First record of Plasmopara sphagneticolae (Peronosporales, Oomycota) in Taiwan

Abstract

Sphagneticola trilobata (Asteraceae) is one of the most common ornamental groundcover plants in public green spaces and also invasive in Taiwan. Downy mildew symptoms were found on this plant in Taipei City. Based on the host, morphology and ribosomal large subunit RNA gene (LSU) sequence analysis, the associated oomycete was identified as Plas-mopara sphagneticolae. The most likely route of introduction of this oomycete together with its host from Hawaii is discussed .

Full text

Fung. Sci. 36(1): 23–32, 2021
*Corresponding author, e-mail: kirschner@ntu.edu.tw
First record of Plasmopara sphagneticolae (Peronosporales,
Oomycota) in Taiwan
Chieh-Ting Wang1, Lang-Dong Lin2, Yu -Wei Yeh3, Roland Kirschner3*
1 The Experimental Forest, National Taiwan University, Jhushan, Taiwan
2 Department of Cultural Heritage Conservation, National Yunlin University of Science and Technology, Yunlin, Taiwan
3 School of Forestry & Resource Conservation, National Taiwan University, Taipei, Taiwan
(Accepted: September 20, 2021)
ABSTRACT
Sphagneticola trilobata (Asteraceae) is one of the most common ornamental groundcover plants in public green spaces
and also invasive in Taiwan. Downy mildew symptoms were found on this plant in Taipei City. Based on the host, mor-
phology and ribosomal large subunit RNA gene (LSU) sequence analysis, the associated oomycete was identified as Plas-
mopara sphagneticolae. The most likely route of introduction of this oomycete together with its host from Hawaii is dis-
cussed.
Key words: Compositae, oomycete-host distribution, plant diseases, rDNA, Wedelia
Introduction
Sphagneticola trilobata (L.) Pruski (Asteraceae),
also known under its older name Wedelia trilo-
bata, originates from the Neotropics. In Taiwan
it was introduced in the early 1980s from Hawaii
and since then has become one of the most com-
mon ornamental groundcover plants in public
green spaces but also one of the most invasive
introduced plants (Hsueh and Yang 2014, Wu et
al. 2004). In a public green space in Taipei City
we found a plantation showing downy mildew
symptoms on the leaves. Several downy mildew
oomycetes (Oomycota) have been recorded from
Asteraceae (Duarte et al. 2014). For these fun-
gus-like eukaryotes, we occasionally use the tra-
ditional term “fungi” referring to the heterotro-
phic filamentous eukaryotic growth form and not
to the systematic group “Fungi”.
Materials and Methods
Collection and morphology
Specimens were collected in a public green space
in Da-an District in Taipei City. Fresh specimens
were put into plastic bags and stored in the re-
frigerator for up to one week during the study.
For photographs, dissecting and light micro-
scopes with Olympus EP50 digital cameras were
used. In order to give an impression of the spo-
rangiophore branching which cannot be resolved
with light microscopic photographs because of
the three dimensions, sketches were done by
hand on scaled paper. Illustrations were com-

24 Fung. Sci. 36(1): 23–32, 2021
Fig. 1. Habitat of Plasmopara sphagneticolae and its host, Sphagneticola trilobata in Taipei City. A. Trapezoid green
space, arrow indicates infected plantation of Sph. trilobata. Note “Taipei 101” in the background. B. Close-up from the
location in A, lower leaves of Sph. trilobata with chlorosis and necrosis.
posed and processed with Photoshop software.
For light microscopy, fresh sporangiophores
were picked up from living leaves with tweezers
and mounted in tap water or ca. 10% aqueous
KOH. Measurements were done at 40× to 1000×
magnification and presented as mean value ±
standard deviation and extreme values in brack-
ets or directly as extreme values. Dried speci-
mens were deposited in the National Museum of
Natural Science, Taichung, Taiwan (TNM).
Phylogenetic analysis
Fresh sporangiophores picked up from living
leaves were also transferred to an Eppendorf cup
with a drop of sterile water, ground with a power
masher pestle and subjected to DNA extraction
with EasyPure Genomic DNA Spin Kit/Plant
(Bioman Scientific Co., Ltd., New Taipei City,
Taiwan). The DNA extract was used for PCR
with primers NL1 and NL4 and PCR conditions
as given in Kurtzman and Robnett (1997) in or-
der to amplify the partial ribosomal large subunit
RNA gene (LSU). The PCR product was se-
quenced by Mission Biotech (Nangang). The for-
ward and reverse sequences were assembled with
CodonCode Aligner and the consensus sequence
deposited in GenBank under MZ959822. A phy-
logenetic analysis was done with MEGAX by as-
sembling an alignment based on most similar se-
quences received from BLAST searches in Gen-
Bank as well as from McTaggart et al. (2015) and
using the default options of MUSCLE (Kumar et
al. 2018). The sequence of Plasmopara majew-
skii Constant. & Thines was much shorter than

Plasmopara sphagneticolae 25
all other sequences and, therefore, excluded from
the dataset. The alignment block was trimmed at
the left and right ends without manual manipula-
tion within the dataset. The phylogenetic rela-
tionships were inferred with Maximum Likeli-
hood in MEGAX using the Hasegawa-Kishino-
Yano model with Gamma distribution as best
model (Hasegawa et al. 1985). The species
names were adopted directly from GenBank
without considering possible new synonymies or
other name changes. The tree was rooted with
Phytophthora tabaci Sawada (according to Index
Fungorum a synonym of Ph. nicotianae Breda de
Haan). In order to infer the relationship of an-
other morphologically similar Plasmopara spe-
cies on Asteraceae, P. invertifolia L.L. Duarte &
R.W. Barreto, for which only partial cytochrome
c oxidase subunit 2 gene (cox2) sequences are
available, we used a cox2 sequence of P. inverti-
folia for a BLAST search.
Results and Taxonomy
The morphology of the oomycete associated with
the abaxial side of the leaf lesions conformed to
Peronosporales, Oomycota (Figs. 1–3). Accord-
ing to the literature and databases, the single
known downy mildew on Sphagneticola hosts is
Plasmopara sphagneticolae (Farr and Rossman
2021). BLAST search with the 773 bp LSU se-
quence yielded two most similar sequences, both
of P. sphagneticolae with 0 or 1 different base
pair, namely from mainland China (773/773 bp,
MW298155) and from the type locality in Aus-
tralia (BRIP 61010, GenBank KM085176,
McTaggart et al. 2015). The next similar se-
quences were 16 sequences from P. halstedii
(Farl.) Berl. & De Toni, all exceeding 650 bp,
with 9–12 different bp (98–99% identity). The
phylogenetic analysis placed the Taiwanese ma-
terial together with the two sequences of P.
sphagneticolae in a strongly supported clade
which was distinctly separated from a sister
clade formed by sequences of P. halstedii (Fig.
4). In our BLAST search with one of the two
cox2 sequences of P. invertifolia, we found that
it shows only up to 91% identity with any other
oomycete, including P. halstedii and P. sphag-
neticolae.
Plasmopara sphagneticolae McTaggart & R.G.
Shivas, in McTaggart, Shuey, McKenna, Davis
& Shivas, Australas. Pl. Path. 44: 84. 2015.
Figs. 1–3
Associated with lesions of living leaves. Leaf
spots amphigenous, angular and vein-limited, 2
mm diam., up to 20 × 10 mm, becoming conflu-
ent, pale yellow, yellow to brown. Mycelium in-
ternal, hyaline, smooth, branched, 4–14 μm wide,
giving rise to ellipsoid intracellular haustoria, 8–
10 × 6–8 μm. Sporangiophores hypophyllous,
penetrating through stomata, forming white felt-
like layer, hyaline, smooth, straight 325–725 μm
long, composed of more than the lower half of an
unbranched stipe, 225–475 μm long and 7–13
μm wide at the slightly bulbous base, branching
mainly monopodial at 3–4 levels of up to four
main branches and angles of 90° at the lower
main branches and angles between 45 and 90° at
the upper and secondary branches, occasionally
2 or 3 branches arising from the same node.
Lower branches predominantly almost as long as
the corresponding distal part of the main axis,
lowermost branch longest, 180–275 μm long,
next distal branch approx. as half as long as more
proximal branch. Retraction septa (“callose

26 Fung. Sci. 36(1): 23–32, 2021
Fig. 2. Disease symptoms associated with Plasmopara sphagneticolae. A. Chlorosis and necrosis of leaves seen from upper
side. B. Leaf lesions on abaxial leaf side with downy mildew symptom. C. Sporangiophores arising from abaxial side. D.
Internal hypha (ca. 10 μm wide) inserting two ellipsoidal haustoria into mesophyll cells.

Plasmopara sphagneticolae 27
plugs”) scattered in the stipe and branches, most-
ly 2–3 μm thick, sometimes irregular and thicker.
Ultimate branchlets 1–3, straight or slightly cur-
ved, 4–22 μm long, 2–4 μm wide at base, nar-
rowing to 1.5–2 μm at apex. Apex appearing
truncate and slightly refractive after dehiscence
of sporangium. Sporangia hyaline, smooth, sub-
globose, ellipsoidal to broadly ovoidal, slightly
broader in the proximal half than in the distal,
with apical papilla and 1.5–2 × 1 μm basal hilum
when fully turgescent, (16–)18–22(–25) × (14–)
15–18(–20) μm (n = 30). Oogonia not found.
Specimens examined. TAIWAN. Taipei City,
Daan District, on living leaves of Sphagneticola
trilobata with downy mildew symptoms, Wo-
long Street, public green, ca. 25.017834,
121.552391, ca. 10 m alt., 22 Aug 2021, Kirsch-
ner R. 5316 (TNM), LSU sequence GenBank
MZ959822; same place, 29 Aug 2021, Kirschner
R. 5316-B.
Known hosts and distribution. Lipochaeta in-
tegrifolia (Nutt.) A. Gray, Sphagneticola trilo-
bata (L.) Pruski (Asteraceae); Australia, Hawaii,
mainland China? (unpublished sequences), Tai-
wan (new record).
Discussion
Species identification
Although appressorium morphology is a diag-
nostic characteristic for the genera of Peronospo-
rales (Voglmayr et al. 2004), internal hyphae and
appressoria were not mentioned in the previous
publications. We found that these characteristics
conformed to the concept of Plasmopara (Shin
and Choi 2006, Voglmayr et al. 2004). Species of
Plasmopara on Asteraceae in the past were con-
sidered as P. halstedii (Spring 2019). Although
lengths of sporangiophores and sporangia are
claimed as distinctive features for the species on
Asteraceae (Duarte et al. 2014, McTaggart et al.
2015, Davis et al. 2020), the sizes strongly over-
lap (Duarte et al. 2014, Table 1). The sporangio-
phores of our specimen were up to 725 μm long,
but only up to 500 μm in McTaggart et al. (2015);
no sizes were given in Davis et al. (2020). The
extreme values for the lengths of the sporangia in
our measurements were identical with those in
Davis et al. (2020) and for the widths with those
of McTaggart et al. (2015). Sporangia were up to
2 μm longer in McTaggart et al. (2015) and up to
1 μm narrower in Davis et al. (2020). These data
indicate that considerable differences of sporan-
giophore lengths and slight variation of sporan-
gial sizes are not very significant. A better reso-
lution of species was obtained with molecular
data, but different DNA regions are preferred by
different authors. Compared to true fungi, the
ITS seems to be less commonly used in oomy-
cetes so that ITS data lack for many species.
Plasmopara invertifolia is a further recently de-
scribed species on Asteraceae, which was con-
sidered as sister to P. halstedii (Duarte et al.
2014). Plasmopara invertifolia was not included
in the phylogenetic analyses for comparison with
P. sphagneticolae by McTaggart et al. (2015) and
Davis et al. (2020), who considered P. sphagnet-
icolae as sister to P. halstedii. Hitherto two cox2
sequences from the original publication of P. in-
vertifolia are the single available DNA data. In
our BLAST search with one of these two se-
quences, it showed only up to 91% identity with
any other oomycete, including P. halstedii and P.
sphagneticolae. These latter two species in deed

28 Fung. Sci. 36(1): 23–32, 2021
Fig. 3. A–E. Sporangiophores and sporangia of Plasmopara sphagneticolae seen with light microscopy. A. Sketch of
sporangiophore. B. Base of sporangiophore arising from stoma in abaxial epidermis of diseased leaf. C. Upper branches of
sporangiophore. Two retraction septa (“callose plugs”) marked with arrows. D. Ultimate branchlets of sporangiophore. E.
Sporangia. Basal hilum indicated with arrow, apical papilla with arrow head. Scale bars A = 100 μm, B = 10 μm, C = 50
μm, D, E = 20 μm.

Plasmopara sphagneticolae 29
Fig. 4. Maximum likelihood phylogenetic analysis of LSU sequences of Plasmopara species. The tree was rooted with
Phytophthora tabaci. GenBank numbers are given behind the species names. Bootstrap values of 1000 replicates lower
than 50% not shown.
seem to have closer relationships to each other
than to P. invertifolia.
Geographical and host distribution
It is most likely that the oomycete originated
from the same area as the host plant in the Neo-
tropics but remained unnoticed there before be-
ing first discovered outside its natural distribu-
tion. Records of the oomycete are published from
Australia, where the host Sph. trilobata is not na-
tive, either, and from Hawaii on a native host,
Lipochaeta integrifolia (Nutt.) A. Gray (Davis et
al. 2020, McTaggart et al. 2015). Davis et al.
(2020) suggested introduction of P. sphagneti-
colae to Hawaii through introduction of Sph. tri-
lobata, where it had been introduced in the 1960s
(Thaman 1999). The Hawaiian endemic genus
Lipochaeta was considered hardly distingui-
shable from Wedelia-like taxa (Thaman 1999),
which are now split into several genera with dis-
puted boundaries (Orchard 2013). Perhaps the
parasitic oomycete may be the better taxonomist?
In Taiwan, the oomycete might have been intro-
duced together with Sph. trilobata from Hawaii
in the early 1980s. Our discovery of the oomy-
cete implies that it has been overlooked in Tai-
wan for over thirty years. Our own recent first
records of two fungi on this host in Taiwan, Po-
dosphaera xanthii (Castagne) U. Braun & Shish-
kof and Pseudocercospora wedeliae (A.K. Kar &
M. Mandal) Deighton (Kirschner 2014, Yeh et al.
2021), support the hypothesis that even many de-
scribed fungal species remain unnoticed for
many years in a given area. We roughly esti-
mated periods between 30 and 300 years between

30 Fung. Sci. 36(1): 23–32, 2021
the introduction of a potential host plant and dis-
covery of its associated introduced oomycete
(Kirschner 2013, 2015a, b, Wang et al. 2020, Yeh
et al. 2021). Knowledge of geographic distribu-
tion of fungi/oomycetes is still far behind of that
of plants. Sph. trilobata spreads over short dis-
tances mainly by clonal reproduction; it grows
and spreads very easily in its new environment
and can be artificially also grown from seeds (Qi
et al. 2014). We, therefore, assume that later in-
troductions after the first one from Hawaii have
not been necessary and after the 1980s not taken
place for the market of ornamental plants. Oo-
spores have not been found in P. sphagneticolae,
whereas long-distance dispersal through oo-
spore-contaminated sunflower seeds is a likely
route in P. halstedii on sunflower (Spring 2019).
The routes of global distribution of P. halstedii
on sunflower from North America in the 20th cen-
tury to other continents could be reconstructed
based on numerous data worldwide (Spring
2019). For P. sphagneticolae, data of host and ge-
ographic distribution are yet too scarce for allow-
ing sound conclusions about the origin and
global spread. The lack of fundamental mycolo-
gical data is a major obstacle for biogeography
and risk assessment in quarantine and nature
conservation.
Acknowledgements
The study was supported by the Ministry of Sci-
ence & Technology, Taiwan, MOST 110-2621-
B-002 -001 -MY2. We thank Dr. Yu-Hung Yeh
for technical assistance with DNA isolation and
PCR and Dr. H.D. Shin for kindly providing his
book about Peronosporaceae from Korea.
References
Davis WJ, Ko M, Ocenar JR, Romberg MK,
Crouch JA. 2020. First report of Plasmo-
para sphagneticolae on the native Hawaiian
plant Lipochaeta integrifolia. Australasian
Plant Disease Notes 15:29, 3 pp.
Duarte LL, Choi Y-J, Soares DJ, Barreto RW.
2014. Plasmopara invertifolia sp. nov. cau-
sing downy mildew on Helichrysum brac-
teatum (Asteraceae). Mycological Progress
13:285–289.
Farr DF, Rossman AY. 2021. Fungal Databases,
Systematic Mycology and Microbiology
Laboratory, ARS, USDA. Accessed Sep-
tember 2021, from http://nt.ars-grin.gov/
fungaldatabases/
Hasegawa M, Kishino H, Yano T. 1985. Dating
of the human-ape splitting by a molecular
clock of mitochondrial DNA. Journal of
Molecular Evolution 22:160–174.
Hsueh C-H, Yang Z-Y. 2014. The scenic plants
in Taiwan (5). United Distribution, Hsin-
dian, Taiwan. (in Chinese).
Kirschner R. 2013. First record of Plasmopara
obducens on Impatiens walleriana in Tai-
wan: a destructive disease or chance of limi-
ting the competitive ability of an invasive
plant? Plant Pathology and Quarantine 3:
35–39.
Kirschner R. 2014. A new species and new
records of cercosporoid fungi from orna-
mental plants in Taiwan. Mycological Pro-
gress 13:483–491.
Kirschner R. 2015a. First record of Cercospora
mikaniicola on the weedy vine Mikania
micrantha (Asteraceae) in Taiwan. Fungal
Science 30:55–60.
Kirschner R. 2015b. New records of Pseudo-
cercospora oenotherae and Synchytrium

Plasmopara sphagneticolae 31
fulgens on the invasive coastal plant Oeno-
thera laciniata in Taiwan. Plant Pathology
& Quarantine 5:26–33.
Kumar S, Stecher G, Li M, Knyaz C, Tamura K.
2018. MEGA X: Molecular Evolutionary
Genetics Analysis across computing plat-
forms. Molecular Biology and Evolution 35:
1547–549.
Kurtzman CP, Robnett CJ. 1997. Identification
of clinically important ascomycetous yeasts
based on nucleotide divergence in the 5'end
of the large-subunit (26S) ribosomal DNA
gene. Journal of Clinical Microbiology 35:
1216–1223.
McTaggart AR, Shuey LS, McKenna SG, Davis
RI, Shivas RG. 2015. Plasmopara sphagne-
ticolae sp. nov. (Peronosporales) on Sphag-
neticola (Asteraceae) in Australia. Austra-
lasian Plant Pathology 44:81–85.
Orchard AE. 2013. The Wollastonia/Melanthe-
ra/Wedelia generic complex (Asteraceae:
Ecliptinae), with particular reference to
Australia and Malesia. Nuytsia 23:337–466.
Shin HD, Choi YJ. 2006. Peronosporaceae of
Korea. National Institute of Agricultural
Science and Technology, Suwon, Korea.
Spring O. 2019. Spreading and global pathogenic
diversity of sunflower downy mildew –
Review. Plant Protection Science 55:149–
158.
Thaman RR. 1999. Wedelia trilobata: Daisy in-
vader of the Pacific Islands. IAS Technical
Report 99/2. Institute of Applied Science,
University of the South Pacific, Suva, Fiji
Islands, 10 pp.
Voglmayr H, Riethmüller A, Göker M, Weiss M,
Oberwinkler F. 2004. Phylogenetic rela-
tionships of Plasmopara, Bremia and other
genera of downy mildew pathogens with
pyriform haustoria based on Bayesian ana-
lysis of partial LSU rDNA sequence data.
Mycological Research 108(9):1011–1024.
Wang CT, Yeh YW, Lin LD, Kirschner R. 2020.
First record of Erysiphe magnifica on the
new host Magnolia × alba in Taiwan indi-
cates high morphological plasticity of the
anamorph under tropical conditions. Plant
Pathology & Quarantine 10:59–65.
Wu S-H, Hsieh C-F, Rejmánek F. 2004. Cata-
logue of the Naturalized Flora of Taiwan.
Taiwania 49:16–31.
Yeh Y-W, Kirschner R, Lu H-F. 2019. First
Record of Erysiphe elevata on Plumeria ru-
bra in Taiwan. Plant Disease 103:371.
Yeh Y-W, Wu T-Y, Wen H-L, Jair H-W, Lee M-
Z, Kirschner R. 2021. Host plants of the
powdery mildew fungus Podosphaera xan-
thii in Taiwan. Tropical Plant Pathology 46:
44–61.

32 Fung. Sci. 36(1): 23–32, 2021
Plasmopara sphagneticolae 於臺灣的首次紀錄
王介鼎 1、林蘭東 2、葉昱緯 3、羅南德 3*
1國立臺灣大學實驗林管理處，竹山鎮，南投縣，臺灣
2國立雲林科技大學文化資產維護系，斗六市，雲林縣，臺灣
3國立臺灣大學森林環境暨資源學系，大安區，臺北市，臺灣
摘 要
南美蟛蜞菊為一種常見於公共綠地的觀賞性地被植物之一，在臺灣同時也是一種外來入侵種。於臺北市，在此植
物上發現了露菌病之病徵，而後基於宿主、形態學以及核糖體大亞基 RNA 基因 (LSU)序列分析，將該菌種鑑定
為Plasmopara sphagneticolae。本文亦針對此卵菌隨其宿主自夏威夷最可能的輸入途徑進行討論。
關鍵詞：菊科、卵菌-宿主分布、植物病、核糖體 DNA、蟛蜞菊屬