Nutrient economy of red pine is affected by interactions between Pisolithus tinctorius and other forest-floor microbes

Authors


Author for correspondence: Roger T. Koide Tel: +1 814 863 0710 Fax: +1 814 863 6139 Email:rxk13@psu.edu

Summary

  • • The influence of interactions between the mycorrhizal fungus, Pisolithus tinctorius , and saprotrophic organisms on nutrient transfer to host red pine ( Pinus resinosa ) seedlings is presented here.
  • Red pine seedlings were grown axenically, and with P. tinctorius and forest-floor microbes (both individually and in combination), in two experiments varying in nitrogen availability. Root and shoot growth, as well as tissue nitrogen and phosphorus content, were analyzed after harvesting.
  • At low nitrogen availability, forest-floor microbes, but not P. tinctorius , significantly reduced seedling nitrogen content. Moreover, P. tinctorius did not ameliorate this negative effect. However, seedling phosphorus content increased with forest-floor microbes and P. tinctorius individually, and these combined to give an additive effect. Forest-floor microbes and P. tinctorius , individually, significantly increased seedling nitrogen and phosphorus contents at high nitrogen availability, interacting to give additive and synergistic effects on nitrogen and phosphorus content, respectively.
  • The effect of ectomycorrhizal fungi on host-plant nutrition might depend strongly on the nutrient status of coexisting saprotrophic soil microbes. When nutrients are not limiting, their beneficial effects on host nutrition may be additive or synergistic.

Introduction

The roots of ectomycorrhizal trees coexist in the soil with many kinds of organisms including ectomycorrhizal fungi and saprotrophic microbes. Both groups of microorganisms are capable of independently influencing plant nutrient acquisition. When litter falls to the forest floor, the N it contains may be primarily organic. Saprotrophic soil microbes secrete enzymes that hydrolyze complex N-containing organic compounds into simpler organic compounds, which may be absorbed and eventually converted into mineral forms that are available for plant absorption (Myrold, 1999). Some ectomycorrhizal fungi may also have a limited capacity to hydrolyze simple organic compounds, such as protein (Smith & Read, 1997). The majority of organic N in forest soils, however, may be largely unavailable to ectomycorrhizal fungi because it is complexed by polyphenolic compounds (Bending & Read, 1996b). Thus, the largest effect of ectomycorrhizal fungi on N acquisition may be due to the additional absorptive surface area they provide. While both ectomycorrhizal fungi and saprotrophic soil microbes clearly play important roles in the nutrition of plants, we know very little of how these two guilds of microorganisms interact to influence plant nutrition.

Interactions between saprotrophs and mycorrhizal fungi may render mycorrhizal fungi either more or less effective in supplying their hosts with nutrients. For example, Lindahl et al. (1999) found that substantial P could be directly transferred from a wood-rotting fungus to a mycorrhizal fungus, possibly via lysis of the hyphae of the wood-rotter. Moreover, because mycorrhizal fungi appear to secrete fewer hydrolytic enzymes than some saprotrophs (Bending & Read, 1996a,b), mycorrhizal fungi may gain access to nutrients in recalcitrant forms of litter if they live in close association with saprotrophs. Through the action of extracellular enzymes, saprotrophic microbes are capable of hydrolysing complex organic compounds into less complex, absorbable organic forms. Mycorrhizal fungi may have access to these. This has been called ‘physiological complementarity’ (Cooke & Whipps, 1993). Mycorrhizal fungi may also have ready access to nutrients mineralized by saprotrophs. In either case, the activity of saprotrophic microbes could significantly increase nutrient availability to mycorrhizal fungi (Colpaert & van Tichelen, 1996), and so increase their capacity to provide nutrients to their hosts.

When the carbon : nutrient ratios of forest litter are sufficiently high, saprotrophic microbes may immobilize rather than mineralize nutrients. Under such conditions, mycorrhizal fungi may be of no particular nutritional benefit to the host plant unless they compete successfully against the saprotrophic microbes for available nutrients. Even if mycorrhizal fungi can obtain nutrients in direct competition with saprotrophs, there may still be a low probability that nutrients will be transferred from mycorrhizal fungi to their hosts. Like saprotrophic microbes, mycorrhizal fungi may retain (immobilize) a nutrient if limited by its scarcity. Whether ectomycorrhizal fungi immobilize nutrients to the same extent as saprotrophic microbes when nutrients are limiting may have important consequences for plant nutrition.

We have performed two experiments in which red pine seedlings were grown axenically, with the ectomycorrhizal fungus Pisolithus tinctorius, with a mixture of other forest floor microbes, or with both. In the first of two experiments the sole source of N was a small amount of sterile forest floor F-layer material. All other nutrients were added as salts. In the second experiment the sole source of P was sterile F-layer material. All other nutrients, including N, were added as salts. We tested the hypothesis that the effect of P. tinctorius on seedling N or P status depends on the N or P status of the other soil microbes.

Other researchers have examined interactions between mycorrhizal fungi and individual isolates of saprotrophic microorganisms (Bowen & Theodorou, 1973, 1979; Shaw et al., 1995; Colpaert & van Laere, 1996; Colpaert & van Tichelen, 1996; Lindahl et al., 1999). A careful examination of specific microbial interactions has been possible with that approach. The forest floor, however, contains a complex, species-rich microbial community. We therefore have taken another approach in our experiments by introducing a mixture of forest floor microorganisms via nonsterile soil organic matter.

Materials and Methods

Host plant, mycorrhizal fungus, and forest floor microbes

Seeds of red pine (Pinus resinosa Ait.) were obtained from F.W. Schumacher Co. (Sandwich, MA, USA). They were surface sterilized for 30 min in 30% H2O2. Following a rinse in sterile distilled water, they were plated out on a complete nutrient medium similar to modified Melin-Norkrans containing Phytagel (Sigma Chemical Co., St. Louis, MO, USA) as the gelling agent. The sucrose concentration was 10 g l−1, the N concentration was 4.7 mM as NH4Cl, and the P concentration was 3.7 mM as KH2PO4. The concentrations of other nutrients are given in Koide et al. (1998). Following germination, seedlings were sterilely transferred into the growth tubes for expts 1 and 2 (see below). Dr Michael Kiernan (Plant Health Care, Inc., Pittsburgh, PA, USA) provided the original culture of Pisolithus tinctorius (Pers) Coker & Couch. We subsequently produced inoculum of P. tinctorius for expts 1 and 2 in Erlenmeyer flasks containing a mixture of vermiculite, perlite and peat in a volume ratio of 6 : 3 : 1, moistened with a complete nutrient solution (see above). The flasks of inoculum were incubated at 27°C in the dark for 8 wk before use. We chose P. tinctorius because it can colonize red pine (Grenville et al., 1985), it produces a rather distinctive morphotype, but it does not naturally occur in the 60-yr-old plantation of red pine located in Center Co., PA, USA, from which we obtained the F-layer material used to introduce forest floor microbes into our microcosms (to do this, 1 ml of nonsterile, air-dried, F-layer material ground to pass through a #20 mesh was added to appropriate growth tubes, see below). Thus, we could more readily identify colonization by mycorrhizal fungi other than our test mycorrhizal fungus.

Experiment 1 – low nitrogen availability

There were 18 replicate growth tubes for each of the four treatment combinations, which included controls (CON, no microorganisms), P. tinctorius only (PT), forest floor microbes only (SAP), and P. tinctorius plus forest floor microbes (PT + SAP). The 125 ml glass growth tubes (40 × 130 mm) were supplied by Corning Glass Works (Corning, NY, USA). All tubes were partially filled (see below) with a 6 : 3 : 1 mixture of vermiculite, perlite, and pine forest F-layer material collected 30 Jul 1997 for experiment 1. The F-layer material when dry has a density of approx. 0.21 g ml−1 and contains a total of approx. 660 µg P g−1, 50 µg P g−1 of which is inorganic P extractable by water. It also contains a total of approx. 10.5 mg N g−1, approx. 0.1 mg N g−1 of which is NH4+ extractable by water. It contains essentially no nitrate.

The PT and PT + SAP tubes were loaded with 52 ml of the 6 : 3 : 1 mixture of vermiculite, perlite, and F-layer material, moistened with 26 ml of nutrient solution (see above), but lacking sucrose and NH4Cl. The CON and SAP tubes were loaded with 65 ml of the vermiculite, perlite and forest floor mixture moistened with 26 ml of the same nutrient solution, plus 6.5 ml of complete nutrient solution containing carbohydrate and N to compensate for the 13 ml of PT inoculum added to PT and PT + SAP tubes following autoclaving (see below). The 13 ml of P. tinctorius inoculum contained 6.5 ml of complete nutrient solution. Prior to autoclaving, an additional 1.0 ml of ground F-layer material was added to each PT and CON tube to compensate for the F-layer material added to the SAP and PT + SAP tubes following autoclaving. All tubes were then autoclaved at 121°C for 1 h on 18 Sep 1997.

All procedures following autoclaving were performed in a sterile, laminar flow hood using standard sterile techniques. Following autoclaving, 13 ml of inoculum of P. tinctorius were added to PT and PT + SAP tubes, bringing the total volume of medium to 65 ml in all tubes. The P. tinctorius inoculum had previously been plated out in Petri dishes to examine for contaminant microorganisms. There were none. Two sterile, 7-d-old red pine seedlings (see above) were placed into each growth tube on 21 Sep 1997. On 5 Oct 1997, 1 ml of nonsterile, air-dried, ground F-layer material was added to each SAP and PT + SAP tube to introduce forest floor microbes. Each tube was capped with a clear plastic lid that permitted gas exchange but prevented microbial contamination.

The growth tubes were placed in a growth chamber with a 16-h day, 8-h night cycle. There were 400–500 µmole m−2 s−1 photosynthetically active radiation during the day. The relative humidity was approximately 65–75% day and night. Air temperatures were 26 and 18°C during the day and night, respectively.

During the course of the experiment, all tubes received an additional 8 ml sterile distilled water to replace the loss by evaporation. Tubes were observed daily from the outside of the glass with a dissecting microscope to check for contaminating microorganisms (in CON and PT tubes), and mycorrhization (in PT and PT + SAP tubes). On 17 Jan 1998 (118 d following transplanting), seedlings were harvested. A small amount of medium from PT and CON tubes were plated on gelled complete medium, and tubes harboring contaminating microorganisms were excluded from further analysis. Roots and shoots were weighed separately, and N (by Nessler method, Jensen, 1962) and P (by molybdo-phosphate method, Watanabe & Olsen, 1965) concentrations were assessed following digestion at 400°C in a mixture of concentrated H2SO4 and 30% H2O2. Two-factor ANOVA were performed for both experiments using the Statgraphics programs (STSC, 1991). The factors were P. tinctorius (+ and −) and other forest floor microbes (+ and −).

Experiment 2 – high nitrogen availability

Experiment 2 was performed in the same manner as experiment 1. The major difference was the nutrient solution used, which lacked KH2PO4 and sucrose, but contained NH4Cl (4.7 mM N). The following are other differences from experiment 1. The F-layer material was collected 10 Aug 1998. The tubes were autoclaved on 27 Oct 1998. Inoculum of P. tinctorius was added to PT and PT + SAP tubes on 30 Oct 1998. Sterile seedlings were placed into each growth tube on 30 Oct 1998. Forest floor microorganisms were introduced to each SAP and PT + SAP tube on 13 November 1998. During the course of the experiment, all tubes received an additional 10 ml sterile distilled water to replace the water lost by evaporation. Seedlings were harvested on 18 Feb 1999 (111 d following transplanting).

Results

Experiment 1 – low nitrogen availability

Mycorrhizal roots were first observed 30 d after transplanting on PT + SAP plants, and 50 d after transplanting on PT plants. No root tips were examined microscopically, or by using molecular techniques, but it was apparent from careful visual inspection that only a single ectomycorrhizal morphotype with golden-brown hyphae was present on either PT or PT + SAP plants. Moreover, neither CON nor SAP plants possessed observable mycorrhizal root tips. We assumed therefore that essentially no viable ectomycorrhizal fungi were introduced by the 1 ml of nonsterile, air-dried, ground F-layer material added to SAP and PT + SAP plants.

The positive effect of P. tinctorius on short root number was reduced in the presence of forest floor microbes as indicated by the significant interaction (Table 1). Either an unbranched short root with one tip or a bifurcated short root with two tips counted as a single short root. There were significantly more ectomycorrhizal short roots for PT + SAP plants than for PT plants (Table 1). There was no significant difference, however, in the proportion of short roots that were mycorrhizal in PT and PT + SAP plants (25% and 29%, respectively). Thus, while forest floor microbes stimulated short root development, they did not apparently increase the infectiveness of P. tinctorius.

Table 1.  Mean (SE) shoot and root system weights, d. wt partitioning, root length, number and mycorrhizal roots of red pine seedlings grown in experiment 1
 Shoot d. wt (mg)Root d. wt (mg)Root : shootRoot length (cm)Total short rootsMycorrhizal short rootsTotal root tipsMycorrhizal root tips
  1. n  = 10, 9, 18, 15 for CON, PT, SAP and PT + SAP, respectively. The number of mycorrhizal short roots and the number of mycorrhizal root tips were significantly increased by the presence of forest floor microbes (SAP) according to the ANOVA and as indicated by different letters.

CON34.8 (2.4) 8.0 (0.8)0.232 (0.025)14.5 (1.0) 44 (4)  44 (4) 
PT43.0 (2.7)14.8 (1.2)0.348 (0.025)25.9 (2.3) 95 (7)24 (4)b107 (9)35 (7)b
SAP42.6 (2.2)25.7 (1.1)0.619 (0.035)56.8 (6.3)183 (13) 184 (13) 
PT + SAP43.6 (1.4)26.4 (1.3)0.614 (0.035)56.5 (3.8)189 (9)53 (5)a211 (9)72 (8)a
ANOVA        
PT0.01620.00390.00580.00720.0001 0.0003 
SAP0.03740.00010.00010.00010.0001 0.0001 
Interaction0.08770.01910.00370.05940.0001 0.1307 

The total number of root tips (a bifurcated short root counted as two tips) was significantly increased by both P. tinctorius and forest floor microbes (Table 1). The PT + SAP plants had significantly more ectomycorrhizal root tips than the PT plants (Table 1). Nevertheless, forest floor microbes did not significantly affect the proportion of tips that were mycorrhizal (32% and 34% for PT and PT + SAP plants, respectively).

Shoot P content was significantly (P = 0.0134) increased by P. tinctorius, and significantly (P = 0.0022) increased by forest floor microbes (Fig. 1a). Overall, P. tinctorius increased shoot P content by 30 µg P (a 22% increase), and forest floor microbes increased shoot P content by 37 µg P (a 27% increase). There was no significant interaction, indicating that the effects of P. tinctorius and forest floor microbes were essentially additive.

Figure 1.

(a) Mean (± 1 SE) shoot and root phosphorus contents for red pine grown in the four treatment combinations in experiment 1. (b) Mean (± 1 SE) shoot and root nitrogen contents for red pine grown in the four treatment combinations in experiment 1. n  = 10, 9, 18, 15 for CON (control), PT ( Pisolithus tinctorius only), SAP (forest floor microbes only) and PT + SAP ( P. tinctorius plus forest floor microbes), respectively. The dotted lines occur at the values for the CON treatment for ease of comparison.

It was not possible to distinguish between P contained in root tissue and P in fungal mantle in the PT and PT + SAP treatments. Thus, root plus mantle P content was assessed in PT and PT + SAP treatments, and root P content was assessed in CON and SAP treatments. Both P. tinctorius and forest floor microbes had positive effects on ‘root’ P content, but there was a significant interaction (P = 0.0184) such that the positive effect of one treatment was reduced by the other. In other words, the effects of P. tinctorius and forest floor microbes were less than additive.

Shoot P concentrations were relatively high in all treatments (Table 2). Shoot P concentration was not significantly affected by P. tinctorius, but was significantly increased by forest floor microbes. There was no significant interaction. ‘Root’ P concentrations were also relatively high in all treatments. P. tinctorius significantly increased ‘root’ P concentration. By contrast, forest floor microbes significantly decreased ‘root’ P concentration. There was no significant interaction.

Table 2.  Mean (SE) shoot and root system N and P concentrations (mg g −1 d. wt) of red pine seedlings grown in experiment 1
 Shoot PRoot PShoot NRoot N
  1. n  = 10, 9, 18, 15 for CON, PT, SAP and PT + SAP, respectively.

CON3.39 (0.11)3.27 (0.11)16.4 (0.7)18.4 (1.1)
PT3.56 (0.05)4.13 (0.25)11.2 (0.6)12.5 (0.5)
SAP3.78 (0.16)2.98 (0.14) 8.2 (0.4) 7.3 (0.3)
PT + SAP4.22 (0.24)3.40 (0.10) 7.5 (0.9) 7.4 (0.2)
ANOVA    
PT0.11070.00010.00020.0001
SAP0.00770.00150.00010.0001
Interaction0.47760.15290.00280.0001

Shoot N content (Fig. 1b) showed the opposite pattern to shoot P content. Overall, forest floor microbes significantly (P = 0.0001) reduced shoot N content by 185 µg N, a 35% reduction. P. tinctorius did not significantly (P = 0.1657) affect shoot N content. There was no significant interaction. ‘Root’ N content was significantly increased by P. tinctorius (P = 0.0149) and by forest floor microbes (P = 0.0137). Overall, P. tinctorius increased ‘root’ N content by 26 µg N (a 16% increase). Overall, forest floor microbes also increased ‘root’ N content by 26 µg N. There was no interaction, indicating that the effects of P. tinctorius and forest floor microbes were essentially additive.

There was a significant interaction between P. tinctorius and forest floor microbes on shoot and ‘root’ N concentrations (Table 2). In both cases, the negative effect of one treatment was reduced by the other.

Both P. tinctorius and forest floor microbes significantly increased shoot dry weight (Table 1), but there was a marginally significant interaction such that the beneficial effect of one treatment was reduced by the other. In other words, the effects were less than additive. The same pattern held for root d. wt and root length. There was large variation in root : shoot ratio among treatment combinations. There was also a significant interaction for root : shoot ratio such that the positive effect of one treatment was reduced by the other. Root : shoot ratio was significantly (P < 0.0001) correlated with shoot N concentration (r2 = 0.508). Root : shoot ratio was not significantly (P = 0.185) correlated with shoot P concentration.

Experiment 2 – high nitrogen availability

Mycorrhizal roots were first observed 32 d after transplanting on PT + SAP plants, and 46 d after transplanting on PT plants. As in experiment 1, no root tips were examined microscopically, or by molecular techniques, but it was apparent from visual inspection that only the P. tinctorius morphotype appeared on either PT or PT + SAP plants. Moreover, neither CON nor SAP plants possessed mycorrhizal root tips. We assumed, as for experiment 1, that essentially no viable ectomycorrhizal fungi were introduced by the 1 ml of nonsterile, air-dried, ground F-layer material added to SAP and PT + SAP plants.

The positive effect of P. tinctorius on short root number was reduced in the presence of forest floor microbes as indicated by the significant interaction (Table 3). There was no significant difference in the number of ectomycorrhizal short roots of PT + SAP and PT plants (Table 3), and there was no significant difference in the proportion of short roots that were mycorrhizal in PT and PT + SAP plants (both approx. 50%). Thus, as in experiment 1, while forest floor microbes stimulated short root development, they did not apparently increase the infectiveness of P. tinctorius.

Table 3.  Mean (SE) shoot and root system weights, d. wt partitioning, root length, number and mycorrhizal roots of red pine seedlings grown in experiment 2
 Shoot d. wt (mg)Root d. wt (mg)Root : shootRoot length (cm)Total short rootsMycorrhizal short rootsTotal root tipsMycorrhizal root tips
  1. n  = 10, 11, 16, 10 for CON, PT, SAP and PT + SAP, respectively. The number of mycorrhizal roots was significantly different according to the analysis of variance as indicated by different letters.

CON37.2 (2.5)13.7 (1.4)0.362 (0.031)47.1 (4.2) 21 (2)  21 (2) 
PT45.0 (1.5)20.1 (0.6)0.451 (0.020)62.2 (4.9)123 (9)61.5 (7.0)156 (11)94.2 (10.1)
SAP51.3 (3.5)19.8 (1.4)0.399 (0.024)71.3 (5.5)138 (12) 138 (12) 
PT + SAP69.3 (7.9)25.1 (1.6)0.380 (0.026)88.9 (5.6)163 (13)78.7 (8.1)197 (13)112.8 (9.3)
ANOVA        
PT0.00520.00010.19160.00450.0001 0.0001 
SAP0.00010.00020.52910.00010.0001 0.0001 
Interaction0.25270.69820.04490.82120.0001 0.0001 

The positive effect of P. tinctorius on the total number of root tips was reduced in the presence of other forest floor microbes (SAP) as indicated by the significant interaction (Table 3). The PT and PT + SAP plants did not differ significantly in the number of ectomycorrhizal root tips, and they did not differ significantly in the proportion of root tips that were mycorrhizal (44% and 45% for PT and PT + SAP, respectively).

Both P. tinctorius and forest floor microbes increased shoot P content (Fig. 2a), but there was a significant (P = 0.0279) interaction; the effects of P. tinctorius and forest floor microbes were synergistic. P. tinctorius alone increased shoot P content by 71.2 µg P (an increase of 186%). Forest floor microbes alone increased shoot P content by 15.8 µg P (an increase of 41%). Forest floor microbes and P. tinctorius together, however, resulted in an increase of 126.9 µg P (an increase of 331%).

Figure 2.

(a) Mean (± 1 SE) shoot and root phosphorus contents for red pine grown in the four treatment combinations in experiment 2. (b) Mean (± 1 SE) shoot and root nitrogen contents for red pine grown in the four treatment combinations in experiment 2. n  = 10, 11, 16, 10 for CON (control), PT ( Pisolithus tinctorius only), SAP (forest floor microbes only) and PT + SAP ( P. tinctorius plus forest floor microbes), respectively. The dotted lines occur at the values for the CON treatment for ease of comparison.

As in experiment 1, it was not possible to distinguish between P contained in root tissue and P in fungal mantle in the PT and PT + SAP treatments. P. tinctorius significantly (P = 0.0001) increased ‘root’ P content by 33.7 µg P (a 174% increase). Forest floor microbes significantly (P = 0.0006) increased ‘root’ P content by 9.7 µg P (a 31% increase). There was no significant interaction (P = 0.6509), indicating that the effects of P. tinctorius and forest floor microbes were essentially additive.

Shoot and ‘root’ P concentrations were significantly increased by P. tinctorius (Table 4). Forest floor microbes did not significantly affect shoot or ‘root’ P concentrations, and the interaction was not significant for either shoot or ‘root’ P concentration.

Table 4.  Mean (SE) shoot and root system N and P concentrations (mg g −1 d. wt) of red pine seedlings grown in experiment 2
 Shoot PRoot PShoot NRoot N
  1. n  = 10, 11, 16, 10 for CON, PT, SAP and PT + SAP, respectively.

CON1.04 (0.04)1.09 (0.06)14.5 (0.6)11.3 (0.6)
PT2.43 (0.11)2.37 (0.06)15.8 (0.4)10.4 (0.5)
SAP1.08 (0.08)1.20 (0.04)13.8 (0.3)11.4 (0.4)
PT + SAP2.40 (0.09)2.32 (0.07)15.7 (0.5) 8.9 (0.3)
ANOVA    
PT0.00010.00010.00100.0006
SAP0.94240.62100.41030.1376
Interaction0.72970.14510.55980.1139

Overall, P. tinctorius significantly (P = 0.0001) increased shoot N content by 284 µg N (a 46% increase, Fig. 2b). Overall, forest floor microbes significantly (P = 0.0001) increased shoot N content by 276 µg N (a 45% increase). There was no significant interaction (P = 0.1327), indicating that the effects of P. tinctorius and forest floor microbes were essentially additive. Forest floor microbes increased ‘root’ N content by 39 µg N (a 17% increase, P = 0.0296). P. tinctorius did not have a significant overall effect on ‘root’ N content (P = 0.1824). There was no significant interaction (P = 0.1546) for ‘root’ N content, indicating that the effects of P. tinctorius and forest floor microbes were essentially additive.

The shoot N concentration was rather uniform. Only P. tinctorius significantly increased it (Table 4). Forest floor microbes did not significantly affect shoot N concentration, and there was no significant interaction. P. tinctorius significantly decreased ‘root’ N concentration. Forest floor microbes did not significantly affect ‘root’ N concentration, and there was no significant interaction.

Both P. tinctorius and forest floor microbes significantly increased shoot and root d. wt, and root length (Table 3). There were no significant interactions between P. tinctorius and forest floor microbes for each of these variables, indicating that the effects of P. tinctorius and forest floor microbes were essentially additive. For root : shoot ratio, there was a significant interaction between P. tinctorius and forest floor microbes such that the positive effect of one treatment was reduced by the other. Thus, the effects of P. tinctorius and forest floor microbes were less than additive. In contrast with experiment 1, the root : shoot ratio in experiment 2 was not significantly correlated with shoot N concentration (P = 0.359) or shoot P concentration (P = 0.968).

Discussion

The N availability was lower (and thus the C : N ratio was higher) in experiment 1 than in experiment 2. The forest floor microbes in experiment 1 significantly decreased seedling N content and N concentration, which is consistent with net microbial immobilization of N. P. tinctorius was unable to ameliorate the negative effect of the other forest floor microbes on seedling N content. Under N-limiting conditions therefore P. tinctorius may be a poor competitor for N against other forest floor microbes. However, even in their absence, P. tinctorius was unable to increase seedling N content. Thus, even if P. tinctorius could absorb some N from the medium, it may fail to transfer significant quantities of N to its host if it remains N-deficient. Under certain conditions, some mycorrhizal fungi have been shown to have a comparatively low propensity to transfer N to their hosts (Abuzinadah et al., 1986; Abuzinadah & Read, 1989).

As already discussed, P. tinctorius did not increase seedling N content in experiment 1. However, in contrast with the other forest floor microbes, P. tinctorius did not significantly reduce seedling N content. Thus, its propensity for immobilizing N appeared to be less than for the other forest floor microbes. One possible explanation for this difference between P. tinctorius and the other forest floor microbes concerns variation in C : N ratio of available materials. Because mycorrhizal fungi are primarily biotrophic for C but not for N (Lindeberg, 1986; Smith & Read, 1997), if the host reduced the rate of C transfer to the mycorrhizal fungus, either chronically or temporarily, the C : N for the mycorrhizal fungus would be reduced. This would reduce the propensity for N immobilization. By contrast, the C : N ratio experienced by saprotrophic microbes is fixed. It is determined by the C : N ratio of the soil substrates. Further investigation of the consequences of this difference appears to be warranted.

In contrast with experiment 1, in experiment 2 (in which we added additional N in the form of NH4Cl) forest floor microbes resulted in an increased seedling N content. This increase in the availability of N to the plant may have been caused by net mineralization of N from the F-layer material, which was possible because more than enough N was available to meet the needs of the saprotrophs. P. tinctorius was by itself also able to increase seedling N content, possibly because of its ability to hydrolyze simple organic N compounds (Abuzinadah & Read, 1986a), which were unavailable to the seedling (Abuzinadah & Read, 1986b), or because of the simple addition by the fungus to the absorptive surface area. The positive effects of P. tinctorius and forest floor microbes on seedling N content were additive. This additivity may have occured in one of two ways. First, if the saprotrophic microbes and P. tinctorius occured in close proximity, P. tinctorius could have absorbed simple organic N compounds released from the organic matter by the hydrolytic enzymes secreted by the saprotrophs. This has been referred to as physiological complementarity (Cooke & Whipps, 1993). Alternatively, P. tinctorius could have absorbed N mineralized by the saprotrophic microbes.

The additive effects of P. tinctorius and forest floor microbes could also have occured because the forest floor microbes caused more rapid mycorrhiza formation, possibly by increasing the number of short roots available for colonization. Some ‘mycorrhization helper bacteria’ increase the likelihood that a mycorrhizal fungus will come into contact with a root by stimulating lateral root formation via the production of IAA (Garbaye, 1994). Positive effects of saprotrophic microbes on mycorrhizal colonization are not universal, however. Some saprotrophs may have neutral or negative effects on growth of mycorrhizal hyphae and on mycorrhizal colonization (Bowen & Theodorou, 1973, 1979; Shaw et al., 1995).

Less P was available in experiment 2 than in experiment 1, but sufficient P was apparently available in both experiments to prevent P immobilization by forest floor microbes. As they did for N, forest floor microbes may have increased host-available P by mineralizing organic P in the F-layer material. Forest floor microbes also increased root length, which could have increased the uptake of soluble P. P. tinctorius alone also increased seedling P content. Like saprotrophic fungi, many mycorrhizal fungi possess extracellular phosphatase activity (Alexander & Hardy, 1981; Antibus et al., 1992) and can thus mineralize P from organic sources. Moreover, hyphae of P. tinctorius may also significantly increase absorptive surface area, making the capture of poorly diffusible ions such as phosphate more likely.

In experiment 1 the effects of P. tinctorius and forest floor microbes on seedling P content were additive. In experiment 2, their effects on seedling P content were synergistic. Again, earlier mycorrhiza formation may have contributed to this. In addition, phosphate liberated by forest floor microbes may have been only poorly available to a nonmycorrhizal host plant due to its low rate of diffusion, while the hyphae of P. tinctorius provided a large absorptive surface area and thus may have been better than roots alone in capturing the phosphate released by saprotrophs.

Our results clearly show that P. tinctorius may not always increase nutrient uptake. Under conditions likely to promote microbial immobilization of nutrients, mycorrhizal colonization was of little help in obtaining nutrients from the soil. At higher nutrient availabilities, interactions between mycorrhizal fungi and other coexisting forest floor microbes were additive or synergistic in nature. Others have shown that interactions between mycorrhizal fungi and other soil biota exist (Bowen & Theodorou, 1973, 1979; Colpaert & van Laere, 1996; Colpaert & van Tichelen, 1996; Lindahl et al., 1999), or have suggested that such interactions exist (Bending & Read, 1996a,b). Taken together, these results indicate that the ectomycorrhizal symbiosis needs to be appreciated in the context of a complex soil community.

In both experiments, P. tinctorius and forest floor microbes increased seedling growth. The mechanism for this is unclear, but apparently it was not related to the N or P status of seedlings. CON seedlings had the highest N concentrations but the smallest weights in experiment 1. In experiment 2, CON seedlings had higher shoot N concentrations but lower shoot weights than those in the SAP treatment. In experiment 2, the shoot P concentration in the PT treatment was equivalent to that in the PT + SAP treatment, but growth was significantly greater in the PT + SAP treatment. Increased CO2 concentrations in the growth tubes as a consequence of microbial respiration may have contributed to increased seedling growth in the treatments with microorganisms. Others have shown that increased CO2 concentrations can lead to greater growth in some cases (Walker et al., 1995; Cotrufo & Gorissen, 1997; Runion, 1999; Gorissen & Kuyper, 2000), but not in all (Hättenschwiler & Körner, 1998). P. tinctorius and forest floor microbes may also produce compounds that stimulate plant growth including auxins, cytokinins and gibberellins (Gruen, 1959; Ulrich, 1960; Miller, 1967; Laloue & Hall, 1973; Crafts & Miller, 1974; Ng et al., 1982; Rudawska, 1983; Strzelczyk et al., 1986; Vancura, 1986). Auxins, for example, are known to increase root production (Garbaye, 1994), and both gibberellins and auxins can increase shoot growth (Salisbury & Ross, 1992).

Root : shoot ratios were influenced by treatment in experiment 1, but not in experiment 2. Because seedling N uptake was reduced by the addition of forest floor microbes in experiment 1, and because N deficiency may affect patterns of allocation (Bloom et al., 1985), we examined the relationship between shoot N concentration and root: shoot ratio. As expected, there was a significant linear inverse relationship in experiment 1. In contrast, the seedlings in experiment 2 were probably not N deficient and, not surprisingly, there was no significant relationship between shoot N concentration and root : shoot ratio.

In the PT and PT + SAP tubes, a layer of P. tinctorius inoculum was placed on top of a layer of sterile medium. The inoculum consisted of 13 ml of a 6 : 3 : 1 mixture of vermiculite, perlite and peat. The sterile medium below the inoculum consisted of 52 ml of a 6 : 3 : 1 mixture of vermiculite, perlite and F-layer material. The peat and the ground F-layer material are both 100% organic, and they have a similar texture and particle size. Thus the two media probably do not differ greatly in physical properties. However, they are likely to differ chemically. Whether this had an effect on the outcome of the experiment is difficult to determine. The layer of inoculum was approximately 20% of the entire medium. Because it was on top, the vast majority of the roots (including mycorrhizas) of the pine seedlings at the time of harvest were below the inoculum layer, and so an effect of this layering was not evident in short root production or mycorrhiza formation.

In our experiments we have attempted to approximate the complexity of natural soil communities by adding a mixture of soil microbes rather than individual saprotrophs. However, we did not characterize the structure of the microbial community in our microcosms, and that structure is likely to depend on the nature of the growth medium, among other factors. While the medium we used was 10% F-layer, the remainder consisted of the artificial substrates vermiculite and perlite. Moreover, autoclaving may have affected the chemistry of the F-layer material. Thus, the structure of the microbial community in our microcosms may have differed substantially from those in natural ecosystems. Despite these limitations, our results suggest that interactions between mycorrhizal fungi and saprotrophic organisms can have large effects on nutrient transfer to host plants, and that the effects may be determined by the prevailing nutrient availability.

Acknowledgements

We thank the A. W. Mellon Foundation for providing funding for this project. We also thank Ylva Besmer, Michelle Bracht, Ian Dickie and two anonymous reviewers for helpful comments.

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