Quantitative High-Throughput, Real-Time Bioassay for Plant Pathogen Growth in vivo

Effective assessment of pathogen growth can facilitate screening for disease resistance, mapping of resistance loci, testing efficacy of control measures, or elucidation of fundamental host-pathogen interactions. Current methods are often limited by subjective assessments, inability to detect pathogen growth prior to appearance of symptoms, destructive sampling, or limited capacity for replication and quantitative analysis. In this work we sought to develop a real-time, in vivo, high-throughput assay that would allow for quantification of pathogen growth. To establish such a system, we worked with the broad host-range, highly destructive, soil-borne oomycete pathogen, Phytophthora capsici. We used an isolate expressing red fluorescence protein (RFP) to establish a microtiter plate, real-time assay to quantify pathogen growth in live tissue. The system was successfully used to monitor P. capsici growth in planta on cucumber (Cucumis sativus) fruit and pepper (Capsicum annuum) leaf samples in relation to different levels of host susceptibility. These results demonstrate usefulness of the method in different species and tissue types, allowing for highly replicated, quantitative time-course measurements of pathogen growth in vivo. Analyses of pathogen growth during initial stages of infection preceding symptom development show the importance of very early stages of infection in determining disease outcome, and provide insight into points of inhibition of pathogen growth in different resistance systems.