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

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.

Culturomics of the plant prokaryotic microbiome and the dawn of plant-based culture media - A review.

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.

Plant Materials are Sustainable Substrates Supporting New Technologies of Plant-Only-Based Culture Media for in vitro Culturing of the Plant Microbiota.

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.