Please read the abstract in the section 00front of this document / Dissertation (Magister Istitutiones Agrariae)--University of Pretoria, 2006. / Plant Production and Soil Science / unrestricted

Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Potato Virus Y (PVY) and Potato Leafroll Virus (PLRV) are two of the most destructive potato viruses capable of drastically diminishing crop yields by up to 80%. The presence of these viruses in planting material namely seed potato stocks are routinely diagnosed by enzyme-linked immunosorbent assay (ELISA) kits. The kits currently used by Potatoes South Africa are obtained from Europe. These kits have produced false positive and false negative results in the past. Potatoes South Africa required an ELISA that was reliable, cheap and specific for the detection of South African strains of the two respective viruses. In this study the viral coat protein genes were amplified by RT-PCR from a South African source of infected plant material. The PVY and PLRV coat protein genes were subsequently cloned into pGEM-T Easy vector and sequenced. The sequences of the two viruses were aligned and compared to corresponding viral coat protein gene sequences obtained from Genbank. Subsequently the two amplified and cloned coat protein genes of PVY and PLRV were sub-cloned into an expression system (pET-14b) to induce and express the respective recombinant viral coat proteins. The induction of the cloned coat protein genes yielded successful production of the recombinant PVY coat protein but the induction and expression of the recombinant PLRV coat protein was unsuccessful. The isolated recombinant PVY CP was then used to immunize a rabbit to produce highly specific anti-PVY CP immunoglobulins. The antiserum obtained from the rabbit was used to develop an ELISA to detect the presence of PVY in seed potato stocks in South Africa. The ELISA kit was subsequently used in preliminary trials to determine if the kit could detect PVY infected plant material. The initial results of the ELISA trials using PVY infected material obtained from Potatoes South Africa yielded positive results. / AFRIKAANSE OPSOMMING: Aartappel Virus Y (PVY) en Aartappel Rolblad Virus (PLRV) is twee van die mees vernietigende aartappel virusse wat ‘n oes tot 80% kan verlaag. Virus infeksie van plant materiaal tewete aartappelmoere word deur “enzyme-linked immunosorbent assay” (ELISA) toetsstelle bevestig. Die toetsstelle wat op die oomblik gebruik word deur Aartappels Suid- Afrika word in Europa vervaardig. Hierdie toetsstelle het vals positiewe en vals negatiewe resultate in die verlede gegee. Aartappels Suid-Afrika benodig toetsstelle wat betroubaar, goedkoop en spesifiek vir Suid-Afrikaanse virus stamme is. In hierdie studie is besmette plantmateriaal vanuit Suid-Afrika gebruik vir die amplifisering van virale mantel proteïen gene met behulp van RT-PCR. Die PVY en PLRV mantel proteïen gene was daarna in die pGEM-T Easy vektor gekloneer en nukleotied volgordes is bepaal. Die nukleotied volgordes is met ander PVY en PLRV gene vanaf Genbank vergelyk. Die twee ge-amplifiseerde en gekloneerde mantel proteïen gene van PVY en PLRV is uitgesny en gekloneer in ‘n ekspressie sisteem (pET-14b) om die mantel proteïen te produseer. Induksie van die gekloneerde mantel proteïen gene het gelei tot die suksesvolle produksie van ‘n PVY mantel proteïen, maar produksie van die PLRV mantel proteïen was onsuksesvol. Die geïsoleerde PVY mantel proteïen is vervolgens gebruik vir die immunisering van ‘n konyn vir die produksie van konyn anti-PVY antiliggame. Die antiserum verkry vanaf die konyn is gebruik vir die ontwikkeling van ‘n ELISA vir die identifisering van PVY infeksies in aartappelmoere. Voorlopige proewe is deurgevoer om te bepaal of hierdie ELISA PVY infeksies in plantmateriaal sou kon opspoor. Aanvanklike resultate toon dat die ELISA suksesvol PVY infeksies in plantmateriaal verkry vanaf Aartappels Suid-Afrika kan opspoor.

Thesis (MSc)--University of Stellenbosch, 2008. / ENGLISH ABSTRACT: Potato virus Y (PVY) is responsible for considerable yield losses in the South African potato industry. The incidence of this virus has greatly increased over the past few years. Even more worrying is the variation of symptoms observed during PVY infection and the recent appearance of the more virulent PVYNTN strain in local fields. This project aimed to investigate the possible genetic variation within the viral genome and to establish the origin of strains. The project also aimed to establish a dependable, area specific enzyme-linked immunosorbent assay (ELISA) to replace the currently used ELISAs. Currently seed potato certification is done using ELISA kits imported from Europe. These kits were developed for the detection of overseas variants of PVY and the use thereof in South Africa has in the past lead to false negatives. Finally, this project set out to develop, optimize and establish a sensitive and reliable real-time reverse transcriptase polymerase chain reaction (qRT-PCR) detection protocol for PVY. In the first part of the study the coat protein (CP) gene of PVY isolates from plant material obtained from various parts of South Africa was amplified using RT-PCR. The resulting cDNA was then sequenced directly or cloned into a vector and then sequenced. The resulting sequences were aligned in a data matrix with international reference sequences, analyzed and grouped according to strain. Examination of the CP gene within this matrix as well as phylogenetic analysis revealed six main groups of PVY. These six groups included the traditional PVYN and PVYO groups and a recombinant group. Furthermore it also revealed variants of PVYN and PVYO. These mutants and recombinants pose a threat as they may lead to South African strains of PVY expressing coat proteins which vary from those found overseas. This may render the currently used European ELISA method of detection less effective and subsequently result in an increase in viral prevalence. This reinforced the need for a detection method based on local viral strains. Phylogenetic and Simplot analysis also confirmed that a recombinant strain between PVYN and PVYO had evolved and that PVYNTN was such a recombinant. The second part of the study aimed to develop and establish detection methods based on local variants of PVY. This included the development of ELISA and qRT-PCR detection methods of PVY. Previously amplified cDNA of the PVY CP gene was cloned into an expression vector and successfully expressed. Antibodies produced against the recombinant protein, when used in ELISA, however, failed to achieve the required levels of sensitivity. This prompted the development of qRT-PCR detection methods for PVY. Primer combinations for PVY were designed using the previously established CP gene data matrix. A reliable and sensitive SYBR® Green I based qRT-PCR assay was developed for the detection of PVY. The assay effectively detected all known South African variants of PVY. Furthermore, a Taqman® assay was developed for the detection of all variants of PVY. The Taqman® assay was 10 fold less sensitive and does not allow for amplicon verification through melting curve analysis, but it does add more specificity due to the addition of the probe. Although these qRT-PCR detection methods are still too expensive to replace the routine diagnostics done with ELISA, they do offer the opportunity to screen valuable mother material and confirm borderline cases in seed certification. / AFRIKAANSE OPSOMMING: Aartappel virus Y (PVY) is verantwoordelik vir aansienlike opbrengsverliese in die Suid-Afrikaanse aartappelindustrie. Die insidensie van infeksie deur die virus het drasties toegeneem oor die afgelope jare. Wat egter meer kommerwekkend is, is die groter variasie in simptome van PVY infeksie en die onlangse voorkoms ‘n meer virulente ras, PVYNTN. Hierdie projek poog om moontlike genetiese variasie van PVY te ondersoek en om die oorsprong van rasse op te spoor. Die projek het ook gepoog ook om ‘n bruikbare, betroubare en area spesifieke “enzyme-linked immunosorbent assay” (ELISA) toets te ontwikkel om die huidige ingevoerde ELISA te vervang. Hierdie toetse is ontwikkel om oorsese variante van PVY op te spoor en die gebruik daarvan het in die verlede gelei tot vals negatiewes. Verder is daar ook ondersoek ingestel na die ontwikkeling van ‘n sensitiewe en betroubare “real-time reverse transcriptase polymerase chain reaction” (qRT-PCR) protokol vir die opsporing van PVY. In die eerste deel van die studie is die mantelproteïen geen van PVY isolate vanuit plant materiaal geamplifiseer deur die gebruik van RT-PCR. Hierdie materiaal is vanaf verskeie streke in Suid-Afrika ontvang. ‘n Volgordebepalingsreaksie is uitgevoer op gekloneerde of ongekloneerde cDNA verkry uit die RT-PCR. DNA volgordes is in ‘n data matriks geplaas en vergelyk met internationale volgordes om die plaaslike isolate te analiseer en te groepeer. Deur vergelyking en filogenetiese ontleding kon ses hoofgroepe van PVY geïdentifiseer word, wat tradisionele PVYN en PVYO, sowel as ‘n rekombinante ras en variante binne die tradisionele PVYN en PVYO groepe ingesluit het. Rekombinante en mutante kan veroorsaak dat Suid-Afrikanse rasse van PVY mantelproteïene uitdruk wat afwyk van die oorsese rasse wat tot gevolg mag hê dat die ELISAs van oorsee minder effektief kan wees en kan lei tot verhoogde virus voorkoms. Die realiteit en gevaar versterk die gedagte dat ‘n deteksie metode gebaseer op plaaslike virusse absoluut krities is. Filogenetiese sowel as Simplot analise het bevestig dat ’n mutante ras tussen PVYN en PVYO ontstaan het en dat PVYNTN ’n rekombinante ras is. Die tweede deel van die studie was daarop gemik om deteksie metodes te ontwikkel wat gebaseer was op plaaslike variante van PVY. Dit sluit die ontwikkeling van ELISA sowel as qRT-PCR deteksie van PVY in. Voorheen geamplifiseerde cDNA is in ‘n ekspressievektor gekloneer en suksesvol uitgedruk. Teenliggaampies teen die rekombinante proteïen, indien in ELISA aangewend, kon egter nie die nodige sensitiwiteit oplewer nie. Dit het aanleiding gegee tot ontwikkeling van qRT-PCR deteksie metodes. Inleier kombinasies vir PVY was ontwikkel deur die gebruik van die bestaande mantelproteïen geen data matrikse. ‘n Betroubare en sensitiewe SYBR® Green I qRT-PCR deteksie protokol was ontwikkel vir die effektiewe deteksie van alle bekende Suid-Afrikanse rasse van PVY. Verder is ‘n sogenaamde “Taqman®” protokol ook ontwikkel vir deteksie van alle rasse. Die “Taqman®” protokol was 10 voudiglik minder gevoelig and laat nie bevestiging deur smeltkurwe analise toe nie, maar verleen meer spesifisiteit deur die toevoeging van die “Taqman® probe”. Hierdie qRT-PCR deteksie metodes is tans te duur om as roetine diagnostiese toetse te gebruik en kan dus nie ELISA vervang nie, maar hulle bied wel die geleentheid om waardevolle moeder materiaal te toets en grensgevalle in aartappelsaad sertifisering te bevestig.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Potato virus Y (PVY) is responsible for considerable yield losses in the South African potato industry. The incidence of this virus has greatly increased over the past 20 years. In previous studies nonrecombinant strains of PVY, PVY N and PVY O, were detected in South African potatoes. In a recent study the occurrence of non-recombinant strains of PVY in South African potatoes was shown to have decreased while infection by more virulent recombinant strains, PVY NTN and PVY N-W, had increased dramatically. Infection of potato plants with PVY may cause stunted growth and mosaic or necrotic leaf symptoms which in turn can lead to a significant reduction in yield. Highly virulent recombinant PVY isolates as well as some of the non-recombinant strains may cause potato tuber necrotic ringspot disease (PTNRD) which may result in losses of 10% to total crop failure. For this reason investigation of infection by local recombinant isolates on local cultivars was important. To this end a representative number of isolates were selected for whole genome sequencing based on the relative occurrence of the various isolates in South Africa. A number of these sequenced isolates were subsequently used to infect local cultivars of potato in order to investigate the influence of genetic variation within the viral genome on symptom expression. In this study 27 South African isolates of PVY were sequenced through overlapping RT-PCR fragments. Seven of these isolates, six PVY NTN and one PVY N-W, were used to mechanically infect four local cultivars of potatoes under greenhouse conditions. The infected plants were monitored to establish the rate of systemic spread using a highly sensitive qRT-PCR and resulting tubers were visually screened for PTNRD. Highly variable recombinant isolates appear to be less virulent than the more conserved recombinant isolates possibly indicating molecular determinants for pathogenicity. For this reason the amino acid sequences of the South African isolates were compared to those of international isolates and scrutinized for variation and substitutions. Some South African isolates displayed amino acid substitutions unique to the specific isolate, making them unlike those found internationally. Substitution rates throughout the amino acid sequences differed greatly, with some isolates displaying hardly any changes whilst others varied a great deal from overseas isolates. Certain regions, many of which had specific functions, were more conserved than others. This study further investigated the recombination events within the PVY genome using reticulate phylogenetic analysis, molecular dating and network construction techniques. Unlike existing approaches, the one described in this study neither assumes an underlying strictly bifurcating species tree nor assumes prior knowledge of processes underlying deviations between individual gene trees. Through the use of the resulting robust time calibrated phylogeny, the patterns of diversification and recombination in PVY may be placed in the historical context of human cultivation of potatoes. Through the use of these techniques the study aimed to test whether diversification of the major strains of PVY and recombination between them occurred within the time frame of the domestication and modern cultivation of potatoes. From these analyses it can be deduced that recombinant strains of PVY were imported into South Africa. / AFRIKAANSE OPSOMMING: Aartappel virus Y (PVY) is verantwoordelik vir aansienlike opbrengs verliese in die Suid-Afrikaanse aartappelbedryf. Die voorkoms van die virus het grootliks toegeneem oor die afgelope 20 jaar. In vorige studies is nie-rekombinante rasse van PVY, PVY N en PVY O, gedokumenteer in Suid-Afrikaanse aartappels. 'n Onlangse studie het gevind dat die voorkoms van nie-rekombinante rasse van PVY in Suid- Afrikaanse aartappels aansienlik gedaal het terwyl infeksie deur virulente rekombinante rasse, PVY NTN en PVY N-W, dramaties toegeneem het. Infeksie van aartappelplante met PVY kan vertraagde groei en mosaïek- of nekrotiese blaarsimptome veroorsaak wat kan lei tot aansienlike vermindering in opbrengs. Hoogs virulente rekombinante PVY isolate, sowel as sommige nie-rekombinante rasse, kan aartappel nekrotiese ring simptome (PTNRD) veroorsaak wat verliese van 10% tot totale misoes tot gevolg kan hê. Om hierdie rede was die ondersoek van infeksie deur plaaslike rekombinante isolate op plaaslike kultivare belangrik. Vir hierdie doel is 'n verteenwoordigende aantal isolate gekies, gebaseer op die relatiewe voorkoms daarvan in Suid-Afrika, vir heelgenoom-volgordebepaling. Van die isolate is vervolgens gebruik om plaaslike kultivare te besmet ten einde die invloed van genetiese variasie binne die virale genoom op simptoom uitdrukking te ondersoek. In hierdie studie is 27 heelgenoomvolgordes van Suid-Afrikaanse PVY isolate bepaal deur oorvleuelende RT-PCR fragmente. Sewe van hierdie isolate, ses PVY NTN en een PVY N-W, is gebruik om vier plaaslike aartappel kultivare, gegroei onder kweekhuis kondisies, meganies te infekteer. Die geïnfekteerde plante is gemonitor om die tempo van sistemiese verspreiding vas te stel deur middel van 'n hoogs sensitiewe qRTPCR en knolle is visueel inspekteer vir PTNRD. Hoogs variante rekombinante isolate blyk om minder virulent te wees as die meer bewaarde rekombinante isolate wat dui op molekulêre determinante van patogenisiteit. Om hierdie rede is die aminosuurvolgordes van die Suid-Afrikaanse isolate vergelyk met die van internasionale isolate en ondersoek vir variasie en substitusies. Sommige Suid-Afrikaanse isolate vertoon aminosuur substitusies wat uniek is tot die spesifieke isolaat en maak hul dus anders as internasionale isolate. Die aantal aminosuursubstitusies in die volgordes verskil grootliks. In vergelyking met internasionale isolate toon sommige isolate skaars enige veranderinge terwyl ander ‘n aantal verskille toon. Sekere gebiede, waarvan baie spesifieke funksies het, was meer gekonserveerd as ander. Hierdie studie ondersoek ook rekombinasie gebeure binne die PVY genoom deur retikulêre filogenetiese analise, molekulêre datering en netwerk konstruksie tegnieke. In teenstelling met bestaande benaderinge, aanvaar die tegniek wat hier beskryf word nie ‘n streng bifurkeerende filogenie, wat onderliggende verdeel, of enige voorafgaande kennis van die prosesse onderliggend aan afwykings tussen individuele filogenieë nie. ‘n Robuuste, tyd gekalibreer filogenie kan diversifikasie patrone en rekombinasie van PVY plaas in die historiese konteks van menslike verbouing van aartappels. Deur gebruik te maak van hierdie tegnieke poog die studie om te toets of diversifikasie en rekombinasie van PVY rasse plaasgevind het binne die tydsbestek van die inburgering en moderne verbouing van aartappels. Van hierdie ontledinge word afgelei dat rekombinante rasse van PVY wat in Suid-Afrika voorkom, ingevoer is.

Sweet potato (Ipomoea batatas Lam.) is ranked as the seventh most important food crop in the world and its large biomass and nutrient production give it a unique role in famine relief. However, multiple virus infection is the main disease limiting factor in sweet potato production worldwide. The main objective of this research project was to develop a transgenic sweet potato cultivar with broad virus resistance in South Africa (SA). A review of current literature assembled background information pertaining to the origin, distribution and importance of the sweet potato crop; viruses and complexes infecting sweet potato; and the strategies used in sweet potato virus detection and control. A survey to determine the occurrence and distribution of viruses infecting sweet potato (Ipomoea batatas Lam.) was conducted in major sweet potato-growing areas in KwaZulu-Natal (KZN). A total of 84 symptomatic vine samples were collected and graft inoculated onto universal indicator plants, Ipomoea setosa Ker. and Ipomoea nil Lam. Six weeks post inoculation, typical sweet potato virus-like symptoms of chlorotic flecking, severe leaf deformation, stunting, chlorotic mosaic, and distinct interveinal chlorotic patterns were observed on indicator plants. Under the transmission electron microscope (TEM), negatively stained preparations of crude leaf sap and ultra-thin sections from symptomatic grafted I.setosa plants revealed the presence of elongated flexuous particles and pinwheel type inclusions bodies‟ that are characteristic to the cytopathology of Potyviruses. Symptomatic leaf samples from graft-inoculated I. setosa and I. nil were assayed for Sweet potato feathery mottle virus (SPFMV), Sweet potato mild mottle virus (SPMMV), Sweet potato chlorotic stunt virus (SPCSV), Sweet potato chlorotic fleck virus (SPCFV), Sweet potato virus G (SPVG), Sweet potato mild speckling virus (SPMSV), Sweet potato caulimo-like virus (SPCaLV), Sweet potato latent virus (SPLV), Cucumber mosaic virus (CMV), and Sweet potato C-6 virus (C-6) using the nitrocellulose membrane enzyme-linked immunosorbent assay (NCM-ELISA). The majority of leaf samples (52%) tested positive for virus disease and showed the occurrence of SPFMV, SPMMV, SPCSV, SPCFV, SPVG, SPMSV, and SPCaLV. Of these 7 viruses, the most frequently detected were SPFMV (39%), SPVG (30%), followed by SPCSV (13%) and SPMMV (12%). SPCaLV and SPCFV at 10% and SPMSV at 7% were found exclusively in samples collected from one area. SPFMV, SPVG, SPCSV, and SPMMV were identified as the most prevalent viruses infecting sweet potato in KZN. The genetic variability of the three major viruses infecting sweet potato (Ipomoea batatas Lam.) in KZN was determined in this study. A total of 16 virus isolates originating from three different locations (Umbumbulu, Umfume and Umphambanyomi River) in KZN were analyzed. These comprised of 10 isolates of Sweet potato feathery mottle virus (SPFMV), five isolates of Sweet potato virus G (SPVG) and one isolate of Sweet potato chlorotic stunt virus (SPCSV). The phylogenetic relationships of the SPFMV, SPVG and SPCSV isolates from KZN relative to isolates occurring in SA and different parts of the world were assessed. The division of SPFMV into four genetic groups (strains) according to the phylogenetic analysis of coat protein encoding sequences revealed mixed infections of the O (ordinary) and C (common) strains in sweet potato crops from KZN. All SPFMV isolates showed close lineage with isolates from South America, East Asia and Africa. The SPVG isolates showed high relatedness to each other and close lineage with other isolates, especially those from China and Egypt. Analysis of the partial sequence of the Heat shock protein 70 homologue (Hsp70h) gene indicated that the SPCSV isolate from KZN belongs to the West African (WA) strain group of SPCSV and showed close relatedness to an isolate from Argentina. The knowledge of specific viral diversity is essential in developing effective control measures against sweet potato viruses in KZN. Multiple virus infections of Sweet potato feathery mottle virus (SPFMV), Sweet potato chlorotic stunt virus (SPCSV), Sweet potato virus G (SPVG) and Sweet potato mild mottle virus (SPMMV) cause a devastating synergistic disease complex of sweet potato (Ipomoea batatas Lam.) in KZN. In order to address the problem of the multiplicity and synergism of sweet potato viruses in KZN, this study aimed to develop transgenic sweet potato cv. Blesbok with broad virus resistance. An efficient and reproducible plant regeneration protocol for sweet potato (Ipomoea batatas Lam.) cultivar Blesbok was also developed in this study. The effect of different hormone combinations and type of explants on shoot regeneration was evaluated in order to optimize the regeneration protocol. Coat protein (CP) gene segments of SPFMV, SPCSV, SPVG and SPMMV were fused to a silencer DNA, the middle half of the nucleocapsid (N) gene of Tomato spotted wilt virus (TSWV) and used as a chimeric transgene in a sense orientation to induce gene silencing in the transgenic sweet potato. Transformation of apical tips of sweet potato cv. Blesbok was achieved by using Agrobacterium tumefaciens strain LBA4404 harboring a modified binary vector pGA482G carrying the plant expressible neomycin phosphotransferase ll gene (nptll), the bacterial gentamycin-(3)-N-acetyl-transferase gene and the expression cassette. A total of 24 putative transgenic plants were produced from the transformed apical tips via de novo organogenesis and regeneration into plants under 50mg/L kanamycin and 200 mg/L carbenicillin selection. Polymerase chain reaction (PCR) and Southern blot analyses showed that six of the 24 putative transgenic plants were transgenic with two insertion loci and that all plants were derived from the same transgenic event. The six transgenic sweet potato plants were challenged by graft inoculation with SPFMV, SPCSV, SPVG and SPMMV- infected Ipomoea setosa Ker. Although virus presence was detected using NCM-ELISA, all transgenic plants displayed delayed and milder symptoms, of chlorosis and mottle of lower leaves when compared to the untransformed control plants. These results warrant further investigation under field conditions. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Thesis (MSc (Genetics))--Stellenbosch University, 2008. / South Africa is one of the top ten wine producing countries in the world. The South African wine industry contributes approximately R16.3 billion to South Africa’s annual gross domestic product with 42.8% of wine being exported. To compete with the top wine producing countries and to ensure a viable export market, South Africa needs to ensure that healthy, virus free propagation material is produced and sold. One of the viruses that need to be tested for is Grapevine fanleaf virus (GFLV). Grapevine fanleaf virus causes degeneration and malformation of berries, leaves and canes and is responsible for significant economic losses by reducing crop yields by as much as 80%, reducing the longevity of the vines and affecting fruit quality. It is widespread in the Breede River Valley of the Western Cape where the nematode vector, Xiphinema index, is prevalent. The Breede River Valley contributes approximately 30% of the total production of the local wine industry, and severe losses in this region could threaten the viticulture. The Plant Improvement Act states that all propagation material sold must be tested for GFLV by a reputable scientific technique. The technique commonly used in South Africa is the Double Antibody Sandwich - Enzyme-linked Immunosorbent Assay (DAS-ELISA) and the kits are imported from Europe at a significant cost to the South African viticulture industry. The objective of this study was to produce a reliable and sensitive diagnostic assay specific for the South African strains of GFLV. This project aimed to develop and optimize a DAS-ELISA, by using recombinant DNA technology to produce antibodies against bacterially expressed viral coat protein. Total RNA was extracted from GFLV infected grapevine material and the viral coat protein (CP) amplified. The CP was cloned into the pGex-6P-2 expression vector, fusing a Glutathione STransferase (GST) partner to the viral coat protein enhancing solubility and protein purification. Insufficient amounts of the soluble protein were expressed and purified, preventing the production of antibodies and thus the development of the DAS-ELISA. An alternative diagnostic rapid-direct-one-tube-RT-PCR assay was developed. This rapid-directone- tube-RT-PCR assay was compared to commercially available DAS-ELISA and ImmunoStrip tests (Agdia) to assess the reliability, sensitivity and specificity of the rapid-direct-one-tube-RTPCR assay. Twelve GFLV isolates from South Africa were sequenced to investigate the variability between the isolates as well as the variability between the South African isolates and GFLV sequences available in Genbank. Sequence identities between clones from different GFLV isolates from South Africa were between 86-99% and 94-99% at the nucleotide and amino acid levels, respectively. Phylogenetic analysis based on the coat protein gene sequences showed that the South African isolates form two distinct clades or sub-populations. No significant correlation was found between geographical origin and symptoms, nor between geographical origin and sequence variability or between grapevine cultivar and symptom expression. Of the 23 samples tested with all three tests, 21 tested positive with rapid-direct-one-tube-RT-PCR, 19 with the ImmunoStrips and 17 with an imported DAS-ELISA kit (Agdia). Rapid-direct-one-tube-RT-PCR was found to be the most reliable technique for GFLV detection. Although the establishment of a DAS-ELISA directed to the South African strain(s) of GFLV was not successful, an alternative PCR based diagnostic system was developed, and proved to be sensitive and reliable. RT-PCR based diagnostic assays are generally accepted to be more sensitive than DAS-ELISA, but the latter is still used as the diagnostic assay of choice for routine testing due to ease of use. This rapid-direct-one-tube-RT-PCR assay is a rapid, sensitive and reliable diagnostic test, reducing the prevalence of false negatives, contributing to a virus free viticulture industry. The rapid-direct-one-tube-RT-PCR assay is as easy to use as DAS-ELISA, faster and can be performed by semi skilled workers, thus providing all the advantages associated with DAS-ELISA.