C. P. Chanway

RESPONSE OF CRESTED WHEATGRASS (AGROPYRON CRISTATUM L.), PERENNIAL RYEGRASS (LOLIUM PERENNE AND WHITE CLOVER (TRIFOLIUM REPENS L.) TO INOCULATION WITH BACILLUS POLYMYXA

Abstract The effect of inoculation of crested wheatgrass ( Agropyron cristatum L.), perennial ryegrass ( Lolium perenne L.) and white clover ( Trifolium repens L.) with the soil diazotroph Bacillus polymyxa was studied. Plant growth responses to inoculation varied from slightly negative (perennial ryegrass) to highly positive (white clover and crested wheatgrass) when root, shoot and plant dry weights were measured. Root-to-shoot ratios were also increased in the latter two species. Seedling emergence in crested wheatgrass was shown to be enhanced by inoculation with the bacterium. Possible mechanisms of the growth response include suppression of pathogenic organisms in the rhizosphere by the inoculant strain, root-associated nitrogen fixation, solubilization of phosphorus compounds or the bacterial production of plant growth-promoting substances. Use of sterilized soil in experiments and a lack of antibiotic production by the Bacillus render the first possibility unlikely. However, in vitro assays showed the bacterium to possess an active nitrogenase, to be capable of solubilizing organic but not inorganic phosphate compounds, and to produce indoleacetic acid, but not detectable amounts of cytokinins or gibberellins. Experimentation with A. cristatum suggested solubilization of P may be involved in enhanced seedling emergence, but neither N fixation nor phosphate solubilization were the primary contributing factors to the observed growth response. The data support the contention that production of growth-promoting compounds similar in activity to indoleacetic acid by the bacterium is the likely stimulus for the observed increase in plant productivity.

INTERACTIVE EFFECTS OF NUTRIENTS AND DISTURBANCE: AN EXPERIMENTAL TEST OF PLANT STRATEGY THEORY'

The relative impacts of nutrient availability, disturbance intensity, and in- terspecific competition on species distribution and growth were studied by applying five levels of nutrients and five intensities of clipping to simple artificial communities (ofDactylis glomerata, Holcus lanatus, Lolium perenne, and Trifolium repens) in a factorial design for 16 mo. This was done to test the hypothesis that the effects of interspecific competition would decline along gradients as the amount of nutrients decreased and the degree of disturbance increased. Nutrients and disturbance, alone and in interaction, produced sig- nificant effects on percent cover of all species, and all species achieved maximum percent cover in the undisturbed regime. The experimental design permitted us to distinguish between percent cover reductions caused directly by nutrients and disturbance from those caused by interspecific competition. When grown in mixture, interspecific competition produced marked changes in the distribution patterns of percent cover that were in addition to the direct impacts of nutrients and disturbance. The absolute reduction in percent cover due to interspecific competition was greatest in the undisturbed regime, and for many mixtures this competitive effect was significantly reduced or even eliminated in the highly disturbed areas. Along the nutrient gradient the responses were variable and neighbor dependent, but for all species, the absolute reduction in percent cover due to interspecific competition was generally most severe where percent cover in pure stands was highest. When the data were standardized for differences in percent cover in pure stands and competition was assessed as the percent reduction in percent cover between pure and mixed stands, then the effect of interspecific competition on Trifolium declined along both gra- dients. The three grasses had a reasonably constant competitive effect across the disturbance gradient, but along the nutrient gradient the highest competitive effects were generally achieved where percent cover in pure stands was highest. We argue that this standardized index is less appropriate because it obscures the obvious differences in interspecific com- petition effects that are clearly operating within these communities.

INOCULATION OF CONIFER SEED WITH PLANT GROWTH PROMOTING BACILLUS STRAINS CAUSES INCREASED SEEDLING EMERGENCE AND BIOMASS

Abstract 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.

Genotypic coadaptation in plant growth promotion of forage species byBacillus polymyxa

The effect of a plant growth promoting strain ofBacillus polymyxa was investigated using genotypically-defined mixtures of white clover (Trifolium repens L.) and perennial ryegrass (Lolium perenne L.). Addition ofB. polymyxa to a mixture of the species did not induce significant yield effects in perennial ryegrass, but resulted in a 23% (P<0.05) yield increase in the clover component. The clover yield advantage increased further when clones of the legume were inoculated with theB. polymyxa genotypes with which they had previously coexisted in the field from which collections were made. The highest white clover yield was attained when clones of all three organisms (T. repens, L. perenne, andB. polymyxa) that had previously coexisted in the field were grown together in the experimental system.

Growth reduction and root deformation of containerized lodgepole pine saplings 11 years after planting

Abstract Root system architecture and field performance of 90 twelve-year-old lodgepole pine (Pinus contorta var. contorta Dougl.) saplings, initially raised in containers for one year (1979) before planting, were compared with 60 naturally regenerated lodgepole pine saplings of the same age. The naturally regenerated saplings were significantly taller, and leader increments for 1989–1990 and 1990–1991 indicated that they performed better than the planted saplings during the two most recent growing seasons. Planted saplings displayed root morphologies which differed markedly from their natural counterparts; these included poor structural lateral root symmetry, fewer first order lateral roots, a greater distance from the ground surface to the first structural lateral root, and a greater root collar diameter and concentration of lateral roots 10 cm below the groundline. In addition, root stocks of planted saplings possessed remnants of vertical container shaping and a number of deformed roots that were either constricted, coiled and/or kinked. Naturally regenerated saplings illustrated elaborate sinker root development, and an assortment of self-grafted roots which were not observed on planted saplings. Possible effects of containerization on root morphology are discussed in relation to difficulties which may arise from artificial regeneration of cut-over sites.

Detection of GFP-labeled Paenibacillus polymyxa in autofluorescing pine seedling tissues

Paenibacillus polymyxa P2b-2R is a bacterium that originated from internal lodgepole pine (Pinus contorta var. latifolia (Dougl.) Engelm.) seedling stem tissue and fixes nitrogen (N) in association with pine and western red cedar (Thuja plicata Donn.). To evaluate endophytic colonization by this microorganism, we generated P. polymyxa P2b-2Rgfp, a green fluorescent protein (GFP)-labeled derivative of P2b-2R, and grew pine seedlings that were inoculated with the marked strain in a N-limited soil. Tissue disintegration during sample preparation precluded examination of needles for the GFP-labeled endophyte but GFP was detected on roots and in stems of 2- to 14-week-old pine seedlings using confocal laser scanning microscopy. Due to excessive autofluorescence of seedling tissues, labeled bacteria were clearly discernible only in stem tissues of 4- and 6-week-old seedlings. P2b-2Rgfp colonized the root surface extensively and was detected inside the stem cortex, primarily intracellularly. Some labeled bacteria appeared to contain endospores and none were detected in vascular tissues. We conclude that P. polymyxa P2b-2R is capable of endophytic colonization of pine seedlings with specific colonization sites that include the stem cortex but that GFP labeling is of limited value for localization of endophytic bacteria in pine seedling tissues.

Potential for Misidentification of a Spore-Forming Paenibacillus polymyxa Isolate as an Endophyte by Using Culture-Based Methods

ABSTRACT While Paenibacillus polymyxa strain Pw-2 has been identified as an endophyte of lodgepole pine (M. Shishido, B. M. Loeb, and C. P. Chanway, Can. J. Microbiol. 41:707-713, 1995), P. polymyxa strain L6 has not, a distinction that could be explained by the differential abilities of these isolates to form spores, rather than the differential abilities to colonize the interior tissues of lodgepole pine. Chemical disinfection was used to destroy bacteria on the root exterior, but bacterial endospores are known for their ability to withstand chemical disinfection, and strain Pw-2 was found to produce 300 to 11,000 times more germinating endospores than strain L6 under the experimental conditions used by Shishido et al. (Can. J. Microbiol. 41:707-713, 1995). Attempts to identify strain Pw-2 within lodgepole pine root tissues by using confocal microscopy techniques failed. We discuss the possibility that spore-forming bacteria can be mistakenly identified as endophytes when culture-based methods alone are used.

Storage effects on indigenous soil microbial communities and PGPR efficacy

We tested the hypothesis that storage-induced changes in the composition of soil microbial communities altered the efficacy of three Bacillus and three Pseudomonas plant growth promoting rhizobacteria (PGPR) strains on spruce seedlings. Forest soils were collected from near Mackenzie, Salmon Arm and Williams Lake, B.C. Soil microbial population sizes were evaluated using dilution plating. Carbon substrate utilization patterns were determined on fresh forest soils and after 32 weeks of storage at 4°C and −10°C using the Biolog system. Small aliquots of fresh and stored soils comprising <2% of seedling growth medium were also used in PGPR assays. The population sizes of viable soil microorganisms, particularly bacteria and mycorrhizal fungi, declined 10–1000 fold during soil storage at both temperatures. Differences in substrate utilization patterns between soils and after storage treatments were also detected by Shannons diversity index (H′), principal component analysis and cluster analysis of BIOLOG data. The three fresh soils formed a distinct cluster when plotted on ordination axes or by Euclidean distance. Williams Lake and Salmon Arm soils maintained a greater degree of substrate utilization diversity when stored frozen, but the opposite was true for Mackenzie soil. In general, the six PGPR strains had similar effects on spruce seedling growth within soil×storage treatments. Pseudomonas root colonization did not differ significantly between strains (Bacillus colonization was not evaluated), forest soils and storage treatments, but PGPR efficacy depended on the origin and treatment of forest soil. For example, spruce growth promotion was greatest in fresh Williams Lake and Salmon Arm soil, but PGPR efficacy in Mackenzie soil was greatest after storage at −10°C. Our results support the hypothesis that storage-induced changes in soil microflora contribute to variability in PGPR efficacy, but we were unable to identify specific characteristics of these forest soils that related directly to plant growth response variability.

Surface colonization of lodgepole pine (Pinus contorta var. latifolia [Dougl. engelm.]) roots by Pseudomonas fluorescens and Paenibacillus polymyxa under gnotobiotic conditions

Indirect immunofluorescence techniques and confocal scanning laser microscopy were used to identify rhizobacterial strains on the root surfaces of pine seedlings, which were grown from seeds under gnotobiotic conditions. Conifer plant growth promoting rhizobacterial strains Paenibacillus polymyxa L6 and Pw-2, and the forest soil isolate Pseudomonas fluorescens M20, were inoculated onto surface-disinfested pine seeds, singly, or in dual combinations: strains L6 + M20, or strains Pw-2 + M20. Segments containing particular root microsites (root tip, root hair zone, or areas of lateral root emergence) were sampled randomly from roots 7 or 13 weeks after inoculation, and the colonization of roots by each bacterium was observed. Root segments were also sampled from individual roots at six different points along the length of the root, and the qualitative colonization of younger areas, closer to the root tip, contrasted with that of older areas, closer to the root base. The ability of strain M20 to colonize root areas adjacent to sites of lateral root emergence improves in the presence of either P. polymyxa strain, while the ability of the P. polymyxa strains to colonize these areas was not affected. More rhizobacteria were also generally observed on younger root tissues than on areas closer to the root base.

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 × 103 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.