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A panel of real-time PCR assays for specific detection of three phytoplasmas from the apple proliferation group
Petra Nikolić1, Natasa Mehle, Kristina Gruden
1National Institute of Biology, Vecna pot 111, SI-1000 Ljubljana, Slovenia. petra.nikolic@nib.si
Abstract:
We report here on the development of combination of assays for fast, reliable, specific and sensitive detection and discrimination of 'Candidatus Phytoplasma mali', 'Ca. P. prunorum' and 'Ca. P. pyri' from the 16Sr-X (apple proliferation - AP) group. These phytoplasmas are causal agents of diseases of fruit trees within the family Rosaceae, namely apple proliferation (AP), European stone fruit yellows (ESFY) and pear decline (PD). The designed panel of assays uses TaqMan minor groove binder probes (MGB). It comprises the same set of primers and specific probes for species-specific amplification within the 16S-23S rRNA intergenic spacer region, a set of primers and probes for amplification of the 16S ribosomal DNA region for the universal phytoplasma detection, and an additional set of primers and probe for 18S rRNA as an endogenous quality control of DNA extraction. The performance characteristics of the panel were evaluated. The advantages of new assays were shown in a comparative study with the conventional PCR, which proved their higher sensitivity combined with three-fold shorter time of testing process; and in comparison with two reported multiplex real-time PCR assays for detection of 'Ca. P. mali' or 'Ca. P. pyri'. New panel of assays were tested on the DNA samples of 'Ca. P. mali', 'Ca. P. prunorum', 'Ca. P. pyri', other phytoplasmas and other bacteria isolated from plant material. Additionally, 198 symptomatic and asymptomatic fruit tree field samples collecting during several growing seasons were tested with new assays as well. The results of this study indicate that the combination of three specific assays may be applied in routine phytoplasma surveys and in the certification programs.
Insights
This study introduces a new, rapid assay panel for detecting and differentiating three key phytoplasma species affecting fruit trees. The developed method offers higher sensitivity and a faster testing process compared to existing techniques.
Area of Science:
- Plant Pathology
- Molecular Diagnostics
- Agricultural Science
Background:
- Phytoplasmas, such as 'Candidatus Phytoplasma mali', 'Ca. P. prunorum', and 'Ca. P. pyri', cause significant diseases in Rosaceae fruit trees, including apple proliferation (AP), European stone fruit yellows (ESFY), and pear decline (PD).
- Accurate and efficient detection of these pathogens is crucial for disease management and certification programs.
Purpose of the Study:
- To develop and validate a combination of assays for the fast, reliable, specific, and sensitive detection and discrimination of 'Candidatus Phytoplasma mali', 'Ca. P. prunorum', and 'Ca. P. pyri'.
- To compare the performance of the new assays against conventional PCR and existing real-time PCR methods.
Main Methods:
- Development of a TaqMan minor groove binder (MGB) probe-based assay panel.
- The panel includes species-specific primers/probes for the 16S-23S rRNA intergenic spacer region, universal primers/probes for 16S ribosomal DNA, and an 18S rRNA internal control.
- Evaluation of assay performance using DNA from known phytoplasma strains, other bacteria, and field samples from symptomatic and asymptomatic fruit trees.
Main Results:
- The new assay panel demonstrated higher sensitivity and a three-fold reduction in testing time compared to conventional PCR.
- The assays showed specificity when tested against various phytoplasmas and other plant-associated bacteria.
- Field trials on 198 fruit tree samples confirmed the reliability of the new diagnostic tool.
Conclusions:
- The developed combination of three specific assays provides a rapid, sensitive, and reliable method for phytoplasma detection.
- This assay panel is suitable for routine phytoplasma surveys and fruit tree certification programs.
- The use of MGB probes enhances specificity and sensitivity in phytoplasma diagnostics.
