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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/96964 |
| Date | 12 1900 |
| Creators | Frazenburg, Lolita |
| Contributors | Burger, Johan T., Souza Richards, Rosineide, Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | xvi, 121 pages : illustrations |
| Rights | Stellenbosch University |
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