Multiplex detection of Phytophthora: Padlock probe based Universal detection Multiplex Array (PUMA)
P. J. BONANTS (1), K. Gaszczyk (2), O. Mendes (1), E. Verstappen (1), C. D. Schoen (1)
(1) Plant Research International, Wageningen, NETHERLANDS; (2) Forest Research Institute, Raszyn, POLAND
Phytopathology 101:S18

Phytophthora spp. is responsible for many diseases worldwide, can occur on a wide range of different crops and the number of species is increasing. To detect and identify those species several molecular methods have been developed for single species only. A uniform method for detection of all Phytophthora species would be very useful for research and regulatory communities. Therefore we developed a diagnostic method to detect a range of Phytophthora species, including P. ramorum. The method includes a generic TaqMan PCR amplification method for all Phytophthora species combined with species specific padlock probe (PLP) detection on a dedicated universal micro-array. Twentythree padlock probes for 22 Phytophthora species relevant for the Netherlands were developed based on sequence differences in the ITS-1 region. After point mutation specific ligation of a mixture of the 23 PLPs on the generic amplicon, exonuclease treatment to degrade the unreacted probes, amplification of the ligated probes and hybridization on a micro-array, a unique signature on the micro-array can be obtained for each Phytophthora species included in the test. In this paper the specificity and sensitivity of a padlock based diagnostic tool is combined with a cost effective microtiter plate array detection device and has been evaluated using reference Phytophthora cultures as well as mixed infected material collected from field surveys, including air-, root-, water- and plant tissue samples.