Mon Dec 18, 2017 2:41 am

Togribase

TOspovirus aGRIcultural DataBASE

Diagnosis of Tospovirus

     Detection based on symptoms of the tospoviruses cannot be used as a definitive diagnosis of infection. The 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. 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. The techniques available for confirmation or detection or diagnosis of tospoviruses are:

  • Electron microscopic view of tospovirus particles.
  • Serological methods:
    • Enzyme linked immunosorbent assay (ELISA)
    • Lateral flow immunostrips
  • Molecular approaches:
    • Reverse transcriptase-polymerase chain reaction (RT-PCR)
  • Biological method:
    • Mechanical sap inoculation on indicator host plants (Nicotiana spp. Petunia sp., and Emilia sonchifolia) and verifying/characterizing the viruses.

DAS-ELISA        
  • 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 verification methods are used to verify the results of the serological testing.

  • IMMUNOSTRIPS
  • 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        
  • 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.