Genotype composition of populations of grapefruit-cross-protecting citrus tristeza virus strain GFMS12 in different host plants and aphid-transmitted sub-isolates

Abstract

Citrus tristeza virus (CTV) causes severe losses in grapefruit production in South Africa and requires mild strain cross-protection to maintain production. Unfortunately cross-protection breakdown of the preimmunizing CTV grapefruit mild source GFMS12 is prevalent in grapefruit in South Africa. The CTV genotype composition of the GFMS12 population inoculated onto different hosts was determined by sequencing part of ORF1a and the p23 gene of multiple clones from each plant. Analysis of the GFMS12 population in Mexican lime, Marsh and Star Ruby grapefruit varieties, revealed that at least four genotypes occur in the GFMS12 population, and that genotype compositions differed amongst the populations in different host plants.Single aphid transmitted sub-isolates derived from the GFMS12 mother population on Mexican lime appeared to contain three populations of a mixture of VT-like and recombinant B165/VT-like genotypes; a mixture of recombinant RB/VT-and B165/VT-like genotypes; and a single recombinant B165/VT-like genotype. This study underlines the importance of determining the genotype composition of a potential CTV pre-immunizing source on a range of inoculated host species before utilization.

Characterization of a novel citrus tristeza virus genotype within three cross-protecting source GFMS12 sub-isolates in South Africa by means of Illumina sequencing

“…The observed population structure for the sub-isolates tested partially supports and expands upon the results of a previous study [7] in which, based on sequencing multiple clones of amplicons of a segment of ORF1a (A-fragment) and the p23 gene, sub-isolate 12-7 was described as containing an B165/VT-recombinant and an RB/VT-like recombinant, 12-8 a B165/VT-like recombinant, and 12-9 a B165/VT-like recombinant and VT. It is most likely that the B165/VT-like recombinant proposed [7] within all three sub-isolates is actually CT-ZA3, which, like its most closely related isolate T68-1, has been shown by recombination analysis in this study (data not shown) and elsewhere [11] to share most of its 3 0 half with VT, while its ORF1a A-fragment region would have been interpreted in the previous study [7] as a B165-like genotype member in the absence of the subsequently proposed T68 genotype [11].…”

“…It is most likely that the B165/VT-like recombinant proposed [7] within all three sub-isolates is actually CT-ZA3, which, like its most closely related isolate T68-1, has been shown by recombination analysis in this study (data not shown) and elsewhere [11] to share most of its 3 0 half with VT, while its ORF1a A-fragment region would have been interpreted in the previous study [7] as a B165-like genotype member in the absence of the subsequently proposed T68 genotype [11]. The reported presence of a RB/VT-like recombinant (ORF1a/p23 gene) in sub-isolate 12-7 in addition to B165/ VT [7] is not supported in the current study. In this study, a number of reads from 12-7 mapped to large portions of the 5 0 half of Taiwan-PUM/SP/T1 (also an RB-like genotype) with at least one genome region in this half having high read coverage (c3450), along with some reads in its 3 0 half.…”

Genotypic diversity of Citrus tristeza virus within red grapefruit, in a field trial site in South Africa

“…The GFMS 35 source was approved as the replacing cross-protecting source for red grapefruit cultivars (Luttig et al 2002) and has been used to pre-immunise red grapefruit budwood mother trees since 1998 (van Vuuren, personal communication).Various molecular techniques have been used to characterise cross-protecting sources and sub-isolates used within the SACIS, which include single stranded conformation polymorphism (SSCP) (Luttig et al 2002) and restriction fragment length polymorphism (RFLP) . Scott et al (2012) used gene amplification and mass cloning to determine the genotype composition of the original GFMS 12 source maintained under glasshouse conditions. Very little data is available regarding the genotype compositions of CTV populations within trees under field conditions.…”

“…Folimonova et al (2010) showed that strains provide an absolute exclusion against secondary introductions of the same strain. In light of this, it has been suggested that mild-strains of each strain, present in a region, will need to be obtained and used as cross-protecting sources, in order to elicit effective protection again severe strains present in the field (Scott et al 2012). The process of selecting effective cross-protecting sources is complicated by factors that can influence CTV populations, such as climate (Broadbent et al 1996), varying replicative capacities of strains in different hosts (Targon et al 2000) and variable transmission of certain strains depending on the regional vector population (Roy and Brlansky 2009).…”

“…These include single-strand conformation polymorphism (Rubio et al 1996), restriction fragment length polymorphism (RFLP), bi-directional polymerase chain reaction (BD-PCR) (Jiang et al, 2008) and nucleotide sequence analysis (Iglesias et al 2008, Scott et al 2012, Wang et al 2013. While techniques such as SSCP and RFLP can indicate differences between CTV populations, the identity of CTV strains can be difficult to determine without actual nucleotide sequencing data.…”