Characterization of a novel asymptomatic isolate of Tomato spotted wilt virus, Infects chrysanthemum plants in Egypt

Ahmed Kamal El-Attar, Manal Ali EL-Shazly, Samah Abdel Salam Mokbel

Abstract
Background: A latent infection by Tomato spotted wilt virus (TSWV) in traded chrysanthemum plants at the local level constitutes a severe threat to the Egyptian farming.
Methods: The TSWV isolate in asymptomatic infected chrysanthemum plants was detected using double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). Host range studies were carried out by mechanical inoculation of biologically purified isolate on a set of different vegetable, ornamental and herbaceous plants. RT-PCR was used to amplify the full length nucleocapsid protein (N) gene of TSWV; the amplified 777 bp fragment was molecularly cloned and sequenced. The phylogenetic tree was constructed using the DNAMAN software.
Results: The obtained results demonstrated that, this isolate of TSWV causes asymptomatic infection for chrysanthemum plants in different locations in Giza governorate. The ELISA was less efficient than the RT-PCR for the diagnoses of the latent infection. RT-PCR accurately identified 26% more positive samples without symptoms than did the DAS-ELISA test. Moreover, RT-PCR was used to confirm TSWV infection related to the development of local lesions and systemic symptoms from mechanical inoculations. The nucleotide sequence of the TSWV-N gene has been deposited in the GenBank with accession number KY569403. Phylogenetic analysis showed that the fragment sequence shared variable levels of similarity and 93.82% max identity with that of N-gene sequences from Japan (AB038341), Pakistan (KX121046), South Korea (AB910533) and Turkey (KM407603).
Conclusion: The relatively low levels of sequence identity (91.7% to 93.8%) to N-gene demonstrated the release of a new isolate of TSWV in Egypt. Moreover, the study showed the susceptibility of onion and sweet potato to TSWV-isolate suggesting the chance of spread of viral infection on cultivated crops in the future. The information generated in this study will be useful for alleviating problems associated with non-specific symptoms and can be used to aid efficient management strategies directed against TSWV.

Key words: Chrysanthemums; Latent infection; Tospovirus; Tomato spotted wilt virus; Nucleocapsid protein; N-gene; DAS-ELISA; RT-PCR; Cloning.

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