Tomato Spotted Wilt Virus: TSWV in Vegetable Crops: Tospoviruses

Tospoviruses In Solanaceae and Other Crops in The Coastal Plain of Georgia

Tospovirus Detection

Stephen Mullis, Claudia Nischwitz

Observation of the symptoms of TSWV and other tospoviruses cannot be used as a definitive diagnosis of infection. TSWV and other tospovirus symptoms are highly variable or may be absent. Necrosis, chlorosis, “oak-leaf” patterns, concentric ring patterns, bronzing, mottling, speckling, stunting, wilting, and local lesions are common symptoms and can occur on most plant tissues. It has been shown that the same strains of a tospovirus vary widely in symptom expression within the same plant. It is not understood why many plants infected with the virus do not show symptoms. For example, during the last three tobacco growing seasons, half of the TSWV infected tobacco exhibited no outward signs of disease. The vast majority of the known plant hosts also show no symptoms. Some of the symptoms that are expressed may be mimics of some other pathogen or environmental stress, which attenuates the need for testing protocols that give a more definitive determination of infection. Three current techniques are double antibody sandwich-enzyme linked immunosorbent assay, reverse transcriptase- polymerase chain reaction, and mechanical sap inoculation.

Double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) is a serological method commonly used to detect tospoviruses. Antibodies specific to the virus are used to screen for that particular virus, and a combination of antibodies is sometimes used. DAS-ELISA is used on a wide scale due to its reliability and ease of screening a large number of samples over short periods of time. Although it is of common use on a day to day basis, other verifica- tion methods are used to verify the results of the serological testing. A new development based on antibodies are immunostrips. Immunostrips can be used directly in the field. A paper strip is coated with antibodies for a particular virus. Plant tissue is ground in a small plastic bag. The immunostrip is then put in the sap and observed for a color reaction. The immuno-strips are not as sensitive as DAS-ELISA, so this may be a good tool for quick diagnosis in the field, but not a replacement for DAS-ELISA.

Reverse transcriptase-polymerase chain reaction (RT-PCR), is a molecular technique that amplifies a specific region of the viral genome. This technique, although time consuming and expensive, is very valuable in verifying results from DAS-ELISA. It can also be used to identify individual viral strains by sequencing the RT-PCR products. A positive response with this method is the most definitive screen for tospoviruses as even a small quantity of virus can be detected. Depending on the primer design, closely related tospoviruses can be confirmed, even if present in the same sample as mixed infections.

Mechanical sap inoculation is another helpful tool in detection as well as culture of the virus. Tissue from a suspected infected plant is used to inoculate a known susceptible indicator plant. These indicator plants include Nicotiana tabacum L., Nicotiana glutinosa L., Nicotiana benthamiana, Petunia X hybrida Vilm., and Emilia sonchifolia (L.) DC. Successful viral transmission to one of the indicator plants is useful in two ways: It aids in the diagnosis of a plant virus and serves as a culture of a virus.

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