Journal of Experimental Botany, Vol. 54, No. 391, pp. 2331-2342,
October 1, 2003
© 2003 Oxford University Press
N capture by Plantago lanceolata and Brassica napus from organic material: the influence of spatial dispersion, plant competition and an arbuscular mycorrhizal fungus
Received 21 February 2003; Accepted 12 June 2003
Department of Biology, Area 2, The University of York, PO Box 373, York YO10 5YW, UK
* Fax: +44 (0)1904 328510. E-mail: ah29{at}york.ac.uk
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Abstract |
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This study investigated N capture by Plantago lanceolata L. and Brassica napus L. from complex organic material (dual-labelled with 15N/13C) added either as a thin concentrated layer (discrete patch treatment) or dispersed uniformly with the background sand:soil mix in a 10 cm band (dispersed treatment) when grown in monoculture or in interspecific competition and in the presence or absence of a mycorrhizal inoculum (Glomus mosseae). No 13C enrichments from the organic material were detected in the plant tissues, but 15N enrichments were present. Total plant uptake of N from the organic material on a microcosm basis was not affected by the spatial placement of the organic material, but Plantago monocultures captured less N than the species in interspecific competition (i.e. 23% versus 38% of the N originally added). N capture from Brassica monocultures was no different to either Plantago monocultures or both species in mixture. However, N capture from the organic material by both individual Plantago and Brassica plants was reduced when grown with Brassica plants (by 10-fold and by more than half, respectively). N capture from the organic material was directly related to the estimated root length produced in the sections containing the organic material: the individual that produced the greatest root length captured most N. Strikingly, when the organic material was added as a discrete patch the N captured by Brassica, a non-mycorrhizal species, actually increased when the G. mosseae inoculum was present compared to when G. mosseae was absent (i.e. 35% versus 19% of the N originally added).
Key words: Arbuscular mycorrhizal fungus, Brassica napus L., decomposition, nitrogen capture, organic material, Plantago lanceolata L., root demography.
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Introduction |
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Nutrient supply in the soil environment is extremely heterogeneous, varying both spatially and temporally at scales relevant to plant roots. Plant root systems are physiologically and morphologically plastic enabling them to respond to such variations in nutrient supply, for example, showing large, transient, increases in uptake kinetics (Jackson et al., 1990; Jackson and Caldwell, 1991) and proliferation of laterals within nitrogen- or phosphorus-rich zones (Robinson and van Vuuren, 1998; Linkohr et al., 2002). Either or both of these responses can occur in the same part of the root system experiencing the nutrient-rich zone with the physiological response usually occurring first (Drew and Saker, 1978; van Vuuren et al., 1996) but, perhaps because it can be so visibly spectacular, it is the proliferation of roots that has received most attention. Although root proliferation in response to inorganic and organic N-rich zones is easily shown, demonstrating a benefit in terms of N capture has proved more troublesome since there is often poor, or no, correlation between root length density produced, and the subsequent N captured, by either individual plants (van Vuuren et al., 1996; Hodge et al., 1998; Fransen et al., 1998) or monocultures (Wiesler and Horst, 1994; Hodge et al., 2000a). Thus the rationale behind why root systems showed such a large and specific response to patches rich in N and, in particular, the highly mobile NO3– ion, seemed obscure (Robinson, 1996). However, it has subsequently been shown both experimentally (Hodge et al., 1999a) and by simulation modelling (Robinson et al., 1999) that when plants are grown in interspecific competition for a common N-rich patch, root proliferation does confer a direct benefit to the plant: the species which proliferate the most, capture most of the available N. Root proliferation responses, however, are probably more important in plant–plant competition than in plant-microbial competitive interactions, because root proliferation generally takes considerable time to occur, during which time the microbial biomass may have turned over many times (Hodge et al., 2000b).
In addition to the response of roots, resource acquisition by the plant can be influenced by interaction with mycorrhizal symbionts. Although most plant species form mycorrhizal associations, their influence on the response of roots to soil heterogeneity and the implications for resource capture from these nutrient-rich patches has seldom been investigated. Tibbett (2000) proposed that the importance of root proliferation has been overestimated because mycorrhizal hyphal proliferation would occur instead. However, while mycorrhizal fungi do proliferate hyphae in organic matter patches (Nicolson, 1959; St John et al., 1983; Hodge et al., 2001) there is currently no evidence to suggest that AM hyphal proliferation replaces that of root proliferation (Hodge, 2001, 2003). AM fungi may enhance nutrient capture for their associated host plant from heterogeneous patches in other ways. For example, by virtue of their size the AM hyphae may be better able to penetrate to the sites of decomposition within the organic patch and thus be better able to compete with the rest of the soil microbial community for the inorganic N released. There is also some evidence to suggest that AM fungi may be able to take up simple forms of organic N, such as amino acids, intact and transfer this organic N to their associated host plant (Cliquet et al., 1997; Näsholm et al., 1998). However, while it has been established that AM fungi can transfer N from inorganic N sources to their host (Ames et al., 1983; Mäder et al., 2000) their ability to take up organic N sources intact remains a matter for debate (Smith and Read, 1997). In addition, colonization by an arbuscular mycorrhizal fungus, Glomus mosseae, has been demonstrated to increase root proliferation within an organic patch (Hodge et al., 2000c). Finally, the AM fungus, Glomus hoi, was found to enhance decomposition of a complex organic patch in soil (Hodge et al., 2001). Thus, AM fungi may enhance N capture for their host directly through being better able to compete with the other members of the microbial community and/or by the capture of simple organic N compounds intact, or indirectly through modifications of the root response. In addition, enhanced decomposition of the organic patch material by AM fungi could potentially benefit other plants present in the patch regardless of their mycorrhizal status.
The aims of this study were to examine how two plant species, one with the ability to form the AM association (Plantago lanceolata L.), the other a non-mycorrhizal species (oil-seed rape, Brassica napus L.) captured N from complex organic material (Lolium perenne L. shoots) added to soil either as a discrete patch or more uniformly dispersed, in the presence or absence of a mycorrhizal inoculum. Nitrogen capture rather than phosphorus (P) was the focus of this study because N is a key limiting nutrient in terrestrial ecosystems and because, unlike enhanced capture of P, the ability of AM fungi to capture N, particularly from organic sources, is still a controversial area. The organic material added was dual-labelled with 13C and 15N to enable the decomposition of the material and its subsequent capture by the plants to 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. The plants were grown in inter- or intraspecific competition. Root demography within the added organic material and the impact of an added mycorrhizal inoculum upon it, was also followed. Specifically, the following hypotheses were tested: (i) in the presence of a potential host plant less 15N would remain in the soil when the mycorrhizal inoculum was present, as decomposition of the organic material would be increased by the AM fungus. This effect would be greater when the organic material was added as a discrete patch due to the proliferation of AM hyphae within the patch. The presence of the AM inoculum would have no effect on the amount of 15N recovered in the soil from B. napus monocultures. (ii) Root length and dry weight in all species combinations would be greatest where the organic material was most concentrated. Specifically, root morphology would alter, resulting in longer thinner roots in response to the organic material and an increase in specific root length (SRL). (iii) The presence of the mycorrhizal inoculum would further stimulate new root production in the sections containing the organic material as a discrete patch, but the non-mycorrhizal plant would be unaffected; in mixed plant cultures new root production would be intermediate between that produced in the monocultures. (iv) Plants would capture more N from the organic material added as a patch than that which had been dispersed, due to the ability of the roots to detect the concentrated N-rich zone and proliferate roots within it. The species that produced most root length in mixed cultures would gain most N from the organic material. (v) In the presence of the mycorrhizal inoculum, N capture from the organic material would increase due to the AM fungus promoting root proliferation within the patch. This enhanced N capture would be greatest for P. lanceolata monocultures as more potential host plants would be present, but would also benefit the P. lanceolata seedling grown in interspecific plant competition. (vi) The AM fungus would enhance N capture from the organic material for its host plant (P. lanceolata) by the direct uptake of simple organic N from the added organic material.
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Materials and methods |
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Microcosm design
Plants were grown in microcosm tubes made out of a section of PVC pipe (length 20 cm, internal diameter 10 cm) which had two holes cut to allow the insertion of a glass minirhizotron tube (25 cm long x 2.2 cm external diameter) diagonally across the centre of the tube at an angle of 45° to the horizontal. The minirhizotron tubes were sealed with rubber bungs. At the top of each microcosm tube, the top 2 cm section of a PVC funnel (internal diameter 10 cm at top and 7 cm at the base) was placed to direct the roots into the middle section of the tube where the organic material was to be inserted. A wooden pole (15.5 cm long x 2.7 cm diameter) fitted with a plastic attachment at its base shaped to fit securely on to the minirhizotron tube was initially placed vertically from the soil surface to a depth of 12 cm and was removed at a later time to allow the organic material to be added. This allowed precise placement of the organic material once the seedlings had developed suitably, whilst ensuring minimal disturbance to the system. Each microcosm tube was filled with a 50:50 mixture of sand:soil (as described by Hodge et al., 1999b) containing the mycorrhizal inoculum (fresh or autoclaved) and the microcosm unit was then ready for planting.
Eight microcosm tubes were contained within six large (60x40x30 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 100 g wet weight of Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe, isolate UY 21 inoculum added to the sand:soil medium. The non-mycorrhizal controls received 100 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 colonized with G. mosseae 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. seeds (supplied by Emorsgate Seeds, Norfolk, UK) were planted into the microcosm tubes on 6 August 2001. Brassica napus L. seeds (spring oilseed rape line, kindly supplied by Professor Ottoline Leyser, University of York) were planted 4 d later on 10 August 2001. All P. lanceolata seeds had germinated after 1 week while all B. napus seeds had germinated after 3 d. Each microcosm unit contained two seedlings in one of three species combinations: either two P. lanceolata or two B. napus seedlings as monocultures (in intraspecific competition) or one seedling of each in a mixed culture (in interspecific competition). Twenty days after the P. lanceolata seeds (16 d after the B. napus seeds) were planted the organic material was added. The experiment ran for 21 d between 26 August and 16 September 2001. The mean daily temperature over the duration of the experiment was 19.3 °C (SE ±0.04) with a mean daily maximum of 31.6 °C (SE ±0.67) and mean daily minimum temperature of 16.6 °C (SE ±0.15). Photosynthetically active radiation (PAR) flux was recorded weekly at noon and averaged 481 µmol m–2 s–1 at plant level.
Organic material addition
The organic material added to the microcosm tubes was 0.6 g oven-dried finely milled L. perenne shoot material labelled with both 15N and 13C produced as described in Hodge et al. (1998). The organic material was placed in the space created by removal of the wooden tube and was added either as a thin, concentrated zone at 12 cm depth (‘patch’ treatment) or dispersed uniformly with the background sand:soil mix in an 8 cm long column starting 4 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 DW of the sand:soil mix. The organic material added to the tubes contained 25 mg N (1.15 mg 15N) and 263 mg C (3.80 mg 13C) with a C:N ratio of 10.5:1. There were four replicate tubes for each combination of species, organic material placement, and mycorrhizal treatment.
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 videocassette recorder (model No.GV-D900E) at 0, 6, 13, and 20 d after the initial addition of organic material. 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 that differences with time could be followed. For this analysis, data were gathered from frame 5 (12 cm depth), the site of ‘discrete patch’ addition and dispersed material. At day 0 no roots were observed after removal of the wooden pole, thus all the data for the roots 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
At harvest, each soil core was removed intact from its tube and then cut into four sections, extreme top 2 cm and top, middle and bottom, 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. As it was impossible to separate out the roots of B. napus from P. lanceolata when grown in interspecific competition the root length produced by each species was estimated by two methods: in both the ratio of total root length (Lr) in the organic material (OM) sections to total shoot DW (Ws) was multiplied by Ws for the individual species in competition. In method 1, the ratio Lr:Ws was calculated from the monoculture data, and in method 2 from the mixed plant culture data. In both cases the fraction of the N captured from the OM and retained in the roots was estimated for the two species from the estimated Lr fraction for that species. This assumes that the root systems of both plant species were equally effective at capturing the N from the organic material, which may not be true, however, most (i.e. >70%) of the N captured from the organic material is detected in the shoots rather than the roots which could be easily identified and separately analysed. To compare the seedlings grown in competition with those grown in monoculture, the root length and total N capture for each of the two seedlings grown in monoculture was also estimated using the same calculation as method 1.
After root length in each of the sections had been determined a subsample of material was taken from the middle 6 cm section only for mycorrhizal assessment. The remaining root material from the different sections was then oven-dried at 60 °C and weighed before being combined for milling. A subsample of the milled root, shoot and soil material from the top and middle sections 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 and McGraw (1982) but without phenol). Although roots from B. napus monocultures were also checked for mycorrhizal colonization in mixed culture, only roots which were obviously from P. lanceolata seedlings were selected to enable comparison with colonization levels when P. lanceolata was grown in monoculture. Mycorrhizal colonization was examined with a Nikon Optiphot-2 microscope using brightfield and epifluorescence (Merryweather and Fitter, 1991) and x200 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.
Statistical analysis
Data were analysed using the General Linear Model (GLM) factorial design command or, for the root data in the individual sections and the root demography data, the GLM repeated measurements command, in SPSS v. 10.0. 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 or the Duncans’ multiple range test was applied. In addition, root birth rates were estimated from linear regressions of mean cumulative root births and significant differences between fitted lines compared using the F-ratio method (Sokal and Rohlf, 1981; Potvin et al., 1990). For the root data monocultures were directly compared with the mixed culture unless otherwise stated. Data for P. lanceolata and B. napus were analysed separately, except for the shoots where data for only one seedling in each case (i.e. P. lanceolata or B. napus) was used and the shoot 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. All data were checked and transformed appropriately to normalize skewed distributions before statistical analysis. In all cases, a randomized block design was used.
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Results |
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Patch decomposition
At harvest, soil from the sections which had originally received the organic material additions still contained 13C and 15N levels higher than background controls. In addition, there was a linear relationship between the amount of mg 13C and mg 15N remaining in the soil from both the discrete patch and dispersed organic material, showing that N and C release were related (Fig. 1). Little 13C was recovered in the soil, but there was more in the patch treatment (7±1.1% of the original quantity) than the dispersed (3±0.3%). By contrast with the low levels of 13C, more 15N was recovered although, again, soil receiving the discrete patch had more remaining (52±3.4%) compared to the dispersed material (25±1.8%), and the relationship between N and C recovery was quite distinct (Fig. 1). The amount of 15N recovered was also influenced by other factors (Table 1), with the main effect being the 3-way interaction (OM placement x mycorrhiza inoculum x plant species composition, Fig. 2) due to the very high 15N recovery from mycorrhizal patch treatments with P. lanceolata present.
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Root length, DW, specific root length (SRL) and mycorrhizal colonization
Root length was highest in the middle section containing the organic material added as a discrete patch, but there were no other treatment differences in root length produced (Fig. 3). Root weight in the sections followed the same pattern as root length; consequently specific root length (SRL), and by implication root morphology, were unaffected.
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P. lanceolata monocultures (1.3±0.10 g) produced less total root weight than either the mixed species culture or B. napus monocultures (mean for the two treatments 2.2±0.10 g). The G. mosseae inoculum reduced the N content of the root material (i.e. 22.3±1.79 mg N without G. mosseae; 17.6±0.97 mg N with G. mosseae). There was also a significant interaction between species composition and mycorrhiza: G. mosseae inoculum reduced the N content of both P. lanceolata and B. napus roots in monoculture, but increased it in the mixed roots from the intraspecific competition treatment (data not shown).
Although AM hyphae could be clearly seen on the surface of B. napus roots, no internal colonization occurred. For P. lanceolata roots grown without G. mosseae inoculum only 7% of root length was colonized compared to 20±1.3% with the inoculum. Neither spatial placement of the organic material nor the identity of the competitor species affected % root length colonized (RLC). Frequency of arbuscules followed the same pattern as RLC, but were more frequent in P. lanceolata roots grown in monoculture (i.e. 3.5±0.65% in monoculture; 1.3±0.37% in interspecific competition). Vesicles were rarely observed.
Root demography
At day 20, P. lanceolata monocultures had produced fewer new roots (2.9±0.40) than the mixed cultures (4.4±0.45). There was a significant interaction between species x mycorrhizal inoculum x OM placement (P=0.048): P. lanceolata monocultures, root birth rates were higher in the patch + mycorrhizal treatment than in any of the other three treatments (slope 0.228 compared to 0.128 for the combined other treatments; Fig. 4A). In the B. napus monocultures, root births were also highest in the patch + mycorrhizal treatment, but the difference only became significant at the last recording date (Fig. 4B). The rate of root production in the mixed culture tubes were not affected either by organic material placement or by the mycorrhizal inoculum (slope=0.236 across all treatments; Fig. 4C).
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Shoot data
Shoot DW was influenced by the seedling and the competitor present (P <0.001). B. napus seedling shoots were heavier than P. lanceolata shoots (i.e. 3.4±0.31 g B. napus shoot DW; 0.6±0.10 g P. lanceolata shoot DW), but when B. napus was the competitor, shoot DW was less than when P. lanceolata was the competitor (i.e. 1.8±0.28 g with B. napus as the competitor cf. 2.1±0.45 g with P. lanceolata as the competitor). The N content of the shoots followed a similar pattern being highest in B. napus seedlings compared with P. lanceolata, but reduced when B. napus was the competitor seedling. N concentration of the shoots, however, was only influenced by the competitor present, being highest when P. lanceolata was the competitor compared with B. napus (i.e. 26.5±1.11 mg N g–1 with P. lanceolata as competitor; 19.5±0.53 mg N g–1 with B. napus as competitor). In addition, for shoot DW there was a significant interaction between the seedling present and the organic material. Although the placement of organic material did not influence B. napus shoot DW, the dry weight of P. lanceolata shoots was almost twice as great in the patch as compared with the dispersed treatment.
Total DW, N content and N concentration
Total plant DW, N content and N concentration were only affected by the species present. DW was lower in P. lanceolata monocultures (2.9±0.23 g) than in either the mixture or B. napus monocultures which did not differ (mean for these treatments 6.9±0.23 g). N concentration was lowest in the B. napus monocultures (16.6±0.63 mg N g–1) compared with the mixed or P. lanceolata monocultures (mean for these treatments 20.9±0.61 mg N g–1). By contrast, total N contents in P. lanceolata monocultures (62.5±5.38 mg N) were less than half those obtained in the mix and B. napus monoculture tubes (mean across these treatments 127.0±6.83 mg N).
N capture from the organic material
15N enrichment was detected in the plant tissue, but 13C enrichment was not. Although there was no difference in the total amount of N captured from the organic material by P. lanceolata and B. napus grown in monoculture, the mixed culture captured more N from the organic material than that by P. lanceolata monocultures (Fig. 5). N capture from the organic material was only affected by the plant species present, and not by its spatial placement, the presence of the mycorrhizal inoculum or total root length in the organic material containing sections.
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The mycorrhizal inoculum did, however, increase the percentage of the total plant N that was derived from the organic material (i.e. 8.5±0.50% with and 6.7±0.35% without G. mosseae). This percentage was also greater for P. lanceolata monocultures (i.e. 9.3±0.64% of the total N) compared with either the B. napus monocultures or the mixed species (mean across these treatments: 6.7±0.27% of the total N).
Total N (14N+15N) capture from the organic material was a simple function of the estimated root length produced, irrespective of the method used to estimate root length for the individual B. napus and P. lanceolata seedlings in interspecific competition (see Materials and methods): data from the second method of estimating root length are shown in Fig. 6. Although B. napus plants generally captured more N from the organic material than P. lanceolata (Fig. 6), there was no significant difference between these two species in the N captured per unit of root length (mean across treatments: 4.7±0.70 mg N m–1); thus B. napus only captured more N because of the greater root length produced.
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Of the N originally added in the organic material c. 38% was captured by the plants grown in intraspecific competition (Fig. 5) of which the great majority (37%) was captured by B. napus seedlings. There were no significant differences in the N captured from the organic material by P. lanceolata or B. napus due to either the spatial placement of the organic material or the presence of the mycorrhizal inoculum.
As there was such a large size difference between P. lanceolata and B. napus seedlings when grown in interspecific competition, these two species could not be compared directly due to the non-normal distribution of the data. Thus, instead, comparisons between individual P. lanceolata or B. napus plants in monoculture and interspecific competition were made. N capture from the organic material by individual P. lanceolata seedlings was only affected by the competitor plant present (P <0.001), being reduced 10-fold when B. napus was the competitor (Fig. 7A). N capture from the organic material by individual B. napus seedlings was also reduced by more than half when B. napus was the competitor (Fig. 7B). In addition, for B. napus seedlings, there was also a significant interaction between organic material placement and the presence of the G. mosseae inoculum (P =0.018), because the presence of the mycorrhizal inoculum increased N capture from the organic material added as a discrete patch compared with when the G. mosseae inoculum was absent (Fig. 7C).
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The C:N ratio of the added substrate has a large influence on its subsequent decomposition and determines if net mineralization or immobilization of N occurs (Berg and Staaf, 1981). In this study, the C:N ratio of the organic material was 10:1, which is considerably lower than the C:N ratio of >20–30:1 where generally net immobilization of N occurs (Bartholomew, 1965). Thus, the organic material added would be expected to be decomposing and releasing N and C throughout the course of this experiment as shown by the reduced levels of 15N and 13C detected at final harvest. However, although the C:N ratio of the substrate was the same, recoveries of 13C and 15N from the organic material added as a discrete patch were twice that from the dispersed material, implying that decomposition of the dispersed material was faster. Presumably this was due to the larger surface area accessible for microbial decomposition as there was no significant difference in the C:N ratio at final harvest.
Hypothesis (i)
Hypothesis (i) predicted that in the presence of a potential host plant, the amount of 15N recovered from the soil would be reduced when G. mosseae was present, as decomposition of the organic material would be increased by the AM fungus, particularly when the organic material was added as a discrete patch, due to AM hyphal proliferation within the organic patch. Further, the presence of the AM inoculum would have no effect on the amount of 15N recovered in the soil from B. napus monocultures. However, there was little support for this hypothesis: although the G. mosseae inoculum did not influence the amount of 15N recovered from the patch when B. napus monocultures were grown, the 15N recovered from the patch soil actually increased when P. lanceolata was present. Thus, rather than increasing N release from the organic material, more N was retained in the soil when G. mosseae was present. Some of this N may have been taken up by the AM fungal hyphae and released back into the soil–plant system when hyphal turnover occurred, which might benefit plants by reducing N losses through leaching and denitrification processes.
Hypothesis (ii)
Hypothesis (ii) predicted that both root length and DW would be greatest where the organic material was most concentrated. Root length was highest in the middle section containing the organic material added as a discrete patch, but there were no other differences in root length produced. Thus root length only responded to the most concentrated organic material and not to that which was more spatially dispersed. Although localized root proliferation to extreme nutrient concentrations has been convincingly demonstrated (Drew and Saker, 1978; Robinson, 1994; Robinson and van Vuuren, 1998) it was surprising that, within the more spatially dispersed organic material, roots did not similarly show an, albeit less spectacular, proliferation response as demonstrated in a previous study using P. lanceolata seedlings (Hodge, 2003). However, Cui and Caldwell (1996) also observed that, in uniformly fertilized pots, root length density was similar to soil containing no nutrient enrichments, but when nutrients were added as a discrete patch localized root length densities increased. Hypothesis (ii) also predicted that SRL would increase, as a change in root morphology to longer thinner roots is frequently observed in nutrient-rich zones (Robinson and Rorison, 1983; Eissenstat and Caldwell, 1988), but this did not occur.
Hypothesis (iii)
The G. mosseae inoculum did not influence root length produced in the sections containing the organic material regardless of its spatial distribution. By contrast, Cui and Caldwell (1996) found AM colonized roots produced less root length density in P and NO3–-rich patches compared to non-mycorrhizal controls. However, G. mosseae did stimulate new root production by P. lanceolata in the discrete patch as demonstrated by the root demography data, thus giving partial support for hypothesis (iii). Similarly, Hodge et al. (2000c) observed that G. mosseae stimulated new root production by individual P. lanceolata seedlings when grown in the presence of an organic N-rich patch. However, the stimulated root production observed in the P. lanceolata monocultures was not observed when P. lanceolata and B. napus were grown together in mixed culture. The rate of root production in the mixed culture tubes were not affected either by organic material placement or by the presence of the mycorrhizal inoculum. Thus, new root production was clearly different under the conditions of interspecific plant competition and not simply intermediate between that produced by B. napus and P. lanceolata monocultures.
Hypothesis (iv)
Plants captured 23% and 38% of the N added in the organic material. Such N capture values are consistent with other studies on similar organic material: at low C:N ratios (i.e. <4) plants captured >50% of the N originally added in the organic substrate after 49 d, but at higher C:N ratios of 21:1 and 31:1 plant N capture was only 11% after 49 d and >6% after 35 d, respectively (Hodge et al., 1998, 2000d). However, despite the differences in 15N recovery from the soil receiving the patch and dispersed organic material and the proliferation of roots within the patch zone, plant N capture from the organic material was not affected by its spatial placement. Thus, there was no support for hypothesis (iv), that more N would be captured from the discrete patch due to the proliferation of roots within the patch zone. This result was not unexpected as previous studies have shown that when plants are grown as individuals (van Vuuren et al., 1996; Hodge et al., 1998; Fransen et al., 1998) or in either monoculture or mixed plant culture (Hodge et al., 2000a) total root length in, and N capture from, N-rich zones are unrelated. When plants are grown as individuals they may capture similar amounts of N from patches of decomposing organic material, irrespective of the root length they produce within the patch, as the NO3– released has a diffusion coefficient of c. 10–10 m2 s–1 (Tinker and Nye, 2000) and thus is highly mobile in soil. Therefore, a small amount of root can absorb all the NO3– released after a few days. Root proliferation is more important in plant–plant than plant–microbial competitive interactions (Hodge et al., 2000b). Thus, where plants are grown as individuals, without competitive interactions with other roots systems, they will capture the majority of the NO3– released and available for uptake, irrespective of the root length produced. Consequently, any increase in root length will not benefit the plant further. In the natural environment, however, plants grow in communities and it is under these conditions that the root proliferation response has evolved. Moreover NO3– concentrations are often lowest in soil containing the most diverse plant communities (Tilman, 1989; Tilman et al., 1996). This may be related to the differing abilities of the community plant members to exhibit a proliferation response as it has been demonstrated that when plants are grown together in interspecific competition for a common organic patch then there was a direct relationship between root length each species produced and the N captured from the complex organic patch by the plant species (Hodge et al., 1999a, 2000a; Robinson et al., 1999). Such a relationship was also demonstrated in this study (Fig. 6), thus giving support to hypothesis (iv): that the species that produced the most root length in the mixed plant culture (i.e. B. napus) subsequently captured most N from the organic material. However, if total root length produced and total N capture by both species in interspecific competition together is considered then no relationship can be demonstrated (Hodge et al., 2000a).
Hypothesis (v)
Although G. mosseae did stimulate root production by P. lanceolata monocultures in the discrete patch treatment (hypothesis (iii); see above), this did not subsequently lead to enhanced N capture from the patch compared to when the G. mosseae inoculum was absent, as predicted by hypothesis (v), and there was no impact upon the root length produced. Similarly, Hodge et al. (2000c) found that G. mosseae did not enhance N capture for its host plant from organic patches even though root numbers increased.
Both B. napus and P. lanceolata seedlings captured less N from the organic material when in competition with B. napus. The detrimental effect on N capture from the organic material due to the presence of B. napus as a competitor, however, was more severe for P. lanceolata, with only 3% of the total N captured from the organic material when in interspecific competition being captured by P. lanceolata compared with 97% captured by the B. napus seedling. Again it was striking that while N capture from the organic material by P. lanceolata in inter- and intraspecific competition was only affected by the neighbouring species, N capture by B. napus was also influenced by the spatial placement of the organic material and the G. mosseae inoculum: N capture from the discrete patch was increased when the G. mosseae inoculum was present, even though B. napus does not form mycorrhizas. This result seems counter-intuitive and the mechanism by which the AM fungus benefited B. napus is not obvious. Although the AM fungus enhanced N retention in the discrete patch soil for P. lanceolata monocultures and the mixed species there was no such enhanced retention in soil under B. napus monocultures. Furthermore, although the AM inoculum increased total N content of the roots in interspecific competition (of which the majority were estimated to be B. napus in origin), it reduced N content of the roots of both species in monoculture. Hyphae similar to AM hyphae were observed on the roots of B. napus when the G. mosseae inoculum was present, but no internal colonization occurred. It may be that the AM fungus was able to grow saprophytically on the organic material when it was most concentrated for a short period (Nicolson, 1959; Mosse, 1959; Hodge et al., 2001), than in the absence of a potential host or, in the case of the plants in interspecific competition, a reduced root system for colonization, then recycled N back into the soil–plant system. However, this does not explain why B. napus in particular benefited, and not P. lanceolata as well, in the interspecific competition units. Alternatively, the AM fungus may have reduced the occurrence of other fungal pathogens in the soil which have been shown to constrain yields of other brassica crops (Tahvonen et al., 1984). Although AM fungi have been shown to increase protection from root pathogens for their host plant (Newsham et al., 1995a) there is currently no evidence to suggest this protection would also occur in non-host species. Thus the mechanism for this increased N capture by B. napus in the presence of the AM fungus is currently unclear and requires further investigation.
Hypothesis (vi)
Some studies have shown that both AM colonized (Cliquet et al., 1997; Näsholm et al., 1998) and uncolonized plant roots (Thornton, 2001; Näsholm et al., 2001) can take up intact simple organic compounds in the form of amino acids. During decomposition of the complex organic material, simple organic compounds including amino acids, would have been released, but although 15N enrichment was detected in the plant tissue, 13C enrichment was not. It is therefore unlikely the plants were taking up simple forms of organic N intact. If they had, 13C enrichments would have been detected and a relationship with 15N enrichment would have been expected (Näsholm et al., 1998, 2000). Thus, uptake of inorganic N released after microbial decomposition of the organic material was more important for the plants in this study and there was no support for the sixth hypothesis that the AM fungus enhanced N capture from the organic material for its host plant (P. lanceolata) by the direct uptake of these simple organic N compounds.
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
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The microcosm approach used in this study was ideal for testing the hypotheses outlined above and encouraging development of the added mycorrhizal inoculum, as demonstrated by the %RLC of the P. lanceolata roots in the mycorrhizal treatment being more than double that of those colonized only by the indigenous AM population. As the inoculum used in this study comprised colonized roots, spores and hyphal fragments it had to undergo a period of establishment before becoming functional. This situation reflects agricultural practice, where tillage and crop removal discourage the long-term establishment of a mycelial network of AM hyphae which would colonize roots upon contact. Thus in arable situations the AM fungus also has to undergo a period of development before becoming fully functional, similar to that in the microcosm units. However, the quantity of mycorrhizal inoculum added in the present study was quite large. Moreover the nutrient status of the soil was purposely reduced by the addition of 50% sand to encourage root and mycorrhizal responses to the added organic material. In the field, under current agricultural practices, the background soil fertility would be expected to be higher and mycorrhizal colonization levels lower, thus the responses reported in this study might be exaggerated.
Remarkably, there was little support for any of the hypotheses tested, although all were based on widely accepted interpretations of current literature. Although P. lanceolata shoot DW doubled in the discrete patch treatment, most of the differences occurred when the G. mosseae inoculum was also present (i.e. stimulated root production by P. lanceolata monocultures and increased 15N retention in the soil). However, despite these differences, N capture from the organic material by P. lanceolata was only affected by its neighbouring plant species. Although N capture from the organic material by B. napus was also influenced by the neighbouring species, N capture in the discrete patch was increased when the G. mosseae inoculum was present. Such a result was unexpected. Furthermore, this increased N capture was seen in the B. napus plants grown in interspecific competition and monoculture implying it was a direct effect of the AM fungus on B. napus rather than any indirect effect mediated through the mycorrhizal host plant. AM fungi are known to be multifunctional (Newsham et al., 1995b), but why N capture from the organic material was enhanced in a non-host plant is unclear and requires further investigation. The particular AM fungus used in this study, G. mosseae, is one of only two AM fungal taxa commonly found in root systems of plants grown in arable soils (Helgason et al., 1998). Thus, it was an appropriate inoculum to use. Moreover, although the AM fungus had such an unexpected influence on B. napus, internal colonization of its host, P. lanceolata, was unresponsive to the spatial placement of the organic material. This suggests that the external phase of the AM fungus was important in this study which was somehow being affected by the presence of the non-host roots. Although AM fungi are generally thought to have little or no saprotrophic ability perhaps the proliferation of AM hyphae seen on the B. napus roots enhanced decomposition and N release by the patch (Hodge et al., 2001); however, this would have also benefited the P. lanceolata seedlings in the interspecific competition units. Moreover, if decomposition of the organic material was enhanced by AM hyphal proliferation this would have been expected to occur predominantly when a host was present to supply carbon and thus to support the cost of hyphal construction. Finally, although there was no evidence that intact forms of organic N were taken up by mycorrhizal or non- mycorrhizal plants, large amounts of N (23–38% of the N originally added) were captured by the plants after a relatively short time.
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Acknowledgements |
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This work is funded by a Biotechnology and Biological Sciences Research Council (BBSRC) David Phillips Fellowship awarded to A Hodge. I thank C Scrimgeour and W Stein (Scottish Crop Research Institute, Invergowrie, Dundee UK) for conducting the mass spectrometry analysis and Alastair Fitter (University of York, UK) for his helpful comments on the manuscript.
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