Abstract
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.
Highlights
Measurement of pathogen growth on plant tissue is an important tool in developing disease management strategies
In the current work we sought to overcome limitations of the above methods by developing a real-time, highthroughput assay to quantify pathogen growth in live tissue. To establish such a system, we worked with the highly destructive, soil-borne oomycete pathogen, P. capsici, which was first identified in pepper, has a broad host range causing severe damage in many solanaceous, cucurbitaceous, and legume crops (Granke et al, 2012; Lamour et al, 2012; Sanogo and Ji, 2012)
Three cucumber cultigens were used for these experiments: susceptible pickling cucumber breeding line, “Gy14”; the slicing cucumber cultivar, “Poinsett 76” (Seedway, LLC, Hall, NY, United States), with age-related resistance (ARR) to P. capsici; and the doubled haploid breeding line “A4-3,” with moderate young-fruit resistance to P. capsici “A4-3” was derived from breeding line MSU 109483-5-3, a selfpollinated line from cucumber PI109483 (Grumet and Colle, 2017)
Summary
Measurement of pathogen growth on plant tissue is an important tool in developing disease management strategies. In the current work we sought to overcome limitations of the above methods by developing a real-time, highthroughput assay to quantify pathogen growth in live tissue To establish such a system, we worked with the highly destructive, soil-borne oomycete pathogen, P. capsici, which was first identified in pepper, has a broad host range causing severe damage in many solanaceous, cucurbitaceous, and legume crops (Granke et al, 2012; Lamour et al, 2012; Sanogo and Ji, 2012). In this study we use a P. capsici isolate expressing red fluorescent protein (RFP) tdTomato gene (Dunn et al, 2013) to establish a microtiter plate, real-time assay to quantify pathogen growth in live tissue We use this system to monitor P. capsici growth in planta on cucumber fruit and pepper leaf samples in relation to different levels of host susceptibility. The highly replicated data are suitable for growth curve modeling to facilitate hypothesis development regarding key aspects of pathogen response
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