Eur. J. For. Path. 29 (1999) 169–188 © 1999 Blackwell Wissenschafts-Verlag, Berlin ISSN 0300–1237 Detection and quantification of Phytophthora species which are associated with root-rot diseases in European deciduous forests by species-specific polymerase chain reaction 1 2 3 3 4 By R. SCHUBERT *, G. BAHNWEG *, J. NECHWATAL ,T.JUNG ,D.E.L.COOKE , 4 1 2 2 J. M. DUNCAN ,G.MU¨LLER-STARCK ,C.LANGEBARTELS ,H.SANDERMANN JR and 3 W. OßWALD 1Faculty of Forest Sciences, Section of Forest Genetics, Ludwig-Maximilians-University Munich, Am Hochanger 13, D-85354 Freising, Germany (R. Schubert for correspondence); 2GSF-National Research Center for Environment and Health, Institute of Biochemical Plant Pathology, Ingoldsta¨dter Landstr. 1, D-85764 Neuherberg, Germany; 3Faculty of Forest Sciences, Institute of Forest Botany, Phytopathology, Ludwig-Maximilians- University Munich, Am Hochanger 13, D-85354 Freising, Germany; 4Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK Summary Oligonucleotide primers were developed for the polymerase chain reaction (PCR)-based detection of selected Phytophthora species which are known to cause root-rot diseases in European forest trees. The primer pair CITR1/CITR2, complementing both internal transcribed spacer regions of the riboso- mal RNA genes, gave a 711 bp amplicon with Phytophthora citricola. The Phytophthora cambivora specific primer pair CAMB3/CAMB4, producing a 1105 bp amplicon, as well as the Phytophthora quercina specific primer pair QUERC1/QUERC2, producing a 842 bp amplicon, were derived from randomly amplified polymorphic DNA (RAPD)-fragments presented in this paper. All three primer pairs revealed no undesirable cross-reaction with a diverse test collection of isolates including other Phytophthora species, Pythium, Xerocomus, Hebeloma, Russula, and Armillaria. Under the PCR conditions described the detection of a well discernable amplicon was possible down to 100 pg (P. cambivora), 4 pg (P. quercina), and 2 pg (P. citricola) target DNA. This diagnostic PCR system was able to detect P. citricola, P. quercina, and P. cambivora in seedlings of pendunculate oak (Quercus robur) and European beech (Fagus sylvatica) which were artificially infected under controlled conditions. 1 Introduction Several episodes of oak decline have been observed in different European regions, starting from 1878 (reviewed by DELATOUR 1983; SIWECKI and LIESE 1991; LUISI et al. 1993). Dis- tinctly visible above and below-ground symptoms, including yellowing, dieback of branches and parts of the crown, the formation of leaf clusters, necrotic rootlets, and a significantly reduced number of living fine root tips are well documented. The phenomenon of oak decline has been recognized for a long time as a multifactorial disease (MANION 1981) with predisposing factors (e.g. inappropriate site, climatic conditions, industrial pollution), inciting factors (e.g. drought, frost, snow, defoliation by insects), and contributing factors (e.g. secondary pathogens). The isolation of single Phytophthora species from Quercus ilex Received: 17.4.1998; accepted: 29.7.1998; editor: C. Delatour * These authors have contributed equally to this work. U. S. Copyright Clearance Center Code Statement: 0300–1237/99/2903–0169 $14.00/0 170 R. Schubert et al. L., Quercus suber L., Quercus cerris L., Quercus rubra L., Quercus pubescens Willd., Quercus petraea (Matt.) Liebl., and Quercus robur L. confirmed the assumption that in some cases Phytophthora spp. are involved in oak decline (BRASIER 1993; BLASCHKE 1994; JUNG 1996; JUNG and BLASCHKE 1996; JUNG et al. 1996). There are more than 60 species in the genus Phytophthora and most of them are wide- spread destructive soil-borne root pathogens infecting crop plants, shrubs, and trees (reviewed by ERWIN and RIBEIRO 1996). The classical taxonomy of Phytophthora is based on sporangial and sexual structures (NEWHOOK et al. 1978; STAMPS et al. 1990). Isozymes, as well as various kinds of nucleic acid analyses have recently been used in order to elucidate the phylogenetic relationships among different Phytophthora species (MILLS et al. 1991; OUDEMANS et al. 1994; CRAWFORD et al. 1996; COOKE and DUNCAN 1997). Detection and quantification of Phytophthora species in soil samples and diseased plant materials is traditionally performed by baiting techniques with selective hosts (for example apple or lemon fruit) or by direct plating onto selective agar media. Species identification is based on morphological and physiological characters. These approaches are time-consuming and require considerable knowledge of the genus (TSAO 1983). The success of such an approach may depend on several factors, including interference from fast-growing sec- ondary microflora (TSAO 1990) and seasonal changes in pathogenic activity (HORNER and WILCOX 1996). Serological methods using enzyme-linked antibodies have been established for the detection of selected Phytophthora species (e.g. MILLER et al. 1997), but cross- reactions with other species and reduced sensitivity, especially in dark-rooted woody plants, still prevent an extensive application (for detailed information see MILLER 1996). Alter- natively, species-specific oligonucleotide hybridization probes were developed in order to differentiate Phytophthora parasitica Dastur, Phytophthora capsici Leonian, Phytophthora cinnamomi Rands, Phytophthora megakarya Brasier and Griffin, and Phytophthora pal- mivora var. heterocystica Babacauh (GOODWIN et al. 1989; LEE et al. 1993; JUDELSON and MESSENGER-ROUTH 1996). Hybridization, however, is a time-consuming and expensive procedure even though radioactively labelled DNA markers can be replaced by non- radioactive probes. Rapid, simple and reliable identification of Phytophthora species has successfully been achieved using the polymerase chain reaction (PCR), i.e. the in vitro synthesis of a diagnostic DNA marker fragment by a bacterial polymerase after the annealing of sequence specific oligonucleotide primers with the fungal target. For this purpose PCR primer pairs comp- lementing the internal transcribed spacer (ITS) regions of ribosomal RNA genes were constructed in an attempt to detect Phytophthora infestans (Mont.) de Bary, Phytophthora erythroseptica Pethybridge, and Phytophthora nicotianae Breda de Haan in infected potatoes (TOOLEY et al. 1997). The internal transcribed spacer (ITS) regions ITS1 and ITS2 of the ribosomal RNA genes were routinely used for the detection and/or identification of plant pathogenic fungi (NAZAR et al. 1991; XUE et al. 1992; JOHANSON and JEGER 1993; TISSERAT et al. 1994). The evolutionary conservation of the 18S RNA, the 5.8S RNA, and the 28S RNA genes supported the cloning of both ITS sequences, which lie between the coding ribosomal RNA genes, over a broad range of organisms using universal PCR primers (WHITE et al. 1990). On the other hand, sequence variation among the untranslated ITS sequences is often high enough in order to distinguish between different species. Further- more, the ribosomal RNA genes are organized in highly repetitive chromosomal units (for review see APPELS and HONEYCUTT 1986). This feature yields increased PCR sensitivity in comparison with target sequences representing single-copy genes. Alternatively, P. nic- otianae was discovered in inoculated tobacco and tomato plants using PCR on the basis of elicitin gene primers (LACOURT and DUNCAN 1997). Phytophthora citricola Sawada, originally isolated from rotting orange fruits in Taiwan (Sawada 1927), is now known to produce root-rot and trunk canker on many economically important shrubs, e.g. Rhododendron catawbiense Michx. (HOITING and SCHMITTHENNER 1969), Humulus lupulus L. (PICHLMAIER and ZINKERNAGEL 1992), and trees, e.g. Persea Identification of Phytophthora species associated with root-rot diseases by species-specific PCR 171 americana Miller (EL-HAMALAWI and MENGE 1994) and Aesculus hippocastanum L. (WERRES et al. 1995). Phytophthora cambivora (Petri) Buisman was first isolated by PETRI (1917) and has been recognized as the causal agent of ink disease and root-rot of Castanea sativa Mill. and Fagus sylvatica L. in Great Britain (DAY 1938). It was further isolated from Aesculus hippocastanum L. (BRASIER and STROUTS 1976) and Malus pumila Miller (JEFFERS and ALD- WINCKLE 1988). Phytophthora quercina Jung was recently discovered in rotting roots of Q. petraea (Matt.) Liebl. and Q. robur L. (JUNG 1996; JUNG et al. 1999). Moreover, the men- tioned Phytophthora spp. have been frequently isolated from both, natural infected oak and beech stands at different sites in Germany, Switzerland, Hungary, Italy, France, and Slovenia (JUNG 1996; JUNG and BLASCHKE 1996; JUNG et al. 1996). In view of the importance of these Phytophthora spp. as the primary cause of root-rot diseases, an extensive screening of European forest stands and nursery material would be an indispensable element in disease control. The new DNA markers for the PCR-based detection and quantification of P. citricola, P. cambivora, and P. quercina, presented in this paper, are a first milestone in the establishment of a diagnostic tool which aims at the verification of contamination in situ by Phytophthora species in complex forest ecosystems. 2 Materials and methods 2.1 Fungal isolates and cultural conditions The isolates of Phytophthora, Pythium, Xerocomus, Hebeloma, Russula, Armillaria, and Heterobasidion used in primer specificity testing, their culture numbers, sources, hosts, and origins are listed in Table 1. A few additional isolates were used in inoculation experiments