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Mycobiology
ISSN: 1229-8093 (Print) 2092-9323 (Online) Journal homepage: https://www.tandfonline.com/loi/tmyb20
Molecular and Morphological Characterization of
Two Novel Species Collected from Soil in Korea
Kallol Das, Seung-Yeol Lee & Hee-Young Jung
To cite this article: Kallol Das, Seung-Yeol Lee & Hee-Young Jung (2019): Molecular and
Morphological Characterization of Two Novel Species Collected from Soil in Korea, Mycobiology,
DOI: 10.1080/12298093.2019.1695717
To link to this article: https://doi.org/10.1080/12298093.2019.1695717
© 2019 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group on behalf of the Korean Society of
Mycology.
Published online: 10 Dec 2019.
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RESEARCH ARTICLE
Molecular and Morphological Characterization of Two Novel Species
Collected from Soil in Korea
Kallol Das
a
, Seung-Yeol Lee
a,b
and Hee-Young Jung
a,b
a
College of Agriculture and Life Sciences, Kyungpook National University, Daegu, Korea;
b
Institute of Plant Medicine, Kyungpook
National University, Daegu, Korea
ABSTRACT
Two fungal species of ascomycetes were discovered during the screening of soil microflora
from the Gangwon Province in Korea: Didymella chlamydospora sp. nov. (YW23-14) and
Microdochium salmonicolor sp. nov. (NC14-294). Morphologically, YW23-14 produces smaller
chlamydospores (8.0–17.0 7.0–15.0 mm) than D. glomerata and D. musae. The strain NC14-
294 was characterized by smaller conidiogenous cells (4.9–8.8 2.0–3.2 mm) compared with
the closest strains M. fisheri and M. phragmitis. The detailed descriptions, illustrations, and
discussions regarding the morphological and phylogenetical analyses of the closely related
species are provided to support the novelty of each species. Thus, YW23-14 and NC14-294
are described here as newly discovered species.
ARTICLE HISTORY
Received 21 June 2019
Revised 6 November 2019
Accepted 14 November 2019
KEYWORDS
Didymella chlamydospora;
Microdochium salmonicolor;
novel species
1. Introduction
Didymellaceae, the largest family established in
Pleosporales of Ascomycota, with more than 5400
taxon names listed in MycoBank and consisting of
three main genera, viz. Ascochyta,Didymella and
Phoma, and other allied Phoma-like genera [1,2].
The first generic level of Didymella was used by
Saccardo in 1880, with the description of Didymella
exigua [3,4]. The limits of Didymellaceae, redefined
the genera Epicoccum,Peyronellaea and
Stagonosporopsis, and established the genus
Boeremia and the taxonomic revision of Didymella
is necessary, especially because of its phytopatho-
logical importance [5]. Recently, a revision has been
published under the family of Didymellaceae,
encompassing 17 well supported monophyletic
clades which were treated as individual genera [6].
The correct species identification in this family has
always proven difficult, chiefly relying on morph-
ology and plant host association [5,6]. However, the
internal transcribed spacer regions intervening 5.8S
nrDNA (ITS), partial 28S large subunit nrDNA
(LSU) sequences, and partial regions of RNA poly-
merase II second largest subunit (RPB2) and
b-tubulin (TUB2) genes provide a relatively robust
phylogenetic backbone for taxonomic determination
[6]. Moreover, the species of Didymellaceae are
cosmopolitan and distributed throughout a broad
range of environments and most of the members in
this family are plant pathogens of a wide range of
hosts, mainly causing leaf and stem lesions; some
are of quarantine significance [5–8].
The genus Microdochium was introduced with
the isolation of species of M. phragmitis from living
leaves of Phragmites australis in Germany [9].
Microdochium species are recognized as Fusarium-
like fungi, nevertheless, the conidiogenous cells are
not phialidic as in true Fusarium species and
the conidia have a truncate base rather than
‘foot-cells’. The sexual morphs of Microdochium
species are known to reside in Monographella
(Amphisphaeriaceae, Xylariales) [10–13]. However,
the close affinity of Microdochium to Idriella has
been discussed and explored that the genus
Microdochium and Idriella are very similar genera
which have polyblastic conidiogenous cells and
hyaline falcate conidia, with the presence of
chlamydospores in culture [14–16]. Nevertheless,
morphological and ecological delimitation of
Microdochium and Idriella is problematic as well as
remains obscure, and taxonomic affinities inferred
from molecular data have not yet been established.
Furthermore, to accommodate genera like
Microdochium,Idriella,andSelenodriella, the taxo-
nomic relationships of Microdochium Syd.,
Monographella Petr., and Idriella Nelson & Wilh
were recently defined based on morphology and
DNA sequence data, and introduced a new family
Microdochiaceae Hern.-Restr., Crous & Groenew
(Sordariomycetes, Xylariales) [17].
CONTACT Hee-Young Jung heeyoung@knu.ac.kr
ß2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of the Korean Society of Mycology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/),
which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
MYCOBIOLOGY
https://doi.org/10.1080/12298093.2019.1695717
During the recent surveying, two novel fungal
species of Ascomycota were collected from soil in
Korea. The purpose of this study was to identify the
newly discovered fungal species based on morpho-
logical and molecular characteristics.
2. Material and methods
2.1. Sample collection and fungal isolation
In 2017, samples were collected in Korea from the
riverside and forest soils of Yeongwol
(3716’25.7”N, 12831’37.3”E) and Pyeongchang
(3737’06.4”N, 12833’03.4”E), respectively. The soils
were taken randomly from a depth of 10–15 cm
using pre-autoclaved sterile spatulas, immediately
transferred into sterile plastic bags, and then stored
at 4 C and then collected soils (1 g) were added to
10 mL of sterile double-distilled water and vortexed
gently until dissolved [18]. The solution was serially
diluted and then plated on potato dextrose agar
(PDA; Difco, Detroit, MI, USA) plates. After incu-
bating the PDA plates at 25 Cfor3–4 days, single
colonies were transferred onto new PDA plates to
obtain a pure culture, followed by incubation at
25 C. Two strains with different morphology were
selected for further molecular analyses. Metabolically
inactive and living culture of two strains (Didymella
chlamydospora: ZEVCFG0000000092 ¼KCTC 56426;
Microdochium salmonicolor: NIBRFG0000501933 ¼
KCTC 56427) were deposited to the National
Institute of Biological Resources (NIBR) and Korean
Collection for Type Cultures (KCTC). The fungal
strains were maintained in 20% glycerol at –80 Cfor
further study.
2.2. Cultural and morphological observations
The cultural and morphological characteristics of
YW23-14 and NC14-294 were studied by growing
the strains on different media. The strain YW23-14
was transferred onto PDA, oatmeal agar (OA), or
malt extract agar (MEA), incubated at 25 C for
7–21 days, and then treated with near-ultraviolet
(UV) light (12 h light/12 h dark) [19,20]. The strain
NC14-294 was transferred on PDA or OA and incu-
bated at 25 C temperature for 7–21 days [17,21].
The fungal growth of each strain was measured, and
the colony characters were recorded. The myco-
logical characteristics were observed using a light
microscope (BX-50; Olympus, Tokyo, Japan).
2.3. Genomic DNA extraction, PCR amplification,
and sequencing
For molecular analyses, the fungal mycelia were
grown on PDA plates for 1 week at 25 C. Next,
total genomic DNA was extracted using the HiGene
Genomic DNA Prep Kit (BIOFACT, Daejeon,
Korea) according to the manufacturer’s instructions
and then stored at –20 C. The following target
genes were amplified for strain YW23-14 according
to previous studies: the internal transcribed spacer
(ITS) regions, b-tubulin (TUB2), 28S rDNA large
subunit (LSU), and the second largest subunit of
RNA polymerase II (RPB2) genes [19]. ITS and LSU
were used to amplify strain NC14-294 [21]. Next,
the amplified PCR products were purified with
ExoSAP-IT (Thermo Fisher Scientific, Waltham,
MA, USA) and sequenced using Solgent (Daejeon,
Korea). The sequence data obtained in this study
were adjusted using the SeqMan software
(Lasergene, DNAStar Inc., Madison,
Wisconsin, USA).
2.4. Molecular phylogenetic analysis
The phylogenetic analyses were conducted using the
sequences retrieved from the National Center for
Biotechnology Information (NCBI) (Table 1). The
ambiguous regions were excluded from the align-
ments, and the evolutionary relationships with the
neighbor-joining algorithm were calculated using
Kimura’s two-parameter model [22]. The alignments
were manually performed for each gene, and then
the sequences were merged by using MEGA7.0 soft-
ware program. The phylogenetic trees were also
inferred by following the maximum likelihood and
maximum parsimony algorithms using the software
program MEGA7.0 with the bootstrap values based
on 1,000 replications [23]. The bootstrap values
were considered as significant once equal to or
more than 70% [24].
3. Results
3.1. Taxonomical analysis of Didymella
chlamydospora YW23-14
3.1.1. Taxonomy
The strain YW23-14 showed distinct morphological
characteristics compared with other Didymella spe-
cies. Therefore, it is proposed as a new species.
Didymella chlamydospora K. Das, S.Y. Lee and
H.Y. Jung, sp. nov. (Figure 1)
MycoBank: MB 830919
Etymology: From Greek chlamydos-, cloak, and
from Latin - spora, spore.
Typus: Yeongwol, Korea (3716’25.7”N,
12831’37.3”E), isolated from riverside soil. The
stock culture (ZEVCFG0000000092 ¼KCTC 56426)
was deposited in the National Institute of Biological
2 K. DAS ET AL.
Resources (NIBR) and Korean Collection for Type
Cultures (KCTC), metabolically inactive culture.
Specimen examined: South Korea, Yeongwol,
from soil, deposited in NIBR Oct. 2017, H.Y. Jung,
(holotype ZEVCFG0000000092, dried and living cul-
ture, ex-holotype living culture KCTC 56426).
Ecology and distribution: Seed-, air- and soil-borne
saprophyte was reported globally by the various mem-
bers of this fungus. The strain was isolated from river-
side soil in South Korea. The soil was sandy to sandy
loam, yellowish brown, medium moisture capacity.
Cultural characteristics: The colonies vary in
color depending on the fungal growth on different
media. The strain cultured on PDA, MEA, and OA
media to study the cultural and morphological
characteristics (Figure 1(A–C)). The average fungal
growth rates on PDA, MEA, and OA were
60.3–70.0, 52.0–59.0, and 58.0–69.0 mm, respect-
ively, at 25 C for 7 days. The upper surface of the
colonies on PDA were white to olivaceous in the
center, with wide olivaceous-colored margins;
reverse pale brown to olivaceous green. The colonies
on MEA were covered by white aerial mycelia, mar-
gin regular, fluffy; reverse became yellowish. And
the colonies on OA showed white to olivaceous
green in center; reverse olivaceous green to black.
Morphological characteristics: The strain pro-
duced a large amount of pycnidia on the surface of
the OA media after 3 to 4 weeks (Figure 1(D)). The
pycnidia were solitary to aggregate, dark brown to
Table 1. List of species used in this study and their GenBank accession numbers for phylogenetic analysis.
GenBank accession numbers
Speceis Strain numbers ITS LSU TUB RPB2
Didymella acetosellae CBS 179.97 GU237793 GU238034 GU237575 KP330415
D. aeria LC:8120 KY742052 KY742206 KY742294 KY742138
D. americana CBS 185.85 FJ426972 GU237990 FJ427088 KT389594
D. anserina CBS 360.84 GU237839 GU237993 GU237551 KT389596
D. anserina CBS 397.65 KT389500 KT389717 KT389797 KT389597
D. anserina CBS 253.80 KT389498 KT389715 KT389795 KT389595
D. anserina UTHSC:DI16-255 LT592926 LN907398 LT592995 LT593064
D. arachidicola CBS 333.75
T
GU237833 GU237996 GU237554 KT389598
D. boeremae CBS 109942
T
FJ426982 GU238048 FJ427097 KT389600
D. coffeae-arabicae CBS 123380
T
NR135965 MH874817 FJ427104 KT389603
D. curtisii PD 92/1460 FJ427041 GU238012 FJ427151 KT389604
D. eucalyptica CBS 377.91 GU237846 GU238007 GU237562 KT389605
D. exigua CBS 183.55
T
GU237794 EU754155 GU237525 EU874850
D. gardeniae CBS 626.68
T
NR135966 GQ387595 FJ427114 KT389606
D. glomerata CBS 528.66
T
NR158225 MH870525 FJ427124 GU371781
D. herbarum CBS 615.75 NR135967 EU754186 KF252703 KP330420
D. heteroderae CBS 109.92
T
FJ426983 GU238002 FJ427098 KT389601
D. lethalis CBS 103.25 GU237729 GU238010 GU237564 KT389607
D. macrostoma CBS 482.95 GU237869 GU238099 GU237626 KT389609
D. maydis CBS 588.69
T
FJ427086 EU754192 FJ427190 GU371782
D. microchlamydospora CBS 105.95
T
FJ427028 GU238104 FJ427138 KP330424
D. musae CBS 463.69 MH859353 GU238011 FJ427136 LT623248
D. nigricans LC:8136 KY742077 KY742231 KY742319 KY742160
D. pinodella CBS 531.66 FJ427052 MH870528 FJ427162 KT389613
D. pinodes CBS 525.77
T
GU237883 GU238023 GU237572 KT389614
D. pomorum CBS 285.76 MH860978 MH872748 FJ427163 KT389615
D. protuberans CBS 377.93 GU237847 GU238014 GU237565 KT389619
D. sancta CBS 281.83
T
NR135973 GU238030 FJ427170 KT389623
D. subglomerata CBS 110.92 FJ427080 GU238032 FJ427186 KT389626
Neoascochyta paspali CBS 560.81
T
NR135970 GU238124 FJ427158 KP330426
D. chlamydospora YW23-14
T
MK836111 MK836109 LC482279 LC480708
Microdochium albescens CBS 290.79 KP859014 KP858950 ––
M. bolleyi CBS 540.92 KP859010 KP858946 ––
M. citrinidiscum CBS 109067
T
KP859003 KP858939 ––
M. colombiense CBS 624.94
T
KP858999 KP858935 ––
M. chrysanthemoides LC5363 KU746690 KU746736 ––
M. fisheri CBS 242.91
T
KP859015 KP858951 ––
M. fisheri NFCCI 4083 KY777595 KY777594 ––
M. lycopodinum CBS 125585 KP859016 KP858952 ––
M. majus CBS 741.79 KP859001 KP858937 ––
M. musae CBS 143500 MH107895 MH107942 ––
M. neoqueenslandicum CBS 108926
T
NR145247 KP858938 ––
M. nivale CBS 116205 KP859008 KP858944 ––
M. novae-zelandiae CPC 29376 LT990655 LT990627 ––
M. phragmitis CBS 423.78 KP859012 KP858948 ––
M. rhopalostylidis CBS 145125 MK442592 MK442532 ––
M. seminicola CBS 122706 KP859007 KP858943 ––
M. sorghi CBS 691.96 KP859000 KP858936 ––
M. tainanense CBS 269.76
T
KP859009 KP858945 ––
M. trichocladiopsis CBS 623.77
T
KP858998 KP858934 ––
Humicola olivacea DTO 319-C7 KX976676 KX976770 ––
M. salmonicolor NC14-294
T
MK836110 MK836108 ––
The isolated strains are indicated in bold of this study.
MYCOBIOLOGY 3
black in color with the immersed texture on the sur-
face of medium. The numerous pycnidia were also
produced on the surface of agar and the pycnidia
were prolific on PDA after 5–6 weeks with the diam-
eter (n¼30) of 93–273 68–222 mm, and with the
average diameter 169 124 mm(Figure 1(E–G)).
The pycnidial wall pseudoparenchymatous, oblong
to isodiametric cells, thick-wall, outer wall 2–3-lay-
ered, pigmented (Figure 1(H)). The micromorpho-
logical structures were analyzed using the culture on
OA media. The hyphae were septate, bent, smooth
with thin walls, hyaline to pale yellow, subglobose,
branched, and width of 2.40–3.60 mm. The chlamy-
dospores were abundant; solitary or in chains;
mostly multicellular, but sometimes unicellular;
partly short-branched, although sometimes having
unbranched chains; usually guttulate; thick-walled;
pale brown to brown; globose to subglobose; and
with diameters (n¼50) of 8.0–17.0 7.0–15.0 mm
and an average size of 12.5 11.80 mm(Figure
1(I–K)); Table 2). Conidiogenous cells were phiali-
dic, hyaline, smooth, ampulliform to doliiform,
6.14–11.0 3.43–7.4 mm(Figure 1(L–N)). The coni-
dia were unicellular, aseptate, hyaline, and globose
to ellipsoidal, with diameters of 4.70–7.40
2.2–4.0 mm(n¼50) and an average size of
6.1 2.9 mm(Figure 1(O)).
Note: The shape and size of chlamydospores dif-
fered extensively. The YW23-14 strain’s chlamydo-
spores were multicellular or unicellular, with
abundant guttules within cells, thick-walled, subglo-
bose globose, and color pale brown to brown.
In contrast, the chlamydospores of D. glomerata
were usually multicellular-dictyosporous, sometimes
solitary, smooth then roughened, and dark brown to
black, compared with the chlamydospores of the
Figure 1. Cultural and morphological characteristics of YW23-14. The colony on potato dextrose agar (PDA) (A); malt extract
agar (MEA) (B); oatmeal agar (OA) (C) for 7 days incubation at 25 C. Pycnidia forming on OA (D); Pycnidia on PDA (E–G);
Pycnidial wall (H); Chlamydospores (I–K); Conidiogenous cells (L–N); Conidia (O). Scale bars: D–G¼100 lm; H–O¼10 lm.
4 K. DAS ET AL.
most closest species of D. anserina were absent; but
some strains had swollen elements [25]. On the
other hand, the chlamydospores of D. musae were
generally solitary, smooth or irregularly roughened,
tanned to dark brown, multicellular-dictyo/phrag-
mosporous, and often discovered to be terminal ele-
ments of short lateral branches, sometimes growing
as constituent cells [26]. The strain YW23-14 gener-
ated numerous smaller-diameter chlamydospores
(8.0–17.0 7.0–15.0 mm) than the closest known
species D. glomerata ((18.0–)30.0–65.0(–80.0)
(12.0–)15.0–25.0(–35.0) mm) and D. musae
(13.0–45.0(–50.0)7.0–20.0(–25.0) mm) (Table 2)
[25,26,28]. The conidial size of the strain YW23-14
varied (4.70–7.40 2.2–4.0 mm) but were almost
similar to that of D. glomerata ((3.5–)4–8.5(–10)
1.5–3.0(–3.5) mm) and D. musae (4.0(3.5–)–7.0(–8.5)
2.0–4.0 mm), although larger than that of D. anserina
(2.4–3.2(–5.5)1.8–2.4(–3.0) mm) (Table 2)[25,26,28].
3.1.2. Phylogenetic analysis of YW23-14
Through sequence analysis, 621 bp of the ITS
region, 761 bp of LSU, 826 bp of RPB2, and 333 bp
of TUB2 were obtained. The BLAST search results
of ITS sequences in the NCBI database revealed that
the strains Didymella americana R63-023, D. pino-
della CBS 110.32, and D. glomerata CBS 127059
each exhibited 99% similarity with the strain YW23-
14. The 28S rDNA large subunit (LSU) exhibited
99% similarity with that of strains Ascochyta herbi-
cola (Phoma herbicola) B-2-13, Phoma odoratissimi
CGMCC 3.17502, and Microsphaeropsis olivacea
CBS 432.71. The RPB2 gene regions revealed simi-
larities with the strains D. musae CBS 463.69 (95%),
D. glomerata UTHSC DI16-205 (94%), D. anserina
UTHSC DI16-255 (93%), and D. americana P-020
(93%). The partial b-tubulin (TUB2) gene was most
similar to the strains P. australis ICMP 7037 (96%)
and D. americana MF-010-003 (96%). The max-
imum likelihood and maximum parsimony trees
were also constructed to determine the exact taxo-
nomic position of the strain and indicated the nodes
with the filled circles in neighbor-joining phylogen-
etic tree, whereas, open circles indicate the corre-
sponding nodes with the maximum likelihood or
maximum parsimony algorithm (Figure 2). In the
phylogram, the strain YW23-14 is placed closely
together with D. anserina (CBS 253.80, CBS 360.84,
CBS 397.65, and UTHSC:DI16-255), D. musae CBS
463.69, and D. glomerata CBS 528.66. The phylogen-
etic analyses revealed, with strong bootstrap support,
that YW23-14 belongs to a distinct cluster than the
previously identified Didymella species. Thus, the
neighbor-joining phylogenetic tree supports that the
phylogenetic position of the strain YW23-14 is dis-
tinct from the other known species of Didymella
(Figure 2).
3.2. Taxonomical analysis of Microdochium
salmonicolor NC14-294
3.2.1. Taxonomy
The strain NC14-294 showed distinct morphological
characteristics compared with other allied species of
Table 2. Morphological characteristics of Didymella chlamydospora sp. nov. and comparison with the closest species
of Didymella.
Sl. No. Strains name Pycnidia (lm) Chlamydospores (lm) Conidia (lm) References
1D. chlamydosporaa
(YW23-14)
93–273 68–222 8.0–17.0 7.0–15.0 4.7–7.4 2.2–4.0 This study
2D. anserina (CBS 364.91) 112–136 112–176 Absent 2.4–3.2(–5.5)1.8–2.4(–3.0) [25]
3D. musae (CBS 463.69) 150–200 13.0–45.0(–50.0)
7.0–20.0(–25.0)
4.0(3.5–)–7.0(–8.5)2.0–4.0 [26]
4D. glomerata
(CBS 528.66
T
)
100–200 (18.0–)30.0–65.0(–80.0)
(12.0–)15.0–25.0(–35.0)
(3.5–)4.0–8.5(–10)1.5–3.0(–3.5) [25]
5D. herbarum (CBS 615.75) 130–265 120–240 N/A 4.5–6.0 2.0–3.0 [6]
6D. subglomerata
(CBS 110.92)
125–225 30.0–65.0 15.0–35.0 (5.0–)7.0–12.0(–15.0)2.0–3.5(–4.0) [25]
7D. prosopidis
(CBS 136550)
up to 200 5.0–9.0 (5.0–)5.5–6.0(–7.0)(2.5–)3.0(–3.5) [27]
8D. americana
(CBS 185.85)
100–220 15.0–25.0 5.0–8.0(–8.5)2.0–3.0(–3.5) [25]
9D. pinodella (CBS 319.90) 96–320 8.0–20.0 8.0–15.0 4.0–6.8 (–7.0–6.0)2.2–3.4 [28]
10 D. heteroderae
(CBS 875.97)
70–250 5.0–8.0 5.0–8.0 3.5–7.5(–12.0)2.0–3.5(–4.5) [29]
11 D. herbicola (CBS 629.97) 120–340 Absent 5.0–7.0 (–8.5)2.0–3.0 [29]
12 D. tanaceti (TAS
041-0055)
85–215 N/A 4.0–8.5 1.5–3.5 [30]
13 D. chenopodii
(CBS 128.93)
100–250 Absent 4.0–6.8(–9.8)1.6–2.6(–4.0) [31]
14 D. aeria
(CGMCC 3.18353
T
)
155–375(–460)
130–340(–460)
N/A 3.0–5.0 2.0–3.0 [19]
15 D. negriana (CBS 358.71) 70–220 Absent 4.5–8.5 (–10.5)2.0–4.0 [29]
N/A: not available in previous references.
MYCOBIOLOGY 5
Microdochium. Therefore, it is described as a
new species.
Microdochium salmonicolor K. Das, S.Y. Lee
and H.Y. Jung, sp. nov. (Figure 3)
MycoBank: MB 830929
Etymology: The specific name “salmonicolor”
referring to the light salmon color colonies
on media.
Typus: Pyeongchang, Korea (3737’06.4”N,
12833’03.4”E), isolated from forest soil. The stock
culture (NIBRFG0000501933 ¼KCTC 56427) was
deposited in the NIBR and KCTC, metabolically
inactive culture.
Specimen examined: South Korea, Pyeongchang,
from soil, deposited in NIBR Oct. 2017, H.Y. Jung,
(holotype NIBRFG0000501933, dried and living cul-
ture, ex-holotype living culture KCTC 56427).
Ecology and distribution: The different members
of this fungi recorded from grasses, cereals, living
leaves, roots, and aquatic (marine) environment.
The strain isolated from forest soil in South Korea.
The soil content plant debris, yellowish brown,
lower moisture capacity.
Cultural characteristics: The strain was cultured
on PDA and OA media to observe the cultural and
morphological characteristics (Figure 3(A–D)). On
PDA and OA media, the colonies reached 54.0–58.0
and 51.0–56.0 mm, respectively, with a 7-days incu-
bation at 25 C. The colonies on PDA were flat,
tightly attached to the media, aerial mycelium aggre-
gated into slimy masses, small pellets, light salmon
to brown in the center with a regular margin, and
the reverse color light salmon to dark brown in the
center (Figure 3(A,B)). Colonies on OA were flat,
Figure 2. Neighbor-joining phylogenetic tree of YW23-14 based on the combined sequences (ITS þLSU þRPB2 þTUB), show-
ing the relationship between Didymella chlamydospora sp. nov. with the closest Didymella spp. Neoascochyta paspali CBS
560.81
T
used as an outgroup. The numbers above the branches represent the bootstrap values obtained for 1,000 replicates
(values smaller than 70% were not shown). The isolated strain of this study is indicated in bold. Bar, 0.01 substitutions per
nucleotide position.
6 K. DAS ET AL.
white cottony, rosy buff, margin entire, slightly
raised in the center, and tightly attached to the
media with several small pellet-like structures on the
colony; the reverse color was light salmon to dark
brown at the center (Figure 3(C,D)).
Morphological characteristics: The micromor-
phological structures were studied using the cultures
on OA medium. The hyphae were hyaline to pale
brown, septate, and smooth, with a width of
2.30–3.10 mm. The conidiophores were hyaline to
pale brown, septate, branched, and born from the
hyphae (Figure 3(E–F)). The conidiogenous cells
were subcylindrical to oval, bent in the center, hya-
line, tapering towards the edge, (0–)1 septate, and
had a diameter (n¼10) of 4.9–8.8 2.0–3.2 mm and
an average size of 7.7 2.6 mm, with the mycelium
reduced to conidiogenous cells that had grown from
the hyphae (Figure 3(G–I)). The conidia were hya-
line to brown, blunted in both apices, fusiform, cla-
vate, sometimes bent in the middle, (0–)1 septate,
with dimensions (n¼20) of 8.0–11.30
2.40–3.70 mm, and an average size of 9.4 2.9 mm;
sometimes the conidia were produced directly from
hyphae (Figure 3(J–M);Table 3). Chlamydospores
were not observed.
Note: The colonies on PDA were flat, tightly
attached to the media, with small pellet-like struc-
tures, light salmon to brown color in the center
with regular margin, and the reverse color was light
salmon to dark brown in center (Figure 3(A) and
Table 3). The closest species, Microdochium fisheri,
produced colonies that were flat, margin entire,
slightly raised to umbonate in the center, and pink-
ish white with a reverse grayish orange color [21].
M. lycopodinum were white cottony, lanose to flo-
cosse, buff to rosy buff, and margin effuse on OA
media, whereas M. phragmitis displayed as floccose,
white in the center, sparse aerial mycelia, buff to the
periphery, margin effuse, and reverse buff on OA
media [17]. The conidiogenous cells of the strain
NC14-294 were subcylindrical to oval, bent in the
center, tapering towards the edge, hyaline, and had
dimensions of 4.9–8.8 2.0–3.2 mm, which is smaller
than that of the previously identified M. phragmitis
(6.0–24.0 1.5–3.0 mm) but close to that of M. lyco-
podinum (4.0–12.0 2.5–3.5 mm) (Table 3)[17]. The
conidiogenous cells of M. phragmitis were terminal,
sympodial, denticulate, hyaline, smooth, cylindrical
to clavate, and sometimes navicular. M. lycopodinum
produced holoblastic conidiogenous cells with per-
current proliferations that were ampulliform to lage-
niform, and subcylindrical. Regarding M. fisheri, the
conidiogenous cells were terminal to intercalary,
cylindrical to denticulate, and tapering towards the
apex, with great variation in length [21]. The com-
parison with certain species of the genus showing
high sequence similarities did not show colonial
similarities such as tightly attached with the cultural
media, tiny pallets like structures, light salmon to
brown in the center with regular margins; reverse
Figure 3. Cultural and morphological characteristics of NC14-294. Colony on potato dextrose agar (A, B) and oatmeal agar (C,
D), respectively, at 25 C in 7 days. Conidiophores (E, F); Conidiogenous cells (G–I); Conidia (J–M). Black arrow indicated coni-
diogenous cells. Scale bars: E–M¼10 lm.
MYCOBIOLOGY 7
light salmon to dark brown in the center. That’s
why, the cultural and morphological characteristics
indicate that the Korean strain NC14-294 is distinct
from previously known species of Microdochium.
3.2.2. Phylogenetic analysis of NC14-294
After the sequence analysis of NC14-294,578 bp
from the ITS regions and 901 bp from the 28S
rDNA gene were obtained. According to the BLAST
search results, the analysis of the ITS sequences in
the NCBI database indicated similarities of 98%,
97%, and 96% with Microdochium lycopodinum CBS
109398 M. phragmitis CBS 423.78, and M. fisheri
CBS 242.91
T
, respectively. The 28S rDNA large sub-
unit (LSU) showed similarities with those of the
strains M. fisheri CBS 242.90 (99%), Arthrobotrys
foliicola CBS 242.90 (99%), M. phragmitis CBS
423.78 (98%), and M. lycopodinum CBS 146.68
(98%). The phylogenetic analysis was conducted
based on a combination of ITS regions with the par-
tial sequences of the 28S rDNA. The exact taxo-
nomic position of the strain NC14-294 was
indicated by the node in the neighbor-joining
Table 3. Cultural and Morphological characteristics of Microdochium salmonicolor sp. nov. and comparison with the closest
species of Microdochium.
Sl. No. Strains name Cultural characteristics Conidiogenous cells (lm) Conidia (lm) References
1Microdochium
salmonicolora
(NC14-294)
Colonies on PDA were flat, tightly
attached with the media, small
pallets, light salmon to brown
color in center with regular
margin; reverse light salmon to
dark brown in center. Colonies on
OA were flat, white cottony, rosy
buff, margin entire, slightly raised
in the center, and tightly attached
to the media with several small
pellet-like structures on the colony;
the reverse color was light salmon
to dark brown at the center.
4.9–8.8 2.0–3.2 8.0–11.3 2.4–3.7 This study
2M. fisheri (NFCCI 4083) Colonies on PDA were flat, margin
entire, slightly raised to umbonate
center, pinkish white with reverse
grayish orange.
N/A 4.8–12.0 1.6–3.6 [21]
3M. lycopodinum
(CBS 109399)
White cottony, lanose to flocosse,
buff to rosy buff, margin effuse on
OA media. PDA:N/A.
4.0–12.0 2.5–3.5 8.0–15.5 2.5–4.0 [17]
4M. phragmitis
(CBS 285.71)
Floccose, white in the center, sparse
aerial mycelium, buff to the
periphery, margin effuse; reverse
buff on OA media. PDA:N/A.
6.0–24.0 1.5–3.0 10.0–14.5 2.0–3.0 [17]
5M. rhopalostylidis
(CPC 34449
T
)
Colonies flat, spreading, with
moderate aerial mycelium and
smooth, lobate margin, covering
dish after 2 wk at 25 C. On MEA
and PDA surface saffron to luteous,
reverse sienna; on OA surface
umber to saffron.
4.0–10.0 3.0–3.5 (13.0–)16.0–20.0(–23.0)
(2.5–)3.0(–4.0)
[32]
6M. trichocladiopsis
(CBS 623.77
T
)
Colonies on OA flat, lacking aerial
mycelium, rosy buff, black near to
the inoculum, margin diffuse,
reverse similar.
4.0–37.5 2.0–3.0 6.0–18.0 2.0–3.5 [17]
7M. citrinidiscum
(CBS 109067
T
)
Colonies on OA cottony, white,
periphery scarce aerial mycelium,
saffron, margin diffuse, reverse
saffron, no exudate or soluble
pigment produced.
11.0–29.0 1.5–2.0 7.0–31.0 2.0–3.0 [17]
8M. colombiense
(CBS 624.94
T
)
Colonies on OA flat, salmon, no
exudate or soluble pigment
produced, margin diffuse or entire;
reverse saffron.
5.0–11.5 2.5–3.5 5.0–8.0 1.5–2.5 [17]
9M. chrysanthemoides
(CGMCC3.17929
T
)
Colonies on PDA felty, compact, erose
or dentate, white initially, then
becoming yellowish with age.
Exudate occasionally appeared on
old sporodochia. Reverse yellowish
to orange, due to the soluble
pigment secreted.
5.0–12.0 3.0–4.5 4.5–7.0 2.0–3.0 [33]
10 M. neoqueenslandicum
(CBS 108926
T
)
Colonies on OA center flat, creamy,
with concentric rings, peach to
salmon, periphery with cottony
aerial mycelium, white, margin
diffuse, entire.
4.5–10.0 2.0–3.5 4.0–9.0 1.5–3.0 [17]
N/A: not available in previous references.
8 K. DAS ET AL.
phylogenetic tree along with the filled nodes in the
maximum likelihood and maximum parsimony
trees. The corresponding nodes were also recovered
using the maximum likelihood or maximum parsi-
mony algorithms, as indicated by the open circles
(Figure 4). The analysis of the combined sequences
of the phylogenetic tree revealed that the position of
NC14-294 was distinct from the other identified
species under the genus Microdochium (Figure 4).
4. Discussion
For the present study, the following two morpho-
logically different strains were isolated in 2017 from
the soil in Yeongwol and Pyeongchang, South
Korea: YW23-14 and NC14-294. The strains exhib-
ited morphological differences from each of the pre-
viously identified closely related species, as is
supported by descriptions of the latter in previous
reports (Tables 2 and 3). The phylogenetic relation-
ships between the Korean strain YW23-14 and pre-
viously published authentic strains were inferred by
using maximum likelihood (ML), neighbor-joining
(NJ) and maximum parsimony (MP) analyses of the
ITS regions, LSU, RPB2, and TUB2 genes. The
boundaries within the Didymella, a combined align-
ment of ITS regions, LSU, RPB2, and TUB2
sequences was created to clarify the species, contain-
ing 30 strains (including the outgroup Neoascochyta
paspali). To determine the exact taxonomic position
of the strain, phylogenetic analysis was also con-
ducted based on a combination of the sequences
with maximum parsimony (tree length ¼985, con-
sistency index ¼0.43, retention index ¼0.57, and
composite index ¼0.30) (Figure 2). In case of
NC14-294, phylogenetic relationships between the
isolated Korean strain and previously identified
strains were inferred by using ML, NJ, and MP
analyses of the ITS regions and LSU. A combined
alignment of ITS regions and LSU sequences was
created to clarify the species which containing 21
strains including the outgroup Humicola olivacea.
During the analysis of a sequence combination with
the maximum parsimony (tree length ¼422, con-
sistency index ¼0.60, retention index ¼0.78, and
composite index ¼0.56) was also used to determine
the precise taxonomic position of the strain (Figure
4). So, the phylogenetic relationship determined
through the sequence analyses and indicated that
the strains YW23-14 and NC14-294 were distinct
from each of those identified species with the
strength of the internal branches of the trees as well
as the bootstrap values using 1,000 replications
(Figures 2 and 4).
Figure 4. Neighbor-joining phylogenetic tree of NC14-294 based on the combined sequences (ITS þLSU), showing the rela-
tionship between Microdochium salmonicolor sp. nov. with the closest Microdochium spp. Humicola olivacea DTO 319-C7 used
as an outgroup. The numbers above the branches represent the bootstrap values obtained for 1,000 replicates (values smaller
than 70% were not shown). The isolated strain of this study is indicated in bold. Bar 0.02 substitutions per nucleo-
tide position.
MYCOBIOLOGY 9
Previous studies have reported that the genus
Didymella is widely distributed in field and orna-
mental crops, wild plants and most saprobes species
are commonly found in living or dead aerial parts
of herbaceous, wooden plants [8], and some act as
mutualistic endophytes [34]. Didymella pinodes (for-
merly known Mycosphaerella pinodes) has been
reported as the main causal agent of Ascochyta
blight, which is one of the most important fungal
diseases of the pea (Pisum sativum) worldwide
[35,36]. D. tanaceti and D. rosea have been identi-
fied as plant pathogens that cause the tan spot in
pyrethrum [30]. In addition, D. americana is the
causal agent of the foliar disease observed on baby
lima bean (Phaseolus lunatus) in fields across west-
ern New York State, USA [21]. The recently estab-
lished family Didymellaceae [2,37] consist of many
taxa previously classified in the genus Phoma and
their related taxa, and includes many important
plant pathogens, some species of which are of quar-
antine concern [5,7,8].
The members of the genus Microdochium are
important plant pathogens, particularly on grasses
and cereals. Some of the species of Microdochium
are terrestrial cause economic damage to important
plants [38,39], nonpathogenic, and also sometimes
endophytes [40]. Many species of Microdochium
were also identified in the aquatic (marine) environ-
ment after evaluating diseased as well as healthy sal-
mon eggs and have been reported as M.
lycopodinum and M. phragmitis [41]. The root
necrosis and decay of grasses caused by M. bolleyi
[42] and M. paspali is responsible for the seashore
paspalum disease of Paspalum vaginatum [39]. The
study of Microdochium opens a new opportunity to
expand the area of research for bioactive com-
pounds such as cyclosporine A, an active compound
that has been isolated from an estuarine M. nivale
and that has the potential to control human and
animal diseases [43]. Another example is that of an
extract of M. phragmitis from Antarctic angio-
sperms, for which the cytotoxic activity against a
human tumoral cell line has been reported [44].
Finally, the systematic exploration of Microdochium
from natural sources is required to evaluate their
bioactive potentiality.
Considering all the aspects of these two new spe-
cies, the classification and ecology are mostly
important, and the potential activities of each of
species should be further investigated. According to
morphological and phylogenetic analyses, the strains
are especially distinct from previously identified
strains of the genera Didymella and Microdochium.
Thus, these two species are proposed as Didymella
chlamydospora sp. nov. and Microdochium salmoni-
color sp. nov.
Disclosure statement
No potential conflict of interest was reported by
the authors.
Funding
The authors are grateful to the Ministry of Environment
(MOE) of the Republic of Korea for the research on sur-
vey data and discovery of indigenous fungal species sup-
ported by a grant from the National Institute of
Biological Resources (NIBR).
ORCID
Seung-Yeol Lee http://orcid.org/0000-0003-1676-0330
Hee-Young Jung http://orcid.org/0000-0002-4254-3367
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MYCOBIOLOGY 11
