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Cultivation-dependent characterization of bacterial diversity from British Columbia forest soils subjected to disturbance
Paige E. Axelrood, Monica L. Chow, Clarke S. Arnold, Karen Lu, Joseph M. McDermott, and Julian Davies

Abstract: Bacteria from forest surface organic matter and mineral soil horizons were cultivated using four methods and characterized by fatty acid methyl ester (FAME) analysis. Soil samples from a British Columbia Ministry of Forests Long-Term Soil Productivity (LTSP) installation were collected during winter and summer from two disturbance treatments (whole-tree harvesting with no soil compaction (plot N) and whole-tree harvesting plus complete surface organic matter removal with heavy soil compaction (plot S)) and from an unlogged reference plot (REF). Seventy-five percent of 1795 bacterial isolates were affiliated with 42 genera representing - and -Proteobacteria, Actinobacteria, the Bacillus/Clostridium group, and the CytophagaFlexibacterBacteroides group. Approximately half of the culture collection represented genetic diversity confined to four bacterial genera: Pseudomonas, Bacillus, Paenibacillus, and Arthrobacter. A significantly higher proportion of bacterial isolates belonging to Actinobacteria, and the member genus Arthrobacter, were isolated from plot S soil samples compared with soil samples from plots N and REF. Twenty-five percent of bacterial isolates were not conclusively identified to genus with FAME analysis. Sherlock Tracker cluster analysis and partial 16S rRNA gene sequence analysis enabled classification of a subset of these isolates. Key words: bacterial diversity, fatty acid methyl ester analysis (FAME), forest soil. Rsum : Des bactries retrouves dans de la matire organique de surface et dans des horizons de sols minralises Axelrood et al. forestiers ont t cultives laide de quatre mthodes et caractrises par une analyse des drivs desters mthyliques dacides gras (FAME). Les chantillons de sol provenant dune installation de Productivit des Sols Long Terme (LTSP) du ministre des Forts de la Colombie-Britannique ont t recueillis pendant lhiver et lt la suite de deux traitements perturbateurs(une rcolte darbres entiers sans compactage du sol (terrains N) et une rcolte darbres entiers plus un enlvement complet de la matire organique de surface accompagn dun compactage du sol lourd (terrains S)) et partir dun terrain de rfrence non coup (REF). Soixante-quinze pour cent de 1795 isolats bactriens ont t rattachs 42 genres appartenant aux groupes des - et -Proteobacteria, des Actinobacteria, de Bacillus/Clostridium, et de CytophagaFlexibacterBacteroides. Environ la moiti de la collection de cultures tait le reflet de la diversit gntique restreinte quatre genres de bactries, soit Pseudomonas, Bacillus, Paenibacillus et Arthrobacter. Une proportion significativement plus leve disolats bactriens appartenant Actinobacteria, et au genre Arthobacter sy rattachant, a t rcupre des chantillons de terrains S comparativement aux chantillons de sol des terrains N et REF. Il fut impossible daccorder un genre 25% des isolats bactriens par lanalyse FAME. Une analyse de groupement MIDI Tracker et un squenage partiel du gne de lARN 16s a permis de classifier une portion de ces isolats. Mots cls : diversit bactrienne, analyse des drivs desters mthyliques dacides gras (FAME), sols forestiers. [Traduit par la Rdaction] 654

Received 19 February 2002. Revision received 13 May 2002. Accepted 4 June 2002. Published on the NRC Research Press Web site at http://cjm.nrc.ca on 6 August 2002. P.E. Axelrood1 and M.L. Chow. BC Research Inc., 3650 Wesbrook Mall, Vancouver, BC V6S 2L2, Canada. C.S. Arnold. MIDI Inc., 125 Sandy Drive, Newark, DE 19713, U.S.A. J. Davies,2 K. Lu,2 and J.M. McDermott.3 TerraGen Discovery Inc. (now Cubist Pharmaceuticals Inc.), Suite 200, 2386 East Mall, Vancouver, BC V6T 1Z3, Canada.
1 2

Corresponding author (e-mail: paxelrood@bcresearch.com). Present address: Department of Microbiology and Immunology, University of British Columbia, #3006174 University Blvd., Vancouver, BC V6T 1Z3, Canada. 3 Present address: Helix Consulting, 3728 Collingwood Street, Vancouver, BC V6S 2M5, Canada.
Can. J. Microbiol. 48: 643654 (2002) DOI: 10.1139/W02-058 2002 NRC Canada

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Table 1. B.C. Ministry of Forests Long-Term Soil Productivity (LTSP) field plots (Holcomb 1996; Sanborn 1996). Plot code Plot Reference, undisturbed forest Intermediate level organic matter removal, no soil compaction Highest level organic matter removal, heavy soil compaction (5 cm impression) Description Mature forest stand Tree trunks and branches removed; conditions similar to whole-tree harvesting with no soil compaction Tree trunks, branches, logging slash, and all surface organic matter (forest floor) removed; conditions similar to land subjected to heavy, repeated logging equipment traffic LTSP study Reference OM2C0 This study REF N Soil samples collected Surface organic matter Mineral soil Surface organic matter Mineral soil Mineral soil

OM3C2

Introduction
Bacteria play key roles in essential soil processes including organic matter decomposition, nutrient cycling, and mineral transformations. Bacteria also form intimate associations with plants. Forest health and sustainability are directly dependent upon the vast soil microbial communities that contain bacterial populations of approximately 1010 cells per gram dry weight of soil (Torsvik et al. 1990). Prior to the 1990s, studies of forest soil bacteria primarily relied on cultivation followed by taxonomic identification (Hendrickson et al. 1982). More recently, cultivation-independent methods have revealed that cultivated bacteria represent a small fraction of the total bacterial diversity present in soil (Amann et al. 1995; Hugenholtz and Pace 1996). Liesack and coworkers (1997) caution that both cultivation-dependent methods and cultivation-independent molecular methods are needed to appropriately characterize microbial diversity in soil. Although the vastness of bacterial populations and their diversity in forest soils is daunting, gaining knowledge about bacterial communities and how they may vary in soils subjected to different types of forestry practices should provide insights regarding managed forest ecosystems. Forests cover 65% of the land in British Columbia (B.C.) (Canadian Council of Forest Ministers 2001), and forestry is one of the most important industries in the province. Forestry practices, including different methods of tree harvesting and site preparation, result in forest soil disturbance. Yet, little detail is known about what phylogenetic groups of bacteria reside in B.C. forest soils and how they respond to different types of forest soil disturbance. Long-Term Soil Productivity (LTSP) installations provide promising field sites to gain this information. The LTSP installations are based on a model that was developed to determine the longterm effects of soil compaction and organic matter removal on soil and forest productivity (Powers 1990; Powers et al. 1990). An underlying hypothesis for the model is that tree harvesting and subsequent forest site preparations change soil porosity (due to soil compaction) and site organic matter, and these factors can affect forest site productivity (Powers et al. 1990). Changes in these factors may also alter bacterial community composition in soil due to fluxes in physical and chemical properties, such as temperature, available oxygen and moisture, and soil nutrients.

This paper is the first in a two part series on the characterization of bacterial diversity from B.C. forest soils from selected LTSP plot treatments. The objectives of this study were to use cultivation-dependent methods to characterize forest soil bacterial communities and to compare the isolated bacterial diversity of different types of forest soil disturbance and soil horizons for winter and summer seasons. Bacterial isolates were identified taxonomically by fatty acid methyl ester (FAME) analysis, and cluster analysis was used to further explore relationships among isolates. FAME analysis is well recognized for bacterial identification. This method involves the conversion of microbial cellular fatty acids into fatty acid methyl esters, analysis by gas chromatography, and comparisons with FAME profiles representing a reference bacterial species library (Sasser 2001). Partial 16S rRNA gene sequence analysis was also used for taxonomic identification of a subset of bacterial isolates. The companion study addresses similar objectives, but diversity characterization was based on direct DNA isolation from forest soil followed by bacterial 16S rRNA gene cloning and DNA sequence analysis (Axelrood et al. 2002a). The companion study also compares and contrasts bacterial diversity assessments from the same soil samples based on cultivation-independent methods and the cultivation-dependent methods used in this study.

Materials and methods

Forest site and plot treatments Bacterial diversity characterizations were made from soil samples collected from the B.C. Ministry of Forests LTSP Skulow Lake site, near the city of Williams Lake, B.C., Canada (referred to in this study as the Williams Lake site). Details regarding the B.C. LTSP study and plot installations are summarized by Holcomb (1996) and are briefly described below. The biogeoclimatic subzone of the site is Sub-Boreal Spruce, with an elevation of 1050 meters and an orthic gray luvisol soil type. The soils have high levels of cations and are not deficient in any micronutrients (Chapman 2001). The site was dominated by lodgepole pine (Pinus contorta Dougl.) and lesser amounts of hybrid spruce (Picea engelmannii P. glauca), Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco), aspen (Populus tremuloides Michx.), and cottonwood (Populus trichocarpa Torr. & Gray) prior to timber harvest in 1994. LTSP plot treatments
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representing defined levels of soil compaction and organic matter removal (40 70 m) were installed in 1994 to 1995, and lodgepole pine seedlings were planted on the site in 1995. The plot treatments selected for our study included (1) the unlogged reference plot (REF), (2) plot OM2C0, which had tree trunks and crowns removed with no soil compaction (designated plot N in this paper), and (3) plot OM3C2, which had tree trunks, crowns, logging slash, and all surface organic matter (forest floor) removed plus heavy soil compaction (designated plot S in this paper) (Table 1). Plot N represents whole-tree harvesting with no soil compaction, and plot S represents the highest level of soil disturbance in the LTSP model, with disturbance similar to landings where logs are processed during timber harvest (Chapman 2001) and where soil is influenced by heavy logging equipment traffic (Sanborn 1996). Soil sample collection Random compass bearings and 40-m pacing distances were used to locate eight soil sampling locations for each of the three forest plots from the LTSP Williams Lake site in January and August 1996. Eight samples each of surface organic matter (forest floor) and the underlying mineral soil were collected from plots REF and N, and eight samples of mineral soil were collected from plot S. In January 1996, only four random samples per soil horizon were collected from the mature forest directly adjacent to the REF plot due to inaccessibility of the plot and severe winter conditions. Snow was removed at each sample location, uncovering frozen surface organic matter and underlying nonfrozen mineral soil. Vegetation was cleared from the soil surface prior to sample collection in the summer. Aseptic techniques were used for soil collections, which included disinfecting soil sampling tools with ethanol, wearing sterile gloves when required, and using sterile plastic sample bags. The surface organic matter sample size dimensions were approximately 10 cm2 to the depth of the forest floor (13 cm), whereas the mineral soil was collected to a depth of 15 cm. Winter samples were kept frozen and thawed overnight at 4C prior to processing, whereas summer soil samples were kept at 4C. Winter and summer composite soil samples were prepared within 14 days and 3 days, respectively, after collection. Each sample was thoroughly mixed, and one subsample of uniform weight from each of the eight soil samples per soil horizon per plot per season was pooled using aseptic techniques to yield 10 composite soil samples. The five winter composite samples included REF1 (reference plot, surface organic matter), N2 (plot N, surface organic matter), N3 (plot N, mineral soil), S4 (plot S, mineral soil), and REF5 (reference plot, mineral soil). The five summer composite samples included REF6 (reference plot, surface organic matter), N7 (plot N, surface organic matter), N8 (plot N, mineral soil), S9 (plot S, mineral soil), and REF10 (reference plot, mineral soil). These soil samples were used to characterize soil physical and chemical properties (done by Norwest Labs, Langley, B.C., Canada, using standard methods) and to establish a culture collection for taxonomic identification and bacterial diversity assessments. Bacterial culture collection Five culture media and several methods were evaluated

for bacterial isolation using 100-g soil samples from each of the five composite winter samples. Soil suspensions (in 500 mL sterile distilled water) were agitated by hand and then placed on a rotary shaker (150 rpm) at room temperature for 45 min prior to dilution plating on culture media. All media were amended with cycloheximide (100 g/mL) and benomyl (30 g/mL active ingredient) to inhibit fungal growth. The media included 50% trypticase soy agar (TSA) with full strength agar (ATCC medium 18, (Atlas 1993); TSA amended with 50 g/mL nalidixic acid, TSA amended with 50 g/mL methicillin, and international Streptomyces project medium 2 (ISP medium 2) (Atlas 1993), and Lochhead soil extract agar (Johnson and Curl 1972). Selective methods to isolate bacteria that were facultative anaerobes, heat tolerant, or desiccation resistant included (1) incubation of TSA dilution plates under anaerobic conditions using the GasPak System (Difco Laboratories, Detroit, Mich.), (2) heating 10 mL of the undiluted soil suspension sample at 80C for 1 h and cooling at 5C for 10 min prior to dilution plating, and (3) desiccating the soil sample under aseptic conditions in a laminar flow biological safety cabinet (Class II A/B3, Forma Scientific, Division of Mallinckrodt, Inc., Marietta, Ohio) for three days prior to preparing soil suspensions, respectively. Dilution plates were incubated aerobically (unless specified otherwise) at 25C in the dark, and the incubation period (420 days) was identical for all samples for a given method. Forty-eight bacterial isolates per method per soil sample were randomly selected from dilution culture plates using a grid system. Bacterial isolates were streaked for single colonies on TSA (or ISP medium 2 for actinomycetes), purified, and stored in tryptic soy broth (Difco Laboratories) containing 20% glycerol at 80C in microtiter plates. Four methods were selected for the characterization of cultivable bacterial diversity. This selection was based on obtaining diverse colony types on culture plates and on the isolation of different bacterial genera based on FAME identification of randomly selected bacterial isolates (results not shown). The four methods chosen for diversity characterization were soil dilution plating on TSA (TSA), TSA amended with 50 g/mL nalidixic acid (NAL), TSA incubated under anaerobic conditions (AN), and heat treatment of soil suspensions followed by soil dilution plating on TSA (HEAT). These methods were also used to select random bacterial isolates from the five summer soil samples. For winter and summer samples, dilution plates were incubated at 25C in the dark for 4 days (TSA, HEAT), 7 days (NAL), or 10 days (AN) prior to selecting approximately 48 random bacterial isolates per method per soil sample and storing strains at 80C. Forty-three to 48 isolates were characterized per isolation method per soil sample (except for two samples of 31 isolates each). Facultative anaerobic isolates were not obtained from the winter mineral soil sample from the reference site (REF5) due to equipment malfunction. FAME analysis Bacterial identification by FAME analysis has been described (Sasser 1990, 2001) and methods for preparing fatty acid methyl esters followed the Sherlock 2.02 Microbial Identification System (MIS) operations manual version 5 (1993) (MIDI Inc., Newark, Del.), with the following change. Actinomycetes were
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incubated at 30C instead of 28C. All samples were analyzed with a Hewlett-Packard HP 6890 series gas chromatograph system (Agilent Technologies, Wilmington, Del.), containing an HP-Ultra 2 column (diphenyldimethyl-siloxane (5:95) copolymer) to identify and quantify the fatty acids present in the sample. The FAME data were analyzed with Sherlock 4.00 MIS software with MIDI TSBA40 (AEROBE) method version 4.00 (2001) (Calibration Max RMS error set to 0.005) and the TSBA40 library version 4.10 (2001). Actinomycetes were identified using the ACTINO method and ACTIN1 library version 3.80 (1994). A similarity index 0.3 was considered to reflect a good match to a genus represented in the respective MIDI libraries, whereas a similarity index <0.3 indicated no match to library members and an inconclusive identification (referred to as unclassified). A bacterial isolate that yielded a FAME profile of unacceptable quality after two analyses was classified as flagged and data were not used for analyses. Dendrogram analyses, plotted with Euclidean distance values, were completed with MIDI software to explore the relationships among isolates obtained from the same soil sample. Sherlock Tracker software by MIDI Inc. (Arnold 2002) (referred to as Tracker) was used for cluster analyses of two FAME profile data sets, representing 1569 and 109 bacterial isolates, which were obtained with the AEROBE method and the ACTINO method, respectively. Tracker software creates clusters (groups) of samples (derived from bacterial isolates) that have similar normalized profiles of the various fatty acid methyl esters in a sample, where normalized refers to relative percentages (rather than absolute profile amounts). Some samples may contain 0% of some fatty acid methyl esters. Any two samples are separated by a certain distance, which is calculated as the Euclidean distance between their normalized profiles. The width of a cluster is the distance between the two sample points in a cluster that are furthest apart. Tracker constructs clusters by grouping samples that are separated by less than a selected Euclidean distance cut-off factor, using a single-linkage method of clustering (Gordon 1981). The selection of a small or large Euclidean distance cut-off factor increases or reduces the number of clusters, respectively. For this study, a Euclidean distance cut-off factor of 6 was selected in order to minimize the number of clusters that contained less than four samples while maintaining an adequate number of clusters to explore cluster composition in relation to taxonomic structure and isolate source. Sequence analysis of 16S rRNA gene fragments Partial sequence analysis of 16S rRNA gene fragments was used for taxonomic identification of selected bacterial isolates that had FAME similarity indices of 0.3 (to validate the identification to genus) or <0.3 (to provide identification). Total DNA was isolated from bacterial cells following methods provided by Li (1996). Cells were lysed by freezethaw and lysozyme treatment followed by digestion with proteinase K in the presence of sodium dodecyl sulphate. One-half volume of chilled ammonium acetate (7.5 M) was added, and cellular material was pelleted by centrifugation. DNA in the supernatant was precipitated with isopropanol, followed by centrifugation, and the pellet resuspended in 50150 L TrisEDTA buffer (10 mM Tris, 1 mM EDTA, pH 8.0). The concentration of genomic DNA from individual bacterial isolates was estimated using standards of known DNA concentration and gel electrophoresis analysis.

Amplification of 16S rRNA gene sequences was carried out in 25-L reactions using universal bacterial primers 16S.0007F.21 and 16S.1511R.21 (Miao et al. 1997), as described for 16S-clones from soil sample N2 (Axelrood et al. 2002a), with the exception that 1.0 ng bacterial genomic DNA was used in one polymerase chain reaction (PCR) representing each bacterial isolate. The PCR products were purified and sequenced as previously described (Axelrood et al. 2002a). The partial 16S rRNA gene sequences were checked for proper base calling, edited as required, and submitted to the BLAST server (Basic Local Alignment Search Tool (Altschul et al. 1997), available from the National Center for Biotechnology Information (NCBI), http://www.ncbi.nlm.nih.gov/), to determine the closest matching GenBank sequences. Statistical analysis Pearsons and log-likelihood chi-square analyses were completed to test for significant differences in the proportion of isolates, from plots REF, N, and S, classified as Arthrobacter, or Firmicutes subdivision Actinobacteria, or members of Tracker analysis cluster 3. Data were summarized in a 2 3 contingency table with Arthrobacter (or Actinobacteria or cluster 3) as the response variable and the plot as the predictor variable (Pearsons chi-square analysis) for mineral soil samples only, as well as a 2 3 2 contingency table with Arthrobacter (or cluster 3 or Actinobacteria) as the response variable and the plot and soil horizon (surface organic matter and mineral soil) as predictor variables (log-likelihood chi-square analysis). Bacterial isolate codes, GenBank accession numbers, and web site information Bacterial isolate codes begin with a number designating the winter soil sample source (1: unlogged REF plot, surface organic matter; 2: plot N, surface organic matter; 3: plot N, mineral soil; 4: plot S, mineral soil; 5: unlogged REF plot, mineral soil). Partial 16S rRNA gene sequences from bacterial isolates were deposited in the GenBank database and assigned accession numbers AY043532AY043604. A listing of bacterial isolate codes and their corresponding GenBank accession numbers are available online at (http://bacterialdiversity. bcresearch.com/accession_lookup.htm) (Axelrood et al. 2002b).

Results
Soil physical and chemical properties The pH of all surface organic matter and mineral soil samples ranged from 5.2 to 6.0. The summer surface organic matter (forest floor) soil samples contained 58% and 54% organic matter for samples REF6 and N7, respectively, compared with 29% and 31% organic matter for the winter surface organic matter samples REF1 and N2. Summer and winter mineral soil samples contained low levels of organic matter (1.35.4%). Mineral soil samples had a loam texture and particle size analyses indicated some variations in sand (4253%), silt (3147%), and clay (614%) content. Cation exchange capacity (mequiv./100 g) ranged from 56 to 70 and 11 to 22 for surface organic matter and mineral soil samples, respectively. Nitrate was low in all soil samples (110 ppm) and ammonium was marginal in surface organic matter samples REF1, REF6, and N2 (3768 ppm) and low in the re 2002 NRC Canada

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Fig. 1. Classification of 1795 bacterial isolates into bacterial divisions and subdivisions or unclassified and flagged categories for combined data from winter and summer surface organic matter and mineral soil samples from B.C. Ministry of Forests Long-Term Soil Productivity plots N, S, and REF. Bacterial isolates were catagorized as unclassified or flagged if the fatty acid methyl ester (FAME) similarity index was <0.3 or the FAME profile was of unacceptable quality, respectively. See Table 1 for a description of plots N, S, and REF.

maining samples (49 ppm). Phosphate levels ranged from 45 to 86 ppm and 11 to 28 ppm for surface organic matter and mineral soil samples, respectively. Potassium levels ranged from 374 to 531 ppm and from 130 to 194 ppm for surface organic matter and mineral soil samples, respectively (potassium results were erroneous for soil sample REF6). Bacterial isolation method The four selected isolation methods facilitated the isolation of different bacterial genera from soil samples. Amending TSA with nalidixic acid inhibited the growth of most Pseudomonas colonies and enhanced the isolation of Cellulomonas, Clavibacter, Curtobacterium, Kocuria, and Streptomyces. Heat treatment enhanced the isolation of Bacillus, Paenibacillus, and Brevibacillus. Anaerobic incubation facilitated the isolation of facultative anaerobic bacterial species. Taxonomic classification of bacterial isolates FAME analysis FAME analysis facilitated the classification of 1339 of 1795 soil bacterial isolates into three bacterial divisions: Proteobacteria (subdivisions and ), Firmicutes (subdivisions Actinobacteria and Bacillus/Clostridium group), and the CytophagaFlexibacterBacteroides (CFB) group (Fig. 1). Actinobacteria and the Bacillus/Clostridium group are also known as high G+C gram positive and low G+C gram positive bacteria, respectively. The majority of bacterial isolates (72%) were affiliated with -Proteobacteria, Actinobacteria, and the Bacillus/Clostridium group, whereas only 2.5% of bacterial isolates were members of -Proteobacteria and the CFB group. Nineteen percent of bacterial isolates could not be classified to genus as the FAME similarity indices were <0.3, indicating that there were no close matches in the MIDI libraries (designated unclassified, Fig. 1). The remaining 6.5% of isolates in the culture collection were classified as flagged samples due to unacceptable quality of the FAME analysis.

Identified bacterial isolates represented 34 and 37 genera from winter and summer soil samples, respectively (FAME similarity index 0.3) (Table 2). Pseudomonas, Bacillus, Paenibacillus, and Streptomyces were the only genera isolated from all ten soil samples. Bacterial isolates identified as Pseudomonas, Bacillus, Paenibacillus, and Arthrobacter represented 47% of all isolates, indicating that approximately half of the culture collection represented genetic diversity confined to four bacterial genera. Pseudomonas was the most frequently isolated genus from 7 of 10 soil samples and represented 23% of all isolates from LTSP soil samples. Dendrogram analyses for isolates obtained from the same soil sample using the same isolation method revealed strain-level relationships among some isolates belonging to the same genus such as Pseudomonas, Bacillus, Paenibacillus, Arthrobacter, Aeromonas, Clavibacter, Klebsiella, and Nocardia (based on Euclidean distance 2.5, results are not shown). In some instances, bacterial isolates were identified to genus based on similarity indices 0.3 to members in the MIDI library, but the first and second matches to the library represented different genera that were not separated by similarity index values >0.1. Thus, these identifications to genus, based on the first match, may have been inconclusive. Examples of first and second matches to genera with similarity indices 0.3 but separated by values <0.1 included, but were not limited to, Pseudomonas and Chromobacterium, Bacillus and Paenibacillus, Arthrobacter and Paenibacillus, Clavibacter and Kocuria, Enterobacter and Klebsiella, and Nocardia and Rhodococcus. Due to the above potential discrepancies, taxonomic identification was further characterized using partial sequence analysis of 16S rRNA genes from a subset of isolates (see below). Partial sequence analysis of 16S rRNA genes from bacterial isolates identified to genus by FAME analysis There was agreement in identifying 34 of 41 bacterial isolates to the genus level using partial sequence analysis of 16S rRNA genes and FAME analysis. These bacterial
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Table 2. Identificationa of bacterial isolates from surface organic matter (ORG) and mineral (MIN) soil samples from B.C. Ministry of Forests Long-Term Soil Productivity plotsb and corresponding cluster analysis classification. Number of isolatesc from soil samples Winter 1996 REF1 ORG N3 MIN Summer 1996

Taxonomic identification

Cluster numberd

5,17 1 D(1),S(2) 1 S(2) S(2)

7 5 5 5 5 1 5,16 5,16 8 4,D(1) 16,S(4)

-Proteobacteria Alcaligenes Aquaspirillum Burkholderia Chromobacterium Comamonas Variovorax -Proteobacteria Aeromonas Enterobacter Erwinia Klebsiella Pantoea Pseudomonas Rahnella Serratia Stenotrophomonas Xanthomonas Yersinia Actinobacteria Arthrobacter Cellulomonas Clavibacter Corynebacterium Curtobacterium Kocuria Microbacterium Micrococcus Nesterenkonia Nocardia Rathayibacter Rhodococcus Streptomyces Bacillus/Clostridium group Bacillus Brevibacillus Brochothrix Carnobacterium Kurthia 5 3 1 1 60 10 3 1 39 1 4 2 32 1 1 12 1 1 2 5 1 4 4 31 17 1 3 3 51 1 46 4 22 1 5 1 6 2 1 6 26 14

3,S(1) 4,19,20,D(1) 4,D(1),S(3) 4,S(1) 3,4,10,S(1) 2,3,4,11,15,27,D(2),S(4) 4,11,D(1) 2,14,S(2) 4,S(1) 9,S(1) S(1) 9,D(1) A1,A2,AD(1),AS(3)

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2,3,6,22,D(4),S(16) 2,6,24,D(1),S(4) D(1) S(1) 6

N2 ORG 69 49 17 2 1 17 1 2 5 1 6 2 18 12

S4 MIN 1 1 45 41 1 2 1 76 66 6 1 3 36 25 1

REF5 MIN 1 1 32 32 22 1 14 4 3 18 15

REF6 ORG 10 6 2 1 1 62 6 12 1 6 1 32 2 1 1 39 1 2 7 1 1 9 1 1 16 35 25 3 1 2

N7 ORG 3 2 1 74 36 6 6 20 2 1 3 44 6 1 2 11 7 1 1 3 5 7 36 10 1

N8 MIN 65 1 1 63 51 9 3 1 17 8 3 2 3 5 35 28 3 1

S9 MIN 42 39 1 2 73 58 2 2 3 1 5 2 28 17 1 1

REF10 MIN 1 1 78 5 5 52 2 13 1 44 19 6 1 1 6 2 2 1 6 33 20 6

Total 24 12 1 4 3 2 2 578 42 35 1 21 2 413 7 22 27 3 5 420 160 9 53 6 33 44 18 11 2 16 1 13 54 296 183 16 1 1 3

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S(1) 2,3,D(1),S(5) S(3)

Listeria Paenibacillus Staphylococcus CFB groupe Chryseobacterium Flavobacterium Pedobacter Sphingobacterium Unclassifiedf Flagged sampleh Total no. of isolates

18,D(1),S(2) 4,S(3) 18,S(1) 13 Clustersg

1 11 1 6 1 1 1 3 30 24 188

5 1 2 1 1 52 31 189

12 2 2 63 19 186

10 1 1 22 7 188

3 48 18 139

4 1 1 35 8 190

25 8 3 1 4 26 1 192

3 1 1 16 3 171

8 1 20 5 168

7 27 1 184

1 88 3 21 4 4 4 9 339 117 1795

a Identification to genus is based upon a fatty acid methyl ester (FAME) similarity index 0.3 to profiles in the Sherlock Microbial Identification System (MIS; MIDI Inc., Newark, Del.) TSBA40 library (version 4.10) or ACTIN1 library (version 3.80). b See Table 1 for a description of plots N, S, and REF. The numbers following N, S, and REF in the table heading designate composite soil samples prepared from the B.C. Ministry of Forests LongTerm Soil Productivity installation near Williams Lake, B.C. c Four methods were used to isolate bacteria: 50% trypticase soy agar, full strength agar (TSA) with aerobic incubation; TSA medium with anaerobic incubation; heat treatment of the soil suspension at 80C for 1 h prior to culturing bacteria on TSA medium with aerobic incubation; TSA medium supplemented with 50 g/mL nalidixic acid with aerobic incubation. The anaerobic method was not available for soil sample REF5. d Sherlock Tracker (Arnold 2002) cluster analysis was used to classify isolates. The isolates in clusters 128, D, and S were identified using the MIS TSBA40 library, and isolates in clusters A1A5, AD, and AS were identified using the MIS ACTIN1 library. D represents clusters containing only two isolates (defined as double), S represents single isolates that did not cluster with any other isolates in the analysis (defined as single), and the number in brackets following D and S indicates the number of double clusters and the number of nonclustering single isolates, respectively. e CytophagaFlexibacterBacteroides group. f Isolates not matching the MIS TSBA40 and ACTIN1 libraries indicate a FAME similarity index <0.3. g Clusters 1, 2, 4, 6, 9, 12, 13, 19, 20, 21, 2326, 28, D(13), S(82), A1A5, AD(2), AS(14). h Isolates for which the quality of the FAME analysis was not acceptable.

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Fig. 2. Classification of 1795 bacterial isolates into bacterial divisions and subdivisions or an unclassified category for soil samples representing winter and summer surface organic matter (ORG) and mineral (MIN) soil horizons from B.C. Ministry of Forests LongTerm Soil Productivity (LTSP) plots N, S, and REF. The unclassified category includes bacterial isolates with a fatty acid methyl ester (FAME) similarity index <0.3 (unclassified) and bacterial isolates with a FAME profile of unacceptable quality (flagged). See Table 1 for a description of plots N, S, and REF. The numbers following N, S, and REF in the figure designate soil sample codes. Pearsons and log-likelihood chi-square analyses indicated that the isolation of bacteria classified as Actinobacteria was significantly influenced by the LTSP plot treatment (P < 0.001).

isolates were identified as Arthrobacter, Bacillus, Burkholderia, Clavibacter, Rhodococcus, Sphingobacterium, Streptomyces, or Variovorax, and also 11 of 12 Pseudomonas isolates, most Paenibacillus and Stenotrophomonas

isolates, and one of three Kocuria isolates. DNA sequence lengths ranged from 394 to 553 nucleotides. These isolates shared 96100% DNA sequence similarity to members in the GenBank. The identification based on the two
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Axelrood et al. Table 3. Percent representation of genera in clustersa that contain 10 or more isolates.
No. of isolates in cluster 449 % of isolates in cluster 92.0 0.7 0.2 7.1 45.1 26.2 1.2 0.4 0.4 26.7 86.5 9.2 2.7 1.1 0.5 36.3 10.6 7.5 6.8 3.8 1.5 0.8 0.8 0.8 31.1 39.0 23.3 23.3 7.8 3.3 2.2 1.1 76.2 11.9 5.1 6.8 100.0 100.0 55.6 40.7 3.7 100.0 81.2 18.8 100.0 90.0 10.0 78.4 21.6 61.8 38.2

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methods differed for seven isolates that were identified by FAME as Alcaligenes, Kocuria, Paenibacillus, Pseudomonas, and Stenotrophomonas (Axelrood et al. 2002b, Table A1). Partial sequence analysis of 16S rRNA genes from bacterial isolates with low similarity indices to members in FAME libraries Partial sequence analysis of 16S rRNA genes from 32 unclassified isolates was completed to determine if a subset of the unclassified isolates represented novel bacterial diversity. Nineteen of these isolates shared 98100% partial 16S rRNA gene sequence similarity with GenBank members from the genera Janthinobacterium, Paenibacillus, Pseudomonas, Rhizobium, Rhodococcus, Serratia, Stenotrophomonas, Streptomyces, or Variovorax; whereas 13 unclassified bacterial isolates shared 9397% partial 16S rRNA gene sequence similarity with GenBank members representing Bacillus, Burkholderia, Frateuria, Herbaspirillum, Leifsonia, and Paenibacillus. Six of the above genera were not represented by any of the bacterial isolates identified to genus by FAME analysis but were within the same bacterial subdivisions represented by the culture collection, with the exception of Rhizobium, which belongs to -Proteobacteria. These results indicated that the unclassified isolates did not necessarily represent novel genera but their FAME profiles were unique and dissimilar to those in the MIDI libraries. Interestingly, there was agreement for the bacterial genera that were most closely related to 19 of the 32 unclassified isolates using both methods, indicating that in some instances, FAME similarity indices <0.3 to members in MIDI libraries provided accurate identification to the genus level (Axelrood et al. 2002b, Table A2). Differences in bacterial community profiles from LTSP plots and soil horizons A higher proportion of bacterial isolates belonging to Firmicutes subdivision Actinobacteria were obtained from winter and summer mineral soil samples from plot S compared with all surface organic matter and mineral soil samples from plots N and REF (Fig. 2). Pearsons and loglikelihood chi-square analyses indicated that the isolation of Actinobacteria from winter and summer seasons was significantly influenced by the LTSP plot treatment (P < 0.001). The most striking plot effect was seen for winter mineral soil samples as 40% of bacterial isolates were classified as Actinobacteria from plot S compared with 12% and 16% from plots N and REF, respectively. Notably, Arthrobacter was commonly isolated from winter and summer mineral soil samples from plot S and represented 35% of isolates from each of soil samples S4 and S9 (Table 2). This is in contrast with obtaining only one Arthrobacter isolate from winter soil samples from plots REF and N and 0.510% Arthrobacter presence among bacterial isolates from summer soil samples from plots REF and N, respectively. Pearsons and log-likelihood chi-square analyses indicated that the isolation of Arthrobacter from winter and summer seasons was significantly influenced by the LTSP plot treatment and the soil horizon (P < 0.001). These results explain the previously mentioned significant
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Cluster no. 1

Genera representedb Pseudomonas Chromobacterium Aquaspirillum Unclassifiedc Bacillus Paenibacillus Brevibacillus Kocuria Micrococcus Unclassifiedc Arthrobacter Paenibacillus Kocuria Bacillus Curtobacterium Clavibacter Microbacterium Curtobacterium Kocuria Corynebacterium Cellulomonas Flavobacterium Nesterenkonia Xanthomonas Unclassifiedc Enterobacter Klebsiella Serratia Alcaligenes Rahnella Pantoea Erwinia Bacillus Brevibacillus Kurthia Unclassifiedc Aeromonas Stenotrophomonas Nocardia Rhodococcus Unclassifiedc Curtobacterium Kocuria Microbacterium Unclassifiedc Sphingobacterium Unclassifiedc Streptomyces Unclassifiedc Streptomyces Unclassifiedc

244

184

132

90

59

7 8 9

42 27 27

10 11 12 13 A1 A2

21 16 13 10 37 34

a Isolates in clusters 113 were analyzed using the Sherlock Microbial Identification System (MIDI Inc., Newark, Del.) TSBA40 library version 4.10, and isolates in clusters A1A2 were analyzed using the MIS ACTIN1 library version 3.8 in combination with Sherlock Tracker (Arnold 2002) cluster analysis. b Identification to genus was based upon a fatty acid methyl ester (FAME) similarity index 0.3 to a profile in the MIS libraries. c Isolates not matching the MIS library database indicate a FAME similarity index <0.3.

652 Fig. 3. Soil origin of 184 bacterial isolates comprising cluster 3 (the third largest cluster) revealed from Sherlock Tracker cluster analysis of 1678 bacterial isolates. Bacteria were isolated from soil samples designated by the sampling season (winter or summer), B.C. Ministry of Forests Long-Term Soil Productivity plot (LTSP) treatment (N, S, or REF), and the soil horizon (ORG, surface organic matter; MIN, mineral soil). Pearsons and loglikelihood chi-square analyses indicated that the isolation of bacteria classified into cluster 3 was significantly influenced by the LTSP plot treatment and the soil horizon (P < 0.001). The taxonomic composition of cluster 3 is detailed in Table 3.

Can. J. Microbiol. Vol. 48, 2002

Cluster analysis also provided a measure of diversity of 243 unclassified isolates that were present in 20 clusters (plus 15 double clusters) and identified closely related isolates, some of which were identified to the genus level (Tables 2 and 3). Cluster 3 was the only cluster that indicated the LTSP plot treatment (plot S) may have influenced the isolated microbial community composition (Fig. 3). Pearsons and loglikelihood chi-square analyses indicated that the isolation of bacteria classified into cluster 3 was significantly influenced by the LTSP plot treatment and the soil horizon (P < 0.001). This cluster contained 184 bacterial isolates, 77% of which were obtained from winter and summer soil samples from plot S. Eighty-seven percent of the isolates in cluster 3 were identified as Arthrobacter (Table 3), thereby supporting the significant results of relative proportions of Actinobacteria presented in Fig. 2 and indicating that plot S soil conditions may have promoted higher populations of Arthrobacter compared with soil conditions present on other plots.

Discussion
Cultivated bacterial community members isolated from LTSP soil samples represented 42 known bacterial genera from - and -Proteobacteria, Actinobacteria, the Bacillus/ Clostridium group, and the CytophagaFlexibacterBacteroides group. The culture collection of 1795 bacterial isolates was well represented by Pseudomonas, Bacillus, Paenibacillus, and Arthrobacter. These bacterial genera are considered common soil inhabitants, and they participate in numerous facets of soil nutrient cycling and transformation of minerals (Alexander 1977; Hendrickson et al. 1982). Tracker cluster analysis and partial 16S rRNA gene sequence analysis were useful to both validate results based on taxonomic identification and explore relationships among isolates, particularity in instances where isolates were not conclusively identified to genus by FAME analysis. Any culture-dependent approach to characterize bacterial diversity will have built-in biases in the isolation of bacteria. In our study, several methods were used for bacterial isolation, and this enhanced the presence of genera from different taxonomic groups in the culture collection. Some studies have used only one medium to characterize cultivable bacterial community members from soil samples (Dunbar et al. 1999; Kloepper et al. 1992), whereas other studies have enhanced the isolation of different bacterial strains by using multiple culture media (Tabacchioni et al. 2000; Germida 2001). A major objective of this study was to explore similarities and differences in the proportion of isolated bacterial community members from undisturbed and disturbed forest soils represented by LTSP plot treatments. FAME profile analysis followed by taxonomic classification revealed that isolates belonging to Firmicutes subdivision Actinobacteria (Fig. 2) and the member genus Arthrobacter (Table 2) were isolated from winter and summer mineral soil samples from the most disturbed plot S at a significantly higher frequency than surface organic matter and mineral soil samples from LTSP plots N and REF. These results were substantiated by Tracker cluster analysis, which revealed that the third largest cluster of isolates, dominated by Arthrobacter, represented significantly more members from
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plot effect for isolates classified as Firmicutes subdivision Actinobacteria, as Arthrobacter is a member of this bacterial group. Differences in the isolation of bacteria classified as Proteobacteria or the Bacillus/Clostridium group from winter and summer soil samples representing LTSP plots N, S, and REF did not follow a consistent trend (Fig. 2). Sherlock Tracker cluster analysis to further classify bacterial diversity and confirm differences in bacterial community profiles from LTSP plots Cluster analysis was used to further characterize bacterial diversity, since some isolates could not be identified conclusively to genus and to confirm differences in the isolation of Arthrobacter from plots N and S. Sherlock Tracker cluster analysis of FAME profile data revealed that 87% of 1678 isolates were classified into 33 clusters, each containing three or more isolates (clusters 128, clusters A1A5, Table 2). Analysis also showed that 27 additional clusters each contained two isolates (designated double (D) clusters) and 160 isolates did not cluster with any other isolates (designated single (S) isolates or singles). It was common for clusters containing 10 or more isolates to represent more than one bacterial genus (Table 3), indicating that different genera can share related FAME profiles. Furthermore, some bacterial genera were represented in more than one cluster (Tables 2 and 3), indicating diverse FAME profiles were produced by members of one genus. For example, Bacillus isolates were present in four clusters with greater than three members and also classified into four double clusters and 16 singles (Table 2). These results suggest that bacterial characterizations based solely on the taxonomic identity of isolates may provide incomplete information regarding diversity.

Axelrood et al.

653

plot S than from plots N and REF (Fig. 3). This indicates a change in a segment of the soil bacterial community due to soil disturbance from plot treatment S compared with plot treatment N and the unlogged reference plot REF. Plot S had the highest level of soil compaction, and all surface organic matter (forest floor) was removed compared with no soil compaction and complete surface organic matter retention on plots N and REF. The removal of surface organic matter resulted in an immediate loss of the forest floor nutrients, as well as a small reduction in mineral soil carbon (Sanborn et al. 2000). Soil temperatures were affected by both compaction and surface organic matter removal, and soil temperatures had wider diurnal ranges and were warmer on plot S compared with plots N and REF (Chapman 2001). Arthrobacter is a nutritionally versatile obligate aerobe, and the temperatures for optimum growth range from 25 to 30C (Keddie et al. 1986). Further research is required to clarify the impact of soil temperature and other soil physical, chemical, and biological properties on Arthrobacter populations and their diversity in LTSP soils. Although Arthrobacter was frequently isolated from LTSP soil samples from plot S, it was not identified by sequence analysis of 198 random bacterial 16S rRNA gene fragments cloned from the same soil samples (Axelrood et al. 2002a). This suggests that Arthrobacter may be an easily cultivated genus but not necessarily a dominant member of the total bacterial soil community from this plot. Rigorous quantitative methods are required to confirm this hypothesis. This study and the companion study (Axelrood et al. 2002a) have characterized bacterial diversity based on individual community members. In contrast, other researchers have explored diversity at the whole community level using methods such as carbon substrate utilization analysis or phospholipid fatty acid analysis (Staddon et al. 1996, 1998) and terminal restriction fragment length polymorphism analysis (Dunbar et al. 2000). Microbial community analysis based on extracted phospholipid fatty acids from soils is not cultivation-dependent, and this method has revealed changes in bacterial communities in forest soils in response to anthropogenic impacts and soil conditions, such as heavy metal pollution (Pennanen et al. 1996), increases in soil pH due to the application of lime (Frostegard et al. 1993), and soil fertility (Pennanen et al. 1999). Whole community fatty acid analysis is useful to compare microbial communities, but Haack and coworkers (1994) caution that detailed taxonomic characterizations should be supported by other methods. Community-level diversity assessments could prove informative for LTSP sites as well as for tracking Arthrobacter diversity and abundance. Bacterial diversity studies are enhanced by using a combined approach of cultivation and molecular (including cultivation-independent) methods (Liesack et al. 1997). This study provides insights into bacterial community members that can be cultivated from different forest soil horizons and disturbance treatments and allows for comparisons with cultivation-independent assessments (Axelrood et al. 2002a). Clearly, bacterial diversity is influenced by soil environmental, physical, and chemical characteristics, and descriptions of both structural and functional diversity are key to understanding microbial ecology and ecosystem function. Future studies

addressing these factors will expand our knowledge of a poorly understood aspect of forest ecology.

Acknowledgements
We gratefully thank the following people: University of British Columbia co-operative student Neal Faraone and Simon Fraser University co-operative student Bonnie Buchanan for excellent technical assistance; Bill Chapman, Shannon Berch, Paul Sanborn, and Marty Kranabetter, B.C. Ministry of Forests, for insightful discussions regarding the B.C. Ministry of Forests LTSP studies; Bill Chapman for providing access to the Williams Lake Long-Term Soil Productivity site, and for assistance with soil sampling; Reed Radley for assistance with soil sampling; Tom Hsiang for his contributions during his sabbatical visit; Ned Glick for statistical consulting; and Bill Chapman for his critical review of this manuscript and for providing thoughtful suggestions and discussion. Funding for this research was provided by Forest Renewal British Columbia (FRBC).

References
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