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

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

Medicinal Plants and Their Bacterial Microbiota: A Review on Antimicrobial Compounds Production for Plant and Human Health

“…This may be due, for example, to a different balance between nutrients in comparison to that found in plant tissues and/or to the missed establishment of bacterial consortia able to support the growth of all species. At this regard, it seems promising the use of in situ similis culturing aiming to mimic the complex environmental composition observed in plant tissues [ 40 ]. Indeed, Nemr and colleagues have demonstrated that using either leaves as solid substrate or media obtained upon infusion of leaves, the diversity of isolated bacteria results increased in comparison to the use of the standard medium R2A as control.…”

Culture Media Based on Leaf Strips/Root Segments Create Compatible Host/Organ Setup for in vitro Cultivation of Plant Microbiota

“…In fact, such plant-only-based culture media proved to be competitive to replace several synthetic culture media, and to increase the cultivability of the plant microbiota. With the aid of culture-dependent and independent techniques, we were able to enrich previously not yet cultured bacteria (Sarhan et al, 2016(Sarhan et al, , 2018(Sarhan et al, , 2020Hegazi et al, 2017;Nemr et al, 2020). Further, we later introduced the intact leaf-based culturing strategy that allowed the direct inoculation and cultivation of microorganisms on the leaf surfaces in the form of culture pads, following the rationale of "natural environments envelop multiplex of nutrients necessary for the growth of their inherent microbiota" (Nemr et al, 2020).…”

“…With the aid of culture-dependent and independent techniques, we were able to enrich previously not yet cultured bacteria (Sarhan et al, 2016(Sarhan et al, , 2018(Sarhan et al, , 2020Hegazi et al, 2017;Nemr et al, 2020). Further, we later introduced the intact leaf-based culturing strategy that allowed the direct inoculation and cultivation of microorganisms on the leaf surfaces in the form of culture pads, following the rationale of "natural environments envelop multiplex of nutrients necessary for the growth of their inherent microbiota" (Nemr et al, 2020). Here, majority of plant nutrients are supplied to the growing microorganisms in their natural/proportionate concentrations and gradients, (Nemr et al, 2020).…”

“…Further, we later introduced the intact leaf-based culturing strategy that allowed the direct inoculation and cultivation of microorganisms on the leaf surfaces in the form of culture pads, following the rationale of "natural environments envelop multiplex of nutrients necessary for the growth of their inherent microbiota" (Nemr et al, 2020). Here, majority of plant nutrients are supplied to the growing microorganisms in their natural/proportionate concentrations and gradients, (Nemr et al, 2020). Exemplarily for sunflower (Helianthus annuus L.) such strategy, named "leaf in situ similis cultivation, " allowed the recovery of microorganisms reluctant for cultivation and extended the cultivable diversity from the endo-phyllosphere and the endo-rhizosphere to members of Firmicutes, Proteobacteria and Actinobacteria.…”

“…Exemplarily for sunflower (Helianthus annuus L.) such strategy, named "leaf in situ similis cultivation, " allowed the recovery of microorganisms reluctant for cultivation and extended the cultivable diversity from the endo-phyllosphere and the endo-rhizosphere to members of Firmicutes, Proteobacteria and Actinobacteria. As well, culturing strains of genera not commonly reported for sunflower, e.g., Aureimonas, Sphingomonas, Paracoccus, Kosakonia, and Erwinia, was achieved using such in situ similis cultivation technique (Nemr et al, 2020). However, there are still numerous genera (158), which already were detected using 16S rRNA metagenomic sequencing techniques in sunflower, but were not yet cultured in vitro (Tamošiune et al, 2020).…”