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.
This is a new project that continues research from expired projects 2034-22000-012-000D and 2034-22000-013-000D. For additional information, see the reports for the expired projects. Under Objective 1, progress was made in completing the whole genome sequence of a phony peach disease (PPD) strain of Xylella fastidiosa (Xf). PPD is a historical peach disease in the southeastern United States and Xf strains causing PPD are extremely difficult to isolate in culture media (in vitro). Therefore, a metagenomic approach, known as high-throughput sequencing, was used to bypass the in vitro culture barrier and acquire the bacterial genome directly from infected peach roots. Under Sub-objective 1B, progress was made to use machine learning technology to explore the use of genome sequence information to resolve research issues with “Candidatus Liberibacter asiaticus” (CLas), the causal agent associated with citrus Huanglongbing. DNA samples of 40 CLas strains from Mexico were obtained through collaborators. The genomes of two strains, one from Baja California Sur and one from Yucatan, were sequenced. The 40 CLas strains were grouped into two genomic groups according to a classification method based on a single prophage gene (terL). The classification system was reevaluated utilizing a machine learning approach with the recently available CLas genome sequences in GenBank. Additionally, 12 chromosomal genes were found to support the terL classification system. Under Sub-objectives 2B and 2C, ARS researchers inoculated the pathogens Armillaria mellea, Diplodia seriata, Eutypa lata, Neofusicoccum parvum, Phaeoacromonium minima, and Xylella fastidiosa into grapevines. Samples to be collected will be examined for changes in metabolism and the microbiome. Under Sub-objective 3A, different scions and rootstock varieties in an established vineyard were inoculated with the pathogens Eutypa lata, Neofusicoccum parvum, or Xylella fastidiosa, to further assess changes in metabolism in mature plants and determine whether these metabolic changes form signatures of resultant diseases. Under Objective 3, progress was made in acquiring DNA samples of CLas from California, Florida, and Texas. Initial microbiome comparisons were conducted on several HiSeq data sets of CLas strains from Texas. Under Sub-objective 3C, progress was made to establish and maintain colonies of glassy-winged sharpshooter (GWSS) and blue-green sharpshooter (BGSS) in greenhouse. The laboratory colonies yielded samples of GWSS and BGSS that were submitted to collaborators for sequencing. Under Objective 4, progress was made in describing feeding of blue-green sharpshooters (BGSS) on Xylella fastidiosa-infected grapevines. BGSS heads colonized by the bacterium were dissected and provided to collaborators who are examining them with scanning electron microscopy, light microscopy, and transmission electron microscopy.
1. The whole genome sequence of a Xylella fastidiosa (Xf) strain causing phony peach disease. Phony peach disease (PPD) is a potentially destructive disease that has been in the southeastern United States for over a hundred years and is caused by the Xf bacterium. Little is known about the biology and genomics of Xf strains causing PPD. ARS researchers at Parlier, California, collaborated with scientists from University of Georgia and USDA-ARS at Byron, Georgia, to collect bacterial samples from Georgia and Alabama and sequenced the genome of a selected strain using a metagenomic approach. The genome sequence provides a valuable resource for scientists studying the biology and taxonomy of Xf, and the information can be useful for PPD management and for regulatory agencies.
2. Machine learning approach to explore “Candidatus Liberibacter asiaticus” (CLas) taxonomic relationships. More than 30 genome sequences of CLas strains are now available in public databases. Genome sequences are useful for identifying genes controlling specific traits in CLas and understanding genetic relationships among different CLas strains but mining information from large sequence databases is a challenging task. ARS researchers at Parlier, California, collaborated with scientists from the National Station of Plant Epidemiology, Quarantine, and Sanitation (ENE CuSaV) in Queretaro, Mexico, to use a machine learning approach to analyze CLas genome sequences. A previously developed CLas strain classification system based on a single phage gene was further substantiated with 12 chromosomal genes, demonstrating that machine learning is a powerful tool for genomic research in CLas.
3. Metabolomic profiles of citrus associated with mild or severe strains of Citrus tristeza virus (CTV). Different CTV strains cause different symptoms in infected citrus. However, the changes in metabolism linked to infection status are not well understood. ARS researchers in Parlier, California, quantified changes in specific metabolites including amino acids, sugars, terpenoids, and phenolics, to find consistent trends associated with healthy, mild, or severely infected plants. These profiles can be used to identify infection status in certain citrus cultivars, which will contribute to improved disease management and resistance breeding.
Wallis, C.M., Gorman, Z.J., Rattner, R., Hajeri, S., Yokomi, R.K. 2022. Amino acid, sugar, phenolic, and terpenoid profiles are capable of distinguishing Citrus tristeza virus infection status in citrus cultivars: Grapefruit, lemon, mandarin, and sweet orange. PLoS ONE. 17(5) Article e0268255. https://doi.org/10.1371/journal.pone.0268255.