Clay chips and beads capture in situ barley root microbiota and facilitate in vitro long-term preservation of microbial strains.

Capturing the diverse microbiota from healthy and/or stress resilient plants for further preservation and transfer to unproductive and pathogen overloaded soils, might be a tool to restore disturbed plant-microbe interactions. Here, we introduce Aswan Pink Clay as a low-cost technology for capturing and storing the living root microbiota. Clay chips were incorporated into the growth milieu of barley plants and developed under gnotobiotic conditions, to capture and host the rhizospheric microbiota. Afterward, it was tested by both a culture-independent (16S rRNA gene metabarcoding) and -dependent approach. Both methods revealed no significant differences between roots and adjacent clay chips in regard total abundance and structure of the present microbiota. Clay shaped as beads adequately supported the long-term preservation of viable pure isolates of typical rhizospheric microbes, i.e. Bacillus circulans, Klebsiella oxytoca, Sinorhizobium meliloti, and Saccharomyces sp., up to 11 months stored at -20°C, 4°C, and ambient temperature. The used clay chips and beads have the capacity to capture the root microbiota and to long-term preserve pure isolates. Hence, the developed approach is qualified to build on it a comprehensive strategy to transfer and store complex and living environmental microbiota of rhizosphere toward biotechnological application in sustainable plant production and environmental rehabilitation.

"In situ similis" Culturing of Plant Microbiota: A Novel Simulated Environmental Method Based on Plant Leaf Blades as Nutritional Pads.

High-throughput cultivation methods have recently been developed to accelerate the recovery of microorganisms reluctant to cultivation. They simulate in situ environmental conditions for the isolation of environmental microbiota through the exchange of growth substrates during cultivation. Here, we introduce leaf-based culture media adopting the concept of the plant being the master architect of the composition of its microbial community. Pre-physical treatments of sunflower plant leaves, namely punching, freezing, and/or autoclavation, allowed the diffusion of electrolytes and other nutrients to configure the leaf surface as a natural pad, i.e., creating an "in situ similis" environment suitable for the growth of rarely isolated microbiota. We used surface inoculation and membrane-filtration methods to assess the culturability of endophytic bacteria from the sunflower phyllosphere and rhizosphere. Both methods supported excellent colony-forming unit (CFU) development when compared to standard R2A medium, with a special affinity to support better growth of epiphytic and endophytic populations of the phyllosphere compared with the rhizosphere. A 16S rRNA gene analysis of >122 representative isolates indicated the cultivation of a diverse set of microorganisms by application of the new methods. It indicated the predominance of 13 genera of >30 potential species, belonging to Firmicutes, Proteobacteria, and Actinobacteria, and especially genera not commonly reported for sunflower, e.g., Rhizobium, Aureimonas, Sphingomonas, Paracoccus, Stenotrophomonas, Pantoea, Kosakonia, and Erwinia. The strategy successfully extended diversity and richness in the endophyllosphere compared to the endorhizosphere, while CFUs grown on the standard R2A medium mainly pertain to Firmicutes, especially Bacillus spp. MALDI-TOF MS analysis clustered the isolates according to their niche and potential functions, where the majority of isolates of the endorhizosphere were clustered away from those of the endophyllosphere. Isolates identified as Gammaproteobacteria and Alphaproteobacteria were distinguishably sub-clustered, which was in contrast to the heterogeneous isolates of Firmicutes (Bacillus spp.). In conclusion, leaf in situ similis cultivation is an effective strategy to support the future application of culturomics of plant microbiota. This is an effort to access novel isolates that are more adapted and competitive in their natural environments, especially those subjected to abiotic stresses like those prevailing in arid/semi-arid zones, and, consequently, to support the application of agro-biotechnologies, among other technologies, to improving agriculture in such zones.