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.
This study presents a sensor strip for user-friendly, naked-eye detection of Xylella fasitdiosa, the bacterial causal agent of Pierce's disease in grapevine. This sensor uses anti- X. fastidiosa antibodies conjugated to a polydiacetylene layer on a polyvinylidene fluoride strip to generate specific color transitions and discriminate levels of the pathogen. The detection limit of the sensor is 0.8 × 10 cells/mL, which is similar to bacterial load in grapevine 18 days following bacterial inoculation. This sensor enables equipment-free detection that is highly desirable for in-field diagnostic tools in resource-limited settings.
Pantoea stewartii subsp. stewartii is the etiological agent of Stewart's wilt and is a serious bacterial pathogen affecting sweet corn. During the leaf blight phase, P. stewartii colonizes the leaf apoplast and causes a characteristic water-soaked lesion. The Hrp type III secretion system has been implicated in the water-soaking phenotype, and the goal of this study was to investigate other potential factors that contribute to the plant cellular disruption associated with these lesions. The P. stewartii genome contains a gene encoding a large repetitive RTX toxin, designated rtx2. RTX toxins comprise a large family of pore-forming proteins, which are widely distributed among gram-negative bacteria. These cytotoxins usually lyse their target host cells and cause significant tissue damage as a consequence. We hypothesized that this RTX-like toxin plays a role in the water-soaking phase of infection due to its predicted cytolytic properties. Based on the data reported here, we conclude that RTX2 contributes significantly to the development of water-soaked lesions and leakage of plant cellular contents and is an important pathogenicity factor for P. stewartii.
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