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Triplex qPCR for Grapevine Yellows Phytoplasmas Combined with a Universal Endogenous Control.

Luca Fontanesi¹, Anna Pedroncelli¹, Mattia Tabarelli¹

¹Functional Genomics, Laimburg Research Centre, Auer/Ora, Italy.

Methods in Molecular Biology (Clifton, N.J.)

|January 13, 2026

Summary

A new triplex qPCR assay detects Flavescence dorée phytoplasma (FDp) and 'Ca. P. solani', common grapevine yellows pathogens. It uses a universal control for reliable detection in grapevines and insect vectors.

Keywords:

28S rRNA Bois noir Diagnostic Endogenous control Flavescence dorée Grapevine yellows Multiplex qPCR Phytoplasma Real-time PCR ‘Candidatus Phytoplasma solani'

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Area of Science:

Plant pathology
Molecular diagnostics
Entomology

Background:

Flavescence dorée and Bois noir are significant grapevine yellows diseases in Europe.
Early detection of Flavescence dorée phytoplasma (FDp) and 'Candidatus Phytoplasma solani' is vital for managing these diseases in grapevines and insect vectors.
Quantitative PCR (qPCR) is a sensitive method for phytoplasma detection, but requires robust internal controls for accuracy.

Purpose of the Study:

To develop a novel triplex qPCR assay for simultaneous detection of FDp, 'Ca. P. solani', and a universal endogenous control.
To enhance diagnostic flexibility and reliability across various eukaryotic hosts, including grapevines and insect vectors.
To validate DNA extraction efficiency and mitigate PCR inhibition in phytoplasma diagnostics.

Main Methods:

Development of a triplex quantitative PCR (qPCR) assay.
Inclusion of a universal endogenous control targeting the eukaryotic 28S rRNA gene.
Simultaneous detection of Flavescence dorée phytoplasma (FDp) and 'Candidatus Phytoplasma solani'.

Main Results:

The triplex qPCR assay successfully detects FDp and 'Ca. P. solani' simultaneously.
A universal endogenous control, based on the 28S rRNA gene, provides broad host applicability.
The assay facilitates robust and flexible diagnostics in a single reaction setup.

Conclusions:

The developed triplex qPCR assay offers a sensitive, specific, and versatile tool for detecting major grapevine yellows pathogens.
The universal endogenous control enhances the reliability of diagnostics in both plant and insect samples.
This assay supports effective monitoring and control strategies for European viticulture.