Department of Genetics

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Characterisation of citrus tristeza virus-induced stem pitting in citrus
(Stellenbosch : Stellenbosch University, 2023-12) Aldrich, Dirk Jacobus; Maree, Hans Jacob; Bester, Rachelle; Burger, Johan Theodorus; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.
ENGLISH ABSTRACT: Citrus tristeza virus (CTV) is the most important viral pathogen of citrus and causes several disease syndromes in different citrus hosts. CTV-induced stem pitting leads to substantial economic losses in sensitive citrus varieties, including grapefruit. The exact mechanisms of stem pitting development in CTV-infected citrus remain unclear. This study aimed to utilise CTV infectious clone mutants in a reverse genetics approach to study stem pitting induction. A panel of recombinant CTV clones was generated focussing on the open reading frames (ORFs) implicated in stem pitting induction and pathogenicity, namely p33, p18, p13 and p23. ORF replacements from severe- and mild-pitting South African CTV isolates were introduced into the mild-pitting infectious clone (CTV-fl6 - genotype T36) to determine if severe stem pitting could be induced. Stem pitting assessments were complemented with the determination of virus concentration ratios in ‘Mexican’ lime and ‘Duncan’ grapefruit to relate stem pitting outcomes to virus concentration. The various infectious clone and wild type CTV infections yielded a broad range of stem pitting outcomes and specific ORF replacements associated with enhanced stem pitting were identified. Plant responses to different stem pitting pressures were further assessed by untargeted metabolite profiling and the quantitation of the stress-responsive phytohormones, abscisic acid, jasmonic acid and salicylic acid. In both citrus hosts, the metabolite profiling yielded fourteen statistically significant compounds that differed between stem pitting groups. These compounds were mainly phenolic acids and phenolic glycosides and are known to function as antioxidant and stress signalling molecules. Significant differences in phytohormone content were also found between test groups, particularly in plants that were severely stunted. Stem pits were also characterised at the molecular anatomical level using a combination of known and novel techniques to better understand the nature of the xylem and phloem tissues impacted by severe pitting. Established methods such as biological staining and fluorescence microscopy were used to visualise CTV-induced stem pitting and virus localisation. The utility of two novel technologies that have not previously been used to study CTV-induced stem pitting, namely high-resolution CT scanning and serial block face scanning electron microscopy was also evaluated. This study contributed to our understanding of CTV-induced stem pitting in citrus. Importantly, the use of CTV infectious clones for South African CTV research was established, which can allow for targeted follow-up experiments to further characterise the local citrus-CTV pathosystem. This can also provide novel opportunities for potentially employing CTV infectious clones as a crop protection tool against other important citrus diseases on the African continent.
The characterization of the association of apple stem pitting virus with pear stony pit disease
(Stellenbosch : Stellenbosch University, 2023-04) Bougard, Kayleigh; Bester, Rachelle; Maree, Hans, Jacob; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.
ENGLISH ABSTRACT: The overall health of the fruit industry is reliant on high-quality planting material that is free of pathogens and pests. Within the plant improvement scheme, several viruses are tested for before the propagation material may enter commercial orchards. In the pear industry, there have been observations over the last few years of pitted and deformed fruits, possible symptoms of Pear stony pit disease (PSPD). In affected orchards these deformed fruits have been the cause for a drastic decrease in crop yield each season, and with these pears being unmarketable, economic loses are also experienced. PSPD is known to be associated with apple stem pitting virus (ASPV), a virus that is only transmitted via propagation material. But recently, local producers have hypothesized that stink bug feeding may be the cause for these deformities. Therefore, this project included surveying pear orchards distributed over two geographically distinct regions in the Western Cape, South Africa. Leaf samples were harvested from pear trees, covering six different cultivars. To determine the association of ASPV with the disease, preliminary reverse transcription polymerase chain reaction (RT-PCR) testing was performed but after high throughput sequencing (HTS) virome profiling, it was identified that there is a divergent variant of this virus present in South Africa and the preliminary assay was insufficient to capture it. A new multiplex RT-PCR (RT-mPCR) was therefore developed during this project which can detect all known ASPV variants present in South Africa. The HTS results allowed for the first full-length genome sequence of this divergent variant (Isolate B173). Virome profiling also allowed for the reporting of two viruses, citrus virus A, which has not been found on pears in South Africa before, and apple rubbery wood virus 2, which has not been found on apples and or pears in South Africa before. A stink bug feeding trial was performed on two pear cultivars, ‘Forelle’ and ‘Rosemarie’, by enclosing several bugs in mesh bags around pear clusters from fruit set, and the fruit health was evaluated at the end of the season. Phylogenetic analyses were performed, evaluating ASPV/apple green crinkle associated virus (AGCaV) sequences to determine the relationships of host, origin, and also whether a distinction can be made between these two viruses. The result did not provide a clear distinction between these viruses sequences. The data generated in this study highlighted the need to re-evaluate the phytosanitary status of propagation material to ensure virus free pear orchards for the future. Both the virus and feeding damage was identified as potential contributing factors to PSPD-like symptoms. The study suggests, with need for further confirmation, that ASPV infection results in pear symptoms with more defined external pits. The orchards where this symptom was found resulted in 100% and 92% association with ASPV on symptomatic and asymptomatic trees, respectively. The orchards exhibiting the less defined pitted symptoms had a lower association (56%) to ASPV infection. Therefore, currently, the best practice recommendation for symptom management will include to plant ASPV free plant material and to eliminate stink bugs from orchards.
Construction and evaluation of citrus tristeza virus-based gene silencing vectors that target Trioza erytreae and Diaphorina citri
(Stellenbosch : Stellenbosch University, 2022-04) De Nicola, Ella Hannan Maria; Maree, H. J.; Bester, Rachelle; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology.
ENGLISH ABSTRACT: Citrus greening is an incurable and devastating bacterial disease caused by species of phloem- limited Candidatus Liberibacters, transmitted by two phloem-sap feeding insects, Diaphorina citri and Trioza erytreae. Citrus greening poses a constant threat to the success and stability of citriculture worldwide. The pathogen-insect combination of Candidatus Liberibacter asiaticus (CLas) and D. citri, responsible for the most severe form of citrus greening, Huanglongbing (HLB), was reported in Northern Africa for the first time in 2010 and 2016 respectively. Confirmation that the endemic insect vector of Candidatus Liberibacter africanus (CLaf), T. erytreae, can also transmit CLas has significantly increased the risk of HLB reaching South Africa. Until a cure is discovered, the development of a reliable insect vector control strategy is critical. This study aimed to convert an infectious clone of citrus tristeza virus (CTVfl6) into a virus-induced gene silencing (VIGS) vector that targets critical genes in the citrus greening insect vectors and bioinformatically profile the virus- induced small interfering RNAs (vsiRNAs) produced in planta. This putative control strategy is uniquely specific due to the sequence specificity of the RNAi machinery and the phloem-limited infection of CTV. Four candidate genes, arginine kinase, abnormal wing disc, chitin synthase and ran GTPase, were selected as RNAi targets, and six insect- and gene-specific silencing inserts were constructed. A set of 24 intermediate plasmids was assembled, harbouring the silencing inserts in the 3’ terminal end of CTVfl6. These intermediates differed by silencing insert orientation and presence or absence of an upstream duplicated CTV coat protein controller element to evaluate the influence of CTV VIGS vector configuration on vsiRNA production in planta. Assembly of the complete CTVfl6 VIGS vectors was attempted by replacing the 3’ end of the original sequence with the newly engineered intermediate 3’ end. Only three of the 24 were successfully assembled, two T. erytreae insect-specific and one abnormal wing disc gene-specific. Systemic CTV VIGS vector infection was observed in 22% to 33% of N. benthamiana plants 5-6 weeks post-Agrobacterium tumefaciens-infiltration, indicating that CTVfl6 can tolerate large exogenous insertions in the 3’ terminal end. Inoculation into Mexican lime was attempted for the T. erytreae specific CTV VIGS vectors. As CTV was not yet detectable in Mexican lime at the time of sampling, small RNA (sRNA) sequencing was only conducted on CTV positive N. benthamiana material. The vsiRNA coverage across the CTV VIGS vector genomes was over >99%. Hotspots in vsiRNA alignment depth were observed at two of CTV’s silencing suppressors, providing detail on host plant-virus interaction. Insert-specific vsiRNAs were produced in planta, covering >99.78% of the silencing insert. The production of insect-specific vsiRNAs provides some proof of concept for this vsiRNA delivery strategy. The influence of RNAi-mediated knockdown on insect development and mortality should be tested using greenhouse trials. This study has generated the first putative RNAi-based control strategy for the endemic South African citrus greening vector. This strategy is applicable to mitigating the spread of CLaf and, in the event of CLas reaching South Africa, HLB in an African setting.
Determining the distribution and genetic diversity of Coguvirus eburi in South African citrus and development of a citrus-infecting coguvirus detection assay
(Stellenbosch : Stellenbosch University, 2022-04) De Bruyn, Rochelle; Maree, H. J.; Bester, Rachelle; Cook, Glynnis; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology.
ENGLISH ABSTRACT: Citrus virus A (CiVA) is a novel negative-sense single-stranded RNA virus discovered with high- throughput sequencing (HTS). CiVA is classified as a member of the species Coguvirus eburi and is closely related to a member of the species Citrus coguvirus, named citrus concave gum-associated virus (CCGaV). CCGaV and CiVA are members of the genus Coguvirus in the family Phenuiviridae in the order Bunyavirales. CiVA has a bipartite genome encoding an RNA-dependant RNA polymerase (RdRp) on RNA 1 and a nucleocapsid protein (NP) and a putative movement protein (MP) on the ambisense RNA 2. The confirmed presence of CiVA in cultivars of grapefruit (Citrus paradisi Macf.), sweet orange (C. sinensis (L.) Osb.) and clementine (C. reticulata Blanco) in South Africa initiated a study to determine the distribution, genetic diversity and symptom association of CiVA in three provinces and seven citrus production regions. CiVA was detected in six citrus production regions in symptomatic and asymptomatic sweet orange trees. In three citrus production regions, CiVA was detected in sweet orange trees displaying a fruit rind symptom similar to citrus impietratura. CiVA was also detected in grapefruit trees with typical citrus impietratura symptoms and in symptomless clementine trees. The three encoded gene regions of CiVA were Sanger sequenced to investigate the genetic diversity between isolates from the six citrus production regions and three citrus species. Phylogenetic analysis of the nucleotide sequences (nt) of each encoded gene region was performed through the construction of Maximum-likelihood (ML) phylogenetic trees and nucleotide identity matrices. Phylogenetic analysis and nt identity matrices indicated a higher genetic diversity within the NP than the MP and RdRp. More genetic diversity was observed between isolates from the three citrus species than between isolates from the different citrus production regions. A real-time RT-PCR detection assay targeting the RdRp was also developed to simultaneously detect CiVA and CCGaV. Two cDNA synthesis methods for reverse transcription (RT) were compared and a degenerate, dual priming oligo (DPO) reverse primer was designed to improve the specificity of the detection assay. Two PCR assays that utilised the DPO reverse primer with two different forward primers were compared. The cDNA synthesis method and choice of primer pair had an influence on the amplification efficiency, specificity and sensitivity of the real-time detection assay. A tissue specificity assay was also performed and CiVA was detected throughout the plant in leaf midribs, leaf lamina, green bark and roots. Lower Ct values were consistently associated with the green bark and leaf midribs. The detection assay was implemented for pathogen screening within the Citrus Improvement Scheme (CIS) of South Africa, ensuring the release of CiVA free budwood to the citrus industry.
Development of a molecular detection assay for accurate identification of five economically important tephritid species of commercial fruit in South Africa
(Stellenbosch : Stellenbosch University, 2022-12) Andrews, Kelsey Jayne; Maree, H. J.; Bester, Rachelle; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
ENGLISH ABSTRACT: South African fresh fruit production and export are plagued by five major fruit fly pests (Diptera: Tephritidae). These fruit flies: Ceratitis capitata (Wiedemann) Mediterranean fly, Ceratitis cosyra (Walker) Marula fly, Ceratitis quilicii (de Meyer, Mwatawala & Virgilio) Cape fly, Ceratitis rosa (Karsch) Natal fly, and Bactrocera dorsalis (Hendel) the Oriental fly, are of quarantine significance in certain export markets. Apart from economic loss incurred due to limitations in export markets, these flies also cause physical damage to fresh fruit through oviposition. Accurate identification of these fruit flies can be tricky as this fruit fly family consists of multiple morphologically cryptic species and species complexes. Morphological identification of these fruit flies is unreliable when larvae or closely related species are intercepted. This has highlighted a need for accurate methods to distinguish between these species that do not rely on morphological traits. Therefore, this study aimed to develop a molecular identification assay that can differentiate between the five fruit flies of concern to South Africa accurately and timeously. To achieve this, two colony-reared insects from each species underwent DNA extraction and high throughput sequencing (HTS). HTS data were subjected to de novo assembly and used to construct ten complete mitochondrial genomes using a combination of de novo and reference-based assembly methods. From this, two identification assays were developed: a sequencing- based assay targeting a mitochondrial intergenic region and a multiplex PCR assay targeting the gene opsin Rh4. Regarding the sequencing-based assay, a single primer set was designed to amplify a mitochondrial region between tRNAⁱˡᵉ and tRNAᵐᵉᵗ. The intergenic region between tRNAⁱˡᵉ and tRNAᵍˡⁿ (designated intergenic region I) within the amplicon is species-specific in size and proposed as a potential tool for species differentiation of the five species of interest in this study. In the multiplex PCR assay, five sets of species- specific primers with varying sizes were designed and optimised for use in a multiplex format. The resulting species-specific amplicons can be separated using a 2% agarose-TAE gel, providing accurate species identification. Both assays were validated using larval stages and wild, trap-collected specimens. The assays developed in this study can be applied in pest surveillance and monitoring activities and during fruit inspection at packhouses and ports of entry (PoE).
Identification of early indicators of CTV induced stem pitting to aid the selection of CTV cross protection sources.
(Stellenbosch : Stellenbosch University, 2024-03) Van Wyk, Luka; Maree, H. J. ; Bester, Rachelle; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.
ENGLISH ABSTRACT: Citrus tristeza virus (CTV) is the most impactful viral pathogen of citrus, having caused the death of more than 100 million citrus trees. CTV elicits several disease syndromes, but continues to affect citrus production through stem pitting disease phenotypes of varying severity. These phenotypes reduce citrus tree productivity and result in smaller, unmarketable fruit. Stem pitting is incompletely characterised, and the mechanisms underlying its induction remain elusive. This poses limitations regarding our understanding of CTV-induced stem pitting and the management thereof. This study aimed to better understand the plant-pathogen interactions underlying stem pitting in CTV-infected citrus over the course of disease onset from an untargeted perspective. To this end, high-throughput sequencing and ultra-performance liquid chromatography-mass spectrometry were used to profile the transcriptome and metabolome of ‘Mexican’ lime and ‘Duncan’ grapefruit plants infected with T3-KB, T68, and RB isolates of CTV. This profiling was carried out prior to infection, early post-infection, and after symptom development, with the goal of identifying determinants of severe CTV-induced stem pitting. It was shown that secondary metabolite profiles of ‘Mexican’ lime plants were able to clearly discern between CTV infections at ten months post-infection. These profiles indicated a common dysregulation of carbohydrate metabolism over disease onset and provided a putative identification of the furcatin molecule as a major driver of differentiation. Similarly, differential gene expression analysis implicated a carbohydrate metabolism gene, along with seven others, as significantly different in severely pitted, T3-KB infected ‘Mexican’ lime plants. These genes were consistently upregulated over the course of disease onset. This provided a candidate metabolic pathway for further study and eight candidate genes to be validated as determinants of CTV-induced stem pitting severity. The characterization of hypersensitive response and systemic acquired resistance in citrus due to CTV infection was also discussed. It was speculated that determinants of stem pitting severity may lie earlier in the infection progression of CTV within the hypersensitive response of citrus. Carbohydrate metabolism was also putatively associated as a factor of systemic acquired resistance in response to CTV infection. This emphasized the importance of the temporal component of citrus’ response to CTV infection that has only been studied in a limited capacity thus far. The utility of complementary transcriptomic and metabolomic analyses was also demonstrated, and the findings therefrom contribute to the understanding of plant responses to CTV over time.
Investigating the interactome of grapevine leafroll-associated virus 3 and Vitis vinifera
(Stellenbosch : Stellenbosch University, 2023-12) Mostert, Ilani; Maree, Hano; Bester, Rachelle; Burger, Johan; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.
ENGLISH ABSTRACT: Grapevine leafroll disease (GLD) is a globally important grapevine disease that affects the yield and fruit quality of affected vines. Grapevine leafroll-associated virus 3 (GLRaV-3; genus Ampelovirus, family Closteroviridae) has been identified as the main causal agent of GLD due to its consistent association with the symptoms of GLD. GLRaV-3 has not been successfully eliminated from mature vines, and no natural source of resistance to GLRaV-3 has been reported. Although the impact of GLRaV-3 infection on the transcriptome and metabolome of infected vines has been investigated, little is known about the exact mechanisms by which these effects occur. Furthermore, the roles of proteins encoded by GLRaV-3 open reading frames (ORFs) have largely been inferred by sequence homology or analogy to related viruses, and functional studies to determine their involvement in virion assembly and movement have not been performed. The aim of this study was to investigate the GLRaV-3 interactome to identify virus or host genes that play a key role in the proliferation and pathogenesis of GLRaV-3. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays were employed to screen GLRaV-3 ORFs for pairwise interactions. The majority of interactions between structural proteins suggest that GLRaV-3 shares a common mechanism of assembly with members of the genus Closterovirus, family Closteroviridae, although some unexpected interactions were also found. Interaction of p20B, a silencing suppressor, with structural proteins has not been reported for other members of the family, indicating its possible involvement in other aspects of the viral replication cycle. The GLRaV-3 transmembrane protein self-interacted; however, the mechanism by which this interaction occurs remains unknown as it lacks a cysteine residue crucial for the dimerisation of the closterovirus homolog of this protein. To identify virus-host interactions, a Vitis vinifera Y2H prey library was constructed and screened against GLRaV-3 ORFs encoding proteins involved in virion assembly, intracellular movement, and suppression of host silencing. BiFC was then used to demonstrate these interactions in planta. Two interactions identified using Y2H could not be demonstrated in planta and involved GLRaV-3 p20A, a protein of unknown function proposed to play a role in suppression of host defence responses and long-distance transport. In yeast, p20A was found to interact with a V. vinifera chlorophyll a-b binding protein and a V. vinifera SMAX1-LIKE 6 protein. Y2H and BiFC assays both demonstrated the interaction of p20A with V. vinifera mitogen-activated protein kinase and a V. vinifera small heat shock protein, as well as the interaction of GLRaV-3 minor coat protein with V. vinifera 3-deoxy-D-arabino- heptulosonate 7-phosphate synthase 02. All five of these host proteins are associated with host defence responses against pathogens. Furthermore, these interactions demonstrate that the symptoms of GLD may be caused by interference with a variety of pathways. This study contributes to our knowledge on the roles of GLRaV-3-encoded proteins in its replication and spread and provides information on cellular responses by grapevine against GLRaV-3. Understanding the proteins involved in the pathogenesis of GLRaV-3 can lead to the development of novel approaches to manage GLD.

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