2012 Annual Report
1a.Objectives (from AD-416):
The overall goals of this project are to reduce losses due to Xf-caused diseases during crop production and to develop effective, sustainable disease and insect vector management strategies by characterizing the host-pathogen-vector-environment interactions of these complex pathosystems. Specific objectives for the project are outlined below:
Objective 1: Determine the epidemiology of exotic, emerging, re-emerging, and invasive diseases in California, including (but not necessarily limited to) Xf-caused diseases of horticultural, agronomic, and ornamental crops.
Objective 2: Determine the nature and mechanism(s) of susceptibility/resistance to Xf infection and subsequent disease development in horticultural and agronomic crops, including (but not necessarily limited to) Vitis species and Prunus species.
Objective 3: Develop effective, economical, environmentally sound strategies to manage exotic, emerging, re-emerging, and invasive diseases, including (but not necessarily limited to) xylella diseases.
Objective 4: Improve sensitivity and specificity of diagnostics utilizing new biomarkers based on the DNA sequence of the Liberibacter bacterium associated with Huanglongbing (citrus greening) and Zebra chip diseases. (NP 303; Component 1; Problem Statement 1A)
1b.Approach (from AD-416):
Determine the epidemiology of Xylellae diseases in California. Characterize the complex host-pathogen-insect vector-environment interactions of these pathosystems. Determine the biochemical, physiological, genetic and mechanistic bases of resistance to XF infection and subsequent disease development in Vitis and selected Prunus species. Utilize genomic sequences of Liberibacter bacteria associated with Huanglongbing and Zebra Chip diseases to develop new PCR-based assays for pathogen detection.
This is a final report for Project 5302-22000-008-00D (terminated April 12, 2012), which was replaced with new project 5302-22000-010-00D. Research progress for FY12 included breeding for resistance to Xylella fastidiosa (Xf), characterization of Xf-host interactions, ecological and biological characterization of insect vectors transmitting Xf, and genetic analysis of ‘Candidatus Liberibacter’ species associated with Huanglongbing (HLB) and zebra chip (ZC) diseases of citrus and potato, respectively.
Crop improvement efforts included screening of backcross generations of table and raisin grape progeny resistant to Pierce’s disease (PD). Marker assisted selection was used to screen 1,265 seedlings; 505 carried the resistance marker and were planted in the field. PD response of the parents and 38 progeny from V. vinifera x V. arizonica populations was validated in greenhouse tests. Advanced selections (3 table, 7 raisin) were planted in second stage (validation) production trials. Hybrids from a peach (Xf resistant) x almond (Xf susceptible) cross were established in the Parlier, CA research orchard.
Research on Xf-host interactions continued. Evaluation of Arabidopsis thaliana as a model host for Xf indicates that A. thaliana may be a better model to study low-titer asymptomatic infections of weeds than high-titer symptomatic infections of grapevine. Using a plant-virus based expression system, 8 of 57 Xf proteins (of unknown function and predicted to be secreted) were identified as potential virulence factors. Two toxin-antitoxin gene systems encoded by the Xf chromosome were identified. Knock-out mutations of each toxin-antitoxin system altered Xf growth and/or biofilm formation in culture, suggesting a possible role in regulation of Xf virulence.
Transcriptional analysis identified 8 genes in PD resistant grapevines differentially expressed in response to Xf. One of the 8 genes (encoding the known defense response factor jasmonic acid methyl esterase) was cloned from PD resistant grapevines for use in genetic/biochemical analyses. Changes in xylem chemistry associated with Xf infection of susceptible grapevines was evaluated. In PD susceptible grapevines, accumulation of phenolic compounds was elevated in presymptomatic plants but was reduced in older, symptomatic plants.
Research on insects that transmit Xf continued. Preference of female glassy-winged sharpshooters (GWSS) for PD-symptomatic or healthy grapevines was evaluated. In choice-tests, females preferred healthy grapevines; in no-choice tests, females fed more on healthy grapevines. Research on GWSS feeding in relation to Xf transmission determined most Xf cells taken up from artificial diets are rapidly expelled in saliva, rather than bound to the insect’s foregut. These results provide a possible explanation for transmission of Xf immediately following acquisition without bacterial attachment/growth. Volatile compounds that serve as attractants of egg parasitoids to plants infested with GWSS eggs were identified.
Complete genome sequence of a Chinese strain of the Huanglongbing (HLB) pathogen. The American citrus industry is threatened by the invasive and devastating HLB disease associated with infection by the unculturable bacterium ‘Candidatus Liberibacter asiaticus’. ARS scientists at Parlier, California, in collaboration with university scientists, determined and annotated the complete genome sequence of a Chinese strain of ‘Ca. L. asiaticus’. Sequence analysis revealed genomic variation between the Chinese strain and a US Florida strain in both coding and non-coding regions. The availability of genome sequence for two "Ca. L. asiaticus' strains will impact basic science by allowing functional genomic approaches to be used to identify pathogen virulence genes and impact applied science by allowing development of improved DNA-based diagnostic assays.
Impact of almond leaf scorch disease (ALSD) on almond production. ALSD, caused by Xylella fastidiosa (Xf), occurs in California but potential for pathogen spread among almond trees and yield losses due to ALSD are poorly defined. ARS scientists at Parlier, CA, in collaboration with university and industry researchers, evaluated long-term and large-scale epidemiology of ALSD in the San Joaquin Valley of California. A six-year study in two orchards confirmed that there is minimal secondary (tree-to-tree) spread of Xf in almond orchards. A survey of 61 almond orchards over two years determined that ALSD was widespread (e.g., prevalence was high), whereas incidence (percentage of infected trees) within individual orchards was typically low. Impact of the research on almond production results from the recommendation that ALSD trees be replaced only in young orchards (to recover yield) as spread of Xf from infected almonds to healthy almonds is minimal.
DNA fingerprinting of the Huanglongbing (HLB) disease pathogen. Identification of source populations of the invasive bacterium associated with citrus HLB disease requires detailed information on genetic diversity within and among populations of the presumptive pathogen, ‘Candidatus Liberibacter asiaticus’. ARS scientists at Parlier, California collaborated with Chinese scientists to determine genetic structure of ‘Ca. L. asiaticus’ populations in southern China, where HLB was first described. DNA fingerprinting of prophage sequences revealed two distinct populations of ‘Ca L. asiaticus’ resident in low and mid-elevation citrus production areas of China. DNA fingerprint data developed for Chinese populations of 'Ca. L. asiaticus' provides a reference for comparison of a 'Ca. L. asiaticus' strain found in California in 2012.
Arabidopsis thaliana as an experimental host for Xylella fastidiosa (Xf). Research on Pierce’s disease is difficult and time-consuming due to long incubation periods required for symptom development in grapevines following inoculation with Xf. ARS scientists at Parlier, California developed an infection system for Xf using the model plant A. thaliana as a host. The results indicated Xf behavior in A. thaliana differed from that in grapevine: infected A. thaliana remained asymptomatic, with low Xf population levels restricted to inoculated leaves. Thus, A. thaliana is not a suitable alternative host for elucidating host-pathogen interactions that occur in grapevine infected with Xf, but may be suitable as a model for annual weeds (serving as Xf reservoirs) that also do not express disease symptoms and support only limited growth of Xf.
Search behavior of glassy-winged sharpshooter (GWSS) egg parasitoids. Successful deployment of effective egg parasitoids as biological control agents of GWSS is hampered by limited knowledge concerning parasitoid ecology. Parasitoids use volatile compounds as cues to locate plants infested with GWSS eggs, but identity and properties of such compounds remain unknown. ARS scientists at Parlier, California identified two compounds released by GWSS-infested grapevines that are attractive to Gonatocerus ashmeadi wasps, the primary egg parasitoid of GWSS in the US. These results are a first step towards development of a complex blend of attractants that can be used to enhance biological control of GWSS through increased parasitoid recruitment and retention on plants infested with GWSS eggs.
Dispersal and movement of adult glassy-winged sharpshooters (GWSS). Effective control of GWSS populations requires detailed knowledge of insect ecology and population dynamics. Identification of factors affecting GWSS movement are needed to provide practical recommendations on methods to reduce GWSS populations. ARS scientists at Parlier, California, along with university collaborators, analyzed patterns and rates of GWSS movement in a deficit-irrigated citrus orchard to quantify movement and describe net dispersal rates. Results suggest GWSS are unable to make long-range decisions regarding suitability of host plants at a distance. Consequently, GWSS movement from citrus orchards into adjacent vineyards and surrounding habitats could be a result of random dispersal rather than oriented movement in response to host plant conditions.
Molecular mechanism of Xylella fastidiosa (Xf) PemK toxin and PemI antitoxin determined. The PemK/PemI toxin/anti-toxin system confers stability of plasmid DNA inheritance in Xf but mode of action was unknown. ARS scientists at Parlier, California determined PemK toxin is an enzyme that cleaves cellular RNA at specific sequences, thereby killing bacterial cells that do not inherit plasmid expressing PemI anti-toxin. PemI antitoxin was shown to directly bind to PemK toxin, thereby blocking RNA degradation activity of PemK toxin. Improved understanding of the PemK/PemI system facilitated development of a stable plasmid shuttle vector for expression of foreign DNA in Xf, thereby allowing genetic complementation assays of Xf pathogenicity mutants to be conducted in inoculated grapevines.
Feeding behavior of glassy-winged sharpshooter (GWSS) may explain absence of a latent period for Xylella fastidiosa vector transmission. Transmission of X. fastidiosa by GWSS has no latent period following acquisition, yet current theory requires attachment and colonization of the bacterium to the vector foregut prior to transmission, processes known to take several days. ARS scientists at Parlier, California identified a feeding behavior of glassy-winged sharpshooter that may explain the conundrum of Xf transmission immediately after acquisition. GWSS were briefly fed an artificial diet containing fluorescently labeled Xf cells and subsequently transferred to artificial diet lacking Xf. Microscopic examination indicated GWSS deposited both saliva and fluorescently labeled Xf into the clean artificial diet. These results provide a possible explanation for transmission of Xf immediately following acquisition in the absence of bacterial attachment and growth in GWSS.
Pierce’s disease resistant table grape selection. All table grapes grown in California are susceptible to Pierce’s disease caused by Xylella fastidiosa. ARS scientists at Parlier, California, have developed the first Pierce’s disease resistant table grape selection with naturally large, white seedless berries. Currently, this selection is being evaluated in production trials for horticultural characteristics and confirmation of resistance to Pierce’s disease under field conditions. Upon completion of field trials, the selection may be considered for release as a table grape cultivar that can be grown in areas with high incidence of Pierce’s disease.
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