3 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
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Regulation of the RCS Phosphorelay by the Redox Responsive Transcription Factor, OxyR and the Role of a RTX-Like Toxin in Biofilm Formation for the Bacterial Plant Pathogen, Pantoea Stewartii
Pantoea stewartii subspecies stewartii (Pnss) causes Stewart’s Wilt in sweet corn and maize, which is characterized by wilting, caused by the formation of exopolysaccharide (EPS)-based biofilms that block the xylem, and the formation of water-soaked lesions. These lesions can form via Pnss-induced expression of a repeat-in-toxin (RTX)-like protein called RTX2. Deleting rtx2 prevents water-soaked lesion formation and effective plant colonization. The rtx2 gene is in an operon with a second gene that encodes another putative rtx-like toxin (rtx1), and two genes that encode the phosphotransferase and the response regulator of the Regulator of Capsular Synthesis (Rcs) phosphorelay, which regulates gene expression via external stimuli. This operon also has a predicted upstream OxyR transcription factor binding site. Water-soaked lesions contain lethal Reactive Oxygen Species (ROS), and previous work by the Roper lab demonstrated that OxyR protects Pnss against ROS while deletion of oxyR caused increased H2O2 sensitivity and negated EPS production. As the operon contains genes for the Rcs phosphorelay, it is hypothesized that ROS promotes activity of OxyR, thereby inducing gene expression of this operon. Indeed, results of this study show OxyR does bind to the predicted binding site of the rtx1/rtx2/rcsD/rcsB operon, and induces its activity. Furthermore, sublethal concentrations of ROS induce gene expression of certain components of the operon in wild type Pnss, but not in an oxyR deletion mutant. Therefore, ROS appears to be a signal that induces expression of key components of the Rcs phosphorelay via OxyR. The Rcs phosphorelay also plays a key role in cell shape and membrane integrity, and results of this study show that RTX2 affects these cellular characteristics as well, wherein the deletion of rtx2 causes decreased cell length, increased sensitivity to Polymyxin B, and increased overall cellular hydrophobicity when compared to wild type. RTX2-dependent alteration of the cell envelope also causes increased biofilm formation activity as Pnss transitions between the apoplastic and xylem phases of Stewart’s Wilt. Furthermore, RTX2 is required for both adhesion and biofilm formation in-planta, and impacts biofilm height in a non-EPS producing background (mimicking the initial stages of biofilm formation) in-vitro
Recommended from our members
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