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Plant microbiota have co-evolved with their associated plants in the entire holobiont, and their assemblages support diversity, health and productivity on our planet. Of importance is the in vitro cultivation and identification of their hub taxa for possible core microbiome modification. Therefore, we recently introduced the in situ-similis culturing strategy, based on the use of plant leaves as platform for in vitro growth of plant microbiota. Here, we augment the strategy by the incorporation of the Most Probable Number (MPN) method, based on the use of leaf strips and root segments immersed in plain semi-solid water agar, as growth milieu. Cultivability of bacterial endophytes of sunflower plants (Helianthus annuus L.) was monitored in terms of MPN population estimates, community composition through PCR-DGGE analysis and 16S rRNA gene sequencing of representative isolates. The method efficiently enriched the plant endophytes as MPN estimates exceeded log 7.0 g-1 leaf/root. Results of PCR-DGGE analyses indicated divergence in community composition of cultivable bacterial endophytes primarily attributed to the type of culture media, signaling a certain degree of plant-organ specificity and compatibility. Based on 16S rRNA gene sequencing of representative bacterial isolates, 20 genera comprising 32 species were enriched in leaf strips- and root segments-based media. They belonged to the three phyla Bacteroidetes, Firmicutes and Proteobacteria, especially Alpha- and Gammaproteobacteria. The MPN strategy furnished a diversified nutritive platform in terms of homologous/heterologous and ambient/limited oxygenic cultivation. As a result, cultivability was extended to more 8 genera - Bosea, Brevundimonas, Caulobacter, Chitinophaga, Pseudoxanthomonas, Scandinavium, Sphingobacterium, Starkeya - among them three genera, namely Caulobacter, Scandinavium and Starkeya, which were not yet reported for Sunflower, even with the use of metagenomics sequencing methods. Possible unknown species or even one new putative genus were enriched. Thus, not only potential members of the major microbiome but also the rare isolates of satellite microbiomes can be isolated using the presented in situ similis MPN-cultivation strategy. It is a feasible addition to traditional cultivation methods to probe deeper for both the major and rare taxa and fill the many cultivation gaps within plant microbiota.

Graphical abstract, Highlights • The plant microbiome culturomics is substantially lagging behind the human microbiome. • Conventional chemically-synthetic culture media recover, Improving cultivability of a wider range of bacterial and archaeal community members, living natively in natural environments and within plants, is a prerequisite to better understanding plant-microbiota interactions and their functions in such very complex systems. Sequencing, assembling, and annotation of pure microbial strain genomes provide higher quality data compared to environmental metagenome analyses, and can substantially improve gene and protein database information. Despite the comprehensive knowledge which already was gained using metagenomic and metatranscriptomic methods, there still exists a big gap in understanding in vivo microbial gene functioning in planta, since many differentially expressed genes or gene families are not yet annotated. Here, the progress in culturing procedures for plant microbiota depending on plant-based culture media, and their proficiency in obtaining single prokaryotic isolates of novel and rapidly increasing candidate phyla are reviewed. As well, the great success of culturomics of the human microbiota is considered with the main objective of encouraging microbiologists to continue minimizing the gap between the microbial richness in nature and the number of species in culture, for the benefit of both basic and applied microbiology. The clear message to fellow plant microbiologists is to apply plant-tailored culturomic techniques that might open up novel procedures to obtain not-yet-cultured organisms and extend the known plant microbiota repertoire to unprecedented levels.

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.

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 &ldquo, in-situ-similis&rdquo, 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.

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.

This study was carried out to monitor the effect of introducing single and multiple strains of diazotrophs to plant systems (wheat and barley) under gnotobiotic conditions. The relative scarcity of nutrient supply and maintenance requirements under the present experimental conditions resulted in slower growth and much longer doubling time compared to those found in their respective batch cultures. Single inoculations significantly enhanced the efficiency of nitrogen fixation in both plant systems compared to non-inoculated controls. Among tested diazotrophic strains, cells of Azospirillum spp. and Azotobacter spp. were superior in their ARAs, being higher with barley than with wheat seedlings. Increases in total dry weight of plants between 16.8% and 78% were achieved in the wheat system and between 54.5% and 68% in the barley system, with the highest-value being for Azospirillum spp. and Pseudomonas spp. treatments, respectively. It was observed that the type of plant host has a pronounced effect on cell growth and the type of interactions between the two diazotrophic candidates inoculated jointly into wheat and barley systems. Similar to dual inoculation, the doubling time of each candidate increased when inoculated as a mixed inoculum of three or four diazotrophs into the wheat system, and decreased in the barley system.