1a. Objectives (from AD-416):
The goal of this Project is to identify tools to reduce losses caused by pathogens and insect pests of grapevine. The Project Plan includes basic and applied research with flexibility for research on new pathogens and insect pests of grape, should the need arise. The overall concept is based on the multi-trophic structure of the Pierce’s disease pathosystem, which includes many host species and pathogen strains, other microorganisms, insect vectors, natural enemies, and a diverse agricultural landscape. Objective 1: Identify and characterize genes involved with pathogenicity of X. fastidiosa. • Subobjective 1A: Examine Xf genomic and phenotypic diversity. • Subobjective 1B: Determine functional activity of Xf toxin-antitoxin (TA) systems. • Subobjective 1C: Elucidate the genetic basis of Xf physiological responses to cold and elimination of Xf from grapevines exposed to cold. • Subobjective 1D: Develop plasmid vector for protein expression/localization and gene complementation. • Subobjective 1E: Develop antivirulence molecules to disrupt functionality of Xf virulence genes. Objective 2: Identify novel plant resistance mechanisms to infection by microorganisms (including X. fastidiosa) and/or feeding by insect vectors. • Subobjective 2A: Identify novel PD resistance genes. • Subobjective 2B: Identify molecular markers of PD resistance in a plant breeding population. • Subobjective 2C: Elucidate plant defense responses to fungal canker, viral, nematode, bacterial infections, and physiological interactions among these pathogens in planta. • Subobjective 2D: Elucidate plant physiological defenses to Xf infection and interaction of Xf with the environment. • Subobjective 2E: Evaluate grapevine germplasm with respect to deterrence of vector probing behaviors, and determine transmission efficiency of Xf by the vector from and to PD-resistant and -susceptible grapevines. Objective 3: Describe the arthropod community in California vineyards and provide new information on the phytobiome of grapevines. • Subobjective 3A: Describe the arthropod community found in and near vineyards. • Subobjective 3B: Determine seasonal changes in the proportion of Xf-inoculative vectors in vineyards. • Subobjective 3C: Assess microbiome variations associated with Xf-infected grapevines and glassy-winged sharpshooter (GWSS). Objective 4: Elucidate reproductive, developmental, and feeding parameters of hemipteran pests of grapevines. • Subobjective 4A: Determine the role of nutrition on GWSS fecundity. • Subobjective 4B: Describe and characterize tremulatory signals used in mating communication of insect pests of grapevine. • Subobjective 4C: Identify vibrational signals that affect GWSS behaviors, and evaluate natural and synthetic signals to disrupt mating communication of GWSS. • Subobjective 4D: Describe and characterize BMSB feeding behaviors on grapevines, and determine the mechanism of damage to the crop.
1b. Approach (from AD-416):
The approach is to synergistically exploit weak links between main components of the Pierce’s disease (PD) pathosystem (pathogen, vector, plant) and insect pests to induce an unstable or neutral interaction that can lead to disruption of destructive processes affecting grape production. Xylella fastidiosa (Xf) diversity will be examined to provide insights on environmental adaptation and host-specific pathogenicity. Xf gene function will be examined to identify genes affecting pathogenicity and virulence. Plasmids will be developed as tools to characterize Xf gene function and expression. Protocols for delivery of antivirulence molecules into grapevines will be evaluated. Grapevine response to infection will be examined to identify molecular and metabolic networks affecting disease severity and resistance. Effects on PD epidemiology due to deployment of grapevines bearing partial resistance to PD will be determined empirically and modeled by computer simulations. Assemblages of arthropods and microorganisms associated with vineyards will be surveyed. Insect vector fecundity will be quantified to identify novel means to suppress vector populations responsible for pathogen spread. Interaction of Xf with diverse pathogens (fungal, viral, nematode) affecting grapevines will be examined. An additional component of the research will focus on new threats to grape production, including but not limited to, invasive insects such as the brown marmorated stink bug.
3. Progress Report:
Under Objective 1, progress was made towards identification and characterization of genes involved with pathogenicity of Xylella fastidiosa to grapevines. To determine functional activity of toxin-antitoxin (TA) systems in Xylella fastidiosa, bacterial mutants in all six identified TA systems have been constructed and preliminary in vitro characterization were completed. The genetic basis of bacterial response to low temperature has been studied by knockout mutation and complementation of a second gene potentially involved in cold stress. Xylella fastidiosa plasmid vectors with various promoters for constitutive and inducible expression were created and validated to facilitate studies on gene expression. Several virulence genes in Xylella fastidiosa were characterized in vitro and phenotypic characterization of mutant and complemented strains of Xylella fastidiosa in planta were initiated. Data from phenotypic characterization of gene functions through in vitro, in planta, and insect hosts combined have indicated that virulence genes associated with signal regulation and twitching motility can be potentially targeted by anti-virulence molecules as a therapeutic approach for controlling Pierce’s disease of grapevine. Under Objective 2, progress was made towards identification of novel grapevine genes that confer resistance to Pierce’s disease. Time-course RNA expression libraries from resistant and susceptible grapevine selections were constructed. In greenhouse experiments, Pierce’s disease-resistant grapevines were identified from a population and plant samples representative of Pierce’s disease resistance and susceptibility in responses to various stages of Xylella fastidiosa infection have been collected and sequenced. Differential RNA expression profiles are currently being analyzed to identify genetic loci that account for Pierce’s disease resistance. Progress was made on identification of novel molecular markers for Pierce’s disease resistance in a plant breeding population. Specifically, screening for disease resistant individuals from populations referred to as “South East United States” (SEUS) were completed and screening for resistance in hybridized progeny was initiated. The best genotypes representing Pierce’s disease-resistant progeny were selected and sequenced using the genotyping by sequencing method. Currently, genotyping data are being analyzed to develop a linkage association map that will facilitate identification of sequence loci linked to Pierce’s disease resistance in SEUS germplasm. Progress was made characterizing grapevine physiological responses to multiple pathogens including bacterial (Xylella fastidiosa), fungal (Neofusicoccum parvum, Diplodia seriata, and Phaeomoniella chlamydospora), viral (Grapevine leaf roll associated virus-3), and nematode (ring and root knot nematodes). Interactions between pathogens in planta were explored with grapevines infected in tandem with the pathogens listed above. Briefly, data have shown that prior infections with the fungus, Diplodia seriata, significantly reduced subsequent infections by other fungal pathogens via increased host phenolic levels that occur after D. seriata infection. In addition, data obtained so far on host physiological responses suggest the possibility for development of novel early detection methods based on spectral characteristics that could be captured through remote sensing technology. Progress was made on evaluation of grapevine germplasm with respect to deterrence of vector probing behaviors. Feeding behaviors of insect vectors carrying Xylella fastidiosa were recorded via electropenetrography (EPG) on four Vitis vinifera genotypes that were recently shown to exhibit a spectrum of resistance to mechanically inoculated Xylella fastidiosa. Genotypes under investigation include ‘Y129-161’ (highly susceptible to Pierce’s disease), ‘Chardonnay’ (moderately susceptible), ’12-6507-56’ (tolerant), and ‘Norris’ (resistant). All insects tested via EPG fed on all four genotypes, but to highly variable degrees. Under Objective 3, processing of samples collected to describe the arthropod community in vineyards in the San Joaquin Valley is complete and results have been analyzed. Monitoring of glassy-winged sharpshooter populations and grapevines chronically infected with Xylella fastidiosa in the southern San Joaquin Valley is complete. About 1,400 glassy-winged sharpshooters and 600 grapevine samples were screened via quantitative polymerase chain reaction for Xylella fastidiosa. Under Objective 4, experiments to compare egg maturation of glassy-winged sharpshooters reared on a single host plant species versus multiple host plant species have been completed. In total, 228 glassy-winged sharpshooters were reared on different diets and dissected to determine effects of diet on adult size and egg production. Recordings of blue-green sharpshooter vibrational signals used in mating communication are ongoing. The signaling activity of about 50 males and 50 females in pairs or individually on plants were recorded using laser vibrometry. As part of a subordinate project, individual black widow spiders have been established in cages in the laboratory and grape bunches in a vineyard to 1) study vibrational signals used in territorial rivalry and 2) evaluate spider senses in relation to anthropomorphic noises such as tractors and insecticide spraying equipment. In another subordinate project, methods for evaluating vine mealybug resistance to the insecticide imidacloprid are being refined. Because imidacloprid is a systemic insecticide, analytical chemistry methods are being refined to confirm that imidacloprid concentration in leaf tissues are being manipulated as expected based on treatment. Adult Brown Marmorated Stink Bug (BMSB) movement on grapevines and berries were video-recorded to determine on which plant part the insects fed, but unexpectedly, there was virtually no feeding despite 24 hours of starvation prior to initiation of laboratory assays. Subsequent monitoring of adult stink bugs via electropenetrography (EPG) supported the same finding. In contrast, stink bug nymphs were found to feed on grapevine leaves and stems after 24 hours of starvation. Categorization of nymphal waveforms generated via EPG recordings began in collaboration with scientists in Belgium.
1. Individual, field-collected glassy-winged sharpshooter vectors harbor mixed infections of two Xylella fastidiosa subspecies. Distinct subspecies of the invasive plant pathogenic bacterium Xylella fastidiosa evolved in geographic isolation from one another but now occur together due to inadvertent movement of infected plant material through international commerce. ARS researchers in Parlier, California, monitored incidence and subspecies identity of X. fastidiosa present in field-collected glassy-winged sharpshooter vectors associated with an outbreak of Pierce’s disease of grapevines in California. Individual glassy-winged sharpshooters often harbored DNA sequences of two X. fastidiosa subspecies: multiplex (native to North America) and fastidiosa (introduced to North America from Central America circa 1880). These results indicate that co-colonized glassy-winged sharpshooters may serve as the arena where genetic material is exchanged among X. fastidiosa subspecies, thereby increasing the risk of emergence of new, recombinant strains of the pathogen with increased virulence and/or expanded host range that adversely affect health and productivity of perennial crops of economic significance.
2. Late summer is an important period for the spread of Xylella fastidiosa in vineyards by glassy-winged sharpshooter. Xylella (X.) fastidiosa is the causal agent of Pierce’s disease of grapevine, which is lethal to grapevines and has caused significant losses to California viticulture, particularly in areas where the glassy-winged sharpshooter vector is present. Knowledge of vector and pathogen population dynamics can improve efficiency of disease management strategies by revealing critical periods for pathogen spread, but information on seasonal abundance of naturally infected glassy-winged sharpshooter in vineyards was not available. ARS researchers in Parlier, California, conducted a three-year field study that determined that glassy-winged sharpshooters were most abundant in vineyards in late summer and more likely to be carrying X. fastidiosa during late summer than in other periods. Results will aid in developing management recommendations designed to target late season populations of glassy-winged sharpshooters in vineyards. Results indicate that new strategies are needed for managing glassy-winged sharpshooter populations present in late summer.
3. Whole genome sequence of Xylella fastidiosa strain ATCC 35879 and evidence for genome rearrangement within X. fastidiosa subspecies fastidiosa. Xylella (X.) fastidiosa is a bacterial pathogen causing Pierce’s disease of grapevine and many economically important plant diseases worldwide. This bacterial species was first described using the strain ATCC 35879 from Florida, which is used worldwide as the species representative (the type species) for further taxonomic arrangements into species and subspecies groups based on sequences of ribosomal RNA genes and 16S-23S rDNA. Despite multinational efforts to characterize X. fastidiosa strains based on genomic analysis, the complete whole genome of strain ATCC 35879 has not been sequenced. ARS scientists in Parlier, California, sequenced the whole genome of the type strain using two technologies: 1) Illumina MiSeq for short but high-quality sequence reads and 2) PacBio for longer sequence reads that, combined, reliably revealed the bacterial genome structure. Further comparison of strain ATCC 35879 with another strain belonging to the same subspecies group (strain M23 from California) showed significant genome structure variations characterized by differences in the order in which genes are placed within each individual genome. Research findings represent the first discovery of genome structure variation within members of the subspecies fastidiosa and provide a complete and accurate reference of the type species genome for future taxonomic studies.
4. Quantitative assay to detect and differentiate strains of Xylella fastidiosa in plant and insect tissue. The bacterial pathogen Xylella (X.) fastidiosa can cause disease in a wide variety of host plants, but disease severity in a given host plant species depends on which strain of the pathogen is present. Because different X. fastidiosa strains may co-exist in an individual host plant, two separate laboratory assays are needed for strain quantification and identification. ARS scientists in Parlier, California, developed a quantitative test to differentiate multiple types of X. fastidiosa that are found in California based on unique DNA sequences. The one-step process developed in this work has facilitated research designed to determine which pathogen strains are present in plants and insect vectors within almond and grape growing regions of California, and to identify other crop species at risk of infection.
5. Prophage diversity in “Candidatus Liberibacter asiaticus” strains in California. Huanglongbing (HLB) is a highly destructive citrus disease threatening the citrus industry in the U.S. and worldwide. The causal agent of the disease, “Candidatus Liberibacter asiaticus” (CLas), has been recently detected in southern California. However, little is known about the origin of this exotic pathogen and whether reported findings are a result of multiple separate introductions or spread from a single introduction event. ARS scientists in Parlier, California, performed research on CLas diversity that could be used to improve efficiency of the HLB control program. Using the next generation sequencing (Illumina MiSeq and HiSeq) approach, CLas strains from California were found to have four prophage typing groups from different geographical locations. This information improved current understanding of CLas prophage biology and established a novel strain typing system useful for the identification of routes of pathogen invasion and formulation of new HLB management strategies, particularly in California.
6. Lygus lineolaris fed less efficiently on transgenic resistant cotton than on non-transgenic cotton. More than 50 percent of insect-related yield loss to cotton bolls is caused by feeding of Lygus bug species, making them the most economically important pests of cotton in the U.S. One approach to identify and evaluate cotton plant resistance to lygus damage is to quantify insect feeding behaviors related to host plant acceptance and use. ARS scientists in Parlier, California, identified the mechanism of resistance of transgenic cotton to L. lineolaris feeding by comparing via AC-DC electropenetrography the suitability of a susceptible genotype (non-transgenic) with a transgenic genotype expressing a protein from Bacillus thuringiensis (Bt). More frequent plant cell rupturing behaviors, longer tasting and plant testing behaviors, and shorter but more frequent swallowing of the resulting slurry, occurred on pin-head squares of transgenic cotton than non-transgenic cotton. Thus, the insects had to work harder to obtain less food from the transgenic plants, supporting that the transgenic cotton was less palatable and/or digestible to L. lineolaris nymphs than non-transgenic cotton. Data from this study were used by collaborators for registration and marketing of a new Lygus-resistant cotton plant, which is expected to reduce usage of insecticides in cotton production.
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