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.

Ascomycetes, Dematiaceous hyphomycetes, phylogeny, Pleosporaceae, taxonomy

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.

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).

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.

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).

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.