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

2021 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 using a genomic/metagenomic approach to study Xylella species and associated insect vectors. Next generation sequencing (NGS) data were collected from Xylella-infected grapevines in California, pear in Taiwan, peach and plum in Georgia, citrus in Brazil, and insect vectors in California. The mitochondrial genome (mitogenome) sequence of the insect vector Kolla paulula was characterized using NGS approaches. The characterized mitogenome of Kolla paulula was 20,299 base pairs long and included 13 different proteins, two ribosomal RNA genes, and 22 transfer RNA genes. The mitogenome sequence provides important information about insect phylogeny and taxonomy and facilitates global efforts on Pierce’s disease research and control. Homologs of restriction-modification systems, potentially regulating genetic exchange and with demonstrated biological activity, were identified in 118 X. fastidiosa strains. The presence of many restriction-modification systems is limited to specific phylogenetic clades, although two are broadly conserved among X. fastidiosa strains and one is conserved in both X. fastidiosa and X. taiwanensis strains. The epigenome of 12 X. fastidiosa strains from three different subspecies was determined using third generation sequencing. Analysis revealed 10 distinct epigenetic patterns, including differences between subspecies and within sequence types. Transcriptional gene expression influenced by X. fastidiosa toxin-antitoxin systems was characterized in vitro, as well as virulence characterization of toxin-antitoxin mutant strains in grapevine. Progress was made on the evaluation of X. fastidiosa mutants in cold-responsive genes for survival in grapevines subjected to cold treatment. Under Objective 2, grapevine physiological responses were characterized in response to infections by bacteria (X. fastidiosa), fungi (Diplodia seriata, Neofusicoccum parvum, and Phaeomoniella chlamydospora), virus (Grapevine leafroll-associated virus-3 and Grapevine red blotch-associated virus), and nematodes (ring and root knot nematodes). The biochemical data generated as part of these characterizations were uploaded to USDA National Agricultural Library (NAL) Ag Data Commons. Preliminary results showed that previous infections by one pathogen reduced initial establishment of a second pathogen infection, but ultimately pathogens were able to overcome initial delays in infection progression. Plant water stress treatments combined with X. fastidiosa infections substantially increased the progression of Pierce’s disease symptoms compared to infected well-watered plants, with severe symptoms and premature plant death observed at two weeks after inoculation. Data analysis was continued from three years of electropenetrography (EPG) studies of blue-green sharpshooter (BGSS), Graphocephala atropunctata, feeding on Vitis vinifera genotypes that exhibit a spectrum of resistance to mechanically inoculated X. fastidiosa. Quantitative polymerase chain reaction (qPCR) of research samples and measurement of EPG waveforms are underway. Preliminary statistical analysis showed significant differences between resistant grape genotypes and ‘Chardonnay’ for variables measuring X waves (X. fastidiosa inoculation). Under Objective 3, NGS data were collected from citrus and Asian citrus psyllids infected with “Candidatus Liberibacter asiaticus” (CLas) in California, Brazil, and China. Metagenomic and genomic analyses continues to reveal new information in Xylella- and CLas-related diseases. Under Objective 4, mathematical and simulation models were developed for future evaluation of the effectiveness of combining disease management strategies such as insecticides, mating disruption, and deployment of partially resistant plants. Research methods were developed for evaluating effects of soil-applied insecticides on glassy-winged sharpshooter and vine mealybug fitness. Progress was made towards description and characterization of tremulatory signals used in mating communication of the BGSS. Analysis of vibrational communication signals revealed that: 1) one female signal and at least two distinct male signals are used in pair formation; 2) the pair formation process in BGSS is divided into three communication phases; and 3) male-male and female-female rivalry occurs in BGSS. Results include detailed descriptions of sharpshooter communication signals that are relevant for development of vibrational disruption as a novel method to suppress populations under field conditions.

Accomplishments
1. Identification of a novel type of grapevine resistance to Xylella fastidiosa. Pierce’s disease of grapevine is caused by the lethal bacterium Xylella fastidiosa (Xf), which is transmitted from plant to plant by vectors such as the glassy-winged sharpshooter (GWSS). The bacteria-injection behavior by vectors can be visualized and quantified as a specific waveform (called the X wave) generated when studying GWSS feeding via electropenetrography (EPG). ARS researchers in Parlier, California, used EPG to compare GWSS feeding on Xf-resistant (wild Vitis champinii) versus Xf-susceptible (V. vinifera cv Chardonnay) grapevines. Results showed that insect vectors performed fewer 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 inoculating behaviors. Future research will use EPG to screen grapevines for this novel type of resistance, leading to more durable resistance and less insecticide use in the future.

2. Characterization of grapevine physiological responses associated with infections by fungal and bacterial pathogens. Fungal diseases and Pierce’s disease of grapevines, caused by the bacterium Xylella fastidiosa (Xf), reduce yields over time. Progression of such diseases depends on specific plant-microbe interactions at the metabolite level, but information on the link between plant metabolism and aggressiveness of different pathogens are lacking. ARS researchers in Parlier, California, determined that phenolic compounds affect Xf behavior and biofilm formation, whereas fungal pathogen lesion lengths are positively correlated with phenolic levels and negatively correlated with terpenoid levels. Results suggest that grapevine varieties with lowered stem phenolic levels but greater terpenoid levels will be less affected by fungal canker pathogens and that reduced phenolic levels are linked with grapevine resistance to Pierce’s disease. Understanding of fungal stem pathogen-grapevine host interactions will be useful for identifying new grapevine varieties that provide improved resistance to fungal pathogens.

3. Vibrational communication of grapevine leafhoppers in California. Leafhoppers are a concern for grape growers in California due to direct feeding damage by piercing the leaves, transmission of plant pathogens, or both. Behavioral mating disruption is a viable option to manage leafhopper populations, but nothing was known about the mating communication and calling activity of most leafhopper species in vineyards. ARS researchers in Parlier, California, identified and described vibrational signals associated with mate selection behavior of Erasmoneura variabilis and Erythroneura ziczac, and evaluated the calling activity of these and another species (Homalodisca vitripennis) on grapevines in the field. Studies revealed that: distinct vibrational signals are used in pair formation; the pair formation process is divided in three communication phases; and leafhoppers in field populations sing at night and during the day. Results provide detailed descriptions of leafhopper communication signals that are relevant for future development of vibrational disruption as a novel method to suppress populations under field conditions.

4. DNA sequence resources for bacterial plant pathogens of economic importance. Xylella taiwanensis causes pear leaf scorch disease in Taiwan and X. fastidiosa is an important bacterial pathogen that causes plum leaf scald disease (PLSD) and phony peach disease in the southeastern United States. ARS researchers in Parlier, California, collaborated with scientists in Taiwan and Georgia to study the bacterial genomes using a metagenomic approach and next generation sequencing technology. A draft whole genome sequence of a PLSD strain was obtained, representing the first report of a whole genome sequence of a X. fastidiosa strain from plum. A small plasmid (a mobile genetic element of 2,104 base pairs) was found in X. taiwanensis which shared over 65% similarities to those from X. fastidiosa and other bacterial species. This is the first evidence of potential exchange of genetic material between bacterial species, which enhances research in bacterial virulence development and environmental adaptation.

5. Detection and description of a novel phage in “Candidatus Liberibacter asiaticus” (CLas). Candidatus Liberibacter asiaticus (CLas), a non-culturable alfa-proteobacterium, is associated with citrus Huanglongbing (HLB), a devastating disease threatening citrus production worldwide. ARS researchers in Parlier, California, collaborated with scientists in China to study the CLas phages that play important roles in the bacterial biology. Through the use of next generation sequencing technology, a novel phage was found which had a circular genome of 8,869 base pairs and eight open reading frames including two known genes coding for a replication initiation protein and a major capsid protein. Phylogenetic analysis revealed that the phage CLasMV1 belongs to the Microviridae family. The discovery and characterization of the CLasMV1 phage expanded the knowledge of CLas phages that have direct impact on HLB research and control.

6. Phenolic induction determined to be a common plant response to herbivory and pathogen infection. Plant production of phenolic compounds was hypothesized to be a common plant response to infection and infestation, but conflicting reports persisted to the contrary. ARS researchers in Parlier, California, utilized meta-statistical analyses on over 100 peer-reviewed research articles from 2008 to 2017 to determine whether induction of phenolic compounds was a consistent plant response to beneficial microbes, pathogens, or insects (either macerating or piercing-sucking). Phenolics were discovered to be induced when plants were exposed to all of these with the exception of piercing-sucking insects. These results provide information about plant physiological responses to infections by different pathogens, which relates to efforts to characterize grapevine and other plant infection processes by bacteria, fungi, nematodes, and viruses. Knowledge gained that phenolic induction is a consistent response to pests and microbes impacts future hypotheses about plant health and guides industrial research that utilizes plant chemical signatures for detection of pests and disease.

U.S. DEPARTMENT OF AGRICULTURE

Crop Diseases, Pests and Genetics Research: Parlier, CA