spatial dispersion, interspecific competition and mycorrhizal colonization

Transcription

1 Research Plant nitrogen capture from organic matter as affected by Blackwell Publishing, Ltd spatial dispersion, interspecific competition and mycorrhizal colonization Angela Hodge Department of Biology, Area 2, The University of York, PO Box 373, York, YO10 5YW, UK Summary Author for correspondence: Angela Hodge Tel: Fax: Received: 6 August 2002 Accepted: 21 October 2002 The capture of nitrogen (N) by plants from N-rich complex organic material differing in spatial (uniform dispersion or discrete patches) heterogeneity was measured, as well as the subsequent impact on N capture of the addition of a mycorrhizal inoculum (Glomus hoi). The organic material was dual-labelled with 15 N and 13 C to follow plant uptake of N (as 15 N) and to determine the amounts of original 13 C and 15 N which remained in the soil at harvest. The organic material was added to microcosm units containing Lolium perenne or Plantago lanceolata in intra or interspecific competition. Plant N capture from the dispersed organic material was more than twice that from the discrete patch (dispersed: 17%; discrete: 8%). There was no effect of species composition or the mycorrhizal inoculum on total plant N capture except when in interspecific plant competition. Here, N capture was dependent on the root length produced and was always higher when the mycorrhizal inoculum was present. Mycorrhizal colonization increased N capture from the organic material when in interspecific plant competition but not in monoculture. Key words: arbuscular mycorrhizal (AM) fungi, decomposition, organic material, spatial heterogeneity, intra- and interspecific competition. New Phytologist (2003) 157: Introduction Within the soil environment organic matter inputs such as leaf litter, dead roots and soil animals, and their subsequent decomposition, help to create temporal and spatial heterogeneity in nutrient availability. Thus, in order to optimize nutrient capture plant roots have to be physiologically and/or morphologically plastic to enable them to respond to such variations in resource supply. It is well established that plants can respond morphologically by the proliferation of roots within localized nutrient-rich zones or patches which is considered to be a foraging response to the heterogeneous nature of the soil environment (Robinson, 1994; Fitter et al., 2000). This root proliferation response however, was thought to have no benefit to plants in terms of nitrogen (N) capture, a key limiting nutrient in terrestrial ecosystems, as no relationship between root proliferation and N capture could be demonstrated (van Vuuren et al., 1996; Fransen et al., 1998; Hodge et al., 1998). However, when plants with differing capabilities for proliferation are competing for the N released then the speed and the extent of the root proliferation response becomes important and the ability to proliferate roots does confer an advantage (Cahill & Casper, 1999; Hodge et al., 1999a, 2000b; Robinson et al., 1999). Thus, if the conditions under which the proliferation response has evolved, such as plant-plant competition, are omitted then the advantage of the response may be obscured. Another factor that is often over looked in root responses to soil heterogeneity is the colonization of the plant roots by mycorrhizal fungi. Mycorrhizal associations are ubiquitous in the natural environment, thus being mycorrhizal is the normal condition for most plant roots. Of the different types of mycorrhizal associations that can form, the arbuscular mycorrhizal (AM) association is the most common, occurring in two thirds of all plant species (Smith & Read, 1997). As with plant roots, fungal hyphae can also proliferate in nutrient-rich zones as has New Phytologist (2003) 157:

2 304 Research been demonstrated for both free-living (Ritz et al., 1996) and symbiont fungi in the ectomycorrhizal (Bending & Read, 1995) and AM associations (Mosse, 1959; St John et al., 1983; Hodge et al., 2001). Proliferation of the mycorrhizal fungal symbiont either instead of, or in addition to, the host roots could confer a number of advantages to the host plant. Firstly, as the fungal hyphae are thinner, proliferation of the fungal hyphae should represent a lower construction cost to the host plant not only in terms of carbon (C; Fitter, 1991), but also N, as fine roots have elevated N concentrations (Pregitzer et al., 1997). Secondly, again by virtue of their size, AM hyphae should be better able to penetrate throughout the decomposing organic material and so increase N capture, either by being more effective at competing with the other soil microbial community for the inorganic N released or by accessing organic forms of N directly. Although the results of some studies suggest that AM fungi may be able to access simple forms of organic N intact (Cliquet et al., 1997; Näsholm et al., 1998), evidence for a direct role of AM hyphae in organic N uptake and transfer is still lacking and remains a matter of some debate in the literature (Smith & Read, 1997). AM fungi can however, transfer NH 4 + to their associated host plant (Ames et al., 1983; Mäder et al., 2000). Furthermore, it has recently been demonstrated that an AM fungus, Glomus hoi, both enhanced decomposition of, and transferred N to its associated host plant from, a complex organic patch in soil (Hodge et al., 2001). In addition to enhanced nutrient capture, mycorrhizal fungi are known to confer a number of other benefits to their host plant including drought resistance and protection against pathogens (Newsham et al., 1995a; Smith & Read, 1997). Newsham et al. (1995b) proposed a model whereby root systems with a poorly branched architecture rely on the AM mycorrhizal symbiont predominately for enhanced nutrient capture, while those root systems that had a highly branched architecture rely on the mycorrhizal symbiont for other benefits such as pathogen resistance. The aims of this study therefore were to examine how two plant species (Plantago lanceolata L. and Lolium perenne L.) of varying root architecture captured N from complex organic material (L. perenne shoots) added to soil either as a discrete patch or more uniformly dispersed. The influence of an AM inoculum upon N capture and the host plants root proliferation response was also followed. Glomus hoi was selected as the AM inoculum as it has previously been shown to enhance N capture from complex organic patches (Hodge et al., 2001). The following hypotheses were tested; (i) Root growth in general would increase in the sections receiving the organic material and would be greatest where the organic material was most concentrated. Specifically, root morphology would alter, resulting in longer thinner roots and so an increase in specific root length (SRL); (ii) Root growth, particularly root length, would be reduced when the mycorrhizal inoculum was present and this would be most apparent in the sections containing the organic material because hyphal, instead of root, proliferation would occur; (iii) Plants would capture more N from organic material added as a patch from organic material that which had been dispersed, and the species which had the most root length in the patch zone would capture the most N overall. More N would be captured from the dispersed organic material treatments when the AM mycorrhizal inoculum was present due to the spatial placement of the fungal hyphae; (iv) Overall Lolium monocultures, by virtue of their increased root length, would capture more N from the organic material than Plantago monocultures; N capture by the mixed culture would be intermediate between these two. However, the addition of a mycorrhizal inoculum would benefit (in terms of N capture from the organic material) Plantago species more than Lolium. The organic material added was dual-labelled with 13 C and 15 N so that the dynamics of N capture by the plants and the decomposition of the organic material could be followed. Although the organic material differed in its spatial distribution each experimental unit was supplied with the same total amount of N and C. Materials and Methods Experimental design Plants were grown in microcosm tubes made out of a section of PVC pipe (length 20 cm, I.D. 10 cm). 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 organic material was to be inserted. Each microcosm tube was filled to a depth of 5 cm with a 50 : 50 mixture of sand : soil (a medium loam as described by Hodge et al., 1999b) and a smaller section of PVC pipe (length 15 cm, I.D. 6.5 cm) placed centrally inside the larger unit, to enable placement of the organic material once the seedlings had developed and to keep disturbance of the unit to a minimum. The area between the outer and inner tube was filled with the sand : soil mixture containing the mycorrhizal inoculum (fresh or autoclaved) and the microcosm unit was then ready for planting. Eight microcosm tubes were contained within six large ( 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. Mycorrhizal treatments received 150 g wet weight of Glomus hoi (Berch & Trappe) isolate UY 110 inoculum added to the sand:soil medium. The non-mycorrhizal controls received 150 g wet weight of the mycorrhizal inoculum which had been autoclaved (121 C; 30 min). The inoculum consisted of Plantago lanceolata L. (ribwort plantain) root medium New Phytologist (2003) 157:

3 Research 305 colonized with G. hoi and included the sand and Terra- Green (a calcined attapulgite clay soil conditioner, Turf- Pro Ltd, Staines, UK) growth medium. The 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 from the mycorrhizal inoculum, passed through a 20-µm mesh twice to remove AM propagules, to prevent initial differences in microbial communities among microcosm units. Plantago lanceolata L. and Lolium perenne L. seeds supplied by Emorsgate Seeds, Norfolk, UK, were planted into each microcosm tube on 16 March 2000 (two seeds in each tube). The microcosm units contained three species combinations: either two Plantago or Lolium seedlings as monocultures (in intraspecific competition) or one seedling of each in a mixed culture (in interspecific competition). All seeds in the microcosm tubes had germinated after one week. Forty-two days after planting the organic material was added. The experiment ran for 22 d between 3 May and 25 May The mean temperature over the duration of the experiment was 19.5 C (SE ± 0.08) with a mean daily maximum of 35.0 C (SE ± 1.19) and mean daily minimum temperature of 15.7 C (SE ± 0.49). Photosynthetically active radiation (PAR) flux was recorded weekly at noon and averaged 502 µmol m 2 s 1 at plant level. Patch addition The organic material added to the microcosm tubes was 0.5 g oven-dried finely milled L. perenne shoot material labelled with both 15 N and 13 C produced as described in Hodge et al. (1998). The material was placed in the space created by removal of the inner PVC tube and was added either as a thin, concentrated layer (c. 6.5 cm diameter, 1 mm depth) at 11 cm depth in the microcosm unit ( patch treatment) or dispersed uniformly with the background sand : soil mix in a 10-cm band starting 3 cm below the surface ( dispersed treatment). The remainder of the space was filled with the sand: soil mix only and each microcosm unit contained 1600 g d. wt. of the sand : soil mix. The organic material added to the tubes contained 9 mg N (1.37 mg 15 N) and 202 mg C (3.55 mg 13 C) with a C : N ratio of 22 : 1. There were four replicate tubes for each combination of species, organic material placement and mycorrhizal treatment. Plant and soil analysis At harvest, each soil core was removed intact from its tube and then cut into four sections the top 2 cm and then a top, middle and bottom section, each of 6 cm thickness. The shoots were oven-dried at 60 C, weighed and analysed as below. The roots extracted from the different sections were washed thoroughly and the total root length from each section measured on a WinRHIZO (Régent Instruments Inc., Québec, Canada) image analysis system (scanned at 300 dpi). As it was impossible to separate roots of Lolium from those of Plantago when grown in interspecific competition, the root length produced by each species was estimated by adding together the root lengths produced by Lolium and Plantago monocultures and obtaining an average value for each of the four mycorrhiza/organic placement combinations separately; the proportion of roots which were Plantago and Lolium was then calculated from this average sum. The approximate root lengths of each species in competition were then estimated from these proportions as described in Hodge et al. (2000b). This method relies on two assumptions: the species that yielded the most in monoculture was assumed to be the most successful in the mixture; and both intra- and inter-specific interactions were the same. Root N capture from the organic material by the plants grown in competition was similarly estimated and summed with that captured by the shoots (which could be easily separated) to obtain a value of plant N capture for each species in competition. As > 70% of the N captured from the organic material was detected in the shoots this estimate was less prone to error. A subsample of root material was taken from the middle section only for mycorrhizal assessment. The root material from the different sections was then oven-dried at 60 C and weighed before being combined for milling. A subsample of the root, shoot and soil material from the middle section was analysed for total N, C, 15 N and 13 C by continuous-flow isotope ratio mass spectrometry (CF-IRMS). Subsamples of the soil from the different sections of each tube were used for gravimetric 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) (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 or other AM fungal structures were present) were recorded for each intersection. External mycorrhizal hyphae were extracted from two 5 g (FW) samples from the middle soil section only (which contained the organic material added as a patch or half of the organic material added in the dispersed treatment) using a modified membrane filter technique (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 125 magnification (using a 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). New Phytologist (2003) 157:

4 306 Research Statistical analysis Data were analysed using the General Linear Model (GLM) factorial design command or, for the root data in the individual sections, the GLM repeated measurements command, in SPSS v For the root data monocultures were directly compared with the mixed culture unless otherwise stated. Data for Plantago and Lolium were analysed separately except for the shoots where data for only one seedling in each case (i.e. Plantago or Lolium) was used and the data for the neighbouring seedling discarded. The shoot data in competition was then compared against the mean of the two seedlings grown in monoculture. The seedling term refers to the plant species examined whereas the competitor term refers to the other plant species present. Thus, when Plantago was the seedling it was compared against Plantago as the competitor in the monoculture tubes and Lolium in the intraspecific competition tubes. Differences referred to in the text were statistically significant with P < 0.05, unless otherwise stated. All data were checked and transformed appropriately to normalize skewed distributions before statistical analysis. In all cases, a randomised block design was used. Results Organic material decomposition At harvest the soil from the middle section contained more 13 C and 15 N than the controls, indicating that some N and C from the organic material remained in the soil. As only the middle soil section was analysed, which contained all of the organic material added as a patch but only half that added in the dispersed treatment, the values recovered from the dispersed treatment were multiplied by 2 to obtain an estimate of the total mg 13 C and 15 N remaining in the tube. The relationship between 13 C and 15 N loss was different depending on how the organic material had been added (Fig. 1) with more N retained in the soil per unit C when the organic material had been added as a discrete patch compared with when it had been more uniformly dispersed. In an analysis of covariance with mg 13 C as the covariate, organic material Fig. 1 The relationship between mg 13 C and 15 N in the dispersed (open symbols) and patch (closed symbols). Data shown are raw values for Plantago monocultures (circles), Lolium monocultures (squares) and mixed culture (triangles). In an analysis of covariance, mg 13 C was a significant covariate (P < 0.001) and O.M. placement a significant factor (P < 0.001). placement, mycorrhiza inoculum and the interaction between organic material placement and plant species were significant (Table 1). The percentage of original 13 C added in the organic material recovered in the soil at harvest was similar (c. 13%) regardless of how the organic material had been placed in the tube. In contrast, the original 15 N recovered was markedly affected by the method of incorporation with c. 63% of the original 15 N recovered in the patch treatment but only c. 35% recovered in the dispersed treatment (Table 2). When the dispersed and patch data were analysed separately species composition was a significant factor (P = 0.015) in the dispersed treatment whereas mycorrhiza inoculum was a significant factor (P = 0.039) in the patch treatment. In both cases mg 13 C was a significant covariate. A one-way ANOVA followed by Fisher s pairwise comparisons confirmed that the mass 13 C : 15 N ratio of the soil from Plantago monocultures was lower than that of the mixed culture soil in the dispersed organic material treatment. In the patch treatment there was a tendency for more 15 N to be retained in the soil when the mycorrhizal inoculum was present (Table 2) but the differences found in the analysis of covariance not confirmed by a one-way ANOVA, suggesting that the differences due to the mycorrhizal inoculum were slight. Covariate d.f. F P mg 13 C Covariate < Species composition O.M. placement < Mycorrhiza inoculum Species composition O.M. placement Species composition Mycorrhiza inoculum O.M. placement Mycorrhiza inoculum Species composition O.M. placement Mycorrhiza inoculum Table 1 Analysis of covariance using species composition, organic material (O.M.) placement, mycorrhizal inoculum and their interaction as the factors for mg 15 N remaining in the soil with mg 13 C remaining in the soil as the covariate. All F-tests have 47 error degrees of freedom New Phytologist (2003) 157:

5 Research 307 Table 2 Percentage of original organic material 15 N and 13 C and the mass ratio of mg 13 C : mg 15 N recovered in the soil at harvest across all treatments 1 and for the factors 2 shown to be significant after an analysis of covariance on each method of organic material (O.M.) incorporation separately. SE are in brackets O.M. placement Patch 15 N % Patch 13 C % 13 C : 15 N mass ratio Patch 1 Across all treatments 62.6 (3.58) 13.7 (0.77) 0.62 (0.062) Patch 2 With mycorrhiza inoculum 67.3 (4.32) 14.1 (1.09) 0.54 (0.02) Without mycorrhiza inoculum 57.9 (5.56) 13.3 (1.13) 0.70 (0.12) Dispersed 1 Across all treatments 34.9 (3.20) 13.3 (1.00) 1.04 (0.070) Dispersed 2 Plantago monocultures 39.8 (8.30) 11.9 (2.08) 0.80 (0.101) a Lolium monocultures 32.7 (2.97) 13.6 (1.53) 1.11 (0.113) ab Mix culture 32.0 (4.19) 14.3 (1.64) 1.20 (0.111) b Differences in the dispersed treatment were confirmed by a one-way ANOVA. Different letters denote significant differences determined by a Fisher s pairwise comparison. The significant differences observed in a ANCOVA due to the mycorrhizal inoculum in the patch treatment were not confirmed by a one-way ANOVA (see text for details). Mycorrhizal colonization and external hyphae production The percentage of root length colonized (RLC) and the numbers of arbuscules were affected by the plant species present and in both cases the species mycorrhizal interaction was significant. In the presence of the mycorrhizal inoculum percentage RLC was in the order Plantago monocultures (75 ± 2.7%) > Mixed culture (43 ± 5.4%) > Lolium monocultures (30 ± 1.9%), whereas in its absence the levels were lower and in the order Plantago monocultures (19 ± 2.9%) > Mixed and Lolium monocultures (mean across these treatments = 7 ± 1.7%). Number of arbuscules followed the same pattern as percentage RLC. Although low levels (c m g 1 soil d. wt) of aseptate hyphae were observed in treatments which did not receive the mycorrhizal inoculum, hyphal length densities were significantly higher (P < 0.001) in the mycorrhizal treatments (1.21 ± m g 1 soil d. wt). Greater hyphal length densities were recovered from Plantago monocultures (0.79 ± m g 1 soil d. wt) compared with Lolium monocultures (0.57 ± m g 1 soil d. wt). When both plant species were grown together hyphal length densities were intermediate and not significantly different from either plant species grown in monoculture (0.69 ± m g 1 soil d. wt). There were however, no differences in hyphal length densities, percentage RLC or arbuscules between patch and dispersed organic matter treatments. Root length, d. wt and specific root length (SRL) Of the 7 possible 2-, 3- and 4-way interactions for each of root length, root d. wt and SRL in the different sections, 5 were significant (P < 0.05) for root length and d. wt and 4 were significant (P < 0.05) for SRL. Of these, the most biologically interesting are presented below. There was never a significant 4 way interactions between section mycorrhiza species organic material. Root lengths followed the pattern predicted by hypothesis (i), being highest in the sections containing the organic material compared with those that had received no additions (Fig. 2a). Root d. wt followed a similar pattern (data not shown). The presence of organic material significantly altered specific root lengths (SRL) but in the opposite direction predicted by hypothesis (i) as SRL was reduced in the patch where the organic material was most concentrated (Fig. 2b), and in both treatments the highest SRL values were produced in the bottom sections which did not receive any organic material (Fig. 2b). Although the mycorrhizal inoculum generally reduced root growth, this reduction was not just confined to the sections containing the organic material, thus only partially supporting hypothesis (ii). Both root length and d. wt decreased in the top section and increased in the bottom section when the mycorrhizal inoculum was present. In the middle section root length was decreased but d. wt was unaffected. Consequently, in the presence of AM inoculum, SRL was unaffected in either the top or bottom sections but decreased in the middle section (Fig. 3). Total specific root length (SRL) was reduced (P = 0.021) by the mycorrhizal inoculum (with G. hoi inoculum: 162 ± 3.4 m g 1 ; without G. hoi inoculum 173 ± 4.0 m g 1 ). Total root length and root d. wt from all the sections together were influenced only by the species composition and were in the order Lolium monocultures > Mixed culture > Plantago monocultures (Table 3). Shoot data There was no difference between the shoot d. wt of Plantago (0.82 ± g) or Lolium (0.85 ± g) seedlings irrespective of whether they were grown in competition or monoculture. However, the presence of a mycorrhizal inoculum, reduced shoot d. wt (with G. hoi inoculum: 0.79 ± g; without G. hoi inoculum 0.88 ± g; averaged across species). Shoot d. wt was unaffected by the method of organic material addition. Shoot N concentration was only affected by the placement New Phytologist (2003) 157:

6 308 Research Fig. 3 Differences in specific root length in the top (shaded fill), middle (solid fill) and bottom (no fill) soil sections due to the presence or absence of the G. hoi mycorrhizal inoculum in the tubes. Data shown are means with standard error bars (n = 24). Different letters denote significant differences as determined by a Duncans Multiple Range test. Fig. 2 Differences in (a) root lengths and (b) specific root lengths in the top (shaded fill), middle (solid fill) and bottom (no fill) soil sections due to placement of the organic material in the tubes. Data shown are means with standard error bars (n = 24). Different letters denote significant differences between sections and organic material placement as determined by a Duncans Multiple Range test. of the organic material, with higher concentrations when the organic material was dispersed (17.4 ± 0.51 mg N g 1 ) than when it was present as a discrete patch (15.9 ± 0.37 mg N g 1 ). The quantity of 15 N derived from the organic material recovered in the shoots was affected by the competitor and the target seedling present as shown by the significant (P = 0.008) Fig. 4 Total N ( 14 N + 15 N) derived from the organic material in shoots of Plantago and Lolium seedlings grown in monoculture or mixture. Different letters denote significant differences among seedlings at P < 0.05 as determined by Fisher s pairwise comparisons. Data shown are means (n = 16 for monoculture data and n = 8 for mixed culture) with standard errors. interaction. Least 15 N was captured by Plantago when Lolium was the competitor (Fig. 4). Organic material placement also affected the amount of 15 N detected in the shoots, with twice the amount of 15 N detected in the shoots of plants which had Species Root length (m) Root d. wt (g) SRL (m g 1 ) Plantago monoculture 88 (4.8) a 0.52 (0.028) a 170 (7.0) a Lolium monoculture 173 (9.8) c 1.01 (0.052) c 170 (3.2) a Mixed culture 131 (5.1) b 0.81 (0.031) b 162 (2.9) a Table 3 Total root length, d. wt and SRL produced by the different species combinations averaged over all other treatments. SE are in brackets Data were log 10 transformed for statistical analysis. Different letters denote significant differences among species and sections at P < 0.05 as determined by a Duncans Multiple Range test. New Phytologist (2003) 157:

7 Research 309 Fig. 5 Total N ( 14 N + 15 N) captured from the organic material in the absence (open bars) or presence (closed bars) of the mycorrhizal inoculum. Different letters denote significant differences among treatments as determined by a Duncans Multiple Range test. Data are means (n = 8) with standard error bars. received the dispersed organic matter compared with those exposed to a discrete patch (dispersed: 0.08 ± mg 15 N; discrete patch: 0.04 ± mg 15 N). The presence of the mycorrhizal inoculum did not affect the amount of 15 N detected in the shoots. Total d. wt, N content and N capture from the organic material Total plant d. wt was affected only by the species present, being lower in Plantago monocultures (2.2 ± 0.06 g) compared with the mixture or Lolium monocultures which did not differ (mean across these treatments = 2.6 ± 0.08 g). On the other hand total N content of the plants was affected only by the mycorrhizal inoculum, being reduced when the inoculum was present (i.e. with G. hoi inoculum: 34 ± 0.90 mg N; without G. hoi inoculum: 38.5 ± 1.16 mg N). Plants captured more than twice as much N from dispersed organic material than from that added as a discrete patch (i.e. dispersed: 16.7%; discrete patch: 7.9% of the N originally added). Thus, hypothesis (iii) which predicted that plants would capture more N form the organic material added as a patch than that which had been dispersed was incorrect. Moreover, there was no significant interaction between the method of organic material addition and the mycorrhizal treatment suggesting that mycorrhizal treatment did not enhance N capture from the dispersed treatment as had originally been hypothesized. The N captured from the organic material, as a percentage of that added, did not differ between plant species treatments nor was it affected by the mycorrhizal inoculum (mean across treatments = 12.3%); however, the interaction between these two terms was significant (P = 0.021). Both Lolium and Plantago monocultures captured more N from the organic material without mycorrhizal inoculum, but when grown together in a mix culture N capture from the organic material was greater with mycorrhizal inoculum (Fig. 5). Fig. 6 Relationship between plant N capture from the organic material and root length. (a) N captured by plants from the organic material in Plantago monocultures (white symbols), Lolium monocultures (black symbols) and mix culture (grey symbols) against root length. (b) Estimated plant N captured from the organic material against root length in Lolium (black symbols) and Plantago (white symbols) grown in competition only. (c) Same as (b) except showing the influence of the presence of the mycorrhizal inoculum (black symbols) compared to its absence (white symbols). Data are means (n = 4). Analysis of covariance results are given in Table 4. In an analysis of covariance of total N capture ( 14 N + 15 N) from the organic material, root length in the sections to which the organic material had been added was a significant covariate and species composition was a significant factor (Fig. 6a, Table 4a). A Bonferroni means comparison test showed that Plantago differed significantly from both the mixed and Lolium monocultures. Thus, although Plantago monocultures produced absolutely less root length than either Lolium monocultures or the mixed culture, they were more efficient at N capture per unit of root produced (Fig. 6a). In contrast hypothesis (iv) predicted that Lolium monocultures, due to their greater root length, would capture more N from the organic material than Plantago monocultures. In addition, plants captured New Phytologist (2003) 157:

8 310 Research Table 4 Analysis of covariance using total ( 14 N + 15 N) N (mg) captured by the plants as the variate, species, organic material (O.M.) placement or mycorrhiza as the factor and root length in the sections containing O.M. as the covariate Comparison d.f. F 1,8 P (a) Comparison of inter and intraspecific competition (b) Comparison of species in interspecific competition only more N from the dispersed organic material than that added as a patch irrespective of root length produced (Table 4a). There was partial support for hypothesis (iv) from the plant interspecific competition data. When Plantago and Lolium were grown together in competition neither species or organic material placement affected N uptake (Fig. 6b, Table 4b). However root length was a significant covariate, demonstrating that the N captured from the added organic material was dependent on the root length produced and not on the manner in which the organic material had been incorporated, nor on the plant species present. Thus, by virtue of its increased root length, Lolium did capture more N from the organic material than Plantago but only when it was grown in direct competition with Plantago. In interspecific competition, mycorrhizal inoculum was a significant factor and root length was a significant covariate as N capture from the organic material was higher when mycorrhizal inoculum was present (Fig. 6c). Thus, while mycorrhizal inoculum enhanced N capture from the organic material this only occurred when the plants were in interspecific competition and the benefit was to both plant species, not just to Plantago as predicted by hypothesis (iv). However, in an analysis of covariance using plant N capture from the organic material as the variate, neither percentage RLC, number of arbuscules nor external hyphal length density were significant covariates (data not shown). Thus, plant N capture from the organic material was not related to either internal or external mycorrhizal parameters. Discussion Covariate < Species Covariate O.M. Placement < Covariate Mycorrhiza Inoculum Covariate Species Covariate O.M. Placement Covariate < Mycorrhiza Inoculum Root proliferation Although total root length and d. wt were only affected by species composition, localized increased root growth occurred in the sections containing the organic material in agreement with hypothesis (i). Such localized proliferation of roots in nutrient-rich zones is commonly observed and, as occurred in the discrete patch treatment in this study, is often compensated for by a reduction in root growth outside the nutrient-rich area (Fig. 2a; Drew, 1975; Gersani & Sachs, 1992; Hodge et al., 1998). Within nutrient-rich zones a change in root morphology to longer thinner roots is also frequently reported resulting in an increase in SRL (Robinson & Rorison, 1983; Eissenstat & Caldwell, 1988). However, in this study root d. wt increased more than length in the sections containing the organic material, thus SRL decreased where the organic material was most concentrated (Fig. 2b), rather than the increase in SRL which had been predicted by hypothesis (i). AM fungi have previously been demonstrated to both reduce (Cui & Caldwell, 1996) and enhance (Hodge et al., 2000a) root proliferation in nutrient-rich zones. However, in this study, although G. hoi did reduce root length in the top and middle sections, this reduction was not just confined to the sections which contained the organic material. Thus, there was only partial support for the hypothesis that root growth, particularly root length, would decrease when the mycorrhizal inoculum was present, particularly in the sections containing the organic material. Although all species combinations responded to the organic material by increased root growth, Plantago monocultures captured more N per unit of root length produced (Fig. 6a). Thus, there was no support for the fourth hypothesis that Lolium monocultures due to their increased root length would capture more N from the organic material than Plantago monocultures. No 13 C enrichment was detected in the plant tissue, indicating that the N from the organic material was being captured in inorganic N form after microbial decomposition of the organic residue had occurred. It has previously been demonstrated that when plants are grown as individuals (van Vuuren et al., 1996; Fransen et al., 1998; Hodge et al., 1998) or in monoculture (Hodge et al., 2000b) root proliferation in, and N capture from, localized N-rich patches are unrelated. Plants grown in monoculture may capture similar amounts of N from a decomposing organic patch irrespective of their proliferation response as nitrates are soluble in water, and are highly mobile in soil (Tinker & Nye, 2000). Thus, a small amount of root can absorb all the NO 3 available in a matter of a few days. Consequently, increased root growth will not benefit the plant. However, when grown in interspecific competition for a common organic patch then proliferation of roots does confer a competitive advantage; the species which proliferates the most captures more N (Hodge et al., 1999a, 2000b; Robinson et al., 1999). Similarly, in this study the species which proliferated the most in monoculture (Lolium), captured the most N from the organic material when in intraspecific competition, presumably as a direct result of the increased root length produced. Thus although there was no support for hypothesis New Phytologist (2003) 157:

9 Research 311 (iv) from the monoculture data, the increased root length apparently produced by Lolium did confer an advantage when in interspecific competition. As part of the fourth hypothesis it was also predicted that the addition of the G. hoi inoculum would benefit N capture from the organic material by Plantago more than Lolium. Plantago monocultures did have higher AM hyphal length densities and internal mycorrhizal colonization levels than Lolium monocultures, suggesting that Plantago had a greater dependency upon the mycorrhizal partner for nutrient acquisition than Lolium, as expected from their differing root architectures (Newsham et al., 1995b). However, the presence of the G. hoi inoculum affected both plant species in the same manner: reducing N capture from the organic material when in monoculture but increasing N capture by both species when in interspecific competition (Fig. 5). Thus, it is unlikely that the influence by G. hoi was a result of the reduction in root length as, for the reasons discussed previously, this would have been expected to reduce N capture when in interspecific competition rather than increase it. Nor was plant N capture from the organic material related to either internal or external mycorrhizal parameters. Thus, the mechanism by which G. hoi exerted this differing effect upon N capture by plants in monoculture and in interspecific competition is currently unknown and requires further investigation. Mycorrhizal parameters The second hypothesis also predicted that in the sections containing the organic material the reduction in root length due to the presence of the mycorrhizal inoculum would be compensated for by AM hyphal proliferation. However, neither internal mycorrhizal colonization nor external AM hyphal length densities responded to the spatial placement of the organic material. AM fungi have been observed to concentrate hyphae in organic materials (Nicolson, 1959; Hepper & Warner, 1983) and preferential growth of G. hoi hyphae into a compartment containing an organic patch rather than into one containing a potential new host has also been demonstrated (Hodge et al., 2001). Thus, it was surprising that hyphal length densities of G. hoi were unresponsive to the spatial distribution of organic material in this study. St John et al. (1983) found that although AM fungi proliferated hyphae in sterilized organic matter, this proliferation was inhibited by the addition of a soil microbial filtrate. In the present study neither the growth medium or the organic patch material were sterilized as the influence of the G. hoi inoculum in modifying N capture by its host plant when in competition with a native soil microbial community was of interest. It is unlikely however, that the lack of proliferation by the AM fungi in this study was solely due to the presence of the native soil microorganisms, as proliferation of AM hyphae in organic matter has been observed in field-collected samples (Mosse, 1959; Nicolson, 1959) while even in sterilized growth media, three different AM fungi failed to proliferate within a simple organic patch (Hodge, 2001). Although the AM external mycelium is the phase which is in contact with the soil, a reduction in internal mycorrhizal colonization is often observed in fertile soils due to the enhanced nutritional status of the plant (Sanders, 1975; Thomson et al., 1986; Braunberger et al., 1991). Duke et al. (1994) also reported a local reduction in arbuscule frequency in field-grown Agropyron desertorum roots present in nutrient-rich patches containing KH 2 PO 4 and NH 4 NO 3. In this study, mycorrhizal colonization in the middle section was not influenced by the spatial distribution of the organic material even though the presence of the G. hoi inoculum reduced root growth in this section and total plant N content overall. Similarly, colonization of P. lanceolata roots by three different AM fungi had differing effects on the N content of their host but internal colonization was no different in roots experiencing a glycine patch, compared to a water control patch even though overall colonization levels differed among the AM fungi tested (Hodge, 2001). These results imply that internal mycorrhizal colonization levels of roots are generally unresponsive to N-rich patches in soil. Decomposition of, and plant N capture from, the organic material The organic material released more N when dispersed than when added as a discrete patch. Of the N originally added only 35% was recovered from the soil receiving the dispersed material compared with nearly twice as much (i.e. 63%) recovered from the patch soil, although levels of 13 C recovery were similar. The 15 N and 13 C recovered from the tubes receiving the dispersed treatment were multiplied by 2 as only the middle section was analysed. This method assumed that decomposition of the organic material in the top and middle soil section was similar and also did not allow for movement of N throughout the soil profile. At harvest, 71% (discrete patch) and 52% (dispersed) of the 15 N originally added was detected in the soil-plant system, suggesting that some of the original N may have been lost via denitrification and leaching. As previously discussed microbial release of N, predominantly as NO 3, from the organic material was probably important in this study, supported by the fact that only 15 N and no 13 C enrichment was detected in the plant tissue. Therefore, the more rapid release of N from the dispersed organic material was presumably due to the larger surface area accessible for microbial decomposition. As NO 3 is highly mobile in soil it is likely that there was movement of this released N through the soil profile, and any NO 3 ions not captured by the plants would be leached and lost from the soil-plant system. The dispersion of the organic material may also have favoured more rapid microbial turnover as once the smaller fragments of the organic material had been utilized the local increase in microbial growth would no longer be supported. In the patch zone due to the greater concentration of nutrients microbial New Phytologist (2003) 157:

10 312 Research population densities would be expected to be higher and sustained for longer, thus delaying inorganic N release into the soil-plant system. It was surprising that the presence of the G. hoi inoculum did not influence N or C release from the organic material as, by virtue of their size, AM hyphae should have been able to penetrate to the sites of inorganic N release and compete with the other soil microorganisms for the released N. Furthermore, the presence of G. hoi has previously been shown to enhance decomposition of an organic patch (Hodge et al., 2001). However, in that study the AM hyphae proliferated extensively throughout the organic material. In close agreement with the decomposition data, but in contrast to the predictions of hypothesis (iii), plant N capture from the dispersed organic material was more than twice that from the discrete patch. Bonkowski et al. (2000) found that L. perenne plants captured more N from organic material added as a discrete patch than that which had been more evenly dispersed throughout the soil, whereas Hodge et al. (2000b) found no difference in plant N capture by either L. perenne or Poa pratensis regardless of the spatial distribution of organic material. Similarly Cui & Caldwell (1996) found no difference in plant 15 NO 3 acquisition due to N distribution patterns. These contrasting results may be related to a wide range of experimental conditions including the degree of competition between plants and microorganisms, the C : N ratio of the material added, the amount of N added relative to the background concentrations and experimental time scales. Plants captured more N from organic patches of varying physical and chemical complexity when the patches had a low C : N ratio; the patches with a higher C : N ratio were still being decomposed by the microbial community (Hodge et al., 2000c,d). The contribution that this captured N makes to total plant N, rather than the amount captured per se, will determine the response of the plant and the sensitivity of roots to the spatial scale of the organic material placement. In the study by Bonkowski et al. (2000) N capture from the organic material was only 9% of the N originally added but this consituted 15% of the total plant N. In contrast, in the study by Hodge et al. (2000b) plant N capture from the organic material was higher (i.e. 26%) but represented only 1% of the total plant N, which may also explain why root growth did not respond to the spatial placement of organic material as levels were insufficiently different to background to be detected as N-rich zones. In the present study, of the N originally added in the organic material, 8% was captured from the patch treatment compared to 17% capture from the dispersed treatment which corresponded to 2.0% and 4.1% of the total plant N, respectively. Although these values appear low, particularly in the case of the patch treatment, they were apparently sufficient to evoke enhanced root growth in response to the organic material (Fig. 2a) and this was related to N capture from the added material (Fig. 6). Hypothesis (iii) also predicted that more N would be captured from the dispersed organic material treatment when the AM inoculum was present due to the spatial placement of the fungal hyphae. It is well established that AM fungi benefit their host plant by capturing immobile phosphate sources by exploiting a larger volume of soil outwith the nutrient depletion zone around the root surface (Smith & Read, 1997). In this study however, neither AM colonization nor external hyphal length densities influenced N capture from the organic material regardless of its spatial placement. As shown by the decomposition data the dispersed organic material was decomposing rapidly and releasing N, presumably as NO 3. As NO 3 is much more mobile in soil it would readily be available for plant N capture regardless of its initial placement in the tube. Similarly, Cui & Caldwell (1996) observed that AM colonization did not influence plant NO 3 acquisition despite differences in N distribution patterns. Conclusions Few of the initial hypotheses tested were supported. Root growth did increase in the sections containing the organic material (hypothesis (i)) and although this was reduced in the presence of the G. hoi inoculum it was not replaced by AM hyphal proliferation (hypothesis (ii)). Contrary to hypothesis (iii) plants captured more than double the amount of N from the dispersed organic material than from that added as a discrete patch, and the presence of G. hoi did not enhance N capture from the dispersed organic material. The final hypothesis was also not supported, as while Lolium monocultures did produce greater root lengths overall, it was only when grown in interspecific competition that increased root length conferred a competitive advantage. The influence of the neighbouring plant upon N capture from the organic material was clearly seen in the shoot data (Fig. 4) with Plantago capturing less N when grown with Lolium. In addition, it was particularly striking that despite the differences in AM internal colonization and external hyphal length densities produced, and the differences between Lolium and Plantago in root architecture, G. hoi had the same effect on both plant species: it decreased N capture from the organic material when plants were grown in monoculture and increased N capture when in interspecific plant competition. The mechanism by which G. hoi exerted this differing influence on plant N capture from the organic material however, is unclear. Tibbett (2000) suggested that root proliferation responses to soil heterogeneity had been overestimated as their associated mycorrhizal partner would proliferate hyphae instead. In this study roots responded to the spatial distribution of the organic material added but AM hyphae did not. Furthermore, the response of the roots was demonstrably more important in N capture from the organic material than that of the fungal partner (Fig. 6). In a previous study, three different AM fungi did not respond to a glycine patch by the proliferation of hyphae but the roots of their host did (Hodge, 2001). In the study by Hodge et al. (2001) where the fungus did benefit its New Phytologist (2003) 157:

11 Research 313 host in terms of N capture from the organic material, only the AM hyphae were allowed access to the organic material. Thus, when both the fungus and its associated host are experiencing an N-rich patch the response of the plant appears more important, whereas when roots are excluded the response of the AM fungus may be more important. In the soil environment both situations may arise. Acknowledgements 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 A. H. Fitter, I. J. Alexander, K. S. Pregitzer and an anonymous referee for their perceptive comments on the manuscript. References Ames RN, Reid CPP, Porter L, Cambardella C Hyphal uptake and transport of nitrogen from two 15 N-labelled sources by Glomus mosseae, a vesicular-arbuscular mycorrhizal fungus. New Phytologist 95: Bending GD, Read DJ The structure and function of the vegetative mycelium of ectomycorrhizal plants. V. Foraging behaviour and translocation of nutrients from exploited litter. New Phytologist 130: Bonkowski M, Griffiths BS, Scrimgeour C Substrate heterogeneity and microfauna in soil organic hotspots as determinants of nitrogen capture and growth of rye-grass. Applied Soil Ecology 14: Braunberger PG, Miller MH, Peterson RL Effect of phosphorus nutrition on morphological characteristics on vesicular arbuscular mycorrhizal colonization of maize. New Phytologist 119: Cahill JF, Casper BB Growth consequences of soil heterogeneity for two old-field herbs, Ambrosia artemisiifolia and Phytolacca americana, grown individually and in combination. Annals of Botany 83: Cliquet JB, Murray PJ, Boucaud J Effect of the arbuscular mycorrhizal fungus Glomus fasciculatum on the uptake of amino nitrogen by Lolium perenne. New Phytologist 137: Cui M, Caldwell MM Facilitation of plant phosphate acquisition by arbuscular mycorrhizas from enriched soil patches. I. Roots and hyphae exploiting the same soil volume. New Phytologist 133: Drew MC Comparison of the effects of a localized supply of phosphate, nitrate, ammonium and potassium on the growth of the seminal root system, and the shoot, in barley. New Phytologist 75: Duke SE, Jackson RB, Caldwell MM Local reduction of mycorrhizal arbuscule frequency in enriched soil microsites. Canadian Journal of Botany 72: Eissenstat DM, Caldwell JP Seasonal timing of root growth in favourable microsites. Ecology 69: Fitter AH Costs and benefits of mycorrhizas: implications for functioning under natural conditions. Experientia 47: Fitter AH, Hodge A, Robinson D Plant response to patchy soils. In: Hutchings MJ, John EA, Stewart AJA, eds. The ecological consequences of environmental heterogeneity. Oxford, UK: Blackwell Science Ltd, Fransen B, de Kroon H, Berendse F Root morphological plasticity and nutrient acquisition of perennial grass species from habitats of different availability. Oecologia 115: Gersani M, Sachs T Developmental correlations between roots in heterogeneous environments. Plant, Cell & Environment 15: Hepper CM, Warner A Role of organic matter in growth of a vesicular arbuscular mycorrhizal fungus in soil. Transactions of the British Mycological Society 81: Hodge A Arbuscular mycorrhizal fungi influence decomposition of, but not plant nutrient capture from, glycine patches in soil. New Phytologist 151: Hodge A, Campbell CD, Fitter AH An arbuscular mycorrhizal fungus accelerates decomposition and acquires nitrogen directly from organic material. Nature 413: Hodge A, Robinson D, Fitter AH. 2000a. An arbuscular mycorrhizal inoculum enhances root proliferation in, but not nitrogen capture from, nutrient-rich patches in soil. New Phytologist 145: Hodge A, Robinson D, Griffiths BS, Fitter AH. 1999a. Why plants bother: root proliferation results in increased nitrogen capture from an organic patch when two grasses compete. Plant, Cell & Environment 22: Hodge A, Robinson D, Griffiths BS, Fitter AH. 1999b. Nitrogen capture by plants grown in N-rich organic patches of contrasting size and strength. Journal of Experimental Botany 50: Hodge A, Stewart J, Robinson D, Griffiths BS, Fitter AH Root proliferation, soil fauna and plant nitrogen capture from nutrient-rich patches in soil. New Phytologist 139: Hodge A, Stewart J, Robinson D, Griffiths BS, Fitter AH. 2000b. Spatial and physical heterogeneity of N supply from soil does not influence N capture by two grass species. Functional Ecology 14: Hodge A, Stewart J, Robinson D, Griffiths BS, Fitter AH. 2000c. Competition between roots and soil micro-organisms for nutrients from nitrogen-rich patches of varying complexity. Journal of Ecology 88: Hodge A, Stewart J, Robinson D, Griffiths BS, Fitter AH. 2000d. Plant N capture and microfaunal dynamics from decomposing grass and earthworm residues in soil. Soil Biology and Biochemistry 32: Kormanik PP, McGraw A-C Quantification of vesicular arbuscular mycorrhizae in plant roots. In: Schenck NC, ed. Methods and principles of mycorrhizal research. St Paul, MN, USA: American Phytopathological Society Mäder P, Vierheilig H, Streitwolf-Engel R, Boller T, Frey B, Christie P, Wiemken A Transport of 15 N from a soil compartment separated by a polytetrafluoroethylene membrane to plant roots via the hyphae of arbuscular mycorrhizal fungi. New Phytologist 146: McGonigle TP, Miller MH, Evans DG, Rairchild GL, Swan JA A new method which gives an objective measure of colonization of roots by vesicular arbscular mycorrhizal fungi. New Phytologist 15: Merryweather JW, Fitter AH A modified method for elucidating the structure of the fungal partner in vesicular arbuscular mycorrhiza. Mycological Research 95: Miller RM, Jastrow JD Extraradical hyphal development of vesicular arbuscular mycorrhizal fungi in a chronosequence of prairie restoration. In: Read DJ, Lewis DH, Fitter AH, Alexander IJ, eds. Mycorrhizas in ecosystems. Wallingford, UK: CAB International, Mosse B Observations on the extramatrical mycelium of a vesicular arbuscular endophyte. Transactions of the British Mycological Society 42: Näsholm T, Ekblad A, Nordin A, Giesler R, Högberg M, Högberg P Boreal forest plants take up organic nitrogen. Nature 392: Newsham KK, Fitter AH, Watkinson AR. 1995a. Arbuscular mycorrhiza protect an annual grass from root pathogenic fungi in the field. Journal of Ecology 83: Newsham KK, Fitter AH, Watkinson AR. 1995b. Multi-functionality and biodiversity in arbuscular mycorrhizas. Trends in Ecology and Evolution 10: Nicolson TH Mycorrhiza in the Gramineae. I. Vesicular-arbuscular endophytes, with special reference to the external phase. Transactions of the British Mycological Society 42: Pregitzer KS, Kubiske ME, Yu CK, Hendrick RL Relationships among root branch order, carbon, and nitrogen in four temperate species. Oecologia 111: New Phytologist (2003) 157: