1a. Objectives (from AD-416):
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.
1b. Approach (from AD-416):
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.
3. Progress Report:
This report documents progress for project 2034-22000-012-00D, which started in April 2017 and continues research from project 2034-22000-010-00D, “Epidemiology and Management of Pierce's Disease and Other Maladies of Grape.” Under Objective 1, progress was made towards the identification and characterization of genes involved in pathogenicity of Xylella fastidiosa to grapevines. The complete whole genome sequence of Xylella fastidiosa strain ATCC 35879 was acquired through a combination of sequencing technologies. Genomic diversity of strain 35879 is being examined and compared to other known strains. Sequence analysis of DNA molecules (plasmids) from Xylella fastidiosa strains Stag’s Leap and BB01 revealed the presence of a potential antibiotic resistance gene and a gene associated with nutrient absorption, respectively. Mutations made by knocking out genes of six different toxin-antitoxin (TA) systems have been constructed in Xylella fastidiosa (Stag’s Leap strain) for evaluation of functional activity and potential role in pathogen virulence. RNA samples have been prepared for these six mutants under varied growth conditions to further study function of TA systems in pathogen adaption and infection by comparing gene expression levels. In addition, construction of mutant and overexpression strains of Xyella fastidiosa were initiated to study the cold shock proteins Csp1 and Csp2. These strains will be used to study bacterial responses to low temperature conditions. Progress also was made towards development of an analytical method to study fatty acids extracted from bacterial cell wall membranes. Fatty acids from eight different strains of Xylella fastidiosa were extracted and used to create fingerprints of two Xylella subspecies. Data suggest that the fatty acid analysis of bacterial cell membranes combined with analysis of DNA sequences described above could be deployed as a technique to sort Xylella fastidiosa isolates by subspecies, strains, and host. Under Objective 2, progress was made towards understanding grapevine defense responses to nematode infestations and elucidating insect vector feeding behaviors needed to further evaluate transmission of Xylella fastidiosa to Pierce’s disease-resistant and -susceptible grapevines. Analysis of defense-related compounds produced by grapevines upon nematode infestation revealed that resistant rootstocks had distinct phenolic compound profiles when compared with susceptible rootstocks, which could be a potential mechanism of resistance to root knot nematodes. Once confirmed, linking this potential plant resistance trait to the genetic profile of breeding populations of grapevines could result in development of new nematode-resistant rootstocks. Experiments were conducted to study vector feeding behaviors associated with transmission of Xylella fastidiosa to grapevines. After a short exposure to artificial diets containing Xylella fastidiosa, insects were allowed to salivate and egest fluid from foregut into grapevine. Microscopy showed no attachment of bacteria to insect mouthparts and yet, bacteria were successfully inoculated to grapevines. Data suggest that free-floating bacterial cells held in fluid within the vector foregut can be inoculated to grapevines without attaching and multiplying in the insect prior to inoculation. Feeding behaviors of insects carrying Xylella fastidiosa were compared to behaviors of insects not carrying the bacteria to determine whether presence of Xyella fastidiosa alters occurrence and duration of such behaviors. Insects carrying bacteria performed a larger number of xylem salivation and egestion events than did bacteria-free insects. Progress made on Objective 3 focused on the analysis of the arthropod community found in and near vineyards, including determination of seasonal changes in the abundance of insect vectors carrying Xyella fastidiosa. Vineyards and citrus orchards in Kern County, California, were sampled on a regular basis to estimate glassy-winged sharpshooter abundance and Xylella fastidiosa population densities in vectors and chronically infected plants. In addition, sweep net samples from vineyards, almond orchards, olive orchards, alfalfa fields, and pastures were taken to describe the arthropod community and monitor seasonal abundances of the insect vector, green sharpshooter, in and around vineyards in the Central Valley of California. Data show that risk of alfalfa fields serving as a source of insect vectors is offset by the benefit of alfalfa fields serving as a source of generalist natural enemies.
1. Description of glassy-winged sharpshooter communication and identification of candidate signals for mating disruption programs. The glassy-winged sharpshooter is a vector of Xylella fastidiosa, an important bacterial pathogen of several crops. To thrive in crops, sharpshooters must communicate by exchanging mating calls, which travel in plants as sound waves. Exploitation of sound signals to interfere with communication and suppress sharpshooter populations could prove to be a useful tool, but existing knowledge on mating behavior is insufficient to implement a control method for this pest. ARS researchers in Parlier, California, in collaboration with researchers at Fondazione Edmund Mach in Italy were the first to describe sharpshooter mating communication, which led to identification of several candidate disruptive signals for playback interference under field conditions. This knowledge is currently being used to develop a novel environmentally safe pest management tool to mitigate the impact of Xylella fastidiosa for the grape production industry.
2. Identification and validation of pilG as a key virulence gene in Xylella fastidiosa. Xylella fastidiosa is a bacterium that inhabits sap-transporting vessels in plants and possesses an array of virulence factors responsible for causing plant diseases. Using a genetic approach, ARS scientists in Parlier, California, identified and functionally validated a key virulence gene (pilG) in Xylella fastidiosa required for development of Pierce’s disease in grapevines. Molecular analysis indicated that pilG plays a pivotal role in orienting bacterial movement within plants; removal of pilG from a wild Xylella fastidiosa strain markedly reduced virulence in grapevines. This study identified a new target to genetically disarm Xyella fastidiosa pathogenicity and reduce the impact of Pierce's disease for the grape production industry.
3. Characterization of feeding damage to cotton by Lygus species. Lygus species are the most economically important pests of cotton in the United States. In addition to direct feeding damage to plant tissues, bug feeding is known to induce production of tannins by cotton plants, but which specific part of the feeding process results in production of tannins is not known. ARS researchers in Parlier, California, defined three Lygus probing behaviors: cell rupturing (combined salivating and moving of stylets), transition (combined tasting and testing of food), and ingestion (swallowing plant fluid). Examination of damaged plant tissues revealed that the cell rupturing behavior induced red-stained deposits composed of tannins. Findings from this study benefit cotton production by providing knowledge required to develop plant resistance mechanisms to Lygus feeding.
Cervantes, F.A., Backus, E.A., Godfrey, L., Wallis, C.M., Akbar, W., Clark, T., Rojas, M.G. 2017. Correlation of EPG waveforms from Lygus lineolaris feeding on cotton squares and chemical evidence of inducible tannins. Annals of the Entomological Society of America. 110:2068-2075.
Kunta, M., Zheng, Z., Wu, F., Da Graca, J., Park, J., Deng, X., Chen, J. 2017. A draft whole genome sequence of “Candidatus Liberibacter asiaticus” strain TX2351 from Asian citrus psyllids in Texas, USA. Genome Announcements. 5(15):e00170-17.
Mazzoni, V., Gordon, S.D., Nieri, R., Krugner, R. 2017. Design of a candidate vibrational signal for mating disruption against the glassy-winged sharpshooter, Homalodisca Vitripennis. Pest Management Science. doi: 10.1002/ps.4618.
Zhu, Y., He, Y., Zheng, Z., Chen, J., Wang, Z., Zhou, G. 2017. Draft genome sequence of rice orange leaf phytoplasma from Guangdong, China. Genome Announcements. 5(22):e00430-17.
Gordon, S.D., Sandoval, N., Mazzoni, V., Krugner, R. 2017. Mating interference of glassy-winged sharpshooters, Homalodisca vitripennis. Entomologia Experimentalis et Applicata. doi: 10.1111.eea.12594.
Shi, X., Lin, H. 2017. The chemotaxis regulator pilG of Xylella fastidiosa is required for virulence in Vitis vinifera grapevines. European Journal of Plant Pathology. doi: 10.1007/s10658-017-1282-x.
Nieri, R., Mazzoni, V., Gordon, S.D., Krugner, R. 2017. Mating behavior and vibrational mimicry in the glassy-winged sharpshooter, Homalodisca vitripennis. Journal of Pest Science. 90(3):887-899.