Plant-fed versus chemicals-fed rhizobacteria of Lucerne: Plant-only teabags culture media not only increase culturability of rhizobacteria but also recover a previously uncultured Lysobacter sp., Novosphingobium sp. and Pedobacter sp.

Abstract

In an effort to axenically culture the previously uncultivable populations of the rhizobacteria of Lucerne (Medicago sativa L.), we propose plant-only teabags culture media to mimic the nutritional matrix available in the rhizosphere. Here, we show that culture media prepared from Lucerne powder teabags substantially increased the cultivability of Lucerne rhizobacteria compared with a standard nutrient agar, where we found that the cultivable populations significantly increased by up to 60% of the total bacterial numbers as estimated by Quantitative Real-time Polymerase Chain Reaction (qRT-PCR). Cluster analysis of 16S rDNA Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) of cultivable Colony-Forming Units (CFUs) revealed a more distinct composition and separation of bacterial populations recovered on the plant-only teabags culture media than those developed on a standard nutrient agar. Further, the new plant medium gave preference to the micro-symbiont Sinorhizobium meliloti, and succeeded in isolating a number of not-yet-cultured bacteria, most closely matched to Novosphingobium sp., Lysobacter sp. and Pedobacter sp. The present study may encourage other researchers to consider moving from the well-established standard culture media to the challenging new plant-only culture media. Such a move may reveal previously hidden members of rhizobacteria, and help to further explore their potential environmental impacts.

G3 PhyloChip Analysis Confirms the Promise of Plant-Based Culture Media for Unlocking the Composition and Diversity of the Maize Root Microbiome and for Recovering Unculturable Candidate Divisions/Phyla

“…Plant materials were successfully introduced as the sole source of nutrients (plant-only-based culture media) for culturing rhizobacteria. These culture media have proved to be more advantageous in the cultivation of rhizobacteria than other standard artificial culture media ( 23 , 43 , 51 , 62 ). One of the main reasons for the success of plant-based culture media to widen the scope of culturing not-yet-cultured rhizobacteria is their particular composition of C and N compounds, and their richness in metal ions, vitamins, and cofactors ( 51 ).…”

“…Our results present conclusive evidence and strong support for the potential of using plant-based culture media to culture the plant microbiome, both for the purpose of increasing culturability and deciphering not-yet-cultured candidate phyla/divisions. In future studies, adopting novel methods associated with naturally-formulated culture media for further experiments is justified ( 23 , 56 ). Rather than focusing on the populating subsets of organism-medium matrices to elucidate the key growth principles of these unculturables ( 45 ), these methods will help to improve the success rate for recovering as-yet-uncultivated divisions in vitro in order to investigate/foster their environmental impacts.…”

“…Therefore, efforts are continually exerted towards bringing these not-yet-cultured organisms into axenic cultures by developing new growth media with various substrate compositions and modifying existing culture media and cultivation methods. These modifications include the addition of nutritional supplements and dilution of nutrient concentrations, together with the prolongation of incubation periods ( 23 , 51 ). The imitation of natural environments within culture media represents another major approach ( 6 , 17 , 19 ).…”

An inoculum-dependent culturing strategy (IDC) for the cultivation of environmental microbiomes and the isolation of novel endophytic Actinobacteria

“…To gain insights into their unknown functions and exploit their potentials, different approaches were recently introduced to culture such not-yet-cultured bacteria [2,3]. In this respect, the plantonly-based culture media were presented as natural culture media to replace myriad formulas of synthetic culture media, and strongly recommended to increase the cultivability of the plant microbiota [4][5][6][7][8][9][10]. To alleviate the stress of disproportionate nutrients, present in common culture media, we aimed at culturing maize and sunflower microbiota on the natural nutrients present in the plant inoculum itself, compared to highly diluted plant-only-based culture media and standard R2A.…”

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

“…The strategy recovered 13 genera representing the three major phyla of Firmicutes, Proteobacteria, and Actinobacteria. Representatives of these phyla are commonly reported in different compartments of plants subjected to various environments, including both biotic and abiotic stresses (Koberl et al, 2011;Marasco et al, 2012;Sarhan et al, 2016Sarhan et al, , 2018Hegazi et al, 2017).…”