Pseudomonas sp. strain F113 was isolated from the rhizosphere of sugar beets and shown to inhibit a range of plant pathogenic fungi by producing an antibioticlike compound. An antibiotic-negative mutant strain, F113G22, was generated by transposon mutagenesis. This mutant has lost the ability to inhibit both bacterial and fungal microorganisms on high-iron medium. The antibioticlike compound was subsequently identified as 2,4-diacetylphloroglucinol (DAPG), and a high-pressure liquid chromatographic assay was developed for to detect it quantitatively in growth culture media and soil. The growth temperature had a direct bearing on DAPG production by strain F113, with maximum production at 12 degrees C. The iron concentration, pH, and oxygen had no influence on DAPG production by strain F113 under the assay conditions used. However, a low ratio of culture volume to surface area available to the microbe in the growth container was critical for optimum DAPG production. Different types of carbon sources influenced DAPG production by strain F113 to various degrees. For example, sucrose, fructose, and mannitol promoted high yields of DAPG by strain F113, whereas glucose and sorbose resulted in very poor DAPG production.

Isolation of 2,4-Diacetylphloroglucinol from a Fluorescent Pseudomonad and Investigation of Physiological Parameters Influencing Its Production

Deleterious rhizosphere bacteria: an integrating perspective

Pseudomonas fluorescens DR54 showed antagonistic properties against plant pathogenic Pythium ultimum and Rhizoctonia solani both in vitro and in planta. Antifungal activity was extractable from spent growth media, and fractionation by semi-preparative HPLC resulted in isolation of an active compound, which was identified as a new bacterial cyclic lipodepsipeptide, viscosinamide, using 1D and 2D 1H-, 13C-NMR and mass spectrometry. The new antibiotic has biosurfactant properties but differs from the known biosurfactant, viscosin, by containing glutamine rather than glutamate at the amino acid position 2 (AA2). No viscosin production was observed, however, when Ps. fluorescens DR54 was cultured in media enriched with glutamate. In vitro tests showed that purified viscosinamide also reduced fungal growth and aerial mycelium development of both P. ultimum and R. solani. Viscosinamide production by Ps. fluorescens DR54 was tightly coupled to cell proliferation in the batch cultures, as the viscosinamide produced per cell mass unit approached a constant value. In batch cultures with variable initial C, N or P nutrient levels, there were no indications of elevated viscosinamide production during starvation or maintenance of the cultures in stationary phase. Analysis of cellular fractions and spent growth media showed that a major fraction of the viscosinamide produced remained bound to the cell membrane of Ps. fluorescens DR54. The isolation, determination of structure and production characteristics of the new compound with both biosurfactant and antibiotic properties have promising perspectives for the application of Ps. fluorescens DR54 in biological control.

Viscosinamide, a new cyclic depsipeptide with surfactant and antifungal properties produced by Pseudomonas fluorescens DR54.

Native bacteria, Pseudomonas and filamentous bacteria were quantified and localized on wheat roots grown in the field using fluorescence in situ hybridization (FISH). Seminal roots were sampled through the season from unploughed soil in a conservation farming system. Such soils are spatially heterogeneous, and many roots grow slowly through hard soil with cracks and pores containing dead roots remnant from previous crops. Root and rhizosphere morphology, and contact with soil particles were preserved, and autofluorescence was avoided by observing sections in the far-red with Cy5 and Cy5.5 fluorochromes. Spatial analyses showed that bacteria were embedded in a stable matrix (biofilm) within 11 microm of the root surface (range 2-30 microm) and were clustered on 40% of roots. Half the clusters co-located with axial grooves between epidermal cells, soil particles, cap cells or root hairs; the other half were not associated with visible features. Across all wheat roots, although variable, bacteria averaged 15.4 x 10(5) cells per mm(3) rhizosphere, and of these, Pseudomonas and filaments comprised 10% and 4%, respectively, with minor effects of sample time, and no effect of plant age. Root caps were most heavily colonized by bacteria along roots, and elongation zones least heavily colonized. Pseudomonas varied little with root development and were 17% of bacteria on the elongation zone. Filamentous bacteria were not found on the elongation zone. The most significant factor to rhizosphere populations along a wheat root, however, was contact with dead root remnants, where Pseudomonas were reduced but filaments increased to 57% of bacteria (P < 0.001). This corresponded with analyses of root remnants showing they were heavily colonized by bacteria, with 48% filaments (P < 0.001) and 1.4%Pseudomonas (P = 0.014). Efforts to manage rhizosphere bacteria for sustainable agricultural systems should continue to focus on root cap and mucilage chemistry, and remnant roots as sources of beneficial bacteria.

Numbers and locations of native bacteria on field-grown wheat roots quantified by fluorescence in situ hybridization (FISH).

Plant-growth-suppressive activity of deleterious rhizobacteria (DRB) may be due to production of metabolites absorbed through roots. Auxins produced in high concentrations in the rhizosphere by DRB contribute to reduced root growth. Selected DRB able to produce excessive amounts of auxin compounds for suppression of weed seedling growth may be effective for biological control of weeds. The objectives to this study were to assess the ability of DRB originating from weed seedlings to synthesize auxins from L-tryptophan (L-TRP), determine effects of DRB with or without L-TRP on seedling root growth, and characterize auxins produced from L-TRP using high performance liquid chromatography (HPLC). Auxins expressed as indole-3-acetic acid (IAA)-equivalents were produced by 22.8% of the DRB tested based on a colorimetric method. Under laboratory conditions, a DRB isolate classified as Enterobacter taylorae with high auxin-producing potential (72 mg L−1 IAA-equivalents) inhibited root growth of field bindweed (Convolvulus arvensis L.) by 90.5% when combined with 10−5M L-TRP compared with non-treated control. Auxin derivatives produced by E. taylorae from L-TRP in broth culture after 24 h incubation identified by HPLC included IAA (102 μg L−1), indole-3-aldehyde (IALD; 0.4 μg L−1), and indole-3-lactic acid (ILA; 7.6 μg L−1). Results suggest that providing L-TRP with selected auxin-producing DRB to increase phytotoxic activity against emerging weed seedlings may be a practical biological control strategy.

Enhanced suppression of plant growth through production of L-tryptophan-derived compounds by deleterious rhizobacteria

We studied the production of a toxin inhibitory to both winter wheat (Triticum aestivum L.) root growth andEscherichia coli that was produced by a rhizobacterial pseudomonad. Of several carbon sources tested, the most rapid growth and highest toxin concentrations were obtained with glucose, glycerol, or trehalose. Toxin production was repressed with L-cysteine as the nitrogen source. Toxin was produced during the late exponential and early stationary phase of growth by the bacterium and, contrary to studies with other toxins, was unaffected by Fe and P concentrations in the growth medium. Toxin production by the bacterium was the same at growth temperatures of 25 and 15°C while it produced less at 5°C. If the bacterium was able to grow, it produced toxin. No compound tested induced an increase in toxin production indicating toxin production is constitutive.

Toxin production by a rhizobacterialPseudomonas sp. that inhibits wheat root growth

A toxin produced by a deleterious rhizobacterial pseudomonad that inhibits both winter wheat (Triticum aestivum L.) root andEscherichia coli growth was characterized. The toxin was rapidly deactivated at pH 2 and 12 and by autoclaving (121°C, 15 minutes). Less toxin was destroyed as the temperature and time of exposure decreased, and at 40°C it was stable for at least 24 hours. The toxin was extremely polar and could not be extracted from culture filtrates with organic solvents. The compound eluted after the void volume from a Sephadex G-10 column indicating a molecular weight of less than 700. The toxin adsorbed to Dowex 50W strong cation exchange resin and eluted with 2M NH4OH. Numerous thin layer chromatography solvent systems were unsuccessful at purifying the toxin. The partially purified toxin inhibited several different microorganisms while the producing strains were resistant. The toxin appears unique to toxins produced by recognized plant pathogenic bacteria.

H. Bolton

Papers

Toxin production by a rhizobacterialPseudomonas sp. that inhibits wheat root growth

Characterization of a toxin produced by a rhizobacterial Pseudomonas sp. that inhibits wheat growth