Characterising the viral and microbial diversity in old and young grapevines

Oosthuizen, Kristin (2017-12)

Thesis (MSc)--Stellenbosch University, 2017.

Thesis

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.

AFRIKAANSE OPSOMMING: Volgens wynkenners produseer ou wingerde wyne van hoër gehalte as jong wingerde. In Suid-Afrika word wingerde oor die algemeen as 'oud' beskou as hulle meer as 35 jaar oud is en ‘jonk’ as hulle minder as tien jaar oud is. Wingerdstokke is vatbaar vir veelvudige patogene wat die gehalte en opbrengs van die druiwe negatief beïnvloed. Hierdie gewas word ook beset deur diverse endofitiese microörganismes wat 'n belangrike rol in plantegroei, gesondheid en produktiwiteit speel. Tot op hede is min molekulêre navorsing uitgevoer om die kompleksiteit van die patogeniese en endofitiese gemeenskappe in ou wingerde te bepaal. Die doel van hierdie studie was om die virus- en swam-profiele van ou en jong Pinotage wingerdstokke te beskryf, deur gebruik te maak van volgende-generasie volgordebepalingstegnologie in 'n metagenomiese benadering. Die virus diversiteit is bepaal deur die suiwering van dubbelstring RNS vanuit floëem om vir virus-spesifieke nukleïensure te verryk, gevolg deur volgordebepaling met ‘n Illumina instrument. Hoë-kwailiteit volgorde-fragmente is saamgestel in langer konstrukte wat deur BLAST analise teen die NCBI databasis geklasifiseer is. Daarbenewens is die volgorde-fragmente vergelyk met 'n databasis bestaande uit genoomvolgordes van bekende wingerdvirusse en -viroïede. Tru-transkripsie amplifiseringsreaksies is uitgevoer om die teenwoordigheid van die geïdentifiseerde virusse te bevestig. Die swamgemeenskappe is beskryf deur die suiwering van DNS vanuit vaatweefsel, gevolg deur amplifisering van die ITS2 lokus, en Illumina amplikon volgordebapling. Die ITS2 volgorde-fragmente is in operasionele taksonomiese eenhede groepeer, gebaseer op 97% identiteit, en taksonomies geklassifiseer deur BLAST analise teen die UNITE databasis. Virusse van die families Closteroviridae, Betaflexiviridae en Tymoviridae, en vier pospiviroïede is geïdentifiseer. Die virusgemeenskap was meer divers in die ou wingerde; 31 en 16 virus variante is onderskeidelik in die ou en jong wingerde geïdentifiseer. Dit was nie onverwags nie, aangesien die ou wingerde vir 'n langer tydperk aan virale patogene blootgestel was. Die ekonomies-belangrike grapevine leafroll-associated virus 3 was die mees dominante spesie teenwoordig in die monsters, in ooreenstemming met vorige opnames van Wes-Kaapse wingerde. Grapevine Syrah virus 1, en moontlik grapevine rupestris vein feathering virus, is vir die eerste keer in Suid-Afrikaanse wingerdstokke geïdentifiseer. Die amplikon data het die teenwoordigheid van verskillende filament- en gisagtige swamme wat met wingerde geassosieer word aangedui, insluitende Alternaria, Aureobasidium, Cladosporium en Epicoccum spesies. Verskeie patogene van wingerdstamsiektes en na-oesverrotting, en endofitiese spesies met biokontrole-eienskappe was teenwoordig in die monsters. Die jong-wingerd steekproefgroep het 'n groter swamdiversiteit getoon, soos bepaal deur drie alfa-diversiteitsmetrieë, alhoewel dit nie statisties betekenisvol was nie. Daar mag gespekuleer word dat die swamgemeenskap van ou wingerdstokke beter aangepas het by die omgewing, en daarom minder divers is. Geen verskille is tussen die ou en jong wingerde waargeneem met betrekking tot die gemeenskap samestelling nie. Die data wat in hierdie studie genereer is, het bygedra tot navorsing oor die komplekse virus en swam gemeenskappe wat in ou wingerde voorkom.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/102827
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