Thesis (Ph.D.) - Cairo University - Faculty of Agriculture - Department of Plant Pathology and Physiology

Citrus Stubborn Disease (CSD) is a serious disease of citrus caused by Spiroplasma citri, which is a helical motile, wall-less and cultivable microorganism belongs to class mollicutes. S. citri was isolated from samples collected from lime trees representing symptoms of CSD from Egyptian lime, lemon and volkamere lemon trees. The presence of Spiroplasma was checked by examination of cross sections stained with Dienes{u2019} stain as blue stained phloem, indicated the presence of Spiroplasma in infected phloem tissues. Culturing on C3-G specific liquid and solid media was also achieved. The pathogenicity of the isolated Spiroplasma on periwinkle plants revealed that stunting and yellowing symptoms were observed after about 30 days by injecting liquid culture of S. citri into the phloem of healthy transplants. In side grafting experiment, the highest recorded average percentage of CSD (82.45%) in lime after 28-30 days from grafting. In insect transmission experiment; the highest average percentage of CSD (80%) was obtained periwinkle plants became infected with S. citri when high populations of leafhopper (Orosius albicinctus) of (40 insects/ plant) were used in transmission tests.The dodder stolons transmitted S. citri from infected periwinkle plants into healthy ones after 30 days. Infected citrus seeds transmission produced infected transplants with CSD.The isolated Spiroplasma was identified as S. citri by ELISA using specific polyclonal antibodies. By transmission electron microscopy the identity and morphology of S. citri was confirmed. S. citri units were wall-less, spiral filamentous or branched long helices in liquid cultures and rounded segments forming the Fried-egg shape colonies on solid culture. Examination of ultrathin sections prepared in infected plants revealed the presence of elementary helices inside the phloem sieve elements. Necrotizaton and clumping of the cytoplasm of phloem parenchyma in infected volkamer lemon leaf samples was obvious. Damaging of nucleoli in companion cells was also an effect of S. citri infection

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