Part I

Virus Inoculation

Introduction I Virus Inoculation

Introductibn I - Virus Inoculation 3

Inoculation (mechanical inoculation, mechanical transmission or sap transmission) means bringing healthy plant parts, usually leaves, into contact with a virus-containing suspension (inoculum) in such a way that infection ensues. This type of virus transmission is a valuable method for disease diagnosis, local-lesion assay (see Part III), propagation and maintenance of viruses, studying virus-host interactions and detecting viruses. Many viruses, but not all, are mechanically transmissible.

To achieve infection, small wounds must be made, as virus particles cannot penetrate an intact leaf surface, with its cuticle and cell walls acting as major barriers. The most effective way to wound the cell is by using abrasives, e.g. Carborundum powder, dusted on the leaves before inocula­tion. Leaves are then inoculated by rubbing their surface with the inocu­lum.

Mechanical inoculation of leaves is not a very efficient way to achieve infection. Although in theory, one virus particle may be sufficient to infect a cell, in practice a large number of particles, usually more than 500, are required to give rise to infection of one cell leading to formation of a visi­ble lesion. This may be due to the fact that only a small proportion of epi­dermis cells have been wounded to such an extent that virus particles can enter the cytoplasm.

Even when infection occurs, virus multiplication is not synchronous, as the infectious agent moves from cell to cell. This makes the studies of dif­ferent stages of the infection process virtually impossible. To overcome this problem, a method has been developed in which protoplasts isolated from mesophyll (mostly palisade parenchyma) cells are used to study virus replication (Takebe and Otsuki 1969). Such protoplasts can be infected synchronously in large proportions (70-90 %).

Although protoplast systems have proved to be very valuable for study­ing early infection processes, including translation of viral RNA and viral replication, isolated protoplasts cannot be used, for obvious reasons, to investigate virus translocation from cell to cell and to establish the effects

4 PART I - VIRUS INOCULATION

of virus on a plant. To overcome this drawback, a method has been devel­oped by which synchronous infection is achieved by exposing the plants to a differential temperature treatment (Dawson and Schlegel1973).

• References

Dawson WO, Schlegel DE (1973) Differential temperature treatment of plants greatly enhances multiplication rates. Virology 53:476-478

Takebe I, Otsuki Y (1969) Infection of tobacco mesophyll protoplasts by tobacco mosaic virus. Proc Natl Acad Sci USA 64:843-848