Title

The host-pathogen association of Exobasidium in Japan inferred from molecularphylogeny of ITS and large subunit rDNA sequences
Sub Title
ITS領域とrDNA D1/D2領域のシークエンスより解析した日本産もち病菌の宿主病原菌関連性
Author長尾, 英幸(Nagao, Hideyuki)
糟谷, 大河(Kasuya, Taiga)
Publisher
慶應義塾大学日吉紀要刊行委員会
Publication year 2023
Jtitle慶應義塾大学日吉紀要. 自然科学 (The Hiyoshi review of naturalscience). No.70 (2023. 9) ,p.1- 21
JaLC DOI
Abstract
Phylogenetic relationships among Exobasidium species were estimated by the
concatenated ITS and LSU sequences of 72 isolates obtained from fresh material.
The phylogenetic tree suggested 6 host-specific groups for Exobasidium spp. withhigh support values. A Rhododendron clade comprises three groups: Group 1
includes two witches' broom causal species, E. nobeyamense and E.
pentasporium, that were phylogenetically distinguished, while E. nobeyamense, E.
otanianum and E. cylindrosporum were unresolved. Group 2 comprised E.
japonicum and three species that are pathogenic to subgenus Hymenanthes
plants. Group 3 comprised species pathogenic to section Rhododendron andTsutsusi plants that cause leaf blister. Group 4 comprised five species pathogenic
to Vaccinioideae. Groups 5 and 6 are characterized by host specificity to Camellia
and Symplocos, respectively. The six groups are independent of morphologicalcharacteristics for basidiospores and colony appearances. The concatenated ITS
and LSU sequences could be used to predict host preference of Exobasidium
isolates.
Notes
原著論文
Genre
Departmental Bulletin Paper
URL
https://koara.lib.keio.ac.jp/xoonips/modules/xoonips/detail.php?koara_id=AN10079
809-20230930-0001
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The Host-Pathogen
Association
Exobasidium Sciencein Japan Inferred from Molecular Phylogeny of ITS and Large Subunit rDNA Sequences
Hiyoshi
Reviewof ofNatural
Keio University No. 70, 1-21（2023）（NAGAO・KASUYA）

The Host-Pathogen Association of Exobasidium in Japan
Inferred from Molecular Phylogeny of ITS andLarge Subunit rDNA Sequences

Hideyuki NAGAO, ＊，1），2）
Taiga KASUYA ＊＊ITS 領域と rDNA D1/D2領域のシークエンスより解析した
日本産もち病菌の宿主―病原菌関連性

長尾英幸・糟谷大河

Summary―Phylogenetic relationships among Exobasidium species were estimated by
the concatenated ITS and LSU sequences of 72 isolates obtained from fresh material.
The phylogenetic tree suggested 6 host-specific groups for Exobasidium spp. with highsupport values. A Rhododendron clade comprises three groups: Group 1 includes two
witches’ broom causal species, E. nobeyamense and E. pentasporium, that were phylogenetically distinguished, while E. nobeyamense, E. otanianum and E. cylindrosporum
were unresolved. Group 2 comprised E. japonicum and three species that are pathogenic to subgenus Hymenanthes plants. Group 3 comprised species pathogenic to section
Rhododendron and Tsutsusi plants that cause leaf blister. Group 4 comprised five species pathogenic to Vaccinioideae. Groups 5 and 6 are characterized by host specificity
to Camellia and Symplocos, respectively. The six groups are independent of morphological characteristics for basidiospores and colony appearances. The concatenated ITS
and LSU sequences could be used to predict host preference of Exobasidium isolates.
Key Words: Basidiomycetes, Exobasidium, Ericaceae, Japan, molecular phylogenetic analysis
農業生物資源ジーンバンク（〒 305-8602 つくば市観音台 2-1-2）：The NARO Genebank, Tsuku：
ba 305-8602, Japan, 現所属 1）神奈川県立地球博物館外来研究員（〒 250-0031 小田原市入生田 499）
Present address: 1）Visiting researcher, Kanagawa Prefectural Museum of Natural History, 499 Iriuda, Odawara 250-0031, Japan, 2）慶應義塾大学自然科学研究教育センター訪問研究員（〒 223-8521 横浜市港北区日吉 4-1-1）：2 ) Visiting researcher, Research and Education Center for Natural Sciences,
Keio University, 4-1-1 Hiyoshi, Kohoku, Yokohama 223-8521, Japan. e-mail: m0ch1_byo@keio.jp＊＊
慶應義塾大学経済学部生物学教室（〒 223-8521 横浜市港北区日吉 4-1-1）：Faculty of Economics, Keio University, 4-1-1 Hiyoshi, Kohoku, Yokohama 223-8521, Japan.
＊

この著作物は著作権法によって保護されています／ This content is protected by the Japanese Copyright Act

1

慶應義塾大学日吉紀要・自然科学

No. 70（2023）

1. Introduction
Members of the Exobasidiales are known as plant parasites and are classified in fourfamilies (Brachbasidiaceae, Exobasidiaceae, Cryptobasidiaceae and Graphiolaceae)
based on mode of sporulation (Bauer et al. 2001). In the Exobasidiaceae, the parasitism
of Exobasidium is recorded on different host plants in the families Escalloniaceae (Gómez and Kisimova-Horovitz, 1997, 1998), Ericaceae, Saxifragaceae, Symplocaceae and
Theaceae (Nannfeldt, 1981 ; Savile, 1959 a; Ezuka, 1990 a, 1990 b, 1991 a, 1991 b). In Oceania and South America, Ericaceae subfamily Styphelioideae (Chlebicki and Chlebická,
2007 ; McNabb, 1962) is also described as including host plants. Exobasidium induces different types of symptoms, such as galls on leaves, buds, flowers, fruits, and trunks, leaf
blisters and blast, shoestring leaf, and witches’ bloom (Fig. 1). A causal relationship is
not found between the plant taxa and symptoms. Rather, the symptom appearance is
specific to the part of the plant infected on different host plants.
Exobasidiales is characterized by morphologically diverse taxa (Begerow et al., 1997 ;Bauer et al., 2001). The taxonomy of Exobasidium has been particularly controversial
due to their simple morphology, the variable symptoms they induce, and their wide hostrange (Burt, 1915 ; Ezuka, 1990 b; Nannfeldt, 1981 ; McNabb, 1962 ; Savile, 1959 a; Sundström, 1964). Savile (1959 a) synonymized many Exobasidium species isolated from different host plants into a few species based on unicellular or multicellular basidiospores.
Nannfeldt (1981), however, based species differentiation on the mode of basidiospore
germination and cultural characteristics, which were previously studied by Sundström(1964). According to Nannfeldt-Sundström’s morphological species concept, Japanese
Exobasidium species were re-assessed by comparing the morphology of basidia, basidiospores and sterigmata, and the mode of basidiospore germination (Nagao et al. 2001,
2003 a, 2003 b, 2004 a, 2004 b, 2006). The successful inoculation tests by the isolates of
Exobasidium on the cultivated plants of Camellia, Rhododendron, and Vaccinium species
were reported (Ezuka, 1955 ; Graafland, 1960 ; Sundström, 1964 ;Nickerson and Vander
Kloet, 1997). Sundström (1964) confirmed difference in pathogenicity of E. vaccinii “vit.
id” to the susceptible V. vitis-idaea L.
Molecular analyses of the nuclear LSU rDNA have supported the monophyly of thisgroup (Begerow et al. 1997 ; Bauer et al. 2001), whereas those from 18 S rDNA did not,even when only three species of Exobasidium were examined (Döring and Blanz 2000). A
controversial species on Saxifraga, Arctiomyces warmingii (Rostr.) Savile, was placed in
Exobasidiaceae using LSU molecular analysis (Begerow et al., 2002). Savile (1959 b) erect2

The Host-Pathogen Association of Exobasidium in Japan Inferred from Molecular Phylogeny of ITS and Large Subunit rDNA Sequences（NAGAO・KASUYA）

Fig. 1. Symptoms caused by Exobasidium. A. Gall on leaves of Rhododendron, B. Gall on flower
of Vaccinium, C. Leaf blister of Rhododendron, D. Leaf blister blight of Camellia, E. Witches’
broom of Rhododendron, F. Shoot blight of Symplocos, G. Fruit deformation of Symplocos, H.
Gall on leaf of Rhododendron.
3慶應義塾大学日吉紀要・自然科学

No. 70（2023）

ed the new genus Arctiomyces based on the intermediate morphological characteristics
between Exobasidium and Kordyana. The legitimate name for this species is Exobasidium warmingii Rostr. according to its morphology (Donk, 1966 ; Müller, 1977). Even
though Begerow et al. (2002) did not refer to these studies, molecular analysis supported
the position of this species in Exobasidiaceae with 78 % bootstrap value by neighbor-joining analysis. On the other hand, Exobasidium lauri Geyl. on Laurus novocanariensis Rivas Martínez, Lousã, Fernández Prieto, Días, Costa & Aguiar was excluded andmoved to Laurobasidium in Cryptobasidiaceae supported by molecular re-assessments(Begerow et al. 2002). Laurobasidium hachijoense (Y. Otani, Kakish. & Iijima) Kakish.,Nagao & Denchev is recently legitimately described as the new combination of E. hachijoense Y. Otani et al. by the morphological evidence and molecular analysis (Kakishima
et al., 2017). The phylogenetic position of this species has been placed in Cryptobasidiaceae (Clinoconidium spp. and L. lauri) by other researchers in the provisional name of‘Laurobasidium hachijoense’(Maier et al., 2006 ; Wang et al., 2015).
Piątek et al. (2012) chose the way of Nannfeldt-Sundström’s morphological analyses(Nannfeldt, 1981 ; Sundström, 1964) and applied the concatenated ITS+LSU sequence
tree for the speciation of Exobasidium darwinii M. Piatek & M. Lutz, which systemicallyinfects Vaccinium reticulatum Sm. The concatenated ITS+LSU sequence tree resolved
the relationships within the cluster of six species that were unresolved using only LSU
sequences. Kennedy et al. (2012) adopted the concatenated ITS+LSU sequence tree forthe identification of new species Exobasidium ferrugineae Minnis, Kenn. & Goldberg on
flowers of Lyonia ferruginea (Michx.) G.S. Torr.
For species identification, Nannfeldt-Sundström’s morphological analyses was supported by the molecular analysis using the concatenated ITS+LSU (Kennedy et al., 2012 ;Piątek et al., 2012), whereas two different pathogenic Exobasidium species were recognized on one host plant either Rhododendron or Vaccinium (Nannfeldt, 1981). Japanese
Exobasidium species on Rhododendron species were much better documented thanthose on Vaccinium species. We picked up the examples on Rhododendron species andSymplocos lucida Sieb. et Zucc.; i.e. E. japonicum Shirai, E. pentasporium Shirai, and E.
japonicum var. hypophyllum Ezuka on R. kampferi Planch., E. yoshinagae P. Henn. andE. nobeyamense Nagao & Ezuka on R. wadanum Makino, E. yoshinagae and E. otanianum Ezuka emend. Nagao on Rhododendron subgen. Tsutsusi, E. cylindrosporum Ezuka
and E. kawaense Ezuka on R. macrosepalum Maxim., and E. symploci-japoniccae Kusano & Tokubuchi and E. symploci-japoniccae var. caprogenum Nagao & S. Ogawa on S.
lucida. The focus of the present study was to clarify the host-parasite and phylogenetic
relationships with Japanese Exobasidium isolates using ITS and LSU sequences adopted
4The Host-Pathogen Association of Exobasidium in Japan Inferred from Molecular Phylogeny of ITS and Large Subunit rDNA Sequences（NAGAO・KASUYA）

by Piątek et al. (2012).

2. Materials and methods
Eighty-eight Japanese Exobasidium isolates and 7 Cryptobasidiales, i.e., four isolatesof Clinoconidium spp. and three isolates of L. hachijoense, were used to extract DNA as
described below. A total of 70 ITS and 82 LSU sequences were prepared (Table 1). Twenty-six sequences were retrieved from the GenBank (Table 2). ITS (Acc. No. AY854090)
and LSU (Acc. No. L20287) data of Ustilago maydis (DC.) Corda were used as an outgroup.
The fungal DNA was extracted using the sodium dodecyl sulfate (SDS) extraction procedure by Suyama et al. (1996) and PCR amplification profiles by Virtudazo et al. (2001)
and Takeuchi and Nagao (2004) were used with slight modifications. Fungal samples
were scraped from the surface of 14-day-old colonies and incubated in 20 μL extraction
buffer containing 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, 0.01 % Proteinase
K, and 0.01 % SDS for 1 h at 37 °C and then held for 10 min at 95 °C. The extract wasthen centrifuged at 6,000 rpm and the supernatant was subsequently used as the template for PCR amplification. PCR was performed using a HotstartTaq master Mix (Qiagen, Hilden, Germany). The primers ITS 1 F (Gardes and Bruns, 1993) and ITS 4 (Whiteet al. 1990) were used for the ITS regions and NL1 and NL4 (O’Donnell, 1993) for the D1 /D2 regions of LSU rDNA (Table 3). The PCR amplification profile was set as follows: an
initial denaturation step for 15 min at 95 °C; followed by 35 cycles of 30 s denaturation at
94 °C, 1 min annealing at 55 °C, and 1 min extension at 72 °C; with a final extension period of 10 min at 72 °C. PCR products were then electrophoresed on 1 % (w/v) agarosegels containing 0.5 µg/mL ethidium bromide in TAE buffer composed of 40 mM Tris, 20
mM sodium acetate, and 1 mM EDTA, pH 7.4, to confirm successful amplification. PCR
products were purified with MicroSpin columns S-400 HR (Amersham Biosciences Corp.,
Piscataway, NJ, USA) according to the manufacture’s protocol. Amplified products weresequenced with a BigDye dye terminator kit (Applied Biosystems, Foster City, CA, USA).
AutoSeq G-50 (Amersham Biosciences Corp., Piscataway, NJ, USA) was used to remove
excess fluorescent dye-terminators from cycle sequencing reactions prior to analysis on
an ABI 377 or ABI 3100 automated DNA sequencer (Perkin Elmer Co., Foster, CA, USA).
Multiple DNA sequences were initially aligned independently for both genes using
Clustal W ver. 2.1 on the BBDJ web site (http://clustalw.ddbj.nig.ac.jp/index.php?lang=ja) with default settings and the results were output to a NEXUS file. Phylogenetic analysis was conducted by PAUP* 4.3. 99.169.0 (Swofford, 1998). Gaps in sequences were treat5

6

Exobasidium otanianum
Exobasidium otanianum
Exobasidium shiraianum
Exobasidium bisporum
Exobasidium reticulatum
Exobasidium reticulatum
Exobasidium japonicum
Exobasidium symploci-japonicae var. symploci-japonicae
Exobasidium bisporum
Exobasidium yoshinagae
Exobasidium otanianum
Exobasidium pieridis-ovalifoliae
Exobasidium kishianum
Exobasidium japonicum
TSH-B0009
Exobasidium japonicum
TSH-B0011
Exobasidium cylindrosporum
TSH-B0127
Exobasidium cylindrosporum
TSH-B0015
Exobasidium pentasporium
Laurobasidium hachijoense
Exobasidium camelliae
Exobasidium cylindrosporum
TSH-B0107
Exobasidium dubium
TSH-B0074
Exobasidium dubium
TSH-B0076
Exobasidium dubium
TSH-B0077
Exobasidium miyabei
TSH-B0017
Exobasidium gracile
TSH-B0108
Exobasidium gracile
TSH-B0069
Exobasidium japonicum
TSH-B0046
Exobasidium japonicum
TSH-B0063
Exobasidium japonicum
TSH-B0072
Exobasidium japonicum
Exobasidium japonicum
Exobasidium japonicum
TSH-B0125
Exobasidium miyabei
TSH-B0075
Exobasidium miyabei
TSH-B0087
Exobasidium nobeyamense
TSH-B0001
Exobasidium nobeyamense
TSH-B0002
Exobasidium nobeyamense
TSH-B0004
Exobasidium nobeyamense
TSH-B0003
Exobasidium pentasporium
Exobasidium pentasporium
Exobasidium shiraianum
TSH-B0023
Exobasidium shiraianum
TSH-B0024
Exobasidium shiraianum
TSH-B0025
Exobasidium symploci-japonicae var. symploci-japonicae TSH-B0040
Exobasidium yoshinagae
Exobasidium yoshinagae
Exobasidium cylindrosporum
TSH-B0012

EOS44 (K44)
EOS59 (K59)
IFO 30151
IFO 30152
IFO 30393
IFO 30394
IFO 30756
IFO 7790
IFO 9942
IFO 9959
IFO 9960
IFO 9961
IFO 9962
MAFF 238175
MAFF 238176
MAFF 238177
MAFF 238178
MAFF 238179
MAFF 238330
MAFF 238578
MAFF 238579
MAFF 238580
MAFF 238581
MAFF 238582
MAFF 238583
MAFF 238585
MAFF 238586
MAFF 238587
MAFF 238588
MAFF 238589
MAFF 238590
MAFF 238591
MAFF 238592
MAFF 238594
MAFF 238595
MAFF 238596
MAFF 238597
MAFF 238598
MAFF 238599
MAFF 238600
MAFF 238601
MAFF 238602
MAFF 238603
MAFF 238604
MAFF 238605
MAFF 238606
MAFF 238607
MAFF 238608

Voucher

Species

IsolateRhododendron dilatatum var. satsumense
Rhododendron dilatatum var. satsumense
Rhododendron degronianum
Eubotryoides grayana var. oblongifolia
Camellia sinensis
Camellia sinensis
Rhododendron lateriticum
Symplocos lucida
Eubotryoides grayana var. glabra
Rhododendron reticulatum
Rhododendron retuculatum f. retuculatum
Lyonia ovalifolia ssp. neziki
Vaccinium hirtum var. pubescens
Rhododendron obtusum var. kaempferi
Rhododendron indicum
Rhododendron macrosepalum
Rhododendron × pulchrum
Rhododendron obtusum var. kaempferi
Cinnamomum japonicum
Camellia japonica
Rhododendron × pulchrum
Rhododendron yedoense var. yedoense f. yedoense
Rhododendron yedoense var. yedoense f. yedoense
Rhododendron yedoense var. yedoense f. yedoense
Rhododendron dauricum
Camellia sasanqua
Camellia sasanqua
Rhododendron obtusum var. kaempferi
Rhododendron obtusum var. kaempferi
Rhododendron obtusum var. kaempferi
Rhododendron obtusum var. kaempferi
Rhododendron obtusum var. kaempferi
Rhododendron kiusianum
Rhododendron dauricum
Rhododendron dauricum
Rhododendron wadanum
Rhododendron wadanum
Rhododendron wadanum
Rhododendron wadanum
Rhododendron obtusum var. kaempferi
Rhododendron obtusum var. kaempferi
Rhododendron degronianum
Rhododendron degronianum
Rhododendron degronianum
Symplocos lucida
Rhododendron wadanum
Rhododendron wadanum
Rhododendron sp.

Host plant

Table 1. List of Japanese Exobasidium examined in this study.

Kagoshima Pref.
Kagoshima Pref.
Japan
Japan
Japan
Japan
Japan
Japan
Japan
Japan
Japan
Japan
Japan
Shizuoka Pref.
Ibaraki Pref.
Shizuoka Pref.
Gunma Pref.
Nagano Pref.
Tokyo Metropol.
Chiba Pref.
Ibaraki Pref.
Hokkaido Pref.
Hokkaido Pref.
Hokkaido Pref.
Tokyo Metropol.
Shizuoka Pref.
Tokyo Metropol.
Nagano Pref
Kyoto Pref.
Hokkaido Pref.
Ibaraki Pref.
Ibaraki Pref.
Kagoshima Pref.
Hokkaido Pref.
Hokkaido Pref.
Nagano Pref.
Nagano Pref.
Nagano Pref.
Ibaraki Pref.
Nagano Pref.
Nagano Pref.
Nagano Pref.
Nagano Pref.
Nagano Pref.
Shimane Pref.
Nagano Pref.
Nagano Pref.
Aomori Pref.

Locality

AB177561
AB177579
AB178247
AB177582
AB177583
AB177585
AB177581
AB177567
AB177549
AB177584
AB178248
AB176711
AB177551
AB177590
AB178245

AB177571
AB178246
AB177568

AB177586

AB178242
AB178250
AB177563
AB177550

AB177566
AB176712

AB177548
AB176713
AB178243

AB177597
AB177598
AB177599
AB177600
AB177601
AB177602

AB178257
AB178256
AB177595
AB177596
AB177569
AB177570

d1 /d2

AB180326
AB180327
AB180328
AB180330
AB180329
AB180331
AB180332
AB180333
AB180334
AB180335
AB180336
AB180337
AB180338
AB180339
AB180340
AB180341

AB180319
AB180320
AB180321
AB180322
AB180323
AB180324
AB180325

AB180317
AB180318

AB180316

AB178941
AB180315

AB180370
AB180363
AB180364
AB180365
AB180366
AB180367

AB180368
AB180369

AB180373
AB180374ITS

GenBank accession No.

慶應義塾大学日吉紀要・自然科学
No. 70（2023）

Rhododendron × pulchrum
Rhododendron brachycarpum
Rhododendron hyugaense
Rhododendron hyugaense
Rhododendron retuculatum f. glabrescens
Rhododendron yedoense var. yedoense f. yedoense
Vaccinium hirtum var. pubescens
Rhododendron brachycarpum
Rhododendron brachycarpum
Vaccinium hirtum var. pubescens
Symplocos lucida
Vaccinium uliginosum
Rhododendron brachycarpum
Vaccinium hirtum var. pubescens
Vaccinium smallii
Rhododendron brachycarpum
Rhododendron macrosepalum
Rhododendron × mucronatum
Rhododendron obtusum var. kaempferi
Cinnamomum japonicum
Rhododendron brachycarpum
Rhododendron brachycarpum
Vaccinium vitis-idaea
Camellia sasanqua
Rhododendron retuculatum f. retuculatum
Symplocos lucida
Symplocos lucida
Rhododendron obtusum var. kaempferi
Rhododendron obtusum var. kaempferi
Rhododendron aureum
Rhododendron wadanum
Rhododendron wadanum
Camellia sinensis
Camellia sinensis
Lyonia ovalifolia ssp. neziki
Lyonia ovalifolia ssp. neziki
Rhododendron oomurasaki
Cinnamomum japonicum
Rhododendron brachycarpum
Rhododendron brachycarpum
Camellia sasanqua
Camellia sasanqua
Camellia sasanqua
Lyonia ovalifolia ssp. neziki
Cinnamomum daphnoides
Cinnamomum daphnoides
Cinnamomum japonicum
Cinnamomum japonicum

Host plant
Gunma Pref.
Nagano Pref.
Miyazaki Pref.
Miyazaki Pref.
Miyazaki Pref.
Hokkaido Pref.
Hokkaido Pref.
Hokkaido Pref.
Hokkaido Pref.
Hokkaido Pref.
Fukuoka Pref.
Tochigi Pref.
Nagano Pref.
Aomori Pref.
Aomori Pref.
Tochigi Pref.
Shizuoka Pref.
Shizuoka Pref.
Nagano Pref.
Tokyo Metropol.
Hokkaido Pref.
Tochigi Pref.
Tochigi Pref.
Ibarak Pref.
Hiroshima Pref.
Fukuoka Pref.
Fukuoka Pref.
Aomori Pref.
Aomori Pref.
Nagano Pref.
Tochigi Pref.
Tochigi Pref.
Shizuoka Pref.
Shizuoka Pref.
Shizuoka Pref.
Shizuoka Pref.
Shizuoka Pref.
Tokyo Metropol.
Yamanashi Pref.
Yamanashi Pref.
Ibarak Pref.
Ibarak Pref.
Ibarak Pref.
Niigata Pref.
Kagoshima Pref.
Kagoshima Pref.
Kagoshima Pref.
Kagoshima Pref.

Locality

AB177592
AB176710
AB178259
AB178260
AB178258
AB177594

AB178255
AB178251
AB178253
AB178254
AB180378
AB180379
AB180380
AB180381
AB177575
AB177552
AB177574
AB176715
AB177591
AB176716
AB176714

AB177559
AB177573
AB178240
AB177577
AB177555
AB177572
AB177589
AB177580
AB177587
AB177562
AB177578
AB177565
AB177560

AB178244
AB177588
AB177576
AB177554
AB177593
AB177564
AB177556
AB177557

d1 /d2

AB180371

AB180377

AB180353
AB180354
AB180355
AB180356
AB180357
AB180358
AB180359
AB180360
AB180361
AB180362
AB180684
AB180683
AB180677
AB180678
AB180679
AB180681
AB180682
AB180375
AB180376

AB180342
AB180343
AB180344
AB180345
AB180346
AB180347
AB180348
AB180349
AB180350
AB180351
AB180352ITS

GenBank accession No.

EOS: Laboratory of Plant Pathology, Faculty of Agriculture, Kagoshima University; IFO: Institute of Fermantation, Osaka; MAFF: Genebank in National Institute of Agrobiological Sciences;
TUK: Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba

Exobasidium cylindrosporum
TSH-B0014
Exobasidium woronichinii
TSH-B0022
Exobasidium otanianum
TSH-B0058
Exobasidium otanianum
TSH-B0059
Exobasidium otanianum
TSH-B0061
Exobasidium dubium
TSH-B0078
Exobasidium inconspicuum
TSH-B0080
Exobasidium woronichinii
TSH-B0081
Exobasidium woronichinii
TSH-B0085
Exobasidium inconspicuum
TSH-B0086
Exobasidium symploci-japonicae var. caprogenum
TSH-B0090
Exobasidium pachysporum
TSH-B0121
Exobasidium woronichinii
TSH-B0018
Exobasidium kishianum
TSH-B0070
Exobasidium kishianum
TSH-B0071
Exobasidium woronichinii
TSH-B0116
Exobasidium cylindrosporum
TSH-B0128
Exobasidium cylindrosporum
TSH-B0129
Exobasidium japonicum
TSH-B0049
Laurobasidium hachijoense
TSH-B0068
Exobasidium woronichinii
TSH-B0083
Exobasidium woronichinii
TSH-B0114
Exobasidium vaccinii
TSH-B0120
Exobasidium gracile
NIAES1471008
Exobasidium otanianum
NIAES20561
Exobasidium symploci-japonicae var. symploci-japonicae NIAES20520
Exobasidium symploci-japonicae var. symploci-japonicae NIAES20521
Exobasidium japonicum
NIAES20571
Exobasidium japonicum
NIAES20572
Exobasidium caucasicum
NIAES20542
Exobasidium nobeyamense
INM2-15278-052254
Exobasidium nobeyamense
INM2-15278-052256
Exobasidium vexans
INM2-15278-052298
Exobasidium reticulatum
INM2-15278-052327
Exobasidium pieridis
Exobasidium pieridis-ovalifoliae
Exobasidium cylindrosporum
Laurobasidium hachijoense
Exobasidium hemisphaericum
Exobasidium hemisphaericum
Exobasidium gracile
Exobasidium gracile
Exobasidium gracile
Exobasidium pieridis-ovalifoliae
Clinoconidium globosum
Clinoconidium globosum
Clinoconidium onumae
Clinoconidium onumae

MAFF 238609
MAFF 238610
MAFF 238611
MAFF 238612
MAFF 238613
MAFF 238614
MAFF 238616
MAFF 238617
MAFF 238618
MAFF 238619
MAFF 238620
MAFF 238621
MAFF 238622
MAFF 238623
MAFF 238624
MAFF 238625
MAFF 238662
MAFF 238663
MAFF 238664
MAFF 238665
MAFF 238666
MAFF 238667
MAFF 238668
MAFF 238674
MAFF 238677
MAFF 238810
MAFF 238811
MAFF 238824
MAFF 238826
MAFF 238830
MAFF 239439
MAFF 239440
MAFF 239441
MAFF 239442
MAFF 306193
MAFF 306194
MAFF 306198
TUK-E02
TUK-E11
TUK-E13
TUK-E21
TUK-E26
TUK-E30
TUK-E44
TUK-MA01
TUK-MA02
TUK-S703
TUK-S720

Voucher

Species

IsolateThe Host-Pathogen Association of Exobasidium in Japan Inferred from Molecular Phylogeny of ITS and Large Subunit rDNA Sequences（NAGAO・KASUYA）

7

慶應義塾大学日吉紀要・自然科学

No. 70（2023）

Table 2. List of the referance species and its accession number of Large subunit rDNA.
Species

Host

Exobasidium arescens
E. bisporum
E. vaccinii
E. japonicum
E. karstenii
E. myrtilli
E. oxycocci
E. pachysporum
E. pieridis-ovalifoliae
E. rhododendri
E. rostrupii
E. shiraianum
E. sundstroemii
E. symploci-japonicae
E. vaccinii
E. vaccinii
E. warmingii
E. yoshinagae

Vaccinium myrtillus
Eubtryoides grayana
Vaccinium vitis-idaea
Rhododendron indicum = Rhododendron lateritium
Andromeda polifolia
Vaccinium myrtillus
Vaccinium oxycoccos
Vaccinium ulginosum
Lyonia ovalifolia= Lyonia neziki
Rhododendron ferrugineum
Vaccinium oxycoccos
Rhododendron degronianum
Andromeda polifolia
Symplocos sp.
Vaccinium vitis-idaea
Vaccinium vitis-idaea
Saxifraga bryoides
Rhododendron reticulatum

DDBJ Acc. No.
AF352057
AF487386
AF487398
AF487388
AF487389
AF487390
AF487391
AF487392
AF487393
AF009856
AF009857
AF487395
AF487396
AF487397
AF009858
AJ406400
AF487380
AF487399

Table 3. The primers used in this study.
ITS1 (White et al., 1990)
ITS1F (Gardes and Bruns, 1993)
ITS4 (White et al., 1990)
D1 /D2
NL-1 (O’Donnelle, 1993)
NL-4 (O’Donnelle, 1993)

TCCGTAGGTGTAACCTGCGG
CTTGGTCATTTAGAGGAAGTAATCCTCCGCTTATTGATATGC
GCATATCAATAAGCGGAGGAAAAGGGTCCGTGTTTCAAGACGG

ed as “missing”. All molecular characters were unordered and given equal weight.
Phylogenetic trees were examined in the default setting by neighbor-joining (NJ) method
and maximum parsimony (MP) analysis under a heuristic search. Bootstrap values forbranch support were assessed with 1000 bootstrap pseudo-replicates with 10 random taxon additions per bootstrap replicate (Felsenstein, 1985). Maximum likelihood was also
applied and evaluated by quartet puzzling analysis with 1000 puzzling steps (Schmidt
and von Haeseler, 2003 ; Reaz et al., 2014). The quality of the data is evaluated by a support value. A higher support value corresponds to a greater confidence of the bipartition(Schmidt and von Haeseler, 2003).

3. Results
3-1 Phylogenetic analysis for LSU (D1 /D2)
The alignment included 635 total characters of which 406 are constant (proportion =0.63937), 76 variable characters are parsimony uninformative, while 153 characters are
8

The Host-Pathogen Association of Exobasidium in Japan Inferred from Molecular Phylogeny of ITS and Large Subunit rDNA Sequences（NAGAO・KASUYA）

parsimony informative. Neighbor-joining analysis for the datasets of D1 /D2 regions indicated that Exobasidium is distinguished from Cryptobasidiaceae (Clinoconidium andLaurobasidium) with high bootstrap value but the resolution among Exobasidium species
is poor (Fig. 2). A group composed of Exobasidium symploci-japonicae pathogenic to S.
lucida was supported by high bootstrap value with 99 %, whereas Exobasidium spp.
pathogenic to Camellia, Rhododendron, Saxifraga, and Vaccinioideae were phylogenetically paraphyletic supported by moderate bootstrap value in the neighbor-joining analysis. These groupings were recognized in both the maximum parsimony and the maximum likelihood analyses (Figure is not presented). Some Exobasidium spp. pathogenic
to Rhododendron appeared to be polyphyletic.
3-2 Phylogenetic analysis for ITS regions
The alignment included 822 total characters of which 369 are constant (proportion =0.448905), 139 variable characters are parsimony uninformative, while 314 characters are
parsimony informative. Twice as many parsimony–informative characters were found in
the ITS regions as in the D1 /D2 regions of LSU. Topology of neighbor-joining, maximum
parsimony and maximum likelihood analyses were similar to those of the D1 /D2 regionof LSU but showed higher bootstrap values (Fig. 3). Exobasidium otanianum pathogenic
to Rhododendron spp. appeared to be polyphyletic. One group was pathogenic to R. dilatatum Miq. var. satsumense T.Yamaz. and R. hyugaense (T. Yamaz.) T. Yamaz., and another was pathogenic to R. reciculatum f. reciculatum D. Don ex G. Don in the maximum
likelihood analysis. However, the quartet-puzzling value didn’t support two groups.
3-3 Phylogenetic analysis for concatenated ITS+LSU sequences
The alignment included 1479 total characters of which 847 are constant (proportion =0.572684), 229 variable characters are parsimony uninformative, while 403 characters are
parsimony informative. As shown in the phylogenetic tree of ITS regions, the concatenated sequences also produced a similar topology of neighbor-joining, maximum parsimony, and maximum likelihood analyses with higher bootstrap and quartet-puzzling values (Fig. 4). From the phylogenetic tree, the following host-specific groups arerecognized; Group 1 composes E. nobeyamense, E. otanianum, E. cylindrosporum and E.
pentasporium, Group 2 E. japonicum, E. shiraianum, E. caucasicum, and E. woronichinii.
Group 3 E. dubium, E. miyabei, and E. yoshinagae, Group 4 E. bisporum, E. pieridis-ovalifoliae, E. pachysporum, E. inconspicuum, E. kishianum, and E. vaccinii, Group 5 E. camelliae and E. reticulatum and Group 6 E. symploci-japonicae. Groups 1 to 4 are pathogenic to Ericaceae and supported by high bootstrap and quartet-puzzling values.
9慶應義塾大学日吉紀要・自然科学

No. 70（2023）
E. otanianum
E. cylindrosporum

E. nobeyamense

E. otanianum

E. nobeyamense
E. cylindrosporum
E. yoshinagae
E. dubium
E. yoshinagae
E. dubium
E. yoshinagae
E. miyabei
E. pentasporium
E. pachysporum
E. bisporum
E. pieridis-ovalifoliae
E. rostrupii
E. arescens
E. inconspicuum
E. pieridis-ovalifoliae
E. sundostroemii

E. japonicum

E. shiraianum
E. japonicum
E. woronichinii

E. hemisphaericum
E. shiraianum
E. caucasicum
E. rhododendri
E. oxycocci
E. myrtilli
E. kishianum
E. vaccinii
E. karstenii
E. pieridis
E. gracile
E. camelliae
E. reticulatum
E. vexans
E. warmingii
E. symploci-japonicae
Laurobasidium
hachijoense
Clinoconidium
onumaeC. globosum
Ustilago maydis

Fig. 2. Topology obtained by neighbor-joining analysis of D1 /D2 domain of LSU rDNA sequences of Japanese isolates of Exobasidium and relatives rooted with Ustilago maydis. NJ bootstrap
values of 1000 replicates are indicated. Values smaller than 50 % are not shown.
10The Host-Pathogen Association of Exobasidium in Japan Inferred from Molecular Phylogeny of ITS and Large Subunit rDNA Sequences（NAGAO・KASUYA）

Phylogeny2021_its_ML_QP1000
14
29

25

9

78
- / 51
27
52

-/-

27

82
-/-

90
-/44

69
-/-

55
-/-

46

63

-/-

58

-/-

93

99 / 10099

45

100 / 100

43

-/-

65
100 / 9970
82 / 853774
89 / -

81
-/-

30

17

66

-/-

32

56
-/-

88

90
92/ 7392- / 6533

91
93 / 81

49

62

76
-/-

-/-

40

69

-/-

68 / -

99

98
100/ 9052
97
84 / 92

100/ 96

96

85
93/ 93- / 9671

66/ 7344

90

100
100/ 9978

73 / 52

77

-/-

49

77
-/71
100/ 100

95
-/-

100
100/ 10098
98 / 99

97 / 84

74

-/-

89
-/-

83
100/ 100
97

100/ 100

84
100/ 99

95
100/ 100
2170- / 70222226

MAFF239439
MAFF238598
MAFF238596
MAFF238597
MAFF239440
MAFF238599
MAFF238611
MAFF238613
MAFF238612
EOS59
EOS44
MAFF238579
MAFF238663
MAFF238662
MAFF238177
IFO9960
MAFF238677
MAFF238179
MAFF238600
MAFF238601
MAFF238603
MAFF238604
MAFF238602
MAFF238830
MAFF238666
MAFF238825
MAFF238618
MAFF238617
MAFF238610
MAFF238625
MAFF238667
MAFF238588
MAFF238176
MAFF238589
MAFF238592
IFO30756
MAFF238824
MAFF238826
MAFF238587
MAFF238591
MAFF238583
MAFF238595
MAFF238594
MAFF238606
MAFF238607
MAFF238582
IFO9959
MAFF238614
MAFF238623
MAFF238624
MAFF238668
MAFF238619
MAFF238616
MAFF238621
IFO30152
IFO9942
IFO9961
MAFF238585
MAFF238586
E26
MAFF238674
MAFF238578
IFO30393
MAFF239442
MAFF238810
IFO7790
MAFF238811
MAFF238605
MAFF238620
MAFF238665
S703
AY854090

E. nobeyamense

E. otanianum

E. cylindrosporum

E. otanianum

E. pentasporium

E. shiraianum
E. caucasicum

E. woronichinii

E. japonicum

E. miyabei

E. yoshinagae
E. dubium
E. yoshinagae

E. dubium
E. kishianum
E. vaccinii
E. inconspicuum
E. pachysporum
E. bisporum
E. pieridis-ovalifoliae

E. gracile

E. camelliae
E. reticulatum

E. symploci-japonicae

L. hachijoense
C. onumae
Ustilago maydis

Fig. 3. Topology obtained by maximum likelihood analysis of ITS sequences of Japanese isolatesof Exobasidium and relatives rooted with Ustilago maydis. Quartet puzzling support value of1000 puzzling steps is indicated above the branch and NJ and MP bootstrap values of 1000 replicates are indicated below the branch from left to right. Values smaller than 50 % are not shown.
11

Phylogeny2021_its+dd_ML_QP1000

慶應義塾大学日吉紀要・自然科学

No. 70（2023）

Puzzling consensus tree

49

55

47

49
94

61

-/-

89

-/-

- /52

57

47

44

61
-/-

83

74

67

-/-

- /74

- /94

100
100/

53

83

68

- /96

98

-/-

96

-/-

100/98

76

-/-

74

64/70

82

48

-/-

93

78/61

82

95

96/90

100/96

100/

96

67/

91

98
94

91
86

81/74

99/100

73/96

100

98

100/100

44

89

-/-

99

100/96

99

99/97

95

97/73

43

64

68/ -

71

77/81

68/

83

82/60

82/76

97

99/97

100

100

100/100

100/99

86

91

-/-

99

- /70

- /10098

95100/ -

100/100

94

100/100

35

MAFF239439
MAFF239440
MAFF238599
MAFF238597
MAFF238596
MAFF238598
EOS44
EOS59
MAFF238612
MAFF238613
MAFF238611
IFO9960
MAFF238177
MAFF238662
MAFF238600
MAFF238601
MAFF238824
MAFF238826
MAFF238589
MAFF238176
MAFF238587
MAFF238591
MAFF238604
MAFF238603
MAFF238602
MAFF238830
MAFF238617
MAFF238666
MAFF238610
MAFF238667
MAFF238625
MAFF238582
IFO9959
MAFF238614
MAFF238607
MAFF238606
MAFF238595
MAFF238583
MAFF238594
IFO30152
IFO9942
IFO9961
MAFF238621
MAFF238616
MAFF238623
MAFF238624
MAFF238668
MAFF238578
IFO30393
MAFF239442
MAFF238811
MAFF238605
IFO7790
MAFF238620
S703
MAFF238665
AY854090

E. nobeyamense

E. otanianum

Group 1

E. cylindrosporum

E. pentasporium

E. japonicum

E. shiraianum

Group 2

E. caucasicum

E. woronichinii

E. dubium

E. yoshinagae
E. dubium
E. yoshinagae

Group 3

E. miyabei

E. bisporum
E. pachysporum
E. inconspicuumGroup 4

E. kishianum
E. vaccinii
E. camelliae
E. reticulatum

E. symploci-japonicae

Group 5

Group 6

C. onumae
L. hachijoense
Ustilago maydis

Fig. 4. Topology obtained by maximum likelihood analysis of the concatenated ITS+LSU sequences of Japanese isolates of Exobasidium and relatives rooted with Ustilago maydis. Quartet
puzzling support value of 1000 puzzling steps is indicated above the branch and NJ and MP
bootstrap values of 1000 replicates are indicated below the branch from left to right. Values
smaller than 50 % are not shown.
12

The Host-Pathogen Association of Exobasidium in Japan Inferred from Molecular Phylogeny of ITS and Large Subunit rDNA Sequences（NAGAO・KASUYA）

4. Discussion
By concatenating the sequences from the LSU and ITS regions, the most parsimony-informative characters are available among these sequence alignments in this study.
Therefore, the phylogenetic analysis of maximum likelihood showed clades depended
on the species with high quartet-puzzling support values except E. nobeyamense, E. otanianum, and E. cylindrosporum. In general, six proposed phylogenetic groups were resolved by neighbor-joining, maximum parsimony and maximum likelihood analyses withhigh bootstrap and quartet-puzzling support values. Previously, concatenated ITS+LSU
data were reported to resolve relationships within clades with high bootstrap values(Piątek et al., 2012 ; Fig. 2, Wang et.al., 2015 ; Fig. 2 D). Even with a concatenated ITS+LSU data tree, missing ITS data may lead to different results or a topology like an LSU
tree (Kennedy et al., 2012). In our concatenated ITS +LSU trees, E. hemisphaericum inGroup 2 and E. vexans in Group 5 were excluded because ITS data for these isolates
were not obtained.
Rapid separation of the Exobasidiomycetidae into several groups was suggested by theshort internal distances and the small bootstrap values obtained from LSU analysis (Begerow et al., 1997). Begerow et al. (2002) proposed a plausible evolutionary scenario for
Exobasidium. In the interpretation of a topology obtained by LSU with 18 Exobasidium
spp., Exobasidium is presumed to arise as a pathogen on the Ericaceae ancestor (Begerow et al., 2002). In their interpretation of an LSU phylogenetic tree, the dichotomy occurred between the Exobasidium species on Theales and on the Ericaceae, and further
between the Exobasidium species on Rhododendron and those on Vaccinioideae. Even
though the two groupings for the species pathogenic to Vaccinioideae were demonstrated by different studies (Begerow et al., 2002 ; Piątek et al., 2012 ; Brewer et al., 2014 ; andWang et al., 2015), we wonder whether the results from less informative sequences arereliable. We agree with the statement of Brewer et al. (2014) that molecular phylogenetic
studies have supported Nannfeldt’s species concept (Begerow et al. 2002 ; Kennedy et al.
2012 ; Piątek et al. 2012) . However, the phylogenetic tree of LSU alone did not resolve
the relationships of Vaccinium parasites (Fig. 2).
As more distinctive grouping can be proposed by the concatenated ITS+LSU, we focused on the concatenated ITS+LSU tree. Our results also suggested a possible dichotomy between Camellia and the Ericaceae but support values were not significant, while adichotomy between Symplocos and the Ericaceae had high support value.
Multiple gene analysis and protein-coding genes including RPB1, RPB2, and TEF1
13

慶應義塾大学日吉紀要・自然科学

No. 70（2023）(Wang et.al., 2015 ;Figs. 1 and 3) didn’t suggest a remarkable difference compared with aconcatenated ITS+LSU. Further examination should be applied with more informaticand protein-coding genes to identify crucial branching points.
Nannfeldt (1981) compiled the available information for Exobasidium in Europe andsifted through the host-specific and omnivorous infections in the Ericales. Referring toSundström (1964), Nannfeldt (1981) proposed nine new species and a new combinationfrom the examinations of E. vaccinii sensu auctt. p.p. and others. Exobasidium pathogenic to Vaccinioideae often attack a few species and different part(s) of their hosts. As
ITS data of Exobasidium pathogenic to Ericaceae was not sufficient to resolve the clades,
we also added LSU data of those Exobasidium species for the further analysis. The concatenated ITS+LSU of those Exobasidium may resolve the phylogenetic distinction. Ezuka (1991 b) disputed the host range of Exobasidium bisporum Sawada ex Ezuka since
Sawada (1950) reported two different genera as host plants; i. e. Vaccinium axillare Nakai, Eubotryoides grayana (Maxim.) H. Hara var. oblongifolia (Miq.) Ohwi, and E. grayana var. glabra (Komatsu ex Nakai) H. Hara. Ezuka (1991 b) added E. grayana var. hypoleuca (Nakai) H. Hara and V. oldhamii Miq. as new host plants. Morphology and culturalcharacteristics of isolates from both Vaccinium spp. and Eubotryoides spp. were indistinguishable (Sawada, 1950 ; Ezuka, 1991 b). In our study E. bisporum from Vaccinium spp.
was not available.
In the Ericaceae host plants, Exobasidium pathogenic to the Vaccinioideae forms
Group 4, which involves at least three genera and causes leaf blister and leaf blight. Basidiospores are 0-1 to multi-septate and germinate or bud. Consequently, colonies arecomposed of pseudo-hyphae or yeast-like cells. Despite such diverse characteristics,
Exobasidium pathogenic to the Vaccinioideae forms a group with high support value. On
the contrary, Exobasidium pathogenic to Rhododendron is divided into three groups.
Groups 1 and 2 may have a common ancestor and show similar morphology in terms of
0-1 to multi-septate basidiospores, germ-tube type, and pseudo-hyphal growth, while thepathogenicity is diverted in Group 1. Four Exobasidium infect four Rhododendron withthree different symptoms. Among them, Exobasidium pentasporium (Group 1) and E. japonicum (Group 2) infect Rhododendron kaempferi var. kaempferi and cause witches’
broom and leaf gall, respectively. The same result by phylogenetic analysis using theconcatenated ITS+LSU was recently published (Shibata and Hirooka, 2022). The phylogenetic tree supported independent causal agents. In Group 2, Exobasidium pathogenic
to the subgen. Hymenathes cause leaf blight. Group 3 shows a common pathogenicity asleaf blister with small round, flat symptom, and similar morphology. Host plants belong
to subgen. Rhododendron and Tsutsusi. Exobasidium pathogenic to Camellia is placed in
14

The Host-Pathogen Association of Exobasidium in Japan Inferred from Molecular Phylogeny of ITS and Large Subunit rDNA Sequences（NAGAO・KASUYA）

Group 5 with higher bootstrap and support values in NJ, MP, and ML analyses. Exobasidium pathogenic to Symplocos is placed in Group 6 with higher values. Both groups involve a variety of modes of infection, modes of basidiospore germination, and colony
growth. Host specificity and multi-septate basidiospores are common features in these
two groups.
The outstanding issue of two different virulence types of Exobasidium species on Rhododendron host plants were explained by placing Exobasidium species in different
clades, i. e. E. japonicum (Group 2) and E. pentasporium (Group 1) on R. kampferi, E.
yoshinagae (Group 3) and E. nobeyamense (Group 1) on R. wadanum, E. yoshinage(Group 3) and E. otanianum (Group 1) on Rhododendron subgen. Tsutsusi. The supporting values on the point of dichotomy for Group 1 and 2 from Group 3 were 95 / 100 / 96
by ML, NJ, and MP, respectively. But those for separating Group 1 from Group 2 wererelatively moderate, 53 /—/ 96 by ML, NJ, and MP, respectively. Hence, two different virulence types of Exobasidium species remain on the same genus of host plant but belong
to different concatenated ITS+LSU clades. The case of E. symploci-japoniccae and E.
symploci-japoniccae var. caprogenum on S. lucida will be investigated whenever Symplocos-specific Exobasidium species are found. The rarely recognized species E. japonicum
var. hypophyllum and E. kawaense will be examined when available.
As Begerow et al. (2002) showed the position of E. warmingii pathogenic to Saxifragain Exobasidiaceae, our LSU tree also supported placing E. warmingii in the Ericaceae
clade, but all clades in Ericaceae were paraphyletic. A homology search retrieved on 13
Nov. 2022 indicated the top five choices as A. warmingii (Acc. No. MT223875), Exobasidium sp. (Acc. No. OP374143), Exobasidium sp. (Acc. No. ON557301), E. rhododendri (Acc.
No. OP763657), and E. cylindrosporum (Acc. No. CP096880). Wang et al. (2015 ; Fig. 4)
and Crous et al. (2020 ; Fig. 1) also presented the position of E. warmingii in Exobasidiaceae in an LSU tree. Crous et al. (2020) picked up the closest hits of ITS, LSU, and tef1
from a megablast search of NCBI’s GenBank nucleotide database and considered thatclosely related to species are Exobasidium and Muribasidiospora.
Exobasidium causes overgrowth symptoms such as gall formation on buds, fruits, andleaves, blistering, blight, and malformation on shoots, including witches’ broom, shoestring, and red shoot in the different host families (Fig. 1). Li and Guo (2010) concluded
that phylogenetic relationships among 22 Exobasidium species corresponded to the hostplants and symptoms. Our studies showed that fruit malformation is caused only by
Exobasidium pathogenic to Vaccinium (Brewer et al., 2014) in Group 4, Camellia inGroup 5, and Symplocos in Group 6. Witches’ broom is also caused on R. wadanum andR. kaempferi var. kaempferi as mentioned above. Leaf gall is caused on R. kaempferi
15

慶應義塾大学日吉紀要・自然科学

No. 70（2023）

var. kaempferi, Rhododendron subsp. Hymenathes, and Vaccinium spp. These symptoms
are neither host specific nor related with the examined sequence groups except Group 3(leaf blister on Rhododendron spp.).
Although basidiospore morphology and mode of basidiospore germination were
thought to be an important taxonomic character within Exobasidium, results of the present study show that they are poor guidelines to support phylogenetic relationships (Fig.
5). For example, basidiospores with the same number of septa were placed into several
different clades. Exobasidium japonicum and E. pentasporium have 0-1-septated basidiospores, while these two species grouped in different clades (Fig. 4). Exobasidium japonicum was erroneously synonymized to E. vaccinii due to basidiospore morphology asstated previously (Savile, 1959 a). Mode of basidiospore germination also distinguishedthese two species (Sundström, 1964 ; Nannfeldt, 1981). Phylogenetic trees showed different positions of these species in Groups 2 and 4, respectively. Therefore, basidiospore
size and the number of septa poorly reflect phylogenetic relationships. In addition, themode of basidiospore germination does not reflect phylogenetic relationships. Exobasidium symploci-japonicae var. symploci-japonicae germinates via a germ-tube, whereas var.
caprogenum does by budding (Nagao et al. 2003 b). These two varieties formed a monophyletic group within the phylogenetic trees (Figs. 3 and 4). In Group 2, E. woronichinii
germinated by a germ-tube, whereas E. caucasicum and E. shiraianum by budding(Nagao et al. 2004 a).
Nannfeldt (1981) discussed the life cycles and symptoms of Exobasidium spp. and considered how to infect the host plant referring to the interior persisting mycelia. Monocarpic and polycarpic infections were explained according to a manner of symptom.
Mode of basidiospore germination, either germ-tube or budding conidia, may be favorable to infection on certain host plants. For symptom development, there is no specificcharacteristics related to infection by budding conidia. Taphrina species germinate fromascospores by budding but grow in the form of pseudo-mycelium in the hypertrophiedtissue (Nagao and Katumoto, 1998). Yeast-like growth of Exobasidium on the surface ofmedia and leaves may be transformed to pseudo-hyphal growth in the host plant tissue.
Nature of budding yeast has been known by the microtubule regulation and β-tubulin
genes are related (Bode et al., 2003). Phylogenetic analysis with β-tubulin gene sequences may give insight to the common ancestor of species of Exobasidium with yeast-likegrowth.

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Fig. 5. Schematic comparison based on maximum likelihood analysis of the concatenated ITS+LSU sequences groupings with host plant, modeof infection, septal number of basidiospores, mode of basidiospore germination, and colony type.

The Host-Pathogen Association of Exobasidium in Japan Inferred from Molecular Phylogeny of ITS and Large Subunit rDNA Sequences（NAGAO・KASUYA）

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慶應義塾大学日吉紀要・自然科学

No. 70（2023）

Acknowledgement
We thank Prof. A. P. Keinath for English revision and valuable comments. We also
thank Prof. M. Kakishima for introducing Exobasidium taxonomical study and Prof. T.
Sato for providing the facility for experiments in the NIAS Genebank. A part of thisstudy was supported by a Post-Doctoral Fellowship 2005, Genebank, National Institute ofAgrobiological Sciences (currently National Agriculture and Food Research Organization, NARO), Japan, and by a research fund of Forest Development Technological Institute.

References
Bauer, R., D. Begerow, F. Oberwinkler, M. Piepenbring, and M. L. Berbee. 2001. Ustilaginomycetes. In: McLaughlin, D.J., E.G. McLaughlin, and P.A. Lemke (eds.) Mycota
VII Part B. Systematics and evolution. pp. 57-83. Springer Verlag, Heidelberg, NewYork.
Begerow, D., R. Bauer, and F. Oberwinkler. 1997. Phylogenetic studies on nuclear large
subunit ribosomal DNA sequences of smut fungi and related taxa. Can. J. Bot.
75 : 2045-2056.
Begerow, D, R. Bauer, and F. Oberwinkler. 2002. The Exobasidiales: an evolutionary hypothesis. Mycol. Prog. 1 : 187-199.
Bode, C. J., M. L. Gupta, K. A. Suprenant, and R. H. Himes. 2003. The two alpha-tubulin
isotypes in budding yeast have opposing effects on microtubule dynamics in vitro.
EMBO Rep 4 (1): 94-9. https://doi.org/ 10.1038 /sj.embor.embor716.
Brewer, M.T., A. N. Turner, P. M. Brannen, W. O. Cline, and E. A. Richardson. 2014.
Exobasidium maculosum, a new species causing leaf and fruit spots on blueberry inthe southeastern USA and its relationship with other Exobasidium spp. parasitic toblueberry and cranberry. Mycologia 106 (3): 415-23. https://doi.org/ 10.3852 / 13-202
Burt, E.A . 1915. The Thelephoraceae of North America. IV Exobasidium. Ann. Mo. Bot.
Gard. 2 : 627-659.
Chlebicki, A. and M. Chlebická. 2007. A new fungus, Exobasidium gomezii, from Chilean
Patagonia. Nova Hedwigia 85 : 145-149.
Crous, P. W., M. J. Wingfield, R. K. Schumacher, A. Akulov, T. S. Bulgakov, A. J. Carnegie, Ž. Jurjević, C. Decock, S. Denman, L. Lombard, D. P. Lawrence, A. J. Stack, T.
R. Gordon, R. M. Bostock, T. Burgess, B. A. Summerell, P. W. J. Taylor, J. Edwards,
18

The Host-Pathogen Association of Exobasidium in Japan Inferred from Molecular Phylogeny of ITS and Large Subunit rDNA Sequences（NAGAO・KASUYA）

L. W. Hou, L. Cai, A. Y. Rossman, T. Wöhner, W. C. Allen, L. A. Castlebury, C. M.
Visagie, and J. Z. Groenewald. 2020. New and Interesting Fungi. 3. Fungal Syst.
Evol. 6 : 157-231 (75). https://doi.org/ 10.3114 /fuse. 2020.06.09
Donk, M. A. 1966. Check list of European hymenomycetous Heterobasidiae. Persoonia
4 : 145-335.
Döring, H. and P. Blanz. 2000. 18 S rDNA-Analysen bei der Gattung Exobasidium. Hoppea 61 : 85-100.
Ezuka, A. 1955. Artificial culture of two species of Exobasidium: E. vexans Massee and E.
japonicum Shirai. Bull. Tea Div. Tokai-Kinki Agri. Exp. Stn. 3 : 28-53 [in Japanese].
Ezuka, A. 1990 a. Notes on some species of Exobasidium in Japan (I). Trans. Mycol. Soc.
Japan 31 : 375-388.
Ezuka, A. 1990 b. Notes on some species of Exobasidium in Japan (II). Trans. Mycol. Soc.
Japan 31 : 439-455.
Ezuka, A. 1991 a. Notes on some species of Exobasidium in Japan (III). Trans. Mycol.
Soc. Japan 32 : 71-86.
Ezuka, A. 1991 b. Notes on some species of Exobasidium in Japan (IV). Trans. Mycol.
Soc. Japan 32 : 169-185.
Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap.
Evolution 6 : 227-242.
Graafland, W. 1960. The parasitism of Exobasidium japonicum Shir. On Azalea. Acta Bot.
Neerl. 9 : 347-379.
Gardes, M. and T.D. Bruns. 1993. ITS primers with enhanced specificity for basidiomycetes - application to the identification of mycorrhizae and rusts. Mol. ecol. 2 (2): 113-118.
Gómez, L. D. and, L. Kisimova-Horovitz. 1997. Basidiomycetes of Costa Rica. Exobasidiales, Cryptobasidiales. Historical notes. Rev. Biol. Trop. 45 : 1293-1310.
Gómez, L. D. and, L. Kisimova-Horovitz. 1998. Basidiomicetes de Costa Rica. Nuevas especies de Exobasidium (Exobasidiaceae) y registros de Cryptobasidiales. Rev. Biol.
Trop. 46 : 1081-1093.
Kennedy, A. H., N. A. Goldberg, and A. M. Minnis. 2012. Exobasidium ferrugineae sp.
nov., associated with hypertrophied flowers of Lyonia ferruginea in the southeastern
USA. Mycotaxon 120 : 451-460.
Li, Z., and L. Guo. 2010. Studies of Exobasidium new to China: E. rhododendri-siderophylli sp. nov. and E. splendidum. Mycotaxon 114 : 271-279. https://doi.
org/ 10.5248 / 114.271
Maier, W., T. Khoza, N. Harmse, B. D. Wingfield and M. J. Wingfield. 2006. A disease19慶應義塾大学日吉紀要・自然科学

No. 70（2023）

epidemic on Zizyphus mucronata in the Kruger National Park caused by Coniodictyum chevalieri. Stud. in Myc. 55 : 279-288. https://doi.org/ 10.3114 /sim. 55.1. 279
McNabb, R. F. R. 1962. The genus Exobasidium in New Zealand. Trans. Roy. Soc. N. Z.
1 : 259-268
Müller, E. 1977. Reflections on the geographical distribution of Exobasidium warmingii.
Kew bulletin. 31 : 545-550.
Nagao, H., and K. Katumoto. 1998. Leaf blister of Quercus phillyraeoides caused byTaphrina caerulescens. Mycoscience 39 (2): 173-178.
Nagao, H., A. Ezuka, H. Ohkubo, and M. Kakishima. 2001. A new species of Exobasidium causing witches’ broom on Rhododendron wadanum. Mycoscience 42 : 549-554.
Nagao, H., M. Akimoto, K. Kishi, A. Ezuka, and M. Kakishima. 2003 a. Exobasidium dubium and E. miyabei sp. nov. causing Exobasidium leaf blisters on Rhododendron
spp. in Japan. Mycoscience 44 : 1-9.
Nagao, H., S. Ogawa, T. Sato, and M. Kakishima. 2003 b. Exobasidium symploci-japonicae var. caprogenum var. nov. causing Exobasidium fruit deformation on Symplocos
lucida in Japan. Mycoscience 44 : 369-375.
Nagao, H., T. Sato, and M. Kakishima. 2004 a.Three species of Exobasidium causing
Exobasidium leaf blight on subgenus Hymenanthes, Rhododendron spp., in Japan.
Mycoscience 45 : 85-95.
Nagao, H., S. Kurogi, A. Ezuka, T. Sato, and M. Kakishima. 2004 b. Emendation of
Exobasidium otanianum causing Exobasidium leaf blight on Rhododendron spp. inJapan. Mycoscience 45 : 245-250.
Nagao, H., A. Ezuka, Y. Harada, T. Sato, and M. Kakishima. 2006. Two new species of
Exobasidium causing Exobasidium diseases on Vaccinium spp. in Japan. Mycoscience 47 (5) : 277-283.
Nannfeldt, J. A. 1981. Exobasidium a taxonomic reassessment applied to the European
species. Symb. Bot. Upsal. 23 (2): 1-72.
Nickerson, N. L. and Vander Kloet, S. P. 1997. Exobasidium leaf spot of lowbush blueberry. Can. J. Plant Pathol. 19 : 66-68.
O’Donnell, K. L. 1993. Fusarium and its near relatives. In: Reynolds, D.R. and J.W. Taylor. (eds.) The fungal holomorph: mitotic, meiotic, and pleomorphic speciation in fungal systematics. pp. 225-233.CAB international, Wallingford.
Piątek, M., M. Lutz, and P. Welton. 2012. Exobasidium darwinii, a new Hawaiian species
infecting endemic Vaccinium reticulatum in Haleakala National Park. Mycol. Progress 11 : 361-371. https://doi.org/ 10.1007 /s11557-011-0751-4
Reaz, R., M. S. Bayzid and M. S. Rahman. 2014. Accurate Phylogenetic Tree Reconstruc20

The Host-Pathogen Association of Exobasidium in Japan Inferred from Molecular Phylogeny of ITS and Large Subunit rDNA Sequences（NAGAO・KASUYA）

tion from Quartets: A Heuristic Approach. PLoS ONE 9 (8): e104008. https://doi.
org/ 10.1371 /journal.pone. 0104008
Savile, D. B. O. 1959 a. Notes on Exobasidium. Can. J. Bot. 37 : 641-656.
Savile, D. B. O. 1959 b. The botany of Somerset Island, district of Franklin. Can. J. Bot.
37 (5): 959-1002.
Sawada, K. 1950. Fungi from the North-Eastern region of Honshu, Japan (2) Basidiomycetes. Tohoku Biological 1 : 95-100.
Schmidt, H.A. and A. von Haeseler. 2003. Maximum-Likelihood Analysis Using Tree-Puzzle. Current Protocols in Bioinformatics 1 : 6.6. 1-6.6. 23. John Wiley & Sons, Inc.
https://doi.org/ 10.1002 / 0471250953.bi0606 s01
Shibata, S. and Y. Hirooka. 2022. Taxonomy and phylogeny of Exobasidium pentasporium causing witches’ broom of Rhododendron species. Mycoscience 63 : MYC582.
https://doi.org/ 10.47371 /mycosci. 2022.07.002
Sundström, K.-R. 1964. Studies of the physiology, morphology, and serology of Exobasidium. Symb. Bot. Upsal. 18 (3): 1-89
Suyama Y., K. Kawamuro, I. Kinoshita, K. Yoshimura, Y. Tsumura, and H. Takahara.
1996. DNA sequence from a fossil pollen of Abies spp. from Pleistocene peat. Genes
Genet. Syst. 71 : 145-149.
Swofford, D.L. 1998. PAUP* 4.0. Phylogenetic analysis using parsimony. Sunderland, Massachusetts: Sinauer Associates.
Takeuchi, K., and H. Nagao. 2004. DNA sequencing for molecular identification of plant
pathogens. MAFF Microorganism Genetic Resources Manual No. 17. 5-22.
Virtudazo, E. V., H. Nakamura, and M. Kakishima. 2001.Phylogenetic analysis of sugarcane rusts based on sequences of ITS, 5.8 S rDNA and D1 /D2 regions of LSU rDNA.
J. Gen. Plant Pathol. 67 (1): 28-36. https://doi.org/ 10.1007 /PL00012983
Wang, Q.-M., D. Begerow, M. Groenewald, X.-Z. Liu, B. Theelen, F.-Y. Bai, and T. Boekhout. 2015. Multigene phylogeny and taxonomic revision of yeasts and related fungiin the Ustilaginomycotina. Stud. in Myc. 81 : 55-83. https://doi.org/ 10.1016 /j.simyco. 2015.10.004
White, T. J., T. Bruns, S. B. Lee, J. Taylor. 1990. Amplification and direct sequencing of
fungal ribosomal RNA genes for phylogenetics. In :Gelfand, M., D. Sninsky, and T.
White. (eds.) PCR protocols: a guide to methods and applications. pp. 315-322. Academic Press, San Diego.

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