A Re-Consideration of Pseudoperonospora Cubensis and P

Mycol. Res. 109 (7): 841–848 (July 2005). f The British Mycological Society 841 doi:10.1017/S0953756205002534 Printed in the United Kingdom.

A re-consideration of Pseudoperonospora cubensis and P. humuli based on molecular and morphological data

Young-Joon CHOI1, Seung-Beom HONG2 and Hyeon-Dong SHIN1* 1 Division of Environmental Science and Ecological Engineering, College of Life and Environmental Sciences, Korea University, Seoul 136-701, Korea. 2 Korean Agricultural Culture Collection, National Institute of Agricultural Biotechnology, Rural Development Administration, Suwon 441-707, Korea. E-mail : [email protected]

Received 27 May 2004; accepted February 2005.

Phylogenetic analysis of the ITS rDNA region was carried out with two economically important downy mildews, Pseudoperonospora cubensis, which infects species of Cucumis, Cucurbita, and Citrullus belonging to Cucurbitaceae, and P. humuli, which infects plants of the genus Humulus belonging to Cannabaceae. Two closely related species, P. cannabina and P. celtidis, were also included to reveal taxonomic relationships with the first two mildews. All four species formed a well-resolved clade when compared with the ITS sequences of other downy mildew genera, using Bayesian inference and maximum parsimony. The P. cubensis isolates obtained from different hosts and (or) geographical origins in Korea, exhibited no intraspecific variability in the ITS sequences. The phylogenetic analyses of P. cubensis and P. humuli showed that they share a high level of sequence homology; the morphology of the sporangiophores, sporangia, and dehiscence apparatus confirmed the similarity of the two species. We therefore reduce P. humuli to the status of a taxonomic synonym of P. cubensis.

INTRODUCTION P. humuli commonly occurs in all of the hop-growing countries of the Northern Hemisphere. The disease was Among the seven accepted species of Pseudoperono- first reported in 1905 in Japan, and within a few dec- spora (Waterhouse & Brothers 1981, Kirk et al. 2001), ades it had spread pandemically throughout Europe, P. cubensis and P. humuli which cause cucurbit and hop North America, and the former USSR (Miyabe & downy mildews respectively, are the most economically Takahashi 1906, Salmon & Ware 1925, Arens 1929, and ecologically important. P. cubensis, the type species Novotel’nova & Pystina 1985), and crop losses were of the genus, is a destructive pathogen on wild and devastating. P. cannabina, which infects species of cultivated cucurbitaceous plants worldwide. Among Cannabis, has become more widespread with the in- the plants attacked by this fungus, Cucumis sativus, creased cultivation of the host. Although the hosts and C. melo, Cucurbita spp., and Citrullus vulgaris are the morphological characters of the two mildews are the most important cultivated hosts. Because the closely related respectively, Jaczewski (1928) suggested variability of morphological characters and of infec- that P. cannabina is considerably different in the mor- tivity of these fungi was shown to be dependent on the phological characteristics of the sporangiophores and host plants and (or) environmental conditions (Iwata sporangia, and therefore it would be impossible to 1942, 1953a, b, Palti 1974, Palti & Cohen 1980, Water- confuse it with P. humuli. house & Brothers 1981), the fungi were not considered During a taxonomic revision of downy mildews as different taxa (Iwata 1942, Waterhouse & Brothers in Korea (Shin & Choi 2003), the morphological 1981). However, no molecular or genetic evidence to characteristics of specimens belonging to P. cubensis, confirm the homogeny of P. cubensis isolates from P. humuli, and P. celtidis, were studied. This study different host genera or species was provided. indicated that P. cubensis and P. humuli might indeed Two other downy mildews, P. humuli and P. canna- be conspecific, although the hosts of the two mildews bina, are recorded on members of Cannabaceae. are somewhat distantly related. Previously, it had been shown that the shape of the haustoria (Fraymouth * Corresponding author. 1959), and the ultrastructure of the dehiscence

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apparatus (Constantinescu 2000), were identical in both morphological characteristics of sporangiophores and species. However, similarities in the morphology of the sporangia, were examined. downy mildews do not necessarily mean that they are conspecific. Therefore, a confirmation of the similarity DNA extraction, PCR amplification, and sequencing of P. cubensis and P. humuli using molecular methods is required. Genomic DNA was extracted from sporangiophores The five species of Pseudoperonospora, including and sporangia formed on the lower surface of the P. celtidis and P. urticae, parasitic on hosts in the infected leaves or using the infected host tissue of Ulmaceae and Urticaceae, are morphologically simi- herbarium specimens. The presence of the sporangio- lar (Waterhouse & Brothers 1981, Constantinescu & phores and sporangia of the downy mildews was Fatehi 2002), and their host families are also closely checked under a dissecting microscope (Olympus SZ). related (Berg 1977, Soltis et al. 1997, Angiosperm The extraction was undertaken according to White et al. Phylogeny Group 1998, Song et al. 2001). Therefore, (1990). The primers DC6 (5k-GAG-GGA-CTT-TTG- molecular analyses could be expected to provide the GGT-AAT-CA-3k) and ITS4 (5k-TCC-TCC-GCT- necessary data to elucidate the relationships among TAT-TGA-TAT-GC-3k) were used for the selective other species of the genus, as well as between P. amplification of the complete ITS region of the rDNA. cubensis and P. humuli. Sequence analysis of the ITS PCRs were conducted in 50 ml reaction volumes. Each region of rDNA has proved to be a very powerful tool reaction tube contained 1.2 ml of template DNA sol- for the comparison of closely related species within ution (approx. 100 ng), prepared as above, and 5 ml the oomycetes (Brasier & Hansen 1992, Lee & Taylor of 10r buffer (50 mM KCl, 100 mM Tris-HCl (pH 8.0), 1992, Mitchell, Roberts & Moss 1995, Crawford et al. 0.1% Triton X-100, 15 mM MgCl2), 3 ml of 2.5 mM 1996, Cooke et al. 2000), and this method has recently dNTP, 0.4 ml (each) of 100 mM primers, 0.4 mlofTaq x1 been applied to resolve the taxonomic and phylogenetic polymerase (5 unit ml ), and 39.6 ml of ddH2O. The problems of some downy mildews (Rehmany et al. following thermal cycling parameters were used; 2000, Constantinescu & Fatehi 2002, Choi, Hong & denaturation for 1 min at 95 xC, annealing for 1 min Shin 2002, 2003, Voglmayr 2003). at 58 x, and extension for 2 min at 72 x; 35 cycles were The purpose of this work was to reconsider the performed, with the first denaturation and the last relationship between P. cubensis and P. humuli in terms extension time extended to 5 min and 10 min, respect- of morphological and molecular characteristics, and ively. The success of the amplification was monitored to find out if P. cubensis isolates from different host by electrophoresis on 1% agarose gels. The PCR plant genera and (or) species were genetically homo- products were then electrophoresed on a 0.8% agarose genous. To reveal the relationships among the species gel, and the ITS regions were purified using a QIAquick within Pseudoperonospora, P. cannabina and P. celtidis gel extraction kit (Qiagen, Hilden). The purified DNAs were also included in the study. were ligated into pGEM-T easy vector (Promega, Madison, WI). Ligated plasmids were then trans- formed into Escherichia coli DH5aFk cells, and the MATERIALS AND METHODS transformants were selected by the standard blue-white screening procedure (Sambrook, Fritsch & Maniatis Fungal isolates 1989). Plasmids containing the ITS regions were iso- 18 collections of Pseudoperonospora cubensis and P. lated using a QIAquick plasmid minikit (Promega). humuli originating from various localities in the world, The purified plasmids were sequenced on an automatic as well as one specimen of P. celtidis from Korea, and sequencer (ABI Prism TM 377 DNA Sequencer). one of P. cannabina from Latvia, were used. For comparison, several fresh isolates and (or) published Sequence alignment and phylogenetic analysis sequences from GenBank of other members of Perono- sporales,suchasHyaloperonospora, Peronospora, Sequences were edited with the DNASTAR computer Perofascia, Phytophthora,andPythium species were package to obtain the complete ITS regions. An also analysed (Table 1). Voucher specimens of the alignment of the sequences was performed using Korean collections are deposited in the Herbarium CLUSTAL W (Thompson, Higgins & Gibson 1994). of Systematic Mycology of Korea (SMK), Korea Both Bayesian inference and maximum parsimony University, Seoul. methods were used for the phylogenetic analysis. Bayesian analysis was performed using MRBAYES, version 2.01 (Huelsenbeck & Ronquist 2001). The Light microscopy general time reversible model (GTR) with a gamma- Both the fresh and older herbarium materials were distributed substitution rates was determined for a examined under a light microscope (Olympus BX-50). given data set using Modeltest 3.06 (Posada & Crandall To rehydrate the shrunken structures in old specimens, 1998) and PAUP* version 4b10 (Swofford 2002). Four a lactic acid technique was used (Shin & La 1993). incrementally heated simultaneous Markov chains were The symptoms produced on the host plants, and the run for one million generations, saving a tree every

Table 1. Summary of information an specimens used in this study.

Source or herbarium number Species Host /geographical origina/year of collection GenBankc

Hyaloperonospora niessleana Alliaria petiolata Sequence from GenBank, Romania, 2000 AF465763 H. parasitica Capsella bursa-pastoris SMK18835, Hongchon, Korea, 2002 AY210988 Perofascia lepidii Lepidium apetalum SMK15690, Seoul, Korea, 1999 AY211012 L. virginicum SMK17250, Seoul, Korea, 2000 AY211013 Peronospora arborescens Papaver rhoeas Sequence from GenBank, Romania, 2000 AF465761 P. astragalina Astragalus membranaceus SMK18200, Samchok, Korea, 2001 AY608608 P. campestris Arenaria serpyllifolia SMK18203, Kangnung, Korea, 2001 AY608609 P. corydalis Corydalis ochotensis SMK17368, Dongduchon, Korea, 2000 AY211015 P. destructor Allium cepa Sequence from GenBank, Japan, 1999 AB021712 P. farinosa Chenopodium album Sequence from GenBank, Romania, 2000 AF465762 C. album SMK17547, Chunchon, Korea, 2000 AY211017 P. manshurica Glycine max Sequence from GenBank, Japan, 1999 AB021711 G. soja SMK17669, Chunchon, Korea, 2000 AY211019 P. rumicis Rumex acetosa Sequence from GenBank, Finland, 1939 AF465758 P. sparsa Rosa sp. Sequence from GenBank, England AF266783 R. multiﬂora SMK19410, Kimhae, Korea, 2002 AY608610 P. trigonotidis Trigonotis peduncularis SMK19287, Wonju, Korea, 2002 AY608611 Phytophthora cambivora Rubus idaeus Sequence from GenBank, Scotland, 1985 AF266763 P. infestans Solanum tuberosum Sequence from GenBank, Netherland AF266797 P. palmivora Theobroma cacao Sequence from GenBank, Papua New Guinea, 1994 AF266780 Pseudoperonospora cannabina Cannabis sativa MZM71018b, Riga, Latvia, 1936 AY608612 P. celtidis Celtis sinensis SMK17780, Dongduchon, Korea, 2000 AY608613 P. cubensis Citrullus vulgaris SMK14235, Chunchon, Korea, 1997 AY608618 Cucumis melo var. makuwa SMK11284, Kangnung, Korea, 1991 AY608614 C. melo var. reticulatus SMK15170, Kimhae, Korea, 1998 AY608615 C. sativus SMK12174, Kangnung, Korea, 1992 AY608616 C. sativus SMK18951, Samchok, Korea, 2002 AY608617 C. sativus Sequence from GenBank, Austria, 1999 AY198306 C. sativus Sequence from GenBank, China, 2004 AY744946 Cucurbita moschata SMK13288, Kangnung, Korea, 1994 AY608619 C. moschata SMK19205, Chunchon, Korea, 2002 AY608620 P. humuli Humulus japonicus SMK11608, Kangnung, Korea, 1992 AY608621 H. japonicus SMK18856, Namyangju, Korea, 2002 AY608622 H. japonicus SMK19582, Pyongchang, Korea, 2003 AY608623 H. lupulus SMK11675, Suwon, Korea, 1992 AY608624 H. lupulus BR82100, Belgium, 1968 – H. lupulus BR82367, Hungary, 1928 – H. lupulus SOMF16105, Bulgaria, 1983 – H. lupulus Sequence from GenBank, Austria, 1999 AY198304 H. lupulus Sequence from GenBank, Austria, 1999 AY198305 Pythium ultimum Euphorbia pulcherrima Sequence from GenBank, USA AF271225

a BR Jardin Botanique National de Belgique; SMK Systematic Mycology of Korea, Korea University, Seoul; SOMF Institute of Botany, Bulgarian Academy of Sciences; and MZM Moravian Museum, Czech Republic. b ex herbarium J. Smarods, Fungi Lativici, Riga, Latvia, 1936. c GenBank accession no.

100th generation. Among these, the ﬁrst 1000 trees trees. Relative robustness of the individual branches were ignored. MRBAYES was used to compute a 50% was estimated by bootstrapping, using 10 000 rep- majority rule consensus of the remaining trees to obtain licates, with heuristic searches, branch swapping by estimates for the posterior probabilities of groups. tree bisection-reconnection (TBR) and MAXTREES Branch lengths were computed as the mean values over set at 100. Trees were rooted using TREEVIEW pro- the sampled trees. To test the reproducibility of results, gram version 1.6.6 (Page 1996) by selecting the Pythium this analysis was repeated four times, starting with ultimum sequence. random trees and default parameter values. A maximum parsimony (MP) heuristic search was performed with ten random sequence additions, branch RESULTS swapping by tree bisection-reconnection (TBR), and Morphological analysis MAXTREES set at 20 000, using PAUP* version 4b10. Gaps were treated as missing data and all nucleotide In Cucurbita moschata infected by Pseudoperono- substitutions were equally weighted and unordered. spora cubensis, spots are polyangular and limited CI and RI values were calculated for all parsimony by veins, and attacked tissues become discoloured.

Table 2. Morphological comparison of Pseudoperonospora cubensis and P. humuli.

Character P. cubensis (this work) P. cubensisa P. humuli (this work) P. humuli b

Host Cucurbita moschata Cucumis sp. Humulus lupulus Humulus lupulus Symptoms Polyangular, limited by veins, Angular, clearly Small or large, angular, limited Small, irregular, limited scattered to confluent limited by veins by veins, scattered by veins, scattered or or confluent, forming large confluent forming large irregular patches irregular patches Sporangiophores Length 120–480 mm 180–400 mm 120–460 mm 200–460 mm Trunk Width 5–10 mm 5–7 mm 5–10 mm6–7mm Base Somewhat swollen Inflated Somewhat swollen – Branches Branching type Monopodial, occasionally Dichotomous Monopodial, occasionally Dichotomous appearing dichotomous appearing dichotomous Branching times 3–5 orders – 3–5 orders 5–6 orders Ultimate branchlets – Shape Straight to substraight – Straight to substraight Straight, slightly arcuate of sometimes deflexed Apex Subtruncate Subacute Obtuse to subtruncate Bluntly pointed Length (4–)10–15 mm – (4–)10–13(–15) mm– Width at the base 2–3.5 mm – (1.5–)2–3.5 mm– Sporangia – Colour Olivaceous brown Pale greyish to Pale olivaceous to olivaceous Light smoky olivaceous-purple Shape Ellipsoidal Ovoid to ellipsoidal Ellipsoidal Broad, elliptical or obovate Size 22–38r15–22 mm 20–40r14–25 mm 22–33r15–19 mm 22–26r15–18 mm Length:width ratio 1.4–1.7 – 1.4–1.7 – Pedicel Short-lived Present Short-lived – Operculum Thin-walled – Thin-walled –

a Palti (1975). b Miyabe & Takahashi (1906).

Sporangiophores emerge in groups of 2–6 from a ratio of sporangia of ca 1.75–2.20, much higher than stomatal opening; they are 120–480 mm long, straight, in other Pseudoperonospora species (1.3–1.7 or less). 5–10 mm wide, and with somewhat swollen base. Another difference is the presence of 2–4 callose plugs Branches are monopodial, of 3–5 orders, occasionally along the trunk and branches of sporangiophores. appearing dichotomous. The ultimate branchlets are The callose plugs, first observed by Shin & Choi (2003), straight to substraight with a subtruncate apex, had not been described in other species of the genus. (4–)10–15 mm long and 2–3.5 mm wide at the base. Interestingly, this character was also reported in an Sporangia are olivaceous brown, ellipsoidal, 22–38r unidentified Pseudoperonospora sp. infecting Grewia 15–22 mm (length:width ratio 1.4–1.7; n=100), with a sp. in the Malvaceae (Waterhouse & Brothers 1981), short or somewhat protruding pedicel. These charac- suggesting that it might be a common character of ters are more or less similar in all specimens from the Pseudoperonospora species parasitic on trees. Cucumis, Cucurbita,andCitrullus species. P. humuli causes similar symptoms on the leaves of Humulus Molecular analysis lupulus and H. japonicus, and morphological characteristics viewed under a light microscope were also PCR products of between 1250 and 1300 bp, including identical to those found on P. cubensis (Table 2). Our a partial 18S and complete ITS region (ITS1, 5.8S observations show that the morphological characters rDNA, and ITS2), were amplified from each isolate of P. cannabina are very similar to those of P. cubensis using the primers DC6 and ITS4. The sequences of the and P. humuli, except that the sporangiophores are 41 isolates from the six genera, including the Pseudo- only branched up to four orders as in the description peronospora collections, were adjusted to the length of Waterhouse & Brothers (1981). In addition, in of the ITS region. The ITS regions of the P. cubensis P. humuli and P. celtidis the dehiscence apparatus is and P. humuli were both 802 bp, while those of the stained by picronigrosin, in contrast to P. cannabina. P. cannabina and P. celtidis were 800 bp and 801 bp, Although Waterhouse & Brothers (1981) indicated that respectively. P. celtidis was morphologically similar to P. cannabina, The phylogenetic relationships of P. cubensis and the former is clearly differentiated from other species P. humuli, as well as of the other species, were inferred within the genus, most significantly by the length:width from Bayesian (MCMC) analysis and heuristic

Pythium ultimum (Euphorbia pulcherrima)* Phytophthora infestancs (Solanum tuberosum)* 100 Perofascia lepidii (Lepidium apetalum) 100 100 P. lepidii (Lepidium virginicum) 100 100 Hyaloperonospora niessleana (Alliaria petiolata)* 100 H. parasitica (Capsella bursa-pastoris)

Pseudoperonospora cannabina (Cannabis sativus) MZM71018 Ps. celtidis (Celtis sinensis) SMK17780 100 Ps. cubensis (Cucumis sativus) SMK12174 100 Ps. cubensis (Cucumis sativus) SMK18951 Ps. cubensis (Cucurbita moschata) SMK19205 85 Ps. cubensis (Cucurbita moschata) SMK13288 86 Ps. humuli (Humulus japonicus) SMK11608 Ps. humuli (Humulus japonicus) SMK19582

100 Ps cubensis (Cucumis melo var. makuwa) SMK11284 100 Ps. cubensis (Citrullus vulgaris) SMK14235 Ps. cubensis (Cucumis melo var. reticulatus) SMK15170 92 59 Ps. humuli (Humulus lupulus) WU22945* Ps. humuli (Humulus lupulus) WU22946*

Ps. humuli (Humulus lupulus) SMK11675 Ps. humuli (Humulus japonicus) SMK18856 Ps. cubensis (Cucumis sativus) WU22944* Ps. cubensis (Cucumis sativus)*

100 Peronospora sparsa (Rosa multiflora) 100 P. sparsa (Rosa sp.)* P. arborescens (Papaver rhoeas)* 92 93 100 P. farinosa (Chenopodium album) 58 100 P. farinosa (Chenopodium album)* 80 94 87 P. destructor (Allium cepa)* P. corydalis (Corydalis ochotensis) 87 P. campestris (Arenaria serpyllifolia) P. rumicis (Rumex acetosa)* 100 P. manshurica (Glycine soja) 100 P. manshurica (Glycinemax)* 78 98 P. trigonotidis (Trigonotis peduncularis) 64 P. astragalina (Astragalus membranaceus) 73 Phytophthora cambivora (Rubus idaeus)* 68 Phytophthora palmivora (Theobroma cacao)*

0.1

Fig. 1. Phylogenetic tree inferred from Bayesian analysis of the complete ITS region (ITS1, 5.8S rDNA, and ITS2), showing mean branch lengths of a 50% majority-rule consensus tree from one-million generations of MCMC analysis. Numbers above the branches are the posterior probability value. The number of nucleotide changes between taxa is represented by branch length and the scale bar equals the number of nucleotide substitution per site. An asterisk (*) shows taxa whose data were obtainel from GenBank. P., Peronospora; and Ps., Pseudopersonospora.

maximum parsimony (MP) analysis of the aligned in six most parsimonious trees of 1077 steps with a CI nucleotide sequences of the ITS rDNA. For Bayesian of 0.5831 and an RI of 0.7165. Since no diﬀerence was inference, all ﬁve analyses resulted in the same tree found between the tree topologies from the MCMC topology and almost identical posterior probability and MP analyses, only a MCMC tree is shown (Fig. 1). values. After running for one million generations, a P. cubensis and P. humuli formed a well-supported 50% majority rule consensus tree from all the trees is group with high posterior probability (100% in shown in Fig. 1. Out of 905 characters, 312 were par- MCMC and MP trees). The P. humuli isolates from simony-informative, and parsimony analysis resulted Humulus japonicus and H. lupulus showed high

nucleotide similarity (more than 99.6%) and P. cubensis Genetic homogeneity within Pseudoperonospora isolates from Citrullus vulgaris, Cucumis spp., and cubensis Cucurbita moschata had also high homologies of 99.5 The reported host range of Pseudoperonospora and 100%, respectively. The sequences of P. cubensis cubensis includes 49 species in approx. 20 genera of and P. humuli were identical, or more highly conserved Cucurbitaceae (Thomas 1986, Lebeda 1992). Among (more than 99.5% homology), which suggests that the them, there are economically important hosts belong- two mildews belong to the same species. This clade ing to Citrullus, Cucurbita,andCucumis. Although further grouped with P. celtidis and then P. cannabina, morphological analysis of cucurbit mildews showed with strong probability value (100% in MCMC and variability depending on the specimens, host genera or MP trees). The phylogenetic distances between these species, season of collecting, and even different parts species and the clade of cucurbit and hop mildews were of the plant, the fungi were not treated as different considerably greater; the genetic distance of P. celtidis morphological taxa (Iwata 1942, Waterhouse & was ca 3% from the clade of hop and cucurbit mildews, Brothers 1981). Cross-inoculation experiments have while in P. cannabina was 6%. Nonetheless, all four also shown variability of infectivity in this fungus mildews formed a highly coherent clade in both types according to host genera or species (Iwata 1953a, b, of phylogenetic analyses used, and were well separated Palti 1974, Palti & Cohen 1980). In our study, the iso- from Hyaloperonospora, Perofascia, and Peronospora. lates of P. cubensis from various hosts were almost identical in terms of sequence analysis of ITS rDNA, suggesting that P. cubensis is a homogenous taxon. DISCUSSION The taxonomic and nomenclatural status of the Pseudoperonospora cubensis vs P. humuli genus Pseudoperonospora was rather controversial, but the studies of its morphological features and host No comparative study of Pseudoperonospora cubensis affinity showed that it is composed of a homogenous and P. humuli using both morphological and molecular group of about five species (Constantinescu 2000, characteristics had previously been performed yet. Our Constantinescu & Fatehi 2002). Recent molecular results show that the morphology of these two species studies based on the analyses of 28S and ITS rDNA is considerably more similar to each other than to sequences reached the same conclusion (Riethmu¨ ller P. cannabina and P. celtidis. Although the dimensions et al. 2002, Go¨ ker et al. 2003, Voglmayr 2003). In of sporangiophores and sporangia given by different our study, the Pseudoperonospora collections formed a authors show some variation, there are no obvious well-supported monophyletic group, clearly segregated morphological differences between P. cubensis and from the genera Hyaloperonospora, Perofascia, and P. humuli (Table 2). In addition, P. cubensis and Peronospora. Thus, our results reconfirm that Pseudo- P. humuli produce similar symptoms on their hosts. peronospora is both homogenous and distinct from The ultrastructure of the dehiscence apparatus other genera of the Peronosporales. (Constantinescu 2000), as well as the similar mor- Traditionally, as exemplified by several monographs phology of haustoria (Fraymouth 1956), also support (Kochman & Majewski 1970, Vanev, Dimitrova & the conspecificity of these fungi. Interestingly, Salmon Ilieva 1993, Yu et al. 1998), the classification of species & Ware (1928, 1929), Hoerner (1940), and Glazewaska within Pseudoperonospora has been based primarily (1971) showed by cross-inoculation trials that P. humuli upon differences in host range, particularly host can infect Urtica, Cannabis,andCeltis species that families, rather than on morphological characteristics. are hosts of P. urticae, P. cannabina, and P. celtidis, Although a few morphological differences were sug- respectively. Riethmu¨ ller et al. (2002) using sequence gested to divide these species (Waterhouse & Brothers analysis of 26S rDNA, and Voglmayr (2003) who se- 1981), the differences are relatively minor, making quenced the ITS region, both reported that P. cubensis it difficult to identify these fungi, particularly if the was closely related to P. humuli and P. urticae. Our host plant from which the fungus originates is not analysis of the ITS rDNA revealed that the sequences known. Our molecular phylogenetic analysis of P. of P. cubensis from cucurbit specimens are mostly cannabina, P. celtidis, P. cubensis, and P. humuli was identical to those of P. humuli from Humulus. The high intended to determine more precisely the relationships level of rDNA sequence identity between these two amongst these morphologically similar related species. mildews exceeds that found among P. cubensis isolates The results show that P. cannabina and P. celtidis are from several cucurbit genera. This result is somewhat separated from the P. cubensis – P. humuli group, with unexpected because these plants are only distantly high nucleotide dissimilarities (6 and 3%, respectively). related to each other: Cucurbitaceae are members of The morphological features of the sporangia and the Cucurbitales, whereas Humulus spp. belong to the sporangiophores also show that these two pairs of Rosales. It has long been believed that most species of species are distinct. Therefore, P. cannabina and P. the Peronosporaceae have narrow host ranges, or are celtidis should be regarded as distinct, independent sometimes restricted to only a particular host plants species. (Ga¨ umann 1918, 1923, Gustavsson 1959, Crute 1981,

Brandenburger 1985). According to Crute (1981), there species deduced from rDNA sequence analysis. Mycological are no authentic records showing that a downy mildew Research 100: 437–443. can infect host plants belonging to different families. Crute, I. R. (1981) The host specificity of peronosporaceous fungi and the genetics of the relationship between host and parasite. In The Our molecular analysis suggests that a downy mildew Downy Mildews (D. M. Spencer, ed.): 237–253. Academic Press, may have a host range beyond the order of the host London. plant for the first time. Interestingly, Okamoto et al. Fitch, W. M. (1971) Toward defining the course of evolution: mini- (2002) showed that host switching beyond the level of mum change for a specific tree topology. Systematic Zoology 20: the plant family also occurs in Erysiphaceae (powdery 406–416. Fraymouth, J. (1956) Haustoria of the Peronosporales. 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