Species-specific identification and detection of Phytophthora pistaciae, the causal agent of pistachio gummosis

Abstract

Gummosis is the most important disease of pistachio trees in Iranian orchards. The principal cause, Phytophthora pistaciae, is a recently described plant pathogenic oomycete from pistachio trees. This species is very similar, probably due to convergence, to some other non-papillate high temperature species, especially P. megasperma. A PCR based method was developed to provide a reliable molecular tool for the identification of P. pistaciae. A collection of isolates from different locations representing a diversity of species was examined for unique coding regions as well as for non-coding gene sequences including the internal transcribed spacers (ITS1 and ITS2) and the 5.8S gene of rDNA, the heat shock protein 90 gene and ß-tubulin. Eight PCR primers specific for P. pistaciae were designed. Annealing temperatures and extension times were optimized for each set of primers to maximize both specificity and amplification efficiency. Each set was tested against purified DNA from other gummosis-inducing Phytophthora species from pistachio trees as well as 28 other Phytophthora species from different hosts. In conventional PCR, the limit of detection of different primer sets ranged from 10 ng to 50 pg of purified DNA. The best candidate for identification of P. pistaciae isolates was the ITS-S1 primer set which was a combination of ITS-SF1 and ITS-SR1. The ITS-S1 set did not amplify purified DNA from other Phytophthora species tested. The combination of ITS-S1 with ITS6 and ITS4 universal primers could detect up to 100 fg DNA in nested PCR and also can amplify the specific band in infected tissues, infested soil and water.