Development and validation of conventional and quantitative polymerase chain reaction assays for the detection of storage rot potato pathogens, Phytophthora erythroseptica, Pythium ultimum and Phoma foveata

The diseases pink rot, watery wound rot and gangrene are important storage rot diseases of potato associated predominantly with Phytophthora erythroseptica (P.), Pythium ultimum (Py.) and Phoma exigua (Phoma) var. foveata respectively. Reliable molecular-based diagnostic tests are required that will not only allow unequivocal identification of symptoms but will further advance epidemiological studies of these potato diseases to increase our understanding and contribute to more effective management and control strategies to the potato industry. Primers and probes were designed in specific regions of the internal transcribed spacer (ITS) regions to develop conventional and real-time quantitative polymerase chain reaction (PCR) assays able to detect all possible fungal and oomycete pathogens causing pink rot, watery wound rot and gangrene. The specificity of each diagnostic assay was rigorously tested with over 500 fungal/oomycete plant pathogen isolates from potato and reference culture collections, and both conventional and real-time PCR methods produced similar results. In terms of sensitivity, the detection limits for real-time PCR went below ag DNA levels compared with pg DNA levels with conventional PCR. The real-time PCR assays developed to detect Phoma foveata and Py. ultimum on tubers were suitable for the comparative C-t method (Delta Delta Ct) of quantification using the cytochrome oxidase gene of potato as a normalizer assay; an advantage as the need for a standard curve is eliminated. Each assay detected Phoma species (var. foveata or exigua) from naturally infected tubers showing symptoms of gangrene, and P. erythroseptica or Py. ultimum were also detected following inoculation of Russet Burbank tubers. Each diagnostic assay developed could reliably detect and distinguish between the pink rot, watery wound rot and gangrene-causing potato pathogens.