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GFP-tagged multimetal-tolerant bacteria and their detection in the rhizosphere of white mustard.

Piotrowska-Seget Z, Beściak G, Bernaś T, Kozdrój J - Ann. Microbiol. (2011)

Bottom Line: In this study, soil of a metal-mine wasteland was analyzed for the presence of metal-tolerant bacterial isolates, and the tolerance patterns of the isolated strains for a number of heavy metals and antibiotics were compared.From among the successfully tagged isolates, we used the transconjugant Pseudomonas putida G25 (pPROBE-NT) to inoculate white mustard seedlings.Despite a significant decrease in transconjugant abundance in the rhizosphere, the gfp-tagged cells survived on the root surfaces at a level previously reported for root colonisers.

View Article: PubMed Central - PubMed

ABSTRACT
The introduction of rhizobacteria that tolerate heavy metals is a promising approach to support plants involved in phytoextraction and phytostabilisation. In this study, soil of a metal-mine wasteland was analyzed for the presence of metal-tolerant bacterial isolates, and the tolerance patterns of the isolated strains for a number of heavy metals and antibiotics were compared. Several of the multimetal-tolerant strains were tagged with a broad host range reporter plasmid (i.e. pPROBE-NT) bearing a green fluorescent protein marker gene (gfp). Overall, the metal-tolerant isolates were predominately Gram-negative bacteria. Most of the strains showed a tolerance to five metals (Zn, Cu, Ni, Pb and Cd), but with differing tolerance patterns. From among the successfully tagged isolates, we used the transconjugant Pseudomonas putida G25 (pPROBE-NT) to inoculate white mustard seedlings. Despite a significant decrease in transconjugant abundance in the rhizosphere, the gfp-tagged cells survived on the root surfaces at a level previously reported for root colonisers.

No MeSH data available.


Photographs of microscopic images of fluorescent gfp-tagged transconjugant P. putida G25 colonising the root surface of a 54-day-old seedling of white mustard (a) and those surviving in the rhizosphere (b). The photographs were obtained with a confocal laser scanning microscope
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Fig4: Photographs of microscopic images of fluorescent gfp-tagged transconjugant P. putida G25 colonising the root surface of a 54-day-old seedling of white mustard (a) and those surviving in the rhizosphere (b). The photographs were obtained with a confocal laser scanning microscope

Mentions: The introduction of metal-tolerant bacterial strains into soil seeded with plants that tolerate increased concentrations of heavy metals has been reported as a promising approach that facilitates the survival and development of these plants in contaminated habitats (Abou-Shanab et al. 2003; Sheng and Xia 2006; Ma et al. 2009). However, the success of this approach is dependent on the potential of the inoculant to colonise plant roots efficiently, which in turn is related to its own survival in the rhizosphere. To estimate the level of adaptation between the released inoculant cells and white mustard, we inoculated plant seedlings growing in a sandy soil. The numbers of gfp-tagged Pseudomonas putida G25 colonising the roots of white mustard decreased from the initial log 7.48 ± 0.28 to log 4.95 ± 0.25 and log 3.62 ± 0.18 CFU g-1 dry soil on days 14 and 54 post-inoculation, respectively. The average counts of the total indigenous heterotrophic bacteria were about log 7.95 ± 0.24 CFU g-1 dry soil in the rhizosphere. However, the natural resistance to Ap, Tc and Km among the indigenous bacteria was below the detection limit of log 1.47 ± 0.15 CFU g-1dry soil. The microscopic observation of root and rhizosphere preparations confirmed the successful survival of the transconjugant in the rhizosphere and the colonisation of the roots of white mustard seedlings on day 7 (Fig. 3). Although the presence of the gfp-tagged transconjugants on the roots was still visible on day 54, only a few bacterial cells were noticeable in the rhizosphere specimen (Fig. 4). A decrease in counts of introduced bacteria over a few days is often observed due to competition for nutrients and space with other rhizosphere microorganisms. They are grazed on by protozoa and exposed to abiotic stress; some cells die or lose culturability following release (van Veen et al. 1997). As a result, the inoculant population ultimately reaches a level reflecting its ability to adapt to conditions prevailing in the rhizosphere of the appropriate plant species (de Weger et al. 1995; Kozdrój et al. 2004). Errampalli et al. (1998) indicated that gfp-marked Pseudomonas sp., introduced into a creosote-contaminated soil, declined over a 26-day period, although the low numbers recovered up to 13 months after inoculation. It can also not be excluded that the decreased numbers of P. putida G25 (pPROBE-NT) may have resulted from the loss of the plasmid over time. However, this vector has been reported to be a stable one in a broad range of bacterial hosts (Miller et al. 2000). Belimov et al. (2004) reported a slight decrease in the numbers of inoculant rhizobacteria between days 10 and 25 during their colonisation of barley roots. By contrast, an introduced population of Bacillus sp. that was resistant to Cd was still detectable at the same density in the rhizosphere of rape 2 weeks after inoculation (Sheng and Xia 2006). In addition, the survival of inoculants associated with a plant host depends on changes in the physiological state of the plant (Lebeau et al. 2008). Wu et al. (2006b) reported that young mustard seedlings are more favourable to an introduced metal-tolerant strain than flowering plants, possibly due to differences in the composition of the root exudates. We obtained similar results for the survival of gfp-tagged P. putida G25. Immobilisation of bacterial inoculants into carriers, such as alginate, clay, peat or methyl cellulose, which protects them against biotic and abiotic environmental stress, can increase both their survival in soil as well as their colonisation of soil (van Veen et al. 1997; Kozdrój et al. 2004; Braud et al. 2009). However, to facilitate colonisation of the entire rhizosphere and roots of growing seedlings by the released bacterial strains, the application of free-cell suspensions, instead of immobilised cells, appears to be useful (Ciccillo et al. 2002; Mazolla et al. 1995).Fig. 3


GFP-tagged multimetal-tolerant bacteria and their detection in the rhizosphere of white mustard.

Piotrowska-Seget Z, Beściak G, Bernaś T, Kozdrój J - Ann. Microbiol. (2011)

Photographs of microscopic images of fluorescent gfp-tagged transconjugant P. putida G25 colonising the root surface of a 54-day-old seedling of white mustard (a) and those surviving in the rhizosphere (b). The photographs were obtained with a confocal laser scanning microscope
© Copyright Policy
Related In: Results  -  Collection

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Fig4: Photographs of microscopic images of fluorescent gfp-tagged transconjugant P. putida G25 colonising the root surface of a 54-day-old seedling of white mustard (a) and those surviving in the rhizosphere (b). The photographs were obtained with a confocal laser scanning microscope
Mentions: The introduction of metal-tolerant bacterial strains into soil seeded with plants that tolerate increased concentrations of heavy metals has been reported as a promising approach that facilitates the survival and development of these plants in contaminated habitats (Abou-Shanab et al. 2003; Sheng and Xia 2006; Ma et al. 2009). However, the success of this approach is dependent on the potential of the inoculant to colonise plant roots efficiently, which in turn is related to its own survival in the rhizosphere. To estimate the level of adaptation between the released inoculant cells and white mustard, we inoculated plant seedlings growing in a sandy soil. The numbers of gfp-tagged Pseudomonas putida G25 colonising the roots of white mustard decreased from the initial log 7.48 ± 0.28 to log 4.95 ± 0.25 and log 3.62 ± 0.18 CFU g-1 dry soil on days 14 and 54 post-inoculation, respectively. The average counts of the total indigenous heterotrophic bacteria were about log 7.95 ± 0.24 CFU g-1 dry soil in the rhizosphere. However, the natural resistance to Ap, Tc and Km among the indigenous bacteria was below the detection limit of log 1.47 ± 0.15 CFU g-1dry soil. The microscopic observation of root and rhizosphere preparations confirmed the successful survival of the transconjugant in the rhizosphere and the colonisation of the roots of white mustard seedlings on day 7 (Fig. 3). Although the presence of the gfp-tagged transconjugants on the roots was still visible on day 54, only a few bacterial cells were noticeable in the rhizosphere specimen (Fig. 4). A decrease in counts of introduced bacteria over a few days is often observed due to competition for nutrients and space with other rhizosphere microorganisms. They are grazed on by protozoa and exposed to abiotic stress; some cells die or lose culturability following release (van Veen et al. 1997). As a result, the inoculant population ultimately reaches a level reflecting its ability to adapt to conditions prevailing in the rhizosphere of the appropriate plant species (de Weger et al. 1995; Kozdrój et al. 2004). Errampalli et al. (1998) indicated that gfp-marked Pseudomonas sp., introduced into a creosote-contaminated soil, declined over a 26-day period, although the low numbers recovered up to 13 months after inoculation. It can also not be excluded that the decreased numbers of P. putida G25 (pPROBE-NT) may have resulted from the loss of the plasmid over time. However, this vector has been reported to be a stable one in a broad range of bacterial hosts (Miller et al. 2000). Belimov et al. (2004) reported a slight decrease in the numbers of inoculant rhizobacteria between days 10 and 25 during their colonisation of barley roots. By contrast, an introduced population of Bacillus sp. that was resistant to Cd was still detectable at the same density in the rhizosphere of rape 2 weeks after inoculation (Sheng and Xia 2006). In addition, the survival of inoculants associated with a plant host depends on changes in the physiological state of the plant (Lebeau et al. 2008). Wu et al. (2006b) reported that young mustard seedlings are more favourable to an introduced metal-tolerant strain than flowering plants, possibly due to differences in the composition of the root exudates. We obtained similar results for the survival of gfp-tagged P. putida G25. Immobilisation of bacterial inoculants into carriers, such as alginate, clay, peat or methyl cellulose, which protects them against biotic and abiotic environmental stress, can increase both their survival in soil as well as their colonisation of soil (van Veen et al. 1997; Kozdrój et al. 2004; Braud et al. 2009). However, to facilitate colonisation of the entire rhizosphere and roots of growing seedlings by the released bacterial strains, the application of free-cell suspensions, instead of immobilised cells, appears to be useful (Ciccillo et al. 2002; Mazolla et al. 1995).Fig. 3

Bottom Line: In this study, soil of a metal-mine wasteland was analyzed for the presence of metal-tolerant bacterial isolates, and the tolerance patterns of the isolated strains for a number of heavy metals and antibiotics were compared.From among the successfully tagged isolates, we used the transconjugant Pseudomonas putida G25 (pPROBE-NT) to inoculate white mustard seedlings.Despite a significant decrease in transconjugant abundance in the rhizosphere, the gfp-tagged cells survived on the root surfaces at a level previously reported for root colonisers.

View Article: PubMed Central - PubMed

ABSTRACT
The introduction of rhizobacteria that tolerate heavy metals is a promising approach to support plants involved in phytoextraction and phytostabilisation. In this study, soil of a metal-mine wasteland was analyzed for the presence of metal-tolerant bacterial isolates, and the tolerance patterns of the isolated strains for a number of heavy metals and antibiotics were compared. Several of the multimetal-tolerant strains were tagged with a broad host range reporter plasmid (i.e. pPROBE-NT) bearing a green fluorescent protein marker gene (gfp). Overall, the metal-tolerant isolates were predominately Gram-negative bacteria. Most of the strains showed a tolerance to five metals (Zn, Cu, Ni, Pb and Cd), but with differing tolerance patterns. From among the successfully tagged isolates, we used the transconjugant Pseudomonas putida G25 (pPROBE-NT) to inoculate white mustard seedlings. Despite a significant decrease in transconjugant abundance in the rhizosphere, the gfp-tagged cells survived on the root surfaces at a level previously reported for root colonisers.

No MeSH data available.