Determination of the virus diversity associated with Grapevine leafroll disease

Molenaar, Nicholas (2015-04)

Thesis (MSc)--Stellenbosch University, 2015.

Thesis

ENGLISH ABSTRACT: Vitis vinifera is the woody crop most susceptible to intracellular pathogens. Currently 70 pathogens infect grapevine, of which 63 are of viral origin. Grapevine leafroll-associated virus 3 (GLRaV-3) is the type species of the genus Ampelovirus, family Closteroviridae. It is considered to be the primary causative agent of Grapevine leafroll disease (GLD) globally; however, the etiology of GLD is not completely understood. Here we report on the viral populations present in GLD symptomatic grapevines across the Western Cape province, South Africa. A widespread survey was performed to screen 315 grapevines for 11 grapevine-infecting viruses using RT-PCR. Additionally, GLRaV-3 variant groups were distinguished with high-resolution melt (HRM) curve analysis used in conjunction with real-time RT-PCR. Members of the family Closteroviridae were detected with the highest frequency, particularly GLRaV-3 that was detected in 87% of tested plants. Nextgeneration sequencing (NGS) is capable of detecting known and novel viruses without prior knowledge of viral sequences and when used in a metagenomic approach is able to detected viral populations within diseased vines. A total of 17 grapevine samples were subjected to NGS using either an Illumina MiSeq or HiSeq 2500 instrument to determine the virome within GLD vines. Collectively, more than 190 million reads were generated through NGS. Read datasets were trimmed and filtered for quality and subjected to both read-mapping and de novo assembly. Contigs assembled de novo were analyzed with BLAST (Basic Local Alignment Search Tool) against the NCBI (National Centre for Biotechnology Information) database and it was determined that GLRaV-3 was the best-represented virus, comprising 97.5% of the assembled contigs. Grapevine virus F (GVF) was detected for the first time in South African vineyards through de novo assemblies and the complete genome sequence validated through direct Sanger sequencing. The complete genome of GVF isolate V5 spans 7 539 nucleotides and shares 89.11% nucleotide identity to existing GVF genomes. The data generated through this study will assist in further understanding the etiology of GLD, support the current hypothesis of GLRaV-3 as the primary contributor to GLD, aid in understanding virus associations in diseased vines and potentially develop systems in which to control disease spread and symptom severity.

AFRIKAANSE OPSOMMING: Vitis vinifera is die houtagtige oes wat die mees vatbaarste is vir intrasellulêre patogene. Tans word wingerde deur 70 patogene geïnfekteer, waarvan 63 van virale oorsprong is. Grapevine leafrollassociated virus 3 (GLRaV-3) is die tipe spesie van die genus Ampelovirus, familie Closteroviridae. Dit word globaal beskou as die primêre oorsaak van Wingerd krulblaar-siekte (GLD), alhoewel die etiologie van GLD nie heeltemal begryp word nie. In hierdie verslag word die virale populasies teenwoordig in GLD simptomatiese wingerde oor die Wes-Kaap provinsie in Suid-Afrika gerapporteer. ‘n Wydverspreide opname was uitgevoer om 315 wingerde met 11 wingerdinfekterende virusse te ondersoek, deur gebruik te maak van tru-transkripsie polimerase ketting reaksie (PKR). Verder is variantgroepe van GLRaV-3 onderskei met hoë-resolusie smeltingskurweanalise, tesame met die gebruik van in-tyd tru-transkripsie PKR. Die hoogste frekwensie was van die lede van die familie Closteroviridae, veral GLRaV-3 wat in 87% van die ondersoekte plante gevind is. Nuwe-generasie volgorderbepaling (NGS) beskik oor die vermoë om bekende en nuwe virusse te herken in virale populasies in geaffekteerde wingerde sonder vorige kennis van virale volgorderbepalings en wanneer dit in ‘n metagenomiese benadering gebruik word kan die virale bevolkings binne siek wingerde ontdek. ‘n Totaal van 17 wingerd-steekproewe was blootgestel aan NGS deur die gebruik van of ‘n Illumina MiSeq of ‘n HiSeq 2500 instrument om die virome te bepaal van GLD wingerde. In totaal is meer 190 miljoen lesings gegenereer deur NGS. Hierdie data lesings was verwerk en gefilter vir kwaliteit om onderwerp te word vir beide kartering en de novo samestellings. Contigs verkry deur de novo samestellings was geanaliseer met BLAST (Basic Local Alignment Search Tool) teenoor die NCBI (National Centre for Biotechnology Information) databasis en dit was vasgestel dat GLRaV-3 was die mees-verteenwoordigende virus, bestaande uit 97.5% van die saamgestelde contigs. Grapevine virus F (GVF) was vir die eerste keer in Suid- Afrikaanse wingerde waargeneem deur de novo samestellings en die volledige genoom volgordger is geverifieer deur middel van direkte Sanger volgorderbepaling. Die volledige genoom van GVF isoleer V5 spanwydte van 7539 nukleotiedes en deel 89.11% nukleotied identiteite van bestaande GVF genome. Die gegenereerde data van hierdie studie sal bykomende begrip van die etiologie van GLD bystaan, die huidige hipotese van GLRaV-3 as die primêre bydraer tot GLD ondersteun, verhoogde begrip van virus-assosiasies in wingerdsiektes verseker en potensiële sisteme ontwikkel om siektes en simptome te beheer.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/97012
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