Strain L5 Research Articles

Competition for oxygen and 3-chlorobenzoate between two aerobic bacteria using different degradation pathways

In nature a significant part of the microbial activity is concentrated at or near oxic/anoxic interfaces, where oxygen concentrations are often low. Bacteria possessing different kinetic characteristics for oxygen and employing distinct metabolic pathways for the degradation of (halo)aromatic substrates for which oxygen is needed as co-substrate may have to compete with each other in such environments. In this study the competitiveness of Pseudomonas sp. strain A3 relative to Alcaligenes sp. strain L6 was tested in batch and in continuous cultures. While both of these strains are able to metabolise 3-chlorobenzoate (3CBA), the former was isolated under air saturating conditions and employs the catechol pathway, whereas the latter was isolated under reduced partial pressures of oxygen and was capable of metabolising 3CBA via the gentisate pathway. Competition experiments in batch culture resulted in pure cultures of Pseudomonas sp. strain A3 under air saturating conditions. However, if reduced partial pressures of oxygen (2%) were used, Alcaligenes sp. strain L6 remained present in substantial numbers after three transfers. Continuous culture experiments demonstrated that Alcaligenes sp. strain L6 was able to outcompete Pseudomonas sp. strain A3 under oxygen- as well as under carbon-limiting conditions as long as the dilution rate remained below 0.136 h−1 (low oxygen) and below 0.178 h−1 (high oxygen). These results support the hypothesis that organisms metabolising chlorobenzoate via the gentisate pathway may play a significant role in natural ecosystems where xenobiotic compounds and naturally produced aromatics occur at very low concentrations and in combination with limiting oxygen tensions.

Isolation of Alcaligenes sp. strain L6 at low oxygen concentrations and degradation of 3-chlorobenzoate via a pathway not involving (chloro)catechols

Isolations of 3-chlorobenzoate (3CBA)-degrading aerobic bacteria under reduced O2 partial pressures yielded organisms which metabolized 3CBA via the gentisate or the protocatechuate pathway rather than via the catechol route. The 3CBA metabolism of one of these isolates, L6, which was identified as an Alcaligenes species, was studied in more detail. Resting-cell suspensions of L6 pregrown on 3CBA oxidized all known aromatic intermediates of both the gentisate and the protocatechuate pathways. Neither growth on nor respiration of catechol could be detected. Chloride production from 3CBA by L6 was strictly oxygen dependent. Cell-free extracts of 3CBA-grown L6 cells exhibited no catechol dioxygenase activity but possessed protocatechuate 3,4-dioxygenase, gentisate dioxygenase, and maleylpyruvate isomerase activities instead. In continuous culture with 3CBA as the sole growth substrate, strain L6 demonstrated an increased oxygen affinity with decreasing steady-state oxygen concentrations.

External and internal root colonization of lodgepole pine seedlings by two growth-promotingBacillusstrains originated from different root microsites

Root colonization and in vitro carbon substrate utilization by two seedling growth-promoting Bacillus strains that originated from different root microsites were studied in greenhouse and growth chamber experiments. Strain L6, identified as Bacillus polymyxa, was previously isolated from rhizosphere soil containing roots of pasture plants, and Pw-2, tentatively identified also as B. polymyxa, was isolated from within surface-sterilized lodgepole pine (Pinus contorta var. latifolia (Dougl.) Engelm.) roots. Rifamycin-resistant strains derived spontaneously from wild-type strains L6 and Pw-2, designated strain L6-16R and Pw-2R, respectively, were used to monitor lodgepole pine root colonization in a closed tube assay system. Three-week-old pine seedlings were inoculated with 105colony-forming units (cfu) of strain Pw-2R or 106 cfu of strain L6-16R, and external and internal root colonization was assessed 2 and 4 weeks later. Strains L6-16R and Pw-2R were both recovered from pine rhizosphere samples with > 5 × 107 cfu/g fresh root tissue 2 weeks after inoculation, but neither strain was detected in the root interior. When root colonization was assessed 4 weeks after inoculation, the rhizosphere populations of both strains had declined slightly to between 5 × 106and 5 × 107 cfu/g fresh root tissue, but strain Pw-2R was also detected within root tissues with 105 cfu/g fresh root tissue. Lateral root formation was abundant 4 weeks after inoculation and may have facilitated colonization of internal root tissues by strain Pw-2R. Both strains possessed pectolytic activity, although differences between the strains were detected in in vitro substrate utilization capabilities using BIOLOG assays. These differences may be related to their abilities to colonize internal root tissues. On the basis of our results, we hypothesize that internal root colonization by Bacillus strains is not a random event and that root-endophytic Bacillus strains possess specific physiological and (or) biochemical characteristics that facilitate colonization of internal root tissues.Key words: Bacillus, PGPR, rhizosphere, endophytes, colonization.

Differential response of western hemlock from low and high elevations to inoculation with plant growth-promoting Bacillus polymyxa

The influence of inoculation with plant growth-promoting Bacillus polymyxa strains L5 and L6-16R was evaluated on the performance of western hemlock [ Tsuga heterophylla (Raf.) Sarg.] using seed that originated from six British Columbia provenances that differed primarily in elevation (from 152 to 1190 m above sea level). Preliminary analysis revealed a tendency for hemlock originating from high and low elevations to respond differently to bacterial inoculation, therefore subsequent analyses were performed after pooling data into two elevational groups: the three hemlock provenances located at the lowest elevations; and the three found at the highest elevations. B. polymyxa strain L6-16R-inoculated seed from the low elevation group produced an average of 2.38-fold ( P < 0.025) more seedlings than uninoculated controls midway through the emergence period, and 1.41-fold ( P < 0.05) more seedlings than uninoculated controls when emergence was complete. Inoculation of low elevation hemlock seed with strain L5 or of high elevation seed with either bacterial strain did not significantly affect seedling emergence. In contrast to the effects observed on emergence, growth of hemlock seedlings in the low elevation group was unaffected by inoculation with either bacterial strain, but strain L6-16R-inoculated high elevation seed produced seedlings that were significantly taller (1.19-fold; P < 0.05) and heavier (1.30-fold; P < 0.05) than uninoculated controls. Strain L5 did not stimulate growth of high elevation seedlings significantly. Hemlock rhizosphere colonization by strain L6-16R was assessed on seedlings that originated from one of the low elevation provenances and was found to be 1.7 × 10 3 cfu g −1 dry root tissue. These results indicate that seed inoculation with B. polymyxa strain L6-16R can result in colonization of western hemlock root systems and in significant increases in seedling emergence, height and biomass accumulation; however, the type of seedling growth response to inoculation with B. polymyxa may depend on the elevation of the provenance from which seed originates.

Variable effects of emergence-promoting rhizobacteria on conifer seedling growth under nursery conditions

Inoculation of white spruce (Picea glauca Voss.) seed with Bacillus polymyxa strain L5 under nursery conditions significantly increased the number of seedlings that emerged after sowing. No significant effects on seedling emergence were detected when white spruce seed was inoculated with Bacillus polymyxa strain L6, or when Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] seed was inoculated with either strain L5 or L6. However, white spruce seedlings originating from L5-inoculated seed had significantly lower root dry weights when measured 13 weeks after sowing, and reduced shoot dry weights 26 weeks after sowing compared with uninoculated controls. Inoculation of white spruce seed with strain L6 also resulted in seedlings with decreased root dry weights compared with uninoculated controls 13 weeks after sowing, but the significant inhibition of root growth was not apparent 26 weeks after sowing. Douglasfir seedlings originating from L5-inoculated seed had significantly lower root and shoot dry weights compared with uninoculated controls 13 but not 26 weeks after sowing. Inoculation of Douglas-fir seed with strain L6 resulted in seedlings with decreased root collar diameters and shoot dry weights 13 weeks after sowing, and lower root dry weights 26 weeks after sowing compared with uninoculated controls. These results demonstrate that the effects of bacterial inoculation on seedling emergence and on plant growth are independent, and that emergence-stimulating bacteria may inhibit subsequent seedling growth.

Biomass increase and associative nitrogen fixation of mycorrhizal Pinus contorta seedlings inoculated with a plant growth promoting Bacillus strain

One-month-old Pinus contorta Dougl. seedlings were inoculated with mycorrhizal fungi (Wilcoxina mikolae isolate R947), plant growth promoting bacteria capable of fixing nitrogen (Bacillus strain L6), or coinoculated with a combination of Wilcoxina and Bacillus. Seedlings were grown with 0.35 mM Ca(15NO3)2 (5% 15N atom excess) as the only nitrogen source. Fungal inoculation alone resulted in the synthesis of ectendomycorrhizae but decreased shoot biomass. Bacterial inoculation alone did not affect seedling biomass or foliar nitrogen content. Coinoculation of seedlings with Wilcoxina and Bacillus resulted in a similar degree of ectendomycorrhizal infection compared with fungal inoculation alone, but shoot biomass was greater than that of seedlings receiving the fungus alone. Root biomass and stem height were not altered by coinoculation. Total foliar nitrogen was lower in seedlings inoculated with Wilcoxina, and coinoculation with Bacillus did not reverse this trend. Based on the 15N to 14N ratio of foliage, associative nitrogen fixation by Bacillus contributed 4% (P &lt; 0.05) of seedling foliar nitrogen whether or not seedlings were mycorrhizal. However, coinoculated seedlings had lower foliar nitrogen (percentage basis) compared with uninoculated controls. These results suggest that growth of mycorrhizal pine can be stimulated by inoculating with beneficial bacteria. Growth promotion does not result from increased formation of ectendomycorrhizae and may, only in part, result from associative nitrogen fixation. Key words: Pinus, microorganisms, growth promotion, nitrogen fixation.

Inoculation of conifer seed with plant growth promoting Bacillus strains causes increased seedling emergence and biomass

Seed inoculation with Bacillus strains LS and L6 significantly increased the rate of seedling emergence of white spruce but did not affect subsequent seedling growth. Lodgepole pine root growth was promoted by strain L5 in sterilized, but not non-sterile growth medium. Strain L6 promoted pine root growth in sterilized medium, but also caused significant increases in seedling emergence, shoot weight and height, root weight and surface area, and root collar diameter when tested in non-sterile pcat-vcrmiculite medium. The positive effects due to a single inoculation of pine with strain L6 at sowing were not detectable after I2 weeks growth. However, root growth was stimulated after 16 weeks growth if seedlings were re-inoculated with strain L6 mid-way through the experiment. Shoot-growth promotion was also detected when I yr old lodgepole pine seedlings were planted in pots and inoculated with strain L6. Douglas-fir seedlings grown from seed inoculated with strain L5 had increased root collar diameters, whereas those inoculated with strain L6 exhibited increased root surface area.

Properties of the transducing phage Ml of Rhizobium meliloti

AbstractA temperate phage Ml, infective towards some R. meliloti strains, was isolated from soil taken from local fields. Its morphology, host range, transducing ability and the locations of restriction endonuclease cleavage sites on the genomes were determined. Phage Ml was classified as belonging to B BRADLEY's group bacteriophages because of the hexagonal outlines of its head and its long, flexible and non‐contractible tail. The molecular weight of phage DNA was estimated to be 54.1 ± 1 kilobases (kb) and it corresponded to the value expected for capsid size – 70 nm. Phage Ml carries out general transduction and forms stable lysogens with the R. meliloti strain L5.30.

Chemotaxis in Rhizobium meliloti strain L5.30

We have found that Rhizobium meliloti strain L5.30 exhibits positive chemotaxis towards some amino acids, sugars, exudates and extracts from roots of legume plants. From the investigated compounds sugars were better chemo-attractants than amino acids, but legume root substances were the best ones. Positive chemotaxis towards legume root compounds was supported by “clouds” of R. meliloti cells observed at the surface of alfalfa roots. A large deletion, in the nodABC region of the symbiotic megaplasmid (Sym), did not eliminate rhizobial chemotaxis.

Symbiotically defective histidine mutant of Rhizobium meliloti strain L5.30

AbstractHistidine auxotroph of Rhizobium meliloti strain L5.30 was isolated by nitrosoguanidine (NTG) mutagenesis. On alfalfa it formed ineffective nodules that contained only a few degenerating bacteroids inside large vacuoles but many starch granules. Prototrophic forms of this mutant (revertants and transductants) formed pink, effective nodules. Effective nodules were also induced by the His− mutant when histidine was supplied to the plant‐growth medium; this supported the supposition of a causal relationship between histidine auxotrophy and ineffectivity in the R. meliloti strain L5.30.The histidine mutation of the strain studied was mapped onto the chromosome of R. meliloti by transduction near earlier localized cys‐246 auxotrophic mutation (4.4% of cotransduction).

Mutants of Rhizobium meliloti defective in nodulation

AbstractUsing nitrosoguanidine (NTG) two nodulation defective (Nod−), auxotrophic mutants of the Rhizobium meliloti strain L5.30 have been isolated. In their Sym plasmid, pRmeL5.30b, a large deletion has been demonstrated. Both mutants were adsorbed into root hairs of lucerne, but were unable to induce root hair curlings. The nodulation of lucerne was restored to both hair‐curling (Hac−) mutants by the introduction of the Sym plasmid pJB5JI of R. leguminosarum and by plasmid pEK25, carrying a 3.6 kb AvaI/EcoRI fragment of “common” nod genes of R. meliloti 41. This led to the conclusion that in these Nod− deletion mutants nod mutations were concerned with “common” nod genes.

Nitrogen fixation efficiency, natural15N abundance, and morphology of mesquite (Prosopis glandulosa) root nodules

The relative nitrogen fixation efficiencies (RE 1-[H2 evolved÷C2H2 reduced]·100) of four mesquite (Prosopis glandulosa var.torreyana) rhizobia (Strains WR 1001, WR 1002, L5, L9) and a cowpea rhizobia (Strain 176A32) on mesquite were evaluated in a glasshouse experiment. Plant yield, shoot N accumulation, and the natural15N abundance (δ15N) of nodule tissue were determined. Strain WR 1002 failed to nodulate mesquite and strain L5 produced ineffective nodules. Among the three effective strains (WR 1001, L9, 176A32) the cowpea strain (176A32) and strain L9 had significantly higher RE than strain WR 1001. Differences in RE, however, were not accompanied by significantly higher plant yield and shoot N accumulation. The difference in15N abundance between foliar tissue and nodules (nodules minus leaves) was 0.47 δ15N for the ineffective L5 nodules, while for the effective WR 1001, L9, and 176A32 nodules, respectively, this difference was 8.35, 7.81, and 8.35 δ15N. This indicates a similar relationship between N2-fixing effectiveness and natural15N enrichment of nodules that was previously observed in soybeans (Glycine max, L. Merr.). Strains WR 1001 and L9 produced elongate, indeterminate nodules typical for mesquite. The ineffective L5 nodules had few infected cells and an abundance of cortical amyloplasts. Mesquite nodules produced by the cowpea strain were spherical and were somewhat more similar in internal morphology to determinate nodules typical of cowpea than indeterminate nodules normally associated with mesquite.