Molecular and Electron Microscope Evidence for an Association of Phytoplasma with Citrus Witches Broom Disease

During April and May 2013 , citrus included sweet orange (C. sinensis) and mandarin (C.reticulate Planco) showing typical symptoms of witches broom disease were detected from different fields located at AL-Sharqia, AL-Qalyubia and Ismalia, governorates in Egypt. The detected phytoplasma from diseased samples was transmitted by grafting to healthy citrus plants and by dodder to healthy periwinkle plants. DNA extracted from symptomatic samples was used as template for amplification of products of 1.8 kb using universal primer pair P1/P7 and 1.2 kb using primer R16F2n/ R16R2 by direct and nested PCR. Pleomorphic bodies typical to phytoplasma structures were observed in phloem sieve elements in ultrathin sections of infected plants using transmission electron microscope (TEM). Phytoplasma infection resulted in ultrastructure changes especially in sieve elements, as obliteration and necrosis were observed. The ultrastructure changes were expanded to the cell wall, cell membrane and callose deposition on the cell wall was also observed. keywords— Phytoplasma, Citrus witches'broom, Grafting, PCR and TEM. ————————————————————