A NEW PROCEDURE FOR RAPID EVALUATION OF THE ABILITY OF CITRUS TRISTEZA VIRUS MILD ISOLATES TO CROSS PROTECT AGAINST SEVERE ISOLATES

Measures to control damage caused by severe stem pitting strains (SP) of Citrus tristeza virus (CTV) include selective eradication and cross protection with mild strains (MS). Implementation of these measures needs sensitive and specific methods to discriminate between SP and MS variants in the viral populations. We developed a quantitative real-time RT-PCR method using Taqman locked nucleic acid (LNA) probes that enabled quantification of SP and MS variants in natural CTV populations. Here we used this method to monitor the evolution of the CTV population in Pineapple sweet orange plants pre-inoculated with a MS (T32) or a SP (T318) isolate, and then challenge-inoculated with budsticks infected with a SP or a MS isolate, respectively. While control plants singly inoculated with T32 or T318 only contained the cognate CTV variant in successive flushes, the plants doubly inoculated contained the two sequence variants in tissues derived from both the receptor plant and the inoculum budstick, with no strain impairing systemic invasion by the other. The relative amount of T32 and T318 variants in the co-infected plants differed between flushes. The co-infected plants showed stem pitting symptoms similar to those of plants inoculated only with T318, whereas control plants inoculated only with T32 or non-inoculated remained symptomless. This procedure enables rapid evaluation of MS for cross protecting ability.