-Heum Eom
66 ∙The Korean Journal of Mycology Vol. 45, No. 1, 2017
Ascomycota). Multi-gene phylogenetic analysis based on DNA sequences of ten loci as
well as morphological characteristics were used to identify the new species. N. rubicola has
been isolated from raspberry (Rubus idaeus) and soil, and it has not been reported to cause
any disease in animals or plants[14].
Licorice has an important use as a medicinal plant. However, endophytic fungi within
licorice have not been studied in Korea. These studies are crucial to secure biodiversity and
fungal resources. In addition, these endophytic fungi may be used as a natural source of
pharmacologically active substances. In fact, some studies report that endophytic fungi
produce secondary metabolites with anti-inflammatory activities. Therefore, further efforts
to identify endophytic fungi in medicinal plants will allow a better understanding of the
symbiotic relationship between plants and fungi.
Acknowledgements
This work was supported by the Project on Survey and discovery of Indigenous Species
of Korea funded by National Institute of Biological Resources of the Ministry of
Environment (MOE), Republic of Korea.
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Neocosmospora rubicola, an Unrecorded Endophytic Fungus Isolated from Roots of Glycyrrhiza uralensis in Korea
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