Cultivar and Metal-Specific Effects of Endophytic Bacteria in Helianthus tuberosus Exposed to Cd and Zn.

Plant growth promoting endophytic bacteria (PGPB) isolated from Brassica napus were inoculated in two cultivars of Helianthus tuberosus (VR and D19) growing on sand supplemented with 0.1 mM Cd or 1 mM Zn. Plant growth, concentrations of metals and thiobarbituric acid (TBA) reactive compounds were determined. Colonization of roots of H. tuberosus D19 by Pseudomonas sp. 262 was evaluated using confocal laser scanning microscopy. Pseudomonas sp. 228, Serratia sp. 246 and Pseudomonas sp. 262 significantly enhanced growth of H. tuberosus D19 exposed to Cd or Zn. Pseudomonas sp. 228 significantly increased Cd concentrations in roots. Serratia sp. 246, and Pseudomonas sp. 256 and 228 resulted in significantly decreased contents of TBA reactive compounds in roots of Zn exposed D19 plants. Growth improvement and decrease of metal-induced stress were more pronounced in D19 than in VR. Pseudomonas sp. 262-green fluorescent protein (GFP) colonized the root epidermis/exodermis and also inside root hairs, indicating that an endophytic interaction was established. H. tuberosus D19 inoculated with Pseudomonas sp. 228, Serratia sp. 246 and Pseudomonas sp. 262 holds promise for sustainable biomass production in combination with phytoremediation on Cd and Zn contaminated soils.

Bioremediation of synthetic fatliquors under microaerobic condition.

Synthetic fatliquors are useful as a fatliquoring agent, flotation agent and emulsifying agent in a wide range of industrial applications such as leather, pharmacy and farm chemicals. These fatliquors remain recalcitrant to natural biota in existing treatment plants. In the present study, the isolated microaerophilic Serratia sp. HA1 strain CSMB3 is capable of utilizing structurally different fatliquors as the sole substrate for their growth under microaerobic conditions. Degradation of vegetable fatliquors was observed from 95 to 97% in terms of lipids, with the production of lipase at 72 h. Degradation of synthetic fatliquors was observed in terms of chemical oxygen demand from 85% to a minimum of 25%. It is in the order of sulfited/sulfated fatliquors > sulfochlorinated fatliquors > chlorinated fatliquors. A thin layer chromatography chromatogram confirmed the degradation of non polar fatliquor to polar compounds. Production of the red pigment prodigiosin in synthetic fatliquors enhanced the growth of the isolate. Fourier transform infrared spectroscopy (FTIR) confirmed the bioremediation of sulfochlorinated fatliquor into lipids and fatty acids and gas chromatography-mass spectrometry (GC-MS) results confirmed that alcohols and esters are the final end products. Thus the isolated strain CSMB3 may be used in the treatment of wastewaters containing vegetable and synthetic fatliquors.

Role of 1-acyl-sn-glycerol-3-phosphate acyltransferase in psychrotrophy and stress tolerance of Serratia plymuthica RVH1.

A mutant with a transposon insertion just upstream of the lysophosphatidic acid acyltansferase gene plsC was isolated in a screen for mutants affected in growth at low temperature of the psychrotroph Serratia plymuthica RVH1. This mutant had lost its ability to grow at 4 °C and was severely affected in growth at 10 °C, but showed only slightly reduced growth at 30 °C. Fatty acid analysis of membrane extracts showed that the ratio of C16:1/C18:1 fatty acids was six-to sevenfold reduced in the mutant, although the ratio of unsaturated to saturated fatty acids was unaffected. The homeoviscous adaptation ability of the mutant was also unaffected. Growth and fatty acid composition were mostly restored by overexpressing plsC on a plasmid. Supplementation of C16:1 (palmitoleic acid) into the growth medium partially rescued low temperature growth, indicating that a balanced ratio of the two main unsaturated fatty acids is required for psychrotrophy. The mutant was significantly more strongly inactivated by high pressure treatment at 250 MPa, but not at higher pressures. It also showed reduced growth at low pH, but not at increased NaCl concentrations. This work provides novel information on the role of membrane fatty acid composition in stress tolerance.

Strain-specific colonization pattern of Rhizoctonia antagonists in the root system of sugar beet.

To develop effective biocontrol strategies, basic knowledge of plant growth promotion (PGP) and root colonization by antagonists is essential. The survival and colonization patterns of five different biocontrol agents against Rhizoctonia solani AG2-2IIIB in the rhizosphere of greenhouse-grown sugar beet plants were analysed in single and combined treatments. The study included bacteria (Pseudomonas fluorescens L13-6-12, Pseudomonas trivialis RE(*) 1-1-14, Serratia plymuthica 3Re4-18) as well as fungi (Trichoderma gamsii AT1-2-4, Trichoderma velutinum G1/8). Microscopic analysis by confocal laser scanning microscopy revealed different colonization patterns for each DsRed2/green fluorescent protein-labelled strain. Bacteria and T. velutinum G1/8 colonized the root surface and the endorhiza in single and co-culture, while for T. gamsii AT1-2-4, only the transfer of spores was observed. Whereas Pseudomonas strains formed large microcolonies consisting of hundreds of cells, S. plymuthica was arranged in small endophytic clusters or clouds around the entire root system. In co-culture, each strain showed its typical pattern and occupied specific niches on the root, without clear evidence of morphological interactions. PGP was only observed for four strains with rhizosphere competence and not for T. gamsii AT1-2-4. The results provide useful information on which combination of strains to test in larger biocontrol experiments directed to applications.

Slurry-phase biodegradation of weathered oily sludge waste.

We assessed the biodegradation of a typical oily sludge waste (PB401) in Mexico using several regimes of indigenous microbial consortium and relevant bioremediation strategies in slurry-phase system. Abiotic loss of total petroleum hydrocarbons (TPH) in the PB401 was insignificant, and degradation rates under the various treatment conditions ranged between 666.9 and 2168.7 mg kg(-1) day(-1) over a 15 days reaction period, while viable cell count peaked at between log(10)5.7 and log(10)7.4 cfu g(-1). Biostimulation with a commercial fertilizer resulted in 24% biodegradation of the TPH in the oily waste and a corresponding peak cell density of log(10)7.4 cfu g(-1). Addition of non-indigenous adapted consortium did not appear to enhance the removal of TPH from the oily waste. It would appear that the complexities of the components of the alkylaromatic fraction of the waste limited biodegradation rate even in a slurry system.

Mineral and carbon usage of two synthetic pyrethroid degrading bacterial isolates.

AIMS: To investigate the biodegrading ability and cometabolism of synthetic pyrethroid (SP) utilizing bacteria in cultures with various minerals and carbon sources. METHODS AND RESULTS: Previously isolated SP-degrading Pseudomonas sp. and Serratia sp. were used in cultures containing either flumethrin SP or cypermethrin SP formulations. The culture media consisted of either (i) water only, (ii) water and sucrose, (iii) mineral broth or (iv) mineral broth and sucrose. The growth of both organisms was greatest in the mineral broth and sucrose medium, but the growth-limiting factor for Pseudomonas sp. strain Circle was the mineral content whereas for Serratia sp. strain White it was the carbon substrate. CONCLUSION: The greatest extent of degradation of both SP-based compounds occurred with Pseudomonas sp. strain Circle but was dependant on the medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This investigation could lead to the development of a relatively inexpensive medium supplement to enhance the microbial biodegradation of undesirable compounds, either in situ or ex situ. In this particular case, for the biodegradation of SPs used in sheep dip.

Strategy to select and assess antagonistic bacteria for biological control of Rhizoctonia solani Kühn.

A screening strategy was developed to assess the potential of plant-associated bacteria to control diseases caused by Rhizoctonia solani Kühn. About 434 already characterized antagonistic bacterial strains isolated from diverse plant species and microenvironments were evaluated for biocontrol and plant growth promotion by a hierarchical combination of assays. Analyzing in vitro antagonism towards different Rhizoctonia isolates resulted in a selection of 20 potential biocontrol agents. The strains were characterized by their antagonistic mechanisms in vitro as well as their production of the plant growth hormone indole-3-acetic acid. The plant growth promoting effect by antagonistic bacteria was determined using a microtiter plate assay on the basis of lettuce seedlings. Lettuce and sugar beet as host plant were included in the biocontrol experiments in which the antagonistic effect of 17 bacterial isolates could be confirmed in vivo. Sequencing of the 16S rDNA gene and (or) fatty acid methyl ester gas chromatography was used to identify the antagonistic isolates. Molecular fingerprints of isolates obtained by BOX-polymerase chain reaction were compared to avoid further investigation with genetically very similar strains and to obtain unique molecular fingerprints for quality control and patent licensing. According to our strategy, an assessment scheme was developed and four interesting biological control agents, Pseudomonas reactans B3, Pseudomonas fluorescens B1, Serratia plymuthica B4, and Serratia odorifera B6, were found. While S. plymuthica B4 was the best candidate to biologically control Rhizoctonia in lettuce, P. reactans B3 was the best candidate to suppress the pathogen in sugar beet.

Evaluation of potential biocontrol rhizobacteria from different host plants of Verticillium dahliae Kleb.

AIMS: A screening approach was developed to assess the potential of rhizobacterial strains to control Verticillium wilt caused by Verticillium dahliae Kleb. METHODS AND RESULTS: Sixty randomly chosen antagonistic bacterial strains originally isolated from rhizosphere of three different host plants of V. dahliae--strawberry, potato and oilseed rape--were evaluated for biocontrol and plant growth promotion by analysing in vitro antagonism towards V. dahliae and other plant pathogenic fungi, production of fungal cell wall-degrading enzymes and plant growth-promoting effects on strawberry seedlings. To test the plant growth-promoting effect, a microplate assay with strawberry seedlings was developed. Although the rhizobacterial strains were isolated from different plants they showed effects on the growth of strawberry seedlings. According to the in vitro biocontrol and plant growth-promoting activity, the three best candidates Pseudomonas putida B E2 (strawberry rhizosphere), Ps. chlororaphis K15 (potato rhizosphere) and Serratia plymuthica R12 (oilseed rape rhizosphere) were selected for greenhouse experiments to verify the in vitro screening results. Under greenhouse conditions the isolates selected according to this strategy were as effective, or more effective than commercial biocontrol agents and may therefore possibly be valuable as antagonists of V. dahliae. CONCLUSIONS: In this study, the screening strategy resulted in a selection of three interesting biocontrol candidates against Verticillium: Ps. putida B E2 (strawberry rhizosphere), Ps. chlororaphis K15 (potato rhizosphere) and Ser. plymuthica R12 (oilseed rape rhizosphere). SIGNIFICANCE AND IMPACT OF THE STUDY: A new combination of in vitro screening methods including a microplate assay with strawberry seedlings to test the plant growth promoting effect which allow to more efficiently select potential biological control agents was developed successfully.

Toxic effect of water-soluble fractions of crude, refined, and weathered oils on the growth of a marine bacterium.

The water-soluble fractions of three crude and two refined oils reduced the growth rate and maximum cell density of the marine bacterium Serratia marinorubra grown in batch culture. The weathering of a crude and a refined oil was simulated in the laboratory. The water-soluble fractions remaining from this process were more toxic to S. marinorubra than were the parent unweathered oils. Increases in the magnitude of toxic effect of 3 to 30 times were observed as a function of decreasing the concentration of yeast extract in the cultures from 0.1 to 0.05 and 0.01%. The toxicity did not correlate with the concentration of total water-soluble fraction or of aromatic hydrocarbons in the water-soluble fraction. Affected cultures did not exhibit a residual toxicity after being back-inoculated into control media.