Curvularia microspora sp. nov. associated with leaf diseases of Hippeastrum striatum in China

Abstract

An undescribed Curvularia sp. was isolated from the leaf spot disease of Barbados Lily (Hippeastrum striatum (Lam.) Moore). Phylogenetic analyses of combined ITS, 28S, GPD1 and TEF1 sequence data place nine strains of this species in the trifolii-clade, but they clustered together as an independent lineage with strong support. This species was morphologically compared with related species in the trifolii-clade. Based on differences in morphology and phylogeny, it is concluded that this species is a new taxon, introduced as Curvularia microspora sp. nov. Pathogenicity testing determined the new species to be pathogenic on H. striatum.

Full text

Curvularia microspora sp. nov. associated with leaf diseases of Hippeastrum striatum... 49
Curvularia microspora sp. nov. associated with leaf
diseases of Hippeastrum striatum in China
Yin Liang1, Shuang-Fei Ran1, Jayarama Bhat4, Kevin D. Hyde5,
Yong Wang1,2, De-Gang Zhao2,3
1 Department of Plant Pathology, College of Agriculture, Guizhou University, Guiyang, Guizhou 550025,
China 2 Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region,
Ministry of Education, Guizhou University, Guiyang, 550025, P. R. China 3 Guizhou Academy of Agri-
cultural Sciences, Guiyang 550006, China 4 No. 128/1–J, Azad Housing Society, Curca, Goa Velha, India
5Centre of Excellence in Fungal Research and School of Science, Mae Fah Luang University, Chiang Rai,
57100, ailand
Corresponding authors: Yong Wang (yongwangbis@aliyun.com); De-Gang Zhao (dgzhao@gzu.edu.cn)
Academic editor: C.L. Schoch|Received25 September 2017|Accepted 2 January 2018|Published 18 January2018
Citation: Liang Y, Ran S-F, Bhat J, Hyde KD, Wang Y, Zhao D-G (2018) Curvularia microspora sp. nov. associated with
leaf diseases of Hippeastrum striatum in China. MycoKeys 29: 49–61. https://doi.org/10.3897/mycokeys.29.21122
Abstract
An undescribed Curvularia sp. was isolated from the leaf spot disease of Barbados Lily (Hippeastrum striatum
(Lam.) Moore). Phylogenetic analyses of combined ITS, 28S, GPD1 and TEF1 sequence data place nine
strains of this species in the trifolii-clade, but they clustered together as an independent lineage with strong
support. is species was morphologically compared with related species in the trifolii-clade. Based on die-
rences in morphology and phylogeny, it is concluded that this species is a new taxon, introduced as Curvularia
microspora sp. nov. Pathogenicity testing determined the new species to be pathogenic on H. striatum.
Keywords
China, hyphomycetes, identify, pathogen, taxonomy
Introduction
e genus Curvularia includes pathogens and saprobes of various plants, as well as
opportunistic pathogens of humans and animals (Sivanesan 1987, Manamgoda et al.
2011, 2012, da Cunha et al. 2013, Hyde et al. 2014) and has been well-studied in
Copyright Yin Liang et al. 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.
MycoKeys 29: 49–61 (2018)
doi: 10.3897/mycokeys.29.21122
http://mycokeys.pensoft.net
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Launched to accelerate biodiversity research
RESEARCH ARTICLE

Yin Liang et al. / MycoKeys 29: 49–61 (2018)
50
recent years. Identication of Curvularia spp. was previously mainly based on mor-
phological descriptions and comparisons, however, the use of molecular taxonomy has
solved many problems of resolving species (Valente et al. 1999, Mendoza et al. 2001).
A multi-gene phylogenetic tree, based on the internal transcribed spacers including
the 5.8S nuclear ribosomal DNA gene (ITS), the 5’ end of the nuclear ribosomal
large subunit (28S), fragments of the glycerol-3-phosphate dehydrogenase (GPD1)
and translational elongation factor EF-1 alpha (TEF1) gene regions, was provided to
identify fresh collections of Curvularia from various hosts and geographic locations
worldwide (Manamgoda et al. 2015).
In this study, DNA sequences of ITS, 28S, GPD1 and TEF1 gene regions were
used for phylogenetic analyses to identify a new Curvularia species. is was conclud-
ed based on the combined morphology and phylogeny. Curvularia microspora sp. nov.,
is introduced here, associated with leaf diseases of Hippeastrum striatum.
Materials and methods
Isolation and morphological studies
All diseased samples were collected from the Medical Plants Herb Garden, in Chong-
qing City, Nanchuan County, China. is garden is located in a region of subtropical
humid monsoon climate and has conserved more than 3000 kinds of medicinal plants.
In this study, all fungal strains were isolated by the single-spore technique in order to
obtain pure cultures following the method of Chomnunti et al. (2014). Single spores
were transferred to potato-dextrose agar (PDA) and incubated at room temperature
(28 °C). After several weeks of incubation, the morphological characters were recorded
following the methods of Manamgoda et al. (2011, 2012). Conidia and conidiophores
were observed using a compound microscope (Nikon Eclipse E600 DIC microscope
and a Nikon DS-U2 camera or a Nikon 80i compound microscope tted with a Canon
450D digital camera). e holotype specimen was deposited in the Herbarium of the
Department of Plant Pathology, Agricultural College, Guizhou University (HGUP).
Ex-type cultures were also deposited in the culture collection at the Department of
Plant Pathology, Agriculture College, Guizhou University, P.R. China (GUCC).
DNA extraction and sequencing
Fungal cultures were grown on PDA until nearly covering the whole Petri-dish
(90mm) at 28 °C. Fresh fungal mycelia were scraped with sterilised scalpels. A BI-
OMIGA Fungus Genomic DNA Extraction Kit (GD2416) was used to extract fungal
genome 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
and 0.25 μL Taq DNA polymerase (Promega, Madison, WI, USA). Primers ITS4 and
ITS5 (White et al. 1990) were used to amplify the ITS region. e thermal cycling

Curvularia microspora sp. nov. associated with leaf diseases of Hippeastrum striatum... 51
programme was: 3 min initial denaturation at 95 °C, followed by 30 cycles of 30 s
denaturation at 94 °C, 30 s primers annealing at 52 °C, 1 min extension at 72 °C and
a total 10 min extension at 72 °C. To amplify the GPD1 gene, the primers gpd1 and
gpd2 were used (Berbee et al. 1999). e amplication programme included an initial
denaturation step at 96 °C for 2 min, followed by 35 PCR cycles with 1 min at 96 °C,
1 min at 52 °C and 45 s at 72 °C with a nal 10 min extension at 72 °C. e TEF1 and
28S regions were amplied using EF-526F/1567R and LR5/LROR primers respec-
tively (Schoch et al. 2009). e 28S amplication programme included an initial de-
naturation step at 95 °C for 3 min followed by 30 cycles of 40 s denaturation at 94 °C,
50 s primer annealing at 52 °C, 1 min extension at 72 °C. e same PCR reaction was
used to amplify TEF1 with the only change being the annealing temperature at 54 °C.
Phylogenetic analysis
DNA sequences from these isolates and reference sequences were downloaded from
GenBank and analysed by maximum parsimony (MP) and maximum likelihood (ML)
(Table 1). Sequences were optimised manually to allow maximum alignment and
maximum sequence similarity, as detailed in Manamgoda et al. (2012). e align-
ment document of four phylogenetic markers has been submitted to TreeBase (https://
treebase.org/; Accession number: 21970). A partition homogeneity test (PHT) was
performed with 1000 replicates via PAUP v. 4.0b10 (Swoord 2003) to evaluate sta-
tistical congruence amongst sequence data of 28S, ITS, GPD1 and TEF1 gene regions.
MP analyses were performed in PAUP v. 4.0b10 (Swoord 2003), using the heuristic
search option with 1,000 random taxa addition and tree bisection and reconnection
(TBR) as the branch swapping algorithm. Maxtrees were set to 10,000. e characters
in the alignment document were ordered accordingly: 28S+ITS+GPD1+TEF1, with
equal weight and gaps were treated as missing data. e Tree Length (TL), Consistency
Indices (CI), Retention Indices (RI), Rescaled Consistency Indices (RC) and Homo-
plasy Index (HI) were calculated for each tree generated. Maximum likelihood (ML)
trees of DNA sequences were obtained by a heuristic search using the TrN + I + G
model, which was deduced as the best t for the data by the likelihood ratio test using
the MODELTEST wer3.7 and MrMTgui version 1.01 (Posada and Crandall 1998).
Pathogenicity test
Pathogenicity of this species was determined by inoculating healthy leaves of Hippeas-
trum striatum and Canna indica L. with 5 mm diameter mycelial plugs, cut from the
margins of 10-day-old actively growing cultures; the control was treated with sterile
agar plugs. Both inoculated and control plants were kept in a moist chamber at 25°C
for 7 days and observed for disease symptom development. Infected leaves were col-
lected and the fungus was re-isolated in PDA medium and compared against the origi-
nal strains. Control plants were sprayed with sterilised distilled water.

Yin Liang et al. / MycoKeys 29: 49–61 (2018)
52
Table 1. GenBank accession numbers of isolates include in this study.
Species Isolate GenBank accesssion numbers and references
ITS 28S GPD1 TEF1
Alternaria alternata EGS 34.0160 AF071346 Berbee et al. 1999 – – AF081400 Berbee et al. 1999 – –
Curvularia akaii CBS 318.86 HF934921 Amaradasa et al. 2014 – – HG779118 Madrid et al. 2014 – –
C. borreriae CBS 859.73 HE861848 da Cunha etal. 2013 – – HF565455 da Cunha et al. 2013 – –
C. borreriae MFLUCC
11-0442 KP400638 Manamgoda et al.
2015 – – KP419987 Manamgoda et al.
2015 – –
C. gladioli ICMP 6160 JX256426 Manamgoda et al.
2012 JX256393 Manamgoda et al. 2012 JX276438 Manamgoda et al.
2012 JX266595 Manamgoda et al. 2012
C. gudauskasil DAOM 165085 AF071338 Berbee et al. 1999 – – AF081393 Berbee et al. 1999 – –
C. heteropogonis CBS 284.91 JN192379 Manamgoda et al.
2011 JN600990 Manamgoda et al. 2011 JN600969 Manamgoda et al.
2011 JN601013 Manamgoda et al. 2011
C. ovariicola BRIP 15882 JN192384 Manamgoda et al.
2011 JN600992 Manamgoda et al. 2011 JN600971 Manamgoda et al.
2011 JN601020 Manamgoda et al. 2011
C. pallescens CBS 156.35 KJ922380 Manamgoda et al.
2014 KM243269 Manamgoda et al. 2014 KM083606 Manamgoda et al.
2014 KM196570 Manamgoda et al. 2014
C. ravenelii BRIP 13165 JN192386 Manamgoda et al.
2011 JN601001 Manamgoda et al. 2011 JN600978 Manamgoda et al.
2011 JN601024 Manamgoda et al. 2011
C. trifolii AR5169 KP400656 Manamgoda et al.
2015 – – KP645345 Manamgoda et al.
2015 KP735694 Manamgoda et al. 2015
C. trifolii ICMP 6149 JX256434 Manamgoda et al.
2012 JX256402 Manamgoda et al. 2012 JX276457 Manamgoda et al.
2012 JX266600 Manamgoda et al. 2012
C. tripogonis BRIP 12375 JN192388 Manamgoda et al.
2011 JN601002 Manamgoda et al. 2011 JN600980 Manamgoda et al.
2011 JN601025 Manamgoda et al. 2011
Curvularia sp. ICMP 10344 JX256444 Manamgoda et al.
2012 – – JX276455 Manamgoda et al.
2012 – –
Curvularia sp. ICMP 13910 JX256445 Manamgoda et al.
2012 – – JX276456 Manamgoda et al.
2012 – –
C. microspora sp.nov GUCC 6272 MF139088 This study MF139106 This study MF139097 This study MF139115 This study
C. microspora sp. nov GUCC 6273 MF139089 This study MF139107 This study MF139098 This study MF139116 This study
C. microspora sp. nov GUCC 6274 MF139090 This study MF139108 This study MF139099 This study MF139117 This study
C. microspora sp. nov GUCC 6275 MF139091 This study MF139109 This study MF139100 This study MF139118 This study
C. microspora sp. nov GUCC 6276 MF139092 This study MF139110 This study MF139101 This study MF139119 This study
C. microspora sp. nov GUCC 6277 MF139093 This study MF139111 This study MF139102 This study MF139120 This study
C. microspora sp. nov GUCC 6278 MF139094 This study MF139112 This study MF139103 This study MF139121 This study
C. microspora sp. nov GUCC 6279 MF139095 This study MF139113 This study MF139104 This study MF139122 This study
C. microspora sp. nov GUCC 6280 MF139096 This study MF139114 This study MF139105 This study MF139123 This study

Curvularia microspora sp. nov. associated with leaf diseases of Hippeastrum striatum... 53
Results
Phylogenetic analyses
Nine isolates of Curvularia were sequenced from two plants in Chongqing Munici-
pality, China (seven from Hippeastrum striatum and two from Canna indica). PCR
products of approximately 900 bp (28S), 540 bp (ITS), 530 bp (GPD1) and 1200
bp (TEF1) were obtained. In the molecular phylogenetic analyses, the partition ho-
mogeneity test (P = 0.06) indicated that the individual partitions were not highly
incongruent (Cunningham 1997) and thus 28S, ITS, GPD1 and TEF1 sequences
were combined for sequence analyses. By alignment with a single gene region and then
combination according to the order of 28S, ITS, GPD1 and TEF1, only 2689 charac-
ters were obtained, viz. 28S: 1–848, ITS: 849–1330, GPD1: 1331–1771 TEF1: 1772–
2689 with 104 parsimony-informative characters and 157 parsimony-uninformative
characters. e analysis produced three equally parsimonious trees, one of which (TL
= 366, CI = 0.81, RI = 0.82, RC = 0.66 and HI = 0.19) is shown in Figure 1 and the
topologies of MP and ML analysis were congruent, thus only MP topology was shown.
Phylogenetic analysis conrmed nine strains (GUCC 6272, GUCC 6273, GUCC
6274, GUCC 6275, GUCC 6276, GUCC 6277, GUCC 6278, GUCC 6279 and
GUCC 6280) with the same DNA sequences in four phylogenetic markers grouped
into an independent clade supported by high bootstrap values (MP: 100%; ML: 99%).
ese strains were placed in trifolii-clade with strong bootstrap support (MP: 95%;
ML: 95%) and had a close relationship with Curvularia gaudauskasii, C. gladioli, C.
trifolii, C. borreriae and C. pallescens with a high MP support (MP: 87%), but its ML
bootstrap value was lower than 50%.
Taxonomy
Curvularia microspora Y. Liang, K.D. Hyde, J. Bhat & Yong Wang, sp. nov.
MycoBank MB 822544
Figure 2
Diagnosis. Characterised by producing four celled, smaller conidia (4.5–11.5 ×
2–6μm), usually curved at the third cell from the base.
Type. China, Chongqing City, Nanchuan, from leaf spots of Hippeastrum stria-
tum, 28 September 2016, Y. Liang, HGUP 6272, holotype, ex-type living culture
GUCC 6272.
Description. Symptoms on Hippeastrum striatum: Fructication mostly epiphyl-
lous, disease spot 3–12 mm, subspherical to oblong ovate, brown to dark brown, euse
(Figure 2a, b). Symptoms on Canna indica: Fructication of the fungus was mostly
epiphyllous, the large blighted, irregular spots near leaf apex to the whole leaves, grey-
ish-brown (Figure 2c).

Yin Liang et al. / MycoKeys 29: 49–61 (2018)
54
Figure 1. e only one parsimonious tree obtained from combined analyses set of ITS, LSU,β-tubulin
and tef1 sequence data. MP values (>50 %) resulting from 1000 bootstrap replicates. e tree is rooted
with Alternaria alternata (EGS 34-0160). e branch of our new Curvularia is shown in blue.

Curvularia microspora sp. nov. associated with leaf diseases of Hippeastrum striatum... 55
Figure 2. Curvularia microspora (HGUP 6272). a–c Leaf diseases symptoms on Hippeastrum rutilum and
Canna indica. d–f Conidiophores, conidiogenous loci and conidia g–j Immature and mature conidia k–l
Upper (k) and lower (l) surface of colony. Scar bars: d, i (10 μm), e–f = 20μm, g–h, j = (5 μm).
Colonies on PDA, vegetative hyphae septate, branched, subhyaline to brown,
smooth to asperulate, 1.5–3 μm, anastomosing. Sexual morph: Undetermined. Asexual
morph: Hyphomycetous. Conidiophores 10.5–77.5 × 1–3.5 μm (av. = 22.2 × 2.1μm,
n = 30), arising singly, simple or branched, exuous, septate, geniculate at spore bearing
part, pale brown, dark brown, paler towards apex. Percurrent proliferation only observed
occasionally. Conidiogenous loci somewhat thickened and darkened, spores up to 0.8–1
μm diam, smooth. Mature conidia always four celled, 4.5–11.5 × 2–6 μm (av. = 8.2 ×
3.8 μm, n = 50), smooth-walled, usually curved at the third cell from the base, sometimes
straight, navicular, bifurcate, obpyriform, tapering towards rounded ends, pale brown to
dark reddish brown. Hilum usually conspicuous or sometimes slightly protuberant.

Yin Liang et al. / MycoKeys 29: 49–61 (2018)
56
Habitat and distribution. Isolated from leaf diseases of H. striatum and Canna
indica in China
Etymology. microspora, referring to this species producing obviously smaller conidia.
Other material examined. China, Chongqing City, Nanchuan, from leaf diseases
of H. striatum, 28 September 2016, Y. Liang (HGUP 6273), living culture GUCC
6273; China, Chongqing City, Nanchuan, from leaf diseases of H. striatum, 28 Sep-
tember 2016, Y. Liang (HGUP 6274), living culture GUCC 6274; China, Chong-
qing City, Nanchuan, from leaf diseases of H. striatum, 28 September 2016, Y. Liang
(HGUP 6275), living culture GUCC 6275; China, Chongqing City, Nanchuan, from
leaf diseases of H. striatum, 28 September 2016, Y. Liang (HGUP 6276), living culture
GUCC 6276; China, Chongqing City, Nanchuan, from leaf diseases of H. striatum, 28
September 2016, Y. Liang (HGUP 6277), living culture GUCC 6277; China, Chong-
qing City, Nanchuan, from leaf diseases of H. striatum, 28 September 2016, Y. Liang
(HGUP 6278), living culture GUCC 6278; China, Chongqing City, Nanchuan, from
leaf diseases of Canna indica, 28 September 2016, Y. Liang (HGUP 6279), living cul-
ture GUCC 6279; China, Chongqing City, Nanchuan, from leaf diseases of C. indica,
28 September 2016, Y. Liang (HGUP 6280), living culture GUCC 6280.
Pathogenicity test
Test plants (Hippeastrum striatum) were inoculated with 5 mm diam mycelial plugs
of Curvularia microspora with two replicates of each plants and the inoculation ex-
periment was repeated two times (with dierent sporulation generations). Hippeastrum
striatum leaves both exhibited brown to dark brown necrotic spots (Figure 3a, b) after
7 days, which were very similar to those of natural infection (Figure 2a, b). e DNA
sequencing result (ITS region), after re-isolation, identied this as C. microspora. e
successful re-isolation of C. microspora from the inoculated leaves of H, striatum estab-
lished a credible proof of pathogenicity. All test plants were covered with polyethylene
bags for 7 days. However, on Canna indica, disease symptoms did not appear again.
Discussion
e nine strains of Curvularia had typical characters of the genus., viz. the produc-
tion of sympodial conidiophores with tretic, terminal and intercalary conidiogenous
cells and elongate, transversely septate conidia with a dark basal scar (Boedijn 1933).
Phylogenetic analyses compared the DNA sequence from four phylogenetic mark-
ers with related species in the trifolii-clade: Curvularia akali, C. borreriae, C. gladioli,
C. gaudauskasii, C. heteropogonis, C. pallescens and C. trifolii (Figure 1, Manamgoda
et al. 2012, 2015, Madrid et al. 2014, Jeong et al. 2015, Su et al. 2015). ese taxa
are morphologically similar in producing a strongly protruding hilum (Madrid et al.
2014). However, the present taxon had bifurcate conidia, which dierentiates it from

Curvularia microspora sp. nov. associated with leaf diseases of Hippeastrum striatum... 57
Figure 3. Curvularia microspora inoculated to Hippeastrum rutilum (7 days). a the rst time for inoculation
b the second time for inoculation.
Table 2. Morphological comparison and pathogenecity of Curvularia microspora and related species in
trifolii-clade.
Species
name
Taxonomic
references
Conidia Conidio-
phores
Patho-
genecity Pathogenic reports
Shape Size range
Curvularia
microspora This study
curved at the third
cell from the base,
sometimes straight,
navicular, bifurcate,
obpyriform, tapering
towards rounded
ends
4.5–11.5 ×
2.0–6.0 μm
10.5–77.5 ×
1.0–3.5 μm Yes This study
Curvularia
akaii
Tsuda and
Ueyama
(1985)
24–34 × 8.7–
13.8 μm Yes Zhang (2004)
Curvularia
borreriae Ellis (1971) 20–32 ×
8–15 μm No
Curvularia
gladioli
Boerema and
Hamers
(1989)
17.5–37.5 ×
6.5–17.5 μm Yes Horita (1995); Torres
etal. (2013, 2015)
Curvularia
gudauskasii
Morgan-Jones
and Karr Jr
(1976)
27–29 ×
15–19 μm
62–98 ×
5–6μm Yes Chinea (2005); Ratón
et al. (2012)
Curvularia
heteropogonis Alcorn (1990) 27–44 ×
11–19 μm
115–620 ×
4–6 μm Yes Alcorn (1990)
Curvularia
pallescens Ellis (1971) 17–32 ×
7–12 μm Yes
Berg et al. (1995);
Dadwal and Verma
(2009); Mabadeje
(1969); Rajalakshmy
(1976)
Curvularia
trifolii
Groves and
Skolko (1945)
20–34 ×
8–14 μm Yes
Falloon (1976); Khadka
(2016); Sarwar and
Srinath (1965);
Sungetal. (2016);
Zamorski(1983);

Yin Liang et al. / MycoKeys 29: 49–61 (2018)
58
all other species in the trifolii-clade. Curvularia microspora also has smaller conidia than
the related species. A synopsis of the characters in the trifolii-clade is given in Table 2.
e phylogenetic analyses (MP and ML) also conrmed these isolates belong to a new
taxon with strong bootstrap support (Figure 1).
Curvularia species can cause severe or opportunistic diseases of dierent plant taxa
and are often a threat to agricultural production by reducing yield and quality. In the
trifolii-clade, all species except for C. borreriae, have been reported as causing plant dis-
ease. is is especially true of C. trifolii and C. pallescens, which cause serious diseases
of Agrostis stolonifera and Gloriosa superba respectively (Table 2). Koch’s postulates were
performed to show that C. microspora causes leaf spot disease of Hippeastrum striatum
(Figure 3), but on Canna indica might only be saprobic or endophytic. Hippeastrum
striatum as an economic ornamental plant is grown in some areas of China, thus there
is a need to continue investigation on the biology of this species in order to determine
whether it can cause serious disease outbreaks.
Acknowledgments
e research is supported by the project of National Natural Science Foundation of
China (No. 31560489), National Key Technology Research and Development Pro-
gramme of the Ministry of Science and Technology of China (2014BAD23B03/03),
Genetically Modied Organisms Breeding Major Projects of China [2016ZX08010-
003-009], Agriculture Animal and Plant Breeding Projects of Guizhou Province
[QNYZZ2013-009], Fundamental Research on Science and Technology, Ministry of
Science and Technology of China (2014FY120100), postgraduate education innova-
tion programme of Guizhou Province (ZYRC[2014]004) and Bijie science and tech-
nology project No. (2015)39.
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