9 research outputs found
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An In Vitro Pipeline for Screening and Selection of Citrus-Associated Microbiota with Potential Anti-"Candidatus Liberibacter asiaticus" Properties.
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
Microbial Turnover and Dispersal Events Occur in Synchrony with Plant Phenology in the Perennial Evergreen Tree Crop Citrus sinensis.
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Pathogens, Plant Phenology, and Microbial Competition Impact the Structure and Function of the Citrus Microbiome
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
Disease-Induced Microbial Shifts in Citrus Indicate Microbiome-Derived Responses to Huanglongbing Across the Disease Severity Spectrum
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Microbial Turnover and Dispersal Events Occur in Synchrony with Plant Phenology in the Perennial Evergreen Tree Crop Citrus sinensis.
Emerging research indicates that plant-associated microbes can alter plant developmental timing. However, it is unclear if host phenology affects microbial community assembly. Microbiome studies in annual or deciduous perennial plants face challenges in separating effects of tissue age from phenological driven effects on the microbiome. In contrast, evergreen perennial trees, like Citrus sinensis, retain leaves for years, allowing for uniform sampling of similarly aged leaves from the same developmental cohort. This aids in separating phenological effects on the microbiome from impacts due to annual leaf maturation/senescence. Here, we used this system to test the hypothesis that host phenology acts as a driver of microbiome composition. Citrus sinensis leaves and roots were sampled during seven phenological stages. Using amplicon-based sequencing, followed by diversity, phylogenetic, differential abundance, and network analyses, we examined changes in bacterial and fungal communities. Host phenological stage is the main determinant of microbiome composition, particularly within the foliar bacteriome. Microbial enrichment/depletion patterns suggest that microbial turnover and dispersal were driving these shifts. Moreover, a subset of community shifts were phylogenetically conserved across bacterial clades, suggesting that inherited traits contribute to microbe-microbe and/or plant-microbe interactions during specific phenophases. Plant phenology influences microbial community composition. These findings enhance understanding of microbiome assembly and identify microbes that potentially influence plant development and reproduction. IMPORTANCE Research at the forefront of plant microbiome studies indicates that plant-associated microbes can alter the timing of plant development (phenology). However, it is unclear if host phenological stage affects microbial community assembly. Microbiome studies in annual or deciduous perennial plants can face difficulty in separating effects of tissue age from phenological driven effects on the microbiome. Evergreen perennial plants, like sweet orange, maintain mature leaves for multiple years, allowing for uniform sampling of similarly aged tissue across host reproductive stages. Using this system, multiyear sampling, and high-throughput sequencing, we identified plant phenology as a major driver of microbiome composition, particularly within the leaf-associated bacterial communities. Distinct changes in microbial patterns suggest that microbial turnover and dispersal are mechanisms driving these community shifts. Additionally, closely related bacteria have similar abundance patterns across plant stages, indicating that inherited microbial traits may influence how bacteria respond to host developmental changes. Overall, this study illustrates that plant phenology does indeed govern microbiome seasonal shifts and identifies microbial candidates that may affect plant reproduction and development
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Genome Sequence and Assembly of 18 Fusarium Isolates from Florida Citrus under High Huanglongbing Disease Pressure and California Citrus under Low Huanglongbing Disease Pressure
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
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Disease-Induced Microbial Shifts in Citrus Indicate Microbiome-Derived Responses to Huanglongbing Across the Disease Severity Spectrum
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
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An In Vitro Pipeline for Screening and Selection of Citrus-Associated Microbiota with Potential Anti-"Candidatus Liberibacter asiaticus" Properties.
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