Graphical 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 biomass in liquid batch cultures. These culture media were as sufficient as artificial culture media for the cultivation and recovery of the in situ rhizobacteria of barley (Hordeum murinum L.). Based on culture-dependent (CFU plate counting) and culture-independent analyses (qPCR), mowed turfgrass, in particular, supported the highest culturable population of barley endophytes, representing >16% of the total bacterial number quantified with qPCR. This accurately reflected the endophytic community composition, in terms of diversity indices (S′, H′, and D′) based on PCR-DGGE, and clustered the plant culture media together with the qPCR root populations away from the artificial culture media. Despite the promiscuous nature of the plant materials tested to culture the plant microbiome, our results indicated that plant materials of a homologous nature to the tested host plant, at least at the family level, and/or of the same environment were more likely to be selected. Plant-only-based culture media require further refinements in order to provide selectivity for the in vitro growth of members of the plant microbiome, particularly difficult-to-culture bacteria. This will provide insights into their hidden roles in the environment and support future culturomic studies.

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.

Plant microbiota support the diversity and productivity of plants. Thus, cultivation-dependent approaches are indispensable for in vitro manipulation of hub taxa. Despite recent advances in high-throughput methods, cultivability is lagging behind other environmental microbiomes, notably the human microbiome. As a plant-based culturing strategy, we developed culture media based on a broth of cooked aqueous mixtures of host plants. This improved the in vitro growth of representative isolates of plant microbiota and extended the in situ recovery of plant microbiota. With clover, 16S rRNA gene sequencing of representative isolates confirmed the predominance of Firmicutes, Alphaproteobacteria and Gammaproteobacteria, and less frequently Bacteroidetes and Actinobacteria. Whereas bovine-based culture media (modified R2A) confined the diversity to Firmicutes, the plant broth-based culture media revealed a wider scope of endophytes beyond rhizobia, i.e., multiple genera such as Chryseobacterium, Cronobacter, Kosakonia, Tsukamurella, and a potentially/presumptive novel species. Matrix-assisted laser desorption/ionization time-of-flight (MADI-TOF) analysis clustered isolates according to their plant niches, the endo-phyllosphere/endo-rhizosphere. We recommend the plant broth for simplicity, reproducibility and perdurable storage, supporting future culturomics applications, good laboratory practice (GLP) and good manufacturing practice (GMP). The strategy creates an “in-situ-similis” vegan nutritional matrix to analyze microbial diversity and reveal novel microbial resources pertinent to biotechnological and environmental applications.

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.