An in vitro pipeline to screen and select citrus-associated microbiota with potential anti-Candidatus Liberibacter asiaticus properties.

Huanglongbing (HLB) is a destructive citrus disease that is lethal to all commercial citrus 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 non-culturable 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 bacteria Pantoea, Bacillus and Curtobacterium. Purified bioactive natural products with anti-CLas activity, were identified from the fungus, C. cladosporiodes. 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 functional roles of members of the microbiome with the long-term goal of developing anti-CLas 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 its associated microbiome. Using Liberibacter crescens, a culturable surrogate for the unculturable HLB-associated bacterium, Candidatus Liberibacter asiaticus (CLas), we tested the hypothesis that members of the citrus microbiome produce potential anti-CLas natural products with potential anti-CLas activity. A sub-set 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.