Science, Irving K. Barber Faculty of (Okanagan)
Biology, Department of (Okanagan)
DSpace
UBCO
Kahl, Dieter
Master of Science - MSc
University of British Columbia
The first data chapter of this research presents a novel approach for identifying potential insect vectors of circulative plant viruses, significantly reducing time-to-results and the resource costs associated with greenhouse transmission experiments. This artificial transmission protocol using a buffered sucrose solution as the virus recipient in place of a living plant, also eliminates plant-to-plant variation in susceptibility to infection, allowing assessment of virus-insect compatibility required for successful transmission of persistent viruses. To validate this approach, species of hemipteran insects, including leafhoppers, froghoppers, aphids, sharpshooters, and treehoppers, were subjected to the artificial feeding system to determine their ability to vector Grapevine red blotch virus (GRBV). Test insects were allowed to feed on a potted grapevine infected with GRBV for three days and then transferred to tubes containing the sucrose solution partitioned by a thinly stretched Parafilm membrane. After three days of feeding through the membrane, viruliferous test insects were stored for species identification and the sucrose solutions were tested by polymerase chain reaction for the presence of GRBV DNA. Of all the insects tested (n = 393), only nine treehoppers from two different species successfully transmitted GRBV to the sucrose solutions, indicating a high-likelihood of vector capability to plants, to be validated by greenhouse or field experiments. The second data chapter presents research that investigated GRBV titres from several grapevine tissues, including roots, buds, cortical scrapings, and leaves, at varying distances from the cordons, and at different times during the growing season and winter dormancy. Two common nucleic acid extraction techniques were compared, a low cost high-throughput nucleic acid extraction method (GES) and a DNA column purification method. Ideal combinations of sampling parameters were determined for summer and winter diagnostics. In dormant samples, cortical scrapings yielded the highest GRBV titres and produced no false-negatives, and basal leaves yielded high GRBV titres throughout the summer months with only one false-negative on the earliest sample date. These two tissues provide the best samples for reliable and reproducible GRBV diagnostics. The crude GES and DNA column purification methods performed comparably well for binary diagnostics, however the GES method performed poorly in quantitative analysis.
Evaluation of the Grapevine red blotch virus Epidemiological Landscape of the Okanagan Valley by Dieter Kahl B.Sc., University of Victoria 2018 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE COLLEGE OF GRADUATE STUDIES (Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) May 2021 © Dieter Kahl 2021 ii The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis/dissertation entitled: Evaluation of the Grapevine red blotch virus Epidemiological Landscape of the Okanagan Valley submitted by Dieter Kahl in partial fulfillment of the requirements of the degree of Master of Science. Dr. Miranda Hart, Department of Biology, Irving K. Barber Faculty of Sciences Supervisor Dr. D. Thomas Lowery, Department of Biology, Irving K. Barber Faculty of Sciences Supervisory Committee Member Dr. José Ramón Úrbez-Torres, Department of Biology, Irving K. Barber Faculty of Sciences Supervisory Committee Member Dr. Jörg Bohlmann, Department of Botany, Faculty of Science, UBC Vancouver University Examiner Click or tap here to enter text. External Examiner Additional Committee Members include: Dr. Robert Lalonde, Department of Biology, Irving K. Barber Faculty of Sciences Supervisory Committee Member Click or tap here to enter text. Supervisory Committee Member iii Abstract The first data chapter of this research presents a novel approach for identifying potential insect vectors of circulative plant viruses, significantly reducing time-to-results and the resource costs associated with greenhouse transmission experiments. This artificial transmission protocol using a buffered sucrose solution as the virus recipient in place of a living plant, also eliminates plant-to-plant variation in susceptibility to infection, allowing assessment of virus-insect compatibility required for successful transmission of persistent viruses. To validate this approach, species of hemipteran insects, including leafhoppers, froghoppers, aphids, sharpshooters, and treehoppers, were subjected to the artificial feeding system to determine their ability to vector Grapevine red blotch virus (GRBV). Test insects were allowed to feed on a potted grapevine infected with GRBV for three days and then transferred to tubes containing the sucrose solution partitioned by a thinly stretched Parafilm membrane. After three days of feeding through the membrane, viruliferous test insects were stored for species identification and the sucrose solutions were tested by polymerase chain reaction for the presence of GRBV DNA. Of all the insects tested (n = 393), only nine treehoppers from two different species successfully transmitted GRBV to the sucrose solutions, indicating a high-likelihood of vector capability to plants, to be validated by greenhouse or field experiments. The second data chapter presents research that investigated GRBV titres from several grapevine tissues, including roots, buds, cortical scrapings, and leaves, at varying distances from the cordons, and at different times during the growing season and winter dormancy. Two common nucleic acid extraction techniques were compared, a low cost high-throughput nucleic acid extraction method (GES) and a DNA column purification method. Ideal combinations of sampling parameters were determined for summer and winter diagnostics. In dormant samples, cortical scrapings yielded the highest GRBV titres and produced no false-negatives, and basal leaves yielded high GRBV titres throughout the summer months with only one false-negative on the earliest sample date. These two tissues provide the best samples for reliable and reproducible GRBV diagnostics. iv The crude GES and DNA column purification methods performed comparably well for binary diagnostics, however the GES method performed poorly in quantitative analysis. v Lay Summary Grapevine red blotch virus (GRBV) is a virus that infects grapevine, causing significant disease that reduces berry yields and quality. Grapevine viruses are typically spread by the feeding of small living things such as insects or microscopic worms. Identifying the biological agents that spread grapevine viruses is key for effective disease management. Furthermore, informed strategies for identifying and diagnosing virus infections are key for effective disease detection. This research presents a new approach for identifying the insect vectors of certain types of grapevine viruses, including GRBV. This new approach is significantly faster and less resource intensive than the traditional method. This research also presents optimized sampling and molecular diagnostic guidelines for reliable GRBV detection from summer leaf and winter wood tissues. Taken together, the findings of this thesis paint a clearer picture of the status of GRBV in the Okanagan and provides future researchers with powerful tools for disease management. vi Preface This research project was developed primarily by myself with revisions and guidance provided by all members of the research committee. The introductory chapter (chapter 1) contains a copyrighted figure (Fig 1.1) that is reprinted with permission of the SIB Swiss Institute of Bioinformatics, which permits use of their pictures in academic theses. For the study relating to artificial transmission of GRBV (chapter 2), significant assistance was provided by an undergraduate co-op student, Amanda Nyirfa (University of British Columbia, Okanagan), with insect collection and the artificial transmission assays. Species identification of the collected insects was done by Dr. Joel Kits, a research scientist at the National Identification Service (Agriculture and Agri-Food Canada, Ottawa). I helped with insect collection and was solely responsible for assay design, data analysis and writing of the manuscript. The manuscript, co-authored by Dr. Tom Lowery, Dr. José Ramón Úrbez-Torres, Dr. Miranda Hart, Dr. Joel Kits, and Amanda Nyirfa, was submitted to the Canadian Journal of Plant Pathology and accepted with minor revisions. For the study relating to optimal diagnostics of GRBV (chapter 3), assistance was again provided by Amanda Nyirfa in the collection and processing of leaf and woody tissues. She also helped conduct the nucleic acid extractions from these tissues. I was solely responsible for RNA depletion, nucleic acid quantification, serial dilutions, virus quantification, data analysis, and writing of the manuscript. The manuscript, co-authored by Dr. José Ramón Úrbez-Torres, Dr. Tom Lowery, and Dr. Miranda Hart, was submitted to the Canadian Journal of Plant Pathology and is currently in peer review. My research stipend and both studies were funded by matching funds from the Canadian Grapevine Certification Network and Agriculture and Agri-Food Canada through the Growing Forward Canadian Agriculture Partnerships (CAP) Program. vii Table of Contents Abstract .................................................................................................................................... iii Lay Summary ............................................................................................................................ v Preface ...................................................................................................................................... vi Table of Contents .................................................................................................................... vii List of Tables ............................................................................................................................. x List of Figures .......................................................................................................................... xi Chapter 1: Introduction ............................................................................................................ 1 1.1 Biological relationships and disease in grapevine ..................................................... 1 1.2 The importance of grapevine virus epidemiological research .................................... 2 1.3 Disease management and sanitation programs ........................................................ 3 1.4 Grapevine virus physiology and replication biology ................................................... 5 1.5 Grapevine virus transmission biology........................................................................ 6 1.6 The economically significant grapevine viruses ........................................................ 8 1.6.1 The Long History of Grapevine Leafroll-associated Virus-3 ................................10 1.6.2 The Short History of Grapevine Red Blotch Virus ...............................................12 1.7 Virus mutation rates, diversity, and adaptations .......................................................15 1.8 Grapevine virus diagnostics .....................................................................................16 1.9 Research objectives ................................................................................................18 Chapter 2: Identification of candidate insect vectors of Grapevine red blotch virus by means of an artificial feeding diet ......................................................................................... 19 2.1 Review .....................................................................................................................19 2.2 Materials and methods ............................................................................................22 2.2.1 Insect collection and identification ......................................................................22 2.2.2 Artificial transmission assays .............................................................................23 2.2.3 Greenhouse transmission assays ......................................................................24 viii 2.2.4 PCR and product visualization ...........................................................................25 2.2.5 Statistical analysis ..............................................................................................26 2.3 Results ....................................................................................................................26 2.3.1 Artificial transmission assays .............................................................................26 2.3.2 Greenhouse transmission experiments ..............................................................28 2.4 Discussion ...............................................................................................................28 Chapter 3: Seasonal dynamics and optimal diagnostics of Grapevine red blotch virus in a British Columbian vineyard ................................................................................................. 33 3.1 Review .....................................................................................................................33 3.2 Materials and methods ............................................................................................36 3.2.1 Site selection .....................................................................................................36 3.2.2 Dormant sample collection .................................................................................36 3.2.3 Leaf sample collection .......................................................................................37 3.2.4 Nucleic acid extraction and quantification ..........................................................38 3.2.4.1 TNA extraction by GES method .................................................................38 3.2.4.2 DNA purification by column method ...........................................................39 3.2.4.3 Qubit quantification of total DNA.................................................................39 3.2.5 Primer design and optimization of droplet digital PCR ........................................39 3.2.6 GRBV quantification by droplet digital™ PCR ....................................................40 3.2.7 Statistical analysis ..............................................................................................41 3.2.7.1 Viral load calculation ..................................................................................41 3.2.7.2 Mixed effects model to predict the effects of sampling date and locus on viral load in leaves .....................................................................................................42 3.2.7.3 Two-way repeated measures ANOVA to determine the effects of locus and section on viral load in dormant buds, cortical scrapings, and roots ...........................43 3.2.7.4 Method comparison by scatter plot .............................................................43 ix 3.3 Results ....................................................................................................................44 3.3.1 Mixed effects model to predict the effects of sampling date and locus on viral load in leaves ................................................................................................................44 3.3.2 Two-way repeated measures ANOVA to determine the effects of locus and section on viral load in dormant buds, cortical scrapings, and roots ...............................45 3.3.3 Comparison of extraction methods .....................................................................46 3.4 Discussion ...............................................................................................................47 Chapter 4: Conclusion ............................................................................................................ 54 4.1 Determining if vectors of GRBV are present in the Okanagan valley .......................54 4.1.1 Traditional greenhouse transmission experiments .............................................54 4.1.2 Artificial transmission experiments .....................................................................55 4.2 Optimization of GRBV sampling and diagnostics .....................................................56 4.2.1 Best practices in sampling leaves ......................................................................56 4.2.2 Best practices in sampling dormant tissues........................................................57 4.2.3 Best nucleic acid extraction method for diagnostics ...........................................57 4.3 Paving the way forward ...........................................................................................57 Bibliography ............................................................................................................................ 59 x List of Tables Table 1.1. The genomic organization of six economically significant grapevine viruses (Hulo et al. 2011). ss=single stranded; poly(A)=polyadenylation; VPg=viral protein genome-linked; ORF=open reading frame…………………………………………………………………………….…9 Table 1.2. The methods of spread of the six economically significant grapevine viruses outlined in table 1.1……………………………………………………………………………………………….10 Table 2.1. Insects collected for use in the ATS, taxonomic ID’s of a subset of collected insects, and ATS PCR results…………………………………………………………………………………...27 Table 3.1. Linear mixed effects model summary statistics…………………………………………44 xi List of Figures Figure 1.1. The general physiology of a hemipteran insect and the points of entry and exit of virus particles. Reprinted with permission of the SIB Swiss Institute of Bioinformatics (Source: ViralZone www.expasy.org/viralzone, Swiss Institute of Bioinformatics)......................................7 Figure 2.1. Transmission assay experimental setups. Standard artificial transmission system (ATS) (a); inoculation access period (IAP) stage of artificial transmission, showing green honeydew as an indicator of treehopper feeding (b), and; IAP stage of greenhouse.................24 Figure 2.2. Example gel segment showing positive (sample 296) and negative (samples 295, 297-299) polymerase chain reaction amplification events..........................................................28 Figure 2.3. Morphological comparison of two collected buffalo treehoppers species that transmitted Grapevine red blotch virus, S. bisonia (a) and S. basalis (b). Scale = 1 mm...........29 Figure 3.1. Grapevine tissue sampling scheme. (a) Dormant buds (circles) and cortical scrapings (rectangles) were collected from three randomly selected shoots at three different sections per vine (the 1st, 3rd, and 5th buds with scrapings directly adjacent) and pooled by section; (b) Leaf samples were collected randomly in threes from any of the shoots at three different loci per vine (the 1st, 3rd, and 4th wire regions) and pooled by locus..........................38 Figure 3.2. Estimated marginal effects of locus and date on viral load in leaves (lines). Ribbons represent 95% confidence intervals. Shaped points represent raw measurements from individual plants (n = 119). Raw data points are jittered to minimize overlap for optimal visualization................................................................................................................................45 Figure 3.3. Mean viral loads in dormant tissues (filled circles). Hollow circles represent raw measurements from individual plants (n = 70). Raw data points are jittered to minimize overlap for optimal visualization..............................................................................................................46 xii Figure 3.4. Plot of paired measures (green dots) of GRBV titres determined by ddPCR of crude DNA (y-axis) versus titres from column purified DNA (x-axis). Comparison of the variance (grey lines) between the paired measures and a hypothetical ideal agreement (black line) produced a corresponding coefficient of correlation value of 0.55 (green line), indicating minimal agreement between the two methods (n = 188)............................................................................................47 Chapter 1: Introduction 1.1 Biological relationships and disease in grapevine Grapevines have complex relationships with the many organisms that share their growing space. While many of these relationships can be neutral, some can be mutually beneficial while others can be negative for plant growth. Weeds and neighboring plants may compete for soil nutrients and water, but they may also recruit insects that prey on pests of grapevine thereby offering a new line of defense. Conversely, the insects that are recruited or preyed upon may also be vectors of more biological competition, such as pathogenic microorganisms like fungi, bacteria and viruses (Keller 2015). A few examples of these microbial competitors are outlined. Biotrophic fungi, such as mildews, colonise the green surfaces of grapevines, imparting potentially unmarketable discoloration of the grapes or off-odours in wines (Meng et al. 2017). Necrotrophic fungi - the rots - opportunistically infest green tissues of grapevine through wounds or directly through the cuticle of immature tissues causing significant necrosis in the process. One of the few significant bacterial pathogens of grapevine, Rhizobium vitis, produces benign tumours in the host called crown gall (Eastwell et al. 2006). This is accomplished by injecting their own DNA into the host genome and hijacking the transcriptional and translational machinery, much like viral pathogens, to induce localised plant proliferation and alter metabolism to favour bacterial expansion (Burr and Otten 1999). A less common but equally significant bacterium, Xylella fastidiosa, is vectored by and injected into the grapevine xylem by sharpshooter insects (Purcell et al. 1999). This bacterium causes Pierce’s disease, which significantly hinders the movement of water through the host’s vascular tissues and results in the eventual death of the affected vine (Meng et al. 2017). Grapevine viruses, the focus of this research, are ubiquitous in cultivated grapevine and the severity of the diseases they produce varies wildly (Engel et al. 2010). Viruses can suppress 2 host gene expression to favour their own replication by host gene shutoff (Havelda et al. 2008), which contributes to disease symptoms. Infection by certain viruses also interferes with plant phloem transport as a result of callose sugar deposition in the vascular tissues, a plant response to infection aimed to limit the movement of virus particles through the plasmodesmata (Martelli et al. 1986). This restricted circulation results in the accumulation of sugars in the leaves, which induces feedback inhibition of photosynthesis and the accumulation of anthocyanins, made apparent by characteristic interveinal reddening of the leaves in red cultivars and yellowing in white cultivars (Espinoza et al. 2007). Disease symptoms can vary wildly between virus species, grape varieties and environmental conditions. Significant virus diseases generally reduce grapevine vigor and berry yield (Burger et al. 2017). Often, dysfunctional photosynthesis in leaves is observed and berry characteristics are significantly altered in diseased plants, such as reduced sugar levels and elevated acidity (Bertamini and Nedunchezhian 2002; Alabi et al. 2016; Sudarshana et al. 2015; Wallis and Sudarshana 2016). While it is evident that grapevines are challenged by a large range of pathogens, this research will only focus on the epidemiology and management of grapevine viruses and the organisms that vector them. 1.2 The importance of grapevine virus epidemiological research An economic impact study found that the Canadian wine industry grew 33% from $6.8 billion to $9 billion between 2011 and 2015 (Rimerman and Eyler 2015). A similar or even greater trend could be assumed between 2015 and the present. At the time of the study, there was an estimated 1,770 grape growers producing for 604 wineries. It is likely that many of these Canadian businesses acquire grapevine stock materials from American or European nurseries and therefore the potential for the introduction of virus-infected plants or viruliferous vectors from these foreign regions should not be overlooked. In this context, epidemiological research 3 must keep up with the growing demand for effective monitoring and diagnosis of viral infections. Grapevine virus research has a rich history and is well established, however our understanding of the biology of virus, host and vector relationships and the technologies we employ are constantly changing. New grapevine viruses are still being discovered and in some cases the significance of their impact on fruit quality and economy has yet to be determined. 1.3 Disease management and sanitation programs As mentioned, grapevine viruses are ubiquitous and the economically significant ones are well-established worldwide. Before we dwell on the biology of the viruses and the organisms that spread them, it is important to first understand how these viruses have become so prevalent and how they are managed. Primary infection is the initial introduction of an infectious agent to an otherwise healthy vineyard sensu stricto (Gilligan and Kleczkowski 1997). In the case of viruses, this primarily takes two forms: the incorporation of virus-infected nursery material, be that whole plants, rootstocks, or scion grafts, or as the unintended co-transportation of viruliferous insect or microbial vectors. The former is more economically significant in the short run since infected crops may not be recognized as such until they reach a certain maturity, and entire vineyards may be established from clones of diseased plants. On the other hand, the unintended transportation of a virus vector, viruliferous or not, may take a long time to present as a significant issue. Without any initial incentive to control or eliminate the vector, such as the knowledge of its vectoring capacity, it may consequently become established and highly resilient to later interventions. In this context, a virus disease present at endemic levels due to the naturally limited distribution of its vectors has the long term potential of becoming an epidemic as a consequence of the unintentional introduction of its vector, newly conferring the potential for secondary spread. This is exemplified by a local large winery that 4 recently took ownership of a nearby smaller winery. The acquired winery had a serious mealybug problem and the vineyard manager is now concerned that the mealybugs may be transported on equipment from the smaller winery’s vineyard to his larger mealybug-free vineyards. As a result, this manager must determine if leafroll virus is present at the smaller vineyard, and if so, must take precautions to prevent the movement of the likely viruliferous mealybug vectors and therefore leafroll disease to the larger vineyards. Preventative strategies focus on the primary and secondary levels of virus spread. Primary prevention is accomplished by sanitation programs driven to certify source nursery crops as pathogen-free. Secondary prevention is accomplished by informed and responsible pesticide use, by diligent screening of imported and exported plant materials for insect vectors by regulatory bodies, and by timely response to newly identified outbreaks by growers and crop managers. Unfortunately, these strategies were proposed and adopted only after it was recognized that virus-infected materials had already been disseminated worldwide, and rectifying this issue has been largely unsuccessful (Ricketts et al. 2015; 2017). Nevertheless, growers are becoming more aware of the prevalence of grapevine virus diseases and are proactive in implementing management programs to prevent spread. However, some of these programs, such as the use of overly-effective insecticides like those of the neonicotinoid family, may prove to be counter-productive in the long run. Heavy handed interventions such as these are not only effective in eliminating pest insects, but in eliminating beneficial insects as well, such as those that naturally prey on and control populations of pest insects (Altieri and Nicholls 2018). We fear that in such an event, if the application of such an effective insecticide is ceased because the pest has seemingly been eliminated, then it is possible for pest populations to recover at a greater rate than the populations of their natural predators, resulting in significant re-emergent outbreaks. 5 1.4 Grapevine virus physiology and replication biology To understand why viruses often cause disease in plants, we need to understand how viruses reproduce. Plant viruses are submicroscopic obligate parasites that often cannot exist long outside of a biological host (Nelson and Citovsky 2005). They can be hosted by insects, nematodes, protozoans, and plants, but often only produce disease in the latter (Stafford et al. 2011). Plant viruses do not encode their own replicative machinery and therefore depend on that of their hosts (Nelson and Citovsky 2005). They usually have small single-stranded RNA genomes, however DNA-viruses have been identified in grapevine (Scholthof et al. 2011). Their genomes are protected by a layer of protective proteins called the capsid; the complete viral particle is called the virion (Alzhanova et al. 2001). Some viruses may acquire a host-derived lipid-envelope upon exiting their host cell (Nelson and Citovsky 2005). RNA viruses are often found in the host cell cytoplasm where translation takes place; DNA viruses must enter the host cell nucleus to access the transcriptional machinery (Cann 2001). Plant viruses often encode a protein called replicase, an RNA-dependent RNA polymerase (Vo et al. 2003). This transcriptional enzyme catalyzes the replication of viral RNA using viral genomic RNA as a template, and is the powerhouse of viral replication (Cann 2001). Once the genome has been sufficiently replicated and the capsid proteins have been sufficiently translated, the viral particles often form spontaneously, though in some cases a host-derived chaperone protein is required (Cann 2001). Plant viruses often encode a movement protein which enables the intracellular movement of viral genomes through the host plasmodesmata (Hong and Ju 2017). The amino acid composition of a virus’ coat protein determines its ability to be retained by biological hosts and therefore its host range, through host-receptor protein interactions (Cann 2001). 6 These metabolically expensive mechanisms are what induce disease in grapevine, as a result of the diverted cellular resources as well as the plant response to such events, as described above. 1.5 Grapevine virus transmission biology While some plant viruses can be spread between hosts through the mechanical transfer of sap, most require an intermediate host, or vector, to facilitate the transfer. Virus-vector relationships are often complex and highly specific, relying on lock-and-key compatibility between the virion coat-protein and vector cell receptors (Stafford 2011). Watson and Roberts (1939) coined the terms “non-persistent” and “persistent” viruses to better categorise virus-vector relationships. These labels are generally assigned based on virus-retention time by the vector, which is simply the window of transmission competency starting from virus acquisition by the vector. Non-persistent viruses have very short retention times, anywhere from minutes to hours. Semi-persistent viruses may be retained for hours to days, but not beyond vector molting (Sylvester 1956). Persistent viruses are often retained for days or throughout the vectors life. These labels can be assigned by experimenting with acquisition access periods (AAP)—the amount of time a vector must feed to become viruliferous—and inoculation access periods (IAP)—the amount of time it takes a vector to transmit the infection—though many variables come into play such as plant and vector fitness, virus titre and variants, and environmental conditions (Nault 1997). Retention time is not random and reveals the nature of the virus-vector relationship. Nault (1997) expanded the terminology to describe the mechanisms of transmission by Hemipteran vectors: these terms are non-persistently transmitted, stylet-borne; semi-persistently transmitted, foregut-borne; persistently transmitted, circulative; and persistently transmitted, propagative (see figure 1.1 for general insect physiology). 7 Figure 1.1. The general physiology of a hemipteran insect and the points of entry and exit of virus particles. Reprinted with permission of the SIB Swiss Institute of Bioinformatics (Source: ViralZone www.expasy.org/viralzone, Swiss Institute of Bioinformatics). For non-persistent and semi-persistent transmission, the virus is retained only in the vector mouthparts (Ng and Falk 2006). Ng and Falk (2006) exemplify this with aphids, which are unable to visually identify their host plants and therefore must land on and probe many plants in search of suitability. The probing may only last minutes, but these minutes are a sufficiently large window for virus-acquisition from an infected plant and virus-transmission to other otherwise healthy plants. Semi-persistence is very similar to non-persistence except acquisition and inoculation mostly occur from and to phloem tissues (Ng and Falk 2006) and transmission success generally depends on longer AAPs. In this case, viral particles may be isolated from the vector’s foregut, but virions attached to the stylet and the cuticular lining of the anterior alimentary canal are lost upon ecdysis/molting (Nault 1997). Persistent viruses are described as circulative because once ingested by the vector the virus particles must pass through the gut 8 wall into the hemolymph, then from the hemolymph into the salivary glands to be discharged through salivary secretions. The delay between acquisition to salivary secretion, and therefore inoculativity, is called the latency period and is unique to persistent viruses. When a virus is able to replicate within its vector it is considered propagative (Nault 1997). 1.6 The economically significant grapevine viruses To date, over 86 distinct virus species belonging to at least 17 families and 27 genera have been reported to infect grapevine (Meng et al. 2017; Fuchs 2020). Most of these viruses do not produce any significantly apparent disease. Six of the most well studied viruses and their biology are outlined in tables 1.1 and 1.2. Grapevine leafroll-associated virus-3 (GLRaV-3) and grapevine red blotch virus (GRBV) will be the focus of this literature review as they are the most economically significant in the Okanagan. 9 Table 1.1. The genomic organisation of six economically significant grapevine viruses (Hulo et al. 2011). ss=single stranded; poly(A)=polyadenylation; VPg=viral protein genome-linked; ORF=open reading frame. Virus; disease Family, (sub-family), genus Virion Genome Grapevine fanleaf virus (GFLV); fanleaf degeneration Secoviridae, (comovirinae), Nepovirus Non-enveloped, T=pseudo3 icosahedral symmetry 28-30 nm in diameter, genomic RNAs encapsidated separately Bipartite, linear ssRNA (+), RNA-1 7.5 kb and RNA-2 3.9 kb, both with 3’ poly(A) tract and 5’ VPg, both strands contain 1 ORF each encoding a polyprotein Grapevine rupestris stem pitting-associated virus (GRSPaV); rugose wood complex Betaflexiviridae, Foveavirus Non-enveloped, long flexuous filamentous particles, >800 nm long , 12-13 nm in diameter Monopartite, linear ssRNA (+), 8.4-9.3 kb, with 3’ poly(A) tract and 5’ methylated cap Grapevine fleck virus (GFkV); fleck disease Tymoviridae, Maculavirus Non-enveloped, T=3 icosahedral symmetry, 30 nm in diameter Monopartite, linear ssRNA (+), 7.5 kb, with 3’ poly(A) tract, no 5’ cap Grapevine leafroll-associated virus-3 (GLRaV-3); leafroll disease Closteroviridae, Ampelovirus Non-enveloped, long flexuous filamentous particles, 1400-2200 nm long , 10-13 nm in diameter Monopartite, linear ssRNA (+), 16.9-17.9 kb, no 3’ poly(A) tract, likely 5’ methylated cap , 13 ORFs Grapevine red blotch birus (GRBV); red blotch disease Geminiviridae, Grablovirus Non-enveloped, geminate, incomplete T=1 icosahedral symmetry , genome per geminate particle Monopartite, circular ssDNA (+), 3.2 kb, coding regions in virion and negative strand, 6 ORFs Grapevine pinot gris virus (GPGV); grapevine leaf mottling and deformation Betaflexiviridae, Trichovirus Non-enveloped, long flexuous filamentous particles, 640-760 nm long, 10-12 nm in diameter Monopartite, linear ssRNA (+), 7.5-8.0 kb, with 3’ poly(A) tract and potentially 5’ cap 10 Table 1.2. The methods of spread of the six economically significant grapevine viruses outlined in table 1.1. Virus Vectors Germplasm dissemination Mechanical transmission GFLV Dagger nematode (Xiphinema index) Primary source of global presence of disease; vector likely spread in the soil of exported plants; not observed in Canada Only observed experimentally in herbaceous recipients (Cadman et al. 1960) GRSPaV None observed Primary source of global presence of disease; may also spread by pollen or seed (Minafra et al. 2000; Lima et al. 2004) Not observed GFkV None observed Primary source of global presence of disease Not observed GLRaV-3 Mealybugs and soft scale insects Primary source of global presence of disease Not observed GRBV Three-cornered alfalfa treehopper, (Spissistilus festinus) Primary source of global presence of disease Not observed GPGV Erineum mite, (Eriophyes vitis) Primary source of global presence Attempts unsuccessful 1.6.1 The Long History of Grapevine Leafroll-associated Virus-3 GLRaV-3 is a single-stranded (+)-sense RNA virus (family Closteroviridae, genus Ampelovirus) and the most significant causative agent of leafroll disease, likely first observed by Ravaz and Roos in 1905. It was demonstrated to be graft transmissible in Vitis vinifera by Schue in 1935, leading to the first hypothesis of its viral origin. The mealybug Pseudococcus maritimus was the first observed vector of GLRaV-3 by Chiarappa in 1961 (Rosciglione et al. 1983). Later, the first scale species, the cottony vine scale Pulvinaria vitis, and another mealybug species, the citrus mealybug Planococcus citri, were shown to effectively spread the virus as well (Belli et al. 1994; Cabaleiro and Segura 1997). Tsai et al. (2010) showed that there likely is no virus-vector specificity in mealybug transmission of GLRaV-3, as transmission has been shown with all tested mealybugs to date. As for scale, transmission has also been demonstrated in the fig wax scale Ceroplastes rusci, the long brown scale Coccus longulus, cottony maple scale Neopulvinaria innumerabilis, the nigra scale Parasaissetia nigra, the European fruit lecanium scale Parthenolecanium corni, and the European peach scale Parthenolecanium persicae (Mahfoudhi et al. 2009; Kruger and Douglas-Smit 2013; Fortusini et al. 1997; Zorloni et al. 2006; 11 Le Maguet 2012; Sforza et al. 2003; Hommay et al. 2008; Bahder et al. 2013; Habili 2015). Most studies to date have concluded that GLRaV-3 is likely spread in a semi-persistent manner (Cabaleiro and Segura 1997; Golino et al. 2002; Le Maguet et al. 2012; Tsai et al. 2008). In the Tsai et al. study (2008), transmission efficiency in P. ficus was found to increase as AAP time increased, with maximum efficiency observed after 24-hours. No latency period was observed, and insect inoculativity was lost after four days post-AAP, all of which support semi-persistent transmission. Though there has been good success to date identifying the vectors of GLRaV-3, some major concerns are presented: firstly, transmission efficiency has been shown to sometimes be significantly lower in greenhouses compared to in vineyards, which could lead researchers to erroneously conclude that unsuccessful transmission studies, unknowingly due to inappropriate environmental conditions, support the notion that the study species is not a vector of GLRaV-3 (Blaisdell et al. 2016). Secondly, there is growing concern that certain species of mealybug or soft scale that are similar in appearance may have been misidentified and therefore misrepresented, especially in research published before the era of DNA barcoding or simply absent of supporting molecular data. Finally, recent studies have shown that certain RNA viruses, including GLRaV-3, exhibit quasi-species characteristics and many dominating genetic variants have been discovered, which may mean that virus-vector compatibilities and transmission efficiencies could differ between virus populations (explained further in the next section) (Maree et al. 2013; Turturo et al. 2005). In this context, it is extremely important that these transmission studies are repeated with strong taxonomic identifications, high quality DNA sequences representing both the vector species and virus strains, and robust transmission experiments to overcome the limitations of greenhouse transmission studies. A major drawback of greenhouse transmission studies is that they take a very long time to produce results. In a conventional transmission study suspected vectors of a virus are allowed 12 to feed on an infected plant to allow those insects to potentially become viruliferous. They are then transferred onto healthy plants so that they may feed and potentially transfer the virus. Those potentially infected test plants must then be maintained, monitored, and tested for up to a year, or longer. Because transmission efficiency can be low, these experiments are highly prone to false-negatives. Strategies have been employed to evaluate insect-vectoring abilities without implementing these arduous greenhouse trials. One such approach, an artificial transmission assay, will be described. Much as for greenhouse transmissions, artificial transmission assays involve the transfer of virus from candidate insects, though the recipient is not a plant. In this case, candidate insects feed on a phloem-sap-like sucrose solution through a Parafilm membrane and it is the sucrose solution that is inoculated with virus particles. This approach is highly effective for circulative viruses, as once they reach their final tissue destination within an insect vector, the salivary glands, they are excreted with salivary secretions into any media in which the insect feeds. This technique has successfully been employed by Dr. Tony Wolf to evaluate and identify a leafhopper vector of the aster yellows phytoplasma (personal communication). Though this technique may be prone to false-positives as a result of transient, transmission-incompatible transfer of virus particles on the mouthparts of vector or non-vector insects, this technique is arguably still more effective than greenhouse trials due to the comparatively low cost and rapid processing time. One objective of this research will be to employ and evaluate the effectiveness of the artificial transmission assay in identifying potential vectors of GRBV. 1.6.2 The Short History of Grapevine Red Blotch Virus GRBV is a single stranded DNA virus (family Geminiviridae, genus Grablovirus) with a monopartite circular genome containing ORF’s in both directions (Rwahnih et al. 2013). Its 13 genome was first described by Krenz et al. in 2012 after observing the decline of Cabernet Franc vines in a vineyard exhibiting symptoms similar to GLRaV-3. A year later, the Virginia creeper leafhopper (VCLH) (Erythroneura ziczac Walsh) was reported as a vector of GRBV by Poojari et al. (2013) in greenhouse conditions. This study was also the first to describe the physiological effects of red blotch disease on grapevines, including reduced fruit yield, reduced pruning weights, elevated sucrose and starch levels in leaves, and altered berry quality attributes such as elevated titratable acidity (a measure of acidity) and lower total soluble solids (a measure of sugar content). To determine the distribution of GRBV in the United States, a study conducted to test grapevine samples collected from seven states concluded that GRBV is widely distributed across North America (Krenz et al. 2014). Furthermore, this study identified two distinct clades of GRBV, clades 1 and 2. Because this virus was determined to be widely established in the United States despite its emerging status, another study set out to test samples in the American National Clonal Germplasm Repository to determine if this was the source of its dissemination. Unsurprisingly, a significant number of these samples tested positive for GRBV, with the majority of these being from clade 2 (Rwahnih et al. 2015a). This group also analyzed preserved historic grapevine specimens collected between 1937 and 1950, one of which tested positive for GRBV. This sample was collected in Sonoma County, California, in 1940, which suggests that GRBV is not emerging but has potentially been present for the greater part of a century (Rwahnih et al. 2015b). By 2015 there was a strong understanding of the primary sources of spread of GRBV, but very little understanding on the degree of secondary spread. Furthermore, doubt was beginning to be casted on the claim that the VCLH is a vector of GRBV, as attempts to replicate this transmission had been unsuccessful (Daane et al. 2015; Bahder et al. 2016). Bahder et al. (2016) evaluated the flora adjacent to GRBV-positive grapevines in an attempt to find natural 14 reservoirs and alternate hosts of the virus. They were able to detect infection in a wild grape growing outside of a study vineyard, demonstrating that there must exist a mechanism for secondary spread, such as by a flying insect vector. That same year, a separate group reinforced these findings by detecting GRBV in free-living grapevine as well (Perry et al. 2016). Taking these findings, Bahder’s group set out to identify the organism that was facilitating the secondary spread of GRBV and to re-evaluate the claim that the VCLH is a vector (Bahder at al. 2016). While they were unable to replicate transmission of GRBV with the VCLH, they confirmed successful transmission by the three-cornered alfalfa treehopper (Spissistilus festinus). As of late, the treehopper is accepted to be the most likely vector while it is also generally accepted that the VCLH is not. Nonetheless, rigid transmissions studies must be conducted to reaffirm or refute these beliefs. Further, though unlikely for persistent viruses, it must be considered that multiple species of treehopper may transmit GRBV. This notion is supported by the observation of high rates of spread of red blotch disease in Oregon vineyards absent of S. festinus populations (Dalton et al. 2019). In recent years in the Okanagan, year-to-year increases in the incidence of red blotch disease symptoms has been observed in some affected vineyards, however it is unclear if these vineyards contain vectors. These vineyards are mapped yearly by symptomology and each individual symptomatic vine’s disease status is determined by molecular diagnostics. It is possible that some infected vines do not display symptoms in some years, resulting in an apparent increase in disease incidence in following years. Conversely, it is possible that our methods of sampling and diagnosis are suboptimal, resulting in unreliable and highly variable results, perhaps as a result of false-negatives. In this context, it is important that we critically evaluate our methodologies and optimize our pipelines to obtain reliable and reproducible diagnostic data. 15 1.7 Virus mutation rates, diversity, and adaptations In order to understand why highly specific diagnostic methods can still be riddled with false-negatives, we must understand how viruses mutate and evade detection. Plant virus populations are genetically heterologous as a result of high mutation rates, and the resulting variation is key for the evolution of these viruses. Primary sources of variation include genetic replication errors and genetic reassortments, the latter of which often produces new variants and sometimes even new virus species (Schalk et al. 1989). It has been shown that not all viruses equally benefit from or tolerate high mutation rates (Pita and Roossinck 2008). Two studies chemically induced mutations in different viruses, one using Venezuelan equine encephalitis virus and the other using vesicular stomatitis virus; one observed a massive increase in viral fitness, made evident by a 220-fold increase in the frequency of large-plaque mutants, while the other study observed a significant decrease in viral yield (Halle 1968; Holland et al. 1990). Many genetic models have shown that genetic insertions and deletions (indels) are generally much more deleterious than substitutions, as the former often disrupts or shifts open reading frames. However, substitutions in RNA viruses can be just as deleterious, even if synonymous, due to the important role of RNA secondary structure in some RNA viruses’ life cycles (Grogan and Moyer 2001). Furthermore, while it is common knowledge that mutation rates are often much higher in RNA replication compared to DNA replication due to the lack of proofreading of RNA-dependent RNA polymerase (replicase), mutations rates in DNA viruses can be just as high as those of RNA viruses, which suggests that there are other significant selection factors at play in virus evolution and adaptation—this set of factors is termed the mutational neighborhood (Burch and Chao 2000). 16 As mentioned before, GLRaV-3 displays quasi-species characteristics. Since RNA viruses produce massive amounts of progeny and given their high replicative error rate, approx. 0.1 to 2 mutations per genome, these progeny contain a high level of genetic diversity (Tromas and Elena 2010). This works as an advantage to virus populations as they are often under selective pressure, for example when moving between vector tissues, between hosts, or in highly variable host environments. In the quasi-species theory, the fitness of the viral infection is determined not at the individual level but at the population level, which means that selective pressures act on populations as a whole and those mutants that are well-adapted to these pressures have the potential to extend their function to the individuals that are not (Bull et al. 2005). The high degree of plant virus genetic diversity must be considered not only when conducting transmission experiments, but also in the context of diagnostics where the employed targeting systems may have varying degrees of effectiveness as a result of serological and genomic variation within species. 1.8 Grapevine virus diagnostics How viruses are able to evade molecular detection through genetic drift was described above. This section will describe the commonly employed molecular assays in detail and outline alternative diagnostic approaches. The symptoms that present on a diseased grapevine are often distinguishable and characteristic of a specific pathogen. However, in many cases, including that of GLRaV-3 and GRBV, the symptoms are very similar and indiscernible to the untrained eye (Krenz et al. 2012). Furthermore, a plant’s response to a pathogen can often look very similar to its response to a non-pathological stress such as a nutritional deficiency. As such, robust methods are necessary for reliable diagnosis of viral infections (Chalker-Scott 1999). 17 Classically, plant virus infections were confirmed by biological indexing; biological material from a test plant would be used to inoculate an indicator plant, such as with a grafted chip bud, and the indicator plant would be monitored for the development of disease symptoms (Rowhani and Golino 1995). This proved to be labor intensive, time-consuming, and was often unreliable, and so alternative techniques were quickly adopted (Maree et al. 2013). Serological techniques are popular for their robustness and cost-effectiveness, though they are not as sensitive as nucleic-acid based techniques (Gugerli 2009). These include enzyme-linked immunosorbent assays (ELISA), immunofluorescence, and immuno-strip tests (Schaad et al. 2003). Drawbacks to these are their high developmental costs and the challenges associated with producing compatible antisera; to date, researchers have been unable to produce antibodies specific to GRBV. Furthermore, it is unknown if industry-recommended ELISA kits are able to detect all of the newly reported genetic variants of their virus targets (Maree et al. 2013). Nucleic acid-based methods are the most commonly employed. These include the polymerase chain reaction (PCR) and all of its derivatives, such as reverse-transcription PCR (RT-PCR), quantitative PCR (qPCR), droplet digital PCR (ddPCR), immunocapture PCR, and more (Maree et al. 2013). The fundamental basis of PCR is the in vitro amplification of nucleic acid targets using complementary oligonucleotide targeting systems and bacteria-derived transcriptional machinery with high specificity and sensitivity. However, the success of these techniques relies on the specificity of the oligonucleotide primers, and as more and more genetic variants of common viruses are being detected and reported, the commonly used primers may become decreasingly useful (Maree et al. 2013). Next-generation sequencing is currently the golden standard for disease diagnostics as it does not rely on specific targeting systems and provides much more information than all other approaches, but is extremely labor extensive and 18 expensive and therefore not compatible with routine or high-throughput programs (Maree et al. 2013). Regardless of the detection method, a key issue in diagnostics is identifying a reliable method of sampling and method of preparing that sample for testing. For example, although a grapevine may exhibit strong disease symptoms and has even tested positive for a specific viral infection, later retesting can often produce contradictory results, such as false-negatives. This is because grapevine viruses are unevenly distributed in affected-plants, especially GLRaV-3 and GRBV (Teliz et al. 1987; Setiono et al. 2018). As such, experimenters must identify and adopt effective sampling strategies to overcome this challenge. Furthermore, even if an effective sampling strategy is established, it must be complimented with equally reliable protocols for sample processing. Crude extractions of nucleic acids or proteins may be preferable for high-throughput programs, but the drawbacks of using highly inhibited samples and their decreased compatibility with sensitive molecular assays must be critically assessed. In this context, the final objective of this research will be to establish reliable sampling and processing techniques for grapevine virus diagnostics to eliminate or reduce type II error. 1.9 Research objectives The general objective of this research is to produce an in-depth report on the Grapevine red blotch virus epidemiological landscape of the Okanagan region. This will be accomplished through a combination of lab and field experiments, and through highly controlled greenhouse experiments. The proposed studies are the following: 1. Using traditional and novel approaches, evaluate candidate insects as potential vectors of GRBV, and; 2. Determine the ideal grapevine tissue for sampling and method of processing for obtaining the lowest amount of GRBV PCR false-negatives. 19 Chapter 2: Identification of candidate insect vectors of Grapevine red blotch virus by means of an artificial feeding diet Dieter Kahl1,3, José Ramón Úrbez Torres1, Joel Kits2, Miranda Hart3, Amanda Nyirfa1, D. Thomas Lowery1 1. Agriculture and Agri-Food Canada, Summerland Research and Development Centre, 4200 Highway 97, Summerland, British Columbia, V0H 1Z0, Canada. 2. Agriculture and Agri-Food Canada, Ottawa Research and Development Centre, 960 Carling Avenue, Ottawa, Ontario, K1A 0C6, Canada. 3. Department of Biology, The University of British Columbia – Okanagan, 3187 University Way, Kelowna, British Columbia, V1V 1V7, Canada. Corresponding author: D. Thomas Lowery; Tom.Lowery@canada.ca 2.1 Review To date, grapevines have the highest number of identified viruses of any crop, 86, many of which produce varying degrees of disease severity (Fuchs 2020). The effects of virus infection can be inconsequential on the health of the vines and quality of fruit (Meng et al. 2017), while in other cases the disease can be severe and economically detrimental, such as for the highly pathogenic infectious degeneration, leaf roll, rugose wood, and graft incompatibility and decline (Wilcox et al. 2015; Meng et al. 2017). Grapevine red blotch virus (GRBV), the causal agent of grapevine red blotch disease (GRBD), is an emerging, widespread grapevine virus that is a concern due to the severity of the disease it causes (Krenz et al. 2014). While the effects on yield of GRBD have yet to be fully elucidated, one recent study found that GRBD reduced yields of seven-year-old Cabernet Franc by 42% (Bowen et al. 2020). The economic losses associated with infection are estimated to be between $2,213 - $68,548 US per hectare of infected vineyard (Ricketts et al. 2017). Most grapevine viruses are spread by the feeding of insect vectors, while others are vectored by mites, nematodes, and possibly chytrid fungi (Andret-Link and Fuchs 2005). For insect-borne plant viruses, there are several modes of transmission: non-persistent transmission, stylet-borne; semi-persistent transmission, foregut-borne; persistent transmission, circulative; and 20 persistent transmission, propagative (Sylvester 1956; Nault 1997). The number of expected insect vectors of a particular plant virus can be inferred by its mode of transmission. Persistent viruses are described as circulative because once ingested by the vector, the virus particles must pass through the gut wall into the haemolymph; then from the haemolymph into the salivary glands to be discharged during salivary secretions. A lengthy latent period between virus acquisition and inoculativity by salivary secretion is unique to persistent viruses. The minimum latent period for whitefly-transmitted geminiviruses is approximately 19 hours (Rosen et al. 2015) and for leafhopper-transmitted geminiviruses, approximately 23 hours ± 4.1 hours (Hull 2014). When a virus is able to replicate within its vector, it is considered propagative (Nault 1997). GRBV is the type species of the genus Grablovirus in the family Geminiviridae (Varsani et al. 2017), which consists of viruses that are vectored in a persistent-circulative manner (Briddon et al. 1990; Nawaz-ul-Rehman and Fauquet 2009). The known insect vectors of geminiviruses are treehoppers (Hemiptera: Membracidae), leafhoppers (Hemiptera: Cicadellidae), whiteflies (Hemiptera: Aleyrodidae), and aphids (Hemiptera: Aphididae) (Briddon et al. 1996; Bosque-Pérez 2000; Hogenhout et al. 2008; Roumagnac et al. 2015; Chen and Gilbertson 2016). Insect vector specificity is determined by the coat protein of geminiviruses (Azzam et al. 1994; Briddon et al. 1996; Soto et al. 2005). It has been suggested that specific amino acid residues of the coat protein are involved in receptor-mediated endocytosis in the insect gut and salivary glands (Noris et al. 1998; Höhnle et al. 2001; Soto et al. 2005). As such, insect vectors compatible with any particular geminivirus are usually limited to a single species or to a small number of closely related species within a genus. GRBV has only recently been discovered and two vectors, the Virginia creeper leafhopper (VCLH) (Erythroneura ziczac Walsh, Hemiptera: Cicadellidae) and the three-cornered alfalfa treehopper (Spissistilus festinus Say, Hemiptera: Membracidae), have been suggested from laboratory transmission experiments (Poojari et al. 2013; Bahder et al. 21 2016). Recent doubt has been cast on VCLH’s potential as a vector of GRBV, as like most monopartite geminiviruses, GRBV is likely phloem-limited (Jeske 2009) and E. ziczac has the propensity to feed on the mesophyll (Saguez et al. 2015). Additionally, attempts to replicate greenhouse transmission of GRBV by VCLH have been unsuccessful (Daane et al. 2015; Bahder et al. 2016). It is possible that S. festinus is the only viable vector of GRBV, although there may be others. Candidate vectors of plant viruses may be suggested through phylogenetic analyses of known virus-vector relationships if the virus of interest is closely related to another well characterised virus (Jones 2003; Bahder et al. 2016). This approach was implemented by researchers seeking to find the vector of GRBV in a vineyard where spread of GRBD was evident, which eventually led to the discovery of S. festinus as a vector of GRBV (Bahder et al. 2016). Observational studies, such as monitoring and collection of insects present in a vineyard exhibiting year-to-year spread of virus infection, may also suggest possible vectors; however, this approach alone is unproductive when the study site has substantial species richness. Currently, a candidate insect can only truly be deemed a vector of a given virus if transmission is proven through highly controlled greenhouse transmission studies with considerable replication. For persistent viruses in particular, care must be taken to ensure that acquisition access period (AAP) and inoculation access period (IAP) feeding times are sufficiently long to overcome the lengthy latent period. For woody perennials such as grapevines, plants must be cultured for many months, possibly including a period of cold for symptom expression and an increase in virus titre. Plants must be maintained, visually inspected for symptoms, and tested for infection periodically using appropriate methodology, such as the polymerase chain reaction (PCR). This method is simple but time and resource intensive. Negative results could occur due to virus-insect incompatibility, condition of the plants, or from other unknown causes. Virus transmission may in some cases be intrinsically lower under greenhouse conditions, increasing 22 the likelihood of falsely concluding a virus-insect relationship as biologically incompatible (Blaisdell et al. 2016). Furthermore, absent of guidance from a well-designed phylogenetic analysis, identifying insects as likely candidates may be difficult. An insect vector of North American Grapevine Yellows phytoplasma was recently elucidated using an artificial transmission approach (Lenzi et al. 2019). This method is similar to traditional greenhouse experiments, except that IAP feeding is facilitated into a sucrose solution partitioned behind a stretched Parafilm™ membrane rather than a virus-free recipient plant. The sucrose solution is then used as template in PCR, which can detect minute amounts of pathogen DNA (or RNA via reverse-transcription PCR). This method has several advantages over the traditional approach: it eliminates the time and resource commitment required to maintain inoculated plants, reduces time-to-results from months to days, and limits the success of the assay to virus-insect compatibility. Accordingly, the purpose of this study was to utilize the artificial transmission system (ATS) of Lenzi et al. (2019) to identify candidate insect vectors of GRBV. 2.2 Materials and methods 2.2.1 Insect collection and identification Hemipteran insects to be used in the ATS were collected by sweep netting in the Okanagan and Similkameen valleys, British Columbia, Canada. Collected insects were placed in large freezer bags and kept at 4°C . Hemipteran insects were identified based on morphology and logged by generic identification (i.e. leafhopper, treehopper, etc.). Preserved specimens were sent to the Canadian National Collection of Insects, Arachnids and Nematodes (CNC), Agriculture and Agri-Food Canada, Ottawa, ON, for identification (by JK). 23 2.2.2 Artificial transmission assays Collected insects were placed in a cage containing a potted grapevine infected with GRBV that was propagated by cuttings from an infected field plant. These GRBV-infected source plants were maintained in the greenhouse at SuRDC as described below. Virus acquisition plants were initially and regularly tested for GLRaV-1, -2, -3, -4, -7, GRBV, Grapevine fanleaf virus, and Arabis mosaic virus by PCR or reverse transcription-PCR to continually confirm its single infection status. Based on previously published latent periods of 19 and 23 hrs for other geminiviruses (Hull 2014; Rosen et al. 2015), insects were given an AAP of 72 hrs then transferred to individual ATS tubes (Figure 1a). A longer AAP was used in consideration of the unknown and possibly variable latency period for insect transmission of GRBV. Each ATS tube consisted of a 2 mL Eppendorf™ tube (Eppendorf, Hamburg, Germany) punctured with a needle to allow air exchange. 200 µL of a 5% w/v buffered sucrose solution (10 mM Tris , mM EDTA, pH 8.0) containing green food colouring was pipetted into each tube cap, and then sealed using ParafilmTM (Bemis Company Inc, Neenah, WI, USA) and slight pressure applied with the fingertip. The food colouring was added to assist with the visualization of honeydew that indicated successful feeding (Figure 1b). Individual insects were placed in these tubes and allowed to feed for an IAP of three days. Follow the IAP, insects were transferred to new tubes and suspended in 95% ethanol for later species identification, and the sucrose solutions were stored at -80°C for later virus detection. 24 Figure 2.1 Transmission assay experimental setups. (a) Standard artificial transmission system (ATS); (b) Inoculation access period (IAP) stage of artificial transmission, showing green honeydew as an indicator of treehopper feeding, and; (c) IAP stage of greenhouse transmission. 2.2.3 Greenhouse transmission assays Collected VCLH’s and treehoppers were placed in separate cages each containing a potted grapevine infected with GRBV. Following an AAP of 72 hr, VCLH’s and treehoppers were separately transferred in cohorts of five for VCLH’s and cohorts of fifteen for treehoppers to healthy, three-month-old potted Cabernet Franc grapevines grown from seed individually contained within ventilated plastic cages (Figure 1c). After an IAP of three days, the VCLH’s and treehoppers were removed and the recipient plants were placed in a greenhouse chamber to be maintained. Control plants never exposed to study insects were also included. After at least three months, the test plants were lightly trimmed and moved into cold storage to induce diapause-dormancy. After at least two months in dormancy, the test plants were placed in a greenhouse chamber to induce vegetative growth. The plants were closely monitored for pests and treated accordingly. Powdery mildew was treated with application of sulfur dust and mite infestations were treated with either foliar applications of Forbid (spiromesifen, 0.03% v/v) or 25 through releases of Neoseiulus fallacis and Phytoseiulus persimilis (Westgrow Biological Solutions Inc, Fort Langley, BC). Greenhouse chambers were kept between 17-25°C and 30-60% relative humidity in the winter, or 17-28°C and 30-75% relative humidity for all other seasons. Plants were maintained under 400-watt high pressure sodium lights set to 16-hour day-lengths, which were turned off in July and August. Recipient plants were tested for GRBV by PCR one- and three-months post-inoculation, and again six months post-dormancy. For each collection for testing, five leaves consisting of both mature basal and immature distal leaves were sampled from each plant. For each plant 250 mg of leaf petiole tissue was placed in an extraction bag (BIOREBA, Kanton Reinach, Switzerland) and suspended in 5 mL of extraction buffer (15 mM sodium carbonate, 35 mM sodium bicarbonate 2% (w/v) polyvinylpyrrolidone-40, 0.2% (w/v) bovine serum albumin), 0.05% (v/v) Tween-20) (Rowhani et al. 2000). The petioles were ground up and homogenized using a Homex 6 (BIOREBA, Kanton Reinach, Switzerland) then placed on ice. 6 µL of lysate was added to 50 µL of stabilization buffer (100 mM glycine, 50 mM sodium chloride , mM EDTA, 0.5% (v/v) Triton X-100 ,% β-mercaptoethanol) (Rowhani et al. 2000), denatured at 95°C for 10 minutes, then snap cooled on ice. This solution was then used as template in PCR. 2.2.4 PCR and product visualization PCR was conducted on the sucrose solutions and leaf extracts to test for the presence of GRBV DNA. ATS tubes were removed from the freezer to allow the sucrose solutions to thaw. Tube caps containing buffered sucrose solution were carefully removed and the ParafilmTM discarded. PCR for both the ATS assays and greenhouse transmission assays were conducted using a Roche™ Taq PCR kit (Sigma-Aldrich Inc, St. Louis, MO, USA) with the forward primer GTAGATTGAGGACGTATTGG (800 nM) and reverse primer CGCAAGAATACCACGTACCATGGG (800 nM), specific for a 660 nucleotide segment of the GRBV C1 gene (Poojari et al. 2013). Exactly 3 µL of sucrose solution or 3 µL of stabilized leaf 26 extract was used as template in each respective PCR. The following thermal cycle settings were used: 95°C denaturation for 3 minutes; 35 cycles of 95°C denaturation for 30 seconds, 54°C annealing for 45 seconds, 72°C elongation for 60 seconds; 72°C elongation for 5 minutes, then held at 4°C until collected. PCR products were electrophoresed through 1% agarose gels containing ethidium bromide for 45 minutes at 100V. Gels were visualized using a Fluorchem 5500 Gel Imaging System (Alpha Innotech Corp, San Leandro, CA, USA) under UV light. PCR of GRBV-positive samples was repeated to ensure the amplification events were not results of contamination during PCR and gel loading. Positive controls were included and were prepared from crude DNA extracted from singly infected GRBV positive greenhouse plant. No-template controls were included which were loaded with water in place of DNA template. 2.2.5 Statistical analysis Fisher’s exact test (two-sided) was applied in R (version 4.0.1) to determine if nonrandom associations exist between the frequencies of the two categorical variables, ATS insect count and frequency of transmission to sucrose. An alpha value of 0.05 was considered significant. 2.3 Results 2.3.1 Artificial transmission assays A total of 393 phloem, xylem, and mesophyll feeding hemipteran insects were included in the ATS assessments; by superfamily this included 356 Membracoidea, 35 Cercopoidea, and 2 Aphidoidea. Of the tested insects, detection of GRBV from the sucrose solutions only occurred with 9 of 82 treehoppers of the genus Stictocephala (Table 1, Figure 2). The incidence of GRBV transmission to sucrose by treehoppers was statistically significant (p = 3.88 x 10-5). 27 Table 2.1. Insects collected for use in the ATS, taxonomic ID’s of a subset of collected insects, and ATS PCR results. Generic name Superfamily, Family Total assessed in ATS Taxonomically identified species Total ID’d PCR +ves Treehopper Membracoidea, Membracidae 82 Campylenchia rugosa 21 0 Stictocephala basalis 1 1 Stictocephala bisonia 58 8 Stictocephala wickhami 1 0 Stictocephala brevitylus 1 0 Leafhopper Membracoidea, Cicadellidae 238 Amblysellus grex 7 0 Athysanus argentarius 1 0 Ceratagallia sp. 15 0 Ceratagallia siccifolia 2 0 Chlorotettix unicolor 1 0 Colladonus montanus 1 0 Colladonus reductus 6 0 Deltocephalinae sp. 2 0 Dikraneura absenta 3 0 Doratura stylata 1 0 Elymana inornata 1 0 Empoasca sp. 1 0 Endria inimicus 1 0 Erythroneura ziczac 2 0 Euscelidius sp. 14 0 Euscelidius maculipennis 11 0 Euscelidius variegatus 8 0 Exitianus exitiosus 8 0 Gyponana sp. 1 0 Gyponana hasta 2 0 Hebecephalus occidentalis 1 0 Kybos exiguae 1 0 Macrosteles quadrilineatus 5 0 Muirodelphax arvensis 1 0 Neocoelidia lineata 4 0 Osbornellus borealis 1 0 Psammotettix 7 0 Psammotettix alienus 3 0 Psammotettix lividellus 2 0 Scaphytopius sp. 2 0 Scaphytopius acutus 4 0 Xerophloea peltata 6 0 Sharpshooter Membracoidea, Cicadellidae 36 Graphocephala confluens 4 0 Froghopper Cercopoidea, Cercopidae 35 Aphrodes sp. 1 0 Athysanus argentarius 1 0 Graphocephala hieroglyphica 1 0 Philaenus spumarius 21 0 Aphid Aphidoidea, Aphididae 2 Acyrthosiphon pisum 1 0 Not ID’d 123 0 0 28 Figure 2.2. Example gel segment showing positive (sample 296) and negative (samples 295, 297-299) polymerase chain reaction amplification events. 2.3.2 Greenhouse transmission experiments In 2018, three recipient grapevines were included in individual greenhouse transmission experiments involving unidentified treehoppers. In 2019, eight more recipient plants were included. For both years, all eleven test plants and control plants tested negative for GRBV one- and three-months post IAP. All plants again tested negative six months post-dormancy (12-15 months post-IAP, depending on dormancy duration). In 2019, 25 recipient grapevines were included in individual greenhouse transmission experiments involving VCLH’s. Five of these recipient plants received VCLH nymphs, and the other twenty received VCLH adults. All twenty five test plants and control plants tested negative for GRBV one-, two-, and three- months post IAP. All plants again tested negative six months post-dormancy. 2.4 Discussion Nine treehoppers successfully transferred GRBV to the sucrose solutions. These transmission events were observed for two of five treehopper species tested: Stictocephala basalis and S. bisonia, both generically called buffalo treehoppers (Figure 3). For S. bisonia, 8 of 58 (14%) successfully transmitted GRBV to the diet solution. In the case of S. basalis, the only specimen 29 evaluated had successful transmission. Interestingly, these Stictocephala species are closely related to the known GRBV vector S. festinus (Bahder et al. 2016); all being members of the tribe Ceresini within the Membracidae family. This phylogenetic relationship, in agreement with Jones (2003) and Bahder et al. (2016), and previous research showing treehoppers as vectors of geminiviruses, supports that S. bisonia and S. basalis likely have the capacity to vector GRBV to grapevines. Figure 2.3. Morphological comparison of two collected buffalo treehoppers species that transmitted Grapevine red blotch virus, (a) S. bisonia and (b) S. basalis. Scale = 1 mm. The observed incidence of transmission by these treehoppers suggests that biologically viable virus-vector relationships exists. These transmission rates should not be interpreted as natural transmission efficiencies. Many variables would influence transmission efficiency under natural conditions, including vine health and treehopper/host plant interactions (Jeger at al. 2011). Nonetheless, these results demonstrate the utility of this method for the rapid screening of large numbers of insects for their capacity to transmit circulative plant viruses. A key consideration for this system is that virus particles might be passively transmitted to the sucrose solution on an insect’s mouthparts absent of any biologically compatible insect-virus relationship, resulting in a positive PCR test. As such, the ATS is likely an unsuitable method for 30 viruses with modes of transmission other than persistent-circulative where salivary injection of the virus into the plant tissue is key to transmission. Even for circulative viruses, we could expect a baseline rate of transient transmission of virus into the sucrose solutions, but this baseline should be evident in all groups of insects tested. In this study we did not record any cases of transmission from non-treehopper insect species that might suggest transient transmission. An attempt to validate the ATS results through traditional greenhouse transmission experiments involving transfers of fifteen viruliferous treehoppers to each of eleven test plants was unsuccessful. GRBV was not detected by PCR one- or three-months post-inoculation, or six months after a period of dormancy. Furthermore, we did not observe any GRBD symptom development on the test plants. These results could reasonably be taken as evidence that Stictocephala treehoppers do not vector GRBV, however with very important considerations. First, virus transmission efficiency can differ significantly based on insect feeding behavior (Jiang et al. 2000). Treehopper feeding behavior on grapevine may differ significantly between grape cultivars and under varying environmental conditions, such as between greenhouse and field experiments. Our study potentially could have overcome the barrier to detection by significantly increasing our replication size in hopes that this would produce at least one subject with detectable amounts of GRBV. However, we were limited in our replication size by the scarcity of treehoppers in the Okanagan and Similkameen valleys. Besides the logistic constraints of our greenhouse experiments, our inability to detect transmission to plants may have been exacerbated by our limited knowledge of the GRBV infection process. Little is known about seasonal titres and virus distribution following inoculation of grapevines. Virus proliferation might occur too slowly to be reliably detected over the course of a typical experimental timeframe. For example, the Bahder et al. (2016) study that identified S. festinus as a vector of GRBV detected GRBV in only three of fifteen recipient plants. In the 31 three cases of detection, visualization of amplified virus DNA by agarose gel electrophoresis produced marginally visible bands, indicative of markedly low virus loads in the sampled vines five months post-inoculation. It is possible that more recipient plants harbored GRBV but at levels too low to be detected. Although our study was of longer duration and involved a period of dormancy, conditions might not have favored high levels of virus expression. A recent study found that GRBV was only detectable in the heads of S. festinus after AAP’s of at least six days, that AAP’s as long as ten days were necessary to facilitate transmission of GRBV to grapevine, and that even then, transmission rates were low (Flasco et al. 2021). For the current study, allowing treehoppers three days feeding on the GRBV-infected grapevines (AAP) and an additional three days feeding on the artificial diet (IAP) provided an equivalent six days for virus to arrive in the salivary glands, as demonstrated by positive tests of the diet. The longer time (10 days) required before plants tested positive in the earlier study might reflect the need for virus replication to produce sufficiently high titres to permit sample dilutions to overcome inhibitory compounds present in the plants; whereas virus could be immediately detected in the artificial diet. Additional research with AAP’s of various times is required to determine optimum timing to maximize transmission to the artificial diet. Considering the above, our AAP’s and IAP’s each of three days may have been too short for a sufficient amount of GRBV to reach the salivary glands, resulting in failed greenhouse transmission. Finally, our greenhouse transmission may have failed due to innate barriers within plants and insects that limit the rate at which certain virus species are acquired or transmitted. For example, some plants may contain resistance mechanisms that block or limit virus replication or movement beyond the regions of inoculation (Palukaitis and Carr 2008). Expression of RNA interference, a defense mechanism against viruses, is influenced by temperature (Velásquez et al. 2018) and the warm greenhouse conditions may have suppressed virus replication and symptom expression. While some of these resistance mechanisms may be partially understood 32 for the well characterized genera of viruses in grapevine (Oliver and Fuchs 2011), there is currently no information available on grapevine resistance to grabloviruses. As a result, the current literature on differential spread of GRBV in vineyards generally only considers vector incidence as an explanatory variable in regions with varying rates of spread (Cieniewicz et al. 2017; Adiputra et al. 2018; Cieniewicz et al. 2019; Dalton et al. 2019). An inherent feature of the ATS is its elimination of plant-specific barriers to transmission, which should result in higher rates of transmission than one might expect in a greenhouse or in nature. The ability of the ATS to screen large numbers of insects for their candidacy as vectors of persistent-circulative viruses such as GRBV greatly aided us in identifying buffalo treehoppers as suitable test subjects for greenhouse transmission assays. Absent of this screening technique, greater amounts of time and resources would have been spent maintaining recipient test plants exposed to unlikely insect vectors of GRBV. Despite our inability to validate the ATS results through greenhouse transmission experiments, our low artificial transmission rate can guide our and future researchers’ expectations and interpretations of negative laboratory transmission results in the context of replication size. Future studies with larger replicative power are required to reliably determine if buffalo treehoppers or other species of treehopper are able to transmit GRBV to grapevine. 33 Chapter 3: Seasonal dynamics and optimal diagnostics of Grapevine red blotch virus in a British Columbian vineyard Dieter Kahl1,2, D. Thomas Lowery1, Miranda Hart2, José Ramón Úrbez-Torres1 1. Agriculture and Agri-Food Canada, Summerland Research and Development Centre, 4200 Highway 97, Summerland, British Columbia, V0H 1Z0, Canada. 2. Department of Biology, The University of British Columbia – Okanagan, 3187 University Way, Kelowna, British Columbia, V1V 1V7, Canada. Corresponding author: José Ramón Úrbez-Torres; joseramon.urbeztorres@canada.ca 3.1 Review Grapes (Vitis Vinifera and Vitis spp. L.) and wine production represent a significant component of the Canadian economy, with the full economic impact estimated at CAD $9.04 billion in 2015 (Rimerman and Eyler 2017). Much as for other grape growing regions of the world, production of grapes in Canada is challenged by economically important virus diseases such as grapevine leafroll disease (GLD), caused primarily by Grapevine leafroll-associated virus 3 (GLRaV-3), and most recently grapevine red blotch disease (GRBD) caused by Grapevine red blotch virus (GRBV) (Poojari et al. 2017a; 2017b). The severity of these viral diseases varies considerably depending on complex biotic and abiotic interactions (Almeida et al. 2013). In the case of GRBD, a characteristic symptom for red cvs. is the initial formation of irregular red-colored blotches in the inter-veinal areas of mature basal leaves, with symptoms later developing acropetally. In both red and white cvs., reduced yields, undesirable berry quality parameters, and decreased plant vigor are also commonly observed (Poojari et al. 2013; Blanco-Ulate et al. 2017; Bowen et al. 2020). A study conducted in a British Columbia (BC) Cabernet franc vineyard further found that GRBD reduced photosynthesis rates by 21% from July to September, reduced soluble solids by at least four °brix, decreased yield by 42% on average, and GRBD-infected vines were less winter hardy than non-GRBD vines (Bowen et al. 2020). GRBV is a monopartite geminivirus (family Geminiviridae) with a 3.2kb circular ssDNA genome and is the type-species of the genus Grablovirus (Rwahnih et al. 2013; Varsani et al. 2017). 34 GRBV was first reported in 2012 by Krenz et al. from a declining Cabernet franc vineyard in New York. Within a few years of its discovery, it had become clear that GRBV is present in most of the major grape growing regions of North America (Rwahnih et al. 2013; Krenz et al. 2014; Poojari et al. 2017b; Yao et al. 2018; Schoelz et al. 2019; Thompson et al. 2019a; Gasperin-Bulbarela et al. 2019; Poojari et al. 2020). GRBV was first reported to be vectored by the Virginia creeper leafhopper (Erythroneura ziczac Walsh) in greenhouse conditions (Poojari et al. 2013). Later attempts to reproduce this finding were unsuccessful, and a new potential vector was reported from a greenhouse study, the three-cornered alfalfa treehopper (Spissistilus festinus Say) (Bahder et al. 2016). While the three-cornered alfalfa treehopper is now generally accepted to be a vector of GRBV, there is still uncertainty regarding its transmission efficiency and whether there are other unknown vectors. Natural spread of GRBV has been reported in several vineyards in California, Oregon, and New York, as well as in free-living V. californica × V. vinifera hybrids adjacent to a V. vinifera Cabernet franc vineyard in California, though the spatial patterns of spread have not always been indicative of transmission by an alate insect vector such as adult treehoppers (Perry et al. 2016; Cieniewicz et al. 2017; Dalton et al. 2019; Cieniewicz et al. 2019). Furthermore, documented spread of GRBV in study vineyards may be exaggerated due to inherent difficulties obtaining reliable diagnostic data, depending on methods and sampling strategies used. Grapevine tissues are notoriously recalcitrant to nucleic acid extraction due to their high levels of endogenous inhibitors of molecular assays such as polyphenols and polysaccharides (Geuna et al. 1998; Zherdev et al. 2018). To complicate matters further, grapevine viruses are unevenly distributed in plant tissues and there is seasonal variation in virus titer in affected tissues, making sampling inherently difficult and unreliable (Setiono et al. 2018). Year-to-year variations in the robustness of molecular testing as a result of variable inhibition and non-standardized sampling strategies may result in observed and reported increases in disease incidence absent 35 of any true spread. Though work is underway, there is currently no antiserum to GRBV, making nucleic-acid based techniques the only practical methods for GRBV diagnosis (Liou 2018). A short report released in 2000 provided grapevine virus researchers with a low cost, high-throughput nucleic acid extraction protocol that utilizes only two simple buffers, an extraction buffer and a stabilization buffer (GES), comprised of inexpensive and relatively safe-to-use reagents (Rowhani et al. 2000). Sample tissues, such as grape leaf petioles, are ground in the extraction buffer then significantly diluted in GES solution to obtain crude extracts of plant and virus total nucleic acids (TNA). Theses crude TNA extracts are then ready to be used as template in polymerase chain reactions (PCR) and reverse transcription (RT)-PCRs. This technique was adopted by many research groups, including some that studied GRBV (Dovas and Katis 2003; Osman and Rowhani 2006; Osman et al. 2007; Kumar et al. 2015; Poojari et al. 2017a; Thompson et al. 2019b; Poojari et al. 2020). While this protocol has undeniable benefits, a major drawback must be considered: using crude grapevine extracts in PCR implies a high risk of reaction inhibition and the potential to interpret the amplification-negative reaction as a true-negative. This dilemma is exemplified in a study by Osman et al. (2007) where purified RNA and crude GES TNA extracts from grapevines infected with GLRaV-1, -2, -3, and -5 were used in TaqMan® RT-PCR to compare the ability to detect these viruses using these two different input templates. In every instance, the purified RNA performed better, with detection rates between 95 and 100%, whereas the GES templates only produced detection rates between 61 and 84%. As such, it is paramount that parameters such as sampled tissue, timing of sampling, and methods of nucleic acid extraction and virus detection are optimized to achieve reliable and reproducible diagnostic data. Taken the considerations above, this study aimed to investigate GRBV titers from various grapevine tissues, including roots, buds, cortical scrapings, and leaves from shoots at varying distances from the cordons, at different times during the growing season and winter dormancy. 36 This study also compared the two nucleic acid extraction techniques - the crude GES method and a commercially available DNA column purification method - to determine the best combination of sampling parameters to achieve reproducible diagnostics. 3.2 Materials and methods 3.2.1 Site selection In 2013, a commercial vineyard located in the South Okanagan, British Columbia, planted a block of ‘Petit Verdot’ clone 400 on V. riparia rootstock ‘Riparia Gloire de Montpellier’. Conventional vineyard practices were employed in the initial year of planting, then organic practices in subsequent years. In 2018 and onward, biodynamic preparations and no till practices were employed to improve soil health. The vines are yearly cane pruned and basally leaf thinned once during bloom, again at fruit set, and fully around harvest. After noticing leafroll virus-like foliar symptoms in a substantial portion of these ‘Petit Verdot’ vines in 2018, leaf samples tested at the Summerland Research and Development Center showed these vines to be negative for grapevine leafroll associated viruses (GLRaV’s) -1 to -4 but positive for GRBV. Due to the limited number of vineyards with known GRBV infection in the Okanagan and Similkameen valleys (Poojari et al. 2017b), this site was selected for this study. Ten randomly selected ‘Petit Verdot’ grapevines confirmed as infected with GRBV and one virus-free vine were selected for use in this study. 3.2.2 Dormant sample collection Three random shoots were collected from each of the eleven study grapevines on January 16 2019. From these three shoots per vine, buds and cortical scrapings were collected in composites with the 1st, 3rd and 5th buds removed and pooled by node number for extractions. Three cortical scraping composites were collected directly adjacent to the collected buds and 37 pooled by scraping number (bud position) for extractions (Figure 1a). Buds and cortical scrapings were ground to a fine dust in liquid nitrogen using sterile pestle and mortars immediately following collection. The ground samples were then immediately stored at -80°C in three aliquots. Root samples were collected from each vine on November 20 2019, using a 30.5 cm dry soil corer with a 1.9 cm core diameter, 5 cm from the base of each vine’s trunk. For all grinding steps, the pestles and mortars were washed before use in a Liquinox® solution, rinsed thoroughly using distilled water, soaked for at least one hour in a 10% bleach solution, rinsed thoroughly again using distilled water, and then air dried. Dormant samples were processed within four hours of collection. 3.2.3 Leaf sample collection Leaf samples were collected at five-week intervals throughout the 2019 growing season on June 26th, July 31st, September 4th, and October 9th. Leaves were collected from random shoots at three distances from the vine cordons: around the 1st, 3rd and 4th trellis wires with designated group locus names of basal, medial, and distal, respec-tively (Figure 1b). For the last two sample dates, basal leaves were collected slightly higher than the 1st wire as these were the only available leaves after the vineyard own-ers conducted extensive leaf removal. Three leaves were collected from each locus per vine, pooled, and placed on ice for transport. For the extractions, leaf petioles were ex-cised from the leaves, ground to a fine dust in liquid nitrogen, and then immediately stored at -80°C in three aliquots. Leaf samples were processed within four hours of collection. 38 Figure 3.1. Grapevine tissue sampling scheme. (a) Dormant buds (circles) and cortical scrapings (rectangles) were collected from three randomly selected shoots at three different sections per vine (the 1st, 3rd, and 5th buds with scrapings directly adjacent) and pooled by section; (b) Leaf samples were collected randomly in threes from any of the shoots at three different loci per vine (the 1st, 3rd, and 4th wire regions) and pooled by locus. 3.2.4 Nucleic acid extraction and quantification Two methods of extraction were used for each sample: a crude TNA extraction designated the GES method, and a commercial column DNA purification method. 3.2.4.1 TNA extraction by GES method The GES method was that described by Rowhani et al. (2000). Approximately 10 mg of sample ground in liquid nitrogen was suspended in 1 mL of extraction buffer (15 mM sodium carbonate, 35 mM sodium bicarbonate 2% (w/v) polyvinylpyrrolidone (PVP-40), 0.2% (w/v) bovine serum albumin (BSA), 0.05% (v/v) Tween-20) in a 1.5 mL Eppendorf tube and vortexed on high for 30 seconds. Then 10 µL of the suspension was added to 90 µL of GES stabilization buffer (100 mM glycine, 50 mM sodium chloride , mM EDTA, 0.5% (v/v) Triton X-100 ,% β- 39 mercaptoethanol). This GES suspension was then incubated at 95°C for 10 minutes to deactivate endogenous deoxyribonucleases (DNase’s). After cooling on the benchtop for one minute, 100 µg of DNase-free ribonuclease A (RNase A) (Thermo Fisher Scientific, Massachusetts USA) was added to the GES suspension and incubated at 37°C for 15 minutes to degrade the contained RNA. The RNase-treated GES suspension was then stored at -80°C. 3.2.4.2 DNA purification by column method DNA column purification was done using the Qiagen DNeasy® Plant Pro Kit (Qiagen Sciences Inc, Maryland USA) following the prescribed protocol (DNeasy® Plant Pro Kit Handbook 2019). As the samples were already homogenized in liquid nitrogen, the initial bead beating step was omitted and instead approximately 10 mg of liquid nitrogen ground sample was suspended in the extraction buffer and vortexed on high for 30 seconds before continuing with the protocol. DNA was eluted in 50 µL of elution buffer and stored at -80°C. 3.2.4.3 Qubit quantification of total DNA The total DNA content of each RNase-treated GES extracted sample and each column purified DNA sample was determined using the Qubit® ssDNA Assay Kit (Qubit, New York USA) following the prescribed protocol which detects ssDNA, dsDNA, ssRNA, and dsRNA (Qubit® ssDNA Assay Kit 2015). Measurements were taken with a Qubit® 1.0 Fluorometer. 3.2.5 Primer design and optimization of droplet digital PCR Forward primer 5’-CGATCTGAGCGCGGAGAGGTGA-3’ and reverse primer 5’-CCTAGCTCCAGGTCCAGACGTC-3’ specific for a 151 bp segment of GRBV V3 open reading frame were selected for use in this study. These were designed using NCBI’s Primer-BLAST and their suitability and stability were confirmed using Premier Biosoft’s Net Primer online tool (http://www.premierbiosoft.com/netprimer/). Ideal primer concentrations of 100 nM were determined by conducting droplet digital™ PCR (ddPCR™) (Bio-Rad Laboratories Inc, 40 California USA) on diluted fully synthesized GRBV (GenBank accession: KX234088) on a pMK-RQ-Ba vector backbone (ThermoFisher Scientific, Massachusetts USA) over a primer concentration gradient of 50 to 250 nM in increments of 50. An ideal ddPCR™ thermal cycler annealing temperature of 58°C was similarly determined over a thermal gradient of 50.7 – 62.2°C to maximize primer specificity and minimize rain. ddPCR™ was conducted using QX200™ ddPCR™ EvaGreen chemistry (Bio-Rad Laboratories Inc, California USA). 3.2.6 GRBV quantification by droplet digital™ PCR Crude GES extracts were serially diluted as 10 µL of extract in 90 µL of DNase-free UltraPure™ Distilled Water (Invitrogen, California USA) twice to a final dilution of 1/100. Purified DNA extracts were similarly serially diluted three times to a final dilution of 1/1000. Working solutions of 2X QX200 ddPCR™ EvaGreen Supermix and primers were prepared to accommodate 96-well plates. Plated ddPCR™ reactions contained 12.5 µL of Supermix ,.25 µL each of forward primer and reverse primer both at final concentrations of 100 nM, and 10 µL of diluted DNA or TNA template, for total reaction volumes of 25 µL. Droplets were generated following manufacturer instructions using a QX200 AutoDG Droplet Digital™ PCR System (Bio-Rad Laboratories Inc, California USA). After droplet generation, sealed plates were loaded into a C1000 Touch Thermal Cycler (Bio-Rad Laboratories Inc, California USA) and cycled using the following settings: 1 cycle of enzyme activation at 95°C for 5 minutes; 40 cycles of denaturation at 95°C for 30 seconds followed by annealing/extension at 58°C for 45 seconds; 1 cycle of signal stabilization at 4°C for 5 minutes then 90°C for 5 minutes; then held at 4°C. Lid temperature was held constant at 105°C during all stages and reaction volume was inputted as 40 µL. Following thermal cycling, sealed plates were loaded into a QX200 Droplet Reader (Bio-Rad Laboratories Inc, California USA) and plate layouts were inputted into QuantaSoft™ Software 41 v1.7.4 (Bio-Rad Laboratories Inc, California USA). Experiment types were set as absolute quantification by EvaGreen® and readings were taken in channel 1:FAM. All plates included three no-template controls and three positive controls. Positive controls contained fully synthesized GRBV genome. Positive droplets generally fell between the fluorescence amplitude range of 18,000 - 25,000 and baseline negative droplets generally fell between 4,000 and 8,000. Each well had on average 18,652 droplets (range 15,699 - 20,399). The threshold for positive droplets was set at 15,000 for all wells on all plates. 3.2.7 Statistical analysis All data transformations, calculations, visualizations, and statistical analyses were carried out in R version 4.0.3 (R Core Team 2013). An alpha level of 0.05 was used for all tests. 3.2.7.1 Viral load calculation Viral load (virus / ng DNA) was calculated using the following equation: 𝑣𝑖𝑟𝑎𝑙 𝑙𝑜𝑎𝑑 =𝑣𝑖𝑟𝑢𝑠 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 × 𝑟𝑒𝑎𝑐𝑡𝑖𝑜𝑛 𝑣𝑜𝑙𝑢𝑚𝑒 × 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 𝐷𝑁𝐴/𝑇𝑁𝐴 𝑣𝑜𝑙𝑢𝑚𝑒 𝑙𝑜𝑎𝑑𝑒𝑑 × 𝐷𝑁𝐴 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 ; where virus concentration is the sample-specific Poisson-adjusted output value obtained by ddPCR, reaction volume is the ddPCR reaction volume, dilution factor is the ratio of undiluted DNA/TNA to the final diluted solution, DNA/TNA volume loaded is the volume of diluted DNA/TNA loaded in each ddPCR reaction, and DNA concentration is the sample-specific value obtained by Qubit quantification. For statistical analysis, viral load was log10 transformed to address the multiplicative nature of viral replication evident on the original scale. Transforming viral load allowed for linear analysis of these now additive values on the log scale. A constant of 1 was added to all viral load values to avoid calculating the undefined log(0). Original and 42 transformed viral load distributions were visually assessed using ‘ggplot2’ strip charts (Wickham 2016) with sample date as a continuous explanatory variable and locus as a grouping variable. 3.2.7.2 Mixed effects model to predict the effects of sampling date and locus on viral load in leaves We fitted a linear mixed model (estimated using REML and nloptwrap optimizer) to predict viral load with sampling date and sampling locus (formula: log.virus.particles ~ date * locus). The model included date, locus and subject as random effects (formula: ~1 + date * locus | subject). The main effects of date, the date for which leaves were collected, and locus, the regions on the vines from which leaves were collected, were included in a linear mixed model analysis to predict spatiotemporal viral loads. Additionally, the interaction between date and locus was included. The model included the maximal random effect structure justified by the data (Barr et al. 2013). The maximal random effect structure was determined by comparing performance indices of the fully specified random effects model to reduced models, following the hierarchy principal. Performance indices were calculated and tabulated using the function ‘compare_performance’ from the R package ‘performance’ (Lüdecke et al. 2020). The best performing model included correlated random intercepts and slopes and allowed the interactions between date and locus to vary by subject plants. No data points were treated as outliers. Normality of model residuals were visually confirmed using a quantile-quantile plot (function ‘qqnorm’). Estimated marginal means were computed for viral load at all levels of date and locus using the function ‘ggpredict’ from the R package ‘ggeffects’ (Lüdecke 2018) with 95% confidence intervals (CI). 95% CI’s and p-values were computed using the Wald approximation. The statistical report for the linear mixed model presented in the results section below was adapted from the R package ‘report’ (Makowski et al. 2020). 43 3.2.7.3 Two-way repeated measures ANOVA to determine the effects of locus and section on viral load in dormant buds, cortical scrapings, and roots Main effects of locus, the regions on the vines from which dormant samples were collected, and section, the sections of each locus that were analysed separately, were included in a two-way repeated measures ANOVA to determine if viral load differences exist between loci and between sections within each loci. The root samples were not included in the analysis as they were collected in a separate year, had a significantly smaller sample size and a different sampling strategy than the other dormant samples. No extreme outliers were found in the data. The assumption of normality of the conditional response variable, viral load, was visually checked using a quantile-quantile plot (function ‘ggqqplot` from the package ‘ggpubr`) (Kassambara 2020a) and tested using the Shapiro-Wilk Normality Test (function ‘shapiro_test’ from the package ‘rstatix’) (Kassambara 2020b). The ANOVA model was created using the function ‘anova_test’ from the package ‘rstatix’ (Kassambara 2020b). This model creation tool automatically checks the assumption of sphericity using Mauchly's Test for Sphericity and by default applies the Greenhouse-Geisser correction to all within-subjects factors for which the assumption of sphericity is not met. In this case, sphericity was not met in the within-subject factor ‘section’ so sphericity corrections were applied. 3.2.7.4 Method comparison by scatter plot Log viral loads (virus particles / ng DNA) as determined by ddPCR of column purified DNA and GES crude TNA were plotted against each other using the R package ‘ggplot2’ (Wickham 2016) to visualize agreement of methods and to compare their coefficients of correlation. As it is assumed to be more reliable than the crude extraction method, log viral loads obtained from purified DNA were plotted on the x-axis, and log viral loads obtained from crude DNA were plotted on the y-axis (Fig. 4). Agreement between the two methods was determined by 44 calculating the variance between the plotted points and a hypothetical line representing perfect agreement between the methods (y ~ x). 3.3 Results 3.3.1 Mixed effects model to predict the effects of sampling date and locus on viral load in leaves The model's explanatory power related to the fixed effects alone (marginal R2) is 0.75. The model's intercept (and reference), is set at date = June 26 and locus = Basal (Table 1). Table 3.1. Linear mixed effects model summary statistics. Standardized parameters were obtained by fitting the model on a standardized version of the dataset (Figure 2). Viral load Predictors Estimates 95% CI p df (Intercept) 1.76 1.35 – 2.17 <0.001 91 Date 0.01 0.01 – 0.02 <0.001 91 Locus [Medial] -1.37 -1.86 – -0.87 <0.001 91 Locus [Distal] -1.26 -1.92 – -0.59 <0.001 91 Date * Locus [Medial] 0.01 0.00 – 0.02 0.021 91 Date * Locus [Distal] -0.01 -0.02 – 0.01 0.358 91 Random Effects σ2 0.24 τ00 Subject 0.27 τ11 Subject.Date 0.00 τ11 Subject.LocusMedial 0.30 τ11 Subject.LocusDistal 0.81 τ11 Subject.Date:LocusMedial 0.00 τ11 Subject.Date:LocusDistal 0.00 ρ01 Subject.Date -0.88 ρ01 Subject.LocusMedial -0.86 ρ01 Subject.LocusDistal -0.97 ρ01 Subject.Date:LocusMedial 0.71 ρ01 Subject.Date:LocusDistal 0.82 N Subject 10 Observations 119 Marginal R2 0.75 σ2 = within-subject variance; τ00 = random-intercept-variance, or between-subject-variance; τ11 = random-slope-variance; ρ01 = random-slope-intercept-correlation 45 Figure 3.2. Estimated marginal effects of locus and date on viral load in leaves (lines). Ribbons represent 95% confidence intervals. Shaped points represent raw measurements from individual plants (n = 119). Raw data points are jittered to minimize overlap for optimal visualization. 3.3.2 Two-way repeated measures ANOVA to determine the effects of locus and section on viral load in dormant buds, cortical scrapings, and roots There was a statistically significant difference in viral loads between dormant nodes and cortical scrapings, F(1, 9) = 298.60, p = 3.28 x 10-8. Results showed no significant differences in viral loads in sections within loci, F(1.09, 9.78) = 2.38, p = 1.54 x 10-1 (Figure 3). As such, no post hoc tests were run. 46 Figure 3.3. Mean viral loads in dormant tissues (filled circles). Hollow circles represent raw measurements from individual plants (n = 70). Raw data points are jittered to minimize overlap for optimal visualization. 3.3.3 Comparison of extraction methods Overall, the crude GES TNA extraction method performed poorly relative to the DNA purification method (Figure 4). The crude method had a variable proportional bias, where ddPCR™ of crude TNA tended to overestimate viral load when the true value was low, and underestimate viral load when the true value was high. The coefficient of correlation of the crude method was 0.55, much lower than the hypothetical coefficient of correlation of 1.0 corresponding to an ideal agreement between methods. 47 In fourteen cases where the purification method detected no virus particles in the extracts, the crude method produced positives values. The majority of these values were near zero, however two of these crude measurements were an order of magnitude higher. Figure 3.4. Plot of paired measures (green dots) of GRBV titres determined by ddPCR of crude DNA (y-axis) versus titres from column purified DNA (x-axis). Comparison of the paired measures and a hypothetical ideal agreement (black line) produced a corresponding coefficient of correlation value of 0.55 (green line), indicating minimal agreement between the two methods (n = 188). 3.4 Discussion When sampling leaves, major differences in viral loads were found between loci at different sample dates. GRBV was most consistently detected and at the highest titres in basal leaves across all sampling dates. One single basal leaf sample tested negative for GRBV on the first 48 sample date, June 26. Conversely, the majority of the sampled medial (6/10) and distal (7/10) leaf samples tested negative for GRBV on the first sample date. These results are in agreement with Setiono et al. (2018), who reported that 37 of 84 (44%) of their field-grown plants falsely tested negative for GRBV in June 2015, when sampling younger distal leaves. Viral loads in the medial leaves sharply increased over the summer, approximately one order of magnitude every five weeks, and had comparable virus titres to basal leaves by the final sampling date, October 9. Viral loads in the distal leaves did not increase significantly throughout the growing season, although false-negatives stopped occurring in the latter two sample dates, September 4 and October 9. A similar spatiotemporal study was conducted on distal leaves from ‘Chardonnay’ and ‘Cabernet franc’ either infected with both GLRaV-2 and -3 or just GLRaV-3 from May to September and titre increases similar to those in our basal leaves were observed (Shabanian et al. 2020), suggesting that a resistance mechanism exists in grapevine that is particularly effective in limited GRBV movement to distal tissues. A resistance mechanism producing a barrier to infection has been described, where absence of virus in distal leaves suggests that the virus’ ability to ingress into the sieve element of the phloem is limited (Waigmann et al. 2004; Ueki and Citovsky 2006). Taken the above, early in a growing season at least until veraison, basal leaves provide the only viable samples for reliable detection of GRBV, and even then replication should be conducted to avoid false-negatives. After veraison, medial leaf sampling becomes just as viable as basal leaf sampling and may become an especially good option if basal leaf thinning has been conducted. Collecting samples from distal leaves is a poor option throughout the growing season and should only be an option of last resort after veraison. The conflicting findings on the spatiotemporal distribution of leafroll infections emphasizes that no one sampling strategy is optimal and strategy must therefore be optimized by target virus. 49 Detection of virus in grapevine is largely conducted using leaf extracts, however cases may arise where one is interested in sampling and testing when leaves are not present on the vines. As such, this study sought to determine if all dormant tissues perform equally well for GRBV detection, and if not, which is the best. Previous studies have focused on GRBV detection from leaf tissues and cortical scrapings (Poojari et al. 2013; Krenz et al. 2014; Rwahnih et al. 2015; Wallis and Sudarshana 2016; Cieniewicz et al 2017; Setiono et al. 2018; Dalton et al. 2019; Poojari et al. 2020). We report for the first time that GRBV is detectable in grapevine roots and dormant buds. The finding of GRBV in buds is not unexpected, as this has been found with many grapevine viruses and has been exploited in chip inoculations for Grapevine fanleaf virus (GFLV) (Krebelj et al. 2015), Rupestris stem pitting associated virus (RSPaV), Grapevine fleck virus, Grapevine viruses A and B, GLRaVs-1, -2, -3, and -9 (Constable et al. 2013), to name a few. Similarly, grapevine viruses have previously been detected in roots, such as for GLRaV-3 (Bell et al. 2009) and GFLV (Krebelj et al. 2015). GRBV was not detected in two dormant samples, both from the lowest sampled buds. With this finding, cortical scrapings and root collection were found to be more reliable for GRBV detection. Viral loads were on average two orders of magnitudes higher in cortical scrapings than in roots or in buds, making this the best dormant tissue from which to sample in respect to virus titres. Collection from any three of these tissues are comparably labour intensive; cortical scrapings require careful removal of the outer lignified tissue to access the vascular tissues, roots requires extensive sifting and rinsing to remove excessive soil and stones, while buds require the least processing but take substantial effort to pulverize as the tissue is considerably dense. Taken the above, cortical scrapings provide the most reliable dormant samples for detection of GRBV. 50 There were no spatial differences in virus titres in the sections within cortical scrapings or buds. These results may have been different if buds and bark scrapings had been sampled from sections spaced farther along the canes, although this extension would not have been of significant practical value as there are generally no external factors present during dormant sampling that would dictate where one can sample from a cane. A similar study looked at detecting RSPaV in buds collected not only from winter dormant canes but also from shoots during the spring and summer months (Stewart and Nassuth 2001). They detected RSPaV in almost all dormant buds, but detection fell off significantly in opening buds in the spring. In the summer, they tested buds from both already lignified shoots and green shoots and found that detection was high in buds from the lignified shoots but low in the green shoots. Future research on GRBV should similarly investigate seasonal variations in detection of virus in buds. When determining the ideal extraction method for reproducible diagnostics, this study did not consider the cheapest or fastest extraction method, but rather the one with the highest fidelity. The spread of GRBV can only truly be confirmed by repeated molecular testing of plants over time, where eventual positive results are indicative of spread. This experimental approach does not tolerate false results. An initial false-negative followed by subsequent positives will be misinterpreted as a new infection, whereas initial true-negatives followed by subsequent false-negatives will be misinterpreted as absence of spread. Similarly, false-positives may be misinterpreted as new infection. In this context, high fidelity takes precedence over cost and efficiency, within reason. Traditional diagnostics do not often rely on quantitative techniques, with the exception of diagnostic qPCR due to its scalability and non-reliance on electrophoresis, but to better evaluate the agreement between our two methods we took a quantitative approach. When comparing the outcomes of the two experimental pipelines, column purification of DNA was considered the golden standard for GRBV extraction. Significant deviations between the two methods’ viral load 51 determinations were assumed to have resulted from inherent flaws of the crude method, such as high levels of inhibitors in the crude extracts that interfere with PCR. Correct calculation of viral loads relies on accurate Qubit quantification of sample DNA concentrations as well as accurate ddPCR™ quantification of sample virus copy numbers. Incompatibility between crude extract matrices and either Qubit or ddPCR™ would result in incorrect viral load calculations and, as a result, non-agreement between the two methods. Extreme care was taken to mitigate random error, such as practice of proper pipetting and adherence to high standards of bench and equipment sanitation. Use of Bio-Rad™’s automated droplet generator produced consistent pipetting volumes and thus, minimized potential for pipetting error and/or contamination from airborne particles. While this study did observe significant differences in viral loads between methods, this aspect merely suggests incompatibility of crude extracts in quantitative but not qualitative experiments. Overall, the crude method exhibited a variable proportional bias where ddPCR™ of crude TNA tended to overestimate viral load when the true value was low, and underestimate viral load when the true value was high. There were fourteen leaf samples for which GRBV was not detected in the purified DNA extracts but was detected in the crude extracts. This anomaly has more severe implications in respect to diagnostics. The presence of high-fluorescence droplets in the ddPCR™ spectra of these samples may be explained by non-specific binding and subsequent photoactivation of the EvaGreen® dye in the reaction solutions, resulting in false-positive droplets. By contrast, it is possible that crude extracts do inherently promote higher detection sensitivity in PCR analysis. One study found that a minimal amount of non-template carrier DNA is required in PCR solutions for effective amplification of template, as the non-template DNA affects the free water volume through water-caging (Ruiz-Villalba et al. 2017). Within this framework, GRBV template DNA might have been relatively overabundant in some column purified extracts for which GRBV 52 was not detected, resulting in suboptimal PCR conditions compared to the crude extracts in which GRBV was detected. In this study, the GES method did not perform as poorly as it did in the study by Osman et al. (2007) where detection of GLRaVs was between 11 and 39% lower in leaf GES extracts compared to column purified RNA. This can likely be attributed to the fact that the GRBV has a ssDNA genome and therefore does not require an initial reverse transcription step, a process that is highly prone to inhibition by complex secondary metabolites, to produce cDNA for use in diagnostic PCR. If our study had similarly been conducted on an RNA virus, we might have expected a higher frequency of false-negatives from the ddPCR™, although potentially at lower levels than the Osman et al. study, as reverse transcriptase technologies and their resilience to inhibitors have improved remarkably since 2007 (Bustin et al. 2015; Zucha et al. 2020). Several studies describe alternative methods for the extraction of high quality RNA or DNA from grapevines (Gambino et al. 2008; Steinmetz et al. 2020), however these methods fall short of the combination of low-cost, efficiency, and safe-to-use reagents of the GES method. For example, Gambino et al. (2008) describe a modified, low-cost cetyltrimethylammonium bromide (CTAB) method that yields high quality RNA, although their approach takes at least three hours, significantly more time than the GES method. Steinmetz et al. (2020) describe a rapid and inexpensive method with safe-to-use reagents for high yield extractions of RNA of sufficient quality for diagnostics, however they only test this method on cortical scrapings and fail to validate its effectiveness with leaf tissues, the most accessible and common tissue sampled. A point-of-use method for the detection of GRBV has also been described that uses loop-mediated isothermal amplification where extraction of nucleic acids is not required (Romero et al. 2019). That study reports high sensitivity and specificity for GRBV, however collection of template is conducted by prodding the grapevine with a pipette tip, an approach that would seem highly likely to produce a false-negative if a region absent of virus particles was probed. 53 The GES method overcomes this barrier to detection by permitting high inputs of sampled tissue, around 25g of tissue per 5 mL of extraction buffer. With all considered, our study does not objectively conclude an optimal extraction method for diagnostics, as both the GES method and column purification method performed similarly well in terms of detection, although not quantification, and the few anomalies between the two methods can be explained in favor of either method. The application of EvaGreen® ddPCR™ in this study provided precise and absolute measurements of GRBV titres in our extracts. This relatively new technology provides counts without the need for standard curves, requiring only that the counts are normalized to the input DNA concentrations. This technology works by partitioning each PCR mix into thousands of nanoliter-sized droplets, allowing for compartmentalized amplification of droplets containing target DNA (Hindson et al. 2013). EvaGreen® within these reactions then binds to product double-stranded DNA and photoactivates, producing a strong fluorescent signal. Following thermal cycling, each PCR mix is individually analysed through a microfluidics process much similar to flow cytometry. Each reaction yields an absolute count of target copies, Poisson-adjusted to account for droplets that may have contained more than one template. In this study, optimized cycling annealing temperature and primer concentrations provided us with high signal-to-noise ratios and significant resolution between explicit positive and negative droplets while also minimizing droplets in the ‘rain’ range. Previous chip-based digital PCR technologies produced only hundreds or thousands of partitions, whereas modern ddPCR™ technology yields approximately 20,000 droplets, providing substantially higher quantification accuracy. The use of BioRad™’s automated droplet generator provided us with sample-to-sample and plate-to-plate consistency, minimizing user-variability and experimental error. 54 Chapter 4: Conclusion The objective of this research was to help elucidate the GRBV epidemiological landscape of the Okanagan valley. More specifically, this research aimed to 1) determine if this region contains insect species that are able to spread GRBV in grapevine, using both traditional and novel experimental approaches, and 2) optimize GRBV sampling and molecular diagnostics to minimize PCR false-negatives, thereby increasing the reliability of infection tracking which ultimately enables researchers to objectively determine if natural spread is occurring. 4.1 Determining if vectors of GRBV are present in the Okanagan valley 4.1.1 Traditional greenhouse transmission experiments Due to the expensive resource and time costs required for conducting greenhouse transmission experiments, GRBV greenhouse transmission trials were only conducted with insect species justified by previous research: VCLH and buffalo treehoppers. No transmission of GRBV to grapevine was observed from either nymph or adult VCLHs, suggesting that they are not true vectors. While this finding is contradictory to the initial report of their vectoring capacity (Poojari et al. 2013), it is in agreement with all follow-up studies which similarly found that they could not vector GRBV in the greenhouse (Daane et al. 2015; Bahder et al. 2016). As this is the third study to report this finding, it is recommended that future researchers avoid repeating this experiment and focus their time and resources on other, more justified prospects. In the case of buffalo treehoppers, no transmission of GRBV to grapevine was observed. As there is currently no other available research on the GRBV vectoring capacity of buffalo treehoppers, our finding should be not accepted as conclusive at this time. Further research on 55 buffalo treehoppers with more extensive replication is required to either validate or contest our results. 4.1.2 Artificial transmission experiments Unlike the traditional greenhouse approach, the ATS enabled us to screen a large and diverse array of insects for the ability to transmit GRBV to a buffered sucrose solution. Transmission was only observed by treehoppers (9 of 82). Of these 82 treehoppers, five species were identified, two of which produced transmission events (1/1 S. basalis and 8/58 S. bisonia). S. bisonia represented the majority of the treehoppers collected and had a transmission rate of 14%. On the other hand, the single specimen of S. basalis collected successfully transmitted GRBV to solution, suggesting that we might expect a significantly higher transmission rate by S. basalis than S. bisonia if more specimens of the former had been found. These rates also have significant implications in our greenhouse transmission experiments. We did not conduct species identification on insects used in the greenhouse experiments and therefore do not know the species ratios in individual replicates. If by chance our greenhouse experiments did not contain a significant amount of S. basalis or S. bisonia, and instead contained some of the other species identified, then our artificial transmission results suggest that these other species likely would not have transmitted GRBV to the test plants. This potential underrepresentation of treehopper species deemed as likely vectors by the ATS in our greenhouse experiments may have resulted in our inability to produce insect-to-plant transmission. Future research seeking to evaluate buffalo treehoppers’ capacity to vector GRBV in greenhouse should take care to establish monospecies experiments to ensure that potential true-vector candidates are not underrepresented in polyspecies setups. It should be noted that transmission rates in the ATS should be inherently higher than in plants due to the elimination of innate plant barriers in the former, and therefore the transmission rates 56 reported in this research should be used to guide the expectations of future researchers seeking to utilize the ATS or conduct greenhouse trials. While not substantiated by our greenhouse trials, our artificial transmission trials strongly suggest the buffalo treehopper species native to the Okanagan are able to vector GRBV. The success of the ATS in this research indicates that it may very well be used for the study of other virus-vector relationships. At the very least, it can be used to help identify the vectors of other circulative plant viruses. 4.2 Optimization of GRBV sampling and diagnostics 4.2.1 Best practices in sampling leaves Leaves from grapevine may be sampled for the detection of GRBV as early as May or June. GRBD foliar symptoms are an invaluable indicator of which grapevines should be sampled for testing, however these symptoms do not present until veraison or later. As such, early season leaf sampling on plants is largely done blindly. Improper sampling of an infected plant can result in false-negative molecular results and therefore a sampling strategy that promotes reliable and robust diagnostics must be adopted. When sampling leaves, the sampler may opt to select leaves from approximately the basal, medial, or distal sections of a vine. In our study, we found that leaf sampling in June should only be conducted from the basal region where leaves had the highest titres and produced minimal false negatives. Sampling later in the season can be done from the basal or medial regions where GRBV titres sharply increase in the latter relative to June. Distal leaves should only be sampled in late fall and only if medial or basal leaves are not available. 57 4.2.2 Best practices in sampling dormant tissues It is often desirable to determine the infection status of one or many grapevines when leaves are not available, such as during winter dormancy. It is therefore important that dormant tissue sampling be conducted in an optimal manner for reliable diagnostics. In our study, GRBV was detected in all dormant tissues tested: canes, roots, and buds. Titres were the highest in the scrapings from the canes and approximately two orders lower in roots and buds. Because all three tissues perform comparably well for detecting GRBV, and extraction from any of these tissues is comparably labor intensive, dormant tissue selection is flexible and may be adapted to one’s research needs. 4.2.3 Best nucleic acid extraction method for diagnostics We found that for binary diagnostics, DNA obtained from the crude GES method and from column purification produced similar results in PCR. However, the GES method yielded extremely different titre values than the column method, indicating its incompatibility in quantitative analyses. In some cases where GRBV was not detectable in column purified DNA, it was detected in GES extracts from the same samples, either as a result of higher ddPCR sensitivity when using GES extracts, or more likely, as a result of non-specific binding of EvaGreen® dye with something in the crude extracts. Future research should aim to determine which of these possible outcomes is true. 4.3 Paving the way forward The objectives of this research were accomplished successfully. Treehoppers native to the Okanagan with high GRBV vectoring potential have been identified and a robust, high fidelity GRBV molecular diagnostics protocol has been described. 58 Previous reports of suspected spread of GRBV in the Okanagan could not be validated due to difficulties in obtaining reproducible diagnostic data and uncertainty of the presence of true vectors. By following our described protocol, future researchers can more confidently report GRBV spread in vineyards and further substantiate these reports by finding buffalo treehopper populations in these areas. This empowerment will not only benefit researchers in the Okanagan but all others in Nearctic regions where GRBV is present and spread may occur. 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Thesis/Dissertation
eng
Biology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Hart, Miranda
Lowery, Thomas
Evaluation of the grapevine red blotch virus epidemiological landscape of the Okanagan valley
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