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

Authors: GUASCHINO, MICOL - University Of Torino; PRENCIPE, SIMONA - University Of Torino; Somera, Tracey; TABONE, GIULIA - University Of Turin; SPADARO, DAVIDE - University Of Torino

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/9/2024
Publication Date: 1/14/2025
Citation: Guaschino, M., Prencipe, S., Somera, T.S., Tabone, G., Spadaro, D. 2025. Development of a qPCR assay for early detection and quantification of Phytopythium vexans in kiwifruit plant and soil affected by Vine Decline Syndrome. Plant Disease. https://doi.org/10.1094/PDIS-09-24-2044-RE.
DOI: https://doi.org/10.1094/PDIS-09-24-2044-RE

Interpretive Summary: Kiwifruit Vine Decline Syndrome (KVDS) is a soil-borne disease affecting kiwifruit trees in perennial cropping systems. An increasing number of studies support the hypothesis that the oomycete Phytopythium vexans is a major causative agent. P. vexans has also been reported to play a role in complex soilborne disease systems affecting other woody perennial crops, including apple and pear. In this study, a new and improved molecular method for the detection and identification of P. vexans was developed using the cytochrome oxidase subunit I (COI) gene, a region previously shown to be ideal for differentiating oomycete species. Assay specificity was evaluated using 29 P. vexans strains as well as closely related off-target oomycetes and fungi associated with pathogenic soil communities of fruit trees. P. vexans was successfully quantified down to 20 fg with no off-target binding detected. In addition, P. vexans was detected in naturally infected field samples collected from both kiwifruit and apple orchards. The assay will be useful for preventing further spread of P. vexans throughout fruit-producing areas worldwide.

Technical Abstract: Kiwifruit Vine Decline Syndrome (KVDS) is a soil-borne disease affecting (Actinidia) fruit trees in perennial cropping systems. Since the rise of KVDS in 2012, an increasing number of studies support the hypothesis that the oomycete Phytopythium vexans is a major causative agent of the disease syndrome. P. vexans has also been reported to play a role in complex soilborne disease systems affecting other woody perennial crops, including replant disease in apple and pear. To date, the majority of molecular assays designed 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 candidate for differentiating oomycete species. The FOR2/REV4RCA primer pair gave the best results with regard to PCR specificity and was selected use in a SYBR Green-based qPCR assay for the detection of P.vexans. The specificity of the qPCR assay was evaluated using a total of 29 P. vexans strains (including different phylogenetic groups belonging to the species) as well as a wide variety of closely related off-target oomycetes and fungi associated with pathogenic soil communities of fruit trees where P. vexans is also present. Using this assay, P. vexans was successfully quantified down to 20 fg in water and in DNA extracted from kiwifruit roots with no off-target binding detected. P. vexans was also detected directly in artificially inoculated Actinidia deliciosa cv. Hayward plant roots as well as in a variety of naturally infected field samples (roots, rhizosphere and bulk soil of symptomatic and asymptomatic trees). Developing tools for early detection of this emerging, major soilborne pathogen is a valuable strategy for improving phytosanitary monitoring and preventing further spread of P. vexans throughout fruit-producing areas worldwide.