Application of light microscopic staining techniques for the detection of phytoplasmas in yellow leaf disease affected arecanut palms in India

Yellow Leaf Disease (YLD) is one of the most drastic diseases affecting the areca nut palms of Kerala and Karnataka causing extensive crop loss. The disease is associated with phytoplasma presence. A classic diagnostic technique was the observation of symptoms on the leaves however microscopic studies can be used for pathogen visualization along with molecular methods. Although the direct observation of phytoplasmas requires the magnification achieved only by electron microscopy, light and fluorescence microscopy can also be used to detect the presence of phytoplasmas in diseased palm tisues. Dienes’ stain and DAPI fluorescent microscopic method developed for confirming the presence of phytoplasma in hand cut or freezing microtome sections of infected tissues were employed. In the present study, YLD affected plants gave positive results for phytoplasma specific staining. Fluorescent particles of the DAPI-stained phytoplasma were limited to sieve tube elements. Phloem of healthy root sections remained unstained with Dienes’ stain, but phloem of the infected root sections contained many dark blue DAPI stained areas. The results from the present study indicated that these methods can be used as preliminary steps in phytoplasma presence detection, and are found to be very effective in highly infected plants. The microscopic observation of phytoplasma cells could be used as a confirmative step for subsequent application of molecular diagnostic techniques.