Cloning, Genetic Diversity of Coat Protein Gene and Detection of Subgroups of Cucumber mosaic virus Strains from Cantaloupe in Varamin

Document Type : Research Article

Authors

• Majid Jafari ¹
• Somayeh Gholizadeh ²
• Shaheen Nourinejhad Zarghani ³

¹ Higher Educational Complex of Saravan

² College of Abouraihan, University of Tehran, Iran

³ College of Abouraihan, University of Tehran

10.22067/jpp.v33i2.72268

Abstract

Introduction: Varamin is one of the major producer regions of cantaloupes which is located in southern Tehran. Cucumber mosaic virus (CMV) is the type species of viral genus Cucumovirus in the family Bromoviridae and is causal agent of economically important losses in cucurbits. The CMV virions are isometric in shape and the genome is composed of three single-stranded positive sense RNAs. Each of the genomic RNAs 1 and 2 encodes a single large ORF and RNA-2 harbors an additional smaller ORF, 2b, involved in cell-to-cell movement and post-transcriptional gene silencing. The RNA-3 has two ORFs, 3a and 3b, encoding a movement protein and coat protein, respectively. Each of genomic RNAs has a Cap-like and tRNA-like structures at its 5ʹ and 3' ends, respectively. The virus is transmitted by different species of aphids in a non-persistent manner and is easily transmissible to the indicator hosts by mechanical inoculation. CMV is reported from several cucurbitaceae plants in Iran. Isolates of CMV are classified in different groups or subgroups based on different methods especially serology, RT-PCR-RFLP, nucleotide sequencing and phylogeny which are useful data for decision making or choosing the strategy during the development of CMV-resistant varieties and producing transgenic plants by RNA silencing strategy.
Materials and Methods: During 2014-2016, a total number of 132 leaf samples were collected from cantaloupes showing viral symptoms such as mosaic, mottling, leaf deformation and plant stunting. These samples were subjected to double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) with specific antibodies (DSMZ, Germany) to detect CMV based on the manufactures’ instructions. Total RNA was extracted from ELISA positive samples as explained elsewhere. Two microliters of total RNA used as template in RT-PCR to amplify a 870 base pair (bp) fragment of partial CMV genomic RNA3 including the coat protein gene and its flanking regions using specific primers CMVCPf (5'-GCTTCTCCGCGAG-3') and CMVCPr (5'-GCCGTAAGCTGGATGGAC-3') corresponded to 1149–1161 nts and 1998–2015 of CMV strain Q, respectively. In order to address the subgroups of CMV isolates, RT-PCR products were digested by MspI (HpaII) restriction enzyme and electrophoresis was done on 1.5% agarose gel. We expected the presence of the bands with 335 and 537 bp in size for the sub-group I and 393 and 559 bp for sub-group II isolates of CMV. Then, based on the geographical distance of collected CMV isolates, the CP gene from four isolates were ligated into pTG19-T, an A\T cloning vector, and the recombinant plasmids were introduced in Escherichia coli. The selected plasmids were extracted via alkaline lyses method and selected recombinant plasmids were sequenced by Macrogen, South Korea. The sequencing data were aligned with those of previously reported CMV isolates. The alignment and pairwise sequencing to estimate similarity between nucleotide sequences were done with BioEdit software. The Phylogeny Inference package (Phylip) version 3.65 was used to draw phylogeny trees.
Results and Discussion: 59 out of 132 samples (%44.69) positively reacted with CMV specific antibodies in DAS-ELISA. Expected DNA fragments of about 870 bp were successfully amplified via RT-PCR from the ELISA positive samples. in RT-PCR-RFLP for addressing the CMV subgroups, all MspI digested RT-PCR products from the tested samples produced two DNA bands of 537 and 335 bp and it was concluded that these isolates belong to the CMV subgroup I. Sequencing data of selected CMV CP clones showed 99-100% and 100% similarity between tested isolates at the nucleotide and amino acid levels, respectively. These isolates shared 93-100% and 97-100% similarity with previously reported CMV isolates from Iran at nucleic acid and amino acid levels, respectively. The reconstructed phylogenetic tree, re-confirmed the results of RT-PCR-RFLP, and Varamin isolates were placed with few other Iranian CMV isolates including E12, Cues, D13, F13, Sh44, Csu and Cud which were collected from non-melon hosts. Isolate LOR-F, the only CMV isolate previously reported from melons, belongs to the IB subgroup of CMV. This is the first report of CMV subgroup I isolates from melons. 
Conclusion: The RT-PCR-RFLP and sequencing data confirmed the presence of subgroup IA of CMV isolates in Varamin, Pishva and Pakdasht cantaloupe fields whose express severe symptoms in melons. However, the change in Zucchini yellow mosaic virus has been reported from these area, the same isolate of CMV were distributed in the entire region. Unlike Zucchini yellow mosaic virus populations which has been changed in the Varamin, Pakdasht and Pishva regions, it seems that there is just one dominant CMV isolate widespread in these regions.

Keywords

• Cucumovirus
• Melon
• phylogeny
• RT-PCR-RFLP
• Varamin

20.1001.1.20084749.1398.33.2.1.4

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References

1- Arafati N., Farzadfar S., and Pourrahim R. 2013. Characterization of coat protein gene of Cucumber mosaic virus isolates in Iran. Iranian Journal of Biotechnology 11: 109-114.

2- Berniak H., Malinowski T., and Kaminska M. 2009. Comparison of elisa and rt-pcr assays for detection and identification of Cucumber mosaic virus (CMV) isolates infecting horticultural crops in Poland. Journal of Fruit and Ornamental Plant Research 17: 5-20.

3- Clark M.F., and Adams A. 1977. Characteristics of the microplate method of Enzyme-linked immunosorbent assay for the detection of plant viruses. Journal of General Virology 34: 475-483.

4- Deyong Z., Willingmann P., Heinze C., Adam G., Pfunder M., Frey B., and Frey J.E. 2005. Differentiation of Cucumber mosaic virus isolates by hybridization to oligonucleotides in a microarray format. Journal of Virological Methods 123: 101-108.

5- Eyvazi A., Dizadji A., Rastgou M., and Habibi M.K. 2015. Bioassay and phylogeny of five Iranian isolates of Cucumber mosaic virus from different hosts based on CP gene sequence. Plant Protection Science 51: 200-207.

6- Farzadfar S., Pourrahim R., and Arafati N. 2013. Molecular identification of Cucumber mosaic virus subgroup IB isolates in south Iran. Journal of Plant Pathology 95: 423-428.

7- Gallitelli D. 2000. The ecology of Cucumber mosaic virus and sustainable agriculture. Virus Research 71: 9-21.

8- Haas A., and Richter J., and Rabenstein F. 1989. Monoclonal Antibodies for Detection and Serotyping of Cucumber mosaic virus. Journal of Phytopathology 127: 129-136.

9- Hosseinzadeh H., Nasrollanejad S., and Khateri H. 2011. First report of Cucumber mosaic virus subgroups I and II on soybean, pea, and eggplant in Iran. Acta Virologica 56: 145-148.

10- King A.M.Q., Adams M.J., Carstens E.B., and Lefkowitz E.J. 2012. Virus Taxonomy, Classification and nomenclature of viruses, Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press. 1327 p.

11- Liu J.C.K., Ng S., and Perry R.L. 2000. Cucumber mosaic virus Mutants with Altered Physical Properties and Defective in Aphid Vector Transmission. Virology 276: 395-403.

12- Mohammadi K., Hajizadeh M., and Koolivand D. 2016. Detection and identification of four vegetable fruit viruses in west and northwest of Iran. Iranian Journal of Plant Pathology 52: 279-288.

13- Nematollahi S., and Rakhshandehroo F. 2012. Phylogenetic analysis of new isolates of Cucumber mosaic virus from Iran on the basis of different genomic regions. The Plant Pathology Journal 28: 381-389.

14- Nitta N., Masuta C., Kuwata S., and Takanami Y. 1988. Comparative studies on the nucleotide sequence of Cucumber mosaic virus RNA3 between Y strain and Q strain. Annals of the Phytopathological Society of Japan 54: 516-522.

15- Palukaitis P., and Zaitlin M. 1997. Replicase-mediated resistance to plant virus disease. Advances in Virus Research 48: 349-378.

16- Palukaitis P., Roosinck M.J., Dietzgen R.G., and Francki R.I.B. 1992. Cucumber mosaic virus. Advances in Virus Research 41: 281-348.

17- Pares R.D., Gillings M.R., and Gunn L.V. 1998. Differentiation of Australian cucumber mosaic cucumovirus isolates: comparison of dsRNA type, polymerase chain reaction-restriction enzyme analysis, DNA hybridisation and serogrouping. Australasian Plant Pathology 27: 36-39.

18- Quiot J.B. 1981. Ecology of Cucumber mosaic virus in the Rhone Valley of France. III Conference on Epidemiology and Control of Virus Diseases of Vegetables, p. 9-22. Italy, Bari, 29 Agu. 1979.

19- Rasoulpour R., and Izadpanah K. 2008. Properties and taxonomic position of hoary cress strain of Cucumber mosaic virus. Journal of Plant Pathology 90: 97-102.

20- Rizos H., Gunn L.V., Pares R.D., and Gillings M.R. 1992. Differentiation of cucumber mosaic virus isolates using the polymerase chain reaction. Journal of General Virology 73: 2099-2103.

21- Roossinck M.J., Zhang L., and Hellwald K.H. 1999. Rearrangements in the 5′ nontranslated region and phylogenetic analyses of Cucumber mosaic virus RNA 3 indicate radial evolution of three subgroups. Journal of Virology 73: 6752-6758.

22- Sambrook J., and Russell D.W. 2001. Molecular cloning: a laboratory manual. 3rd ed., Cold Spring Harbor Laboratory Press, New York.

23- Schneider W.L., and Roossinck M.R. 2001. Genetic diversity in RNA virus quasispecies is controlled by host-virus interactions. Journal of Virology 75: 6566-6571.

24- Singh Z., Jones R.A., and Jones M.G.K. 1995. Identification Cucumber mosaic virus subgroup I isolates from banana plants affected by infectious chlorosis disease using RT-PCR. Plant Disease 79: 713-716.

25- Sokhandan Bashir N., Kalhor M.R., and Zarghani S.N. 2006. Detection, differentiation and phylogenetic analysis of Cucumber mosaic virus isolates from cucurbits in the northwest region of Iran. Virus Genes 32: 277-288.

26- Wilson A.D., and Halliwell R.S. 1985. Characterization and field studies of a Cucumber mosaic virus isolate from Spinach in the winter garden area of Texas. Plant Disease 69:751-754.

27- Yordanova A., Hristova D., Stoimenova E. 2002. Serological and electrophoretic characterization of the necrotic strain CMV-NB of Cucumber mosaic virus. Journal of Culture Collections 3: 84-91.