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ARS Home » Pacific West Area » Parlier, California » San Joaquin Valley Agricultural Sciences Center » Crop Diseases, Pests and Genetics Research » Research » Research Project #442038

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

2023 Annual Report

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.

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
Progress was made under Objective 1 in developing genomic resources and application of multi-omic approaches for microbial systematics and pathogenesis. In support of Sub-objective 1A, researchers at Parlier, California, collected grapevine leaf samples showing Pierce’s disease (PD) symptoms from Georgia and pecan leaf samples showing leaf scorch symptoms from Georgia and Texas. Sample DNA was extracted and Xylella fastidiosa (Xf) infection was confirmed by polymerase chain reaction (PCR). Xf pure cultures were obtained from PD grape leaf samples. In support of Sub-objective 1B, researchers at Parlier California, evaluated Illumina sequence data from Xf-infected pecan, and ‘Candidatus Liberibacter asiaticus’ (CLas)-infected citrus (both root and leaf samples from the same tree) from Texas. Microbiome structures were revealed using the Kaiju metagenomic classifier. Xf and CLas genome sequences were extracted. Significant microbiome variations within and between samples were evaluated. In support of Sub-objectives 1C and 1D, researchers at Parlier, California, mutated Xf strains randomly to create collections of mutants for further screening in plant disease experiments. Random mutant collections were validated to confirm that enough separate mutations were made to target all non-essential genes in the Xf genome. RNA obtained from Xf cultivated in vitro under different temperature conditions was sequenced for evaluation of gene expression. Progress was made under Objective 2 in developing phenomic approaches to identify environmental and plant determinants of pathogen infection. In support of Sub-objective 2A, researchers at Parlier, California, evaluated CLas and Spiroplasma citri infected citrus at the Contained Research Facility in Davis, California, to ensure infection was still detectable. Several plants were reinoculated to obtain dually-infected citrus plants. Citrus plants exhibited typical symptoms of HLB and stubborn disease. In support of Sub-objective 2B, researchers at Parlier, California, inoculated greenhouse-grown grapevines with fungal pathogens or Xf to obtain samples for metabolite analyses. Additionally, different field-grown grapevines were inoculated with fungal pathogens because California regulations do not permit field inoculations with Xf. In support of Sub-objective 2C, researchers at Parlier, California, inoculated wine grape cultivars Cabernet Sauvignon, Sauvignon Blanc, and Zinfandel, and table grape cultivars Flame Seedless, Scarlet Royal, and Thompson Seedless with Xf strain Stag’s Leap in a greenhouse. Samples were collected from these plants before inoculation and at 24, 48, and 96 hours, 1 week, and 4 weeks after inoculation to characterize response of different grapevine cultivars to Xf infection using RNAseq. Progress was made under Objective 3 in the characterization of microbiomes of pathogen-infected grapevine and citrus as well as associated insect vectors. In support of Sub-objective 3A, researchers at Parlier, California, collected leaf, stem, and root samples from fungal pathogen- or Xf-inoculated grapevines. Phenolic compounds were extracted and analyzed by high-performance liquid chromatography. Additionally, phospholipid fatty acid (PLFA) profiles were obtained from tissues to observe overall in planta microbial community composition. In support of Sub-objective 3B, researchers at Parlier, California, were permitted to collect Citrus yellow vein clearing virus (CYVCV) from field sources, and established CYVCV in a containment greenhouse in Parlier, California. Research continues to examine CYVCV symptomatology in various citrus plants and to conduct vector transmission experiments. In support of Sub-objective 3C, Xf-infected and healthy grapevines maintained in a greenhouse by researchers at Parlier, California, allowed confirmation of the infection of Xf in grapevines by PCR. Grapevines will be used for microbiome studies. Researchers also maintained colonies of glassy-winged sharpshooters (GWSS) and blue-green sharpshooters (BGSS). Blue-green sharpshooter sequence data were analyzed for draft genome assembly. In support of Sub-objective 3D, researchers at Parlier, California, collected Asian citrus psyllid (ACP) from a commercial lemon orchard near Goleta, California, and preserved adults and nymphs in 95% ethyl alcohol for sequencing. Additionally, researchers at Parlier, California, established laboratory colonies of the spirea aphid (Aphis spiraecola) and cotton aphid (A. gossypii) to conduct vector transmission tests with CYVCV. Both aphid species transmitted CYVCV to test citrus plants. A laboratory colony of the citrus whitefly (Dialeurodes citri) is being established for further transmission studies. Progress under Objective 4 involved elucidation of vector-pathogen-crop interactions to disrupt pathogen transmission. In support of Sub-objective 4A, researchers at Parlier, California, completed Xf colonization experiments. Insect vector heads were dissected and provided to a collaborator for scanning electron microscopy (SEM) examination of the insect foregut. Results indicate that colonization of Xf differs by acquisition access period and Xf strain. In support of Sub-objective 4B, insect vector acquisition access periods for Xf colonization were completed to determine whether microscopic detection of bacteria is time-dependent. Insect vector heads were dissected and provided to collaborators for microscopic examination. Results are consistent with previous anatomical observations of the precibarium and support hypotheses that, 1) bacteria can be observed microscopically above, but not below, the valve, and 2) an invagination is present in sharpshooters, as it is in spittlebugs.

1. DNA modification in Xylella fastidiosa (Xf) provides new insight into strain recombination. Xf infects hundreds of plant species and causes severe disease in several important crops including grape, almond, citrus, and olive. Specific strains of Xf will only cause disease in some host plant species and not others, but the mechanism for the host specificity cannot always be determined based on DNA sequence alone. ARS researchers in Parlier, California, identified DNA methylation patterns present in a wide range of Xf strains and found that closely related strains maintained similar patterns to each other. This suggests that DNA modification patterns could prevent recombination between less closely related strains, which is important for understanding the emergence of new strains and adaption of Xf to new hosts. This will benefit regulators such as the USDA Animal and Plant Health Inspection Service by allowing more informed decisions regarding which strains of Xf require increased scrutiny and the likelihood of new strains emerging.

2. Complete genome sequence of Citrus yellow vein clearing virus CA1 isolate provides insight to its origin. Citrus yellow vein clearing virus (CYVCV) is an emerging exotic disease in California that poses a threat to the citrus industry in the United States. CYVCV is mechanically and graft transmissible and vectored by aphids and whiteflies when established in other parts of world. Most citrus cultivars are susceptible but asymptomatic. However, lemons are highly symptomatic, and infection cause significant loss of production. CYVCV was detected in 2022 in urban citrus trees in the city of Tulare, California. ARS researchers at Parlier, California, sequenced the entire genome of the California strain of CYVCV. Phylogenetic analysis revealed that the closest strain is from India. This suggests that the pathway of entry of CYVCV-CA1 was by introduction of infected plants or budwood. Results will be useful for USDA Animal and Plant Health Inspection Service, Plant Protection & Quarantine and California Department of Food and Agriculture to target entrance pathways of these exotic pathogens and raise awareness of demographics of potential higher risk.

3. Biomarkers of Xylella fastidiosa identified through proteomics. Xylella fastidiosa (Xf) causes diseases in many important crops around the world. There are currently over 140 Xf whole genome sequences deposited in the GenBank database, but proteomic information of Xf is limited. ARS scientists in Parlier and Albany, California, employed a top-down proteomic technique to identify biomarkers of Xf strains using matrix-assisted laser desorption ionization-mass spectrometry. One marker, an outer membrane/hypothetical protein, was found to have a potential capacity to differentiate closely related Xf strains which is critical for regulatory issues and biological studies. This research will assist USDA Animal and Plant Health Inspection Service and other regulatory agencies in improved diagnostics for identifying dangerous Xf strains.

4. Transcriptomic analysis reveals new biological features of Microviridae phage in CLas. “Candidatus Liberibacter asiaticus” (CLas) is associated with citrus Huanglongbing (HLB), a devasting and deadly disease that severely limits citrus production wherever it is present. Knowledge of CLas biology is important for HLB management. One recent research development is the discovery of a Microviridae phage through genome sequence analysis, but evidence of phage activity is lacking. ARS scientists in Parlier, California, and collaborators in South China Agricultural University, further investigated the activity of Microviridae phage in the plant host with transcriptomic tools. Lytic activity of the phage was detected which may involve regulation of CLas genes related to cell surface components and Sec pathway. This could benefit phage infection. Results expanded current knowledge of the Microviridae phage and will benefit further CLas phage research and HLB control.

5. New DNA sequence resources for Xylella fastidiosa (Xf) were established. Xylella fastidiosa (Xf) is an economically important bacterial pathogen of many crops. Whole genome sequences are critical resources for research to understand the biology of Xf. ARS scientists in Parlier, California, Byron, Georgia, and researchers at University of Georgia, used next-generation sequencing technology to create DNA sequence resources for Xf strains causing phony peach disease and blueberry bacterial leaf scorch disease. This genomic information filled gaps in Xf genome sequence databases, which are critical for genetic and taxonomic studies of Xf. Both researchers and regulatory agencies such as USDA Animal and Plant Health Inspection Service benefit from sequence data to improve diagnostic technologies.

6. Grapevine fungal trunk pathogen infection affects the development of subsequent infections in grapevines. Grapevines are exposed to infection sources through various organisms simultaneously but most studies examining pathogen infections on plant hosts consider only one infection at a time. ARS researchers in Parlier, California, investigated grapevines previously infected with one of three fungal trunk pathogens that were then inoculated with another subsequent pathogen. Prior infections significantly reduced the development of a trunk pathogen known as Diplodia seriata, but the other pathogens in the study (Neofusicoccum parvum and Phaeomoniella chlamydospora) were unaffected by prior infections. Infections by Diplodia seriata also resulted in greater grapevine phenolic levels than controls or infections by the other pathogens. These results showed that fungal trunk pathogens not only vary in the ability to induce changes in host metabolites, presumably as a host defense response, but also vary in the capacity to affect other infections. Additional research to explore the interactions of multiple infections on the same plant hosts is currently ongoing to fully understand how the disease develops.

7. Machine learning elucidates the genomic diversity of “Candidatus Liberibacter asiaticus” strains from North America. Huanglongbing (HLB) is a highly destructive citrus disease associated with the nonculturable bacterium “Candidatus Liberibacter asiaticus" (CLas). Understanding the population diversity of CLas is critical for HLB management. ARS scientists in Parlier, California, and collaborators at the National Station of Plant Epidemiology, Quarantine and Sanitation (SENASICA), Mexico, collected and analyzed 40 CLas samples from 20 states in Mexico using a machine learning approach. Results showed there were at least two different introductions of CLas into Mexico, and that CLas strains from Mexico and the United States are closely related. Results can inform efforts to manage HLB in North America by improving understanding of how HLB has historically moved around the continent, which will be useful information for USDA Animal and Plant Health Inspection Service and other regulatory agencies.

8. Pest categorization of Xylella taiwanensis by the European Food Safety Authority. Xylella taiwanensis is a recently described bacterial pathogen causing pear leaf scorch disease in Taiwan. This pathogen is currently known to occur only in Taiwan. ARS scientists in Parlier, California, participated in a scientific panel that produced a reference guide to prevent invasion of X. taiwanensis in Europe and United States where pears are commercially grown. This guide is available to the public and will be especially useful for U.S. and European Union regulatory agencies.

9. Exploring the role of grapevine-produced phenolic compounds in root resistance to nematode feeding. Ring and root knot nematodes attack grapevine roots and may reduce productivity over time. Grapevines produce compounds known as phenolics presumably to thwart nematode feeding and infection. ARS researchers in Parlier, California, examined the production of phenolics in response to ring nematode feeding, and the effects of phenolics on survival of root knot nematodes. Results suggest a limited role of phenolics in preventing nematode feeding. Other defense mechanisms are likely present to explain observed differences in rootstock cultivar resistance to these pests, and this results in informing ongoing and future research efforts to be conducted accordingly to conserve resources.

10. Characterization of grapevine host responses to Grapevine leafroll-associated virus-3 (GLRaV-3) and Grapevine red blotch virus (GRBV). Grapevine productivity is negatively affected by infections of different viruses including GLRaV-3 and GRBV. However, effects of these viruses on host physiology remains poorly understood. ARS researchers at Parlier, California, observed phenolic and amino acid levels in grapevine leaves throughout the growing season in healthy and virus-infected vines. Hydroxycinnamic acids (HCAs) were present in greater levels in healthy compared to infected grapevines. Because HCAs are positively associated with plant tissue maturity, reduced levels in infected plants suggest that plant tissues in infected grapevines mature at an increased rate. These results highlight changes that viral pathogens induce in grapevine hosts, which could be used to develop biomarkers that plant diagnosticians may use to identify a potentially infected vine.

Review Publications
O'Leary, M.L., Burbank, L.P. 2023. Natural recombination among Type I restriction-modification systems creates diverse genomic methylation patterns among Xylella fastidiosa strains. Applied and Environmental Microbiology. 89(1).
Huang, J., Alanis-Martinez, L., Kumagai, L., Dai, Z., Zheng, Z., Perez de Leon, A.A., Chen, J., Deng, X. 2022. Machine learning and analysis of genomic diversity of “Candidatus Liberibacter asiaticus” strains from 20 citrus production states in Mexico. Frontiers in Plant Science. 13. Article 1052680.
Bragard, C., Baptista, P., Chatzivassiliou, E., Di Serio, F., Gonthier, P., Jaques Miret, J., Justesen, A.F., MacLeod, A., Magnusson, C., Milonas, P., Navas-Cortes, J.A., Parnell, S., Potting, R., Stefani, E., Thulke, H., Van der Werf, W., Civera, A., Yuen, J., Zappala, L., Chen, J., Migheli, Q., Vloutoglou, I., Streissl, F., Reignault, P. 2023. Pest categorisation of Xylella taiwanensis. European Food Safety Authority (EFSA) Journal. 21(1). Article e07736.
Oliver, J.E., Lewis, K.J., Taylor, S.J., Chen, J. 2023. Complete genome sequences of Xylella fastidiosa subsp. fastidiosa and X. fastidiosa subsp. multiplex strains causing blueberry bacterial leaf scorch disease in Georgia, USA. Microbiology Resource Announcements. 12(6). Article e00114-23.
Wang, C., Fang, F., Li, Y., Zhang, L., Wu, J., Li, T., Zheng, Y., Xu, Q., Fan, S., Chen, J., Deng, X., Zheng, Z. 2022. Biological features and in planta transcriptomic analyses of a Microviridae phage (CLasMV1) in “Candidatus Liberibacter asiaticus”. International Journal of Molecular Sciences. 23(17). Article 10024.
Hajeri, S., Yokomi, R.K. 2023. Citrus tristeza virus. In: Gaur, R.K., Patil, B.L., Selvarajan, R., editors. Plant RNA Viruses. 1st edition. Cambridge, MA: Academic Press. p.117-133.
Wallis, C.M., Gorman, Z.J., Galarneau, E.R., Baumgartner, K. 2022. Mixed infections of fungal trunk pathogens and induced systemic phenolic compound production in grapevines. Frontiers in Fungal Biology. 3. Article 1001143.