G3 PhyloChip Analysis Confirms the Promise of Plant-Based Culture Media for Unlocking the Composition and Diversity of the Maize Root Microbiome and for Recovering Unculturable Candidate Divisions/Phyla

Abstract

The rapid development of high-throughput techniques and expansion of bacterial databases have accelerated efforts to bring plant microbiomes into cultivation. We introduced plant-only-based culture media as a successful candidate to mimic the nutritional matrices of plant roots. We herein employed a G3 PhyloChip microarray to meticulously characterize the culture-dependent and -independent bacterial communities of the maize root compartments, the endo- and ecto-rhizospheres. An emphasis was placed on the preference of the growth of unculturable candidate divisions/phyla on plant-only-based culture media over standard culture media (nutrient agar). A total of 1,818 different operational taxonomic units (OTUs) were resolved representing 67 bacterial phyla. Plant-only-based culture media displayed particular affinity towards recovering endophytic over ectophytic rhizobacteria. This was shown by the slightly higher recovery of CFUs for endophytes on plant-only-based culture media (26%) than on standard culture media (10%) as well as the higher taxa richness and numbers of exclusive families of unculturable divisions/phyla. Out of 30 bacterial phyla (comprising >95% of the whole population), 13 were of a significantly higher incidence on plant-only-based culture media, 6 phyla of which were not-yet-cultured (Atribacteria, OP9; Dependentiae, TM6; Latescibacteria, WS3; Marinimicrobia, SAR406; Omnitrophica, OP3; BRC1). Furthermore, plant-only-based culture media significantly enriched less abundant and/or hard-to-culture bacterial phyla (Acidobacteria, Gemmatimonadetes, and Tenericutes). These results present conclusive evidence of the ability of plant-only-based culture media to bring the plant-fed in situ microbiome into the status of plant-fed in vitro cultures, and to widen the scope of cultivation of heretofore-unculturable bacterial divisions/phyla.

Keywords: Candidatus Phytoplasma, candidate phyla; Plant microbiome; divisions; plant-based culture media; unculturable bacteria.

Fig. 1

Culture-dependent and -independent recovery of rhizobacteria associated with maize root compartments. Log numbers of CFUs of culturable rhizobacteria in the endorhizosphere (En) and ectorhizosphere (Ec) as developed on nutrient agar (EnN, EcN), and plant-based culture medium (EnC, EcC). Total numbers of rhizobacteria measured by qPCR in maize root compartments of the endorhizosphere (EnR) and ectorhizosphere (EcR). Significant differences are indicated by different letters at P≤0.05 (Tukey’s HSD).

Fig. 2

Analysis of culture-dependent (CFUs) and culture-independent (maize roots) bacterial and archaeal community compositions of maize root compartments based on G3-PhyloChip technology. A, Principal Co-ordinates Analysis (PCoA); B, Hierarchical clustering, bootstrap probabilities (%) are indicated in green and approximately unbiased p-values are shown in red; C, Bacterial richness at different taxonomic levels (phyla, classes, orders, families). En, Endorhizosphere; Ec, ectorhizosphere; R, culture-independent root sphere; C, plant-based culture media; N, nutrient agar culture media.

Fig. 3

Overlapping of culture-dependent (on plant-based culture medium and nutrient agar) and -independent bacterial communities of the maize root endorhizosphere. A, Venn diagram at the family level for bacterial communities displaying unique and overlapping families; families exclusively grown on only one of our tested media are shown in the linked boxes, and not-yet-cultured candidate divisions are marked in bold; B, Heatmap of weighted abundance of the OTUs that displayed significant differences among the 206 families commonly grown on both culture media (EnN, nutrient agar; EnC, plant-based culture medium, three replicates shown for each medium). Please refer to Data S1 for detailed information.

Fig. 4

Heatmap representing significant differences between tested culture media (nutrient agar and plant-based culture media) in respect of the weighted abundance of 30 phyla, representing more than 95% of all detected OTUs, in maize compartments of the endorhizosphere and ectorhizosphere. The phyla indicated in bold are those lacking even a single representative isolate.