Project Number: 2034-22000-015-000-D
Project Type: In-House Appropriated
Start Date: Apr 9, 2022
End Date: Apr 8, 2027
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.