Diseases of Cymbopogon citratus (Poaceae) in China: Curvularia nanningensis sp. nov

Abstract

Five Curvularia strains isolated from diseased leaves of lemongrass ( Cymbopogon citratus ) in Guangxi Province, China, were examined. NCBI-Blast searches of ITS sequences suggested a high degree of similarity (99–100%) to Curvularia akaii , C. akaiiensis , C. bothriochloae , C. heteropogonis and C. sichuanensis . To accurately identify these strains, we further analysed their morphology and phylogenetic relationships based on combinations of ITS, GAPDH, and tef 1 gene sequences. Morphological observations indicated that the key character differing from similar species was conidial size, whereas phylogenetic analyses indicated that the five strains represent one species that is also distinct from C. akaii , C. akaiiensis and C. bothriochloae by conidial size and conidiophore length. Thus, the strains examined are found to represent a new species described herein as Curvularia nanningensis . The pathogenicity test on the host and detached leaves confirmed the new species to be pathogenic on Cymbopogon citratus leaves. Standardised requirements for reliable identification of Curvularia pathogens are also proposed.

Full text

Curvularia nanningensis sp. nov 49
Diseases of Cymbopogon citratus (Poaceae) in China:
Curvularia nanningensis sp. nov.
Qian Zhang1, Zai-Fu Yang1, Wei Cheng2, Nalin N. Wijayawardene3,
Kevin D. Hyde4, Zhuo Chen5, Yong Wang1
1 Department of Plant Pathology, Agriculture College, Guizhou University, Guiyang, Guizhou Province,
550025, China 2 Department of Practaculture Science, Animal Science College, Guizhou University, Guiyang,
Guizhou 550025, China 3 Center for Yunnan Plateau Biological Resources Protection and Utilization, College
of Biological Resource and Food Engineering, Qujing Normal University, Qujing, Yunnan 655011, China
4 Center of Excellence in Fungal Research and School of Science, Mae Fah Luang University, Chiang Rai,
57100, ailand 5 Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education,
Guizhou University, Guiyang 550025, China
Corresponding author: Zhuo Chen (gychenzhuo@aliyun.com), Yong Wang (yongwangbis@aliyun.com)
Academic editor: Huzefa Raja|Received11 December 2019|Accepted 30 January 2020|Published 13 February2020
Citation: Zhang Q, Yang Z-F, Cheng W, Wijayawardene NN, Hyde KD, Chen Z, Wang Y (2020) Diseases of
Cymbopogon citratus (Poaceae) in China: Curvularia nanningensis sp. nov. MycoKeys 63: 49–67. https://doi.
org/10.3897/mycokeys.63.49264
Abstract
Five Curvularia strains isolated from diseased leaves of lemongrass (Cymbopogon citratus) in Guangxi Prov-
ince, China, were examined. NCBI-Blast searches of ITS sequences suggested a high degree of similarity
(99–100%) to Curvularia akaii, C. akaiiensis, C. bothriochloae, C. heteropogonis and C. sichuanensis. To ac-
curately identify these strains, we further analysed their morphology and phylogenetic relationships based
on combinations of ITS, GAPDH, and tef1 gene sequences. Morphological observations indicated that
the key character diering from similar species was conidial size, whereas phylogenetic analyses indicated
that the ve strains represent one species that is also distinct from C. akaii, C. akaiiensis and C. bothrioch-
loae by conidial size and conidiophore length. us, the strains examined are found to represent a new
species described herein as Curvularia nanningensis. e pathogenicity test on the host and detached leaves
conrmed the new species to be pathogenic on Cymbopogon citratus leaves. Standardised requirements for
reliable identication of Curvularia pathogens are also proposed.
Keywords
Cymbopogon, phylogeny, plant disease, Pleosporaceae, taxonomy
Copyright Qian Zhang et al. This is an open access ar ticle 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.
MycoKeys 63: 49–67 (2020)
doi: 10.3897/mycokeys.63.49264
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RESEARCH ARTICLE

Qian Zhang et al. / MycoKeys 63: 49–67 (2020)
50
Introduction
Cymbopogon citratus Stapf (lemongrass), believed to be a native of Malaysia, is now
widely distributed in all continents and particularly in America, China, Guatemala
and Southeast Asia. Essential oil from lemongrass is often used in aromatherapy (Wil-
liamson et al. 1996; Noel et al. 2002; Yang and Lei 2005; Shah et al. 2011). As a
traditional Chinese medicine, lemongrass is known to provide relief from a variety
of ailments including eczema, cold, headache and stomach-ache (Zhou et al. 2011).
Guatemala is known to be the main exporter of lemongrass with about 250 tons per
year. China produces 80 to 100 tons of lemongrass annually and the USA and Russia
each imports about 70 tons per year (DAFF 2012). Depending on climatic conditions,
lemongrass can be severely infected with a rust disease caused by Puccinia nakanishikii
Dietel in Hawaii and California (Gardner 1985; Koike and Molinar 1999). In Brazil,
a rust on lemongrass caused by another Puccinia species named P. cymbopogonis Massee
has been reported (Vida et al. 2006). Joy et al. (2006) summarised the various disease
symptoms and their causal agents of lemongrass.
Curvularia spp. infect many herbaceous plants including Cymbopogon Spreng.
(Smithet al. 1989). Helminthosporium cymbopogi C.W. Dodge (≡ Curvularia cym-
bopogonis (C.W. Dodge) J.W.Groves & Skolko) is responsible for a severe disease of
lemongrass in the lowlands of Guatemala (Dodge 1942). Barua and Bordoloi (1983)
discovered C. verruciformis causing disease on Cymbopogon exuosus Stapf. Curvu-
lariaandropogonis (Zimm.) Boedijn led to foliage blight of Cymbopogon nardus (L.)
Rendle in the Philippines (Sato and Ohkubo 1990). akur (1994) reported C. lunata
(Wakker) Boedijn as the causal agent of a new blight disease of Cymbopogon martini
(Roxb.) Wats. var. motia Burk. Chutia et al. (2006) discovered that a leaf blight of
Cymbopogon winterianus Jowitt is caused by Curvularia spp., resulting in a dramatic
change in oil yield and its constituents. Recently, Santos et al. (2018) characterised
the morphological and molecular diversity of the isolates of C. lunata, associated with
Andropogon Linn. seeds.
Starting in 2010, there have been outbreak reports of pathogenic Curvularia in
Asian countries, especially India and Pakistan (Pandey et al. 2014; Avasthi et al. 2015;
Majeed et al. 2015). As China is a neighbouring country, we felt obligated to evaluate
the potential threat of Curvularia to our crops. A severe Curvularia leaf blight disease
was observed in three farms of Curcuma aromatica Salisb. in Hainan Province during
2010 (Chen et al. 2013).Gao et al. (2012) reported a new rice black sheath spot dis-
ease caused by C. fallax Boedijn in Hunan Province. Our research group is also con-
ducting a disease survey on the occurrence of Curvularia diseases in Southwest China
since 2017. Two new pathogens (C. asianensis Manamgoda, L. Cai & K.D. Hyde and
C. microspora Y. Liang, K.D. Hyde, J. Bhat & Yong Wang bis), which aected Epiprem-
num pinnatum (L.) Engl. and Hippeastrum rutilum Herb. (Liang et al. 2018; Wang et
al. 2018), respectively, were found.
Meanwhile, a severe leaf blast disease on lemongrass was found in Guangxi Prov-
ince, China, that rst appeared on the tips of leaves. As the infection progressed, more
than 30% of leaves showed dierent degrees of abnormalities, while in the later stages

Curvularia nanningensis sp. nov 51
more than 50% of the upper leaves appeared diseased and disease incidence reached
80% or above in the lower leaf blades. We provide a detailed morphological description
and phylogenetic analyses of the pathogen conrming it as a new Curvularia species.
Koch’s postulates (see later text) have been carried out to conrm its pathogenicity. Our
study provides a further understanding of Curvularia disease on lemongrass in China.
Materials and methods
Isolation
Leaves of Cymbopogon citratus showing leaf blast symptoms were collected from
Guangxi Medicinal Botanical Garden in Nanning, China, during 2017. Diseased leaf
pieces were surface disinfected with 70% ethanol for 30 s, 1% NaClO for 1 min and
repeatedly rinsed in sterile distilled water for 30 s. For isolation of Curvularia, conidia
were removed from the diseased tissue surface using a sterilised needle and placed in
a drop of sterilised water followed by microscopic examination. e spore suspension
was drawn with a Pasteur pipette and transferred to a Petri dish with 2% water agar
(WA) or 2% malt extract agar (MEA) and 100 mg/l streptomycin to inhibit the growth
of bacteria. e plates were incubated for 24 h in an incubator (25°C) and examined
for single spore germination under a dissecting microscope. Germinating conidia were
transferred separately to new 2% MEA plates (Chomnunti et al. 2014).
Morphological studies
Single germinated spores were transferred to PDA or MEA and incubated at 28°C in a
light incubator with 12 h light/12 h darkness. Ten days later, the colony and morpho-
logical characters were recorded according to Manamgoda et al. (2011, 2012). Colony
diameters on PDA and MEA were measured at 1, 3, 5 and 7 days post-inoculation and
average growth rates were calculated. Conidia and conidiophores were examined using
a compound microscope tted with a digital camera (Olympus BX53). e holotype
specimen is deposited in the Herbarium of the Department of Plant Pathology, Ag-
ricultural College, Guizhou University (HGUP). An ex-type culture is deposited in
the Culture Collection of the Department of Plant Pathology, Agriculture College,
Guizhou University, China (GUCC) and Mae Fah Luang University Culture Collec-
tion (MFLUCC) in ailand (Table 1).
DNA Extraction and Sequencing
Fungal cultures were grown on PDA at 28°C until the entire Petri dish (90 mm)
was colonised. Fresh fungal mycelia were scraped o the surface of the PDA using
a sterilised scalpel. A BIOMIGA Fungus Genomic DNA Extraction Kit (GD2416,

Qian Zhang et al. / MycoKeys 63: 49–67 (2020)
52
Table 1. Sequences used for phylogenetic analysis.
Species name Strain number GenBank Accession numbers
ITS GAPDH tef1
Curvularia aeria CBS 294.61THE861850 HF565450 –
C. anis CBS 154.34TKJ909780 KM230401 KM196566
C. ahvazensis CBS 144673TKX139029 MG428693 MG428686
C. akaii CBS 317.86 KJ909782 KM230402 KM196569
C. akaiiensis BRIP 16080TKJ415539 KJ415407 KJ415453
C. alcornii MFLUCC 10-0703TJX256420 JX276433 JX266589
C. americana UTHSC 08-3414THE861833 HF565488 –
C. asiatica MFLUCC 10-0711TJX256424 JX276436 JX266593
C. australiensis BRIP 12044TKJ415540 KJ415406 KJ415452
C. australis BRIP 12521TKJ415541 KJ415405 KJ415451
C. bannonii BRIP 16732TKJ415542 KJ415404 KJ415450
C. beasleyi BRIP 10972TMH414892 MH433638 MH433654
C. beerburrumensis BRIP 12942TMH414894 MH433634 MH433657
C. boeremae IMI 164633TMH414911 MH433641 –
C. borreriae CBS 859.73 HE861848 HF565455 –
MFLUCC 11-0422 KP400638 KP419987 KM196571
C. bothriochloae BRIP 12522TKJ415543 KJ415403 KJ415449
C. brachyspora CBS 186.50 KJ922372 KM061784 KM230405
C. buchloes CBS 246.49TKJ909765 KM061789 KM196588
C. carica-papayae CBS 135941THG778984 HG779146 –
C. chiangmaiensis CPC 28829TMF490814 MF490836 MF490857
C. chlamydospora UTHSC 07-2764THG779021 HG779151 –
C. clavata BRIP 61680b KU552205 KU552167 KU552159
C. coatesiae BRIP 24261TMH414897 MH433636 MH433659
C. coicis CBS 192.29TJN192373 JN600962 JN601006
C. colbranii BRIP 13066TMH414898 MH433642 MH433660
C. crustacea BRIP 13524TKJ415544 KJ415402 KJ415448
C. cymbopogonis CBS 419.78 HG778985 HG779129 –
C. dactyloctenicola CPC 28810TMF490815 MF490837 MF490858
C. dactyloctenii BRIP 12846TKJ415545 KJ415401 KJ415447
C. deightonii CBS 537.70 LT631356 LT715839 –
C. ellisii CBS 193.62TJN192375 JN600963 JN601007
C. eragrosticola BRIP 12538TMH414899 MH433643 MH433661
C. eragrostidis CBS 189.48 HG778986 HG779154 –
C. geniculata CBS 187.50TKJ909781 KM083609 KM230410
C. gladioli CBS 210.79 HG778987 HG779123
C. graminicola BRIP 23186TJN192376 JN600964 JN601008
C. gudauskasii DAOM 165085 AF071338 – –
C. harveyi BRIP 57412TKJ415546 KJ415400 KJ415446
C. hawaiiensis BRIP 11987TKJ415547 KJ415399 KJ415445
C. heteropogonicola BRIP 14579TKJ415548 KJ415398 KJ415444
C. heteropogonis CBS 284.91TJN192379 JN600969 JN601013
C. hominis CBS 136985THG779011 HG779106 –
C. homomorpha CBS 156.60TJN192380 JN600970 JN601014
C. inaequalis CBS 102.42TKJ922375 KM061787 KM196574
C. intermedia CBS 334.64 HG778991 HG779155 –
C. ischaemi CBS 630.82TJX256428 JX276440 –
C. kenpeggii BRIP 14530TMH414900 MH433644 MH433662
C. kusanoi CBS 137.29TJN192381 –JN601016
C. lamingtonensis BRIP 12259TMH414901 MH433645 MH433663
C. lunata CBS 730.96TJX256429 JX276441 JX266596
C. malina CBS 131274TJF812154 KP153179 KR493095
C. mebaldsii BRIP 12900TMH414902 MH433647 MH433664
C. micropus CBS 127235T HE792934 LT715859 –
C. microspora GUCC 6272TMF139088 MF139106 MF139115
C. miyakei CBS 197.29TKJ909770 KM083611 KM196568
C. mosaddeghii IRAN 3131CTMG846737 MH392155 MH392152
C. muehlenbeckiae CBS 144.63THG779002 HG779108 –

Curvularia nanningensis sp. nov 53
Species name Strain number GenBank Accession numbers
ITS GAPDH tef1
C. neergaardii BRIP 12919TKJ415550 KJ415397 KJ415443
C. nanningensis sp. nov. GUCC 11000 MH885316 MH980000 MH980006
GUCC 11001 MH885317 MH980001 MH980007
GUCC 11002 MH885318 MH980002 MH980008
GUCC 11003 MH885319 MH980003 MH980009
GUCC 11005TMH885321 MH980005 MH980011
C. neoindica BRIP 17439 AF081449 AF081406 –
C. nicotiae CBS 655.74T = BRIP 11983 KJ415551 KJ415396 KJ415442
C. nodosa CPC 28800TMF490816 MF490838 MF490859
CPC 28801 MF490817 MF490839 MF490860
CPC 28812 MF490818 MF490840 MF490861
C. nodulosa CBS 160.58 JN601033 JN600975 JN601019
C. oryzae CBS 169.53TKP400650 KP645344 KM196590
C. ovariicola CBS 470.90TJN192384 JN600976 JN601020
C. pallescens CBS 156.35TKJ922380 KM083606 KM196570
C. palmicola MFLUCC 14-0404 MF621582 – –
C. papendori CBS 308.67TKJ909774 KM083617 KM196594
C. perotidis CBS 350.90TJN192385 KJ415394 JN601021
C. petersonii BRIP 14642TMH414905 MH433650 MH433668
C. pisi CBS 190.48TKY905678 KY905690 KY905697
C. platzii BRIP 27703bTMH414906 MH433651 MH433669
C. portulacae CBS 239.48T = BRIP 14541 KJ415553 KJ415393 KJ415440
C. prasadii CBS 143.64TKJ922373 KM061785 KM230408
C. protuberata CBS 376.65TKJ922376 KM083605 KM196576
C. pseudobrachyspora CPC 28808TMF490819 MF490841 MF490862
C. pseudolunata UTHSC 09-2092THE861842 HF565459 –
C. pseudorobusta UTHSC 08-3458 HE861838 HF565476 –
C. ravenelii BRIP 13165TJN192386 JN600978 JN601024
C. reesii BRIP 4358TMH414907 MH433637 MH433670
C. richardiae BRIP 4371TKJ415555 KJ415391 KJ415438
C. robusta CBS 624.68TKJ909783 KM083613 KM196577
C. rouhanii CBS 144674TKX139030 MG428694 MG428687
C. ryleyi BRIP 12554TKJ415556 KJ415390 KJ415437
C. senegalensis CBS 149.71 HG779001 HG779128 –
C. sesuvii Bp-Zj 01TEF175940 – –
C. shahidchamranensis IRAN 3133CTMH550084 MH550083 –
C. soli CBS 222.96TKY905679 KY905691 KY905698
C. sorghina BRIP 15900TKJ415558 KJ415388 KJ415435
C. spicifera CBS 274.52 JN192387 JN600979 JN601023
C. sporobolicola BRIP 23040bTMH414908 MH433652 MH433671
C. subpapendori CBS 656.74TKJ909777 KM061791 KM196585
C. trifolii CBS 173.55 HG779023 HG779124 –
C. tripogonis BRIP 12375TJN192388 JN600980 JN601025
C. tropicalis BRIP 14834TKJ415559 KJ415387 KJ415434
C. tsudae ATCC 44764TKC424596 KC747745 KC503940
C. tuberculata CBS 146.63TJX256433 JX276445 JX266599
C. uncinata CBS 221.52THG779024 HG779134 –
C. variabilis CPC 28813 MF490820 MF490842 MF490863
CPC 28814 MF490821 MF490843 MF490864
CPC 28815TMF490822 MF490844 MF490865
CPC 28816 MF490823 MF490845 MF490866
C. verruciformis CBS 537.75 HG779026 HG779133 –
C. verruculosa CBS 150.63 KP400652 KP645346 KP735695
CPC 28792 MF490825 MF490847 MF490868
CPC 28809 MF490824 MF490846 MF490867
C. warraberensis BRIP 14817TMH414909 MH433653 MH433672
Bipolaris drechsleri MUS0028 KF500532 KF500535 KM093761
B. maydis CBS 136.29TAF071325 KM034846 KM093794
Ex-type isolates were labeled with “T”.

Qian Zhang et al. / MycoKeys 63: 49–67 (2020)
54
BIOMIGA, Inc., San Diego, California, USA) was used to extract the genomic DNA.
DNA amplication was performed in a 25 μl reaction volume which contained 2.5 μl
10 × PCR buer, 1 μl of each primer (10 μM), 1 μl template DNA, 0.25 μl Taq DNA
polymerase (Promega, Madison, WI, USA) and 18.5 μl ddH2O. Primers used and
thermal cycling programme for PCR amplication of the ITS (ITS4/ITS5), GAPDH
(gpd1/gpd2) and tef1 (EF-526F/1567R) genes were followed as described previously
(White et al. 1990; Berbee et al. 1999; Schoch et al. 2009; Liang et al. 2018).
Phylogenetic analyses
DNA sequences originated from ve strains (GUCC 11000, GUCC 11001, GUCC
11002, GUCC 11003 and GUCC 11005) and reference sequences of ex-type or
representative sequences of Curvularia species were downloaded from GenBank da-
tabase (Table 1) with strains of Bipolaris maydis (Y. Nisik. & C. Miyake) Shoemaker
(CBS 136.29) and B. drechsleri Manamgoda & Minnis (MUS0028) as outgroup
taxa. Alignments for each locus were performed in MAFFT v7.307 online version
(Katoh and Standley 2016) and manually veried in MEGA 6.06 (Tamura et al.
2013). Phylogenetic analyses were performed by Maximum Parsimony (MP), Maxi-
mum Likelihood (ML) and Bayesian methods. Sequences were optimised manually
to allow maximum alignment and maximum sequence similarity as detailed in Ma-
namgoda et al. (2012). MP analyses were performed in PAUP v. 4.0b10 (Swoord
2003) using the heuristic search option with 1,000 random taxa additions and tree
bisection and reconnection (TBR) as the branch-swapping algorithm. Five thousand
maxtrees were set to build up the phylogenetic tree. e characters in the alignment
matrix were ordered according to ITS+GAPDH+tef1 with equal weight, and gaps
were treated as missing data. e MP phylogenetic analysis of Curvularia ITS se-
quences included pathogens from China, India and Pakistan and the wrong sequence
(KN879930), actually belonging to Alternaria alternata (taxon:5599), was selected
as the outgroup. e Tree Length (TL), Consistency Indices (CI), Retention Indices
(RI), Rescaled Consistency Indices (RC) and Homoplasy Index (HI) were calculated
for each tree generated. e resulting PHYLIP le was used to generate the ML tree
on the CIPRES Science Gateway (https://www.phylo.org/portal2/login.action) us-
ing the RAxML-HPC2 black box with 1000 bootstrap replicates and GTRGAMMA
as the nucleotide substitution model. For Bayesian inference analysis, the best model
of evolution (GTR+I+G) was determined using MrModeltest v2 (Nylander 2004).
Bayesian inference analysis was done using MrBayes v 3.2.6 (Ronquist et al. 2012).
Bayesian analyses were launched with random starting trees for 2 000 000 genera-
tions and Markov chains were sampled every 1000 generations. e rst 25% result-
ing trees were discarded as burn-in. Alignment matrices are available in TreeBASE
under the study ID 25080.

Curvularia nanningensis sp. nov 55
Koch’s Postulate test
To conrm the pathogenicity of the fungus, ve healthy plants of Cymbopogon citratus
were inoculated with 5 mm diameter mycelial plugs of the ve isolates (GUCC 11000,
GUCC 11001, GUCC 11002, GUCC 11003 and GUCC 11005) cut from the mar-
gins of 10-day-old actively growing cultures; the control was treated with sterile agar
plugs. e plants were kept for two days in an illuminating incubator at 28° ± 3°C. Ad-
ditionally, two plants were sprayed with distilled water and kept as control under the
same conditions. Both inoculated (host and detached leaves) and control plants were
kept for two days in an illuminating incubator at 28 ± 3°C. After four days of incuba-
tion, the inoculated plants and leaves were observed for the development of symptoms
(Zhang et al. 2018). Infected leaves were collected and the fungus was re-isolated using
PDA medium and the ITS sequence was compared with original strains.
Results
Phylogenetic analyses
First, we compared the DNA sequence identity of ITS, GAPDH and tef1 gene regions
(Table 2). Among our ve strains, there was only one base dierence. In the ITS gene
region, for C. akaiiensis, the base sequence was identical to our strains; only 1 dier-
ence for C. bothriochloae; base dierences were 8 for C. akaii, 9 for C. deightonii and
5 for C. sichuanensis. Only C. heteropogonis had noticeable (25) base dierences with
our strains. In the GAPDH and tef1 gene regions, the mutation rate of DNA bases
was apparently faster than the ITS region. ere were between 9 to 19 base dierences
in GAPDH and 3 to 8 in tef1. is means that in Curvularia, GAPDH has a faster
Table 2. DNA sequence dierences between Curvularia nanningensis and related species in three gene
regions.
Species Strain number ITS (1–547 bp) GAPDH (550–1031bp) tef1 (1034–1899 bp)
C. nanningensis GUCC11000 0 1 0
GUCC11001 0 0 0
GUCC11002 0 1 0
GUCC11003 0 1 0
GUCC11005T000
C. akaii CBS 317.86 8 9 4
C. akaiiensis BRIP 16080 T 0 10 5
C. bothriochloae BRIP 12522 T 1 19 8
C. deightonii CBS 537.70 9 13 –
C. heteropogonis CBS 284.91 T 25 12 3
C. sichuanensis HSAUP II.2650-1 T 5––
T = ex-type

Qian Zhang et al. / MycoKeys 63: 49–67 (2020)
56
Figure 1. Maximum Parsimony (MP) topology of Curvularia generated from a combination of ITS,
GAPDH and tef1 sequences. Bipolaris maydis (CBS 136.29) and B. drechsleri (MUS0028) were used as
outgroup taxa. MP and ML above 50% and BPP values above 0.90 were placed close to topological nodes
and separated by “/”. e bootstrap values below 50% and BPP values below 0.90 were labelled with “-”.
Our main research clade was labelled with green colour.

Curvularia nanningensis sp. nov 57
Figure 2. Maximum Parsimony (MP) analysis of Curvularia pathogens in China, India and Pakistan
based on ITS sequences. Alternaria alternata (taxon:5599) was used as outgroup taxon. Bootstrap values
(≥ 50%) of the MP method are shown near the nodes.
evolutionary rate than ITS and tef1 and therefore some mycologists have suggested the
use of ITS+GAPDH for phylogenetic analysis and GAPDH as a secondary barcode
marker for accurate identication.

Qian Zhang et al. / MycoKeys 63: 49–67 (2020)
58
e alignment of Curvularia combining three gene fragments (ITS, GAPDH and
tef1) comprised 116 strains belonging to 104 taxa. In order to accurately identify our
strains, phylogenetic analysis included all ex-type and published strains of all Curvu-
laria spp. described recently (Hyde et al. 2017; Marin-Felix et al. 2017; Dehdari et al.
2018; Heidari et al. 2018; Hernández-Restrepo et al. 2018; Mehrabi-Koushki et al.
2018; Tan et al. 2018; Jayawardena et al. 2019) which are listed in Table 1. e nal
alignment comprised 2032 characters (each gene fragment was separated with 2 “N”)
including gaps (ITS: 1−600, GAPDH: 603−1162 and tef1: 1165−2032). Among these
characters, 2032 are constant, 125 variable characters are parsimony-uninformative
and 503 are parsimony-informative. e parameters of the phylogenetic trees are TL
= 2590, CI = 0.38, RI = 0.72 and HI = 0.62. In the Curvularia phylogenetic tree
(Figure 1), all isolates grouped together with 100% (MP and ML) bootstrap support.
Our strains (GUCC 11000, 11001, 11002, 11003 and 11005) formed a strongly
supported group (MP: 100%; ML: 100%; BPP: 1.00) with a close relationship to
C. akaii, C. akaiiensis, C. bothriochloae, C. deightonii and C. heteropogonis with high
bootstrap support (MP: 94%; ML: 97%; BPP: 1.00). In this group, the ve examined
strains were closer to C. akaii, C. akaiiensis and C. bothriochloae and also showed high
bootstrap support (MP: 82% and ML: 94%; BPP: 0.98).
e phylogenetic analysis of the ITS gene region evaluated all new Curvularia
pathogens recently described from China, India and Pakistan. e aligned matrix con-
sisted of fty-four ITS sequences and included ex-type sequences of 13 Curvularia
species (Supplementary Table 1). e phylogenetic tree (Figure 2) indicated that ITS
BLAST searches only provided limited value for pathogenic identication. In Curvu-
laria lunata, only one sequence WCCL (MG063428) showed a very close relationship
with the ex-type strain sequence of C. lunata CBS 730.96 (MG722981). e other
eight sequences were grouped into two branches, e.g. taxon:5503 (LN879926) which
might belong to C. aeria, while the other seven formed an independent lineage. ITS
sequences did not separate Curvularia anis, C. asianensis and C. fallax and some of
their sequences even clustered with C. australiensis HNWB9-1 (KT719300). After
multi-gene analysis, the phylogenetic distance was shown to be unreliable and may
suggest whether they belong perhaps to dierent species.
Taxonomy
Curvularia nanningensis Qian Zhang, K.D. Hyde & Yong Wang bis, sp. nov.
MycoBank No: 829056
Facesoungi number: FoF 05596
Figure 3A–I
Diagnosis. Characterised by the size of conidia.
Type. China, Guangxi Province, Nanning City, Guangxi Medicinal Botanical
Garden, 22°51’N, 108°19’E, on blighted leaves of Cymbopogon citratus, 30 Septem-

Curvularia nanningensis sp. nov 59
Figure 3. Curvularia nanningensis (GUCC11005, holotype) A, B diseased symptom C colony on PDA
from above D colony on PDA from below E−G conidia and conidiophores H−I conidia. Scale bars: 50
μm (E), 20 μm (F), 10 μm (G−I).
ber 2017, Q. Zhang, ZQ0091 (HGUP 11005, holotype, MFLU19-1227, isotype),
GUCC 11005 and MFLUCC 19-0092, ex-type.
Description. Pathogenic on Cymbopogon citratus. Fungus initially producing
white to grey lesions with dark borders on all parts of the shoot, later enlarging and
coalescing over entire leaf.
Colonies on PDA irregularly circular, with mycelial growth rate = 1.0 cm/day, vege-
tative hyphae septate, branched, subhyaline to brown, smooth to verruculose, 2–3 μm,
anastomosing. Aerial mycelium dense, felted, initially pale grey, becoming darkened
and greyish-green at maturity, producing black extracellular pigments. On MEA, the
colony morphology similar to PDA, with growth rate = 1.35 cm/day. Sexual morph:
Undetermined. Asexual morph: Hyphomycetous. Conidiophores macronematous,
arising singly, simple or branched, exuous, 8–10 septate, geniculate, pale brown to
dark brown, paler towards apex, 120–200 × 2–3 μm (av. = 170 × 2.5 μm, n = 30). Co-
nidiogenous cells polytretic, sympodial, terminal, sometimes intercalary, cicatrised, with
thickened and darkened conidiogenous loci up to 1.0–1.2 μm diam., smooth. Mature
conidia 3 to rarely 4 septa, acropleurogenous, obovoid, usually straight to curved at the
slightly wider, smooth-walled, larger third cell from the base, 24.5–36.0 × 14.0–20.5
μm (av. = 29.5 × 17.5 μm, n = 50), sub-hyaline to pale brown end cells, pale brown to
dark brown at intermediate cells, with conspicuous or sometimes slightly protuberant
hilum. Germination of conidia bipolar.
Distribution. China, Guangxi Province, Nanning City.
Other material examined. China, Guangxi Province, Nanning city, Guangxi
Medicinal Botanical Garden, on blight leaves of C. citratus, 30 September 2017,

Qian Zhang et al. / MycoKeys 63: 49–67 (2020)
60
Q. Zhang, ZQ0087 (HGUP 11000); ZQ0088 (HGUP 11001); ZQ0089 (HGUP
11002); ZQ0090, (HGUP 11003).
Etymology. With reference to the location, Nanning City where the fungus was
isolated.
Pathogenicity test
Four days after inoculation, blast symptoms appeared on all inoculated plants, which
were similar to symptoms of plants in the eld (Figures 3A, B, 4A, B). Non-treated
control plants remained healthy without any symptoms (Figure 4C). Curvularia nan-
ningensis was re-isolated from the lesions of inoculated plants and the identity of the
fungus was conrmed by sequencing the ITS region. Meanwhile, a detached leaf-ex-
periment was also conducted in an illuminated incubator at 28 ± 3°C, where similar
symptoms appeared on healthy inoculated leaves of Cymbopogon citratus after four days
(Figure 4 D right), while the control leaf (Figure 4 D left) did not show symptoms.
Discussion
Phylogenetic analysis based on combined DNA sequences of ITS, GAPDH and tef1
showed that our strains were related to three Curvularia species named C. akaii (Tsuda
& Ueyama) Sivan., C. akaiiensis Sivan. and C. bothriochloae Sivan., Alcorn & R.G.
Shivas. e main morphological characters that discriminate our strains from related
species are the size-range of conidia and length of conidiophores. Curvularia bothrioch-
loae produced conidia measuring 30–47 × 15–25 μm (Sivanesan et al. 2003) while C.
akaiiensis produced the smallest conidia (22.5–27.5 × 7.5–15.5 μm). Conidial length
of C. nanningensis was very close to C. akaii (24–34 μm) (Tsuda and Ueyama 1985)
but the conidia of our species were broader than those of C. akaii (8.7–13.8 μm). Co-
nidiophores of C. nanningensis were shorter than those of C. bothriochloae (360–425
μm) (Alcorn 1990). In the case of C. sichuanensis Meng Zhang & T.Y. Zhang, only
one ITS sequence AB453881 was available in GenBank for analysis. While examining
our sequences, only 4–5 bp dierences were revealed in 499 bp characters between
C. nanningensis and C. sichuanensis, thus indicating a close relationship between the
two strains based on ITS sequence data and likely between the two species. However,
according to Zhang et al. (2007), the conidial width of C. sichuanensis (10–15 μm) is
smaller than C. nanningensis (14–20.5 μm) on PDA. For C. sichuanensis, the conidial
wall of the median cell is deepened and thickened while C. nanningensis obviously does
not have these characters. Meanwhile, the hilum of conidia in C. sichuanensis is obvi-
ously protuberant while C. nanningensis lacked this character.
e pathogenicity test based on natural inoculation and detached leaves (Figure 3)
conrmed that Curvularia nanningensis is a pathogen of Cymbopogon citratus blast dis-
ease. We previously named our strains as C. cymbopogonis following a previous report
of the species by Groves and Skolko (1945) as a seed-borne pathogen of Cymbopogon

Curvularia nanningensis sp. nov 61
Figure 4. Pathogen inoculation and symptom (4 days). A Cymbopogon citratus inoculated and disease
symptom B inoculation point and disease symptom C control D detached experiment. Left. Control.
Right. Inoculation point and disease symptoms.
nardus. Curvularia cymbopogonis is a common pathogen which also causes diseases of
sugar-cane, rice, seedlings of itchgrass, Agrostis palustris Huds. and Dactylis glomerata
L. (Santamaria et al. 1971; Walker and White 1979; Olufolaji 1996; Yi et al. 2002). A
single strain named C. cymbopogonis (CBS 419.78) included in our analyses grouped
distant from C. nanningensis but its reliability seems questionable and apparently be-
longs to a dierent species (Fig. 1). We further checked the original description of this
species (Groves and Skolko 1945) and found that dierences in conidial shape mainly
resulted from conidial width (C. cymbopogonis: 11–13 μm vs C. nanningensis: 14–20.5
μm). Additionally, Groves and Skolko (1945), Hall and Sivanesan (1972) and Yi et
al. (2002) reported that C. cymbopogonis produced 4 to 5-septate conidia, whereas
conidia of C. nanningensis only had 3-septa. Curvularia spp. are important pathogens
of lemongrass. Morphological studies together with phylogenetic analyses provided
evidence that C. nanningensis is a new pathogen distinct from all hitherto reported dis-
eases on lemongrass. Our ndings expanded the documented diversity of Cymbopogon

Qian Zhang et al. / MycoKeys 63: 49–67 (2020)
62
pathogens within the genus Curvularia and further claried the taxonomy of this novel
pathogen, Curvularia nanningensis.
Moreover, 29 rst reports of Curvularia diseases on dierent plants in China, India
and Pakistan were found in the literature from 2010 to the present. It is evident that
in this vast geographical area, Curvularia spp. have maintained a close association with
plant diversity and thereby possess a rich fungal diversity that is aected by crops distri-
bution. Among them, six reports only provided morphological data and more than half
(16) only referred to ITS sequence data and morphological description (Suppl. Table 1).
For unknown reasons, Iftikhar et al. (2016) misidentied the Curvularia pathogen with
an Alternaria sequence (LN879930.1). Our phylogenetic tree, based on 54 reported ITS
sequence data of Curvularia diseases in these countries (Figure 2), also indicated that
this approach is not eective for identifying these pathogens, especially in the case of C.
lunata as a prevalent species. However, identication of Curvularia isolates by multi-gene
phylogenetic analyses has withstood scrutiny (Liang et al. 2018; Wang et al. 2018; Zhang
et al. 2018). Additionally, nearly all reports, even for severe diseases, are based on a single
isolate, which preclude an objective evaluation. We, therefore, propose the following
standardised steps as required for the reliable identication of Curvularia diseases: 1) col-
lect several isolates from diseased samples, 2) obtain sequences of the ITS, GAPDH and
tef1 or at least ITS+GAPDH for phylogenetic analysis, 3) perform BLAST searches with
sequences originated from ex-type or representative strains in GenBank, and 4) combine
morphological comparison and phylogenetic analysis for accurate identication.
Acknowledgments
is research is supported by the following projects: National Natural Science Foun-
dation of China (No. 31972222, 31560489), Program of Introducing Talents of
Discipline to Universities of China (111 Program, D20023), Science and Technol-
ogy basic work of MOST [2014FY120100], National Key Technology Research
and Development Program of the Ministry of Science and Technology of China
(2014BAD23B03/03), Talent project of Guizhou Science and Technology Coopera-
tion Platform ([2017]5788-5 and [2019]5641) and Guizhou Science, Technology De-
partment International Cooperation Base project ([2018]5806). Nalin Wijayawardene
thanks National Natural Science Foundation of China (No. NSFC 31950410558).
We thank Mr Mike Skinner for linguistic editing.
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Curvularia nanningensis sp. nov 67
Supplementary material 1
Table S1. Disease occurrence caused by Curvularia spp. in China, India and Pa-
kistan
Authors: Qian Zhang, Zai-Fu Yang, Wei Cheng, Nalin N. Wijayawardene, Kevin D.
Hyde, Zhuo Chen, Yong Wang
Data type: occurrence
Copyright notice: is dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). e Open Database License
(ODbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.
Link: https://doi.org/10.3897/mycokeys.63.49264.suppl1