molecular characterization of aster yellows phytoplasma associated with citrus varieties, using multiplex pcr

a survey was carried out in citrus nurseries in mazandaran and kerman provinces as the major citrus growing regions of iran. different varieties of orange and tangerine with abnormal symptoms were examined for the occurrence of candidatus liberobacter asiaticus, spiroplasma citri and phytoplasma as the most important vascular limited pathogens in citrus. by visual inspection and molecular examination of 360 samples for the presence of fastidious prokaryotes only an occasional occurrence of 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. pcr products of phytoplasmas were digested with msei, alui, bamhi and taqi restriction enzymes and the digestion results showed phytoplasma related to aster yellows phytoplasma group. no amplicon was obtained for candidatus liberobacter asiaticus. the findings suggest when pathogen’s titer is low; symptoms of vascular limited diseases are almost confused with disorder and deficiency signs. so for sanitary selection and certification program, use of sensitive detection methods is recommended. multiplex pcr assay used in this study successfully detected candidatus liberibacter asiaticus, spiroplasma citri and phytoplasma in one reaction and is suggested for health certification programs.