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pathogens
Article
New Species of the Genus Curvularia:
C. tamilnaduensis and C. coimbatorensis from Fungal
Keratitis Cases in South India
Noémi Kiss 1, Mónika Homa 1,2, Palanisamy Manikandan 3,4 , Arumugam Mythili 5,
Krisztina Krizsán6, Rajaraman Revathi 7, Mónika Varga 1, Tamás Papp 1,2 , Csaba Vágvölgyi 1,
LászlóKredics 1, * and Sándor Kocsubé1, *
1Department of Microbiology, Faculty of Science and Informatics, University of Szeged, 6726 Szeged,
Hungary; kissnoemi621@gmail.com (N.K.); homamoni@gmail.com (M.H.);
varga.j.monika@gmail.com (M.V.); pappt@bio.u-szeged.hu (T.P.); csaba@bio.u-szeged.hu (C.V.)
2MTA-SZTE “Lendület” Fungal Pathogenicity Mechanisms Research Group, 6726 Szeged, Hungary
3Department of Medical Laboratory Sciences, College of Applied Medical Sciences, Majmaah University,
Al Majmaah 11952, Saudi Arabia; manikandanpalanisamy@gmail.com
4Greenlink Analytical and Research Laboratory India Private Ltd., Coimbatore, Tamil Nadu 641014, India
5Department of Microbiology, Dr. G.R. Damodaran College of Science, Coimbatore, Tamil Nadu 641014,
India; mythilia1689@gmail.com
6Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre,
Hungarian Academy of Sciences, 6726 Szeged, Hungary; krizsank@gmail.com
7
Aravind Eye Hospital and Postgraduate Institute of Ophthalmology, Coimbatore, Tamil Nadu 641014, India;
revathi@aravind.org
*Correspondence: kredics@bio.u-szeged.hu; (L.K.); shigsanyi@gmail.com; (S.K.)
Received: 6 December 2019; Accepted: 18 December 2019; Published: 20 December 2019
Abstract:
Members of the genus Curvularia are melanin-producing dematiaceous fungi of increasing
clinical importance as causal agents of both local and invasive infections. This study contributes to the
taxonomical and clinical knowledge of this genus by describing two new Curvularia species based on
isolates from corneal scrapings of South Indian fungal keratitis patients. The phylogeny of the genus
was updated based on three phylogenetic markers: the internal transcribed spacer (ITS) region of the
ribosomal RNA gene cluster as well as fragments of the glyceraldehyde-3-phosphate dehydrogenase
(gpdh) and translation elongation factor 1-
α
(tef1
α
) genes. The maximum likelihood phylogenetic tree
constructed from the alignment of the three concatenated loci revealed that the examined isolates are
representing two new, yet undescribed, Curvularia species. Examination of colony and microscopic
morphology revealed differences between the two species as well as between the new species and
their close relatives. The new species were formally described as Curvularia tamilnaduensis N. Kiss
& S. Kocsub
é
sp. nov. and Curvularia coimbatorensis N. Kiss & S. Kocsub
é
sp. nov. Antifungal
susceptibility testing by the broth microdilution method of CLSI (Clinical & Laboratory Standards
Institute) revealed that the type strain of C. coimbatorensis is less susceptible to a series of antifungals
than the C. tamilnaduensis strains.
Keywords:
Curvularia; keratitis; taxonomy; antifungal susceptibility; Curvularia coimbatorensis;
Curvularia tamilnaduensis
1. Introduction
The fungal genus Curvularia (Ascomycota, Pleosporales, Pleosporaceae) comprises of
dematiaceous, melanin-producing molds with various lifestyles including saprophytism, plant
endophytism [1], plant parasitism [2], and human pathogenicity [3].
Pathogens 2020,9, 9; doi:10.3390/pathogens9010009 www.mdpi.com/journal/pathogens
Pathogens 2020,9, 9 2 of 14
The genus-level identification of Curvularia was performed traditionally by the examination of
pigmentation, as well as the morphology of the septate conidia and hyphae [
3
]. The first sequence-based
species-level identification attempts targeted the internal transcribed spacer (ITS) region of the
ribosomal RNA gene cluster, which alone, however, proved inappropriate, either for the purposes
of exact diagnosis [
4
] or for the phylogenetic resolution of the genus and the clarification of its
relationship to the closely related genera Bipolaris,Cochliobolus, and Drechslera [
3
]. Multilocus sequence
typing (MLST) involving fragments of the nuclear ribosomal large subunit RNA (LSU) as well as
the glyceraldehyde-3-phosphate dehydrogenase (gpdh) and translation elongation factor 1-
α
(tef1a)
genes in addition to ITS had resulted in the recently accepted phylogenetic concept of the genus
Curvularia [
5
], which was applied in more recent works [
6
–
8
]. Recently, the genus involves more than
100 described species, which can be divided into six clades (americana, eragrostidis, hominis, lunata,
spicifera, and trifolii) according to Madrid et al. [
7
] based on MLST of four loci (ITS, LSU, gpdh, and the
RNA polymerase II subunit rpb2).
Krizs
á
n et al. [
3
] reviewed the clinical importance of the genus Curvularia, and identified
Curvularia australiensis,Curvularia geniculata,Curvularia hawaiiensis,Curvularia lunata,Curvularia pallescens,
and Curvularia spicifera as the species most frequently isolated from clinical samples. Further members
of the genus with confirmed clinical relevance include Curvularia americana,Curvularia chlamydospora,
Curvularia hominis,Curvularia muehlenbeckiae,Curvularia pseudolunata [
7
], Curvularia brachyspora [
9
],
Curvularia senegalensis [
10
,
11
], Curvularia clavata [
12
], Curvularia tuberculata [
13
], and
Curvularia inaequalis [
14
–
16
]. A Curvularia infection in humans is designated as curvulariosis,
a subtype of phaeohyphomycoses (i.e., fungal infections caused by dematiaceous fungi) [
3
]. The
resulting diseases include deep and disseminated infections [
3
,
17
–
19
], infections complicating
peritoneal dialysis [
14
,
20
,
21
], respiratory infections including sinusitis and bronchopulmonary
mycosis [
3
,
10
,
22
], urinary tract infections [
23
], as well as localized infections affecting the skin,
nail [
4
,
24
,
25
], and the eye. Among eye infections, the involvement of Curvularia spp. is most
frequent in keratitis—a suppurative, ulcerative disease of the cornea, but endophthalmitis and chronic
dacryocystitis cases have also been reported [3,26].
In this study, we describe two new species of the genus Curvularia, the type strains of which
were isolated from corneal scraping samples derived from South Indian patients diagnosed with
fungal keratitis.
2. Results
2.1. Strain Selection and Case Details
About two thirds of the dematiaceous fungi isolated from corneal ulcers in the Aravind Eye
Hospital, Coimbatore, Tamil Nadu, India belong to the genus Curvularia (unpublished data). The
four strains involved in this study were selected retrospectively based on the inability of reliable
species-level identification of some Curvularia isolates by ITS sequence analysis. Details available of the
cases are presented in Table 1. All four patients were diagnosed with fungal corneal ulcer. The corneal
scrapings from the ulcers were in all cases positive for fungal filaments in direct microscopy (both
10% KOH and Gram staining). None of the cases had a history of contact lens wear. History of falling
dust (2) and mud (1) into the eye was recorded as predisposing factors. Based on the typical clinical
picture and the KOH report, topical antifungal therapy was started with natamycin (5% suspension)
and econazole drops (2%) every half an hour, along with homatropine (1%) administered three times a
day. Unfortunately, the patients were lost to follow up after one or two visits.
Pathogens 2020,9, 9 3 of 14
Table 1. Case details of the fungal keratitis infections.
Strain Age Sex Clinical
Diagnosis
Corneal
Scraping Therapy Outcome
SZMC 22225 80 Male Fungal
corneal ulcer 11 July 2012 NAT, ECZ,
HTR
Lost to follow up
after two visits
SZMC 22226 66 Male Fungal
corneal ulcer 2 March 2013 NAT, ECZ,
HTR
Lost to follow up
after one visit
SZMC 26758 40 Male Fungal
corneal ulcer 21 March 2011 NAT, ECZ,
HTR
Lost to follow up
after one visit
SZMC 26759 NA NA Fungal
corneal ulcer NA NA Lost to follow up
NAT: natamycin (5%); ECZ: econazole (2%), HTR: homatropine (1%); NA: data not available.
2.2. Updated Phylogeny of the Genus Curvularia
Table 2shows the strains and sequences involved in the phylogenetic analysis of the genus
Curvularia, including four isolates derived from cases of fungal keratitis diagnosed and treated in the
Aravind Eye Hospital, Coimbatore, Tamil Nadu, India. The tef1
α
dataset consisted of 902 characters
of nucleotide alignment without binary characters. The gpdh dataset contained 684 characters with
601 characters of nucleotide alignment and 63 binary characters derived from indel coding. The
length of the ITS alignment was 1193 characters long, containing 896 bp of nucleotide data and
297 binary characters.
Table 2. Sequences used for the phylogenetic analysis.
Curvularia Species Strain GenBank Accession Number
ITS tef1a gpdh
Bipolaris maydis CBS 136.29 TKJ909780 KM093794 KM034846
Curvularia aeria BRIP 61232b KX139029 KU552155 KU552162
Curvularia affinis CBS 154.34 TKJ909782 KM196566 KM230401
Curvularia ahvazensis SCUA-1bi TKJ415539 MG428686 MG428693
Curvularia akaii CBS 317.86 JX256420 KM196569 KM230402
Curvularia akaiiensis BRIP 16080 THE861833 KJ415453 KJ415407
Curvularia alcornii
MFLUCC 10-0703
TJX256424 JX266589 JX276433
Curvularia americana UTHSC 08-3414 TKJ415540 - HF565488
Curvularia asiatica
MFLUCC 10-0711
TKJ415541 JX266593 JX276436
Curvularia australiensis BRIP 12044 TKJ415542 KJ415452 KJ415406
Curvularia australis BRIP 12521 TMH414892 KJ415451 KJ415405
Curvularia bannonii BRIP 16732 TMH414894 KJ415450 KJ415404
Curvularia beasleyi BRIP 10972 TMH414911 MH433654 MH433638
Curvularia beerburrumensis BRIP 12942 TKP400638 MH433657 MH433634
Curvularia boeremae IMI 164633 TKJ415543 - MH433641
Curvularia borreriae MFLUCC 11-0422 KJ922372 KM196571 KP419987
Curvularia bothriochloae BRIP 12522 TKJ909765 KJ415449 KJ415403
Curvularia brachyspora CBS 186.50 HG778984 KM230405 KM061784
Curvularia buchloës CBS 246.49 TMF490814 KM196588 KM061789
Curvularia carica-papayae CBS 135941 THG779021 - HG779146
Curvularia chiangmaiensis CPC 28829 TMH275055 MF490857 MF490836
Curvularia chlamydospora UTHSC 07-2764 TKU552205 - HG779151
Curvularia chonburiensis
MFLUCC 16-0375
TMH414897 - MH412747
Curvularia clavata BRIP 61680b AF081447 KU552159 KU552167
Curvularia coatesiae BRIP 24261 TMH414898 MH433659 MH433636
Curvularia coicis CBS 192.29 TLT631357 JN601006 AF081410
Curvularia colbranii BRIP 13066 TLT631310 MH433660 MH433642
Pathogens 2020,9, 9 4 of 14
Table 2. Cont.
Curvularia Species Strain GenBank Accession Number
ITS tef1a gpdh
Curvularia comoriensis CBS 110673 KJ415544 - LT715841
Curvularia crassiseptum CBS 503.90 THG778985 - LT715882
Curvularia crustacea BRIP 13524 TMF490815 KJ415448 KJ415402
Curvularia cymbopogonis CBS 419.78 KJ415545 - HG779129
Curvularia dactyloctenicola CPC 28810 TLT631356 MF490858 MF490837
Curvularia dactyloctenii BRIP 12846 TJN192375 KJ415447 KJ415401
Curvularia deightonii CBS 537.70 MH414899 - LT715839
Curvularia ellisii CBS 193.62 THG778986 JN601007 JN600963
Curvularia eragrosticola BRIP 12538 TKJ909781 MH433661 MH433643
Curvularia eragrostidis CBS 189.48 HG778987 - HG779154
Curvularia geniculata CBS 187.50 JN192376 KM230410 KM083609
Curvularia gladioli CBS 210.79 KJ415546 - HG779123
Curvularia graminicola BRIP 23186a TKJ415547 JN601008 JN600964
Curvularia harveyi BRIP 57412 TKJ415548 KJ415446 KJ415400
Curvularia hawaiiensis BRIP 11987 TKJ415549 KJ415445 KJ415399
Curvularia heteropogonicola BRIP 14579 THG779011 KJ415444 KJ415398
Curvularia heteropogonis CBS 284.91 TJN192380 JN601013 JN600969
Curvularia hominis CBS 136985 TKJ922375 - HG779106
Curvularia homomorpha CBS 156.60 THG778991 JN601014 JN600970
Curvularia inaequalis CBS 102.42 TMH861533 KM196574 KM061787
Curvularia intermedia CBS 334.64 MH414900 - HG779155
Curvularia ischaemi CBS 630.82 TMH855025 - LT715790
Curvularia kenpeggii BRIP 14530 TMH414901 MH433662 MH433644
Curvularia kusanoi CBS 137.29 JX256429 JN601016 LT715862
Curvularia lamingtonensis BRIP 12259 TJF812154 MH433663 MH433645
Curvularia lunata CBS 730.96 TMH414902 JX266596 JX276441
Curvularia malina CBS 131274 THE792934 KR493095 KP153179
Curvularia mebaldsii BRIP 12900 TMF139088 MH433664 MH433647
Curvularia micropus CBS 127235 KJ909770 - LT715859
Curvularia microspora GUCC6272 TMG846737 MF139115 MF139106
Curvularia miyakei CBS 197.29 TKP400647 KM196568 KM083611
Curvularia mosaddeghii IRAN 3131C TKJ415550 MH392152 MH392155
Curvularia muehlenbeckiae CBS 144.63 TMH414910 KM196578 KP419996
Curvularia neergaardii BRIP 12919 TKJ415551 KJ415443 KJ415397
Curvularia neoindica IMI 129790 TMF490816 MH433667 MH433649
Curvularia nicotiae BRIP 11983 TJN601033 KJ415442 KJ415396
Curvularia nodosa CPC 28800 TKP400650 MF490859 MF490838
Curvularia nodulosa CBS 160.58 JN192384 JN601019 JN600975
Curvularia oryzae CBS 169.53 TKJ922380 KM196590 KP645344
Curvularia ovariicola CBS 470.90 TMH275056 JN601020 JN600976
Curvularia pallescens CBS 156.35 TKJ415552 KM196570 KM083606
Curvularia pandanicola
MFLUCC 15-0746
THG778995 MH412763 MH412748
Curvularia papendorfii CBS 308.67 TMH414905 KJ415441 KJ415395
Curvularia perotidis CBS 350.90 TKY905678 KM230407 HG779138
Curvularia petersonii BRIP 14642 TMH414906 MH433668 MH433650
Curvularia pisi CBS 190.48 TKJ415553 KY905697 KY905690
Curvularia platzii BRIP 27703b TKJ922373 MH433669 MH433651
Curvularia portulacae BRIP 14541 TKJ922376 KJ415440 KJ415393
Curvularia prasadii CBS 143.64 TMF490819 KM230408 KM061785
Curvularia protuberata CBS 376.65 THE861842 KM196576 KM083605
Curvularia pseudobrachyspora CPC 28808 THE861838 MF490862 MF490841
Pathogens 2020,9, 9 5 of 14
Table 2. Cont.
Curvularia Species Strain GenBank Accession Number
ITS tef1a gpdh
Curvularia pseudolunata UTHSC 09-2092 TJN192386 - HF565459
Curvularia pseudorobusta UTHSC 08-3458 MH414907 - HF565476
Curvularia ravenelii BRIP 13165 TKJ415555 JN601024 JN600978
Curvularia reesii BRIP 4358 TKJ909783 MH433670 MH433637
Curvularia richardiae BRIP 4371 TKX139030 KJ415438 KJ415391
Curvularia robusta CBS 624.68 TKJ415556 KM196577 KM083613
Curvularia rouhanii SCUA-2bi-2 THG779001 MG428687 MG428694
Curvularia ryleyi BRIP 12554 TKY905679 KJ415437 KJ415390
Curvularia senegalensis CBS 149.71 KJ415558 - HG779128
Curvularia soli CBS 222.96 TMH414904 KY905698 KY905691
Curvularia sorghina BRIP 15900 TKP400655 KJ415435 KJ415388
Curvularia sp. BRIP 17068b KP400654 MH433666 MH433648
Curvularia sp. AR5117 HE861826 KP735698 KP645349
Curvularia sp. MFLUCC 120177 JN192387 KP735697 KP645348
Curvularia sp. UTHSC 8809 MH414908 - HF565477
Curvularia spicifera CBS 274.52 KJ909777 JN601023 JN600979
Curvularia sporobolicola BRIP 23040b TMH275057 MH433671 MH433652
Curvularia subpapendorfii CBS 656.74 THG779023 KM196585 KM061791
Curvularia thailandicum
MFLUCC 15-0747
TJN192388 MH412764 MH412749
Curvularia trifolii CBS 173.55 KJ415559 - HG779124
Curvularia tripogonis BRIP 12375 TKC424596 JN601025 JN600980
Curvularia tropicalis BRIP 14834 TJX256433 KJ415434 KJ415387
Curvularia tsudae ATCC 44764 THG779024 KC503940 KC747745
Curvularia tuberculate CBS 146.63 TMF490822 JX266599 JX276445
Curvularia uncinate CBS 221.52 THG779026 - HG779134
Curvularia variabilis CPC 28815 TKP400652 MF490865 MF490844
Curvularia verruciformis CBS 537.75 MH414909 - HG779133
Curvularia verruculosa CBS 150.63 MH275058 KP735695 KP645346
Curvularia warraberensis BRIP 14817 TAF071338 MH433672 MH433653
Curvularia xishuangbannaensis KUMCC 17-0185 TKJ909780 MH412765 MH412750
Curvularia gudauskasii DAOM 165085 KX139029 KM093794 AF081393
Curvularia tamilnaduensis sp. nov. SZMC 22226 T*MN628311 MN628303 MN628307
SZMC 26758 * MN628308 MN628300 MN628304
SZMC 26759 * MN628309 MN628301 MN628305
Curvularia coimbatorensis sp. nov. SZMC 22225 T*MN628310 MN628302 MN628306
T
type strain; * Strains examined during the present study. Sequences derived from the present study are set in bold.
On the phylograms obtained from each of the three loci, the four keratitis isolates of this study
were resolved as two new species with over 80% of confidence values (data not shown), one of them
represented by the single isolate SZMC 22225, while the other one by isolates SZMC 22226, SZMC
26758, and SZMC 26758. As the individual inferences were largely congruent, the three loci were
concatenated and partitioned. The phylogenetic tree obtained from the concatenated dataset is shown
in Figure 1.
Pathogens 2020,9, 9 6 of 14
Pathogens 2020, 9, x FOR PEER REVIEW 6 of 13
Figure 1.
Maximum likelihood phylogeny of the genus Curvularia inferred from the
concatenated internal transcribed spacer (ITS), translation elongation factor 1-
α
(tef1a), and
glyceraldehyde-3-phosphate dehydrogenase (gpdh) sequences. The isolates examined in this study are
Pathogens 2020,9, 9 7 of 14
shown as the new species Curvularia tamilnaduensis and Curvularia coimbatorensis (highlighted in color).
Sequences of the reference Curvularia strains were collected from the GenBank Nucleotide database
(Table 1). Bootstrap support values greater than 60% are shown above the branches. Bipolaris maydis
CBS 136.29 was used to root the tree. Abbreviations of culture collections: BRIP: Plant Pathology
Herbarium, Queensland, Australia; CBS: Westerdijk Fungal Biodiversity Institute culture collection,
The Netherlands; CPC: Cultures of Pedro Crous, housed at Westerdijk Fungal Biodiversity Institute;
DAOM: Canadian National Mycological Herbarium, Ottawa, Canada; GUCC: Guizhou University
Culture Collection, Guizhou, China; IMI: CABI Bioscience, Eggham, UK; IRAN: Iranian Fungal Culture
Collection, Iranian Research Institute of Plant Protection, Tehran, Iran; KUMCC: Culture Collection
of Kunming Institute of Botany, Kunming, China; MFLUCC: Mae Fah Luang Culture Collection,
Chiang Rai, Thailand; SCUA: Collection of Fungal Cultures, Department of Plant Protection, Shahid
Chamran University of Ahvaz, Iran; SZMC: Szeged Microbiology Collection, Szeged, Hungary; UTHSC:
University of Tennessee Health Science Center, Memphis, USA. T: type strain.
2.3. Taxonomy and Related Information
Curvularia coimbatorensis N. Kiss & S. Kocsub
é
sp. nov. (Figure 2). MycoBank accession number:
MB 833656. The etymology is referring to the city in Tamil Nadu, South India where the type strain
was isolated.
Pathogens 2020, 9, x FOR PEER REVIEW 7 of 13
Figure 1. Maximum likelihood phylogeny of the genus Curvularia inferred from the concatenated
internal transcribed spacer (ITS), translation elongation factor 1-α (tef1a), and glyceraldehyde-3-
phosphate dehydrogenase (gpdh) sequences. The isolates examined in this study are shown as the
new species Curvularia tamilnaduensis and Curvularia coimbatorensis (highlighted in color). Sequences
of the reference Curvularia strains were collected from the GenBank Nucleotide database (Table 1).
Bootstrap support values greater than 60% are shown above the branches. Bipolaris maydis CBS 136.29
was used to root the tree. Abbreviations of culture collections: BRIP: Plant Pathology Herbarium,
Queensland, Australia; CBS: Westerdijk Fungal Biodiversity Institute culture collection, The
Netherlands; CPC: Cultures of Pedro Crous, housed at Westerdijk Fungal Biodiversity Institute;
DAOM: Canadian National Mycological Herbarium, Ottawa, Canada; GUCC: Guizhou University
Culture Collection, Guizhou, China; IMI: CABI Bioscience, Eggham, UK; IRAN: Iranian Fungal
Culture Collection, Iranian Research Institute of Plant Protection, Tehran, Iran; KUMCC: Culture
Collection of Kunming Institute of Botany, Kunming, China; MFLUCC: Mae Fah Luang Culture
Collection, Chiang Rai, Thailand; SCUA: Collection of Fungal Cultures, Department of Plant
Protection, Shahid Chamran University of Ahvaz, Iran; SZMC: Szeged Microbiology Collection,
Szeged, Hungary; UTHSC: University of Tennessee Health Science Center, Memphis, USA. T: type
strain.
Figure 2. Morphological features of Curvularia coimbatorensis SZMC 2225. (a) Colony morphology on
PDA (potato dextrose agar) medium after 7 d at 25 °C; (b,c) conidiophores with septate conidia; (d)
branching conidiophores; (e) swollen cells; (f–l) septate conidia. Scale bars: (b–e) 20 µm; (f–l) 10 µm.
Curvularia tamilnaduensis N. Kiss & S. Kocsubé sp. nov. (Figure 3). MycoBank accession number:
MB 833657. The etymology is referring to the state of South India where the type strain and the other
two examined strains were isolated.
Vegetative hyphae septate, subhyaline to brown, branched, smooth walled, but often heavily
asperulate, 2–3 µm in width. Colonies on PDA reaching approximately 6–7 cm in diameter after 7 d
at 25 °C, surface lanose, aerial mycelium abundant, margin fimbriate, olivaceous green.
Conidiophores erect, usually unbranched, in most cases uniformly brown, sometimes with paler tip,
seminematous, septate, slightly flexuous, rarely geniculate towards the apex, up to 125 µm long, 2.5–
4 µm wide. Conidiogenous cells integrated, terminal or intercalary, smooth, pale brown to brown,
mono- or polytretic, proliferating sympodially. Chlamydospores present, subglobose, terminal and
intercalary, 8–22 µm in diameter. Conidia ellipsoidal to clavate to obovoid, asymmetrical with paler
basal and apical cells, usually curved at the third cell from the base which is darker than the other
cells, (15-)20–23(-28) × (7-)8–10(-11) µm, (2-)3-distoseptate with non-protuberant, thickened, and
darkened hila.
Specimens examined: India, Coimbatore, human corneal scraping from corneal ulcer, 2013,
(holotype: freeze dried culture specimen in the Szeged Microbiological Collection (SZMC) at the
Figure 2.
Morphological features of Curvularia coimbatorensis SZMC 2225. (
a
) Colony morphology on
PDA (potato dextrose agar) medium after 7 days at 25
◦
C; (
b
,
c
) conidiophores with septate conidia;
(
d
) branching conidiophores; (
e
) swollen cells; (
f
–
l
) septate conidia. Scale bars: (
b
–
e
) 20
µ
m; (
f
–
l
) 10
µ
m.
Vegetative hyphae septate, subhyaline to brown, branched, smooth, 3–4
µ
m in width. Colonies
on PDA reaching approximately 4–6 cm in diameter after 7 days at 25
◦
C, surface funiculose, margin
fimbriate, olivaceous black to olivaceous grey, velutinous with sparse aerial mycelium. Conidiophores
erect, often branched, in most cases uniformly brown, sometimes pale brown at apex, seminematous,
septate, flexuous, in most cases geniculate towards the apex, up to 210
µ
m long, 3–4
µ
m wide, basal
cells sometimes swollen. Conidiogenous cells integrated, terminal, or intercalary with sympodial
proliferation, smooth, brown, mono- or polytretic. Chlamydospores not observed. Conidia ellipsoidal
to clavate to obovoid, asymmetrical with paler end cells, usually curved at the third cell from the base,
(13-)16–18(-23) ×(7-)8–9(-10) µm, 3-distoseptate, hila slightly protuberant, thickened and darkened.
Specimens examined: India, Coimbatore, human corneal scraping from corneal ulcer, 2012,
(holotype: freeze dried culture specimen in the Szeged Microbiological Collection (SZMC) at the
Pathogens 2020,9, 9 8 of 14
Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Hungary,
SZMC 22225, includes ex-type culture).
Curvularia tamilnaduensis N. Kiss & S. Kocsub
é
sp. nov. (Figure 3). MycoBank accession number:
MB 833657. The etymology is referring to the state of South India where the type strain and the other
two examined strains were isolated.
Pathogens 2020, 9, x FOR PEER REVIEW 8 of 13
Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Hungary,
SZMC 22226, includes ex-type culture); India, Coimbatore, human corneal scraping from corneal
ulcer, 2011, (SZMC 26758); India, Coimbatore, human corneal scraping from corneal ulcer, 2011–2013,
(SZMC 26759).
Figure 3. Morphological features of Curvularia tamilnaduensis SZMC 2226. (a) Colony morphology on
PDA medium after 7 d at 25 °C; (b) conidiophores with septate conidia; (c) subglobose intercalary
chlamydospore; (d–f) septate conidia. Scale bars: (b,c) 20 µm; (d–i) 10 µm.
2.4. Antifungal Susceptibilities of Curvularia Strains Isolated from Fungal Keratitis
The minimum inhibitory concentrations (MIC) of nine antifungal agents towards C.
coimbatorensis SZMC 22225, C. tamilnaduensis SZMC 22226, SZMC 26758, and SZMC 26759, as well as
the type strains of C. australiensis (CBS 172.57), C. hawaiiensis (CBS 173.57), and C. spicifera (CBS 274.52)
are shown in Table 3. The MIC of natamycin was 2 µg mL−1 for both new species and all other strains
tested, while substantial differences between them could be observed in the case of clotrimazole,
econazole, miconazole, and terbinafine, with the type strain of C. coimbatorensis having 4, 8, 4, and 4–
8 times higher values, respectively. Among the tested isolates, the type strain of C. spicifera proved to
be the less susceptible to clotrimazole, econazole, fluconazole, ketoconazole, and miconazole. Notable
strain-to-strain variations between the C. tamilnaduensis strains could be observed only in the case of
itraconazole and ketoconazole with detected MIC ranges of 0.03–0.25 and 0.06–0.25, respectively.
3. Discussion
The phylogenetic tree obtained from the concatenated dataset of three loci presents an update
about the phylogeny of Curvularia, which is mostly in agreement with the recently published
phylogenies of this genus (Figure 1). C. ischaemi formed a clade with C. coicis, which is in contradiction
with the results of Tan et al. [8] and Tibpromma et al. [27], where C. ischaemi formed a sister clade to
C. gladioli, but in agreement with the phylogram obtained by Madrid et al. [7] and Manamgoda et al.
[28]. Our analysis placed C. perotidis as a sister clade to C. australiensis, however, other studies
[7,8,27,29] suggested that this species is closer to C. spicifera. The placement of C. variabilis was also
different from previously published articles [8,29]. According to the analyses of Tan et al. [8] and
Marin-Felix et al. [29], C. variabilis forms a clade with C. hawaiiensis, C. nodosa, C. dactyloctenicola, and
C. beasleyi, however, in this study we found C. variabilis as a sister clade of C. tsudae and C. mebaldsii.
The same authors found C. tripogonis, C. pseudorobusta, C. robusta, C. alcornii, C. protuberata, and C.
inaequalis as members of two distinct monophyletic clades, while our results indicate that these
species are closely related and paraphyletic, however, none of the topologies have strong statistical
supports. The observed slight differences between the previous inferences and our analyses did not
Figure 3.
Morphological features of Curvularia tamilnaduensis SZMC 2226. (
a
) Colony morphology on
PDA medium after 7 days at 25 ◦C; (b) conidiophores with septate conidia; (c) subglobose intercalary
chlamydospore; (d–f) septate conidia. Scale bars: (b,c) 20 µm; (d–i) 10 µm.
Vegetative hyphae septate, subhyaline to brown, branched, smooth walled, but often heavily
asperulate, 2–3
µ
m in width. Colonies on PDA reaching approximately 6–7 cm in diameter after 7 days
at 25
◦
C, surface lanose, aerial mycelium abundant, margin fimbriate, olivaceous green. Conidiophores
erect, usually unbranched, in most cases uniformly brown, sometimes with paler tip, seminematous,
septate, slightly flexuous, rarely geniculate towards the apex, up to 125
µ
m long, 2.5–4
µ
m wide.
Conidiogenous cells integrated, terminal or intercalary, smooth, pale brown to brown, mono- or
polytretic, proliferating sympodially. Chlamydospores present, subglobose, terminal and intercalary,
8–22
µ
m in diameter. Conidia ellipsoidal to clavate to obovoid, asymmetrical with paler basal and apical
cells, usually curved at the third cell from the base which is darker than the other cells, (15-)20–23(-28)
×(7-)8–10(-11) µm, (2-)3-distoseptate with non-protuberant, thickened, and darkened hila.
Specimens examined: India, Coimbatore, human corneal scraping from corneal ulcer, 2013,
(holotype: freeze dried culture specimen in the Szeged Microbiological Collection (SZMC) at the
Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Hungary,
SZMC 22226, includes ex-type culture); India, Coimbatore, human corneal scraping from corneal
ulcer, 2011, (SZMC 26758); India, Coimbatore, human corneal scraping from corneal ulcer, 2011–2013,
(SZMC 26759).
2.4. Antifungal Susceptibilities of Curvularia Strains Isolated from Fungal Keratitis
The minimum inhibitory concentrations (MIC) of nine antifungal agents towards C. coimbatorensis
SZMC 22225, C. tamilnaduensis SZMC 22226, SZMC 26758, and SZMC 26759, as well as the type strains
of C. australiensis (CBS 172.57), C. hawaiiensis (CBS 173.57), and C. spicifera (CBS 274.52) are shown
in Table 3. The MIC of natamycin was 2
µ
g mL
−1
for both new species and all other strains tested,
while substantial differences between them could be observed in the case of clotrimazole, econazole,
miconazole, and terbinafine, with the type strain of C. coimbatorensis having 4, 8, 4, and 4–8 times
higher values, respectively. Among the tested isolates, the type strain of C. spicifera proved to be
the less susceptible to clotrimazole, econazole, fluconazole, ketoconazole, and miconazole. Notable
Pathogens 2020,9, 9 9 of 14
strain-to-strain variations between the C. tamilnaduensis strains could be observed only in the case of
itraconazole and ketoconazole with detected MIC ranges of 0.03–0.25 and 0.06–0.25, respectively.
Table 3.
Antifungal susceptibilities of the Curvularia coimbatorensis and Curvularia tamilnaduensis
strains in comparison with the type strains of Curvularia australiensis,Curvularia hawaiiensis, and
Curvularia spicifera determined by the CLSI (Clinical & Laboratory Standards Institute) broth
microdilution method (minimum inhibitory concentrations (MIC) values in µg mL−1).
Strain Antifungal Agent
AMB CLT ECN FLC ITC KTC MCZ NTM TRB
C. australiensis CBS 172.57 T0.25 0.25 0.125 16 0.03 0.25 0.25 2 0.25
C. hawaiiensis CBS 173.57 T0.25 0.06 0.06 4 0.03 0.06 0.125 2 0.25
C. spicifera CBS 274.52 T0.5 4 2 >32 0.25 2 2 2 1
C. coimbatorensis SZMC 22225 T0.5 0.5 1 32 0.25 0.25 1 2 1
C. tamilnaduensis SZMC 22226 T1 0.125 0.125 8 0.03 0.06 0.25 2 0.25
C. tamilnaduensis SZMC 26758 0.5 0.125 0.125 16 0.03 0.25 0.25 2 0.125
C. tamilnaduensis SZMC 26759 1 0.125 0.125 16 0.25 0.25 0.25 2 0.25
T
: type strain; AMB: amphotericin B; CLT: clotrimazole; ECN: econazole; FLC: fluconazole; ITC: itraconazole; KTC:
ketoconazole; MCZ: miconazole; NTM: natamycin; TRB: terbinafine.
3. Discussion
The phylogenetic tree obtained from the concatenated dataset of three loci presents an update about
the phylogeny of Curvularia, which is mostly in agreement with the recently published phylogenies of
this genus (Figure 1). C. ischaemi formed a clade with C. coicis, which is in contradiction with the results
of Tan et al. [
8
] and Tibpromma et al. [
27
], where C. ischaemi formed a sister clade to C. gladioli, but in
agreement with the phylogram obtained by Madrid et al. [
7
] and Manamgoda et al. [
28
]. Our analysis
placed C. perotidis as a sister clade to C. australiensis, however, other studies [
7
,
8
,
27
,
29
] suggested that
this species is closer to C. spicifera. The placement of C. variabilis was also different from previously
published articles [
8
,
29
]. According to the analyses of Tan et al. [
8
] and Marin-Felix et al. [
29
], C. variabilis
forms a clade with C. hawaiiensis,C. nodosa,C. dactyloctenicola, and C. beasleyi, however, in this study we
found C. variabilis as a sister clade of C. tsudae and C. mebaldsii. The same authors found C. tripogonis,
C. pseudorobusta,C. robusta,C. alcornii,C. protuberata, and C. inaequalis as members of two distinct
monophyletic clades, while our results indicate that these species are closely related and paraphyletic,
however, none of the topologies have strong statistical supports. The observed slight differences
between the previous inferences and our analyses did not affect the validity of any of the previously
described species, and some of them might be the result of the slightly broader taxon sampling.
One of the newly described species, C. coimbatorensis is only known from the type specimen
isolated from corneal ulcer. Phylogenetic analysis based on three loci placed C. coimbatorensis as a sister
clade to the other newly described species C. tamilnaduensis. The two species are closely related, but can
be distinguished by tef1a,gpdh, and ITS sequences, with percentage identities of 99%, 98%, and 99%,
respectively. C. petersonii [
8
] is also closely related and can be distinguished by all three loci (98% in
tef1a, 93% in gpdh and 96% in ITS). C. coimbatorensis differs from C. tamilnaduensis in colony morphology,
the lack of chlamydospores, and the size of conidia. C. petersonii is very similar in colony morphology,
however, has significantly shorter (up to 110
µ
m) and only slightly geniculate conidiophores bearing
narrower (5-)5.5–6(-7) conidia [8]. C. coimbatorensis has longer conidiophores.
The phylogenetic analysis based on three loci placed the other newly described species,
C. tamilnaduensis as a sister clade to the recently described species C. petersonii.C. tamilnaduensis
can be reliably distinguished from the ex-type of C. petersonii by tef1a,gpdh and ITS sequences with
percentage identities of 99%, 95%, and 96%, respectively. The two species also differ by morphology, as
C. petersonii has not been reported to produce chlamydospores and has different conidial dimensions
(17–19
×
5.5–6) [
8
]. C. americana [
7
] and C. verruculosa [
30
] are also related species with considerable
amount of genetic distances and none of these species have been reported before to have chlamydopores.
Pathogens 2020,9, 9 10 of 14
C. americana has 4(-5)-distoseptate and wider (7–15
µ
m) conidia, while C. verruculosa has mostly
3-distoseptate conidia, but also wider (12–17 µm) than those of C. tamilnaduensis.
The antifungal susceptibilities of the examined strains of C. coimbatorensis and C. tamilnaduensis
to amphotericin B, clotrimazole, econazole, fluconazole, itraconazole, ketoconazole, miconazole,
natamycin, and terbinafine were within the MIC ranges reported for other clinically relevant Curvularia
species in the study of Guarro et al. [
11
] and the review of Krizs
á
n et al. [
3
]. The type strain of
C. coimbatorensis proved to be less susceptible than the strains of C. tamilnaduensis to all antifungals
except for natamycin. For itraconazole and ketoconazole our results are in agreement with the study
of Guarro et al. [
11
], who reported that amphotericin B, itraconazole, miconazole and ketoconazole
are highly effective against a series of Curvularia species known from fungal keratitis (C. brachyspora,
C. clavata,C. geniculata,C. lunata,C. pallescens,C. senegalensis, and C. verruculosa).
4. Materials and Methods
4.1. Curvularia Strains, Culture Conditions, and Morphological Examination
The Curvularia strains involved in this study derived from corneal scrapings from fungal
corneal ulcers of keratitis patients attending the Aravind Eye Hospital and Postgraduate Institute of
Ophthalmology, Coimbatore, India. All cases were initially screened by experienced ophthalmologists,
and the corneal scrapings were collected following the clinical diagnosis of fungal keratitis. The
samples were initially processed microbiologically for the isolation of the causative agents as described
earlier [
31
]. The corneal scrapings of all patients were subjected to Gram stain, Giemsa stain, and 10%
KOH wet mount. Culture methods involved direct inoculation of specimens onto 5% sheep blood agar,
chocolate agar, non-nutrient agar, potato dextrose agar, thioglycolate broth, and brain–heart infusion
broth. The microbial cultures were considered positive only if the growth of the same organism was
demonstrated on two or more solid media, or there was confluent growth at the site of inoculation
on one solid medium with consistent direct microscopic findings. The isolates were deposited in
the Szeged Microbiology Collection (SZMC, Szeged, Hungary) under the accession numbers SZMC
22225, SZMC 22226, SZMC 26758, and SZMC 26759. Colony morphology of the isolates was examined
on PDA (BioLab, Budapest, Hungary) medium after 7 days of incubation at 25
◦
C under normal
day/night light conditions. Micromorphological characters were examined with a Leica DMI 4000B
(Leica, Wetzlar, Germany) microscope equipped with a Leica DFC 295 camera. Microscopic features
were examined in lactic acid (100% v/v) on glass slides. Conidiophores were studied in the same
mounting fluid with the transparent tape method. Conidiophores and conidia were measured using
the software ImageJ v2.52a (National Institute of Mental Health, Bethesda, MD, USA). Size ranges of
the conidia were derived from 50 measurements. Lengths and widths are given as (minimum value)
mean size minus SD-mean size plus SD (maximum value).
4.2. DNA Extraction, Amplification, Sequencing, and Phylogenetic Analysis
Genomic DNA was isolated from the examined Curvularia strains SZMC 22225, SZMC 22226, SZMC
26758, and SZMC 26759 with the Masterpure
™
Yeast DNA Purification Kit (Epicentre Biotechnologies,
Madison, WI, USA) according to the manufacturer’s instructions. Fragments of tef1a and gpdh were
amplified as described previously [
5
,
32
,
33
]. The ITS region of the ribosomal RNA gene cluster was
amplified according to White et al. [
34
]. Sequencing of the amplicons was carried out on a 3500 Genetic
Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) by the sequencing service of the Biological
Research Centre, Szeged, Hungary. Resulting sequences were deposited in the GenBank Nucleotide
database (www.ncbi.nlm.nih.gov) under the accession numbers shown in Table 2.
Sequences of the four clinical isolates were aligned with publicly available sequences of
108 previously described Curvularia species, as well as Bipolaris maydis as the outgroup (Table 2).
Phylogenetic analyses were conducted using three loci (tef1
α
,gpdh and ITS). Sequences of all three loci
were aligned with the phylogeny-aware sequence alignment tool Canopy v0.1.4 using RAxML as tree
Pathogens 2020,9, 9 11 of 14
estimator and PRANK [
35
] with the -F option as the aligner with 10 iterations and seed decomposition
strategy. Alignments of the three loci were concatenated and partitioned by region. The tef1
α
sequences
formed one partition while in the case of gpdh sequences the dataset was partitioned to exons and
introns. The ITS dataset was divided to rDNA and ITS1-ITS2 regions. Alignments of gpdh and ITS
datasets contained high number of indels with important phylogenetic signal, therefore gaps were
coded as absence/presence characters by SequenceMatrix v1.8 [
36
] using the simple indel coding
algorithm [
37
]. The two indel matrices were concatenated and added as a single partition to the dataset.
Maximum likelihood analysis was performed using RAxML-NG v0.9.0 [
38
] under the GTR model with
gamma-distributed rate heterogeneity using empirical base frequencies. As indel-based datasets do
not contain constant sites, the ascertainment bias correction described by Lewis [
39
] was used for this
partition. Statistical support of the best ML tree was obtained with 1000 thorough bootstrap replicates.
4.3. Antifungal Susceptibility Testing
In vitro
antifungal susceptibility tests were carried out according to the CLSI M38-A2 broth
microdilution method [
40
]. Nine antifungal agents: amphotericin B, clotrimazole, econazole,
fluconazole, itraconazole, ketoconazole, miconazole, natamycin and terbinafine (Sigma-Aldrich,
Budapest, Hungary) were examined. Microtiter plates were incubated at 35
◦
C for 72 h. Plates were
evaluated both spectrophotometrically with a Spectrostar Nano microplate reader (BMG Labtech,
Ortenberg, Germany) and by visual examination.
5. Conclusions
The present study demonstrates, that although the phylogeny of the genus Curvularia is resolved
and well established, further expansion can be expected both in the list of described Curvularia species
and in the known spectrum of clinically relevant members of the genus. The collection of further
keratitis isolates from the genus Curvularia and gaining data about their antifungal susceptibilities are
therefore tasks of increasing importance. Furthermore, comparing the infectivity of various Curvularia
species causing keratitis—including the recently described ones—in animal keratitis models would be
an intriguing topic for future research.
Author Contributions:
Conceptualization, S.K., L.K., T.P. and C.V.; methodology, N.K., A.M., M.H. and S.K.;
software, S.K.; validation, R.R., P.M., M.H., C.V., M.V. and S.K.; formal analysis, K.K., T.P. and M.V.; investigation,
N.K., A.M., P.M., K.K., M.H., M.V. and S.K.; resources, R.R., P.M., A.M., T.P. and C.V.; data curation, N.K., S.K.,
K.K., L.K. and M.H.; writing—original draft preparation, N.K., S.K., L.K.; writing—review and editing, N.K., S.K.,
L.K., P.M., T.P. and C.V.; visualization, N.K., M.H. and S.K.; supervision, S.K.; project administration, S.K., T.P.,
L.K., and P.M.; funding acquisition, S.K., T.P., L.K. and P.M. All authors have read and agreed to the published
version of the manuscript.
Funding:
This research was funded by grants NKFI PD-116609 (National Research, Development and Innovation
Office, Hungary), GINOP-2.3.2-15-2016-00035 (Sz
é
chenyi 2020 Programme) and also supported by the COST
action HUPLANTcontrol (Control of Human Pathogenic Micro-organisms in Plant Production Systems, CA16110).
LK is grantee of the J
á
nos Bolyai Research Scholarship (Hungarian Academy of Sciences) and the Bolyai Plus
Scholarship (New National Excellence Programme). TP and MH are supported by the grants LP2016-8/2016 and
by the FIKP program (TUDFO/4738-1/2019 ITM) of the Ministry of Human Capacities.
Acknowledgments:
The authors wish to thank Venkatapathy Narendran (Aravind Eye Hospital and Postgraduate
Institute of Ophthalmology, Coimbatore, Tamil Nadu, India), Coimbatore Subramanian Shobana (Department of
Microbiology, PSG College of Arts and Science, Coimbatore, Tamil Nadu, India) and Kanesan Panneer Selvam
(Department of Microbiology, M.R Government Arts College, Mannargudi, Tamil Nadu, India) for constantly
supporting the research efforts on fungal keratitis within the frames of the Indo-Hungarian Fungal Keratitis
Research Group.
Conflicts of Interest: The authors declare no conflict of interest.
Pathogens 2020,9, 9 12 of 14
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