Abstract
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.
Highlights
Cultivation-dependent approaches are still indispensable for unlocking the treasure of environmental microbiomes for further elucidation of the ecology, physiology, and biotechnological applications of their major players (Aoi et al, 2009)
Physical pre-treatments of leaves, by punching, pressing, and/or scratching, were exercised to allow outside release of nutrients and electrolytes to support the growth of superposed bacterial inocula
There was no statistical difference in colony-forming units (CFUs) counts between the different leaf pre-treatment conditions (Figure 1); the combined effect of punching, pressing, and scratching resulted in a higher diversity of colony morphologies
Summary
Cultivation-dependent approaches are still indispensable for unlocking the treasure of environmental microbiomes for further elucidation of the ecology, physiology, and biotechnological applications of their major players (Aoi et al, 2009). Several attempts have been reported to improve the culturability of different environmental microbiota based on the modification of conventional cultivation methods and growth conditions Among such attempts were the optimization of substrate compositions and concentrations (oligotrophic media), gelling agents, incubation time, cell density, trace additives of syntrophic growth factors, as well as signaling molecules. These efforts successfully improved the cultivation of rarely isolated bacterial phyla (Nichols et al, 2008, 2010) This is in addition to the highthroughput methods that have improved cultivation capabilities and recovered uncultivated microorganisms, including diffusion chambers (Kaeberlein et al, 2002; Bollmann et al, 2007), gel microdroplet-based microfluidic systems (Manome et al, 2001; Zengler et al, 2002), and microfluidic streak plates (Jiang et al, 2016), combined with a number of micro-devices (Ingham et al, 2005, 2007). These tools allowed the characterization of new ecologically important isolates (Morris et al, 2002; Zengler et al, 2002; Ferrari et al, 2005; Nichols et al, 2008)
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