Determining the distribution and genetic diversity of Coguvirus eburi in South African citrus and development of a citrus-infecting coguvirus detection assay

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dc.contributor.advisorMaree, H. J.en_ZA
dc.contributor.advisorBester, Rachelleen_ZA
dc.contributor.advisorCook, Glynnisen_ZA
dc.contributor.authorDe Bruyn, Rochelleen_ZA
dc.contributor.otherStellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology.en_ZA
dc.date.accessioned2022-02-18T12:04:16Z
dc.date.accessioned2022-04-29T09:34:56Z
dc.date.available2022-02-18T12:04:16Z
dc.date.available2022-04-29T09:34:56Z
dc.date.issued2022-04
dc.descriptionThesis (MScAgric)--Stellenbosch University, 2022.en_ZA
dc.description.abstractENGLISH 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.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Sitrus virus A (CiVA) is ontdek met die gebruik van hoë-deurset volgordebepaling gebaseerde opsporing (HTS). CiVA is ‘n lid van die spesie Coguvirus eburi en is nouverwant aan sitrus-konkaaf gom- geassosieerde virus (CCGaV), ‘n lid van die spesie Citrus coguvirus. Beide virusse is lede van die genus Coguvirus wat deel is van die familie Phenuiviridae in die orde Bunyavirales. CiVA is ‘n negatiewe-string RNA virus en besit twee genome. Die RNA 1 genoom kodeer vir die RNA-afhanklike RNA polimerase (RdRp) en die RNA 2 genoom kodeer vir ‘n nukleokapsiedproteïen (NP) en ‘n vermeende bewegingsproteïen (MP). Die bevestiging van die teenwoordigheid van CiVA in variëteite van pomelo (Citrus paradisi Macf.), soetlemoene (C. sinensis (L.) Osb.) asook clementine (C. reticulata Blanco) in Suid-Afrika, het gelei na ‘n opname in drie provinsies en sewe sitrus produksie gebiede. In drie sitrus produksie gebiede is CiVA in soetlemoen bome met vrugskil simptome soortgelyk aan sitrus- impietraturasiekte geïdentifiseer. CiVA is ook in soetlemoen bome met geen vrugsimptome geïdentifiseer. Pomelo bome met tipiese sitrus-impietraturasiekte simptome was geïnfekteer met CiVA, maar pomelo bome met geen simptome in dieselfde sitrus boord was vry van CiVA. Clementine bome met geen vrugskil simptoom was ook geïnfekteer met CiVA. Die nukleotied (nt) volgorde van die drie proteïne van CiVA was bepaal met behulp van Sanger-volgorde bepaling om die genetiese diversiteit tussen die isolate van die ses produksie gebiede en drie sitrus tipes te ondersoek. Filogenetiese analises van die nt volgorde van die drie proteïne was uitgevoer deur die konstruksie van ‘n maksimum waarskynlikheid (ML) filogenetiese boom. Additionele nukleotied identiteit matrikse was ook uitgevoer. Filogenetiese analise en nt identiteit matrikse het 'n hoër genetiese diversiteit binne die NP aangedui as in die MP en RdRp. Meer genetiese diversiteit is waargeneem tussen isolate afkomstig van die drie sitrus spesies as tussen isolate afkomstig van die verskillende sitrus produksie gebiede. ‘n Tru-transkripsie polimerase-kettingreaksie (RT-PCR) opsporings toets, wat in werklike tyd waargeneem kan word, is ook ontwikkel vir die gelyktydige identifikasie van CCGaV en CiVA. Twee cDNA sintese metodes vir tru-transkripsie was gevergelyk en ‘n gedegenereerde omgekeerde inleier met dubbele inleier aktiwitiet (DPO) is ontwerp om die spesifisiteit van die opsporingstoets te verbeter. Twee PCR toetse wat gebruik maak van die DPO omgekeerde inleier en twee verskillende vorentoe inleiers was gevergelyk tydens amplifikasie. Die cDNA sintese metode en keuse van inleier paar het 'n invloed gehad op die amplifikasie doeltreffendheid, spesifisiteit en sensitiwiteit. 'n Weefselspesifisiteitstoets is uitgevoer en CiVA is regdeur die plant opgespoor in die hoofaar van die blaar, blaarskyf, groen bas en wortels. Laer Ct-waardes was geassosieer met die groen bas en hoofaar van die blaar. Die opsporingstoets is geïmplementeer in die sitrusverbeteringskema (CIS) van Suid- Afrika om die vrystelling van CiVA vrye plant materiaal aan die sitrusbedryf te verseker.af_ZA
dc.description.versionMastersen_ZA
dc.format.extentix, 87 pages : illustrations (some color)en_ZA
dc.identifier.urihttp://hdl.handle.net/10019.1/124816
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subjectCitrus -- Breeding -- South Africaen_ZA
dc.subjectCitrus -- Virus diseasesen_ZA
dc.subjectCitrus virus A (CiVA)en_ZA
dc.subjectCitrus fruits -- Disease and pest resistance -- Genetic aspectsen_ZA
dc.subjectCoguvirus eburi -- Virus -- Detectionen_ZA
dc.subjectCitrus Improvement Schemeen_ZA
dc.subjectUCTDen_ZA
dc.titleDetermining the distribution and genetic diversity of Coguvirus eburi in South African citrus and development of a citrus-infecting coguvirus detection assayen_ZA
dc.typeThesisen_ZA
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