Pseudomonas syringae pv. syringae (Pss) is the primary causal agent of bacterial canker in sweet cherry and represents one of the most significant phytosanitary challenges for cherry orchards worldwide.
Effective disease management requires diagnostic tools capable of accurately quantifying bacterial inoculum in susceptible tissues during the most critical phenological stages.
A recent study from Chile validated an absolute qPCR assay based on the syrB gene, demonstrating how this methodology can overcome the limitations of traditional culture-based methods and support more targeted and sustainable control strategies.
The aim of the study was to develop and validate a quantitative real-time PCR protocol for the absolute determination of Pss in three key phenological stages: tight cluster buds, open cluster buds, and full bloom.
The selected target gene, syrB, encodes a lipopeptide toxin (syringomycin) associated with pathogen virulence, ensuring high analytical specificity.
Cherry tissues contain high concentrations of phenolic compounds and polysaccharides, which are known PCR inhibitors.
To address this issue, the CTAB DNA extraction protocol was modified by adding PVPP (polyvinylpolypyrrolidone), allowing to obtain linear standard curves and specific amplifications, which had not been achieved in preliminary tests without PVPP.
The standard curves showed excellent linearity (R2 > 0.99) and amplification efficiencies ranging from 103.6% to 107.3%, with intra- and inter-assay variations below 0.17 Cq cycles, confirming high reproducibility.
The limits of detection (LOD) and quantification (LOQ) were suitable for detecting bacterial concentrations associated with the development of flower blast under conditions favorable to disease development, such as cool and wet periods or late frost events.
Comparison with the conventional drop plate method on King’s B medium revealed important differences: while 77.1% of the samples showed fluorescent colonies on plates, only 18.8% were confirmed as positive for Pss by the specific qPCR assay.
The correlation between the two methods was low but significant (ρ = 0.35), with statistically significant differences.
In particular, at the open cluster stage qPCR did not detect any positive samples, probably due to bactericide treatments applied the day before sampling, whereas the culture-based method reported bacterial loads ranging from 8.8 × 103 to 8.8 × 106 CFU per bud.
This discrepancy highlights a structural limitation of traditional methods, which cannot distinguish between pathogenic strains and non-pathogenic fluorescent populations or biological control agents such as P. protegens.
The assay specificity was tested against 23 bacterial strains, including several Pseudomonas, Bacillus spp., and Xanthomonas arboricola pv. juglandis, with no non- specific amplifications observed.
An in silico analysis confirmed the high specificity of the primers for the P. syringae complex, although caution is recommended in regions where P. fragariae is present.
It should also be noted that qPCR detects total DNA and does not distinguish between viable and non-viable cells; therefore, the researchers propose the possible future use of v-qPCR techniques with PMA to discriminate metabolically active cells.
Operationally, the assay allows the processing of up to 300 samples per day, making it a high-throughput tool suitable for farm-level or regional monitoring programs.
The conclusions highlight that qPCR represents a more precise alternative to the drop plate method, particularly within integrated disease management programs.
This approach makes it possible to focus control measures against Pss during periods of real epidemiological risk, thereby reducing unnecessary treatments.
In a context of increasing attention to sustainability and the reduction of synthetic pesticide use, the integration of quantitative molecular tools such as this can improve decision-making accuracy, optimize disease control strategies, and provide a foundation for extending molecular diagnostics to other Pseudomonas species associated with cherry bacterial canker.
Source: Lovera, Y., San Martin, J., Ruiz, B., Bastías, R. M., Gerding, M., Hirzel, J., & Moya-Elizondo, E. (2025). A qPCR assay for the detection and quantification of Pseudomonas syringae pv. syringae in susceptible tissues of sweet cherry. Journal of Microbiological Methods, 107351. https://doi.org/10.1016/j.mimet.2025.107351
Image source: Bugiani, SFR RER
Andrea Giovannini
PhD in Agricultural, Environmental and Food Science and Technology - Arboriculture and Fruitculture, University of Bologna, IT
28 Aug 2024
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