Jooste, Anna Elizabeth Catharina (2011-03)
Thesis (PhD (Genetics))--University of Stellenbosch, 2011.
important diseases of grapevines, occurring in all grape-producing countries worldwide.
Grapevine leafroll-associated virus 3 (GLRaV-3) is known to be closely associated with GLR
disease and occurs commonly in South African vineyards. In this study three genetic variants
of GLRaV-3 were identified in vineyards of the Western Cape, South Africaby single strand
conformation polymorphism (SSCP) profiles generated from a region amplified in ORF5. A
specific SSCP profile could be assigned to each variant group and these wereconfirmed by
sequencing of the ORF5 regions.These results demonstrated that SSCP analysis on this region
in ORF5 provides a fast and reliable indication of the GLRaV-3 variant status of a plant,
which in many instances showed mixed infections. The full genome sequence of one
representative of each variant group i.e. isolates 621 (group I), 623 (group II) and PL-20
(group III), was determined by sequencing overlapping cloned fragments of these isolates.
The sequences of genomic 5’ ends of these isolates were determined by RLM-RACE.
Sequence alignment of the 5’UTRs indicated significant sequence and length variation in this
region, between the three South African variant groups. Nucleotide sequence alignment of the
Hsp70h and CP gene regions of these isolates with those of isolates from elsewhere in the
world, followed by phylogenetic analysis, further supported the presence of three GLRaV-3
variants in South Africa, and that two or three additional variant groups occurs elsewhere in
the world. We further investigated the prevalence of these three GLRaV-3 variants in mother
blocksof different cultivars and from different vine growing regions, using SSCP analysis.
The majority of the plants studied, were infected with the group II variant, similar to isolates
623 and GP18. The distribution of the three GLRaV-3 variants within a spatio-temporally
recorded cluster of diseased plants was studied by means of SSCP profile analysis. We
showed that different GLRaV-3 variants are transmitted to adjacent plants in an infection
cluster. Results showed that, in some leafroll disease clusters, the variant that was present in
the original GLRaV-3 infected plant of a cluster was transmitted to adjacent plants in a row
and across rows. Some plants in the cluster were also infected with variants not present in the
original plant. These infections could have been caused by mealybug vectors feeding on
plants from surrounding areas and then infecting these plants.
The scientific information generated on GLRaV-3 variants in this project contributed to the
advancement of our knowledge of genetic variability and provides a basis of further
epidemiology and vector-virus studies. The study showed for the first time that different
GLRaV-3 variants were transmitted to adjacent plants in a row and across rows in a GLR
disease cluster. The diversity detected in the 5’UTR between variants from the three genetic
groups provides a platform for the further study of the biological characteristics of GLRaV-3
AFRIKAANSE OPSOMMING: Wingerdsiektes, veral virus siektes, bedreig wingerd industrieë wêreldwyd, asook die Suid
Afrikaanse wingerdbedryf. Rolbladsiekte is een van die belangrikste siektes op wingerd en
kom wêreldwyd voor. Die virus, grapevine leafroll-associated virus 3 (GLRaV-3), word sterk
geassosieer met Rolbladsiekte en kom wydverspreid voor in Suid Afrikaanse wingerde.
Tydens hierdie studie is drie genetiese variante van GLRaV-3 geïdentifiseer in wingerd
moederblokke in die Wes-Kaap. Die GLRaV-3 variante is geïdentifiseer met ‘n tegniek wat
‘single-strand conformation polymorphism (SSCP)’ genoem word. Die SSCP profiele was
gegenereer vanaf PKR produkte van die ORF5 area op die genoom van GLRaV-3. Die
geamplifiseerde produk van die ORF5 gebied is gebruik om die SSCP profiele te verkry en
DNA-volgorde data in die gebied het die drie SSCP profiele gestaaf. Hierdie metode om virus
variasie te bestudeer in plante is vinnig en betroubare resultate is verkry. Gemengde infeksies,
wat gereeld in wingerd voorkom, kon ook met die tegniek opgespoor word. Die volledige
nukleotied-volgorde van elkeen van die drie GLRaV-3 genome is volledig bepaal. Die isolate
wat die drie variant groepe verteenwoordig is isolaat 621 (groep I), 623 (groep II) en PL-20
(groep III). Die nukleotiedvolgorde in die 5’UTR is bepaal met die RLM-RACE tegniek.
Wanneer die 5’UTRs van die drie variante vergelyk is, het dit getoon dat daar verskille is in
die volgordes en lengtes voorgekom het. Ander dele van die genoom, o.a. die dopproteïen
(CP) en Hsp70 areas, is filogeneties vergelyk met isolate van regoor die wêreld. In die
filogenetiese analise is bevind dat die drie GLRaV-3 variante saamgegroepeer het met ander
isolate in die wêreld en dat daar elders ook twee to drie addisionele variant groepe van
GLRaV-3 voorkom. Die verspreiding van die drie GLRaV-3 variante in wingerde is bestudeer
in verskillende kultivars en in verskillende verbouingsgebiede. Die meerderheid van die
plante in die studie was geïnfekteer met die groep II variant wat dieselfde is as isolate 623 en
GP18. Die voorkoms van die drie variante in ‘n siekte cluster is bestudeer d.m.v SSCP. Die
studie het gewys dat verskillende GLRaV-3 variante versprei word na aangrensende plante in
‘n ry en tussen rye. In sommige gevalle is die variant wat in die oorspronklik geïnfekteerde
plant voorkom, oorgedra na naasliggende plante. Sommige van die plante in the infeksie area
was ook met ander GLRaV-3 variante geïnfekteer wat moontlik deur wolluise oorgedra is
vanaf naburige geïnfekteerde plante.
Die wetenskaplike inligting wat tydens hierdie studie beskryf word aangaande die
identifikasie van GLRaV-3 variante, dra by tot die molekulêre kennis van GLRaV-3 en
verskaf ‘n basis vir verdure epidemiologiese -en insek oordragingstudies. Die studie het vir
die eerste keer bewys dat verskillende GLRaV-3 variante na aanliggende plante in ‘n ry asook
oor rye oorgedra word. Die diversiteit tussen die GLRaV-3 variant groepe in die 5’UTR moet
verder ondersoek word en die deel van die genoom kan ‘n belangrike rol speel in die
biologiese eienskappe van die variante.
Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/6655
This item appears in the following collections: Doctoral Degrees (Genetics)43