Reciprocal N (¹⁵NH₄⁺ or ¹⁵NO₃⁻) transfer between nonN₂-fixing Eucalyptus maculata and N₂-fixing Casuarina cunninghamiana linked by the ectomycorrhizal fungus Pisolithus sp.

Summary

• • 
  Two-way N transfers mediated by Pisolithus sp. were examined by excluding root contact and supplying ¹⁵NH₄⁺ or ¹⁵NO₃⁻ to 6-month-old Eucalyptus maculata or Casuarina cunninghamiana grown in two-chambered-pots separated by 37 m screens.
• • 
  Mycorrhizal colonization was 35% in Eucalyptus and 66% in Casuarina (c. 29% N₂-fixation). Using an environmental scanning electron microscope, living hyphae were observed to interconnect Eucalyptus and Casuarina. Biomass and N accumulation was greatest in nodulated mycorrhizal Casuarina/mycorrhizal Eucalyptus pairs, less in nonnodulated mycorrhizal Casuarina/mycorrhizal Eucalyptus pairs, and least in nonnodulated nonmycorrhizal Casuarina/nonmycorrhizal Eucalyptus pairs.
• • 
  In nonnodulated mycorrhizal pairs, N transfers to Eucalyptus or to Casuarina were similar (2.4–4.1 mg per plant in either direction) and were 2.6–4.0 times greater than in nonnodulated nonmycorrhizal pairs. In nodulated mycorrhizal pairs, N transfers were greater to Eucalyptus (5–7 times) and to Casuarina (12–18 times) than in nonnodulated mycorrhizal pairs. Net transfer to Eucalyptus or to Casuarina was low in both nonnodulated nonmycorrhizal (< 0.7 mg per plant) and nonnodulated mycorrhizal pairs (< 1.1 mg per plant). In nodulated mycorrhizal pairs, net transfer to Casuarina was 26.0 mg per plant.
• • 
  The amount and direction of two-way mycorrhiza-mediated N transfer was increased by the presence of Pisolithus sp. and Frankia, resulting in a net N transfer from low-N-demanding Eucalyptus to high-N-demanding Casuarina.

Introduction

Plants with the same fungal species can be linked to one another below-ground by a common mycorrhizal network (CMN) in either intra- or interspecific combinations (Newman, 1988; Newman et al., 1994). CMNs provide pathways for movement of nutrients such as carbon, nitrogen (N) and phosphorus. Unidirectional transfer of NH₄⁺ or NO₃⁻ has been observed for arbuscular mycorrhizal (AM) CMNs (Bethlenfalvay et al., 1991; Frey & Schuepp, 1993; Johansen & Jensen, 1996), and of NH₄⁺ for ectomycorrhizal (EM) CMNs (Arnebrant et al., 1993; Ekblad & Huss-Danell, 1995). Such one-way N transfer between plants may occur directly through fungal hyphae in a CMN without entering the soil solution. Differences in N concentration between N-rich donors and N-poor receivers may drive N transfer (Bethlenfalvay et al., 1991; Frey & Schuepp, 1993). However, these studies did not determine net N transfer because only one-way N transfer was measured.

In Australia, the native trees, Casuarina and Eucalyptus, grow as pure or mixed stands and are mycorrhizal (Attiwill & Adams, 1996). N₂-fixing Casuarina can contribute N to soil and indirectly to other adjacent nonN₂-fixing plants including Eucalyptus (Subbarao & Rodriguez-Barrueco, 1995; Attiwill & Adams, 1996). However, the role of mycorrhizas in one- or two-way N transfer between these two species has not been investigated. Mycorrhiza-mediated N transfer may be determined by interactions among plant roots, mycorrhizal fungi, N₂ fixation, and plant N requirements. We traced N movement between plants using ¹⁵NH₄⁺ or ¹⁵NO₃⁻ and asked four questions: first, do mycorrhizas mediate N transfer from Casuarina to Eucalyptus? Second, do mycorrhizas mediate N transfer from Eucalyptus to Casuarina? Third, is N transfer altered by mycorrhization or by N₂ fixation? Fourth, what is the amount and direction of net N transfer?

Materials and Methods

Experimental pots and plant growth conditions

Experimental pots were 5-l plastic boxes (300 × 12 × 150 cm). Boxes were separated by two perforated Perspex plates (0.25 cm wide) to form a 5 mm air gap between the two chambers. A similar system was used by Johansen & Jensen (1996). The purpose of the air gap was to prevent water and solute movement between the two chambers. Each perforated plate had 312 holes (6 mm in diameter). The Perspex plates had no holes in the bottom 10 mm to prevent water flow at the bottom of the chambers. On one side of the plates, there was a 37 m nylon mesh that allowed only hyphal connections. The structure of the experimental boxes resembled a multiple layer sandwich.

Each chamber contained 3 kg potting mix (sand: peat = 2 : 1, v/v, pH 6.5). Each m³ of potting mix contained 8.3 kg of stock fertilizer mix (1.5 kg cattle blood and bone powders, 0.3 kg K₂SO₄, 1.5 kg superphosphate, 2.5 kg dolomite, 1.5 kg hydrated lime, and 1.0 kg gypsum). In addition to the air gap, to further minimize water movement through nylon meshes within and between the two chambers, 0.5% (w/w) high water holding capacity crystals (RainSaver®, Hortex Australia Pty. Ltd, Seven Hills, NSW, Australia) were mixed with the potting mix. To prevent transformation of NH₄⁺ to NO₃⁻, a nitrification inhibitor, N-serve [2-chloro-6-(trichloromethyl) pyridine, Sigma-Aldrich Pty Ltd, Castle Hills, NSW, Australia] was applied. N-serve was dissolved in ethanol, diluted in distilled water and mixed with potting mix (5 mg kg⁻¹ potting mix). N-serve retards the first step in nitrification of NH₄⁺ to hydroxylamine by Nitrosomonas sp. (Slangen & Kerkhoff, 1984) and has little effect on other soil microorganisms when < 10 mg kg⁻¹ soil is used (Laskowski et al., 1975). The experimental pot contained one Casuarina seedling in one chamber and one Eucalyptus seedling in another chamber. Plants were grown for 6 months (September 1998 to February 1999) in a temperature-controlled glasshouse (28/23°C, day/night) located at the St Lucia campus of the University of Queensland. Plants were watered weekly with 5.0 ml nutrient solution (Brundrett et al., 1996) containing 2.0 mmol (¹⁴NH₄)₂SO₄ or 4.0 mmol K¹⁴NO₃.

Experimental design

There were three treatments: first, nonnodulated nonmycorrhizal (Nod⁻Myc⁻); second, nonnodulated mycorrhizal (Nod⁻Myc⁺); and third, nodulated mycorrhizal (Nod⁺Myc⁺) (Table 1). The Nod⁻Myc⁻ treatment was used to measure N transfer through the growth medium (soil pathway). The Nod⁻Myc⁺ treatment was used to determine effects of mycorrhizas on N transfer. The Nod⁺Myc⁺ treatment was used to study effects of both mycorrhization and N₂ fixation on N transfer.

Table 1.  Experimental design for two-way N transfer between Casuarina and Eucalyptus seedlings grown in two-chambered pots. The mycorrhizal fungus was Pisolithus sp. and the N₂-fixing symbiont was Frankia

Pair name	N donor	N receiver	Treatment status
			Myc	Nod	Code
^15N was supplied to the growth medium of donor plant side. Myc or M, mycorrhizal status; Nod or F, nodulation status; C, Casuarina; E, Eucalyptus.
Nonnodulated	Casuarina	Eucalyptus	–	–	C^M–F–→E^M–
nonmycorrhizal pairs	Eucalyptus	Casuarina	–	–	E^M–→C^M–F–
Nonnodulated	Casuarina	Eucalyptus	+	–	C^M+F–→E^M+
mycorrhizal pairs	Eucalyptus	Casuarina	+	–	E^M+→C^M+F–
Nodulated	Casuarina	Eucalyptus	+	+	C^M+F+→E^M+
mycorrhizal pairs	Eucalyptus	Casuarina	+	+	E^M+→C^M+F+

When plants were 5 months old, 6 mg ¹⁵N [(¹⁵NH₄)₂SO₄, c. 98.8 atom percentage ¹⁵N or K¹⁵NO₃, c. 97.6 atom percentage ¹⁵N], was dissolved in 200 ml deionized water and added to the growth media of the donor side for a 4-wk-period without further N supplement. No N was supplied to the receiver plants during this period. Either Casuarina or Eucalyptus was the donor. Thus net N transfer could be calculated as the difference between ¹⁵N translocation from Casuarina to Eucalyptus and from Eucalyptus to Casuarina.

Production of aseptic ectomycorrhizal seedlings

A sporocarp of Pisolithus sp. was collected by JW Cairney in 1988 at North Head, New South Wales, Australia. The sporocarp isolate was maintained by PA McGee and WG Allaway, University of Sydney, Australia. The isolate was cultured on nutrient agar (Ahmad & Hellebust, 1991). N was supplied as (¹⁴NH₄)₂SO₄ or as K¹⁴NO₃. Seeds of Casuarina cunninghamiana Miq. (river she-oak) and Eucalyptus maculata Hook (spotted gum) were purchased from the Queensland Institute of Forestry, Australia. Seeds were surface sterilized (70% ethanol, 30 s; 15% H₂O₂, 5 min), rinsed with sterile water, and germinated on the media of Ahmad & Hellebust (1991) (minus biotin and vitamin B₁₂). Axenically germinated seeds were placed on agar plates with or without Pisolithus sp. in a growth chamber (23/18°C, day/night) (Brundrett et al., 1996). Mycorrhizal formation occurred after 6 wk.

Germinated Casuarina seeds were also placed on sterilized agar plates with or without Frankia (two strains UGL020604 and UGL020605, from CT Wheeler, University of Glasgow, UK). Plates were incubated in a growth chamber (23/18°C, day/night); nodules formed after 6 wk. To make Frankia inoculums, c. 5-mm diameter fresh Frankia nodules were surface sterilized (70% ethanol, 30 s), rinsed, ground, filtered and diluted with sterile water. To produce nodulated Casuarina seedlings, seedlings roots were dipped in fresh diluted Frankia suspension for 20 min before transplanting into plastic bags (90 cm × 150 cm). Five ml of this suspension was added to the growth media near tap roots at wk 1 and 3 after transplanting. In the Nod⁻ treatment, seedlings of Casuarina and Eucalyptus seedlings received boiled nodule suspension. In the glasshouse, seedlings were grown for 2 months in plastic bags, then transplanted into experimental pots and grown for 4 months until harvest.

Plant harvest

Plants were harvested 4 wk after ¹⁵N addition to the growth media on the N donor side. Shoots, roots and nodules were oven-dried, weighed, grounded in a ball-mill (Retsch GmbH & Co. KG, 5657 Haan 1, Germany) and analyzed for total N and ¹⁵N. Fresh Casuarina needles were collected before ¹⁵N treatment for estimation of N₂ fixation. Subsamples of fresh roots were preserved in 50% ethanol for determination of mycorrhizal colonization (Brundrett et al., 1996). Using an ElectroScan E3 environmental scanning electron microscope (Electro-Scan Corp., Wilmington, MA, USA), living fungal hyphae and CMN formation were observed on mesh between the two chambers of experimental pots for mycorrhizal Casuarina/Eucalyptus and Eucalyptus/Casuarina treatments (Fig. 1; He, 2002).

Figure 1. Environmental scanning electron micrographs of hyphae of Pisolithus sp. (a) hyphae ramified on 37 m nylon mesh (× 225); (b) hyphae crossing mesh (× 395); (c) higher magnification showing hyphae within a hole of mesh (× 650).

Nitrogen assays, N₂ fixation and N transfer calculations

Samples were analyzed for ¹⁵N (Automated ¹⁵N : ¹³C analyser-mass spectrometers, Europa Scientific, Crewe, UK). N₂ fixation was expressed as specific nodule activity (g tissue N mg⁻¹ d. wt nodule). Percent plant nitrogen derived from fixation (% N_fixed) was calculated (Knowles & Blackburn, 1993; Unkovich et al., 2001):

• % N_fixed = (δ¹⁵N_{nonfixing plant} − δ¹⁵N_{fixing plant})/ (δ¹⁵N_{nonfixing plant} − B) × 100(Eqn 1)

where ‘B’ is the δ¹⁵N value of nodulated N₂-fixing plants grown without added N.

N transfer was expressed as percentage N_transfer (eqn 2), amount of N transferred (mg per plant, eqn 4) and percentage NDFT (N in the N receiver derived from transfer, eqn 5) (Johansen & Jensen, 1996):

• % N_transfer = ¹⁵N content_receiver × 100/ (¹⁵N content_receiver + ¹⁵N content_donor)(Eqn 2)

where

• ¹⁵N content_plant = atom %¹⁵N excess_plant × total N_plant/ atom % ¹⁵N excess_{labeled N}(Eqn 3)

Amount of N transferred (mg per plant):

• N_transfer = % N_transfer × total N_donor/(100 − % N_transfer)(Eqn 4)

% of N in receiver derived from transfer (% NDFT):

• % NDFT = N_transfer × 100/total N_receiver(Eqn 5)

% NDFT values were also calculated as follows (Tomm et al., 1994):

• % NDFT_donor = (atom %¹⁵N excess_receiver/ atom %¹⁵N excess_donor) × 100(Eqn 6)

Statistical analyses

Each treatment had nine replicates. Data were arc-sine transformed if zero values existed, then analyzed by anova procedures for N donor and N receiver separately. Differences in means were compared (Tukey's Honestly Significant Difference Method) and were considered significant at P ≤ 0.05 or 0.01 level (Sokal & Rohlf, 1995).

Results

Biological nitrogen fixation in Casuarina plants

Shoot δ¹⁵N values in Nod⁺Myc⁺Casuarina seedlings were similar when either (NH₄)₂SO₄ or KNO₃ was the N-source (Table 2). There were no differences in percent N₂ fixation and specific nodule activity for either N source. Casuarina provided more than a quarter of its total N needs by N₂ fixation (Table 2).

Table 2.  Effect of N source on N₂ fixation by 5-month-old mycorrhizal Casuarina seedlings grown in two-chambered pots

N-source	Shoot δ^15N (‰)	Nfixed (%)	Specific Nodule Activity (µg N mg^−1 d. wt nodule)
Data are means ± SE (n = 9). For each parameter, values followed by the same letter are not significantly different (P ≤ 0.05). Percent Nfixed is calculated by eqn 1. Shoot δ^15N value of Casuarina grown without added N was − 6.44 ± 0.78.
(NH4)2SO4	−0.33 ± 0.19a	27.0 ± 2.9a	22.2 ± 0.4a
KNO3	−0.84 ± 0.11a	28.7 ± 1.6a	22.1 ± 2.1a

Formation of mycorrhizas and common mycorrhizal networks

In the nonmycorrhizal treatments, colonization was < 5% (Table 3). No hyphae were observed on the nylon mesh and very few mycelia were seen in the growth media. Ectomycorrhizas were formed on mycorrhizal roots in mycorrhizal treatments. Colonization differed significantly between Myc⁻ and Myc⁺ plants in both species. Eucalyptus had nearly double the colonization of Casuarina. Both Nod⁻Myc⁺ and Nod⁺Myc⁺Casuarina had similar infection levels (Table 3). Observations with an environmental scanning electron microscope showed that living fungal mycelia ramified over nylon mesh (Fig. 1a), penetrated the mesh between chambers and established links between roots of the two plants in mycorrhizal treatments (Fig. 1b,c) in mycorrhizal treatments. No roots crossing the nylon meshes were observed in any treatment.

Table 3.  Mycorrhizal colonization and percentage N of Casuarina and Eucalyptus seedlings grown in two-chambered pots

N-source	Treatment		Colonization (%)		N (%)
	Myc	Nod	Casuarina	Eucalyptus	Casuarina	Eucalyptus
Plants were fertilized with (^14NH4)2SO4 or K^14NO3 for 5 months, and then with (^15NH4)2SO4 or K^15NO3 (N donor only) for 1 month before harvest. Data are means ± SE (n = 18). For each parameter, values followed by different letters designate significant differences (P ≤ 0.05) between treatments for a given N form and a given species within columns (a, b, c), and between species for a given N form and a given treatment within rows (x, y, z), respectively. Myc, mycorrhizal status; Nod, nodulation status.
(^15NH4)2SO4	–	–	5 ± 1(b, x)	5 ± 1(b, x)	0.85 ± 0.01(c, x)	0.83 ± 0.04(b, x)
	+	–	34 ± 4(a, y)	62 ± 3(a, x)	1.06 ± 0.06(b, x)	0.90 ± 0.02(ab, x)
	+	+	34 ± 7(a, y)	66 ± 3(a, x)	1.69 ± 0.06(a, x)	0.96 ± 0.03(a, y)
K^15NO3	–	–	2 ± 1(b, x)	3 ± 1(b, x)	0.92 ± 0.03(c, x)	0.82 ± 0.05(b, x)
	+	–	33 ± 4(a, y)	63 ± 4(a, x)	1.02 ± 0.05(b, x)	0.91 ± 0.05(ab, x)
	+	+	35 ± 5(a, y)	64 ± 6(a, x)	1.66 ± 0.10(a, x)	0.96 ± 0.06(a, y)

Biomass production

Biomass production of Casuarina and Eucalyptus followed the following pattern: Nod⁺Myc⁺ treatment (1.4–1.8 times) > Nod⁻Myc⁺ treatment (1.2–1.4 times) > Nod⁻Myc⁻ treatment (Fig. 2). Biomass production was not affected by whether Casuarina or Eucalyptus served as N donor or N receiver (Fig. 2), so donor and receiver biomass data were combined.

Figure 2. Biomass of Casuarina (solid bars) and Eucalyptus (open bars) seedlings as affected by mycorrhizas, nodulation and N source. Plants were fertilized with ¹⁴N for 5 months and ¹⁵N (N donor only) for 1 month before harvest. Since the identity of the donor plant did not affect biomass, data (means ± SE, n = 18) were combined. Values followed by the same letter for each plant species and each treatment are not significantly different (P ≤ 0.05). Abbreviations: C, Casuarina; E, Eucalyptus; M, mycorrhizal status; F, nodulation status.

Plant N concentration and N content

Nod⁺Myc⁺Casuarina had significantly greater %N than either Nod⁻Myc⁺Casuarina or Nod⁻Myc⁻Casuarina (Table 3). There were no statistical differences in N concentrations between Myc⁻ and Myc⁺Eucalyptus, if Eucalyptus was paired with Nod⁻Myc⁺Casuarina. When Myc⁺Eucalyptus was paired with Nod⁺Myc⁺Casuarina, plant %N was greater than Myc⁻Eucalyptus (Table 3). N content in Casuarina and Eucalyptus was greater in the Nod⁺Myc⁺ treatment (3–4 times, Casuarina; 1.5–2 times, Eucalyptus) than in the Nod⁻Myc⁺ treatment (1.5 times, Casuarina; 1.2–1.5 times, Eucalyptus) or in the Nod⁻Myc⁻ treatment (Fig. 3). N content was not affected by N source or the genus of donor and receiver. Biomass production increased with tissue N content in both Casuarina and Eucalyptus (Fig. 4, r² ranged from 0.83 to 0.99). Nod⁻Myc⁺ plants had significantly higher biomass and N content than Nod⁻Myc⁻ controls. The greatest biomass and N content was found in the combination – Nod⁺Myc⁺Casuarina and Myc⁺Eucalyptus.

Figure 3. Nitrogen content of Casuarina and Eucalyptus seedlings (solid bars for donors and open bars for receivers) as affected by mycorrhizas, nodulation, N source, and identity of the N-donor or N-receiver. Plants were fertilized with ¹⁴N for 5 months and with ¹⁵N (N donor only) for 1 month before harvest. Data are means ± SE (n = 9). Values followed by the same letter for each plant species and each treatment are not significantly different (P ≤ 0.05). Abbreviations: C, Casuarina; E, Eucalyptus; M, mycorrhizal status; F, nodulation status.

Figure 4. Relationship between biomass and N content of Casuarina (circles) and Eucalyptus (triangles) seedlings. The ¹⁵N-donors are indicated by solid symbols, ¹⁵N-receivers by open symbols. Data are means ± SE (n = 9). Regressions are shown for Casuarina (solid lines) and for Eucalyptus (dashed lines). Abbreviations: C, Casuarina; E, Eucalyptus; M, mycorrhizal status; F, nodulation status.

¹⁵N atom % excess and ¹⁵N content

As expected, ¹⁵N atom percentage excess was much greater in N donors than in N receivers, regardless of mycorrhizal status and N source (Table 4). For both Casuarina and Eucalyptus donors, whether ¹⁵NH₄⁺ or ¹⁵NO₃⁻ was the N source, plants in the Nod⁻Myc⁻ treatment had significantly greater ¹⁵N atom percentage excess, followed by plants with Nod⁻Myc⁺ treatment, then by plants with Nod⁺Myc⁺ treatment. By contrast, in the Nod⁻Myc⁻ and Nod⁻Myc⁺ treatments, receivers had similar values for ¹⁵N atom % excess. When Myc⁺Eucalyptus was grown with Nod⁺Myc⁺Casuarina, both plant species had significantly higher ¹⁵N atom % excess than their Nod⁻Myc⁻ counterparts. In addition, ¹⁵N atom % excess was generally higher when ¹⁵NO₃⁻ was the N source.

Table 4. ¹⁵N atom % excess and total ¹⁵N content of Casuarina and Eucalyptus seedlings grown in two-chambered pots. Plants were fertilized with (¹⁴NH₄)₂SO₄ or K¹⁴NO₃ for 5 months, and then with (¹⁵NH₄)₂SO₄ or K¹⁵NO₃ (N donor only) for 1 month before harvest

N-source	Codes	N donor	^15N (atom % excess)	^15N (mg per plant)	N receiver	^15N (atom % excess)	^15N (mg per plant)
Data are means ± SE (n = 9). For each parameter, values followed by different letters designate significant differences (P ≤ 0.05) between treatments for a given N form and a given species within columns (a, b, c), and between species for a given N form and a given treatment within rows (x, y, z), respectively. C, Casuarina; E, Eucalyptus; M, mycorrhizal status; F, nodulation status.
(^15NH4)2SO4	C^M–F–→E^M–	Casuarina	0.475 ± 0.006(a, x)	1.041 ± 0.048(c, x)	Eucalyptus	0.004 ± 0(b, y)	0.006 ± 0(c, y)
	E^M–→C^M–F–	Eucalyptus	0.846 ± 0.054(a, x)	1.771 ± 0.076(a, x)	Casuarina	0.003 ± 0(b, y)	0.005 ± 0(c, y)
	C^M+F–→E^M+	Casuarina	0.387 ± 0.006(b, x)	1.398 ± 0.098(b, x)	Eucalyptus	0.008 ± 0(b, y)	0.018 ± 0.001(b, y)
	E^M+→C^M+F–	Eucalyptus	0.479 ± 0.002(b, x)	1.273 ± 0.073(b, x)	Casuarina	0.004 ± 0(b, y)	0.011 ± 0.001(b, y)
	C^M+F+→E^M+	Casuarina	0.243 ± 0.002(c, x)	1.710 ± 0.069(a, x)	Eucalyptus	0.014 ± 0.001(a, y)	0.042 ± 0.003(a, y)
	E^M+→C^M+F+	Eucalyptus	0.276 ± 0.021(c, x)	0.945 ± 0.121(c, x)	Casuarina	0.019 ± 0.004(a, y)	0.123 ± 0.025(a, y)
K^15NO3	C^M–F–→E^M–	Casuarina	0.995 ± 0.045(a, x)	2.205 ± 0.115(a, x)	Eucalyptus	0.011 ± 0.002(b, y)	0.014 ± 0.001(c, y)
	E^M–→C^M–F–	Eucalyptus	1.037 ± 0.072(a, x)	2.124 ± 0.187(a, x)	Casuarina	0.010 ± 0.001(b, y)	0.015 ± 0.001(c, y)
	C^M+F–→E^M+	Casuarina	0.551 ± 0.025(b, x)	1.733 ± 0.068(b, x)	Eucalyptus	0.012 ± 0.001(b, y)	0.025 ± 0.002(b, y)
	E^M+→C^M+F–	Eucalyptus	0.701 ± 0.063(b, x)	1.818 ± 0.007(b, x)	Casuarina	0.015 ± 0.001(b, y)	0.036 ± 0(b, y)
	C^M+F+→E^M+	Casuarina	0.281 ± 0.038(c, x)	1.918 ± 0.371(b, x)	Eucalyptus	0.025 ± 0.002(a, y)	0.068 ± 0.010(a, y)
	E^M+→C^M+F+	Eucalyptus	0.496 ± 0.047(c, x)	1.667 ± 0.021(b, x)	Casuarina	0.035 ± 0.002(a, y)	0.243 ± 0.011(a, y)

N donors had significantly greater ¹⁵N content than N receivers. As a donor when ¹⁵NH₄⁺ as the N-source, Casuarina showed a different pattern of ¹⁵N accumulation from Eucalyptus (Table 4). All donor and receiver plants fertilized with ¹⁵NH₄⁺ had lower ¹⁵N content than plants fertilized with ¹⁵NO₃⁻ (Table 4). When ¹⁵NH₄⁺ was supplied and either plant species was the N donor, ¹⁵N content in Casuarina (donor or receiver) was lowest in Nod⁻Myc⁻ treatment, greater in Nod⁻Myc⁺ treatment and greatest in Nod⁺Myc⁺ treatment. When ¹⁵NH₄⁺ was supplied and either plant species was the N donor, ¹⁵N content decreased in Eucalyptus donor from Nod⁻Myc⁻ to Nod⁻Myc⁺, then to Nod⁺Myc⁺ plants, while ¹⁵N content increased in Eucalyptus receiver from Nod⁻Myc⁻ to Nod⁻Myc⁺, then to Nod⁺Myc⁺ plants. By contrast, when ¹⁵NO₃⁻ was supplied and either plant species was the N donor, ¹⁵N accumulation was similar in Nod⁺Myc⁺ and Nod⁻Myc⁺Casuarina and Eucalyptus donors, but the greatest ¹⁵N accumulation was displayed in Nod⁻Myc⁻Casuarina and Eucalyptus donors. On the other hand, for both Casuarina and Eucalyptus N receiver, ¹⁵N content consistently increased as follows: Nod⁺Myc⁺ > Nod⁻Myc⁺ > Nod⁻Myc⁻ plants, when either ¹⁵NH₄⁺ or ¹⁵NO₃⁻ was supplied (Table 4).

Nitrogen transfer between Casuarina and Eucalyptus

In general, N transfer with either (¹⁵NH₄)₂SO₄ or K¹⁵NO₃ as the tracing N source was bi-directional in all Casuarina/Eucalyptus pairs (Table 5). There were three parameters of N transfer: first, percent N transfer; second, percent N in the receiver derived from transfer (% NDFT); and third, amount of N transferred. In Nod⁻Myc⁻ pairs, these three parameters were similar in either direction – from Casuarina to Eucalyptus or from Eucalyptus to Casuarina (Table 5). In Nod⁻Myc⁻ pairs, both % N transfer and % NDFT were very low (1.0%); amount of N transferred was < 1.4 mg N per plant. In Nod⁻Myc⁺ pairs, these three N transfer parameters were also similar in either direction, but they were 2–4 times greater than those in Nod⁻Myc⁻ pairs (Table 5).

Table 5.  Total N transfer and net N transfer between Casuarina and Eucalyptus seedlings grown in two-chambered pots. Plants were fertilized with (¹⁴NH₄)₂SO₄ or K¹⁴NO₃ for 5 months, and then with (¹⁵NH₄)₂SO₄ or K¹⁵NO₃ (N donor only) for 1 month before harvest

N-source	Plant pairs	N transfer (%)		NDFT (%)		N transfer (mg per plant)		Net N transfer
		Casuarina↓Eucalyptus	Eucalyptus↓Casuarina	Casuarina↓Eucalyptus	Eucalyptus↓Casuarina	Casuarina↓Eucalyptus	Eucalyptus↓Casuarina	Sink plant	mg per plant
Data are means ± SE (n = 9). For each parameter, values followed by different letters designate significant differences (P ≤ 0.05) between directions of transfer in pairs for a given N form within rows (a, b, c), and between pairs for a given N form within columns (x, y, z), respectively. NDFT, N in the receiver derived from transfer; C, Casuarina; E, Eucalyptus; M, mycorrhizal status; F, nodulation status.
(^15NH4)2SO4	C^M–F–↔E^M–	0.6 ± 0 (a, z)	0.3 ± 0 (a, z)	0.9 ± 0 (a, z)	0.3 ± 0 (a, z)	1.3 ± 0 (a, z)	0.6 ± 0 (a, z)	Eucalyptus	+0.7
	C^M+F–↔E^M+	1.3 ± 0 (a, y)	1.0 ± 0.1(a, y)	2.1 ± 0 (a, y)	1.0 ± 0.1(a, y)	3.5 ± 0.7(a, y)	2.4 ± 0.2(a, y)	Eucalyptus	+1.1
	C^M+F+↔E^M+	2.4 ± 0.1(b, x)	11.3 ± 1.1(a, x)	5.9 ± 0.3(a, x)	7.0 ± 1.1(a, x)	17.4 ± 1.2(b, x)	43.7 ± 6.2(a, x)	Casuarina	+26.3
K^15NO3	C^M–F–↔E^M–	0.7 ± 0.1(a, z)	0.7 ± 0.1(a, z)	1.0 ± 0.1(a, z)	0.7 ± 0 (a, z)	1.4 ± 0.1(a, z)	1.4 ± 0.1(a, z)	Neither	0
	C^M+F–↔E^M+	1.4 ± 0.1(a, y)	2.0 ± 0 (a, y)	2.1 ± 0.1(a, y)	1.7 ± 0.1(a, y)	3.6 ± 0.7(a, y)	4.1 ± 0.8(a, y)	Casuarina	+0.5
	C^M+F+↔E^M+	3.5 ± 0.3(b, x)	12.7 ± 0.7(a, x)	8.6 ± 0.2(a, x)	7.3 ± 0.3(a, x)	24.1 ± 1.0(b, x)	50.1 ± 5.8(a, x)	Casuarina	+26.0

In Nod⁺Myc⁺ pairs, N transfer expressed as % NDFT was similar in either direction, while % N transfer and amounts of N transfer were different and depended on the direction from Casuarina to Eucalyptus or from Eucalyptus to Casuarina (Table 5). In Nod⁺Myc⁺ pairs, compared with Nod⁻Myc⁺ pairs, more N was transferred to Eucalyptus (5–7 times) or to Casuarina (12–18 times). When Eucalyptus was paired with Nod⁺Myc⁺Casuarina, nonN₂-fixing Myc⁺Eucalyptus donated 2.1–2.5 times more N to Nod⁺Myc⁺Casuarinas than the reverse (Table 5). In addition, Nod⁻Myc⁻ and Myc⁺ pairs showed little net N transfer, while Nod⁺Myc⁺ pairs showed much greater net N transfer to Casuarina (Table 5).

Discussion

Biological nitrogen fixation

We used two strains of Frankia, UGL020604 and UGL020605, which are known to be highly effective on C. cunninghamiana (Wheeler et al., 2000). In our study, N₂-fixation by C. cunninghamiana (27–29%) was generally comparable with data from other studies with Casuarina species including C. cunninghamiana (Mariotti et al., 1992; Parrota et al., 1994, 1996; Subbarao & Rodriguez-Barrueco, 1995). We used ¹⁵N natural abundance to calculate both percent of total plant N supplied by N₂-fixation and the amount of N in the nodules expressed as g N mg⁻¹ dry weight nodule. It is difficult to compare our values with Reddell & Bowen (1985) and Reddell et al. (1988, 1997). Reddell and coworkers calculated the amount of N in the nodule that was derived from fixation as mg N fixed mg⁻¹ nodule dry weight; they did not calculate total nodule N, which includes N from other sources such as soils. In our study, plants received only 5 ml per pot of 4 mmol N as either (¹⁴NH₄)₂SO₄ or K¹⁴NO₃ once a week as a fertilizer. This low N load did not reduce fixation. Other studies reported that low N fertilization even stimulated N₂-fixation (Huss-Danell, 1997; Wheeler et al., 2000). There was no effect of N source on N₂-fixation in our study, but Zhang and Torrey (1985) found that NH₄⁺ inhibited nodulation of C. cunninghamiana more than NO₃⁻. Other studies with Casuarina species did report reduction of fixation by either NH₄⁺ or NO₃⁻ (Kohls & Baker, 1989; Arnone et al., 1994; Baker et al., 1994).

Mycorrhizal colonization

Mycorrhizal colonization in Casuarina (c. 35%) and in Eucalyptus (c. 65%) was generally higher than previously reported by other researchers. With a variety of EM fungi, Casuarina had 10–40% EM (Gardner, 1986; Theodorou & Reddell, 1991; Subbarao & Rodriguez-Barrueco, 1995); and Eucalyptus formed 10–60% EM colonization (Chilvers et al., 1987; Bellei et al., 1992; Burgess et al., 1993, 1994; May & Simpson, 1997; Reddell et al., 1999; Chen et al., 2000). Our seedlings were both EM and nodulated. Gardner (1986) and Subbarao & Rodriguez-Barrueco (1995) also worked with EM and nodulated Casuarinas species. We did not observe any increase in mycorrhization with nodulation, nor did Gardner (1986). Well-controlled aseptic and suitable glasshouse environments may have favored higher mycorrhizal colonization rates.

Common mycorrhizal networks between plants

In our experiment, fungal mycelia grew through nylon mesh and linked the roots of Casuarina and Eucalyptus, establishing a common EM network (He, 2002). We demonstrated that the CMN functioned in two-way N transfer. Mycorrhizal links between plants have been observed in transparent microcosms (Francis & Read, 1984; Newman, 1988; Newman et al., 1994) and by carbon and phosphorus isotope autoradiography (Chiariello et al., 1982; Francis & Read, 1984; McKendrick et al., 2000). Nitrogen movement through mycorrhizal linkage or CMNs from plant to plant has been reported (Haystead et al., 1988; Eissenstat, 1990; Bethlenfalvay et al., 1991; Frey & Schuepp, 1992, 1993; Arnebrant et al., 1993; Johansen et al., 1993a,b; Ikram et al., 1994; Tobar et al., 1994a,b; Ekblad & Huss-Danell, 1995; Ek et al., 1996; Johansen & Jensen, 1996). Read (1997) discussed the ecological importance of nutrient transfer through a CMN and speculated that nutrient transfer affects plant growth and biogeochemical cycling. Two-way nutrient transfer could reduce competition for soil resources between plants.

Responses of plant growth to mycorrhizal colonization and nodulation

Both biomass and N content in Casuarina and Eucalyptus were highest when Casuarina was Nod⁺Myc⁺, lower in Nod⁻Myc⁺ plants, and lowest in Nod⁻Myc⁻ plants. Although mycorrhizal infection was double in Eucalyptus compared with Casuarina, this had a slight effect on N accumulation by Eucalyptus. Mycorrhization did, however, enhance N accumulation by Nod⁻Myc⁺Casuarina. Compared with Nod⁻Myc⁺Casuarina, N content doubled in Nod⁺Myc⁺Casuarina, especially when Casuarina was the receiver. As the receiver, Casuarina experienced a 1-month-period with no N fertilizer, perhaps creating nutrient stress. The magnitude of increase in N content in mycorrhizal Eucalyptus was much less when they were partnered with Nod⁺Myc⁺Casuarina, rather than with Nod⁻Myc⁺Casuarina. Thus, the combination of Frankia and a mycorrhizal fungus had the greatest effect on growth of Casuarina. Both Casuarina and Eucalyptus receivers obtained substantial benefit via CMNs from their donors, when Casuarina was nodulated and mycorrhizal. Biomass production by Casuarina and Eucalyptus was similar whether either species acted as donor or receiver. This suggested a physiological benefit to receivers from N transfer. In our study, Nod⁻Myc⁺ seedlings were generally taller, had larger root systems, greater shoot and/or root biomass and higher N content than Nod⁻Myc⁻ seedlings. Similar results were observed for Nod⁻Myc⁺ and Nod⁺Myc⁺ seedlings. This has been reported by others (Smith & Read, 1997; Jonsson et al., 2001; Rogers et al., 2001).

Below-ground N-transfer between plants

Our results demonstrated that ectomycorrhizas mediated bidirectional N transfer between Casuarina and Eucalyptus. In the Nod⁻Myc⁻ treatment, there was almost no N transfer (< 1% in both N transfer and NDFT) to Casuarina or to Eucalyptus, and very low N transfer (c. 2% in both N transfer and NDFT) to Casuarina or to Eucalyptus in the Nod⁻Myc⁺ treatment. By contrast, N transfers were greatest in the Nod⁺Myc⁺ treatment. More N moved to Nod⁺Myc⁺Casuarina than to Myc⁺Eucalyptus, for both ¹⁵NH₄⁺ and ¹⁵NO₃⁻. About twice as much N was transferred from Eucalyptus to Nod⁺Myc⁺Casuarinas than the reverse, resulting in increased growth by Casuarina due to interspecific N transfer. Since there was virtually no N transfer in the nonmycorrhizal treatment, but significant N transfer in the mycorrhizal treatment, N transfer was mediated by mycorrhizal fungi. The much higher N transfer between nodulated mycorrhizal plants indicated that mycorrhizas and Frankia together enhanced N fluxes between N₂-fixing Casuarina and nonN₂-fixing mycorrhizal Eucalyptus. The amount of N transfer was significantly greater from Eucalyptus to Nod⁺Myc⁺Casuarina than the reverse, contradicting the general hypothesis that N flows from N₂-fixing to nonN₂-fixing plants (Bethlenfalvay et al., 1991; Frey & Schuepp, 1993). However,% NDFT was similar for Casuarina and Eucalyptus even in Nod⁺Myc⁺ treatment, regardless of the direction of N transfer.

Nitrogen transfer from legume to non-legume can take place by indirect or direct routes (McNeill & Wood, 1990). Indirect N transfer via soil solution comes from dead and decayed nodules and roots (Dubach & Russelle, 1994), exudates such as ammonium, amino acids and amides from legume roots (Ta et al., 1989; Paynel et al., 2001), and sloughed-off root-cortical cells (Brophy & Heichel, 1989). These materials are deposited in soil and could be absorbed by adjacent plants. This is the soil pathway (Stern, 1993; Chalk, 1996). Direct N transfer may be mediated by mycorrhizal hyphae where CMNs connect plants. One-way N transfer via CMNs has been demonstrated using fine nylon mesh barriers that allow passage of hyphae but not roots (Bethlenfalvay et al., 1991; Frey & Schuepp, 1992; Arnebrant et al., 1993; Johansen & Jensen, 1996). The one-way N transfer hypothesis states that N flows from an N-rich legume source (N donor) to an N-poor nonlegume receiver (sink) (Bethlenfalvay et al., 1991; Frey & Schuepp, 1993). AM-mediated (¹⁵NH₄⁺ or ¹⁵NO₃⁻) or EM-mediated (¹⁵NH₄⁺ only) one-way N transfer from N₂-fixing to nonN₂-fixing plants has been reported (Bethlenfalvay et al., 1991; Frey & Schuepp, 1992; Arnebrant et al., 1993; Ekblad & Huss-Danell, 1995; Johansen & Jensen, 1996; Martensson et al., 1998). None of these studies measured bidirectional transfer.

We calculated net N transfer as the difference between N transferred from Casuarina to Eucalyptus and from Eucalyptus to Casuarina. Net N transfer was low in both the Nod⁻Myc⁻ and the Nod⁻Myc⁺ treatments (Table 5). Surprisingly, in the Nod⁺Myc⁺ treatment, there was greater net N transfer from nonN₂-fixing Eucalyptus to N₂-fixing Casuarina (Table 5). A similar phenomenon was reported for grass to legume pairs, resulting in a two-way N-flow between N₂-fixing and nonN₂-fixing plants (Brophy et al., 1987; Tomm et al., 1994; Hogh-Jensen & Schjoerring, 2000). Transfer was not significant under N-limited conditions and effects of mycorrhization were not examined in those studies. N transfer from barley to pea seedlings connected by Glomus intraradices was very low (Johansen & Jensen, 1996). No N transfer was found between clover and ryegrass through AM hyphal links (Rogers et al., 2001).

Inorganic N in soil is available to plants as NH₄⁺ or NO₃⁻ (Forde & Clarkson, 1999). Most EM fungi can assimilate NH₄⁺ (Jongbloed et al., 1991; Martin & Botton, 1993; Martin & Plassard, 2001), but few can efficiently reduce NO₃⁻ (Scheromm et al., 1990; Martin & Botton, 1993; Martin & Plassard, 2001). Apparently most AM fungi can assimilate both NH₄⁺ (Smith et al., 1985; Cliquet & Stewart, 1993; Johansen et al., 1996) and NO₃⁻ (Ho & Trappe, 1975; Johansen et al., 1996; Faure et al., 1998). The less mobile NH₄⁺ is adsorbed to soil colloids and moves toward plant roots by diffusion. The highly mobile NO₃⁻ is transported toward plant roots by mass flow (Brady & Weil, 2002). Mycorrhizal hyphae may play a greater role in transport of NH₄⁺ than of NO₃⁻ to plant roots, except in water-deficient soil (Tobar et al., 1994b; Wu et al., 1999).

Our results showed that mycorrhization increased biomass production and N accumulation and that plant biomass increased with N content for both species. Mycorrhizas enhanced N accumulation, biomass production and N transfer between plants, and may thus affect plant fitness. Mycorrhizas had less impact on N accumulation in nonN₂-fixing Eucalyptus than in N₂-fixing Casuarina, despite the fact that Eucalyptus had nearly double the mycorrhizal infection compared with Casuarina. When Casuarina was nodulated and mycorrhizal and when Eucalyptus was mycorrhizal, biomass yield and N content were maximized in both plants. Our data corroborate previous reports that the main effect of mycorrhization on plant N status was in N₂-fixing plants (Hayman, 1986). All these data suggest that N transfer has biological significance. Mycorrhization, together with N availability, plays a vital role in re-distribution of N between plants. Considering that N supply is crucial for plant growth in most terrestrial ecosystems (Vitousek et al., 1997; Moffat, 1998), and that the potential benefits of N transfer are great (CSIRO Publishing, 1998), two-way N transfer may be very significant.

Acknowledgements

For valuable comments on the manuscript, we thank two anonymous reviewers and Dr Francis Martin, the New Phytologist Section Editor; Dr D Southworth, Department of Biology, Southern Oregon University; and Dr AJ Bloom, Department of Vegetable Crops, University of California at Davis, USA. This research was supported by Postgraduate Research Scholarships from the Department of Education, Science and Training, and the University of Queensland, Australia; and a Postdoctoral Fellowship from NSF grant ‘Biocomplexity Phase I’ (DEB-9981711 to Dr CS Bledsoe). XH He is grateful to the Education Department of Yunnan Province and the Yunnan Normal University, China for permission to study overseas.