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Mycobiology
ISSN: 1229-8093 (Print) 2092-9323 (Online) Journal homepage: https://www.tandfonline.com/loi/tmyb20
Cladophialophora lanosa sp. nov., a New Species
Isolated from Soil
Kallol Das, Seung-Yeol Lee & Hee-Young Jung
To cite this article: Kallol Das, Seung-Yeol Lee & Hee-Young Jung (2019):
Cladophialophora�lanosa sp. nov., a New Species Isolated from Soil, Mycobiology, DOI:
10.1080/12298093.2019.1611242
To link to this article: https://doi.org/10.1080/12298093.2019.1611242
© 2019 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group on behalf of the Korean Society of
Mycology
Published online: 17 May 2019.
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RESEARCH ARTICLE
Cladophialophora lanosa sp. nov., a New Species Isolated from Soil
Kallol Das, Seung-Yeol Lee and Hee-Young Jung
School of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National University, Daegu 41566, Korea
ABSTRACT
Cladophialophora is characterized by simple hyphomycetes with brown hyphae that give rise
to branched chains of pale brown conidia and shows affinities with the Herpotrichiellaceae.
A fungal strain belonging to the genus Cladophialophora was isolated from soil in Daegu,
Korea. This strain produces numerous greenish to dark black lanose aerial mycelia with hair
like structures. It is morphological similar to C.chaetospira,C.inabaensis, and C.multiseptata;
however, the conidiophores and conidia sizes of the newly isolated strain (KNU16-032) are
clearly different from them. The novelty of the strain was also confirmed based on phylo-
genetic analysis using the data sets of the internal transcribed spacer region of and the par-
tial sequence of 28S ribosomal DNA region along with the cultural characteristics. Because
morphological observations and phylogenetic analysis indicated that the strain is distinct
from previously known Cladophialophora species, we propose this species as a new species
Cladophialophora lanosa sp. nov., and provide the detailed descriptions in this study.
ARTICLE HISTORY
Received 22 November 2018
Revised 18 February 2019
Accepted 8 April 2019
KEYWORDS
Cladophialophora lanosa;
herpotrichiellaceae; soil
inhabiting fungi
1. Introduction
Cladophialophora Borelli are relatively simple
hyphomycetes with brown hyphae that give rise to
branched chains of pale brown conidia.
Cladophialophora is an asexual, morphologically one-
celled, and ellipsoidal to fusiform, dry conidia arising
through blastic, acropetal conidiogenesis, branched
chains and the chains are usually coherent and coni-
dial scars are nearly unpigmented [1]. Currently,
Cladophialophora comprises fewer than 30 species,
which are opportunistic human pathogens, phyto-
pathogens or are isolated from environmental sources
and so far 11 species have been shown to cause dis-
ease in humans [2–6].
Moreover, the genus was initially erected to
accommodate fungal species exhibiting both
Cladosporium and Phialophora-like conidiogenesis.
Cladophialophora includes species that show affin-
ities with the Herpotrichiellaceae, as revealed by
molecular data such as the internal transcribed spa-
cer (ITS) and the large subunit ribosomal DNA
region (28S rDNA) [7–9] and by the production of
Cladophialophora-like anamorphs in some species of
Capronia, the only known teleomorphic genus in
this family [10,11]. A series of molecular phylogen-
etic studies revealed that Cladophialophora is poly-
phyletic in the order Chaetothyriales and the genus
is closely related to members of several anamorphic
genera, including Exophiala,Cyphellophora,
Fonsecaea,Knufia,Phialophora and the teleomor-
phic genus Capronia. Particularly, two species of
the genus Cladophialophora were presented as new
species, the ITS and partial 28S rDNA data revealed
the relationship with other species [12].
During an extensive investigation on the
unreported fungi in Korea, strain KNU16-032 was
isolated from soil. Based on morphological character-
istics and molecular analysis, the fungus represents
an undescribed species belonging to the genus
Cladophialophora. In this study, the isolated fungus is
described and illustrated as a novel species.
2. Materials and methods
2.1. Collection of soil and isolation
During the investigation of unrecorded fungal spe-
cies in 2016, soil was collected from Daegu, Korea
(3553041.600N, 12835010.100 E). Afterward, the col-
lected soil (1 g) was diluted with 10 mL of sterile
distilled water and vortexed gently to mix with the
sterile water. Then, it was diluted serially and spread
on the potato dextrose agar (PDA; Difco, Detroit,
MI) plates. The plates were incubated at 25 C for
3 days without any disturbance. After 3 days, numer-
ous single colonies were observed growing on the
plates. Then, the single colonies were transferred to
new PDA plates and again put into incubation at
25 C to favor the growth of fungal mycelia. The
CONTACT Hee-Young Jung heeyoung@knu.ac.kr School of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National
University, Daegu 41566, Korea
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/10.1080/12298093.2019.1611242
strain KNU16-032 was selected based on cultural
characteristics and molecular phylogenetic analyses.
2.2. Morphological characterization
The morphological characteristics of the strain were
determined on PDA, oatmeal agar (OA; Difco), and
malt extract agar (MEA; Difco) [13]. All the three
media were used to investigate the morphological
characteristics of the strain KNU16-032 followed by
an incubation period of 21 days at a temperature of
25 C. After incubation, the diameter of the colonies
of each medium was measured, and the colony color
of the strain was observed. A light microscope (BX-
50, Olympus, Tokyo, Japan) was used to observe the
mycological characteristics of the strain.
2.3. DNA extraction, PCR, and sequencing
Genomic DNA was extracted from mycelia using
the HiGene Genomic DNA prep kit (Biofact,
Daejeon, Korea). Molecular identification of the
strain at genus and species levels was conducted
using sequences of the ITS and 28S rDNA region.
The ITS regions, including 5.8S, were amplified
using the ITS primers ITS1/ITS4 [14] and PCR
amplification was initiated with 2 min denaturation
at 94 C, followed by 35 cycles of denaturation at
94 C for 30 s, annealing at 55 C for 45 s, and
extension at 72 C for 1 min and 30 s, followed by a
final extension at 72 C for 5 min. In case of 28S
ribosomal DNA gene, the PCR amplification was
performed using LROR/LR5 primer pair [15]. The
amplified PCR products were purified with
EXOSAP-IT (Thermo Fisher Scientific, Waltham,
MA) and sequenced by Solgent (Daejeon, Korea).
The obtained sequences from KNU16-032 were
deposited in NCBI GenBank under accession num-
bers LC387460 and LC387461 for ITS region and
the partial 28S ribosomal DNA genes, respectively.
2.4. Phylogenetic analyses
The reference sequences were retrieved from the
National Center for Biotechnology Information
(NCBI). Evolutionary matrices for the maximum
likelihood, neighbor-joining and maximum-
parsimony were constructed using Kimura’stwo-
parameter model [16]. Tree topology was inferred
by the maximum likelihood, neighbor-joining and
maximum-parsimony method using the program
MEGA7 with bootstrap values based on 1,000 repli-
cations [17].
3. Results
3.1. Taxonomy
The collected Cladophialophora species KNU16-032
showed distinct morphological structures compared
with other allied species. Therefore, it is described
as a new species.
Cladophialophora lanosa K. Das, S.Y. Lee and
H.Y. Jung, sp. nov. (Figures 1 and 2).
MycoBank: MB826874
Etymology: The specific name lanosa, derived
from Latin lana, meaning woolly, refers to the
fleece-like appearance of aerial mycelia.
Typus: Daegu, Korea (3553041.600N,
12835010.100E), isolated from soil. The stock culture
(NIBRFG0000499862 ¼KCTC 56424) was deposited
in the National Institute of Biological Resources
(NIBR) and Korea Collection for Type Cultures
(KCTC), metabolically inactive culture.
Habitat: On soil. The soil was yellowish brown,
fine gravelly clay loam, lower moisture capacity.
Cultural characteristics: The average diameters
of the strain colonies in PDA, OA, and MEA were
24.4, 21.2, and 25.5 mm, respectively. The growth of
mycelial colonies was round, and there were also
variations in the diameter of the colonies of across
the different media (Figure 1). They produced
greenish to dark black mycelia with dark brown to
black color mycelia appeared in opposite side of the
PDA medium (Figure 1(a,b)). The strain was
slow growing and developed greenish to dark black
lanose aerial mycelia with hair-like structures.
The produced numerous lanose aerial mycelia, could
be clearly differentiated from aerial mycelia
(Figure 2(a,b)).
Morphological characteristics: Hyphae were
irregularly septate, straight or bent, smooth, thin-
walled, hyaline to brown in color, guttulate,
branched, with a size of width 1.4–1.7 lm, with for-
mation of hyphal strands, and differentiated subglo-
bose conidiophores, as well as numerous conidia.
Conidiophores were solitary, macronematous,
well distinguishable under the light microscope
from aerial mycelium, pale to brown, subcylindrical,
straight to somewhat curved and erect with the
wide 1.2–2.5 lm(Figure 2(f)). Conidiogenous
cells were branched, smooth-walled, and round
(Figure 2(g–h)). Conidia were one-celled, smooth,
acropetal, catenulate, hyaline to pale brown with a
size in the range of 2.1–4.4 2.0–3.2 lm, ramoconi-
dia subcylindrical, guttulate (Figure 2(c–e)).
Notes: The new species is morphologically simi-
lar to C.chaetospira,C.inabaensis, and C.multisep-
tata.Cladophialophora lanosa produces numerous
greenish to dark black lanose aerial mycelia with
hair like structures, whereas C. inabaensis produces
2 K. DAS ET AL.
dark gray to dark brown in color, felty, with lanose
aerial mycelia on PDA media [18]. The average size
of hyphae of C. lanosa is 1.4–1.7 lm, while hyphae
of C. inabaensis, C. multiseptata and C. chaetospira
are 1.6–3.6 lm, 2.0–4lm, 2.0–3.5 lm, respectively
(Table 1). The average size of conidia of C. lanosa
(3.4 2.7 lm) is shorter than C. inabaensis
(4.6 3.9 lm). In case of C. multiseptata, the coni-
dial size ranges 4.5–18 3–5lm[18] and the coni-
dial size of C. chaetospira is 25–30 3–4lm
(Table 1)[2].
3.2. Phylogenetic analyses
After analyzing the sequences, 581 and 764 bp were
obtained from the ITS regions and 28S ribosomal
DNA gene, respectively. The BLAST search results
of ITS region of C. lanosa revealed 92.3% and 92.9%
similarities with that of C. chaetospira CC 14-28
(KF359558) and C. inabaensis EUCL1 (LC128795),
respectively. In case of the partial 28S ribosomal
DNA gene showed 98.4% and 98.8% similarities
with C. chaetospira CBS 514.63 (MH869959) and C.
inabaensis EUCL1 (LC128795), respectively. The
lodged sequences of existing Cladophialophora spe-
cies from GenBank were used to compare the ITS
region and partial 28S rDNA region to explore this
fungal study (Table 2). Maximum-likelihood phylo-
genetic tree showed the relationship between the
strain KNU16-032 and other Cladophialophora spe-
cies based on the ITS and the partial 28S rDNA
regions (Figure 3). The neighbor-joining and max-
imum-parsimony were also constructed to deter-
mine the exact taxonomic position of the strain and
indicated with the nodes in maximum-likelihood
phylogenetic tree. Filled circles indicate that the cor-
responding nodes were also recovered in trees gen-
erated with the neighbor-joining and maximum-
parsimony algorithms. Open circles indicate that the
corresponding nodes were also recovered in the tree
generated with the neighbor-joining or maximum-
parsimony algorithms (Figure 3). As the results, the
phylogenetic tree revealed that the phylogenetic pos-
ition of KNU16-032 was clearly separated from the
C. inabaensis. In this reason, the KNU16-032 origi-
nating from soil was phylogenetically distinct from
the other species of Cladophialophora.
4. Discussion
Availability of DNA sequencing technology in the
past two-to-three decades has generated an enor-
mous amount of DNA sequence data, allowing fun-
gal taxonomy through phylogenetic revolution. To
assess the phylogenetic position of C. floridana and
C. tortuosa, ITS and partial 28S rDNA regions were
used previously [12]. The reason for using the gene
is to classify the strain KNU16-032 to determine the
Figure 1. Cultural characteristics of KNU16-032 (a–f), Colony on PDA (a, b), OA (c, d) and MEA (e, f) and in front and reverse,
accordingly.
MYCOBIOLOGY 3
Figure 2. Morphological characteristics of KNU16-032. Lanose aerial mycelia on stereo microscope (a, b); conidia and conidial
chains (c, d, e); conidiophores (f), and conidiogenous cells (g, h). Arrows indicated conidiogenous cells. Scale bars: a,
b¼1000 lm; c–h¼10 lm.
Table 1. Morphological characteristics of Cladophialophora lanosa sp. nov. in this study and comparison with the closest spe-
cies of the genus Cladophialophora.
Characteristics
Cladophialophora lanosa
a
Cladophialophora
inabaensis
b
Cladophialophora
multiseptata
c
Cladophialophora
chaetospira
d
(KNU16-032)
Cultural characteristics Greenish to dark black lanose
aerial mycelia with hair
like structures and round
brown margin at the
edge; reverse black
on PDA.
Dark gray to dark brown in
color, felty, with lanose
aerial mycelia on PDA.
Olive-gray with olivaceous-
black, slightly lobate mar-
gin; reverse black on PDA.
Irony-gray, olivaceous-black;
reverse olivaceous-black
on PDA
Hyphae wide (lm) 1.4–1.7 1.6–3.6 2.0–4.0 2.0–3.5
Conidiophore wide (lm) 1.2–2.5 N/A 2.0–4.0 3.0–4.0
Conidia (lm) 2.1–4.4 2.0–3.2 3.4–7.2 4.5–18 3–525–30 3–4
N/A: not available in previous references.
a
Fungal strain studied in this paper.
b
Sources of the descriptions [18].
c
Sources of the descriptions [23].
d
Sources of the descriptions [2].
4 K. DAS ET AL.
Table 2. List of species used in this study and their GenBank accession numbers for the phylogenetic analysis.
GenBank Accession Numbers
Speceis Strain Numbers ITS þLSU ITS LSU
Cladophialophora arxii CBS 306.94
T
–NR 111280 KX822320
C. australiensis CBS 112793
T
EU035402 ––
C. bantiana UM 956 –KU928131 KU928133
C. boppii CBS 126.86 –NR 131297 FJ358233
C. chaetospira CBS 114747 –KF928450 KF928514
C. devriesii CBS 147.84
T
–NR 111279 KC809989
C. floridana SR1004 AB986344 ––
C. immunda CBS 834.96
T
–NR 111283 KC809990
C. inabaensis EUCL1
T
LC128795 ––
C. matsushimae MFC-1P384 –FN549916 FN400758
C. minourae CBS 556.83
T
–AY251087 FJ358235
C. multiseptata FMR 10591 –HG003668 HG003671
C. mycetomatis CBS 454.82 –LC192112 LC192077
C. parmeliae CBS 129337 –JQ342180 JQ342182
C. pseudocarrionii CBS 138591 –KU705827 KU705844
C. samoensis CBS 259.83
T
–NR 111282 KC809992
C. saturnica CBS 102230 –AY857508 KC809993
C. subtilis CBS 122642
T
–NR 111363 KX822322
C. tortuosa BA4b006
T
AB986424 ––
C. yegresii CBS 114405 –EU137322 KX822323
C. lanosa KNU16-032
T
–LC387460 LC387461
Phialophora reptans CBS 113.85
T
–NR 121346 JQ766493
Figure 3. Maximum-likelihood phylogenetic tree based on the combined internal transcribed spacer (ITS) sequences and the
partial sequence of 28S ribosomal DNA genes, showing the relationship between Cladophialophora lanosa sp. nov. with the
closest Cladophialophora spp. Phialophora reptans CBS 113.85 was used as an outgroup. The numbers above the branches rep-
resent the bootstrap values obtained for 1000 replicates (values smaller than 60% were not shown). The isolated strain of this
study is indicated in bold. Bar, 0.01 substitutions per nucleotide position.
MYCOBIOLOGY 5
taxonomic position. The phylogenetic trees revealed
that the strain KNU16-032 was distinct from the
other known Cladophialophora species, confirmed
with the results of neighbor-joining, maximum-
parsimony and maximum-likelihood phylogenetic
trees (Figure 3).
There are numerous fungi from different habitats
and diverse geographic regions. C. inabaensis iso-
lated from eggplant roots using the baiting method
in Japan [18]. Several studies have reported on
plant-associated Cladophialophora species such as C.
hostae,C. scillae,C. proteae, and C. sylvestris,
whereas, the species C. australiensis and C. potulen-
torum were found in soft drinks [4]. C. chaetospira
found on the roots of Chinese cabbage [19] and also
in association with bamboo, on roots of Picea abies
and from soil collected in a wheat field [2].
Moreover, the genus Cladophialophora comprises a
number of environmental saprobes such as C. sat-
urnica from plant debris in the environment. And
the members of the genus can also be isolated from
diverse environment conditions [5]. C. carrionii was
particularly isolated in arid and semiarid climates of
e.g. South and Central America and Australia
[20–21]. C. abundans was isolated from muddy bur-
rows of the mangrove-land crab (Ucides cordatus)in
Brazilian mangrove habitats [22]. The current study
identified a novel species (C. lanosa sp. nov.) from
soil in Korea. The precise ecological niches of
Cladophialophora varies from different habitat,
hosts and diverse geographic regions. So, this might
be a clue to explore their behavior as rare environ-
mental oligotrophs as well as invaders of human
tissue containing aromatic neurotransmitters. The
understanding of the phylogeny and ecology of
Cladophialophora is therefore essential.
Disclosure statement
No potential conflict of interest was reported by
the authors.
Funding
We are grateful to the Ministry of Environment (MOE)
of the Republic of Korea for the research on survey
data and discovery of indigenous fungal species supported
by a grant from the National Institute of Biological
Resources (NIBR).
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