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Keywords:

  • bacterial growth;
  • fungal growth;
  • competition;
  • selective inhibition;
  • antibiotic;
  • decomposer ecology

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

It is important to know the contributions of bacteria and fungi to decomposition in connection with both the structure of the food web and the functioning of the ecosystem. However, the extent of the competition between these groups of organisms is largely unknown. The bacterial influence on fungal growth in a soil system was studied by applying three different bacterial inhibitors – bronopol, tylosin and oxytetracycline – in a series of increasing concentrations, and comparing the resulting bacterial and fungal growth rates measured using leucine and acetate-in-ergosterol incorporation, respectively. Direct measurements of growth showed that fungi increased after adding inhibitors; the level of increase in fungal growth corresponded to that of the decrease in bacterial growth, irrespective of the bacterial inhibitor used. Similar antagonistic effects of the bacteria on fungal growth were also found after adding the bacterial inhibitors together with additional substrate (alfalfa or straw plant material). The resulting responses in bacterial and fungal growth indirectly indicated that the negative interaction between fungi and bacteria was mostly attributable to exploitation competition. The results of this study also emphasize the increased sensitivity of using growth-related, instead of biomass-based, measurements when studying bacterial and fungal interactions in soil.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Fungi and bacteria together dominate the decomposition of organic material in soil. The fungal and bacterial biomass produced during decomposition is then transferred through different energy pathways in the soil ecosystem (Hunt et al., 1987; Hedlund et al., 2004; Moore et al., 2005). Furthermore, the two groups are thought to have different efficiencies in the sequestering of carbon (Six et al., 2006), although this has recently been questioned (Thiet et al., 2006). Consequently, the bacterial and fungal contributions to soil decomposition should be separated to determine the structure of the food web and the functioning of the ecosystem. There is increasing evidence that environmental factors affect fungi and bacteria differently. These include variables such as substrate composition (Bittman et al., 2005; de Boer et al., 2005; Georgieva et al., 2005; Engelking et al., 2007; Rousk & Bååth, 2007b), pH (Blagodatskaya & Anderson, 1998; Pennanen et al., 1998; Bååth & Anderson, 2003), salinity (Sardinha et al., 2003; Rasul et al., 2006), temperature (Ley & Schmidt, 2002; Lipson et al., 2002; Schadt et al., 2003; Pietikäinen et al., 2005) and heavy metals (Rajapaksha et al., 2004). Another major determinant of the composition of the decomposer community, which has been less thoroughly studied, is their interaction.

The interaction of organisms can be studied in two ways. One alternative is to combine the organisms and study the response of one to the addition of the other. Using this approach, Mille-Lindblom & Tranvik (2003) studied fungal and bacterial interaction on leaves submerged in water based on the biomass production of the separate groups. Starting with uncolonized plant material, they added single strains of fungi, a natural bacterial community, or a combination of them. They found a reciprocal negative interaction between the decomposer groups. Other studies using this approach report both antagonistic (Møller et al., 1999; Mille-Lindblom et al., 2006) and synergistic interactions (Bengtsson, 1992). Although this additive approach is often easy to interpret, it can only be used to study interactions during colonization of a substrate. Furthermore, it depends on the ability of the scientist to introduce a representative composition of the inoculated organisms, often consisting of single or a few strains.

The other alternative that can be used to study the bacterial–fungal interaction is a removal experiment, where one group is experimentally removed and the effect on the other group is monitored. Studies of the fungal–bacterial interaction in soils are often based on the application of selective inhibitors. There have been reports of fungal inhibition by bacterial presence (Thiele-Bruhn & Beck, 2005; Feeney et al., 2006), of bacterial biomass increasing after fungal suppression without reciprocity (Ingham & Coleman, 1984), and of no interaction between the decomposer groups (Lin & Brookes, 1999; Verma et al., 2007). The removal approach is, however, sensitive to the selectivity of the applied inhibitor, which should have no nontarget effects.

The studies cited above all rely on changes in fungal and bacterial biomass to elucidate the interaction. Using biomass measurements to investigate the decomposer ecology in soil may yield ambiguous results, because these measurements will be susceptible to confounding factors including predation (Cotner et al., 1997) and potential recalcitrance of biomass proxies (Mille-Lindblom et al., 2004; Zhao et al., 2005). Assessing the fungal and bacterial growth rates directly, however, can circumvent these problems (Rousk & Bååth, 2007b) and will thus be more sensitive to detect effects, both positive and negative.

In the present study one microorganism group of an intact decomposer community in a natural soil, bacteria, was inhibited and the response of the other, fungi, was studied using direct measurements of growth. Three possible outcomes were predicted following the application of bacterial inhibitors: (1) inhibiting bacteria would benefit fungal growth, indicating niche overlap and thus competition; (2) inhibiting bacteria would not affect fungal growth, indicating complete partitioning of the fundamental niches of the decomposers; or (3) inhibiting bacteria also decreases fungal growth, indicating that bacteria facilitate fungal growth (synergistic effects). To avoid possible imperfect inhibitor selectivity, three different bacterial inhibitors, oxytetracycline, tylosin and bronopol, were applied to soil and the resulting bacterial and fungal growth were measured with the leucine (Leu) incorporation technique (Kirchman et al., 1985; Bååth, 1994) and the acetate-in-ergosterol (Ac-in-erg) incorporation technique (Newell & Fallon, 1991; Bååth, 2001), respectively. The effects of these three inhibitors on soil organisms have previously been studied by e.g. Anderson & Domsch (1973), Westergaard et al. (2001), Bailey et al. (2003), Thiele-Bruhn & Beck (2005) and Feeney et al. (2006).

If decreasing bacterial growth was shown to benefit fungi (indicating competitive release), it was predicted that the strength of this effect would increase with the level of bacterial growth inhibition during the experiment. Therefore a range of concentrations was applied, resulting in little to severe inhibition of bacterial growth, for each inhibitor. The three bacterial inhibitors were furthermore applied using three different kinds of substrate amendments, one where alfalfa had been added to the soil, one where straw had been added, and one where no substrate had been added. Rousk & Bååth (2007b) showed that applying straw and alfalfa benefited fungi and bacteria differently; the fungi benefited more than bacteria when straw was added, and vice versa when alfalfa was added. This gave a gradient of bacterial colonization, lowest in the unamended soil, higher in the straw-amended and highest in the alfalfa-amended soil. Hence, the bacterial inhibitor should benefit the fungi increasingly ranging from the unamended to the alfalfa-amended soil.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Three bacterial inhibitors (oxytetracycline, tylosin and bronopol) were added to a sandy-loam grassland soil (pH 5.9, water content 27%, organic matter content 17%, sieved <2.8 mm), creating gradients of increasing inhibitor concentrations in a series of microcosms. Oxytetracycline hydrochloride (CAS 2058-46-0), tylosin tartrate (CAS 74610-55-2) and bronopol (CAS 52-51-7) were purchased from Sigma Aldrich, Steinheim, Germany. A pilot study was performed to find suitable concentrations of the bacterial inhibitors (data not shown). Based on this, oxytetracycline and tylosin were added at 0, 30, 60, 120, 240, 480, 960 and 1920 μg g−1 soil, and the more potent bronopol at 0, 10, 20, 40, 80, 160, 320 and 640 μg g−1 soil, using inert talcum powder to equalize the amount of dry material added to each soil sample, and to facilitate mixing of the substances into soil. This resulted in 24 samples, which were duplicated, giving 48 microcosms consisting of 5 g soil in closed 50-mL polyethylene Saerstedt tubes.

Three initial soil treatments, (1) unamended, (2) straw-amended and (3) alfalfa-amended, were investigated in three independent experiments. Dried, ball-milled (<0.25 mm) alfalfa (C/N=15) or straw (C/N=75) was added at 2 mg g−1 soil. The unamended and straw-amended soil samples were incubated for 5 days and the alfalfa-amended soil samples were incubated for 3 days, because previous experiments had indicated maximal fungal and bacterial growth at these times (Rousk & Bååth, 2007b).

Bacterial growth was estimated using the Leu incorporation technique (Bååth, 1994; Bååth et al., 2001). Briefly, 1 g soil was mixed with 20 mL water and treated on a multivortex shaker at maximum intensity for 3 min. Following this, low-speed centrifugation at 1000 g for 10 min created a bacterial suspension in the supernatant. Aliquots of this suspension (1.5 mL) were transferred to 2-mL microcentrifugation tubes, and 2 μL labelled Leu, [3H]Leu (37 MBq mL−1, 5.74 TBq mmol−1, Amersham) and 2 μL 200 mM nonlabelled Leu were added to each tube, resulting in 275 nM Leu in the bacterial suspensions. After 2 h incubation, growth was terminated using 75 μL 100% trichloroacetic acid. Washing and subsequent measurement of radioactivity were performed as described by Bååth et al. (2001). The amount of incorporated radioactivity was determined using a scintillator, and the amount of Leu incorporated per mL bacterial suspension and per hour was used as a measure of bacterial growth.

Fungal growth was assessed using the Ac-in-erg method (Newell & Fallon, 1991) adapted for soil (Pennanen et al., 1998; Bååth, 2001). Briefly, 1 g of soil was transferred to testtubes to which 0.025 mL 1,2-[14C]acetic acid (sodium salt, 7.4 MBq mL−1, 2.04 GBq mmol−1, Amersham), 0.475 mL 1 mM unlabelled acetate (pH=6) and 1.5 mL distilled water were added, resulting in a final acetate concentration of 0.22 mM. The resulting soil slurry was incubated at room temperature (22 °C) without light for 16 h, after which 1 mL 5% formalin was added to terminate growth. Ergosterol was then extracted, separated and quantified using HPLC and a UV detector (282 nm) according to Rousk & Bååth (2007b). The ergosterol peak was collected. The amount of incorporated radioactivity was determined using a scintillator, and the amount of Ac incorporated into ergosterol per hour and per gram of soil was used as a measure of fungal growth.

Basal respiration was measured by transferring 1 g of soil to a 20-mL glass vial, and purging with pressurized air. The vial was sealed and incubated for 24 h, after which the CO2 concentration was determined using GC.

The relative fungal and bacterial growth rates and respiration (Figs 1 and 2) were calculated as the value derived from a sample divided by the mean value obtained from the zero concentration of the bacterial inhibitors of that sample's soil (unamended, with straw or alfalfa). The relationships between relative fungal and bacterial growth were analysed using log–log (power-curve) regression. The changes (Δ values) were calculated as the difference in means between the four samples of the two lowest inhibitor concentrations (with no effect) and the four samples of the two highest concentrations of that soil (unamended, with straw or alfalfa).

image

Figure 1.  Response of fungal (□) and bacterial (◊) growth and respiration rate (○) to increasing concentration of bacterial inhibitors in soils with different substrate amendments. The responses are relative to the mean value of the zero and lowest concentrations of bacterial inhibitors. (a–c) show the effects of bronopol, (d–e) those of tylosin and (g–i) oxytetracycline application. (a), (d) and (g) show the results in unamended soil, (b), (e) and (h) alfalfa-amended soil, and (c), (f) and (i) straw-amended soil. Bronopol was added at 0, 10, 20, 40, 80, 160, 320 and 640 μg g−1 soil and tylosin and oxytetracycline at 0, 30, 60, 120, 240, 480, 960 and 1920 μg g−1 soil. Lines were drawn using smoothing functions.

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image

Figure 2.  Log–log relationships between relative fungal and bacterial growth resulting from the application of bacterial inhibitors to soils with different substrate amendments. Bronopol (a) resulted in negative log–log regressions in unamended soil (•, R2=0.84, P<0.0001), alfalfa-amended soil (◆, R2=0.92, P<0.0001) and straw-amended soil (▪, R2=0.91, P<0.0001). Tylosin (b) resulted in negative log–log relationships in unamended soil (•, R2=0.73, P<0.0001), alfalfa-amended soil (◆, R2=0.85, P<0.0001) and straw-amended soil (▪, R2=0.81, P<0.0001). Oxytetracycline (c) application resulted in negative log–log regressions in unamended soil (•, R2=0.44, P=0.0069), alfalfa-amended soil (◆, R2=0.79, P<0.0001) and straw-amended soil (▪, R2=0.19, P=0.036).

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To calculate the inhibitor concentration inhibiting 50% of the activity (effective concentration, EC50), a logistic model was fitted to the data, Y=c/[1+e b(X−a)], where Y is the measured level of Leu incorporation, X is the logarithm of the concentration of the inhibitor, a is the value of log EC50, c is the growth rate in the control sample and b is a slope parameter indicating the inhibition rate. Nonlinear regression analysis was performed using statsoft statistica 7.0 statistics software.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial growth was clearly inhibited by the addition of inhibitors. Values of EC50 were between 40 and 80 μg g−1 for bronopol soil, between 570 and 690 μg g−1 for tylosin soil and between 810 and 1420 μg g−1 for oxytetracycline soil (Table 1). Significantly higher EC50 values were observed in the straw-amended than in the unamended soil following bronopol addition, with alfalfa-amended soils having the lowest EC50 values (P<0.05). The trends for the other two inhibitors were similar in that alfalfa-amended soils had lowest EC50 values.

Table 1.   The effective concentrations of bacterial inhibitors inhibiting 50% of bacterial growth (EC50-values) in soil samples with different substrate amendments
 BronopolTylosinOxytetracycline
  1. Mean values and the 95% confidence interval are given within parentheses. Inhibitor concentrations are given in μg g−1 soil.

Unamended506901010
(45–59)(430–1110)(790–1300)
Straw-amended796601420
(70–88)(500–870)(1120–1800)
Alfalfa-amended37570810
(30–46)(410–790)(530–1230)

Bronopol addition inhibited the bacterial growth similarly in all three soils. After adding bronopol concentrations above 10 μg g−1 soil, the bacterial growth was clearly inhibited, reaching a minimum of <1% of the growth in the control samples when 320 μg bronopol g−1 soil or higher was added (Fig. 1a–c). The effect on the fungal growth mirrored the effect on the bacteria, in that at bronopol concentrations higher than 10 μg g−1, the fungal growth rate started to increase, reaching maximum rates of about 25 times that in the controls in the unamended soil (Fig. 1a), about 20 times in the alfalfa-amended soil (Fig. 1b) and about 35 times in the straw-amended soil (Fig. 1c). The respiration showed no clear pattern in any of the three soils, and was largely unaffected by the addition of bronopol.

The bacterial growth rate decreased at about 20 μg g−1 tylosin in the unamended and straw-amended soils (Fig. 1d and f), and at about 50 μg g−1 in the alfalfa-amended soil (Fig. 1e). Bacterial growth was also similar at increasing concentrations in the unamended and straw-amended soils, both treatments resulting in bacterial growth of about 0.3 times that in the controls at the highest tylosin concentration studied. Bacterial growth in the alfalfa-amended soil decreased to about 0.1 times that in the controls at the highest tylosin concentration. Fungal growth in the unamended and alfalfa-amended soil was similar. Starting at tylosin concentrations of 50–100 μg g−1, fungal growth increased gradually, reaching a maximum of about 1.5 times that in the controls in the unamended (Fig. 1d), and about two times that in the controls in the straw-amended soil (Fig. 1f). Fungal growth in the alfalfa-amended soil deviated from that of the controls above 100 μg g−1 tylosin, and increased to a maximum of about four times that in the controls (Fig. 1e). The respiration rate was largely unaffected by tylosin application regardless of the concentration (Fig. 1d–f).

Bacterial growth was inhibited by oxytetracycline in the unamended soil, starting at a concentration of about 100 μg g−1 soil and reaching a minimal bacterial growth of about 0.4 times that in the controls at the maximum concentration added (Fig. 1g). Bacterial growth was retarded at similar concentrations in both the alfalfa- and straw-amended soils (Fig. 1h and i). The effects on fungal growth (Fig. 1g–i) were somewhat more ambiguous than when adding the other two inhibitors. However, at concentrations exceeding 100 μg oxytetracycline g−1 soil, the fungal growth rates in the alfalfa- and straw-amended soils were elevated, gradually reaching a maximal fungal growth three times that in the controls (Fig. 1h and i). In the unamended soil, the fungal growth rate increased to almost twice that in the controls at the highest oxytetracycline concentration. The respiration did not deviate from that in the controls in the unamended or straw-amended soil (Fig. 1g and i), while the alfalfa-amended soil (Fig. 1h) exhibited a minor increase compared with the controls at the highest oxytetracycline concentration.

The relative fungal growth and the relative bacterial growth showed an inverse relationship, indicating that a certain decrease in bacterial growth resulted in a corresponding increase in fungal growth, irrespective of the inhibitor or soil amendment (Fig. 2). Bronopol application resulted in significant negative log–log relationships (Fig. 2a) for unamended soil (R2=0.84, P<0.0001), alfalfa-amended soil (R2=0.92, P<0.0001), and straw-amended soil (R2=0.91, P<0.0001). Tylosin also resulted in negative log–log relationships between bacterial and fungal growth (Fig. 2b) in the unamended (R2=0.73, P<0.0001), alfalfa-amended (R2=0.85, P<0.0001) and straw-amended soils (R2=0.81, P<0.0001). Although oxytetracycline application also inhibited bacterial growth and stimulated fungal growth in all three soils, resulting in negative log–log relationships, they were weaker (Fig. 2c, R2=0.44, P=0.0069 for unamended, R2=0.79, P<0.0001 for alfalfa-amended and R2=0.19, P=0.036 for straw-amended soils).

Estimation of absolute levels of fungal growth in the samples without inhibitors revealed higher levels in the straw- and alfalfa-amended soils, of about 1.6 and 5.2 times the unamended soil, respectively (Table 2). The estimated bacterial growth showed a similar pattern, with absolute levels about 1.6 and 3.3 times those in the unamended soil in the straw- and alfalfa-amended soils, respectively. The respiration increased similarly in the amended soils, to about 2.5 times in the straw-amended soil and about 2.4 times in the alfalfa-amended soil, compared with the unamended soil.

Table 2.   Fungal and bacterial growth and respiration rate in soils with different substrate amendments (mean ± SE)
 Bacterial growth (Leu)Fungal growth (Ac-in-erg)Respiration
  1. Bacterial growth (measured as Leucine incorporation) is given as mol × 10−12 Leu incorporated into bacteria, fungal growth (measured as acetate incorporation into ergosterol) as mol × 10−12 Ac incorporated into ergosterol, and respiration rate as μg C h−1 g−1 soil.

Unamended2.4 ± 0.0681.9 ± 0.100.7 ± 0.004
Straw-amended3.8 ± 0.0742.9 ± 0.241.7 ± 0.018
Alfalfa-amended7.8 ± 0.299.7 ± 0.521.6 ± 0.082

The change resulting from application of bacterial inhibitors, measured as the difference between the average of the two highest concentrations and the average of the zero and lowest bacterial inhibitor concentration, with no effect on the measurements, differed between substrate amendments (Table 3). For each inhibitor applied, the alfalfa-amended soil showed the largest negative changes in bacterial growth (ΔLeu incorporation) and the largest positive changes in fungal growth (ΔAc-in-erg incorporation), while differences in the straw-amended soil were intermediate, and the unamended soil showed the smallest absolute changes in bacterial and fungal growth rates. For instance, bronopol treatment decreased bacterial growth by 2.3, 3.8 and 7.8 mol × 10−12 Leu incorporated, in unamended, straw- and alfalfa-amended soil, respectively, while fungal growth increased correspondingly by 44, 92 and 166 mol × 10−12 Ac-in-erg incorporated. There were only small differences in ΔRespiration, although straw amendment led to negative values (indicating a decrease in respiration rate at the highest bacterial inhibitor concentrations) while both the unamended and alfalfa-amended soils had slightly increased values.

Table 3.   The change in bacterial and fungal growth and respiration rate after adding bacterial inhibitors to soil samples with different substrate amendments
 ΔBacterial growth (ΔLeu)ΔFungal growth (ΔAc-in-erg)ΔRespiration
  1. The means of the control without inhibitor and lowest inhibitor concentration, both of no effect, were subtracted from the means of the two highest inhibitor concentrations. Bacterial growth (measured as Leu incorporation) is given as mol × 10−12 Leu incorporated into bacteria, fungal growth (measured as Ac-in-erg incorporation) as mol × 10−12 Ac incorporated into ergosterol, and respiration rate as μg C h−1 g−1 soil.

Unamended
 Oxytetracycline−1.40.690.026
 Tylosin−1.20.740.37
 Bronopol−2.3440.021
Straw-amended
 Oxytetracycline−1.93.4−0.18
 Tylosin−2.42.1−0.022
 Bronopol−3.892−0.4
Alfalfa-amended
 Oxytetracycline−5.2160.33
 Tylosin−5.3170.023
 Bronopol−7.81660.21

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

All the three inhibitors repressed bacterial growth without negatively affecting the fungi, showing that they were selective in inhibiting bacteria. This study also found clear dose-response effects on bacterial growth, which were modelled well by a logistic equation. Fungal growth increased in response to decreased bacterial growth, the increase corresponding to that of bacterial growth inhibition. Consequently, the results of this study showed a negative relationship between fungal and bacterial growth in the soils studied, indicating that antagonistic effects were exerted by the bacteria on the fungi (Figs 1 and 2).

The negative relationship between fungal and bacterial growth was confirmed when contrasting the effects of the differently amended soils. The unamended soil showed lowest fungal and bacterial growth rates. Both substrates stimulated all microbial growth, but it was hypothesized that the straw would benefit fungal growth more than bacterial, while the alfalfa-amended soil would benefit bacteria more than fungi. This means that the bacteria should have been least established in the unamended soil, intermediately established in the straw-amended soil, and most established in the alfalfa-amended soil, indicating that the fungi should have benefited from inhibited bacterial growth in the same order. The largest absolute effects on fungal and bacterial growth induced by the application of bacterial inhibitors were indeed found in the alfalfa-amended soil; the differences were intermediate in the straw-amended soil and smallest in the unamended soil (Table 3), thus confirming the predictions.

Competition can take place through two main mechanisms. In interference competition one group negatively affects the other through direct inhibition by, for example, exudation of allelochemical substances. In exploitation competition one group negatively affects the other by exploiting a common resource, thus depriving the other group of this resource. The difference between interference and exploitation competition would, however, be indistinguishable if the total mineralization was unaffected by the mode of competition. If, however, interference competition resulted in one of the decomposer groups denying the other's access to a resource without fully exploiting it themselves, the outcome would be a reduction in gross decomposition. The mode of interaction thus has the potential to influence the ecosystem functioning profoundly. Although this experiment was not primarily designed to differentiate between these two mechanisms, there was clear evidence of exploitation competition. The changes in fungal (ΔAc-in-erg) and bacterial growth (ΔLeu, Table 3) clearly showed that a new resource (straw or alfalfa) was readily exploited and used by the fungi (positive ΔAc-in-erg with substrate) when an inhibitor reduced its colonization by bacteria. In addition to making a resource available to the fungi (increasing growth as shown in Fig. 1 and positive ΔAc-in-erg with substrate in Table 3), the inhibitor also reduced the bacterial utilization of the resource (seen as decreasing growth in Fig. 1 and negative ΔLeu with substrate in Table 3). The bacteria thus out-competed the fungi for the availability of resources when no inhibitor was added, to exploit them for their own growth. Furthermore, no indication that the interaction between the decomposers was different in the unamended soil (Figs 1 and 2, negative ΔLeu and correspondingly positive ΔAc-in-erg in Table 3) was noticed. Thus, when inhibitors decreased the bacterial utilization of resources, this led to corresponding increases in fungal growth, both in the colonization of new substrates (straw- and alfalfa-amended soils) and in unamended soil. The most parsimonious explanation of the results is therefore that exploitation competition dominated the interaction between fungi and bacteria in all the soils studied, although this study is not able to exclude interference competition.

Although few studies have addressed the fungal and bacterial interaction during decomposition in soil, some recent studies have indicated fungal inhibition by the presence of bacteria (Thiele-Bruhn & Beck, 2005; Feeney et al., 2006). In both studies, the addition of bacterial inhibitors (oxytetracyline and bronopol) resulted in an increase in fungal biomass (ergosterol). However, the extent of bacterial inhibition was not measured in either study. The increased fungal growth, and decreased bacterial growth, following application of heavy metals (Rajapaksha et al., 2004; Tobor-Kapłon et al., 2005) or NaCl (Tobor-Kapłon et al., 2005), which was interpreted as the fungi being more resistant to metal and salt stress, might also, at least partly, be an effect of competitive release.

Antagonism between bacteria and single strains of fungi on decomposing plant litter has also been shown previously in limnic microcosms (Mille-Lindblom & Tranvik, 2003). Contrary to the findings of the present study, the authors claimed that the detected antagonism was most probably not due to exploitation competition, because there was a negative correlation between fungal and bacterial biomass accumulation before the resources appeared to be exhausted. Similar conclusions were also reached in other studies of aquatic microcosm experiments (Wohl & McArthur, 2001; Mille-Lindblom et al., 2006). In addition, it has been established that bacteria can produce antifungal substances, and this has been proposed as one explanation of fungistasis in soil (Lockwood, 1977; Zou et al., 2007). Hence, the mode of interaction between the decomposer groups may vary, calling for more research aimed at exploring this.

Inhibitor addition had only minor effects on soil respiration, despite the considerable effects on fungal and bacterial growth. The straw- and alfalfa-amended soils respired more than the unamended soil, but the application of bacterial inhibitors did not affect the total respiration. One explanation of this could of course be that any decline in bacterial contribution to respiration was compensated for by the immediate increase in fungal contribution, further indicating that exploitation competition was indeed the major mechanism of interaction. Discrepancies between the combined bacterial and fungal growth compared with respiration have, however, been reported previously (Rousk & Bååth, 2007b), suggesting potential uncoupling between microbial growth and respiration. This could be due, for example, to different growth efficiencies (Six et al., 2006). To determine growth efficiencies, there is often a need for reliable conversion factors to calculate bacterial and fungal production from growth rate data. The conversion factor determined to estimate the production of fungal biomass from Ac-in-erg measurements in unamended soil (Rousk & Bååth, 2007a) was a step in achieving this, although more work is still needed to determine conversion factors in different soils, and to determine conversion factors for bacterial growth in soil. Estimation of bacterial and fungal biomass production would enable comparisons of the relative contributions of fungi and bacteria to decomposition, finally revealing how the composition of the soil microbial community affects the carbon budget of different ecosystems.

Using the Ac-in-erg and Leu incorporation techniques this study was able to estimate fungal and bacterial responses to bacterial inhibitors with high sensitivity. It has often been claimed that to study the effects of inhibiting substances such as antibiotics in soil one has to add a substrate in order to observe an effect. Anderson & Domsch (1973), for example, found that when using selective inhibition to measure the respiration contribution of fungi and bacteria, a substantial addition of glucose (1 mg g−1 soil) was required to measure a negative effect of the added inhibitor. Furthermore, Thiele-Bruhn & Beck (2005) concluded that activation of microbial activity by adding substrate [in their case 2 mg C g−1 of milled maize and glucose (9 : 1)] was required to resolve any influence of the bacterial inhibitors oxytetracycline and sulfapyridine, using respiration and dehydrogenase activity to assess the activity. Feeney et al. (2006) found that adding bronopol to soil had no effect in unamended soil, and that increased fungal biomass was only detected after adding straw to the soil. In contrast, the present study showed clear suppression of bacterial growth by both oxytetracycline and bronopol without having to add additional nutrients, emphasizing the difference in sensitivity in direct measurements of growth compared with biomass or gross measurements of activity such as respiration.

In conclusion, this study has demonstrated the antagonistic effect of bacteria on fungal growth in a natural soil. This was achieved by measuring the direct growth of the decomposer groups using the Leu and Ac-in-erg incorporation techniques, which allowed the study of the decomposer interaction in soil without the requirement of substrate addition. Furthermore, the relationship between bacteria and fungi did not differ during the colonization of new substrate compared with native soil conditions, suggesting that the interaction was primarily characterized by exploitation competition. Finally, there is a call for further studies to investigate if the interaction between the soil decomposers is reciprocal, i.e. how the bacterial growth will react to a selective suppression of fungi.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This study was supported by grants from the Swedish Research Council and FORMAS (the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning) to E.B.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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