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:
FY13 research focused on Xylella fastidiosa (Xf), the bacterium responsible for Pierce’s disease (PD) of grapes, and sharpshooter insects that transmit Xf. The research was performed in the context of four objectives dealing with genetic analyses of Xf and grapes, Xf-host plant interactions, sharpshooter ecology/biology/transmission, and PD resistance/control. Progress on Objective 1 (Characterize genomic and biological diversity of Xf strains) included genome sequencing of Xf strains from red oak and muscadine grapes. Comparison of these two Xf genomes with previously sequenced strains is ongoing. Greenhouse experiments designed to evaluate the potential of Xf strains from olive to cause PD were initiated. Genome sequencing of Xf strains from olive has commenced. Progress on Objective 2 (Elucidate molecular interactions of Xf with horticultural and model plant hosts that result in disease susceptibility or resistance) focused on i) Xf genes associated with virulence/pathogenicity, and ii) genetic differences among grapevines that are resistant or susceptible to PD. Gene structure/function studies were conducted with two toxin-antitoxin systems that regulate Xf growth and biofilm formation. Xf proteins of unknown function were evaluated as potential virulence factors in plants using a transient expression system. A seedling population derived from a cross between PD-susceptible and PD-resistant grapes was evaluated for differences in gene expression. Resulting profiles were compared to identify expression patterns specific to PD-resistance. Gene sequences specific to PD-resistant seedlings were used to design DNA-based assays for marker-assisted selection. Progress on Objective 3 (Determine biological/ecological/transmission properties of Xf vectors) focused on sharpshooters. A blue green sharpshooter colony was established to use as a model system for Electrical Penetration Graph (EPG) monitoring of feeding in relationship to Xf inoculation. Preliminary EPG assays identified waveforms representing key behaviors. A protocol to estimate egg maturation rates of individual glassy-winged sharpshooter (GWSS) females was developed. Experiments to determine the effects of plant sap chemistry on GWSS egg maturation rates were initiated. Two compounds released by GWSS-infested grapevines were shown to be attractive to a GWSS egg parasitoid. Chemicals emitted by red-tip photinia, an alternative GWSS host, also are being analyzed to identify parasitoid attractants. Progress on Objective 4 (Develop and assess strategies to manage diseases caused by Xf) focused on breeding and cultural control. Crosses were performed in both table grapes and raisins to combine fruit quality with PD resistance. Additional crosses combined PD resistance with powdery mildew (PM) resistance; resistant seedlings were transplanted to the field. A new PD-evaluation field plot was established in Monte Alto, TX with 13 advanced table grape selections. Rootstock effects on susceptibility of scions to PD were evaluated. PD-susceptible scions grafted onto rootstock ‘101-14MG’ were more tolerant to Xf infection than when grafted onto other rootstocks.
1. Pierce’s disease-resistant table grapes are on the way. Grapevine productivity decreases and vines typically die after infection with Xylella fastidiosa, the bacteria responsible for Pierce’s disease of grapes. ARS scientists at Parlier, California, have succeeded in combining high fruit quality from domesticated grapes with Pierce’s disease resistance from Vitis arizonica, a non-domesticated relative. Fruit from resulting hybrids of successive backcrosses now resemble table grapes that consumers are accustomed to seeing in supermarkets. Numerous selections are currently in production trials to identify Pierce’s disease-resistant vines capable of sustained production with high fruit quality and storage ability. The incorporated resistance will allow table grape production in areas where Pierce’s disease incidence is recurrent.
2. Genetic diversity of Xylella fastidiosa causing Pierce’s disease. Advanced grape selections bearing resistance to Pierce’s disease are being field tested in Texas but are intended for deployment in California. Therefore, relationships among X. fastidiosa strains causing Pierce’s disease in Texas and California need to be defined. ARS scientists at Parlier, California, determined that X. fastidiosa strains from California and Texas are highly similar, albeit not identical. These results suggest recent gene flow among bacterial populations resident in the two regions and that Pierce’s disease challenge of grape selections in Texas is suitable for evaluation of resistant material intended for deployment in California.
3. Molecular characterization of Xylella fastidiosa toxin-antitoxin systems. Bacteria frequently utilize toxin-antitoxin systems to regulate growth, often in response to specific environmental cues. ARS scientists in Parlier, California, characterized two toxin-antitoxin systems that regulate growth and/or affect biofilm formation of the bacterium (X. fastidiosa) causing Pierce’s disease of grape. Toxins MqsR and RelE were shown to be ribonucleases that degrade RNA; YgiT and DinJ were shown to be antitoxins that bind to and inhibit toxin ribonuclease activity. Although both toxins reduced bacterial growth in liquid culture, only MqsR toxin stimulated biofilm formation. As biofilm formation is a critical attribute of X. fastidiosa with respect to disease induction and transmission by insects, MqsR was identified as a target for reducing virulence and/or insect transmission of X. fastidiosa to grapevines.
4. Genome sequences of two Xylella fastidiosa strains determined. Xylella fastidiosa infects a wide variety of perennial crops and landscape plants. Previous genome analyses indicate X. fastidiosa exists as multiple subspecies that vary in host range. ARS scientists at Parlier, California, have determined genome sequences for two southeast U.S. strains of X. fastidiosa isolated from red oak or muscadine grapes. Bioinformatic analyses indicated that the red oak strain belonged to subspecies multiplex, whereas the muscadine grape strain was identified as subspecies fastidiosa. Availability of the two genome sequences will facilitate basic research on pathogen host range and genome variation.
5. Complete sequence of a conjugative plasmid of Xylella fastidiosa. Bioinformatics analyses indicate that Xylella fastidiosa exists as multiple subspecies that, until recently, were geographically isolated from one another. In the past 130 years, exotic X. fastidiosa subspecies have been introduced to North America. ARS scientists at Parlier, California, isolated and sequenced a large plasmid encoding genes controlling exchange of genetic material. Nearly identical plasmids were identified in strains of subspecies multiplex (native to the U.S.) and subspecies fastidiosa (introduced to the U.S. circa 1880). These results indicate that gene flow has occurred between subspecies formerly isolated from one another, such that appearance of new X. fastidiosa strains (via genetic exchange among subspecies) must be considered in development of robust management strategies designed to minimize economic impact of diseases caused by X. fastidiosa.
6. Plant water stress effects on Pierce’s disease epidemiology. In California, models predict reduced water reservoir carryover storage and increased groundwater pumping. Consequently, strategies to improve water-use efficiency and sustainability in numerous perennial crop systems are necessary. How water deficit will affect incidence and severity of Pierce’s disease of grapevine is unknown. ARS researchers at Parlier, California, determined that water stress reduced insect vector transmission of the causal agent (Xylella fastidiosa) of Pierce’s disease. These results suggest that application of water-savings strategies to vineyards may reduce incidence of Pierce’s disease.
7. Host-finding behavior of glassy-winged sharpshooter (GWSS) egg parasitoids. Parasitoids use chemical volatile compounds as cues to locate plants infested with GWSS eggs. However, little information is available on the identity of such compounds. ARS researchers at Parlier, California, identified two compounds released by GWSS-infested grapevines that are attractive to Gonatocerus ashmeadi, the primary egg parasitoid of GWSS in the United States. The volatile compounds identified may be used to enhance effectiveness of biocontrol programs for GWSS.
8. A protocol to estimate glassy-winged sharpshooter (GWSS) egg maturation rates. Reproductive capacity is a key parameter of pest population growth that, for GWSS, is poorly understood. ARS scientists at Parlier, California, developed and tested a protocol that measures GWSS egg maturation rate. Development of this protocol will enable additional studies designed to understand the effects of nutrition on GWSS egg maturation. Accordingly, identification of factors affecting egg maturation rate will aid in predicting GWSS population growth, thereby contributing to more efficient pest population control in perennial crops infested with GWSS.
Voegel, T.M., Doddapaneni, H., Cheng, D., Lin, H., Stenger, D.C., Kirkpatrick, B., Roper, C. 2013. Identification of a response regulator involved in surface attachment, cell-cell aggregation, exopolysaccharide production and virulence in the plant pathogen, Xylella fastidiosa. Molecular Plant Pathology. 14:256-264.