Uptake and transport of organic and inorganic nitrogen ... - Springer Link

Plant and Soil 226: 275–285, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.

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Uptake and transport of organic and inorganic nitrogen by arbuscular mycorrhizal fungi Heidi-Jayne Hawkins1,∗ , Anders Johansen2 & Eckhard George1 1 Institute

of Plant Nutrition, Hohenheim University, Stuttgart 70593, Germany. 2 Department of Ecology and Molecular Biology, The Royal Veterinary and Agricultural University, Thorvaldsenvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark. Received 27 August 1999. Accepted in revised form 27 February 2000

Key words: arbuscular mycorrhiza, Daucus carota, Glomus mosseae, Glomus intraradices, monoxenic culture, N uptake, Triticum aestivum

Abstract New information on N uptake and transport of inorganic and organic N in arbuscular mycorrhizal fungi is reviewed here. Hyphae of the arbuscular mycorrhizal fungus Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe (BEG 107) were shown to transport N supplied as 15 N-Gly to wheat plants after a 48 h labelling period in semi-hydroponic (Perlite), non-sterile, compartmentalised pot cultures. Of the 15 N supplied to hyphae in pot cultures over 48 h, 0.2 and 6% was transported to plants supplied with insufficient N or sufficient N, respectively. The increased 15 N uptake at the higher N supply was related to the higher hyphal length density at the higher N supply. These findings were supported by results from in vitro and monoxenic studies. Excised hyphae from four Glomus isolates (BEG 84, 107, 108 and 110) acquired N from both inorganic (15 NH4 15 NO3 , 15 NO3 − or 15 NH4 + ) and organic (15 NGly and 15 N-Glu, except in BEG 84 where amino acid uptake was not tested) sources in vitro during short-term experiments. Confirming these studies under sterile conditions where no bacterial mineralisation of organic N occurred, monoxenic cultures of Glomus intraradices Schenk and Smith were shown to transport N from organic sources (15 N-Gly and 15 N-Glu) to Ri T-DNA transformed, AM-colonised carrot roots in a long-term experiment. The higher N uptake (also from organic N) by isolates from nutrient poor sites (BEG 108 and 110) compared to that from a conventional agricultural field implied that ecotypic differences occur. Although the arbuscular mycorrhizal isolates used contributed to the acquisition of N from both inorganic and organic sources by the host plants/roots used, this was not enough to increase the N nutritional status of the mycorrhizal compared to non-mycorrhizal hosts. Abbreviations: CCCP – carbonyl cyanide m-chlorophenyl hydrazone; AM – arbuscular mycorrhiza(l) Introduction Several studies have demonstrated the transport of inorganic nitrogen (N) by arbuscular mycorrhizal (AM) hyphae to plants (Ames et al., 1983; Frey and Schüepp, 1993; George et al., 1992; Hawkins and George, 1999; Johansen et al., 1992, 1993, 1994). Most studies have used NH4 + as a N source (Johansen et al., 1992, 1994; Tobar et al., 1994a) because NH4 + is less mobile than NO3 − in the soil. Uptake ∗ FAX No: 711 459 3295. E-mail: [email protected]

of NO3 − has also been examined under drought conditions (Tobar et al., 1994b) in an attempt to reduce NO3 − movement in the soil, which simplifies measurements of N from a localised supply of N. Since NO3 − is the predominant N form in most agricultural soils (see Marschner, 1995), it is important to quantify the transport of NO3 − by AM fungi under non-drought conditions. Amino acids in soils exist in amounts that could make a considerable contribution to N nutrition of plants. This is true not only in mineralisation-limited arctic, boreal (Väre et al., 1997) and heathland

276 (Read, 1996) ecosystems but also typical of nutrientpoor sites in general, such as wet mires and sand plains (Chen et al., 1999), and even agricultural sites (Scheller et al. 1996). Glutamate, Gln, Asp and Ala are amongst the most prevalent free amino acids in soil, with concentrations ranging between 1 and 10 µg g−1 dry soil (Abuarghub and Read, 1988). Amino acid concentrations in soil solution have been reported to be between 50 µM (Monreal and McGill, 1985) and 5 mM (Schobert and Komor, 1987). Free solution diffusion rates of NO3 − and amino acids with a net zero charge (75% at pH 5–7) are comparable (1.9 × 10−5 cm2 s−1 and 9.7 × 10−6 cm2 s−1 , respectively) and plant uptake rates of inorganic and organic N (Gly, Met, Asp) were in the same range in solution culture (Jones and Durrah, 1993) so that amino acids in the field are possibly a viable source of N for plants. It is well-known that various mycorrhizal fungi utilise organic N, e.g. ectomycorrhizal fungi utilise amino acids (Lipson et al., 1999) and peptides (Abuzinadah and Read, 1989); ericoid mycorrhizal fungi utilise even recalcitrant organic sources of N (Read, 1996); and certain dark septate mycorrhizal endophytes of the Australian Epacridaceae also utilise amino acids (Chen et al., 1999). In contrast to arctic sedge (Chapin et al., 1993) and some boreal forest plants (Näsholm et al., 1998), most plants in temperate areas and especially agricultural species are thought to utilise only very small amounts of amino acids and grow better on inorganic N sources (e.g. barley, Chapin et al., 1993). Since there is indirect evidence for amino acid uptake by arbuscular mycorrhizal plants in the field (Näsholm et al., 1998), it is of interest to determine whether arbuscular mycorrhizal fungi, which are associated with plants in temperate, tropical and mediterranean agricultural or natural soils alike are able to take up and translocate amino acids to the host plants. Irrespective of N form, little is known about N transfer mechanisms (transporters) at the biotrophic interface between plants and AM fungi or whether new transport mechanisms are switched on as a result of the symbiosis. The N form translocated within the extraradical hyphae is also unknown. For higher plants, there is convincing evidence that the energy source driving NO3 − uptake is derived from a coupling to the proton electrochemical gradient across the plasma membrane, i.e. uptake occurs via a NO3 − :2H+ symport mechanism (McClure et al., 1990). Although no kinetic or biochemical data are available, a study by Bago et al. (1996) indicated that the AM fungus G. intraradices in symbiosis with tomato can acquire NO3 −

as a N source, probably coupled to a H+ -symport mechanism. Amino acid transport systems have been widely studied in higher plants (Frommer et al., 1994), yeasts and non-mycorrhizal fungi (Sophianopoulou and Diallinas, 1995). More recently, the kinetics and energetics of a general amino acid transporter with a proton symport mechanism similar to that reported for plants (Bush, 1993) has been reported for the ectomycorrhizal fungus, Paxillus involutus, in pure culture (Chalot et al., 1996). From these and other studies of eukaryotes, several amino acid transporters have been described exhibiting different properties concerning substrate affinity, specificity, capacity and regulation (Bush, 1993; Fischer et al., 1998; Roos, 1989). For instance, in higher plants, algal and fungal tissues, the presence of separate amino acid permeases for basic, neutral and acidic amino acids has been proposed (Bush, 1993; Schobert and Komor, 1987). In contrast, a permease with a broad specificity for L- and Damino acids, non-protein amino acids and also amino acid analogues has been identified for Saccharomyces cerevisiae (Sophianopoulou and Diallinas, 1995). It still remains for amino acid uptake in arbuscular mycorrhizal fungi to be shown directly before any biochemical characterisation of the putative carrier(s) can take place. In various studies, the direct transport of N via the extraradical hyphae of AM fungi has been shown in systems where the mass flow and diffusion of N from a compartment containing root-distant hyphae to another compartment containing roots and rootnear hyphae was reduced or prevented (Hawkins and George, 1999; Johansen et al., 1992, 1994). In these cases, between 1 and 7% of the 15 NH4 15 NO3 supplied to Glomus mosseae hyphae was recovered in the wheat host plant after 48 h (Hawkins and George, 1999) and more (27–49%) over 27 d with cucumber and G. intraradices (Johansen et al., 1994). However, there has been no evidence provided for amino acid transport from extraradical hyphae to the host plant. The aims of this paper are: 1. to summarise what is known about uptake and transport of N by AM fungi; 2. to present new data on uptake and transport of inorganic and organic N in the intact symbiosis, excised hyphae in vitro and in monoxenic culture; 3. to discuss the suitability of these techniques for determining N uptake in mycorrhizal systems.

277 Materials and methods

(iii) Set-up for use of excised hyphae (BEG 84)

(i) Set-up for whole plant culture with AM

External hyphae of G. mosseae (strain H03-M, BEG 84) were grown as described by Joner and Johansen (1999) and incubated in 5 ml of medium (pH = 6.0) where N was supplied as either K15 NO3 , NH4 15 NO3 , (15 NH4 )2 SO4 or 15 NH4 NO3 . The atom % 15 N excess in all labelled N forms was 99.1% and the final concentration of the 15 N-labelled NH4 + and NO3 − was 3.3 mM N in the incubation medium in all treatments. The hyphal material was sampled after 2 or 4 h of incubation (three replicates), after which the hyphae were washed three times (15 s) in 6.6 mM N as NH4 NO3 to remove labelled N compounds on the hyphal surface. After drying, the total content of N and 15 N in the fungal material was determined simultaneously using an automated N analyser (Carlo Erba NA1500) interfaced with a Finnigan MAT Delta continuous-flow isotope-ratio mass spectrometer (ANA-MS method, Jensen, 1991).

Summer wheat (Triticum aestivum L. cv. Hano) was cultivated in a Perlite drip-irrigation system for 7 weeks with or without the mycorrhizal fungus Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe (BEG 107) in containers with a central root compartment and two distal hyphal compartments as described by Hawkins and George (1999). Nitrogen was supplied to plants as either 0.2 mM or 2 mM NO3 − ; hyphae received 2 mM NO3 − . Two days before harvesting, 15 N was supplied to hyphal compartments as 20 ml of 10 mM 95% atom enriched 15 N-Gly in nutrient solution (Hawkins and George, 1999). Transport of 15 N to the plant was measured by determining shoot and root 15 N concentration. The percentage atom enrichment of 15 N in dried, pulverised samples was determined by dry oxidation and reduction (Roboprep CN Biological Sample Converter, Europa Scientific Ltd, Crewe, UK) and subsequent mass spectrometry (Tracermass Sample Isotope Analyser, Europa Scientific Ltd.). (ii) Set-up for use of excised hyphae (BEG 107, 108 and 110) Wheat plants were cultured as in (i) except that 2-mm glass beads replaced Perlite in the hyphal compartments. After six weeks, the hyphae of G. mosseae (BEG 107, BEG 108) and G. intraradices Schenk and Smith (BEG 110) were harvested over two stacked sieves (1 mm and 40 µm) as described by Redecker et al. (1995) and used to determine the in vitro uptake rate of 15 NO3 − , 15 NH4 + , 15 NH4 15 NO3 , 15 N-Gly and 15 N-Glu. Excised hyphae and attached spores were resuspended, incubated and agitated in 10 ml assay medium in the dark at 25 ◦ C for 2 h. The 15 N assay medium comprised: 4 mM 95% atom enriched 15 N; 1.5 mM MES-KOH (pH 6) and 10 µM P in half-concentrated, modified Long Ashton nutrient solution (Hewitt, 1966) and with or without the proton gradient uncoupler, carbonyl cyanide mchlorophenyl hydrazone, CCCP (4 µM). Uptake in the presence of CCCP (+CCCP) was termed passive uptake and uptake without CCCP minus the passive uptake ([-CCCP] – [+CCCP]) was termed active uptake. Harvested hyphae were subsequently rinsed in distilled water and frozen in liquid N2 before being dried at 60 ◦ C for 48 h and analysed for 15 N content as in (i).

(iv) Set-up for monoxenic root and fungal cultures Cultures of Ri T-DNA transformed carrot (Daucus carota L.) roots colonised or not colonised by G. intraradices Schenck and Smith (Bécard and Fortin, 1988, no BEG number available) were kindly provided by Prof. G. Bécard (Laboratoire de Mycologie Végétale, Centre de Biologie et Physiologie Végétales, Université Paul Sabatier, Toulouse, France) and maintained as described by Chabot et al. (1992). For the experiment, -AM and +AM roots were placed in undivided Petri dishes or divided Petri dishes as described by St.-Arnaud et al. (1996) except that sucrose was present in both compartments. In the divided Petri dishes, colonised or non-colonised roots were placed only on one side (root compartment). After 4–6 weeks, hyphae crossed the division into the medium in the second compartment (physically separate from the root compartment), thereafter referred to as the hyphal compartment. Sucrose, Ca and K were supplied so that the respective ratios remained constant regardless of the N treatment (1:1 or 1:20 inorganic:organic N). When the roots started growing, the same amount of medium (25 ml) was available to roots in divided (25 ml in each of the two compartments) and undivided (25 ml in the single compartment) Petri dishes. The 15 N-labelled amino acids (95 atom% 3.13 or 1.65 mM 15 N-Gly or 15 N-Glu) were added to the hyphal compartments only, in the case of the divided Petri dishes (+Hyp),

278 and were present in all undivided Petri dishes (-AM and +AM). Roots and hyphae were harvested after 10 weeks. Hyphae and spores in the hyphal and root compartments were cut out of the solid medium in blocks of about 10 mm2 . The blocks were placed into 5 ml 10 mm Na-citrate buffer (pH 6) in test tubes at 30 ◦ C and agitated gently for 30 min. The buffer solubilised the gellan gum (Doner and Bécard, 1991; Doner and Douds, 1995). The test tubes were then gently inverted until all the remaining pieces of the gel block had dissolved, leaving the fungal material behind (as controls for the buffer treatment, some samples were simply washed over a sieve to dissolve the gel resulting in no significant differences in 15 N content). Spores and hyphae were then decanted onto a 40 µm sieve and rinsed with deionised water. Fungal material was frozen in liquid N2 and then dried at 60 ◦ C for 48 h before analysing for 15 N. Roots were lifted onto a 1 mm sieve, rinsed under tap water to remove adhering gel and then rinsed in deionised water before removing an approximately 0.1 g sub-sample to determine the percentage colonisation of the root. The remaining roots were frozen in liquid N2 and freeze-dried before pulverising and analysing for 15 N. Hyphal/root length determination and root staining In Experiments (i) and (iv) hyphal lengths were determined at the end of the experimental period using the gridline-intersect method (Giovannetti and Mosse, 1980) with a block size of 5 mm according to the modified agar film technique described by Li et al. (1991). Hyphal length in monoxenic cultures was determined by counting intact hyphae in situ. In Experiment (iv), the increase in root length in monoxenic culture was expressed as a percentage increase compared to the starting length of root pieces transferred from the parent culture. This starting root length was designated as 100%. In all experiments, the percentage of root length colonised by mycorrhizal fungi was determined on roots stained in trypan blue (Koske and Gemma, 1989) using the gridline-intersect method (Giovannetti and Mosse 1980). Results in (iv) were not presented or compared on an uptake rate basis, since an uptake rate could not be calculated for the +Hyp treatment where hyphae crossed into the 15 N-labelled compartment at different times.

Results Experiment (i) There was no significant difference between the shoot or root dry weight (Figure 1A) or N concentration (data not shown) of non-mycorrhizal and mycorrhizal plants. Plants grown at 2.0 mM NO3 − were larger than those grown at 0.2 mM NO3 − (P< 0.05, Student’s ttest, Figure 1A) and had a significantly (P = 0.003, Student’s t-test) greater percentage of colonised root length (91% versus 70%). The hyphal length density in the hyphal compartment was also significantly increased (P = 0.001, Student’s t-test) for those plants supplied with the higher NO3 − level (16.4 m cm−3 Perlite) compared to those supplied with the lower N level (0.6 m cm−3 Perlite). Mycorrhizal roots grown at the higher NO3 − level also had an increased 15 N concentration compared to those at the lower level (P = 0.045, Student’s t-test). There was no significant difference between the specific uptake rate of 15 N per unit hyphal length of plants supplied with 0.2 mM N (12.2 nmol 15 N m−1 hyphae) or 2.0 mM N (10.7 nmol 15 N m−1 hyphae). Between 0.2 and 6.0% of the 15 N supplied was taken up by hyphae in 0.2 mM and 2.0 mM NO3 − treatments, respectively. Practically no 15 N was detected in non-mycorrhizal plants (Figure 1B). Experiments (ii) and (iii) The N concentration in the hyphae of the different AM fungi from glass bead cultures was similar to that found in plant tissues, i.e. 3.88 ± 0.41% (G. mosseae, BEG 107), 3.0 ± 0.25% (G. mosseae, BEG 108), 4.0 ± 0.11% (G. intraradices, BEG 110). The percentage root colonisation of wheat by these fungi was not significantly different: 67 ± 2% (BEG 107), 69 ± 13% (BEG 108), 66 ± 0.4% (BEG 110). Uptake of 15 N (from NO3 − , NH4 + , NH4 NO3 , Gly or Glu) into excised hyphae could be demonstrated for all four fungal isolates (Figures 2A, B and 3). The active uptake was greatest for NH4 + and NH4 NO3 , followed by the amino acids and less (Figure 3) or almost no (Figure 2A, B) NO3 − was taken up. Except for the NO3 − and Gly uptake rates, the uptake of the other N forms by G. mosseae (BEG 108) was higher than the uptake rates of the other G. mosseae (BEG 107) or of G. intraradices (BEG 110) (P< 0.05, Tukey test, Figure 2A, B). Passive uptake (Figure 2A) was comparable to active uptake and even higher in the case of NO3 − . Hyphal uptake of N from inorganic sources by G. mosseae (BEG 84) depended markedly on the

279

Figure 1. Experiment (i): Dry weight (A) and 15 N uptake rate (B) of non-mycorrhizal and mycorrhizal T. aestivum plants supplied with 10 mM 15 N-Gly to the hyphal compartments 48 h before harvesting. Different letters indicate a statistically significant difference between -AM and +AM treatments (P< 0.05, Student’s t-test). Values are means of four replicates. The symbols (-/+) represent plants without/with AM, respectively.

Figure 2. Experiment (ii): Passive (A) and active (B) uptake rate of inorganic and organic N into excised hyphae of G. mosseae (BEG 107), G. mosseae (BEG 108) and G. intraradices (BEG 110). Different letters indicate a statistically significant difference between the uptake rate of different N forms (P< 0.05, Tukey test). Values are means of four replicates.

280 Table 1. Experiment (iv): Dry weight of Ri T-DNA transformed D. carota roots colonised (+AM) or not colonised (-AM) by G. intraradices with varying N supplies and cultured in undivided or divided Petri dishes for 10 weeks. Different letters indicate a statistically significant difference between -AM and +AM treatments (P< 0.05, Student’s t-test) at the same type of N supply. Values are means ± SE of 6 replicates. n.a. = not applicable Ratio of applied N forms -AM

Dry weight (mg) +AM

Undivided Petri dishes 1:1 NO3 − :15 N-Gly 1:20 NO3 − :15 N-Gly 1:1 NO3 − :15 N-Glu 1:20 NO3 − :15 N-Glu

65.9 ± 3.5a 66.6 ± 5.2a 64.9 ± 2.1a 70.7 ± 3.9a

57.0 ± 0.7b 57.8 ± 2.3a 49.0 ± 6.5b 58.2 ± 1.3b

Divided Petri dishes 1:1 NO3 − :15 N-Gly 1:20 NO3 − :15 N-Gly 1:1 NO3 − :15 N-Glu 1:20 NO3 − :15 N-Glu

n.a. n.a. n.a. n.a.

52.5 ± 6.0 47.1 ± 3.7 52.0 ± 2.9 58.9 ± 5.7

Figure 3. Experiment (iii): 15 N concentration in excised external hyphae of G. mosseae (BEG 84) 2 and 4 h after application of K15 NO3 , NH4 15 NO3 , (15 NH4 )2 SO4 or 15 NH4 NO3 . Values are means ± SE of three replicates. Bars represent SE of the mean.

type of inorganic N source supplied (Figure 3). When N was supplied as (15 NH4 )2 SO4 or 15 NH4 NO3 , the uptake of 15 N was 12–15 µmol g−1 dry mass after 2 h of incubation. In comparison, the 15 N uptake was fifteen times lower when K15 NO3 or NH4 15 NO3 were supplied. The hyphal 15 N uptake was similar for (15 NH4 )2 SO4 and 15 NH4 NO3 and was roughly doubled when the incubation time was extended to 4 h. The same pattern was observed for K15 NO3 and NH4 15 NO3 . Using the values for N uptake at the start of the labelling period (zero) and after 2 and 4 h, an average uptake rate for NH4 + (5.8 µmol N g−1 dry weight h−1 ) and NO3 − (0.4 µmol N g−1 dry weight h−1 ) could be calculated. Total uptake (passive plus active uptake in ii) of 15 NH4 + and 15 NO3 − and was similar in the two experiments (ii) and (iii), i.e. between 6 and 20 µmol 15 N g−1 dry weight of hyphae for 15 NH4 + and between 0.5 and 4 µmol 15 N g−1 dry weight of hyphae for 15 NO3 − . Experiment (iv) In sterile root and root-fungus cultures, the N treatment within each mycorrhizal and compartment treatment did not affect root dry weight while mycorrhizal colonisation decreased root dry weight (Table 1).

The relative increase in root length was highest in the NO3 − :Gly/ 1:20 treatment in mycorrhizal roots (Table 2). The N treatments had no significant effect on the hyphal length of mycorrhiza in either the undivided or divided Petri dishes (Table 2). Although it would appear that Glu inhibited hyphal growth, any effects of N treatment on hyphal growth across the division were masked by high intrinsic standard errors (Table 2), which could be attributed to the effect of the partition in the Petri dish. The partition prevented hyphal growth into the hyphal compartment, apparently at random, in some Petri dishes. The 15 N uptake into -AM and +AM roots in the undivided Petri dishes did not differ significantly except for the 1:20 NO3 − :15 NGlu treatment where there was significantly more 15 N in the +AM versus the -AM roots (Figure 4A, P = 0.020, Student’s t-test). The absolute 15 N uptake into +Hyp roots via the hyphae only (divided system) was relatively small compared to roots grown in the undivided system. One reason for this is that the hyphae were exposed to the 15 N source in the hyphal compartment only after having grown into this compartment. Hyphae transported more 15 N from 15 N-Gly compared to 15 N-Glu in the divided system (Figure 4A) in both 1:1 or 1:20 treatments (P< 0.05, Student’s t-tests). Not surprisingly, more 15 N was taken up when the 15 Namino acids were supplied as 1:20 NO3 − :amino acid compared to the 1:1 NO3 − :amino acid treatment. This

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Figure 4. Experiment (iv): 15 N uptake of (A) Ri T-DNA transformed roots of D. carota colonised (+AM) or not colonised (-AM) by G. intraradices or (B) hyphae of G. intraradices. Both A and B were supplied with varying N sources and cultured in undivided or divided Petri dishes for 10 weeks. Different letters indicate a statistically significant difference between the different treatments (P< 0.05, Tukey test after a one-way anova) within each N treatment. Values are means ± SE of six replicates.

was also reflected in the 15 N uptake into the hyphae (Figure 4B). There was also a significantly greater amount of 15 N in hyphae in the hyphal compartment (except for the 1:20 NO3 − :Gly treatment) compared to those in the root compartment (P< 0.05, Student’s t-tests). The uptake of 15 N was in the same order of magnitude for the roots and hyphae within each N treatment but uptake per gram hyphae was higher than for roots (Figure 4A, B).

Discussion Advantages and disadvantages of the experimental systems Various methods were used to study N uptake by mycorrhizal hyphae or plants since each method had advantages that compensated partially or completely for the disadvantages of the other methods. The choice of methods was based on the following prerequisites for studying N uptake in mycorrhizal systems: (a) demonstration of N uptake via hyphae into the whole plant, (b) demonstration of N uptake by the hyphae separately from the plant roots and (c) demonstration of N uptake from organic sources with minimal or no mineralisation of this N prior to uptake. Experiment (i)

with whole plants satisfied prerequisite (a) and (b) but, as a disadvantage, probably some mineralisation of organic N occurred. The Experiments (ii) and (iii) with excised hyphae satisfied prerequisite (b), i.e. demonstrated uptake by hyphae alone, and due to the limited uptake period, probably very little N was mineralised. Also, excised hyphae represent a simple and convenient system since these can be obtained rapidly and used for uptake experiments in small volumes. A general disadvantage of this system is that excised hyphae tolerate only short storage times and the removal of the sink (the plant) would markedly affect uptake rates once the uptake requirements of the fungus had been met and/or storage structures for N were filled. For this reason, excised hyphae are suitable for short uptake or experimental periods only. A check of hyphal vitality (e.g. exclusion of Evan’s blue stain (Sabbah and Tal, 1990)) for each isolate and experiment would improve validity and reproducibility of such experiments. Pertaining to N uptake in particular, it is preferable to rinse hyphae in an unlabelled solution containing N to remove adhering 15 N from the hyphal surfaces as performed in Experiment (iii). Experiment (iv), using monoxenic root cultures satisfied the prerequisite (c), i.e. mineralisation of the amino acids was excluded. This system is suitable for any long-term labelling with organic compounds. Although the system is ar-

282 Table 2. Experiment (iv): Percentage increase in root length, percentage root colonisation and length of external hyphae associated with Ri T-DNA transformed roots of D. carota grown with (+AM) or without (-AM) G. intraradices in undivided or divided Petri dishes and supplied with varying N supplies for 10 weeks. Different letters indicate a statistically significant difference between the different N treatments (P< 0.05, Tukey test after one-way anova) within each mycorrhizal and compartmentalised system. Values are means ± SE of 6 replicates. n.a. = not applicable Ratio of applied N forms

Increase in root length (%)

Root colonisation (%)

Hyphal length (cm)

+AM: Undivided Petri dishes 1:1 NO3 − :15 N-Gly 1:20 NO3 − :15 N-Gly 1:1 NO3 − :15 N-Glu 1:20 NO3 − :15 N-Glu P (Tukey test)

102 ± 11a 181 ± 18b 115 ± 11a 109 ± 23a 0.010

55 ± 4a 63 ± 3a 55 ± 3a 52 ± 2a 0.104

235 ± 46a 401 ± 23a 315 ± 54a 264 ± 45a 0.067

+AM: Divided Petri dishes 1:1 NO3 − :15 N-Gly 1:20 NO3 − :15 N-Gly 1:1 NO3 − :15 N-Glu 1:20 NO3 − :15 N-Glu P (Tukey test)

115 ± 4a 178 ± 21b 126 ± 16a 107 ± 15a 0.016

29 ± 4a 25 ± 5a 21 ± 4a 14 ± 3a 0.091

287 ± 148a 669 ± 354a 27 ± 25a 0 ± 0a 0.080

-AM: Undivided Petri dishes 1:1 NO3 − :15 N-Gly 1:20 NO3 − :15 N-Gly 1:1 NO3 − :15 N-Glu 1:20 NO3 − :15 N-Glu P (Tukey test)

140 ± 18a 159 ± 20a 120 ± 15ab 67 ± 6b 0.003

n.a.

n.a.

tificial, it offers the advantage over excised hyphae that the root-fungus culture is growing and intact. Disadvantages are that the sink of the plant shoot is not present and the roots are growing in an artificial medium – these would probably affect the uptake of N or any other element. In addition, the system is restricted to a few fungal isolates at present and requires a greater investment of time and materials compared to whole plant culture or the use of excised hyphae. The significance of the results obtained, with the advantages and limitation of the various methods in mind, are discussed below. Uptake and transport of organic and inorganic N by hyphae Colonisation of wheat plants with G. mosseae (BEG 107) in Experiment (i) did not significantly affect the growth or N concentration of the NO3 − -fed mycorrhizal plants relative to the non-mycorrhizal plants,

although growth responses in wheat have been observed previously (Hawkins and George, 1999) using the same AM fungal isolate but another inorganic N source. As previously reported using NH4 NO3 as the N source (Hawkins and George, 1999), however, plants that were supplied with a higher level of N in the root compartment had mycorrhiza with a greater hyphal length. Since the specific 15 N uptake rate per unit hyphal length was similar at both N levels, the greater uptake rate was apparently related to the greater hyphal length at the higher N level. The present results also indicated that AM hyphae are able to take up the amino acid Gly and transport N from this source to the plant roots in a form that is presently unknown. This supports indirect evidence of amino acid uptake by mycorrhizal fungi in the field (Näsholm et al., 1998). The 15 N uptake rate from 15 NGly per unit root mass was higher than was previously reported for inorganic N while the amount transported to the plant within 48 h was comparable (Hawkins and

283 George, 1999) but less than reported for inorganic N uptake in other studies (Frey and Schüepp, 1993; Johansen et al., 1994). These differences are not large and can be attributed to differences in experimental conditions (see Hawkins and George, 1999). The possibility exists that during the 15 N-Gly supply period in the first experiment, 15 N-Gly was mineralised to some extent and absorbed as mineral N by the hyphae, making a definitive statement about organic N uptake uncertain in this experiment. For this reason, excised hyphae were used for in vitro uptake experiments over a much shorter time period. Results of these experiments confirmed that hyphae took up 15 N not only when supplied as inorganic N but also when supplied as the amino acids Gly and Glu. Hyphae of three Glomus isolates were shown to take up the amino acid N in the same order of magnitude as inorganic N. Generally, 15 N from 15 NH4 + and 15 NH 15 NO was actively taken up in larger amounts 4 3 than 15 N from 15 N-amino acids and 15 NO3 − . The relatively high passive uptake of N, and in particular from those N forms requiring active uptake, is possibly related to increased adsorbtion due to cell surface modifications by the metabolic inhibitor CCCP, as has been proposed to explain enhanced metal adsorption in micro-organisms using other chemical killing agents (Huang and Huang, 1996). It is unlikely that high passive uptake is related to gross unrestricted uptake at damaged surfaces of hyphae since hyphae were mostly intact and exhibited cytoplasmic streaming. The uptake rate of the three isolates, counting both inorganic and organic N, could be ranked as follows: BEG 108 > BEG 110 > BEG 107. The isolate BEG 108, which originated from a organically managed, unfertilised field, had approximately twice the uptake rate of amino acids compared to BEG 107, which originated from a conventionally managed agricultural field. The isolate BEG 110 from a nutrient poor site in Syria was intermediate between the two. In order to correlate the origin of the fungi with the respective N uptake capacities, similar experiments with more isolates and additional parameters are needed. In Experiment (iii), G. mosseae (BEG 84) showed broadly similar uptake behaviours, with respect to NH4 + and NO3 − , as the AM-fungal isolates in Experiment (ii), where NH4 + was taken up more efficiently than NO3 − . Incubating the hyphae with NH4 NO3 , labelled with 15 N in either of the N positions, clearly showed a hyphal preference for the NH4 + , which may be energetically more favourable when assimilated into organic N than in the case with

NO3 − . Assimilation probably occurs via a glutamine synthetase-glutamate synthase pathway (Johansen et al., 1996). The above mentioned results pertaining to N uptake from organic sources were confirmed by an experiment in sterile culture, which avoided bacterial mineralisation of amino acids. Transformed carrot roots, both colonised and uncolonised by G. intraradices, were able to acquire 15 N supplied as either Gly or Glu. When both roots and hyphae were present in the same compartment (the undivided system), the amount of 15 N taken up into roots (including internal AM structures) was not different from the uptake of 15 N into non-mycorrhizal roots. This showed that root-near hyphae did not contribute significantly to the uptake of the amino acids tested. When the mycorrhizal roots were physically separated from the 15 N-amino acid source in the hyphal compartment, the hyphae growing into this compartment did acquire this 15 N and transport it to the roots, although only in small amounts compared to the amounts taken up by roots directly in the undivided system. This can be at least partly explained by the shorter duration of 15 N uptake by the hyphae, which were exposed to the 15 N source only after having grown over the Petri dish partition. Alternatively, the transformed roots may not have constituted a sink for the amino acid N transport. There was significantly less 15 N taken up into hyphae in the root compartment compared to hyphae in the hyphal compartment in the divided system. Possibly, the presence of roots inhibited or competed with root-near hyphae for the uptake of 15 N. Also, it was observed that the hyphal length in the root compartment was less than that in the hyphal compartment and therefore the growth of root-near hyphae also appeared to be suppressed. The proximity of roots and hyphae is apparently important in regulating the balance in P uptake between hyphae and roots (Pearson and Jakobsen, 1993) and the same may be true for amino acid uptake. In this culture, a similar mass of roots was present in a smaller volume in the divided (although the amount of nutrient medium was the same) compared to the undivided system and hyphae were in closer proximity to the roots. This may have accounted for the lower colonisation rates determined, i.e. was due to pH changes in the rhizosphere, or phenolic or other compounds exudated by roots. It was also observed that the hyphal growth rate increased as soon as the hyphae grew over the Petri dish partition away from the medium in the root compartment, which had turned a light yellow colour after 8 weeks.

284 Conclusion and future prospects Results showed that mycorrhizal hyphae can take up and translocate inorganic and organic N to transformed roots as well as under non-sterile conditions using whole plants and excised hyphae. Clearly, however, the amounts of N transported via hyphae do not constitute a large contribution to the N nutrition of the plant. The results nevertheless indicate that rootdistant hyphae could contribute to inorganic N and amino acid uptake in plants if these hyphae have access to a N source spatially unavailable to the plant. In soils, the distribution of N and other elements is heterogeneous at a scale relevant to plants and mycorrhizal fungi (Ozinga et al., 1997). Arbuscular mycorrhizal fungi, by increasing the absolute volume of soil available to the plant, may indeed access N, including organic N, not available to the plant alone. Such a capacity may be especially important in ecosystems with impoverished inorganic N sources. An additional contribution by AM fungi could be that N is immobilised into external hyphae before other microorganisms (or roots) can take up this N. In this way, the hyphae can conserve the N in the plant/fungal system, thus representing a competitive acquisition of N. The uptake capacity for amino acids differed between isolates, even of the same species, in this study. This supports the idea of ecotypes emerging depending on the local edaphic and/or nutrient conditions (Kielland, 1995). Supporting experiments using many different isolates would be needed to confirm this suggestion. As yet, little is known about the mechanisms of N uptake by AM fungi. In addition, nothing is known about the N transfer form from fungus to plant although Smith et al. (1994) suggest that N-rich amides (probably Asn or Gln) are possible candidates for the major form in which N is transported. The existence of mycorrhizal fungus ecotypes, the mechanisms of N uptake and the transport forms within the hyphae and fungus-root interface are all areas in N nutrition of AM fungi that require further investigation.

Acknowledgements The first author thanks the Hochschulsonderprogramm III, Universität Hohenheim, Germany for the award of a bursary during the tenure of this work.

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