Detection and Identification of Phytophthora drechsleri in Soil, Water and Infected Pistachio Tissue through Conventional PCR

Pistachio root and crown rot disease is one of the most important soil-borne diseases. Rapid detection of pathogens in different sources is a crucial stage in effective disease management strategies. Detection and molecular identification of Phytophthora drechsleri from infected water, soil and plant tissue were performed based on specific amplification fragments of rDNA-ITS region using the primer set DF2/DR2. Sampling and isolation were done during the year 2010-2011 from pistachio orchards in Rafsanjan and Anar regions of Kerman province, Iran. A total number of 46 isolates of P.drechsleri and 17 isolates of Phytophthora citrophthora were obtained and identified using selective media method. Sensitivity and specificity of the primer sets were studied using 15 predominant P. drechsleri isolates, 25 Phytophthora spp. and other closely pistachio soil-borne fungi. The results showed, this primer set is specific for detection of P. drechsleri and produced 567bp amplicon but there was no cross-reaction with other pistachio soil-borne fungal tested. In sensitivity test, the detection limit was 100pg of P. drechsleri pure genomic DNA in conventional PCR. As few as 17×103 ml-1 pure P. drechsleri zoospores (85 zoospores) could be detected by the primer set DF2/DR2. Amplification of genomic DNA extracted DNA from infected soil samples were also resulted in production of a 567 bp species specific band. The primers could not detect the target directly from infected pistachio tissue due to the presence of inhibitory compounds but the detection was successful from baiting method using apple fruit inoculated with infected soil and plant tissue after three days. However, the detection system proved to be accurate and sensitive and could help in pathogen detection but also could be used as part of disease control strategy.