RESEARCH ARTICLE
Mycobiology 40(1) : 14-19 (2012)
© The Korean Society of Mycology
http://dx.doi.org/10.5941/MYCO.2012.40.1.014
pISSN 1229-8093
eISSN 2092-9323
New Record of Mariannaea elegans var. elegans in Korea
Longqing Tang1, Min Woo Hyun1, Yeo Hong Yun1, Dong Yeon Suh1, Seong Hwan Kim1,2* and Gi Ho Sung3
1
Department of Microbiology, Dankook University, Cheonan 330-714, Korea
Institute of Basic Sciences, Dankook University, Cheonan 330-714, Korea
3
Mushroom Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Suwon 404707, Korea
2
(Received March 13, 2012. Accepted March 16, 2012)
A Mariannaea fungus was isolated during investigation of an elm tree infested with unidentified beetles. Based on morphological characteristics and molecular analysis of the internal transcribed spacer rDNA sequence, the fungus was identified
as Mariannaea elegans var. elegans. Fungal growth was better on malt extract agar than on potato dextrose agar and oatmeal
o
agar. Optimal temperature and pH for growth of the fungus were 30 C and pH 7.0, respectively. The fungus was found
to have the ability to produce extracellular enzymes such as amylase, β-glucosidase, cellulase, and protease. This is first
report on M. elegans var. elegans in Korea.
KEYWORDS : Elm Tree, ITS rDNA, Mariannaea elegans var. elegans
Introduction
Materials and Methods
The genus Mariannaea is comprised of eight species.
Mariannaea species is widespread and has frequently
been isolated from soil, decaying bark of pine, submerged
wood in freshwater streams, and from insects [1-3].
M. elegans is the representative species of the genus
Mariannaea. This species was described in 1974 by
Samson as the type species of the genus [1]. M. elegans
has two varieties, M. elegans var. elegans and M. elegans
var. punicea. In 1991, the teleomorph of M. elegans was
reported as Nectria mariannaeae by Samuels and Seifert
[2]. In Korea, M. elegans was reported in 2004 as the
anamorph stage of Cordyceps pruinosa, an entomopathogenic
mushroom [4]. However, in 1991, the anamorph of C.
pruinosa was reported as Mariannaea pruinosa by Liang
[5]. In a recent study, Sung et al. [6] reported on their
acceptance of M. pruinosa as the anamorph of C.
pruinosa. Thus, currently, no official report has been
published on M. elegans in Korea.
A species of Mariannaea was recently isolated during
investigation of an elm tree infested with unidentified
beetles. In this study, we report on identification of the
isolated Mariannaea species and analysis of the growth
properties and ability of its mycelia to produce extracellular
enzymes.
Isolation of fungi. Several pieces of wood were taken
as samples from an elm tree infested with unidentified
beetles in Asan City in the Chungchungnamdo (province)
of Korea in September, 2010 (Fig. 1A and 1B). Several
unidentified beetle larvae were captured from the egg
gallery of the beetle found in the sample wood pieces.
One example is shown in Fig. 1C. For isolation of fungi,
a few of the captured beetle larvae were washed with
sterile water and dried for few minutes in a bio-safety
cabinet. They were then placed on malt extract agar (MEA;
Difco, Detroit, MI, USA) supplemented with streptomycin
(200 µg/mL). Some of the captured beetle larvae were
placed directly on MEA supplemented with streptomycin
(200 µg/mL). All larvae containing MEA were incubated
at 25oC for 3~7 days. Using sterile needles, mycelia
grown out from larvae containing MEA were transferred
to new MEA plates, followed by incubation. Single
spore isolates were obtained from fungi grown on the
incubated MEA. Pure cultures of the isolates were
maintained on MEA and 10% glycerol stock cultures
were prepared. Following comparison of the colony
pattern on MEA and microscopic examination of
microstructure of the pure cultures, an isolate coded as
DUCC400 with morphology resembling that of Mariannaea
*Corresponding author <E-mail : piceae@dankook.ac.kr>
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium,
provided the original work is properly cited.
14
Mariannaea elegans var. elegans in Korea
was used for subsequent study. The DUCC400 isolate was
deposited in the Dankook University Culture Collection
(DUCC), Cheonan, Korea.
Microscopic analysis. A phase-contrast microscope
(Axioskop 40; Carl Zeiss, Jena, Germany) and scanning
electron microscope (SEM, Hitachi S-430; Hitachi, Tokyo,
Japan) were used for observation of morphological
characteristics of the isolate DUCC400. Examination of
fungal structures was based on fresh materials prepared on
o
MEA and potato dextrose agar (PDA) at 25 C for 7~14
days. For observations using a SEM, culture agar blocks
were cut from MEA plates and fixed with 2% glutaraldehyde
in 0.1 M cacodylate buffer for 12 hr and then 1% osmic
acid for 1 hr. Fixed samples were washed with 0.05 M
cacodylate buffer, followed by dehydration in a series of
different concentrations of ethanol from 50% to 100% for
20 min each. The samples were subsequently dried with a
Hitachi critical point dryer and coated with platinumpalladium for 50 sec using a Hitachi E-1030 ion sputter,
followed by observation using a SEM.
Molecular analysis. Using the drilling method described
by Kim et al. [7], mycelia of the isolate DUCC400
freshly grown on PDA for 7 days were collected for DNA
extraction. Fungal specific primers internal transcribed
spacer (ITS) 1F and ITS4 were used for amplification of
the ITS rDNA sequence of the isolate DUCC400 [8, 9].
PCR amplification was performed in a 50 µL reaction
mixture containing 100 ng fungal genomic DNA, 20 pmol
of each primer, 10 mM of the four deoxynucleotide
triphosphates (dNTPs), 1× PCR buffer (10 mM Tris-Cl
[pH 8.0], 1.5 mM MgCl2, 50 mM KCl), and 1.0 unit Taq
DNA polymerase (Promega, Madison, WI, USA). PCR
o
conditions were as follows: denaturation at 94 C for
o
10 min, followed by 30 cycles of denaturation at 94 C for
o
o
1 min, annealing at 56 C for 1 min, and extension at 72 C
o
for 1 min, and one final cycle of extension at 72 C for
10 min. A PCR clean-up kit (Qiagen, Hilden, Germany)
was used for purification of the PCR product, followed
subsequently by ligation into T&A cloning vectors (RBC,
Taipei, Taiwan). The ligated vectors were transformed into
competent Escherichia coli DH5α cells, according to the
manufacturer’s instructions (RBC).
DNA Sequencing was performed at Macrogen Inc.
(Seoul, Korea) using an ABI 3700 automated sequencer
(Perkin-Elmer Inc, Waltham, MA, USA). The ITS
sequence obtained was blasted in the GenBank database
(http://www.ncbi.nlm.nih.gov). Phylogenetic analysis based
on the ITS sequence was performed using the neighborjoining method implemented in the PAUP* 4.0 program
[10], and bootstrap values were determined through
heuristic searches with 1,000 replicates. Nectria radicicola
was used as an out-group.
15
Assessment of mycelial growth and the capacity for
extracellular enzyme secretion. Mycelial growth on
solid medium was assessed on PDA, MEA, and oatmeal
agar (OA). Optimal temperature and pH for mycelial
growth were tested on MEA. The capacity for secretion of
extracellular enzymes was evaluated on chromogenic
reaction medium containing 0.5% CM-cellulose (SigmaAldrich, St. Louis, MO, USA), D-cellobiose (SigmaAldrich), polygalacturonic acid (MP Biomedicals, Sanata
Ana, CA, USA), starch (Sigma-Aldrich), xylan (SigmaAldrich), and Avicel (Fluka, Cork, Ireland) as a carbon
source, 0.1% yeast nitrogen base without amino acid (BD,
Franklin Lakes, NJ, USA) as a nitrogen source, 1.5%
agar, and 0.5% Congo Red (Sigma-Aldrich) as a dye.
Protease production was measured on skim milk (Fluka,
Buchs, Switzerland) agar [11]. After cultivation for 14
days at 25oC, colony diameter and the clear zone formed
by fungal growth were measured for determination of the
capacity for extracellular enzyme production.
Results and Discussion
Morphology. Differences were observed in colony
morphology of DUCC400 grown on MEA, PDA, and OA
(Fig. 1D~1F). First, colony color on MEA was white, and
later became yellow and yellow brown with age; the
reverse plate was yellow brown due to diffusion of the
exudate into the surrounding agar. Colonies on PDA were
strongly zonate and mat like. Colonies on OA were
velutinous in appearance but more weekly zonate than
those on PDA. The reverse of colonies on PDA and OA
were yellow brown to dark brown. Vegetative aerial hyphae
of the isolate DUCC400 were hyaline and thin on MEA
(Fig. 1D).
Conidiophores were erect and formed zonation on MEA,
PDA, and OA. Conidiophore structures were irregular and
complex, but usually verticillate, having a stalk that
measured up to 300 µm in length and 3~7 µm in width,
bearing short branches with whorls of 3~6 phialides (Fig.
2A and 2D). Phialides, which measured 9~25 × 2~3 µm,
were flask shaped, hyaline, smooth walled, and sometimes
inflated. Conidia, which measured 4~9 × 2~3.5 µm, were
hyaline, ellipsoidal to fusiform, smooth walled, divergent
in size, measuring from 4 × 2 to 9 × 3.5 µm, widest in the
middle of the cell, and usually formed imbricate chains
having contact with phialides (Fig. 2B, 2D and 2E).
Chlamydospores, which were rarely present in intermediary
chains, were thick walled, roughened, yellow-brown, and
ellipsoidal in shape, and measured 30 × 10 µm (Fig. 2C).
No microstructure of sexual stage was observed, indicating
that the isolate DUCC400 was in anamorphic stage. These
observed morphological properties were compared with
those of M. elegans varieties and M. aquaticola (Table 1).
The isolate DUCC400 was found to be most similar to M.
16
Tang et al.
Fig. 1. Wilted and debarked elm tree branches infested with unidentified beetles (A), a sample piece of elm wood with the gallery
and holes (B), and an unidentified beetle larva captured from a hole in the sample piece of elm wood (C). Morphology of
colonies formed on potato dextrose agar (D), malt extract agar (E), and oatmeal agar (F) after growth of the isolate DUCC
o
400 obtained from the larva shown in (C) for 14 days at 25 C.
Fig. 2. Morphological features of the isolate DUCC400. Conidia and conidiophore structures (A), conidia imbricate chain (B), and
chlamydospores (C) observed using a phase contrast light microscope. Structures of conidiophore (D) and conidia (E)
observed by scanning electron microscope (scale bars: A~E = 10 µm).
elegans var. elegans.
Molecular characterization. The ITS rDNA sequence
of the isolate DUCC400 was determined as 601 bp and
deposited in GenBank with the accession no. JQ690354.
Sequence homology search through the Genbank DNA
database revealed that the isolate DUCC400 showed
100% homology with Nectria mariannaeae (AB099509)
and 99% homology with Marianaea elegans (AY787717).
M. elegans is the anamorph of N. mariannaeae [2];
Mariannaea elegans var. elegans in Korea
17
Table 1. Comparison of morphological characteristics of the isolate DUCC400 with three Mariannaea species
Characteristics
DUCC400 (present study)
M. elegans var. elegansa
M. elegans var. puniceaa
M. aquaticolab
Colony color on MEA
Conidia size (µm)
Conidia shape
Yellow
4~9 × 2~3.5
Ellipsoidal to fusiform,
widest in the middle
of the cell
Present
Yellow-brown
4~6 × 1.5~2.5
Ellipsoidal to fusiform,
widest in the middle
of the cell
Present
Red-purple
4~6 × 1.5~2.5
Ellipsoidal to fusiform,
widest at the 1/4 part
from the apex
Present
Yellowish to dark brown
5~10 × 2~4.5
Ellipsoidal to fusiform
Chlamydospores
Absent
a
Data from Samson [1] in Mycobank (http://www.mycobank.org).
Data from Cai et al. [12] in Mycobank (http://www.mycobank.org).
b
Fig. 3. Phylogenetic position of DUCC400 among Nectira (N) and Mariannaea (M) species. The tree was generated by the
neighbor-joining method based on internal transcribed spacer rDNA sequences. Numbers at nodes represent the percentage
of bootstrap re-samplings based on 1,000 replicates. The bar represents the number of nucleotide substitutions per site.
Nectria radicicola was used as an out-group.
therefore, we could say that the ITS rDNA sequence of
the isolate DUCC400, which did not show its telemorph
in this study, is most homologous to that of M. elegans.
Phylogenetic analysis of ITS rDNA sequences also
positioned the isolate DUCC400 with M. elegans and its
telemorph N. mariannaeae (Fig. 3). M. aquaticola, a
species with morphological similarity to M. elegans, diverged
from the isolate DUCC400. The molecular divergence of
M. elegans from M. aquaticola is in agreement with the
report by Cai et al. [12]. So far, two varieties of M.
elegans, M. elegans var. elegans and M. elegans var.
punicea, have been reported. These two varieties can be
clearly separated based on their capacity for production of
reddish-purple colonies on MEA. M. elegans var. punicea
produced reddish-purple colonies on MEA, but M. elegans
var. elegans did not [13, 14]. The isolate DUCC400 did
not show a reddish-purple color, but rather a yellow color
on MEA (Table 1, Fig. 1D). The isolate DUCC400 was
well separated from M. elegans var. punicea in the
phylogram (Fig. 4). These molecular data support that the
isolate DUCC400 is not M. elegans var. punicea. Therefore,
based on morphological (Table 1) and molecular data
(Fig. 4), we identified the isolate DUCC400 as M. elegans
var. elegans.
Growth and biochemical properties. Results of the
mycelia growth test showed that the isolate DUCC400
was moderately faster on MEA than on PAD and OA.
The optimal temperature and pH for mycelial growth of
o
the isolate DUCC400 on MEA was 30 C and pH 7,
respectively (Fig. 4). Fungal growth was slightly retarded
at pH 4; however, it grew moderately well at pH 5 to 10.
Results from assessment of the capacity for production
of extracellular enzyme demonstrated that the isolate
DUCC400 possessed diverse extracellular enzyme activity
for all substrates used in the study. In particular, the
isolate DUCC400 showed relatively higher β-glucosidase
and amylase activity, when compared with other extracellular
enzymes, such as CM-cellulase, Avicelase, and xylanase
(Fig. 5). So far, there has been no report on the capacity
for production of extracellular enzymes by M. elegans var.
elegans. Considering that the isolate DUCC was isolated
from a beetle’s larva found in elm wood, it is agreeable
that the fungus shows the ability to degrade wood
components. Questions remain with regard to how M.
elegans var. elegans can occur in an elm tree infested with
18
Tang et al.
Fig. 4. Mycelial growth of the isolate DUCC400 on different media at a temperature of 30oC (A), on malt extract agar (MEA) at
o
different temperature (B) and pH at 30 C for 10 days (C). PDA, potato dextrose agar; OA, oatmeal agar.
Fig. 5. Extracellular enzyme activities of the isolate DUCC400 shown on chromogenic reaction medium containing each enzymatic
substrate. A, CM-cellulase; B, xylanase; C, Avicelase; D, pectinase; E, β-glucosidase; F, amylase; G, protease.
beetle’s larva, where the DUCC was isolated. Therefore,
further study of the ecological role of M. elegans var.
elegans in association with the elm tree is needed.
In conclusion, by combining analyses of its morphological
characteristics and phylogenetics, this study indentified
and characterized the isolate DUCC as M. elegans var.
Mariannaea elegans var. elegans in Korea
elegans. This is first report on its description in Korea.
Acknowledgements
This study was supported by a grant from the National
Institute of Biological Resources, the Ministry of
Environment, and by a grant from the Next Generation
BioGreen 21 program (Project No. PJ0081542011), Rural
Development Administration, Republic of Korea.
7.
8.
9.
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