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

2018 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 examining Xylella fastidiosa genomic and phenotypic diversity through next generation sequencing (NGS) and cell membrane fatty acid profiling approaches, respectively. A single copy plasmid with two toxin-antitoxin systems was discovered and plasmid variation was detected between Xylella fastidiosa grown in vitro versus in planta. In addition, NGS analysis revealed that two different subspecies of Xylella fastidiosa are associated with pecan leaf scorch disease in Georgia. Eight distinct isolates of Xylella fastidiosa were analyzed by profiling the fatty acid membrane composition to allow bacterial characterization at the subspecies, strain, and host plant association levels. Progress was made towards the identification and characterization of genes involved in pathogenicity of Xylella fastidiosa in grapevines. Xylella fastidiosa knockout-mutant strains in several toxin-antitoxin gene systems were evaluated for growth, survival, and altered gene expression patterns under varied nutrient conditions in the laboratory. In greenhouse experiments, four Xylella fastidiosa toxin-antitoxin knockout mutants were inoculated into grapevines and are currently being evaluated for effect on disease progression. To elucidate Xylella fastidiosa responses to low temperatures, a knockout mutant was created in the pnp gene, which encodes an RNA-modifying enzyme important for Xylella fastidiosa temperature response. Pnp protein is being analyzed for enzyme activity. Plasmid vectors were constructed and validated for use in gene studies that require expression control in Xylella fastidiosa. Also under Objective 1, comparative genome sequence analyses identified several key virulence genes in Xylella fastidiosa. Knockout mutations were created for genes involved in Xylella fastidiosa twitching motility, biofilm formation, and signal regulation. These genes are being considered as candidate targets for development of anti-virulence therapeutic tools for diseases caused by Xylella fastidiosa. Under Objective 2, progress was made towards identifying and tracking disease resistant traits in breeding grapevine populations. A new backcross hybrid population between Vitis arizonica (Pierce’s disease-resistant) and Vitis romanetii (Powdery mildew-resistant) was created as an attempt to combine the two resistance traits into one grapevine accession. Molecular screening for resistance in hybrids identified 40 individuals that carry powdery mildew-resistance genes. While molecular screening of these hybrids for resistance to Pierce’s disease is still in progress, greenhouse pathogenicity assays and molecular screening of individuals from another population (SEUS FL183) identified 12 Pierce’s disease-resistant progeny that could be used in future Vitis spp. hybridizations. From a physiological perspective, progress has been made towards understanding shared and divergent grapevine responses to infections by different bacterial, fungal, viral, and nematode pathogens. Specifically, grapevines were inoculated with Xylella fastidiosa, Neofusicoccum parvum, Grapevine leaf roll associated virus-3, or root knot nematode. Infected grapevines are currently being evaluated by chromatography to describe alterations in chemical composition. Infection of grapevines by pathogens in tandem (i.e., one pathogen followed by another) were initiated to determine whether and how host plant physiological changes induced by one pathogen affects plant response to another pathogen. Feeding behaviors of insect vectors carrying Xylella fastidiosa were compared on Vitis vinifera ‘Chardonnay’ (Pierce’s disease-susceptible) versus Vitis arizonica (Pierce’s disease-resistant) to determine whether Vitis arizonica also confers resistance to insect feeding behaviors responsible for inoculation of Xylella fastidiosa. Preliminary analysis of electropenetrography data showed that insects performed less salivation and egestion in Vitis arizonica when compared to Vitis vinifera, suggesting that Xylella fastidiosa inoculation events are reduced in Vitis arizonica when compared to Vitis vinifera. Under Objective 3, progress was made towards describing the arthropod community in California vineyards. About 240,000 specimens were collected from vineyards and adjacent crops such as almonds, olives, alfalfa, and pastures in the California Central Valley. Specimens were identified to the class or family level. A Pierce’s disease epidemic occurring near Arvin, California, continues to be monitored by testing of insect and plant samples for presence of Xylella fastidiosa. Samples have been collected from vineyards and citrus orchards every three weeks to determine time of year that insect vectors are most likely to acquire and inoculate Xylella fastidiosa. To date, more than 1,000 insect vectors were collected and subjected to quantitative polymerase chain reaction (PCR) to detect Xylella fastidiosa. Multi-Locus Sequencing Typing (MLST) of Xylella fastidiosa present in field-collected insect vectors revealed most Xylella fastidiosa-positive insects harbored mixed infections of subspecies fastidiosa (causing Pierce’s disease) and subspecies multiplex (causing disease of other perennial crops and landscape plants but not Pierce’s disease). This observation is ecologically important, as mixed infections in insect vectors likely represent the arena where Xylella fastidiosa subspecies exchange genetic material via transformation and homologous recombination. Such genetic exchange poses a risk for the emergence of new pathotypes with altered virulence and/or host range. In addition, samples from grapevines chronically infected with Xylella fastidiosa were collected to describe seasonal changes in Xylella fastidiosa population levels that may affect vector acquisition. Preliminary analyses indicate that population levels of Xylella fastidiosa in chronically infected grapevines were highest in late summer, the period when the greatest percentage of insect vectors collected from vineyards tested positive for presence of Xylella fastidiosa. To assess microbiome variations associated with Xylella fastidiosa-infected grapevines and insect vectors, a metagenomic study based on next generation sequencing technology was initiated with the vectors glassy-winged sharpshooter and Kolla paulula, and Xylella fastidiosa-infected grapevines generated in greenhouse. Under Objective 4, progress was made towards identification and characterization of mating communication signals used in intraspecific communication of the variegated leafhopper. Vibrational communication signals were recorded from about 150 trials conducted with males and females alone on the plant and in groups of two or more individuals per plant. Spectral and temporal features of communication signals are being analyzed to describe the mating behavior and to identify potential weak links in the insect’s communication system that may be exploited for development of control methods. A colony of brown marmorated stink bugs was established to describe and characterize feeding behaviors on grape clusters. Preliminary video recordings were made for stink bugs on grape clusters, and will be continued in the next year when larger numbers of insects will be available.

Accomplishments
1. Mating disruption of glassy-winged sharpshooter by playback of vibrational signals through vineyard trellis. Glassy-winged sharpshooter (GWSS) is an important vector of the bacterium Xylella fastidiosa, the causal agent of Pierce’s disease of grapevine. GWSS communicate by exchanging mating calls that are transmitted through host plants as vibrational signals. Interference with GWSS communication by playback of disruptive signals should lead to reduced population growth, but research was needed to evaluate efficacy of a novel vibrational signal playback method in disrupting GWSS mating under field conditions. ARS scientists in Parlier, California, showed that playback of vibrational signals through vineyard trellis significantly reduced mating of GWSS on grapevines compared to control. Although further studies are needed prior to method implementation, data from this study continue to support application of vibrational mating disruption as a novel method to control GWSS populations.

2. Identification of a new plasmid and a plasmid transfer system in Xylella fastidiosa. The plant pathogenic bacterium, Xylella fastidiosa, can transfer large circular pieces of DNA (plasmids) to cells of different strains. This type of DNA transfer mechanism, known as conjugation, is an important mechanism controlling the spread of antibiotic resistance and virulence-associated genes in bacterial pathogens. ARS scientists in Parlier, California, used next generation sequencing technology to detect and describe a new plasmid that occurred as a single copy per bacterial cell and encoded 27 genes, including four genes forming two toxin-antitoxin systems. In another study, a set of genes responsible for transfer of plasmids between different strains of Xylella fastidiosa was identified. Findings from this study are important for understanding how DNA is transferred between pathogen strains and is expected to improve DNA-based pathogen identification and diagnostics.

3. Predicting the outcome of deploying a cultivar of a perennial crop that is partially resistant to an insect-transmitted plant pathogen. Cultivars that are partially resistant to a pathogen have lower pathogen population levels than susceptible cultivars, but remain hosts for the pathogen. While plant breeders aim to develop cultivars that are immune to the pathogen, often the only available resistance traits confer partial resistance. ARS researchers in Parlier, California, developed a mathematical model that determined the conditions under which deployment of a partially resistant cultivar will not exacerbate disease problems in susceptible cultivars planted in proximity. The model indicates that plant breeders and industry regulators may evaluate the risk of deploying a partially resistant cultivar by quantifying the rate at which insect vectors acquire the pathogen from the partially resistant cultivar and by determining the extent to which pathogen spread is suppressed among susceptible cultivars.

4. Different electropenetrograph settings, adhesives, and electrical signals affect feeding behaviors and waveforms for blue-green sharpshooters and lygus bugs. The blue-green sharpshooter and lygus bugs are economically important insect pests of grapevines and cotton, respectively. Modern methods to study insect feeding in real time, such as electropenetrography (EPG), can aid development of insect-resistant plants, but the most recent design of electropenetrograph has not been calibrated for use with these insect pests. ARS scientists in Parlier, California, determined the best methods and equipment settings for monitoring sharpshooter and lygus bug feeding behaviors by comparing various wiring methods, adhesive types, applied voltage levels, and amplifier sensitivities. The use of handmade silver glue produced the most detailed waveforms, low voltages of alternating current (AC) minimized behavioral abnormalities, and direct current (DC) voltages negatively affected feeding at all amplifier settings and any voltage level above two millivolts. These findings indicate that EPG settings and methods tailored to each species will allow better understanding of insect feeding, which can lead to identification of plant traits that confer resistance to specific damaging feeding behaviors.

5. Nutritional resources required for glassy-winged sharpshooter egg production are acquired via adult feeding during the first week after adult emergence. The glassy-winged sharpshooter (GWSS) is a vector of Xylella fastidiosa, a pathogen lethal to grapevine and the causal agent of Pierce's Disease. Describing patterns of resource acquisition affecting GWSS reproduction could aid in predicting population growth, but the role of nymphal and adult diet on GWSS egg production was poorly understood. ARS scientists in Parlier, California, determined that nymphal diet affected development time and adult size, but did not affect timing of egg production. Results indicate that quality of the adult diet can override nutritional deficiencies during immature stages. Results provide insight into GWSS population growth, a requirement for developing environmentally friendly strategies for suppressing GWSS populations.

6. Discovery of a Type 3 prophage of “Candidatus Liberibacter asiaticus” carrying a restriction-modification system. “Candidatus Liberibacter asiaticus” is a bacterial pathogen associated with citrus Huanglongbing (HLB), a highly destructive disease threatening citrus industries worldwide. Manipulation of bacterial viruses such as Type 3 prophages could be exploited as a novel approach to reduce the impact of HLB in the citrus industry, but little is known about phages in “Candidatus Liberibacter asiaticus”. ARS scientists in Parlier, California, studying bacterial viruses of “Candidatus Liberibacter asiaticus” discovered and described a new phage, called P-JXGC-3. Through a next generation sequencing approach, analyses of 523 DNA samples collected from HLB-affected trees revealed that the phage carried a unique bacterial defense system, called restriction-modification system, which could be responsible for enhancing pathogen survival under field conditions. The new phage information facilitates understanding of phage biology of the HLB pathogen that will help formulation of new HLB management strategies.

U.S. DEPARTMENT OF AGRICULTURE

Crop Diseases, Pests and Genetics Research: Parlier, CA