Project : USDA ARS

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Research Project: Identification of Novel Management Strategies for Key Pests and Pathogens of Grapevine with Emphasis on the Xylella Fastidiosa Pathosystem

Location: Crop Diseases, Pests and Genetics Research

2022 Annual Report

Objectives
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.

Approach
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.

Progress Report
This is the final report for project 2034-22000-012-000D, Identification of Novel Management Strategies for Key Pests and Pathogens of Grapevine with Emphasis on the Xylella fastidiosa Pathosystem, which has been replaced by new project 2034-22000-014-000D, Development of Applied Management Systems for Diseases of Perennial Crops with Emphasis on Vector-Borne Pathogens of Grapevine and Citrus. For additional information, see the new project report. In support of Objective 1, genome sequence analyses revealed evolutionary relationships among Xylella fastidiosa (Xf) strains. Complete genome sequences for three Xf strains from olive in California were generated. Further analyses of Xf from different hosts and geographic locations provided insights into Xf adaptation to new habitats and revealed differences among Xf with respect to rates of horizontal transfer of genetic material. Data suggest that different Xf were brought into proximity recently through human activities. Studies targeting Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus Huanglongbing (HLB), improved genome-based detection of CLas from plant material. Next Generation Sequencing technology was used to examine CLas strains and highly variable prophage regions were identified. A prophage-based system was developed to differentiate CLas strains in California. Analysis detected an error in the primer sequence of a commonly used polymerase chain reaction (PCR) method. A corrected primer was proposed, improving CLas detection and classification. Genomic investigations led to discovery of a new CLas prophage. Prophages and plasmids often encode virulence and environmental adaptation factors. A new Type 3 prophage of CLas and a new Xf circular plasmid were discovered. Results add new information to further research in Xf and CLas genome diversity and possible virulence factors. Knowledge obtained from genome data helped development of molecular tools for Xf detection and biology. For example, native Xf plasmids were found to carry genes for conjugative transfer, allowing Xf to obtain new genetic material. Plasmid vectors were engineered for genetic studies in Xf and other bacteria, and a quantitative PCR assay was developed to differentiate Xf subspecies found in California. Results facilitate Xf diagnosis, functional genetic research on Xf virulence determinants, and studies on pathogen-plant-vector interactions. Identification of Xf genes and physiological processes involved in host infection help identify targets for antimicrobial activity. In that regard, Xf growth factors were studied and the bacterial toxin-antitoxin DinJ/RelE system was found to attenuate Xf growth in grapevine. Knockout mutants of this gene system in Xf led to more rapid disease progression and plant death. Identification of disease severity factors facilitate development of resistant plants and screening for highly virulent strains. Results contribute to further identification of other determinants of Xf infection. Similarly, characterization of Xf virulence genes affecting pathogenicity in grapevines and transmission by the insect vector glassy-winged sharpshooter (GWSS) may lead to novel methods to control disease and pathogen transmission. Deliberate mutations in the Xf pilG and popP genes resulted in a remarkable reduction in Xf virulence and showed that PilG plays a key role in orienting Xf movement. Mutations of the pilG, popP and other genes altered transmission processes of Xf by the GWSS including adhesion, multiplication, and retention of Xf in insects, suggesting these as new targets to disarm Xf pathogenicity. In support of Objective 2, efforts to decode host physiological responses to infections by multiple pathogens revealed plant defense-related compounds (e.g., phenolics and primary metabolites) in grapevines infected by fungal cankers, Grapevine red blotch-associated viruses, and root knot nematodes. Physiological changes associated with disease development led to hypotheses about how pathogens affect overall plant health. Knowledge gained improves understanding how infection by one pathogen impacts another and overall plant health, with implications for Integrated Pest Management programs. Further studies focused on identification of novel plant resistance mechanisms to infection by microorganisms revealed the role of phenolic compounds on Xf behaviors and Pierce’s disease (PD) progression. A comparison of healthy and Xf-infected grapevines determined that those with the PdR1 locus had far lower phenolic levels and that phenolics affect Xf virulence. Tolerance to PD was associated with reduced innate grapevine phenolic levels, which may be used as a desirable trait in resistance breeding programs. Electropenetrography (EPG) was used to compare Xf inoculation behaviors (X waves) performed by blue-green sharpshooters (BGSS) on healthy grapevines after prior probing on basil, uninfected or Xf-infected grapevines. Fewest and shortest X waves were performed on basil, most frequent and longest on uninfected grapevines, and intermediate on Xf-infected grapevines. Because effects of prior probing were portable, they were likely caused by blockage of the precibarial valve due to, respectively, absence of microbes, or acquisition of non-Xf microbes or Xf. Thus, blockage was greater for non-Xf microbes from grapevine xylem than Xf. EPG can detect effects of microbial acquisition on vector feeding, increasing uses of EPG for studies of grapevine resistance. In support of Objective 3, studies revealed important aspects of PD epidemiology in the San Joaquin Valley of California. A multiyear field study showed that the abundance and number of Xf-positive GWSS increase in late summer, suggesting that late summer is a crucial time for Xf spread in vineyards. Most Xf-positive GWSS carried the same Xf subspecies, but 25% of GWSS were colonized by two subspecies. Co-colonization suggests GWSS visits multiple host species infected with different Xf strains and can acquire Xf multiple times from different hosts. Additional insect surveys in vineyards, almond orchards, olive orchards, alfalfa fields, and pastures revealed that hemipteran and dipteran insects were the most abundant and diverse orders, respectively. Alfalfa fields had the greatest abundance of insects from these and other insect orders, suggesting that alfalfa may play an important role in the metapopulation dynamics of many insect species. Results indicate the important role of landscape composition on abundance and diversity of insects within crop fields. In support of Objective 4, investigations revealed the effects of GWSS nutrition on population growth. GWSS nymphs are able to feed on hundreds of plant species and can move considerable distances. Studies determined that nymphal diet affects developmental time and adult size, adult feeding increases lipid reserves depending on host plant quality, and increased lipid reserves are linked to greater egg production. Results indicate that habitat diversity and quality play a role in GWSS population growth. A novel control method that interferes with GWSS behavior was developed. Development of pest control methods based on behavioral interference relies heavily on knowledge of mating communication signals that confuse, stimulate, or suppress sender and receiver behaviors. Vibrational communication signals of GWSS were identified, characterized, and candidate disruptive signals were tested via playback to individuals in a vineyard. Playback of the disruptive signal reduced mating of GWSS compared to a silent test group, providing a novel method of GWSS control that is compatible with other pest management approaches.

Accomplishments
1. Glassy-winged sharpshooters avoid plants treated with a soil applied systemic insecticide. The glassy-winged sharpshooter is a vector of the bacterial pathogen Xylella fastidiosa, which causes numerous economically important plant diseases including Pierce’s disease of grapevine. The neonicotinoid insecticide imidacloprid is used to suppress glassy-winged sharpshooter populations. Imidacloprid may be applied via the irrigation system and is taken up by the plant roots and circulated through the plant via the vascular system. ARS researchers in Parlier, California, determined that glassy-winged sharpshooters cease feeding on imidacloprid-treated plants and avoid them, suggesting that application of soil-applied imidacloprid is more likely to push sharpshooters out of treated habitats rather than kill them. Results can be used by growers and regulators to ensure the most effective means of controlling glassy-winged sharpshooter are implemented.

2. Grapevine immune response recognizes Xylella fastidiosa cold shock protein. Bacterial cell components can trigger immune response in plants including production of peroxides. The specific proteins that induce the plant immune response can then be exploited for identifying components of plant disease resistance and for understanding resistant versus susceptible plant-pathogen interactions. ARS researchers in Parlier, California, identified a cold shock protein in Xylella fastidiosa (Xf) that induces production of peroxides in grapevine tissue, indicating recognition by the plant immune response. Removal of this protein from Xf shows that the bacterium is unable to attach to cuticular surfaces without it, which would be essential for binding to insect mouthparts and functional foregut. This information will facilitate development of novel disease management strategies by providing a potential target for disruption of the disease or transmission process.

3. New understanding of plant water deficit effects on development of Pierce’s disease of grapevine. Pierce’s disease is a lethal bacterial disease of grapevines caused by Xylella fastidiosa, which can rapidly spread within vineyards, requiring complete replacement in a matter of years. Water availability is increasingly challenging for grape growers in California and one solution is to periodically reduce irrigation to save water. However, the effects of deficit-regulated irrigation on the development of Pierce’s disease is unclear. ARS scientists in Parlier, California, determined that a month-long period of water stress prior to inoculation of Xylella fastidiosa greatly increased Pierce’s disease development and plant mortality. These findings suggest that the practice of deficit irrigation in grapevine orchards where Pierce's disease is endemic requires increasing other management techniques to control the spread of Xylella fastidiosa.

4. Glassy-winged sharpshooters perform more Xylella fastidiosa-inoculating behaviors on susceptible, rather than resistant grapevines. Grape breeders are developing new varieties that are resistant to Xylella fastidiosa (Xf), the bacterium that causes lethal Pierce’s disease. Sharpshooter vectors inoculate Xf into grapevines during their xylem cell acceptance process, represented by the X wave using electropenetrography (EPG). ARS scientists in Parlier, California, used EPG to compare glassy-winged sharpshooter feeding on Xf-resistant, wild Vitis champinii versus susceptible, cultivated grape ‘Chardonnay.’ Results showed that sharpshooters performed X waves in fewer xylem cells, of overall shorter duration, on V. champinii than on Chardonnay. Thus, EPG can detect a novel type of grapevine resistance to vector inoculation of bacteria. Future research could add this novel type of resistance to bacteria-resistant grapevines for greater durability of resistance.

5. New knowledge about grapevine response to single and multiple pathogen infections. Grapevines encounter many different pathogens simultaneously throughout their lifetimes but understanding of changes in metabolism and how one infection impacts another remains poorly understood. ARS researchers in Parlier, California, infected grapevines with bacterial, fungal, viral, and nematode pathogens to examine the effects of one infection on another. Overall, an initial infection caused changes in the amount and type of plant defense compounds, but these changes only slowed down disease progression of subsequent infections and did not prevent eventual disease. These findings suggest that integrated management programs implemented to control specific grapevine diseases do not need to consider other, previously existing infections to be successful.

6. Genome rearrangements revealed in Xylella fastidiosa strains that infect blueberry. Xylella fastidiosa (Xf) causes major diseases in a wide range of agricultural crops. Different strains of Xf infect different crops, but the genetic components that dictate which plant species are most affected are not well understood, making detection and surveillance difficult. Strains of Xf that infect blueberry are much more diverse than strains found in grapevine. Comparing diverse strains can provide key insights into pathogen genetics impacting disease. ARS scientists in Parlier, California, assembled new high-quality genome sequences of Xf strains AlmaEM3 and BB08-1 from blueberry that show chromosomal rearrangement. This provides valuable information to better understand the genetic basis for association of different Xf strains with different host plants.

7. Genomic technology expands knowledge of Xylella fastidiosa (Xf) and Candidatus Liberibacter asiaticus (CLas) biology. Diseases caused by fastidious prokaryotic pathogens such as Xylella fastidiosa (Xf) and Candidatus Liberibacter asiaticus (CLas) are threatening fruit crop production worldwide. No effective control methods are available for these pathogens. In-depth understanding of the biology of these pathogens is critical, but it is limited due to the difficulty in obtaining in vitro culture. ARS researchers at Parlier, California, used genomic and metagenomic approaches to break barriers associated with inability to grow these organisms in culture media to explore and characterize the genome of Xf and CLas. The new information, along with the new Xf and CLas detection technologies developed, provide valuable resources for researchers, regulatory agencies, and growers for disease management.

U.S. DEPARTMENT OF AGRICULTURE

Crop Diseases, Pests and Genetics Research: Parlier, CA