A new Curvularia lunata variety discovered in Huanghuaihai Region in China

Abstract

The purpose of this study was to identify the dominant pathogens of Curvularia leaf spot and their pathogenicity variation in Huanghuaihai Region of China in recent years. In 2013 and 2016–2017, the occurrences of Curvularia leaf spots on maize were investigated in fields located in Henan, Hebei, Shandong, and Anhui provinces, and 292 fungi were isolated from diseased leaves. These fungal isolates were subjected to morphological identification, and 232 isolates were found to have about 70% uncurved conidia and were identified as Curvularia lunata var. Most of the conidia of 2 representative isolates, namely, HNWB-131 and HNWB-185, were oblong with parallel septations and were distinctly different from a reference isolate CX-3. For further determination, the internal transcribed spacer (ITS), glyceraldehyde 3-phosphate dehydrogenase (GPDH), the large subunit (LSU), and translation elongation factor 1-alpha (EF1-α) sequences of HNWB-131, HNWB-185, and CX-3 were amplified and sequenced. The results of sequence analysis showed that the 4 gene sequences from the 3 isolates had a similarity of more than 99% to C. lunata. Based on the sequences of ITS and the combined data of the 4 genes, neighbor-joining trees were constructed for phylogenetic analysis. The results indicated that these 3 isolates were clustered together with C. lunata. The expression of Clg2p and ClUrase genes in mycelia and conidia was significantly (P<0.05) higher in CX-3 than in HNWB-131 and HNWB-185. This study found that the dominant pathogen of Curvularia leaf spot was a new variety of C. lunata with morphological variations in Huanghuaihai Region from 2013 to 2017. The pathogenicity of the C. lunata var. was not significantly enhanced, and the expression of Clg2p and ClUrase genes of C. lunata var. was decreased.

Full text

Journal of Integrative Agriculture 2020, 19(2): 551–560
RESEARCH ARTICLE
Available online at www.sciencedirect.com
ScienceDirect
A new Curvularia lunata variety discovered in Huanghuaihai Region
in China
CHANG Jia-ying1, LIU Shu-sen1, SHI Jie1, GUO Ning1, ZHANG Hai-jian1, CHEN Jie2
1 Plant Protection Institute, Hebei Academy of Agricultural and Forestry Sciences/Key Laboratory of IPM on Crops in Northern
Region of North China, Ministry of Agriculture and Rural Affairs/IPM Centre of Hebei Province, Baoding 071000, P.R.China
2 School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, P.R.China
Abstract
The purpose of this study was to identify the dominant pathogens of Curvularia leaf spot and their pathogenicity variation in
Huanghuaihai Region of China in recent years. In 2013 and 2016–2017, the occurrences of Curvularia leaf spots on maize
were investigated in elds located in Henan, Hebei, Shandong, and Anhui provinces, and 292 fungi were isolated from
diseased leaves. These fungal isolates were subjected to morphological identication, and 232 isolates were found to have
about 70% uncurved conidia and were identied as Curvularia lunata var. Most of the conidia of 2 representative isolates,
namely, HNWB-131 and HNWB-185, were oblong with parallel septations and were distinctly different from a reference
isolate CX-3. For further determination, the internal transcribed spacer (ITS), glyceraldehyde 3-phosphate dehydrogenase
(GPDH), the large subunit (LSU), and translation elongation factor 1-alpha (EF1-α) sequences of HNWB-131, HNWB-185,
and CX-3 were amplied and sequenced. The results of sequence analysis showed that the 4 gene sequences from the
3 isolates had a similarity of more than 99% to C. lunata. Based on the sequences of ITS and the combined data of the 4
genes, neighbor-joining trees were constructed for phylogenetic analysis. The results indicated that these 3 isolates were
clustered together with C. lunata. The expression of Clg2p and ClUrase genes in mycelia and conidia was signicantly
(P<0.05) higher in CX-3 than in HNWB-131 and HNWB-185. This study found that the dominant pathogen of Curvularia
leaf spot was a new variety of C. lunata with morphological variations in Huanghuaihai Region from 2013 to 2017. The
pathogenicity of the C. lunata var. was not signicantly enhanced, and the expression of Clg2p and ClUrase genes of
C. lunata var. was decreased.
Keywords: Curvularia lunata, Curvularia leaf spot, morphological identication, phylogenetic analysis
1. Introduction
Curvularia leaf spot is a common disease in the maize leaf
worldwide. It occurs in Europe, North and South America,
Asia, and Africa and results in signicant reductions in
maize yields, with serious losses of up to 60% (Yan et al.
1999). The disease was rst observed in the coastal areas
of Shandong, China in the late 1970s to the early 1980s and
was initially termed “unknown spot” because the pathogen
Received 21 September, 2018 Accepted 18 March, 2019
CHANG Jia-ying, E-mail: cjy198908@163.com; Correspondence
SHI Jie, E-mail: shij99@163.com; CHEN Jie, E-mail: jiechen59@
sjtu.edu.cn
© 2020 CAAS. Published by Elsevier Ltd. This is an open
access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
doi: 10.1016/S2095-3119(19)62655-9

552 CHANG Jia-ying et al. Journal of Integrative Agriculture 2020, 19(2): 551–560
had not been identied (Dai et al. 1995). In the mid-1980s,
the disease occurred in succession on maize plants in
Henan, Hebei, Shanxi, and other regions of China. The
yield was reduced by 20–50% when the epidemics were
severe and sometimes resulted in total crop loss (Zhang
et al. 2010). Curvularia leaf spot has spread widely from
the northeast to the southwest of China. Since the end of
the 20th century, it has become a serious disease because
of its effects on maize yield.
To date, more than 10 species of Curvularia that can
infect maize, including Curvularia lunata, C. clavata,
C. pallescens, C. intermedia, C. senegalensis, C. inaequalis,
C. australiensis, and C. tuberculate, have been reported.
Among these pathogens, C. lunata is the dominant
aggressive species in China (Shi et al. 2000; Dou and Jin
2007; Liu et al. 2008; Li et al. 2013). The genetic variability,
which is associated with the cultivation of disease-resistant
maize varieties, has been suggested to have happened to
this pathogen. Lu et al. (1997) found that a maize variety,
NewIron 10, which is resistant to C. lunata, had lost disease
resistance in the eld, presumably from the emergence of a
new, more aggressive strain of the fungus. Indeed, genetic
diversity studies on a limited number of fungal isolates have
demonstrated a signicant level of variability among them
(Chen et al. 2003; Yan et al. 2005; Xu et al. 2007). In 22
C. lunata isolates, 100 genetic polymorphisms were detected
using the random amplied polymorphic DNA technique (Yan
and Chen 2002). In addition, 110 reproducible bands and
75 specic proteins associated with virulence differentiation
were identied from 6 isolates of C. lunata through one- and
two-dimensional gel electrophoresis, suggesting signicant
biodiversity among these isolates (Xu et al. 2007). Recent
studies have focused on breeding highly resistant maize
varieties, differentiation of virulence, and identication of
virulence factors in this fungus, such as cell wall-degrading
enzymes, melanin, and toxins (Feng et al. 2002; Xu et al.
2007; Liu et al. 2009; Liu et al. 2011). Nevertheless, the
maize cropping system has changed substantially in China,
with increased cultivation of disease-resistant varieties
and/or the cultivation of single, high-yield maize varieties
(Chen et al. 2011). These changes may have affected
maize pathogen populations, including those of C. lunata.
However, reports on the effects caused by these changes
on C. lunata populations are limited.
Normally, Curvularia leaf spot is a small spot leaf disease,
which has less impact on maize when it occurs in the late
growth stages. In the present study, the symptoms on some
leaves were found to be different from those in previous
observations, with some spots expanding to 2–3 times,
their initial size on susceptible maize varieties (for example
a local cultivar Zhengdan 958). The spots were oval with
an irregular edge, and the leaves often withered because
of the coalescing spots. We investigated the occurrence
of Curvularia leaf spot in Huanghuaihai Region (Henan,
Anhui, Hebei, and Shandong provinces) in China and
isolated the pathogens from maize leaves with displayed
symptoms. The dominant population was identified
according to the morphological characteristics and was
further conrmed by DNA sequences and phylogenetic
analysis based on the internal transcribed spacer (ITS),
glyceraldehyde 3-phosphate dehydrogenase (GPDH),
the large subunit (LSU), and translation elongation factor
1-alpha (EF1-α) genes. Moreover, the pathogenicity of
the representative isolates were veried in the eld and
the expression of Clg2p and ClUrase genes of C. lunata
var. were measured.
The occurrence of Curvularia leaf spot disease, its
dominant pathogens and pathogenic changes were
identied, as well as the variation mechanism of pathogens
was claried, which would provide a basis for the epidemic,
prevention and control of the disease. We recommend
screening new target-site agents for the disease control.
2. Materials and methods
2.1. Field investigation of Curvularia leaf spot
Maize leaves displaying symptoms of Curvularia leaf spot
were investigated and collected from 111 locations in Henan,
Hebei, Shandong, and Anhui provinces of China in 2013 and
2016–2017 (Appendix A). At least 20 plants in each eld
plot were arbitrarily surveyed for disease severity evaluation.
We used the disease severity index as follows:
Disease severity index=(∑Di×Dd)/(Mi×Md)×100
where Di represents the number of unhealthy plants at
each grade; Dd indicates the representative value of each
grade; Mi represents the total number of plants studied;
and Md indicates the representative value of the highest
grade. The entire experiment was performed 3 times with
similar results.
2.2. Collection and isolation of fungal isolates
The diseased samples were placed in paper bags and stored
at 4°C until they were processed for isolation. Fungi were
isolated according to the following method: small pieces
(2 mm×4 mm) of infected leaves were excised from the
junction of diseased and healthy tissues; the collected leaf
material was surface-disinfected with 75% ethanol for 30 s,
rinsed 3 times with sterile water, transferred to potato
dextrose agar (PDA) medium, and incubated in the dark at
28°C for 3–5 days; the isolated fungi were puried through
single-spore separation and subcultured on PDA, yielding
pure cultures (Zhang et al. 2008; Chomnunti et al. 2011).

553
CHANG Jia-ying et al. Journal of Integrative Agriculture 2020, 19(2): 551–560
2.3. Morphological identication
All puried isolates were characterized based on macro-
and micromorphology. All isolates were segregated into
their species based on the description of Ellis (1971) and
Manamgoda et al. (2012) and compared with a reference
C. lunata isolate CX-3, which has high virulence and kindly
provided by Professor Chen Jie’s laboratory, Shanghai Jiao
Tong University, China. Conidial morphology is dened as
the percentage of conidia that are crescent-shaped on PDA
and observed on PDA, oatmeal agar (OA), and potato carrot
agar (PCA) media (Manamgoda 2015).
2.4. DNA extraction and PCR amplication
Two representative isolates (Table 1) and the reference
isolate CX-3 were inoculated on PDA and cultured for 3
days at 28°C in the dark. Then, small pieces of fungal
colonies were excised from the Petri dishes and transferred
into potato dextrose medium for further incubation at 28°C
with shaking at 180 r min–1 in the dark. Mycelia were
collected 3 days later and used to extract genomic DNA
by using hexadecyltrimethylammonium bromide (Stewart
and Vai 1993). The DNA fragments of ITS, GPDH, LSU,
and EF1-α were amplied in an automated thermal cycler
(SimpliAmp Thermal Cycler; Thermo Fisher Scientic Inc.,
USA). Primers ITS1 and ITS4 (Glass and Donaldson 1995)
were used to amplify the 5.8S and anking ITS regions. To
amplify the GPDH gene, primers gpd1 and gpd2 (Berbee
et al. 1999) were used. The EF1-α and LSU regions were
amplied using EF983F/2218R and LR5/LROR primers,
respectively (Schoch et al. 2009).
2.5. Sequence alignment and phylogenetic analysis
The sequences of ITS, GPDH, LSU, and EF1-α, which were
generated from 3 isolates, were deposited in GenBank
(Table 1) and were confirmed by performing BLASTn
algorithm-based searches at the NCBI website (https://
blast.ncbi.nlm.nih.gov). Multiple sequence alignment was
performed using the ClustalW program in MEGA5. The
sequences of the ITS region from these 3 isolates were
aligned with 25 related Curvularia isolates listed in Table 1,
and a concatenated four-locus dataset of each isolate
was further aligned with 21 reference isolates (Table 1).
For the phylogenetic analysis, the neighbor-joining tree
was generated using the aligned sequences with default
parameters and substitution models proposed by the
program. Alternaria alternata was regarded as the outgroup.
Statistical condence for the phylogeny tree was evaluated
by bootstrap method with 1
000 replicates (Tamura et al.
2011).
2.6. Clg2p and ClUrase gene expression in mycelia
and conidia
Real-time quantitative PCR (Q-PCR) was used to
analyze Clg2p and ClUrase gene expression in mycelia
and conidia as previously described by Liu et al. (2014,
2016). Primers 2F/2R (Liu et al. 2016) and CIUrase-F
(5´-TCGCTACGGCAAGGACAATGTTC-3´)/CIUrase-R
(5´-GGTGGTCTGCTTCTGCGTGTC-3´) were used for
Clg2p and ClUrase gene amplication, respectively. GAPDH
(Liu et al. 2016) was used as an endogenous control. The
relative quantitative transcripts were calculated using the
2−ΔΔCt method.
2.7. Pathogenicity assay
Pathogenicity assays of the representative C. lunata var.
isolates HNWB-131 and HNWB-185 were conducted on
maize inbred lines Chang 7-2, A3, Ye 478, Huangzao 4,
and Zheng 58. The puried isolates were cultured on PDA
for 5 days at 28°C in the dark and transferred to sterile corn
seed medium for 7 days. For inoculation, the conidia were
washed from the medium using sterile distilled water. Then,
the conidial suspensions (1×106 CFU mL–1 plus 0.1% (v/v)
Tween 20) were sprayed onto the leaves of healthy plants by
watering pot during the 7th-leaf stage, ~100 mL for one plant.
Three healthy plants sprayed with sterile distilled water
containing 0.1% (v/v) Tween 20 were used as the negative
controls. The experimental eld was under normal farming
management. The disease severity index was estimated
after 20 days from inoculation according to Liu et al. (1999).
The pathogens were re-separated and re-identied using
the above method for further determination.
3. Results
3.1. Occurrence and characteristics of Curvularia
leaf spot in Huanghuaihai Region
In the summer of 2013, maize Curvularia leaf spot was
observed in southeastern Henan Province, specically, to
the east of Suiping and to the south of Fugou and Minquan.
Among these areas, the most severe disease symptoms
were observed in Shangshui, Shenqiu, Xiayi and Zhecheng
counties. Once the disease severity index reached 56 to
76, severe leaf senescence occurred. The disease was
also observed in other places in Henan Province, but with
a lower severity (disease severity index was 15 to 40) and
less impact. Curvularia leaf spot was the main disease in
Anhui Province in 2013. The disease severity index was
between 76 and 93 in Xiao, Xutong, Mengcheng, Suntong,
and Suixi counties. The disease occurred in other places

554 CHANG Jia-ying et al. Journal of Integrative Agriculture 2020, 19(2): 551–560
Table 1 Details of isolates subjected to multi-gene DNA sequence analysis
Species Accession no. Host Location GenBank no. Reference
ITS GPDH 28sRNA EF1-α
Curvularia lunata CX-3 Zea mays China This study
C. lunata HNWB-131 Z. mays China KX100874 KX100869 KX100879 KX100864 This study
C. lunata HNWB-185 Z. mays China KX100875 KX100870 KX100880 KX100865 This study
Curvularia hawaiiensis comb.
nov
BRIP 109711) Chloris gayana Australia JN601030 JN600967 JN600989 JN601011 Manamgoda et al. (2011)
MFLUCC 10-07301) Panicum sp. Thailand JX256427 JX276439 Manamgoda et al. (2012)
BRIP 109721) C. gayana Australia JN192377 JN600968 JX256394 JN601012 Manamgoda et al. (2011)
Curvularia spicifera CBS 274.521) Soil Spain JN192387 Jn600979 JX256400 JN601023 Manamgoda et al. (2011)
C. spicifera CBS 198.31 Capsicum anuum Cyprus HF934916 Madrid et al. (2014)
Curvularia ellisii CBS 193.621) Air Pakistan JN192375 JN600963 JN600985 JN601007 Manamgoda et al. (2011)
Curvularia ovariicola comb. nov CBS 470.901) Eragrostis interrupta Australia JN192384 JN600976 JN600998 JN601020 Manamgoda et al. (2011)
BRIP 158821) JN601031 JN600971 JN600992 JN601015 Manamgoda et al. (2011)
Curvularia ravenelii comb. nov BRIP 131651) Sporobolus fertilis Australia JN192386 JN600978 JN601001 JN601024 Manamgoda et al. (2011)
Curvularia tripogonis comb. nov BRIP 123751) Unknown Australia JN192388 JN600980 JN601002 JN601025 Manamgoda et al. (2011)
Curvularia heteropogonis CBS 284.91 Heteropogon contortus Australia JN192379 JN600969 JN600990 JN601013 Manamgoda et al. (2011)
Curvularia gladioli ICMP 6160 Gladiolus sp. New Zealand JX256426 JX276438 JX256393 JX266595 Manamgoda et al. (2012)
Curvularia trifolii ICMP 6149 Setaria glauca New Zealand JX256434 JX276457 JX256402 JX266600 Manamgoda et al. (2012)
C. lunata MFLUCC 10-0706 Oryza sativa Thailand JX256431 JX276443 JX256398 JX266598 Manamgoda et al. (2012)
CBS 730.96 (Neo type) Human lung biopsy USA HF934911 Madrid et al. (2014);
Manamgoda et al. (2012)
CBS 730.96 (Neo type) Human lung biopsy USA JX256429 JX276441 JX256396 JX266596 Manamgoda et al. (2012)
CBS 157.34 Unknown Indonesia JX256430 JX276442 JX256397 JX266597 Manamgoda et al. (2012)
Curvularia alcornii MFLUCC 10-0703 Z. mays Thailand JX256420 JX276433 JX256387 JX266589 Manamgoda et al. (2012)
MFLUCC 10-0705 Panicum sp. Thailand JX256421 JX276434 JX256388 JX266590 Manamgoda et al. (2012)
Curvularia coicis CBS 192.29 Coix lacryma Japan AF081447 AF081410 JN600984 JN601006 Berbee et al. (1999);
Manamgoda et al. (2011)
Curvularia asianensis MFLUCC 10-0687 Oryza sativa Thailand JX256422 JX276435 JX256389 JX266591 Manamgoda et al. (2012)
MFLUCC 10-0711 Panicum sp. Thailand JX256424 JX276436 JX256391 JX266593 Manamgoda et al. (2012)
Curvularia sp.MFLUCC 10-0686 O. sativa Thailand JX256435 JX276446 JX256403 JX266601 Manamgoda et al. (2012)
Curvularia sp.MFLUCC 10-0690 O. sativa Thailand JX256437 JX276448 JX256405 JX266602 Manamgoda et al. (2012)
Curvularia senegalensis CBS 149.71 Substrate unknown Nigeria HG779001 Madrid et al. (2014)
Curvularia verruciformis CBS 537.75 Lobibyx sp. feather New Zealand HG779026 Madrid et al. (2014)
Curvularia gudauskasii DAOM 165085 AF071338 Berbee et al. (1999);
Manamgoda et al. (2012)
Curvularia ischaemi ICMP6172 Ischaemum indicum Solomon Islands JX256428 Manamgoda et al. (2012)
Cochliobolus cymbopogonis 88109-1 AF071351 Berbee et al. (1999);
Manamgoda et al. (2012)
Curvularia tuberculata CBS 146.63 Z. mays Rajasthan JX256433 Manamgoda et al. (2012)
Curvularia robusta CBS 624.68 Dichanthium annulatum leaf USA HG779000 Madrid et al. (2014)
Curvularia perotidis comb.nov CBS 350.90 Perotis rara Australia JN192385 Manamgoda et al. (2011)
Curvularia prasadii CBS 143.64 Jasminum sambac India HG778996 Madrid et al. (2014)
Curvularia graminicola BRIP 231861) Australia JN192376 JN600964 JN600986 JN601008 Manamgoda et al. (2011)
Alternaria alternata EGS 34.0160 AF071346 AF081400 Berbee et al. (1999);
Manamgoda et al. (2012)
1) Species transferred from Bipolaris to Curvularia.

555
CHANG Jia-ying et al. Journal of Integrative Agriculture 2020, 19(2): 551–560
in Anhui Province, with a disease severity index of 32 to 68.
Sporadic outbreaks occurred in a few areas of Shandong
and Hebei provinces.
In 2014–2015, the Curvularia leaf spot was not discovered
during the investigation of the corn planting area in Henan,
Anhui, Shandong, and Hebei provinces. In the summer
of 2016, maize Curvularia leaf spot was observed in the
south of Henan Province and the north of Anhui Province,
specically, in Shangqiu, Huaibei, and Suzhou cities. Once
the disease severity index reached 56 to 89, severe leaf
senescence occurred. Sporadic outbreaks (disease severity
index was 18) occurred in a few areas of Shandong, Hebei,
and the north of Henan provinces.
During the summer of 2017, maize Curvularia leaf spot
was observed in north Anhui Province, specifically, in
Bozhou, Suzhou, and Huaibei cities. Among these areas,
the most severe disease symptoms were observed in
Mengcheng, Lingbi, and Suixi counties. Once the disease
indices reached to 25 to 76, severe leaf senescence
occurred. Sporadic outbreaks occurred in a few areas of
Shandong, Hebei, and Henan provinces.
This symptom of bigger spots, which were oval with
an irregular edge, and the leaves often withered because
of the coalescing spots (Fig. 1), occurred distinctly and
generally in Zhumadian, Zhoukou, and Shangqiu cities in
Henan Province, as well as less frequently in other places
in Henan Province. This symptom occurred in almost the
whole of Anhui Province, with the most affected area being
Bozhou, Suzhou, and Huaibei cities.
3.2. Morphological identication of C. lunata isolates
Single spores were separated and puried from diseased
leaves collected from Henan, Hebei, Shandong, and Anhui
provinces of China in 2013 and 2016–2017. In 2013, 142
C. lunata isolates were separated, of which approximately
80.99% isolates have about 70% uncurved conidia. In
2016–2017, 41 and 109 C. lunata isolates were separated,
and approximately 73.17 and 79.82% isolates have about
70% uncurved conidia, respectively.
After the pathogenicity pre-experiment, we chose
2 representative isolates HNWB-131 (about 92.00%
uncurved conidia) and HNWB-185 (about 93.14%
uncurved conidia) to be further compared with a reference
C. lunata isolate CX-3 and found that they differed in
macro- and micromorphologies (Fig. 2-A and B; Table 2).
The main difference was that the conidia size of HNWB-
131 and HNWB-185 were smaller than that of CX-3, and
most conidia of the 2 isolates were oblong with parallel
septations, whereas those of CX-3 were bent. Base on this
result, we suggest that the isolated fungus is a C. lunata
variety.
3.3. Sequence analysis of C. lunata var. isolates
Sequences of ITS region, GPDH, LSU, and EF1-α were
amplied from genomic DNA of Curvularia isolates HNWB-
131, HNWB-185, and CX-3, and the fragments with lengths
of approximately 500, 500, 800, and 900 bp were obtained
for the 4 genes, respectively. The BLAST algorithm-based
analysis demonstrated that the 4-gene sequences from the
3 isolates all had a similarity of more than 99% to C. lunata
in GenBank, supporting the morphological identication of
these isolates in this study. Based on the sequences of
the ITS region, we constructed a neighbor-joining tree to
determine the phylogenetic similarities of the 25 Curvularia
isolates (Fig. 3-A). The ITS sequences of HNWB-131,
HNWB-185, and CX-3 were clustered together with C. lunata
and had a close genetic relationship with C. hawaiiensis. A
further phylogenetic analysis was carried out by combining
the data from ITS, GPDH, LSU, and EF1-α, and we found
that these 3 isolates were also in the same cluster as
C. lunata (Fig. 3-B).
3.4. Expression analysis of Clg2p and ClUrase genes
in mycelia and conidia
To investigate the Clg2p and ClUrase gene expression
patterns in the mycelia and conidia of different isolates, total
RNA was extracted from the vegetative growth mycelium and
subjected to real-time PCR analysis. The Q-PCR analysis
A B
C D
Fig. 1 Images of maize Curvularia leaf spots. A and B,
connected large lesions. C and D, typical lesions.

556 CHANG Jia-ying et al. Journal of Integrative Agriculture 2020, 19(2): 551–560
indicated that the expression level of Clg2p and ClUrase
genes were significantly (P<0.05) different in different
isolates. The expression was signicantly higher in the
mycelia and conidia of CX-3 than the other isolates in this
study, indicating that the expression of Clg2p and ClUrase
correlates with the changes in conidial morphology of the
isolates (Fig. 4-A and B).
3.5. Pathogenicity of C. lunata var.
The pathogenicity of C. lunata var. isolates HNWB-131
and HNWB-185 were tested by using the maize inbred
lines Chang 7-2, A3, Ye 478, Huangzao 4, and Zheng 58
that were growing in the eld. The result showed that the
isolates were able to infect maize leaves and led to typical
symptoms similar to those observed in the eld. Twenty
days after inoculation, typical symptoms were detected on
the leaves inoculated with CX-3, HNWB-131, and HNWB-
185, and the pathogenicity difference between HNWB-131
and HNWB-185 were obvious (Table 3 and Fig. 5). The
pathogenicity of HNWB-185 was similar to that of the
control strain CX-3, with no signicant difference; the
highest disease index was found in Huangzao 4, 82 and
80, respectively. HNWB-131 was signicantly different
from HNWB-185 and control strain CX-3, and its disease
index was 12–22 on different maize varieties. The negative
control did not show any disease symptoms. Subsequently,
the same fungus was suc-cessfully re-isolated and re-
identied from the diseased leaves following the above
methods, but not from the healthy controls, thereby
satisfying Koch’s postulates. The isolate was shown to be
the causal agent for the maize Curvularia leaf spot.
4. Discussion
According to our recent study, we investigated the
occurrence of maize Curvularia leaf spot in Henan, Hebei,
Shandong, and Anhui provinces of China in 2013 and
2016–2017. The disease severity index of this disease
was generally recorded at 56 to 93 in Henan and Anhui
a
h
g
f
e
d
c
b
A
h
g
f
e
d
c
b
a
B
20 μm
20 μm 20 μm
20 μm
20 μm
20 μm
20 μm
20 μm
Fig. 2 Morphological identication. A, Curvularia lunata CX-3. B, C. lunata var. HNWB-185. a–c, colonies on PDA, OA, and PCA
media, respectively, at 28°C after 5 days; d–h, conidiophores and conidia. Scale bars are 20 µm.
Table 2 Colonial and conidia morphologies of Curvularia lunata CX-3 and C. lunata var. HNWB-131 and HNWB-185
C. lunata CX-3 C. lunata var. HNWB-131 and HNWB-185
Colonial White on the edge, black in the middle, black in the back,
no aerial mycelium and water soluble pigment
White on the edge, light to dark brown in the middle,
black in the back, ocky mycelium, no water soluble
pigment
Conidiophores Grow singly or in a cluster, brown, unbranched, septate,
produced apically in a sympodial manner, obvious scar
Grow singly or in a cluster, brown, unbranched, septate,
produced apically in a sympodial manner, obvious scar
Conidia morphology Circle triangle, bent Spindle, unbending
Conidia color Dark brown, light color at both ends Brown, light color at both ends
Cell morphology Quite expanded at the third cell, smaller at both ends A little expanded at the third cell, smaller at both ends
Conidia size (μm) (17.9–33.1)×(8.3–15.6) (16.3–33.0)×(3.0–11.7)
Septation 3-Septate, unparallel 3-Septate, parallel
Hilum Scarce protuberant hilum Scarce protuberant hilum

557
CHANG Jia-ying et al. Journal of Integrative Agriculture 2020, 19(2): 551–560
provinces, which indicated that the damage degree of this
disease was more severe than that in previous years. In
the survey, in addition to the typical Curvularia spots on
individual maize cultivars, irregular oval lesions formed
by the fusion of several typical spots were observed on
common cultivar leaves in most elds. This change in the
type of lesion was evident and might be the reason for the
aggravation of the disease.
Identication of the dominant aggressive species causing
maize Curvularia leaf spot was done using morphological
methods, and these species were nally determined to be
C. lunata but had morphological characteristics inconsistent
with previous studies (Shi et al. 2000; Dou et al. 2007;
Liu et al. 2008; Li 2013). Morphological characteristics,
such as spore shape, size, color, septation, and colony
characteristics in traditional classications can be variable,
which may reect the inuences of environmental factors
(Dou et al. 2006; Dou and Jin 2007; Yan et al. 2009). Most
of the morphological characters of Curvularia spp. are
0
0.2
0.4
0.6
0.8
1.0
1.2
Clg2p CIUrase
Relative expression
0
0.2
0.4
0.6
0.8
1.0
1.2
Clg2p CIUrase
Relative expression
CX-3 HNWB-131 HNWB-185
A B
Fig. 4 Expression patterns of Clg2p and CIUrase by real-time quantitative PCR (Q-PCR) analysis. A, mycelial growth in potato
dextrose (PD) medium for 3 days at 28°C in the dark. B, conidia collected from 7-day culture on PDA plates at 28°C in the dark.
Data are mean±SE (n=3).
Curvularia asianensis MFLUCC 10-0687
Curvularia senegalensis CBS149.71
Curvularia tripogonis BRIP 12375
a
Curvularia verruciformis CBS537.75
Curvularia ravenelii CBS537.75
Curvularia gudauskasii DAOM165085
Cochliobolus cymbopogonis 88109-1
Curvularia ovariicola CBS 470.90
a
Curvularia coicis (Cochliobolus nisikadoi) CBS 192.29
Curvularia trifolii ICMP 6149
Curvularia gladioli ICMP 6160
Curvularia ischaemi ICMP6172
Curvularia alcornii MFLUCC 10-0703
Curvularia robusta CBS 624.68
Curvularia heteropogonis CBS 284.91
Curvularia perotidis CBS 350.90
Curvularia ellisii CBS 193.62
a
Curvularia spicifera CBS 274.52
a
Curvularia spicifera CBS198.31
Curvularia hawaiiensis MFLUCC10-0730
Curvularia lunata HNWB-131 (This study)
Curvularia lunata HNWB-185 (This study)
Curvularia lunata CBS 730.96 (Neo type)
Curvularia lunata CX-3 (This study)
Curvularia lunata CBS 730.96 (Neo type)
Curvularia Prasadii
CBS 143.64
Curvularia graminicola BRIP23186
a
Curvularia tuberculata CBS 146.63
Curvularia sp. MFLUCC10-0686
Alternaria alternata EGS 34.0160
61
100
95
97
76
58
66
36
19
51
47
69
28
31
59
30
28
29
16
10
4626
8
9
0.01
Cochliobolus hawaiiensis BRIP 10971
a
Curvularia hawaiiensis BRIP 10972
a
Curvularia spicifera CBS 274.52
a
Curvularia ellisii CBS 193.62
a
Curvularia ovariicola CBS 470.90
a
Cochliobolus ovariicola BRIP 15882
a
Curvularia ravenelii BRIP 13165
a
Curvularia heteropogonis CBS 284.91
Curvularia gladioli ICMP 6160
Curvularia trifolii ICMP 6149
Curvularia tripogonis BRIP 12375
a
Curvularia coicis CBS 192.29
Curvularia asianensis MFLUCC 10-0687
Curvularia asianensis MFLUCC 10-0711
Curvularia alcornii MFLUCC 10-0703
Curvularia alcornii MFLUCC 10-0705
Curvularia lunata MFLUCC 10-0706
Curvularia lunata CBS 730.96 (Neo type)
Curvularia lunata CBS 157.34
Curvularia lunata HNWB-185 (This study)
Curvularia lunata CX-3 (This study)
Curvularia lunata HNWB-131 (This study)
Curvularia sp.MFLUCC10-0686
Curvularia sp. MFLUCC 10-0690
Alternaria alternata EGS 34.0160
100
88
99
100
100
100
100
97
76
66
78
41
20
43
27
0.000.010.020.030.040.05
BA
Fig. 3 Molecular biological identication. A, the phylogenetic tree of internal transcribed spacer (ITS) sequences from Curvularia
lunata and related species. B, the phylogenetic tree based on the combined ITS, GPDH, LSU, and EF1-α sequence data. The tree
is rooted with Alternaria alternata. Bootstrap values of more than 40 are shown in the tree. a, species transferred from Bipolaris
to Curvularia.
Table 3 Disease severity index caused by different isolates on
different maize inbred lines
Isolate Disease severity index
Ye 478 Huangzao 4 Chang 7-2 A3 Zheng 58
HNWB-131 16 22 16 20 12
HNWB-185 64 82 56 64 64
CX-3 68 80 62 68 66
CK 0 0 0 0 0

558 CHANG Jia-ying et al. Journal of Integrative Agriculture 2020, 19(2): 551–560
CX-3 CX-3HNWB-131 HNWB-131HNWB-185 HNWB-185
A3 Chang 7-2
Fig. 5 Pathogenicity analysis of Curvularia lunata var. Disease symptoms on maize inbred line A3 and Chang 7-2 leaves sprayed
with conidial suspensions of CX-3, HNWB-131 and HNWB-185.
overlapping. The differences between morphologically
dened species are based on subtle characters that may
vary depending on culture conditions, life modes, and
hosts (Manamgoda 2015). In the present study, we found
that the representative C. lunata var. isolates HNWB-131
and HNWB-185 had different properties, such as colony
morphology, spore color, and conidial production, when
cultured on PDA and even showed sector mutations,
indicating that a high level of genetic diversity exists in
C. lunata. The morphogenesis of C. lunata during the
evolutionary process was affected by various factors and
was unstable. It is incorrect, doubtful, or remain ambiguous
to identify species on the basis of morphology (Revankar and
Sutton 2010; Vermeire et al. 2010; da Cunha et al. 2013).
Several recent studies have shown that the morphological
identication of Curvularia spp. do not always correlate
with phylogenetic species recognition (Yanagihara et al.
2010; Manamgoda et al. 2012; da Cunha et al. 2013).
In summary, molecular identication is highly important
for species identification. Furthermore, strengthening
morphological identication for morphologically mutable
strains is necessary and conducive to the separation of
special morphological variants.
Based on these reasons, traditional classications should
be combined with molecular methods to identify Curvularia
spp. Manamgoda et al. (2012) classified Curvularia,
Bipolaris, and Cochliobolus and analyzed their phylogenies
using ITS, GPDH, LSU, and EF1-α sequences. Madrid
et al. (2014) identied new species using morphology and
molecular biology. Among the molecular biology methods,
they established a phylogenetic tree using ITS, GPD, LSU,
and RPB2 sequences and then carried out a systematic
analysis. Here, the classication of HNWB-131 and HNWB-
185 was further determined comprehensively and accurately
by combining the two methods. The results were consistent
with the morphological results.
Liu et al. (2016) have shown that the Clg2p gene in
C. lunata is similar to the Ras gene in other phytopathogenic
fungi. Their results suggested that Clg2p interact with
Clf to regulate appressorium formation, morphology, and
pathogenicity. However, Clg2p interacted with ClUrase
to regulate conidial morphology without affecting the
pathogenicity of C. lunata. In our study, the morphology
of the conidia of C. lunata var. was changed, and the
expression of Clg2p and ClUrase genes in C. lunata var. was
lower than that of CX-3 in the same condition. Furthermore,
no significant change in pathogenicity was observed.
Therefore, the morphological variations of C. lunata var. in
this study may be related to the expression of Clg2p and
ClUrase genes.
The pathogenicity of C. lunata var. demonstrated to be
the causal agent of Curvularia leaf spot and might be a
potential threat to maize production. However, there were
differences in pathogenicity between strains, which might be
due to the selectivity of different host varieties and agents.
We performed ISSR-PCR analysis on the isolates and
found that there were some differences in genetic diversity
among the years, but no signicant differences among
geographical populations. Meanwhile, according to the
results of overwintering test, we speculated that C. lunata in
this area were not mainly from local sources but mainly from
maize growing areas in southern China or Southeast Asia
(Chang et al. 2019). Therefore, the variation of pathogens
was caused by various radiations during high-altitude drift.
In the future, we will continue to monitor the occurrence
of this type of C. lunata and determine its host biotypes and

559
CHANG Jia-ying et al. Journal of Integrative Agriculture 2020, 19(2): 551–560
resistance levels, which will provide a more comprehensive
understanding of this pathogen, and it will be tested to nd
out whether there is any new drug target site to control
pathogens. Furthermore, we will resequence the C. lunata
var. genomes to elucidate the mechanism of genetic
evolution and pathogenesis.
5. Conclusion
This study found that the dominant pathogen of Curvularia
leaf spot was a new variety of C. lunata with morphological
variations in Huanghuaihai Region of China in 2013 and
2016–2017. The pathogenicity of the C. lunata var. was
not signicantly enhanced, and the expression of Clg2p and
ClUrase genes of C. lunata var. were decreased.
Acknowledgements
This work was supported by the earmarked fund for the
China Agriculture Research System (CARS-02) and the
National Key Research and Development Program of China
(2017YFD0200400). We thank Prof. Yang Wenxiang,
College of Plant Protection, Agricultural University of Hebei/
Biological Control Center of Plant Diseases and Plant Pests
of Hebei Province) for critical reading of the manuscript and
useful discussions.
Appendix associated with this paper can be available on
http://www.ChinaAgriSci.com/V2/En/appendix.htm
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