Establishment of a new method for rapid and precise estimation of apple proliferation phytoplasma concentration in periwinkle.

Quantification of a plant pathogen is essential to study its population dynamic in various conditions and to relate symptom expression with pathogen concentration. Up to now, very few methods have been published to quantify phytoplasmas. So, the objective of this work was to establish a method able to quantify the Apple Proliferation (AP) phytoplasma populations in periwinkles. The present work was based on a method previously published to detect AP phytoplasma. This method was optimized to transform it into a quantitative method. First, a new probe specific for AP detection was applied. This probe successfully detected only AP isolates (versus closely related ESFY and PD phytoplasmas). Secondly, the method was adapted to allow the quantification of phytoplasma in periwinkle leaves. For quantification, the calibration curve was built on serial dilutions of a plasmid containing the amplified fragment (phytoplasma 16Sr gene). The limit of detection of the method was one copy of cloned phytoplasma DNA in the reaction while the lower and upper limits of quantification were 102 and 108. Sample DNA extracts were diluted 100X before amplification and standards were prepared in 100x diluted DNA extract from healthy plant. Using the calibration curve, the concentrations in the tested samples were calculated at 2 x 10(5) to 10(6) individuals per mg of fresh midrib. This work is a preliminary step to study the interaction of phytoplasmas with their hosts in relation to symptoms expression.