عنوان مقاله [English]
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.