Plant pathogens pose significant threats to agricultural ecosystems, directly impacting global food security and economic stability. Effective management and control of plant diseases depend on the rapid and precise diagnosis of such pathogens. In this context, the integration of advanced molecular diagnostic tools plays a key role in enhancing the capabilities of researchers and diagnostic laboratories in the field of agriculture and phytopathology.
Robust nucleic acid extraction methods and amplification reagents are essential for the detection of plant pathogens such as Phytoplasmas. These are wall-less bacteria that can cause diseases in a wide variety of plant species leading to severe yield losses. We detail optimized protocols for both manual and automated extraction of Phytoplasma DNA from infected plant tissue. Our method includes a reliable qPCR detection of Phytoplasma with no amplification inhibition. Additionally, we propose an approach for direct amplification of Phytoplasma DNA from plant leaf punches with minimal qPCR inhibition.
We also show that our automated extraction technique based on paramagnetic beads allows isolation of high-quality pathogenic DNA suitable for long-read sequencing. Phytoplasma DNA was successfully sequenced and differentiated from other sequencing reads, offering more detailed studies and management of phytoplasma-induced plant diseases.
We propose thus reliable and efficient molecular diagnostic tools, crucial in the fight against plant pathogens.