Characterization of Paecilomyces variotii and Talaromyces amestolkiae in Korea Based on the Morphological Characteristics and Multigene Phylogenetic Analyses

Abstract

During fungal diversity surveys of the order Eurotiales in Korea, two fungal strains, EML-DG33-1 and EML-NCP50, were isolated from samples of rat dung and fig tree leaf collected at a garden located in Gwangju in 2014. To complete the National Species List of Korea, it is a prerequisite to verify whether many questionable species, which were previously recorded but not confirmed, indeed present in Korea. Herein, the isolates were confirmed as undescribed species, Paecilomyces variotii and Talaromyces amestolkiae based on the combination of morphological and phylogenetic analyses of multigenes including the rDNA internal transcribed spacer, β-tubulin, and RNA polymerase II subunit 2.

Full text

248
Mycobiology
Characterization of Paecilomyces variotii and
Talaromyces amestolkiae in Korea Based on the
Morphological Characteristics and Multigene
Phylogenetic Analyses
Thi Thuong Thuong Nguyen , Narayan Chandra Paul and Hyang Burm Lee *
Division of Food Technology, Biotechnology & Agrochemistry, College of Agriculture & Life Sciences, Chonnam National University,
Gwangju 61186, Korea
Department of Herbal Crop Research, National Institute of Horticultural and Herbal Science, Eumseong 27709, Korea
Abstract During fungal diversity surveys of the order Eurotiales in Korea, two fungal strains, EML-DG33-1 and EML-NCP50,
were isolated from samples of rat dung and fig tree leaf collected at a garden located in Gwangju in 2014. To complete the
National Species List of Korea, it is a prerequisite to verify whether many questionable species, which were previously recorded
but not confirmed, indeed present in Korea. Herein, the isolates were confirmed as undescribed species, Paecilomyces variotii
and Talaromyces amestolkiae based on the combination of morphological and phylogenetic analyses of multigenes including the
rDNA internal transcribed spacer, β-tubulin, and RNA polymerase II subunit 2.
Keywords Fig tree leaf, Morphology, Multigene phylogenetic analysis, Paecilomyces variotii, Rat dung, Talaromyces amestolkiae
The order Eurotiales described by Beny and Kimbrough [1]
consists of three families: Trichocomaceae, Thermoascaceae,
and Eremomycetaceae. Especially, the Trichocomaceae
contains species which are important to both industry and
medicine and as mycotoxin producer on various foods [2].
The most well-known genera include Aspergillus, Penicillium,
Paecilomyces, and Talaromyces [2-4].
The genus Paecilomyces (teleomorph, Byssochlamys) in
the Eurotiales was originally described by Bainier [5], based
on a single species, Paecilomyces variotii. This species was
characterized by verticillate conidiophores with divergent
whorls of phialides, which have a cylindrical or inflated
base, tapering to a long and distinct neck. The genus was
revised by Brown and Smith [6] and modified by Samson
[7], who defined 31 species and divided the genus into two
sections, Paecilomyces and Isarioidea. To date, more than
100 species of the genus Paecilomyces have been recognized
[8]. The type species of Paecilomyces, P. variotii, has a
sexual Byssochlamys state [9]. This species is frequently
found in soils, animals, indoor environments, and food
products [7, 10]. Some species of Paecilomyces have been
isolated from insects, and some can even cause infections
in humans [11-13]. Most members of the genus Paecilomyces
have optimum growth temperatures ranging from 30~37
o
C
[9]. Several studies have demonstrated the importance of
Paecilomyces species in various biotechnological applications,
including the production of tannase [6-10] and secondary
metabolites, some of which have useful biological activities
[14, 15].
On the other hand, the genus Tal a r om y c e s in the Eurotiales
was described by Benjamin [16] with Tal a r o m y c e s ve r m i cul a t u s
as the type species. This genus was characterized by soft
cleistothecial ascomata with a wall of interwoven hyphae. It
was re-defined and restricted to species producing asci
in chains by Stolk and Samson [17]. Samson et al. [18]
successfully transferred species of Penicillium subgenus
Biverticillium to Talaromyc e s by following the one fungus
one name concept. On the other hand, Yilmaz et al.
[19] provided a monograph and accepted 88 species of
Research Article
Mycobiology 2016 December, 44(4): 248-259
https://doi.org/10.5941/MYCO.2016.44.4.248
pISSN 1229-8093 • eISSN 2092-9323
© The Korean Society of Mycology
*Corresponding author
E-mail: hblee@jnu.ac.kr
Received December 7, 2016
Revised December 12, 2016
Accepted December 15, 2016
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.

Characterization of Paecilomyces variotii and Talaromyces amestolkiae in Korea 249
Talaro m y ces . Based on internal transcribed spacer (ITS), β-
tubulin, and RBP2 multigene phylogeny, Yilmaz et al. [19]
divided those species in seven sections including: Bacillispori,
Helici, Islandici, Purpurei, Subinflati, Tal ar o m y c e s , and
Tra chysp e r mi. Especially, several species in Tal a r o m y ces
such as T. thermophilus, T. funiculosum, and T. marneffei
show biotechnological and medical important properties
[20].
In Korea, seven species of Paecilomyces and seventeen
species of Talaromyc e s have been reported in Korea, of
which only three Paecilomyces and five Talaromyc e s species
have been well described, excluding P. variotii and T.
amestolkiae [21]. In the Korean fungal species list published
by the National Institute of Biological Resources (NIBR),
the Paecilomyces variotii species is listed as an undescribed
record which does not match any references.
During fungal diversity surveys of the order Eurotiales
in Korea, two fungal strains, EML-DG33-1 as a dung
fungus and EML-NCP50 as an endophyte, were isolated
from samples of rat dung and fig tree leaf collected at a
garden located in Gwangju in 2014. The objective of the
present study was to clarify the phylogenetic status of the
polyphyletic species of P. variotii and T. amestolkiae and to
confirm them as undescribed species in Korea based on
the morphological and multigene phylogenetic analyses.
MATERIALS AND METHODS
Isolation of fungal strains from rat dung and fig tree
leaves. Rat dung samples were collected using sterile
forceps, from the garden of Chonnam National University,
Gwangju, Korea, in 2014. The samples were transferred to
the laboratory in plastic bags, placed on sterile moist
Whatman’s filter paper in Petri dishes, and incubated in
a moist chamber at 25
o
C for 7 days. Hyphal tips were
transferred to potato dextrose agar (PDA; Difco, Franklin
Lakes, NJ, USA) plates using a stereomicroscope. To isolate
pure cultures, individual colonies of varied morphologies
were transferred to PDA plates. On the other hand, leaf
samples of fig tree (Ficus sp.) were collected from the campus
of Chonnam National University, Gwangju, Korea, in 2014.
Collected samples were stored in sterile polyethylene bags
where they were cleaned under running tap water to
remove debris before use, air dried, and then processed for
isolation of endophytic fungi. The leaves were cut into
small, 1 cm long and 0.5 cm wide pieces. Tissue pieces were
surface-sterilized with 2% sodium hypochlorite for 3 min
and with 70% ethanol for 1min, and then washed three times
with sterile distilled water. The surface-sterilized samples
were allowed to dry on sterile paper towel in a laminar
airflow chamber. Ten fragments from each leaf were placed
onto PDA and Rose Bengal chloramphenicol agar (Difco)
supplemented with the antibiotic, streptomycin sulfate (0.4
mg/mL; Sigma-Aldrich, Munich, Germany), to suppress
bacterial growth. After incubation at 25
o
C for 5 days,
individual hyphal tips of the developing fungal colonies
were removed and placed onto in PDA medium to be
incubated for 5~10 days.
To isolate pure cultures, individual colonies of varied
morphologies were transferred to PDA plates. Pure isolates
of P. variotii (EML-DG33-1 and EML-DG33-2) and T.
amestolkiae (EML-NCP50) were maintained on PDA
slant tubes and also stored in 20% glycerol at −80
o
C at
the Environmental Microbiology Laboratory Fungarium,
Chonnam National University, Gwangju. Dry cultures
(EML-DG33-1 and EML-NCP50) were preserved at
Chonnam National University Fungal Collection (CNUFC),
Division of Food Technology, Biotechnology & Agrochemistry,
College of Agriculture & Life Sciences, Gwangju. Duplicates
of EML-DG33-1 and EML-NCP50 were also deposited as
glycerol stock at −80
o
C at the Culture Collection of NIBR,
Incheon, as KOSPFGC000002024 and KOSPFGC000002029,
respectively.
Morphological studies. To obtain samples for microscopic
examination and growth rate determination, EML-DG33-1
and EML-NCP50 were cultured on each of the three
different media. The media used were malt extract agar
(MEA; 30 g malt extract, 1 g peptone, 20 g glucose, 0.005 g
CuSO
4
·5H
2
O, 0.01 g ZnSO
4
·7H
2
O, and 20 g agar, in 1 L of
deionized water), Czapeck yeast autolysate agar (CYA; 3 g
NaNO
3
, 5 g yeast extract, 30 g sucrose, 1.3 g K
2
HPO
4
·3H
2
O,
0.5 g MgSO
4
·7H
2
O, 0.5 g KCl, 0.01 g FeSO
4
·7H
2
O, 0.005 g
CuSO
4
·7H
2
O, 0.01 g ZnSO
4
·7H
2
O, and 20 g agar, in 1 L of
deionized water), and yeast extract sucrose agar (YES; 20 g
yeast extract, 150 g sucrose, 0.5 g MgSO
4
·7H
2
O, 0.005 g
CuSO
4
·7H
2
O, 0.01 g ZnSO
4
·7H
2
O, and 20 g agar, in 1 L of
deionized water). Plates were incubated at 20
o
C, 25
o
C,
30
o
C, 35
o
C, and 40
o
C in the dark for 7 days. Samples were
mounted in lactophenol solution (Junsei Chemical Co.
Ltd., Tokyo, Japan) and examined under a light microscope
(DFC290; Leica Microsystems, Wetzlar, Germany). Fine
fungal structures were observed using scanning electron
microscopy (SEM, Hitachi S4700; Hitachi, Tokyo, Japan).
Samples were cultured in PDA medium in the dark at 27
o
C
for 7 days, fixed in 2.5% paraformaldehyde-glutaraldehyde
in 0.05 M phosphate buffer (pH 7.2) for 1 hr, and then
washed with cacodylate buffer (Junsei Chemical Co. Ltd.).
Cellular membranes were preserved by fixing the samples
in 1% osmium tetroxide (Electron Microscopy Sciences,
Hatfield, PA, USA) diluted with cacodylate buffer for 1 hr,
washing again in cacodylate buffer, dehydrating in graded
ethanol (Emsure, Darmstadt, Germany) and isoamyl acetate
(Junsei Chemical Co. Ltd.), and drying in a fume hood.
Finally, samples were sputter-coated with gold and observed
under a Hitachi S4700 field emission scanning electron
microscope at the Korea Basic Science Institute, Gwangju.
DNA extraction, PCR, and sequencing. Total genom ic
DNA was extracted directly from the mycelia using the
HiGene Genomic DNA Prep Kit (BIOFACT Corp., Daejeon,
Korea). The gene sequences of EML-DG33-1 and EML-

250 Nguyen et al.
NCP50-1, consisting of the ITS, Tub2, and RPB2 genes,
were amplified with the primer pairs ITS1, ITS4 [22]; Bt2a,
Bt2b [23]; and RPB2-5F, RPB2-7Cr [20], respectively. The
PCR amplification mixture (total volume of 20 μL) contained
10 ng of fungal genomic DNA template, 5 pmol/μL of each
primer, and Accupower PCR Premix (Taq DNA polymerase,
dNTPs, buffer, and a tracking dye; Bioneer Corp., Daejeon,
Korea). PCR products were purified using the Accuprep
PCR Purification Kit (Bioneer Corp.) according to the
manufacturer’s instructions. DNA sequencing was performed
on an ABI 3700 Automated DNA sequencer (Applied
Biosystems Inc., Foster City, CA, USA).
Phylogenetic analysis. Sequences (Table 1) were subjected
to phylogenetic analysis using Clustal_X v.1.83 [24] and
Bioedit v. 5.0.9.1 software [25]. Phylogenies were assessed
using MEGA 6 software [26]. Maximum likelihood (ML)
phylogenetic trees were constructed for combined datasets
Table 1 . Sequences used in this study, including GenBank accession numbers
Taxon name Collection No.
(isolate No.)
GenBank accession No.
ITS Tub2 RPB2
Aspergillus ochraceoroseus CBS 101887 - - JN121416
A. sydowii CBS 264.81 - - JN121476
A. versicolor CBS 245.65 - - JN121468
Byssochlamys fulva CBS 146.48 FJ389940 FJ389986 -
B. lagunculariae CBS 373.70 FJ389944 FJ389995 -
B. nivea CBS 100.11 FJ389934 FJ389999 JF417414
B. spectabilis CBS 101075
HT
- - JF417446
B. verrucosa CBS 604.74 DQ073329 DQ073328 -
B. zollerniae CBS 374.70 FJ389933 FJ390008 -
Coccidioides immitis RS - - XM_001240649
Eupenicillium crustaceum CBS 344.61
IsoT
- - JF417428
Geosmithia viridis CBS 252.87
HT
- - JF417422
Hamigera avellanea CBS 295.48
IsoT
- - JF417424
Paecilomyces brunneolus CBS 370.70
T
EU037050 EU037068 -
P. d iv ar ic at us CBS 284.48
T
FJ389931 FJ389992 -
P. f or mo su s CBS 990.73E FJ389929 FJ389993 -
P. f or mo su s DTO 45H8 GU968650 GU968683 -
P. f or mo su s DTO 63F1 GU968670 GU968686 -
P. f or mo su s DTO 49D6 GU968655 GU968691 -
P. f or mo su s CBS 372.70 FJ389926 FJ389990 -
P. saturatus CBS 323.34 FJ389947 FJ390005 -
P. v ar io ti i DTO 63D7 GU968659 GU968693 -
P. v ar io ti i CBS 102.74
T
EU037055 EU037073 -
P. v ar io ti i DTO 63D9 GU968661 GU968695 -
P. v ar io ti i CBS 101075 EU037051 EU037069 -
P. v ar io ti i DTO 63E6 GU968667 GU968680 -
P. v a ri ot i i EML-DG33-1 KX060750 KX060751 KY350194
P. v a ri ot i i EML-DG33-2 KX060753 KX060752 KY350195
Penicillium citrinum CBS 139.45
NT
- - JN606604
P. e xp an su m CBS 325.48 - - JF417427
P. g la br um CBS 125543
NT
- - JF417447
P. namyslowskii CBS 353.48 - - JF417430
Sagenomella diversispora CBS 398.69 - - JF417436
S. humicola CBS 427.67
IsoT
- - JF417439
S. griseoviridis CBS 426.67 - - JF417438
S. striatispora CBS 429.67
IsoT
- - JF417440
Rasamsonia argillacea CBS 101.69 - - JF417415
R. byssochlamydoides CBS 413.71 - - JF417437
R. eburnea CBS 100538 - - JN406532
Talaromy ces a m e s t o l k i a e CBS 132696
T
JX315660 JX315623 JX315698
T. am esto l kiae CBS 263.93 JX315669 JX315625 JX315707
T. am esto l kiae CBS 252.31 JX315668 JX315624 JX315706
T. a m e s t o l k i ae EML-NCP50 KU985307 KU985308 KX363885
T. at r o r o s e u s CBS 133442
T
KF114747 KF114789 KF114763
T. bacillisporus CBS 296.48
T
JN899329 AY753368 JN121634

Characterization of Paecilomyces variotii and Talaromyces amestolkiae in Korea 251
of the ITS rDNA, Tub2, and RPB2 gene sequences. The
nearest-neighbor-interchange was selected for the ML
heuristic method, and the initial ML tree was set
automatically. Sequences of Byssochlamys verrucosa and
Trich o c oma para d oxa and Coccidioides immiti were used as
outgroups. The percentage of sequence identity was obtained
using the basic local alignment search tool (BLAST) of the
National Center for Biotechnology Information (NCBI) for
each isolate.
RESULTS
Phylogenetic analysis. The groups of Eurotiales within
ascomycete fungi has been known to be polyphyletic. In
this study, the phylogenetic status of EML-DG33-1 and-2
isolates belonging to Paecilomyces & Byssochlamys clade
and EML-NCP50 isolate belonging to Ta l a romyc e s s. str.
and Penicillium subg. Biverticillium clade within Eurotiales
are shown in the ML tree based on RPB2 sequence analysis
(Fig. 1). The RPB2 sequence of EML-DG33-1 had 99.8%
(971/973 bp) sequence similarity with the known sequence
of a teleomorph of Paecilomyces, Byssochlamys spectabilis
(GenBank accession No. JF417446).
A BLAST search of ITS sequences via the NCBI database
indicated that the ITS sequence of EML-DG33-1 is most
closest to P. variotii DTO 63E (GenBank accession No.
GU968667), with 98.7% (473/479 bp) homology. The Tub2
sequence of P. variotii DTO 63D9 (GenBank accession No.
GU968695) showed 99.1% (447/451 bp) homology to that
of EML-DG33-1. Based on this multigene analysis, the
isolate was identified as P. variotii (Fig. 2). The ITS, Tub2,
and RPB2 sequences of EML-DG33-1 and EML-DG33-2
Table 1 . Continued
Taxon name Collection No.
(isolate No.)
GenBank accession No.
ITS Tub2 RPB2
T. coalescens CBS 103.83
T
JN899366 JX091390 KM023277
T. derx ii CBS 412.89
T
JN899327 JX494306 KM023282
T. ducl auxii CBS 322.48
T
JN899342 JX091384 JN121491
T. emodensis CBS 100536
HT
- - JF417445
T. f l a v u s CBS 310.38
NT
- - JF417426
T. heli c us CBS 335.48
T
JN899359 KJ865725 KM023273
T. leycettanus CBS 398.68
HT
- - JF417435
T. lut e u s CBS 371.87 - - JF417431
T. lut e u s CBS 348.51
NT
- - JF417429
T. min i o l u t e u s CBS 642.68
NT
- - JF417443
T. islandi cus CBS 338.48
T
KF984885 KF984655 KF985018
T. marneffei CBS 388.87
T
JN899344 JX091389 KM023283
T. pseudostromat i c u s CBS 470.70
T
JN899371 HQ156950 KM023298
T. purpureogenus CBS 286.36
T
JX315671 JX315639 JX315709
T. pur pureus CBS 475.71
T
JN899328 GU385739 JN121522
T. ruber CBS 132704
T
JX315662 JX315629 JX315700
T. rugulosu s CBS 371.48
T
KF984834 KF984575 KF984925
T. scorte u s CBS 340.34
T
KF984892 KF984684 KF984916
T. stolli i CBS 408.93
T
JX315674 JX315633 JX315712
T. subi n f latus CBS 652.95
T
JN899397 JX494288 KM023308
T. thai l a n d e n sis CBS 133147
T
JX898041 JX494294 KM023307
T. thermophilus CBS 236.58
HT
- - JF417420
T. tra c h y s p e r mus CBS 373.48
T
JN899354 AY753371 JF417432
T. varians CBS 386.48
T
JN899368 KJ865731 KM023274
T. wortmannii CBS 391.48
NT
- - JF417433
Thermoascus aurantiacus CBS 891.70 - - JF417444
T. crustaceus CBS 181.67
T
- - JF417417
T. thermophilus CBS 528.71
NT
- - JF417442
Thermomyces lanuginosus CBS 218.34 - - JF417418
T. lanu g i n o s u s CBS 224.63 - - JF417419
Tric h o coma p arad o x a NRRL 28276 EU021600 EU021675 EU021633
T. para d o x a CBS 247.57 - - JF417421
Warcupiella spinulosa CBS 512.65
NT
- - JF417441
Bold letters indicate accession numbers determined in our study.
ITS, internal transcribed spacer; CBS, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; HT, holotype; IsoT, isotype; T,
ex-type strain; NT, neotype; EML, Environmental Microbiology Laboratory, Fungarium, Chonnam National University, Gwangju, South
Korea; NRRL, ARS Culture Collection, Peoria, Illinois, USA.

252 Nguyen et al.
were deposited in the NCBI database. The accession numbers
for ITS, Tub2 and RPB2 sequences were KX060750,
KX060751 and KY350194 for EML-DG33-1 isolate, and
KX060753, KX060752, and KY350195 for EML-DG33-2
isolate, respectively. Analysis of the multigene genes placed
the strains of EML-DG33-1 and EML-DG33-2 within the
variotii group (Fig. 2).
BLAST search analysis indicated that the ITS (accession
No. KU985307), Tub2 (accession No. KU985308), and
RPB2 (accession No. KX363885) sequences of EML-
NCP50 had 99.8% (524/525 bp), 100% (427/427 bp), and
100% (1,038/1,038 bp) sequence similarity, respectively,
with the known sequences of T. amestolkiae DTO179F5
(GenBank accession Nos. JX315660, JX315623, and JX315698,
respectively). Analysis of the multigene genes placed the
EML-NCP50 strain within the section Tal a r om y c es (Fig. 3).
Taxono my of E M L-DG 33-1.
Paecilomyces variotii Bainier, Bull. Soc. Mycol. Fr. 23:27
(1907) (Table 2, Fig. 4)
≡Penicillium variotii (Bainier) Sacc., Syll. Fung. 22:1273
(1913)
=Penicillium aureocinnamomeum Biourge, La Cellule
33:213 (1923)
Description: The colonies of EML-DG33-1 exhibited fast
growth on MEA, covering the Petri dish within 7 days at
25
o
C. The color of the colonies was pale yellow and white
at the margins. The color of the colony reverse was greenish
Fig. 1. Phylogenetic relationships within genera Paecilomyces (teleomorph, Byssochlamys) and Tal a ro my c e s of the Trichocomaceae
(Eurotiales) based on Maximum likelihood analysis of RPB2 sequence data. The corresponding sequence of Coccidioides immiti
was used as the outgroup. Bootstrap support values > 50% are indicated at the nodes. The bar indicates the number of
substitutions per position. Classification system presented by Houbraken et al. [3].

Characterization of Paecilomyces variotii and Talaromyces amestolkiae in Korea 253
yellow (Fig. 4A and 4D). Mycelial growth on MEA was
sparse; however, sporulation was extensive. Conidiophores
were mostly short and irregularly branched (Fig. 4G and
4I). Phialides were club-shaped with a long neck, with up
Fig. 2. Phylogenetic tree of the Paecilomyces variotii EML-DG33-1 and EML-DG33-2 within the variotii group, based on
maximum likelihood analysis of combined datasets for internal transcribed spacer rDNA, and Tub2. The corresponding
sequence of Byssochlamys verrucosa was used as the outgroup. Bootstrap support values > 50% are indicated at the nodes. The
bar indicates the number of substitutions per position. Classification presented by Houbraken et al. [27]. *Asterisk indicates
classification system suggested by authors.
Fig. 3. Phylogenetic tree of the Ta l a r o m y ces amestolkiae EML-NCP50 within the Sect. Ta l a romyce s based on maximum
likelihood analysis of combined datasets for internal transcribed spacer rDNA, Tub2, and RPB2. The corresponding sequence of
Tri c h ocom a p ara d oxa was used as the outgroup. Bootstrap support values > 50% are indicated at the nodes. The bar indicates
the number of substitutions per position.

254 Nguyen et al.
to seven in a whorl, and measured 8.5~20.5μm in length
(Fig. 3H). Conidia were ellipsoidal to cylindrical in chains,
yellow in color, and measured 4.0~6.0 ×2.5~4.5 μm (Fig. 4K).
Colonies on YES grew faster than those on CYA, and
attained a diameter of 53~56 mm after 7 days at 25
o
C. The
color of the colonies was deep yellow, and the color of the
colony reverse was dull brown. Conidia grown on YES
were often slightly larger than those grown on CYA (Fig.
4B and 4E). Colonies on CYA grew very slowly, and
attained a diameter of 40~42 mm after 7 days at 25
o
C. The
color of the colonies was light yellow, and the color of the
colony reverse was brownish yellow in the center and
white at the margins (Fig. 4C and 4F).
Culture characteristics: The isolate was observed to grow
over a wide range of temperatures with varying growth
rates on MEA, YES, and CYA. The average growth rates on
MEA, YES, and CYA were 13.5, 8.0, and 6.0 mm/day,
respectively. The optimum growth temperature range was
30~35
o
C, slow growth was observed at below 20
o
C, and
could grow well at temperatures as high as 40
o
C.
Specimen examined: Republic of Korea, Jeonnam Province,
garden of the Chonnam national University located in
Fig. 4. Morphological and conidial characteristics of the Paecilomyces variotii isolate, EML-DG33-1. A, D, Colonies on malt
extract agar; B, E, Colonies on yeast extract sucrose agar; C, F, Colonies on Czapeck yeast autolysate agar. A~C, Obverse view;
D~F, Reverse view; G~J, Branched conidiophores and club-shaped phialides (arrows); K, Conidial chain (scale bars: G = 50 µm,
H~J = 20 µm, K = 10 µm).
Table 2 . Morphological comparison of the isolate EML-DG33-1 and closely related species
Characteristic Present isolate Paecilomyces variotii
a
Colony Fast-growing, covering the Petri dish within 7 days
at 25
o
C, pale yellow; reverse: greenish yellow
Fast-growing, attaining a diameter of 40 mm after 7 days,
light yellowish olive; reverse: yellow to yellow-brown
Phialide 8.5~20.5 μm long, club-shaped showing long neck,
up to 7 in a whorl
12~20 × 2.5~5.0 μm, cylindrical to ellipsoidal
Conidia 4.0~6.0 × 2.5~4.5 μm, ellipsoidal to cylindrical in
chains, light yellow
3.2~5.0 × 2.0~4.0 μm, ellipsoidal to cylindrical, hyaline to
yellow
a
From the description by Samson [7].

Characterization of Paecilomyces variotii and Talaromyces amestolkiae in Korea 255
(Fig. 5C and 5F). On the YES agar, good sporulation on
colonies was observed, whereas pigments and exudates
were absent. The colonies were 34~36 mm in diameter.
The mycelium on the YES agar was white; it exhibited
moderately dense sporulation, narrow margins, and tufts
(Fig. 5B and 5E). On the MEA agar, the mycelium was
white, sometimes red in the center. Diameter of colonies
that had regular wide margins varied within 34~53 mm.
Sporulation varied from moderately dense to dense.
Soluble pigment was absent (Fig. 5A and 5D).
The texture, conidiophores, conidia, metulae, phialide,
and stripes were examined under a compound light
Gwangju (35
o
10' N, 126
o
55' E), from a rat dung, 10 Sep
2014 (EML-DG33-1), deposited at the Culture Collection
of NIBR, Incheon, as KOSPFGC000002024.
Taxonomy of EML-NCP50.
Talaromyces amestolkiae Yil m az, Houbra k e n, Fris v ad &
Samson, Persoonia 29: 48 (2012) (Table 3, Fig. 5)
Description: Colonies were characterized after 7 days at
25
o
C. On the CYA agar, colonies were 21~24 mm in
diameter and raised in the center. They had wide, regular
margins (2~3 mm), white or yellow mycelium, which was
reddish in the center, and moderately dense sporulation
Fig. 5. Morphological characteristics of the Tal a r o m y ces amestolkiae isolate, EML-NCP50. Colonies on malt extract agar; B, E,
Colonies on yeast extract sucrose agar; C, F, Colonies on Czapeck yeast autolysate agar. A~C, Obverse view; D~F, Reverse view;
G~K, Conidiophores; L, Conidiophore and conidia (scale bars: G = 50 µm, H~J = 20 µm, K, L = 10 µm).
Table 3 . Morphological comparison of the isolate EML-NCP50 and closely related species
Characteristic Present isolate Talaromyces amestolkiae
a
Colony on CYA Slow-growing, attaining a diameter of 21~24 mm after 7
days, mycelium white and yellow; reddish in the center.
Slow-growing, attaining a diameter of 29~30 mm after
7 days, mycelium white and yellow; red in the center.
Phialide 3~4 in total, acerose, 9.0~13.5 ×1.5~2.5 μm 3~6 in total, acerose, 9.5~12.0 × 2.5~3.0 μm
Metulae In verticils of 3~5, 9.5~13.0 × 2.0~3.5 μm In verticils of 3~5, 9.5~14.0 × 3.0~4.0 μm
Conidia Smooth-walled, single-celled, ellipsoidal, 2.5~4.0 × 1.0~
3.5 μm
Smooth or some rough-walled, ellipsoidal, 2.0~3.0 ×
1.5~2.5 μm
a
From the description by Yilmaz et al. [19].

256 Nguyen et al.
microscope and SEM. Densely textured conidiophores were
borne solitary or in small fascicles, simple or branched,
smooth-walled, and pale to light brown in color (Fig. 5G and
5J). Conidia were smooth-walled, single-celled, ellipsoidal,
and their size varied in the range of 2.5~4.0 × 1.0~3.5 μm
(Fig. 5L). Metulae were arranged in verticils of 3~5; their
length ranged from 9.5~13.0 μm and the width varied
from 2.0~3.5 μm. There were 3~4 acerose phialides in total
and their size varied in the range of 9.0~13.5 × 1.5~2.5 μm.
Branches of stripes (2~3) were observed; they were smooth-
walled, 86~201 μm long, and 2.0~3.0 μm wide.
Culture characteristics: The isolate was observed to grow
over a wide range of temperatures with varying growth
rates on MEA, YES, and CYA. The average growth rates
on MEA, YES, and CYA were 6.5, 5.0, and 4.8 mm/day,
respectively. Optimal growth was observed around 25~
30
o
C, slow growth was observed at below 20
o
C, and no
growth at 40
o
C (Fig. 6).
Specimen examined: Republic of Korea, Jeonnam Province,
garden of the Chonnam national University located in
Gwangju (35
o
10' N, 126
o
55' E), from a figs leaf, June 2014
(EML-NCP50), and deposited at the Culture Collection of
NIBR, Incheon, as KOSPFGC000002029.
DISCUSSION
In this study, we combined a morphological description
with a multigene analysis to assess the phylogenetic placement
of a poorly reported species, Paecilomyces variotii in Korea.
Although the species of Paecilomyces variotii has been only
mentioned in several studies in Korea [15, 21, 28, 29], there
were no phylogenetic placement analyses or morphological
descriptions or official record. Especially, the species of P.
variotii has been used as a test microorganism in Korea,
but it has not been described as undescribed species [30].
Because of lack of clear information about the species of P.
variotii, confirmation of this species as undescribed species
in Korea based on the detailed descriptions of the morphology
and multigene phylogenetic analyses are required.
Dung is not only a rich source of nutrients, including
carbohydrates, nitrogen, vitamins, minerals, and growth
factors, but also contains a high amount of water with a
neutral pH of around 6.5 [31, 32]. Thus, it is considered a
good substratum for fungal growth in a niche. A number
of studies on the diversity of fungi have been reported with
regard to different animal dung substrates [33-35]. Nyberg
and Persson [36] observed 24 species in 14 genera in
mouse dung, whereas Richardson [37] found 32 species in
17 genera from the dung of sheep, deer, cattle, rabbit, hare,
and grouse. Although species of rat are among the most
common animals worldwide, there is lack of information
regarding the Paecilomyces species occurring in rat dung.
Recently, the species P. v ar io ti i was isolated from the hair
of golden hamster by Bagy et al. [38]. Notably, their study
indicated that P. v ar io t ii and Aspergillus niger were the
dominant groups of fungi recovered from the hair of golden
hamsters. Furthermore, many species of fungi that have
been isolated from dung produce secondary metabolites
[32, 39, 40], such as appenolides A~C and coniochaetone-
A, produced by Podospora appendiculata isolated from deer
dung and Coniochaeta saccardoi isolated from lemming
dung, respectively.
The ability of different species of the genus Paecilomyces
to produce mycotoxin and other biological metabolites has
been reported in previous studies [9]. According to Bokhari
et al. [41], P. v ar i o ti i produces the genotoxic mycotoxin,
patulin. Peptide mycotoxins known as leucinostatins, with
activity against fungi, are extracted from P. li la c in u s and
possess high toxicity to experimental animals [42]. In
addition, Paecilomyces was used as a biological control.
Perveen et al. [43] reported that the Paecilomyces species
exhibit antifungal activity against Sclerotium rolfsii and
Pythium aphanidermatum; whereas, P. li la ci nu s affects
nematodes that attack plant roots [44].
In recent years, endophytes have received considerable
scientific attention because they have been recognized as
biological control agents. Vaz et al. [45] screened endophytic
fungi from the leaves of Myrciaria floribunda, Alchornea
Fig. 6. Effect of temperature and culture medium on mycelial growth of Paecilomyces variotii EML-DG33-1 and Tal a r o myce s
amestolkiae EML-NCP50. Mycelia were grown on malt extract agar (MEA), Czapeck yeast autolysate agar (CYA), and yeast
extract sucrose agar (YES) at different temperatures, as indicated.

Characterization of Paecilomyces variotii and Talaromyces amestolkiae in Korea 257
castaneifolia, and Eugenia aff. bimarginata to examine their
antimicrobial activity against pathogenic microorganisms.
Thirty-eight fungal extracts exhibited antimicrobial activity
against at least one of the target microorganisms tested.
Similar results were obtained by Paul et al. [46, 47], who
showed that non-pathogenic endophytic fungi might reduce
the growth of pathogenic C. acutatum, F. ox y s po r um , and
Phytophthora capsici. In this study, the isolate EML-DG33-
1 showed antifungal activity against A. alternata and F.
oxysporum. The endophytic isolate EML-NCP50, weakly to
moderately inhibited the growth of eight fungal pathogens
(data not shown). This result is consistent with previous
reports demonstrating that many endophytes exhibit
antifungal activity against different pathogenic fungi [45].
Our study suggests that the strains, EML-DG33-1 and
EML-NCP50, might be a source of biologically active
secondary metabolites and biological control agents.
In addition, in the present study, we demonstrated the
thermal tolerance of this EML-DG33-1 strain. In comparison
with previous results [7], the colony morphology and
cultural characteristics of the present isolate on MEA
medium was similar to that of P. variotii.
Based on the sequences of the 18S and ITS rDNA
regions, Luangsa-ard et al. [48] and Inglis and Tigano [49]
demonstrated that Paecilomyces is polyphyletic across two
Ascomycetes orders, the Eurotiales and the Hypocreales.
Luangsa-ard et al. [48] analyzed the 18S rDNA and showed
that the species P. v ar io ti i and its thermophilic relatives
belong to the order Eurotiales (Trichocomaceae), and the
mesophilic species related to P. f ar in os us are in the order
Hypocreales (Clavicipitaceae and Hypocreaceae). In another
aspect, Samson et al. [9] studied the genus Byssochlamys and
its Paecilomyces anamorphs using a polyphasic approach to
differentiate the species by analyzing each of ITS region,
parts of the Tub2 and calmodulin gene. Our results showed
that the strains EML-DG33-1 and EML-DG33-2 belonged
to a variotii group containing P. v ar i ot i i species. There
was only two RPB2 sequences data in the Paecilomyces &
Byssochlamys clade available in GenBank (Fig. 1). The
results of phylogenetic analysis showed that this species
belongs to the same clade presented by Houbraken et al.
[27].
To our k nowl edg e , th e genu s Tal a r o myce s has been
frequently isolated from soil and root aquatic in Korea [50,
51]. Figs trees are rarely found in Korea. There have been
no studies of fungal endophytes on the host in Korea.
Thus, this finding suggest that figs tree may be useful
source of fungal endophytics. Endophytic microorganisms
have been discovered in all plant families, representing
various species in different climatic regions of the world
[52]. Species of fig tree (Ficus sp.) are native throughout
the tropical region with several species extending into the
semi-warm temperate zones. In 2001, Suryanarayanan and
Vijaykrishna [53] isolated the endophytes from the aerial
root of fig tree (Ficus benghalensis) in India. Wang et al.
[54] investigated the fungal diversity on fallen leaves of
Ficus in northern Thailand in 2008.
So far, several studies to evaluate and compare the
phylogenetic relationships between some species belonging
to the genus Ta l a ro m yc e s have been carried out, using the
ITS region, β-tubulin, and RPB2 [18, 19]. Phylogenetic
analyses of three genes showed that EML-NCP50 belonged
to the genus Talar o my c e s , including T. amestolkiae, T. stollii,
and T. ruber. Morphological characteristics of the isolate
EML-NCP50 were similar to those of T. amestolkiae described
by Yilmaz et al. [19]. Therefore, these results confirmed
that the isolate EML–NCP50 belongs to the species T.
amestolkiae within sect. Tal a r o m y ces.
Some genera including Paecilomyces and Tal a r o myc e s
belonging to Eurotiales within class Ascomycetes are
polyphyletic (Fig. 1). Thus, to include unrecorded species
in a national species list, it is very important to confirm
the first records of poorly known species based on the
detailed morphological descriptions and the current
molecular phylogenetic placement analyses.
ACKNOWLEDGEMENTS
This work was supported by the Project on Survey and
Discovery of Indigenous Species of Korea funded by NIBR
of the Ministry of Environment, and in part by a fund
from the National Institute of Animal Science under Rural
Development Administration, Republic of Korea. We are
grateful to Dr. Paul Kirk from Royal Botanic Gardens, Kew,
United Kingdom, for kind review of the manuscript.
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