Soil and litter organisms in Pacific northwest forests under different management practices

Applied Soil Ecology 18 (2001) 159–175

Soil and litter organisms in Pacific northwest forests under different management practices Katherine K. Donegan a,∗ , Lidia S. Watrud b , Ramon J. Seidler b , Sharon. P. Maggard a , Tamotsu Shiroyama b , L. Arlene Porteous b , George DiGiovanni c,1 a

Dynamac Corporation, National Health and Environmental Effects Research Laboratory, Western Ecology Division, NHEERL-WED, 200 SW 35th Street, Corvallis, OR 97333, USA b Environmental Protection Agency, National Health and Environmental Effects Research Laboratory, Western Ecology Division, 200 SW 35th Street, Corvallis, OR 97333, USA c National Research Council, National Health and Environmental Effects Research Laboratory, Western Ecology Division, 200 SW 35th Street, Corvallis, OR 97333, USA Received 2 November 2000; accepted 30 May 2001

Abstract Soil and litter organisms are important indicators of stress and disturbance to forest ecosystems. In this study, soil and litter organisms were monitored for their response to different forest management practices. Litter and soil cores (0–10, 10–20 cm) were collected at approximately 8 week intervals over a 19 month period from a low elevation 110–140-year-old Douglas-fir forest and adjacent 8-year-old clearcut and from a high elevation 200–250-year-old Douglas-fir forest and adjacent 5-year-old clearcut. The low elevation clearcut had been broadcast burned and replanted with Douglas-fir trees and a grass–legume mixture whereas the high elevation clearcut was not burned, large woody debris was left, and it was replanted with Douglas-fir, Noble-fir, Grand-fir, and western white pine. The litter and soil cores were analyzed for types of microarthropods and numbers of nematodes, fungi, culturable, aerobic bacteria, spore-forming bacteria, and chitin-degrading bacteria. Microbial community metabolic profiles, using the BiologTM method, were also generated for the 0–10 cm soil samples. Populations of Pseudomonas spp. were analyzed in the litter and soil samples using 16S rDNA fingerprints. Plant surveys were conducted to identify potential relationships of soil organisms to plant community composition. At both elevational field sites, there were significantly (P < 0.05) higher levels of nematodes and microarthropods in litter and soil in forest plots than in clearcut plots. Bacterial and fungal populations were also significantly higher in litter in forest plots than in clearcut plots at the high elevation site. In the litter and soil at the low elevation site and the soil at the high elevation site, however, microbial levels were higher in clearcut plots than in forest plots. The Pseudomonas spp. populations and the microbial community metabolic profiles in the 0–10 cm soil differed significantly between the forest and clearcut plots at the low elevation site but not at the high elevation site. At both elevational field sites, the plant cover (%) and plant density were significantly higher in clearcut plots than in forest plots. These observed differences in the population size and composition of organisms between mature

∗ Corresponding author. Tel.: +1-541-754-4809; fax: +1-541-754-4799. E-mail address: [email protected] (K.K. Donegan). 1 Present address: Quality Control and Research Laboratory, American Water Works Service Company Inc., 1115 South Illinois Street, Belleville, IL 62220, USA.

0929-1393/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 9 - 1 3 9 3 ( 0 1 ) 0 0 1 5 5 - X

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forests and both low management and high management clearcuts demonstrated the impacts forest management practices may have on the soil ecosystem. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Forest management; Clearcutting; Soil biology; Soil ecology

1. Introduction In forest ecosystems, litter and soil organisms and their processes can serve as valuable indicators of stress and disturbance resulting from anthropogenic causes, including forest management practices. In undisturbed forests, litter accumulates on the forest floor and provides storage of essential plant resources, particularly nitrogen and water (Berg and Tamm, 1994). The decomposition of this litter by soil organisms (bacteria, fungi, microarthropods, nematodes, and protozoa) contributes to the soil organic matter and maintains soil productivity. Clearcutting may accelerate the turnover of detrital and soil carbon pools, including litter, roots, and soil organic matter. Thus, the surface organic matter may be reduced due to the increased decomposition rates following clearcutting (Covington, 1981). Clearcutting in coniferous forests has been shown to result in increased CO2 efflux from the forest soil (Gordon et al., 1987). Disturbance of the forest floor, by activities such as clearcutting, has a direct effect on nitrogen cycling in forest soils. One typical effect is a short-term increase in mineralization rates followed by a longer period of recovery during which net mineralization decreases (Matson and Vitousek, 1981; Vitousek and Andariese, 1986). Declines in the densities of microarthropods and nematodes have been noted for extended periods following clearcutting (Seastedt and Crossley, 1981; Sohlenius, 1982; Blair and Crossley, 1988). Nematodes and microarthropods, due to their large numbers and biomass, wide distribution in soil, and the many functions they perform in the soil ecosystem, are valuable measures of soil health and indicators of stress and disturbance (Parmelee, 1995). Nematodes and microarthropods play major roles in the decomposer food web and are key regulators of decomposition and nutrient mineralization/immobilization (Wood, 1989). The microbial mineralization of carbon and nitrogen is influenced by nematodes and microarthropods in several ways: through their direct feeding on microorganisms; their

transport of microbial propagules to new substrates; their excretion of inorganic nitrogen which becomes available for further microbial or plant uptake; their fragmentation of organic matter which increases the surface area available to microorganisms and; by their movement through soil and release of fecal pellets which affects soil aggregate size, pore space and overall stability (Anderson, 1995). Changes in the population dynamics and composition of nematodes and microarthropods have been used as bioindicators of effects from heavy metals, herbicides, insecticides, fungicides, nutrient cycling rates, and agricultural and forest management practices (Parmelee, 1995; Larink, 1997; Zunke and Perry, 1997). Microorganisms also play a crucial role in forest ecosystems, including decomposition, nutrient cycling, and plant symbioses (Cromack and Caldwell, 1992; Paul and Clark, 1989). Pseudomonas species of bacteria are widely distributed in soil, often in close association with plants as rhizobacteria, and have been shown to be impacted by soil management practices such as fire and clearcutting (Atlas and Bartha, 1987; Bääth et al., 1995). The effect of forest management practices on microorganisms has been examined in several studies; results from clearcutting have included observations of higher total bacterial populations (Sundman et al., 1978; Lundgren, 1982), and reduced total and active fungal biomass (Bååth, 1980). Traditional microbiological plating methods, and also the BiologTM method, have demonstrated that different plant species may have distinctive microbial populations (Grayston and Campbell, 1996; Grayston et al., 1998; Latour et al., 1996; Lemanceau et al., 1995). The BiologTM method has been used to demonstrate microbiological effects of forest management practices and detected effects of silvicultural practices and differences in microbial community structure along a forest climatic gradient (Staddon et al., 1997, 1998). Community DNA fingerprints generated from specific primers are useful for providing qualitative molecular assessments of microbial communities. They have been applied to evaluate effects

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of forest management practices and have demonstrated differences between forests and clearcuts in microbial species composition (Shaffer et al., 2000). It has recently been proposed that changes in the functional diversity and composition of plant communities resulting from land management practices may affect ecosystem processes (Beare et al., 1995; Hooper and Vitousek, 1997; Tilman et al., 1997). Plant functional diversity has also been proposed as an indicator of environmental change (Hobbs, 1997). Therefore, studies of plant community composition can provide information on the effects of land management on plant communities and the subsequent effects on soil microbial and invertebrate communities. In this study, we evaluated the response of nematodes, microarthopods, and microorganisms to different forest management practices at two sites with mature forests and adjoining clearcuts. Differences in the numbers and composition of organisms between the mature forests and the clearcuts were evaluated in respect to the forest management practices used. 2. Methods 2.1. Field sites The low elevation field site, at an elevation of 537 m, is located in the western hemlock (Tsuga heterophylla) vegetation zone in the south Santiam drainage of the Oregon Cascades. The soil texture is mostly silt loam with bits of very coarse sand and very fine gravel and can be classified as coarse-loamy, mesic, Typic Hapludand. The Douglas-fir (Pseudotsuga menziesii) forest at the low elevation site is ca. 110–140 years old based on tree-ring counts. Historical records indicate there was a large stand-replacement fire in 1856 followed by a 100 year storm in 1861. The even-age structure of the Douglas-fir trees probably resulted from natural regeneration after the 1856 stand-replacement fire. Douglas-fir is the dominant tree in the overstory and vine maple (Acer circinatum) and western hemlock are found in the understory. The stand density for trees >20 cm dbh is ca. 238 trees/ha and the forest seral stage is late successional. The clearcut area at the low elevation site was commercially thinned in 1969, 40 acres were clearcut

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in 1988, the area was broadcast burned in 1990, and replanted to P. menziesii in 1993. The clearcut area is managed as an elk wintering and calving habitat; the area was aerially seeded with legumes (Trifolium and Lotus) and grasses (Dactylis and Lolium) and fertilizer was applied (N:P:K, 20:16:0) in the spring of 1994 and 1996 at a rate of 250 lb/acre (the fertilizer was applied in the areas surrounding, but not within, the clearcut plots used in this study). The low elevation site typically experiences a wet, mild maritime climate. Precipitation occurs primarily during the winter and summers are relatively dry and contribute only 6–9% of the total precipitation. The high elevation field site is in the Pacific silver-fir (Abies amabalis) vegetation zone in the upper reaches of the McKenzie River drainage and is at an elevation of 1220 m. The sandy loam soil has a medium granular structure and can be classified as a coarse-loamy, mixed, frigid, Typic Hapludand. The forest at the high elevation site is thought to be ca. 200–250 years of age based on tree-ring counts. Douglas-fir is the dominant overstory tree species in the high elevation site forest and western hemlock and Pacific silver-fir occur in the understory. As with the low elevation site, the even-age structure of the dominant Douglas-fir in this forest is consistent with a pattern of natural regeneration following high intensity stand-replacement fires. The stand density for trees >20 cm dbh is ca. 267 trees/ha and the seral stage for the forest is late successional. The clearcut area at the high elevation site results from a 1991 harvest of ca. 46 acres, followed by a 1994 replanting of a mixture of Douglas-fir (15.3%), Noble-fir (73.1%), Grand-fir (7.5%), and western white pine (4.2%) at a density of 523 trees/acre. Following the 1991 clearcutting, no slash burning was done and much of the large woody debris was left at the site. The climate at the high elevation site is wetter and cooler than at the low elevation site. At the high elevation site, considerably more of the precipitation is in the form of snow. The site commonly receives winter snow packs of up to 1–3 m. 2.2. Field research plot design and sampling The sample plots were 1 m×1 m. Within the forests, a total of 24 sample plots were established at the low

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elevation and at the high elevation field sites. These 24 (1 m × 1 m) plots were placed within eight randomly chosen 15 m × 15 m plots that were also randomly isolated within a 0.6 ha area of forest at each site. Thus, three 1 m×1 m sample plots were systematically placed within each of the eight 15 m × 15 m areas, i.e. one sample plot was placed centrally within each of the NW, NE, and SE quadrants of the 15 m×15 m area. Within the clearcuts, a total of 21 (1 m×1 m) sample plots were established at the low elevation and at the high elevation field sites. At the low elevation field site, the plots were located 5 m apart along a 105 m transect that was laid out to randomly sample only the control (unfertilized) area of the clearcut. At the high elevation field site, the plots were located 20 m apart along two parallel EW transects, with 20 m between transects. The sampling began in May of 1996 at the low elevation field site and June of 1996 at the high elevation field site. The sampling at each site continued at approximately 8 week intervals until September 1997 for the low elevation site and November of 1997 for the high elevation site. Due to site access problems from weather conditions, the low elevation site was not sampled during the months of November, December, and January, and the high elevation site was not sampled from December through May. Consequently, a total of nine sampling events were done at the low elevation site as compared to a total of eight sampling events at the high elevation site. Litter samples were collected with a gloved hand using a 20 cm circular template in the forest plots and a 58 cm circular template in the clearcut plots. Due to a thinner and non-continuous litter layer in the clearcut plots, the larger template was necessary in order to collect adequate litter quantities needed for the various assays. Soil cores for the biological and molecular assays were collected with a 3 cm diameter soil core and divided into 0–10 and 10–20 cm samples. Litter and soil samples were pooled in threes so that out of a total of 21 clear cut plots, seven samples each for litter, 0–10 cm soil, and 10–20 cm soil were collected and out of a total of 24 forest plots, eight samples each of litter, 0–10 cm, and 10–20 cm were collected. 2.3. Microarthropod extraction and identification Subsamples of 20 g of the litter and the 0–10 cm soil samples were placed the same day they were

collected in modified Tullgren funnels for extraction of microarthropods into beakers containing water according to Edwards (1991) and Tullgren (1918). After 96 h, the microarthropods were collected and identified to class (Diplopoda, Chilopoda and Symphyla) or to order (Acari, Protura, Collembola, Diplura, and order of insect) according to Borror et al. (1992). The total number of each class or order of microarthropods was converted to the number per 20 g of DW litter or soil. 2.4. Nematode extraction and quantification Subsamples of 20 g of the litter and the 0–10 and 10–20 cm soil samples were weighed and placed the same day they were collected in modified Baermann funnels according to Baermann (1917) and Edwards (1991). Nematodes were collected after 96 h. The total number of nematodes per sample were counted and expressed as the number per 20 g of DW litter or soil. 2.5. Microorganism culturing and quantification Litter and soil samples were stored after field collection for 24 h at 5◦ C. Subsamples of 10 g of litter, 0–10 cm soil, and 10–20 cm soil were placed in dilution bottles with 90 ml extraction buffer (0.2% sodium hexametaphosphate and 6 uM Zwittergent detergent) and shaken for 5 min at a setting of 8 on a Multi-Wrist Shaker (Lab-line Instruments, Inc., Melrose Park, IL). Ten-fold dilutions were spread-plated in duplicate to determine total bacteria (1/10 tryptic soy agar, Difco Manual, 1984, with 100 mg/ml cycloheximide); total fungi (dichloran-rose bengal agar, King et al., 1979, with 50 ␮g/ml chlortetracycline and 200 ␮g/ml streptomycin); chitin-utilizing bacteria (chitin agar, Hsu and Lockwood, 1975, with 100 ␮g/ml cycloheximide); and spore-forming bacteria (5 min heat treatment at 85◦ C before plating on 1/10 tryptic soy agar with 100 ␮g/ml cycloheximide). Plates were incubated for 5–7 days at 25◦ C and the colony-forming units (CFUs) were counted. For the chitin-utilizing bacteria plates, only CFUs surrounded by clearing of the agar (indicating enzymatic breakdown of the chitin) were counted. CFUs were converted to log CFU/g DW of litter or soil.

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2.6. Microbial community metabolic profiles Metabolic profiles of the soil microbial communities were based on patterns of utilization of a series of 95 single substrates on microtitre plates (Garland, 1996; Di Giovanni et al., 1999). The dilutions of the 0–10 cm soil prepared for the microorganism culturing and quantification were also used for determining microbial community metabolic profiles. Samples from three dates (May 1996, May 1997 and July 1997) for the low elevation site and two dates (June 1997 and August 1997) for the high elevation site were analyzed. The soil dilutions were centrifuged for 10 min at 170 g and adjusted to a transmittance value of 85% (±2%). BiologTM GN plates (BiologTM Inc., Hayward, CA) were inoculated with 150 ␮l of extract per well and incubated at 27◦ C. Plate incubations were stopped when a standardized metabolic activity reference point (average well color development (AWCD) A590 ) value of 0.75 ± 0.05 was reached on a Titertek Multiscanner (Titertek Instruments, Inc., Huntsville, AL). 2.7. Microbial DNA fingerprints Bulk DNA (20–23 kb) was directly extracted and purified from litter and 0–10 cm soil samples, amplified by polymerase chain reaction (PCR), and digested by restriction enzyme analysis to obtain microbial community DNA fingerprints (Porteous et al., 1997). PCR products (990 bp) specific for Pseudomonas species were amplified using primers based on 16S rRNA (Widmer et al., 1998), digested with 5 units Hae III (Boeringer Mannheim, Indianapolis, IN) and visualized using agarose gel electrophoresis. Bulk DNA fingerprints were compared to restriction fragment length polymorphisms (RFLPs) of published Genbank Pseudomonas sequences (Benson et al., 1999) cut to the 990 bp target region and restricted with Hae III using MacDNASIS version 3.6 (Hitachi Software Engineering America, Ltd.; San Bruno, CA). Community DNA fingerprints and Pseudomonas spp. RFLPs were presented as fragment sizes on a logarithmic scale. 2.8. Litter and soil dry weight determination Dry weights for litter and soil samples were determined gravimetrically by subsampling ca. 1 g of litter

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or soil and drying in a VWR 1300U drying oven (Sheldon Manufacturing, Inc., Cornelius, OR) at 110◦ C to constant weight. 2.9. Plant surveys Estimations of plant and plant species percent cover and density at ground level in clearcut and forested areas were based on plot photographs taken in 1996 and 1997. Areas of the plots (m2 ) were demarcated by a portable PVC quadrat frame. Due to parallax, the quadrat appeared as a trapezoid in plot photos. Measurements (mm) were made of the lengths of the top (a), bottom (b) and height (h) of the trapezoid, in order to calculate total plot areas (A = 1/2[(a + b)h]), as they appeared in photographs. A clear plastic overlay containing a 5 mm × 5 mm grid was placed over individual plot photographs; the area in which plant material appeared were counted to determine the percent of the plot area (vegetation/total plot × 100, where both vegetation and total plot are expressed in mm2 ) that was covered by vegetation. Species richness (total species number) determinations were made for each plot and cumulatively, for each site, in each of 2 years (1996/1997) of the study. Species identifications were made visually in the field and confirmed based on standard taxonomic descriptions (Hitchcock and Cronquist, 1973). 2.10. Meteorological data collection Meteorological data were collected during the field experiment with a 21× Micrologger (Campbell Scientific, Logan, UT). Air temperature, relative humidity, solar radiation, precipitation, wind speed, and soil temperature and moisture were measured within the forest and the clearcut plots at each elevation. Snow depth was also measured in the clearcut plots at both elevations. 2.11. Statistical analysis of data Data were analyzed for differences between treatments (forested and clearcut) both within and across sample types (litter, 0–10 cm soil, 10–20 cm soil) and sample dates using two-way analysis of variance (ANOVA) procedures in SAS (SAS, 1990). Means were compared using Tukey’s Student’s range test

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when the ANOVA detected significant (P < 0.05) effects. For the statistical analysis of the microbial community metabolic profile data, principal components (PC) scores were calculated, and MANOVAs were performed on the PC scores, in SAS. Values of P ≤ 0.05 were considered to be statistically significant.

3. Results 3.1. Microarthropod populations Numbers of microarthropods in litter and soil were generally much greater in the forest than in the clearcut plots at both elevational sites (Table 1). When the data were grouped across all sample dates and sample types, the numbers of total microarthropods, and the subsets of hexapods, arachnids, insects, and

myriapods, were significantly higher (P = 0.0001) in the forest than in the clearcut plots at both the low and high elevation sites. These differences between the forest and clearcut plots were much more pronounced for the litter samples. When the data for the litter samples were grouped across all sample dates, the total microarthropod numbers, and the microarthropod subset numbers, in the forest plots were significantly higher (P = 0.0001) at both sites. In the 0–10 cm soil samples, only the hexapods at the low elevation site (P = 0.0007), and the myriapods at the high elevation site (P = 0.0343), were significantly greater in the forest than in the clearcut plots. Despite these differences in numbers, the forest and clearcut plots at both the low and high elevation sites were similar in their taxonomic composition of microarthropods; arachnids were most prevalent (61–87% — Acari, Pseudoscorpiones, Araneae), followed by hexapods (10–39% — Collembola, Diplura, Protura), then insects (3%

Table 1 Mean population levels of microarthropods and nematodes (number per 20 g DW litter or soil)

Across all sample types and days Low elevation High elevation By sample type, across all sample days Litter; low elevation Litter; high elevation 0–10 cm soil; low elevation 0–10 cm soil; high elevation 10–20 cm soil; low elevation 10–20 cm soil; high elevation

Nematodes

Microarthropods

Forest Clearcut Forest Clearcut

193a,b 174 249∗ 140∗

145∗ 79∗ 103∗ 33∗

Forest Clearcut Forest Clearcut Forest Clearcut Forest Clearcut Forest Clearcut Forest Clearcut

457c 408 499∗ 228∗ 94 88 151∗ 116∗ 27 26 96∗ 75∗

267∗ 138∗ 192∗ 55∗ 23 20 15 12 NA NA NA NA

a Mean number per 20 g DW litter or soil, calculated from 56–72 samples (seven clearcut, or eight forest, treatment replicates per sample day × 8 high elevation, or 9 low elevation, sample days). b Data were grouped by treatment (forest or clearcut), regardless of sample type (litter, 0–10 cm soil, and 10–20 cm soil) and analyzed across all sample days by ANOVA. Tukey’s Student’s range test indicated that the forest and clearcut treatment means with an asterick (∗) were significantly (P < 0.05) different from each other. c Data were grouped by treatment (forest or clearcut) and by sample type (litter, 0–10 cm soil or 10–20 cm soil) and analyzed across all sample days by ANOVA. Tukey’s Student’s range test indicated that the forest and clearcut treatment means with an asterick (∗) were significantly (P < 0.05) different from each other. NA: population levels of microarthropods were not determined for the 10–20 cm litter and soil samples.

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— Coleoptera, Diptera, Hymenoptera, Thysanoptera, Homoptera, Neuroptera, Lepidoptera, Pscoptera), then myriapods (0.3–0.7% — Diploploda, Chilopoda, Symphyla). At both the low and high elevation sites, in both the forest and clearcut plots, numbers of microarthropods were significantly higher (P = 0.0001) in litter samples than in 0–10 cm soil samples. 3.2. Nematode populations Nematode numbers at the high elevation site were substantially higher in the forest compared to the clearcut plots whereas such pronounced differences were not observed at the low elevation site (Table 1). When the data were grouped across all sample dates and sample types (litter, 0–10 cm soil; 10–20 cm soil), nematode numbers were significantly higher in the forest plots than in the clearcut plots at the high elevation site (P = 0.001) but not at the low elevation site. When the data were grouped across all sample dates and analyzed within each sample type, the nematode numbers at the high elevation site were greater in the forest plots than in the clearcut plots for litter (P = 0.0001), 0–10 cm soil (P = 0.002), and 10–20 cm soil (P = 0.0156) but significant differences were not found for the low elevation site. When the data were analyzed within each sample date and sample type, however, some significant differences were observed between the forest and clearcut plots at the low elevation site and generally were due to higher nematode numbers in the forest plots. The nematode numbers at the low elevation site in the forest plots were significantly higher than in the clearcut plots in litter samples in July 1996 (P = 0.0001), September 1996 (P = 0.0009), May 1997 (P = 0.0011) and July 1997 (P = 0.0126); in 0–10 cm soil samples on July 1996 (P = 0.0456), September 1996 (P = 0.0110) and September 1997 (P = 0.0001); and in 10–20 cm soil samples on March 1997 (P = 0.0472). On two sample dates, the nematode numbers for the low elevation site were significantly higher in the clearcut plots: in 0–10 cm soil samples on March 1997 (P = 0.0449) and in 10–20 cm soil samples on May 1997 (P = 0.0209). At both the low and high elevation sites, for both the forest and clearcut plots, nematode numbers were significantly different (low elevation site P = 0.001; high elevation site P = 0.001)

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among the sample types and had the ranking of litter >0–10 cm soil >10–20 cm soil. 3.3. Microbial populations The microbial populations did not generally follow the same trends as those observed for the nematodes and microarthropods, being higher in numbers in the clearcut plots rather than the forest plots in litter samples at the low elevation site and in soil samples from both sites (Table 2). When the data were grouped across all sample dates and sample types, the numbers at the low elevation site were significantly higher in the clearcut than in the forest plots for the total fungi (P = 0.0001), total bacteria (P = 0.0001), spore-forming bacteria (P = 0.0006) and chitin-degrading bacteria (P = 0.0001). In contrast, for the high elevation site the only significant difference between the forest and clearcut plots was higher numbers of total bacteria in the forest plots (P = 0.0037). When the data were grouped across all sample dates and analyzed within sample types, the numbers in the litter samples from the clearcut plots at the low elevation were significantly higher for total fungi, total bacteria, spore-forming bacteria, and chitin-degrading bacteria (P = 0.0001, 0.0001, 0.0005 and 0.0015, respectively). The opposite result was observed at the high elevation site; microbial numbers in litter samples from the forest plots were significantly higher than in the clearcut plots for total fungi, total bacteria, spore-forming bacteria, and chitin-degrading bacteria (P = 0.0001, 0.0210, 0.0003, and 0.0093, respectively). In the 0–10 and 10–20 cm soil samples, results were more similar for the low and high elevation sites and microbial numbers were highest in the clearcut plots. At the low elevation site, significantly higher numbers were measured in the clearcut plots than in the forest plots in the 0–10 cm soil samples for the total bacteria (P = 0.0001), spore-forming bacteria (P = 0.0042), and chitin-degrading bacteria (P = 0.0001). At the high elevation site, significantly higher numbers were measured in the clearcut plots than in the forest plots in 0–10 cm soil for spore-forming bacteria (P = 0.0001) and chitin-degrading bacteria (P = 0.0001). For the 10–20 cm soil samples, numbers were significantly higher in the clearcut plots than in the forest plots at the low elevation site for the chitin-degrading bacteria (P = 0.0001) and at

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Table 2 Mean population levels of culturable total fungi, total bacteria, spore-forming bacteria, and chitin-utilizing bacteria (log CFU/g DW litter or soil)

Across all sample types and days Low elevation High elevation

Forest Clearcut Forest Clearcut

By sample type, across all sample days Litter; low elevation Forest Clearcut Litter; high elevation Forest Clearcut 0–10 cm soil; low elevation Forest Clearcut 0–10 cm soil; high elevation Forest Clearcut 10–20 cm soil; low elevation Forest Clearcut 10–20 cm soil; high elevation Forest Clearcut

Total fungi

Total bacteria

Spore-forming bacteria

Chitin-utilizing bacteria

5.68a,b,∗ 5.79∗ 5.79∗ 5.71∗

7.34∗ 7.51∗ 6.88 6.86

6.49∗ 6.60∗ 5.21 5.21

6.56∗ 6.69∗ 6.25 6.25

6.05c,∗ 6.45∗ 6.32∗ 6.06∗ 5.72 5.70 5.74 5.75 5.27 5.21 5.32 5.32

7.96∗ 8.28∗ 7.30∗ 7.18∗ 7.26∗ 7.42∗ 6.86 6.86 6.78 6.83 6.46 6.54

6.36∗ 6.60∗ 4.99∗ 4.73∗ 6.67∗ 6.78∗ 5.32∗ 5.44∗ 6.44 6.42 5.31∗ 5.45∗

6.94∗ 7.06∗ 6.47∗ 6.33∗ 6.59∗ 6.77∗ 6.28∗ 6.37∗ 6.13∗ 6.25∗ 6.00∗ 6.06∗

a Mean log CFU/g DW litter or soil, calculated from averaged duplicate plates for bacteria and fungi, of 56–72 samples (7, clearcut, or 8, forest, treatment replicates per sample day × 8, high elevation or 9, low elevation, sample days). b Data were grouped by treatment (forest or clearcut), regardless of sample type (litter, 0–10 cm soil, and 10–20 cm soil) and analyzed across all sample days by ANOVA. Tukey’s Student’s range test indicated that the forest and clearcut treatment means with an asterick (∗) were significantly (P < 0.05) different from each other. c Data were grouped by treatment (forest or clearcut) and by sample type (litter, 0–10 cm soil or 10–20 cm soil) and analyzed across all sample days by ANOVA. Tukey’s Student’s range test indicated that the forest and clearcut treatment means with an asterick (∗) were significantly (P < 0.05) different from each other.

the high elevation site for the spore-forming bacteria (P = 0.0025) and the chitin-degrading bacteria (P = 0.0417). 3.4. Microbial community metabolic profiles Metabolic profiles of microorganisms differed significantly (P = 0.0001) between the forest and clearcut plots at the low elevation site across all sample dates, based on principal components (PC) scores from the BiologTM GN plates. The PC scores for the July 1997 sampling illustrate the significant differences between the forest and clearcut plots at the low elevation site that were seen for each sample date (Fig. 1). The percent variance explained by PC1 was 44.5% and by PC2 was 10%. The substrates contributing to the PC1 variance were mono-methyl succinate, cis-aconitic acid, urocanic acid, methyl pyruvate, and ␣-keto butyric acid. The substrates contributing to the

PC2 variance were d-melibiose, maltose, l-fucose, d-raffinose, and d-sorbitol. At the high elevation site, PC scores were not significantly different between the forest and clearcut plots (data not shown). There were no significant differences between the AWCD values of forest and clearcut plots for either the low or the high elevation site. 3.5. Microbial DNA fingerprints Pseudomonas community fingerprints in the 0–10 cm soil samples were different between the clearcut and forest at the low elevation site (Fig. 2). For example, for the low elevation site September 1997 samples, Fig. 2 shows fingerprint regions (boxes ‘a’ and ‘c’) where fragment bands (325 and 525 bp) are present in the forest soil (lanes 18–21) but absent in the clearcut soil (lanes 26–29). Very few of these bands (325 and 525 bp) were observed in the soil or litter from the

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Fig. 1. PC scores from BiologTM analyses of 0–10 cm soil samples from forest (䊉) and clearcut (䊏) plots collected from the low elevation site on July 1997. Each clearcut data point represents one of seven samples that were pooled in threes from a total of 21 plots, and each forest data point represents one of eight samples that were pooled by threes from a total of 24 plots. MANOVA indicated that the PC scores of the forest and clearcut plots were significantly different (P < 0.05).

high elevation site (Fig. 3, absence of bands just above and below box ‘b’). A computer search of Genbank found no similar known Pseudomonas species RFLP matches for the 325 and 525 bp fragments. The Pseudomonas fingerprints also indicated differences between litter and 0–10 cm soil at both elevational sites. For example, for the high elevation site, November 1996 samples, the 600 and 200 bp fragments (Fig. 3, boxes ‘a’ and ‘c’) were generally more intense in the forest and clearcut litter (Fig. 3, lanes 1–4 and 9–12) than in the corresponding 0–10 cm soil samples (Fig. 3, lanes 4–8 and 13–16). Additionally, two fragments (about 500 and 170 bp) were regularly present in the 0–10 cm soil samples from the forest and clearcut sites at both elevations. These bands (Fig. 2, boxes ‘b’ and ‘d’, lanes 18–20 and 26–29) were infrequently and less intensely observed in the litter from the forest and clearcut sites (Fig. 2, lanes

14–17 and 22–25). The 500 and 170 bp bands were also commonly observed in fingerprints from the high elevation site 0–10 cm soil samples (Fig. 3, boxes ‘b’ and ‘d’, lanes 5–8 and 13–16). Computer generated models of community DNA fingerprints (Fig. 2, lanes 14–29) and simulated Pseudomonas spp. RFLPs (Fig. 2, lanes 1–13) indicated that the 500 and 170 bp fragment bands were similar to P. marginalis (Fig. 2, lane 12). 3.6. Plant surveys The percent ground cover was highest in the clearcut plots at both elevations in each of the 2 years of the study (Table 3). The percent ground cover was lowest in the high elevation forest. At both elevations, plant density and species density was highest in the clearcut plots (Table 3). Plant density was lowest in the low

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Fig. 2. Hae III RFLPs of 13 published control Pseudomonas spp. sequences and 16 community Pseudomonas fingerprints of litter and 0–10 cm soil samples from the low elevation forest and clearcut samples collected on 15 September 1997. Phylotype names and GenBank accession numbers are: P. mendocina, D84016; P. putida, L28676; P. stutzeri, D84024; P. aeruginosa, X06684; P. alcaligenes, D84006; P. aureofaciens, D84008; P. corrugata, D84012; P. syringae, L24786; P. fluorescens, D11286; P. fluorescens, L24788; P. syringae, D84026; P. marginalis, Z76663; P. agarici, D84005. Boxes ‘a’ and ‘c’ designate sample regions where typical fragments were not detected in the low elevation clearcut soil. Boxes ‘b’ and ‘d’ show unique DNA fragments appearing primarily in the soil samples of both the forest (lanes 18–21) and clearcut (lanes 26–29) that are similar in size to those of P. marginalis (lane 12). Lanes marked M, Hae III digested phi X 174 DNA.

Fig. 3. Pseudomonas spp. community DNA fingerprints from the high elevation site 12 November 1996 forest and clearcut samples shown as an inverted electrophoretic scan. Bulk DNA directly extracted, amplified and restricted with the endonuclease Hae III from forest litter (lanes 1–4), forest soil (lanes 5–8), clearcut litter (lanes 9–12) and clearcut soil (lanes 13–16). Boxes ‘a’ and ‘c’ designate Hae III fragments representative of many pseudomonads detected in soil and litter. Boxes ‘b’ and ‘d’ designate fragments primarily detected in soil that are similar to those of P. marginalis. Lanes marked M, Hae III digested phi X 174 DNA.

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Table 3 Plant species richness, percent cover, and density in experimental plots at the field sites Low elevation clearcut Species 1996 1997

High elevation forest

16 14

26 28

26 24

26

15

27

26

99 ± 5.0 100 ± 0.0

52 ± 4.7 59 ± 4.7

78 ± 5.0 38 ± 5.0

42 ± 4.7 31 ± 4.7

99 ± 2.5

56 ± 4.7

58 ± 5.0

36 ± 4.7

6 ± 0.4 10 ± 0.4

3 ± 0.4 4 ± 0.4

9 ± 0.4 8 ± 0.4

8 ± 0.4 10 ± 0.4

8 ± 0.4

4 ± 0.4

8 ± 0.4

9 ± 0.4

89 ± 8.3 134 ± 8.2

12 ± 7.8 31 ± 7.7

90 ± 8.3 103 ± 8.2

62 ± 7.8 68 ± 7.7

112 ± 8.3

22 ± 7.8

96 ± 8.3

65 ± 7.8

Average (species/m2 )c

Average Density 1996 1997

High elevation clearcut

26 26

Average Percent coverb 1996 1997

Density 1996 1997

Low elevation forest

richnessa

(plants/m2 )d

Average a

Cumulative numbers of plant species found in all 21 clearcut or 24 forest plots at the low or high elevation site in 1996 and 1997. Means of estimated percent cover at ground level for all 21 clearcut or 24 forest plots at the low or high elevation site in 1996 and 1997. c Means of estimated density of species/m2 for all 21 clearcut or 24 forest plots at the low or high elevation site in 1996 and 1997. d Means of estimated density of plants/m2 for all 21 clearcut or 24 forest plots at the low or high elevation site in 1996 and 1997. b

elevation forest plot. Plant species richness was equivalent among both of the clearcut sites and the high elevation forest site but was considerably lower at the low elevation forest site (Table 3). Plant community composition differed between the forest and clearcut plots, and also between the low and high elevation sites (Table 4). 3.7. Meteorological conditions and sample moisture measurements Mean annual precipitation was 239 ± 55 cm at the high elevation site and 201 ± 46 cm the low elevation site. The mean annual maximum snowpack was 0 cm at the low elevation site and 162 ± 32 cm at the high elevation site. Mean annual temperature was 10.2 ± 0.2◦ C at the low elevation site and 7.6 ± 0.1◦ C at the high elevation site. The mean annual soil temperature at 5 cm depth was 9.1 ± 0.2◦ C at the low elevation site and 5.8 ± 0.1◦ C at the high elevation site. At both elevations, litter and soil temperatures were typically greater in the clearcut plots as compared to the forest

plots. The mean annual soil moisture at 0–20 cm was 26.5 ± 0.7% at the low elevation site and 23.6 ± 0.4% at the high elevation site. The percent moisture measured in the litter and soil samples used for the biological analyses, averaged over the sample dates and treatment replicates, for the low elevation site were forest litter 62 ± 4.57%, clearcut litter 55 ± 6.95%, forest 0–10 cm soil 40 ± 2.30%, clearcut 0–10 cm soil 38 ± 3.27%, forest 10–20 cm soil 29 ± 1.15%, clearcut 10–20 cm soil 29 ± 2.07% and for the high elevation site were forest litter 53 ± 6.45%, clearcut litter 38 ± 6.62%, forest 0–10 cm soil 25 ± 2.35%, clearcut 0–10 cm soil 27 ± 3.55%, forest 10–20 cm soil 21 ± 1.62%, clearcut 10–20 cm soil 23 ± 1.94%. At the high elevation site, the percent moisture levels in the forest litter samples were consistently higher than those in the clearcut litter samples whereas at the low elevation site there were not consistent differences between the forest and clearcut litter samples. At both elevations, the litter samples had higher percent moisture than the soil samples.

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Table 4 Plant species located in experimental plots at the field sitesa Low elevation clearcut

Low elevation forest

High elevation clearcut

High elevation forest

Acer circinatum Berberis nervosa Cerastium arvense Cirsium vulgare Dactylis glomerata Epilobium minutum Gallium oreganum Gaultheria shallon Hieracium albiflorum Iris tenax Lolium perenne Lotus corniculatus Polystichum munitum Pseudotsuga menziesii Pteridium aquilinum Rhamnus purshiana Rubus ursinus Rumex acetosella Trifolium pratense Trifolium repens Trientalis latifolia Trillium ovatum Tsuga heterophylla Vaccinium membranaceum Vicia sativa Viola orbiculata

Acer circinatum Adenocaulon bicolor Anemone deltoidea Asarum caudatum Berberis nervosa Epilobium minutum Gallium oreganum Gaultheria shallon Mitella pentandra Oxalis oregana Polystichum munitum Pseudotsuga menziesii Rubus ursinus Trillium ovatum Tsuga heterophylla Viola orbiculata

Abies amabilis Abies procera Achlys triphylla Asarum caudatum Berberis nervosa Ceanothus velutinus Chimaphila umbellata Cirsium vulgare Clintonia uniflora Corallorhiza maculata Cornus canadensis Epilobium minutum Festuca idahoensis Fragaria virginiana Gallium oreganum Hieracium albiflorum Linnaea borealis Navarretia divaricata Pachystima myrsintes Pteridium aquilinum Rosa gymnocarpa Rubus ursinus Smilacina stellata Symphoricarpos mollis Trientalis latifolia Trillium ovatum Vaccinium membranaceum Viola orbiculata

Abies amabilis Achlys triphylla Adenocaulon bicolor Anemone deltoidea Asarum caudatum Berberis nervosa Chimaphila umbellata Clintonia uniflora Cornus canadensis Festuca idahoensis Gallium oreganum Goodyera oblongifolia Linnaea borealis Listera caurina Mitella pentandra Navarettia divaricata Pseudotsuga menziesii Pyrola picta Rosa gymnocarpa Smilacina stellata Trientalis latifolia Tiarella trifoliata Trillium ovatum Tsuga heterophylla Vaccinium membranaceum Viola orbiculata

a

A cumulative list of plant species identified in all 21 clearcut or 24 forest plots at the low or high elevation site in 1996 and 1997.

4. Discussion The finding of higher levels of microarthropods in the forest plots, compared to the clearcut plots, is consistent with other studies. In one such study, 8 years following clearcutting, mean annual densities of litter microarthropods were 28% lower in the clearcut area relative to the forested area (Blair and Crossley, 1988). A major contributing cause is thought to be the difference in litter quantity, and also quality, between forested and clearcut areas (Wallwork, 1976; McBrayer et al., 1977). In our study, the clearcut plots contained a very thin, non-continuous litter layer whereas the forest plots had a very thick (up to 8 cm), continuous litter layer that was often permeated with fungal mycelia. The observation that the numbers of microarthropods in the forest and clearcut plots differed far more for the litter samples than for the soil

samples supports the importance of the amount and type of litter in determining numbers of microarthropods. Densities of microarthropods have been shown to be positively related to standing stocks of organic matter and moisture content (Blair et al., 1992). This was demonstrated in our study by the substantially lower levels of microarthropods in all plots during the dry season at the field sites and also the consistently higher levels of microarthropods in litter as compared to soil. The observation that higher population levels of nematodes in the forest plots relative to the clearcut plots was pronounced at the high elevation site but less extreme and consistent at the low elevation site may have resulted from management differences between the low and high elevation clearcut sites which affected moisture levels and microbial populations. The seeding of grasses and legumes at the low elevation

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clearcuts produced a very high percent plant cover which may in turn have promoted greater litter moisture levels and microbial populations. This possibility is supported by the finding that at the high elevation site, litter moisture levels were significantly higher in the forest plots than in the clearcut plots but there were not major differences between forest and clearcut plots at the low elevation site in litter moisture levels. These higher litter moisture levels in the low elevation clearcut plots, along with the greater production of plant exudates, due to the high percent plant cover, may have also promoted the growth of microbial populations. The finding that microbial populations were highest in the low elevation clearcut plots, relative to all other plots, supports this. Because many nematodes are microbivores, it would be expected that their populations would be positively correlated with microbial populations. As with the microarthropods, nematode numbers were generally higher in litter relative to soil. Nematode density is generally negatively correlated with microarthropod density due to predation (some Collembola and mites consume nematodes) and also competition for their common food source of microorganisms (Moore and Walter, 1988). This negative correlation was also observed in our study; nematode populations were slightly larger at the high elevation site whereas microarthropod levels were greatest at the low elevation site. Litter moisture levels and also plant density and species composition may have contributed to the difference in litter samples in populations of culturable total fungi and total bacteria between the low and high elevations in forest and clearcut populations (levels were highest in the clearcut at the low elevation site but at the high elevation site were highest in the forest). Greater moisture levels in the forest litter compared to clearcut litter was very pronounced at the high elevation site but was not observed at the low elevation site. Other studies on effects of clearcutting have demonstrated significant positive correlation of microbial numbers with forest floor moisture content (Hendrickson et al., 1985). Another factor contributing to the higher levels of microorganisms in the clearcut at the low elevation site may be that at the low elevation site there was a much greater difference between the clearcut and forest plots in plant density and species richness than was measured at the high elevation site. The almost 100% plant cover in the low

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elevation clearcut plots, primarily due to seeding of grasses and legumes, favored microbial populations by providing higher moisture levels, protection from extreme temperatures, and also provided plant exudates from a diversity of plants species that could be utilized by the microorganisms as a nutrient source. The fact that the highest populations of both total fungi and total bacteria, for all experimental plots, were observed in the low elevation clearcut, supports the likely favorable influence on microorganisms of the high percent plant cover. The Pseudomonas community, however, did not appear to be favored by these factors and actually showed lower diversity in the clearcut than in the forest at the low elevation. Similarly, the microbial community metabolic profiles indicated differences between the clearcut and the forest at the low elevation site. These microbial community results suggest that the post-harvest intensive management practices used at the low elevation site promoted microbial growth but not necessarily recovery of the original, forest microbial community. The intensive management practices may in fact have favored fast growing, competitive microbial species rather than forest-adapted species. The observed higher populations in litter as compared to soil, for all the plots and sites, of culturable, aerobic total fungi, total bacteria, and chitin-degrading bacteria, and the more intense bands in the Pseudomonas DNA fingerprints has been found in other studies and is likely due to the higher organic matter content and moisture levels of the litter (Paul and Clark, 1989). Similarly, the lower populations of spore-forming bacteria in soil is also typical and is probably due to the lower moisture and organic matter content in soil and the consequent greater impetus for spore formation (Doyle et al., 1995). Therefore, it is interesting that the levels of spore-forming bacteria were approximately 10-fold higher at the low elevation site than at the high elevation site. Management practices have been more extensive at the low elevation site (slash burning and seeding of the clearcut, and fertilization of the area surrounding the clearcut plots) and have resulted in greater disturbance of the low elevation site. The populations of total and chitin-degrading bacteria were also approximately five-fold greater at the low elevation site as compared to the high elevation site. This may in part be explained by predation due to the higher levels of nematodes at the high elevation site and the fact that

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bacterial-feeding nematodes are usually the dominant nematode trophic group in forests (Ruess, 1995). Different woody and herbaceous plant species, cultivars, and genotypes have been shown to have different rhizosphere microbial community populations (Azad et al., 1985; Lemanceau et al., 1995; Grayston and Campbell, 1996; Latour et al., 1996; Grayston et al., 1998; Di Giovanni et al., 1999). Our results similarly suggest that differences in plant community composition between forests and clearcuts may be reflected by differences in soil microbial community composition and metabolic profiles. The metabolic profiles of soil microbial communities differed significantly between the clearcut and forest plots at the low elevation site while no such differences were found at the less intensively managed high elevation site, the same as was observed with the plate counts for total bacterial populations. Potential reasons for the observed difference between forest and clearcut soils at the low elevation site, but not at the high elevation site, include larger differences in plant community composition and soil moisture between the low elevation clearcut and forest, than in the less intensively managed plots at the high elevation site. A longer post-harvest elapsed time, 7 years, at the low elevation site, versus 5 years at the high elevation site, may also have contributed to observations of microbial community metabolic profile differences at the low elevation site, but not at the high elevation site. Following clearcutting, decades may be required before restoration to the original forest vegetation occurs and the years immediately following clearcutting may favor colonization of pioneer invasive species, rather than re-establishment of the original vegetation (Dyrness, 1973; Halpern and Spies, 1995). The difference between 5 and 7 years post-clearcutting could affect the amount of time available for colonization by pioneer invasive plant species and by the grasses and legumes sown at the low elevation site. This is reflected in our data where there is a much greater difference in plant species composition between the forest and clearcut plots at the low elevation site than at the high elevation site. The clearcuts at both elevations had a higher percent ground cover and plant density than in the corresponding forested areas. The low plant density, along with the low plant species richness, at the low elevation forested site may be due to the limiting light conditions associated with the dense vine maple

(A. circinatum) understory in the forest. Other studies have shown that clearcutting or thinning can lead to increased percent cover and species richness relative to old growth stands and is, in part, due to increased light (Bailey et al., 1998). The extremely high percent plant ground cover in the low elevation clearcut is likely due in part to the seeding of orchard grass (D. glomeratus) and perennial rye (L. perenne), and legumes such as bird’s foot trefoil (L. corniculatus), and clover (T. repens and T. pretense). Fertilization of the adjacent area uphill from the low elevation clearcut, done as management of the site as elk habitat, may also have contributed to the higher percent ground cover measured in the plots. The high elevation clearcut was neither seeded nor were any adjacent areas fertilized. In addition, the high elevation clearcut was much less disturbed than the low elevation clearcut; slash was left on the ground at the high elevation clearcut, whereas it was burned at the low elevation clearcut. The larger difference in plant community composition between clearcut and forest areas at the low elevation site is suggested by a lower coefficient of community (30%), as compared to the high elevation site (45%). The plant species list indicates that the clearcut and forest plots at the low elevation site differed more than at the high elevation site. For example, 42.3% of the species found in the low elevation site clearcut were invader or sown species: Cirsium (thistle), Cerastium (chickweed), Epilobium (fireweed), Hieracium (hawkweed), Pteridium (bracken), and Rumex (sorrel) were invaders, and Lolium, Dactylis, Trifoloium, Lotus, and Vicia were sown. At the high elevation site, only 17.9% of the clearcut species were invader type species: Ceanothus (snow bush), Cirsium (thistle), Epilobium (fireweed), Hieracium (hawkweed), and Pteridium (bracken). These observed differences in plant community composition and percent cover can in part be attributed to differences between the low and high elevation clearcuts in management practices, stand age, and elapsed time after clearcutting.

5. Conclusions The different forest management practices at our field sites influenced the litter and soil organisms we monitored. Our findings suggest that management

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practices that compensate for the loss of forest litter that occurs with clearcutting and the consequent decrease in litter and soil moisture and nutrients and increase in temperature, such as seeding of grasses and forbs, may favor microbial and plant populations but does not necessarily lead to re-establishment of the original litter and soil biota communities that existed prior to clearcutting. Post-harvest practices such as types and numbers of species seeded, plant density, and fertilization have been proposed to have a greater effect on plant community composition than either logging or burning (Halpern and Spies, 1995). In studies of early plant succession following logging in the western Cascades of Oregon, invader species were observed to increase in the 5 years after burning, before residual herbaceous forest species regained dominance (Dyrness, 1973). In our studies, in which the clearcut at the low elevation site was burned, seeded with grasses and legumes and fertilized to provide forage for elk, the plant community was dominated by the seeded grass and legume species, and percent cover approached 100%. The combination of post-harvest practices carried out at the low elevation clearcut plots (burning, seeding and fertilizing) did successfully produce high culturable microbial populations and high plant percent cover and density. However, these plant communities, and also the microbial communities, as indicated by the metabolic profiles and the Pseudomonas species DNA fingerprints, were not similar in numbers and composition to those measured in the forest. In addition, the microarthropod populations within the litter layer in the clearcut remained substantially lower than those in the forest. The management practices did not produce re-establishment of the pre-clearcut plant community and soil biota, at least following a period of 7 years. Longer term monitoring may indicate which management practices, the intensive practices at the low elevation site, or the less intensive practices at the high elevation site, will be most successful in promoting re-establishment of forest plant communities and soil biota.

Acknowledgements We thank Drs. Robert Mckane, David Tingey, and Mark Johnson for providing background information

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