Browsing by Author "Oosthuizen, Kristin"

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    Characterising the viral and microbial diversity in old and young grapevines
    (Stellenbosch : Stellenbosch University, 2017-12) Oosthuizen, Kristin; Burger, Johan T.; Maree, H. J.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
    ENGLISH ABSTRACT: There is anecdotal evidence suggesting that old vines produce wines of higher quality than young vines. In South Africa, vines are generally regarded ‘old’ when they reach 35 years of age, while ‘young’ vines are less than ten years old. Grapevines are susceptible to a large spectrum of pathogens that have negative impacts on grape quality and yield. This crop is also colonised by diverse endophytic microorganisms that play an important role in plant growth, health and productivity. To date, limited molecular research has been performed to determine the complexity of the pathogenic and endophytic communities in old vines. This study aimed to characterise the viral and fungal profiles of old and young Pinotage grapevines, using next-generation sequencing in a metagenomics approach. To determine the viral diversity, double-stranded RNA was extracted from phloem to enrich for virus-specific nucleic acids, and sequenced on an Illumina platform. High-quality reads were assembled into contigs and classified through BLAST analysis against the NCBI database. Additionally, the reads were mapped to a database consisting of known grapevine virus and viroid genome sequences. Reverse-transcription PCR detection assays were performed to validate the presence of the identified viruses. The fungal communities were characterised by extracting total DNA from the vascular tissues of the cane, followed by amplification of the ITS2 region, and deep amplicon sequencing. The ITS2 sequences were clustered into operational taxonomic units at a 97% identity threshold and taxonomically classified through BLAST analysis against the UNITE database. Viruses of the families Closteroviridae, Betaflexiviridae and Tymoviridae, and four pospiviroids were detected. The virus community was more diverse in the old vines, with 31 and 16 virus variants detected in the old and young vines, respectively. This was expected, since old vines have been exposed to viral pathogens for a longer period. The economically important grapevine leafroll-associated virus 3 was the most abundant species present in the samples, consistent with previous surveys of vineyards in the Western Cape. Grapevine Syrah virus 1, and possibly grapevine rupestris vein feathering virus, was identified for the first time in South African grapevines, expanding the global distribution of the virus(es). The amplicon data revealed the presence of different filamentous and yeast-like fungal taxa commonly associated with grapevines, including species of Alternaria, Aureobasidium, Cladosporium and Epicoccum. Several pathogens of grapevine trunk diseases and postharvest rot, and endophytic species with biocontrol properties were detected. The young-vine sample group showed greater fungal diversity, as determined by three alpha diversity metrics, although not statistically significant. It may be speculated that the fungal community of old vines is more accustomed to the environment, and therefore less diverse. No differences were observed between the old and young vines, with regards to the community composition. The data generated in this study has contributed to research on the complex viral and fungal communities inhabiting old vines.
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    Phylogenomic analysis reveals deep divergence and recombination in an economically important grapevine virus.
    (Public Library of Science, 2015) Maree, Hans J.; Pirie, Michael D.; Oosthuizen, Kristin; Bester, Rachelle; Rees, D. Jasper G.; Burger, Johan T.
    The evolutionary history of the exclusively grapevine (Vitis spp.) infecting, grapevine leafroll-associated virus 3 (GLRaV-3) has not been studied extensively, partly due to limited available sequence data. In this study we trace the evolutionary history of GLRaV-3, focussing on isolate GH24, a newly discovered variant. GH24 was discovered through the use of next-generation sequencing (NGS) and the whole genome sequence determined and validated with Sanger sequencing. We assembled an alignment of all 13 available whole genomes of GLRaV-3 isolates and all other publicly available GLRaV-3 sequence data. Using multiple recombination detection methods we identified a clear signal for recombination in one whole genome sequence and further evidence for recombination in two more, including GH24. We inferred phylogenetic trees and networks and estimated the ages of common ancestors of GLRaV-3 clades by means of relaxed clock models calibrated with asynchronous sampling dates. Our results generally confirm previously identified variant groups as well as two new groups (VII and VIII). Higher order groups were defined as supergroups designated A to D. Supergroup A includes variant groups I-V and supergroup B group VI and its related unclassified isolates. Supergroups C and D are less well known, including the newly identified groups VII (including isolate GH24) and VIII respectively. The inferred node ages suggest that the origins of the major groups of GLRaV-3, including isolate GH24, may have occurred prior to worldwide cultivation of grapevines, whilst the current diversity represents closely related isolates that diverged from common ancestors within the last century.