How do you diagnose a virus infection in a plant?

Hopefully by a combination of one or more of the following...

1.) Inclusions

Staining properties: There are two stains used in looking for viral inclusions. One is Azure A (AA) which stains RNA pink (top left and right) and DNA blue. The second stain is the orange-green stain(OG-bottom left) which stains protein.

Some inclusions, such as the cylindrical inlcusions of most potyviruses stain only in OG. (Some will stain lightly in AA if heated in the stain) Others like cucumber mosaic on the top right will stain with both stains. Tobacco mosaic inclusions like the one on the bottom left will stain with both stains but it will only stain in AA when it heated. This differential staining can help in diagnosis.

Location: Many inclusions are found in the epidermis (left) but others can only be found in the vascular tissue like Citrus tristeza virus on the top right. Those of gemini virues and rhabdoviruses (bottom right) are found in the nucleus. The location of the inclusion also helps to narrow down the type of virus infecting the plant.

2.) Host Range Inocluations

Over the years plant virologists have inoculated plant viruses to many different plants. Each known plant virus has a list of susceptible and insuseptible plants.

By grinding a sample of an infected plant in a buffer (left), you can manually inoculate other plants (right). Those that become infecrted can be compared with the list of plants suseptible to a suspected virus.

If the host range of your unknown virus is the same as that of some known virus you have another clue as to what virus you may have.

3.) Electron Microscopy

Virus particles can be seen only with the use of a very powerful microscope called and electron microscope (EM). Plant sap containing virus particles can be prepared and put into an electron microsope and examined for virus particles. There are several basic shapes that virus particles take . By viewing the particle in the EM you can narrow down the type of virus you have.

Four differnet shapes are shown on the left. A rhabdovirus will have a bullet-like shape (top left). Tobamoviruses like tobacco mosaic have rigid rods (top right). Potyviruses (bottom right) and clostrovirues have flexous rods. Many other viruses have a spherical shape (bottom left).

Plant tissue can also be fixed, thinly sectioned, and viewed under the EM. If you were to see something like that on the right you would know you had a potyvirus!

Immunodiffusion Test

4.) Serological Tests

Viruses can be purified and the purified virus injected into a mammal such as a rabbit. The inoculated animal will make antibodies to protein coat of this viurs. The animal is bled and the serum (antiserum) that results can used to detect plant viruses.

On the left antiseum was put into the middle well. The two wells on the far upper left contain virus identical to the virus used to make the antiserum. The two wells on the top right are a related virus. The two bottom wells are healthy controls.

On right is an example of test called an ELISA test. There are several different procedures for this type of test but the end results are the same. The wells that are yellow are positive for the virus (they reacted with the antiserum). The other wells are either healthy controls or negative for the virus.

ELISA Test

5.) RNA/DNA tests

Tests can also be done that will detect the nucleic acid of the virus.

On the left is an example of a potyvirus detected in a sample by PCR (polymerase chain reaction)

On the right is a test using a DNA probe which attaches to the sample when the virus is present. The dark spots are positive for the virus and those that do not react are negative. This test requires radioactive material and so it is not used in routine diagnosis. However non-radiaoactive probes may be available soon.

There is another type of test which can detect the double stranded form or replicating form of some RNA viruses. Using appropriate controls, the size of the band on a gel can tell you what type of virus you have