Host Specificity and Spatial Distribution Preference of Three Pseudomonas Isolates

Plant hosts recruit and maintain a distinct root-associated microbiota based on host and bacterium traits. However, past studies disregarded microbial strain-host specificity and spatial micro-heterogeneity of the root compartment. Using genetic manipulation, confocal laser scanning microscopy, real-time quantitative PCR, and genome sequencing we characterized the colonization patterns of three Pseudomonas spp. isolates native to wheat roots, on the micro-scale. Namely, isolates P. fluorescens NT0133, P. stutzeri NT124, and P. stutzeri NT128. All three isolates preferentially colonized wheat over cucumber roots that served as control for host specificity. Furthermore, not only had the isolates strong host specificity but each isolate had a distinct spatial distribution on the root, all within a few millimeters. Isolate P. stutzeri-NT0124 preferentially colonized root tips, whereas P. fluorescens-NT0133 showed a preference for zones distant from the tip. In contrast, isolate P. stutzeri-NT0128 had no preference for a specific niche on the root. While all isolates maintained genetic potential for motility and biofilm formation their phenotype varied significantly and corresponded to their niche preference. These results demonstrate the importance of spatial colonization patterns, governed by both niche and bacterial characteristics which will have great importance in future attempts to manipulate the plant microbiome by constructing synthetic microbial consortia

Ecology of Root Colonizing <em>Massilia</em> (Oxalobacteraceae)

<div><h3>Background</h3><p>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 <em>Massilia</em> (Oxalobacteraceae), a major group of rhizosphere and root colonizing bacteria of many plant species.</p> <h3>Methodology/Principal Findings</h3><p>The ecology of <em>Massilia</em> was explored in cucumber root and seed, and compared to that of <em>Agrobacterium</em> population, using culture-independent tools, including DNA-based pyrosequencing, fluorescence <em>in situ</em> hybridization and quantitative real-time PCR. Seed- and root-colonizing <em>Massilia</em> were primarily affiliated with other members of the genus described in soil and rhizosphere. <em>Massilia</em> colonized and proliferated on the seed coat, radicle, roots, and also on hyphae of phytopathogenic <em>Pythium aphanidermatum</em> infecting seeds. High variation in <em>Massilia</em> abundance was found in relation to plant developmental stage, along with sensitivity to plant growth medium modification (amendment with organic matter) and potential competitors. <em>Massilia</em> 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 <em>Agrobacterium</em> was moderate and their dominance increased at later stages of succession.</p> <h3>Conclusions</h3><p>In accordance with contemporary models for microbial ecology classification, copiotrophic and competition-sensitive root colonization by <em>Massilia</em> is suggested. These bacteria exploit, in a transient way, a window of opportunity within the succession of communities within this niche.</p> </div

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

<p>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, <i>Massilia</i> spp. and <i>Agrobacterium</i> spp. Means and standard deviations are presented (n = 5). Different letters indicate significant differences between the means according to factorial ANOVA (<i>P</i><0.05) followed by the post-hoc Tukey HSD test.</p

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

<p>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 <i>Pythium aphanidermatum</i> (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.</p

Best-fit analysis of the interaction between relative and absolute abundance of seed- and root-colonizing <i>Massilia</i> (a) and <i>Agrobacterium radiobacter</i> (b).

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

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.

<p>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.</p

Quantitative assessment of <i>Massilia</i> spp. population size.

<p>Cucumber seed- and root-associated <i>Massilia</i> spp. and total bacteria targets were quantified using specific qPCR assays and normalized to the plant tef gene. The averages of the absolute and the relative (to general 16S rRNA gene targets, in brackets) abundances are presented (n = 4). The sampling days represent 4 plant developmental stages: Germination (1 day), primary root formation (2 days), first leaf (7 days) and vine-tip over (21 days).</p>*<p>Different letters indicate significant difference between samples after 1 day of germination. Determined by one-way ANOVA and post-hoc Tukey test (<i>P</i><0.05).</p>**<p>Determined by factorial ANOVA and post-hoc Newman-Keuls test (For all samples excluding <i>Pythium</i> inoculated ones).</p