LABORATORY PROCEDURES FOR PLANT CELL VIRUSES
PVC/1998/2.03 Appendix 3
CLEAN UP OF VIRUS ISOLATES IN MIXED INFECTIONS BY MECHANICAL TRANSMISSION TO DIFFERENTIAL HOSTS
Separation of mixed infections to obtain pure cultures is most conveniently done, with viruses that are mechanically transmissible and that have different susceptible and immune hosts. A cleaning up of mixed virus infections can also be reached using a host that supports systemic invasion and restricting the contaminating virus to the initial sites of infection – inoculated leaves – by sustaining only localised virus infection or hypersensitive reaction. A further separation of different viruses or virus strains within the same plant forming a mixed infection can be achieved by local lesion transfer.
Essentially, methods described inPVC/1998/2.02 Appendix 2 are followed. All efforts for cleaning up are followed by EM (PVC/1998/2.02 Appendix 5) and IEM (PVC/1998/2.03 Appendix 2), where applicable. Differential hosts, plant parts invaded with only one virus or, local lesions are used to prepare an inoculum and to transmit the separated virus by mechanical inoculation on adequate host plants. To separate viruses and their strains by local lesion transfer, the local lesion are excised at the earliest stage possible when just visible at the beginning of necrotisation. The smallest area possible is taken and rubbed onto leaves of healthy plants using 1 local lesion per plant. This approach is repeated for at least 3-5 passages all followed by EM examination to guarantee purity of strain/isolate. When different local lesion types are formed, then all types are excised and transmitted to separate plants.
Guidelines prepared for CABRI by DSMZ, 3 Feb. 1998
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