Nabil Hegazi

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.

Although plant-based culture media enhances in vitro cultivation of rhizobacteria, studies assessing their biomass potential for large-scale applications are lacking. Here, we advance plant pellets (PPs) as a novel technology to unlock the potential of such vegan culture media for biomass production of Rhizobium leguminosarum. PP formulations were based on mixtures of Egyptian clover powder and the agro-byproducts glycerol and molasses. These mixtures were either contained or not contained in teabags during culture media preparation. Metrics of biomass included colony forming units, optical density (OD600nm), and cell dry weight (DW). Biomass comparisons between culture media based on PPs and standard yeast extract mannitol (YEM) revealed that the following PPs composition, contained in teabags, cultivated rhizobia at levels comparable to YEM: 16 g clover powder, 5% molasses, and 0.8% glycerol. This PPs composition enabled shorter generation times of rhizobia (PP: 3.83 h, YEM: 4.28 h). Strikingly, PPs mixtures supplemented with 10% molasses and not contained in teabags promoted rhizobia without apparent lag phases and produced 25% greater DW than YEM. PPs potentiate the use of dehydrated vegan feedstocks for both plant microbiota cultivation and biomass production and appear as cost- and labor-effective tools, easy to handle and store for plant-based culture media preparation.