Abstract:
Citrus tristeza virus (CTV) is present in almost all of the major citrus production
areas where it continues to reduce the profitability of citriculture worldwide. Severe
stem-pitting strains are endemic to Southern Africa. In addition to making use of
decline-tolerant rootstocks, the implementation of a mild-strain cross-protection
program has sought to increase the citrus production in the region. Of all the
commercially cultivated citrus species, grapefruit cultivars are among the most
susceptible to the severe stem-pitting strains and infections often lead to a
breakdown of cross-protection. Recent research has shown that CTV crossprotection
operates through a strain-specific mechanism, which relies on a virusspecific
protein, expressed from the p33 gene. The specificity of this mechanism has
highlighted the need for determining the CTV diversity within production areas as
well as accurately characterising CTV cross-protection sources that will be capable
of preventing secondary inoculations of severe strains in the field. The accurate
characterisation of CTV populations, which are usually made up of a number of
disparate strains, requires the use of robust PCR protocols. Mismatches between
primers and their corresponding binding sites may introduce primer-associated bias
during amplification. The primer-associated bias of four sets of CTV specific primers,
targeting the A and F regions and the p23 and p33 genes, were evaluated. This was
done through the amplification of defined templates followed by their characterisation
using the sequencing of multiple clones, as well as Illumina next generation
sequencing. High levels of bias were found to be associated with the primer pairs
targeting the A and F regions. The p33 gene primers were found to be biased
against two genotypes and suggestions for preventing this apparent bias are
discussed. The primer pair targeting the conserved p23 gene was found to have very
little associated bias. The second major aspect of this study was the large scale
survey of CTV diversity of pre-immunised Star Ruby grapefruit orchards in Southern
Africa. Samples were collected from eight Star Ruby production sites throughout
Southern Africa, namely Hoedspruit 1, Hoedspruit 2, Malelane 1, Malelane 2,
Swaziland (Mananga), Northern Cape (Kakamas), Sundays River Valley and
Nkwalini Valley, where between 16 and 32 samples per site were collected. The p33
gene was amplified for each of the collected samples and subjected to direct Sanger
sequencing. A protocol making use of Illumina MiSeq sequencing was established and used to sequence 96 samples. A subset of six samples was selected for cloning,
which resulted in a total of 218 sequenced clones and compared with that of the
Illumina sequencing data. High levels of CTV diversity were observed between
orchards, as well as between different trees within the same orchard. Most of the
populations were made up of a single dominant group, sometimes with several minor
sequence types. Sequence reads corresponding to strains within the Resistance
Breaking (RB) genotype were most numerous, especially in the most recently
planted orchards and were present within all of the populations analysed. The
Kpg3/SP/T3 group appeared to be represented the second most prevalent genotype
and seems to become more common as the orchard ages. Genotypes of the HA 16-
5, VT, AT-1, T36, Taiwan-Pum/M/T5 and T30 types, were represented sporadically
and at variable levels in populations from numerous collection sites. This has been
one of the most extensive diversity studies of CTV to date and has provided
unprecedented baseline knowledge of CTV diversity in Southern Africa.