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Abstract
During April and May 2013, typical symptoms of witches- broom disease were detected in citrus, orchards of sweet orange (C. sinensis) and mandarin (C.reticulate Planco) located at Sharqia, Qalyubia and Ismalia , governorates in Egypt. DAS-ELISA technique was used to detect phytoplasma citrus- witches broom in doubtful samples. Data showed that the number of infected orange was 7 out of 55 samples from Al-Sharqia with rate of infection 12.7%, 10 out of 93 samples from Al-Qalyubia with rate of infection 10.7%, and 4 out of 32 samples out of Ismalia with rate of infection 12.5%. The number of infected mandarin was 5 out of 40 samples from Al-Sharqia with rate of infection 12.5%, 7 out of 63 samples from Al-Qalyubia with rate of infection 11.1%, and 2 out of 20 samples from Ismalia with rate of infection 10%. 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) ranged in size from 400-800 nm in diameter. Phytoplasma infection resulted in ultrastructural changes especially in sieve elements, obliteration and necrosis were observed. The ultrastructural changes were expanded to the cell wall and the cell membrane