Molecular Identification of Potato virus Y (PVY) Strain C isolated from Tomato in Mazandaran Province

Document Type : Research Article


1 دانشگاه فردوسی مشهد

2 Ferdowsi University of Mashhad

3 دانشگاه مازندران


Introduction: Potato virus Y (PVY) is the type member of the genus Potyvirus, which is responsible for serious diseases in a wide range of plant species, mostly from the family Solanaceaesuch as potato, tomato, tobacco and pepper worldwide. The virus is transmitted by at least 40 aphid species (family Aphididae) in a nonpersistent manner. The level of damage to crop is determined by the strain of PVY infecting the plants, the viral load, the time at which infection occurs as well as the tolerance the host possesses toward the virus. Infection of a potato field with PVY may ultimately result in 10-100% loss in yield. Several distinct strains of PVY have been identified according to their biological and serological properties and their genome sequences. These include the ordinary strain (PVYO), the tobacco veinal necrosis strain (PVYN), the stipple streak strain (PVYC). In addition, two most prominent recombinant strains, PVYNTN and PVYN-Wi (PVYN:O) have been generated by recombination between PVYO and PVYN. Like other potyviruses, PVY has non-enveloped flexuous filamentous virions of 730-740 nm long and 11–12 nm wide, which encapsidate a single-stranded, positive-sense RNA molecule of approximately 10,000 nt long. PVY causes severe crop losses and consequently economic damage in the world as well as in Iran. Hence, identification of the virus in different regions of the country is a concern. The aim of this research was to study the existence of PVY in tomato fields of Mazandaran province and determining the type of the strain and finally finding their phylogenetic relationship. Polymerase chain reaction (PCR) with degenerate primers for conserved sequences of the viral genomes has facilitated the rapid detection of many potyviruses and enabled partial genomic sequencing. According to previous studies comparisons using the CI-coding region most accurately reflected those for the complete genome, and this region was deemed to be the most suitable for diagnostic and taxonomy purposes if the complete sequence could not be obtained.
Materials and Methods: From July to September 2013, a total of 38 leaf samples of tomato showing virus symptoms were collected from different tomato fields in Mazandaran province. Total RNA was extracted from all samples. RT-PCR assay was performed using potyvirus degenerate primers corresponding to the virus CI gene. Expected PCR products were purified from 1% agarose gels, cloned into the pTZ57R/T vector and then sequenced. Sequences were compared to data available in GenBank. Multiple alignments of the nucleotide (nt) and amino acid (aa) sequences were performed with Clustal W implemented in MEGA6 or in BioEdit v.7.2.5. Phylogenetic tree for grouping was constructed by MEGA6 using neighbor-joining method.
Results and Discussion: An amplicon of the expected size (680 bp) was generated from 9 out of 38 plant samples. Specific amplification using the potyvirus degenerate primers in infected samples, but not from healthy sample, confirmed the presence of a potyvirus. All PCR-positive samples were cloned, sequenced and submitted to BLASTn to identify the best matching sequences recorded in GenBank. BLASTn analysis showed that the PCR-amplified fragments of four samples belonged to Potato virus Y strainC. The most typical symptoms in PVY-positive leaf samples were mosaic, mottling, distortion and rugosity. Among them two isolates namely GB and GRA were selected for further analyses. Phylogenetic tree based on multiple sequence alignment of 680 nt of CI gene divided 117 PVY isolates into three main groups: I, II and III. Group I included a wide range of isolates from Europe, Asia, Australia, America and Africa. Members of group I were divided into six subgroups. Iranian isolates (GB and GRA) were classified in the group IF with isolates nnp (Italy), Foggia (Italy), CN1 (Australia), PRI-509 (Netherland), LYE84.2 (Spain) and Tannat (Uruguay), which all were belonging to strain C. Isolate of GB showed the highest (96.5%) nt sequence identity with isolate LYE84.2 and the lowest (81.3%) with isolates T13 (Japan), Tu_660 (Canada), ME162 (China) and ID20 (USA). Also, GRA displayed the highest (95.7%) nt sequence identity with LYE84.2 and the lowest (80.9%) withT13 and Tu_660 isolates. The two Iranian isolates had the highest (99.1%) aa sequence identity with isolates LYE84.2, Foggia and Tannat and the lowest (93.4%) with T13. The identities between isolates GB and GRA were 98.4% and 100% at nt and aa levels, respectively.
Conclusions: PVY is one of the most destructive and widespread plant pathogen. In this study, for the first time we reported the occurrence of PVY in tomato fields in Mazandaran province and determining its phylogenetic relationship with other isolates of the virus available in the GenBank. Also, to our knowledge this is the first report of PVY strain C in tomato in Iran. Because the use of CI is more accurate in defining orders in potyvirus taxonomy and in evolutionary relationships, detection of the virus was performed by RT-PCR using potyvirus degenerate primers based on CI coding region. Phylogenetic analyses based on CI gene indicates that the Iranian PVYC isolates are much close to European isolates plus one Australian (CN1) and one Uruguayan (Tannat) isolates. One possible explanation for such sequence similarities is that most of seed potatoes in Iran originated from seed tubers imported from Europe, and not directly from its original habitat, South America. So, it is possible that the Iranian C strain originated potentially from an ancestral European PVY strains and then spread in the country via aphid vectors from infected to healthy plants. Resistance to PVY infection by hosts is low in many cases. On the other hand, PVV is transmitted by aphids in a non-persistent manner, which makes the control of the virus vectors inefficient. Therefore, cultivation of resistant varieties is the most suitable approach for control of the virus. The data obtained in this study will be beneficial to improve control strategies for this virus in Iran. Further studies on PVY isolates from different geographic regions and hosts of Iran and genetic diversity evaluation of the virus will be useful for breeders to make more efficient and durable resistant cultivars.


1- Adams M.J., Antoniw J.F., and Fauquet C.M. 2005. Molecular criteria for genus and species discrimination within the family Potyviridae. Archives of Virology, 150:459–479.
2- Adams M.J., Zerbini F.M., French R., Rabenstein F., Stenger D.C., and Valkonen J.P.T. 2012. Family Potyviridae. p. 1069˗1089. In: King A.M.Q., Adams M.J., Carstens E.B., Lefkowitz E.J. (eds) Virus taxonomy: ninth report of the International Committee on Taxonomy of Viruses. Elsevier, Academic Press, San Diego, CA, USA.
3- Barker H., and Dale M.F.B. 2006. Natural resistance mechanisms of plants to viruses. Springer, Resistance to viruses in potato, p. 341–366.
4- Blanco-Urgoiti B., Tribodet M., Leclere S., Ponz F., Perez de San Roman C., Legorburu F.J., and Kerlan, C. 1998. Characterization of potato potyvirus y isolates from seed potato batches. Situation of the NTN, Wilga and Z isolates. European Journal of Plant Pathology, 104:811-819.
5- Boonham N., Walsh K., Preston S., North J., Smith P., and Barker I. 2002. The detection of tuber necrotic isolates of Potato virus Y, and the accurate discrimination of PVYO, PVYN and PVYC strains using RT-PCR. Journal of Virological Methods, 102:103-112.
6- Cuevas J.M., Delaunay A., Visser J.C., Bellstedt D.U., Jacquot E., et al. 2012. Phylogeography and molecular evolution of Potato virus Y. PLoS ONE, 7(5): e37853.
7- De Bokx J.A., and Huttinga H. 1981. Potato virus Y: description of plant viruses. Kew, England: Common wealth Mycology Institute. Association of Applied Biology.
8- Edwardson J.R., and Christie R.G. 1997. Potyviruses. In: Florida Agricultural Experiment Station Monograph Series 18-II-Viruses infecting pepper and other solanaceous crops. Gainesville (FL): University of Florida. p. 424-524.
9- FAO. 2014. FAOSTAT-Agriculture, Production Statistics, Food and Agriculture Organization of the United Nations Statistical Databases.
10- Ghasemzadeh A., Sokhandanbashir N., and Khakvar R. 2012. Molecular detection of Potato virus Y using universal primers from Ardabil province. Journal of sustainable agriculture and production science, 22:67-80. (In Persian with English abstract)
11- Gholami S., Koohihabibi M., Bushehri A.A., and Naghavi M.R. 2007. Detection of Potato virus Y by reverse transcription polymerase chain reaction (RT-PCR). Iranian journal of agricultural sciences, 38:399-405. (In Persian)
12- Ha C., Coombsi S., Revill P.A., Harding R.M., Vu M., and Dale J.L. 2008. Design and application of two novel degenerate primer pairs for the detection and complete genomic characterization of potyviruses. Archives of Virology, 153:25-36.
13- Hameed A., Iqbal Z., Asad S., and Mansoor S. 2014. Detection of multiple potato viruses in the field suggests synergistic interactions among potato viruses in Pakistan. Plant Pathology Journal, 30(4):407–415.
14- Hosseini A., Massumi H., Heydarnejad J., Hossein pour A., and Varsani A. 2011. Characterisation of Potato virus Y isolates from Iran. Virus Genes, 42:128-140.
15- Jones R.A.C. 1990. Strain group specific and virus specific hypersensitive reactions to infection with potyviruses in potato cultivars. Annals of Applied Biology, 117:93–105.
16- Kerlan C., and Moury B. 2008. Potato virus Y. p. 287-296. In: Mahy BWJ, Van Regenmortel MHV, editors. Encyclopedia of Virology. Third Edition vol. 4. Elsevier; Oxford.
17- Lacroix C., Glais L., Kerlan C., Verrier J.L., and Jacquot E. 2010. Biological characterization of French Potato virus Y (PVY) isolates collected from PVY-susceptible or -resistant tobacco plants possessing the recessive resistance gene va. Plant Pathology, 59 (6):1133-1143.
18- Lee K.C., Wong S.M., Mahtani P.H., and Chng C.G. 1997. Sequence and phylogenetic analysis of the cytoplasmic inclusion protein gene of Zucchini yellow mosaic potyvirus: its role in classification of the Potyviridae. Virus Genes, 14:41–53.
19- Lorenzen, J. H., Meacham, T., Berger, P. H., Shiel, P. J., Crosslin, J. M., Hamm, P. B., and Kopp, H. 2006. Whole genome char‌acterization of Potato virus Y isolates collected in the west‌ern USA and their comparison to isolates from Europe and Canada. Archives of Virology, 151:1055-1074.
20- Majdabadi Farahani S., Jafarpour B., Falahati Rastegar M., and Sabokkhiz M. 2011. Detection of (PVYNTN) strain in potato fields of Khorasan Razavi province. Journal of plant protection, 24 (4): 385-390. (In Persian with English abstract)
21- Mostafae S., Mosahebi G., Koohihabibi M., and Ansari Dezfouli E. 2008. Study of biological and molecular characterization of pepper-PVY isolates. Iranian Journal of Virology, 1(4): 31-34.
22- Nie X., and Singh R.P. 2003. Specific differentiation of recombinant PVYN:O and PVYNTN isolates by multiplex RT-PCR. Journal of Virological Methods, 113:69-77.
23- Ogawa T., Nakagawa A., Hataya T., and Ohshima K. 2012. The genetic structure of populations of Potato virus Y in Japan; Based on the analysis of 2120 full genomic sequences. Journal of Phytopatholy, 160:661-673.
24- Pourrahim R., Farzadfar Sh., Golnaraghi A. R., and Ahoonmanesh A. 2007. Incidence and distribution of important viral patho‌gens in some Iranian potato fields. Plant Disease, 91:609-615.
25- Pourrahim R., and Farzadfar S. 2016. Population analysis of Iranian Potato virus Y isolates using complete genome sequence. The Plant Pathology Journal, 32(1):33-46.
26- Przybys M., Doroszewska T., and Berbec A. 2013. Point mutations in the viral genome-linked protein (VPg) of Potato virus Y probably correspond with ability to overcome resistance of tobacco. Journal of Food, Agriculture & Environment, 11(3&4):986-989.
27- Sadeghi M.S., Bejatnia S.A.A., Masumi M., and Izadpanah K. 2008. Characterization of a strain of Potato virus Y causing eggplant mosaic in southern Iran. Australasian Plant Pathology, 37(1):79-86.
28- Scholthof K.B., Adkins S., Czosnek H., Palukaitis P., Jacquot E., Hohn T., Hohn B., Saunders K., Candresse T., Ahlquist P., Hemenway C., and Foster G. 2011. Top 10 plant viruses in molecular plant pathology. Molecular Plant Pathology, 12:938–954.
29- Schubert J., Fomitcheva V., and Sztangret-Wi´sniewska J. 2007. Differentiation of Potato virus Y strains using improved sets of diagnostic PCR-primers. Journal of Virological Methods, 140:66 –74.
30- Shukla D.D., Ward C.W., and Brunt A.A. 1994. The Potyviridae. CAB International, Walling ford, UK. p. 74-112.
31- Singh R.P., Valkonen J.P.T., Gray S.M., Boonham N., Jones R.A.C., Kerlan C., and Schubert J. 2008. Discussion paper: the naming of Potato virus Y strains infecting potato. Archives of Virology, 153:1–13.
32- Smith KM. 1931. Composite nature of certain potato viruses of the mosaic group. Nature, 127:702.
33- Tamura K., Stecher G., Peterson D., Filipski A., and Kumar S. 2013. MEGA6: Molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution, 30:2725-2729.
34- Warren M., Cruger K., and Schoeman A.S. 2005. Potato virus Y (PYV) and Potato leaf roll virus (PLRV). PhD. thesis. Faculty of Natural and Agriculture Sciences, University of Pretoria.
35- Yardımc N., Çulal Kılıç H., and Demir Y. 2015. Detection of PVY, PVX, PVS, PVA, and PLRV on different potato varieties in Turkey using DAS-ELISA. Journal of Agriculture Science and Technology, 17:757-764.
36- Younesi B., Shams Bakhsh M., Safaie N., and Khelghatibana F. 2011. Simultaneous detection of several important viruses in naturally infected potato plants using multiplex RT-PCR in comparing with ELISA. Modern Genetics Journal, 6 (1):17-26. (In Persian with English abstract)