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Molecular characterisation of South African isolates of grapevine fanleaf virus and a new, associated satellite RNALamprecht, Renate Luise 12 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Grapevine fanleaf virus (GFLV) is one of the oldest, most widespread and devastating
viruses infecting grapevine, and occurs globally where Vitis vinifera is grown. In South
Africa (SA) GFLV is predominant in the Breede River Valley, one of the highest wine
producing regions in SA. To date, only three GFLV isolates have been completely
sequenced internationally, and limited sequence information is available for SA GFLV
isolates. In this study, the first full-length GFLV genome sequence from a South African
isolate, GFLV-SAPCS3, was determined. Full-length sequences were used for
phylogenetic analysis and revealed that the SA isolates are separate from other
sequenced GFLV isolates. Full-length sequences were also used to investigate putative
intra- and interspecies recombination events involving GFLV-SAPCS3 RNA1 and RNA2
between GFLV and Arabis mosaic virus (ArMV) isolates. Using two different
recombination analysis software packages, the most notable of the putative
recombination events involving GFLV-SAPCS3 indicated that the GFLV-SAPCS3 RNA2
5’ UTR might have evolved from an interspecies recombination event between GFLVF13-
type and ArMV Ta-type isolates. The presence of satellite RNAs (satRNA)
associated with South African GFLV isolates was also investigated. In a collaborative
study (see Chapter 4 for details), more than a 100 GFLV- infected grapevine plants
were screened for satRNAs. SatRNAs were present in only two plants, containing
isolates GFLV-SACH44 and GFLV-SACH47. The full-length nucleotide sequences of
the GFLV-SACH44 genomic RNAs 1 and 2, and the associated satRNA were
determined. No significant sequence variation could be detected between the GFLV
isolates that had the presence of a satRNA and those that had not. The GFLV-SACH44
RNA2 5’ UTR also had the same conserved sequence that was found in GFLVSAPCS3,
which suggests that GFLV-SACH44, like GFLV-SAPCS3, may have arisen
from a common ancestor, which may have originated from an interspecies
recombination event. The GFLV-SACH44 satRNA was found to be more closely related
to the ArMV large satRNA than to the satRNA associated with GFLV-F13. A full-length
cDNA clone of GFLV-SACH44 satRNA was constructed and its replication and systemic spread in herbaceous hosts, when mechanically co-inoculated with two GFLV isolates
as helper viruses, was demonstrated. Replication of the GFLV-SACH44 satRNA cDNA
clone was however abolished when co-inoculated with an ArMV helper virus, even
though it is phylogenetically more closely related to ArMV satRNAs. The full-length
satRNA clones were modified to be used as vectors for expression and/or silencing of
foreign genes, by inserting the green fluorescence protein (GFP) full-length or partial
sequences downstream of the open reading frame of the satRNA. These constructs
were cloned into a binary vector to allow for agro-infiltration into plants. Full-length
cDNA clones of GFLV-SAPCS3 RNA1 and RNA2 were constructed to be used in
conjunction with modified GFLV-SACH44 satRNA full-length clones. The full length
GFLV-SAPCS3 RNA1 and RNA2 clones were however not infectious in Nicotiana
benthamiana after agro-infiltration and therefore the evaluation of the modified satRNA
expression and silencing constructs had to be aborted. Attempts to understand this
failure revealed that, among other point mutations, four frameshifts had occurred in the
RNA1 full-length clone, rendering the transcripts untranslatable, and hence noninfectious.
Strategies to correct the mutations are discussed. Once these mutations
have been corrected this study can continue in evaluating the use of the satRNA
component for expression and silencing analysis. / AFRIKAANSE OPSOMMING: Grapevine fanleaf virus (GFLV) is een van die oudste, mees wydverspreide en mees
verwoestende virusse wat wingerd affekteer en word wêreldwyd waar Vitis vinifera
verbou word, gevind. In Suid Afrika (SA) kom GFLV veral in die Breederivier vallei, een
van die mees produktiewe wyn-produserende areas in SA, voor. Tot dusver is daar net
drie GFLV isolate waarvan die volledige nukleïensuurvolgorde internasionaal bepaal is.
Die nukleïensuurvolgorde informasie vir SA GFLV isolate is redelik beperk. In hierdie
studie was die eerste volledige nukleïensuurvolgorde van ‘n SA GFLV isolaat, GFLVSAPCS3,
bepaal. Die volledige nukleïensuurvolgordes was vir filogenetiese analise
gebruik, en vermeende intra- en interspesie rekombinasie gebeurtenisse, wat GFLVSAPCS3
RNA1 en RNA2 betrek, tussen GFLV en Arabis mosaic virus (ArMV) isolate
was ondersoek. Twee verskillende rekombinasie-analise sagteware programme was
gebruik en die noemenswaardigste van die vermeende rekombinasie gebeurtenisse,
met betrekking tot GFLV-SAPCS3, het aangedui dat die GFLV-SAPCS3 RNA2 5’
ontransleerde streek (UTR) waarskynlik van ‘n interspesie rekombinasie gebeurtenis
tussen ‘n GFLV-F13-tipe en ‘n ArMV-Ta-tipe isolaat ontwikkel het. Die teenwoordigheid
van satelliet RNAs (satRNAs), wat met SA GFLV isolate geassosieer is, was ook
ondersoek. Meer as ‘n 100 GFLV ge-infekteerde wingerd plante was in ‘n
samewerkingsprojek (sien Hoofstuk 4 vir besonderhede) getoets vir die
teenwoordigheid van satRNAs. SatRNAs was net in twee plante teenwoordig, in isolate
GFLV-SACH44 en GFLV-SACH47. Die vollengte nukleïensuurvolgordes van GFLVSACH44
RNA1, RNA2 en geassosieerde satRNA was bepaal. Geen beduidende
volgorde variasie tussen die GFLV isolate wat satRNAs bevat het, en die GFLV isolate
sonder satRNA was waargeneem nie. Die GFLV-SACH44 RNA2 5’ UTR het ook die
gekonserveerde volgorde, wat in GFLV-SAPCS3 teenwoordig was, gehad en dit dui
daarop dat GFLV-SACH44, soos GFLV-SAPCS3, van dieselfde stamvader, wat tydens
‘n vorige rekombinasie gebeurtenis ontstaan het, mag ontwikkel het. Die GFLVSACH44
satRNA was meer naverwant aan die ArMV satRNAs as aan die satRNA, wat
met GFLV-F13. ‘n Vollengte cDNA kloon van die GFLV-SACH44 satRNA was ontwikkel en die replisering en sistemiese verspreiding in sagte plante, nadat dit met twee GFLV
isolate as helper virusse saam ge-inokuleer was, was gedemonstreer. Replisering van
die GFLV-SACH44 satRNA cDNA kloon was egter ontwrig toe dit saam met ‘n ArMV
helper virus saam ge-inokuleer was, al is dit filogeneties meer verwant aan ArMV
satRNAs. Die vol-lengte satRNA klone was gemodifiseer om as vektore vir uitdrukking
en/of uitdowing van transgene te dien, deur om vol-lengte of gedeeltelike groen
fluoressensie proteïen (GFP) nukleïensuurvolgordes aan die einde van die satRNA
leesraam te koppel. Hierdie konstrukte was in ‘n binêre vektor gekloon om agroinfiltrasie
in plante toe te laat. Vol-lengte cDNA klone van GFLV-SAPCS3 RNA1 en
RNA2 was ontwikkel om in samewerking met die gemodifiseerde GFLV-SACH44
satRNA konstrukte gebruik te word. Die vol-lengte GFLV-SAPCS3 RNA1 en RNA2
klone het egter nie in Nicotiana benthamiana gerepliseer na agro-infiltrasie nie, daarom
was die evaluasie van die gemodifiseerde satRNA konstrukte gestaak. Pogings om die
mislukking te verstaan, het daarop gewys dat, behalwe punt mutasies, vier leesraam
versteurings in die RNA1 vollengte kloon voorgekom het, wat ontransleerbare
transkripte, en dus nie-repliserende konstrukte tot gevolg gehad het. Strategieë om die
mutasies te korrigeer is bespreek. Sodra die mutasies gekorrigeer is, kan die studie
voortgaan om te evalueer of die satRNA komponent vir uitdrukking en uitdowing analise
gebruik kan word.
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The development of a diagnostic assay for nepoviruses in grapevineFrazenburg, Lolita 12 1900 (has links)
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.
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Etude de l'embryogenèse somatique et transformation génétique de différentes variétés de porte-greffes de vigne en vue d'induire la résistance au Grapevine Fanleaf Virus / Somatic embryogenesis and genetic transformation of different varieties of grapevine rootstocks to induce resistance to Grapevine fanleaf virusBenard-Gellon, Mélanie 24 November 2011 (has links)
Dans cette étude, nous avons dans un premier temps adapte le protocole d'embryogenèse somatique primaire a différentes variétés d'hybrides porte-greffes (3309C, 110R, Fercal, 41B et SO4) en nous appuyant sur l'expérience acquise au laboratoire sur Vitis vinifera cv Chardonnay. Les résultats montrent que le génotype, le type d'explant (étamine, fleur ou nœud), le type et la dose d'auxine utilisés dans le milieu d’induction (2,4-D ou 2,4,5-T) ont une influence sur les efficacités d'embryogenèse somatique. En effet, pour le 3309C, l'utilisation du 2,4,5-T dans le milieu d'induction a montré une efficacité embryogène supérieure à partir de nœuds par rapport à celle obtenue à partir d'étamines. Cependant la meilleure efficacité a été obtenue à partir de fleurs de cette variété, sur un milieu d'induction contenant du 2,4-D. De plus, le protocole d'embryogenèse somatique secondaire utilise de manière récurrente au laboratoire nous a permis d'obtenir des masses embryogènes ainsi que des embryons somatiques secondaires de ces porte-greffes. Le protocole de conversion des embryons en plantes, en présence de 4,5 uM de cytokinine (BAP) s'est avère efficace pour le 11OR et le 41B. Dans un second temps, nous avons co-cultivé le matériel embryogène obtenu pour quatre de ces génotypes (110R, 3309C, Fercal et 41B), avec Agrobacterium tumefaciens contenant trois constructions génétiques : (i) une copie d'une séquence partielle (1020 pb) du gène de la coque protéique du virus en orientation sens; (ii) une partie courte en sens et en anti-sens (280 pb) de cette même séquence formant une structure en épingle a cheveux (hpRNA = hairpin RNA) ; (iii) un amiRNA ciblant une séquence virale. Le gène bactérien codant la néomycine phosphotransférase et conférant la résistance à un antibiotique, la kanamycine, a été utilisé comme gène de sélection. Les conditions de sélection a la kanamycine ont nécessité des adaptations expérimentales telles que l’ajustement de la concentration en antibiotique puisque la sélection avec 75 mg.L-1 de kanamycine s'avère insuffisamment drastique dans Ia plupart de nos expériences de co-cullture. Les résultats d'analyse moléculaire par PCR ont montré l'amplification probable des fragments d'intérêt (CPGFLV et amiRI1TA-71) dans des échantillons de 11OR et de 41B résistants à la kanamycine. Cependant des analyses moléculaires supplémentaires par AL-PCR ne nous ont pas renseignées sur une éventuelle intégration du transgène amiRATA-71 dans des masses embryogènes de 41B. / In this study, we initially adapted the protocol of primary somatic embryogenesis in different varieties of hybrid rootstocks (3309C, 110R, Fercal, 41B and SO4) building on the experience gained in the laboratory on Vitis vinifera cv Chardonnay. The results show that the genotype, the explant type (stamen, flower or node), the type and the dose of auxin used in the induction medium (2,4-D or 2,4,5-T) influence the efficiency of somatic embryogenesis. Indeed, for the 3309C, the use of 2,4,5-T in the induction medium showed a higher efficiency from embryogenic nodes compared to that obtained from stamens. However, the better efficiency was obtained from the flowers of this variety on an induction medium containing 2,4-D. In addition, a protocol used in the laboratory for secondary somatic embryogenesis allowed us to obtain embryogenic masses as well as secondary somatic embryos from these rootstocks. The protocol conversion of embryos into plants, in the presence of 4.5 [tM of cytokinin (BAP), was effective for the 110R and 41B. In a second step, we co-cultivated embryogenic material obtained for four of these genotypes (110R, 3309C, Fercal and 41B), with Agrobacteriwn tumefaciens containing three genetic constructs: (i) a copy of a partial sequence (1020 bp) of the coat protein gene of the virus in the sense orientation, (ii) a short part-way and antisense (280 bp) of the same sequence forming a hairpin structure (hairpin RNA = hpRNA) (iii) one amiRNA targeting a viral sequence. The nptll bacterial gene encoding neomycin phosphotransferase and conferring resistance to the antibiotic kanamycin, was used as the selection gene. The selection conditions to kanamycin have required experimental adaptations such as adjusting the concentration of antibiotic because the selection with 75 mg.L-1 of kanamycin was not enough drastic in most of our experiments of co-culture. The results of molecular analysis by PCR showed probable amplification of fragments of interest (CPGFLV and amiRNA-71) in samples of 11OR and 41B resistant to kanamycin. However, additional molecular analysis by AL-PCR did not inform us about a possible integration of the transgene amiRNA-71 in embryogenic masses of 41B.
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Výskyt virových patogenů na klonech révy vinné (Vitis vinifera L.) českého a zahraničního původuZávodský, Pavel January 2015 (has links)
The thesis deals with the occurrence of viral pathogens on grape - Chardonnay clones. Monitored and evaluated clones were 8, 95, 96 (foreign) on rootstocks 1103 Paulsen, SO4, Kober 5 BB and 110 Richter and VP-155/6-VP 161/6, PO-158/7 and PO-160 / 1 (Czech) on the rootstock Kober 5 BB. All plants have a controlled origin. The experiment was conducted in 2013 on the test sites in the cadastral Perná. At the beginning of vegetation were recorded values on 1 herbaceous plant -- sprouting and not-sprouting buds. During vegetation were the plants observed. From the monitored plants were harvested grapes and following parameters were checked: number and weight of the grapes, weight of berries and the stem. Furthermore, before leaf the leaves were sampled for subsequent ELISA test for viral diseases Grapevine fanleaf virus, Arabis mosaic virus, Grapevine fleck virus, Grapevine leafroll-associated virus 1 and 3, Grapevine virus A. All values were evaluated by statistical program Statistica 10.
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The development of an enzyme linked immunosorbent assay for the detection of the South African strain(s) of grapevine fanleaf nepovirusLiebenberg, Annerie 12 1900 (has links)
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.
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