An In Vitro Pipeline for Screening and Selection of Citrus-Associated Microbiota with Potential Anti-“ Candidatus Liberibacter asiaticus” Properties

Abstract

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.

Defeating Huanglongbing Pathogen Candidatus Liberibacter asiaticus With Indigenous Citrus Endophyte Bacillus subtilis L1-21

“…However, biocontrol of plant diseases can be the most promising alternative to continuously failing treatments and endophyte-mediated biocontrol offers consistent results due to intimate association with its host and shared niche with the pathogen where endophyte could fully manifest their antagonistic potential, whereas the non-endophytic biocontrol agents often fail under field conditions due to fierce competition in the rhizosphere and harsh environmental conditions (Ahmed et al, 2020;Blacutt et al, 2020;Trivedi et al, 2020). In biocontrol programs, native endophytes could be used to maintain long-term colonization inside the citrus host and eliminate CLas through niche and nutrient competition (Munir et al, 2018b).…”

Microbial turnover and dispersal events occur in sync with plant phenology in the perennial evergreen tree crop, Citrus sinensis

“…The copyright holder for this preprint this version posted February 12, 2022. ; https://doi.org/10.1101/2022.02.10.480024 doi: bioRxiv preprint a perennial arboreal crop system [21][22][23][24][25] . Due to its well-defined phenology, citrus is an ideal system to investigate the interplay between host phenology and microbial community composition.…”

Bacteriomic Analyses of Asian Citrus Psyllid and Citrus Samples Infected With “Candidatus Liberibacter asiaticus” in Southern California and Huanglongbing Management Implications

“…Similarly, citrus-associated bacteria (CABacts) were also reported (Blaustein et al, 2017;Ginnan et al, 2018). CABacts identified after surface sterilization are referred to as endophytes, e.g., Pantoea, Bacillus, and Curtobacterium (Blacutt et al, 2020). The biological roles of most endophytic bacteria vary from beneficial to detrimental (Lodewyckx et al, 2002).…”