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

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.