First report of a 16SrII group phytoplasma associated with die‐back diseases of papaya and citrus in Ethiopia

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Citrus orange (Citrus aurantifolia) and papaya (Carica papaya) are amongst the most common fruits grown in Ethiopia and the area devoted to their cultivation has increased considerably in the last six decades (Seifu, 2003). Recently decline disorders of both citrus and papaya have been reported as causing significant yield losses amongst the major farms growing these crops in the country. Symptoms in citrus include leaf interveinal chlorosis, mosaic or mottling, a reduction in the size and curling of leaves. In severe cases, premature fruit fall, twig drying and die-back, and death of the tree. In papaya, the first symptom is a bright yellowing of the upper young leaves; later, symptoms of mosaic, crinkling and leaf tip necrosis develop, followed by a drying of the upper leaves, which progresses to death of the whole plant. Leaf samples showing symptoms from 12 papaya and 6 orange plants were collected from farms in the Melkassa area of east Shewa, during September 2005. Citrus tristeza virus (CTV) has been previously reported in Ethiopia (Khassaye et al., 1977), so citrus samples were tested for CTV by DAS-ELISA, yielding 4/6 positives. DNA was extracted from all plants and indexed in both a nested PCR with phytoplasma generic primers P1/P7 and R16F2n/R16R2, and a simple PCR for papaya bunchy top (PBT) rickettsia using primers PBTF1/PBTR1 (Davis et al., 1998). Nested PCR amplicons were obtained for 11/12 papaya and six citrus samples. Following RFLP analysis, all exhibited identical HaeIII, RsaI and AluI profiles, which were typical of a 16SrII phytoplasma. None of the papaya and citrus samples evaluated was positive for the PBT rickettsia. Sequencing was done by The Sequencing Service (Univ. Dundee, Scotland; http://www.dnaseq.co.uk) using Applied Biosystems Big-Dye Ver 3·1 chemistry, on an Applied Biosystems model 3730 sequencer. The 16S rDNA phytoplasma sequences of a representative sample of papaya (GenBank Acc. No. DQ285659) and citrus (GenBank Acc. No. DQ286576) were identical and their highest similarity (98%) was with papaya yellow crinkle in Australia (GenBank Acc. No. Y10097), a member of the 16SrII group (Candidatus Phytoplasma aurantifolia). This is the first record of phytoplasmas associated with die-back diseases of papaya and citrus in Ethiopia. The finding of a mixed infection of phytoplasma and CTV in some citrus samples will require further studies before any recommendations for disease management can be made. Work in the UK was done under Defra plant health license No. PHF 174D/5185(08/2005). Rothamsted Research receives grant aid from the Biotechnology and Biological Sciences Research Council of the UK.