ISOLATION AND IDENTIFICATION OF PHYTOPHTHORA SPECIES FROM DISEASED PLANTS IN ORNAMENTAL NURSERIES IN POLAND. Abstract

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1 *Research Institute of Pomology and Floriculture, Skierniewice, Poland **State Plant Health and Seed Inspection Service, Central Laboratory, Toruń, Poland ISOLATION AND IDENTIFICATION OF PHYTOPHTHORA SPECIES FROM DISEASED PLANTS IN ORNAMENTAL NURSERIES IN POLAND *L.B. Orlikowski, *K. Wiejacha, *A. Trzewik, **G. Szkuta and *T. Orlikowska Abstract Usefulness of potato dextrose agar (PDA) and unripe apple baits in the isolation of Phytophthora citricola and P. cinnamomi from diseased plants was evaluated. Both species were isolated from at least 72% of diseased Lawson cypress and rhododendron plants on PDA. Unripe apple fruits gave possibilities of P. cinnamomi isolation from the most of analysed cypress and rhododendron diseased stem parts and from 2/3 of soil samples. In the case of controversial results of morphological analyses or isolation of Phytophthora sp. from new host, isoenzymatic tests are advisable. The only procedure, which allows pathogen identification in diseased tissues, was PCR with species-specific primers. A combination of classical and molecular methods is possible when DNA is extracted from apple baits or from colonies grown on original samples. Key words: Phytophthora, isolation, stem, soil, identification, isoenzyme, PCR Introduction Since 1989 a strong increase in ornamental nursery production has been observed in Poland (Marosz 2004). It has been accompanied by a growing incidence of Phytophthora root and stem rot, and dieback of shoots in container-grown plants (Orlikowski et al. 1995, Orlikowski and Szkuta 2002 a, b). Nine Phytophthora species (Table 1) have been found in Polish ornamental nurseries. Phytophthora cinnamomi Rands and P. citricola Sawada have been the most often isolated species, especially from Chamaecyparis lawsoniana (Andr.) Parl., Calluna vulgaris (L.) Salisb. Phytopathol. Pol. 35: The Polish Phytopathological Society, Poznań 2005 ISSN

2 184 L.B. Orlikowski, K. Wiejacha, A. Trzewik, G. Szkuta and T. Orlikowska Phytophthora species isolated from different plant hosts in Polish nurseries during the period Table 1 Species Host plants P. cactorum Rhododendron spp. P. cambivora Acer pensylvanicum, Chamaecyparis lawsoniana, Cotoneaster horizontalis P. cinnamomi Calluna vulgaris, Chamaecyparis lawsoniana, Ilex aquifolia, Microbiota decussata, Pinus nigra, Podocarpus alpinus, Rhododendron spp. P. citricola Abies alba, Calluna vulgaris, Chamaecyparis lawsoniana, Picea omorica, Rhododendron spp., Thuja occidentalis, Vaccinum vitis-idaea P. citrophthora Picea abies, Pieris japonica, Podocarpus alpinus, Rhododendron spp., Syringa vulgaris P. cryptogea Chamaecyparis lawsoniana, Sempervivum sp. P. nicotianae Skimmia japonica P. palmivora Hedera helix P. ramorum Calluna vulgaris,photinia frasei,rhododendron spp. and Rhododendron spp. (Orlikowski et al. 1995, 2004, Orlikowski and Szkuta 2002 a, b, 2003 a, b, Szkuta 2004). Shoot blight caused by P. ramorum Werres, de Cock, Man in t Veld (Werres et al. 2001) has been sporadically observed on rhododendron (Orlikowski and Szkuta 2003 a). In integrated nursery management quick information on causal disease agents is necessary for the elimination of infected plants before planting them in a nursery and for choosing an effective control method. The purpose of this study was to find out the optimal methods of isolation and identification of Phytophthora species. Materials and methods Traditional methods The effectiveness of traditional methods in isolation of P. cinnamomi and P. citricola based on the growth of mycelium and the morphology of vegetative and generative structural organs was assessed in ericaceous and coniferous plants. Diseased plants were collected during summer time from more than 10 nurseries in different parts of Poland. 292 heather, 488 Lawson cypress and 367 rhododendron plants were analysed in the study (Table 2). After disinfection (Szkuta 2004), tissues of diseased plants were transferred onto a potato dextrose agar (PDA) and fungi-like organisms were isolated on PDA slants. The percentage of plants invaded by Phytophthora spp. was calculated. Additionally, unripe apples were used as a medium for isolating Phytophthora species according to Szkuta (2004). After four six days of incubation at 24 C, the browning parts of flesh around holes were transferred onto PDA slants.

3 Isolation and identification of Phytophthora species Isoenzymatic analyses The tests used were those described by Man in t Veld et al. (1998) and Werres et al. (2001), which employed malate dehydrogenase and malic enzyme. DNA analyses Identification and/or confirmation of classification to species was performed by DNA analyses. Wiejacha et al. (2002) had described the DNA extraction from mycelium and from infected plant tissue. We established a procedure in which PCR with non-specific primers RAPD and ISSR, were used to generate DNA profiles characteristic of 10 Phytophthora species, including P. cinnamomi, P. citricola, P. cryptogea Pethybr. & Laff. and P. ramorum. The primers RAPD C85, C92 (Lee et al. 1996), OPC-02 (Operon) and ISSR 808, 809, 810, 818, 889, 890, 895 (UBC) were the most useful. This first step was followed by confirmation of results using PCR with species-specific primers: P. cinnamomi DC9 and DC5, P. cryptogea CRYF2 and CRYR2 (Duncan, personal communication 2003), P. citricola CITR1 and CITR2 (Schubert et al. 1999), P. ramorum Phyto1 and Phyto4 (Garbelotto et al. 2002). The alternative way of confirmation was the use of the PCR-RFLP method, as described by Cooke and Duncan (1997). This method involves digesting of amplified DNA fragment obtained with primers ITS4 and ITS6 (PhytID 2002) with restriction enzymes, which produces bands of marker value. An alternative procedure was the sequential use of two PCR rounds, so called nested PCR. In the first PCR round, a pair of primers amplifies a longer product from the DNA template extracted from a natural sample. This product was then used as the template in the second round, in which internal primers are used (Nechwatal et al. 2001). Results Traditional methods The use of PDA resulted in the isolation of P. citricola from at least 72% of diseased plants (Table 2) with the exception of heather, in the case of which the Table 2 Isolation of Phytophthora species from diseased plants on potato dextrose agar Plant species Phytophthora citricola analysed plants positive isolations (%) Phytophthora cinnamomi analysed plants positive isolations (%) Calluna vulgaris Chamaecyparis lawsoniana Rhododendron spp

4 186 L.B. Orlikowski, K. Wiejacha, A. Trzewik, G. Szkuta and T. Orlikowska Table 3 Frequency of Phytophthora cinnamomi isolation from diseased stems and naturally infested peat on unripe apple baits (n = 15) Source of the pathogen Diseased stems Naturally infested peat Chamaecyparis lawsoniana Rhododendron spp pathogen was isolated only from 11% of plants. Phytophthora cinnamomi was isolated from at least 68% of the plants analysed, but especially from Lawson cypress. The use of apple baiting with diseased stem parts of cypress and rhododendron was as effective as the use of PDA in the isolation of P. cinnamomi (Table 3). The effectiveness of the isolation from baiting on PDA from infested substratum was around 66% of the analysed samples (Table 2). The isolate species were initially identified on the basis of the morphology of their organs. Isoenzymatic analyses When controversial results of morphological analyses were obtained or when a pathogen was isolated from a new host for the first time, isoenzymatic tests were carried out. This was the case, for example, when P. ramorum was found on Calluna vulgaris. DNA analyses The RAPD-PCR and ISSR-PCR (Phot. 1), as well as PCR-RFLP (Phot. 2), do need DNA extraction from pure cultures of pathogens, which makes them useless for the detection of pathogens in natural samples plant tissue, soil or water. They also did not hasten the identification procedure, although they ensured and verified the classification. The only procedure, which allows pathogen identification in diseased tissue, and also pathogen detection when no symptoms of disease are yet Phot. 1. DNA bands amplified by the use of ISSR primer, characteristic of four Phytophthora species (photo by K. Wiejacha)

5 Isolation and identification of Phytophthora species Phot. 2. Restriction DNA fragments characteristic of four Phytophthora species: CR P. cryptogea, CN P. cinnamomi, CT P. citricola, R P. ramorum. AluI, TaqI and MspI restriction enzymes (photo by K. Wiejacha) Phot. 3. DNA bands confirming the presence of DNA of four Phytophthora pathogens, amplified by the use of PCR with species-specific primers (photo by K. Wiejacha) visible, was PCR with species-specific primers (Phot. 3). Nested PCR had been helpful when the ratio of pathogen to plant DNA was too low to produce PCR products large enough to be visible after electrophoresis. A combination of classical and molecular methods had been used, in which DNA was extracted from apple baits or from colonies grown on the original samples placed on PDA. Discussion The isolation and identification of Phytophthora species from more than one thousand of diseased plants grown in ornamental nurseries was accomplished by different techniques traditional microbiological, isoenzymatic assay and DNA markers based on non-specific and specific primers. The most often were isolated P. citricola and P. cinnamomi and the most invaded plants were Rhododendron sp. and C. lawsoniana (Szkuta 2004).

6 188 L.B. Orlikowski, K. Wiejacha, A. Trzewik, G. Szkuta and T. Orlikowska In our hands the isolation of P. citricola and P. cinnamomi was equally effective with and without baiting on unripe apples which was in contrary to Hansen et al. (1980) results obtained when four Phytophthora species were isolated from Douglas fir. However, apple baiting was very useful for isolation of P. ramorum, which grows slowly on the PDA medium and its colonies can be quickly overgrown by species of Mucor, Trichoderma and Fusarium. We purified cultures of our isolates to built a collection for genetic and population studies (Wiejacha et al. 2004, 2005) and used DNAs extracted from such cultures. Purification of cultures is time consuming procedure, which usually takes days. However, for the screening of nursery plantations or plant material at a trade turnover, methods giving a quick answer are necessary. These methods are based on the PCR techniques with species-specific primers rdna (Cooke and Duncan 1997), coxi, coxii and others (Martin and Tooley 2004), which can be done from natural plant, soil or water samples. A new techniques and assays, as quantitative PCR and microarrays, are now under developing. The first one amplifies DNA of pathogen giving a possibility to quantify it at each cycle, which supplies information on initial number of copies (Böhm et al. 1999). The second one enabled hybridisation of labelled sample DNA or RNA to specific pathogen oligonucleotic sequences spotted on small surface, which enables detection of different pathogens in a single test (Blohm and Guiseppi-Elie 2001, Kamiński 2002, Bonants et al. 2005). In our work, the combination of traditional techniques a short incubation of original samples on PDA or baiting them in apple and isolation of DNA from growing hyphae was beneficial for all, time and labour needed and quality of identification results. Literature Blohm D.H., Guiseppi-Elie A., 2001: New developments in microarray technology. Curr. Opin. Biotechnol. 12: Böhm J., Hahn A., Schubert R., Bahnweg G., Adler N., Nechwatal J., Oehlmann R., Oßwald W., 1999: Real-time quantitative PCR: DNA determination in isolated spores of the mycorrhizal fungus Glomus mosseae and monitoring of Phytophthora infestans and P. citricola in their respective host plants. J. Phytopathol. 147: Bonants P.J.M., Schoen C.D., Szemes M., Speksnijder A., Klerks M.M., Boogert van den P.H.J.F., Waalwijk C., Van der Wolf J.M., Zijlstra C., 2005: From single to multiplex detection of plant pathogens: puma, a new concept of multiplex detection using microarrays. Phytopathol. Pol. 35: Cooke D.E.L., Duncan J.M., 1997: Phylogenetic analysis of Phytophthora species based on ITS1 and ITS2 sequences of the ribosomal RNA gene repeat. Mycol. Res. 101: Garbelotto M., Rizzo D.M., Hayden K., 2002: Phytophthora ramorum and sudden oak death in California: III. Pathogen genetics. USDA For. Serv. Gen. Tech. Rep. 184: Hansen E.M., Roth L.F., Hamm P.B., Julis A.J., 1980: Survival, spread, and pathogenicity of Phytophthora spp. on Douglas fir trees planted on forest sites. Phytopathology 70: Kamiński S., 2002: DNA microarrays a methodolical breakthrough in genetics. J. Appl. Genet. 43:

7 Isolation and identification of Phytophthora species Lee J.S., Lee P.O., Roh M.S., 1996: Classification of lilies using Random Amplified Polymorphic DNA (RAPD) analysis. Acta Hortic. (The Hague) 414: Man in t Veld W.A., Veenbaas-Rijks W.J., Ilieva E., Cock de A.W.A.M., Bonants P.J.M., Pieters R., 1998: Natural hybrids of Phytophthora nicotianae and P. cactorum demonstrated by isozyme analyses and random amplified polymorphic DNA. Phytopathology 88: Marosz A., 2004: Analiza szkółkarstwa ozdobnego w Polsce na tle wybranych krajów Unii Europejskiej. Typescript. Research Institute of Pomology and Floriculture, Skierniewice. Martin F.N., Tooley P.W., 2004: Identification of Phytophthora isolates to species level using restriction fragment length polymorphism analysis of a polymerase chain reaction-amplified region of mitochondrial DNA. Phytopathology 94: Nechwatal J., Schlenzig A., Jung T., Cooke D.E.L., Duncan J.M., Oßwald W.F., 2001: A combination of baiting and PCR techniques for the detection of Phytophthora quercina and P. citricola in soil samples from oak stands. For. Pathol. 31: Orlikowski L.B., Gabarkiewicz R., Skrzypczak Cz., 1995: Phytophthora species in Polish ornamental nurseries. I. Isolation and identification of Phytophthora species. Phytopathol. Pol. 9, 21: Orlikowski L.B., Sroczyński M., Szkuta G., 2004: First notice of Phytophthora tip blight of Calluna vulgaris. Phytopathol. Pol. 31: Orlikowski L.B., Szkuta G., 2002 a: Dieback of Pieris japonica caused by Phytophthora citrophthora. Acta Mycol. 36, 2: Orlikowski L.B., Szkuta G., 2002 b: Occurrence of Phytophthora cinnamomi on ericaceous plants in container grown ornamental nurseries in Poland. J. Plant Prot. Res. 42: Orlikowski L.B., Szkuta G., 2003 a: Phytophthora citricola on Rhododendron spp. in Polish nurseries. J. Plant Prot. Res. 43, 1: Orlikowski L.B., Szkuta G., 2003 b: Studies on the occurrence and colonisation of plants by Phytophthora ramorum in Poland. Acta Mycol. 38, 1/2: PhytID identification of plant pathogenic Phytophthora species by ITS fingerprinting Schubert R., Bahnweg G., Nechwatal J., Jung T., Cooke D.E.L., Duncan J.M., Müller-Starck G., Langebartels C., Sandermann H.Jr., Oßwald W.F., 1999: Detection and quantification of Phytophthora species which are associated with root-rot diseases in European deciduous forests by species-specific polymerase chain reaction. Eur. J. For. Pathol. 29: Szkuta G., 2004: Występowanie, izolacja, identyfikacja i szkodliwość gatunków z rodzaju Phytophthora w szkółkach ozdobnych roślin iglastych. Typescript. Department of Plant Protection AU, Cracow. Werres S., Marwitz R., Man in t Veld W.A., Cock de A.W.A.M., Bonants P.J.M., Weerdt de M., Themann K., Ilieva E., Baayen R.P., 2001: Phytophthora ramorum sp. nov., a new pathogen on Rhododendron and Viburnum. Mycol. Res. 105: Wiejacha K., Szkuta G., Orlikowska T., 2002: Optimization of DNA isolation procedure as the first step in identification of Phytophthora spp. Bull. Pol. Acad. Sci. Biol. Sci. 50: Wiejacha K., Trzewik A., Orlikowski L., Szkuta G., Orlikowska T., 2004: Genotypic differences of isolates Phytophthora cinnamomi and P. citricola obtained from ten nursery plants species. J. Plant Prot. Res. 44: Wiejacha K., Trzewik A., Orlikowska T., 2005: Genetic differences between isolates of Phytophthora citricola obtained from ornamental nurseries in Poland. Phytopathol. Pol. 35: Authors addresses: Prof. dr hab. Leszek B. Orlikowski, Katarzyna Wiejacha, M.Sc., Aleksandra Trzewik, M.Sc., Prof. dr hab. Teresa Orlikowska, Research Institute of Pomology and Floriculture, ul. Pomologiczna 18, Skierniewice, Poland lorlikow@insad.pl

8 190 L.B. Orlikowski, K. Wiejacha, A. Trzewik, G. Szkuta and T. Orlikowska Dr Grażyna Szkuta, State Plant Health and Seed Inspection Service, Central Laboratory, ul. Żwirki i Wigury 73, Toruń, Poland Accepted for publication: