Molecular characterization of bacterial diversity in Lodgepole pine (Pinus contorta) rhizosphere soils from British Columbia forest soils differing in disturbance and geographic source (original) (raw)

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Present address: Xenon Genetics Inc., 3650 Gilmore Way, Burnaby, BC, Canada V5G 4W8.

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Present address: Helix Consulting, 3728 Collingwood St., Vancouver, BC, Canada V6S 2M5.

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Present address: Department of Microbiology and Immunology, University of British Columbia, #300-6174 University Blvd., Vancouver, BC, Canada V6T 1Z3.

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Monica L. Chow, Christopher C. Radomski, Joseph M. McDermott, Julian Davies, Paige E. Axelrood, Molecular characterization of bacterial diversity in Lodgepole pine (Pinus contorta) rhizosphere soils from British Columbia forest soils differing in disturbance and geographic source, FEMS Microbiology Ecology, Volume 42, Issue 3, December 2002, Pages 347–357, https://doi.org/10.1111/j.1574-6941.2002.tb01024.x
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Abstract

Rhizosphere bacteria from Lodgepole pine (Pinus contorta) seedlings were characterized from forest soils which differed in disturbance and geographic source. Soil disturbance treatments included whole-tree harvesting with and without heavy soil compaction and whole-tree harvesting with complete surface organic matter removal and heavy soil compaction from British Columbia (BC) Ministry of Forests Long-Term Soil Productivity installations in three biogeoclimatic subzones in central BC, Canada. Bacterial community members were characterized by DNA sequence analysis of 16S rRNA gene fragments following direct DNA isolation from soil, polymerase chain reaction amplification and cloning. Phylogenetic analyses revealed that 85% of 709 16S rDNA clones were classified as α-, β-, γ-, and δ-Proteobacteria, Actinobacteria, _Cytophaga_-_Flexibacter_-Bacteroides group, Acidobacterium, Verrucomicrobia, and candidate divisions OP10 and TM6. Members of the Proteobacteria and Acidobacterium represented 55% and 19% of the clone library, respectively, whereas the remaining bacterial divisions each comprised less than 4% of the clone library. One hundred and six 16S rDNA clones could not be classified into known bacterial divisions. No significant differences were detected for soil disturbance treatment or site effects on the proportions of 16S rDNA clones affiliated with Proteobacteria and Acidobacterium. Phylogenetic analyses revealed that it was common for 16S rRNA gene fragments from different soil disturbance treatments and geographic locations to be closely related.

1 Introduction

Rhizosphere microbiology has received significant attention during the last century due to the recognized importance of rhizosphere microorganisms to plant growth and health [1,2]. Root exudates, mucilage, and sloughed-off root cells provide a nutritional source for microbial cell multiplication and colonization of root surfaces and adjacent soil [3]. The rhizosphere is a dynamic niche containing complex microbial communities and microbial members may participate in a variety of beneficial and detrimental interactions with plants [4]. Beneficial interactions include the roles microorganisms play in enhancing nutrient uptake by plants, stimulating plant growth by a variety of mechanisms, and offering biological disease control. In contrast, major and minor pathogens can impair plant health and decrease productivity in agricultural and forested environments.

Culture-dependent and culture-independent methods have been used to explore rhizosphere bacterial communities in relation to variables such as the host plant species and soil properties [5–9]. These factors have been found to influence rhizosphere bacterial communities but it is difficult to draw general conclusions due to differences in the methods used for soil sampling [10] and bacterial community assessment, as well as the multitude of interactions that can result from different plant/soil/environmental systems. To address questions of ecological concern, studies must be designed using defined parameters linking a host plant and soil factors with a specific environment or ecosystem.

It was of interest to us to compare rhizosphere bacterial communities in soils disturbed by methods similar to operational forestry practices. Between 1975 and 1998, approximately 16 million hectares of Crown (publicly owned) forested land in Canada was harvested primarily by clear-cut forestry practices and the most common method of forest regeneration was by planted seedlings (National Forestry Database Program: http://nfdp.ccfm.org/frames2_e.htm). Forestry practices, including different methods of tree harvesting and site preparation, result in forest soil disturbance. Two important types of soil disturbance that can affect forest site productivity include changes in soil porosity (due to soil compaction) and changes in organic matter content [11]. These factors also have potential to alter bacterial communities through changes in soil physical and chemical properties such as available oxygen and moisture, temperature fluxes, and soil nutrients. The Long-Term Soil Productivity (LTSP) model was developed to determine the long-term effects of soil compaction and organic matter removal on soil and forest productivity [11] and 62 LTSP studies have been established in the USA and Canada [12]. LTSP studies provided unique field sites for us to explore rhizosphere bacterial diversity as related to different types of forest soil disturbance.

Previously, we used cultivation-dependent and cultivation-independent methods to characterize soil bacterial diversity from two British Columbia (BC) Ministry of Forests LTSP plot treatments representing two types of forest soil disturbance: whole-tree (clear-cut) harvesting with no soil compaction (plot N); and whole-tree (clear-cut) harvesting plus complete surface organic matter removal with heavy soil compaction (plot S) [13,14]. The relative abundance of Actinobacteria and the member genus Arthrobacter in a collection of 1492 bacterial isolates was greater from composite mineral soil samples from plot S compared to plot N [13]. Cultivation-independent methods revealed that the relative abundance of γ-Proteobacteria and the member genus Pseudomonas in clone libraries was greater from composite mineral soil samples from plot N compared to plot S [14]. These studies showed significant effects indicating that the examined LTSP soil disturbance treatments may have given rise to differences in the bacterial community composition in mineral soil.

In this study, we examined bacterial communities in the rhizosphere of planted Lodgepole pine seedlings, as little is known about this niche in Canadian temperate forests and rhizosphere bacterial communities are critical to plant health. Our major objectives were (1) to characterize Lodgepole pine rhizosphere bacterial communities using partial DNA sequence analysis of 16S rRNA genes and (2) to determine if rhizosphere bacterial community profiles differ in three types of forest soil disturbance treatments replicated in three geographic locations in BC.

2 Materials and methods

2.1 LTSP sites, treatment plots and rhizosphere soil sample collections

The BC Ministry of Forests LTSP studies, site conditions, and plot treatments have been described elsewhere [15–18]. Rhizosphere soil samples were collected from three BC Ministry of Forests LTSP sites in three distinct subzones of the Sub-boreal Spruce biogeoclimatic zone in central BC, Canada (Fig. 1). The sites included the LTSP Skulow Lake Installation (north-east of the city of Williams Lake); the LTSP Log Lake Installation (north of the city of Prince George); and the LTSP Topley Installation (south-east of the town of Smithers) (designated in this study, the Williams Lake, Prince George and Smithers sites, respectively). All sites have deep, medium textured loam soils and trees were 112–140 years old prior to harvest. Plot treatments were installed 1 to 2 years after tree harvest and container-grown Lodgepole pine seedlings were planted on the Prince George and Smithers sites in 1994 and the Williams Lake site in 1995 (1 year after plot installation).

1

The plot treatments selected for this study represented three types of soil disturbance (Table 1). Plot treatments OM2C0 and OM2C2 (designated plots N and C, respectively, in this study) had tree trunks and crowns removed and were similar to whole-tree harvesting with no soil compaction and with heavy soil compaction, respectively. The plot treatment OM3C2 had trunks, crowns, logging slash and all forest floor (humus) surface organic matter removed with heavy soil compaction (designated plot S in this study). Plot treatment S represents the highest level of soil disturbance in the LTSP model and has disturbance similar to landings where logs are processed during timber harvest (B. Chapman, personal communication) and where soil is influenced by heavy logging equipment traffic [16].

The plot treatments selected for this study represented three types of soil disturbance (Table 1). Plot treatments OM2C0 and OM2C2 (designated plots N and C, respectively, in this study) had tree trunks and crowns removed and were similar to whole-tree harvesting with no soil compaction and with heavy soil compaction, respectively. The plot treatment OM3C2 had trunks, crowns, logging slash and all forest floor (humus) surface organic matter removed with heavy soil compaction (designated plot S in this study). Plot treatment S represents the highest level of soil disturbance in the LTSP model and has disturbance similar to landings where logs are processed during timber harvest (B. Chapman, personal communication) and where soil is influenced by heavy logging equipment traffic [16].

1

A description of treatment plotsa sampled from BC Ministry of Forests LTSP sites near Williams Lake, Smithers, and Prince George, BC, Canada

1

A description of treatment plotsa sampled from BC Ministry of Forests LTSP sites near Williams Lake, Smithers, and Prince George, BC, Canada

Four Lodgepole pine seedlings (3–4 years old) were randomly selected for rhizosphere soil sampling during September, 1997 from each of three plot treatments (N, C and S) from each of three LTSP sites (Williams Lake, Prince George and Smithers) (Table 1). Each seedling was gently lifted from the ground with the intact roots surrounded by abundant soil and the soil was gently shaken from the roots. Random egressed roots plus clinging soil were aseptically cut from the seedling, approximately 3 cm from the root plug, and placed in a 500-cc sterile bottle. Each composite root sample was comprised of roots plus adhered rhizosphere soil from two seedlings. These samples were maintained at approximately 4°C and processed within 72 h to isolate DNA (described below). Composite root samples were saved after DNA isolation and the roots were used for microscopic assessment of mycorrhizae (approximately 400 root tips per composite sample) and then entire root samples were heated at 60°C for 72 h to determine dry weights.

2.2 Rhizosphere soil DNA isolation and purification

Rhizosphere soil was collected by submerging roots plus clinging soil from two seedlings in 250 ml of sterile TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) for 20–25 min at room temperature with intermittent mixing by hand (one sample). The soil suspension was filtered through a sterile mesh screen (1 mm pore size) to remove roots, small rocks and large pieces of organic matter. An additional 50 ml of TE was added to rinse the screen and the suspension was transferred to a sterile Waring blender chamber and blended for 30 s, at high speed. The liquid portion was decanted to a sterile centrifuge tube and spun at 300×g at 4°C for 10 min. The supernatant was centrifuged at 5000×g at 4°C for 10 min to pellet the microbial cell fraction. The pellet was washed once in TE, homogenized with a sterile toothpick, and stored at −20°C until DNA extraction. The FastDNA™ Kit MH (Qbiogene, Carlsbad, CA, USA) was used for DNA isolation from 100 mg cell pellet per sample as previously described [14]. Purified DNA samples were stored at −20°C.

2.3 DNA amplification with universal bacterial primers

Polymerase chain reaction (PCR) amplification of 16S rRNA gene sequences from rhizosphere soil DNA (0.5 ng) was carried out in 25 μl reactions using universal bacterial primers 16S.0007F.21 and 16S.1511R.21 [19] as previously described for 16S rDNA clones from surface organic matter and mineral forest soil samples [14]. A reaction without DNA was prepared as a control. A RoboCycler® Gradient 96 Temperature Cycler (Stratagene, La Jolla, CA, USA) was used to generate a gradient of four annealing temperatures (50.7°C, 51.6°C, 52.5°C, 53.4°C) and two reactions were done at each temperature for each soil sample as previously described [14]. The PCR product size was visualized by gel electrophoresis and ethidium bromide staining. PCR products from different annealing temperatures were pooled for each soil DNA sample and purified with QIAquick PCR purification spin columns (Qiagen, Missisauga, ON, Canada). DNA concentrations were quantified using a fluorometer. PCR products were stored at −20°C.

2.4 DNA cloning

The PCR products amplified with the universal bacterial primers were ligated into the plasmid vector pGEM®-T (3:1 insert to vector ratio) as per the pGEM®-T and pGEM®-T-Easy Vector System technical manual (Promega, Madison, WI, USA). The ligation products were purified and concentrated two-fold using Ultrafree-MC Millipore Filters (30 000 nominal molecular mass limit polysulfone membrane, Millipore, Bedford, MA, USA). An aliquot of the purified ligation product (25 ng) was transformed into the Escherichia coli host strain ElectroMAX DH10B™ (Gibco BRL, Rockville, MD, USA) using electroporation as previously described [14]. Serial dilutions were plated as described by the pGEM®-T manual, and approximately 40 clones were randomly selected to represent each of 18 composite rhizosphere soil samples. Clones were grown overnight in Terrific Broth [20] with ampicillin (100 μg ml−1) and stored at −80°C in 20% glycerol.

2.5 Plasmid DNA isolation, DNA sequencing and analysis

Methods used for plasmid DNA isolation from 16S rDNA clones and partial sequencing of the 16S rRNA gene inserts with the primer 16S.0007.F21 have been described [14]. Plasmid DNA was stored at −20°C. Partial 16S rRNA gene sequences were submitted to the BLAST server (Basic Local Alignment Search Tool, National Center for Biotechnology Information (NCBI): http://www.ncbi.nlm.nih.gov/[21]) to determine the closest matching sequences in the GenBank and to infer possible phylogenetic affiliations. Closely related 16S rRNA gene sequences originating from the same rhizosphere soil sample were aligned to determine similarity using Geneworks 2.4 alignment software (Accelrys, Cambridge, UK). The partial sequence of a 16S rDNA clone was considered unique, included in phylogenetic analyses and deposited in the GenBank if it had greater than four nucleotide differences within the region sequenced (approximately 450 nucleotide bases; <99% sequence similarity) compared to other 16S rDNA clones from the same rhizosphere soil sample.

2.6 16S rDNA clone sequence designations and phylogenetic analyses

Partial 16S rRNA gene sequences of 16S rDNA clones were designated by a source code to identify the LTSP plot (N, C, or S, Table 1), rhizosphere soil sample number, followed by a period (.), the clone number, and the LTSP site location (WL=Williams Lake; PG=Prince George; SM=Smithers). Partial 16S rRNA gene sequences from this study and from selected GenBank members (designated with the corresponding accession number) were edited to start and end with the corresponding positions in E. coli for a 5′ start at nucleotide position 38 (beginning at GGCGG) and a 3′ end at position 432 (ending with GTAAA) prior to preparing multiple alignments with ClustalW version 1.73 [22].

Phylogenetic analysis included building a series of trees based on variations in 16S clone sequence sets and GenBank sequences representing known and candidate bacterial divisions. Initial neighbor-joining phylogenetic trees were built with 100 bootstraps using PHYLIP software (Phylogeny Inference Package) version 3.5c [23] as previously described [14]. Neighbor-joining phylogenetic trees referred to in this paper were constructed with 100 bootstraps using TreeCon for Windows 1.3b [24]. All alignment positions were included in the analysis but insertions and deletions were not taken into account when calculating the Jukes and Cantor distance. Final classification of 16S rDNA clones to a phylogenetic division or subdivision, or to an unclassified category, was based on combined results from the phylogenetic group represented by the closest matching sequences in the GenBank and phylogenetic tree analyses.

2.7 Additional analyses

A three-way contingency table analysis (using a log-linear model for categorical data [25], analyzed with GLIM software) was used to test for LTSP plot (soil disturbance) effects and for LTSP site (geographic location) effects with respect to the relative abundance of 16S rDNA clones belonging to specific bacterial divisions or subdivisions. Analyses were done independently for clone members of Proteobacteria, α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, and Acidobacterium. Statistical analyses could not be done for other bacterial groups since clone member numbers were too low.

2.8 GenBank accession numbers and web site information

Partial 16S rRNA gene sequences from unique clones were deposited in the GenBank database and assigned accession numbers AF431057 to AF431610 for clones classified as α-, β-, γ-, and δ-Proteobacteria, Actinobacteria, Cytophaga-Flexibacter-Bacteroides (CFB) group, Acidobacterium and Verrucomicrobia; and AF432612 to AF432629 and AF432699 to AF432781 for unclassified 16S clones. A listing of 16S rDNA clones and their corresponding GenBank accession numbers are available (http://bacterialdiversity.bcresearch.com/accession_lookup.htm) and 16S rDNA sequences for 16S clones are also available (http://bacterialdiversity.bcresearch.com/dna_sequences.htm).

3 Results and discussion

3.1 Mycorrhizae, seedling root dry weights, and DNA yields from rhizosphere soil samples

Seedling roots were collected after rhizosphere soil processing to examine mycorrhizae and quantify root biomass. Mycorrhizal fungal colonization levels of Lodgepole pine root tips were similar for seedlings harvested from three LTSP plot treatments (N, C and S) from each of three sites (75–95% root tip colonization per sample). Mycorrhizal fungi identified included Amphinema, Cenococcum and tuberculate mycorrhizae. Mean root dry weights for the six samples from each of Williams Lake, Smithers and Prince George LTSP sites were 2.59 g (S.D.=0.51 g), 3.13 g (S.D.=1.08 g) and 2.56 g (S.D.=0.64 g), respectively. DNA yields from the 18 rhizosphere soil samples were variable and ranged from 2.0 to 23.1 ng DNA mg−1 cell pellet (mean DNA yield=11.7 ng DNA mg−1 cell pellet; S.D.=7.4 ng DNA mg−1 cell pellet). DNA template concentrations were standardized for all PCR reactions to 0.5 ng soil DNA per 25 μl reactions to minimize potential sources of variability such as the amount of rhizosphere soil sampled and DNA yields from samples. We followed methods commonly used for sampling rhizosphere soil [10] which included collection of soil clinging to plant roots after plant excavation from soil.

3.2 Characterization of 16S rDNA clones

Thirty-six to 40 16S rDNA clones for each of 18 rhizosphere soil samples, for a total of 709 clones, were partially sequenced at the 5′ end of the 16S rRNA gene. The length sequenced ranged from 368 to 632 nucleotides (three sequences were less than 400 nucleotides). Overall, 7% of the 16S rDNA clone sequences (50 clones) shared ≥99% sequence similarity with another clone from the same soil sample. Identical or nearly identical bacterial 16S rRNA gene fragments have been recovered from non-rhizosphere forest soil samples from the Williams Lake LTSP site [14], grassland rhizosphere soil [26] and agricultural soil [27]. Retrieval of identical sequences from the same soil sample may indicate species abundant in soils or an artefact due to PCR. Full-length sequence analysis would be required to accurately determine the identical nature of cloned gene fragments.

Phylogenetic analyses revealed that 85% of 709 rhizosphere soil 16S rDNA clones were classified as α-, β-, γ-, and δ-Proteobacteria, Actinobacteria, CFB group, Acidobacterium, Verrucomicrobia, and candidate divisions OP10 and TM6 (Fig. 2). Members of Proteobacteria represented 55% of the clone library and α-Proteobacteria had the greatest representation followed by β-, γ-, and δ-Proteobacteria. Acidobacterium comprised 19% of the clone library whereas the other bacterial divisions each comprised less than 4% of the clone library. Other studies have indicated that α-Proteobacteria members were most abundant in 16S rDNA clone libraries from non-rhizosphere LTSP forest soil samples from BC, Canada [14], Australian forest soil [28,29], Scotland grassland rhizosphere soil [26], and maize roots [30]. In contrast, Acidobacterium members were most abundant in clone libraries from Arizona pinyon pine rhizosphere and bulk soil samples [31]. The relative abundance of Actinobacteria in the rhizosphere rDNA clone library in this study was substantially less compared to clone libraries from mineral soil from Williams Lake LTSP plots N and S [14] and rhizosphere soil from grassland plant species [26] and _Lolium perenne_[32].

2

Classification of 16S rDNA clones (_n_=709) from rhizosphere soil samples from three BC Ministry of Forests LTSP plots from three LTSP installations into bacterial phylogenetic groups.

One hundred and six 16S rDNA clones (15% of the clone library) could not be classified into known bacterial divisions based on publicly available 16S rRNA gene sequence information and phylogenetic analyses. Many of these clones formed bootstrap-supported clusters with each other and GenBank member sequences were not closely related (results not shown). Unclassified clones with chimeric structures were not detected upon examination of long-distance base pairing in the secondary DNA structure (J.K. Harris, unpublished results).

Detailed phylogenetic trees were not included in this paper due to their size and complexity but these trees are presented in detail on a web site (http://bacterialdiversity.bcresearch.com/rhizosphere_soil_16S/phylogenetic_trees.htm, Fig. R1, α-Proteobacteria; Fig. R2, β-Proteobacteria; Fig. R3, γ-Proteobacteria; Fig. R4, δ-Proteobacteria; Fig. R5, Actinobacteria; Fig. R6, CFB group; Fig. R7, Acidobacterium; and Fig. R8, Verrucomicrobia). Phylogenetic analyses revealed that 80% of 16S rDNA clone sequences formed bootstrap-supported clusters that contained GenBank member sequences originating from diverse soil sources, sludge and aquatic environments. The remaining clones either were represented in 21 bootstrap-supported clusters which excluded GenBank member sequences (64 clones which represented α-, β-, γ-Proteobacteria and Acidobacterium) or were single sequences not contained in clusters (54 clones classified as α-, β-, γ-Proteobacteria) (Table 2). These 16S rDNA clone sequences may represent members of novel lineages within Proteobacteria and Acidobacterium that have not been previously reported. A summary of the cluster affiliation from all phylogenetic trees is presented in Table 2 and the highlights for Proteobacteria and Acidobacterium are discussed below.

2

Summary of the 16S rDNA clone affiliation in phylogenetic tree clusters based on the most closely related GenBank member sequences included in treesa and present in clusters

b

Numbers in parentheses indicate the number of 16S rDNA clones which had less than five nucleotide differences compared to other 16S rDNA clones from the same soil sample.

c

Classification based on Hugenholtz and coworkers [34].

2

Summary of the 16S rDNA clone affiliation in phylogenetic tree clusters based on the most closely related GenBank member sequences included in treesa and present in clusters

b

Numbers in parentheses indicate the number of 16S rDNA clones which had less than five nucleotide differences compared to other 16S rDNA clones from the same soil sample.

c

Classification based on Hugenholtz and coworkers [34].

3.2.1 Proteobacteria

Three hundred and ninety-two 16S rDNA clone sequences were affiliated with α-, β-, γ- and δ-Proteobacteria and 43% of these clone sequences were related to 15 bacterial families and groups representing cultivated bacterial genera (Table 2). In contrast, 27% of the Proteobacteria 16S rDNA clone sequences were present in phylogenetic tree clusters containing only uncultivated GenBank representatives. The largest cluster of related Proteobacteria 16S rRNA gene sequences consisted of 46 clones affiliated with Burkholderia (Fig. 3) (also represented as cluster 1 in the β-Proteobacteria tree, Fig. R2, http://bacterialdiversity.bcresearch.com/rhizosphere_soil_16S/phylogenetic_trees.htm). These 16S clone sequences were recovered from all LTSP plots and sites and represented 6.5% of the clone library indicating Burkholderia may play a functionally important role in Lodgepole pine rhizosphere ecology. Burkholderia species are nutritionally versatile, common residents of rhizosphere soil, and beneficial attributes of some members include nitrogen fixation, plant growth promotion and biological disease control [33]. Twelve of the 46 16S rDNA clone sequences shared ≥99% sequence similarity with other clones originating from the same soil sample. A notable feature of this cluster is that member sequences were identical or nearly identical to partial 16S rRNA gene sequences from different LTSP plots and sites indicating the conserved nature of the 5′ end of the 16S rRNA gene in the recovered Burkholderia group sequences.

3

Bootstrap-supported cluster of Burkholderia group partial 16S rRNA gene sequences extracted from a phylogenetic tree containing β-Proteobacteria 16S rDNA clone sequences from BC Ministry of Forests LTSP installations and NCBI GenBank sequences (http://bacterialdiversity.bcresearch.com/rhizosphere_soil_16S/phylogenetic_trees.htm, Fig. R2). 16S rDNA clone names reveal source information designated by the LTSP plot (N, C, or S; see Table 1 for a description), composite rhizosphere soil sample number followed by a period (.), the clone code number, and ending with the city closest to the LTSP installation (WL=Williams Lake; SM=Smithers; or PG=Prince George). Numbers in parentheses following 16S rDNA clone names indicate the number of 16S rDNA clones which had less than five nucleotide differences compared to other 16S rDNA clones from the same soil sample. Partial 16S rRNA gene sequences from the GenBank are designated by the accession number. The distance scale represents 0.05 fixed mutations per site.

Although Pseudomonas is also recognized as a common rhizosphere colonist [2], only six of the 709 rhizosphere 16S rDNA clone sequences formed a bootstrap-supported cluster with GenBank member Pseudomonas sequences (http://bacterialdiversity.bcresearch.com/rhizosphere_soil_16S/phylogenetic_trees.htm, Fig. R3, cluster 12). Cloned 16S rRNA genes affiliated with Pseudomonas were also infrequently recovered from grassland rhizosphere soil samples [26]. In contrast, 11 of 67 clones from L. perenne and Trifolium repens rhizosphere soil samples were affiliated with _Pseudomonas_[32]. Pseudomonas was readily cultivated from mineral soil samples from plots N and S from the Williams Lake LTSP site [13] but a significantly greater proportion of Pseudomonas 16S rRNA gene sequences were cloned from mineral soil samples from plot N compared to plot S [14]. The six rhizosphere clones affiliated with Pseudomonas were all obtained from plot N from all three LTSP sites. Further research is warranted to examine rhizosphere-associated Pseudomonas abundance and diversity from LTSP plots and sites.

Rhizobiaceae group sequences (26 clones) were recovered from Lodgepole pine rhizosphere soil from all LTSP plots and sites except for plot C from the Smithers LTSP site (http://bacterialdiversity.bcresearch.com/rhizosphere_soil_16S/phylogenetic_trees.htm, Fig. R1, clusters 5, 6, and 8). Vegetation present on LTSP plots included leguminous plants (B. Chapman, personal communication) and this may have influenced the recovery of Rhizobiaceae DNA. Similarly, 16S rRNA gene fragments related to Rhizobium were also recovered from maize roots and it was noted that pea was grown in the field site prior to maize cultivation [30]. It is also possible that Rhizobium is a native member of non-legume rhizospheres.

3.2.2 Acidobacterium

One hundred and thirty-one 16S rDNA clone sequences were affiliated with seven of the eight monophyletic Acidobacterium subdivisions proposed by Hugenholtz and coworkers [34] indicating broad diversity and potential ecological significance of this bacterial division in Lodgepole pine rhizospheres (Table 2,Fig. 4; http://bacterialdiversity.bcresearch.com/rhizosphere_soil_16S/phylogenetic_trees.htm, Fig. R7). Seventy-four percent of the clone sequences were affiliated with Acidobacterium subdivisions 1, 2 and 3. The sequences did not cluster with GenBank member Holophaga and Geothrix 16S rRNA gene sequences which belong to Acidobacterium subdivision 8. Phylogenetic analyses revealed that two 16S rDNA clone sequences (C47.23PG and N42.38PG) were members of Acidobacterium but they formed their own bootstrap-supported cluster without other GenBank member representatives of Acidobacterium (Fig. 4). Full-length sequence analysis is needed to further explore the affiliation of C47.23PG and N42.38PG with Acidobacterium. Although Acidobacterium has few cultivated members, DNA sequences from uncultivated representatives have been frequently recovered from soil [34,35] including forest environments [14,36].

4

Phylogenetic tree of Acidobacterium partial 16S rRNA gene sequences representing 133 rhizosphere 16S rDNA clones from three BC Ministry of Forests LTSP installations and 26 reference sequences from the NCBI GenBank representing Acidobacterium subdivisions 1–8 (labelled to the right of each cluster) as per the classification proposed by Hugenholtz and coworkers [34]. The number of rhizosphere 16S rDNA clones represented in each cluster is: cluster 1: 41 clones; cluster 2: 29 clones; cluster 3: 29 clones; cluster 4: 10 clones; cluster 5: two clones; cluster 6: 13 clones; cluster 7: seven clones; and cluster 8: no clones, the cluster is represented by two GenBank member sequences. Two clones, C47.23PG and N42.38PG, were not affiliated with the eight proposed subdivisions of Acidobacterium. The tree was rooted with a Fibrobacter/Acidobacterium group partial 16S rRNA gene sequence, Fibrobacter succinogenes (M62696), as the outgroup. The distance scale represents 0.05 fixed mutations per site. The same phylogenetic tree is presented in detail on a web site where the clusters are not collapsed into triangles (http://bacterialdiversity.bcresearch.com/rhizosphere_soil_16S/phylogenetic_trees.htm, Fig. R7).

3.3 Comparison of bacterial community profiles of different LTSP soil disturbance treatments and different LTSP sites

Similar trends in community profiles were apparent among 16S rDNA clones from all three soil disturbance plot treatments from the three LTSP sites and the relative abundance of Proteobacteria was greatest for each plot at each site, followed by Acidobacterium (Table 3). Within the Proteobacteria, the trend from highest to lowest relative abundance was α-, β-, γ-, and δ-Proteobacteria, respectively, for the majority of the plots for each of the sites. Statistical analyses revealed that there were no significant LTSP plot (soil disturbance) effects and no significant LTSP site (geographic location) effects on the relative abundance of the clones belonging to Proteobacteria, α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, or Acidobacterium. All likelihood ratio P values exceeded 0.2 when analyses were completed for Proteobacteria at the division level and for individual analyses of α-, β-, and γ-Proteobacteria. For Acidobacterium, both P values (testing for plot effects and site effects) were approximately 0.06 and the magnitudes of differences in the number of Acidobacterium clones among plots or among sites were not dramatic. The numbers of 16S rDNA clones belonging to other bacterial groups were too low for statistical analyses. Felske and Akkermans [37] also found more similarities than differences in community profiles when they examined temperature gradient gel electrophoresis fingerprints from 160 soil samples representing three grassland field sites. The sites varied in their fertilization history and were within 2 km of each other.

3

Classification of rhizosphere 16S rDNA clones from BC Ministry of Forests LTSP plot treatmentsa N, C and S from LTSP sites Williams Lake (WL), Smithers (SM) and Prince George (PG) into bacterial phylogenetic groups

a LTSP plot treatments, N: intermediate level organic matter removal, no soil compaction; C: intermediate level organic matter removal, heavy soil compaction; S: highest level organic matter removal, heavy soil compaction.

b The number of rhizosphere 16S rDNA clones represents pooled numbers from approximately 40 clones from each of two composite rhizosphere soil samples per plot treatment per site. No significant LTSP plot effects or site effects were detected with respect to the relative abundance of 16S rDNA clone members of Proteobacteria, α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, and Acidobacterium (_P_>0.05). Statistical analyses could not be done for other bacterial groups since 16S clone member numbers were too low.

3

Classification of rhizosphere 16S rDNA clones from BC Ministry of Forests LTSP plot treatmentsa N, C and S from LTSP sites Williams Lake (WL), Smithers (SM) and Prince George (PG) into bacterial phylogenetic groups

a LTSP plot treatments, N: intermediate level organic matter removal, no soil compaction; C: intermediate level organic matter removal, heavy soil compaction; S: highest level organic matter removal, heavy soil compaction.

b The number of rhizosphere 16S rDNA clones represents pooled numbers from approximately 40 clones from each of two composite rhizosphere soil samples per plot treatment per site. No significant LTSP plot effects or site effects were detected with respect to the relative abundance of 16S rDNA clone members of Proteobacteria, α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, and Acidobacterium (_P_>0.05). Statistical analyses could not be done for other bacterial groups since 16S clone member numbers were too low.

Examination of the source of 16S rRNA gene sequences within individual bootstrap-supported clusters from phylogenetic trees provided a better comparison of the bacterial community profile from different LTSP soil disturbance treatments and sites. This approach may have more biological relevance as opposed to a general analysis of the relative abundance of 16S rDNA clones belonging to individual bacterial divisions or subdivisions. Clusters were selected that contained seven or more members (a minimum of 1% of the clone library) and that were supported by bootstrap values ≥80 to allow for comparisons of related 16S rRNA gene fragments. Twenty-six tree clusters fit these criteria and each cluster contained seven to 46 clones from trees representing each of eight bacterial groups (http://bacterialdiversity.bcresearch.com/rhizosphere_soil_16S/phylogenetic_trees.htm: Fig. R1, α-Proteobacteria, nine clusters; Fig. R2, β-Proteobacteria, three clusters; Fig. R3, γ-Proteobacteria, two clusters; Fig. R4, δ-Proteobacteria, one cluster; Fig. R5, Actinobacteria, one cluster; Fig. R6, CFB group, two clusters; Fig. R7, Acidobacterium, six clusters; Fig. R8, Verrucomicrobia, two clusters). Twenty-four of the 26 clusters each contained one or more 16S rRNA gene fragments originating from each of the three LTSP sites and 25 of the 26 clusters each contained one or more 16S rRNA gene fragments originating from each of the three LTSP plots N, C and S. Therefore, it was common for phylogenetic tree clusters to contain related 16S rRNA gene fragments representing 16S rDNA clones from different LTSP sites and plots. Only six clusters had more than 15 clones, and three of these clusters (including the Burkholderia group cluster, Fig. 3) each contained one or more 16S rRNA gene fragments originating from all three LTSP plots from all three LTSP sites.

The above results indicate that the rhizosphere environment generated by Lodgepole pine may have exerted a stronger selective pressure in determining the rhizosphere bacterial community than soil conditions due to LTSP soil disturbance treatments and geography. It is important to note that the LTSP plot treatments of surface organic matter removal and soil compaction influenced soil chemical and physical properties and vegetation cover at the Williams Lake, Smithers and Prince George LTSP sites during the time frame of our study [17,18,38,39]. Specifically, heavy soil compaction (plots C and S) significantly increased soil bulk density by approximately 15% compared to plots with no soil compaction (plot N) [17]. The removal of all surface organic matter (plot S) resulted in loss of forest floor nutrients, a small reduction in mineral soil carbon [39], and an increase in soil temperature to a depth of 10 cm (B. Chapman, personal communication) compared to soil disturbance treatments which retained surface organic matter (plots N and C). Total surface organic matter removal (plot S) also caused changes in forest vegetation including reductions in total plant species richness and changes in species composition compared to plots N and C [38].

Historically it has been thought that rhizosphere microbial populations are directly or indirectly related to root exudates [2], indicating the importance of the host plant. Numerous studies under field and controlled conditions have reported the importance of the host plant and/or soil factors in influencing the composition of rhizosphere bacterial communities [5–9,40,41]. PCR-based methods have proved valuable in generating cultivation-independent microbial diversity data. Yet, it is recognized that PCR artefacts may cause biases [42] and the relative proportions of different bacterial groups represented in clone libraries may not reflect the relative proportions present in template DNA samples [43,44]. The analysis of multiple soil samples representing host plant or soil types strengthens the conclusions which can be drawn from bacterial diversity studies.

4 Conclusions

Forestry practices such as tree harvesting, site preparation and subsequent planting of seedlings can result in changes in the soil environment. In this study we addressed two main questions: what is the bacterial community profile in Lodgepole pine rhizosphere soils; and does this profile differ among three types of forest soil disturbance treatments from LTSP studies replicated in three subzones of the Sub-boreal Spruce biogeoclimatic zone in central BC? The 16S rDNA clone library from Lodgepole pine rhizosphere soils represented seven known bacterial divisions. There were no significant LTSP plot effects or LTSP site effects on the relative abundance of 16S rDNA clones belonging to Proteobacteria or Acidobacterium which together comprised 74% of the clone library. Phylogenetic analyses indicated that it was common for 16S rRNA gene fragments from different soil disturbance treatments and different LTSP sites to be grouped together in bootstrap-supported clusters. These results suggest that the Lodgepole pine rhizosphere is a resilient niche supporting extremely diverse microbial communities which share similar profiles across the examined types of soil disturbance and geographic regions.

Torsvik and coworkers [45] stress that information about bacterial communities and their diversity is required to explore questions regarding the impact of environmental factors on ecosystem function. However, Staddon and coworkers [46] note that information is lacking about the variability of soil microbial communities in forest ecosystems. This study established a library of diverse 16S rRNA gene fragments from Lodgepole pine rhizosphere soil which can be used to construct specific DNA primers and probes to target bacterial groups of interest. Microbial indicators could prove valuable for assessments of soil quality [47] relating to ecological forest management [46]. Future studies on LTSP sites can be strengthened by using combinations of molecular methods to profile and fingerprint whole rhizosphere bacterial communities. Furthermore, a unique opportunity exists for future investigations addressing the relationship between soil bacterial communities and forest ecosystem functions through integration with other LTSP sites across climatic gradients in North America.

Acknowledgements

We gratefully thank many people including Saima Kassam and Simon Fraser University co-operative student Millie Mung for excellent technical assistance; Bill Chapman, Paul Sanborn, Marty Kranabetter and Shannon Berch for providing helpful suggestions and information regarding the BC Ministry of Forests LTSP studies and access to LTSP sites; Bill Chapman, Paul Sanborn, Marty Kranabetter and Reed Radley for assistance with soil sampling; Guoping Xiao for assessment of mycorrhizae; Ned Glick for statistical consulting; Agata Wodzynska for formatting phylogenetic trees; and Bill Chapman for his critical review of the manuscript. Funding for this research was provided by Forest Renewal British Columbia (FRBC) to P.E.A.

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Author notes

1

Present address: Xenon Genetics Inc., 3650 Gilmore Way, Burnaby, BC, Canada V5G 4W8.

2

Present address: Helix Consulting, 3728 Collingwood St., Vancouver, BC, Canada V6S 2M5.

3

Present address: Department of Microbiology and Immunology, University of British Columbia, #300-6174 University Blvd., Vancouver, BC, Canada V6T 1Z3.

© 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

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