EPIDEMIOLOGY AND MANAGEMENT OF PIERCE'S DISEASE AND OTHER MALADIES OF GRAPE

Pierce�s disease (PD) is an economically important disease affecting US grape production. PD is caused by the xylem-limited bacterium Xylella fastidiosa (Xf), a generalist pathogen also causing disease in numerous horticultural crops and landscape ornamentals. In California, prevalence and incidence of PD increased following introduction and establishment of the glassy-winged sharpshooter (GWSS), Homalodisca vitripennis. Presently, PD is managed in California via an area-wide surveillance and insecticide application program aimed at suppressing GWSS populations in citrus (the most common feeding/oviposition host) and urban landscape plants. Development of an integrated management program for PD requires detailed knowledge of host-pathogen-vector-environment interactions. Such knowledge is limited for this complex pathosystem involving multiple hosts and vectors, a genetically diverse pathogen, and a non-uniform agro- ecosystem. The project objectives are designed to address knowledge gaps in the biology, ecology, and genetics of Xf, sharpshooter vectors, and host plants. Genomic diversity, as well as the evolutionary, biological and epidemiological relationships among Xf strains will be characterized. The genetic and molecular basis of host-pathogen-vector interactions will be determined to better understand PD development and epidemiology. Biotic and abiotic factors that affect sharpshooter ecology and pathogen transmission mechanisms/efficiency will be identified. The nature, basis, and mechanism(s) of host resistance to Xf will be identified, characterized, and incorporated into advanced grape selections. New information and products will facilitate mitigation of PD losses, with the ultimate goal of developing an environmentally friendly, integrated management strategy that may augment or replace the area-wide surveillance and insecticide application program. This is the final report for project 2034-22000-010-00D, which expired in April 2017 and has been replaced by new project 2034-22000-012-00D, "Identification of Novel Management Strategies for Key Pests and Pathogens of Grapevine with Emphasis on the Xylella Fastidiosa Pathosystem." For additional information see the new project report. Substantial accomplishments were obtained from research objectives identified to address knowledge gaps in the biology, ecology, and genetics of Xylella fastidiosa (Xf), sharpshooter vectors, and host plants. Under Objective 1, genomic diversity, as well as the evolutionary, biological, and epidemiological relationships among Xf strains were characterized. Genetic and biological properties of Xf strains recovered from olive revealed that the California olive strains were closely related to endemic North American strains causing almond leaf scorch disease but not Pierce's disease (PD) of grapevine. Although laboratory assays indicated that Xf infection does not cause disease in olive, olive may serve as a source of Xf for insect vectors. Research on Xf plasmids indicated that gene flow has occurred between Xf subspecies, formerly isolated from one another, such that appearance of new strains via genetic exchange must be considered in future studies on Xf genomic diversity, bioinformatics, and development of disease management strategies. Genome sequences obtained from Xf strains originating from California, Texas, Florida, Georgia, and Taiwan facilitated basic research on pathogen host range and genome variation. Evaluation of the Taiwanese strain resulted in the identification and description of X. taiwanensis, a new species in the genus Xylella. These genome sequence data combined with data being collected in the current project plan will provide a defined and standardized taxonomy of Xylella. The first procedure to study small RNAs (sRNAs) in Xf was developed and used for identification of over 20 candidate sRNA genes with potential roles in Xf gene regulation. Because disruption of such sRNAs may lead to reduced virulence in plants, the approach has been considered for identification of novel targets for biological manipulation. Under Objective 2, host-pathogen-environment interactions were studied by functional analysis of pathogen genes to better understand PD development. Genes MqsR-YgiT were identified as important for Xf biofilm formation, an essential part of PD development, suggesting that blocking MqsR activity may represent a new target for reducing virulence and/or spread of Xf. Functional study of other similar genes (toxin-antitoxin systems) in Xf virulence is an objective of the current project. Eight Xf- secreted proteins that increased bacterial virulence also were identified as potential targets for pathogen control. Xf cold shock protein 1 (Csp1) was found to be important for plant infection and bacterial survival under cold conditions. Csp1 functions as an RNA-binding protein that is produced independent of growth temperature. When studying the Xf genes PilG and popP, deliberate mutations followed by pathogenicity assays in grapevines reduced bacterial virulence when compared to a wild type of Xf. Because popP is a key virulence factor in Xf, these data are being used in the current project to expedite design of a target gene-based therapeutic approach for disease control. Determination of the function of Xf genes required development of a method to generate stable genetically modified cells. In this project, development of DNA vectors greatly simplified the process for generating mutant cells for current research aimed at increasing understanding of Xf virulence. By providing a better understanding of how Xf causes disease in plants and withstands unfavorable environmental conditions, this research identified new molecular targets for disarming Xf virulence. Regarding grapevine physiology, as it relates to disease development, defense-related chemical compounds produced by different scion � rootstock combinations of grapevines in response to pathogen infections were identified and quantified. Analysis revealed that disease symptoms developed faster on varieties that exhibited stronger response to Xf infection, whereas early senescence, likely linked to reduced fruit quality, were associated with changes in plant chemistry triggered by Grapevine red blotch associated virus. Under Objective 3, biotic and abiotic factors that affect insect vector fitness, behaviors, and pathogen transmission mechanisms were identified. Research demonstrated that 1) Glassy-winged Sharpshooter (GWSS) feeding behaviors related to transmission of Xf were less frequent on host plants exposed to either cold temperatures or deficit irrigation, compared with controls, and 2) plant water stress negatively affected GWSS abundance. Because environmental conditions vary greatly among grape growing regions, results support the use of regional climate modelling and application of regulated deficit irrigation regimens to reduce incidence of diseases caused by Xf. Research on mechanisms of Xf transmission showed that Xf is injected into grapevine xylem cells by a vector �spitting up� bacterial cells loosened from binding sites on the walls of the vector�s mouth cavity, likely by enzymatic activity of saliva swished around in the mouth and spit out. Research also showed that beta 1, 4 glucanase, the enzyme required to loosen bacterial cells, is indeed �spit up� into xylem cells during vector feeding. This knowledge may be used to develop grapevines resistant to the vector�s transmission behavior. A protocol to estimate egg maturation rates of GWSS was developed and used to determine that egg production is affected by lipid availability and that certain nutritional components of xylem sap correlated with egg production. Knowledge of GWSS nutritional requirements are facilitating current studies designed to elucidate effects of nutrition on GWSS fecundity, which in the future may enable identification and breeding of crop cultivars with suboptimal xylem sap chemistries that do not support GWSS population growth. Natural enemies of GWSS are known to use volatile chemical compounds as cues to locate plants infested with GWSS eggs. Exploitation of such compounds could enhance effectiveness of biological control programs for GWSS, but the identity of compounds was not available. Two compounds released by GWSS-infested grapevines were identified and shown to be attractive to the primary natural enemy of GWSS. Under Objective 4, a spatially-explicit simulation model was developed to evaluate factors affecting successful implementation of a remove-and- replant disease control strategy. Model simulations indicated efficient tree removal and coordinated action among farms suppressed pathogen spread with few trees removed. This model forms the basis of a new model being constructed in the current project for evaluating spread of Xf in an agroecosystem consisting of resistant and susceptible plants. Models on the spread of insect-transmitted plant pathogens often ignore the role of vector reproduction and mortality. A new model analysis indicated that identification of factors that prevent eruptive vector population growth is critical for reducing disease incidence. Efforts to breed PD-resistant grapevines succeeded in combining high fruit quality from domesticated grapes with PD resistance from Vitis arizonica, a non-domesticated relative. Numerous selections are currently in production trials to identify vines capable of sustained production and storage ability. A screening method was developed to compare Xf motility and colony development in xylem sap from PD-resistant and -susceptible grapevines. Significant reductions in Xf aggregation and motility were observed in sap from PD-resistant compared to PD-susceptible grapevines, suggesting that a rapid screening method may facilitate identification of traits responsible for PD resistance. Altogether, the incorporated resistance will allow grape production in areas where PD incidence is recurrent. Concurrent research showed that grapevine scions grafted onto rootstocks �101-MG�, �Freedom�, and �Salt Creek� developed less severe PD symptoms than scions grafted onto other rootstocks, suggesting that certain rootstocks could increase the likelihood of Xf disinfection by pruning or winter-curing. Research on almond rootstock improvement in California made new rootstocks available, including several peach � almond (PEAL) hybrids that combine desirable traits such as tree anchorage and resistance to soil pathogens. While a previously developed lineage of a PEAL rootstock was resistant to Xf, in another PEAL hybrid lineage the bacteria grew to high population densities, indicating differences in the genetic basis of resistance to Xf. In a five-year field study, �Nemaguard�, �Okinawa�, �Nonpareil�, and Y119 (a PEAL hybrid) were used to determine rootstock effects on incidence and severity of almond leaf scorch disease (caused by Xf). Only �Nemaguard� promoted complete pathogen elimination and remission of leaf scorching symptoms, indicating that a Xf-resistant trait in the rootstock can be valuable for maintaining low incidence of disease in California and that understanding the degree of susceptibility to Xf in complex hybrids should be an important part of rootstock development. Accomplishments 01 Genome sequence analysis of Xylella fastidiosa strains including description of a new species. Integration of population genetic studies and epidemiology can provide important insights into the sources and patterns of pathogen spread. However, strain and species differentiation of Xylella fastidiosa, a bacterial pathogen causing diseases in many crops such as grape, in relation to disease epidemics are not fully understood. ARS researchers in Parlier, California, in collaboration with researchers at National Chung Hsing University and Taiwan Agricultural Research Institute in Taichung, Taiwan, used a whole genome sequence analysis technique that resulted in the characterization and description of a new species in the genus Xylella, X. taiwanensis. In addition, the draft genome sequence of an Xf strain causing blueberry bacterial leaf scorch in Georgia was acquired and archived in the GenBank data repository. A defined and standardized taxonomy of Xylella based on DNA sequences may lead to discovery of genes that contribute to disease development under different environmental conditions and host plant associations. 02 Xylella fastidiosa toxin-antitoxin system DinJ/RelE suppresses bacterial proliferation and virulence in grapevine affected by Pierce�s disease. Pierce�s disease progression is characterized by widespread distribution of Xylella fastidiosa (Xf) cells within sap-transporting vessels, leading to scorching symptoms and eventual grapevine decline and death. Excessive blockage of plant vessels by bacterial aggregations does not appear to be advantageous to the pathogen; Xf has genetic mechanisms to regulate population levels conducive to survival and spread in plants, but many of which are still poorly understood. ARS researchers in Parlier, California, identified DinJ (antitoxin protein) and RelE (toxin) as genetic factors likely to be involved in regulation of Xf growth. Disease symptoms and pathogen populations progressed more rapidly in grapevines inoculated with an Xf mutant strain deficient in the DinJ/RelE toxin-antitoxin system than in grapevines inoculated with wild type Xf, resulting in premature plant death. Knowledge of genetic factors controlling pathogen growth in the plant host will lead to identification of novel control targets. 03 Elucidating grapevine physiological responses to Xylella fastidiosa and Grapevine red blotch associated virus. Plants respond to pathogen infection by producing defense-related chemical compounds, but pathogen- grapevine interactions leading to changes in plant chemical profile are poorly understood. ARS researchers in Parlier and Davis, California, characterized and quantified defense-related phenolic compounds produced by grapevines in response to Xylella fastidiosa and Grapevine red blotch associated virus infection, the causal agents of Pierce�s disease and Red Blotch disease of grapevines, respectively. Contrary to expectation, Pierce�s disease symptoms developed faster and Red Blotch disease symptoms were more severe on varieties that produced more plant defense compounds. Characterization of grapevine physiological processes altered by viral and bacterial infection may aid in identification of disease-resistant plants to reduce the impact of pathogens for the grape production industry. 04 Developed a method to estimate energy reserves available to live glassy- winged sharpshooters. ARS researchers in Parlier, California, validated a body-mass index for live glassy-winged sharpshooters. Females with a high body-mass index were shown to mature more eggs over a six-day feeding period than females with a low body-mass index. This method will allow researchers to control for variation in energy reserves available to females during egg maturation experiments, thereby providing a more accurate estimate of the response of females to experimental conditions. Knowledge of the reproductive biology of the glassy-winged sharpshooter is needed to predict population dynamics of this insect vector, an invasive species capable of transmitting the pathogen Xylella fastidiosa to grapevine. 05 Elucidating the molecular mechanism underlying thermotherapy of huanglongbing (HLB)-affected citrus. Huanglongbing (HLB), also known as Citrus Greening disease because fruit tends to turn green after ripening, poses a serious threat that the citrus industry. Thermotherapy has been proposed as a practice to mitigate the impact of HLB under field conditions, but little is known about how heat affects the bacterial pathogen associated with the disease. ARS researchers in Parlier, California, analyzed the protein profile of HLB-affected trees exposed to heat treatments and discovered that certain heat-induced proteins in HLB-affected citrus play an active role in suppressing bacterial growth and reversing disease progression processes. Identification of genes responsible for production of such proteins may lead to breeding of disease-resistant plants to reduce the impact of the disease for the citrus production industry. 06 Post-harvest storage conditions and plant physiological characteristics that limit progression of potato zebra chip disease. Zebra chip disease, putatively caused by �Candidatus Liberibacter solanacearum� (CLso), is a major threat to potato production in North America and elsewhere. Long-term approaches for production of marketable potato tubers rely on understanding of factors that limit disease symptom development, but little is known about post-harvest practices and plant physiological characteristics that can limit disease symptom progression. ARS researchers in Parlier, California and collaborators at the University of Idaho, University of California-Riverside, and the Mexican National Institute for Agriculture and Forest Research (INIFAP) reported reduced symptom development when late-season CLso-infected tubers were kept in storage at 6 or 9 degrees Celsius versus 3 degrees Celsius. Also, various Mexican-bred potato cultivars exhibiting tolerance to CLso were observed to have reduced host changes in chemistry upon infection by CLso when compared to susceptible cultivars, with the end result being tubers that remained marketable. Results from these studies support the potential use of higher storage temperatures or tolerant cultivars to mitigate the impact of pathogen infections on tuber quality.