Development and Optimization of a Real Time Detection Assay for Xanthomonas fragariae in Strawberry Crown Tissue with Receiver Operating Characteristic (ROC) Curve Analysis

Authors: Turechek, William; Hartung, John; McCallister, Jennifer

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/6/2007
Publication Date: 6/11/2008
Citation: Turechek, W., Hartung, J.S., Mccallister, J.E. 2008. Development and Optimization of a Real Time Detection Assay for Xanthomonas fragariae in Strawberry Crown Tissue with Receiver Operating Characteristic (ROC) Curve Analysis. Phytopathology. 98(3):359-368.

Interpretive Summary: Angular leaf spot of strawberry is caused by a systemic bacterium and the disease is transmitted primarily through infected nursery stock, which often does not have symptoms of the disease. This creates problems for the strawberry industry because of quarantine restrictions and legal liabilities. To detect the bacterium in the absence of symptoms, we previously developed a set of specific DNA-based tests. In this study, we modified the previous tests so that they can be completed in less than an hour. Our new, faster tests also proved to be both more sensitive and specific than the previous version. Moreover, the detection of the bacterium in strawberry crowns was possible with the new, but not with the older test. This is a significant improvement since strawberry crowns are sold commercially. The information on sensitivity and specificity of our new assays was used to evaluate the performance of these assays with receiver operating characteristic (ROC) curve analysis. ROC curve analysis is widely used in the biomedical field, but this is the first application of this methodology to infected plant material. The results of this research will be useful to the strawberry industry plant inspectors who must certify that plants offered for sale meet high standards of health.

Technical Abstract: Angular leaf spot of strawberry is caused by the systemic bacterium Xanthomonas fragariae. The disease is transmitted primarily through infected nursery stock. This creates problems for nurseries wishing to export plants to Europe because of quarantine restrictions. Currently, field inspections for symptoms are used to certify plants free of X. fragariae, but visual inspection is not useful for detecting plants infected systemically. To detect systemic infections, PCR is the desired tool because of its sensitivity, specificity, and ease of use. In this study, we developed three sets of real-time PCR primers and probes and determined optimal reaction conditions for use of these primers for the detection of the bacterium X. fragariae. Real time detection proved to be both more sensitive and specific than standard PCR. Moreover, the detection of X. fragariae in crown tissue extract was possible with real time PCR but not with standard PCR which is a significant improvement over standard PCR. The information on sensitivity and specificity of the primer sets was used to evaluate the performance of these primers with receiver operating characteristic (ROC) curve analysis under different tolerances. The results of this analysis can be used to provide guidance on threshold selection to manage disease below unacceptable levels. The results of this research may be useful to regulators and inspectors who must certify that plants meet EPPO standards.