Geneviève L. Grundmann

Potential of a 16S rRNA-based taxonomic microarray for analyzing the rhizosphere effects of maize on Agrobacterium spp. and bacterial communities.

ABSTRACT Bacterial diversity is central to ecosystem sustainability and soil biological function, for which the role of roots is important. The high-throughput analysis potential of taxonomic microarray should match the breadth of bacterial diversity. Here, the power of this technology was evidenced through methodological verifications and analysis of maize rhizosphere effect based on a 16S rRNA-based microarray developed from the prototype of H. Sanguin et al. (Environ. Microbiol. 8:289-307, 2006). The current probe set was composed of 170 probes (41 new probes in this work) that targeted essentially the Proteobacteria. Cloning and sequencing of 16S rRNA amplicons were carried out on maize rhizosphere and bulk soil DNA. All tested clones that had a perfect match with corresponding probes were positive in the hybridization experiment. The hierarchically nested probes were reliable, but the level of taxonomic identification was variable, depending on the probe set specificity. The comparison of experimental and theoretical hybridizations revealed 0.91% false positives and 0.81% false negatives. The microarray detection threshold was estimated at 0.03% of a given DNA type based on DNA spiking experiments. A comparison of the maize rhizosphere and bulk soil hybridization results showed a significant rhizosphere effect, with a higher predominance of Agrobacterium spp. in the rhizosphere, as well as a lower prevalence of Acidobacteria, Bacteroidetes, Verrucomicrobia, and Planctomycetes, a new taxon of interest in soil. In addition, well-known taxonomic groups such as Sphingomonas spp., Rhizobiaceae, and Actinobacteria were identified in both microbial habitats with strong hybridization signals. The taxonomic microarray developed in the present study was able to discriminate and characterize bacterial community composition in related biological samples, offering extensive possibilities for systematic exploration of bacterial diversity in ecosystems.

Comparison of rhizobacterial community composition in soil suppressive or conducive to tobacco black root rot disease.

Work on soils suppressive to Thielaviopsis basicola-mediated tobacco black root rot has focused on antagonistic pseudomonads to date. The role of non-Pseudomonas rhizosphere populations has been neglected, and whether they differ in black root rot-suppressive versus -conducive soils is unknown. To assess this possibility, tobacco was grown in a suppressive and a conducive soil of similar physicochemical properties, and rhizobacterial community composition was compared using a 16S rRNA taxonomic microarray. The microarray contains 1033 probes and targets 19 bacterial phyla. Among them, 398 probes were designed for Proteobacteria, Firmicutes, Actinomycetes, Cyanobacteria and Bacteroidetes genera/species known to include strains relevant for plant protection or plant growth promotion. Hierarchical clustering as well as principal component analysis of microarray data discriminated clearly between black root rot-suppressive and -conducive soils. In contrast, T. basicola inoculation had no impact on rhizobacterial community composition. In addition to fluorescent Pseudomonas, the taxa Azospirillum, Gluconacetobacter, Burkholderia, Comamonas and Sphingomonadaceae, which are known to comprise strains with plant-beneficial properties, were more prevalent in the suppressive soil. Mycobacterium, Bradyrhizobium, Rhodobacteraceae, Rhodospirillum and others were more prevalent in the conducive soil. For selected taxa, microarray results were largely corroborated by quantitative PCR and cloning/sequencing. In conclusion, this work identified novel bacterial taxa that could serve as indicators of disease suppressiveness in soil-quality assessments, and it extends the range of bacterial taxa hypothesized to participate in black root rot suppression.

Rhizosphere bacterial communities associated with disease suppressiveness stages of take‐all decline in wheat monoculture

The decline of take-all disease (Gaeumannomyces graminis var. tritici), which may take place during wheat monocropping, involves plant-protecting, root-colonizing microorganisms. So far, however, most work has focused on antagonistic fluorescent pseudomonads. Our objective was to assess the changes in rhizobacterial community composition during take-all decline of field-grown wheat. The study was based on the development and utilization of a taxonomic 16S rRNA-based microarray of 575 probes, coupled with cloning-sequencing and quantitative PCR. Plots from one experimental field grown with wheat for 1 yr (low level of disease), 5 yr (high level of disease) or 10 yr (low level of disease, suppressiveness reached) were used. Microarray data discriminated between the three stages. The outbreak stage (5 yr) was mainly characterized by the prevalence of Proteobacteria, notably Pseudomonas (Gammaproteobacteria), Nitrosospira (Betaproteobacteria), Rhizobacteriaceae, Sphingomonadaceae, Phyllobacteriaceae (Alphaproteobacteria), as well as Bacteroidetes and Verrucomicrobia. By contrast, suppressiveness (10 yr) correlated with the prevalence of a broader range of taxa, which belonged mainly to Acidobacteria, Planctomycetes, Nitrospira, Chloroflexi, Alphaproteobacteria (notably Azospirillum) and Firmicutes (notably Thermoanaerobacter). In conclusion, take-all decline correlated with multiple changes in rhizobacterial community composition, far beyond the sole case of pseudomonads.

Is diversification history of maize influencing selection of soil bacteria by roots

A wide range of plant lines has been propagated by farmers during crop selection and dissemination, but consequences of this crop diversification on plant‐microbe interactions have been neglected. Our hypothesis was that crop evolutionary history shaped the way the resulting lines interact with soil bacteria in their rhizospheres. Here, the significance of maize diversification as a factor influencing selection of soil bacteria by seedling roots was assessed by comparing rhizobacterial community composition of inbred lines representing the five main genetic groups of maize, cultivated in a same European soil. Rhizobacterial community composition of 21‐day‐old seedlings was analysed using a 16S rRNA taxonomic microarray targeting 19 bacterial phyla. Rhizobacterial community composition of inbred lines depended on the maize genetic group. Differences were largely due to the prevalence of certain Betaproteobacteria and especially Burkholderia, as confirmed by quantitative PCR and cloning/sequencing. However, these differences in bacterial root colonization did not correlate with plant microsatellite genetic distances between maize genetic groups or individual lines. Therefore, the genetic structure of maize that arose during crop diversification (resulting in five main groups), but not the extent of maize diversification itself (as determined by maize genetic distances), was a significant factor shaping rhizobacterial community composition of seedlings.

Development of a 16S rRNA gene-based prototype microarray for the detection of selected actinomycetes genera

Actinomycetes are known for their secondary metabolites, which have been successfully used as drugs in human and veterinary medicines. However, information on the distribution of this group of Gram-positive bacteria in diverse ecosystems and a comprehension of their activities in ecosystem processes are still scarce. We have developed a 16S rRNA-based taxonomic microarray that targets key actinomycetes at the genus level. In total, 113 actinomycete 16S rRNA probes, corresponding to 55 of the 202 described genera, were designed. The microarray accuracy was evaluated by comparing signal intensities with probe/target-weighted mismatch values and the Gibbs energy of the probe/target duplex formation by hybridizing 17 non-actinomycete and 29 actinomycete strains/clones with the probe set. The validation proved that the probe set was specific, with only 1.3% of false results. The incomplete coverage of actinomycetes by a genus-specific probe was caused by the limited number of 16S rRNA gene sequences in databases or insufficient 16S rRNA gene polymorphism. The microarray enabled discrimination between actinomycete communities from three forest soil samples collected at one site. Cloning and sequencing of 16S rRNA genes from one of the soil samples confirmed the microarray results. We propose that this newly constructed microarray will be a valuable tool for genus-level comparisons of actinomycete communities in various ecological conditions.

Actinobacterial community dominated by a distinct clade in acidic soil of a waterlogged deciduous forest

Members of the Actinobacteria are among the most important litter decomposers in soil. The site of a waterlogged deciduous forest with acidic soil was explored for actinobacteria because seasonality of litter inputs, temperature, and precipitation provided contrasting environmental conditions, particularly variation of organic matter quantity and quality. We hypothesized that these factors, which are known to influence decomposition, were also likely to affect actinobacterial community composition. The relationship between the actinobacterial community, soil moisture and organic matter content was assessed in two soil horizons in the summer and winter seasons using a 16S rRNA taxonomic microarray and cloning-sequencing of 16S rRNA genes. Both approaches showed that the community differed significantly between horizons and seasons, paralleling the changes in soil moisture and organic matter content. The microarray analysis further indicated that the actinobacterial community of the upper horizon was characterized by high incidence of the genus Mycobacterium. In both horizons and seasons, the actinobacterial clone libraries were dominated (by 80%) by sequences of a separate clade sharing an ancestral node with Streptosporangineae. This relatedness is supported also by some common adaptations, for example, to soil acidity and periodic oxygen deprivation or dryness.

Assessment of the relationship between geologic origin of soil, rhizobacterial community composition and soil receptivity to tobacco black root rot in Savoie region (France)

Background and aimsIn Morens (Switzerland), soils formed on morainic deposits (which contain vermiculite clay and display particular tobacco rhizobacterial community) are naturally suppressive to Thielaviopsis basicola-mediated tobacco black root rot, but this paradigm was never assessed elsewhere. Here, we tested the relation between geology and disease suppressiveness in neighboring Savoie (France).MethodsTwo morainic and two sandstone soils from Savoie were compared based on disease receptivity (T. basicola inoculation tests on tobacco), clay mineralogy (X-ray diffraction), tobacco rhizobacterial community composition (16S rRNA gene-based taxonomic microarray) and phlD+Pseudomonas populations involved in 2,4-diacetylphloroglucinol production (real-time PCR and tRFLP).ResultsUnlike in Morens, in Savoie the morainic soils were receptive to disease whereas T. basicola inoculation did not increase disease level in the sandstone soils. Vermiculite was not present in Savoie soils. The difference in rhizobacterial community composition between Savoie morainic and sandstone soils was significant but modest, and there was little agreement in bacterial taxa discriminating soils of different disease receptivity levels when comparing Morens versus Savoie soils. Finally, phlD+ rhizosphere pseudomonads were present at levels comparable to those in Morens soils, but with different diversity patterns.ConclusionsThe morainic model of black root rot suppressiveness might be restricted to the particular type of moraine occurring in the Morens region, and the low disease receptivity of sandstone soils in neighboring Savoie might be related to other plant-protection mechanisms.