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

2020 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
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, whole transcriptome RNA libraries were created for bacterial mutants in all six identified TA systems under multiple growth conditions. The genetic basis of bacterial response to low temperature has been studied by in vitro characterization of knockout mutants in cold responsive genes and plant virulence assays. Xylella fastidiosa plasmid vectors designed for expression of tagged proteins were constructed to expand studies of gene function. A library of anti-virulence molecules was constructed for functional analysis of virulence genes. In vitro and in planta assays suggested virulence genes, pilG and popP, are good targets for phenotypic disruption. A target gene-based transcriptional inhibition assay was designed, and more than 2,000 putative anti-virulence compounds were screened. Seven anti-virulence compounds were identified as candidates for mitigating pathogenicity of Xylella fastidiosa. Under Objective 2, progress was made towards identification of grapevine resistance to pathogen infection and feeding by insect vectors. Transcriptome analysis of differential expression profiles of Pierce’s disease-resistant and -susceptible genotypes revealed several pathways involved in defense responses to Xylella fastidiosa infection including jasmonic acid (JA) and salicylic acid (SA)-mediated signaling pathways, and genes coded for biosynthesis of secondary metabolites such as phytoalexins, chitinases, and protease inhibitors. A list of candidate genes identified in Pierce’s disease-resistant grapevines will facilitate development of host resistance against disease. Screening of hybrid grapevines from crosses of Pierce’s disease-resistant and -susceptible genotypes was completed. A total of 94 progenies with 47 from resistant and 47 from susceptible genotypes and parent grapevines were subjected to genotyping by sequencing. Genome-wide association analysis identified 122 Single Nucleotide Polymorphism (SNP) markers linked to Pierce’s disease resistant traits. Molecular markers developed facilitated marker-assisted selection for Pierce’s disease-resistant breeding lines. Grapevine physiological responses to 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) pathogens were characterized. A database of grapevine physiological responses to infection by these pathogens is under preparation as a resource for remote, plant physiological-based detection of infection. Briefly, grapevines infected previously with a fungal pathogen exhibited physiological changes that were fungistatic to subsequent infections, albeit the second infection ultimately was able to successfully establish. Methods were developed to trigger water stress responses in grapevines grown in the greenhouse for physiological experiments. In a preliminary study, Pierce’s disease symptoms were observed to progress faster in grapevines under water stress conditions than those that were well-watered. For electropenetrography (EPG) studies, analysis began from two years of feeding behavior data for blue-green sharpshooter on Vitis vinifera genotypes that exhibit a spectrum of resistance to mechanically inoculated Xylella fastidiosa. Behaviors are being analyzed in conjunction with quantitative polymerase chain reaction (qPCR) to identify plant traits (or genotypes) that can reduce inoculation of Xylella fastidiosa. Progress was also made on evaluation of grapevine germplasm with respect to transmission of Xylella fastidiosa by the glassy-winged sharpshooter. Pathogen transmission from susceptible to resistant genotypes, and vice-versa, were completed in greenhouse experiments. Plant and insect samples are being tested by qPCR for presence of Xylella fastidiosa to evaluate differences in pathogen transmission efficiencies between treatments. Progress was made in the elucidation of glassy-winged sharpshooter feeding behaviors associated with transmission of Xylella fastidiosa. An X-ray synchrotron was used to video record movements of food pump muscles in sharpshooter heads while feeding was being recorded via EPG. Results have shown that there are two functional groups of muscles that control fluid uptake, and either egestion or swallowing of plant xylem sap. An early part of the EPG X wave represents small undulations of the lower muscle group to take up sips of fluid while a later X wave part represents large, repeated jerks of both muscles to pull gulps of fluid into the food pump. Results include association of food pump muscle movements with behaviors that control inoculation of X. fastidiosa, which can be used in grapevine breeding programs to identify novel traits for resistance to Pierce’s disease. Under Objective 3, significant progress was made using a next generation sequencing (NGS) approach to study Xylella spp. and their insect vectors, and “Candidatus Liberibacter asiaticus” (CLas) associated with citrus Huanglongbing (HLB). NGS data have been collected from Xylella fastidiosa-infected grapevine, pear, pecan, citrus and associated insect vectors from California, Taiwan, and Brazil. NGS data were also collected from CLas-infected citrus and Asian citrus psyllids (ACPs) from California, Brazil, and China. Metagenomic and genomic analyses have been progressing rapidly. For Xylella fastidiosa, the major focus has been on metagenome of pecan bacterial leaf scorch disease in Georgia. Two subspecies of Xylella fastidiosa were detected in pecan and their draft genome were acquired. Analyses of the metagenome of a Xylella fastidiosa insect vector (Kolla paulula) from Taiwan led to the discovery of a new bacterial endosymbiont (an organism that lives within another organism). For citrus HLB samples (both citrus and ACP), in addition to CLas, several bacteria in different phylogenetic groups were identified. The microbiome information resolved an issue of PCR detection accuracy when CLas titer was low (high threshold cycle (Ct) values). Some members of the microbiomes of grapevine, pecan, and citrus could serve as candidates for future biocontrol research. Under Objective 4, nymphs of the brown marmorated stink bug (BMSB) were acquired from ARS scientists in Wapato, Washington. Initial recordings of BMSB nymphal feeding were conducted at various amplifier sensitivity levels in EPG studies, but data are not sufficient to complete a waveform library. Progress was made towards description and characterization of tremulatory signals used in mating communication of the variegated leafhopper, Erasmoneura variabilis. Analysis of vibrational communication signals revealed that: 1) one female signal and two distinct male signals are used in pair formation, 2) the pair formation process is divided into three communication phases, 3) pre-copulatory communication is longer in the presence of male-male rivalry, and 4) Erasmoneura variabilis and other grapevine leafhoppers coexisting in vineyards sing at night and during the day. Results include detailed descriptions of leafhopper communication signals that are relevant for development of vibrational disruption as a novel method to suppress populations under field conditions.

Accomplishments
1. DNA sequence resources for bacterial plant pathogens of economic importance. The pathogens Xylella fastidiosa and ‘Candidatus Liberibacter spp.’ have significant impacts on crop production worldwide. DNA sequences from these pathogens can be used for detection, surveillance, and disease tracking. ARS scientists in Parlier, California, Byron, Georgia, and collaborators in Brazil, used next-generation sequencing technology to create DNA sequence resources for Xylella fastidiosa strains associated with olive in California and pecan in Georgia, and to fully sequence for the first time a ‘Candidatus Liberibacter asiaticus’ strain from Brazil. This new DNA sequence information is valuable for development of new disease management and diagnostic strategies, and for pathogen detection and quarantine applications.

2. Identification of cultural and biological control methods for insect vectors of grapevine red blotch virus. The threecornered alfalfa hopper was recently shown to be a vector of grapevine red blotch virus, an economically important pathogen of wine grapes. There are no pest management recommendations targeting the vector. Risk of threecornered alfalfa hoppers establishing in vineyards may be reduced by planting cover crops that are not hosts for the pest and by releasing commercially available biological control agents that consume threecornered alfalfa hoppers. ARS researchers in Parlier, California, determined that orchard grass, creeping red fescue, fawn tall fescue, hard fescue, and California poppy did not support adult threecornered alfalfa hopper survival or reproduction and determined that green lacewing larvae readily consumed all juvenile life stages of the threecornered alfalfa hopper. Results can be used for grape growers when selecting cover crop species and biological control agents that may aid in reducing threecornered alfalfa hopper populations in vineyards.

3. Evaluation of blueberry as a host for Xylella fastidiosa in California. Xylella fastidiosa (Xf) is a bacterium that causes disease on numerous crops. Xf strains commonly found in California can infect a number of crops including grapevine and almond. Although strains of X. fastidiosa found in the southeastern United States also cause significant disease in highbush blueberry, risk to blueberry had not been assessed for the strains present in California. ARS scientists in Parlier, California, evaluated susceptibility of southern highbush blueberry cultivars to X. fastidiosa strains isolated locally, as well as the ability of glassy-winged sharpshooter to transmit the pathogen between blueberry plants and grapevine. This study identified blueberry cultivar ‘Emerald’ to be more susceptible to California strains of X. fastidiosa than four other cultivars tested and found that glassy-winged sharpshooter can acquire X. fastidiosa from infected blueberry plants. This information is essential for understanding X. fastidiosa epidemiology in California, and in evaluating disease risk for expansion of highbush blueberry acreage in areas where this pathogen is problematic.

4. Functional characterization of virulence genes of Xylella fastidiosa affecting its transmission by the glassy-winged sharpshooter. Xylella fastidiosa, the causal agent of Pierce’s disease of grapevine, is known to be transmitted between grapevines by the glassy-winged sharpshooter. While extensive information is available on X. fastidiosa-grapevine interactions at the biological and molecular levels, nothing is known about X. fastidiosa-vector interactions, especially as it pertains to transmission efficiency of the pathogen. ARS researchers in Parlier, California, created several virulence gene mutants of X. fastidiosa to investigate the role of virulence genes affecting feeding and transmission efficiency by the glassy-winged sharpshooter. Results indicated that mutations altered transmission processes including initial adhesion, multiplication, and long-term retention of X. fastidiosa in insect mouthparts in comparison to a wild type strain of X. fastidiosa. This study provided new information important for understanding X. fastidiosa-vector interactions during transmission process, which facilitate identification of key target genes that may serve as a gene-based control method to mitigate pathogen spread in vineyards.

5. Identification of microorganisms associated with insect vectors of plant pathogens. Kolla paulula and Diaphorina citri are major insect vectors of plant pathogenic bacteria of international concern (Xylella fastidiosa and ‘Candidatus Liberibacter spp.’, respectively). Microorganisms associated with insect vectors can impact pathogen transmission and insect survival and growth. ARS scientists in Parlier, California, developed a computational protocol to detect and identify microorganisms associated with Kolla paulula in Taiwan, and Diaphorina citri and diseased citrus trees in California, based on DNA sequences. This approach identified a symbiotic bacterial species associated with Kolla paulula, and several bacteria associated with diseased citrus. This information will facilitate research on biological control strategies for bacterial plant diseases and associated insect vectors.

6. Development of plasmids for genetic studies of pathogenic bacteria. Research on the functional biology of pathogens relies on molecular biology tools such as plasmids (modified circular DNA molecules). The vast majority of plasmids constructed for research are designed for use in model systems or human biology applications and are not effective in the diverse range of pathogenic bacteria relevant to agriculture. ARS scientists in Parlier, California, developed a set of plasmids that are stable in environmental systems and can be used to study gene functions in a wide range of pathogenic bacteria including Xylella fastidiosa, a slow-growing bacterium that is difficult to manipulate in the laboratory. These plasmids are important tools for research development aimed at understanding pathogen dynamics in environmental and agricultural systems and have been requested by researchers in the United States, Spain, France, Japan, and India.

U.S. DEPARTMENT OF AGRICULTURE

Crop Diseases, Pests and Genetics Research: Parlier, CA