Therefore, the committee identified research areas it considers high priority (labeled HP) and medium priority (labeled MP). These research areas are also presented in Table 4-1, at the end of this chapter.

GLD KNOWLEDGE GAPS

Basic Research to Generate Foundational Knowledge for Resolving the Complex Biology of GLD

Several distinct but taxonomically related closteroviruses (GLRaVs) have been reported in association with GLD, each of which may also have divergent strains or molecular variants (Martelli et al., 2002, 2012). It is generally understood that there is a stronger expression of symptoms in response to GLD in red or black-fruited cultivars than in white-fruited cultivars. Though widely reported, color-based symptoms of GLD in white-fruited varieties are often subtle and may be unrecognizable (Maree et al., 2013; Naidu et al., 2015). In addition, symptoms such as the patterns of interveinal chlorosis are unreliable because they mimic the symptoms of nutritional deficiencies. While it is true that leaf rolling in certain white-fruited varieties is diagnostic for GLD, this symptom occurs mostly in the advanced stage of GLD and often in chronically infected vines. There are also varietal differences; while GLD-induced leaf rolling at harvest is often conspicuous in Chardonnay, this is not always the case for Sauvignon blanc or Thompson Seedless (Maree et al., 2013). The reasons underlying these differences have not been well elucidated.

Because GLD is an exceptionally complex viral disease, molecular and genomic approaches are needed to better understand the many dimensions of the disease biology. Experimental systems are needed for studying viral gene functions, virus-vector-host interactions, and the transmission biology of GLRaV-3. The availability of complementary DNA (cDNA) clones for genetic variants of GLRaV-3 (Jarugula et al., 2018; Shabanian et al., 2023) can provide the critical reagents needed to apply synthetic biology approaches, including the de novo synthesis of viral genomes (Wimmer et al., 2009), for understanding the role of genetically divergent variants in different aspects of GLD. These synthetic biology approaches could enable additional studies to determine the relative pathogenicity of genetic variants of the virus(es), their relative transmission efficiencies, and disease outcomes of their co-occurrences in grapevine, among other insights.

Conclusion 4-1: Despite decades of research, knowledge on the genetic and phenotypic complexity of GLD-associated viruses remains limited.

Vitis species would also be useful for applying genome editing to generate GLRaV-3-resistant grape cultivars. Finally, non-coding RNAs have been discovered in grapevines, and the GLRaV-3 genome also contains noncoding genomic regions (Alabi et al., 2012). Non-coding RNAs are known to play significant roles in plant defense against viruses or cooperation with viruses in symptom and disease development (Wang et al., 2015; Yang et al., 2019; Ahmed et al., 2020; Shrestha and Bujarski, 2020; Javaran et al., 2021; Kumar and Chakraborty, 2021; Prasad and Prasad, 2021), but the roles of these non-coding RNAs in GLRaV-3 infection and symptom development remain largely unexplored. Understanding the role of non-coding RNAs in virus-grapevine and vector interactions could lead to important insights to inform RNAi-based biocontrol strategies for GLRaV-3.

In addition, further studies are required to understand why different cultivars show different levels and patterns of GLD susceptibility and symptom expression and why GLD symptoms are expressed only during the post-veraison stage of the crop in red or black-fruited cultivars even though GLRaV-3 can be detected in infected vines throughout the season (Naidu et al., 2015). Elucidating the cascade of molecular events occurring during asymptomatic pre-veraison stages and symptomatic post-veraison stages could advance knowledge of the virus-host interactions that lead to symptom expression at a specific phenological stage in red or black-fruited cultivars or the lack thereof in white-fruited cultivars. The knowledge derived from these fundamental studies could also support the development of novel strategies to fight the disease, such as through the application of RNAi and CRISPR/Cas-based genome-editing technologies (see Host Plant Resistance to Viruses and Vectors section in Chapter 5).

Conclusion 4-3: Host factors required for GLRaV-3 infection and resistance in Vitis hosts have not been discovered, yet knowledge of these factors could create opportunities for developing novel control strategies.

Conclusion 4-4: The grapevine and GLRaV-3 genomes contain regions for generating non-coding RNAs whose role in infection and symptom development has not been explored.

Conclusion 4-5: Further investigations into the extent of GLRaV-3 host range within (and beyond) Vitis may generate valuable information that could be exploited for GLD management.

Recommendation 4-3 (MP): Support research to identify host factors required for GLRaV-3 infection and resistance in Vitis hosts and to investigate the role of non-coding regions of grapevine and GLRaV-3 genomes in infection and symptom development.

• What are the consequences of co-infections of different GRBV variants?

Virus-Host Interactions and Host Defense Mechanisms

GRBV is different from other members of the Geminiviridae family in some important ways (Gilbertson, 2024), and the discovery of GRBV and later ratification of this new virus species (Grablovirus vitis) spurred the formation of a novel genus named Grablovirus (Varsani et al., 2017; Fiallo-Olivé et al., 2021). Most of the putative open reading frames (ORFs) in the GRBV genome have no ascribed function to date. Transient expression of the GRBV C2 and V2 ORFs in Nicotiana benthamiana line 16c green fluorescent protein marker plants suggests a role for these genes in overcoming RNA silencing (Weligodage et al., 2023). Evidence for alternative splicing has been demonstrated in both the viral and complementary sense ORFs, and a novel ORF was discovered in the viral sense (V0) (Vargas-Asencio et al., 2019).

Although GRBV protein products of the V1 ORF (coat protein) and the V2 ORF (unknown function) have been physically detected in infected grapevine tissues (Buchs et al., 2018), no virions have ever been observed in GRBV-infected plants. This lack of observed virions is a gap in basic GRBV biology and an opportunity for future study, since filling this gap would have practical implications for understanding transmission and improving diagnostics. Visualizing virions may be aided by discovering a suitable herbaceous model host, which has also eluded researchers thus far. Infectious GRBV clones have been inoculated into various herbaceous hosts (Solanum lycopersicum cv. Florida Lanai, Nicotiana benthamiana, and Phaseolus vulgaris cv. HyStyle) and GRBV replication was confirmed in inoculated leaves, but not in apical leaves (Flasco et al., 2021). The lack of systemic tractable model hosts for GRBV limits the study of virus-host interactions. Although the exact features of a pathosystem such as latency, susceptibility, and symptomatology may not be identical to what happens in grapevines, the development of an appropriate herbaceous model host can allow research to happen more quickly and in smaller spaces compared with research conducted in most grapevine varieties (Roy and Fuchs, 2024). If virus replication is higher in the herbaceous host, this would also improve the likelihood of visualizing virions. Therefore, a model herbaceous host would facilitate research on the basic biology of GRBV infection and help to identify features of interest. However, while an herbaceous host may be ideal for studying virus-host interactions, insights gained from such studies would ultimately need to be confirmed in Vitis spp. For this reason (and because an appropriate herbaceous model host has not been identified so far), it may be more practical to use Pixie grapevine, a Pinot Meunier mutant

with a dwarfing and shortened internode phenotype released by the U.S. Department of Agriculture for unrestricted use, as a woody model host to study virus-host interactions. The Pixie grapevine’s small size and production of clusters when cultivated in a greenhouse or growth chamber can enable a broader scale or scope of research uses compared with commercial grapevine varieties, assuming Pixie becomes infected with vitiviruses like other Vitis spp.

Conclusion 4-7: Despite some progress in determining GRBV gene function, there are still major gaps in understanding the function of the GRBV genome with regard to specific roles of GRBV proteins in plant cells.

Conclusion 4-8: To date, virions have not been observed in GRBV-infected plants using microscopy; the lack of a tractable herbaceous model host that becomes systemically infected with GRBV limits the study of virus gene functions and virus-host interactions.

Recommendation 4-6 (MP): Support research to determine optimal model hosts (e.g., Pixie grapevine and/or herbaceous hosts) to facilitate the study of molecular GRBV-plant interactions and direct research efforts to transfer this knowledge to wine grape cultivars.

Research questions that need to be addressed include the following:

• What functionally equivalent host factors are required for GRBV infection of plants?
• What is the virion structure of GRBV?
• Which varieties of herbaceous and/or Vitis hosts are the best model systems for studying virus-host interactions?

The lack of knowledge on the length of the latency period following GRBV inoculation is another major gap that has direct implications for epidemiology and management. In particular, there is a need to refine questions regarding latency and incubation periods to focus on determining the time intervals between vector-mediated inoculation and systemic GRBV infection, between inoculation and GRBV acquisition by the vector, and between inoculation and symptom expression. It would be reasonable to expect variability in each type of latency and incubation period among different cultivars and under different environmental conditions. Insights into these factors could directly impact management recommendations by helping to inform virus testing procedures and elucidating how asymptomatic infections may contribute to virus spread.

et al., 2018; Xiao et al., 2018; Yao et al., 2018; Arnold et al., 2019; Diaz-Lara et al., 2019; Jones and Nita, 2019; Soltani et al., 2020; Xiao and Meng, 2023), and, in some cases, aggregated diseases such as sudden vine collapse (Bolton, 2020) are associated with co-infection of vitiviruses and leafroll viruses (Rowhani et al., 2018). With respect to GRBV, geminiviruses in other systems have demonstrated synergism in mixed infections (Moreno and López-Moya et al., 2020); however, it is not yet clear whether any synergistic effects are associated with the co-infection of GRBV with other viruses. Synergy has been reported for co-infections of vitiviruses with GLRaVs that cause changes in symptom expression, death of the vine, or changes in virus replication (Rosa et al., 2011; Rowhani et al., 2018; Čarija et al., 2022). Four studies have examined the co-transmission of GLRaV genetic variants, or GLRaV with other virus species, and using different mealybug vectors. One study included transmission of co-infections of GLRaV-3-I and GLRaV-3-VI by Planococcus ficus and Pseudococcus viburni (Blaisdell et al., 2015). Other studies examined transmission of GLRaV-1 + grapevine virus A (GVA), GLRaV-3 + GVA, GLRaV-1 + GLRaV-3, and GLRaV-1 + GLRav-3 + GVA by Heliococcus bohemicus (Bertin et al., 2016a) and by Pl. ficus and Planococcus citri (Bertin et al., 2016b), and co-infections of GLRaV-3 + GVA, GVA + grapevine rupestris stem pitting-associated virus, or GVA + grapevine virus B by Pl. ficus (Blaisdell et al., 2020). The results from these studies showed that the presence of multiple viruses can increase or decrease the transmission of one or more of the viruses, but changes in transmission were not observed in every study. In some studies, changes in transmission appeared to be influenced by virus-vector interactions, and in others, changes in the frequency of transmission were due to virus-plant interactions after vector inoculation. Together these results highlight the complexity of virus-vector-host plant interactions that can influence transmission and host infection outcomes. The implications of the background virome (i.e., mixed infections with other viruses) for the co-transmission of GLRaVs and GRBV, expression of GLD and GRBD symptoms, fruit quality, or for GLRaV-3 and GRBV fitness have not yet been investigated. The influence of mixed infections on the evolution and epidemiology of these viruses remains poorly understood.

Effects of Environmental Factors

There are also knowledge gaps regarding the potential influence of environmental factors such as temperature, humidity, carbon dioxide, ozone, drought, and vineyard management practices on the vector, virus, plant, and interactions among them. Laboratory or greenhouse studies can be used to investigate how these influence within-plant factors related to transmission efficiency, virus replication, and disease severity. Broader landscape-level

effects also need to be understood, which would require studies at the field level or modeling studies to examine regional shifts in degree days (temperatures) that regulate insect generations, plant growth, and geographic distributions of vector and plant hosts for viruses (Trebicki, 2020; Mangang et al., 2024, and references within). Understanding the effects of changing climatic conditions and other biotic and abiotic factors that modulate the disease cycle in the field will be important for current and future research and control strategies.

Conclusion 4-10: Infection of grapevines with multiple viruses has been reported, but how mixed infections affect disease severity and evolution of GLRaVs and GRBV (or GLD and GRBD) has not been thoroughly investigated.

Conclusion 4-11: The effects of changing climatic conditions and other factors (biotic and abiotic) that modulate disease cycles, including temperature, humidity, carbon dioxide, ozone, drought, and vineyard management practices, on virus-vector-host interactions have not been determined.

Recommendation 4-8: Support research on the effects of mixed infections on GLRaV and GRBV evolution and the diseases they cause, as well as research on the effects of environmental factors, grapevine management practices, and changing climatic conditions on GLD and GRBD virus-vector-host interactions and epidemiology. Industry trends and stakeholder input could be used as a guide for prioritizing scion-rootstock combinations to use in experiments.

Research questions that need to be addressed include:

• Do co-infections of GLRaV-3 or GRBV with specific classes of grapevine viruses facilitate disease establishment or enhance its severity?
• What are the consequences of mixed infections of GLRaV-3 with other viruses (e.g., synergism, antagonism, neutral)?
• What are the consequences of mixed infections of GRBV with other viruses (e.g., synergism, antagonism, neutral)?
• How do abiotic factors, other stresses, and non-viral diseases influence disease caused by GLRaV-3 and GRBV?

the collective impact of multiple, co-infecting viruses on vineyard performance and longevity as well as the potential for a synergistic enhancement of disease symptoms.

It would also be informative to investigate whether there are differences in virus titer between vinifera scion cultivars and rootstocks and whether scion-rootstock combinations influence symptom expression and virus titer. Such insights would inform approaches for testing samples from the scion of grafted vines for GLRaV-3 or GRBV and could help to determine whether there is a delay in virus movement across the graft union (in contrast to own-rooted vines) leading to delayed symptom expression. Another question that is important to growers is whether delayed symptom expression in post-planting grafted vines is due to delayed expression in infected, non-certified vines or to vector-mediated transmission of the virus after planting. Understanding the relative contribution of infected, non-certified vines to the spread of GLD and GRBD compared with vector-mediated spread would help guide management by identifying efforts needed for vine certification programs versus in-field management activities.

Conclusion 4-12: A variety of factors, including the scion cultivar, genetic background of rootstock, rootstock-scion interactions, virus profile in individual grafted vines, synergistic interactions between co-infecting viruses, and environmental conditions, could contribute to the presence and severity of symptoms from GLD and GRBD.

Conclusion 4-13: Resistant rootstocks along with other control strategies could help to mitigate negative effects of viral diseases in vineyards.

Recommendation 4-9 (MP): Support research on the presence and diversity of viral resistance in grapevine rootstocks with different genetic backgrounds in order to inform the incorporation of resistant rootstocks into virus control strategies.

Recommendation 4-10: Support research to determine the contribution of planting with infected, non-certified vines on virus spread.

KNOWLEDGE GAPS IN GLRaV-3 AND GRBV DIAGNOSTICS AND DETECTION

Visual scouting for GLRaV-3- or GRBV-infected vines in vineyards is unreliable due to the variability of symptoms in different types of wine grape cultivars, because symptoms may not always be expressed clearly in affected grapevines, and because typical virus symptoms are easily confused with other maladies. In particular, white-fruited grapevine cultivars often do

not show discernible symptoms when infected with either virus. Moreover, red or black-fruited wine grape cultivars can show red and reddish-purple leaf symptoms in response to many factors other than viral infections, such as nutrient deficiencies, physiological disorders, mechanical injuries, infection with crown gall bacterium, or insect herbivory, making it challenging to discern the impacts of these stressors from true symptoms of GLD or GRBD. When symptoms are present, initial testing and troubleshooting must be conducted to eliminate non-viral stress factors before proceeding with using symptoms to guide site-specific management of grapevine diseases. The following section describes potential testing methods that offer diagnostics for different situations, with each offering different scales of testing across a vineyard. Investments to develop these should be made based on stakeholder input and prioritized based on which one(s) will help growers and the industry accomplish site-specific and area-wide virus management goals most effectively and economically.

Cost-Effective Field-Deployable or Laboratory-Based Tools for Large-Scale Detection of GLRaV-3 and GRBV

Simple Plant-Based Assays for Detection

Although there have been significant strides in GLRaV-3 and GRBV diagnostics (see Diagnostics sections in Chapters 2 and 3), there is still a need for additional diagnostic tools, especially those that could improve the early detection of GLRaV-3 and GRBV and allow for affordable, high-throughput testing of commercial vineyards. The lack of affordable diagnostic methods for on-site detection delays timely disease diagnosis and management efforts, allowing the viruses to continue to spread and lead to substantial economic losses.

To date, the feasibility of developing serological methods, such as enzyme-linked immunosorbent assay (ELISA) or squash-blot, for detecting GRBV has not been determined. Virions have not been observed in infected tissues (Buchs et al., 2018), nor have virions been purified for producing antisera. Researchers have attempted to express and produce viral proteins in experimental host plants (R. Gilbertson, personal communication, March 5, 2024) but have yet to successfully generate the quality and quantity of viral proteins necessary to produce antisera against GRBV, hindering the application of serological assays in the diagnosis of GRBV. However, despite these challenges, developing portable serological assays for on-site testing is a realistic and worthwhile goal. Using recombinant or synthetic virus proteins could lead to the production of GRBV-specific antibodies, i.e., by engineering viral proteins such as coat protein in vector plasmids to be expressed in cultured cells for making antigens. Since coat protein

was detected and quantified in proteomic profiling of GRBV-infected leaf and petiole tissues (Buchs et al., 2018), coat protein is a good candidate for producing a GRBV-specific antigen for developing a serological method for detecting GRBV. This could open the door to developing affordable rapid and on-site detection assays, such as lateral flow assays, that would be accessible to growers without requiring specialized laboratory equipment.

For GLRaV-3, current diagnostic techniques often rely on either visual scouting (which is unreliable), or laboratory-based assays such as ELISA, reverse-transcription polymerase chain reaction (RT-PCR), or HTS, which are more reliable but not practical for real-time field testing due to their cost and dependence on specialized equipment and trained personnel (Bester et al., 2012; Blouin et al., 2017; Rowhani et al., 2017a; Galvan et al., 2023). ELISA methods for laboratory-based detection are among the most scalable testing techniques that could be developed for large-scale testing if automation capacity can be improved. Recently, a simple crude plant extract-based reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed to detect GLRaV-3 efficiently (Kishan et al., 2024). This tool can offer a cost-effective solution that could be validated and made commercially available for on-site vineyard detection.

Conclusion 4-14: There is a need for additional affordable diagnostic tools that can detect GLRaV-3 and GRBV 3 infections early and are suitable for extensive use in commercial vineyards.

Recommendation 4-11 (HP): Support research to develop new, simple, and affordable high-throughput tests for GLRaV-3 and GRBV.

Research may include the following:

• Producing GRBV-specific antigens that could enable development of a serological assay.
• Validating a simple crude plant extract-based LAMP and RPA assays for GLRaV-3 and GRBV to determine the suitability of isothermal assays for large scale and/or on-site detection.
• Improving the automation testing capacity for existing GLRaV-3 ELISAs to improve throughput and reduce costs.

Volatile Organic Compound Detection

Disease detection using dogs, electronic noses (ENs), or micro-electromechanical systems could help with early detection (i.e., during latency periods or when visual symptoms are absent) and could also address the problem of uneven distribution of pathogens in the host, which is an issue with detection methods such as PCR (Gottwald et al., 2020).

___________________

¹ This estimate was provided in the article available at https://www.goodfruit.com/sniffing-out-diseases/.

citrus greening disease with 90-99.9 percent accuracy (Aksenov et al., 2014; McCartney et al., 2024), and this approach may have applications for detection of other plant pathogens.

Conclusion 4-15: Canine olfactory capacity could be used for GLRaV-3 and GRBV field detection, but the most effective, practicable, and cost-effective way to employ dogs for monitoring and early detection has yet to be determined. Canine detection may be best suited for nurseries rather than commercial vineyards.

Conclusion 4-16: Research to profile plant responses to GLRaV-3 and GRBV (and their vectors) may reveal unique VOC profiles that could establish a basis for the development of handheld EN or DMS devices for pathogen detection in the field.

Recommendation 4-12: Support research to identify VOCs unique to GLRaV-3 and GRBV infection or relevant vector infestations and determine the detection efficiency of VOC-based methods compared with other diagnostic tools.

Remote Sensing

Remote sensing technologies, including imaging spectroscopy, hyperspectral imaging, and RGB imaging, share a common feature of capturing detailed information about plant health status from a distance. These technologies can significantly aid in grapevine virus detection by enabling the identification of subtle changes in leaf color, texture, and reflectance patterns that may indicate viral infections. Remote sensing has significant potential as a tool for disease diagnosis in white-fruited grapevine cultivars, which do not exhibit conspicuous symptoms, and in infected red or black-fruited cultivars at the pre-veraison stage. If feasible, using imaging devices for in-field diagnosis of GLRaV-3 and GRBV would likely be attractive to growers and crop consultants because it would eliminate the need to collect plant tissue samples, and could be used to screen large areas or entire vineyards. However, due to the complexity of the symptoms associated with these viral diseases, remote sensing data collected from a vineyard would still require validation of the infection status of a large number of vines using a reliable laboratory diagnostic tool, such as PCR or ELISA. Although several research groups (MacDonald et al., 2016; Bendel et al., 2020; Nguyen et al., 2021; Galvan et al., 2023; Sawyer et al., 2023; Wang et al., 2023a,b, 2024; Lee at al., 2024; Wang and Pagay, 2024; Žibrat and Knapič, 2024) are studying the suitability of various remote sensing devices, to date this research has focused on detecting grapevine viruses

based on only the leaf (or canopy) reflectance without any studies examining this method’s applicability to other tissue types such as fruit berries, young twigs, branches, or trunks. Further research could help to elucidate the method’s applicability to other tissue types and its feasibility for field deployment.

Conclusion 4-17: Remote sensing technology has the potential for remote or in-field diagnosis of GLD and GRBD in individual vines; however, testing the efficacy of this approach will require scalable deployment of remote sensing devices for detection of infected vines in a large-scale area.

Conclusion 4-18: Remote sensing technology can be a part of a multi-layered system to guide sampling efforts by taking advantage of different spectra and resolutions to address specific goals.

Conclusion 4-19: In addition to leaves, remote sensing devices can also potentially be used on other visible parts of the vines to detect grapevine viruses.

Recommendation 4-13 (HP): Support studies on the use of remote sensing technology to facilitate large-scale and early detection of GLD and GRBD in various tissues of commercial cultivars (including white-fruited cultivars) to increase the reliability, specificity, and sensitivity of detection with this technology.

Improved Methods for Detection of New GLRaV-3 and GRBV Variants

Nucleic Acid-Based Assays

GLRaV-3 and GRBV will continue to evolve in cultivated vines, wild vines, and other refuge plants in the vineyard ecosystem, and it is important to continually monitor for the occurrence of new GLRaV-3 and GRBV variants in vineyards and riparian habitats. Since the primers used in existing nucleic acid-based assays may not detect these newly emerging variants, nucleic acid-based detection assays need to be improved frequently by upgrading primer sequences. In addition, rolling circle amplification (RCA), which has been used to amplify the whole genome of GRBV, could be another method to detect GRBV at very low concentrations (e.g., in nursery settings). Although the feasibility of this method for diagnosing GRBV is yet to be determined, if used, sequencing the RCA products may be particularly useful for universal detection of emerging GRBV variants.

vineyards, and the logistical feasibility of various steps in the process. For example, Meyers et al. (2011) have suggested that stratified sampling based on vineyard blocks or rows may improve accuracy by capturing spatial heterogeneity. Statistical methods such as power analysis can be employed to determine the minimum sample size needed to achieve a desired level of accuracy in prevalence estimation (McDonald, 2008; Hajian-Tilaki, 2014). However, these two approaches may not fully account for the complex dynamics of GLRaV-3 and GRBV spread within nurseries and vineyards.

Another strategy that could be considered is to detect GLRaV-3 and GRBV in insects feeding on grapevine. These phloem-restricted viruses could be a part of insects’ diets when feeding on phloem of virus-infected grapevines, and phloem contents (and any microbes present) may accumulate in the insects’ gut. Detecting viral markers in insects offers a unique and relatively targeted way to sample the phloem contents of a vine, and this strategy has been successfully employed to detect citrus viruses in vector and non-vector phloem-feeding insects (Saponari et al., 2008; Britt-Ugartemendia et al., 2022). Several studies have already documented the presence of GRBV in the TCAH and other insects of unknown vector status (Cieniewicz et al., 2018; LaFond et al., 2022; Wilson et al., 2022), but additional research would be needed to determine the sensitivity of this method for detecting GLRaV-3 and GRBV in insects and to define best practices for sampling insects from grapevines. Since sampling insects will generally be more time consuming than foliage sampling, it will also be important to determine the feasibility and best application of this method for virus detection.

Conclusion 4-21: Consensus is lacking on the most effective sampling technique and minimum sample size for accurately estimating GLRaV-3 and GRBV prevalence across different vineyard settings, regions, and nursery increase blocks.

Conclusion 4-22: Virus detection in vectors and other phloem-feeding insects may be an alternative to testing grapevines for viruses.

Recommendation 4-16 (HP): Support research evaluating optimal sampling methods and minimum sample size for accurate estimation of GLVaV-3 and GRBV prevalence in vineyards to inform the development of best practices for adopting new technologies and for integrating multiple detection methods to improve accuracy and scale (i.e., using both molecular methods and remote sensing technology).

Wilson et al., 2022). Although this proves they acquired the virus by feeding on an infected plant, it does not prove the pathogen can move across insect membranes, enter the salivary glands, and be delivered to a recipient grapevine for successful transmission. Of the studies that have investigated transmission potential with vectors besides TCAH, not all experiments were replicated, some are missing critical details about how samples were handled for processing, and some leave open the possibility of false positive results due to the viral contamination in honeydew excreted by both vector and non-vector insects (Rosell et al., 1999). While artificial diet assays provide a way to test for transmission quickly, additional experiments are needed to confirm transmission and infection in live plants (Kahl et al., 2021).

No studies have examined sex-related differences in transmission or behavior of TCAH, even though males and females may transmit pathogens with different efficiencies or respond to environmental stimuli differently (Sakurai et al., 1998; van de Wetering et al., 1998, 1999; Beanland et al., 1999; Ghanim and Czosnek, 2000; Ning et al., 2015; Ogada and Poehling, 2015; Zhao et al., 2016; Lu et al., 2017). In the southeastern United States, TCAH males and females are present in overwintering populations, but males die soon after mating, whereas females live for an average of 38 days post-copulation (Mitchell and Newsom, 1984b). In California, both males and females have been found to be present in vineyards, but the ratios of males and females can fluctuate (Preto et al., 2019). Additional studies on TCAH population dynamics would help determine the frequency at which males and females are present in vineyards when transmission occurs and help to determine whether any sex-related differences in GRBV transmission efficiency or host feeding behaviors may be relevant to improving management approaches (see further discussion in the Virus-Vector Interactions section of this chapter).

Conclusion 4-25: While there are reports about potential additional insect vectors of GRBV, there has not been definitive evidence that other insects in addition to TCAH can transmit GRBV to grapevines.

Recommendation 4-19 (MP): Support research to identify additional vectors of GRBV using rigorous experimental approaches.

Research to identify additional vectors should employ the following best practices:

• Select vector candidates for study based on field data suggesting an association between the insect and virus spread.
• Replicate controlled laboratory transmission experiments, including replicating experimental units (insects and plants) each time

• transmission is tested under a given set of conditions and replication of experiments to draw verifiable conclusions.
• Allow for a minimum time of 10 days for the acquisition access period (AAP), 10 days for the latent period, and 4 days for the inoculation access period (IAP) based on the minimum times reported for TCAH. Males and females should be tested separately.
• Because plant viruses can be excreted and detected in honeydew, it is necessary to use a cleaning procedure to remove honeydew from plant tissue prior to virus testing. Methods designed to detect a viral RNA transcript could also prevent false positives due to contaminated honeydew.
• Testing transmission using artificial diets represents one way to demonstrate vector competence, but transmission to grapevines is needed to confirm the epidemiological significance of vector transmission in the field.

Additional Vectors of GLRaVs

Understanding the transmission of GLRaVs remains an ongoing area of research with several important knowledge gaps. Currently, mealybugs and scale insects are recognized as the vectors of GLRaVs, but the knowledge of the full spectrum of potential vectors and their distribution in California may be incomplete. The vine mealybug, Pl. ficus, is the major vector identified in commercial vineyards. The predominant role of this vector is partially attributed to its high reproductive capacity, which increases spread over time. The grape mealybug is also a prominent vector; however, the relative contributions of individual species to GLRaV transmission in the field are unknown. The epidemiological relevance of each vector or community of vectors will be impacted by differences in abundance, distribution, life cycle, and other life history characteristics. There does not appear to be specificity or fidelity among the leafroll viruses and mealybug vectors, but most studies have focused on GLRaV-3 transmission by Pl. ficus. Reports suggest that GLRaVs are semi-persistently transmitted with no latent period (Cabaleiro and Segura, 1997; Tsai et al., 2008). Characteristics such as the durations of acquisition, retention, and inoculation periods have not been determined for all mealybug and scale vectors, and few transmission assays have been conducted to quantify vector acquisition and inoculation efficiencies for different species of GLRaVs or genetic variants of particular GLRaV species.

Conclusion 4-26: There are gaps in the understanding of GLRaV-3 transmission, particularly with regard to the role of different vector species and their distribution in California; the mechanisms of GLRaV-3

acquisition and transmission; the transmission efficiency of diverse GLRaV-3 isolates; the acquisition, retention, and inoculation periods of all vector species; and how environmental factors influence GLRaV-3 transmission dynamics.

Recommendation 4-20 (HP): Support research on the mechanisms and timing of acquisition, retention, and transmission of all GLRaV vector species, as well as the influence of environmental conditions and host genotype on GLRaV transmission dynamics.

Research to identify additional vectors should employ the following best practices:

• Conduct transmission assays that individually assess acquisition, retention, and inoculation.
• Healthy vectors should be caged on infected plants for AAPs that range from several hours to several days to assess acquisition efficiency.
• For inoculation assays, infected insects should be isolated in groups on healthy plants to assess virus transmission. Inoculation assays should utilize insects of similar developmental stage. IAPs can range from several hours to days, as longer IAPs yield higher transmission efficiencies.
• Transmission experiments in which insects feed on artificial media through a membrane can also be used to assess vector capacity, but ultimately this approach may not provide an accurate indicator of vector transmission capacity or efficiency.
• Transmission differences between vector species may be specific to grape cultivars and environment; therefore, comparisons of efficiency should be evaluated in controlled assays to assess the contributions of these factors to the epidemiology of vector transmission.
• Differences in transmission efficiency among clones or populations of vector species should be evaluated using comparable AAPs and IAPs to effectively assess the epidemiological importance of particular vector species or phenotypic variation in transmission efficiency that exists in pathogen transmission.

Virus-Vector Interactions

Additional knowledge gaps that exist for both GLRaVs and GRBV include the mechanisms of virus-vector interactions, the effect of the environment on epidemiology, and how mixed infections with multiple viruses might impact transmission. The time required to acquire and transmit these viruses has been examined, but virus localization in the vectors has not been

confirmed, and the precise viral retention sites have not been thoroughly characterized. The genetic, cellular, and physiological mechanisms underlying vector transmission remain unknown. Virus localization would confirm the mode of transmission of GLRaVs by mealybugs and scales; transmission is reported as semi-persistent, but definitive transmission studies of GLRaV-3 are generally lacking. Studies examining GRBV localization in TCAH may help explain the long latent period required for the virus to circulate through the vector and generate new hypotheses about transmission. In other pathosystems, vector or endosymbiont proteins have been reported to bind to virions circulating in insects, and endosymbionts can alter transmission of plant viruses and insect-plant interactions (Gonella et al., 2019; Wilson et al., 2019; Ghosh and Ghanim, 2021; Wu et al., 2022; Sanches et al., 2023, and references within these). These factors have not been studied for GLRaV-3 or GRBV, and identification of endosymbionts, genes, proteins, and metabolites responsible for virus transmission may help generate novel control strategies designed to block these interactions (Heck, 2018; Milenovic et al., 2022; Ali and Ume-Farwa, 2024).

Conclusion 4-27: Knowledge of virus localization in the vectors and the precise role of viral retention sites in vector transmission would improve knowledge about the mode of transmission for GLRaV-3 and GRBV.

Conclusion 4-28: The roles of vector endosymbionts, genes, proteins, and metabolites mediating transmission have not been studied for GLRaVs or GRBV. This information is needed to understand transmission dynamics and to develop novel tools for disrupting transmission for the management of GLD and GRBD.

Recommendation 4-21: Support studies to identify interactions between GLRaVs and GRBV and their vectors that are required for transmission, as well as studies to identify genes, proteins, and metabolites involved in virus transmission to develop control strategies based on interference of virus-vector interactions.

Vector Plant Preference and Behavior Manipulation by GLRaVs and GRBV

Impact of Known Hosts on Disease Spread and Insect Behavior Manipulation

The reported host range of GLRaV-3 and GRBV is limited to Vitis and non-cultivated grapevines, but the relative contributions of different species or varieties in GLRaV-3 or GRBV spread are not well understood. The

variation in host utilization by vectors and the virus prevalence in host plants are also not clearly defined. Knowledge about variation in vector behavior and virus susceptibility among Vitis and non-cultivated grapevines may help explain patterns of spread and guide management decisions. This includes whether vector behavior changes in response to vine health or different species and varieties of Vitis and non-cultivated grapevines. No study to date has comprehensively compared vector preferences for plant host species. Molecular gut content analyses could help identify which plants vectors are feeding on before they move into vineyards (Cooper et al., 2016, 2019, 2022, 2023; Hepler et al., 2021, 2023; Reyes Corral et al., 2021a,b; Dorman et al., 2024; Pitt et al., 2024). Knowledge on host plant preference or suitability can also be studied by evaluating behavioral responses, such as through host choice experiments, olfactometer assays, or electrophysiological studies, to assess vector responses to host VOCs. Feeding assays, in the form of electrical penetration graph assays, laboratory assays, or greenhouse assays, may provide information about host preference, host suitability of different grape cultivars for specific vectors, and virus transmission, which would provide information to assess the epidemiological importance of individual vector species (Fereres and Collar, 2001; Tjallingii and Prado, 2001; Fernandez-Calvino et al., 2006; Sandanayaka et al., 2013, 2014; Boquel et al., 2015; Mustafa et al., 2015; Muturi et al., 2016; Obok et al., 2018).

In addition, a growing body of work has shown that virus infection can alter insect vector behavior or fitness of host plants in ways that promote the acquisition and transmission of the viruses, a concept known as the vector manipulation hypothesis (Ingwell et al., 2012; Stafford et al., 2011; Su et al., 2015; Chen et al., 2013; Eigenbrode et al., 2018). This can occur via changes induced in the host plants that alter visual cues, volatile profiles, palatability, host defense, or nutritional quality and influence insect settling and feeding behaviors. In some pathosystems, the fitness of the vectors is improved due to metabolite profile changes in infected plants, which leads to increased reproductive or developmental rates. All of these changes may influence vector settling behaviors, feeding behaviors, and dispersal patterns related to the acquisition and spread of viruses, but changes in vector behavior (and biology) are not consistent or generalizable across or within pathosystems (Jones, 2014). This underscores the need to study how GLRaV-3 and GRBV specifically alter the behavior of the insect vector(s), which could have important epidemiological ramifications for understanding and modeling their spread.

Conclusion 4-29: GLRaV-3 and GRBV have only been reported to occur on Vitis and non-cultivated grapevines, but the relative contributions of different host species or varieties in GLRaV-3 or GRBV spread are not known.

tested (Cieniewicz et al., 2018). Overwintering of pathogens in adult vector populations contributed to the early-season spread of Pierce’s disease by the glassy-winged sharpshooter (Purcell, 1975; Almeida et al., 2005). It is unknown whether GRBV can persist in the overwintering TCAH adults that acquired GRBV before they left vineyards in the fall or whether overwintering adults acquire GRBV from cultivated or wild Vitis spp. during the spring. Studies have identified wild Vitis spp. as an alternate host for GRBV; however, surveying these hosts for TCAH and assessing their importance as a source for GRBV spread into vineyards is difficult, and even large research efforts may not be sufficient to draw meaningful conclusions.

The movement of TCAH into vineyards that is responsible for GRBV spread will be influenced by generation times, seasonal host plant availability, host plant attraction cues, host preferences, and movement behavior of TCAH. Information about TCAH movement dynamics between grapevines and alternative hosts may improve monitoring efforts and help scouts, growers, and consultants understand how to interpret observations of TCAH populations that are only transiently using vineyards while alternative hosts are largely absent during the summer (Cieniewicz et al., 2018; Preto et al., 2019). A better understanding of host preference and timing of movement by the more mobile adults of TCAH may also inform the implementation of trap cropping strategies to intercept, concentrate, and kill TCAH on alternative hosts that are more attractive than grapevines along borders of vineyards before these insects encounter grapevines.

In California, most GRBV-TCAH-grapevine studies have been conducted in the Napa Valley. However, given the marked differences in distribution and abundance of the TCAH in other states where GRBV is spreading, and consequently differences in disease epidemiology, there are also expected differences across regions in California. This underscores the need for studies to determine the impacts of geographic factors on TCAH abundance and seasonal dynamics, which may include differences in regional viticultural practices, landscape composition, and climate patterns. At this time, there is no optimized sampling methodology for accurate estimation of TCAH populations in vineyards. Accuracy in sampling methodology is critical for developing population models. In addition, initial temperature-based degree-day population development models have been developed for TCAH (Bick et al., 2020), and additional efforts can be made to refine these models. This may include modeling vector populations as a function of local factors, including grapevine phenological stages, which may help result in more region-specific information.

Conclusion 4-32: There are major knowledge gaps regarding TCAH overwintering behavior, seasonal GRBV spread to grapevines, and differences among distinct grapevine-growing regions in California.

This page intentionally left blank.