1a. Objectives (from AD-416):
The overall goal of this project is to mitigate losses due to Xylella fastidiosa-caused diseases during crop production and to develop effective, sustainable disease and insect-vector management strategies by characterizing host-pathogen-vector-environment interactions of these complex pathosystems. Specific objectives for the project are outlined below: Objective 1: Characterize genomic and biological diversity of Xylella fastidiosa strains. Objective 2: Elucidate molecular interactions of Xylella fastidiosa with horticultural and model plant hosts that result in disease susceptibility or resistance. Objective 3: Determine biological/ecological/transmission properties of Xylella fastidiosa vectors. Objective 4: Develop and assess strategies to manage diseases caused by Xylella fastidiosa.
1b. Approach (from AD-416):
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.
3. Progress Report:
This new project was implemented in April, 2012 and replaces former project 5302-22000-008-00D entitled “Epidemiology and Management of Xylella fastidiosa (Xf) and other Exotic and Invasive Diseases and Insect Pests”. The new project plan focuses on continuation of basic and applied research on Pierce’s disease of grapevine and other vascular diseases of fruit, nut, and landscape plants caused by Xf. Production of additional backcross generations to combine high quality table and raisin grapes with Pierce's disease (PD) resistance is continuing. Eleven crosses to combine PD resistance with high quality table and raisin grapes were made. The crosses consisted of 44,187 pollinations. In addition, 15 crosses to combine powdery mildew (PM) and PD resistance were made, consisting of 47,347 pollinations. An example of increased fruit quality is this year’s selection of 6 table and 11 raisin grape selections propagated into preliminary stage production trials. The 12 grape selections planted to advanced field trails at Weslaco, TX were inoculated with Xf and will be sampled after 13 months. DNA sequence analyses were conducted using publicly available sequences of V. vinifera (‘Pinot Noir’) and other grape genomic sequences to identify genetic regions containing simple sequence repeats and putative resistance gene analogs. These genetic regions were assembled into a chromosome-based data set for marker-assisted mapping. Composition of xylem sap from 6 grapevine cultivars inoculated with Xf was assessed at 2, 4, and 6 months post-inoculation. Results indicate initial induction of multiple host defense-associated phenolic compounds ceased as Pierce's disease symptoms became severe. Grape xylem sap was collected from PD-resistant grape species/hybrids (V. arizonica, V. shuttleworthii, V. candicans and V. arizonica hybrid #67) and PD-susceptible grapes (V. astetivalis, V. chardonnay, and V. arizonica hybrid #94) using a pressure chamber. In vitro bioassays to evaluate the effect of xylem sap source on Xf growth are in progress. Protocols for quantification of Xf in glassy-winged sharpshooter (GWSS) vectors have been developed and validated. New qPCR primers to green fluorescent proten (GFP) allow discrimination of Xf (marked with GFP) that was introduced via artificial diet versus unmarked Xf, acquired naturally. Two olive groves in Fresno County within the GWSS infected zone were identified as potential sites to determine GWSS population dynamics in olive. Sampling to evaluate GWSS activity in olives was initiated. Preliminary experiments to correlate GWSS egg maturation rates with compounds found in xylem sap were initiated. Experimental protocols for estimating GWSS egg maturation rates are underway. Similarly, methods for extracting and analyzing amino acid and carbohydrate profiles of xylem sap are being refined. A spatially-explicit simulation model was developed to evaluate factors affecting successful implementation of a rogue-and-replace disease control strategy in a perennial cropping system, with particular emphasis on Pierce’s disease of grapevines.
1. Modeling roguing as a disease control strategy. Parameters effecting disease control via removal and replacement of infected plants are poorly understood for large-scale, perennial cropping systems. ARS scientists at Parlier, California developed a spatially-explicit simulation model to evaluate factors affecting successful implementation of a remove-and-replant disease control strategy. Simulations indicated efficient tree removal and coordinated action among farms suppressed pathogen spread with few trees removed. This resulted in high yields. In contrast, with low rates of infected tree removal and/or low compliance among farms, large numbers of trees were replaced with little yield benefit. The model impacts efforts to control plant diseases by providing insight into effective cultural control of plant disease on an agro-ecosystem scale that would otherwise be impractical to determine empirically.
2. Detection of small RNAs (sRNAs) in Xylella fastidiosa (Xf). Non-coding sRNAs are regarded as ubiquitous regulatory elements in bacteria, yet knowledge of sRNAs in X. fastidiosa does not exist. ARS scientists at Parlier, California identified over 20 candidate sRNA genes from whole genome sequence analyses and developed a new technique to verify the presence of sRNAs in bacterial samples. Analyses of a selected number of sRNA genes showed their potential roles in bacterial gene regulation. Disruption of Xf sRNAs represents a potentially novel means of disease control by altering expression levels or timing of Xf genes required for pathogenicity and/or virulence.