Real-Time PCR and Droplet Digital PCR Are Accurate and Reliable Methods To QuantifyPseudomonas syringaepv.actinidiaeBiovar 3 in Kiwifruit Infected Plantlets

Abstract

Pseudomonas syringae pv. actinidiae (Psa) 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 is important in applying effective measures of disease control. Psa 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 Psa quantification methods in two infected kiwifruit cultivars: start of growth time, quantitative qPCR (qPCR), and droplet digital PCR (ddPCR). Method performance in each case was compared to the colony counting method. Methods were validated using calibration curves obtained with serial dilutions of Psa3 inoculum and standard growth curves obtained from kiwifruit samples infected with Psa3 inoculum. All three alternative methods showed high correlation (r > 0.85) with the colony counting method. qPCR and ddPCR were very specific, sensitive (5 × 102 CFU/cm2), highly correlated to each other (r = 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.

Rapid Methodologies for AssessingPseudomonas syringaepv.actinidiaeColonization and Effector-Mediated Hypersensitive Response in Kiwifruit

“…This latter issue would require an internal normalization process to facilitate adjustment for variation between cultivars, but is unavailable for classical growth assays. DNA-based detection as an alternative to classical infection quantification has been deployed for plant-pathogen studies in other pathosystems but has not yet been deployed in the Psa-kiwifruit system until recently (Abdullah et al, 2018;Barrett-Manako et al, 2020;Brouwer et al, 2003;Gallelli et al, 2014;Narusaka et al, 2009;Ross and Somssich, 2016;Weßling and Panstruga, 2012). The use of nucleic acid extraction tools for DNA-based detection has typically been the rate-limiting step for this approach.…”

“…Notably, though, this traditional approach to assessing resistance was achieved in 2 weeks, with significant limitations on the number of plant genotypes (6-12) able to be assessed per run because of labour requirements and time restrictions (Table 1). To increase the throughput of assessing resistance in infected plants, the quantification of Psa3 colonization of the host tissues was also undertaken using a novel DNA extraction method (PDQeX), which simplified and streamlined extraction (Stanton et al, 2019), coupled to previously reported qPCR-based detection (Barrett-Manako et al, 2020). This coupled approach of PDQeX-qPCR for plants sampled at 7 dpi reproduced the resistance in AA07_03 seen by traditional growth assays (Fig.…”

“…1C). This infection-sampling-PDQeX-qPCR approach reduced assessment times to half that of the traditional plating approach, with the added benefit of allowing around 24 individual plant genotypes to be assessed per run (Table 1), and is an improvement on the previous throughput possible ( Barrett-Manako et al, 2020).…”

“…Real-time quantitative PCR (qPCR) was carried out on an Illumina Eco Real-Time PCR platform using the SsoFast™ EvaGreen Supermix (BioRad, NZ) with 2 μL of PDQeX template DNA. qPCR was performed over 40 cycles using Psa ITS (target) and AcEF1α (reference) primers and protocol outlined in Barrett-Manako et al (2020), but with a lower melting temperature (T m ) of 57°C. qPCR results were reported as relative expression log 10 values of 2 for each sample with means and standard errors calculated for four pseudobiological replicates.…”

“…These assays, based on axenically grown plantlets in tissue culture, are reproducible but are nevertheless laborious and time consuming. Recently, a robust methodology for qPCR-mediated quantification of Psa infection was described for kiwifruit (Barrett-Manako et al, 2020;Gallelli et al, 2014). Sampling and DNA extraction issues were noted as limitations.…”