Characterization of citrus tristeza virus variants and their influence on symptom expression in grapefruit

Cook, G. , van Vuuren, S. P., Breytenbach, J. H. J., Steyn, C. , Burger, J. T. , andMaree, H. J. 2016. Characterization ofCitrus tristeza virus single-variant sources in grapefruit in greenhouse and field trials. Plant Dis. 100:2251-2256. Citrus tristeza virus (CTV) is endemic to southern Africa and the stem pitting syndrome that it causes was a limiting factor in grapefruit production prior to the introduction of cross-protection in the Citrus Improvement Scheme. This disease mitigation strategy, using various field-derived CTV sources, has significantly extended the productive lifespan of grapefruit orchards in South Africa. CTV commonly occurs as a population of various strains, masking the phenotypic effect of individual strains. Likewise, current South African CTV cross-protection sources are strain mixtures, obscuring an understanding of which strains are influencing cross-protection. The severity of various CTV strains has mostly been assessed on sensitive indicator hosts, but their effect on commercial varieties has seldom been investigated. Single-variant CTV isolates were used to investigate the phenotypic expression of CTV strains in commercial grapefruit varieties as well as CTV indicator hosts. They were biologically characterized for their ability to cause stem pitting and their rate of translocation and titer in the different hosts, monitored by enzyme-linked immunosorbent assay. Complete genome sequences for three CTV strain variants were generated. Isolates of CTV strains VT, T68, RB, and HA16-5 did not induce severe stem pitting in four grapefruit hosts in a glasshouse trial. Viral titers of the strains differed in the grapefruit hosts, but the RB isolate reached a higher titer in the grapefruit hosts compared with the VT, T68, and HA16-5 isolates. Additionally, horticultural assessment of two grapefruit varieties inoculated with the RB isolate in two field trials demonstrated that mild stem pitting did not negatively influence the horticultural performance of the grapefruit trees over an eight-year assessment period. ‘Star Ruby’ trees containing the CTV source GFMS35 showed less stem pitting than trees inoculated with the RB isolate, but had smaller canopy volumes and lower yields than trees containing the RB isolate. This suggests that the influence of CTV sources on tree performance is not limited to the effect of stem pitting. Citrus tristeza virus (CTV) is a member of the family Closteroviridae, with an approximately 19.3-Kb genome (Karasev et al. 1995) that displays significant sequence variation among currently known strains (Harper 2013). The genotypic characterization of CTV variants has progressed significantly, but their phenotypic expression in various citrus varieties is less defined. Fundamental work in CTV is mainly done on sensitive biological indicator hosts in which symptom expression is faster and more definitive. However, these results cannot be extrapolated to commercial citrus varieties with varying CTV susceptibility, as symptom expression can be host-dependent. CTV can cause a stem pitting syndrome in the trunks and limbs of grapefruit (Citrus paradisi) trees that are established on rootstocks other than sour orange (C. aurantium). The resulting disruption of vascular flow leads to a gradual tree decline and is often associated with lower yield and a decrease in fruit size, impacting exportability of the fruit. This is contrasted to the quick decline syndrome or ‘Tristeza’ associated with trees grafted on sour orange rootstocks. This stem pitting syndrome necessitates earlier tree rotation resulting in financial losses associated with tree replacement and production lag. Different phenotypes of stem pitting are found in citrus, including large grooves visible on tree trunks and limbs, ‘honey-comb’ stem pitting, referring to a high density of small pits, which is more damaging to the tree, and a spectrum between these two phenotypes (Moreno and Garnsey 2010). The requirements for stem pitting development are not fully understood, but expression of the CTV p33, p13, and possibly the p18 genes appear to be involved (Tatineni and Dawson 2012). Expression of the same genes by CTV strains in varying combinations is required for infection of certain citrus species (Tatineni et al. 2011). These findings imply very specific interactions of strains with various citrus hosts that influence host susceptibility. Apart from South Africa, ‘mild-strain’ cross-protection is successfully applied to reduce the effect of CTV stem pitting on commercial citrus by a number of citrus-producing countries, including Australia (Broadbent et al. 1991), Brazil (Salibe et al. 2002), and Peru (Bederski et al. 2005). CTV control by eradication of infected trees is not feasible in southern Africa due to the endemic presence of the brown citrus aphid Toxoptera citricida, the most efficient aphid vector of CTV. The South African Citrus Improvement Scheme implemented crossprotection to minimize losses incurred due to stem pitting, primarily in grapefruit. This management strategy has significantly extended the productive lifespan of grapefruit orchards in South Africa by minimizing losses incurred due to stem pitting (Marais 1994). A strain-specific exclusion mechanism, superinfection exclusion (SIE), has been demonstrated and proposed as a possible mechanism for cross-protection (Folimonova et al. 2010). It was established that, once a plant is infected with a certain strain, a challenge virus of the same strain is not able to infect the plant. This exclusion is not applicable to dissimilar strains and relies on a homologous p33 viral protein of the same strain (Folimonova 2012). Additionally, it was demonstrated that two leader proteases, L1 and L2, are also involved in SIE (Atallah et al. 2016). This mechanism has not been completely resolved on a biological level, as the existence of a ‘mild’ variant of a pathogenic strain is required to prevent infection of ‘severe’ variants. If this is the mechanism at work in cross-protection, ultimate Corresponding author: G. Cook; E-mail: glynnis@cri.co.za Nucleotide sequence data is available in the GenBank database under accession numbers KU883265, KU883266 and KU883267. *The e-Xtra logo stands for “electronic extra” and indicates that a supplementary figure and a supplementary table are published online. Accepted for publication 22 May 2016. http://dx.doi. org/10.1094/PDIS-03-16-0391-RE © 2016 The American Phytopathological Society Plant Disease /November 2016 2251 Stellenbosch University https://scholar.sun.ac.za