Exocortis is one of the most important citrus diseases which has spread to several citrus growing areas of the world. Typical symptoms of the disease are bark scaling and dwarfing of citrus trees budded on trifoliate orange rootstock and stunting of the entire citrus trees. Exocortis-like symptoms have been observed in several citrus orchards in Mazandaran province. The possible viroid etiology of the disease has been demonstrated through graft and mechanical transmission of the agent into Etrog citron, Lycopersicon esculentum (cv. Rutgers) and Gynura sarmentosa as well as observed viroid-like RNA bands following gel electrophoresis. However, the identity of the viroids occurring in citrus trees in Iran has remained undetermined. Therefore, molecular characterization and identification of exocortis disease agents in the north of Iran would help in better understanding the disease. In response to presence of Citrus tristeza virus (CTV) in Mazandaran, there is an ongoing trend toward the use of Poncirus trifoliata and its hybrids as rootstock in the north of Iran. On the other hand, these rootstocks are sensitive to exocortis disease. Thus, development of rapid and sensitive methods for detecting the exocortis and similar viroid in citrus trees would be very useful for implementation of control measures. In 2002, bark chips from Thompson navel and Sanguinella sweet orange trees grafted on P. trifoliata rootstocks with exocortis symptoms were budded on Etrog citron (Arizona 861-S1) seedlings and maintained in the greenhouse at 25-30°C. After 6 months, the inoculated citron plants showed variable (Mild to severe) symptoms. Ribonucleic acids were extracted from young symptomatic leaves and subjected to Reverse transcription - polymerase chain reaction using specific primer pairs for Citrus exocortis viroid (CEVd). Amplified products were analysed through electrophoresis on 2% agarose gels. Although some samples didn’t show any relevant DNA band, most did. The CEVd DNA bands were purified using High Pure PCR Product Purification Kit (Roch, Germany) and subjected to Single-strand conformation polymorphism (SSCP) analysis. Five samples from each of the 5 different isolates which had different SSCP pattern were cloned in pBluescript SK. At least one clone of each of the five CEVd isolates were sequenced in both directions. Alignment of the nucleotide sequences by BLASTn (NCBI) program revealed marked identity with other CEVd isolates deposited in GenBank. A further study was undertaken to recognize the nature of the variation observed for exocortis-like symptoms, amid the fact that CEVd was not found in some of the samples. RT-PCR was performed using specific primer pairs for Citrus bent leaf viroid, Citrus viroid OS, Citrus viroid I-LSS, Hop stunt viroid, Citrus viroid III and Citrus viroid IV. PCR products were electrophoresed on 2%
