Cucumber mosaic virus identification in pumpkin plants

Cucumber mosaic virus identification in pumpkin plants

2011

E04967

Cucumber mosaic virus (CMV) causing viral diseases of many important plants worldwide has been isolated from pumpkin (Cucurbita pepo L.) plant leaves collected in Ukraine. Diseased pumpkin plant samples were collected in Ternopol region (Zalischyky district) and Chernivtsi region (Khotyn district) in 2009. Diseased plants had light green mottled foliage. Leaves were smaller than normal, yellow mottled and crinkled. The diagnostic study of the pathogen was done in 2009-2010 at the Plant Virus Laboratory of the Institute of Botany, Nature Research Centre. The determination of causal agent has been based on host range, symptom expression in the test plant species and morphological properties of the virus particles using transmission electron microscopy (EM), and using specific oligonucleotide primers in reverse transcription-polymerase chain reaction (RT-PCR). An experimental host range and induced symptoms for causal agent were determined by mechanically inoculating the group of herbaceous hosts representing seven botanical families (Fabaceae Lindl., Solanaceae Juss., Cucurbitaceae Juss., Aizoaceae Rudolphi, Chenopodiaceae Vent., Amaranthaceae Juss., Asteraceae Dumort.). The virus infected a wide experimental host range and developed specific symptoms for CMV. EM investigation revealed presence of isometric virions with hollow centre about 28-30 nm in diameter characteristic of CMV. Total nucleic acids from virus infected plants were isolated and purified by TRIzol Reagent method. Primers to CMV1 and CMV2 complementary to conserved region of CMV RNA sequences were designed to produce amplicons of different lengths of CMV isolates belonging to subgroups I or II (Singh et al., 1995). The RT-PCR was directly performed in crude sap extracts of CMV infected pumpkin and test plants. All PCR procedures were carried out in "T-Gradient Thermocycler" ("Biometra", Germany). Cycling parameters for DNA amplification were used as developed by Parrella and Sorrentino (2009). The RT-PCR assay of infected pumpkin plants and test plants yielded a band of 499 to 502 base pairs specific to CMV subgroup II and confirmed that virus isolates from pumpkin samples collected in Ternopil and Chernivtsi regions of Ukraine belong to subgroup II of CMV.

98(4):421-426, ZEMDIRBYSTE (AGRICULTURE)

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