The use of nested primers in the polymerase chain reaction for the detection of Phytophthora fragariae and P-cactorum in strawberry

Phytophthora fragariae var. fragariae Hickman, which causes red core disease of strawberry, is a quarantine organism on which a nil tolerance is placed. Detection of the fungus is by a root tip bait test which is highly specific and sensitive but time-consuming (5-6 weeks), has to be done at 12 degrees C and can require some taxonomic experience. Even with the test, detecting red core can still be difficult, especially with early primary infection of propagation beds or infected symptomless resistant cultivars. Moreover, P. cactorum, a closely related fungus, causes crown rot of strawberry. It is not a quarantine organism, typical tolerances of 1% infection are allowed, and can confuse diagnosis. Techniques based to the Polymerase Chain Reaction (PCR) are promising alternatives. Various parts of the ribosomal RNA gene repeat (rDNA) have been used to design primers with different specificities: specific PCR primers that can differentiate between P. fragariae and P. cactorum and others designed to detect a wider range of Peronosporales, to which Phytophthora belongs. In nested PCR, combinations of these primers can detect both pathogens with high specificity and sensitivity. Competitive PCR has also been developed to allow quantification of P. cactorum infection in strawberry crowns. With these techniques, P. fragariae was detected in strawberry plants one day after inoculation whereas the symptoms were only visible after one week. Moreover, very low numbers of zoospores could be detected in water samples. The test has been evaluated on samples provided by the inspection services of Scotland and The Netherlands.