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Keywords:

  • Arbuscular mycorrhizal fungi (AMF);
  • decomposition;
  • nutrient capture;
  • organic patches;
  • glycine;
  • root proliferation

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References
  •  The contribution of different arbuscular mycorrhizal fungi (AMF) to nutrient capture from an organic patch, and the subsequent impact on root proliferation was investigated.
  •  Organic patches were created with glycine labelled with 15N and 13C. This allowed decomposition (as 13CO2 release) and uptake of nutrients (as 13C and 15N enrichments in the plant tissues) to be followed. Changes in root responses were followed in situ by the use of minirhizotrons and compared to responses in control (H2O) patches.
  •  Although there were differences in internal colonization and external mycelium production among the three AMF tested, none of the fungi responded to the presence of the glycine patch, and N and C capture was no different to uncolonized controls. However, the presence of glycine affected the manner in which colonized roots responded, particularly below the patch. The presence of AMF affected the decomposition of glycine.
  •  Root responses to the presence of N-rich patches appear more important than AMF responses.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

The distribution of nutrients in the natural environment is inherently patchy both spatially and temporally. It is well established that plant roots, upon encountering such nutrient-rich zones or patches, can proliferate roots within them and or alter their rates of nutrient uptake (Robinson, 1994; Fitter et al., 2000). However, the possible role of mycorrhizal fungi in both patch exploitation and modification of root responses to patches has been less well studied. Mycorrhizal associations are ubiquitous in the natural environment and being mycorrhizal is therefore the normal condition for most plant species. Mycorrhizal fungi play a key role in nutrient (particularly phosphate) acquisition for the plant (Smith & Read, 1997). Of the seven different types of mycorrhizal association, the arbuscular mycorrhiza (AM) is by far the most common. Proliferation of AM fungal hyphae within nutrient-rich organic patches has also been demonstrated (St. John et al., 1983a,b; Ravnskov et al., 1999). Thus, when both external AM hyphae and colonized roots encounter a patch, root proliferation might be replaced by hyphal proliferation which would carry a lower carbon cost for the plant. However, although several studies have demonstrated that AM fungi can transport inorganic N, particularly NH4+, to their host plant (Ames et al., 1983; Mäder et al., 2000) their ability to capture organic N sources directly remains controversial (Smith & Read, 1997) with some evidence from controlled culture experiments (Cliquet et al., 1997) and indirect evidence from field studies (Näsholm et al., 1998).

The aim of this investigation was to examine the influence of three different AM fungi (Glomus mosseae, G. hoi and Scutellospora dipurpurescens) on the decomposition, and plant N capture from, a simple organic material (15N and 13C labelled glycine) added as a discrete patch to microcosm units containing Plantago lanceolata. The influence of the different AM fungi on root responses in the patch and the zones above and below the patch was monitored by the use of in situ minirhizotron tubes. Glycine was added as an organic patch because amino acids comprise a large fraction of hydrolysed organic matter and are often present in the soil solution. Furthermore, although AM colonized roots were found not to take up N directly from a complex organic patch added to soil (Hodge et al., 2000) there is indirect evidence that such roots may be able to take up glycine intact (Näsholm et al., 1998). Although the concentrations of glycine added in this study were higher than those typically found in soil solution (i.e. between 20 and 200 µM; Monreal & McGill, 1985) this was because the aim was to add a nutrient-rich zone compared to the bulk soil in order that root and hyphal responses could be assessed. The following hypotheses were tested: root proliferation occurs in the organic patch but is reduced when a mycorrhizal inoculum is present; AM fungal hyphae proliferate in the organic patch to a greater extent than roots; nitrogen capture is related to external hyphal mycelium produced in the patch; and addition of a mycorrhizal inoculum enhances N uptake of simple intact organic compounds from the added patch.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Experimental design

Full details of the experimental design are given in Hodge et al. (1999a). Briefly, plants were grown in microcosm tubes made out of a section of PVC pipe (length 20 cm, I.D. 10 cm), which had two holes cut to allow insertion of a glass minirhizotron tube (25 cm long × 2.2 cm E.D.) at an angle of 45° to the horizontal. In addition, a smaller hole was cut to allow placement of a solution injection/gas sampling tube (15 cm long × 0.3 cm I.D.) directly parallel above the minirhizotron tube. At the top of each tube, the top 2 cm section of a PVC funnel (I.D. 10 cm at top and 7 cm at base) was placed to direct the roots into the middle section of the tube where the patch was to be inserted. The minirhizotron and gas sampling tubes were sealed with rubber bungs. Each microcosm tube was filled with 1600 g d. wt of a 50 : 50 mixture of autoclaved (121°C; 30 min) sand : soil as described in Hodge et al. (1999a) which had been left for 2 wk after autoclaving to reduce any potential phytotoxic effect of the heating process (Rovira & Bowen, 1966).

Eight microcosm tubes were contained within seven large (60 × 40 × 30 cm) freely draining insulated boxes containing a mixed turf of Trifolium repens L. (white clover) and Lolium perenne L. cv. Fennema (perennial rye-grass) to buffer the microcosm tubes against fluctuations in external temperature and to produce a realistic microclimate around the tubes. The boxes were maintained in a glasshouse and watered daily.

Three different mycorrhizal fungi were tested: Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe, isolate UY 21; Glomus hoi (Berch & Trappe) isolate UY 110; and Scutellospora dipurpurescens (Morton & Koske) INVAM isolate WV109A. Mycorrhizal treatments received 110 g wet weight of inoculum added to the autoclaved sand : soil medium. The nonmycorrhizal controls received 110 g wet weight of the combined mycorrhizal fungi inoculum which had been autoclaved (121°C; 30 min). The inoculum consisted of Plantago lanceolata L. (ribwort plantain) root medium colonized with the mycorrhizal fungus and included the sand and Terra-Green® (a calcined attapulgite clay soil conditioner, Turf-Pro Ltd, Staines, UK) growth medium. All inoculum was checked to confirm the presence of both root colonization and spores before addition to the experimental microcosm units. In addition, all microcosm units received 10 ml of filtered washings of the combined mycorrhizal fungi, passed through a 20-µm mesh twice to remove AM propagules, to prevent initial differences in microbial communities among microcosm units (van der Heijden et al., 1998).

Plantago lanceolata L. seeds supplied by Emorsgate Seeds, Norfolk, UK, were planted into each microcosm tube on 16 March 2000 (two seeds in each tube). All seeds in the microcosm tubes had germinated after 1 wk, after a further week seedlings were thinned to one per microcosm tube. After (33 d) planting the experimental and control patches were added. The experiment ran for 41 d between 18 April and 29 May 2000. The mean temperature over the duration of the experiment was 18.9°C (SE ± 0.05) with a mean daily maximum of 28.5°C (SE ± 0.74) and mean daily minimum temperature of 17.1°C (SE ± 0.41). Photosynthetically active radiation (PAR) flux was recorded weekly at noon and averaged 475 µmol m−2 s−1 at plant level.

Patch addition

Patches were added as either 1 ml of H2O (control patches) or 1 ml of a 400-mM glycine solution (organic patch). Thus, each of the seven boxes contained one replicate of each treatment (i.e. three different mycorrhizal fungi inoculum and one autoclaved inoculum (control) with H2O or glycine patches). The glycine solution consisted of a 1-M labelled glycine stock solution containing a 1 : 1 mix of 15N-labelled (98 atom%15N; Promochem, Hertfordshire, UK) and 13C-labelled (2–13C, 99 atom%13C; Promochem, Hertfordshire, UK) glycine added to unlabelled glycine (Sigma-Aldrich, Dorset, UK) to obtain the final required concentration of 400 mM. Thus, each 1 ml of the 400 mM glycine added as patches contained 5.78 mg N (2.82 mg 15N; 48.7 atom%15N) and 9.80 mg C (2.52 mg 13C; 25.7 atom%13C). The patches were added by injecting the H2O or glycine solution down the gas sampling tube at a depth of 10 cm.

Measurements and harvests

To monitor the respiratory 13C release from the patch, soil gas samples were taken by inserting a syringe needle (11.5 cm long) into the gas sampling tube and removing 10 ml of the soil air from the patch zone. The sample was then injected into an evacuated gas sample container (Europa Scientific, Crewe, UK) and analysed for 13C as described by Hodge et al. (1999a). Soil gas was sampled from four boxes at 4, 10, 24, 48, 72, 120, 168, 240, 336, 432 and 528 h after patch addition.

At harvest, the microcosm tubes were lifted out from the boxes containing the bulk turf to allow the minirhizotron and gas sampling tube to be removed. Each soil core could then be removed intact from its tube. The core was then cut into three sections – top, middle (containing the patch zone) and bottom – each of 6 cm thickness. The soil from the middle section was further divided so that the inner 6 cm core containing the point of patch addition could be removed for N, 15N, C, 13C and hyphal length determination (see below). The shoots were oven-dried at 60°C, weighed and analysed as below.

Root demography

Images of roots were recorded using an Olympus OES swing-prism borescope (Olympus Industrial, KeyMed, Southend-on-Sea, UK) with fibre-optic light source and a Sony Video Walkman digital video cassette recorder (model No. GV-D900E) at 0, 12, 23, 27, 31, 35 and 40 d after initial patch addition. The video images were captured onto a PC fitted with a PYRO video digitizer card and software for digital video capture and playback (Ulead Videostudio v. 3; ADS Technologies, Cerritos, CA, USA). This enabled a series of images in temporal sequence to be viewed simultaneously. Roots were identified on screen with a unique code number so differences with time could be monitored. For this analysis, data were gathered from frame 5 (10 cm depth), the site of ‘patch’ addition and frame 4 and 6 (2 cm above and below the patch, respectively). At day 0 low numbers of roots were present in the patch zone (mean across treatments = 1.0 ± 0.16), there were no differences due to the type of AM inoculum present (P = 0.211). These roots were subtracted from those used for the data analysis. Thus, the data for net numbers of roots in the patch zone are only for roots produced after the patches had been added. The term ‘root births’ refers to when new roots appeared in the field of view while ‘root deaths’ refers to when roots disappeared from the field of view.

Plant and soil analysis

The roots extracted from the different sections (top, middle, bottom) at harvest were washed thoroughly, oven-dried at 60°C, weighed and milled. Before drying the total root length of the roots recovered from the middle (patch addition) section were measured on a WinRHIZO (Régent Instruments Inc., Québec, Canada) image analysis system and then a subsample removed for mycorrhizal assessment (see below). A subsample of the root, shoot and patch soil material was analysed for total N, C, 15N and 13C by continuous-flow isotope ratio mass spectrometry (CF-IRMS). Subsamples of the soil from the different sections of each tube were used for moisture content determinations (105°C).

For mycorrhizal assessment, roots were cleared in KOH (90°C, 10 min), acidified in HCl (room temperature, 1 min) and stained with acid fuchsin (90°C, 20 min) (as Kormanik & McGraw (1982) but without phenol). Mycorrhizal colonization was examined with a Nikon Optiphot-2 microscope using brightfield and epifluorescence (Merryweather & Fitter, 1991) and × 200 magnification. Mycorrhizal scoring, using 100 intersections, was by the method of McGonigle et al. (1990). Numbers of arbuscules, vesicles and root length colonized (RLC; the percentage of total intercepts where hyphae were present) were recorded for each intersection. External mycorrhizal hyphae (EMH) were extracted from two 5 g (f. wt) samples from the middle soil section (the site of patch addition) using a modified membrane filter technique (as described in Staddon et al., 1999). Assessment of hyphal length was carried out using the gridline intercept method (Miller & Jastrow, 1992) for a minimum 50 fields of view at X 125 magnification (using a 10 × 10 grid of 1 cm side lengths, obtained from Graticules Ltd, UK). The hyphal lengths were then converted to hyphal densities (m hyphae g−1 soil : sand d. wt).

Statistical analysis

Data on samples taken over the duration of the experiment (i.e. soil gas samples and root demography) were analysed using the General Linear Model (repeated measurements) command in SPSS v 7.0. In addition, root birth rates were estimated from linear regressions of mean cumulative root births and significant differences between slopes of the fitted lines compared using the F-ratio method (Sokal & Rohlf, 1981; Potvin et al., 1990). Data recorded at the destructive harvest only were analysed using a General Linear Model (general factorial) command in SPSS v 7.0. Differences referred to in the text were statistically significant with P < 0.05, unless otherwise stated. For comparisons between treatments, the Bonferroni mean comparison test was applied. All data were checked and transformed appropriately to normalize skewed distributions before statistical analysis. In all cases, a randomised block design was used.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Patch decomposition

Initially (i.e. after 24 h) the patches in the tubes containing the mycorrhizal fungi decomposed more rapidly as shown by the higher amounts of 13C (as 13CO2) recovered in the soil gas (Fig. 1), although only tubes containing G. mosseae were significantly different to the control. At 48 h when the 13C fraction peaked in all treatments there were no differences among the treatments. At 72 h 13C recovered from the control (autoclaved inoculum) was higher than all the mycorrhizal treatments because the peak in this treatment was sustained for a longer period. Thereafter amounts of 13C recovered declined sharply in all patches (Fig. 1).

image

Figure 1. Amount (µg) of 13C as 13CO2 recovered from the soil atmosphere from the glycine patches with time. Note the log scale on the x-axis. Data are means with SE bars (n = 4). Control, circles; Scutellospora dipurpurescens, diamonds; Glomus hoi, triangles; Glomus mosseae, squares.

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At harvest the soil which had received the glycine patches contained more 15N and 13C than the corresponding background H2O patches showing that some of the glycine was still present, probably in the microbial biomass (data not shown). In the treatments receiving glycine patches, the relationship between 13C and 15N loss was different in the control (autoclaved inoculum) and mycorrhizal treatments (Fig. 2) with more C retained in soil per unit N in the mycorrhizal treatments. In an analysis of covariance both mg 15N as the covariate and inoculum type were significant (Table 1).

image

Figure 2. The relationship between mg 15N and 13C in the glycine patch soil at harvest in the mycorrhizal treatments (squares) and the control (circles). Data shown are raw values. In an analysis of covariance, mg 15N was a significant covariate (P < 0.001) using inoculum type as the main factor (P = 0.004).

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Table 1.  Analysis of covariance using inoculum type as the factor for all inoculum types (including autoclaved inoculum control) a , for the control and all mycorrhizal fungi grouped together b and for the mycorrhizal fungi separately for mg 13C with mg 15N as the covariate c
VariateCovariate PF1 ,23
mg 13Cmg 15NCovariate< 0.00165.29
  aInoculum type  0.004 5.92
   PF1 ,25
mg 13Cmg 15NCovariate< 0.00145.56
  bInoculum type  0.042 4.62
   PF1 ,17
mg 13Cmg 15NCovariate< 0.00161.26
  cInoculum type  0.014 5.53

The percentage of original patch 15N recovered in the soil at harvest was within the range 10–12% in all treatments. By contrast, the range of original patch 13C detected in the soil at harvest was much wider (10–24%, Table 2). The glycine added contained 2.52 mg 13C and 2.82 mg 15N (mass ratio of 13C : 15N, 0.89) but the 13C : 15N mass ratio from the soil at harvest was higher than this particularly in the mycorrhizal treatments (Table 2). A one-way ANOVA followed by Fisher’s pairwise comparisons confirmed the mass 13C : 15N ratio in the soil containing a S. dipurpurescens inoculum was significantly higher than all the other treatments suggesting a greater tendency to retain patch carbon in the presence of this fungus. There were no significant differences between the other treatments.

Table 2.  Percentage of original glycine patch 15N and 13C and the mass ratio of mg 13C : mg 15N recovered in the soil at harvest. SE are in brackets
Inoculum typePatch 15N %Patch 13C %13C : 15N mass ratio
Control 9.9 (1.54)10.3 (1.85)0.92 (0.081)
Glomus mosseae11.3 (1.87)15.2 (3.94)1.13 (0.123)
Glomus hoi11.6 (1.46)18.2 (4.95)1.30 (0.262)
Scutellospora dipurpurescens10.9 (1.66)23.6 (4.16)1.90 (0.110)

Mycorrhizal colonization and external hyphae production

The percentage of root length colonized (RLC) was higher in treatments containing a G. hoi inoculum (49 ± 2.5%) than the other two mycorrhizal treatments which did not differ (37 ± 2.2%). Arbuscule frequency was the same among all the mycorrhizal treatments (18 ± 1.3%). Vesicles were very infrequent (2 ± 0.4%) and only observed in roots grown with a G. hoi or G. mosseae inoculum. No mycorrhizal structures were observed in the control roots grown in the presence of an autoclaved mycorrhizal inoculum. There were no differences in colonization due to the type of patch added.

Although low levels (≈ 0.15 m g−1) of aseptate hyphae were observed in the controls, hyphal length densities were higher in all the mycorrhizal treatments (Fig. 3). G. hoi treatments produced c. 2.5 times greater hyphal length density than the two other mycorrhizal fungi. There was no difference among treatments due to patch addition and the patch × fungus interaction was not significant. In an analysis of covariance of external hyphal length density, neither percentage RLC or percentage arbuscules were significant covariates although the type of mycorrhizal fungus was always a significant factor (Table 3), which implies the variation could be accounted for by differences among the fungi, but that within the individual AM fungi types production of internal and external mycorrhizal structures were unrelated (Fig. 4).

image

Figure 3. Hyphal length density (m hyphae g−1 soil d. wt) in the patch zones. Data are means with SE bars (n = 14). Significant differences between arbuscular mycorrhiza (AM) inoculum treatments are indicated by letters. There were no significant differences due to the type of patch added and the fungus × patch interaction was not significant.

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Table 3.  Analysis of covariance using external hyphal length density from the mycorrhizal treatments minus control hyphal length density as the variate, mycorrhizal fungi, patch type or their interaction as the factor and root length colonized (RLC) or arbuscules as the covariate
CovariatedfPF1 ,35
% RLCCovariate1  0.456 0.57
 AM fungi2< 0.00143.81
 Patch1  0.283 1.19
 AM fungi × Patch2  0.276 1.33
% arbusculesCovariate1  0.588 0.30
 AM fungi2< 0.00160.68
 Patch1  0.267 1.27
 AM fungi × Patch2  0.262 1.39
image

Figure 4. The relationship between external mycelium and percentage root length colonized (RLC). Hyphal length density (m hyphae g−1 soil d. wt) data are differences between arbuscular mycorrhiza (AM) inoculum – controls (autoclaved inoculum). Raw data are shown (n = 7). Treatments receiving a glycine patch are shown by the closed symbols while those receiving a H2O patch are shown by the open symbols. Scutellospora dipurpurescens, diamonds; Glomus mosseae, squares; Glomus hoi, triangles. Differences between the AM fungi occurred but within the individual AM fungi; there was no relationship between hyphal length density and percentage RLC.

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Root demography within the patch

Net numbers of roots in the patch were greater after glycine compared to H2O addition (Fig. 5a). There were no differences within patch treatments due to the type of mycorrhizal inoculum present. Cumulative births in the glycine patches were also higher than those from the H2O patches. In the H2O patches, roots grown with a G. hoi inoculum showed lower birth rates than any of the other treatments (slope 0.067 for G. hoi compared to 0.088 for the combined other treatments; Fig. 5b). In the glycine patches however, cumulative root birth rate was higher from the tubes containing a G. hoi inoculum than the other mycorrhizal fungi (slopes are 0.28 for G.hoi and 0.21 for G. mosseae and S. dipurpurescens combined data). Cumulative root birth rate in the non-mycorrhizal control (slope = 0.28) receiving glycine did not differ from any of the mycorrhizal treatments (Fig. 5c).

image

Figure 5. (a) Net numbers of roots in treatments receiving a glycine (closed symbols) or a H2O (open symbols) patch. All treatments receiving a glycine patch had greater net numbers of roots compared to those receiving a H2O patch as indicated by the different letters. There were no other differences among treatments. Cumulative births in the either the (b) H2O patch (open symbols) or (c) glycine patch (closed symbols) with time. Different letters indicate significant (P < 0.05) differences determined by the F-ratio method for statistically comparing fitted lines (Potvin et al., 1990). Mean values are shown (n = 7). Control, circles; Glomus mosseae, squares; Glomus hoi, triangles; Scutellospora dipurpurescens, diamonds.

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After 40 d cumulative root deaths were lower in the G. hoi + H2O treatment than in either the G. hoi or control tubes receiving glycine (data not shown). There were no significant differences in either instantaneous root births or deaths in the patches over the experimental period.

Root demography outside the patch

There were no differences in root demography in the area directly above the patch. Directly below the patch however, differences in cumulative birth rate occurred. All the tubes containing the mycorrhizal fungi showed different birth rates below a glycine patch compared to under a H2O patch. Only the birth rate in the control (autoclaved inoculum) did not differ due to the type of patch added in the zone above (Fig. 6a). Birth rate of roots in the presence of a G. mosseae (Fig. 6b) and, in particular, a G. hoi (Fig. 6c) inoculum were enhanced below a glycine patch compared to below a H2O patch. By contrast, birth rate of roots in tubes containing a S. dipurpurescens inoculum were slower below glycine (Fig. 6d). Differences among the treatments also occurred. Below a H2O patch root birth rate was in the order: control = G. mosseae > S. dipurpurescensG. hoi. Below a glycine patch however, root birth rate was in the order G. hoi > control = G. mosseaeS.dipurpurescens. The rate of root births in the control and G. mosseae treatments never differed. In addition, below a glycine patch roots colonized by G. hoi died faster than either the G. hoi or nonmycorrhizal (control) roots receiving a water patch.

image

Figure 6. Cumulative births for (a) control (b) Glomus mosseae (c) Glomus hoi and (d) Scutellospora dipurpurescens treatments below either the H2O (open symbols) or glycine (closed symbols) patch zone. Where different lines are drawn the cumulative birth rate was statistically (P < 0.05) different between patch treatments as determined by the F-ratio method.

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Plant growth and N content

The type of patch added to the microcosms had no effect on shoot d. wt (mean across treatments = 3.0 ± 0.15 g). Shoots of plants grown with a S. dipurpurescens inoculum were heavier than those grown with a G. mosseae inoculum: 3.3 ± 0.19 g compared with 2.4 ± 0.13 g. There were no other fungal effects on shoot d. wt. Neither the type of patch nor the mycorrhizal inoculum present affected length, d. wt or specific root length of roots recovered from the middle soil section. The total root d. wt recovered was also unaffected by either patch or mycorrhizal status (mean across treatments = 1.3 ± 0.07 g). When root d. wt in the middle section was expressed as a fraction of the total root d. wt however, there were significant differences between the treatments (Fig. 7). Plants grown in the presence of a glycine patch produced relatively more root in the middle section than plants with a H2O patch. In addition, plants grown with a G. mosseae inoculum placed a higher fraction of their roots in the middle section than those grown with G. hoi. The fungus × patch interaction however, was not significant (P = 0.337).

image

Figure 7. D. wt of roots in the middle section as a fraction of the total root d. wt. Different letters indicate significant differences (P < 0.05) due to the fungal species. In addition, the treatments receiving a glycine patch (closed columns) were significantly (P < 0.05) greater than those receiving a H2O patch (open columns). Data shown are means with standard error bars (n = 7).

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Plant N contents were higher from the glycine patch than from the H2O patch treatment (61 ± 2.5 mg N compared with 52 ± 2.1 mg N). Plants grown with G. mosseae had lower N contents than those grown with G. hoi or the autoclaved inoculum (i.e. 47 ± 2.3 mg N compared to 60 ± 2.7 mg). However, in no case was there a significant interaction between inoculum type and patch. N concentrations and the C : N ratio of the plant tissues were not different among any of the treatments.

Carbon and Nitrogen uptake from the glycine patches

At harvest the shoot and root material of the treatments receiving glycine were enriched with 15N and 13C compared to the H2O controls. However, the amount of C captured did not differ due to the type of fungal inoculum present and overall levels were low (c. 0.8% of the C originally added). By contrast, the plants had captured 72 ± 2.0% of the N added in the glycine patches regardless of the added mycorrhizal inoculum. Most (c. 60 ± 1.9%) of this N was detected in the shoots. There were no differences among treatments in the amount of N from glycine detected in the shoots but control (nonmycorrhizal) roots contained more patch N than S. dipurpurescens colonized roots (c. 14 v 9%). There were no other differences among treatments. Although plants captured a large proportion of the N from the patch, it only constituted c. 7% of the plants’ total N content and this value did not vary among treatments.

Although there was an apparent relationship between mg 15N and mg 13C from glycine in the roots of plants colonized with G. hoi (Fig. 8) there was no relationship between total plant N or C capture from an organic patch and percentage RLC, percentage arbuscules or length of mycorrhizal hyphae (m g−1 soil) in the patch, irrespective of which fungus was involved, suggesting that the relationship was spurious. Similarly root length in the patch and N capture from it were unrelated.

image

Figure 8. The apparent relationship in Glomus hoi colonized roots between mg 13C and mg 15N captured from a glycine patch. The relationship however, was not related to overall levels of Glomus hoi internal colonization or external mycelium production. Control, circles; Scutellospora dipurpurescens, diamonds; Glomus hoi, triangles; Glomus mosseae, squares.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Patch decomposition

The glycine patches decomposed rapidly as shown by the release of 13CO2, which peaked after only 48 h (Fig. 1). The presence of the mycorrhizal fungi influenced patch decomposition by promoting initially more rapid 13C, release as shown by the pattern of 13CO2 recovered. This contrasts with the pattern of 13CO2 release from a complex organic patch (Lolium perenne shoot material) where decomposition of the patch was unaffected by the presence of an AM inoculum (Hodge et al., 2000). In addition, the presence of plant roots in a complex organic patch resulted in enhanced N loss from the patch irrespective of the type of plant species present (Hodge et al., 1998) whereas in the present study more C from glycine was retained in the soil, particularly when a S. dipurpurescens inoculum was present. The proportion of this retained C present in the external mycorrhizal hyphae is unknown.

Mycorrhizal parameters

Mycorrhizal colonization can decrease in fertile soil (Thomson et al., 1986) and a decline in arbuscule frequency was observed in field grown Agropyron desertorum roots experiencing a nutrient-rich microsite (Duke et al., 1994). However, in this study mycorrhizal colonization was unaffected by the presence of a glycine patch. It was striking that the length of external mycelium produced by the AM fungi was unaffected by the addition of glycine. Thus, there was no support for the second hypothesis that AM hyphae would proliferate in the organic patch to a greater extent than roots. Glycine has previously been shown to stimulate hyphal growth from resting spores in vitro (Hepper & Jakobsen, 1983). Proliferation of arbuscular mycorrhizal fungi (AMF) hyphae in sterilized organic matter has also been reported by St John et al. (1983a,b), however, when a filtrate containing microorganisms was applied inhibition of AM fungal proliferation was observed (St John et al., 1983a). Similarly the addition of yeast and bovine serum albumin enhanced hyphal growth of Glomus intraradices whereas addition of carbon sources, such as cellulose, depressed hyphal growth, probably as a result of competitive interactions with other soil microorganisms stimulated by the added resources (Ravnskov et al., 1999). In this study, although glycine has a low C : N ratio (1.7 : 1) and has been shown to be a poor substrate for other soil microorganisms (Lipson et al., 1999), AM hyphal proliferation did not occur.

Root proliferation

The first hypothesis, that root proliferation would occur in the organic patch compared with the control patch but would be less in the presence of mycorrhizal inoculum was only partly supported. Roots clearly proliferated in the glycine patch compared to the control (H2O) patches. However, this proliferation was most marked in the G. hoi colonized roots, which showed the lowest root birth rate in the control patches, but the greatest increase among the mycorrhizal treatments in the glycine patches. Hodge et al. (2000) observed enhanced root proliferation in a complex organic patch after addition of a G. mosseae inoculum compared with indigenously AM colonized roots. Although G. mosseae did not respond in the same manner in the present study, colonization was much lower (37%) than either the added inoculum treatment (c. 80%) or the indigenously colonized control (c. 60%) in the previous experiment. In G. hoi treatments colonization was higher than with both the other AM fungi tested and so was the rate at which roots were produced in a glycine patch. By contrast, Cui & Caldwell (1996) reported that proliferation of Agropyron desertorum roots in nutrient-rich patches decreased when colonized by AM fungi, although the difference was only weakly significant (P = 0.07). In the present study rates of root proliferation in the glycine patch were never different from the uncolonized controls regardless of the AM fungi present. Similarly, Farley & Fitter (1999) reported that mycorrhizal colonization did not alter the manner in which roots of herbaceous perennials foraged for nutrients. Furthermore, the influence of mycorrhizal colonization on root production was most marked not in the patch itself but in the zone below the patch. Both G. hoi and G. mosseae showed enhanced root production below a glycine patch compared to a H2O patch whereas the rate at which roots were produced when colonized by S.dipurpurescens was reduced. Although Torrisi et al. (1999) observed an overall increase in local cotton root density following mycorrhizal colonization, in the present study the mycorrhizal roots were clearly responding to being below a glycine patch presumably because some of the products of patch decomposition had diffused to this zone. Furthermore the mycorrhizal inoculum present was causing the roots to respond in different ways. Root deaths were higher in the treatments where root birth rates were faster (e.g. for G. hoi and controls in the glycine patch and G. hoi colonized roots below a glycine patch) and slower in the G. hoi colonized roots in a H2O patch. This contrasts with the decrease in root longevity of poplar fine roots as a result of mycorrhizal colonization reported by Hooker et al. (1995).

Plant parameters and N and C capture from the organic patch

Plant N contents were higher in microcosms containing the glycine as opposed to H2O patches, although there was no effect on plant N concentrations. Similarly, after 41 d growth in a complex organic patch N concentrations of mycorrhizal plants were no different to controls (Hodge et al., 2000). Shoot growth was also unaffected by a glycine patch although more root biomass was allocated to the patch zone than above or below the patch. Such co-ordinated responses within root system to nutrient-rich zones have been reported previously (reviewed by Robinson, 1994). Plant N capture was not related to any of the mycorrhizal parameters measured. In a previous study, N capture from a complex organic patch was found to be related to the production of vesicles, thought to be important storage organs, in AM colonized roots (Hodge et al., 2000). By contrast, in the present study N capture from roots colonized by S. dipurpurescens, which does not produce vesicles, was no different to the other mycorrhizal treatments or to the uncolonized controls. The low 13C enrichment observed in the plant tissues suggest that some intact glycine was taken up from the patch. Uptake of intact amino acids by roots and mycorrhizal roots has been reported previously (Jones & Darrah, 1994; Cliquet et al., 1997) although in this study this uptake was not related to mycorrhizal colonization. More N than C was retained in plants from the glycine patches (i.e. 72% cf. 0.8%) suggesting that inorganic N was the dominant form in which N was being taken up by the plants. Mycorrhizal hyphae have been shown to transfer both NH4+ (Ames et al., 1983) and, under drought conditions, NO3 (Tobar et al., 1994) to their associated hosts but this seems unlikely to have been an important transfer mechanism in the present study as total patch N capture mycorrhizal colonized plants did not differ from the nonmycorrhizal controls.

Conclusions

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

In this study AM colonization had no effect on N capture from a simple organic patch and none of the initial hypotheses were supported. This was despite the three AM fungi tested differing in their internal colonization, external mycelium production and their impact on root proliferation. It was striking that hyphal proliferation did not occur within the organic patch zone. However root proliferation did occur. Although root proliferation was unrelated to N capture from the patch this was not unexpected because it has been previously demonstrated that root proliferation in, and N capture from, organic patches are only related when plants are competing for the resources (Hodge et al., 1999b; Robinson et al., 1999). Tibbett (2000) recently suggested that the significance of root responses to nutrient patches in soil has been overestimated because mycorrhizal hyphal proliferation would occur instead. The results of this study contrast sharply with this view point. Mycorrhizal fungal hyphae may have a role to play in accessing nutrient-rich zones at a distance from the root system. However, when both roots and mycorrhizal symbionts are experiencing a N-rich patch it appears root responses play the dominant role, although their response may be modified by the fungal partner.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

A. H. is funded by a BBSRC David Phillips Fellowship. I thank C. Scrimgeour and W. Stein (Scottish Crop Research Institute, Invergowrie, Dundee UK) for conducting the mass spectrometry analysis and Alastair Fitter for his comments on the manuscript.

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  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References
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