Real-Time PCR and Droplet Digital PCR Are Accurate and Reliable Methods To Quantify <i>Pseudomonas syringae</i> pv. <i>actinidiae</i> Biovar 3 in Kiwifruit Infected Plantlets.

Abstract

<i>Pseudomonas syringae</i> pv. <i>actinidiae</i> is the etiological agent of kiwifruit canker disease, causing severe economic losses in kiwifruit production areas around the world. Rapid diagnosis, understanding of bacterial virulence, and rate of infection in kiwifruit cultivars are important in applying effective measures of disease control. <i>P. syringae</i> pv. <i>actinidiae</i> load in kiwifruit is currently determined by a labor-intense colony counting method with no high-throughput and specific quantification method being validated. In this work, we used three alternative <i>P. syringae</i> pv. <i>actinidiae</i> quantification methods in two infected kiwifruit cultivars: start of growth time, quantitative PCR (qPCR), and droplet digital PCR (ddPCR). Method performance in each case was compared with the colony counting method. Methods were validated using calibration curves obtained with serial dilutions of <i>P. syringae</i> pv. <i>actinidiae</i> biovar 3 (Psa3) inoculum and standard growth curves obtained from kiwifruit samples infected with Psa3 inoculum. All three alternative methods showed high correlation (<i>r</i> > 0.85) with the colony counting method. qPCR and ddPCR were very specific, sensitive (5 × 10² CFU/cm²), highly correlated to each other (<i>r</i> = 0.955), and flexible, allowing for sample storage. The inclusion of a kiwifruit biomass marker increased the methods' accuracy. The qPCR method was efficient and allowed for high-throughput processing, and the ddPCR method showed highly accurate results but was more expensive and time consuming. While not ideal for high-throughput processing, ddPCR was useful in developing accurate standard curves for the qPCR method. The combination of the two methods is high-throughput, specific for Psa3 quantification, and useful for research studies (e.g., disease phenotyping and host-pathogen interactions).