ENGLISH ABSTRACT: South Africa is regarded as one of the top wine producing countries in the world.
One of the threats to the sustainability of the wine industry is viral diseases of which
Grapevine leafroll-associated virus 3 (GLRaV-3) and Grapevine virus A (GVA) are
considered to be the most important and wide spread. Scion material is regularly
tested for viruses; however scion material is often grafted onto rootstocks that have
questionable phytosanitary status. Virus detection in rootstocks is challenging due to
low and varying titres, but is imperative as a viral control mechanism. An additional
viral control mechanism is the use of transgenic grapevine material which offers
resistance to grapevine infection.
The objective of this project was to establish a detection system using real time PCR
(qPCR) techniques, to accurately and routinely detect GLRaV-3 and GVA in
rootstock propagation material. qPCR would furthermore be used to perform
molecular characterisation of transgenic plants containing a GLRaV-3 antiviral
ΔHSP-Mut construct.
A severely infected vineyard (Nietvoorbij farm) in the Stellenbosch area was
screened throughout the grapevine growing season to investigate virus prevalence
throughout the season and to determine the optimal time for sensitive virus detection.
A large scale screening of nursery propagation material for GLRaV-3 infection was
also conducted. The qRT-PCR results were compared to DAS-ELISA results to
compare the efficacy and sensitivity of the two techniques. For the severely infected
vineyard, the ability to detect GLRaV-3 increased as the season progressed towards
winter. qRT-PCR was more sensitive and accurate in detecting GLRaV-3 than DASELISA,
as the latter technique delivered numerous false positive results later in the
season. The best time to screen for GLRaV-3 in the Western Cape region was from
the end of July to September. For the nursery screenings, our qRT-PCR results were
compared to the results of the DAS-ELISA performed by the specific nurseries. No
GLRaV-3 infection was detected in the specific samples received from the two
different nurseries. The results for all the samples correlated between the two techniques. This confirms that the propagation material of these nurseries has a
healthy phytosanitary status with regards to GLRaV-3.
However, the detection of GVA in the severely infected vineyard yielded inconsistent
results. Detection ability fluctuated throughout the season and no specific trend in
seasonal variation and virus titre fluctuation could be established. The highest
percentage of GVA infected samples were detected during September, April and the
end of July. Previously published universal primers were used for the detection of
GVA, but further investigation indicated that they might not be suitable for sensitive
detection of specific GVA variants present in South Africa.
Vitis vinifera was transformed with a GLRaV-3 antiviral construct, ΔHSP-Mut.
SYBR Green Real time PCR (qPCR) and qRT-PCR were utilised as alternative
methods for molecular characterisation of transgenic plants. The qPCR and Southern
blot results correlated for 76.5% of the samples. This illustrated the ability of qPCR
to accurately estimate transgene copy numbers. Various samples were identified
during qRT-PCR amplification that exhibited high mRNA expression levels of the
transgene. These samples are ideal for further viral resistance studies.
This study illustrated that the versatility of real time PCR renders it a valuable tool for
accurate virus detection as well as copy number determination.
AFRIKAANSE OPSOMMING: Suid Afrika word geag as een van die top wyn produserende lande ter wereld. Die volhoubaarheid van die wynbedryf word onder andere bedreig deur virus-infeksies.
Grapevine leafroll associated virus 3 (GLRaV-3) en Grapevine virus A (GVA) is van
die mees belangrike virusse wat siektes veroorsaak in Suid-Afrikaanse wingerde.
Wingerd bo-stok materiaal word gereeld getoets vir hierdie virusse, maar hierdie
materiaal word meestal geënt op onderstokmateriaal waarvan die virus status
onbekend is. Virus opsporing in onderstokke word egter gekompliseer deur baie lae
en variërende virus konsentrasies, maar opsporing in voortplantingsmateriaal is ‘n
noodsaaklike beheermeganisme vir virus-infeksie.
Die doel van die projek was om ‘n opsporingsisteem te ontwikkel via kwantitatiewe
PCR (qPCR) tegnieke vir akkurate en gereelde toetsing van GLRaV-3 en GVA in
onderstokmateriaal. qPCR sal ook verder gebruik word vir molekulêre
karakterisering van transgeniese plante wat ‘n GLRaV-3 antivirale ΔHSP-Mut
konstruk bevat.
‘n Hoogs geïnfekteerde wingerd was regdeur die seisoen getoets om seisoenale
fluktuasies in viruskonsentrasie te ondersoek en om die optimale tydstip vir
sensitiewe virus opsporing te bepaal. ‘n Grootskaalse toetsing van kwekery
voortplantingsmateriaal vir GLRaV-3 infeksie was ook uitgevoer. Die qRT-PCR
resultate is met die DAS-ELISA resultate vergelyk om die effektiwiteit en
sensitiwiteit van die twee tegnieke te vergelyk. Vir die hoogs geïnfekteerde wingerd
het die GLRaV-3 opsporing toegeneem met die verloop van die seisoen tot en met
winter. qRT-PCR was meer sensitief en akkuraat as DAS-ELISA in die opsporing
van GLRaV-3, weens verskeie vals positiewe resultate wat later in die seisoen deur
die laasgenoemde tegniek verkry is. Die beste tyd om vir GLRaV-3 te toets is vanaf
einde Julie tot September. Tydens die kwekery toetsings was qRT-PCR resultate met
die DAS-ELISA resultate van die spesifieke kwekerye vergelyk. Geen GLRaV-3
infeksie was waargeneem in die spesifieke monsters wat vanaf die kwekerye ontvang
is nie. Die resultate van die twee tegnieke het ooreengestem vir al die monsters wat
v
getoets is. Dit het bevestig dat die voortplantingsmateriaal van hierdie kwekerye
gesonde fitosanitêre status met betrekking tot GLRaV-3 gehad het.
Die opsporing van GVA in die geïnfekteerde wingerd het egter wisselvallige resultate
gelewer. Opsporing van die virus het ook regdeur die seisoen gefluktueer en geen
spesifieke neiging in seisoenale opsporingsvermoë kon gemaak word nie. Die
hoogste persentasie GVA geïnfekteerde monsters was waargeneem tydens
September, April en die einde van Julie. Voorheen gepubliseerde universele inleiers
was gebruik vir die opsporing van GVA, maar verdere ondersoeke het getoon dat
hierdie inleiers nie noodwendig geskik is vir sensitiewe opsporing van GVA variante
wat teenwoordig is in Suid-Afrika nie.
Vitis vinifera was getransformeer met ‘n GLRaV-3 antivirale konstruct, ΔHSP-Mut.
SYBR Green Real time PCR (qPCR) en qRT-PCR was ingespan as alternatiewe
metodes vir molekulêre karaterisering van transgeniese plante. Die qPCR en
Southern-klad resultate het ooreengestem vir 76.5% van die monsters. Dit illustreer
die vermoë van qPCR om akkurate kopie-getalle van transgene te bepaal. Verskeie
plante is geïdentifiseer tydens qRT-PCR amplifisering wat hoë vlakke van transgeen
mRNA uitdrukking getoon het. Hierdie monsters is ideaal vir verdere virus
weerstandbiedendheids studies.
Hierdie studie het die veelsydigheid van real time PCR bewys en getoon dat dit ‘n
kosbare tegniek is vir akkurate virus opsporing sowel as kopie-getal bepaling.
