Date of Graduation

12-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Agricultural, Food and Life Sciences (PhD)

Degree Level

Graduate

Department

Entomology and Plant Pathology

Advisor/Mentor

Tzanetakis, Ioannis E.

Committee Member

Goggin, Fiona L.

Second Committee Member

McWhirt, Amanda L.

Third Committee Member

Srivastava, Vibha

Keywords

Blackberry yellow vein disease; Crinivirus; Dodder; Ilarvirus; Infectious clones; Rubus

Abstract

Since blackberry yellow vein disease (BYVD) was first described, the blackberry virome has expanded significantly. BYVD is a complex disease associated with over ten viruses, yet the role of each virus in its etiology remains understudied. Studying the effect of each virus in disease development requires the identification of single infections; however, this task has been difficult for BYVD, with all field collected material over the past two decades consistently found infected by multiple viruses. Given the diversity of viruses associated with the disease and their different modes of transmission, obtaining pure virus cultures necessitate transmission of individual viruses via diverse vectors including whiteflies, mealybugs, thrips, hoppers, and eriophyid mites, making this highly challenging. To address this, we developed infectious clones for individual viruses starting with two of the most prevalent viruses in the complex, blackberry yellow vein associated virus (BYVaV) and blackberry chlorotic ringspot virus (BCRV). Both infectious clones induced systemic infection and symptoms in the indicator host, Nicotiana benthamiana. Additionally, we demonstrated that resulting viruses were transmissible to healthy hosts, with BYVaV transmitted using the whitefly vector Trialeurodes vaporariorum, and BCRV via mechanical inoculation. Then, we modified both infectious clones into virus-induced gene silencing (VIGS) vectors, effectively inducing gene silencing in N. benthamiana. Next, we delivered the infectious clones to Rubus spp.; for this, we developed a novel dodder-based approach. This approach facilitated the transmission of BYVaV and BCRV infectious clones from an agroinoculated herbaceous host to Rubus spp. The transmission rates were significantly higher than those of traditional direct agroinoculation methods. Finally, we utilized the BCRV infectious clone to explore why 'Munger' black raspberry (Rubus occidentalis) is consistently used as the preferred indicator for Rubus viruses. We used the BCRV infectious clone to establish single infections in two Rubus genotypes: 'Munger' and 'Natchez' (blackberry, Rubus L. subgenus Rubus Watson). Virus expression levels were monitored over six months, revealing different patterns of accumulation and detection amongst hosts. More consistent and higher RNA levels were observed in ‘Munger’ than ‘Natchez’, which may explain its use as the standard indicator for Rubus viruses for the past century and enhances our understanding of virus-host dynamics of BCRV and Rubus spp. Taken together, the developed infectious clones, VIGS vectors, and dodder-based approach provide the required tools to initiate studies of individual virus roles in BYVD etiology. This knowledge will assist in the development of more effective disease management strategies that can help minimize the impact of BYVD. With the availability of the blackberry genome, developed VIGS vectors enable genome-wide association studies, accelerating the breeding process by identifying genes of interest. Additionally, these vectors can be modified to control devastating diseases in perennial crops through RNA interference (RNAi) vaccination, providing an alternative to chemical control. The dodder-based approach will facilitate the study of virus biology and host interactions across various plant species, contributing to developing more resilient crops. Finally, differences in virus accumulation on both hosts indicate that further research into host mechanisms could deepen our understanding of virus tolerance and persistence in Rubus spp.

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Available for download on Friday, February 06, 2026

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