In vitro domestication of halophyte microbiota for future SynCom application

Abstract

Background and aims: Microbiome-mediated strategies for future stressed-agriculture entail exploration of repertoires of halophyte microbiota. Culturomics strategies are advanced to improve culturability and extend diversity of microbiota of Salicornia europaea L. Methods: The plant broth-based-seawater-culture medium (PBSW) was advanced for in vitro domestication of microbiota of endo-rhizosphere/endo-phyllosphere of S. europaea. Populations (Colony Forming Units, CFUs) and biomass production (Optical Density, OD) were monitored throughout successive steps of in vitro cultivation/domestication in liquid batch cultures. Culture-dependent methods were applied to cultivate and identify (16S rRNA gene sequencing) representative isolates; and culture-independent analyses (DGGE/qPCR) for community composition. Results: PBSW supported higher CFUs counts; and related to 16S rRNA gene copy numbers (qPCR), increased (> 40 fold) culturability compared to NaCl-salted-standard culture medium. Successive in vitro domestication/batch cultures boosted bacterial growth, diminished differences among tested culture media and shortened doubling times (DT). PCR-DGGE showed divergence in culturable community composition primarily attributed to culture media. 16S rRNA gene sequencing of representative isolates indicated: a) greater diversity in endo-phyllosphere than endo-rhizosphere; b) abundant phyla were Pseudomonadota/Bacillota /Actinomycetota; c) dominance of Halomonas among 15 genera identified; d) Gracilibacillus, Metabacillus, Mixta, Salinicoccus, Zhihengliuella, Marinobacter, Marinimicrobium and Planomicrobium were first reported/cultivated for S. europaea. In vitro domestication resulted in dominance of genera of Pseudomonadota/Bacillota for endo-phyllosphere and Halomonas sp. of Pseudomonadota for endo-rhizosphere. Conclusion: PBSW created in situ similis milieu for cultivation of halophyte bacteria, and enabled in vitro domestication for propagating microbiota, instead of laborious construction of consortia of single isolates, for future SynCom applications.