The New Zealand perspective of an ecosystem biology response to grapevine leafroll disease.

Grapevine leafroll-associated virus 3 (GLRaV-3) is a major pathogen of grapevines worldwide resulting in grapevine leafroll disease (GLD), reduced fruit yield, berry quality and vineyard profitability. Being graft transmissible, GLRaV-3 is also transmitted between grapevines by multiple hemipteran insects (mealybugs and soft scale insects). Over the past 20 years, New Zealand has developed and utilized integrated pest management (IPM) solutions that have slowly transitioned to an ecosystem-based biological response to GLD. These IPM solutions and combinations are based on a wealth of research within the temperate climates of New Zealand's nation-wide grape production. To provide context, the grapevine viruses present in the national vineyard estate and how these have been identified are described; the most pathogenic and destructive of these is GLRaV-3. We provide an overview of research on GLRaV-3 genotypes and biology within grapevines and describe the progressive development of GLRaV-3/GLD diagnostics based on molecular, serological, visual, and sensor-based technologies. Research on the ecology and control of the mealybugs Pseudococcus calceolariae and P. longispinus, the main insect vectors of GLRaV-3 in New Zealand, is described together with the implications of mealybug biological control agents and prospects to enhance their abundance and/or fitness in the vineyard. Virus transmission by mealybugs is described, with emphasis on understanding the interactions between GLRaV-3, vectors, and plants (grapevines, alternative hosts, or non-hosts of the virus). Disease management through grapevine removal and the economic influence of different removal strategies is detailed. Overall, the review summarizes research by an interdisciplinary team working in close association with the national industry body, New Zealand Winegrowers. Teamwork and communication across the whole industry has enabled implementation of research for the management of GLD.

Scalable Early Detection of Grapevine Viral Infection with Airborne Imaging Spectroscopy.

The U.S. wine and grape industry loses $3B annually due to viral diseases including grapevine leafroll-associated virus complex 3 (GLRaV-3). Current detection methods are labor-intensive and expensive. GLRaV-3 has a latent period in which the vines are infected but do not display visible symptoms, making it an ideal model to evaluate the scalability of imaging spectroscopy-based disease detection. The NASA Airborne Visible and Infrared Imaging Spectrometer Next Generation was deployed to detect GLRaV-3 in Cabernet Sauvignon grapevines in Lodi, CA in September 2020. Foliage was removed from the vines as part of mechanical harvest soon after image acquisition. In September of both 2020 and 2021, industry collaborators scouted 317 hectares on a vine-by-vine basis for visible viral symptoms and collected a subset for molecular confirmation testing. Symptomatic grapevines identified in 2021 were assumed to have been latently infected at the time of image acquisition. Random forest models were trained on a spectroscopic signal of noninfected and GLRaV-3 infected grapevines balanced with synthetic minority oversampling of noninfected and GLRaV-3 infected grapevines. The models were able to differentiate between noninfected and GLRaV-3 infected vines both pre- and postsymptomatically at 1 to 5 m resolution. The best-performing models had 87% accuracy distinguishing between noninfected and asymptomatic vines, and 85% accuracy distinguishing between noninfected and asymptomatic + symptomatic vines. The importance of nonvisible wavelengths suggests that this capacity is driven by disease-induced changes to plant physiology. The results lay a foundation for using the forthcoming hyperspectral satellite Surface Biology and Geology for regional disease monitoring in grapevine and other crop species. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A Metagenomic Investigation of the Viruses Associated with Shiraz Disease in Australia.

Shiraz disease (SD) is an economically important virus-associated disease that can significantly reduce yield in sensitive grapevine varieties and has so far only been reported in South Africa and Australia. In this study, RT-PCR and metagenomic high-throughput sequencing was used to study the virome of symptomatic and asymptomatic grapevines within vineyards affected by SD and located in South Australia. Results showed that grapevine virus A (GVA) phylogroup II variants were strongly associated with SD symptoms in Shiraz grapevines that also had mixed infections of viruses including combinations of grapevine leafroll-associated virus 3 (GLRaV-3) and grapevine leafroll-associated virus 4 strains 5, 6 and 9 (GLRaV-4/5, GLRaV-4/6, GLRaV-4/9). GVA phylogroup III variants, on the other hand, were present in both symptomatic and asymptomatic grapevines, suggesting no or decreased virulence of these strains. Similarly, only GVA phylogroup I variants were found in heritage Shiraz grapevines affected by mild leafroll disease, along with GLRaV-1, suggesting this phylogroup may not be associated with SD.

Grapevine Leafroll-Associated Virus 3 Genotype Influences Foliar Symptom Development in New Zealand Vineyards.

Grapevine leafroll disease (GLD) constrains wine production worldwide. In New Zealand, the main causal agent of GLD is grapevine leafroll-associated virus 3 (GLRaV-3). To control GLD, an integrated management program is used and includes removing (roguing) GLRaV-3-infected vines from the vineyard. The classical foliar symptoms from virus-infected red-berry cultivars are leaves with dark red intervein, green veins, and downward rolling of margins. Growers use these phenotypic cues to undertake visual symptom identification (VSI) for GLD. However, the influence of the known large genetic variation among GLRaV-3 isolates on the foliar symptoms from different grapevine cultivars remains undescribed, especially in cool-climate growing environments, such as New Zealand. Over three vintages (2015, 2016, and 2017), VSI for GLD was undertaken at three field sites in New Zealand (Auckland, Hawke's Bay, and Marlborough), each including four cultivars (Merlot, Pinot noir, Sauvignon blanc, and Pinot gris) infected with three GLRaV-3 genotypes (Groups I, VI, and X) or GLRaV-3-uninfected control plants. Throughout this study, no visual symptoms were observed on white-berry cultivars infected with GLRaV-3. For red-berry cultivars, the greatest variability in observed foliar symptoms among regional study sites, cultivars, and GLRaV-3 genotypes was observed early in the growing season. In particular, Group X had significantly delayed symptom expression across all three sites compared with Groups I and VI. As the newly infected, young vines matured in years 2 and 3, the GLRaV-3 genotype, cultivar, region, and environmental conditions had minimal influence on the accuracy of VSI, with consistently high (>95%) within-vintage identification by the end of each vintage. The results from this study strongly support the use of VSI for the GLD management of red-berry cultivar grapevines, Merlot and Pinot noir, as a reliable and cost-effective tool against GLD.

Potential effects of Grapevine leafroll-associated virus 3 (genus Ampelovirus; family Closteroviridae) or Grapevine red blotch virus (genus Grablovirus; family Geminiviridae) infection on foliar phenolic and amino acid levels.

OBJECTIVE: Grapevine (Vitis spp.) viral infections, including those by Grapevine leafroll-associated virus 3 (GLRaV-3) and Grapevine red blotch virus (GRBV), greatly reduce fruit yields and quality. Evidence exists that host chemistry shifts result in reductions in fruit quality. However, changes over the season in foliar chemistry has not been well examined. Therefore, phenolic and amino acid levels were examined in leaves collected in grapevines with different rootstocks that were healthy or were infected with GLRaV-3 or GRBV. This was part of an effort to assess changes that different pathogens cause in grapevine tissues. RESULTS: Month and year appeared to account for the greatest variability in grapevine foliar phenolic or amino acid levels, followed by differences in rootstock, and then differences in infection status. GLRaV-3 infection significantly lowered levels of total and individual hydroxycinnamic acid derivatives, and GRBV lowered total phenolic levels, total and individual hydroxycinnamic acids. Amino acid levels were increased over controls in vines infected by GLRaV-3, but not with GRBV. Overall, changes within grapevine leaves due to viral infection were likely too small to overcome variability due to sampling time or rootstock cultivar, and therefore such factors should be considered in determining infection effects on plant foliar chemistry.

Genome-wide screening of novel RT-qPCR reference genes for study of GLRaV-3 infection in wine grapes and refinement of an RNA isolation protocol for grape berries.

BACKGROUND: Grapevine, as an essential fruit crop with high economic values, has been the focus of molecular studies in diverse areas. Two challenges exist in the grapevine research field: (i) the lack of a rapid, user-friendly and effective RNA isolation protocol for mature dark-skinned berries and, (ii) the lack of validated reference genes that are stable for quantification of gene expression across desired experimental conditions. Successful isolation of RNA with sufficient yield and quality is essential for downstream analyses involving nucleic acids. However, ripe berries of dark-skinned grape cultivars are notoriously challenging in RNA isolation due to high contents of polyphenolics, polysaccharides, RNase and water. RESULTS: We have optimized an RNA isolation protocol through modulating two factors at the lysis step that could impact results of RNA isolation - 2-ME concentration and berry mass. By finding the optimal combination among the two factors, our refined protocol was highly effective in isolating total RNA with high yield and quality from whole mature berries of an array of dark-skinned wine grape cultivars. Our protocol takes a much shorter time to complete, is highly effective, and eliminates the requirement for hazardous organic solvents. We have also shown that the resulting RNA preps were suitable for multiple downstream analyses, including the detection of viruses and amplification of grapevine genes using reverse transcription-polymerase chain reaction (RT-PCR), gene expression analysis via quantitative reverse transcription PCR (RT-qPCR), and RNA Sequencing (RNA-Seq). By using RNA-Seq data derived from Cabernet Franc, we have identified seven novel reference gene candidates (CYSP, NDUFS8, YLS8, EIF5A2, Gluc, GDT1, and EF-Hand) with stable expression across two tissue types, three developmental stages and status of infection with grapevine leafroll-associated virus 3 (GLRaV-3). We evaluated the stability of these candidate genes together with two conventional reference genes (actin and NAD5) using geNorm, NormFinder and BestKeeper. We found that the novel reference gene candidates outperformed both actin and NAD5. The three most stable reference genes were CYSP, NDUFS8 and YSL8, whereas actin and NAD5 were among the least stable. We further tested if there would be a difference in RT-qPCR quantification results when the most stable (CYSP) and the least stable (actin and NAD5) genes were used for normalization. We concluded that both actin and NAD5 led to erroneous RT-qPCR results in determining the statistical significance and fold-change values of gene expressional change. CONCLUSIONS: We have formulated a rapid, safe and highly effective protocol for isolating RNA from recalcitrant berry tissue of wine grapes. The resulting RNA is of high quality and suitable for RT-qPCR and RNA-Seq. We have identified and validated a set of novel reference genes based on RNA-Seq dataset. We have shown that these new reference genes are superior over actin and NAD5, two of the conventional reference genes commonly used in early studies.

Retention and Transmission of Grapevine Leafroll-Associated Virus 3 by Pseudococcus calceolariae.

Grapevine leafroll-associated virus 3 (GLRaV-3), an economically significant pathogen of grapevines, is transmitted by Pseudococcus calceolariae, a mealybug commonly found in New Zealand vineyards. To help inform alternative GLRaV-3 control strategies, this study evaluated the three-way interaction between the mealybug, its plant host and the virus. The retention and transmission of GLRaV-3 by P. calceolariae after access to non-Vitis host plants (and a non-GLRaV-3 host) White clover (Trifolium repens L. cv. "Grasslands Huia white clover"), Crimson clover (T. incarnatum), and Nicotiana benthamiana (an alternative GLRaV-3 host) was investigated. For all experiments, P. calceolariae first instars with a 4 or 6 days acquisition access period on GLRaV-3-positive grapevine leaves were used. GLRaV-3 was detected in mealybugs up to 16 days on non-Vitis plant hosts but not after 20 days. GLRaV-3 was retained by second instars (n = 8/45) and exuviae (molted skin, n = 6/6) following a 4 days acquisition period on infected grapevines leaves and an 11 days feeding on non-Vitis plant hosts. Furthermore, GLRaV-3 was transmitted to grapevine (40-60%) by P. calceolariae second instars after access to white clover for up to 11 days; 90% transmission to grapevine was achieved when no alternative host feeding was provided. The 16 days retention period is the longest observed in mealybug vectoring of GLRaV-3. The results suggest that an alternative strategy of using ground-cover plants as a disrupter of virus transmission may be effective if mealybugs settle and continue to feed on them for 20 or more days.

Exploring Grower-sourced Data to Understand Spatiotemporal Trends in the Occurrence of a Vector, Pseudococcus maritimus (Hemiptera: Pseudococcidae) and Improve Grapevine Leafroll Disease Management.

Grapevine leafroll disease is a significant concern in the wine grape industry, as it spreads rapidly and contributes to economically significant reductions in yield and grape quality. Our objective was to utilize 5 yr of grower-sourced data from Napa (California, USA) to improve local and regional disease management efforts. Specifically, we applied a spatially integrated multivariate clustering technique to improve understanding of spatiotemporal trends in Pseudococcus maritimus (Ehrhorn) male populations-the primary vector in the region. We also implemented generalized linear mixed models to evaluate the effects of two key practices, insecticide sprays and roguing, on disease incidence. Results show P. maritimus has a biannual flight pattern in the study area, with the first flight peaking in early May and the second between early August and early September. Clusters of P. maritimus flight data fall largely within the vineyard footprints of individual growers, but also showed clear neighborhood effects. We found that when disease incidence within a block is <1%, consistent monitoring and removal of diseased vines is required to contain within-block spread. As within-block disease incidence grows to 1-20%, both insecticide applications and roguing are effective practices to reduce spread. At incidence levels >20%, roguing is a critical practice. Our results emphasize the importance of individual management efforts, but also the value of programs that engage the wider neighboring community and highlight the power of community data collection to guide decision-making.

Studies on the Occurrence of Viruses in Planting Material of Grapevines in Southwestern Germany.

Viral diseases in viticulture lead to annual losses in the quantity and quality of grape production. Since no direct control measures are available in practice, preventive measures are taken to keep the vines healthy. These include, for example, the testing of propagation material for viruses such as Arabis mosaic virus (ArMV), Grapevine fanleaf virus (GFLV) or Grapevine leafroll-associated virus 1 (GLRaV-1) and 3 (GLRaV-3). As long-term investigations have shown, GLRaV-1 (2.1%) occurs most frequently in southwestern German wine-growing regions, whereas GLRaV-3 (<0.1%) is almost never found. However, tests conducted over 12 years indicate that there is no general decline in virus-infected planting material. Thus, it can be assumed that a spread of the viruses via corresponding vectors still takes place unhindered. Beyond the examinations regulated within the German Wine Growing Ordinance, one-time tests were carried out on Grapevine Pinot gris virus (GPGV). This analysis showed that GPGV was found in 17.2% of the samples.

A Lectin Disrupts Vector Transmission of a Grapevine Ampelovirus.

Grapevine leafroll disease is one of the most important virus diseases of grapevines and occurs in every major grape-growing region of the world. The vector-transmission mechanisms of the causative agent, Grapevine leafroll-associated virus 3 (GLRaV-3), remain poorly understood. We show that the vine mealybug, Planococcus ficus, feeds through a membrane feeding system on GLRaV-3 viral purifications from both V. vinifera and N. benthamiana and transmits the virus to test plants from plants from both species. Building on this strategy, we used an immunofluorescence approach to localize virions to two retention sites in P. ficus mouthparts. Assays testing molecules capable of blocking virus transmission demonstrated that GLRaV-3-transmission by P. ficus could be disrupted. Our results indicate that our membrane feeding system and transmission-blocking assays are a valid approach and can be used to screen other candidate blocking molecules.

Combined drought and virus infection trigger aspects of respiratory metabolism related to grapevine physiological responses.

In the Mediterranean region, grapevines usually deal with drought during their summer growth season. Concurrently, grapevines are hosts to a large number of viruses from which grapevine leafroll associated virus-3 is one of the most widespread and provokes considerable economic losses in many vineyards. However, information concerning grapevine metabolic responses to the combination of drought and viral infection is scarce. Gas-chromatography coupled to mass-spectrometry based metabolite profiling was used in combination with growth analysis, viral loads and gas exchange data to perform an integrative study of the effects of individual and combined stress in two Majorcan grapevine varieties at two experimental years. Metabolic responses of both varieties to the combination of water stress and virus infection were specific and not predicted from the sum of single stress responses. Correlations between respiration, biomass and key metabolites highlight specific adjustments of respiratory and amino acid metabolism possibly underlying the maintenance of carbon balance and growth in grapevines under stress combination.

Development of infectious cDNA clones of Grapevine leafroll-associated virus 3 and analyses of the 5' non-translated region for replication and virion formation.

Infectious cDNA clones were developed for Grapevine leafroll-associated virus 3 (GLRaV-3, genus Ampelovirus, family Closteroviridae). In vitro RNA transcripts generated from cDNA clones showed replication via the production of 3'-coterminal subgenomic (sg) mRNAs in Nicotiana benthamiana protoplasts. The detection of sgRNAs and the recovery of progeny recombinant virions from N. benthamiana leaves agroinfiltrated with full-length cDNA clones confirmed RNA replication and virion formation. The 5' non-translated region (5' NTR) of GLRaV-3 was exchangeable between genetic variants and complement the corresponding cognate RNA functions in trans. Mutational analysis of the 5' NTR in minireplicon cDNA clones showed that the conserved 40 nucleotides at the 5'-terminus were indispensable for replication, compared to downstream variable portion of the 5' NTR. Some of the functional mutations in the 5' NTR were tolerated in full-length cDNA clones and produced sgRNAs and virions in N. benthamiana leaves, whereas other mutations affected replication and virion formation.

Expression of disease resistance in genetically modified grapevines correlates with the contents of viral sequences in the T-DNA and global genome methylation.

Increased tolerance to pathogens is an important goal in conventional and biotechnology-assisted grapevine breeding programs worldwide. Fungal and viral pathogens cause direct losses in berry production, but also affect the quality of the final products. Precision breeding strategies allow the introduction of resistance characters in elite cultivars, although the factors determining the plant's overall performance are not fully characterized. Grapevine plants expressing defense proteins, from fungal or plant origins, or of the coat protein gene of grapevine leafroll-associated virus 3 (GLRaV-3) were generated by Agrobacterium-mediated transformation of somatic embryos and shoot apical meristems. The responses of the transformed lines to pathogen challenges were investigated by biochemical, phytopathological and molecular methods. The expression of a Metarhizium anisopliae chitinase gene delayed pathogenesis and disease progression against the necrotrophic pathogen Botrytis cinerea. Modified lines expressing a Solanum nigrum osmotin-like protein also exhibited slower disease progression, but to a smaller extent. Grapevine lines carrying two hairpin-inducing constructs had lower GLRaV-3 titers when challenged by grafting, although disease symptoms and viral multiplication were detected. The levels of global genome methylation were determined for the genetically engineered lines, and correlation analyses demonstrated the association between higher levels of methylated DNA and larger portions of virus-derived sequences. Resistance expression was also negatively correlated with the contents of introduced viral sequences and genome methylation, indicating that the effectiveness of resistance strategies employing sequences of viral origin is subject to epigenetic regulation in grapevine.

Infection and Colonization of Nicotiana benthamiana by Grapevine leafroll-associated virus 3.

Grapevine leafroll disease is an increasing problem in all grape-growing regions of the world. The most widespread agent of the disease, Grapevine leafroll-associated virus 3 (GLRaV-3), has never been shown to infect species outside of the genus Vitis. Virus transmission to several plant species used as model systems was tested using the vine mealybug, Planococcus ficus. We show that GLRaV-3 is able to infect Nicotiana benthamiana. Working with GLRaV-3 infected N. benthamiana revealed distinct advantages in comparison with its natural host Vitis vinifera, yielding both higher viral protein and virion concentrations in western blot and transmission electron microscopy observations, respectively. Immunogold labelling of thin sections through N. benthamiana petioles revealed filamentous particles in the phloem cells of GLRaV-3 positive plants. Comparison of assembled whole genomes from GLRaV-3 infected V. vinifera vs. N. benthamiana revealed substitutions in the 5' UTR. These results open new avenues and opportunities for GLRaV-3 research.

Interactive effects of grapevine leafroll-associated virus 3 (GLRaV-3) and water stress on the physiology of Vitis vinifera L. cv. Malvasia de Banyalbufar and Giro-Ros.

Among several biotic and abiotic stress combinations, interaction between drought and pathogen is one of the most studied combinations in some crops but still not in grapevine. In the present work, we focused on the interaction effects of biotic (GLRaV-3) and abiotic (drought) stresses on grapevine photosynthetic metabolism on two cultivars (cvs. 'Malvasia de Banyalbufar and Giro-Ros'). Non-infected and GLRaV-3 infected potted plants were compared under water stress conditions (WS) and well-watered (WW) conditions. Under WW condition, the results showed that photosynthesis (AN) in both cultivars was decreased by the presence of GLRaV-3. The stomatal conductance (gs) was the main factor for decreasing AN in Malvasia, meanwhile reductions in Giro-Ros were closely related to decreases in gm. The observed differences in gm between both cultivars might result from variation in their leaf anatomical, Giro-Ros having higher values of gm and leaf porosity (in all treatments). Moderate water deficit resulted in a closure of stomata and a decrease in gm accompanied by a decrease in AN in both cultivars. The maximum velocity of carboxylation (Vcmax) and electron transport rate (Jmax) were also reduced under water stress. Moreover, the combined stress resulted in a reduction of most physiological parameters compared to healthy irrigated plants. However, no considerable differences were found between non-infected and virus infected (GLRaV-3) plants under water stress. Most of the results could be explained by the difference of virus concentration between cultivars and treatments.

Interactions Within Susceptible Hosts Drive Establishment of Genetically Distinct Variants of an Insect-Borne Pathogen.

Coinfections are common, leading to pathogen interactions during transmission and establishment in a host. However, few studies have tested the relative strengths of pathogen interactions in vectors and hosts that determine the outcome of infection. We tested interactions between two genetically distinct variants of the mealybug-transmitted Grapevine leafroll-associated virus 3. The transmission efficiency of each variant in single variant inoculations by two vector species was determined. The effects of vector species, a coinfected source, and simultaneous inoculation from multiple hosts to one host on variant establishment were examined. Within-vector interactions could have a role in transmission from hosts containing mixed infections, but not when vectors were moved from separate singly infected source plants to a single recipient plant. The invasive Planococcus ficus (Signoret) was a more efficient vector than Pseudococcus viburni (Signoret). Transmission efficiency of the two variants did not differ in single variant inoculations. Overall infections were the same whether from singly or coinfected source plants. In mixed inoculations, establishment of one variant was reduced. Mixed inoculations from two singly infected source plants resulted in fewer mixed infections than expected by chance. Therefore, the observed outcome was determined subsequent to host inoculation rather than in the vector. The outcome may be due to resource competition between pathogens. Alternatively apparent competition may be responsible; the pathogens' differential ability to overcome host defenses and colonize the host may determine the final outcome of new infections. Detailed knowledge of interactions between pathogens during transmission and establishment could improve understanding and management of disease spread.