Overcoming bias in Citrus tristeza virus (CTV) genotype detection and a population study of CTV within Southern African Star Ruby grapefruit orchards

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.