LABORATORY PROCEDURES FOR PLANT CELL VIRUSES
PVC/1998/2.02 Appendix 2
MECHANICAL TRANSMISSION OF VIRUSES TO INDICATOR PLANTS AND PROPAGATION HOSTS
Infectivity tests are performed by rubbing homogenates of virus-infected plants to a set of susceptible and non susceptible – immune – herbaceous host plants to induce virus infection. For mechanical inoculation, the set of plants is chosen according to the CMI/AAB Descr. Pl. Viruses, and/or the descriptions and lists from the VIDE Database. The set comprises a minimum experimental host range of plants that are diagnostically susceptible or unsusceptible and react with systemic infections or localised infections. Symptoms in these indicator plants can be used in differential diagnosis indicating for a particular virus and also for presence of a contaminating virus in mixed virus infections. Suitable hosts reacting with systemic infections are used for virus propagation.
Differential Diagnosis using indicator plants is a significant component of quality control for mechanically transmissible viruses and, by testing of plant samples presumably infected with a non-mechanically transmissible virus, for purity of a particular sample.
Standard homogenisation/inoculation buffer. Unless plant viruses require specific inoculation buffers, this buffer is used for all mechanical transmission experiments.
Phosphate buffer (standard)
0.1 M Na2H/KH2PO4 buffer pH 7.0
2% (w/v) polyvinyl pyrollidone (PVP, MW 11.000)
0.2 % (w/v) Na2SO3
Using a sterile scalpel, a small piece of a symptomatic plant part – either fresh of desiccated leave - is excised and homogenised in a small amount of cold homogenisation buffer (1:2-1:5 w/v) using a sterile pestle and mortar to yield a finely ground virus suspension. The homogenate is rubbed immediately onto carborund dusted leaves of respective test plants using sterile glass rods or cotton swaps. After mechanical inoculations the inoculated leaves are sprayed with water to remove inoculum and to reduce excessive evaporation.
For inoculation experiments either seedlings or plants in the 3-5 leave stage are most susceptible, hence this stage is most commonly used for differential diagnosis. Homogenates of virus infected plants are rubbed onto fully expanded cotyledons – e.g. cucurbitaceae -, on the first true leaves – e.g. leguminosae – or, on three fully expanded leaves – e.g. solanaceae - , depending on the host plant used.
Guidelines prepared for CABRI by DSMZ, 3 Feb. 1998
© The CABRI Consortium 1999 -