Mycol Progress (2016) 15: 36 DOI 10.1007/s11557-016-1178-8

ORIGINAL ARTICLE

Genetic diversity and host range of powdery mildews on Papaveraceae

Katarína Pastirčáková1 & Tünde Jankovics2 & Judit Komáromi3 & Alexandra Pintye2 & Martin Pastirčák4

Received: 29 September 2015 /Revised: 19 February 2016 /Accepted: 23 February 2016 /Published online: 10 March 2016 # German Mycological Society and Springer-Verlag Berlin Heidelberg 2016

Abstract Because of the strong morphological similarity of of papaveraceous hosts. Although E. macleayae occurred nat- the powdery mildew fungi that infect papaveraceous hosts, a urally on Macleaya cordata, Macleaya microcarpa, M. total of 39 samples were studied to reveal the phylogeny and cambrica,andChelidonium majus only, our inoculation tests host range of these fungi. ITS and 28S sequence analyses revealed that the fungus was capable of infecting Argemone revealed that the isolates identified earlier as Erysiphe grandiflora, Glaucium corniculatum, Papaver rhoeas, and cruciferarum on papaveraceous hosts represent distinct line- Papaver somniferum, indicating that these plant species may ages and differ from that of E. cruciferarum sensu stricto on also be taken into account as potential hosts. Erysiphe brassicaceous hosts. The taxonomic status of the anamorph cruciferarum originating from P. somniferum was not able to infecting Eschscholzia californica was revised, and therefore, infect A. grandiflora, C. majus, E. californica, M. cordata, a new species name, Erysiphe eschscholziae, is proposed. The and P. rhoeas. The emergence of E. macleayae on M. taxonomic position of the Pseudoidium anamorphs infecting microcarpa is reported here for the first time from the Glaucium flavum, Meconopsis cambrica, Papaver dubium, Czech Republic and Slovakia. The appearance of chasmothecia and Stylophorum diphyllum remain unclear. This study re- of E. macleayae on C. majus in Slovakia was reported, as well. vealed that Erysiphe macleayae exhibits a specific host range Erysiphe cruciferarum was identified on G. corniculatum and different from that of E. cruciferarum, the common pathogen reported here for the first time from Slovakia.

Keywords Erysiphaceae . Eschscholzia . Macleaya . ITS . Section Editor: Franz Oberwinkler 28S nrDNA . Morphology Taxonomic novelty Erysiphe eschscholziae Pastirč. & Jankovics Katarína Pastirčáková and Tünde Jankovics contributed equally to this work. Introduction

* Katarína Pastirčáková Asexual morphs of powdery mildews belonging to the same [email protected] genus are, in many cases, morphologically indistinguishable from each other. Therefore, their identification is often com- plicated, especially in the absence of the sexual morphs 1 Institute of Forest Ecology, Branch for Woody Plant Biology, Slovak Academy of Sciences, Akademicka 2, SK-94901 Nitra, Slovakia (chasmothecia), and sometimes questionable based exclusive- ly on morphological data. The identification might be more 2 Plant Protection Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, P.O. Box 102, problematic in those particular cases when one host plant spe- H-1525 Budapest, Hungary cies is infected by more than one asexual powdery mildew 3 Agricultural Institute, Centre for Agricultural Research, Hungarian fungi with highly similar morphologies (Cook et al. 1997). Academy of Sciences, Brunszvik u. 2, On the other hand, morphologically indistinguishable asexual H-2462 Martonvásár, Hungary morphs may represent genetically distinct lineages that seem 4 Research Institute of Plant Production, National Agricultural and to be specialized to one or a few host plant species belonging Food Centre, Bratislavska cesta 122, SK-92168 Piestany, Slovakia even to different plant families. Recently, phylogenetic 36 Page 2 of 18 Mycol Progress (2016) 15: 36 relationships among various closely related powdery mildew this study were (i) to investigate the phylogenetic relationships species have been revealed based on the nucleotide sequences and taxonomic positions of the powdery mildew pathogens of the internal transcribed spacer (ITS) and 28S regions of occurring naturally on papaveraceous hosts and exhibiting nuclear ribosomal DNA (nrDNA) (e.g. Cunnington et al. morphologically indistinguishable asexual morphs of the 2005; Kiss et al. 2006; Inuma et al. 2007; Jankovics et al. Pseudoidium type, based on the nucleotide sequences of the 2008; Takamatsu et al. 2008, 2013). ITS and 28S nrDNA regions; and (ii) to determine whether the To date, three Erysiphe species exhibiting asexual two powdery mildew species E. cruciferarum and E. Pseudoidium stages have been reported from the family macleayae, both of which occur on hosts native to Europe, Papaveraceae, namely Erysiphe cruciferarum Opiz ex L. can infect papaveraceous plant species other than their origi- Junell, E. hylomeci H.D. Shin & Y.J. La, and E. macleayae nal hosts. R.Y. Zheng & G.Q. Chen (Braun and Cook 2012). Erysiphe cruciferarum is the most common causal agent of the powdery mildew on various papaveraceous hosts in Asia and Europe (Braun 1987; Braun and Cook 2012), while E. hylomeci was Materials and methods recorded only on Hylomecon vernalis in Korea (Shin 2000), and E. macleayae haslongbeenconsideredtobeendemicto Fungal material and morphological observations Asia (Park et al. 2012). Recently, asexual powdery mildew morphs identified as E. macleayae have been reported to oc- In order to find fruiting bodies of the powdery mildew patho- cur on Macleaya cordata in Germany (Ale-Agha et al. 2008; gen infecting C. majus, diseased leaves of this host were col- Schmidt and Scholler 2011)andMacleaya microcarpa in lected in Slovakia and Hungary, where the fungus is known to Poland and Ukraine (Park et al. 2012; Heluta and Kravchuk occur only in its asexual stage. For the precise identification of 2015). In Germany, the asexual morph of this species has been the fungus causing symptoms on Glaucium corniculatum,the observed on Chelidonium majus as BOidium sp.^ since 2003 powdery mildew infected leaves and seed pods of this plant (Jage et al. 2010), while both the asexual and sexual morphs species were also collected in Slovakia in 2013. The speci- were found to be present on a new host, Meconopsis cambrica mens were examined using a stereo binocular microscope (Schmidt and Scholler 2011). Since the first record of pow- (SZ61, Olympus, Japan) and a standard light microscope dery mildew infection on C. majus in Italy (Ciferri and (BX51, Olympus, Japan). Fresh powdery mildew mycelium Camera 1962), only Oidium/Pseudoidium sp. has been iden- was scraped off the leaves and mounted in distilled water for tified on this host in several European countries until recently morphological characterization and measurements using oil (Pastirčáková and Pastirčák 2013; Heluta and Kravchuk immersion at 1000× magnification. The morphological struc- 2015). In 2014, chasmothecia have been collected on C. majus tures of the fungi were photographed using a digital camera in Germany several times by H. Jage and were distributed as (SP350, Olympus, Japan). Representative specimens of the U. Braun, Fungi selecti exsiccati 213 (Braun 2014). In Asia, a new collections were deposited in the herbarium NR. A du- powdery mildew, indistinguishable from the one occurring in plicate of the specimen of E. macleayae on C. majus Europe and identified as either E. cruciferarum or Oidium sp., (with mature chasmothecia) from Slovakia was deposit- hasbeenreportedtooccuronChelidonium spp. in the Russian ed at BPI, as well. Far East (Bunkina 1991), Japan (Nomura 1997), and South In order to re-examine the powdery mildew fungi collected Korea (Shin 2000). Recently, Jiang et al. (2015)havefound previously from various papaveraceous hosts, 29 herbarium chasmothecia on C. majus in China and identified the causal specimens were borrowed from G, HMAS, KR, and SOMF agent as E. macleayae confirmed by morphological and (abbreviations of herbaria according to Thiers [continuously molecular analyses. However, although the above men- updated]). The specimens from Japan and Romania were pro- tioned European records suggest the recent emergence vided by S. Takamatsu (Mie University, Tsu, Japan) and V. of E. macleayae in Europe, exhaustive attempts to iden- Iacob (University of Applied Life Sciences and Environment, tify these primarily asexual powdery mildew morphs Iasi, Romania), respectively. The herbarium specimens repre- have not been made so far, particularly by means of sented powdery mildew fungi occurring naturally on 13 dif- molecular approaches. ferent host plant species, i.e. C. majus, Eschscholzia Because of their morphological similarity, as well as the californica, G. corniculatum, G. flavum, M. cordata, M. close affinities of their host species, many powdery mildews cambrica, Papaver aurantiacum, P. croceum, P. dubium, P. on papaveraceous hosts have been assigned to E. nudicaule, P. rhoeas, P. somniferum,andStylophorum cruciferarum or Oidium sp./Pseudoidium sp. Such powdery diphyllum. Herbarium specimens of E. cruciferarum on type mildew species might easily be misidentified, above all when host Alyssum spp. (Brassicaceae) were borrowed from PRM only morphological patterns and hosts are considered; there- and SAV. The lactic acid technique (Shin and La 1993)was fore, a more complex approach is required. The objectives of used for microscopic examination of herbarium material. Mycol Progress (2016) 15: 36 Page 3 of 18 36

DNA extraction, PCR amplification, and sequencing Foster City, CA) according to the manufacturer’s instructions. of the ITS and 28S nrDNA regions Both strands were sequenced using the primers ITS1F and ITS4, and NL1 and TW14 for ITS and 28S regions, respec- To identify the powdery mildew pathogens occurring natural- tively. Electrophoresis was carried out on an ABI PRISM ly on papaveraceous hosts more precisely, DNAwas extracted 3100 Genetic Analyzer. The sequences were compiled from from the diseased leaves of C. majus, E. californica, G. electrophoregrams using Pregap4 and Gap4 (Staden et al. corniculatum, G. flavum, M. cordata, M. cambrica, P. 2000) and deposited in GenBank. aurantiacum, P. croceum, P. dubium, P. nudicaule, P. rh oe as, P. somniferum,andS. diphyllum collected in this work and Sequence analyses borrowed from herbaria (Table 1). Herbarium specimens of E. cruciferarum on the brassicaceous hosts Alyssum alyssoides The ITS dataset consisting of 55 sequences and 28S nrDNA (including type specimen), A. hirsutum,andBerteroa incana dataset consisting of 49 sequences were aligned with PRANK were also included. Small pieces of leaves covered by pow- (Löytynoja and Goldman 2008) using the PRANKSTER in- dery mildew mycelia were cut from the dried specimens using terface. The final alignment was 613 characters long for the sterile razor blades, then collected in Eppendorf tubes and ITS and 593 characters long for the 28S dataset. The ITS and stored at −18 °C until whole-cell DNA was extracted using a 28S sequences of E. glycines F.L. Tai served as outgroups. All Qiagen DNeasy Plant Mini Kit (Qiagen GmbH, Hilden, indels were coded with GapCoder (Young and Healy 2003) Germany). The ITS region of the fungal nrDNAwas amplified because indels can improve the phylogenetic potential of fun- by a nested polymerase chain reaction (PCR) using the pow- gal ITS sequences (Nagy et al. 2012). Bayesian (MCMC) dery mildew specific primer set PMITS1/PMITS2 analyses were performed on the combined datasets with (Cunnington et al. 2003). The reaction components for the MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003)basedon first PCR were 2 μL of total genomic DNA, 2 μLofincuba- the GTR nucleotide substitution model. Markov chains were tion buffer (Fermentas, Lithuania), 2 μLofMgCl2 (25 mM; run over 5,000,000 generations; one tree was sampled every Fermentas), 0.5 μL of an equivalent mixture of each dNTP 500 generations with a burn-in at 3000 trees. To test the con- (10 mM each; Fermentas), 0.2 μL of each of the primers vergence of runs, the results were analysed with AWTY PMITS1 and PMITS2 at 50 μM each, 0.8 units of Taq poly- (Nylander et al. 2008); no indication of lack of convergence merase (Fermentas), and milliQ water up to a final volume of was detected. In addition, maximum likelihood (ML) phylo- 20 μL. The PCR was performed with the following cycling genetic analyses were carried out with raxmlGUI (Silvestro parameters: an initial denaturation step at 94 °C for 5 min, 2012) using GTR nucleotide substitution model. followed by 35 cycles consisting of a denaturation step of Bootstrapping were performed with 1000 replicates. 45 s at 94 °C, primer annealing for 45 s at 62 °C, and extension Phylogenetic trees were viewed and edited by Tree Explorer for 1 min at 72 °C. The final extension step was performed at of the MEGA 5 program (Tamura et al. 2011) and a text editor. 72 °C for 10 min. A negative control, where template DNA was replaced by milliQ water, was included for each set of Plant and fungal material and experimental design reactions. The components of the second PCR were identical of the inoculation tests with E. cruciferarum to those of the first one, except that the fungal-specific primer and E. macleayae pair ITS1F/ITS4 (Gardes and Bruns 1993) was used instead of PMITS1 and PMITS2 primers. The cycling parameters were Inoculation tests were carried out to determine whether the also identical, except that the primer annealing temperature powdery mildew species E. cruciferarum and E. macleayae, was 55 °C. The 28S nrDNA was amplified by a nested PCR occurring on P. somniferum and C. majus, respectively, can using the primer sets PM3/TW14 (Mori et al. 2000; infect papaveraceous plants other than their original hosts. Takamatsu and Kano 2001) and NL1/TW14 (Mori et al. The test plants of Argemone grandiflora, C. majus, E. 2000) for the first and second amplifications, respectively. californica, G. corniculatum, M. cordata, M. cambrica, P. The other components and cycling parameters of the first rhoeas,andP. somniferum were included in these experi- and second reactions were identical to those described for ments. The seeds of common native plant species (C. majus the amplification of ITS region, except that the primer anneal- and P. rhoeas) were collected in their natural habitats in ing temperature was 54 °C in both reactions. PCR products Slovakia. The seeds of P. somniferum cv. Sokol were obtained were detected by electrophoresis in agarose gel containing from a commercial source. The seeds of non-native or uncom- ethidium bromide and visualized over a UV light source. mon plant species (A. grandiflora, E. californica, G. The PCR products were purified using a High Pure PCR corniculatum, M. cordata,andM. cambrica)wereprovided Product Purification Kit (Roche Applied Science, by the curators of the following five botanical gardens: Mannheim, Germany), then sequenced using a BigDye Botanical Garden in Teplice, Medicinal Herbs Centre of Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Faculty of Medicine and Botanical Garden of Faculty of 36 Page 4 of 18 Mycol Progress (2016) 15: 36

Table 1 List of herbarium specimens of powdery mildew fungi used for nucleotide sequence analysis

Host plant Powdery mildew Location and year of collection Reproductive stage Herbarium GenBank accession no. fungus of the fungusa specimen no.b ITS 28S

Brassicaceae Alyssum alyssoides Erysiphe cruciferarum Prague, Czech Republic, 1841 T PRM 194779 KU672364 KU672344 A. alyssoides E. cruciferarum Dealul Allah Bair, Romania, 1973 T PRM 736620 KU672365 KU672345 A. hirsutum E. cruciferarum Tuzla, Romania, 1977 T PRM 821146 – KU672346 Berteroa incana E. cruciferarum Devínska Kobyla, Slovakia, 1984 A SAV 11 KU672366 KU672347 Papaveraceae Chelidonium majus Oidium chelidoniic Iasi, Romania, 2012 A NR 5087 KT588619 KU672348 C. majus Oidium sp. c Potsdam, Germany, 2005 A KR 21933 KT588620 – C. majus E. cruciferarumc Nyon, Switzerland, 2004 A G 111703 KT588621 – C. majus E. macleayae Karlsruhe, Germany, 2009 A KR 4850 KT588622 KU672349 C. majus E. macleayae Karlsruhe, Germany, 2011 A KR 29748 KT588623 KU672350 C. majus E. macleayae Nitra, Slovakia, 2014 T NR 5186 KU672367 – C. majus E. macleayae Nitra, Slovakia, 2015 T NR 5220 KU672368 KU672351 C. majus subsp. Pseudoidium sp.c Tokyo, Japan, 2012 A NR 5088 KT588624 KU672352 asiaticum Eschscholzia E. cruciferarum Stellenbosch, South Africa, 1999 A G 111707 KT588625 KU672353 californica E. californica E. cruciferarumd Nyon, Switzerland, 2001 A G 111708 KT588626 – E. californica E. cruciferarumd Gland, Switzerland, 2002 A G 111709 KT588627 KU672354 Glaucium corniculatum E. cruciferarum Piestany, Slovakia, 2013 T NR 5092 KT588628 KU672355 G. flavum E. cruciferarum Geneve, Switzerland, 1999 A G 111710 KT588629 KU672356 G. flavum E. cruciferarum Geneve, Switzerland, 2000 A G 111711 KT588630 KU672357 Macleaya cordata E. macleayae Potsdam, Germany, 2005 A KR 21935 KT588631 KU672358 Meconopsis cambrica E. cruciferarum Geneve, Switzerland, 1996 A G 111712 KT588632 KU672359 Papaver aurantiacum E. cruciferarum Geneve, Switzerland, 1998 A G 111713 –– P. croceum E. cruciferarum L’Orient, Switzerland, 1998 T G 111715 KT588633 KU672360 P. croceum E. cruciferarum Le Brassus, Switzerland, 2003 T G 111717 KT588634 – P. dubium E. cruciferarum Geneve, Switzerland, 1998 A G 111721 KT588635 KU672361 P. nudicaule E. cruciferarum Geneve, Switzerland, 2002 A G 111716 KT588636 KU672362 P. nudicaule E. cruciferarum Geneve, Switzerland, 2004 T G 111718 KT588637 KU672363 P. rhoeas E. cruciferarum Geneve, Switzerland, 1997 T G 111719 –– P. somniferum E. cruciferarume Pirin Mts., Bulgaria, 1982 T SOMF 17662 KT588638 – Stylophorum diphyllum E. cruciferarum Geneve, Switzerland, 2003 A G 111722 KT588639 – a A anamorph, T teleomorph b Sources: G = Herbarium of the Conservatoire et Jardin botaniques de la Ville de Geneve, Geneve, Switzerland; KR = Herbarium of State Museum of Natural History, Karlsruhe, Germany; NR = Plant Pathology Herbarium of the Institute of Forest Ecology of Slovak Acad. Sci., Nitra, Slovakia; PRM = Mycological Herbarium of the National Museum, Prague, Czech Republic; SAV= Herbarium of the Institute of Botany of Slovak Acad. Sci., Bratislava, Slovakia; SOMF = Mycological Collection of the Institute of Biodiversity and Ecosystem Research of Bulgarian Acad. Sci., Sofia, Bulgaria c The powdery mildew fungi were identified as E. macleayae in this study d Erysiphe eschscholziae sp. nov. e The herbarium material was revised as E. macleayae by its collector V.I. Fakirova in Mar 1984 and identified as E. cruciferarum in this study

Science of the Masaryk University in Brno, Czech Republic; provided by the Medicinal Herbs Centre of the Botanical Garden of University of Latvia, Riga, Latvia; and MasarykUniversityinBrnowerealreadyinfectedwith Botanical Garden of Parma, University of Parma, Parma, Italy. powdery mildew, and thus the material was deposited as The test plants were grown from seed in pots, in isola- the voucher specimen in NR. tion in a greenhouse, until three to five fully expanded The test plants were inoculated in both a whole plant assay leaves developed. The seed pods of M. microcarpa and a detached leaf assay at the Research Institute of Plant Mycol Progress (2016) 15: 36 Page 5 of 18 36

Production (RIPP) in Slovakia. In the case of the whole plant anamorphic powdery mildew fungus occurring naturally assay, the experiments were carried out in separate greenhouse on C. majus in Martonvásár, Hungary. The test plants compartments. Three potted plants were used for each test were grown from the same seed collections as those plant species and for each powdery mildew species. The in- grown in Slovakia. The experimental design and the oculation was carried out by gently pressing the naturally in- evaluation of powdery mildew infections were carried fected leaves of C. majus and P. somniferum cv. Sokol, the out as described above for the whole plant assay per- original hosts of E. macleayae and E. cruciferarum collected formed in Slovakia. in Slovakia, onto the leaves of healthy test plants. Two plants representing the original hosts were included in the tests as positive controls. Two non-inoculated plants per each plant Results species served as negative controls. All plants were covered with plastic bags and maintained in the greenhouse compart- ITS and 28S sequence analyses ments at a temperature of 20 °C under 16-h natural and artifi- cial illumination and were occasionally sprayed with sterile ITS and 28S sequences were identified in a total of 26 and 20 distilled water. Ten days after inoculation, the test plants were powdery mildew samples, respectively. The samples repre- visually examined for the presence or absence of powdery sented mainly herbarium specimens collected from 11 mildew colonies. If symptoms appeared, the colonies were papaveraceous hosts, i.e. C. majus, E. californica, G. removed from the leaves using a piece of a clear adhesive tape corniculatum, G. flavum, M. cordata, M. cambrica, P. and were examined under a light microscope. The susceptibil- croceum, P. dubium, P. nudicaule, P. somniferum,andS. ity of the test plants were evaluated by the presence of fungal diphyllum (Table 1) in different parts of Europe and a single colonies and intensity of sporulation, where B+++^ represent- specimen of C. majus subsp. asiaticum from Japan. The DNA ed the colonies with heavy sporulation, B++^ the colonies with extraction from the herbarium specimens of P. aurantiacum sparse sporulation, B+^ restricted hyphal growth without spor- and P. rhoeas failed. ITS and 28S sequences were also deter- ulation, and B−^ the absence of colonies. The test plants with minedinthesamplesofE. cruciferarum infecting scores of B+++^ and B++^ were considered susceptible and brassicaceous hosts, A. alyssoides, A. hirsutum,andB. incana. moderately susceptible hosts, respectively. The test plants The sequences were deposited in GenBank (for GenBank with non-sporulating colonies and those with no colony de- accession numbers see Table 1). The alignment of the ITS velopment were considered non-hosts in these tests. and 28S datasets were deposited in TreeBASE under the ac- In the case of the detached leaf assay, fully expanded leaves cession numbers S18880 and S18882, respectively. from healthy test plants were cut using sterile scissors and The ITS sequences included in the analysis were divided transferred to empty, sterile Petri dishes. Before inoculation, into two main groups with strong bootstrap support (Fig. 1). the leaves were checked for the presence of powdery mildew The first group (clade 1) consisted of the isolates that showed conidia under the stereo binocular microscope. The detached either identical ITS sequences or differed in one or a few leaves were then placed horizontally in plastic Petri dishes nucleotide positions only. These isolates originated from a (three dishes/plant species/mildew species) containing water wide range of host plant species belonging to various distinct agar, 10 mg/L AgNO3, 1 mL/L Wuxal Super mineral nutrient plant families, e.g. Apocynaceae, Crassulaceae, Dipsacaceae, solution and 40 mg/L of benzimidazole as a senescence inhib- Hydrangeaceae, Onagraceae, Papaveraceae, Ranunculaceae, itor. The heavily infected individuals of the original host plant and Solanaceae. Within this group, the sequences obtained species (80–90 % of the leaf surface was covered with sporu- from isolates from C. majus and M. cordata in Europe were lating powdery mildew mycelia) were used for the inocula- identical (ITS haplotype 1), except for the one collected on C. tion. The inoculum was brushed directly from the infected majus subsp. asiaticum in Japan, which differed in one nucle- leaves of the original host onto the healthy detached leaves otide position only. The ITS haplotype 1 was identical to the of the test plant species in Petri dishes using a sterile paint- ITS sequence identified earlier as E. macleayae infecting C. brush. The leaves in the Petri dishes were inoculated individ- majus (Jiang et al. 2015) as well and differed in one to three ually. The detached leaves in two Petri dishes for each test nucleotide positions from the isolates collected earlier on C. plant species were kept non-inoculated and served as negative majus and Macleaya spp. (Takamatsu et al. 1999;Kovácsetal. controls. Then the Petri plates were placed in a growth cham- 2011;Parketal.2012). The isolates from P. croceum, P. ber at a temperature of 20 °C (±2 °C) under continuous illu- nudicaule , P. somniferum , M. cambrica,andG. corniculatum mination. The presence or absence of powdery mildew colo- were identical (ITS haplotype 2) and differed from ITS nies were evaluated ten days after inoculation as described haplotype 1 in one nucleotide position across the ITS region. above. The ITS haplotype 2 was found to be identical to the ITS In Hungary, another set of inoculation tests were performed sequences determined earlier in specimens of E. aquilegiae in a whole plant assay using three local isolates of the DC. collected from various ranunculaceous hosts worldwide 36 Page 6 of 18 Mycol Progress (2016) 15: 36

(Takamatsu et al. 1999, 2015; Cunnington et al. 2004; Fig. 1 A Bayesian tree based on the nrDNA ITS region of 54 powdery„ Jankovics et al. 2008), and from Catharanthus roseus mildew sequences. The powdery mildew isolates obtained from the hosts (Apocynaceae) (Liberato and Cunnington 2006); and to those of Papaveraceae and Brassicaceae and sequenced in this work are shown in bold type. The lineages, which include the isolates with a taxonomic obtained from E. catalpae Simonyan, the powdery mildew status confirmed by morphology and/or inoculation tests in this study, are fungus infecting Catalpa bignonioides (Bignoniaceae) (Cook shaded. The ITS sequence of Erysiphe glycines (AB015934) served as et al. 2006)andE. knautiae Duby infecting Knautia arvensis outgroup. The bootstrap values presented as percentages are above the (Dipsacaceae) (Jankovics et al. 2009). Two other ITS haplo- branches, while posterior probabilities are below. Bootstrap values below 70 % and posterior probabilities below 0.90 are not shown. The data set types (3 and 4) were also identified in this clade, each comprised 613 characters. Bar indicates 0.2 expected change per site per representing Pseudoidium anamorphs collected from G. branch flavum and S. diphyllum, respectively. The ITS haplotypes 3 and 4 differed in three and two, and two and one nucleotide positions from the ITS haplotypes 1 and 2, respectively. branched, septate, hyaline, smooth, thin-walled, 4–8 μmwide; The second group (clade 2) included powdery mildews hyphal appressoria moderately lobed, single or opposite in occurring on hosts belonging to the families Berberidaceae, pairs; conidiophores erect, simple, 65–120 × 7–10 μm, foot- Brassicaceae, Capparaceae, Fabaceae, Nyctaginaceae, cells straight, followed by 1–2(3) shorter or sometimes longer Onagraceae, Plumbaginaceae, Rhamnaceae, and cells, producing conidia singly, Pseudoidium type. Conidia Papaveraceae. Within this clade, E. cruciferarum on the type formed singly, cylindrical to doliiform, hyaline, 28– host A. alyssoides and that on B. incana formed a subgroup 46 × 10.5–20 μm, without fibrosin bodies, conidial germ tubes together with the asexual morph of Erysiphe infecting P. terminal or subterminal, ending in a moderately lobed appres- dubium in Switzerland. The anamorph infecting E. californica sorium. Mature chasmothecia dark brown to black, scattered, in the same region grouped into a distinct subgroup. globose, 90–150 μm in diameter, peridial cells irregularly po- Meanwhile, a single collection of a Pseudoidium collected lygonal. Appendages 10–25 per chasmothecium, mycelioid, on E. californica in South Africa grouped in a third subgroup. rarely branched, septate, brownish, 150–400 μm long. Asci 3– The isolates collected on P. dubium in Switzerland and E. 7(10) per chasmothecium, short stalked, 48–75 × 31–50 μm, californica in South Africa exhibited ITS sequences identical containing (2)3–6(8) ascospores. Ascospores ellipsoid to to those of E. cruciferarum on Raphanus sativus, Brassica ovoid, hyaline, 15– 24×9–14.5 μm. pekinensis (Brassicaceae) and a Cleome sp. (Capparaceae) (Baiswar et al. 2013; Takamatsu et al. 2015;Zhaoetal. Fresh specimen examined 2014)andE. trifoliorum (Wallr.) U. Braun on Pisum sativum, Vicia nigricans,andLathyrus magellanicus (Fabaceae) On Glaucium corniculatum – SLOVAKIA, Piešťany, (Attanayake et al. 2010; Takamatsu et al. 2015), respectively. Research Institute of Plant Production, field, 17 Oct 2013, However, only 97 % similarity was found when the ITS se- leg. M. Pastirčák,det.K.Pastirčáková, teleomorph (NR quences retrieved from the two isolates on E. californica col- 5092). lected in Switzerland were compared to those deposited pre- viously in NCBI database. Herbarium specimens examined The 28S sequences included in the analysis were divided into two main groups (clade 1 and clade 2) similar to those On Glaucium flavum – SWITZERLAND, Geneve, 4 Oct identified in ITS analysis (Fig. 2). Within the clade 1 the 1999, leg. et det. A. Bolay, anamorph (G 111710); Geneve, sequences obtained from isolates from C. majus, G. flavum, 7 Aug 2000, leg. et det. A. Bolay, anamorph (G 111711). M. cordata,andM. cambrica were identical (28S haplotype 1) On Meconopsis cambrica – SWITZERLAND, Geneve, 24 and differed in two nucleotide positions from the isolates ob- Jun 1996, leg. et det. A. Bolay, anamorph, mycelium only on tained from G. corniculatum, P. croceum, and P. nudicaule the stems (G 111712). (28S haplotype 2). Within clade 2, E. cruciferarum on the type On Papaver aurantiacum – SWITZERLAND, Geneve, 29 host A. alyssoides and that on A. hirsutum and B. incana and Jun 1998, leg. et det. A. Bolay, anamorph (G 111713). the asexual morphs infecting P. dubium and E. californica On Papaver croceum – SWITZERLAND, L’Orient, grouped similarly as it was described in the ITS analysis. 30 Sep 1998, leg. et det. A. Bolay, teleomorph (G 111715); Le Brassus, 13 Oct 2003, leg. et det. A. Taxonomy Bolay, teleomorph (G 111717). On Papaver dubium – SWITZERLAND, Geneve, 14 Jul Erysiphe cruciferarum Opiz ex L. Junell, Sv. Bot. Tidskr. 1998, leg. et det. A. Bolay, anamorph, parasitized by 61(1): 217 (1967), Fig. 3 Ampelomyces quisqualis (G 111721). Mycelium on stems, leaves, and pods, on leaves On Papaver nudicaule – SWITZERLAND, Geneve, 1 Sep amphigenous, effuse, or forming white patches; hyphae 1998, leg. et det. A. Bolay, teleomorph (G 111714); Geneve, 17 Mycol Progress (2016) 15: 36 Page 7 of 18 36 36 Page 8 of 18 Mycol Progress (2016) 15: 36 Mycol Progress (2016) 15: 36 Page 9 of 18 36

ƒFig. 2 Phylogenetic tree based on the nrDNA 28S region of 49 powdery Additional specimens examined mildew sequences. The powdery mildew isolates obtained from the hosts of Papaveraceae and Brassicaceae and sequenced in this work are shown – in bold type. The lineages, which include the isolates with a taxonomic On Alyssum alyssoides CZECH REPUBLIC, Prague, status confirmed by morphology and/or inoculation tests in this study, are Lieben, 1841, leg. et det. F.M. Opiz as Alphitomorpha alyssi, shaded. The 28S sequence of Erysiphe glycines (LC009910) served as rev. L. Junell as Erysiphe martii, 1963, teleomorph (PRM outgroup. Numbers above the branches denote bootstrap values from 194779); − ROMANIA, Constanta District, Dealul Allah 1000 replications. Percentage values below branches are posterior prob- abilities. Bootstrap values below 70 % and posterior probabilities below Bair, 3 Jun 1973, leg. G. Negrean, det. O. Constantinescu, 0.90 are not shown. The data set comprised 593 characters. Bar indicates teleomorph (PRM 736620). 0.1 expected change per site per branch On Alyssum hirsutum – ROMANIA, Constanta District, Tuzla, 10 Jun 1977, leg. G. Negrean, det. O. Constantinescu, teleomorph (PRM 821146). Sep 2002, leg. et det. A. Bolay, anamorph (G 111716); Geneve, On Berteroa incana (syn. Alyssum incanum) – 5Oct2004,leg.etdet.A.Bolay,teleomorph(G111718). SLOVAKIA, Devínska Kobyla, 29 Sep 1984, leg. et det. C. On Papaver rhoeas – SWITZERLAND, Geneve, 8 Sep Paulech, anamorph, according to the first present author the 1997, leg. et det. A. Bolay, teleomorph (G 111719); between host plant was incorrectly identified as A. alyssoides (SAV11). Echichens and Lonay, 14 Sep 1998, leg. et det. A. Bolay, Notes: Two Glaucium species, G. corniculatum and G. anamorph, parasitized by A. quisqualis (G 111720). flavum, grow on rubble, vineyards, and scrublands of sunny On Papaver somniferum – BULGARIA, Bankya, Sofia hillsides in the warm-climate areas of Slovakia (Dostál and District, 29 Sep 1981, leg. et det. V.I. Fakirova, teleomorph, Červenka 1991). In October 2013, leaves, stems, and seed pods revised by the collector as E. macleayae, Mar 1984 (SOMF of G. corniculatum growinginafield(RIPP,Piešťany, 15923); Pirin Mts., 26 Aug 1982, leg. et det. V.I. Fakirova, Slovakia) were found to be infected by the fungus E. teleomorph, revised by the collector as E. macleayae, Mar cruciferarum (both the asexual and sexual morphs were pres- 1984 (SOMF 17662). ent). This record represents a new host of E. cruciferarum in On Stylophorum diphyllum – SWITZERLAND, Geneve, 7 Slovakia. Previous records of E. cruciferarum on G. Jul 2003, leg. et det. A. Bolay, anamorph, sparse mycelium (G corniculatum are known from Romania, Turkey, and Ukraine, 111722); Geneve, 5 Aug 2003, leg. et det. A. Bolay, and on G. flavum from France (Braun 1995) and Switzerland anamorph, sparse mycelium (G 111706). (Bolay 2005). Amano (1986 ) listed BErysiphe communis^ on

Fig. 3 Erysiphe cruciferarum on Glaucium corniculatum: a, b Chasmothecium; f Ascus with ascospores; g Ascospores. Source: NR Conidiophores bearing conidia; c Conidia; d Hyphal appressoria; e 5092. Scale bars: a, b, f =20μm; c, d =15μm; e =50μm; g =10μm 36 Page 10 of 18 Mycol Progress (2016) 15: 36

G. flavum from France and Oidium sp. on G. flavum and Petzwalova Street, 26 Sep 2014, 31 Oct 2014, leg. et det. K. Glaucium sp. from Corsica and Italy. Pastirčáková, teleomorph (NR 5187, NR 5188); Nitra, Zobor The Bulgarian specimens of powdery mildew on P. Hill, near the Institute of TB and Respiratory Diseases, 19 Oct somniferum (SOMF 15923, 17662) were originally identified 2014, leg. et det. K. Pastirčáková, teleomorph (NR 5186, BPI as E. cruciferarum and later revised to E. macleayae by the 892983); Nitra, Jesenskeho Street, park around Nitra Castle, 2 collector. The Bulgarian material is confirmed to be E. Nov 2014, leg. et det. K. Pastirčáková, teleomorph (NR cruciferarum in this study. 5189); Nitra, Dolnozoborská Street, near Wine Enterprise, The examined herbarium specimens of E. cruciferarum on 11 Oct 2015, leg. et det. K. Pastirčáková, teleomorph (NR B. incana, G. flavum, M. cambrica, S. diphyllum, and on two 5220). Papaver species, P. aurantiacum and P. dubium, contained On Macleaya microcarpa – SLOVAKIA, Piešťany, only the Pseudoidium stage. Mature chasmothecia corre- Research Institute of Plant Production, experimental field, sponding with E. cruciferarum were found on A. alyssoides, 20 Nov 2014, leg. M. Pastirčák, anamorph (NR 5193). A. hirsutum, and on the following Papaver species: P. croceum, P. nudicaule, P. rh oe as,andP. somniferum. Herbarium specimens examined Amano (1986) listed an Oidium sp. on Meconopsis betonicifolia and Meconopsis sp. from the USA and On Chelidonium majus – GERMANY, Potsdam, 25 Sep England, respectively. A herbarium specimen of E. 2005, leg. et det. V. Kummer as Oidium sp. (KR 21933); cruciferarum on Meconopsis sp. [K(M) 181632, not seen] Jena, 18 Oct 2005, leg. et det. V. Kummer as Oidium sp. collected by R.W.G. Dennis in 1951 is also from England. (KR 21934); Karlsruhe, 13 Sep 2009, leg. et det. M. BErysiphe cichoracearum DC.^ (with catenescent conidia) Scholler as E. macleayae, anamorph (KR 4850); Karlsruhe, on M. betonicifolia found in the USA by Gardner et al. 1Jul2011,leg.etdet.H.JageasE.cf.macleayae,anamorph (1970) probably represents Golovinomyces orontii (KR 29748); − JAPAN, Tokyo, Chiyoda, Imperial Palace, 12 (Castagne) V.P. Heluta. Oct 2012, leg. et det. S. Takamatsu and T. Yoshimura as Erysiphe macleayae R.Y. Zheng & G.Q. Chen, Sydowia Pseudoidium sp. (NR 5088); − ROMANIA, Iasi, 15 Oct 34: 290 (1981), Fig. 4 2012, leg. et det. V. Iacob as Oidium chelidonii (NR 5087); = Oidium chelidonii Iacob, Lucrari stiintifice USAMV Iasi, − SWITZERLAND, Nyon, 10 Nov 2004, 25 Nov 2004, leg. Seria Horticultura 51: 1050 (2008), nom. inval. et det. A. Bolay as E. cruciferarum, anamorph (G 111705, G Mycelium on stems, leaves, and infructescence, on leaves 111703). amphigenous, forming circular to irregular white patches, later On Macleaya cordata – CHINA, Moganshan, Zhejiang confluent; vegetative hyphae branched, septate, hyaline, Prov., 17 Oct 1979, leg. H. Huang, det. R.Y. Zheng and G.Q. smooth, thin-walled, 4–7 μm wide; hyphal appressoria soli- Chen, teleomorph (HMAS 40100); GERMANY, Potsdam, 15 tary, lobed to nipple-shaped; conidiophores erect, simple, 55– Oct 2005, leg. et det. V. Kummer as E.cf.macleayae, 120 × 7.5–9.5 μm, foot-cells straight or often flexuous in the anamorph (KR 21935); − JAPAN, Mt. Fujiwara, Mie Pref., 5 basal half, followed by 1–2 shorter cells, producing conidia Oct 2002, leg. et det. S. Takamatsu, teleomorph (NR 5208). singly, Pseudoidium type. Conidia cylindrical to ellipsoid- On Macleaya microcarpa – CZECH REPUBLIC, Brno, doliiform, hyaline, 30–50 × 12–18 μm, without fibrosin bod- Medicinal Herbs Centre, Faculty of Medicine, Masaryk ies, conidial germ tubes subterminal, septate, short to long, University, 2012, det. M. Pastirčák, anamorph on the seed short germ tubes ending in a lobed appressorium, germinating pods (NR 5191). conidia form single or multiple germ tubes. Mature Notes: Naturally infected C. majus plants have been chasmothecia dark brown to black, scattered, globose, checked in the selected localities in Slovakia from late sum- (65)75–100(115) μm in diameter, peridial cells irregularly po- mer to winter every year since 2006. Until recently, no lygonal. Appendages 12–20 per chasmothecium, mycelioid, chasmothecia have been found to develop on these plants. rarely branched, tortuous, septate, brown to olivaceous- Fruiting bodies of this powdery mildew on C. majus have brown, 250–480(550) μm long. Asci (2)3–4(6) per recently been reported from China (Jiang et al. 2015) and chasmothecium, ellipsoid to obovoid, 55–68 × 32–46 μm, Germany (Braun 2014). Immature chasmothecia on the leaves stalked, containing (2)3–5 ascospores. Ascospores ellipsoid of C. majus were found in late September 2014 in Slovakia. In to ovoid, hyaline, often with oil drops, 21–30 × 11.5–16.5 μm. October, numerous mature chasmothecia were observed. The morphological characteristics of chasmothecia, asci, and asco- Fresh specimens examined spores of the Slovak specimen corresponded very well to those of the chasmothecia obtained during the re- On Chelidonium majus – HUNGARY, Budapest, Szilágyi examination of E. macleayae on M. cordata from China Erzsébet Avenue, near hotel Danubius, 7 Nov 2013, leg. K. (HMAS 40100, holotype) and Japan (NR 5208). The Pastirčáková, anamorph (NR 5192); − SLOVAKIA, Nitra, holomorph of the fungus has been reported on M. cordata in Mycol Progress (2016) 15: 36 Page 11 of 18 36

Fig. 4 Erysiphe macleayae on Chelidonium majus: a White powdery tubes; i Chasmothecia on the leaf surface; j, k Crushed mature colonies on the leaf surface; b Conidiophore with primary conidium; c chasmothecia; l, m Asci with ascospores; n ascospores. Source: NR Conidiophore with secondary conidium; d Conidia; e Hyphal 5186. Scale bars: b–d, f–h, l, m =20 μm; e =10 μm; i = 200 μm; j, appressoria; f–h Germinating conidia form single or multiple germ k =100μm; n =15μm

China and Japan (Zheng and Chen 1981;Nomura1997)and diagnosis and a holotype, the name Oidium chelidonii Iacob on M. cambrica in Germany (Schmidt and Scholler 2011). was not validly published (ICBN Art. 36.1, Art. 37.6). The The finding of the asexual stage of this fungus on infected name must be considered to be a nomen invalidum because it seed pods of M. microcarpa collected in the Czech Republic was not in accordance with the rules of the International Code confirms its occurrence in that country. The fungus was also of Nomenclature for algae, fungi, and plants (ICN) at the time found for the first time on M. microcarpa plants grown in a of its publication. Latin diagnoses were required for the valid field in Slovakia (Fig. 5). From the recent findings of E. publication of all new taxa prior to 2012. macleayae on Macleaya spp. in front gardens, parks, and Erysiphe eschscholziae Pastirč. & Jankovics, sp. nov., botanical gardens in Germany, Poland, Ukraine, and Fig. 6 Czech Republic (Ale-Agha et al. 2008;Parketal.2012; MycoBank, MB814056. Heluta and Kravchuk 2015; this paper), we expect this Genetically distinct on species level from other species of fungus will spread on this host genus into other European Erysiphe sect. Erysiphe infecting Papaveraceae and other countries as well. hosts confirmed by ITS and 28S sequence data (Figs. 1 Iacob and Drobota (2008) recorded an asexual powdery and 2) and inoculation tests (Table 2). Morphologically close mildew on C. majus in Romania and proposed a new name to, but distinguishable from the asexual morphs of E. for this fungus. Because of the absence of both a Latin cruciferarum, E. hylomeci,andE. macleayae in having longer 36 Page 12 of 18 Mycol Progress (2016) 15: 36

Table 2 The results of the inoculation tests of eight test plant species belonging to Papaveraceae with the powdery mildew species Erysiphe cruciferarum and E. macleayae, performed in whole plant and detached leaf assays

Non-host species. No symptoms appeared in the inoculation tests (−); or a restricted hyphal growth without sporulation was observed following the penetration into the host tissue, which was followed by the necrosis of the plant epidermal cells and arrested fungal development (+) Host species. Obvious powdery mildew symptoms appeared in the inoculation tests involving the formation of colonies with heavy sporulation (+++) or colonies with sparse sporulation (++) Original host (positive control). Colonies with heavy sporulation were formed conidiophores (up to 140 μm) and somewhat larger conidia. Notes: ITS and 28S sequence analysis showed that the Chasmothecia absent. Swiss Pseudoidium collections on E. californica formed a Holotype (designated here): on Eschscholzia californica separate group, indicating an isolated position among the (Papaveraceae), SWITZERLAND, Nyon, 6 Oct 2001, leg. Erysiphe species (Figs. 1 and 2). The inoculation tests of E. A. Bolay, anamorph (G 111708). californica with E. cruciferarum and E. macleayae originating ITS sequence ex holotype: GenBank accession number from P. somniferum and C. majus, respectively (Table 2), KT588626. showed that this plant species remained symptomless and thus Etymology: Epithet derived from the name of the host plant may be a host of powdery mildew fungi other than E. genus, Eschscholzia. cruciferarum and E. macleayae. Based on the congruent re- Mycelium on leaves amphigenous, forming circular to ir- sults of morphological observations, molecular analyses and regular white to greyish patches; vegetative hyphae branched, inoculation tests a new species, Erysiphe eschscholziae on E. septate, hyaline, smooth, thin-walled, 4–8 μm wide; hyphal californica, is proposed. The generic affinity of the fungus on appressoria lobed or simple, opposite in pairs or single; conid- Eschscholzia and its status as species of its own have been iophores erect, simple, 70–120(140) × 7.5–10 μm, foot-cells confirmed which warrants an assignment of this species to straight or flexuous in the basal half, followed by 1–2shorter Erysiphe, based on the new ICN, Art. 59, and the new Bone cells or longer cells (the basal septum formed at the branching fungus, one name^ principle, since Pseudoidium is now a point of the mycelium), producing conidia singly, heterotypic synonym of Erysiphe. Pseudoidium type; conidia cylindrical to ellipsoid-doliiform, The anamorph of E. cruciferarum on E. californica has hyaline, 30–52(58) × 12–18 μm, without fibrosin bodies, co- been recorded from Romania (Eliade 1990), Switzerland nidial germ tubes subterminal, ending in a lobed appressori- (Bolay 2005), Germany (Schmidt and Scholler 2011), um. Chasmothecia not seen. USA (Glawe 2006),andSouthAfrica(Crousetal. Additional collection examined (paratype): on E. 2000), and Oidium sp. in Australia, Tasmania (Amano californica, SWITZERLAND, Gland, 9 Oct 2002, leg. A. 1986), and Japan (Nomura 1997). Recently, Yanez- Bolay, anamorph (G 111709). ITS and 28S sequences ex Morales et al. (2009)alsorecordedanOidium sp. on E. paratype: GenBank accession numbers KT588627 and californica in Mexico and pointed to slight morphological KU672354, respectively. differences against E. cruciferarum. The specimen of E. The herbarium specimens were originally designated as E. cruciferarum on E. californica [K(M) 64373, anamorph] cruciferarum. collected by A. Henrici in 1999 is from the territory of Mycol Progress (2016) 15: 36 Page 13 of 18 36

Fig. 5 Erysiphe macleayae on Macleaya microcarpa: a Powdery mildew symptoms on a leaf of M. microcarpa following the natural infection at the experimental field in Piešťany, Slovakia; b Infected seed pods collected in the Czech Republic; c Powdery mildew mycelium on a seed pod; d Healthy seeds. Sources (b–d): NR 5191. Scale bars = 1 mm

England. The powdery mildew (without chasmothecia) on mentioned countries. It is worth mentioning here that dif- E. californica occurs frequently in the Royal Botanical ferent spellings of the genus name Eschscholzia may Gardens, Kew (B. M. Spooner and A. Henrici, pers. cause a false variability therein or some herbarium speci- comm.). Further ITS analyses are needed to reveal the mens may be overlooked. The misspelled names relationship between E. eschscholziae and the Eschscholtzia, Eschholtzia, Escholtzia,orEscholzia were Pseudoidium reported on E. californica from the above- found in the literature, as well as on herbarium labels.

Fig. 6 Erysiphe eschscholziae on Eschscholzia californica: a Two adjacent conidiophores bearing conidia; b Hyphal appressoria; c Conidia; d Germinating conidium. Source: G 111708. Scale bars: a =20μm; b =10μm; c, d =15μm 36 Page 14 of 18 Mycol Progress (2016) 15: 36

Another species of powdery mildew, Leveillula taurica (Howe) U. Braun & S. Takam. on plane trees was observed by (Lév.) G. Arnaud has been reported to occur on this host in Ranković (2003) about 90 years after the first report of the Africa (Riley 1960;Amano1986), but it is easily distinguish- Pseudoidium stage in Europe (Sprenger 1916). In both path- able from the species described here in having internal myce- ogens, a significant delay in sexual reproduction, and in the lium with conidiophores emerging through stomata and lan- appearance of chasmothecia, was observed after the expan- ceolate primary conidia (asexual morph of Oidiopsis type). sion of their area of distribution. Erysiphe macleayae regularly completes its life cycle on its original hosts (Macleaya spp.) in Inoculation experiments Asia. Recently, the chasmothecia of this species have also been found on M. cambrica in Germany (Schmidt and The results of both the detached leaf and whole plant assays Scholler 2011) and on C. majus in China, Germany, and performed in Slovakia and Hungary indicated that E. Slovakia (Jiang et al. 2015;Braun2014; this paper). Recent macleayae on C. majus conclusively infected A. grandiflora, records, however, from different regions of Europe demon- M. cordata, P. rhoeas,andP. so m nif eru m by forming colonies strated that infections on C. majus, M. cordata,andM. with heavy sporulation (Table 2) similarly to the symptoms on microcarpa were usually confined to the asexual morph of C. majus plants used as positive controls in the experiments. an Erysiphe species (Jankovics 2007;Jageetal.2010; Erysiphe macleayae was able to infect, to some extent, G. Schmidt and Scholler 2011; Park et al. 2012; Pastirčáková corniculatum and M. cambrica by forming colonies that pro- and Pastirčák 2013; Heluta and Kravchuk 2015). Other stud- duced conidiophores sporadically. No sporulating colonies, ies provided evidence that the Pseudoidium collected from C. however, were observed on the leaves of E. californica follow- majus at various sites in Europe (i.e. Hungary, Slovakia, ing the inoculations with E. macleayae. Only small, undevel- Czech Republic, Ukraine) represent a single anamorphic spe- oped colonies without conidiophores were found, and the pen- cies, which was found to be phylogenetically closely related to etrations were always followed by necroses of the plant epider- E. macleayae (Jankovics 2007; Jankovics et al. 2008;Kovács mal cells and arrested fungal development. et al. 2011). In the present study, the powdery mildew fungus Erysiphe cruciferarum maintained on P. somniferum con- on C. majus was clearly identified as E. macleayae based on clusively infected its original host only in our experiments. both nrDNA sequence analysis (Figs. 1 and 2) and the mor- Meanwhile, this fungus developed colonies with sparse spor- phology of its asexual and sexual stages. The new and rare ulation on G. corniculatum and M. cambrica. Erysiphe appearance of chasmothecia on C. majus suggests that E. cruciferarum was not able to infect A. grandiflora, C. majus, macleayae most probably expanded its area of distribution E. californica, M. cordata,andP. rh oe as. to Europe only several decades ago. The recent host range Both powdery mildew species formed colonies with abun- expansion of E. macleayae to C. majus might also have oc- dant sporulation on their original hosts used as positive con- curred, as chasmothecia on this host have only been found trols in both assays, while all the non-inoculated control plants recently in Asia (Jiang et al. 2015), where the fungus was and detached leaves used as negative controls remained pow- known to be native to Macleaya species only. dery mildew-free. The pathogens developing colonies on the Erysiphe macleayae may be capable of infecting a wider inoculated test plants were always identical in their morpho- range of hosts than was explored in previous studies, which logical patterns to those maintained on their original hosts and considered M. cordata, M. microcarpa, C. majus,andM. used as inocula. In addition, the ITS sequences, which were cambrica as the hosts of this fungus (Schmidt and Scholler determined in the powdery mildews appearing on the test 2011; Braun and Cook 2012; Park et al. 2012; Jiang et al. plants in the experiments, always confirmed the identity of 2015). This was clearly shown by the inoculation experiments the pathogens. using the isolates obtained from C. majus in Slovakia and Hungary, which indicated that A. grandiflora, G. corniculatum, P. rhoeas,andP. s omn if er um must also be tak- Discussion en into account as potential hosts. The present re-examination of herbarium materials revealed, however, that E. macleayae Some powdery mildew species are much more widespread in occurs naturally on M. cordata and M. microcarpa only in their asexual forms, especially those introduced to new geo- addition to C. majus, while A. grandiflora, G. corniculatum, graphic areas or expanding their host ranges to new hosts or P. rhoeas,andP. somniferum have never been found to serve ranges of hosts. The most well-known example is the grape- as natural hosts of E. macleayae. Nevertheless, it is worth vine powdery mildew fungus Erysiphe necator Schwein., mentioning that the number of herbarium specimens was lim- which was surviving in the asexual stage exclusively for al- ited in this study, and thus the possibility of natural occurrence most five decades following its first introduction to Europe in of this pathogen on other hosts cannot be ruled out. It was not the middle of the nineteenth century (Bulit and Lafon 1978). possible, for instance, to obtain collections of Oidium Likewise, the formation of chasmothecia in Erysiphe platani papaveracearum Bappamm., Hosag. & Udaiyan with Mycol Progress (2016) 15: 36 Page 15 of 18 36

Pseudoidium conidiophores found on Argemone mexicana in despite the slight differences revealed in their nrDNA se- India (Bappammal et al. 1995) and deposited in HCIO (Indian quences and morphology. Similar results were published in Agricultural Research Institute, New Delhi, India; V.B. the cases of the most derived groups of the genus Hosagoudar, pers. comm.). Based on morphology, Braun Golovinomyces (U. Braun) V.P. Heluta (Matsuda and and Cook (2012)consideredO. papaveracearum asynonym Takamatsu 2003; Takamatsu et al. 2013) leading to the con- of O. matthiolae Rayss with an E. cruciferarum teleomorph. clusion that the phylogeny of powdery mildews was not al- However, one can assume, based on the susceptibility of A. ways consistent with that of the host plants. The present study grandiflora to E. macleayae revealed in our tests, that the repeatedly revealed that the use of ITS sequences in the precise Indian collection may represent the anamorph of E. identification of closely related powdery mildew fungi, espe- macleayae. We also could not include E. hylomeci, the species cially those with Pseudoidium stages, is limited as it was point- exhibiting morphological features very similar to those of E. ed out by Kovács et al. (2011) and Takamatsu et al. (2015). macleayae (Braun and Cook 2012) in this study, because the Both E. cruciferarum and E. macleayae were able to infect type specimen was not available. Further phylogenetic and M. cambrica, G. corniculatum,andP. somniferum in our in- host range studies are needed to confirm the identity of the oculation experiments. This suggests that these host species powdery mildew fungus on A. mexicana and to investigate may be infected by at least two powdery mildew species de- the relationship between E. hylomeci and other powdery mil- spite not being discovered in the herbarium specimens exam- dews infecting Papaveraceae, especially E. macleayae,of ined in this study. Therefore, the natural occurrence of these which E. hylomeci may represent just a variety (Braun and two pathogens on the hosts mentioned above cannot be ex- Cook 2012). cluded. This might explain why the anamorph on M. cambrica The re-examination of herbarium specimens also suggested could not be clearly identified in the present study, although that P. croceum, P. nudicaule,andP. somniferum were natu- Schmidt and Scholler (2011)foundE. macleayae (both the rally infected by E. cruciferarum. In addition, this fungus was asexual and sexual morphs) on this host in Germany. In addi- identified on G. corniculatum and was reported here for the tion, morphological characterization of the asexual stages ex- first time from Slovakia. The E. cruciferarum isolates collect- clusively does not provide enough data for a well-founded ed from the above-mentioned papaveraceous hosts represent a identification of these pathogens. Many examples of the hosts distinct lineage (ITS haplotype 2, 28S haplotype 2) of E. that can be infected by more than one powdery mildew cruciferarum sensu lato recognized in clade 1 (Figs. 1 and anamorphs are known in the literature, such as rhododendron 2). Although P. croceum and P. nudicaule were not included (Inman et al. 2000), tomato (Kiss et al. 2001), soybean in the inoculation tests, successful infections were observed (Takamatsu et al. 2002), hornbeam (Braun et al. 2006), catalpa on G. corniculatum and M. cambrica following the inocula- (Cook et al. 2006), mango (Limkaisang et al. 2006), peony tions with E. cruciferarum isolates obtained from P. (Takamatsu et al. 2006), and lilac (Seko et al. 2008). These somniferum. The isolates in this lineage possessed nrDNA conidial stages are, in most cases, morphologically indistin- sequences identical to some of those of E. aquilegiae, a dis- guishable from each other. tinct species infecting ranunculaceous hosts worldwide. These The host range of E. cruciferarum s. l. consist of numerous were also highly similar to E. macleayae and many other host plant species belonging to the plant families known herb-parasitic species belonging to a recently recog- Capparidaceae, Cleomaceae, Brassicaceae, Resedaceae, nized homogenous clade spanning an exceptionally wide Papaveraceae, and Fumariaceae (Braun and Cook 2012). In range of host families (Takamatsu et al. 2015). The present this study, the isolates identified earlier as E. cruciferarum on study supports that E. macleayae on C. majus exhibits a spe- the papaveraceous hosts were shown to represent several lin- cific host range different from that of E. cruciferarum, the eages, which were clearly distinguishable based on the ITS common pathogen of papaveraceous hosts. In addition, this and 28S sequences (Figs. 1 and 2) and were distinct from the fungus was not able to infect plant species outside lineage consisted of the isolates of E. cruciferarum on the type Papaveraceae, e.g. Aquilegia vulgaris (Ranunculaceae), host A. alyssoides and other brassicaceous hosts, i.e. A. Passiflora caerulea (Passifloraceae), Sedum alboroseum hirsutum and B. incana (E. cruciferarum sensu stricto). (Crassulaceae), and Solanum lycopersicum (Solanaceae), However, the identity of this species could be confirmed only which represent the hosts of several closely related powdery in the case of the isolates belonging to lineage characterized mildew fungi (Jankovics et al. 2008). The natural occurrence by ITS haplotype 2 and 28S haplotype 2 (clade 1, Figs. 1 of E. macleayae in the sexual stage has been recorded from and 2). The anamorphs collected from P. dubium and E. only one non-papaveraceous host, namely Torenia fournieri californica grouped together with the powdery mildew fungi (Linderniaceae) (Men et al. 2014). In general, the two pow- infecting hosts of Brassicaceae, Cleomaceae, Nyctaginaceae, dery mildew species, E. macleayae and E. cruciferarum,show Fabaceae and Plumbaginaceae (clade 2, Fig. 1 and 2). These markedly distinct host ranges among the numerous species fungi, among many others, have recently been found to form a recognized as closely related by Takamatsu et al. (2015) genetically much more diverse clade (Takamatsu et al. 2015), 36 Page 16 of 18 Mycol Progress (2016) 15: 36 which was mixed concerning the tree-parasitic or herb- Attanayake RN, Glawe DA, McPhee KE, Dugan FM, Chen W (2010) — parasitic nature as well as the chasmothecium morphology. Erysiphe trifolii a newly recognized powdery mildew pathogen of pea. Plant Pathol 59:712–720. doi:10.1111/j.1365-3059.2010. Although the number of specimens was limited in this study 02306.x to reveal the genetic diversity among the powdery mildew Baiswar P, Takamatsu S, Ngachan SV, Chandra S, Harada M (2013) isolates infecting papaveraceous hosts within clade 2, the dif- Molecular and morphological characterization of three Oidium ferences in nrDNA sequences suggest that this group may be spp. on Ocimum basilicum, Brassica pekinensis and Crotalaria sp. from India. Environ Ecol 31:1480–1485 genetically more diverse than clade 1. The present results also Bappammal M, Hosagoudar VB, Udaiyan K (1995) Powdery mildews of suggest that powdery mildew fungi identified earlier as E. Tamil Nadu, India. New Botanist 22:81–175 cruciferarum s. l. might represent a species complex Bolay A (2005) Les oïdiums de Suisse (Erysiphacées). 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Nauka, Leningrad, pp 11–142 therefore the introduction of a new species, E. eschscholziae. Ciferri R, Camera C (1962) Tentativo di elencazione dei funghi italiani. I. Erisifali. Quad Ist Bot Univ Pavia 21:1–46 Acknowledgments The authors are grateful to the curators of the Cook RTA, Inman AJ, Billings C (1997) Identification and classification herbaria G, HMAS, KR, PRM, SAV, and SOMF for the loans of the of powdery mildew anamorphs using light and scanning electron specimens. Susumu Takamatsu (Mie University, Tsu, Japan) and microscopy and host range data. Mycol Res 101:975–1002. doi: Viorica Iacob (University of Applied Life Sciences and Environment, 10.1017/S095375629700364X Iasi, Romania) are thanked for kindly providing some of the powdery Cook RTA, Henricot B, Henrici A, Beales P (2006) Morphological and mildew samples used in this work. Alick Henrici and Brian M. Spooner phylogenetic comparisons amongst powdery mildews on Catalpa in (Royal Botanic Gardens, Kew, Surrey, UK) are thanked for information the UK. Mycol Res 110:672–685. doi:10.1016/j.mycres.2006.02. on British record of Eschscholzia powdery mildew. We are much obliged 005 to the curators of Botanical Garden in Teplice, Medicinal Herbs Centre of Crous PW, Phillips AJL, Baxter AP (2000) Phytopathogenic fungi from Faculty of Medicine and Botanical Garden of Faculty of Science of the South Africa. University of Stellenbosch Printers, Department of Masaryk University in Brno (Czech Republic), Botanical Garden of Plant Pathology Press, Stellenbosch University of Latvia in Riga (Latvia), and Botanical Garden of Parma, Cunnington JH, Takamatsu S, Lawrie AC, Pascoe IG (2003) Molecular University of Parma, Parma (Italy) for providing Papaveraceae seeds. 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