عنوان مقاله [English]
Introduction: Citrus tristeza virus (CTV), the causal agent of the most important viral disease of citrus, is transmitted by infected reproductive materials and several aphid species. Tristeza has been reported from the north and south of Iran and Aphis gossypii has been known as the vector of Tristeza. Detection of the virus in the vectors has been before carried out based on biology, serology and electron microscopy. Access to rapid and sensitive molecular techniques for epidemiological studies is the aim of the study. Reverse transcription polymerase chain (RT-PCR) and RT-nested PCR techniques were applied in the present study.
Materials and Methods: The SD4-infected Mexican lime (Citrus aurantifolia) seedlings acquired from the virus collection with origin of declining trees of Sari region were used as donor host for virus transmission assays. Pure colony of A. gossypii after rearing on cucumber (Cucumis sativus) was placed on the infected seedlings. 20 seedlings were considered for biological indexing. 20 aphids were placed carefully on healthy Mexican lime seedlings by brush. The acquisition and transmission feeding time of virus were 48 hours and 20 aphids were considered for each test plant. The seedlings were transferred to the conditioned greenhouse (23/16°C, day/night). Indirect virus detection was carried out by monitoring of symptoms, direct tissue blot immuonoassay with Biorba antiserum and two step RT-PCR with RNA extracted from indicator plants using SDS-Potassium acetate method and T36CPF/T36CPR primers. In direct detection virus of aphid, RNA extracted by Trizol method and one step RT-PCR reaction using AMV Reverse Transcriptase enzyme by T36CPF/T36CPR primers were used. The final product of this reaction was introduced in the nested-PCR using T36CPF/P25R primers. RT-nested-PCR assay was applied also with PexF/PexR primers in the first stage and PinF/PinR in the second stage.
Results and Discussion: Indirect virus detection in melon aphid was carried out by survey of vein clearing and vein corking symptoms in the receptor indicator plants. A total of 4 of 20 Mexican lime seedlings showed the symptoms during 3 months after aphid transmission assay. The presence of virus in these seedlings was confirmed by direct tissue blot immunoassay and RT-PCR techniques. Also a product of about 672 bp was amplified using specific primers of coat protein gene. Direct virus detection with one-step RT-PCR and RNA extracted from aphids by Trizol method using T36CPF/T36CPR primers resulted 672 bp weak bands. By performing the second stage of PCR using T36CPF/P25R primers, 362 bp specific bands were obtained. On the other hand, RT-nested PCR with PinF/PinR and PexF/PexR primers was not able to detect the virus in the infected aphids due to false positive reactions. The purpose of this study was to evaluate the effectiveness of different methods in selecting an appropriate method for detection of virus in melon aphid, the most effective vector of CTV in Iran. The detection of virus-associated targets in vectors that are capable of transmitting viruses is crucial for both the studies of viral replication and the optimization of control strategies. Biological indexing is the earliest test of detection of virus in aphids, which has the advantages and disadvantages, including the objective measurement of biological activities such as ability to transmit and reproduce based on production of symptoms, the long time required, and the specific greenhouse conditions. Although RT-PCR method has been used to detect a number of plant viruses in vectors, the detection of virus by this method is possible if extraction efficiency of RNA is increased by use of materials such as Gene Releaser and Trizol. Nevertheless, more sensitive methods are required for detection of semi-persistently and non-persistenly transmitted viruses. Detection of CTV by nested RT-PCR was directly related to appropriate primers and efficient extraction procedure.
Conclusion: According to this study, RNA extraction using Trizol method is the most appropriate method for extracting nucleic acid from aphids, so it has been shown that detection of CTV by nested RT-PCR technique was directly related to the used extraction procedure. In this research, detection of Citrus tristeza virus in melon aphid for the first time in Iran was performed using RT-PCR and RT-nested-PCR molecular methods. The results showed that RT-nested-PCR based on the efficient extraction method and the selection of appropriate primers is a reliable technical for detection Tristeza virus in its aphid vector, A. gossypii.