Plant Materials are Sustainable Substrates Supporting New Technologies of Plant-Only-Based Culture Media for <i>in vitro</i> Culturing of the Plant Microbiota
Abstract:In order to improve the culturability and biomass production of rhizobacteria, we previously introduced plant-only-based culture media. We herein attempted to widen the scope of plant materials suitable for the preparation of plant-only-based culture media. We chemically analyzed the refuse of turfgrass, cactus, and clover. They were sufficiently rich to support good in vitro growth by rhizobacteria isolates representing Proteobacteria and Firmicutes. They were also adequate and efficient to produce a cell bio… Show more
“…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.…”
The recent introduction of plant-only-based culture media enabled cultivation of not-yet-cultured bacteria that exceed 90% of the plant microbiota communities. Here, we further prove the competence and challenge of such culture media, and further introduce "the inoculum-dependent culturing strategy, IDC". The strategy depends on direct inoculating plant serial dilutions onto plain water agar plates, allowing bacteria to grow only on the expense of natural nutrients contained in the administered inoculum. Developed colonies are successively transferred/subcultured onto plant-only-based culture media, which contains natural nutrients very much alike to those found in the prepared plant inocula. Because of its simplicity, the method is recommended as a powerful tool in screening programs that require microbial isolation from a large number of diverse plants. Here, the method comfortably and successfully recovered several isolates of endophytic Actinobacteria represented by the six genera of Curtobacterium spp., Plantibacter spp., Agreia spp., Herbiconiux spp., Rhodococcus spp., and Nocardioides spp. Furthermore, two of the isolates are most likely novel species belonging to Agreia spp. and Herbiconiux spp.
“…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.…”
The recent introduction of plant-only-based culture media enabled cultivation of not-yet-cultured bacteria that exceed 90% of the plant microbiota communities. Here, we further prove the competence and challenge of such culture media, and further introduce "the inoculum-dependent culturing strategy, IDC". The strategy depends on direct inoculating plant serial dilutions onto plain water agar plates, allowing bacteria to grow only on the expense of natural nutrients contained in the administered inoculum. Developed colonies are successively transferred/subcultured onto plant-only-based culture media, which contains natural nutrients very much alike to those found in the prepared plant inocula. Because of its simplicity, the method is recommended as a powerful tool in screening programs that require microbial isolation from a large number of diverse plants. Here, the method comfortably and successfully recovered several isolates of endophytic Actinobacteria represented by the six genera of Curtobacterium spp., Plantibacter spp., Agreia spp., Herbiconiux spp., Rhodococcus spp., and Nocardioides spp. Furthermore, two of the isolates are most likely novel species belonging to Agreia spp. and Herbiconiux spp.
“…Our previous research introduced and documented the challenge of plant-only-based culture media for increasing in vitro cultivability of rhizobacteria [32][33][34][35]37]. Here, the developed plant pellets represent a novel application of plant-only-based culture media potentiating the use of plant materials for value-added biomass production of rhizobacteria on a large scale.…”
Section: Discussionmentioning
confidence: 99%
“…In view of our previous publications [32][33][34][35]37], we set up a number of preliminary experiments to assess the growth of Rhizobium leguminosarum and biomass production on the expense of increasing concentrations of dehydrated clover plant powder and boosting effects of supplementation with rich and available agro-byproducts of local molasses and glycerol in various concentrations. Then, the main experiments were designed to measure the suitability of supplemented concentrations of molasses and glycerol not only to enrich the plant material but also to formulate stable and consistent plant pellets.…”
Section: Experimental Designmentioning
confidence: 99%
“…In particular, our recent work demonstrated that culture media prepared from the juices or slurries of Trifolium alexandrinum, Paspalum vaginatum, and Opuntia ficus-indica support in vitro growth and biomass production of a number of model rhizobacteria [32]. In addition, innovations to facilitate the application of plant-only-based culture in the form of plant powder teabags resulted in improving in vitro cultivability of rhizobacteria and also allowed investigations on their diversity and, to a lesser extent, the biomass potential [33][34][35][36]. Taken together, utilization of plant materials for culture media preparation offers a promising and relatively inexpensive strategy towards biomass production of agro-biopreparates.…”
Section: Introductionmentioning
confidence: 99%
“…While plant materials in the forms of juices, slurries, and powders in teabags have been used for the application of plant-only-based culture media to improve in vitro cultivability of rhizobacteria [33][34][35][36], no studies have (1) directly assessed the potential of such media to produce biomass and (2) integrated the rich nutrient stores of plant materials with agro-byproducts to produce value-added biomass.…”
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.
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