11 urn:lsid:arphahub.com:pub:C004A564-9D6A-5F9F-B058-6A3815DFE9C3 urn:lsid:zoobank.org:pub:A4FD9303-3F01-45D9-B438-FCCFE465D2F7 MycoKeys MC 1314-4057 1314-4049 Pensoft Publishers 10.3897/mycokeys.120.156570 156570 Research Article Ascomycota Microsporidia DNA barcoding Taxonomy Asia Morphological and phylogenetic analyses reveal two novel species of Curvularia (Pleosporales, Pleosporaceae) from southern China Guo Zhuan-Jun https://orcid.org/0009-0006-5760-4536 1 Amenyogbe Mawuli Korsi https://orcid.org/0000-0002-3180-7726 1 Chen Shi-Qiang https://orcid.org/0009-0003-7533-921X 1 Rashad Younes M. https://orcid.org/0000-0002-7702-8023 2 Deng Jian-Xin djxin555@yangtzeu.edu.cn https://orcid.org/0000-0001-7304-5603 3 Luo Huan luohuan_0813@163.com https://orcid.org/0000-0002-5503-2507 4 Department of Plant Protection, College of Agriculture, Yangtze University, Jingzhou 434025, China Yangze University Jingzhou China MARA Key Laboratory of Sustainable Crop Production in the Middle Reaches of the Yangtze River (Co–Construction by Ministry and Province), Yangtze University, Jingzhou 434025, China Arid Lands Cultivation Research Institute Alexandria Egypt Plant Protection and Biomolecular Diagnosis Department, Arid Lands Cultivation Research Institute (ALCRI), City of Scientific Research and Technological Applications (SRTA-City), New Borg El-Arab, Alexandria 21934, Egypt Yangtze University Jingzhou China College of Resources and Environment, Southwest University, Chongqing 400715, China Southwest University Chongqing China

Corresponding authors: Jian-Xin Deng (djxin555@yangtzeu.edu.cn); Huan Luo (luohuan_0813@163.com)

Academic editor: Nalin Wijayawardene

 2025 29 07 2025 120 139 156 CE6C3C2C-A9E6-58CD-9727-6A57416DDD4C 16629135 22 04 2025 26 06 2025 Zhuan-Jun Guo, Mawuli Korsi Amenyogbe, Shi-Qiang Chen, Younes M. Rashad, Jian-Xin Deng, Huan Luo This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Members of Curvularia are of widespread clinical and agricultural importance, but species identification remains challenging due to morphological similarities and environmental variation. In this study, nine Curvularia strains were isolated from diseased leaves of Nelumbo nucifera and Nicotiana tabacum in Guangxi and Guizhou provinces, China. Morphological characteristics and phylogenetic analyses based on ITS, GAPDH, and tef1-α sequences revealed two novel species of Curvularia s. str., described here as C. nuciferaesp. nov. and C. tabacisp. nov. This study enhances understanding of Curvularia taxonomy and expands the known species diversity.

 Dematiaceous hyphomycetes morphology multi-locus phylogeny taxonomy two new taxa The National Natural Science Foundation of China (32270022). Citation

Guo Z-J, Amenyogbe MK, Chen S-Q, Rashad YM, Deng J-X, Luo H (2025) Morphological and phylogenetic analyses reveal two novel species of Curvularia (Pleosporales, Pleosporaceae) from southern China. MycoKeys 120: 139–156. https://doi.org/10.3897/mycokeys.120.156570

 Introduction

The genus Curvularia encompasses species functioning as either pathogens or saprobes on various plant hosts, separated from the related genera and monographed by Manamgoda et al. (2015) and typified by C. lunata. Species of Curvularia predominantly target plants within the Poaceae family, posing significant threats to grasses and staple crops such as rice, maize, wheat, and sorghum (Pintos et al. 2019; van Vuuren et al. 2024; Adomako et al. 2025). However, they are not exclusively reported from Poaceae and also infect or are reported from a broad range of host families, including Amaryllidaceae, Cactaceae, Caricaceae, Convolvulaceae, Cyperaceae, Fabaceae, Moringaceae, Nyctaginaceae, and Portulacaceae (Marin-Felix et al. 2017a, 2020; Tan et al. 2018; Hernãndez-Restrepo et al. 2018; Cui et al. 2020; Crous et al. 2020; Safi et al. 2020). Beyond their agricultural importance, some Curvularia species cause opportunistic infections in humans. For instance, C. chlamydospora and C. lunata have been implicated in respiratory, skin, brain, and corneal infections, particularly in immunocompromised individuals (Carter and Boudreaux 2004; Madrid et al. 2014; Manamgoda et al. 2015; Kiss et al. 2020; Iturrieta-González et al. 2020). Some species have also been reported from aquatic habitats, including C. lunata, C. pallescens, C. senegalensis, and others (Pratibha et al. 2013).

Historically, Bipolaris, Curvularia, Drechslera, and Exserohilum were grouped under the genus Helminthosporium (Sivanesan 1987). Morphological identification within Curvularia and related genera has primarily relied on morphological characters (Manamgoda et al. 2011). However, distinguishing these genera can be challenging due to overlapping features influenced by environmental factors, host type, and growth medium (Tsuda et al. 1978; Dama Ram et al. 2024; Jayawardena et al. 2025). Key differences have been noted in their sexual and conidial traits. For example, the conidia are characterized by their distinctive curvature due to disproportionately larger intermediate cells and by a length usually less than 100 μm, a feature absent in Bipolaris, which exhibits evenly distributed curvature and longer conidia (Manamgoda et al. 2012; Marin-Felix et al. 2017a). Despite these morphological markers, identification based on morphology alone remains unreliable, as demonstrated by discrepancies in five species – C. borreriae, C. catenulata, C. prasadii, C. trifolii, and C. tuberculata – where molecular findings conflicted with traditional descriptions (Dama Ram et al. 2024).

The integration of molecular techniques, such as ITS, GAPDH, and tef1-α gene sequencing, has significantly advanced the classification of Curvularia species (Manamgoda et al. 2014; Tan et al. 2014; Marin-Felix et al. 2017b). These methods have provided a stable framework for resolving taxonomic complexities, revealing extensive genetic diversity within the genus (Madrid et al. 2014; Manamgoda et al. 2015). GAPDH has been suggested as a promising secondary barcode marker to distinguish Curvularia species (Ahmadpour et al. 2025). Through DNA sequence analyses, 29 species previously classified under Bipolaris have been reassigned to Curvularia s. str. (Manamgoda et al. 2012; Tan et al. 2014; Marin-Felix et al. 2020). The use of multigene phylogenetics has thus proven invaluable in differentiating species and validating classifications based on morphology. Recent research has broadened our knowledge of the diversity within the genus Curvularia (Jayawardena et al. 2025). Madrid et al. (2014) determined six major clades of Curvularia based on a combined sequence analysis of the ITS, LSU, GAPDH, and tef1-α gene regions. Kusai et al. (2015) revised Ellis’s (1971) taxonomic key, identifying four major species – C. eragrostidis, C. geniculata, C. hawaiiensis, and C. lunata – and incorporating three additional species: C. australiensis, C. catenulata, and C. spicifera. Ahmadpour et al. (2025) established a new distinct clade using phylogenetic analyses. Over the past six years, numerous new species have been identified worldwide (Hyde et al. 2017; Marin-Felix et al. 2017a, 2017b, 2020; Dehdari et al. 2018; Ferdinandez et al. 2021, 2023). The genus comprises 59 species from China listed in the USDA Fungal Databases (https://fungi.ars.usda.gov/; accessed 19 June 2025).

In this study, two novel Curvularia species, C. nuciferae and C. tabaci, were identified through a combination of morphological traits and multigene sequence analyses. This research adds to the expanding corpus of literature on Curvularia taxonomy and diversity.

 Materials and methods Isolation and morphological studies

Curvularia isolates were obtained from Nelumbo nucifera leaves with brown spots collected from a pond in Liuzhou City (24°16′49″N, 109°21′85″E), Guangxi Province, in May 2023, and from Nicotiana tabacum leaves with dry spots harvested from a plantation in Zunyi City (26°00′74″N, 106°44′36″E), Guizhou Province, in September 2022. The samples were placed in clean bags and transported to the laboratory for processing. Fungal isolation was conducted using two methods. In the first method, infected tissues were cut into small segments (2 × 2 mm, including both diseased and healthy portions) and placed on moist filter paper inside Petri dishes, which were incubated at 25 °C to induce sporulation (Zhao et al. 2023). Emerging fungal conidia were examined under a stereomicroscope, and a single conidium was isolated and transferred to fresh potato dextrose agar (PDA) plates (24 g/L Difco, 20 g/L agar) using a sterilized glass needle. For the second method, tissue segments were surface-sterilized by sequential immersion in 70% ethanol for 30 seconds, 2% sodium hypochlorite for 1 minute, and three washes in sterile water (Wei et al. 2022). These segments were then placed on PDA and incubated at 25 °C. Hyphal tips from the emerging fungal colonies were transferred to fresh PDA plates to obtain pure cultures. All purified cultures were stored as test-tube slants at 4 °C and in cryovials containing 20% glycerol at -80 °C. Specimens (holotypes) and their ex-types were deposited in the Fungi Herbarium of Yangtze University (YzU), Jingzhou, Hubei, China (434025). The newly introduced species were registered in MycoBank following the guidelines of Crous et al. (2004).

For cultural characterization, isolates were incubated on PDA and TWA (water agar supplemented with sterile wheat straws) at 25 °C for seven days in complete darkness. Colony size, texture, and color were documented, with color descriptions following Rayner’s (1970) color charts. To evaluate morphological features such as conidial size, shape, color, and sporulation patterns, fresh mycelia were transferred onto PCA (potato carrot agar) and TWA plates and incubated under the same conditions (Madrid et al. 2014; Guo 2016). After seven days, 80 randomly selected conidia were mounted in water, and digital images were captured using a Nikon ECLIPSE Ni–U microscope system (Nikon, Japan). Sporulation patterns were also documented at the same time.

 DNA extraction, PCR amplification, and sequencing

Genomic DNA was extracted from fresh mycelia cultured on PDA for 3–5 days using the CTAB method described by Watanabe et al. (2010). The ITS, GAPDH, and tef1-α gene regions were amplified using the primer pairs ITS5/ITS4 (White et al. 1990), gpd1/gpd2 (Berbee et al. 1999), and EF983/2218R (Schoch et al. 2009), respectively. Each PCR reaction consisted of a 25 μL mixture containing 21 μL of 1.1× Taq PCR Star Mix (TSINGKE), 2 μL of template DNA, and 1 μL of each primer. Amplification was performed using a BIORAD T100 thermocycler with the following conditions: an initial denaturation at 94 °C for 5 minutes, followed by 30 cycles of denaturation at 94 °C for 30 seconds, annealing at 55 °C (for ITS and GAPDH) or 53 °C (for tef1-α) for 30 seconds, extension at 72 °C for 1 minute, and a final extension step at 72 °C for 10 minutes. PCR products were verified by electrophoresis on 1% agarose gels. Successfully amplified products were purified and sequenced by TSINGKE Biotechnology (Beijing, China).

 Phylogenetic analyses

Preliminary sequence verification was conducted using BioEdit v.7.2.3 (Hall 1999), and sequences were assembled with manual adjustments using PHYDIT v.3.2 (Chun 1995). Relevant ITS, GAPDH, and tef1-α gene sequences of Curvularia strains were downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/genbank/). Sequence alignment and manual editing were performed using MEGA 11 (Tamura et al. 2021). Phylogenetic analyses were carried out on a combined dataset of ITS, GAPDH, and tef1-α gene regions using maximum likelihood (ML) and Bayesian inference (BI) methods. ML analysis was executed with RAxML v.7.2.8 (Stamatakis 2006), while the best-fit evolutionary model (GTR+I+G) for each partition was determined using MrModeltest v.2.3 (Posada and Crandall 1998) based on the Akaike Information Criterion (AIC). For BI analysis, two Markov Chain Monte Carlo (MCMC) chains were run for 1,000,000 generations, sampling every 100 generations. The first 25% of samples were discarded as burn-in, and a 50% majority-rule consensus tree was constructed to calculate posterior probability values. The resulting phylogenetic trees were visualized and edited in FigTree v.1.3.1 (Rambaut and Drummond 2010). Branch support values greater than 60% for ML bootstrap and 0.6 for posterior probability (PP) were indicated on the resulting phylogram.

 Results Phylogenetic analyses

Four representative strains, out of nine isolated Curvularia strains, were preliminarily identified based on morphological characterization. Following BLAST searches in GenBank, 114 relevant strains more closely related to the unidentified isolates were retrieved (Table 1). Bipolaris zeae (BRIP 11512) and Exserohilum rostratum (CBS 325.87) were selected as the out-group. The combined dataset of three gene sequences (ITS, GAPDH, and tef1-α) comprised 1,532 bp (375 bp from ITS, 420 bp from GAPDH, and 737 bp from tef1-α), including alignment gaps. The phylogenetic analyses using maximum likelihood (ML) and Bayesian inference (BI) produced congruent tree topologies. The final consensus tree (Fig. 1) included RAxML bootstrap support (BS) and Bayesian posterior probability (PP) at the nodes. The clade containing YzU 231509 and YzU 231510 was phylogenetically close to C. lonarensis and C. mosaddeghii and was found to be independent with PP and BS values of 1.0/100%. Another clade, YzU 221481 and YzU 221482, was closely related to C. chlamydospora and C. pseudolunata, forming a distinct branch (1.0/100%). These results suggest that the present strains represent two new taxa.

Curvularia strains and their GenBank accession numbers used in the phylogenetic analysis.

Species Strains Country Host/Substrate ITS GAPDH tef1-α
Bipolaris zeae BRIP 11512 Australia Zea mays KJ415538 KJ415408 KJ415454
Curvularia aeria CBS 294.61 Brazil Air HE861850 HF565450 –
C. ahvazensis CBS 144673 Iran Zinnia elegans rotten roots KX139029 MG428693 MG428686
C. akaiiensis BRIP 16080 India Unknown KJ415539 KJ415407 KJ415453
C. alcornii MFLUCC 10-0703 Thailand Zea sp. JX256420 JX276433 JX266589
C. arcana CBS 127224 Unknown Unknown MN688801 MN688828 MN688855
C. asiatica MFLUCC 10-0711 Thailand Panicum sp. JX256424 JX276436 JX266593
C. australiensis BRIP 12044 Australia Oryza sativa KJ415540 KJ415406 KJ415452
C. australis BRIP 12521 Australia Sporobolus sp. KJ415541 KJ415405 KJ415451
C. austriaca CBS 102694 Austria Nasal cavity of patient with sinusitis MN688802 MN688829 MN688856
C. bannonii BRIP 16732 USA Jacquemontia tamnifolia KJ415542 KJ415404 KJ415450
C. beasleyi BRIP 10972 Australia Chloris gayana MH414892 MH433638 MH433654
C. beerburrumensis BRIP 12942 Australia Eragrostis bahiensis MH414894 MH433634 MH433657
C. boeremae IMI 164633 India Portulaca oleracea MH414911 MH433641 –
C. bothriochloae BRIP 12522 Australia Bothriochloa bladhii KJ415543 KJ415403 KJ415449
C. cactivora CBS 580.74 Republic of Suriname Member of Cactaceae MN688803 MN688830 MN688857
C. canadensis CBS 109239 Canada Overwintered grass MN688804 MN688831 MN688858
C. caricae-papayae CBS 135941 India Carica papaya HG778984 HG779146 –
C. chiangmaiensis CPC 28829 Thailand Zea mays MF490814 MF490836 MF490857
C. chlamydospora UTHSC 07-2764 USA Toe nail HG779021 HG779151 –
C. clavata BRIP 61680b Australia Oryza rufipogon KU552205 KU552167 KU552159
C. coatesiae BRIP 24261 Australia Litchi chinensis MH414897 MH433636 MH433659
C. coicis CBS 192.29 Japan Coix lacryma JN192373 JN600962 JN601006
C. colbranii BRIP 13066 Australia Crinum zeylanicum MH414898 MH433642 MH433660
C. crassiseptata CBS 503.90 Nigeria Plant material LT631310 LT715882 MN688859
C. crustacea BRIP 13524 Indonesia Sporobolus sp. KJ415544 KJ415402 KJ415448
C. dactylocteniicola CPC 28810 Thailand Dactyloctenium aegyptium MF490815 MF490837 MF490858
C. dactyloctenii BRIP 12846 Australia Dactyloctenium radulans KJ415545 KJ415401 KJ415447
C. deserticola CN025B5 Namibia: Far East Stipagrostis ciliata ON074982 ON355397 ON355358
CN025B7 Namibia: Far East Stipagrostis ciliata ON074983 ON355398 ON355359
CN027A5 Namibia: Far East Stipagrostis ciliata ON075008 ON355400 ON355361
C. ellisii CBS 193.62 Pakistan Air JN192375 JN600963 JN601007
CBS 127083 Australia Dactyloctenium aegyptium MN688805 MN688832 MN688860
C. eragrostidicola BRIP 12538 Australia Eragrostis pilosa MH414899 MH433643 MH433661
C. gladioli CBS 210.79 Romania Gladiolus sp. HG778987 HG779123 –
C. gobabebensis CN013C4 Namibia: Mirabib Stipagrostis ciliata ON074797 ON355381 ON355347
CN010F9 Namibia: Mirabib Stipagrostis ciliata ON332848 ON355373 ON355344
C. graminicola BRIP 23 186 Australia Unknown JN192376 JN600964 JN601008
C. harveyi BRIP 57412 Australia Triticum aestivum KJ415546 KJ415400 KJ415446
C. hawaiiensis BRIP 11987 USA Oryza sativa KJ415547 KJ415399 KJ415445
C. heteropogonicola BRIP 14579 India Heteropogon contortus KJ415548 KJ415398 KJ415444
C. heteropogonis CBS 284.91 Australia Heteropogon contortus JN192379 JN600969 JN601013
C. hominis CBS 136985 USA Homo sapiens sapiens HG779011 HG779106
C. homomorpha CBS 156.60 USA Air JN192380 JN600970 JN601014
C. ischaemi CBS 630.82 New Zealand Ischaemum indicum JX256428 JX276440 –
C. kenpeggii BRIP 14530 Australia Triticum aestivum MH414900 MH433644 MH433662
C. kusanoi CBS 137.29 Japan Eragrostis major JN192381 – JN601016
C. lamingtonensis BRIP 12259 Australia Microlaena stipoides MH414901 MH433645 MH433663
C. lonarensis CBS 140569 India Lonar lake KT315408 KY007019 –
USJCC–0082 Sri Lanka Saccharum officinarum OQ275224 OQ269635 OQ332411
C. lunata CBS 730.96 USA Lung biopsy JX256429 JX276441 JX266596
C. maraisii CN037F7 Namibia: Far East Soil OR471647 ON355439 OR486044
CN021G3 Namibia: Far East Stipagrostis ciliata ON074886 ON355385 ON355351
C. mebaldsii BRIP 12900 Australia Cynodon transvaalensis MH414902 MH433647 MH433664
C. micropus CBS 127235 USA Paspalum notatum HE792934 LT715859 –
C. mosaddeghii IRAN 3131C Iran Syzygium cumini leaf spot MG846737 MH392155 MH392152
C. muehlenbeckiae CBS 144.63 India Muehlenbeckia sp. HG779002 HG779108 –
C. namibensis CN027C4 Namibia: Far East Stipagrostis ciliata ON075010 ON355402 ON355363
CN027A9 Namibia: Far East Stipagrostis ciliata ON075009 ON355401 ON355362
C. neergaardii BRIP 12919 Ghana Oryza sativa KJ415550 KJ415397 KJ415443
CBS 276.91 Australia Unknown MN688806 MN688833 MN688861
CBS 277.91 Australia Unknown MN688807 MN688834 MN688862
C. neoindica IMI 129790 India Brassica nigra NR_158450 MH433649 MH433667
C. nicotiae CBS 655.74 Algeria Desert soil KJ415551 KJ415396 KJ415442
C. nodosa CPC 28800 Thailand Digitaria ciliaris MF490816 MF490838 MF490859
C. nodulosa CBS 160.58 USA Eleusine indica JN601033 JN600975 JN601019
C. nuciferae YzU 231509 China Nelumbo nucifera OR888819 PP066825 2827452
YzU 231510 China Nelumbo nucifera OR888818 PP066826 2827459
C. oryzae-sativae CBS 127725 Argentina Oryza sativa MN688808 MN688835 MN688863
C. ovariicola CBS 470.90 Australia Eragrostis interrupta MN688809 MN688836 –
C. stipagrostidicola CN060H5 Namibia: Reverse Stipagrostis ciliata ON332838 ON355415 ON355368
C. stipagrostidicola CN060G4 Namibia: Reverse Stipagrostis ciliata ON332837 ON355414 ON355367
C. patereae CBS 198.87 Argentina Triticum durum seed MN688810 MN688837 MN688864
C. penniseti CBS 528.70 Unknown Pennisetum sp. seed MN688811 MN688838 –
C. perotidis CBS 350.90 Australia Perotis rara JN192385 KJ415394 JN601021
C. pisi CBS 190.48 Canada Pisum sativum KY905678 KY905690 KY905697
C. platzii BRIP 27703b Australia Cenchrus clandestinum MH414906 MH433651 MH433669
C. portulacae BRIP 14541 USA Portulaca oleracea KJ415553 KJ415393 KJ415440
C. pseudobrachyspora CPC 28808 Thailand Eleusine indica MF490819 MF490819 MF490819
CBS 207.59 Unknown Unknown MN688812 MN688839 MN688865
CBS 533.70 Denmark Pennisetum sp. seed MN688813 MN688840 MN688866
HNWN001 China Areca catechu leaf MH516132 MH516133 MH516134
CBS 337.64 USA Agropyron repens MN688815 MN688842 MN688867
CBS 339.64 USA Pennisetum glaucum MN688816 MN688843 MN688868
MFLUCC 10–0739 Thailand Oryza sativa JX256443 JX276454 JX266603
C. pseudoclavata CBS 539.70 Denmark Oryza sativa seed MN688817 MN688844 MN688869
C. pseudoellisii CBS 298.80 Sudan Sorghum bicolor seed MN688818 MN688845 MN688870
C. pseudointermedia CBS 553.89 Brazil Cultivated pasture soil MN688819 MN688846 MN688871
CBS 188.61 Guadeloupe Decaying grass MN688820 MN688847 MN688872
C. pseudolunata UTHSC 09-2092 USA Nasal sinus HE861842 HF565459 –
C. pseudoprotuberata CBS 385.69 Canada Soil under Thuja occidentalis MN688821 MN688848 MN688873
CBS 550.69 Canada Soil under Pinus strobus MN688822 MN688849 MN688874
C. pseudorobusta UTHSC 08-3458 USA Nasal sinus HE861838 HF565476 –
C. ravenelii BRIP 13165 Australia Sporobolus fertilis JN192386 JN600978 JN601024
C. reesii BRIP 4358 Australia Air MH414907 MH433637 MH433670
C. richardiae BRIP 4371 Australia Richardia brasiliensis KJ415555 KJ415391 KJ415438
C. rouhanii CBS 144674 Iran Blighted leave of Syngonium vellozianum KX139030 MG428694 MG428687
C. ryleyi BRIP 12554 Australia Sporobolus creber KJ415556 KJ415390 KJ415437
C. senegalensis CBS 149.71 Nigeria Unknown HG779001 HG779128 –
C. sesuvi Bp-Zj 01 Unknown Sesuvium sp. EF175940 – –
C. shahidchamranensis IRAN 3133C Iran Soil MH550084 MH550083 –
C. soli CBS 222.96 Papua New Guinea Soil KY905679 KY905691 KY905698
C. sorghina BRIP 15900 Australia Sorghum bicolor KJ415558 KJ415388 KJ415435
C. spicifera CBS 274.52 Spain Soil JN192387 JN600979 JN601023
C. sporobolicola BRIP 23040b Australia Sporobolus australasicus MH414908 MH433652 MH433671
C. tabaci YzU 221481 China Nicotiana tabacum OR888817 PP066824 OR818398
C. tabaci YzU 221482 China Nicotiana tabacum PP601257 PP779503 PP779504
C. tribuli CBS 126975 South Africa Tribulus terrestris leaf MN688825 MN688852 MN688875
C. trifolii CBS 173.55 USA Trifolium repens HG779023 HG779124 –
C. tripogonis BRIP 12375 Australia Unknown JN192388 JN600980 JN601025
C. tropicalis BRIP 14834 India Coffea arabica KJ415559 KJ415387 KJ415434
C. tsudae ATCC 44764 Japan Chloris gayana KC424596 KC747745 KC503940
C. tuberculata CBS 146.63 India Zea mays JX256433 JX276445 JX266599
C. uncinata CBS 221.52 Vietnam Oryza sativa HG779024 HG779134 –
C. variabilis CPC 28815 Thailand Chloris barbata MF490822 MF490844 MF490865
C. verrucosa CBS 422.93 Cuba Air MN688826 MN688853 MN688876
C. warraberensis BRIP 14817 Australia Dactyloctenium aegyptium MH414909 MH433653 MH433672
Exserohilum rostratum CBS 325.87 USA Homo sapiens HE664035 LT715898 HE664082

CBS: Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands; CPC: Culture Collection of Pedro Crous, housed at the Westerdijk Fungal Biodiversity Institute; IMI: International Mycological Institute, CABI–Bioscience, Egham, UK; IRAN: Iranian Fungal Culture Collection, Iranian Research Institute of Plant Protection, Tehran, Iran; MFLUCC: Mae Fah Luang University Culture Collection, Chiang Rai, Thailand; UTHSC: Fungus Testing Laboratory, Department of Pathology, University of Texas Health Science Center, San Antonio, Texas, USA; GUCC: Culture Collection at the Department of Plant Pathology, College of Agriculture, Guizhou University, China; YzU: Yangtze University, China; ITS: internal transcribed spacer region; GAPDH: partial glyceraldehyde-3-phosphate dehydrogenase gene; tef1-α: partial translation elongation factor 1-alpha gene.

 10.3897/mycokeys.120.156570.figure1 16629175 66B42603-7DE5-592B-8F6A-B2F57E0C3E6F

Phylogenetic tree of Curvularia based on the combined ITS, GAPDH, and tef1-α sequences. Bayesian posterior probabilities > 0.60 (PP) and maximum likelihood bootstrap support values > 60 (BS) are indicated at the nodes (PP/BS). The novel species are highlighted in bold.

https://binary.pensoft.net/fig/1384196 Taxonomy Fungi Pleosporales Pleosporaceae CB71E48B-6561-5EB9-9AB9-AB895D3652CD Curvularia nuciferae 851620 J.X Deng & Z.J Guo sp. nov. Fig. 2 Holotype.

China • Guangxi Province, Liuzhou City, 24°16′49″N, 109°21′85″E, from leaf spot of Nelumbo nucifera, 8 June 2023 (holotype: YzU-H-2023138, ex-type YzU 231510 was stored in cryovials containing 20% glycerol at -80 °C).

 Etymology.

The term “nuciferae” refers to the host species on which it was collected, Nelumbo nucifera.

 Description.

Colonies on PDA circular and flat shapes, vinaceous buff (no. 86) in the center with lighter margins on the plate. Reverse plates have olivaceous buff pigmentation with a diameter of 70–75 mm (Fig. 2). Colonies on TWA: Asexual morph—Hyphae 2.5–4 μm wide, subhyaline to brown, septate, branched. Conidiophores 105–328 × 4–6 μm (mean ± SD = 238.6 ± 68.2 × 5.1 ± 0.7 μm), solitary, flexuous, unbranched, septate, hyaline to brown, and lighter toward apex. Conidiogenous cells 9–15 × 4–7 μm (mean ± SD = 12.8 ± 2.1 × 5.2 ± 1 μm), terminal or intercalary, subcylindrical to slightly swollen, pale brown. Conidia 23–29(–32) × 12–16 μm (mean ± SD = 26.9 ± 2.6 × 13.6 ± 1.1 μm), smooth-walled, with the middle cells dark brown and the terminal ends paler, 2–3 septa. Additionally, the conidia were asymmetrically swollen and curved at the third cell from the base, with a protruding, darkened, and thickened hilum (2–3 μm). Sexual morph undetermined.

10.3897/mycokeys.120.156570.figure2 16629177 86C7B696-5A7B-5119-91FB-633397F70F42

Curvularia nuciferae sp. nov. (strain: YzU 231510). A–C. Colony phenotypes on PDA, WA, and TWA; D. Conidiophores; E Sporulation patterns; F–G. Conidia on TWA and PCA at 25 °C. Scale bars: 50 μm (E); 20 μm (D, F, G).

https://binary.pensoft.net/fig/1384197

On PCA: Hyphae 2.5–4 μm wide, subhyaline to brown, septate, branched. Conidiophores 68–360 × 3–5 μm (mean ± SD = 222.3 ± 98.6 × 4.1 ± 0.9 μm), solitary, curved, ranging from nearly clear to dark brown, unbranched, erect, sometimes bent at the apex, thick-walled. Conidiogenous cells 9–16 × 4–6 μm (mean ± SD = 13 ± 2.5 × 5.2 ± 0.9 μm), terminal, proliferated sympodially, subcylindrical to irregular. Conidia 21–33(–40) × 10–18 μm (mean ± SD = 28.4 ± 4.6 × 14.1 ± 2.1 μm), slightly curved, smooth-walled, brown to dark brown, and swollen at the fourth cell from the base, 2–3 septate. No chlamydospores, microconidiation, or sexual morphs were observed.

 Additional material studied.

China • Guangxi Province: Liuzhou City, on leaves of Nelumbo nucifera (Nelumbonaceae, Proteales), 8 June 2023, collected by Z.J. Guo, S.Q. Chen & J.X. Deng (ex-type living culture: YzU 231510, ex-isotype: YzU 231509).

 Notes.

Curvularia nuciferae is phylogenetically close to C. lonarensis and C. mosaddeghii but can be distinguished from these species by its wider, lighter brown conidia, absence of bifurcate conidia, and larger conidia. Under the culturing conditions described by Sharma et al. (2016) (at 28 °C in darkness on PDA) and Ferdinandez et al. (2023) (at 25 °C with a 12 h/12 h photoperiod on PDA), conidia measuring 25–31(–35) × 12–18 µm and 22–30(–32) × 11–15 µm, respectively. Curvularia nuciferae is similar to C. lonarensis but differs by having wider and lighter brown conidia and lacking the formation of bifurcate conidia (Ferdinandez et al. 2023). Additionally, it is easily distinguished from C. mosaddeghii by producing larger conidia (21–30(–34) × 9–13 µm) at 28 °C with a 12 h/12 h photoperiod (Heidari et al. 2018). After a nucleotide pairwise comparison, C. nuciferae can be readily differentiated from the other two related novel species based on ITS, GAPDH, and tef1-α gene regions, which have 0 bp and 3 bp differences in the ITS region, 8 bp and 8 bp in GAPDH, and 4 bp and 5 bp in tef1-α when compared with C. lonarensis and C. mosaddeghii, respectively. Strains YzU 231509 and YzU 231510 were placed in a single lineage within the same clade as C. lonarensis and C. mosaddeghii, supported by posterior probability and bootstrap values, but with sufficient genetic distance to be considered distinct species.

 Fungi Pleosporales Pleosporaceae 65135A3B-75DE-57E2-B29C-D603E6545896 Curvularia tabaci 851619 J.X Deng & Z.J Guo sp. nov. Fig. 3 Holotype.

China • Guizhou Province, Zunyi City, 26°00′74″N, 106°44′36″E, from leaf spot of Nicotiana tabacum. 19 September 2022 (holotype: YzU-H-2022101, ex-type YzU 221481 was stored in cryovials containing 20% glycerol at -80 °C)

 Etymology.

The epithet “tabaci” refers to the host species on which it was collected, Nicotiana tabacum.

 Description.

Colonies on PDA circular, pale vinaceous grey (no. 115) structures with cottony mycelia in the center. The reverse side of the plate with ochreous pigmentation, with colonies measuring 51–56 mm in diameter (Fig. 3). Colonies on TWA: Asexual morph—Hyphae 2–4 μm wide, pale brown to brown, septate, branched. Conidiophores 52–247 × 4–6 μm (mean ± SD = 152.9 ± 69.6 × 5.2 ± 0.9 μm), straight to flexuous, septate, predominantly geniculate at the upper part, unbranched, pale brown to brown, paler toward apex. Conidiogenous cells 8–16 × 3–6 μm (mean ± SD = 10.8 ± 2.7 × 4.3 ± 1.7 μm), terminal or intercalary, proliferating sympodially, subcylindrical to slightly swollen. Conidia straight or slightly curved, 3-distoseptate, slightly constricted near the septa, brown to dark brown, apical and basal cells paler than the central cells. The basal third cell variably inflated, a punctate wall, measuring 23–29(–33) × 10–15 μm (mean ± SD = 26.2 ± 2.9 × 12.2 ± 1.7 μm). Hilum 2–3 μm, protruding, darker, and thickened. Sexual morph undetermined.

10.3897/mycokeys.120.156570.figure3 16629179 0D1E68CE-5B7C-5D12-A1E0-FE99294FBF04

Curvularia tabaci sp. nov. (strain: YzU 221481). A–C. Colony phenotypes on PDA, WA, and TWA; D. Conidiophores; E. Sporulation patterns; F–G. Conidia on TWA and PCA at 25 °C. Scale bars: 50 μm (E); 20 μm (D, F, G).

https://binary.pensoft.net/fig/1384198

On PCA: Hyphae 2–4 μm wide, pale brown to brown, septate, branched. Conidiophores formed as solitary or flexuous, septate, pale brown to brown, becoming paler towards the apex, unbranched, 90–315 × 4–6 μm (mean ± SD = 222.2 ± 76.9 × 4.9 ± 0.7 μm). Conidiogenous cells terminal or intercalary, proliferating sympodially, and ranged from subcylindrical to irregular in shape, measuring 8–16 × 3–6 μm (mean ± SD = 12.8 ± 2.8 × 4.6 ± 1.2 μm). Conidia mostly curved, 2–4 celled, brown to dark brown, apical and basal cells paler than the central cells, and smooth-walled, measuring 20–26 × 10–13 μm (mean ± SD = 23.2 ± 2.2 × 11.8 ± 1.2 μm). Hilum 2–3 μm, protruding, darker, and thickened. No chlamydospores, microconidiation. Sexual morph undetermined.

 Additional material studied.

China • Guizhou Province: Zunyi City, on leaves of Nicotiana tabacum (Solanaceae, Lamiales), 19 September 2022, collected by Z.J. Guo, H. Luo & J.X. Deng (ex-type living culture: YzU 221481, ex-isotype: YzU 221482).

 Notes.

Phylogenetic analysis revealed that Curvularia tabaci is closely related to C. chlamydospora and C. pseudolunata (Fig. 1). Pairwise DNA sequence comparison revealed that strains YzU 221481 and YzU 221482 exhibit identical sequences and are distinct from C. chlamydospora and C. pseudolunata. After a nucleotide pairwise comparison, C. tabaci differs from C. chlamydospora and C. pseudolunata by 5 and 4 bp in the ITS region and 13 and 9 bp in GAPDH, respectively. Furthermore, C. chlamydospora and C. pseudolunata have been reported as human pathogens in the USA (Madrid et al. 2014). Morphologically, C. tabaci (10–15 μm) differs from C. chlamydospora (7–12 μm) and C. pseudolunata (8–12 μm) by forming slightly wider, 2–4-celled conidia without observed chlamydospores or microconidiation. This branch represents a novel species more closely related to C. chlamydospora and C. pseudolunata (PP/BS = 1.00/83%) than to C. aeria, C. caricae-papayae, C. homomorpha, C. namibensis, C. pseudobrachyspora, and C. warraberensis.

 Discussion

In this work, nine fungal isolates with morphological characteristics similar to Curvularia were obtained from plant leaves belonging to the Nymphaeaceae and Solanaceae families. The identification of Curvularia species was frequently insufficient based on micromorphology, as many species have overlapping conidial dimensions (Manamgoda et al. 2015; Tan et al. 2018). In this study, it was found that standard conditions are quite important for Curvularia taxonomy based on morphological characteristics. Standardized morphological criteria should be adopted to minimize taxonomic inconsistencies arising from variations in temperature and substrates. To minimize potential misidentification of Curvularia species, we followed recommendations by Madrid et al. (2014) and Guo (2016), using PCA and TWA media for seven days at 25 °C. These media effectively promoted typical morphological characteristics. To enhance species identification within Curvularia and related genera, molecular techniques have become increasingly essential. According to Ram et al. (2024), approximately 25% of Curvularia species identified through morphological analysis are inconsistent with molecular findings – an issue that needs to be addressed through the exploration of more powerful gene fragments.

Previous studies have shown that the ITS region, while lacking clear discriminatory power, remains necessary for Curvularia identification (e.g., Manamgoda et al. 2014, 2015; van Vuuren et al. 2024). In the individual alignments of the three loci, the two new species – Curvularia nuciferae and C. tabaci – were clearly distinguished from their related species using the GAPDH gene. In addition, the tef1-α region is recommended as a barcode for Curvularia, although sequence information for some species remains unavailable (Manamgoda et al. 2015). The GAPDH gene shows high resolution in distinguishing closely related species within Curvularia, which is consistent with previous research (Manamgoda et al. 2015; Ferdinandez et al. 2021; Ahmadpour et al. 2025). Currently, a polyphasic approach – integrating morphological characteristics with molecular phylogenetic data – is used to characterize and differentiate these taxa.

This study identified two new species, Curvularia nuciferae and C. tabaci, using both morphological traits and multi-locus phylogenetic analyses of ITS, GAPDH, and tef1-α gene regions. The isolates were collected from Nelumbo nucifera (lotus) and Nicotiana tabacum (tobacco) in Guangxi and Guizhou provinces, respectively. This research contributes to the expanding diversity of Curvularia by describing two novel species. In studies on the diversity of endophytic fungi in lotus (Nelumbo nucifera), Chen and Kirschner (2018) reported 33 genera, with 71% parasitizing leaves, but did not detect Curvularia. However, C. lunata has been found on lotus leaves in earlier studies (Cui and Sun 2012) and in Thailand (Techaoei et al. 2018). In tobacco production, Curvularia, along with Alternaria, Aspergillus, Cladosporium, and Penicillium, negatively affects both yield and quality (Chen et al. 2017, 2021). Additionally, notable species such as C. australiensis, C. clavata, and C. trifolii have been reported in tobacco (Chen et al. 2017; He et al. 2021; Motlagh et al. 2023). Hence, this research contributes to the expanding diversity of Curvularia by describing two novel species: C. nuciferae from N. nucifera and C. tabaci from N. tabacum.

 Additional information Conflict of interest

The authors have declared that no competing interests exist.

 Ethical statement

No ethical statement was reported.

 Use of AI

No use of AI was reported.

 Funding

This study was financed by the National Natural Science Foundation of China (32270022).

 Author contributions

All authors contributed to the design of the study. Z.J. Guo conceived and designed the study and conducted the experiments; Z.J. Guo, J.X. Deng, S.Q. Chen, and H. Luo collected the samples; and Z.J. Guo drafted the manuscript. Y.M. Rashad, M.K. Amenyogbe, and J.X. Deng provided critical revisions to the manuscript.

 Author ORCIDs

Zhuan-Jun Guo https://orcid.org/0009-0006-5760-4536

Mawuli Korsi Amenyogbe https://orcid.org/0000-0002-3180-7726

Shi-Qiang Chen https://orcid.org/0009-0003-7533-921X

Younes M. Rashad https://orcid.org/0000-0002-7702-8023

Jian-Xin Deng https://orcid.org/0000-0001-7304-5603

Huan Luo https://orcid.org/0000-0002-5503-2507

 Data availability

All of the data that support the findings of this study are available in the main text.

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