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Three new Curvularia species from clinical and environmental sources
expand article infoIsabel Iturrieta-González, Josepa Gené, Nathan Wiederhold§, Dania García
‡ University Rovira i Virgili, Reus, Spain
§ University of Texas Health Science Center, San Antonio, United States of America
Open Access

Abstract

Curvularia is a Pleosporalean monophyletic genus with a great diversity of species, including relevant phytopathogenic, animal and human pathogenic fungi. However, their microscopic identification is difficult due to overlapping morphological features amongst species. In recent years, multi-locus sequence analysis using the ITS region of the rDNA and fragments of the genes gapdh and tef1 revealed numerous cryptic species, especially in isolates that commonly produced 3-septate conidia. Therefore, based on sequence analysis of the above-mentioned DNA barcodes recommended for species delineation in Curvularia, we propose three novel species, C. paraverruculosa, C. suttoniae and C. vietnamensis, isolated from soil, human clinical specimens and plant material, respectively, collected in different countries. These new species are morphologically characterised and illustrated in the present study. Curvularia paraverruculosa differs from its counterparts, C. americana and C. verruculosa, mainly by its narrower conidia. Curvularia suttoniae and C. vietnamensis are closely related to C. petersonii, but the former two have larger conidia.

Keywords

Ascomycetes, Dematiaceous hyphomycetes, phylogeny, Pleosporaceae, taxonomy

Introduction

The genus Curvularia Boedijn (1933), typified by C. lunata (Wakker) Boedijn, belongs in Pleosporaceae, Pleosporales (Wijayawardene et al. 2018). Members of Curvularia show different life modes, i.e. saprophytic, endophytic and also pathogenic on plants and animals (Marin-Felix et al. 2017a). Phytopathogenic species can affect wild grasses and staple crops, such as rice, maize, wheat or sorghum and give rise to serious losses in agricultural production (Gautam et al. 2013, Manamgoda et al. 2015, Marin-Felix et al. 2017a, Tan et al. 2018). The endophytic species have garnered interest in recent years for their use in the production of bio-based products that are beneficial to living organisms and the environment (Bengyella et al. 2019). Since the first report of Curvularia as a human pathogen in a patient with mycetoma (Baylet et al. 1959), other clinical presentations have been reported, such as superficial and deep infections that mainly affect the respiratory tract but can even cause cerebral phaeohyphomycosis with an extremely poor prognosis (de Hoog et al. 2000).

The genus is morphologically distinguished mainly by its asexual morph, which shows sympodial conidiophores with mono- to polytretic conidiogenous cells and transversally septate conidia. Typically, the conidia in Curvularia are curved due to the hypertrophy of one of the intermediate cells and they are euseptate (Ellis 1971), although other authors opine that the conidia in Curvularia are distoseptate (Sivanesan 1987, Seifert et al. 2011, Madrid et al. 2014). The species of Bipolaris and Exserohilum have typically straight and distoseptate conidia; however, some of them have been transferred to Curvularia, based on their DNA sequence analyses (Manamgoda et al. 2012, Hernández-Restrepo et al. 2018, Tan et al. 2018). Furthermore, due to the overlapping of morphological characters amongst certain species of Curvularia, such as conidial size, shape and septation, an accurate identification at the species level is difficult without a DNA sequence analysis (da Cunha et al. 2013, Madrid et al. 2014, Manamgoda et al. 2015). Several cryptic species have been described recently using only multi-locus sequence analyses of the recommended DNA barcodes for species delimitation, i.e. the internal transcribed spacer (ITS) region of the rDNA and the protein-coding loci glyceraldehyde-3-phosphate dehydrogenase (gapdh) and translation elongation factor 1-a (tef1) (Marin-Felix et al. 2017a, Tan et al. 2018). Nearly 130 species have so far been accepted in Curvularia, including the species classified previously in the teleomorphic genera Cochliobolus and Pseudocochliobolus after applying the current criteria for fungal nomenclature (Manamgoda et al. 2012, 2015, Madrid et al. 2014, Hyde et al. 2017, Marin-Felix et al. 2017a, 2017b, Dehdari et al. 2018, Heidari et al. 2018, Hernández-Restrepo et al. 2018, Liang et al. 2018, Mehrabi-Koushki et al. 2018, Tan et al. 2018, Tibpromma et al. 2018, Kiss et al. 2019, Raza et al. 2019, Zhang et al. 2020).

Based on a polyphasic approach, combining morphological and phylogenetic analyses, three novel Curvularia species are proposed here, isolated from human clinical specimens in the USA, soil in Mexico and seed and plant debris in Vietnam and Indonesia, respectively.

Material and methods

Origin of isolates

Five unidentified Curvularia isolates, maintained in the fungal collection of the Medical School of the Rovira i Virgili University (FMR; Reus, Spain), were included in the study. Two of these (FMR 10992, FMR 11690) were isolated from human specimens in the USA by Deana A. Sutton of the Fungus Testing Laboratory at the University of Texas Health Sciences Center (UTHSC; San Antonio, USA) and the other three (FMR 11956, FMR 17656, FMR 17659) were isolated from environmental samples; the first from sorghum seeds collected in Indonesia, the second from soil collected in the Mexican region of Michoacán and the third from unidentified plant material collected in the north-east of Vietnam.

DNA extraction, PCR, sequencing and phylogenetic analysis

The fungal DNA was extracted from colonies growing on potato dextrose agar (PDA; Pronadisa, Madrid, Spain) for 7 to 10 days at 25 °C in darkness and following the protocol of Müller et al. (1998). The ITS barcode, including the 5.8S gene and the genes gapdh and tef1 were analysed following Marin-Felix et al. (2017a). Amplification was carried out using the primer pairs ITS5/ITS4 for the ITS region (White et al. 1990), gpd1/gpd2 for gapdh (Berbee et al. 1999) and EF983/2218R for tef1 (Schoch et al. 2009). The PCR products were purified and stored at -20 °C until sequencing. The same pairs of primers used for the amplification were also used to obtain the DNA sequences, which were processed at Macrogen Europe (Macrogen Inc., Madrid, Spain). The sequences of each isolate were edited using SeqMan v. 7.0.0 (DNAStar Lasergene, Madison, WI, USA) to obtain the consensus sequences.

We made a preliminary comparison of gapdh sequences generated from our isolates with those of the National Center for Biotechnology Information (NCBI) using the Basic Local Alignment Search Tool (BLASTn) for their molecular identification. To establish the phylogenetic position of unidentified isolates with respect to the most accepted species in Curvularia, we carried out individual (data not shown) and combined alignments of the three loci complemented by all available sequences of the ex-type and reference strains of Curvularia species retrieved from NCBI (Table 1). Based on this first phylogeny of the genus, a more restricted multi-locus analysis was carried out, including only those Curvularia species most related to the isolates under study. The alignments were made in the MEGA (Molecular Evolutionary Genetics Analysis) software v.6.0. (Tamura et al. 2013), using ClustalW algorithm (Thompson et al. 1994), refined with MUSCLE (Edgar 2004) in the same platform and manually adjusted as necessary. Phylogenetic reconstructions were made using Maximum Likelihood (ML) and Bayesian Inference (BI) approaches under RAxML-HPC2 on XSEDE v.8.2.12 (Stamatakis et al. 2014) in CIPRES Science gateway portal (Miller et al. 2010) and MrBayes v. 3.2.6 (Ronquist et al. 2012), respectively.

Table 1.

Species included in this study, their substrate, origin and GenBank accession numbers.

Species Strain no1 Substrate Country Genbank accession no.2
ITS gapdh tef1
Bipolaris maydis CBS 136.29 T Zea mays USA AF071325 KM034846 KM093794
B. saccharicola CBS 155.26 T Unknown Unknown KY905674 KY905686 KY905694
Curvularia aeria CBS 294.61 T Air Brazil HF934910 HG779148
C. affinis CBS 154.34 T Unknown Indonesia KJ909780 KM230401 KM196566
C. ahvazensis CBS 144673 T Zinnia elegans Iran KX139029 MG428693 MG428686
C. akaii CBS 317.86 Themada triandra subsp. japonica Japan KJ909782 KM230402 KM196569
C. akaiiensis BRIP 16080 T Unknown India KJ415539 KJ415407 KJ415453
C. alcornii MFLUCC 10-0703 T Z. mays Thailand JX256420 JX276433 JX266589
C. americana UTHSC 08-3414 T Human ankle USA HE861833 HF565488
UTHSC 07-2649 Human toe tissue USA HE861834 HF565486
UTHSC 08-84 Human nasal sinus USA HG779015 HG779115
UTHSC 08-278 Human peritoneal dialysis fluid USA HE861832 HF565487
UTHSC 08-2697 Human leg USA HG779016 HG779117
C. annelliconidiophori CGMCC3.19352 T Roots of Saccharum officinarum China MN215641 MN264077 MN263935
C. asiatica MFLUCC 10-0711 T Panicum sp. Thailand JX256424 JX276436 JX266593
C. australiensis BRIP 12044 T Oryza sativa Australia KJ415540 KJ415406 KJ415452
CBS 172.57 O. sativa seeds Vietnam JN601026 JN601036 JN601003
C. australis BRIP 12521 T Sporobolus caroli Australia KJ415541 KJ415405 KJ415451
C. bannonii BRIP 16732 T Jacquemontia tamnifolia USA KJ415542 KJ415404 KJ415450
C. beasleyi BRIP 10972 T Chloris gayana Australia MH414892 MH433638 MH433654
BRIP 15854 Leersia hexandra Australia MH414893 MH433639 MH433655
C. beerburrumensis BRIP 12942 T Eragrostis bahiensis Australia MH414895 MH433634 MH433657
C. boeremae IMI 164633 T Portulaca oleracea India MH414911 MH433641
C. borreriae CBS 859.73 Volcanic ash soil Chile HE861848 HF565455
C. bothriochloae BRIP 12522 T Bothriochloa bladhii Australia KJ415543 KJ415403 KJ415449
C. brachyspora CBS 186.50 Soil Indonesia KJ922372 KM061784 KM230405
C. buchloes CBS 246.49 T Buchloë dactyloides USA KJ909765 KM061789 KM196588
C. carica-papayae CBS 135941 T Carica papaya India HG778984 HG779146
C. chiangmaiensis CPC 28829 T Z. mays Thailand MF490814 MF490836 MF490857
C. chlamydospora UTHSC 07-2764 T Human toe nail USA HG779021 HG779151
C. chonburiensis MFLUCC 16-0375 T Dead leaf of Pandanus sp. Thailand MH275055 MH412747
C. clavata BRIP 61680 Oryza sp. Australia KU552205 KU552167 KU552159
C. cymbopogonis CBS 419.78 Yucca leaf spot Netherlands HG778985 HG779129
C. coatesiae BRIP 24261 T Litchi chinensis Australia MH414897 MH433636 MH433659
C. coicis CBS 192.29 T Coix lacryma-jobi Japan AF081447 AF081410 JN601006
C. coimbatorensis SZMC 22225 T Human corneal scraping India MN628310 MN628306 MN628302
C. colbranii BRIP 13066 T Crinum zeylanicum Australia MH414898 MH433642 MH433660
C. comoriensis CBS 110673 Unknown Unknown LT631357 LT715841
C. crassiseptum CBS 503.90 T Plant material Nigeria LT631310 LT715882
C. crustacea BRIP 13524 T Sporobolus sp. Indonesia KJ415544 KJ415402 KJ415448
C. dactyloctenicola CPC 28810 T Dactyloctenium aegyptium Thailand MF490815 MF490837 MF490858
C. dactyloctenii BRIP 12846 T Dactyloctenium radulans Australia KJ415545 KJ415401 KJ415447
C. deightonii CBS 537.70 Sorghum vulgare Denmark LT631356 LT715839
C. determinata CGMCC3.19340 T Leaves of S. officinarum China MN215653 MN264088 MN263947
C. elliptiformis CGMCC3.19351 T Roots of S. officinarum China MN215656 MN264091 MN263950
C. ellisii CBS 193.62 T Air Pakistan JN192375 JN600963 JN601007
C. eragrosticola BRIP 12538 T Eragrostis pilosa Australia MH414899 MH433643 MH433661
C. eragrostidis CBS 189.48 Sorghum seed Indonesia HG778986 HG779154
C. falsilunata CGMCC3.19329 T Roots of S. officinarum China MN215660 MN264093 MN263954
C. flexuosa CGMCC3.19447 T Roots of S. officinarum China MN215663 MN264096 MN263957
C. gladioli CBS 210.79 Gladiolus leaf Romania HG778987 HG779123
C. geniculata CBS 187.50 Andropogon sorghum seed Indonesia KJ909781 KM083609 KM230410
C. graminícola BRIP 23186 T Aristida ingrata Australia JN192376 JN600964 JN601008
C. guangxiensis CGMCC3.19330 T Roots of S. officinarum China MN215667 MN264100 MN263961
C. gudauskasii DAOM 165085 Unknown Unknown AF071338 AF081393
C. harveyi BRIP 57412 T Triticum aestivum Australia KJ415546 KJ415400 KJ415446
C. hawaiiensis BRIP 11987 T O. sativa USA KJ415547 KJ415399 KJ415445
C. heteropogonicola BRIP 14579 T Heteropogon contortus India KJ415548 KJ415398 KJ415444
C. heteropogonis CBS 284.91 T H. contortus Australia KJ415549 JN600969 JN601013
C. hominis CBS 136985 T Human cornea USA HG779011 HG779106
C. homomorpha CBS 156.60 T Air USA JN192380 JN600970 JN601014
C. inaequalis CBS 102.42 T Soil France KJ922375 KM061787 KM196574
C. intermedia CBS 334.64 Avena versicolor USA HG778991 HG779155
C. ischaemi CBS 630.82 T Ischaemum indicum Solomon Islands MH861533 JX276440
C. kenpeggii BRIP 14530 T Triticum aestivum Australia MH414900 MH433644 MH433662
C. kusanoi CBS 137.29 Eragrostis major Japan JN192381 LT715862 JN601016
C. lamingtonensis BRIP 12259 T Microlaena stipoides Australia MH414901 MH433645 MH433663
C. lunata CBS 730.96 T Human lung biopsy USA JX256429 JX276441 JX266596
C. malina CBS 131274 T Zoysia matrella USA JF812154 KP153179 KR493095
C. manamgodae CGMCC3.19446 T Roots of S. officinarum China MN215677 MN264110 MN263971
LC13495 Roots of S. officinarum China MN215678 MN264111 MN263972
C. mebaldsii BRIP 12900 T Cynodon transvaalensis Australia MH414902 MH433646 MH433664
BRIP 13983 Cynodon dactylon x C. transvaalensis Australia MH414903 MH433647 MH433665
C. micropus CBS 127235 ET Paspalum notatum Georgia HE792934 LT715859
C. microspora GUCC 6272 T Hippeastrum striatum China MF139088 MF139106 MF139115
C. miyakei CBS 197.29 T Eragrostis pilosa Japan KJ909770 KM083611 KM196568
C. mosaddeghii IRAN 3131C T Syzygium cumini Iran MG846737 MH392155 MH392152
C. muehlenbeckiae CBS 144.63 T Sorghum sp. USA MH858242 HG779108 KM196578
C. neergaardii BRIP 12919 T O. sativa Ghana KJ415550 KJ415397 KJ415443
CBS 276.91 Unknown Australia LT631362 LT715848
C. neoindica IMI 129790 T Brassica nigra India MH414910 MH433649 MH433667
C. nicotiae BRIP 11983 T Soil Algeria KJ415551 KJ415396 KJ415442
C. nodosa CPC 28800 T Digitaria ciliaris Thailand MF490816 MF490838 MF490859
CPC 28801 Brachiaria reptans Thailand MF490817 MF490839 MF490860
C. nodulosa CBS 160.58 Eleusine indica Unknown JN601033 JN600975 JN601019
C. oryzae CBS 169.53 T O. sativa Vietnam KP400650 KP645344 KM196590
C. ovariicola CBS 470.90 T Eragrostis interrupta Australia JN192384 JN600976 JN601020
C. pallescens CBS 156.35 T Air Indonesia KJ922380 KM083606 KM196570
C. palmicola MFLUCC 14-0404 T Dead branches of Acoelorrhaphe wrightii Thailand MF621582
C. pandanicola MFLUCC 15-0746 T Dead leaf of Pandanus sp. Thailand MH275056 MH412748 MH412763
C. papendorfii CBS 308.67 T Acacia karroo South Africa KJ909774 KM083617 KM196594
C. paraverruculosa FMR 17656 T Soil Mexico LR736641 LR736646 LR736649
C. petersonii BRIP 14642 T D. aegyptium Australia MH414905 MH433650 MH433668
C. perotidis CBS 350.90 T Perotis rara Australia JN192385 KJ415394 KM230407
C. phaeospara CGMCC3.19448 T Roots of S. officinarum China MN215686 MN264118 MN263980
C. pisi CBS 190.48 T Pisum sativum Canada KY905678 KY905690 KY905697
C. plantarum CGMCC3.19342 T Roots of S. officinarum China MN215688 MN264120 MN263982
C. platzii BRIP 27703b T Cenchrus clandestinum Australia MH414906 MH433651 MH433669
C. polytrata CGMCC3.19338 T Roots of S. officinarum China MN215691 MN264123 MN263984
C. portulacae BRIP 14541 T Portulaca oleracea USA KJ415553 KJ415393 KJ415440
C. prasadii CBS 143.64 T Jasminum sambac India KJ922373 KM061785 KM230408
C. protuberans CGMCC3.19360 T Leaves of S. officinarum China MN215693 MN264125 MN263986
C. protuberata CBS 376.65 T Deschampsia flexuosa UK KJ922376 KM083605 KM196576
C. pseudobrachyspora CPC 28808 T Eleusine indica Thailand MF490819 MF490841 MF490862
C. pseudolunata UTHSC 09-2092 T Human nasal sinus USA HE861842 HF565459
C. pseudorobusta UTHSC 08-3458 Human nasal sinus USA HE861838 HF565476
C. radici-foliigena CGMCC3.19328 T Roots of S. officinarum China MN215695 MN264127 MN263988
LC11956 Roots of S. officinarum China MN215698 MN264130 MN263991
C. radicicola CGMCC3.19327 T Roots of S. officinarum China MN215699 MN264131 MN263992
LC11953 Roots of S. officinarum China MN215700 MN264132 MN263993
C. ravenelii BRIP 13165 T Sporobolus fertilis Australia JN192386 JN600978 JN601024
C. reesii BRIP 4358 T Air Australia MH414907 MH433637 MH433670
C. richardiae BRIP 4371 T Richardia brasiliensis Australia KJ415555 KJ415391 KJ415438
C. robusta CBS 624.68 T Dichanthium annulatum USA KJ909783 KM083613 KM196577
C. rouhanii CBS 144674 T Syngonium vellozianum Iran KX139030 MG428694 MG428687
C. ryleyi BRIP 12554 T Sporobolus creber Australia KJ415556 KJ415390 KJ415437
C. saccharicola CGMCC3.19344 T Roots of S. officinarum China MN215701 MN264133 MN263994
C. sacchari-officinarum CGMCC3.19331 T Leaves of S. officinarum China MN215705 MN264137 MN263998
C. senegalensis CBS 149.71 Unknown Nigeria HG779001 HG779128
C. shahidchamranensis IRAN 3133C T Crude oil contaminated soil Iran MH550084 MH550083
C. soli CBS 222.96 T Soil Papua New Guinea KY905679 KY905691 KY905698
C. sorghina BRIP 15900 T Sorghum bicolor Australia KJ415558 KJ415388 KJ415435
C. spicifera CBS 198.31 Capsicum anuum Cyprus HF934916 HG779136
CBS 274.52 Soil Spain JN192387 JN600979. JN601023
C. sporobolicola BRIP 23040b T Sporobolus australasicus Australia MH414908 MH433652 MH433671
C. subpapendorfii CBS 656.74 T Soil Egypt KJ909777 KM061791 KM196585
C. suttoniae FMR 10992 T Human leg wound USA HE861828 HF565479 LR736651
FMR 11690 Human sphenoid sinus USA HE861826 HF565477 LR736650
C. tamilnaduensis SZMC 22226 T Human corneal scraping India MN628311 MN628307 MN628303
SZMC 26758 Human corneal scraping India MN628308 MN628304 MN628300
SZMC 26759 Human corneal scraping India MN628309 MN628305 MN628301
C. thailandicum MFLUCC 15-0747 T Decaying leaves of Pandanus sp. Thailand MH275057 MH412749 MH412764
C. trifolii CBS 173.55 Trifolium repens USA HG779023 HG779124
C. tripogonis BRIP 12375 T Tripogon loliiformis Australia JN192388 JN600980 JN601025
C. tropicalis BRIP 14834 T Coffea arabica India KJ415559 KJ415387 KJ415434
C. tsudae ATCC 44764 T Chloris gayana Japan KC424596 KC747745 KC503940
BRIP 10967 Leaf tip blight of C. gayana Australia KC424604 KC747754 KC503949
C. tuberculata CBS 146.63 T Z. mays India JX256433 JX276445 JX266599
C. umbiliciformis CGMCC3.19346 T Roots of S. officinarum China MN215711 MN264142 MN264004
C. uncinata CBS 221.52 T O. sativa Vietnam HG779024 HG779134
C. variabilis CPC 28815 T Chloris barbata Thailand MF490822 MF490844 MF490865
CPC 28816 Imperata cylindrica Thailand MF490823 MF490845 MF490866
C. verruciformis CBS 537.75 Lobibyx sp. feather New Zealand HG779026 HG779133
C. verruculosa CBS 149.63 Elaeis guineensis Nigeria HF934909 HG779110
CBS 150.63 Punica granatum leaf India KP400652 KP645346 KP735695
CPC 28792 C. dactylon Thailand MF490825 MF490847 MF490868
CPC 28809 E. indica Thailand MF490824 MF490846 MF490867
C. vietnamensis FMR 17659 T Unidentified dead leaves Vietnam LR736642 LR736644 LR736647
FMR 11956 Sorghum seed Indonesia LR736652 LR736643 LR736648
C. warraberensis BRIP 14817 T D. aegyptium Australia MH414909 MH433653 MH433672
C. xishuangbannaensis KUMCC 17-0185 T Decaying leaves of Pandanus amaryllifollus China MH275058 MH412750 MH412765

For the ML analysis, the best nucleotide substitution model for the combined analysis of ITS, gapdh and tef1, determined using the MEGA programme, was Kimura 2-parameters with Gamma distribution (K2+G); the combined analysis of these three phylogenetic markers was tested through Incongruence Length Difference (ILD) implemented in the Winclada programme (Farris et al. 1994). ML bootstrap values (bs) ≥ 70% were considered significant.

For the BI phylogenetic analysis, the best nucleotide substitution model was determined using jModelTest (Posada 2008). For the ITS region, we used Kimura 2-parameter with Invariant sites (K80+I), for gapdh General Time Reversible with gamma distribution (GTR+G) and for tef1 General Time Reversible with invariant sites (GTR+I). The parameter settings used were two simultaneous runs of 5M generations, four Markov chains, sampled every 1000 generations. The 50% majority-rule consensus tree and posterior probability values were calculated after discarding the first 25% of the samples. A posterior probability (pp) value of ≥ 0.95 was considered significant.

Sequence data generated in the present study were deposited in GenBank (Table 1) and the alignments in TreeBASE (http://treebase.org).

Phenotypic study

Macroscopic characterisation of the colonies was made on PDA, oatmeal agar (OA; oatmeal 30 g, agar 13 g, distilled water 1 litre) and potato carrot agar (PCA; potato 20 g, carrot 20 g, agar 13 g, distilled water 1 litre), after 7 days at 25 °C in darkness. Colours of the colonies in descriptions were based on Kornerup & Wanscher (1978). Cardinal temperatures for growth were obtained on PDA after 7 days in darkness.

Microscopic features were studied from the specimens mounted in Shear’s solution growing on the same media (Madrid et al. 2014). At least 30 measurements were taken for the calculation of conidial and conidiophores length and width ranges, which are also reported as the mean plus or minus standard deviation in the descriptions. Photomicrographs were taken using a Zeiss Axio-Imager M1 light microscope (Zeiss, Oberkochen, Germany) with a DeltaPix Infinity X digital camera.

Nomenclatural novelties and descriptions were deposited in MycoBank (Crous et al. 2004). Ex-type cultures and holotypes, which were dried cultures, were deposited at the Westerdijk Fungal Biodiversity Institute from Utrecht (CBS, The Netherlands).

Results

BLASTn results with gapdh sequences showed that the isolate FMR 17656 was ≤ 97.6%, similar to C. verruculosa CGMCC 28792; FMR 11956 and FMR 17659 showed a similarity of 93.31% and 93.6%, respectively, with C. spicifera CBS 198.31; and isolates FMR 10992 and FMR 11690 both exhibited a similarity of 94.7% with the ex-type strain of C. petersonii (BRIP 14642). Sequence similarity with this marker between FMR 11956/17659 and FMR 10992/11690 was 97%. These values suggested that the unidentified isolates represented putative new species for the genus, which were then confirmed by multi-locus sequence analysis of ITS, gapdh and tef1 barcodes. The combined analysis included 128 sequences representing 126 taxa in the genus Curvularia and these were rooted with Bipolaris maydis (CBS 136.29) and B. saccharicola (CBS 155.26) (Suppl. material 1: Fig. S1). The alignment comprised a total of 1928 bp (ITS 432, gapdh 573 bp and tef1 923 bp), including 546 variable sites (ITS 119 bp, gapdh 253 bp and tef1 174 bp) and 445 phylogenetically informative (ITS 83 bp, gapdh 233 bp and tef1 129 bp). The unidentified isolates were allocated to three single lineages in the same clade (74/0.99) together with sequences of the ex-type strains of C. americana (UTHSC 08-3414), C. petersonii (BRIP 14642) and C. verruculosa (CBS 150.63), but with enough distance to be considered distinct species. The two clinical isolates (FMR 10992 and FMR 11690) formed a fully-supported clade closely related to isolates FMR 11956 and FMR 17659, which were collected in Indonesia and Vietnam, respectively and to C. petersonii. The fifth isolate (FMR 17656) was related to C. verruculosa and C. americana, but formed an independent and distant branch from the previously-mentioned species.

In order to evaluate possible intra- and inter-specific variability within the species and to confirm the novelty of these fungi, we performed a multi-locus analysis, including only those sequences of the species that were more related to the unidentified Curvularia isolates (Fig. 1). The alignment comprised a total of 1894 bp (ITS 409, gapdh 562 bp and tef1 923 bp), with 298 variable sites (ITS 66 bp, gapdh 135 bp and tef1 97 bp) and 240 being phylogenetically informative (ITS 51 bp, gapdh 117 bp and tef1 72 bp). The phylogenetic analyses show that these isolates indeed represent three new species, which are described and illustrated in the Taxonomy section. The species can be morphologically differentiated mainly by features of their conidia (Table 2).

Table 2.

Conidial features of the novel Curvularia species proposed here and of their closest relatives.

Species Size (µm) Septum no. Ornamentation References
C. americana 13–28 × 7–15 3–4 Smooth upper cells, verruculose basal cell Madrid et al. (2014)
C. palmicola 23.9–34.7 × 9.3–15.7 3 Smooth Hyde et al. (2017)
C. paraverruculosa 11–37 × 8–12 3(–4) Verruculose to verrucose Present study
C. petersonii (15–)17–19(–21) × (5–)5.5–6(–7) 3 Smooth Tan et al. (2018)
C. suttoniae 8–22 × 5–9 (2–)3 Smooth upper cells, verruculose basal cell Present study
C. verruculosa 20–40 × 12–17 3 Rough to verruculose Sivanesan (1987)
C. vietnamensis 15–28 × 5–12 (1–)3(–4) Smooth Present study
Figure 1. 

Phylogenetic tree of the Curvularia species most related to the new taxa based on Maximum Likelihood analysis obtained by RAxML, using the combined analysis of ITS, gapdh and tef1 and rooted with Bipolaris maydis CBS 136.29 and Bipolaris saccharicola CBS 155.26. Bootstrap values (bs) greater than 70% and Bayesian posterior probabilities (pp) greater than 0.95 are given at the nodes (bs/pp). Bold branches indicate bs/pp of 100/1. The novel species are highlighted in bold. Ex-type isolates are marked with a superscript T.

Taxonomy

Curvularia paraverruculosa Iturrieta-González, Gené & Dania García, sp. nov.

MycoBank No: 833024
Fig. 2

Etymology

Name refers to the phylogenetic closeness to Curvularia verruculosa.

Type

Mexico, Michoacán, Villa Jiménez, from soil, Sept 2016, E. Rosas de Paz. (holotype CBS H-24293, culture ex-type FMR 17656, CBS 146220).

Description

(PDA at 25 °C). Mycelium composed of branched, septate, subhyaline to pale brown, thin- and smooth-walled hyphae, 2–4 μm wide. Conidiophores semi- to macronematous, mononematous, septate, straight or flexuous, geniculate at upper part, unbranched or slightly branched, smooth-walled, yellowish-brown to brown, 19–85(–145) × 3–6 μm (av. (±SD) 49.6 (±43.8) × 4.6 (±0.69)). Conidiogenous cells terminal or intercalary, polytretic, proliferating sympodially, yellowish-brown, with darkened scars, subcylindrical, 4–6 μm wide. Conidia 3(–4)-septate, mostly curved at the third cell from base which is usually larger than the others, sometimes apically bifurcate, verruculose to verrucose, apical and basal cells subhyaline to pale brown, middle cells brown, 11–37 × 8–12 μm (av. (±SD) 24 (±18.38) × 9.58 (±1.66)); hila slightly protuberant, thickened and darkened. Sexual morph not observed.

Culture characteristics

(7 d at 25 °C). Colonies on PDA reaching 45 mm diam., dark green (30F8), final edge whitish, velvety, flat, margin regular and fimbriate; reverse dark green (30F8). On PCA and OA, reaching 58–60 mm diam., dark green (30F8), final edge whitish, slightly floccose, flat, margin regular and fimbriate; reverse dark green (30F8). Sporulation was abundant on the three media.

Cardinal temperature for growth

Optimum 30 °C, maximum 37 °C, minimum 15 °C.

Distribution

Mexico.

Notes

Curvularia paraverruculosa is allocated phylogenetically to a strongly-supported clade (100/1) with C. verruculosa and C. americana (Fig. 1). All three species commonly have 3-septate conidia, but these can be distinguished by their size and ornamentation. Although conidia in C. verruculosa, the closest phylogenetic species and C. paraverruculosa are entirely verruculose, they are larger in the former (20–40 × 12–17 μm) (Sivanesan 1987). Furthermore, C. paraverruculosa also produces apically bifurcate conidia (Fig. 2), which have not been described in C. verruculosa. The conidia of C. americana are smaller (13–28 × 7–15 μm) and smooth-walled with a slightly verruculose basal cell (Madrid et al. 2014). In addition, microconidiation, described in C. americana, has not been observed in C. paraverruculosa.

Figure 2. 

Curvularia paraverruculosa sp. nov. (ex-type FMR 17656). A–C Colonies on PDA, PCA and OA, respectively, at 25 °C after 7 d D–H conidiophores and conidia. Scale bars: 10 μm.

Curvularia suttoniae Iturrieta-González, Wiederhold, Gené & Dania García, sp. nov.

MycoBank No: 833025
Fig. 3

Etymology

Named in honour of the American mycologist Deanna A. Sutton for her contribution to the body knowledge of microfungi.

Type

USA, Texas, from a human leg wound, 2009, D.A. Sutton (holotype CBS H-24294, culture ex-type UTHSC 09-3575, CBS 146221, FMR 10992).

Description

(PDA at 25 °C). Mycelium consisting of branched, septate, pale brown, smooth-walled to verruculose hyphae, 1–4 µm wide. Conidiophores mononematous, semi- to macronematous, erect to slightly flexuous, geniculate at the apex, unbranched or branched, smooth-walled to verruculose, pale brown, 43–103 × 3–5 µm (av. (±SD) 80 (±32.35) × 3.7 (±0.67)). Conidiogenous cells terminal, subterminal or intercalary, polytretic, proliferating sympodially, pale brown, darkened scars, subcylindrical to slightly swollen, 3–5 µm wide. Conidia (2–)3-septate, straight or curved, with the third cell often larger than the rest, apical and middle cells smooth-walled, basal cell verruculose, pale brown to brown, apical and basal cells paler than the middle cells, 8–22 × 5–9 µm (av. (±SD) 15 (±9.89) × 6.88 (±1.18)); hila protuberant, thickened and darkened. Sexual morph not observed.

Figure 3. 

Curvularia suttoniae sp. nov. (ex-type FMR 10992). A–C Colonies on PDA, PCA and OA, respectively, at 25 °C after 7 d D–I conidiophores and conidia with verruculose basal cells (arrows). Scale bars: 10 μm.

Culture characteristics

(7 d at 25 °C). Colonies on PDA reaching 66–68 mm diam., yellowish-grey (4B2), velvety, flat, margin slightly irregular and fimbriate; reverse black to brownish-orange (5C4); soluble pigment brown (6E6) present in cultures between 30–37 °C. On PCA, reaching 67 mm diam., olive grey (3D2), slightly floccose at the centre, flat, margin regular and whitish; reverse olive grey (3D2), whitish towards periphery. On OA, reaching 64 mm diam., olive grey (3F2), slightly floccose at the centre, flat, margin regular and whitish; reverse olive grey (3F2). Scarce sporulation on the three media.

Cardinal temperature for growth

Optimum 25–30 °C, maximum 37 °C, minimum 5 °C.

Distribution

USA.

Additional specimen examined

USA, South Carolina, from human sphenoid sinus, 2008, D.A. Sutton (UTHSC 08-809, FMR 11690).

Notes

Curvularia suttoniae is included in a well-supported clade with C. petersonii and C. vietnamensis, the latter also described here. Although the three species are clearly differentiated phylogenetically (Fig. 1), they can be distinguished only by subtle morphological features. While the conidia of C. petersonii and C. vietnamensis are entirely smooth, those of C. suttoniae show verruculose basal cells. Furthermore, the conidia in C. petersonii are narrower (5–7 µm wide) (Tan et al. 2018) and, in C. vietnamensis, they are larger (15–28 × 5–12 μm) than those of C. suttoniae (8–22 × 5–9 µm). In addition to these morphological features, gapdh sequences easily distinguish the two latter species.

Curvularia vietnamensis Iturrieta-González, Gené & Dania García, sp. nov.

MycoBank No: 833027
Fig. 4

Etymology

Name refers to the country where the species was collected.

Type

Vietnam, north-east region, on an unidentified dead leaf, Aug 2011, J. Guarro (holotype CBS H-24295, culture ex-type CBS 146222, FMR 17659).

Description

(PDA at 25 °C). Mycelium composed of branched, septate, subhyaline to pale brown, thin and smooth-walled to verruculose hyphae, 2–4 μm wide. Conidiophores macronematous, mononematous, septate, straight or flexuous, sometimes slightly geniculate at upper part, unbranched to slightly branched, smooth to verruculose, pale brown to brown, 11–136(–194) × 3–6 μm (av. (±SD) 92.2 (±72.86) × 4.21 (±0.85)). Conidiogenous cells terminal or intercalary, mono- or polytretic, proliferating sympodially, pale brown, with darkened scars, subcylindrical to swollen, 3–7 μm wide. Conidia (1–)3(–4)-septate, curved, with the third cell from base unequally enlarged, some apically bifurcate, smooth-walled, apical and basal cells pale brown, middle cells brown, 15–28 × 5–12 μm (av. (±SD) 21.38 (± 3.44) × 9.34 (±1.83)); hila slightly protuberant, thickened and darkened. Sexual morph not observed.

Figure 4. 

Curvularia vietnamensis sp. nov. (ex-type FMR 17659). A–C Colonies on PDA, PCA and OA, respectively, at 25 °C after 7 d D–H conidiophores and conidia. Scale bars: 10 μm.

Culture characteristics

(7 d at 25 °C). Colonies on PDA reaching 62 mm diam., greenish-grey to dark green (28C2/29F8), final edge white, umbonate, densely floccose, margin regular; reverse grey (29F1), final edge pale grey (1B2). On PCA, reaching 58 mm diam., olive grey to grey (3F2/3B1), slightly floccose at the centre, margin regular, final edge whitish; reverse olive grey to grey (3F2/3B1). On OA, reaching 74 mm diam., olive (2F3) slightly floccose at the centre, margin regular, flat; reverse olive to greenish-grey (2F3/1C2). Sporulation abundant mainly on PCA and OA.

Cardinal temperature for growth

Optimum 30 °C, maximum 37 °C, minimum 15 °C.

Distribution

Indonesia and Vietnam.

Additional specimen examined

Indonesia, from Sorghum seed, 1948, J. van der Vecht (CBS 188.48 = FMR 11956).

Notes

See C. suttoniae described above.

Discussion

As in other Pleosporalean genera, Curvularia is currently a well-delineated genus on the basis of molecular data (Manamgoda et al. 2015, Marin-Felix et al. 2017a). However, morphological features and analyses of the ITS barcode are insufficient to accurately identify Curvularia species. Thus, the multi-locus sequence analysis of different gene markers (i.e. LSU, ITS, gapdh, rpb2 and tef1) has been used to study the species diversity in Curvularia and phylogentic relationships with other similar genera (Hernández-Restrepo et al. 2018, Manamgoda et al. 2012, 2015, Madrid et al. 2014, Marin-Felix et al. 2017a, 2017b, Tan et al. 2018). Marin-Felix et al. (2017a) regarded ITS, gapdh and tef1 as the DNA barcodes for species delineation in the genus. During the last three years, numerous new Curvularia species have been introduced (Hyde et al. 2017, Marin-Felix et al. 2017a, 2017b, Dehdari et al. 2018, Heidari et al. 2018, Liang et al. 2018, Mehrabi-Koushki et al. 2018, Tan et al. 2018, Tibpromma et al. 2018, Kiss et al. 2019, Raza et al. 2019, Zhang et al. 2020). Novel species are found, not only on fresh material collected in various geographical regions, but also in re-evaluation of Curvularia isolates deposited in fungal collections and earlier identified by morphological features or ITS sequence analysis.

The five isolates, studied here, showed morphological similarity with C. americana or C. lunata (Sivanesan 1987, Madrid et al. 2014), but they also showed subtle variations that did not match with these species. Multi-locus analysis of the recommended barcodes facilitated the delineation of the novel species C. paraverruculosa, C. suttoniae and C. vietnamensis, which were closely related to the known species C. americana, C. petersonii and C. verruculosa (Fig. 1).

As in the case of C. suttoniae, other related species, such as C. americana and C. verruculosa, have also been associated with clinical specimens previously (da Cunha et al. 2013, Madrid et al. 2014). However, the role of all these fungi in human diseases has never been proven. Contrary to that, the recently described species C. coimbatorensis and C. tamilnaduensis were shown to be causal agents of fungal keratitis in India (Kiss et al. 2019). These two latter species, as with C. suttoniae and C. vietnamensis in our case, could only be molecularly differentiated by gapdh and tef1 loci; ITS sequence similarity between C. coimbatorensis and C. tamilnaduensis was 99% (Kiss et al. 2019) and between C. suttoniae and C. vietnamensis, it was 100%. Therefore, considering clinical laboratories commonly use ITS barcode for fungal diagnosis, not only will the diversity of Curvularia species remain obscure in the clinical setting, but also, subsequently, the epidemiology of its species associated with human or animal diseases. Our results suggest that gapdh and tef1 loci could be good alternatives as barcodes for Curvularia identification, since both have a high discriminatory power amongst species. However, gapdh would be the recommended locus because there are more sequences available for different species in the genus.

The ITS analysis revealed that C. palmicola, only known for its type specimen found on dead branches of Acoelorrhaphe wrightii in Thailand (Hyde et al. 2017), is also closely related to the novel species described here. However, this fungus was not included in our concatenate analysis since sequences of gapdh and tef1 were not available for comparison. Nevertheless, C. palmicola can be distinguished morphologically from our species mainly by having conidia with constricted wall at the septum level. Furthermore, C. palmicola has longer conidia (23.9–34.7 µm) than C. suttoniae (8–22 µm) and C. vietnamensis (15–28 µm) and it differs from C. paraverruculosa by its smooth-walled conidia.

Despite the fact that DNA sequence analysis is currently mandatory for Curvularia identification, two species were recently characterised exclusively, based on morphological data and host association, i.e. C. tremae on living leaves of Trema orientalis (Haldar 2017) and C martyniicola on Martynia annua (Kumar and Singh 2018), both from India. Curvularia tremae produces up to 4-septate and larger conidia (average length 152.21 μm and 67.75 μm wide at the broadest part) than those described here. Despite the conidia being mostly 3-sepetate, as in our species, C. martyniicola differs by having longer conidiophores (95–200 μm) than those of C. paraverruculosa (19–85(–145) μm) and C. suttoniae (43–103 μm) and by larger conidia (25–45 × 10–15 μm) than those observed in C. vietnamensis (15–28 × 5–12 μm).

Acknowledgements

The authors are grateful to Sándor Kocsubé of the University of Szeged (Hungary) for sending the sequences of C. tamilnaduensis and C. coimbatorensis used in this study. The study was supported by the Spanish Ministerio de Economía y Competitividad, grant CGL2017-88094-P.

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