To date, use of standardised host range of four Citrus species and a scion-rootstock combination is the only method available to determine the biological properties of a given CTV isolate. However, glasshouse indexing is time consuming and labour intensive. Clearly, a more rapid and specific methods of identifying individual CTV isolates and predicting their biological properties are needed to screen budwood source trees, for epidemiological studies and to evaluate cross-protection trials. Cross-protection is a phenomenon whereby prior infection with one (protecting) plant virus will prevent or interfere with superinfection by another, usually related virus (challenge). It has been used as a practical means of protecting citrus plants against CTV infections in those countries where tristeza is endemic due to the presence of the vector.

Mild CTV isolates used in cross-protection assays might contain a mixture of strains and a different balance of strains could be established depending on the host and/or the environmental conditions. Quick and specific procedures to monitor the viral population and for periodical evaluation of cross-protection trials will be developed.

Single-strand conformation polymorphism (SSCP) analysis has proved a very appropriate technique for the study of viral populations because it allows the separation of complex mixtures of equal molecular weight DNA with different nucleotide sequence. Sequence variations as small as single base point mutations can be identified in PCR (polymerase chain reaction) products amplified from a variety of genomic or cDNA sources.

We have applied SSCP analysis of viral p27 gene to screen the genetic heterogeneity of CTV isolates. The product of p27 gene is the minoritary (5%) viral coat protein, present only in one end of the particle, for which it is postulated a function in the transmissibility by aphid vectors. CTV isolates used in this study belong to the five different biogroups defined on the basis of symptoms induced on indicator plants.

The results obtained, applying the SSCP analysis have allowed us to discern subpopulations of the CTV genome present in the isolates studied in a proportion as low as 3%. SSCP analysis, which is less time consuming than biological assays, seems to be a convenient tool for detecting mixed infections.

On the other hand, there may also be strains within an isolate that are "hidden" through interference mechanisms. These strains could be detected by a method like to SSCP but probably not by a biological assay.

Moreover, SSCP analysis of CTV p27 gene seems to be a convenient tool for assigning CTV isolates to a certain biogroup as well as for detecting mixed infections. To confirm this finding, we are conducting SSCP analysis of p27 gene in a higher number of CTV isolates belonging to the different biogroups above mentioned.