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

[摘要] ENGLISH ABSTRACT: Grapevine diseases, in particular virus and virus-like diseases, are threatening grapevineindustries worldwide; also in South Africa. Grapevine leafroll (GLR) is one of the mostimportant diseases of grapevines, occurring in all grape-producing countries worldwide.Grapevine leafroll-associated virus 3 (GLRaV-3) is known to be closely associated with GLRdisease and occurs commonly in South African vineyards. In this study three genetic variantsof GLRaV-3 were identified in vineyards of the Western Cape, South Africaby single strandconformation polymorphism (SSCP) profiles generated from a region amplified in ORF5. Aspecific SSCP profile could be assigned to each variant group and these wereconfirmed bysequencing of the ORF5 regions.These results demonstrated that SSCP analysis on this regionin 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 onerepresentative 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 thisregion, between the three South African variant groups. Nucleotide sequence alignment of theHsp70h and CP gene regions of these isolates with those of isolates from elsewhere in theworld, followed by phylogenetic analysis, further supported the presence of three GLRaV-3variants in South Africa, and that two or three additional variant groups occurs elsewhere inthe world. We further investigated the prevalence of these three GLRaV-3 variants in motherblocksof 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 isolates623 and GP18. The distribution of the three GLRaV-3 variants within a spatio-temporallyrecorded cluster of diseased plants was studied by means of SSCP profile analysis. Weshowed that different GLRaV-3 variants are transmitted to adjacent plants in an infectioncluster. Results showed that, in some leafroll disease clusters, the variant that was present inthe original GLRaV-3 infected plant of a cluster was transmitted to adjacent plants in a rowand across rows. Some plants in the cluster were also infected with variants not present in theoriginal plant. These infections could have been caused by mealybug vectors feeding onplants from surrounding areas and then infecting these plants.The scientific information generated on GLRaV-3 variants in this project contributed to theadvancement of our knowledge of genetic variability and provides a basis of furtherepidemiology and vector-virus studies. The study showed for the first time that differentGLRaV-3 variants were transmitted to adjacent plants in a row and across rows in a GLRdisease cluster. The diversity detected in the 5'UTR between variants from the three geneticgroups provides a platform for the further study of the biological characteristics of GLRaV-3variants.

[发布机构] Stellenbosch University