Preferential accumulation of severe variants of Citrus tristeza virus in plants co-inoculated with mild and severe variants

The viral population in sweet orange plants, either healthy or pre-inoculated with the asymptomatic isolate of Citrus tristeza virus (CTV) T32, and then graft- or aphid-inoculated with the stem-pitting isolate T318, was characterized with respect to symptom expression, reaction with monoclonal antibody MCA13, single-strand conformation polymorphism (SSCP) of genes p18 and p20, bi-directional RT-PCR, and dot-blot hybridisation. All plants inoculated with T318, with or without pre-inoculation, showed stem pitting, reacted with MCA13, had the SSCP profile characteristic of this isolate, and in bi-directional RT-PCR yielded a 450-bp DNA product associated with severe isolates, indicating that T32 afforded no protection against T318. The latter isolate had two main sequence variants, the minor one of which was indistinguishable from the main T32 sequence, and both were detected in most plants that were graft-inoculated with T318. However, the T32 variant was not detected in plants that were aphid-inoculated only with T318 and also showed stem pitting. This suggested an association of symptoms with the major T318 sequence and preferential transmission of this variant by aphids. The T318-specific variant accumulated more than the T32 variant in plants in which both were replicating, suggesting a higher fitness of the former. Our results clearly emphasize the potential threat of severe CTV variants in areas where mild isolates are presently predominant.