Using Real time PCR Technique for the Detection of Xanthomonas citri subsp. citri, Causal Agent of Citrus Canker for Certification of Canker Free Orchards

Abstract

To export pomelo (Citrus maxima Merr.) to the European Union (EU), exporter is obliged to follow the International Standards for Phytosanitary Measures No.6 and No.10 (ISPM No. 6 and ISPM No.10) Beside, Guidelines for Surveillance Requirements for the Establishment of Pest Free Places of Production and Pest Free Production Sites must be followed. Canker disease of pomelo caused by Xanthomonas citri subsp. citri is a quarantine pest of the EU. Therefore, Pomelo orchards certification for canker free is necessary and the effective detection method for X. citri subsp. citri needs to be  eveloped. This work was to utilize the Real time PCR, the rapid and high accuracy technique, to detect X. citri subsp. citri for the canker-free certification of pomelo orchards. The primers 2/3 and D1/D2 were designed from avirulence/pathogenicity gene (pthA gene) of X. citri subsp. citri which had high specificity and could detect as minimum as 5 picrograms of DNA or 81 CFU/ml of the bacterial cells. The method showed high accuracy of detection of X. citri subsp. citri in both symptom and symptomless samples. In 2014, this Real time PCR method was used for the establishment of canker-free production sites in pomelo orchards at Wiang Kaen Districts, Chiang Rai Province. From 130 orchards, 84 canker-free production orchards were successfully established. The Real time PCR method developed in this study was eventually accepted by the EU for the pomelo production in Thailand for export to the EU and representing as a model for the cankerfree certification of others crops for countries where X. citri subsp. citri is a quarantine pests