Development of a qPCR assay for early detection and quantification of Phytopythium vexans in kiwifruit plant and soil affected by Vine Decline Syndrome

Kiwifruit Vine Decline Syndrome (KVDS) is a soilborne disease affecting Actinidia fruit trees in perennial cropping systems. Since its emergence in 2012, studies have increasingly identified the oomycete Phytopythium vexans as a major causative agent of the disease. P. vexans is also implicated in complex soilborne disease systems of woody perennial crops, including replant disease in apple and pear. To date, most molecular assays for the detection of P. vexans target the nuclear ribosomal internal transcribed spacer (ITS), a region which is ill-suited for distinguishing between closely related oomycete species. The cytochrome oxidase subunit I (COI) mitochondrial gene was targeted for the design of new primers because it was previously identified as a better marker for differentiating oomycete species. The FOR2/REV4RCA primer pair gave the best results regarding PCR specificity and was selected for use in a SYBR Green-based qPCR assay. The specificity of the qPCR assay was evaluated using 29 P. vexans strains (including different phylogenetic groups) as well as a wide variety of closely related off-target species associated with pathogenic soil communities of fruit trees. P. vexans strains were successfully quantified down to 20 fg in water and in DNA extracted from kiwifruit roots. P. vexans was also detected in artificially inoculated Actinidia plant roots as well as in a variety of naturally infected field samples of both kiwi and apple trees. These results suggest that the qPCR assay developed in this study is highly sensitive and specific to target pathogen, regardless of sample matrix.