Optimizing Efficient RNAi-Mediated Control of Hemipteran Pests (Psyllids, Leafhoppers, Whitefly): Modified Pyrimidines in dsRNA Triggers

The advantages from exogenously applied RNAi biopesticides have yet to be realized in through commercialization due to inconsistent activity of the dsRNA trigger, and the activity level of RNAi suppression. This has prompted research on improving delivery methods for applying exogenous dsRNA into plants and insects for the management of pests and pathogens. Another aspect to improve RNAi activity is the incorporation of modified 2′-F pyrimidine nucleotides into the dsRNA trigger. Modified dsRNA incorporating 32–55% of the 2′-F- nucleotides produced improved RNAi activity that increased insect mortality by 12–35% greater than non-modified dsRNA triggers of the same sequence. These results were repeatable across multiple Hemiptera: the Asian citrus psyllid (Diaphorina citri, Liviidae); whitefly (Bemisia tabaci, Aleyroididae); and the glassy-winged sharpshooter (Homalodisca vitripennis, Cicadellidae). Studies using siRNA with modified 2′-F- pyrimidines in mammalian cells show they improved resistance to degradation from nucleases, plus result in greater RNAi activity, due to increase concentrations and improved binding affinity to the mRNA target. Successful RNAi biopesticides of the future will be able to increase RNAi repeatability in the field, by incorporating modifications of the dsRNA, such as 2′-F- pyrimidines, that will improve delivery after applied to fruit trees or crop plants, with increased activity after ingestion by insects. Costs of RNA modification have decreased significantly over the past few years such that biopesticides can now compete on pricing with commercial chemical products.

Improved draft reference genome for the Glassy-winged Sharpshooter (Homalodisca vitripennis), a vector for Pierce's disease

Homalodisca vitripennis (Hemiptera: Cicadellidae), known as the glassy-winged sharpshooter, is a xylem feeding leafhopper and an important agricultural pest as a vector of Xylella fastidiosa, which causes Pierce’s disease in grapes and a variety of other scorch diseases. The current H. vitripennis reference genome from the Baylor College of Medicine's i5k pilot project is a 1.4-Gb assembly with 110,000 scaffolds, which still has significant gaps making identification of genes difficult. To improve on this effort, we used a combination of Oxford Nanopore long-read sequencing technology combined with Illumina sequencing reads to generate a better assembly and first-pass annotation of the whole genome sequence of a wild-caught Californian (Tulare County) individual of H. vitripennis. The improved reference genome assembly for H. vitripennis is 1.93-Gb in length (21,254 scaffolds, N50 = 650 Mb, BUSCO completeness = 94.3%), with 33.06% of the genome masked as repetitive. In total, 108,762 gene models were predicted including 98,296 protein-coding genes and 10,466 tRNA genes. As an additional community resource, we identified 27 orthologous candidate genes of interest for future experimental work including phenotypic marker genes like white. Further, as part of the assembly process, we generated four endosymbiont metagenome-assembled genomes (MAGs), including a high-quality near complete 1.7-Mb Wolbachia sp. genome (1 scaffold, CheckM completeness = 99.4%). The improved genome assembly and annotation for H. vitripennis, curated set of candidate genes, and endosymbiont MAGs will be invaluable resources for future research of H. vitripennis.

Improved draft reference genome for the Glassy-winged Sharpshooter (Homalodisca vitripennis), a vector for Pierce's disease

Homalodisca vitripennis (Hemiptera: Cicadellidae), known as the glassy-winged sharpshooter, is a xylem feeding leafhopper and an important agricultural pest as a vector of Xylella fastidiosa, which causes Pierce's disease in grapes and a variety of other scorch diseases. The current H. vitripennis reference genome from the Baylor College of Medicine's i5k pilot project is a 1.4-Gb assembly with 110,000 scaffolds, which still has significant gaps making identification of genes difficult. To improve on this effort, we used a combination of Oxford Nanopore long-read sequencing technology combined with Illumina sequencing reads to generate a better assembly and first-pass annotation of the whole genome sequence of a wild-caught Californian (Tulare County) individual of H. vitripennis. The improved reference genome assembly for H. vitripennis is 1.93 Gb in length (21,254 scaffolds, N50 = 650 Mb, BUSCO completeness = 94.3%), with 33.06% of the genome masked as repetitive. In total, 108,762 gene models were predicted including 98,296 protein-coding genes and 10,466 tRNA genes. As an additional community resource, we identified 27 orthologous candidate genes of interest for future experimental work including phenotypic marker genes like white. Further, as part of the assembly process, we generated four endosymbiont metagenome-assembled genomes (MAGs), including a high-quality near complete 1.7-Mb Wolbachia sp. genome (1 scaffold, CheckM completeness = 99.4%). The improved genome assembly and annotation for H. vitripennis, curated set of candidate genes, and endosymbiont MAGs will be invaluable resources for future research of H. vitripennis.

Xylella fastidiosa (Pierce's disease of grapevines).

Xylella fastidiosa has a wide plant host range and spectrum of insect species capable of serving as vectors which should increase the bacterium's invasiveness and make it difficult to prevent introduction via live plants from the tropical or subtropical Americas. The spread of phony disease of peach within the south-eastern USA from the 1890s until about 1930 and of citrus variegated chlorosis disease of orange throughout Brazil in the 1990s indicates that new strains of X. fastidiosa have the potential to spread over a few years to cause increasing damage. Intersubspecific homologous recombination of strains is implicated in the potential for invasion of new host plants (Nunney et al., 2014). X. fastidiosa represents a very serious threat for the EPPO region. In 2013 the bacterium was reported causing serious damage to olive trees in Puglia, Italy. It was also detected in numerous other host plants (mainly ornamentals). Colonisation of a host with X. fastidiosa does not always equate to disease development and an endophytic life stage has been suggested (Chatterjee et al., 2008). These non-symptomatic hosts and hosts which are slow to develop symptoms can limit the effectiveness of quarantine procedures and may provide a reservoir for maintenance of the pathogen in the wider environment. According to the EFSA Panel on Plant Health (European Food Safety Authority, 2015), establishment and spread of X. fastidiosa in the EU is very likely. The consequences are considered to be major because yield losses and other damage would be high and require costly control measures. X. fastidiosa is included in the EPPO A1 list of pests recommended for regulation as quarantine pests. Among potential insect vectors, only Homalodisca vitripennis, Xyphon fulgida, Draeculacephala minerva and Graphocephala atropunctata are also listed in the EPPO A1 list. The European Food Safety Authority suggests that all xylem sap feeder insects should be regarded as potential vectors of X. fastidiosa. Elbeaino et al. (2014b) detected X. fastidiosa in the phloem feeder Euscelis lineolatus in Italy, suggesting that potential vectors may include phloem feeding insects. Further studies are needed to confirm transmission in E. lineolatus. Newly introduced isolates of X. fastidiosa are likely to be transmitted by endemic vector species even without the introduction of non-native vectors (Almeida et al., 2005).In summary, X. fastidiosa may have the potential to invade agro-ecosystems in Mediterranean regions wherever suitable vectors (overwintering in the adult stage and thus able to inoculate vines during spring) are endemic or become established. The same may be true for tropical-subtropical Asia and Africa.

Models Applied to Grapevine Pests: A Review

This paper reviews the existing predictive models concerning insects and mites harmful to grapevine. A brief conceptual description is given on the definition of a model and about different types of models: deterministic vs. stochastics, continuous vs. discrete, analytical vs. computer-based, and descriptive vs. data-driven. The main biological aspects of grapevine pests covered by different types of models are phenology, population growth and dynamics, species distribution, and invasion risk. A particular emphasis is put on forecasting epidemics of plant disease agents transmitted by insects with sucking-piercing mouthparts. The most investigated species or groups are the glassy-winged sharpshooter Homalodisca vitripennis (Germar) and other vectors of Xylella fastidiosa subsp. fastidiosa, a bacterium agent of Pierce’s disease; the European grape berry moth, Lobesia botrana (Denis and Schiffermuller); and the leafhopper Scaphoideus titanus Ball, the main vector of phytoplasmas agents of Flavescence dorée. Finally, the present and future of decision-support systems (DSS) in viticulture is discussed.

Control of Pierce’s Disease Through Areawide Management of Glassy-Winged Sharpshooter (Hemiptera: Cicadellidae) and Roguing of Infected Grapevines

The General Beale Pilot Project serves as a case study for the use of areawide monitoring and treatment programs for glassy-winged sharpshooter (GWSS), Homalodisca vitripennis (Germar), and monitoring and roguing programs for grapevines infected with Xylella fastidiosa, to achieve regional management of Pierce’s disease. The Project is located in southeast Kern County, CA, and contains ~2,800 ha of citrus and grapevines grown within approximately 50 km2. For nearly 20 yr, an average of 470 traps have been used to monitor GWSS populations regionally by the California Department of Food and Agriculture, and to inform coordinated, areawide treatments by the USDA-APHIS Areawide Treatment Program to overwintering GWSS in citrus. Grape growers were responsible for treating their own vineyards, and for the roguing of infected grapevines based on surveys provided by the University of California. Herein, we provide a history of the General Beale Pilot Project, broken down into six eras based on levels of Project success, which incorporate data on GWSS captures, pesticide use, and disease incidence. We describe patterns of success related to the regional coordination of effective treatment and roguing programs that can be used by grape and neighboring citrus growers for areawide management of Pierce’s disease. We conclude by describing current and future challenges for Pierce’s disease management, including pesticide availability and resistance, GWSS refuges, the inability to detect and rogue infected vines in the year they become infected, and the sustainability of voluntary programs that rely on public funding.

Xylella fastidiosa and Glassy-Winged Sharpshooter Population Dynamics in the Southern San Joaquin Valley of California

Xylella fastidiosa is a vector-transmitted bacterial plant pathogen that affects a wide array of perennial crops, including grapevines (Pierce’s disease). In the southern San Joaquin Valley of California, epidemics of Pierce’s disease of grapevine were associated with the glassy-winged sharpshooter, Homalodisca vitripennis. During the growing season, rates of X. fastidiosa spread in vineyards are affected by changes in pathogen distribution within chronically infected grapevines and by vector population dynamics. Grapevines chronically infected with X. fastidiosa rarely tested positive for the pathogen prior to July, suggesting vector acquisition of X. fastidiosa from grapevines increases as the season progresses. This hypothesis was supported by an increase in number of X. fastidiosa-positive glassy-winged sharpshooters collected from vineyards during July through September. Analysis of insecticide records indicated that vineyards in the study area were typically treated with a systemic neonicotinoid in spring of each year. As a result, abundance of glassy-winged sharpshooters was typically low in late spring and early summer, with abundance of glassy-winged sharpshooter adults increasing in late June and early July of each year. Collectively, the results suggest that late summer is a crucial time for X. fastidiosa secondary spread in vineyards in the southern San Joaquin Valley, because glassy-winged sharpshooter abundance, number of glassy-winged sharpshooters testing positive for X. fastidiosa, and grapevines with detectable pathogen populations were all greatest during this period.

Targeted Mutations in Xylella fastidiosa Affect Acquisition and Retention by the Glassy-Winged Sharpshooter, Homalodisca vitripennis (Hemiptera: Cicadellidae)

Xylella fastidiosa (Wells) is a xylem-limited bacterium that causes Pierce’s disease of grapevines. The bacterium is transmitted by insect vectors such as the glassy-winged sharpshooter (GWSS), Homalodisca vitripennis (Germar). Experiments were conducted to compare the role of selected X. fastidiosa genes on 1) bacterial acquisition and retention in GWSS foreguts, and 2) transmission to grapevines by GWSS. Bacterial genotypes used were: mutants Xf-ΔpilG, Xf-ΔpilH, Xf-ΔgacA, and Xf-ΔpopP; plus wild type (WT) as control. Results showed that Xf-ΔpilG had enhanced colonization rate and larger numbers in GWSS compared with WT. Yet, Xf-ΔpilG exhibited the same transmission efficiency as WT. The Xf-ΔpilH exhibited poor acquisition and retention. Although initial adhesion, multiplication, and retention of Xf-ΔpilH in GWSS were almost eliminated compared with WT, the mutation did not reduce transmission success in grapevines. Overall, Xf-ΔgacA showed colonization rates and numbers in foreguts similar to WT. The Xf-ΔgacA mutation did not affect initial adhesion, multiplication, and long-term retention compared with WT, and was not significantly diminished in transmission efficiency. In contrast, numbers of Xf-ΔpopP were variable over time, displaying greatest fluctuation from highest to lowest levels. Thus, Xf-ΔpopP had a strong, negative effect on initial adhesion, but adhered and slowly multiplied in the foregut. Again, transmission was not diminished compared to WT. Despite reductions in acquisition and retention by GWSS, transmission efficiency of genotypes to grapevines was not affected. Therefore, in order to stop the spread of X. fastidiosa by GWSS using gene-level targets, complete disruption of bacterial colonization mechanisms is required.

Infection of Blueberry Cultivar ‘Emerald’ with a California Pierce’s Disease Strain of Xylella fastidiosa and Acquisition by Glassy-Winged Sharpshooter

Bacterial leaf scorch disease caused by Xylella fastidiosa occurs in southern highbush blueberry varieties in the southeastern United States. Susceptibility to X. fastidiosa varies by blueberry cultivar, and these interactions are often strain-specific. Xylella fastidiosa subsp. fastidiosa is the causal agent of Pierce’s disease in grapevines, and it has been problematic in the San Joaquin Valley of California since the introduction of the glassy-winged sharpshooter (Homalodisca vitripennis). The glassy-winged sharpshooter is known to feed on blueberry, a crop that is expanding in the San Joaquin Valley. Currently, little is known about the potential for the spread of X. fastidiosa between grape and blueberry in this region. The ability of a Pierce’s disease strain of X. fastidiosa from the San Joaquin Valley to cause disease in southern highbush blueberry and the potential for the glassy-winged sharpshooter to transmit X. fastidiosa between blueberry and grapevine were investigated. Experimental inoculations showed that the X. fastidiosa subsp. fastidiosa strain Bakersfield-1 can cause disease in blueberry cv. Emerald, and that the glassy-winged sharpshooter can acquire X. fastidiosa from artificially inoculated blueberry plants under laboratory conditions. Understanding the possibility for X. fastidiosa strains from the San Joaquin Valley to infect multiple crops grown in proximity is important for area-wide pest and disease management.