Application of Molecular Methods for Detection of Citrus tristeza virus in Aphis gossypii

Document Type : Research Article


1 Guilan

2 Citrus and Subtropical Fruits Research Center Horticultural Science Research Institute Agricultural Research, Education and Extension Organization


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.


1- Ahmadi S., Afsharifar A., Niazi A., and Izadpanah K. 2008. Distribution and genetic diversity of Citrus tristeza virus isolates in Kerman province. Iranian Journal of Plant Pathololgy 43: 353-371. (In Persian with English abstract)
2- Alavi V., and Rezvani A. 2007. Seasonal fluctuations of citrus aphid in the East of Mazandaran and Citrus tristeza virus transmissibility by the major species. Plant Pests and Diseases 75: 49-53. (In Persian with English abstract)
3- Bahri B., Bani Hashemian S.M., Koolivand D., and Radmand O. 2013. Application of a simple nucleic acid extraction method for detection of citrus tristeza virus. In: 8th national biotechnology congress of Iran and 4th congress on biosafety and genetic engineering July 6-8, Tehran. (In Persian with English abstract)
4- Bahri B. 2014. Tolerance of Bakraii rootstock to Citrus tristeza Virus in greenhouse condition. M. Sc. thesis, Islamic Azad University, Malekan Branch. (In Persian with English abstract)
5- Bani Hashemian S.M., Gol Mohammadi M., Mohammad Alian Y., Golein B., and Moreno P. 2013. Decline of citrus trees in Iran. P. 39. In: 19th Conference of the International organization Of Citrus Virologist. 28th July–2nd Aug, South Africa.
6- Barzegar A., Rahimian H., and Hashemi H. 2010. Comparison of the minor coat protein gene sequences of aphid-transmissible and non-transmissible isolates of Citrus tristeza virus. Journal of General Plant Pathology 76:143-151.
7- Bertolini E., Moreno A., Capote N., Olmos A., Eduardo Vidal A., Perez-Panades J., and Cambra M. 2008. Quantitative detection of Citrus tristeza virus in plant tissues and single aphids by real-time RT-PCR. European Journal of Plant Pathololgy 120: 177-188.
8- Cambra M., Hermoso de Mendoza A., Moreno P., and Navarro L. 1981. Use of enzyme-linked inmunosorbent assay (ELISA) for detection of Citrus tristeza virus (CTV) in different aphid species. P. 444-448 .In: International Citrus Congress, November 9-12, Japan.
9- Cambra M., Gorris M.T., Roman M.P., Terrada E., Garnsey S.M., Camarasa E., and Colomer M. 2000. Routine detection of Citrus tristeza virus by direct Immunoprinting-ELISA method using specific monoclonal and recombinant antibodies. P. 34-41. In: Proceedings of the 14th Conference of the International Organization of Citrus Virologists, 13-18 September, Brazil.
10- Cambra M., Bertolini E., Olmos A., and Capote N. 2006. Molecular methods for detection and quantitation of virus in aphids. Virus Diseases and Crop Biosecurity 81-88.
11- Campolo O., Chiera E., Malacrinò A., Laudani F., Fontana A., Albanese G.R., and Palmeri V. 2014. Acquisition and transmission of selected CTV isolates by Aphis gossypii. Journal of Asia-Pacific Entomology 493-498.
12- Ghorbani S. 1983. Detection of Citrus tristeza virus in its aphid vector in northern Iran by Immunosorbent electron microscopy. P. 8. In: Proceedings of the 7th Iranian Plant Protection Congress. (In Persian with English abstract)
13- Hilf M.E., and Garnsey S.M. 2000. Characterization and classification of Citrus tristeza virus isolates by amplification of multiple molecular markers. P. 18-27. In: Proceedings of 14th Conference of the International Organization of Citrus Virologists.
14- Kim J.J. 2007. Influence of Lecanicillium attenuatum on the development and reproduction of the cotton aphid, Aphis gossypii. Biocontrol 52: 789-799.
15- Marroquin C., Olmos A., Gorris M.T., Bertolini E., Martinez M.C., Carbonell E.A., Hermoso deMendoza A., and Cambra M. 2004. Estimation of the number of aphids carrying Citrus tristeza virus that visit adult citrus trees. Virus Research 100: 101-108.
16- Mehta P., Brlansky R.H., Gowda S., and Yokomi R.K. 1997. Reverse-transcription polymerase chain reaction detection of Citrus tristeza virus in aphids. Plant Disease 8: 1066-1069.
17- Minasian V. 1983. Use of enzyme-linked immunosorbent assay (ELISA) for detection of Citrus tristeza virus (CTV) in aphids from the North of Iran. P. 82-83. In: Proceedings of the 7th Iranian Plant Protection Congress. (In Persian with English abstract)
18- Mohamed M.E., Bani Hashemian S.M., Dafalla G., Bove J.M., and Duran-Vila N. 2009. Occurrence and identification of citrus viroid from of Sudan. Journal of Plant Pathology 91: 185-190.
19- Moreno P., Ambros S., Albiach-Marti M.R., Guerri J., and Pena L. 2008. Citrus tristeza virus: a pathogen that changed the course of the citrus industry. Molecular Plant Pathology 9: 251-268.
20- Naidu R.A., Robinson D.J., and Kimmins F.M. 1998. Detection of each of the causal agents of groundnut rosette disease in plants and vector aphids by RT-PCR. Journal of Virological Methods 9-18.
21- Olmos A., Dasi M.A., Candresse T., and Cambra M. 1996. Print capture PCR: a simple and highly sensitive method for the detection of Plum pox virus (PPV) in plant tissues. Nucleic Acids Research 24: 2192-2193.
22- Olmos A., Cambra M., Esteban O., Gorris M.T., and Terrada E. 1999. New device and method for capture, reverse transcription and nested PCR in a single closed tube. Nucleic Acids Research 27: 1564-1565.
23- Pirone T.P., and Harris K.F. 1977. Nonpersistent transmission of plant viruses by aphids, Annual Review of Phytopathology 15: 55-73.
24- Rahimian H., Alavi V., Shaygan J., and Hadizadeh A. 2000. Spread of Citrus tristeza virus by Aphis gossypii in the north of Iran. Iranian Journal of Plant Pathology 36: 103. (In Persian with English abstract)
25- Roberts P.D. 1996. Survival of Xanthomonas fragariae on strawberry in summer nurseries in Florida detected by specific primers and nested polymerase chain reaction. Plant Disease 80: 1283-1288.
26- Saponari M., Manjunath K., and Yokomi R.K. 2008. Quantitative detection of Citrus tristeza virus in citrus and aphids by real-time reverse transcription-PCR. Journal of Virological 147: 43-53.
27- Satyanarayana T., Gowda S., Ayllon M.A., and Dawson W.O. 2004. Closterovirus bipolar virion: evidence for initiation of assembly by minor coat protein and its restriction to the genomic RNA 5′ region. P. 799-804. In: Proceeding of National Academy of Science, USA.
28- Shafiee V., and Izadpanah K. 1998. Distribution of Citrus tristeza virus in southern Iran. P. 256. In: 13th Iranian Plant Protection Congress. (In Persian with English abstract)
29- Singh R.P., Dilworth A.D., Singh M., and McLaren D.L. 2004. Evaluation of a simple membrane-based nucleic acid preparation protocol for RT-PCR detection of potato viruses from aphid and plant tissues, Journal of Virological Methods 121: 163-170.
30- Timmer L.W., Garnsey S.M., and Graham J.H. 2000. Compendium of Citrus Diseases. St Paul, MN: APS Press.
31- Yokomi R.K., and Garnsey S.M. 1987. Transmision of Citrus tristeza virus by A. gossypii and A. citricola in Florida. Phytophylactica 19: 169-172.
32- Yokomi R.K., and DeBorde R.L., 2005. Incidence, transmissibility, and genotype analysis of Citrus tristeza virus (CTV) isolates from CTV eradicative and noneradicative districts in central California. Plant Disease 89: 859-866.
33- Zafari H., Bani Hashemian S.M., Ruhibakhsh A., and Bahri B. 2014. Detection of Citrus tristeza virus in Guilan province. Iranian Journal of Plant Pathology 49: 463-464. (In Persian with English abstract)