Identification and Prevalence of Potato VirusM and PotatoVirus Sin Some Provinces of Iran

Introduction: Potato virus M (PVM) and Potato virus S (PVS) are members of the genus Carlavirus in the family Betaflexiviridae. They have worldwide distribution and potato is a major host of the viruses. They have flexuous filamentous virions with a linear, single-stranded, positive-sense RNA genome of about 8.5 kb. The RNA genome contains a 5´- cap structure, six open reading frames (ORFs), and a poly-A tail at the 3´-terminus. Potato virus M was first isolated from Solanum tuberosum in 1923 in the United States by Schulz et al. It has since been found in all potato production areas worldwide, alone or in mixed infection with PVS. Despite evolutionary adaptation to broad geographical niches, PVM and PVS have a narrow host range limited to solanaceous species. In naturally infected plants, PVM causes mottling, mosaic, crinkling, leaflet deformation and stunting of shoots. The symptoms of PVM on potato vary from latency to severe and are similar to those caused by some other potato-infecting viruses, including potato virus S (PVS, Carlavirus), potato virus X (PVX, Potexvirus) and the common strain of potato virus Y (PVY-O, Potyvirus). PVS has a severe symptom of mosaic and yellowing on potato plants. Most carlaviruses, including PVM and PVS, are naturally transmitted by aphids in a non-persistent manner. PVM and PVS are also transmissible experimentally by mechanical inoculation, however, the common route for the spread of PVM and PVS within the crop is through activities such as seed cutting operations and machinery movements. All of the potato plants grown in a field may become infected, depending on the potato cultivar and environmental conditions. Materials and Methods: In the present study the prevalence of PVM and PVS were surveyed in potato fields from different fields of major potato producing area in Hamedan, Northern Khorasan, Khorasanrazavi, Esfahan, Kerman provinces of Iran. Specific antibody against PVM and PVS (DSMZ, Germany) were used to detect the viruses in fresh leaf crude extracts by DAS-ELISA. Antigen samples were prepared by grinding 500 milligrams of leaf tissue in 200 mMTris-HCl, pH 8.2, 140 mMNaCl, 2 % polyvinylpyrrolidone 40, and 0.05 % Tween 20. Leaf samples from healthy potatoes were also included as negative controls. DAS-ELISA was performed as described by Clark and Adams, according to the instructions of the antibody supplier. The rate of substrate hydrolysis was measured at 405 nm using a microplate reader. A number of samples were also tested by mechanical inoculation on test plants Solanum tuberosum, Lycopersicum escolentum and Nicotiana glutinosa by using 0.1M sodium phosphate buffer. The presence of infection was confirmed about two weeks later by the symptoms and DAS-ELISA results. Total RNA was extracted from fresh young leaf tissue by Accuzol, total RNA extraction kit (Bioneer). Polymerase Chain Reaction (PCR) was carried out using specific primers, corresponding to a complete coat protein gene of PVM and PVS. Amplified fragments were ligated in pTZ57R/T plasmid and cloned in DH5α isolate of Escherichia coli using InsT/A clone PCR product cloning kit (Fermentas) as manufacturer’s protocol. Recombinant plasmids extracted using Prime prep Plasmid DNA isolation Kit (Genetbio) then sequenced bidirectional using pUC-M13 universal primers with an ABI PRISM system (Bioneer company, Korea). Consensus sequences were compared with those deposited in the GenBank using BLAST program from the National Center for Biotechnology Information (NCBI). Results and Discussion: Results showed that 10.5% and17.1% of the total 970 samples were infected with the PVM and PVS, respectively. The highest abundance of infection was among Kerman and then Esfahan samples. PVS prevalence as greater than PVM; however, most of the samples had mixed infection. The PVM-inoculated test plants developed mottling and leaf deformation symptoms, whereas mosaic, leaf necrosis and leaf malformation were the characteristic symptoms induced by PVS. Mechanical inoculation of PVS on Lycopersicum escolentum resulted no symptoms but in PVM produced mosaic and yellowing. However, a number of symptomless samples were found to be infected by the viruses. The symptoms on test plants and the results of their ELISA confirmed the previous ELISA results. RT-PCR was amplified a 915 and 1118 bp DNA fragment from the ELISA positive samples of PVM and PVS, respectively. However rarely amplification was carried out in ELISA negative samples, indicated DAS-ELISA has low sensitivity to identify PVM infection in the samples. Blast search showed that 915 and 1118 bp fragments amplified by RT-PCR were corresponding to full length sequence of coat protein gene of PVM and PVS, respectively. The coat protein gene of PVM and PVS isolates analyzed in this study had 73.3-100 and 70-96 percent nucleotide identity with the corresponding sequences in GenBank, respectively