• soil-borne plant pathogens
• fungi
• Oomycota
• diagnostic methods
• duplex and multiplex PCR
• nucleic acid lateral flow devices

Strawberry crown and root rot diseases are caused by soil-borne pathogens including Macrophomina phaseolina (Mp), Verticillium dahliae (Vd) and Phytophthora cactorum (Pc). The symptoms caused by these pathogens are very similar and difficult to distinguish, and traditional culture-based detection methods are laborious and time consuming. The aim of the work was the development of duplex and multiplex PCR-NALFIA assays using three pairs of species-specific labelled primers able to amplify the target sequences of the pathogens. The NALFIA devices used in this study consisted of a commercial device for the detection of two analytes (Mp+Vd) and a prototype device for the detection of three analytes (Mp+Vd+Pc). Detection methods were tested by single, duplex and multiplex PCR using DNA extracted from pure cultures of the pathogens, and DNA extracted from artificially inoculated melon stems and strawberry crowns. PCR reactions were used for both agarose gel electrophoresis (AGE) and NALFIA assays. Results from single PCRs demonstrated the ability of all primer pairs to amplify target sequences using the chosen PCR program. The proper functioning of duplex PCRs using primers pairs for Vd and Mp was confirmed by AGE and NALFIA assays. Multiplex PCRs using primer pairs for Vd, Mp and Pc, resulted in successful amplifications when visualized by AGE. The NALFIA assay, has led to some false positives in the control reactions, indicating that the prototype device needs to be refined.