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Notes on powdery mildews (Erysiphales) in Japan:II. Erysiphe sect. Microsphaera

Jamjan Meeboon a,b, Susumu Takamatsu a,*

a Department of Bioresources, Graduate School, Mie University, 1577 Kurima-Machiya, Tsu 514-8507, Japanb Department of Agriculture, Ministry of Agriculture and Cooperatives, 50 Phaholyothin Rd., Ladyao, Chatuchak,

Bangkok, Thailand

a r t i c l e i n f o

Article history:

Received 12 May 2014

Received in revised form

26 June 2014

Accepted 26 June 2014

Available online 14 August 2014

Keywords:

Erysiphaceae

Molecular phylogeny

Morphology

New host

Pseudoidium

* Corresponding author. Tel.: þ81 59 231 949E-mail address: takamatu@bio.mie-u.ac.j

http://dx.doi.org/10.1016/j.myc.2014.06.0071340-3540/© 2014 The Mycological Society of

a b s t r a c t

Several novelties of powdery mildews in Japan are reported, including the first Japanese

collection of the teleomorph of Erysiphe platani on Platanus � acerifolia, the first description

of ascomata of E. viburni on Viburnum sieboldii var. obovatifolium, and the first morphological

descriptions and molecular data for E. wallrothii on Leucotho€e grayana and Pseudoidium

hortensiae on Hydrangea serrata var. megacarpa based on Japanese specimens.

© 2014 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.

This is the second part of a series dealing with new records

and data on powdery mildews in Japan. In this report, four

powdery mildew species belonging to Erysiphe sect. Micro-

sphaera and its Pseudoidium anamorphs are described and

illustrated, and supplementary molecular data were obtained.

The nucleotide sequences of the 50-end of the 28S rRNA

gene (including domains D1 and D2) and the internal tran-

scribed spacer (ITS) regions were determined according to the

procedure of Meeboon and Takamatsu (2014). Representative

new sequences determined in this study were deposited in

DNA Data Base of Japan (DDBJ) under the accession numbers

AB926018eAB926022. Sequences generated from the 28S rRNA

gene and ITS region were aligned with other sequences of the

7; fax: þ81 59 231 9540.p (S. Takamatsu).

Japan. Published by Else

Erysiphaceae that were retrieved from DNA databases (DDBJ,

EMBL, NCBI) using MUSCLE (Edgar 2004) implemented in

MEGA 5 (Tamura et al. 2011). Alignments were then manually

refined using the MEGA 5 program and were deposited in

TreeBASE (http://www.treebase.org/) under the accession

number S15710. Phylogenetic trees were obtained from the

data using maximum parsimony (MP) method and maximum

likelihood (ML) method as described in Meeboon and

Takamatsu (2014).

The 28S rRNA gene for three specimens of Erysiphe and

Pseudoidium were determined in this study. The three se-

quences were aligned with 28 Erysiphe sequences retrieved

from the DNA databases. Erysiphe glycines F.L. Tai (AB022397)

vier B.V. All rights reserved.

Erysiphe trifoliorum ex Trifolium AB103078

Erysiphe multappendicis ex Berberis AB103076

Erysiphe cruciferarum ex Cardaria AB102944

Erysiphe lycopsidis ex Anchusa AB103072

Erysiphe heraclei ex Chaerophyllum AB103067

Erysiphe heraclei ex Daucus AB022391

Erysiphe bremeri ex Alhagi AB103077

Erysiphe friesii ex Rhamnus AB022382

Erysiphe wallrothii ex Vaccinium AB015930

Erysiphe wallrothii ex Leucothoe AB926020

Erysiphe alphitoides ex Quercus AB257431

Erysiphe monascogera ex Styrax AB331645

Erysiphe nomurae ex Symplocos AB331648

Erysiphe quercicola ex Quercus AB292694

Erysiphe hypophylla ex Quercus AB292716

Erysiphe syringae ex Syringa AB015920

Erysiphe platani ex Platanus AB926022

Erysiphe abbreviata ex Quercus AB271785

Erysiphe epigena ex Quercus AB292722

Erysiphe hypogena ex Quercus AB292727

Erysiphe corylopsidis ex Corylopsis AB478988

Erysiphe pulchra var. pulchra ex Cornus AB015935

Erysiphe paeoniae ex Paeonia AB257438

Erysiphe ligustri ex Ligustrum AB015917

Erysiphe aquilegiae ex Cimicifuga AB022405

Pseudoidium hortensiae ex Hydrangea AB926018

Pseudoidium sp. ex Medicago AB102942

Erysiphe staphyleae ex Staphylea AB015922

Erysiphe vanbruntiana ex Sambucus AB015925

Erysiphe pulchra var. japonica ex Cornus AB022389

Erysiphe glycines ex Desmodium AB022397

5 changes

70/75

75/84-/95

-/76

76/87

92/89

87/80

92/99

28S rRNA gene31 sequences773 characters191 stepsCI = 0.5550RI = 0.6458RC = 0.3584

Fig. 1 e Phylogenetic analysis of the divergent domains D1 and D2 sequences of the 28S rRNA gene for 31 sequences from

the genus Erysiphe. This tree is a phylogram of one of the 1107 most parsimonious trees with 191 steps, which was found

using a heuristic search. Horizontal branch lengths are proportional to the number of substitutions that were inferred to

have occurred along a particular branch of the tree. BS (>70%) values by the maximum parsimony (MP) and maximum

likelihood (ML) methods were shown on the respective branch. Sequences determined in this study are shown in boldface.

The GenBank number for each sequence is presented after species name on the tree.

myc o s c i e n c e 5 6 ( 2 0 1 5 ) 2 3 0e2 3 6 231

was used as outgroup according to Mori et al. (2000). The

alignment data matrix consisted of 31 sequences and 773

characters, of which 91 (11.8%) characters were variable and

55 (7.1%) characters were informative for parsimony

analysis. A total of 1107 equally parsimonious trees with

191 steps were constructed using MP analysis. One of the

1107 trees with the highest log likelihood value is shown in

Fig. 1.

Erysiphe platani ex Platanus AB926022

Erysiphe platani ex Platanus KF499270

Erysiphe platani ex Platanus JX997805

Erysiphe syringae ex Syringa AB015920

Erysiphe syringae ex Ligustrum AB295461

Erysiphe magnifica ex Magnolia AF011312

Erysiphe magnifica ex Magnolia GU195046

Erysiphe liriodendri ex Liliodendron AF011302

Erysiphe abbreviata ex Quercus AB271785

Erysiphe castaneigena ex Castanea AF298545

Erysiphe quercicola ex Quercus AB292691

Erysiphe quercicola ex Quercus AB292694

Erysiphe alphitoides ex Quercus AB237783

Erysiphe pseudolonicerae ex Cocculus AB015915

Erysiphe wallrothii ex Vaccinium AB015930

Erysiphe wallrothii ex Leucothoe AB926021

Erysiphe epigena ex Quercus AB292720

Erysiphe hypogena ex Quercus AB292724

Erysiphe monascogera ex Stylax AB331646

Erysiphe hypophylla ex Quercus AB292715

Pseudoidium neolycopersici ex Lycopersicon AB032484

Pseudoidium neolycopersici ex Lycopersicon AB034722

Pseudoidium hortensiae ex Hydrangea AB926019

Erysiphe sedi ex Sedum JX173290

Pseudoidium hortensiae ex Hydrangea JQ669944

Erysiphe aquilegiae ex Cimicifuga AB000944

Erysiphe aquilegiae ex Clematis AB015929

Erysiphe macleayae ex Macleaya AB016048

Erysiphe aquilegiae ex Clematis AF154322

Erysiphe circaeae ex Circaea AB104517

Erysiphe paeoniae ex Paeonia AB257437

Erysiphe paeoniae ex Paeonia AB257436

Erysiphe glycines ex Desmodium AB015927

5 changes

100/100

93/90

100/100

100/100

100/99

100/100

-/78

ITS33 sequences579 characters217 stepsCI = 0.7604RI = 0.8632RC = 0.6563

Fig. 2 e Phylogenetic analysis of the ITS region for 33 sequences from the genus Erysiphe. This tree is a phylogram of one of

the 1414 most parsimonious trees with 217 steps, which was found using a heuristic search. Horizontal branch lengths are

proportional to the number of substitutions that were inferred to have occurred along a particular branch of the tree. BS

(>70%) values by the maximum parsimony (MP) and maximum likelihood (ML) methods were shown on the respective

branch. Sequences determined in this study are shown in boldface. The GenBank number for each sequence is presented

after species name on the tree.

my c o s c i e n c e 5 6 ( 2 0 1 5 ) 2 3 0e2 3 6232

For the ITS analysis, the three sequences determined in

this study were aligned with 30 Erysiphe sequences retrieved

from the DNAdatabases. Erysiphe glycines (AB015927)was used

as outgroup according to Takamatsu et al. (1999). The align-

ment data matrix consisted of 33 sequences and 579

characters, of which 139 (24.0%) characters were variable and

77 (13.3%) characters were informative for parsimony anal-

ysis. A total of 1414 equally parsimonious trees with 217 steps

were constructed using MP analysis. One of the 1414 trees

with the highest log likelihood value is shown in Fig. 2. The

Fig. 3 e Teleomorph of Erysiphe platani on Platanus £ acerifolia (MUMH5657). A: Chasmothecium. B: Asci. C, D: Appendages.

Bars: A 100 mm; B 60 mm; C, D 50 mm.

myc o s c i e n c e 5 6 ( 2 0 1 5 ) 2 3 0e2 3 6 233

phylogenetic trees generated using ML analysis were almost

identical to the MP trees in both 28S rRNA gene and ITS ana-

lyses, and thus only the bootstrap values were shown on the

MP trees.

Erysiphe platani (Howe) U. Braun & S. Takam., Schlechtendalia

4: 12, 2000. Fig. 3.

Description: Mycelium on leaves, amphigenous, dense, white,

forming patches, confluent, evanescent to persistent. Chas-

mothecia scattered to gregarious, (90e)97e119(e126) mm

diam. Peridium composed of irregularly polygonal cells,

10e20 mm diam. Appendages 9e16 per chasmothecium,

equatorial, straight to curved, 1e2 times as long as the chas-

mothecial diam., 7e10 mm wide, 0e1-septate, hyaline, brown

at the base, walls smooth to rough, thin in the upper half,

thick towards the base. Apices 4e6 times densely and regu-

larly dichotomously branched, rarely trichotomously, occa-

sionally forked in the lower half, tips recurved. Asci 3e6 per

chasmothecium, broadly ellipsoid-obovoid, saccate, (47e)

52e60(e64) � (38e)41e43(e46) mm, short-stalked, 4e6-spored.

Ascospores ellipsoid-ovoid, hyaline, (16e)18e21(e23) � (9.5e)

11e12(e14) mm.

Collection examined: on Platanus � acerifolia (Aiton) Willd.

(Platanaceae), Japan, Mie Pref., Mie University, 1 Dec 2012, S.

Takamatsu, MUMH5657, GenBank accession number:

AB926022 (ITS and 28S rRNA gene).

Remarks: Erysiphe plataniwas first described byHowe (1874)

as Microsphaera platani based on a specimen found on Pl. occi-

dentalis L. from U.S.A. It is likely that the fungus was then

introduced to South America, South Africa, Europe, and Asia

(Braun and Cook 2012). In Japan, powdery mildew on Platanus

spp. was first found in Tokyo in 1997 (Tanda 1999; Horie et al.

2002), but was found only in anamorphic stage. The ITS

sequence obtained from E. platani on Pl. � acerifolia was iden-

tical to that of E. platani (JX997805) collected in Korea and

differed only one base from KF499270 collected in Brazil.

These three sequences formed a distinct clade with high

bootstrap supports (100% in both MP and ML analyses; Fig. 2).

Erysiphe viburni Duby, Bot. Gall. 2: 872, 1830. Fig. 4.

Description: Mycelium amphigenous, evanescent. Chas-

mothecia (79e)84e101(e108) mm diam., scattered, globose to

subglobose, dark brown. Peridium composed of dark brown,

irregularly polygonal cells, 15e25 mm diam. Appendages 4e9

per chasmothecium, 1e1.5 times as long as the chasmothecial

diam., ± equatorial, flaccid or loosely curved, 0(e1)-septate,

completely hyaline or pigmented light brown at the base, thin-

walled, smooth. Apices 3e5 times irregularly to dichoto-

mously branched, often loosely branched, primary branches

somewhat elongated, tips straight. Asci 2e4 per chasmothe-

cium, (32e)38e63(e70) � (35e)47e54(e60) mm, 3e5-spored,

broadly obovoid to subglobose, short-stalked or subsessile.

Ascospores (18e)19.5e24(e25.5) � 10e15(e16.5) mm, ellipsoid-

ovoid, hyaline. Anamorph not observed.

Collection examined: on Viburnum sieboldii Miq. var. obo-

vatifolium (Yanagita) Sugimoto (Adoxaceae), Japan, Niigata

Pref., Mt. Yahiko, 18 Oct 1996, S. Takamatsu, MUMH263.

Remarks: Erysiphe viburni is a powdery mildew species

commonly found on Viburnum spp. (Adoxaceae) in the

Northern Hemisphere. Occurrence of powdery mildew on V.

sieboldii var. obovatifolium was reported by Hirata and Wada

(1973) and Sato (1980) as Microsphaera sp. but without a

morphological description. Thus, the morphology of chas-

mothecia of this species occurring on this host was examined

in detail. In comparison to the description of Braun and Cook

(2012), the specimen occurring on V. sieboldii var. obovatifolium

has smaller chasmothecia and ascospores, and less number of

asci. DNA sequencing of this collection failed.

Erysiphe wallrothii (U. Braun & Tanda) U. Braun & S. Takam.,

Schlechtendalia 4: 15, 2000. Fig. 5.

Description: Mycelium amphigenous, in patches or effuse,

white, evanescent to persistent. Hyphae 3e6 mm wide. Chas-

mothecia (114e)118e120(e141) mm diam., globose, dark

brown, scattered. Peridium 9e16 mm thick, composed of dark

brown, irregularly polygonal cells, 17e30 mm diam. Append-

ages 5e8 per chasmothecium, (56e)97e148(e226) � (5.5e)

Fig. 4 e Teleomorph of Erysiphe viburni on Viburnum sieboldii var. obovatifolium (MUMH263). A, B: Chasmothecium. CeF:

Ascus. GeJ: Appendages. Bars: A, B 100 mm; CeF 25 mm; GeJ 30 mm.

my c o s c i e n c e 5 6 ( 2 0 1 5 ) 2 3 0e2 3 6234

7e10 mm, 1e2 times as long as the chasmothecial diam.,

equatorial, stiff to flexuous, aseptate or with a single septum

at the base, hyaline, walls thick at the base, thin towards the

apex, smooth to somewhat rough. Apices 4e6 times regularly

and tightly dichotomously branched, rarely forked in the

lower half, tips distinctly recurved. Asci 4e6 per chasmothe-

cium, (35e)46.5e63(e67) � (37e)40e47(e50.5) mm, 5e7-spored,

broadly ellipsoid-obovoid to saccate, sessile or short-stalked.

Ascospores 17e24(e26) � 8e13 mm, ellipsoid to ovoid, hyaline.

Collections examined: on Leucotho€e grayana Maxim. (Erica-

ceae), Japan, Mie Pref., Mt. Miike, Happu Pass, 28 Oct 2007, S.

Takamatsu, MUMH4752, GenBank accession number:

AB926021 (ITS), AB926020 (28S rRNA gene); Mie Pref., Mt.

Kama-ga-take, 27 Oct 2002, S. Takamatsu, MUMH2152.

Remarks: Powderymildew occurring on L. grayanawas first

reported by Takamatsu (1982) as Microsphaera sp. without a

morphological description. Thus, morphological characteris-

tics of E. wallrothii collected from this host were examined in

detail, supplemented with rRNA gene sequence data. Molec-

ular analyses showed that the powdery mildew found on L.

grayana is 100% identical to E. wallrothii occurring Vaccinium

hirtum (AB015930) in both 28S rRNA gene and ITS sequences

(Figs. 1, 2).

Pseudoidium hortensiae (Jørst.) U. Braun & R.T.A. Cook, Taxo-

nomic Manual of the Erysiphales (Powdery Mildews): 606,

2011. Fig. 6.

Description:Myceliumamphigenous,white, indense or thin

patches, irregular, evanescentorpersistent.Hyphalappressoria

lobed, solitary or in opposite pairs. Conidiophores arising cen-

trally from the upper surface of mother cells, erect, (48.5e)

56e75.5(e91) mm long. Foot cells cylindrical, straight, (21e)

Fig. 5 e Teleomorph of Erysiphe wallrothii on Leucotho€e grayana (MUMH4752). A: Chasmothecium. BeE: Ascus. FeJ:

Appendages. Bars: A 120 mm; BeE 20 mm; FeJ 30 mm.

Fig. 6 e Pseudoidium hortensiae on Hydrangea serrata var. megacarpa (MUMH281). A: Conidia. B: Conidiophores. C:

Appressoria. Bar: 20 mm.

myc o s c i e n c e 5 6 ( 2 0 1 5 ) 2 3 0e2 3 6 235

my c o s c i e n c e 5 6 ( 2 0 1 5 ) 2 3 0e2 3 6236

23e25(e33.5) � 7e10 mm, followed by 0e3 shorter cells, with a

basal septum at the branching point of the mycelium, 1e3 mm,

forming conidia singly. Conidia ellipsoid-ovoid, (25.5e)

29.5e36.5(e39.5) � 12e16(e18) mm. Germ tube not observed.

Collections examined: on Hydrangea serrata (Thunb. ex

Murray) Ser. var. megacarpa (Ohwi) H. Ohba (Hydrangeaceae),

Japan, Niigata Pref., Mt. Yahiko, 8 Oct 1996, S. Takamatsu,

MUMH221; Niigata Pref., Mt. Yahiko, 18 Oct 1996, S. Taka-

matsu, MUMH281, GenBank accession number: AB926019

(ITS), AB926018 (28S rRNA gene).

Remarks: Hirata and Wada (1973) reported occurrence of

Microsphaera sp. on H. serrata var. megacarpa without a

morphological description. Molecular analyses showed that

the ITS sequence of the powdery mildew occurring on H. ser-

rata var.megacarpa is identical to that ofOidium hortensiae (¼Ps.

hortensiae) collected from H. macrophylla (JQ669944) (Fig. 2).

Acknowledgments

This work was financially supported in part by a Grant-in-Aid

for Scientific Research (No. 23580061) from the Japan Society

for the Promotion of Science, a grant from the Institute for

Fermentation, Osaka, Japan to ST, and MONBUKAGAKU SHO:

MEXT (Ministry of Education, Culture, Science, and Technol-

ogy) Scholarship of the Japanese Government awarded to JM.

r e f e r e n c e s

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