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

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.
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.
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.
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.