Skip to main content
eScholarship
Open Access Publications from the University of California

Scholarly Works (11 results)

  • Thesis
  • Peer Reviewed
UC Riverside Electronic Theses and Dissertations (2020)

Pivotal advances in sequencing and bioinformatics revealed that extensive microbial communities inhabit terrestrial plants. Microbiota extend beyond the plant host genetics to provide vital traits and services, including microbiome-mediated disease resistance, nutrient acquisition, and tolerance to changing climates. The development of engineered microbial based plant therapies could help sustainably enhance plant health and improve yield. To gain insight on plant associated microbiomes and contribute to evolving agrosystem management strategies we focused on connecting microbial populations with plant disease outcomes, phenological stages, and community self-regulation.

Considering the urgent demand for novel treatments to combat citrus Huanglongbing (HLB) disease, which continues to devastate the global citrus industry, we directed our efforts on perennial tree crop hosts from the Citrus genus. Using empirical dataset produced through metabarcoding amplicon-based sequencing we concluded that disease status and phenological stage are significant driving factors of bacterial and fungal leaf and root communities.

We provide evidence that pathogens can induce plant dysbiosis followed by a microbiome-mediated immune response and that cyclical community assembly is governed by classic community ecological mechanisms: selection, dispersal, and speciation. Correlations between community structure and host phenotypes, suggest that microbiota functionally contribute to the rate of HLB symptom development and host phenological plasticity. Additionally, laboratory based gnotobiotic experimentation with a synthetic community indicated that competition influences microbial niche selection and the structure of the microbiome.

Furthermore, we have built avenues for discovering and delivering beneficial citrus associated microbiota. Specifically, we established a citrus microbial culture collection, a microbial active metabolite discovery pipeline, compared product delivery methods, and developed a microbiota-free citrus system in an ongoing effort to unravel host-microbiome and inter-microbiome interactions. These tools and our identification of beneficial microbial candidates encourage future research projects. Overall, this dissertation advances basic knowledge of plant microbiome assembly and symbiotic relationships between perennial plants and microbial communities.

    Cover page: Pathogens, Plant Phenology, and Microbial Competition Impact the Structure and Function of the Citrus MicrobiomeCreative Commons 'BY' version 4.0 license
    • Thesis
    • Peer Reviewed
    UC Riverside Electronic Theses and Dissertations (2016)

    Xylella fastidiosa is a xylem-limited, insect transmitted bacterium that causes fatal diseases in economically important crops, most notably, Pierce’s disease of grapevine. Due to its unique dual lifestyle, X. fastidiosa utilizes different mechanisms to facilitate colonization of its plant and insect hosts. While research has focused heavily on the role of surface-associated proteins, we establish the major cell surface carbohydrate, lipopolysaccharide (LPS), as an important bacterial virulence factor and modulator of innate immunity in grapevine.

    We focused our attention on the terminal O antigen moiety of the LPS molecule, which serves as a key point of contact between potential hosts and the environment. Biochemical and structural analysis identified the major O antigen polysaccharide as a linear α1-2 linked rhamnan polymer. By targeting an O antigen polymerase (Wzy), we found that loss of the rhamnose-rich O antigen considerably altered bacterial adhesion dynamics in the blue-green sharpshooter vector. Scanning electron microscopy confirmed that this defect in initial attachment compromised subsequent biofilm formation within vector foreguts, thus impairing pathogen acquisition. During the plant host-pathogen interaction, successful pathogens must overcome plant immune responses to establish and cause disease. Due to the fact that X. fastidiosa lacks the quintessential type III secretion system, and its arsenal of effectors, we sought to determine the contribution of O antigen to this complex biological process. Using RNAseq, we found that in intact cells, O antigen functions as a shield; this allows X. fastidiosa to delay early recognition by the grapevine immune system, thus promoting bacterial survival and the establishment of infection.

    The results presented in this dissertation provide a comprehensive understanding of the composition and structure of this major cell surface polysaccharide and offer valuable insight into its diverse functions. Specifically, the studies detailing the implication of O antigen in the evasion of basal defense responses in grapevine present a novel and unprecedented mechanism employed by this unique xylem-dwelling bacterial pathogen.

      Cover page: Structural and Functional Characterization of the Xylella fastidiosa O Antigen: Contribution to Bacterial Virulence, Insect Transmission, and Modulation of Innate Immune Recognition in Grapevine
      • Article
      • Peer Reviewed
      UC Davis Previously Published Works (2017)
      Bacterial plant pathogens often encounter reactive oxygen species (ROS) during host invasion. In foliar bacterial pathogens, multiple regulatory proteins are involved in the sensing of oxidative stress and the activation of the expression of antioxidant genes. However, it is unclear whether xylem-limited bacteria, such as Xylella fastidiosa, experience oxidative stress during the colonization of plants. Examination of the X. fastidiosa genome uncovered only one homologue of oxidative stress regulatory proteins, OxyR. Here, a knockout mutation in the X. fastidiosa oxyR gene was constructed; the resulting strain was significantly more sensitive to hydrogen peroxide (H2 O2 ) relative to the wild-type. In addition, during early stages of grapevine infection, the survival rate was 1000-fold lower for the oxyR mutant than for the wild-type. This supports the hypothesis that grapevine xylem represents an oxidative environment and that X. fastidiosa must overcome this challenge to achieve maximal xylem colonization. Finally, the oxyR mutant exhibited reduced surface attachment and cell-cell aggregation and was defective in biofilm maturation, suggesting that ROS could be a potential environmental cue stimulating biofilm development during the early stages of host colonization.
        Cover page: Tolerance to oxidative stress is required for maximal xylem colonization by the xylem‐limited bacterial phytopathogen, Xylella fastidiosa
        • Article
        • Peer Reviewed
        UC Riverside Previously Published Works (2023)
        The citrus root and rhizosphere microbiomes have been relatively well described in the literature, especially in the context of Huanglonbing disease. Yet questions addressing the assembly of root microbial endophytes have remained unanswered. In the above ground tree tissues, leaves and stems have been the research focus point, while flush and flower microbiomes, two important tissues in the vegetative and reproductive cycles of the tree, are not well described. In this study, the fungal and bacterial taxa in five biocompartments (bulk soil, rhizosphere, root endosphere, flower and flush) of citrus trees grown in a single California orchard were profiled using an amplicon-based metagenomic Illumina sequencing approach. Trees with no observable signs of abiotic or biotic stresses were sampled for two consecutive years during the floral development phase. The rhizosphere was the most biodiverse compartment compared to bulk soil, root endosphere, flower and flush microbiomes. In addition, the belowground bacteriome was more diverse than the mycobiome. Microbial richness decreased significantly from the root exosphere to the endosphere and was overall low in the above ground tissues. Root endophytic microbial community composition shared strong similarities to the rhizosphere but also contained few taxa from above ground tissues. Our data indicated compartmentalization of the microbiome with distinct profiles between above and below ground microbial communities. However, several taxa were present across all compartments suggesting the existence of a core citrus microbiota. These findings highlight key microbial taxa that could be engineered as biopesticides and biofertilizers for citriculture.
          Cover page: Microbiome diversity, composition and assembly in a California citrus orchard
          • Article
          • Peer Reviewed
          UC Davis Previously Published Works (2021)
          Pierce's disease (PD) in grapevine (Vitis vinifera) is caused by the bacterial pathogen Xylella fastidiosa. X. fastidiosa is limited to the xylem tissue and following infection induces extensive plant-derived xylem blockages, primarily in the form of tyloses. Tylose-mediated vessel occlusions are a hallmark of PD, particularly in susceptible V. vinifera. We temporally monitored tylose development over the course of the disease to link symptom severity to the level of tylose occlusion and the presence/absence of the bacterial pathogen at fine-scale resolution. The majority of vessels containing tyloses were devoid of bacterial cells, indicating that direct, localized perception of X. fastidiosa was not a primary cause of tylose formation. In addition, we used X-ray computed microtomography and machine-learning to determine that X. fastidiosa induces significant starch depletion in xylem ray parenchyma cells. This suggests that a signalling mechanism emanating from the vessels colonized by bacteria enables a systemic response to X. fastidiosa infection. To understand the transcriptional changes underlying these phenotypes, we integrated global transcriptomics into the phenotypes we tracked over the disease spectrum. Differential gene expression analysis revealed that considerable transcriptomic reprogramming occurred during early PD before symptom appearance. Specifically, we determined that many genes associated with tylose formation (ethylene signalling and cell wall biogenesis) and drought stress were up-regulated during both Phase I and Phase II of PD. On the contrary, several genes related to photosynthesis and carbon fixation were down-regulated during both phases. These responses correlate with significant starch depletion observed in ray cells and tylose synthesis in vessels.
            Cover page: Xylella fastidiosa causes transcriptional shifts that precede tylose formation and starch depletion in xylem.
            • Article
            • Peer Reviewed
            UC Riverside Previously Published Works (2020)
            Plant microbiomes are critical components to plant health and can influence disease outcomes. We provide empirical data describing disease-induced shifts within the citrus microbiome at different levels of huanglongbing (HLB) disease severity. HLB is associated with an invasive phloem-limited bacterium, ‘Candidatus Liberibacter asiaticus’, that is introduced into the aerial portions of the tree by an insect vector. Disease manifests as aboveground foliar and fruit symptoms and significant root decline belowground. During the early phase of disease, there were depletions of putative keystone taxa in leaves and roots, followed by enrichments of putative beneficial taxa, suggesting a microbially derived immune response involved in plant protection that is ancillary to immune components encoded in the plant’s genome. In the late phase of disease, we observed enrichments of parasitic and saprophytic microorganisms, particularly in the roots. The community shifts within the root compartment are emblematic of a disease-induced dysbiosis where pathogens other than ‘Ca. L. asiaticus’ begin to dominate the community. Furthermore, we define key taxa enriched in trees with a slower rate of disease development, referred to as survivor trees, that are hallmarks of those found in trees in the early phase of disease that may be drivers of the survivor tree phenotype. We propose a disease ecology model that illustrates the relationship between the pathogen, the microbiome, and the host plant that highlights microorganisms that may serve as disease facilitators or antagonists.
              Cover page: Disease-Induced Microbial Shifts in Citrus Indicate Microbiome-Derived Responses to Huanglongbing Across the Disease Severity Spectrum
              • Article
              • Peer Reviewed
              UC Riverside Previously Published Works (2015)
              Hemipteran insect vectors transmit the majority of plant pathogens. Acquisition of pathogenic bacteria by these piercing/sucking insects requires intimate associations between the bacterial cells and insect surfaces. Lipopolysaccharide (LPS) is the predominant macromolecule displayed on the cell surface of Gram-negative bacteria and thus mediates bacterial interactions with the environment and potential hosts. We hypothesized that bacterial cell surface properties mediated by LPS would be important in modulating vector-pathogen interactions required for acquisition of the bacterial plant pathogen Xylella fastidiosa, the causative agent of Pierce's disease of grapevines. Utilizing a mutant that produces truncated O antigen (the terminal portion of the LPS molecule), we present results that link this LPS structural alteration to a significant decrease in the attachment of X. fastidiosa to blue-green sharpshooter foreguts. Scanning electron microscopy confirmed that this defect in initial attachment compromised subsequent biofilm formation within vector foreguts, thus impairing pathogen acquisition. We also establish a relationship between O antigen truncation and significant changes in the physiochemical properties of the cell, which in turn affect the dynamics of X. fastidiosa adhesion to the vector foregut. Lastly, we couple measurements of the physiochemical properties of the cell with hydrodynamic fluid shear rates to produce a Comsol model that predicts primary areas of bacterial colonization within blue-green sharpshooter foreguts, and we present experimental data that support the model. These results demonstrate that, in addition to reported protein adhesin-ligand interactions, O antigen is crucial for vector-pathogen interactions, specifically in the acquisition of this destructive agricultural pathogen.
                Cover page: O Antigen Modulates Insect Vector Acquisition of the Bacterial Plant Pathogen Xylella fastidiosa
                • Article
                • Peer Reviewed
                UC Davis Previously Published Works (2018)
                Lipopolysaccharides (LPS) are among the known pathogen-associated molecular patterns (PAMPs). LPSs are potent elicitors of PAMP-triggered immunity (PTI), and bacteria have evolved intricate mechanisms to dampen PTI. Here we demonstrate that Xylella fastidiosa (Xf), a hemibiotrophic plant pathogenic bacterium, possesses a long chain O-antigen that enables it to delay initial plant recognition, thereby allowing it to effectively skirt initial elicitation of innate immunity and establish itself in the host. Lack of the O-antigen modifies plant perception of Xf and enables elicitation of hallmarks of PTI, such as ROS production specifically in the plant xylem tissue compartment, a tissue not traditionally considered a spatial location of PTI. To explore translational applications of our findings, we demonstrate that pre-treatment of plants with Xf LPS primes grapevine defenses to confer tolerance to Xf challenge.
                  Cover page: Lipopolysaccharide O-antigen delays plant innate immune recognition of Xylella fastidiosa
                  • Article
                  • Peer Reviewed
                  UC Riverside Previously Published Works (2023)
                  The genomes of eighteen Fusarium isolates cultured from diseased and healthy citrus trees were sequenced, assembled, and annotated. Isolate species identification was confirmed using single marker (TEF1-alpha) phylogenetic assessment. Studies of the traits and genotypes of plant-associated isolates are important to understanding the fungal contribution to phytobiomes of citrus.
                    Cover page: Genome Sequence and Assembly of 18 Fusarium Isolates from Florida Citrus under High Huanglongbing Disease Pressure and California Citrus under Low Huanglongbing Disease PressureCreative Commons 'BY' version 4.0 license
                    • Article
                    • Peer Reviewed
                    UC Riverside Previously Published Works (2020)
                    Huanglongbing (HLB) is a destructive citrus disease that is lethal to all commercial citrus plants, making it the most serious citrus disease and one of the most serious plant diseases. Because of the severity of HLB and the paucity of effective control measures, we structured this study to encompass the entirety of the citrus microbiome and the chemistries associated with that microbial community. We describe the spatial niche diversity of bacteria and fungi associated with citrus roots, stems, and leaves using traditional microbial culturing integrated with culture-independent methods. Using the culturable sector of the citrus microbiome, we created a microbial repository using a high-throughput bulk culturing and microbial identification pipeline. We integrated an in vitro agar diffusion inhibition bioassay into our culturing pipeline that queried the repository for antimicrobial activity against Liberibacter crescens, a culturable surrogate for the nonculturable "Candidatus Liberibacter asiaticus" bacterium associated with HLB. We identified microbes with robust inhibitory activity against L. crescens that include the fungi Cladosporium cladosporioides and Epicoccum nigrum and bacterial species of Pantoea, Bacillus, and Curtobacterium Purified bioactive natural products with anti-"Ca. Liberibacter asiaticus" activity were identified from the fungus C. cladosporioides Bioassay-guided fractionation of an organic extract of C. cladosporioides yielded the natural products cladosporols A, C, and D as the active agents against L. crescens This work serves as a foundation for unraveling the complex chemistries associated with the citrus microbiome to begin to understand the functional roles of members of the microbiome, with the long-term goal of developing anti-"Ca Liberibacter asiaticus" bioinoculants that thrive in the citrus holosystem.IMPORTANCE Globally, citrus is threatened by huanglongbing (HLB), and the lack of effective control measures is a major concern of farmers, markets, and consumers. There is compelling evidence that plant health is a function of the activities of the plant's associated microbiome. Using Liberibacter crescens, a culturable surrogate for the unculturable HLB-associated bacterium "Candidatus Liberibacter asiaticus," we tested the hypothesis that members of the citrus microbiome produce potential anti-"Ca Liberibacter asiaticus" natural products with potential anti-"Ca Liberibacter asiaticus" activity. A subset of isolates obtained from the microbiome inhibited L. crescens growth in an agar diffusion inhibition assay. Further fractionation experiments linked the inhibitory activity of the fungus Cladosporium cladosporioides to the fungus-produced natural products cladosporols A, C, and D, demonstrating dose-dependent antagonism to L. crescens.
                      Cover page: An In Vitro Pipeline for Screening and Selection of Citrus-Associated Microbiota with Potential Anti-“Candidatus Liberibacter asiaticus” Properties
                      Top