3.3.79
DETECTION OF PHYTOPHTHORA IN PLANTS BY THE POLYMERASE CHAIN REACTION

JM DUNCANl, DC COOKEl I LACOURTl, PJM BONANTS2 and JA MURPHY4

lScottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK; 2Research Institute for Plant Pathology, PB 9060, Binnenhaven 5, Wageningen, NL-6700 GW, NL; sup>3Mylnefield Research Services Ltd, Invergowrie, Dundee DD2 5DA, UK

Background and objectives
Many important phytophthora diseases of horticultural and perennial crops are spread through planting vegetatively propagated stocks cryptically infected with the pathogen. Highly specific, sensitive and rapid tests for planting material are needed to prevent such spread but the three principal methods of detection (visual inspection and isolation of the fungus; immunological; and baiting) have limitations. Visual inspection and isolation require a large amount of time and trained staff; immunological tests lack species-specificity; and bait tests require time. The polymerase chain reaction (PCR) offers both speed and specificity and has been tested extensively for its utility for the detection of plant pathogens. In this paper, the value of ITS spacer regions of RDNA for the detection by PCR of many Phytophthora species in plant material was tested.

Materials and methods
A forward PCR primer (DC6) designed to amplify a region of RDNA from all species within the orders Pythiales (including Phytophthora) and Peronosporales of the Oomycota was used in the first round of nested PCR with the 'Universal' reverse primer ITS4 [1]. The resultant amplicon was designed to contain part of the 18S and 28S subunits and the whole of the ITS1 and ITS2 spacer regions and the 5.8S subunit of RDNA. An aliquot of this product was then used in a second round of PCR with primers specifically designed for P. fragariae var. fragariae, P. fragariae var. rubi, P. cambivora, P. cinnamomi, P. cryptogea, P. cactorum or P. nicotianae; these being the main species affecting soft fruit, glasshouse and hardy ornamental crops in UK. The specific primers were designed by comparing the DNA sequences of the ITS1 and ITS2 regions of RDNA of the target species with those of c. forty other species. Six forward and four reverse PCR primers were used in various combinations to give a highly specific set of primers for each target species.

Results and conclusions
In extensive tests vvith plant and fungal DNA, the DC6 'Peronosporales' primer only amplified RDNA from species belonging to the Pythiales and Peronosporales of the Oomycota, including the obligate downy mildews and white blister rusts, even when present in a much larger background of plant DNA. Each of the species-specific Phytophthora primer combinations was indeed highly specific. Cross-reactions with other Phytophthora spp. were few and easily eliminated by increasing the stringency of the reaction conditions. In nested PCR, sensitivity was very high (<10 femtograms of target DNA detected) and each of the target species was detected in plant material. The protocol has been used to detect more than one target species in infected root material from field grown plants, simply by testing the product of the first round of amplification vvith the different sets of specific primers in the second round. Nested PCR has also detected zoospores of the target species in water [2]. The tests for P. fragariae and P. cactorum have been used on a wide range of commercial field and glasshouse propagation material of raspberry and strawberry. Both have detected their respective targets at least as well as existing bait tests but within 1-2 days rather than 3-5 weeks, and in a number of examples they have detected very low levels of cryptic infection where bait tests have failed. The test for P. fragariae in strawberry is now being developed as the statutory standard for strawberry stocks within Europe under a collaborative European Union programme. Various formats for testing plants, water and soil will be examined in this programme.

References
1. Lee SB, Taylor JW, 1992. Journal of Molecular Biology and Evolution 9, 636-653.
2. Bonants PJM, Hagenaar-De Veerdt M, Van Gent-Peizer MP, Lacourt L, Cooke DEL, Duncan JM, 1997. European Journal of Plant Pathology 103, 345-355.