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

Abstract

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.

Fig. 1

Culture-dependent bacterial community analysis of maize rhizosphere and phyllosphere. a Log CFU counts developed on the tested culture media over time. Statistically significant differences are designated by different letters based on Tukey’s Honestly Significant Differences (HSD, P ≤ 0.05, n = 4). b DGGE analysis of 16S rRNA gene profiling of culturable bacterial community recovered on the tested culture media after 32 days of incubation, clustered with UPGMA method based on Euclidean distances (totallab.com/home/cliqs). c Morphologies of colonies grown on WA together with zoom in section of macro- and micro-colonies. Scaling was carried out using ImageJ software (imagej.nih.gov/ij). WA water agar, R2A 1/100 diluted (v/v) R2A, AJ autoclaved 1/200 diluted (v/v) maize juice agar, and FJ filtered 1/200 diluted (v/v) maize juice agar culture media

Fig. 2

Maximum likelihood (ML) phylogenetic tree of the bacterial isolates recovered from WA (15 isolates) and FJ (13 isolates) culture media, based on 16S rRNA gene sequences. The subtree includes the isolates F19 and F20 with all reported species of the genera Agreia and Herbiconiux, including Microbacterium oxydans as an outgroup. Bootstrapping was performed for each tree with 1000 replicates; the percentage of trees in which the associated taxa clustered together is shown next to the branches. Phylogenetic analyses were conducted in MEGA X. WA water agar, R2A 1/100 diluted (v/v) R2A, AJ autoclaved 1/200 diluted (v/v) sunflower juice agar, and FJ filtered 1/200 diluted (v/v) sunflower juice agar culture media