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 Downloaded from https://www.cambridge.org/core. University of Athens, on 02 Oct 2021 at 23:55:17, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0953756205002534 Pseudoperonospora cubensis 842 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 com- parison, 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