JOURNA L O F PLAN T PROTECTIO N RESEARC H Vol. 55, No.

4 (2015)

R api d c o m municatio n

Characterization of Phyllosticta hostae causing Phyllosticta

leaf spot on spider lily in China

Run Hua Yi*, Luo Jun Gan, Jing Chen, Xiao Ling
Xu

Department of Biotechnology, Agricultural College, Guangdong Ocean University, Zhanjiang 524088, Guangdong Province,
China

Received: May 5, 2015

Accepted: November 3, 2015

Abstract: Leaf spot disease on the spider lily [Hymenocallis littoralis (Jacq.) Salisb.] continues to cause serious problems in China. To
con- firm the pathogen, the pathogenicity of isolates from diseased leaves was tested according to Koch’s postulates. The isolates
were tenta- tively identified using morphological characteristics and confirmation was done by phylogenetic analysis of the
translation elongation factor 1-alpha gene (TEF1), the actin gene (ACT), and internal transcibed spacer (ITS) sequences using
neighbor-joining (NJ), maximum parsimony (MP), and Bayesian inference (BI) methods. The pathogen was identified as Phyllosticta
hostae. Molecular analysis indicated very little diversity in the TEF1, ACT, and ITS gene. This is the first report of P. hostae causing leaf
spot disease on spider lily in China.

Key words: Hymenocallis littoralis, leaf spot disease, Phyllosticta hostae, spider lily

Introduction ince. Specimen which had typical symptoms were dried

and preserved. To isolate the pathogen, sections from
Hymenocallis littoralis (Jacq.) Salisb (members of the Ama-
ryllidaceae), commonly named the spider lily, is a
tropical bulbous herb with the staminal membrane
surrounded by long and narrow tepals that characterize
spider-like flowers (Martín et al. 2012). As a horticultural
and medici- nal plant, the spider lily was introduced to
China in the early nineteen-eighties, and is widely
cultivated in the provinces of Guangdong, Fujian,
Yunnan, and Guangxi. The natural compounds of the
spider lily display anti- neoplastic, antiviral properties,
and potent cytotoxic- ity against human tumor cell
lines (Renard-Nozaki et al.
1989; Griffin et al. 2007; Yew et al. 2010).
In June 2011, a new fungal disease, Phyllosticta
leaf spot disease found on the spider lily, was
noted
on the Guangdong Ocean University campus, Zhanji-
ang, Guangdong Province, China. The disease occurred
throughout the year and almost 100% of the plants were
infected, which led to the decline of ornamental and me-
dicinal value. Considering the seriousness and the lack
of knowledge concerning the disease in China, the objec-
tive of this paper was to determine and characterize the
pathogen of this disease based on morphology, and
phy-
logenetic analyses.

Materials and Methods

From June 2011 to December 2014, leaves with spots
were collected from different sites of Guangdong Prov-
 stored at a 4°C re- frigeration for the pathogenicity test,
diseased leaf tissues were cut into pieces approximately
microscopic obser- vation, and DNA extraction.
0.5 × 0.5 cm, surface-sterilised with 70% ethanol and
Isolates were incubated on PDA plates for 7 days at
0.1%
25°C in the dark. Artificial inoculation was conducted to
acid mercuric chloride, rinsed with sterile distilled wa-
confirm the pathogenicity test, using the spore suspen-
ter, blotted dry on sterile paper, and transferred to plates
sion and mycelial plugs according to Koch’s postulate.
containing 20 ml Potato Dextrose Agar (PDA) medium
Forceps were used to pick up the pycnidia and put them
amended with 20 µg ∙ ml–1 ampicillin. Isolation plates
into a mortar. The pycnidia were squashed with sterile
were incubated at 28°C in the dark, for 3 days. The
distilled water to release the pycnidiospores, the suspen-
hyphae tips growing from the tissues were transferred
sion was adjusted so the concentration was approximate-
onto PDA plates to get the axenic cultures. To obtain
ly 1 × 105 spores ∙ ml–1. Approximately 0.5 ml of spore
single spored isolates, conidia from pure cultures were
suspension was sprayed on the leaves, which were sur-
suspended in sterile water and streaked onto PDA plates.
face-disinfected with a 70% ethanol tampon three times,
The single-co- nidia forming colonies were obtained after
washed three times with the sterile distilled water, and
an in-the-dark incubation at 28°C for 24 h. The isolates
acupunctured with sterile needles. Sterile distilled water
were incubated at 28°C in the dark for 7–10 days and
was sprayed as a control. To keep the humidity level
high, the leaves were covered with plastic bags for 48 h.

*Corresponding address:
scibyrh@163.com
439 Journal of Plant Protection Research
Characterization
55 (4), of Phyllosticta hostae causing Phyllosticta leaf spot on spider lily in China
Six millimeters mycelial plugs from the colony MrBayes ver- sion 3.1.2 (Ronquist and Huelsenbeck
margin were inoculated onto the sterile surface of the 2003), respectively.
leaves after the leaves had been acupunctured with
sterile needles. The absorbent tampons were laid aside,
and covered with transparent cellophane that was
perforated with a needle for aeration. Sterile PDA plugs
were used as the controls. The disease development was
observed everyday.
Morphological characteristics on PDA were observed
periodically until sporulation, by the methods described
by Wulandari et al. (2009). Mycelial discs (5 mm diam-
eter) were cut from the edges of the growing areas of the
7-day-old colony, and transferred to the center of Corn-
meal Agar (CMA), Malt Extract Agar (MEA), PDA, and
Oatmeal Agar (OA) plates, and incubated at 25°C under
dark conditions. The mycelial growth rates were mea-
sured after incubation for two weeks. The pycnidia on
the diseased leaf were examined and crushed to release
spores in water mounts on a glass slide, under light mi-
croscope (Olympus BX51). Images were acquired with
a digital camera DXM 1200F (Nikon). All microscopic
characteristics were measured for at least 50 individuals,
to calculate the mean size.
Pathogens were grown on Potato Dextrose Broth
(PDB) and incubated in a 180 rpm rotatory shaker for 5
days at 25°C. Mycelia collection and genomic DNA exac-
tion followed the protocol of Yi et al. (2003). Internal tran-
scribed spacer (ITS) and small subunit (SSU) sequences
were respectively amplified using the primer pair ITS1/
ITS4 and NS1/NS4 (White et al. 1990). Primer pair EF1-
728F (Carbone and Kohn 1999) and EF-2 (O’Donnell et al.
1998) were used to amplify the partial translation elonga-
tion factor 1-alpha gene (TEF1). The primer pair ACT-
512F
(Carbone and Kohn 1999) and ACT2Rd (Quaedvlieg et al.
2011) were used to amplify the partial actin gene
(ACT).
To amplify the different loci, the polymerase chain
reaction (PCR) mixture that was used consisted of tem-
plate DNA (5–10 ng), 1.5 U Taq DNA polymerase, 1X
PCR buffer, 0.25 mM of each dNTP, and 0.2 µM of
each
primer, and made up to a total reaction volume of 50 µl
with sterile double-distilled water (ddH2O). The cycling
parameters were started with a long initial denaturation
step at 95°C for 4 min, 35 cycles of denaturation at 94°C
for 60 sec, annealing at 50°C for SSU primers and 55°C
for ITS, ACT, and TEF primers for 60 sec, extension at
72°C for 60 sec followed by a final extension at 72°C for
10 min, and a soaking at 4°C for 3 h. Amplified
fragments were purified and sequenced by the Sangon
Biotech (Shanghai, China) Co., Ltd.
The sequences from the forward and reverse primers
were assembled and deposited in the GenBank. The
novel sequences of three isolates in this study, together
with the additional reference sequences of representative
Phyl- losticta taxa were retrieved from GenBank (Table 1).
The alignment of the obtained sequences was performed
and truncated at the 5′- and 3′-ends using BioEdit (Hall
1999).
Phylogenetic trees were constructed using neighbor join-
ing (NJ), maximum parsimony (MP), and Bayesian infer-
ence (BI) methods. It should be noted, that the NJ, MP,
and BI analyses were performed using MEGA5.0
(Tamura et al. 2011), PAUP4.0 (Swofford 2003), and
440 ForJournal
NJ analyses, the evolutionary
of Plant Protection Research 55 distances
Characterization
(4), were
of Phyllosticta hostae causing Phyllosticta leaf spot on spider lily in China
com- puted using the Kimura 2-parameter model with
1,000 bootstrap replicates. Maximum-parsimony
analyses were estimated using the heuristic searches
with tree bisection reconnection (TBR) branch swapping
and 1,000 random addition sequences. Branches of zero
length were col- lapsed. Statistics calculated for
parsimony included tree length (TL), consistency index
(CI), retention index (RI), and the rescaled consistence
index (RC). For Bayesian analyses, the best fit nucleotide
substitution model was evaluated using jModelTest
v2.1.4 (Santorum et al. 2014) according to the Bayesian
information criterion (BIC). The model (GTR+I+G) and
parameters lset apply to = (1/DNA) nst = 6 rates =
invgamma; unlink shape = (all) pinvar = (all) statefreq =
(all) revmat = (all); prset ratepr
= variable statefreqpr = dirichlet (1,1,1,1); were applied.
Posterior probabilities (PP) were determined by the Mar-
kov Chain Monte Carlo (MCMC) method (Mossel and
Vi- goda 2005), four MCMC chains were run
simultaneously from random trees for ten million
generations, sampled every 1,000 generations, and
repeated twice. The first 25 percentages of trees were
discarded as the burn-in phase of each analysis. The
remaining trees were used for de- termining posterior
probabilities and were shown in the majority rule
consensus tree. The aligned data and phylo- genetic trees
were deposited in TreeBase (http://purl.org/
phylo/treebase/phylows/study/TB2:S14438).

Results
Phyllosticta leaf spot on spider lily occurred all year but
became most serious during late September to early Oc-
tober in Guangdong Province. Small, circular or oval,
brown-reddish to black spots occurred on the leaves in
the early period. Spots sometime formed to be concave,
oval spots, occasionally surrounded by a water-soaked,
discolored halo. The spots gradually enlarged to be big
patches. Subsequently, the diseased leaves became
yellow with blighting at the tip (Fig. 1A). Pycnidia
appeared on the infected positions or at the leaf tip (Fig.
1B).
The leaves with typical symptoms and with pyc-
nidia were collected from the campus of the Guangdong
Ocean University, Zhanjiang, Guangdong Province, Chi-
na (E110°17’47” N21°08’58”). The collected leaves were
dried at 40°C for a week and deposited in the
Mycological Herbarium of Institute of Microbiology,
Chinese Acade- my of Sciences, Beijing, China (HMAS).
The numbers of the voucher herbarium specimens were
HMAS 244379 and HMAS 244380.
From different sites, leaves with various degrees of
se- verity and water-soaked discolored patches, brown-
black patches, concave oval spots, and irregular big
patches, were sampled so as to isolate the pathogens. The
same morphological characteristics of isolates were
obtained from all the diseased leaf tissues. On PDA,
these isolates displayed the Phyllosticta-like
characteristics of being lo- bate, and irregular with
feathery margins.
Two representative isolates, CGMCC 3.15214 and
CGMCC 3.15215, were selected for the pathogenicity
test and deposited in the China General Microbiological
Culture Collection Center (CGMCC). Three days after
 441 Journal of Plant Protection Research
Characterization
55 (4), of Phyllosticta hostae causing Phyllosticta leaf spot on spider lily in China

Table 1. Sources of Guignardia and Phyllosticta isolates and GenBank accession numbers used in this study
Strain voucher Host Locality GenBank accession number
Speci number
ITS TEF ACT
Botryosphaeria obtusa CMW 8232 Conifers South Africa AY972105 DQ280419 AY972111
Guignardia bidwellii CBS 111645 Parthenocissus quinquefolia USA JN692542 JN692530 JN692518
(Vitaceae)

G. mangiferae IMI 260576* Mangifera indica India JF261459 JF261501 JF343641

G. vaccinii CBS 126.22 Vaccinium macrocarpon USA FJ538353 FJ538411 FJ538469
(Ericaceae)

Phyllosticta aloeicola CPC 21020 Aloe ferox South Africa KF154280 KF289193 KF289311
P. capitalensis CBS 128856* Stanhopea sp. Brazil JF261465 JF261507 JF343647
P. citriasiana CBS 120486* Citrus maxima (Rutaceae) Thailand FJ538360 FJ538418 FJ538476
P. citribraziliensis CBS 100098* Citrus sp. (Rutaceae) Brazil FJ538352 FJ538410 FJ538468
P. citricarpa CBS 127454* Citrisx limon (Rutaceae) Australia JF343583 JF343604 JF343667
P. citrichinaensis CBS 130529* Citrus maxima (Rutaceae) China JN791597 JN791452 JN791526
P. citrimaxima CPC 20276* Citrus maxima Thailand KF170304 KF289222 KF289300
P. concentrica CBS 937.70* Hedera helix Italy FJ538350 FJ538408 KF289257
P. cordylinophila CPC 20261* Cordyline fruticosa (Asparagaceae) Thailand KF170287 KF289172 KF289295
P. cussonia CPC 14875* Cussonia sp. (Araliaceae) South Africa JF343579 JF343600 JF343663
P. ericarum CBS 132534* Erica gracilis (Ericaceae) South Africa JX069865 KF289227 KF289291
P. foliorum CBS 447.68 * Taxus baccata Netherlands KF170309 KF289201 KF289247
P. gaultheriae CBS 447.70* Gaultheria humifusa USA JN692543 JN692531 KF289248
P. hostae CGMCC 3.14355* Hosta plantaginea (Liliaceae) China JN692535 JN692523 JN692511
P. hostae CGMCC 3.14356 Hosta plantaginea (Liliaceae) China JN692536 JN692524 JN692512
P. hostae CGMCC 3.15214 Hymenocallis littoralis China JX524169 KJ094498 KJ094496
(Amaryllidaceae)

P. hostae CGMCC 3.15215 Hymenocallis littoralis China KC995113 KJ094499 KJ094495

(Amaryllidaceae)

P. hostae GDOU 201203 Hymenocallis littoralis China KC995114 KJ094500 KJ094497

(Amaryllidaceae)

P. hubeiensis CGMCC3.14986* Viburnum odoratissimum China JX025037 JX025042 JX025032

(Adoxaceae)

P. hymenocallidicola CBS 131309* Hymenocallis littoralis Australia JQ044423 KF289211 KF289242

(Amaryllidaceae)

P. hypoglossi CBS 434.92* Ruscus aculeatus Italy FJ538367 FJ538425 FJ538483

P. ilicis-aquifolii CGMCC3.14358* Ilex aquifolium (Aquifoliaceae) UK JN692538 JN692526 JN692514
P. paxistimae CBS 112527* Paxistima mysinites USA KF206172 KF289209 KF289239
P. philoprina CBS 587.69 Ilex aquifolium (Aquifoliaceae) Spain KF154278 KF289206 KF289250
P. podocarpicola CBS 728.79* Podocarpus maki USA KF206173 KF289203 KF289252
P. rubra CBS 111635* Acer rubrum USA KF206171 KF289198 KF289233
P. spinarum CBS 292.90 Chamaecyparis pisifera France AF312009 JF343606 JF343669
(Cupressaceae)

P. styracicola CGMCC3.14985* Styrax grandiflorus (Styracaceae) China JX025040 JX025045 JX025035

P. telopeae CBS 777.97* Telopea speciosissima Tasmania KF206205 KF289210 KF289255
P. vacciniicola CPC 18590* Vaccinium macrocarpum USA KF170312 KF289229 KF289287
P. yuccae CBS 117136 Yucca elephantipes (Asparagaceae) New Zealand JN692541 JN692529 JN692517

*indicates the ex-type cultures; CGMCC – China General Microbiological Culture Collection Center, China; CBS – Centra albureau
voor Schimmel cultures, Utrecht, The Netherlands; MUCC – Lab. of Plant Pathology, Mie University; GDOU – Lab. of Plant
Pathology, Guangdong Ocean University; CPC – Culture collection of P.W. Crous, housed at CBS
ITS – internal transcribed spacer; TEF – translation elongation factor; ACT – actin gene
442 Journal of Plant Protection Research
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55 (4), of Phyllosticta hostae causing Phyllosticta leaf spot on spider lily in China

Fig. 1. Typical foliar symptom of Phyllosticta leaf spot on Hymenocallis littoralis: A – small, circular or oval, brown-reddish to black
spots on leaves and the yellowing and blighting leaves at the tip; B – black pycnidia on diseased leaf; C – leaf with artificial
inoculation
inoculation with spore suspension, tiny water-soaked appendage, 5.8–48.1 (av. 15.9±6.6) × 1.3–2.1 (av.
spots appeared on the artificially inoculated positions. 1.7±0.2)
Gradually, the same symptoms developed as in nature
(Fig. 1C), whereas the control leaves did not develop any
symptoms. The water-soaked and discolored symptoms
occurred later when using the mycelial plus inoculation,
than with the spores suspension. The same morphologi-
cal fungi were re-isolated from these diseased tissues.
The isolates morphology characteristics were de-
scribed as below:
Phyllosticta hostae Y.Y. Su & L. Cai, Persoonia 28, 76–
84 (2012) (Fig. 2).
Conidiomata pycnidial, solitary, black, erumpent,
globose, separate or in small groups, embedded in a sub-
epidermal stroma on host, stromata globose to ampulli-
form, forming an irregularly folded crust on PDA, exud-
ing a colorless to opaque glossy conidial mass; pycnidia
41.6–150.3 (av. 92.3±22.4) µm in diam; pycnidial wall
consisting of several layers of brown textura angularis,
8.7–24.7 (av. 16.1±3.3) µm thick; ostiole single,
central,
15.1–36.0 (av. 25.5±5.2) µm wide, consisting of thickened,
brown cells. Conidiophores subcylindrical to doliiform,
frequently reduced to conidiogenous cells, coated in
a mucoid layer. Conidiogenous cells termina terminal,
subcylindrical to doliiform, hyaline, smooth, 6.3–23.8
(av. 14.3±3.3) × 2.3–6.8 (av. 4.4±0.9) µm. Conidia 5.2–
12.5
(av. 8.5±2.5) × 3.8–8.6 (av. 6.4±1.8) µm, solitary,
hyaline,
aseptate, thin- and smooth- walled, smoothly guttulate,
ellipsoid to obovoid, tapering toward a narrowly trun-
cate base, enclosed in a mucilaginous sheath, 1.8–4.0 (av.
2.1±0.5) µm thick, and bearing a hyaline, mucoid apical
 µm, straight
443 to Plant
Journal of flexible, unbranched,
Protection tapering
Research 55 (4), towards
Characterization an
of Phyllosticta hostae causing Phyllosticta leaf spot on spider lily in China
acute apex; conidia length/width ratio 1.2–2.0 (av.
1.6±0.2).
Culture characteristics (in the dark, 25°C after 2
weeks): colonies reaching 74.2 mm diam. after 2 weeks
on MEA, 76.8 mm on PDA and 71.3 mm on CMA, but
only
55.3 mm on OA. Colonies on PDA were flat, the surface
was black in the centre, spreading, lobate, irregular with
feathery margin, sparse aerial mycelium, surface oliva-
ceous grey, and reverse black, olivaceous at margin.
Colo- nies on MEA were flat, spreading, irregular, with
smooth, lobate margin, and sparse aerial mycelium. The
surface was leaden to leaden-gray in colour. Colonies on
OA were flat, with feathery, lobate margins and sparse
aerial my- celium, olivaceous-black in the centre, pale
olivaceous in the outer region. Colonies on CMA were
flat, with feath- ery margins. The surface was dark in the
centre, and an olive colour in the outer regions. No
teleomorph was ob- served in agar (OA, PDA, MEA, and
CMA).
Based on a megablast search of NCBIs GenBank
nucleotide database, the closest hit using the ITS se-
quence of isolates CGMCC 3.15214 and CGMCC 3.15215
is P. hostae CGMCC 3.14357 [causing leaf spot of Hosta
plantaginea in China, GenBank JN692537; Identities =
= 549/552 (99.457%), Gaps = 2/552 (0.362%)]. A megablast
search of the NCBIs GenBank nucleotide sequence da-
tabase using the SSU sequence (GenBank JX524170) of
CGMCC 3.15214 confirms its placement in the genus;
closest hits included Guignardia sp. IFB-GLP-4 [Gen-
Bank GU380271; Identities = 1040/1063 (97.856%), Gaps =
= 19/1063 (1.787%)].
The thirty-five combined sequences from 30 taxa of
Phyllosticta (teleomorph: Guignardia) were phylogeneti-
 444 Journal of Plant Protection Research
Characterization
55 (4), of Phyllosticta hostae causing Phyllosticta leaf spot on spider lily in China

Fig. 2. Morphological characteristics of pathogen causing Phyllosticta leaf spot disease on spider lily: A – symptoms with pycnidia
forming on leaf of Hymenocallis littoralis; B – an overlook of pycnidia with an ostiole in leaf tissue; C – vertical section of
pycnidium in leaf tissue; D – colony on PDA in the dark, 25°C, after 15 d; E–F – pycnidia forming on PDA; G – vertical
section of initial pycnidium on PDA; H–J – conidia with mucoid sheath and apical mucilaginous appendage, H–I – conidia
forming on host; J – conidia forming on PDA; K – spermatium. Scale Bars: C = 20 µm; F = 500 µm; G = 50 µm; H = 10 µm; I, J,
K = 20 µm
cally analysed using the combined ITS, ACT, and TEF bootstrap value and Bayesian posterior probability
dataset. Botryosphaeria obtusa CMW8232 was used as an (Fig. 3). The isolates also formed a heterogenous group
outgroup. The combined dataset comprised 1106 total with P. hymenocallidicola CBS 131309, which was isolated
characters including gaps, of which 548 characters were from H. littoralis and caused leaf spot and tip blight dis-
constant, 338 were parsimony informative, and 220 were ease (Crous et al. 2011).
variable and parsimony-uninformative. Phylogenetically, the isolates of CGMCC 3.15214,
Phylogenetic analysis using NJ, MP, and Bayes- CGMCC 3.15215, and GDOU201203 clustered together
ian inference yielded evolutionary trees with identical with Phyllosticta hostae isolates based on all three of the
topology, which represented the evolutionary history gene regions sequenced. Three fixed nucleotide changes
of Phyllosticta taxa. The isolates of CGMCC 3.15214, were observed over 548 nucleotides for ITS; whereas
CGMCC 3.15215, and GDOU201203 formed a mono- TEF1 only contained one fixed nucleotide change over
phyletic group with P. hostae CGMCC3.14355 and 258 nucleotides, and ACT had only 3 fixed nucleotide
CGMCC3.14356，which was strong supported by the changes and 3 indels over 232 nucleotides (Table 2).
445 Journal of Plant Protection Research
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55 (4), of Phyllosticta hostae causing Phyllosticta leaf spot on spider lily in China

Fig. 3. Phylogenetic tree of Phyllosticta species reconstructed by neighbor-joining (NJ), maximum parsimony (MP) and Bayesian
inference (BI) using the combined internal transcribed spacer (ITS), partial actin gene (ACT) and translation elongation factor
(TEF) dataset. The numbers along the branches indicate posterior probability values (%) or bootstrap percentages resulting
from different analyses in the order: BI/MP/NJ. The values lower than 50 are given as “–” except for NJ

Table 2. Nucleotide differences and their base positions observed in three loci among the isolates of Phyllosticta hostae. Sequences of
CGMCC3.14355 were used as references to calculate base positions, which do not include spaces caused by alignment gaps

ITS ACT TEF

Isolates References
33 150 151 102 111 186 187 188 232 253

CGMCC3.14355 A C T A C C G C G T Crous et al. 2011

CGMCC3.14356 A C T A C C G C G T Crous et al. 2011

CGMCC3.15215 G T C G T – – – A C this study

CGMCC3.15214 G T C G T – – – A C this study

GDOU 201203 G T C G T – – – A C this study

ITS – internal transcribed spacer; ACT – partial actin gene; TEF – translation elongation factor
 446 Journal of Plant Protection Research
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55 (4), of Phyllosticta hostae causing Phyllosticta leaf spot on spider lily in China

Table 3. Phyllosticta spp. described from Amaryllidaceae and Hosta plantaginea

Pycnidia size Conidiogenous Conidia Appendage size

Phyllosticta species Host plant References
[µm] cells [µm] [µm] [µm]

P. amaryllidicola Amaryllis sp. 120–150 2–6 × 1.5–4 9–12 × 5.5–7.5 4–7 Van der Aa 1973

P. cliviae Clivia nobilis 105–150 – 3–6 × 2–3 – Saccardo 1972

Hymenocallis (8–)9–10(–11) ×
P. hymenocallidicola littoralis 200 7–15 × 3–4 × (6–)6.5–7 3–5(–8) × 1.5(–2) Crous et al. 2011

P. hostae Hosta plantaginea 40–150 7–22 × 2–5 8–15 × 5–9 4–8 × 1–3 Su and Cai 2012

Hymenocallis 6.3–23.8 × 5.2–12.5 ×

P. hostae littoralis 41.6–150.3 × 2.3–6.8 × 3.8–8.5 5.8–48.1 × 1.3–2.1 this study

Discussion and Conclusions on H. littoralis, namely P. hymenocallidis and P.

hymeno-
Recognition of the Phyllosticta species was based on host callidicola. Phyllosticta hymenocallidis, formerly named as
and morphological characteristics. Because of its narrow P. narcissi, P. oudemansii, and Phoma narcissi, was a
host range, if two or more taxa were obtained from the syn-
same host, then the symptoms, ecological and pure cul- onym of Peyronellaea curtisii (Berk.) Aveskamp, Gruyter
ture characteristics under well-defined conditions, and and Verkley 2010, which causes leaf scorch, neck rot, red
molecular phylogenetic method were used for classifica-
tion (Van der Aa 1973; Wikee et al. 2011).
The three isolates: CGMCC3.15214, CGMCC3.15215,
and GDOU201203 can be distinguished morphologically
or/and phylogenetically from other Phyllosticta spp. on
Amaryllidaceae (Table 3). Nine Phyllosticta species had
been recorded on Amaryllidaceae. The nine were: P.
ama- ryllidicola, P. amaryllidis, P. cliviae, P. crinicola, P.
gemmip- ara, P. hymenocallidicola, P. hymenocallidis, P.
narcissi, and P. oudemansii. Six species, i.e. P.
amaryllidis, P. crinicola, P. gemmipara, P. hymenocallidis, P.
narcissi, and P. oudeman- sii, did not belong to
Phyllosticta. Van der Aa and Vanev (2002) treated P.
amaryllidis as a synonym of Asteromella amaryllidis.
Phyllosticta crinicola, as a homotypic syn- onyms of
Phoma crinicola, was renamed as Boeremia crini- cola
(Boerema 1978; Aveskamp et al. 2010). Phyllosticta
gemmipara was a heterotypic synonym of Phoma exigua
var. exigua and was transferred to Boeremia exigua var.
exigua (Boerema 1993; Aveskamp et al. 2010). Phyllosticta
oudemansii was a competing homonym of P. narcissi,
while P. narcissi and P. hymenocallidis, as basionym of
Phoma nar- cissi, whose current name is Peyronellaea
curtisii (Boerema
1993; Boerema et al. 2004; Aveskamp et al.
2010).
Phyllosticta amaryllidicola, with the teleomorph Guig-
nardia sansevieriae was changed to G. mangiferae (Ana-
morph: Phyllosticta capitalensis) (Punithalingam 1974;
Wu-
landari et al. 2009), while P. capitalensis occurred on
Stanho-
pea (Orchidaceae) causing leaf spot disease in
glasshouses
(Hennings 1908), and belonged to a different branch
with
CGMCC 3.15214, CGMCC 3.15215, and GDOU201203 in
the phylogenetic tree (Fig. 3). Phyllosticta cliviae, on host
of Clivia nobilis, produced smaller conidia than the three
isolates, 3–6 × 2–3 µm vs. 5.2–12.5 (av. 8.5±2.5) × 3.8–8.6
(av. 6.4±1.8) µm (Saccardo
1972).
Only two species of Phyllosticta were known to occur
 447 Journal of Plant Protection Research
Characterization
55 (4), of Phyllosticta hostae causing Phyllosticta leaf spot on spider lily in China
spot disease, red leaf spot disease of Narcissus spp., Hip-
peastrum spp. and other Amaryllidaceae (Boerema 1993;
Boerema et al. 2004; Aveskamp et al. 2010). Phyllosticta
hy- menocallidicola, which causes brown leaf spots and
leaf tip blight of H. littoralis, differs from the characters of
CGMCC
3.15214, CGMCC 3.15215, and GDOU201203. The
pycnid- ia is smaller than P. hymenocallidicola, 41.6–150.3
(av. 92.1) µm vs. 200 µm in diameter, the ostiole is wider,
15.1–36.0 (av. 25.5) µm vs. 20 µm, the size of the
conidiogenous cells are bigger, 6.3–23.8 (av. 14.3) × 2.3–
6.8 (av. 4.4) µm vs. 7–15 × 3–4 µm, and the appendage is
longer, 5.8–48.1 (av. 15.9±6.6) × 0.5–1.0 µm vs. 3–5(–8) ×
1.5(–2) µm (Crous et al. 2011). The ITS sequence
(GenBank JX524169) of CGMCC 3.15214 showed
significant non-similarity with P. hymenocallidicola CBS
131310 [GenBank JQ044424, Iden- tities = 584/630
(92.698%), Gaps = 13/630 (2.063%)]. Phylo- genetic
analysis using NJ, MP, and Bayesian showed that P.
hymenocallidicola CBS 131310 was clustered into differ-
ent groups with CGMCC 3.15214, CGMCC 3.15215, and
GDOU201203.
Morphologically and phylogenetically, the isolates
CGMCC 3.15214, CGMCC 3.15215, and GDOU201203
were closest to P. hostae on H. plantaginea (Liliaceae)
(Table 3, Fig. 3). However, they were different from P.
hos- tae by the longer appendage [5.8–48.1 (av.15.9±6.6) ×
0.5–
1.0 µm vs. 4–8 × 1–3 µm in P. hostae], by the smaller
conid- ia, 5.2–12.5 (av. 8.5±2.5) × 3.8–8.6 (av. 6.4±1.8) µm
vs. 8–15 (av. 10.9±1.4) × 5–9 (av. 7.6±0.8) µm in P. hostae,
and by the color difference in the colony centre and at
the margin on PDA, the black in the centre and the
olivaceous color at the margin vs. P. hostae with a leaden-
grey colour in the centre and lavender-grey at the
margin (Su and Cai 2012).
In conclusion, Phyllosticta leaf spot disease on spider
lily was caused by P. hostae Y.Y. Su & L. Cai (Su and
Cai
2012).

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