Detection of Zucchini yellow mosaic virus from Cucurbits of Guilan Province and Urmia County

Document Type : Research Article

Authors

• Mohammad Ahmadzadeh ¹
• Ahmad Rouhibakhsh ¹
• mina rastgou ²

¹ Guilan University

² Urmia University

10.22067/jpp.v32i2.67619

Abstract

Introduction: Cucurbit plays an important role in human nutrition because of an ingredient of vitamin and other dietary substances. Cucurbit crops are very sensitive to viral infection. Over 35 viruses from Cucurbitaceae family have been reported. Plant viral diseases are major limiting factors for successful cultivation causing major yield losses in agricultural crops worldwide. Zucchini yellow mosaic virus (ZYMV) is a member of the genus Potyvirus, in the family Potyviridae has flexuous filamentous particles of about 750 nm in length and consists of a single-stranded RNA about 9.5kb. It was first reported in Italy in 1981. ZYMV is transmitted by certain species of aphids, by plant sap containing the virus and through infected seeds. ZYMV is one of the major pathogens of the family Cucurbitaceae. The common symptoms of ZYMV infection are local lesions, chlorotic mosaic, vein banding, yellowing, and stunting. Thrips-transmitted tospoviruses are economically important viruses affecting a wide range of field and horticultural crops worldwide. Tomato spotted wilt virus (TSWV) is the type member of the Orthospovirus genus in the family Tospoviridae with a broad host range of more than 900 plant species. TSWV has tripartite, ambisense, and single-stranded (ss) RNA genome transmitted by thrips in propagative and persistent manner and is ranked second among the top ten economically important viruses in the world. The aim of this research was to serological and molecular detection of ZYMV and TSWV on cucurbits of Guilan province and Urmia county and concerning their phylogenetic relationship with other isolates of the world.
Materials and Methods: In order to detect ZYMV and TSWV during the growing season of 2014, a total of 457 leaf samples of squash, watermelon, cucumber, melon and cantaloupe showing virus symptoms were collected from different fields of Guilan province and Urmia vicinity. DAS-ELISA was carried out on all samples using specific polyclonal antisera and samples with values at least twice the value of the negative control value according to the absorbance at 405 nm was accepted as positive. The samples which showed positive result in ELISA test were inoculated to Chenopodium amaranticolor Coste et Reyn, C. quinoa Wild., Cucumis melo L., Cucurbita pepo L., Lycopersicon esculentum L., Nicotiana rustica, N. tabacum var. Samsun and Gomphrena globosa L. Total RNA were extracted from positive samples in ELISA (for ZYMV) using RNXTM (-Plus) kit and RT-PCR was performed using ZYMV and TSWV specific primers corresponding to the CP and L-gene of these viruses, respectively. PCR positive products were sent for sequencing from both sides. Sequences were compared to data available in GenBank and multiple alignments of the nucleotide sequences and a phylogenetic tree were constructed by MEGA6 using the neighbor-joining method with a 1000 replicate bootstrap search.
Results and Discussion: Among 457 symptomatic samples collected from cucurbit fields of Guilan province and Urmia vicinity showing viral symptoms, 39 squash samples were infected by ZYMV but none of them by TSWV in ELISA test. Almost all inoculated plants showed symptoms of infection by ZYMV described in literature them. But this was different symptoms. An amplicon of the expected size (458 bp) was generated from ELISA positive samples during RT-PCR using specific primers but not from the healthy sample and no positive result for TSWV. Two PCR-positive samples of Guilan province and one Urmia isolate were sequenced and submitted to BLASTn. The results revealed that the PCR-amplified fragments of all samples belonged to ZYMV. The Kiashahr and Astaneh isolates showed maximum similarity (98%) with Czech Republic and Iran (Fars) isolates, and Urmia isolates showed the highest homology (99%) with Slovakia isolate in group A.
Conclusions: Viruses are known to greatly reduce cucurbitaceous yield. ZYMV is one of the most destructive and widespread plant pathogens. In this study, for the first time, molecular detection of ZYMV in squash fields of Guilan province and Urmia vicinity has been reported concerning its phylogenetic relationship with other isolates of the virus available in the GenBank. Phylogenetic analyses based on CP gene indicated that the Iranian ZYMV isolates are closer to European isolates plus one Iranian isolate and clustered in group a clade. The data obtained in this study will be beneficial to improve control strategies for this virus in Iran and are the base for further work on ecology, epidemiology, and diversity of these viruses in these two regions. Further studies on ZYMV isolates from different geographic regions and hosts of these two regions and genetic diversity evaluation of the virus will be useful for breeders to make more efficient and durable resistant cultivars. TSWV was not detected in cucurbits of these two regions during this study. It has been reported only in Malaysia on cucurbits.

Keywords

• DAS-ELISA
• RT- PCR
• Sequencing
• TSWV
• ZYMV

20.1001.1.20084749.1397.32.2.5.1

---

References

1- Azarfar A., Izadpanah K., Afsharifar A., and Masumi M. 2012. Purification and the complete genome sequence of Zucchini yellow mosaic virus- Fars isolate. Iranian Journal of Plant Pathology, 48, 403-409. (In Farsi with English abstract).

2- Bananej K., Shahraeen N., and Ahoonmanesh A. 1996. Distinguishing and determining the characteristics of Tomato spotted wilt virus in Varamin farms. Iranian Journal of Plant Pathology, 32 (1-2): 42-45.

3- Brittlebank C.C. 1919. Tomato diseases. Journal of Agriculture, Victoria, 27:231-235.

4- Briese T., Alkhovskiy S., Beer M.H., Calisher C., Charrel R., Ebihara H., Jain R., Kuhn J., Lambert A., Maes P., Nunes M., Plyusnin A., Schmaljohn C.B. Tesh R., Yeh S., Elbeaino T., Digiaro M., Martelli G., Muehlbach H., and Junglen S. 2016. Create a new order, Bunyavirales, to accommodate nine families (eight new, one renamed) comprising thirteen genera.

5- Clark M. F., and Adams S. A. N. 1997. Characteristics of microplates method of enzyme-linked-immunosorbent assay for detection of plant viruses. Journal of General Virology, 34: 475-483.

6- Desbiez C., and Lecoq H. 1997. Zucchini yellow mosaic virus. Plant Pathology, 46: 809-829.

7- Desbiez C., Wipf-Scheibel C., and Lecoq H. 2002. Biological and serological variability, evolution and molecular epidemiology of Zucchini yellow mosaic virus with special reference to Caribbean Islands. Virus Research, 85: 5-16.

8- Gholamalizadeh R., Vahdat A., Keshavarz T., Elahinia S. A., Shahraeen N., and Bananej K. 2008. Occurrence and distribution of ten viruses infecting cucurbit plants in Guilan province, Iran. Acta Virologica, 52:113-118.

9- Gholizadeh-Roshanagh S., Nourinejhad Zarghani Sh, Aminian H., Jafari M., and Ramshini H. 2017. Imported infected cucurbit seeds cause of establishment and distribution of central Europe isolates of Zucchini yellow mosaic virus in Varamin. Genetic Engineering and Biosafety Journal, 5 (2):91-99.

10- Ghorbani S. 1988. Isolation of Zucchini yellow mosaic virus in the Tehran province. Iran Journal of Plant pathology, 24:25-34.

11- Lecoq H., and Desbiez C. 2012. Viruses of cucurbit crops in the Mediterranean region: an ever-changing picture. Advances in Virus Research, 84: 67-126.

12- Lecoq H., Wipf-Scheibel C., Chandeysson C., Le Van A., Fabre F., and Desbiez C. 2009. Molecular epidemiology of Zucchini yellow mosaic virus in France: an historical overview. Virus Research, 141:190–200.

13- Massumi H., Shaabanian M., Heydarnejad J., Hosseinipour A., and H. Rahimian H. 2011. Host range and phylogenetic analysis of Iranian isolates of Zucchini yellow mosaic virus. Journal of Plant Pathology, 93: 187-193.

14- Nascimento L. C., Pensuk V., Costa N. P., Deom C. M., and Sherwood J. 2006. Evalution of peanut genotypes for resistance to Tomato spotted wilt virus by mechanical and thrips inoculation. Pesquisa Agropecuaria Brasileira, 41: 937-942.

15- Rastgou M., Koohi Habibi M., Izadpanah K., and Mosahebi Gh. 2010. Frequency of cucurbit viruses around Urmia and first report of Zucchini yellow fleck virus. Proceeding of 19th Iranian Plant Protection Congress .31 July-3 August. p.755.

16- Shaabanian M., Massumi H., Hosseinipour A., Heydarnejad J., and Poramini N. 2005. Detection and distribution of Zucchini yellow mosaic virus based on Immunocapture RT-PCR in some parts of Iran, The Fourth National Biotechnology Conference Islamic Republic of Iran, Kerman. P. 2098.

17- 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.

18- Zitter T. A., Hopkins D. L., and Thomas C. E. 1996. Compendium of cucurbit diseases. APS press. 87pp.