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ARS Home » Southeast Area » Fort Pierce, Florida » U.S. Horticultural Research Laboratory » Subtropical Plant Pathology Research » Research » Publications at this Location » Publication #365090
Research Project: Mitigating High Consequence Domestic, Exotic, and Emerging Diseases of Fruits, Vegetables, and Ornamentals

Location: Subtropical Plant Pathology Research

Title: Detection of viable Xanthomonas fragariae cells in strawberry using propidium monoazide and long-amplicon quantitative PCR

Author
item WANG, HEHE - Clemson University
item Turechek, William

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/12/2019
Publication Date: 2/10/2020
Citation: Wang, H., Turechek, W. 2020. Detection of viable Xanthomonas fragariae cells in strawberry ssing propidium monoazide and long-amplicon quantitative PCR. Plant Disease. 104(4):1105-1112. https://doi.org/10.1094/PDIS-10-19-2248-RE.
DOI: https://doi.org/10.1094/PDIS-10-19-2248-RE

Interpretive Summary: Xanthomonas fragariae causes angular leaf spot in strawberry. The pathogens’ association with its host tissue is thought to be a condition for its survival. Consequently, transmission of the pathogen to field production sites occurs almost exclusively through the movement of contaminated planting stock. In this study, we developed and validated a PMA-qPCR protocol that allowed accurate quantification of viable- and dead-cell populations of X. fragariae cells. This assay will assist in the identification of inoculum sources in the strawberry production cycle and may serve in a certification protocol to satisfy quarantine regulations imposed on nursery stock.

Technical Abstract: Xanthomonas fragariae causes angular leaf spot in strawberry. The pathogens’ association with its host tissue is thought to be a condition for its survival. Consequently, transmission of the pathogen to field production sites occurs almost exclusively through the movement of contaminated planting stock. The aim of this study was to develop a PMA-qPCR protocol for specific detection of viable X. fragariae cells. The qPCR procedure was developed for two different primer pairs: one producing a long amplicon (863 bp) and the other a short amplicon (61 bp). Both pairs were tested with mixtures of viable and heat-killed bacteria cells and with bacteria-spiked strawberry petiole samples. Results showed that long-amplicon PMA-qPCR enabled specific and sensitive detection of X. fragariae with a detection limit of 10^3 CFU/ml, and it significantly improved the PMA efficiency on differentiating viable from dead bacterial cells relative to short-amplicon PMA-qPCR. PMA-qPCR with long amplicon was able to completely suppress the detection of dead X. fragariae cells from 10^4-10^7 CFU/ml in pure bacterial suspensions and from 10^3-10^7 CFU/ml in bacteria-spiked strawberry petiole samples. The quantification results from PMA-qPCR for mixtures of viable and dead cells were highly correlated with the predicted bacterial concentrations in a linear relationship (R2 = 0.981). This assay will assist in the identification of inoculum sources in the strawberry production cycle and may serve in a certification protocol to satisfy quarantine regulations imposed on nursery stock.