Detection of Xanthomonas citri subsp. citri from field samples using single‐tube nested PCR

• Published in: Plant pathology 2011-06, Vol.60 (3), p.436-442
• Main Authors: Kositcharoenkul, N., Chatchawankanphanich, O., Bhunchoth, A., Kositratana, W.
• Format: Article
• Language: English
• Subjects: bacterial citrus canker; Bacterial plant pathogens; Biological and medical sciences; Canker; Citrus; Citrus maxima; ELISA; Enzyme-linked immunosorbent assay; Fundamental and applied biological sciences. Psychology; Leaves; Orchards; Pathogens; Phytopathology. Animal pests. Plant and forest protection; Plant tissues; Polymerase chain reaction; pomelo; Primers; Temperature effects; Xanthomonas
• ISSN: 0032-0862; 1365-3059
• DOI: 10.1111/j.1365-3059.2010.02390.x

A single‐tube nested PCR was developed for detection of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. The assay targets the pthA gene of Xcc and utilizes different annealing temperatures for the two primer pairs. It reliably detected as few as 1·0 × 102 Xcc cells, and was unaffected by the presence of PCR inhibitors. It was 10‐fold and 8500‐fold more sensitive than standard PCR and ELISA, respectively. Increased sensitivity was also achieved via the use of a washing method for DNA extraction, as opposed to direct extraction from leaf tissue. When evaluated for Xcc detection in 90 samples collected from affected pomelo orchards, the single‐tube nested PCR was superior to standard PCR, detecting the pathogen in 67 vs. 54 samples. It was also able to detect Xcc from samples with and without symptoms. This assay can be used as a rapid and sensitive technique for routine Xcc detection in field samples for surveillance of citrus canker.