Epitypification and molecular confirmation of Erysiphe cucurbitacearum as a synonym of Golovinomyces tabaci

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Epitypification and molecular confirmation of Erysiphe cucurbitacearum as a synonym of Golovinomyces tabaci Peng-Lei Qiu a, Xiao-Fan Qi a, Yu Li a, Uwe Braun b, Shu-Yan Liu a, * a b

Laboratory of Plant Pathology, Institute of Plant Pathology, Jilin Agricultural University, Changchun 13, 130118, Jilin Province, PR China €t, Institut für Biologie, Bereich Geobotanik und Botanischer Garten, Neuwerk 21, 06099, Halle, Saale, Germany Martin-Luther-Universita

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 June 2019 Received in revised form 9 September 2019 Accepted 10 September 2019 Available online xxx

Recently published phylogenetic analyses of the Golovinomyces orontii complex revealed that three different species of the genus Golovinomyces may occur on cucurbitaceous hosts, viz., G. bolayi, G. orontii s. str., and G. tabaci. Owing to its morphological characteristics, Erysiphe cucurbitacearum (≡ G. cucurbitacearum) was tentatively reduced to synonym with G. tabaci. However, final conclusions on the identity and status of E. cucurbitacearum, described from China on Cucumis sativus, as putative synonym of G. tabaci required a phylogenetic confirmation and epitypification with ex-epitype sequences. Therefore, a sample of G. cucurbitacearum collected on C. sativus in Xinjiang Uygur Autonomous Region, China, in 2014 (HMJAU-PM91862) has been sequenced and is designated as epitype of the latter species, since its holotype material (HMAS 40016, collected in 1954) turned out to be too old for molecular examinations. As a result of morphological examinations and phylogenetic analyses based on ex-epitype ITS and 28S rDNA sequences, the recently assumed synonymy of G. cucurbitacearum, found on C. sativus in China, with G. tabaci could be confirmed. In addition, two new host species of G. tabaci, viz., Trigonotis peduncularis (Boraginaceae) and Rubia cordifolia (Rubiaceae), were concurrently identified. Trigonotis peduncularis is the first verified boraginaceous host for G. tabaci. © 2019 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.

Keywords: Cucumis sativus ITS Powdery mildew Rubia cordifolia Trigonotis peduncularis 28S rDNA

1. Introduction Golovinomyces orontii (Castagne) V.P. Heluta (≡ Erysiphe orontii Castagne) was previously treated as a single plurivorous species complex (Braun & Cook, 2012). First phylogenetic analyses of the genus Golovinomyces raised serious doubts on the conjectural monophyly of G. orontii s. lat. and suggested a splitting of this complex into three distinct clusters (groups) based on nucleotide sequences of the internal transcribed spacer (ITS) and 28S rDNA  naite, _ 2013). Braun et al. regions (Takamatsu, Matsuda, & Grigaliu (2019) recently published a phylogenetic analysis of G. orontii s. lat., based on a much broader sampling, confirmed the separation of this complex into the three previously detected clusters and an additional one constituted by sequences retrieved from G. orontii s. lat. on Vinca spp. As a result, Braun et al. (2019) split G. orontii s. lat. €tz, into four distinct species, viz., G. bolayi S. Takam., Lebeda & M. Go G. orontii s. str., G. tabaci (Sawada) H.D. Shin, S. Takam. & L. Kiss and G. vincae U. Braun & S. Takam. Erysiphe cucurbitacearum R.Y. Zheng

* Corresponding author. E-mail address: [email protected] (S.-Y. Liu).

& G.Q. Chen [≡ G. cucurbitacearum (R.Y. Zheng & G.Q. Chen) Vakal. & Kliron.] and E. rubiae H.D. Shin & Y.J. La [≡ G. rubiae (H.D. Shin & Y.J. La) U. Braun] were reduced to synonym with G. tabaci(Braun et al., 2019). However, in contrast to E. rubiae, E. cucurbitacearum was only tentatively considered a synonym of G. tabaci (with question mark), just based on its morphological traits, but without confirmation by sequence data. Zheng and Chen (1981) introduced E. cucurbitacearum as a new powdery mildew species on the basis of type material found on Cucumis sativus L. in Xinjiang Uygur Autonomous Region, China (holotype: HMAS 40016, collected in 1954). In 2001, E. cucurbitacearum was recognized as species of its own, morphologically clearly distinct from G. orontii on cucurbitaceous hosts, and reallocated to Golovinomyces (Vakalounakis & Klironomou, 2001). Braun and Cook (2012) accepted the taxonomy proposed in the latter publication and recognized two Golovinomyces species on hosts of the Cucurbitaceae, viz., G. orontii and G. cucurbitacearum. However, in the light of the recent phylogenetic splitting of G. orontii s. lat. into three species that may all occur on cucurbitaceous host, the true identity and status of E. cucurbitacearum (≡ G. cucurbitacearum) and its putative synonymy with G. tabaci urgently require an epitypification of this

https://doi.org/10.1016/j.myc.2019.09.002 1340-3540/© 2019 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.

Please cite this article as: Qiu, P.-L et al., Epitypification and molecular confirmation of Erysiphe cucurbitacearum as a synonym of Golovinomyces tabaci, Mycoscience, https://doi.org/10.1016/j.myc.2019.09.002

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species with a corresponding powdery mildew collection on C. sativus from China and ex-epitype sequences (Braun et al., 2019). A sample of G. cucurbitacearum on C. sativus, the type host of this species, with catenescent conidia and chasmothecia, collected in Xinjiang Uygur Autonomous Region in 2014, has been genetically examined. This specimen is an appropriate candidate for the epitypification of this species, aiming to confirm the synonymy of G. cucurbitacearum with G. tabaci. Previous phylogenetic examinations suggested a close coevolution between Golovinomyces species and their host plants, such as G. cichoracearum (DC.) V.P. Heluta supposed to be confined to hosts of tribe Cichorioideae within Asteraceae (Braun & Cook, 2012; Matsuda & Takamatsu, 2003). However, several plurivorous species have to be taken into consideration, like G. tabaci, for instance. In total, host species pertaining to six plant family (Asteraceae, Brassicaceae, Cucurbitaceae, Papaveraceae, Rubiaceae and Solanaceae) have been confirmed to be infected by G. tabaci (Braun et al., 2019). In the course of the present examinations, two additional hosts of G. tabaci have been found, namely Trigonotis peduncularis (Trev.) Benth. (Boraginaceae) and Rubia cordifolia L. (Rubiaceae), confirmed by morphological examinations and phylogenetic analyses. 2. Materials and methods 2.1. Morphological examination Several powdery mildew samples recently collected in China have been microscopically examined, using a light microscope with phase contrast (Axio Scope. A1, ZEISS, Germany): on C. sativus, Xinjiang Uygur Autonomous Region, Altay Prefecture, Sep 2014 (HMJAU-PM91862); on T. peduncularis, Jilin Province, Changchun, laboratory of Jilin Agricultural University, Nov 2016 (HMJAUPM91761); on R. cordifolia, Jilin Province, Changchun, campus of Jilin Agricultural University, Jun and Aug 2018 (HMJAU-PM91863 and HMJAU-PM91864); on R. cordifolia, Beijing, Oct 2018 (HMJAUPM91865). The dried leaves of C. sativus and living leaves of T. peduncularis and R. cordifolia were used for morphological examinations. Piece of the infected living leaves were dipped in a drop of lactic acid, and then some superficial structures of the fungus (mycelium, conidiophores and conidia) was scraped off using a clean scalpel. Dried leaves were immersed in a drop of lactic acid and gently boiled before parts of the asexual morph were scraped off for microscopic examinations. Fresh conidia were used to examine the presence or absence of fibrosin bodies. At least 30 conidia, conidiophores and other structures were measured for each sample. The samples of the infected host plants were pressed and dried at room temperature and deposited in the Herbarium of Mycology of Jilin Agricultural University. 2.2. DNA sequencing and data analyses Genome DNA was extracted by the Chelex-100 method (Hirata & Takamatsu, 1996; Walsh, Metzger, & Higuchi, 1991). The complete ITS region including 5.8S rDNA and 50 end of the 28S rDNA including D1 and D2 regions of the pathogens were amplified by polymerase chain reaction (PCR) with the primers ITS5/ITS4 and LSU1/LSU2 separately (Scholin, Herzog, Sogin, & Anderson, 1994; White, Bruns, Lee, & Taylor, 1990, pp. 315e322). The reaction components and conditions were determined as described by Qiu et al. (2018). The Nucleotide sequences were obtained for both strands using direct sequencing methods. The ITS and 28S rDNA sequences obtained in this study were separately aligned with some sequences used in Braun et al. (2019) and a few sequences retrieved from the NCBI database (Table 1)

using MUSCLE implemented in MEGA 7 programme (Kumar, Steche, & Tamura, 2016). The alignments were deposited in TreeBASE (http://www.treebase.org/) under the accession number S24446. Phylogenetic trees were constructed with the aligned sequence data using the maximum parsimony (MP) in PAUP 4.0a with heuristic search option using the tree bisections reconnection (TBR) algorithm. All sites were treated as unordered and unweighted, with the gaps treated as missing data. The strength of the internal branches from the resulting trees was tested by bootstrap (BS) analysis using 1000 replications (Felsenstein, 1985) in parsimony analyses. Tree scores, including tree length, consistency index (CI), retention index (RI) and rescaled consistency index (RC), were also calculated. The initial trees were obtained by the random addition of sequences (100 replicates). BS supports of 60% or higher are shown. 3. Results 3.1. Phylogenetic analyses The obtained five ITS sequences from the powdery mildews on Cucumis sativus, T. peduncularis and R. cordifolia were identical to each other. The alignment dataset comprised thirty-seven sequences. Two sequences of G. magnicellulatus (U. Braun) V.P. Heluta (accession numbers: AB077621 and AB077647) were used as outgroup. The alignment data matrix contained 492 characters, of which 5 characters were variable but not informative, and 58 characters were informative for parsimony analysis. A most parsimonious tree (tree length ¼ 84, CI ¼ 0.8452, RI ¼ 0.9182, RC ¼ 0.7761) with the highest likelihood value is shown in Fig. 1. The ITS sequences obtained from Golovinomyces found on C. sativus, T. peduncularis and R. cordifolia clustered within the G. tabaci clade [group 1 in Braun et al. (2019)], which is genetically clearly separated from the closely allied species G. bolayi and G. orontii, and also morphologically distinguishable. The position of the sequence from the fungus on T. peduncularis is surprising and represents the first genetically proven record of a boraginaceous host for G. tabaci because Braun, Bradshaw, Zhao, Cho, and Shin (2018) recently showed that G. asperifolii (Erikss.) U. Braun & H.D. Shin that is distant from G. tabaci is the main powdery mildew species on Trigonotis spp. in Asia. The five 28S rDNA sequences obtained in this study are also identical to each other. Twenty-seven 28S rDNA sequences were retrieved and aligned. Two sequences of G. magnicellulatus (accession numbers: AB077620 and AB077646) were used as outgroup. The alignment data set consisted of 541 characters, of which 11 characters were variable but not informative, and 13 characters were informative for parsimony analysis. A most parsimonious tree (tree length ¼ 30, CI ¼ 0.8667, RI ¼ 0.9231, RC ¼ 0.8000) with the highest likelihood value is shown in Fig. 2. The five 28S rDNA sequences obtained from the samples collected for this study formed an independent group together with two sequences of G. tabaci (AB077674 and AB077678), which is almost identical to the structure in the ITS tree. 3.2. Morphological studies Golovinomyces tabaci (Sawada) H.D. Shin, S. Takam. & L. Kiss, Mycol. Progr. 18: 347, 2019. Fig. 3. ≡ Erysiphe tabaci Sawada, Rep. Dept. Agric. Gov. Res. Inst. Formosa 35: 23, 1928. ¼ Erysiphe cucurbitacearum R.Y. Zheng & G.Q. Chen, Sydowia 34: 258, 1981 [holotype: on C. sativus, China, Xinjiang Uygur, Urumqi, 1954, Zhang et al. (HMAS 40016); epitype (designated here, MycoBank: MBT 387182): on C. sativus, China, Xinjiang Uygur

Please cite this article as: Qiu, P.-L et al., Epitypification and molecular confirmation of Erysiphe cucurbitacearum as a synonym of Golovinomyces tabaci, Mycoscience, https://doi.org/10.1016/j.myc.2019.09.002

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Table 1 Vouchers, hosts and sequences used in this study. Species

Voucher

Host family

Host species

Accession number ITS

Golovinomyces asperifolii G. asperifolii G. asperifoliorum G. bolayi G. bolayi G. bolayi G. bolayi G. bolayi G. bolayi G. calceolariae G. calceolariae G. cichoracearum

KUS-F29281

Boraginaceae

Trigonotis peduncularis

MH189708 e

KUS-F29206 KUS-F28744 MUMH672 MUMH676 HMJAU91769 HMJAU91770 MUMH1264 MUMH1978 MUMH1934 MUMH1879 MUMH937

Boraginaceae Boraginaceae Asteraceae Asteraceae Asteraceae Asteraceae Asteraceae Cucurbitaceae Scrophulariaceae Rubiaceae Asteraceae

T. peduncularis Symphytum officinale Cichorium intybus C. intybus Lactuca sativa L. sativa L. scariola Cucumis sativus Calceolaria polyrrhiza Galium aparine Tragopogon pratensis

MH189712 MH189692 AB077666 AB077669 MG148337 MG263993 AB077688 AB427187 AB430810 AB430812 AB769449

e e AB077667 AB077668 MG148338 MG263994 AB077687 e e e e

Braun et al. (2018) Braun et al. (2018) Braun et al. (2019) Braun et al. (2019) Braun et al. (2019) Braun et al. (2019) Braun et al. (2019) Braun et al. (2019) Takamatsu, Heluta, Havrylenko, and Divarangkoon (2009) Takamatsu et al. (2009) Takamatsu et al. (2013)

G. G. G. G. G. G. G. G. G. G. G. G. G.

KAR529 VPRI20383 MUMH759 VPRI20429 MUMH1880 MUMH2527 DNAno.18 MUMH441 MUMH930 MUMH938 KAR516 MGH Ecr1

Asteraceae Asteraceae Asteraceae Boraginaceae Asteraceae Asteraceae Polemoniaceae Solanaceae Caprifoliaceae Caprifoliaceae Caprifoliaceae Cruciferae Cucurbitaceae

T. buphthalmoides Scorzonera hispanica S. hispanica Myosotis sp. Leuceria thermarum L. thermarum Phlox paniculata Physalis alkekengi Valerianella locusta V. officinalis V. cf. uncinata Arabidopsis thaliana Cucurbita pepo

AB077694 GQ183946 AB077682 AB769455 AB246765 AB246766 AB077621 AB077647 AB769470 AB769471 AB077693 AF009176 AF229016

e e AB077681 e e e AB077620 AB077646 e e AB077692 e e

Matsuda and Takamatsu (2003) Cunnington, Lawrie, and Pascoe (2010) Matsuda and Takamatsu (2003) Takamatsu et al. (2013) Takamatsu, Matsuda, Niinomi, and Havrylenko (2006) Takamatsu et al. (2006) Matsuda and Takamatsu (2003) Matsuda and Takamatsu (2003) Braun et al. (2019) Braun et al. (2019) Matsuda and Takamatsu (2003) Braun et al. (2019) Braun et al. (2019)

VPRI19066 MUMH683 MUMH952 MUMH2433 MUMH661 HMJAUPM91862 HMJAUPM91761 HMJAUPM91863 HMJAUPM91864 HMJAUPM91865 MUMH677 MUMH711

Plantaginaceae Asteraceae Asteraceae Plantaginaceae Plantaginaceae Cucurbitaceae

Penstemon serrulatus Sonchus arvensis S. arvensis Plantago sp. P. lanceolata Cucumis sativus

GQ183941 AB077673 AB453762 AB769467 AB077665 MK937797

e AB077672 e e AB077664 MK937802

Braun et al. (2019) Matsuda and Takamatsu (2003) Takamatsu et al. (2013) Takamatsu et al. (2013) Matsuda and Takamatsu (2003) This study

Boraginaceae

MK937796 MK937801 This study

Rubiaceae

Trigonotis peduncularis Rubia cordifolia

Rubiaceae

R. cordifolia

MK937799 MK937804 This study

Rubiaceae

R. cordifolia

MK937800 MK937805 This study

Cucurbitaceae Asteraceae

Cucurbita pepo Dahlia pinnata

AB077670 AB077671 Braun et al. (2019) AB077677 AB077678 Braun et al. (2019)

cichoracearum cichoracearum cichoracearum cynoglossi leuceriae leuceriae magnicellulatus magnicellulatus orontii orontii orontii orontii orontii

G. orontii G. sonchicola G. sonchicola G. sordidus G. sordidus G. tabaci G. tabaci G. tabaci G. tabaci G. tabaci G. tabaci G. tabaci a

Reference

28Sa Braun et al. (2018)

MK937798 MK937803 This study

“e” represents the sequences are not available.

Autonomous Region, Altay Prefecture, Sep 2014, Wen-Tao Jiang (HMJAU-PM91862)]. ≡ Golovinomyces cucurbitacearum (R.Y. Zheng & G.Q. Chen) Vakal. & Kliron., Mycotaxon 80: 490, 2001. ¼ Erysiphe rubiae H.D. Shin & Y.J. La, Korean J. Pl. Pathol. 5: 182, 1989. ≡ Golovinomyces rubiae (H.D. Shin & Y.J. La) U. Braun, Schlechtendalia 3: 51, 1999. Description (based on type material of E. cucurbitacearum): Mycelium amphigenous, thin, effuse, white, covering both sides of the leaves; hyphae straight to sinuous, thin-walled, smooth, hyaline, 3.2e7.7 mm wide; hyphal appressoria nipple-shaped, occasionally forked, mostly solitary or several in series (Fig. 3A and B); conidiophores 73.9‒169.2  7.6e11 mm (without conidia) (mean 127  9.6 mm), arising from the upper surface of hyphal mother cells or occasionally laterally, most towards one end of the cell (Fig. 3C and D); foot-cells straight or curved (but not abruptly curved at the very base as in conidiophores that arise laterally from mother cells), 31.5e98.2(‒125.6)  8.2e11.2 mm (mean 74  9.8 mm), followed by 2e4(‒5) shorter cells, basal septum usually at the junction with the supporting hypha; conidia

catenescent, without fibrosin bodies, ellipsoid-ovoid or cylindrical, 21.6e37.3(‒47.4)  9.4e16 mm (mean 28.4  13 mm) with a length/ width ratio varying from 1.5 to 3 (Fig. 3E‒G); germ tubes short, clavate, aseptate or with a single septum, Euoidium type (Fig. 3H and I). Chasmothecia amphigenous, mostly immersed in the mycelium, (87.3‒)100.7e154.0(‒165.8) mm diam (mean 122 mm) (Fig. 3J); peridium cells rounded to irregularly polygonal, 6.7e23.3 mm diam; appendages mycelioid, mostly arising from the lower half, occasionally branched, interwoven with each other and with the mycelium, 0.4e2.9 times as long as the chasmothecial diam, up to 250 mm, brown below and paler towards the apex, septate, thin-walled, smooth or almost so; asci 8e20, broad obovoid-saccate or almost clavate, short-stalked or sessile, 49.4‒ 82.5  27.5e49.4 mm (mean 66  34 mm), 2-spored (Fig. 3K‒M); ascospores ovoid or ellipsoid, 15.8‒28  11.7e17.9 mm (mean 21  14 mm) (Fig. 3N, O). Golovinomyces tabaci on T. peduncularis. Fig. 4. Description: Mycelium effuse, thin, white, covering both sides of the leaves and petioles; hyphae straight to sinuous, thin-walled, smooth, hyaline, 3.3e7.6 mm wide; hyphal appressoria nippleshaped, mostly several in a series, occasionally solitary (Fig. 4A

Please cite this article as: Qiu, P.-L et al., Epitypification and molecular confirmation of Erysiphe cucurbitacearum as a synonym of Golovinomyces tabaci, Mycoscience, https://doi.org/10.1016/j.myc.2019.09.002

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Fig. 1. Phylogenetic analysis of Golovinomyces tabaci found on Cucumis sativus, Trigonotis peduncularis and Rubia cordifolia based on ITS sequences. Golovinomyces magnicellulatus (accession numbers: AB077621 and AB077647) is used as an outgroup taxon. Bootstrap values (>60%) by the maximum parsimony are shown on the respective branches. Sequences determined in this study are highlighted in boldface.

and B); conidiophores 119.8e218.3(‒233)  8.1e12.3 mm (without conidia) (mean 163  9.8 mm), arising from the upper surface of hyphal mother cells or sometimes laterally, mostly towards one end of the cell (Fig. 4C‒E); foot-cells mostly straight or a few curved (but not abruptly curved at the very base as in conidiophores that arise laterally from mother cells), 43.4e122.6(‒135)  8.5e11.5 mm (mean 79  10 mm), followed by 1e3 shorter cells, basal septum at the junction with the supporting hypha; conidia catenescent, without fibrosin bodies, ellipsoid-ovoid or a few cylindrical, 23.6‒ 43.1  13e19.2 mm (mean 31  16 mm) with a length/width ratio varying from 1.3 to 2.6(‒3.2) (Fig. 4F and G); germ tubes short, clavate, Euoidium type, rarely with a distinct terminal appressorium (Fig. 4H). Golovinomyces tabaci on R. cordifolia. Fig. 5. Description: Mycelium effuse, thin, white, covering both sides of the leaves and stems; hyphae straight to sinuous, thin-walled, smooth, hyaline, 2.8e7.2 mm wide; hyphal appressoria nippleshaped, mostly several in series or occasionally solitary (Fig. 5A); conidiophores (59.7‒)67.7e159.4(‒175.4)  7.5e12 mm (without conidia) (mean 108  9.8 mm), arising from the upper surface of hyphal mother cells or sometimes laterally, mostly towards one end of the cell (Fig. 5B); foot-cells mostly straight or a few curved in the middle (but not abruptly curved at the very base as in

Fig. 2. Phylogenetic analysis of Golovinomyces tabaci found on Cucumis sativus, Trigonotis peduncularis and Rubia cordifolia based on the 28S rDNA sequences (including domains D1 and D2). Golovinomyces magnicellulatus (accession numbers: AB077620 and AB077646) is used as outgroup taxon. Bootstrap values (>60%) by the maximum parsimony are shown on the respective branches. Sequences determined in this study are highlighted in boldface.

conidiophores that arise laterally from mother cells), 30.6e122(‒ 130.4)  8.7e12.3 mm (mean 66  10 mm), followed by (0‒)1e4 shorter cells, basal septum usually at the junction with the supporting hypha; conidia catenescent, without fibrosin bodies, ellipsoid-ovoid or almost cylindrical, 22.3‒35.1  11.3e16.4 mm (mean 27.6  14.2 mm) with a length/width ratio varying from 1.4 to 2.6 (Fig. 5C and D); germ tubes short, solitary or in opposite pairs, mostly clavate, a few are forked, Euoidium type, with (0)1e5 appressoria at the tip (Fig. 5E‒I). 4. Discussion Erysiphe cucurbitacearum was described on the basis of Chinese type material from Xinjiang Uygur Autonomous Region collected on C. sativus in 1954, characterized by having catenescent conidia without fibrosin bodies and ascomata with mycelioid appendages and several 2-spored asci (Zheng & Chen, 1981). Vakalounakis and Klironomou (2001) reallocated E. cucurbitacearum to Golovinomyces based on the new taxonomic generic concept of the Erysiphe (s. lat.) complex published in Braun (1999). They recognized this powdery mildew as a separate species morphologically distinguished from

Please cite this article as: Qiu, P.-L et al., Epitypification and molecular confirmation of Erysiphe cucurbitacearum as a synonym of Golovinomyces tabaci, Mycoscience, https://doi.org/10.1016/j.myc.2019.09.002

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Fig. 3. Golovinomyces tabaci found on Cucumis sativus in China (epitype: HMJAU-PM91862). A: Nipple-shaped hyphal appressoria, several formed in series. B: Slightly lobed hyphal appressorium. C, D: Conidiophores. EeG: Conidia. H, I: Conidia with germ tubes. J: Chasmothecium. KeM: Asci. N, O: Ascospores. Bars: 10 mm.

G. orontii. Recently, comprehensive phylogenetic analyses and reexaminations of the morphology of G. orontii s. lat. led to a splitting of this complex into several species, including G. tabaci, a species which is genetically and morphologically clearly distinct from G. orontii s. str. (Braun et al., 2019). On account of its morphological traits, G. cucurbitacearum has been tentatively reduced to synonym with G. tabaci, emphasizing the need for epitypification and ex-epitype sequences in order to confirm this synonymy. A Chinese Golovinomyces specimen on C. sativus, type host of E. cucurbitacearum, from Xinjiang Uygur Autonomous Region, collected in 2014, is suitable to overcome the uncertainty with regard to the tentatively proposed synonymy of E. cucurbitacearum with G. tabaci. The morphological characters of the present powdery mildew collection on C. sativus agree well with the circumscription of G. tabaci, but all structures are somewhat larger in comparison to the original description of E. cucurbitacearum in Zheng and Chen (1981) and Zheng and Yu (1987), although all measurements are overlapping, reflecting a certain degree of variation, such as the number of the asci originally specified to be (5‒)7e11(‒13) per chasmothecium, which is on average lesser than in common G. tabaci ascomata (5‒20). The molecular analyses of ITS and 28S rDNA data confirmed that the powdery mildew found on C. sativus pertains to G. tabaci, supporting the assumed taxonomic status of E. cucurbitacearum as synonym of G. tabaci (Braun et al., 2019). In order to establish this synonymy, the sequenced specimen from Xinjiang Uygur Autonomous Region has been designated as epitype of the latter species. In any case, Golovinomyces collections on cucurbitaceous hosts have to be treated and identified with utmost care, whenever possibly by mean of sequence analyses, since all three species of G. orontii s. lat. (G. bolayi,

G. orontii s. str., and G. tabaci) may occur on hosts of this plant family. Morphological differences between these species exist, but they are only gradual and not easily discernible. Samples of Golovinomyces on boraginaceous hosts were previously assigned to a single species, G. cynoglossi s. lat. (Braun & Cook, 2012), which has turned out to represent a genetically and morphologically heterogeneous complex, recently divided into G. cynoglossi s. str. (Wallr.) V.P. Heluta, G. asperifoliorum (Grev.) U. Braun & H.D. Shin and G. asperifolii. Trigonotis spp. are common hosts of the plurivorous, widespread species G. asperifolii (Braun et al., 2018). The genetic verification of G. tabaci on T. peduncularis in the course of the present examinations, based on rDNA sequence data, was astounding and represent the first record of G. tabaci on a host of the family Boraginaceae, emphasizing that simple “identifications” just based on the host identity are insufficient and unreliable. Chasmothecia of G. tabaci are different from those of G. asperifolii mainly by having 5e20 2-spored asci per fruiting body, versus 5e15 2‒4-spored asci per ascoma in G. asperifolii. There are also slight differences in the asexual morphs (the basal septum of the conidiophores is usually formed at the junction with the supporting hypha and not elevated, whereas in G. asperifolii they are somewhat elevated, 5e25 mm, see Braun et al. (2018)). Golovinomyces tabaci found on T. peduncularis produced longer conidiophores compared to the description published in Braun et al. (2019), which might be explained by the fact that this sample was collected from an infected plant growing under room conditions in a laboratory. In China, R. cordifolia was reported as host of E. cichoracearum (s. lat.) but without molecular support (Tai, 1979). Braun et al. (2019) reduced G. rubiae to synonym with G. tabaci and verified the occurrence of the latter species on various Rubia spp.,

Please cite this article as: Qiu, P.-L et al., Epitypification and molecular confirmation of Erysiphe cucurbitacearum as a synonym of Golovinomyces tabaci, Mycoscience, https://doi.org/10.1016/j.myc.2019.09.002

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Fig. 4. Golovinomyces tabaci found on Trigonotis peduncularis (voucher: HMJAU-PM91761). A: Nipple-shaped hyphal appressoria, several formed in series. B: Nipple-shaped hyphal appressorium formed singly. CeE: Conidiophores. F, G: Conidium. H: Conidium with germ tube. Bars: 20 mm.

Fig. 5. Golovinomyces tabaci found on Rubia cordifolia (voucher: HMJAU-PM91863). A: Nipple-shaped hyphal appressoria, several formed in series. B: Conidiophore. C, D: Conidia. EeI: Conidia with germ tubes. Bars: 20 mm.

Please cite this article as: Qiu, P.-L et al., Epitypification and molecular confirmation of Erysiphe cucurbitacearum as a synonym of Golovinomyces tabaci, Mycoscience, https://doi.org/10.1016/j.myc.2019.09.002

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supplemented by the present confirmation of R. cordifolia as host of this species. Disclosure The authors declare no conflicts of interest. All the experiments undertaken in this study comply with the current laws of the country where they were performed. Acknowledgements This research was supported by the National Natural Science Foundation of China (31470153, 31670022). We are much obliged to Prof. Susumu Takamatsu for his review of the manuscript. References Braun, U. (1999). Some critical notes on the classification and generic concept of the Erysiphaceae. Schlechtendalia, 3, 49e55. Braun, U., Bradshaw, M., Zhao, T. T., Cho, S. E., & Shin, H. D. (2018). Taxonomy of the Golovinomyces cynoglossi complex (Erysiphales, Ascomycota) disentangled by phylogenetic analyses and reassessments of morphological traits. Mycobiology, 46, 192e204. https://doi.org/10.1080/12298093.2018.1509512. Braun, U., & Cook, R. T. A. (2012). Taxonomic manual of the Erysiphales (powdery mildews). CBS Biodiversity series No. 11. Utrecht: CBS-KNAW Fungal Biodiversity Centre. Braun, U., Shin, H. D., Takamatsu, S., Meeboon, J., Kiss, L., Lebeda, A., et al. (2019). Phylogeny and taxonomy of Golovinomyces orontii revisited. Mycological Progress, 18, 335e357. https://doi.org/10.1007/s11557-018-1453-y. Cunnington, J. H., Lawrie, S. C., & Pascoe, I. G. (2010). Genetic characterization of the Golovinomyces cichoracearum complex in Australia. Plant Pathology, 59, 158e164. https://doi.org/10.1111/j.1365-3059.2009.02175.x. Felsenstein, J. (1985). Confidence limits on phylogenetics: An approach using the bootstrap. Evolution, 39, 783e791. https://doi.org/10.2307/2408678. Hirata, T., & Takamatsu, S. (1996). Nucleotide diversity of rDNA internal transcribed spacers extracted from conidia and cleistothecia of several powdery mildew

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Please cite this article as: Qiu, P.-L et al., Epitypification and molecular confirmation of Erysiphe cucurbitacearum as a synonym of Golovinomyces tabaci, Mycoscience, https://doi.org/10.1016/j.myc.2019.09.002