Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: The nepoviruses are a group of nematode-transmitted plant viruses that are distributed worldwide and infect a wide range of plant species, including grapevine. Most of the nepoviruses are foreign to South Africa and to date, only Grapevine fanleaf virus (GFLV) is present. The Department of Agriculture, Forestry and Fisheries (DAFF), as the official National Plant Protection Organisation (NPPO) of South Africa, is committed to prevent the importation and spread of plant pathogens by administering the Agricultural Pests Act, 1983 (Act No. 36 of 1983). Effective measures are implemented by which the introduction of agricultural pests may be prohibited to safeguard the agricultural environment. One of the core functions of DAFF is to render a routine plant health diagnostic service for imported plants and plant products to prevent exotic pathogens from entering the country. The objective of this study was to develop a diagnostic assay for the detection of nepoviruses in grapevine. The project aimed to produce antibodies by recombinant DNA technology against bacterially expressed viral coat protein of a specific nepovirus [Tomato ringspot virus (ToRSV)] and subsequently develop a DAS-ELISA (Double Antibody Sandwich Enzyme-linked Immunosorbent) assay for the detection of the virus. The coat protein (CP) was successfully isolated from imported ToRSV-infected grapevine material. Two expression systems were utilised for expression of the ToRSV-CP, the GST gene fusion system and an Agrobacterium-mediated expression system. The GST gene fusion system was unsuccessful as insufficient soluble protein expression prevented the production of antibodies and thus the development of the DAS-ELISA assay. Tissue print immunoassay (TPIA) initially showed positive results for transient expression of the fusion protein in tobacco plants, but further confirmation proved to be inconclusive. The project also aimed to develop a real-time PCR assay for the specific detection and relative quantification of GFLV, based on a conserved region of the RNA-2 genome. A partial GFLV-RNA-2 from a South African isolate of grapevine was sequenced and used for the design of specific primers. The quantitative real-time PCR assay based on SYBR green technology proved to be sensitive in detecting levels as low as 0.11ng/reaction in infected plants, making it a highly effective diagnostic tool for the detection of GFLV. / AFRIKAANSE OPSOMMING: Die nepovirusse is 'n groep van nematode-oordraagbare plant virusse wat wêreldwyd versprei word en 'n wye verskeidenheid van plantspesies infekteer, insluitend wingerd. Die meeste van die nepovirusse is uitheems aan Suid-Afrika en tot op datum is net Wingerd netelblaar virus (GFLV) teenwoordig. Die Departement van Landbou, Bosbou en Visserye (DAFF), as die amptelike Nasionale Plant Beskermings Organisasie (NPBO) van Suid-Afrika, is daartoe verbind om die invoer en verspreiding van plantpatogene te voorkom deur administrasie van die Wet op Landbouplae, 1983 (Wet No. 36 van 1983). Doeltreffende maatreëls word geïmplementeer waardeur die invoer van landbouplae verbied word om sodoende die landbou-omgewing te beskerm. Een van die kernfunksies van DAFF is om 'n roetine plant gesondheid diagnostiese diens vir ingevoerde plante en plantprodukte te lewer om te verhoed dat eksotiese patogene die land binnedring. Die doel van hierdie studie was om 'n diagnostiese toets vir die opsporing van nepovirusse in wingerd te ontwikkel. Die projek was daarop gemik om antiliggame te vervaardig deur rekombinante DNA-tegnologie teen bakterieël-uitgedrukte virale mantelproteïen van 'n spesifieke nepovirus [Tomato ringspot virus (ToRSV)] en vervolgens ‘n DASELISA (Double Antibody Sandwich Enzyme-linked Immunosorbent) toets vir die opsporing van die virus te ontwikkel. Die mantelproteïen (CP) is met sukses geïsoleer vanaf ingevoerde ToRSV-besmette wingerdmateriaal. Twee uitdrukking stelsels is gebruik vir uitdrukking van die ToRSV-CP, die “GST gene fusion” stelsel en 'n Agrobacterium-bemiddelde uitdrukking stelsel. Die “GST gene fusion” stelsel was egter onsuksesvol aangesien onvoldoende oplosbare proteïen uitdrukking die produksie van antiliggame en dus die ontwikkeling van die DAS-ELISA toets verhoed het. “Tissue print immunoassay” (TPIA) het aanvanklik positiewe resultate getoon vir tydelike uitdrukking van die fusie proteïen in tabakplante, maar verdere bevestiging was onoortuigend. Die projek was ook daarop gemik om ‘n in-tyd polimerase ketting reaksie (PKR) toets vir die spesifieke opsporing en relatiewe kwantifisering van GFLV, gebaseer op 'n gekonserveerde volgorde van die RNA-2 genoom, te ontwikkel. 'n Gedeeltelike GFLV-RNA-2 nukleïensuurvolgorde van 'n Suid-Afrikaanse wingerd isolaat is bepaal en gebruik vir die ontwerp van spesifieke inleiers. Die kwantitatiewe in-tyd PKR toets gebaseer op SYBR groen tegnologie was sensitief genoeg om vlakke van so laag as 0.11ng/reaksie in geïnfekteerde plante op te spoor, wat dit 'n hoogs effektiewe diagnostiese hulpmiddel vir die opsporing van GFLV maak.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: The aim of this study was to combine two common diagnostic tools: serological kits and genetic fingerprinting to identify cherry leaf-roll nepovirus (CLRV), and to establish a marker system to characterize walnut germplasm. The detection of plant viruses is difficult. Restrictions are imposed for quarantine purposes on the importation of plant material from foreign countries. Modern techniques such as a PCR based screening method for CLRV are required to ensure material do not harbour viruses. A primer pair was designed to amplify a 430 bp non-coding homologous region. For the choice of primers, consensus sequences were considered and areas where the sequence data shared 98.5% homology, were chosen. The sensitivity of this detection method was 100-fold higher when compared to the ELISA. The PCR fragment was verified by nucleotide sequencing. AFLP technology was used to identify polymorphic fragments for 6 walnut cultivars and a rootstock, and SCARs were developed from AFLP specific bands. The AFLP technique distinguished all the walnut cultivars and the rootstock. However, conversion of AFLP fragments to SCAR markers for the development of a simple robust technique for cultivar discrimination, was not successful. Using 27 AFLP primer combinations, polymorphic fragments as high as 47.8% were scored. The reason for the lack of efficient conversion was as the result of the AFLP technique. The SCAR primers were generated from sequences internal to the AFLP primers but the specificity of the markers was in the AFLP primers not the internal sequence. In this study using AFLP, walnut cultivars were found to be closely related. The AFLP primer pairs used, provided polymorphic fragments. From these fragments, 7 SCAR markers were developed. It was expected that these SCARs derived from the AFLP markers would detect slight differences between cultivars. The Paradox SCAR marker was the only one that could divide the cultivars into two groups. When Chandler SCAR products were digested with the restriction enzyme Rsal, the same banding pattern as that of Paradox SCAR products was observed. / AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om twee algemene opsporingstegnieke te kombineer: serologiese toetsstelle en genetiese vingerafdrukke om cherry leaf-roll nepovirus (CLRV) te eien en om In merkersisteem te ontwikkel wat okkerneut kiemplasma kan karakteriseer. Die opsporing van plant virusse is baie moeilik. As gevolg van kwarantyn vereistes, word daar beperkinge geplaas word op die invoer van plant materiaal vanuit die buiteland. Moderne tegnieke soos hierdie een wat op PKR berus, word benodig om te verseker dat CLRV nie in plantmateriaal teenwoordig is nie. In Stel inleiers is ontwerp wat In 430 bp nie-koderende homoloë area amplifiseer. Hiervoor is konsensus volgordes bestudeer en slegs die volgordes wat 98,5% homologie getoon het, is gekies. In vergelyking met ELISA was die sensitiwiteit van hierdie deteksie metode 100 maal beter. DNA volgordebepaling is op die resulterende fragment gedoen om die PKR produk te verifieer. AFLP tegnologie is gebruik om polimorfiese fraqmente vir 6 okkerneut kultivars en 'n onderstok te identifiseer en SCARs is uit hierdie fragmente ontwikkel. Die AFLP tegniek kon tussen al die okkerneut kultivars en die onderstok onderskei. Die omskakeling van die AFLP fragmente in SCAR merkers om sodoende In eenvoudige kragtige tegniek vir kultivar onderskeiding te ontwikkel, was egter nie suksesvol nie. Met die gebruik van 27 AFLP inleier kombinasies, kon polimorfiese fragmente van so hoog as 47.8% verkry word. Die rede hoekom omskakeling onsuksesvol was lê by die aard van die AFLP tegniek. Die SCAR inleiers is ontwikkel uit volyordes intern tot die AFLP inleiers, maar die spesifisiteit van die merkers het juis in die AFLP inleiers gelê en nie in die interne volgordes nie. In hierdie studie, met die gebruik van AFLP, is gevind dat okkerneut kultivars baie naby verwant is. Die AFLP inleierstelle wat gebruik is, het polimorfiese fragmente gelewer. Uit hierdie fragmente is 7 SCAR merkers ontwikkel. Daar is verwag dat die SCARs wat uit die AFLP merkers ontwikkel is, klein verskille tussen kultivars sou opspoor. Dit was egter net die Paradox SCAR merker wat die kultivars in twee groepe kon verdeel. Restriksie ensiem vertering met Rsalop die Chandler SCAR produkte het dieselfde bandpatrone as die van die Paradox SCAR produkte gelewer.