A survey was carried out on citrus nurseries in south and north of Iran as the major citrus growing regions. Different varieties with abnormal symptoms were examined for the occurrence of Candidatus Liberibacter asiaticus, Spiroplasma citri, Xylella fastidiosa and phytoplasma as most important citrus vascular limited pathogens. By visual inspection and molecular examination of 360 samples for the presence of fastidious prokaryotes only an occasional occurrence of a Candidatus phytoplasma and Spiroplasma citri were detected, using phytoplasma universal primer pairs P1/P7 and fU5/rU3 (in single tube nested PCR assay) and Spiroplasma specific primers P89-r/f and P58-6f/4r. The phytoplasma infected samples showed yellowing and deficiencies symptoms. PCR products of phytoplasmas were digested with MseI, AluI, BamHI and TaqI restriction enzymes and the digestion results showed phytoplasma is phylogenetically related and belong to aster yellows phytoplasma group. No amplicons was obtained for Candidatus Liberibacter asiaticus and Xylella fastidiosa. The finding suggest, symptoms of vascular limited disease when pathogens titre is low, are almost confused with disorder and deficiencies signs so for sanitary selection and certification program, using sensitive detection method is recomended. Multiplex PCR assay used in this study successfully detected Candidatus Liberibacter asiaticus, Spiroplasma citri and phytoplasma in one reaction and suggest for health certification programs.

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