Ecology of root colonizing Massilia (Oxalobacteraceae)

Abstract

Background: Ecologically meaningful classification of bacterial populations is essential for understanding the structure and function of bacterial communities. As in soils, the ecological strategy of the majority of root-colonizing bacteria is mostly unknown. Among those are Massilia (Oxalobacteraceae), a major group of rhizosphere and root colonizing bacteria of many plant species.

Methodology/principal findings: The ecology of Massilia was explored in cucumber root and seed, and compared to that of Agrobacterium population, using culture-independent tools, including DNA-based pyrosequencing, fluorescence in situ hybridization and quantitative real-time PCR. Seed- and root-colonizing Massilia were primarily affiliated with other members of the genus described in soil and rhizosphere. Massilia colonized and proliferated on the seed coat, radicle, roots, and also on hyphae of phytopathogenic Pythium aphanidermatum infecting seeds. High variation in Massilia abundance was found in relation to plant developmental stage, along with sensitivity to plant growth medium modification (amendment with organic matter) and potential competitors. Massilia absolute abundance and relative abundance (dominance) were positively related, and peaked (up to 85%) at early stages of succession of the root microbiome. In comparison, variation in abundance of Agrobacterium was moderate and their dominance increased at later stages of succession.

Conclusions: In accordance with contemporary models for microbial ecology classification, copiotrophic and competition-sensitive root colonization by Massilia is suggested. These bacteria exploit, in a transient way, a window of opportunity within the succession of communities within this niche.

Figure 1. Composition of cucumber (Cucumis sativus) seed- and root-colonizing bacterial (a) and Oxalobacteraceae community (b), based on 454-pyrosequencing of general bacterial 16S rRNA gene fragments.

Sequences were obtained from samples of seeds (1 day) and roots (2, 7 and 21 days old) grown in perlite and compost-amended perlite, with and without inoculation with Pythium aphanidermatum (1day only). Numbers indicate the relative abundance of the indicated taxon (% of total bacteria). Families for which relative abundance was ≥1% are included in panel a.

Figure 2. Neighbor-joining tree depicting representatives of the most abundant Oxalobacteraceae OTUs (in bold; number of sequences represented are in brackets) and related sequences from the NCBI database.

The tree was calculated using the Kimura two-parameter model. The scale bar represents number of substitutes per site. Numbers at the nodes indicate bootstrap values (1000 replicates). Symbols indicate sequence origin: brown square- soil; green triangle- rhizosphere; yellow circle- human and mouse skin swabs; blue diamond- fresh water; red star- air and dust; and purple cross- clean room. Sequences from other origins are underlined.

Figure 3. Cucumber seed and root colonization by Massilia.

FISH-CLSM analyses of the plant samples: blue- DAPI stain; red- total bacteria; green- Massilia. a,b) Seed radicle after 24 h in compost-amended perlite; c,d) seed coat after 24 h in compost-amended perlite; e,f) seed coat after 24 h in Pythium aphanidermatum-inoculated perlite; g) root tip after 48 h in perlite; h) mature zone of primary root after 48 h in perlite; colony on root after 48 h in perlite: bright-field image on the right, Massilia probe and DAPI stain on the left. Arrows indicate Pythium hyphae.

Figure 4. Best-fit analysis of the interaction between relative and absolute abundance of seed- and root-colonizing Massilia (a) and Agrobacterium radiobacter (b).

Relative and absolute abundances, normalized to the plant tef gene, were determined by qPCR assay. df: degrees of freedom; MSE: mean square error.

Figure 5. Relative abundance of Massilia (a) and Agrobacterium spp. (b) on cucumber seedling roots as determined by real-time quantitative PCR.

Cucumber seeds were germinated and grown under greenhouse conditions in sandy soil for 6 days and then transplanted into the same soil (bright grey) or wild rocket amended soil (dark grey). Roots were sampled 3 and 6 days after transplantation and DNA extracted from the samples was used for quantification of total bacteria, Massilia spp. and Agrobacterium spp. Means and standard deviations are presented (n = 5). Different letters indicate significant differences between the means according to factorial ANOVA (P<0.05) followed by the post-hoc Tukey HSD test.