Research Project: Identifying Vulnerabilities in Vector-host-pathogen Interactions of Grapevine and Citrus Pathosystems to Advance Sustainable Management Strategies

Location: Crop Diseases, Pests and Genetics Research

2025 Annual Report

Objectives
Objective 1: Develop genomic resources and application of multi-omic approaches for understanding microbial systematics and pathogenesis [NP303 C1, C2, PS1A, PS1B, PS2A, PS2B]. Sub-objective 1A: Expand whole genome sequence databases of Xylella fastidiosa (Xf) and “Candidatus Liberibacter asiaticus” (CLas) strains. Sub-objective 1B: Characterize metagenomes of Xylella spp. and “Ca. Liberibacter spp.” infected samples using machine learning (ML) focusing on improvement of taxonomic identification. Sub-objective 1C: Identify genetic determinants of Xf host range using a transposon mutagenesis and high-throughput sequencing approach. Sub-objective 1D: Identify genetic determinants of Xf persistence and survival under different climatic conditions. Objective 2: Develop phenomic approaches to identify environmental and plant determinants of pathogen infection [NP303, C1, C2, C3, PS1A, PS1B, PS2B, PS3A]. Sub-objective 2A: Characterize host response of citrus to S. citri infection that can influence co-infection of CLas by the ACP. Sub-objective 2B: Develop simplified metabolomic profiles for different grapevine cultivars and associate them with observed resistance to fungal pathogens, Xf, and associated diseases. Sub-objective 2C: Characterize response of different grapevine cultivars to Xf infection using RNAseq. Objective 3: Characterize microbiomes of pathogen-infected grapevine and citrus as well as associated insect vectors [NP303, C1, C2, PS1A, PS1B, PS2A, PS2C]. Sub-objective 3A: Describe the phytobiome of healthy, Xf-infected, and fungal canker pathogen-infected grapevines, and relate to host physiological status. Sub-objective 3B: Describe the phytobiome of healthy and CLas-infected citrus plants. Sub-objective 3C: Describe the microbiomes of sharpshooter vectors of Xf. Sub-objective 3D: Describe the microbiome in the Asian citrus psyllid vectors of CLas. Objective 4: Elucidate vector-pathogen-crop interactions to disrupt pathogen transmission [NP303, C2, C3, PS2B, PS2D, PS3A, PS3B]. Sub-objective 4A: Elucidate a time course of Xf bacterial colonization and exopolysaccharide attachment formation in functional foregut of sharpshooters. Sub-objective 4B: Characterize ultrastructure of the precibarial valve in the functional foregut of sharpshooters, and its possible role in Xf transmission over time.

Approach
Objective 1. The genomic underpinnings of pathogenesis can be determined for diseases caused by Xf and CLas by use of multi-omic approaches. Next Generation Sequencing (NGS)technologies will be used to generate giga bp level DNA sequence data sets which will be subjected to datamining through machine learning (ML) approaches to develop new and unique biological information. Genomic determinants of host susceptibility will be examined by mutagenesis and bioassays of tolerant versus susceptible host cultivars. Persistence of Xf will be studied under ambient and low temperature conditions using transcriptome sequencing and mutational validation of gene functions. Objective 2. Through measurements of growth, performance, and composition of grapevines and citrus under different pathogen challenge conditions, environmental and host susceptibilities to pathogen infection can be identified. Because citrus stubborn disease and huanglongbing are caused by phloem-restricted insect-vectored bacteria (Spiroplasma citri and CLas, respectively), pre-infection of S. citri will be examined to test if pathogen competition can reduce plant infectivity and/or susceptibility to CLas. Metabolomics of grapevines inoculated with Xf and fungal pathogens will be studied for specific chemical profiles and molecular attributes that could be help identify host susceptibility or resistance traits. Similarly, transcriptome analysis will be conducted on susceptible, tolerant, and resistant cultivars of grapevines challenged by Xf to better understand host plant resistance and improve disease mitigation of Xf diseases. Objective 3. An exploration of the microbiomes of grapevines and citrus infected by Xf and CLas, respectively, along with their insect vectors, will identify microorganisms and insect endosymbionts that may be used or developed to mitigate or reduce spread of Xf and CLas. Phytobiomes of grapevines inoculated by Xf and fungi will be examined by NGS to determine microbial community shifts correlated to host physiology. Phytobiomes of citrus infected by CLas will be examined by NGS to identify prophage(s) that can be used to differentiate and identify CLas populations and other microbes. Microbiomes of insect vectors of Xf and CLas will be examined by NGS technologies to identify insect endosymbionts. This information will be used in studies to reduce vector fitness and/or propensity of transmission. Objective 4. Xf attachment in the foregut of the blue green sharpshooter (BGSS) depends on exo-polysaccharide adhesives secreted by Xf and the ultrastructure of the functional foregut, especially the precibarial valve, in the vector. Functional foregut of BGSS exposed to grapevines infected by a mild versus a virulent strain of Xf will be examined by scanning electron microscopy (SEM) to determine if extent of bacterial colonization is correlated with disease virulence. Time course acquisition access periods and light and transmission microscopy will be used to ascertain if foregut morphology (grooves and invaginations) and bacterial adhesion to the cuticular lining of the functional foregut influence Xf transmission.

Progress Report
This report documents FY 2025 progress for project 2034-22000-015-000D, “Identifying Vulnerabilities in Vector-host-pathogen Interactions of Grapevine and Citrus Pathosystems to Advance Sustainable Management Strategies”, which began in April 2022. In support of Sub-objective 1A, ARS researchers in Parlier, California, acquired samples of pecans, elderberries, and grapevines collected from Oklahoma to determine presence of the bacterium Xylella fastidiosa (Xf). The genome of newly acquired Xf strains from Oklahoma were sequenced and determined to belong in the subspecies multiplex. Under Sub-objective 1B, progress was made using Large Language Models (LLMs), operated by USDA SCINet’s high-performance computer clusters, to decipher genomic variations of “Candidatus Liberibacter asiaticus” (CLas) and Xf. LLMs also were used to summarize peer-reviewed publications as part of an effort to make a literature review. For Sub-objective 1C, ARS researchers sequenced and analyzed the genome of Xf mutant pools that were passed through grapevines and almond trees. This allowed the selection of several specific Xf mutants that had relevant alterations in the ability to infect grapevines and almond trees. In support of Sub-objective 2A, ARS researchers in Parlier, California, tested the ability of Citrus yellow vein clearing virus (CYVCV) to infect both citrus and herbaceous hosts. Citrus and citrus relatives such as sweet orange, grapefruit, mandarins, lemons, tangelos, pummelo, citron, limes, sour orange, and Kumquat all became systemically infected and supported high levels of CYVCV, but only lemon and sour orange expressed strong symptoms of yellow vein clearing, leaf distortion, and water soaking appearance. Virus infection in herbaceous plants remained restricted to the inoculated leaves (non-systemic infection) and virus became undetectable by polymerase chain reaction (PCR) after two weeks, suggesting a smaller host range for the virus than reported in the literature. Under Sub-objective 2B, ARS researchers in Parlier, California, processed plant samples to collect phenolics and volatile organic compounds (VOCs) from CYVCV-infected trees to determine if the virus affects the metabolite levels of the plant and if the VOCs produced by the infected plants could affect the behavior of the virus’ insect vector. The research showed that emission of key VOCs was reduced by CYVCV infection, which was associated with increased whitefly vector attraction to infected plants. For Sub-objective 2C, ARS researchers in Parlier, California, continued RNA sequencing analyses of susceptible and resistant grapevine genotypes infected with Xf. In support of Sub-objective 2D, research continued on establishing embryonic callus propagation lines using immature grapevine leaves (cv. Thompson Seedless) for the next-step protoplast preparation, transfection, and genome editing. The grapevine gene CYP734A15 was demonstrated to code for a valid brassinosteroid (BR)-inactivating enzyme, and as such this would be an ideal genome editing target for elevating endogenous BR levels in grapevines to result in improved broad-spectrum disease resistance and abiotic stress tolerance. Under Sub-objective 3A, ARS researchers characterized differences in grapevine responses to pathogen infections by fungal canker pathogens including Diplodia seriata, Eutypa lata, and Neofusicoccum parvum. The microbiome within leaf, stem, and root tissues of fungi-infected grapevines were determined to understand how pathogen-induced shifts in grapevine physiology impacted the microbiomes. This resulted in understanding about members of the microbiome important in plant health, which could be utilized as biostimulant products to increase grapevine yields. For Sub-objective 3B, microbiome and metagenomic analyses were performed on multiple samples including citrus infected with CLas from Texas and almond samples infected with Xf from California. In support of Sub-objective 3C, glassy-winged sharpshooters were collected from infested regions in California and DNA was extracted for high-throughput sequencing. Under Sub-objective 3E, research continued on extracting DNA from soil samples collected from around different grapevine rootstock cultivars. Ribosomal 16S DNA sequencing was performed to reveal and compare soil bacterial microbiomes unique to the microbial communities present around different rootstocks. For Objective 4, ARS researchers in Parlier, California, made progress in understanding the time course of Xf colonization of sharpshooter foreguts and how it may differ by Xf strain. In support of Sub-objective 4A, ARS researchers investigated colonization of Xf in sharpshooter heads by quantitative polymerase chain reaction (qPCR) and scanning electron microscopy. Differences by acquisition access period (AAP) day and strain were observed. These differences were likely due to insect behaviors related to removal of Xf from mouthparts. Under Sub-objective 4B, ARS researchers characterized the role of the precibarial valve in Xf transmission by examining insect vector heads via light microscopy. Results confirmed the role of anatomy and operation of the precibarial valve with Xf bacteria being present below, but not above, the valve. An invagination was present and determined to be involved in both valve operation and likely Xf transmission.

Accomplishments
1. Insect pest feeding behavior influences retention of vector-borne plant pathogen. The insect-transmitted bacterial plant pathogen Xylella fastidiosa (Xf) is widespread in the U.S. and causes significant economic damage to California’s grape industry. Xf cells colonize the foregut of insect vectors, but little is known about the process of bacterial injection into the plant vascular system during insect feeding. ARS scientists in Parlier, California, examined bacterial quantity and placement within the foregut of insect vectors over time and found bacterial biofilms at multiple different stages and structures. Patterns of bacterial colonization within the insect suggest that detachment and dispersal of bacterial cells are influenced by insect behaviors during feeding on plants. This enhanced understanding of pathogen transmission rates in grapevines based on observing insect vector behavior can be exploited in plant breeding programs aimed at reducing Xf spread.

2. Host range studies of newly emerging citrus yellow vein clearing virus show severe disease potential is limited to lemons. Citrus yellow vein clearing virus (CYVCV) is a new invasive virus in California and is a threat to the citrus industries in the United States. ARS scientists in Parlier, California, inoculated the virus in a broad range of citrus cultivars, citrus relatives, and herbaceous plants to examine disease severity. Results indicated that all citrus cultivars and citrus relatives tested were susceptible to systemic infection and supported viral replication, although severe symptoms developed only in lemon and sour orange. Herbaceous plants were found not to be viable hosts. Results suggest that the virus affects only lemon production, which is important to both growers and regulatory agencies for making decisions about inoculum removal and insect vector control to limit spread of the virus.

3. Development of a method to use next-generation genome sequencing for diagnostics of Xylella fastidiosa in woody plants. Different strains of the bacterial pathogen Xylella fastidiosa (Xf) may cause disease in various woody plants. Detecting Xf infection in woody plants is difficult because commonly used molecular methods for bacterial detection from plants yield ambiguous results, and in some cases, such as with pecan trees, pathogen isolation in artificial media is difficult. ARS scientists in Parlier, California, and Byron, Georgia, collaborated to develop a whole genome-based high-throughput DNA sequencing protocol that unambiguously detected Xf in hickory and pecan trees, with genomes obtained. This approach to use next-generation genomic sequencing to identify and characterize Xf strains to subspecies level from plant material could be used for quick and accurate disease diagnosis.

4. Discovery of indicators for resistance in grapevines to Eutypa dieback. Eutypa dieback, caused by the fungus Eutypa lata, is a grapevine disease distributed worldwide and yet, little is known about how grapevines naturally resist disease development. An ARS scientist in Parlier, California, and collaborators measured differences in plant physiological and biochemical characteristics in resistant and susceptible grapevine cultivars infected with E. lata. Results demonstrated that downregulating physiological functions in response to infection in resistant grapevines led to improved long-term resistance. These responses are indicators of resistance that could be used in plant breeding efforts to develop new grapevine cultivars resistant to Eutypa dieback.

5. Citrus yellow vein clearing virus infection makes lemon trees more attractive to the whitefly vector pest. Citrus yellow vein clearing virus (CYVCV) is an emerging viral disease that negatively impacts lemon production where present and is spread by insect vectors including the citrus whitefly. However, little was known about how plant infection by CYVCV might impact vector behaviors. ARS scientist in Parlier, California, showed that CYVCV-infected lemon trees had increased whitefly settling behaviors compared to noninfected trees. The increased settling behaviors of the citrus whitefly were associated with changes in the composition of volatile organic compounds (VOCs) that occurred when lemon trees were CYVCV-infected. Results could be utilized to develop novel methods that use VOCs to affect whitefly behaviors that, in turn, could reduce the spread of CYVCV.

Review Publications
Dutra, M.F., Silva, P.A., Chen, J., Wulff, N.A. 2024. The complete genome sequence of "Candidatus Liberibacter asiaticus" strain 9PA and the characterization of field strains in the Brazilian citriculture. Plant Disease. 9(12). Article e00376-24. https://doi.org/10.1128/msphere.00376-24.
Cardenas-Amaya, C., Romero-Salas, D., Aguilar-Dominguez, M., Alonso-Diaz, M., Rosas-Saito, G., Perez de Leon, A.A. 2025. Report on the presence of Amblyomma inornatum in Michoacán, Mexico. La Revista Mexicana de Ciencias Pecuarias. 16(4):150-157. https://doi.org/10.22319/rmcp.v16s4.6699.
Sinclair, G.C., Travadon, R., Eschen, P., Wallis, C.M., Baumgartner, K., Delmas, C.E., Hnizdor, J.F., Bartlett, M. 2025. Differential physiological responses of resistant and susceptible grape cultivars to Eutypa dieback. Journal of Experimental Botany. Article eraf103. https://doi.org/10.1093/jxb/eraf103.
Hidalgo, D., Ramirez, J.L., Navarrete, M., Cevallos, V., Ramos, M., Bravo, B., Carranza, K., Montes, V., Perez De Leon, A.A. 2025. Research advances in Ecuador on use of entomopathogenic fungi for control of the cattle tick, Rhipicephalus microplus: The case of Beauveria bassiana sensu lato strain INIAP L3B3. Frontiers in Fungal Biology. 6. Article 1492395. https://doi.org/10.3389/ffunb.2025.1492395.
Bock, C.H., Hotchkiss, M.H., Hilton, A.E., Chen, C., Chen, J. 2025. First report of Xylella fastidiosa subsp. multiplex infecting southern shagbark hickory (Carya carolinae-septentrionalis)in Georgia, U.S.A. Plant Disease. 109(3):711. https://doi.org/10.1094/PDIS-09-24-1990-PDN.
Sun, Y., Wallis, C.M., Krugner, R., Yokomi, R.K. 2025. Citrus yellow vein clearing virus infection in lemon influences host preference of the citrus whitefly by affecting the host metabolite composition. Plants. 14(2). Article 288. https://doi.org/10.3390/plants14020288.
Hilton, A.E., Bock, C.H., Brenneman, T., Chatwin, W.B., Chen, J. 2025. Whole genome resource for Xylella fastidiosa subsp. multiplex strain GaT2 causing pecan bacterial leaf scorch in Georgia, United States. PhytoFrontiers. https://doi.org/10.1094/PHYTOFR-09-24-0103-A.
Peng, H., Zhai, Y. 2025. Grapevine CYP734A15 is a brassinosteroid-inactivating cytochrome P450 enzyme. Biochemical and Biophysical Research Communications. 775. Article 152128. https://doi.org/10.1016/j.bbrc.2025.152128.
Sun, Y., Yokomi, R.K. 2024. The discovery of Citrus yellow vein clearing virus Hacienda Heights isolate diversifies the geological origins of the virus in California, United States. Viruses. 16(9). Article 1479. https://doi.org/10.3390/v16091479.
Sun, Y., Yokomi, R.K., Folimonova, S. 2024. Citrus tristeza virus: A century-long challenge for the world's citrus industries. Annals of Applied Biology. 185:304-322. https://doi.org/10.1111/aab.12939.
Demard, E.P., Backus, E.A., Diepenbrock, L.M. 2025. Comparison of electropenetrography waveform libraries for Nipaecoccus viridis (Hemiptera: Pseudococcidae) using different tethering materials and monitor settings. Journal of Insect Science. 25(3). Article 23. https://doi.org/10.1093/jisesa/ieaf063.