Identification and molecular characterization of three genetic variants of Grapevine leafroll-associated virus 3 (GLRaV-3) from South African vineyards and their spread in local vineyards

Jooste, Anna Elizabeth Catharina (2011-03)

Thesis (PhD (Genetics))--University of Stellenbosch, 2011.

Includes bibliography

Thesis

ENGLISH ABSTRACT: Grapevine diseases, in particular virus and virus-like diseases, are threatening grapevine industries worldwide; also in South Africa. Grapevine leafroll (GLR) is one of the most 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 variants.

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.

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