Visualization of grapevine root colonization by the Saharan soil isolate Saccharothrix algeriensis NRRL B-24137 using DOPE-FISH microscopy

Background and aim There is currently a gap of knowledge regarding whether some beneficial bacteria isolated from desert soils can colonize epi- and endophytically plants of temperate regions. In this study, the early steps of the colonization process of one of these bacteria, Saccharothrix algeriensis NRRL B-24137, was studied on grapevine roots to determine if this beneficial strain can colonize a non-natural host plant. An improved method of fluorescence in situ hybridization (FISH), the double labeling of oligonucleotide probes (DOPE)-FISH technique was used to visualize the colonization behavior of such bacteria as well as to determine if the method could be used to track microbes on and inside plants. Methods A probe specific to Saccharothrix spp. was firstly designed. Visualization of the colonization behavior of S. algeriensis NRRL B-24137 on and inside roots of grapevine plants was then carried out with DOPE-FISH microscopy. Results The results showed that 10 days after inoculation, the strain could colonize the root hair zone, root elongation zone, as well as root emergence sites by establishing different forms of bacterial structures as revealed by the DOPE-FISH technique. Further observations showed that the strain could be also endophytic inside the endorhiza of grapevine plants. Conclusions Taking into account the natural niches of this beneficial strain, this study exemplifies that, in spite of its isolation from desert soil, the strain can establish populations as well as subpopulations on and inside grapevine plants and that the DOPE-FISH tool can allow to detect it

Root colonization by Pseudomonas sp. DSMZ 13134 and impact on the indigenous rhizosphere bacterial community of barley.

Over the last few decades, the ability of rhizosphere bacteria to promote plant growth has been considered to be of scientific, ecological, and economic interest. The properties and mechanisms of interaction of these root-colonizing bacteria have been extensively investigated, and plant protection agents that are based on these bacterial strains have been developed for agricultural applications. In the present study, the root colonization of barley by Pseudomonas sp. DSMZ 13134, that is contained in the commercially available plant protection agent Proradix, was examined using the fluorescence in situ hybridization method with oligonucleotide probes and specific gfp-tagging of the inoculant strain in combination with confocal laser scanning microscopy. In the first phase of root colonization, the inoculant strain competed successfully with seed and soil-borne bacteria (including Pseudomonads) for the colonization of the rhizoplane. Pseudomonas sp. DSMZ 13134 could be detected in all parts of the roots, although it did not belong to the dominant members of the root-associated bacterial community. Gfp-tagged cells were localized particularly in the root hair zone, and high cell densities were apparent on the root hair surface. To investigate the impact of the application of Proradix on the structure of the dominant root-associated bacterial community of barley, T-RFLP analyses were performed. Only a transient community effect was found until 3 weeks post-application

Response of barley to root colonization by <em>Pseudomonas</em> sp. DSMZ 13134 under laboratory, greenhouse, and field conditions.

Beneficial rhizobacteria strains are of substantial interest as biological plant protection agents in agriculture. Bacteria of the genus Pseudomonas have been studied for many years for their role in plant growth and biocontrol. In this study, we analyzed the influence of the commercially available agent Proradix&reg;, which contains the strain Pseudomonas sp. DSMZ 13134, on barley. In controlled infection experiments, we showed that Pseudomonas sp. DSMZ 13134 induces resistance to the barley leaf pathogen Rhynchosporium secalis and inhibits the growth of the barley root pathogen Gaeumannomyces graminis. In greenhouse experiments, Pseudomonas sp. DSMZ 13134-treated plants showed enhanced growth and yield under nutrient deprivation. In field trials, an increase of yield and straw weight was observed. While the quality of grains, as determined by starch and protein content, was not affected, the yield increased by up to 20%. Our results demonstrate the value of Pseudomonas sp. DSMZ 13134 in agronomical applications for barley

Effects of glyphosate on the bacterial community associated with roots of transgenic Roundup Ready^&reg; soybean.

Introduction of glyphosate-resistant soybean plants into agricultural systems has greatly increased the application frequency of glyphosate. Because glyphosate is able to inhibit 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) not only in plants but also in different microorganisms, its application could lead to shifts in rhizosphere microbial communities in farming soils. In this study, greenhouse experiments were conducted with the objective to evaluate the effects of glyphosate on the composition and diversity of rhizosphere bacterial communities of transgenic soybean. This was especially relevant, because foliar applied glyphosate is transported down to the roots and exuded into the rhizosphere. After two foliar herbicide applications, root samples of treated and untreated plants were analysed by 16S rRNA gene T-RFLP analysis. Multivariate statistical analysis of the data and diversity indices were used to assess changes in the microbial populations in response to glyphosate applications. A comparison of rhizosphere communities revealed that the abundance of a T-RF representing microbes related to Burkholderia sp. significantly decreased under glyphosate application, while the abundance of a T-RF representing uncultured Gemmatimonadetes significantly increased. Interestingly, the bacterial community associated with soybean roots after glyphosate application not only demonstrated effective resilience after the disturbance but in addition, T-RF diversity also increased in comparison to the untreated control samples. The results suggest that bacterial diversity was even stimulated in the rhizosphere after glyphosate application

Dynamic regulation of N-acyl-homoserine lactone production and degradation in Pseudomonas putida IsoF.

The biocontrol strain Pseudomonas putida IsoF, which was isolated from a tomato rhizosphere, is a known N-acyl-homoserine lactone (AHL) producer with only one LuxI/LuxR-like quorum-sensing (QS) system. The production and degradation of AHLs were analysed in different growth phases of the bacterium. Using the analytical tools of ultra performance liquid chromatography and high resolution MS, it was possible to determine not only the various AHLs synthesized over time but also their degradation products. 3-oxo-decanoyl-homoserine lactone was found to be the dominant AHL, which reached its maximum in the early logarithmic growth phase. Although the pH of the medium was neutral, the AHLs were degraded thereafter rapidly to the corresponding homoserines and other metabolites. The proposed lactonase gene of P. putida IsoF could not be identified, because it is apparently quite different from hitherto described lactonases. The analytical data were used to calculate the rates and thresholds of AHL production by mathematical modelling, allowing quantitative predictions and a further understanding of the QS-based regulations in this bacterium. This study, combining microbiological, chemical and mathematical approaches, suggests that AHL degradation is an integral part of the whole autoinducer circuit of P. putida IsoF

Analysis of <em>N</em>-acylhomoserine lactone dynamics in continuous cultures of <em>Pseudomonas putida</em> IsoF by use of ELISA and UHPLC/qTOF-MS-derived measurements and mathematical models.

In this interdisciplinary approach, the dynamics of production and degradation of the quorum sensing signal 3-oxo-decanoylhomoserine lactone were studied for continuous cultures of Pseudomonas putida IsoF. The signal concentrations were quantified over time by use of monoclonal antibodies and ELISA. The results were verified by use of ultra-high-performance liquid chromatography. By use of a mathematical model we derived quantitative values for non-induced and induced signal production rate per cell. It is worthy of note that we found rather constant values for different rates of dilution in the chemostat, and the values seemed close to those reported for batch cultures. Thus, the quorum-sensing system in P. putida IsoF is remarkably stable under different environmental conditions. In all chemostat experiments, the signal concentration decreased strongly after a peak, because emerging lactonase activity led to a lower concentration under steady-state conditions. This lactonase activity probably is quorum sensing-regulated. The potential ecological implication of such unique regulation is discussed

Membrane vesicle-mediated bacterial communication

The classical quorum-sensing (QS) model is based on the assumption that diffusible signaling molecules accumulate in the culture medium until they reach a critical concentration upon which expression of target genes is triggered. Here we demonstrate that the hydrophobic signal N-hexadecanoyl-L-homoserine lactone, which is produced by Paracoccus sp., is released from cells by the aid of membrane vesicles (MVs). Packed into MVs, the signal is not only solubilized in an aqueous environment but is also delivered with varying propensities to different bacteria. We propose a novel MV-based mechanism for binary trafficking of hydrophobic signal molecules, which may be particularly relevant for bacteria that live in open aqueous environments