Directory UMM :Data Elmu:jurnal:T:Tree Physiology:Vol16.1996:

Tree Physiology 16, 941--948
© 1996 Heron Publishing----Victoria, Canada

Root adaptation and nitrogen source acquisition in natural ecosystems
MATTHEW H. TURNBULL,1 SUSANNE SCHMIDT,2 PETER D. ERSKINE,2
SUANNE RICHARDS2 and GEORGE R. STEWART2,3
1
2
3

Department of Plant and Microbial Sciences, University of Canterbury, Christchurch, New Zealand
Department of Botany, The University of Queensland, Brisbane, Queensland 4072, Australia
Author to whom correspondence should be addressed

Received October 25, 1995

Summary The capacity for nitrate reduction, as measured by
nitrate reductase activity (NRA), was generally low for a range
of plant communities in Australia (coastal heathland, rainforest, savanna woodland, monsoon forest, mangrove, open Eucalyptus forest, coral cay open forest) and only a loose
relationship existed between NRA and leaf nitrogen concentration. This suggests that nitrate ions are not the sole nitrogen
source in these communities. Based on 15N labeling experiments, we found a range of tree species exhibiting a pronounced preference for uptake of ammonium over nitrate.

Analysis of soil solutions from several forest and heathland
communities indicated that ammonium ions were more prevalent than nitrate ions and that soluble forms of organic nitrogen
(amino acids and protein) were present in concentrations similar to those of mineral nitrogen. To determine the extent to
which root adaptations and associations might broaden nitrogen source utilization to include organic nitrogen, we assessed
the effects of various nitrogen sources on seedling growth in
sterile culture. Non-mycorrhizal seedlings of Eucalyptus grandis W. Hill ex Maiden. and Eucalyptus maculata Hook. grew
well on mineral sources of nitrogen, but did not grow on organic
sources of nitrogen other than glutamine. Mycorrhizal seedlings grew well on a range of organic nitrogen sources. When
offered a mixture of inorganic and organic nitrogen sources at
low concentrations, mycorrhizal seedlings derived a significant
proportion of their nitrogen budget from organic sources. We
also demonstrated that a species of the obligately non-mycorrhizal genus Hakea, a heathland proteaceous shrub possessing
cluster roots, had the ability to incorporate 15N-labeled organic
sources (e.g., glycine). We conclude that mycorrhizal associations and root adaptations confer the ability to substantially
broaden the nitrogen source base on some plant species.
Keywords: ammonium, cluster root, mycorrhiza, nitrate, nitrate reductase.

Introduction
Several root characteristics are reported to enhance mineral
nutrition, including various kinds of mycorrhizal roots (Harley

and Smith 1983), cluster roots (Lamont 1982) and N2-fixing

nodulated roots (Sprent and Sprent 1990). With the exception
of nodulated roots, the significance of these root specializations for nitrogen nutrition of plants in natural ecosystems is
less clear than for phosphorus nutrition (Alexander 1983,
Dinkelaker et al. 1995). Nitrogen availability may be a key
factor determining photosynthetic capacity of natural plant
communities (Field and Mooney 1986), especially in plant
communities for which acute deficiencies of nitrogen are common.
Vesicular-arbuscular, ericoid, orchid and ecto-mycorrhizae
all play a role in plant nitrogen acquisition (Read 1991).
Alexander (1983) showed that ectomycorrhizae enhance nitrate and ammonium uptake by plants, because the fungal
hyphae increase the effective volume of soil exploited by the
roots. Mycorrhizal fungi can also utilize organic forms of
nitrogen (Abuzinadah and Read 1986, Finlay et al. 1992), thus
making forms of nitrogen available to the host that would
otherwise be unavailable to it. This capacity may be of particular importance for woody plants growing in nutrient-poor soils
(Read 1991).
Root clusters are aggregations of hairy rootlets that are
produced on the root systems of many plants (Lamont 1982).

Species with cluster roots are able to grow on poor soils,
especially those low in phosphorus and nitrogen (Lamont
1993). Cluster roots improve the nutrition of P, Fe, and Mn by
altering rhizosphere conditions through excretion of organic
acids and phenolics (Dinkelaker et al. 1995). Cluster roots are
induced in species of the genus Hakea under N-limiting conditions and are mainly produced in soil horizons rich in organic
matter (Lamont 1973). The possible significance of proteoid
roots for N nutrition was noted by Pate and Jeschke (1993) who
found higher concentrations of amino acids in the xylem sap
of proteoid roots than of non-proteoid roots of Banksia
prionotes Lindley.
Three distinct groups of N2-fixing root associations can be
identified: the rhizobial associations of leguminous species,
the actinorrhizal associations of the genus Casuarina and the
coralloid roots of cycads (Lamont 1984). The haustoria of root
hemiparasites constitute another potentially important root
specialization implicated in the uptake of organic nitrogen

942

TURNBULL ET AL.

(e.g., in the genera Exocaropus, Anthobolus, Santalum and
Striga) (Lamont 1984).
To date, the importance of mycorrhizal associations and
other root adaptations to the nitrogen nutrition of species from
forest systems in sub-tropical and tropical regions has received
little attention. Because the supply of nitrogen in these systems
may be limiting to plant growth (Bowen 1981), we have
explored the impact of root specializations on the potential to
utilize various nitrogen sources and their subsequent metabolism in several subtropical species. We have analyzed enzymatic activities and metabolism in plants from a range of
sub-tropical and tropical communities, and demonstrated that
different plant species utilize different nitrogen sources and
that the type of root specialization strongly influences the
characteristics of nitrogen assimilation.

Materials and methods

bosa) excavated from the field and transferred to liquid culture.
Hakea sp. seedlings were fed either a single 15N-labeled nitrogen source (NH +4 , NO −3 or glycine) or were offered the three

nitrogen sources simultaneously, with one being 15N labeled
and the other two at natural abundance. At the end of the
incubation period, leaves and roots were extracted in methanol
and analyzed as described below.
Gas chromatography--mass spectrometry
Methanolic extracts from labeling experiments were prepared
and derivatized as described by Kershaw and Stewart (1992).
The amount of 15N incorporated into each amino acid (including glutamine and asparagine) was determined by GC--MS
analysis as described by Turnbull et al. (1995). The presence
of free amino acids in the methanol-soluble fraction of fungal
mycelium was determined by a post-column ninhydrin derivitization, HPLC-based amino acid analyzer (Model 6300,
Beckman Instruments, Palo Alto, CA).

Study sites and plant material

Soil solution analysis

Measurements were made in nine plant communities from
sub-tropical and tropical northern Australia: (1) sub-tropical
coastal heathland (Beerwah State Forest, 60 km north of Brisbane, Queensland); (2) semi-arid ‘‘mulga’’ woodland (Currawinya National Park, 1000 km west of Brisbane, Queensland);

(3) sub-tropical rainforest (Lamington National Park, Green
Mountains, 120 km south of Brisbane, Queensland); (4) savanna woodland (Kakadu National Park, 200 km east of Darwin, Northern Territory); (5) monsoon open forest (Kakadu
National Park, 200 km east of Darwin, Northern Territory); (6)
eucalypt open forest (Brisbane Forest Park, Brisbane, Queensland); (7) tropical mangrove forest (Kakadu National Park, 200
km east of Darwin, Northern Territory); (8) coral cay forest
(Heron Island, Great Barrier Reef Marine Park, Queensland);
and (9) tropical rainforest (Kakadu National Park, 200 km east
of Darwin, Northern Territory). Data for other plant communities have been included where appropriate and are sourced
from previous works as indicated in the text.

Soils from the coastal heathland (Site 1) and eucalypt open
forest (Site 6) were sampled. At Site 6, soils from two distinct
open-forest types were analyzed, a moist fertile forest dominated by Eucalyptus grandis W. Hill ex Maiden. and a ridge
top dominated by Eucalyptus maculata Hook. Soluble components were extracted from the soil samples (0--5 cm depth) in
distilled water or in 1 mol m −3 KCl, and nitrate, ammonium,
amino acids and soluble protein were determined as described
by Turnbull et al. (1995). At the coastal heathland site (Site 1),
we compared the immediate availability of ammonium, nitrate
and amino acids in the soil solution of the rooting zone by
means of mixed-bed ion exchange resin bags (Stewart et al.

1993). Ammonium and nitrate in the eluate were assayed as
described by Stewart et al. (1993). Amino acid concentration
was determined by HPLC.

Nitrate reductase assays
Terminal leafy material from the principal overstory and understory species was collected from each community. The suite
of species studied generally represented over 80% of the community biomass--ground cover at the respective sites. Nitrate
reductase activity (NRA) was determined on freshly harvested
leaf material by an in vivo assay (Stewart et al. 1986). Leaf
nitrogen concentration was determined in oven-dried and
finely ground material by automated combustion.
15

N Labeling experiments

The pathway of incorporation and metabolism of 15N-labeled
substrates (NH +4 , NO −3 , glycine) was determined after the seedlings had been incubated for 8 h in a fresh aerated liquid
medium containing 1 mol m −3 nitrogen (99 or 65 atom%
excess 15N). Experiments were conducted on Eucalyptus sp.
seedlings grown in sterile culture and on Hakea seedlings

(Hakea sp. Mt. Coolum P.R. Sharpe 3338; formerly H. gib-

Growth assessments and production of mycorrhizal seedlings
Seeds of Eucalyptus grandis and Eucalyptus maculata were
surface sterilized and germinated on agar as described by
Turnbull et al. (1995). For production of mycorrhizal seedlings, a small amount of homogenate (sterilized distilled water
and mycelium from the edge of an actively growing culture of
Elaphomyces sp. (isolate NQ732)) was added to the petri dish
in which the seeds were germinating. Seedlings were incubated at 24 °C for 7--10 days after germination. The seedlings
were then cultured for 42 days in sealed, sterilized plastic
vessels containing sterile agar and a range of inorganic and
organic nitrogen sources (Turnbull et al. 1995). To determine
the extent to which mycorrhizal plants utilize organic forms of
nitrogen in a mixed nitrogen source, E. grandis seedlings were
grown in sterile agar to which was added 15N-labeled nitrate or
ammonium (10 atom% excess) either as the sole source or in
combination with unlabeled asparagine or bovine serum albumin (BSA). To ensure that seedlings would ‘‘scavenge’’ for
nitrogen, the sources were added to provide a total of 10 mg l −1
nitrogen. After 42 days, seedlings were harvested and leaf
samples oven-dried at 60 °C. The samples were ground to a

N SOURCE ACQUISITION AND SPECIALIZED ROOT ADAPTATION

943

fine powder in a vibratory ball mill (Retsch MM-2, Haan,
Germany) and nitrogen concentration of the leaf material was
determined by automated combustion. Duplicate samples of
approximately 120 µg nitrogen were analyzed for 15N with a
continuous flow isotope ratio mass spectrometer (CF-IRMS,
Tracer Mass, Europa Scientific, Crewe, U.K.) set to the single
nitrogen mode. Precision of the instrument, based on multiple
analysis (n = 133) of a laboratory standard (Eucalyptus crebra
F.J. Muell. leaves) was 0.21 SD. The percentage of plant
nitrogen derived from the unlabeled organic source (Forg ) was
calculated as:

range of 1200--2400 pkat gfw−1 (Figure 2), indicating that
nitrate ions are the primary source of nitrogen. In contrast, in
a range of largely vesicular-arbuscular mycorrhizal (VAM) and

vesicular-arbuscular and ecto-mycorrhizal (VAM-ECM) herbaceous and woody species, the potential for nitrate reduction
was much lower, with average values in the range of 50--350
pkat gfw−1. Of the species sampled from families with very
specialized mycorrhizal associations (the Ericaceae and Epacridaceae), all displayed low capacities for nitrate reduction.



Atom% 15 N excess in plant
Forg = 1 − 
100.
15
Atom%
N
excess
in
labeled
source



Putative N2-fixing species often displayed the highest nitrogen
concentrations, indicative of the importance of nitrogen fixation to the nitrogen status of species in nutrient-poor ecosystems. For example, in the tropical savanna woodland (Site 4),
nitrogen-fixing species had an average nitrogen concentration

Results and discussion

Contribution of nitrogen fixation

Nitrate reduction
Figure 1a describes the relationship between community-averaged leaf NRA and leaf nitrogen concentration for nine distinct
plant communities from northern Australia. The capacity for
reduction of nitrate ions was generally low compared with
values reported for plants for which nitrate ions are an important source of nitrogen (Smirnoff et al. 1984). Average NRA
ranged from 22 pkat gfw−1 for plants in the coastal heathland
(Site 1) to 117 pkat gfw−1 for plants in the semi-arid ‘‘mulga’’
woodland (Site 2). For eight of the nine communities, average
NRA was below 100 pkat gfw−1. A few species appeared to be
predominantly nitrate utilizers based on their relatively high
NR activities and high concentrations of nitrate in leaf extracts
and xylem sap (data not shown). Although NRA was generally
low in the ecosystems studied, it was not always associated
with low leaf nitrogen concentration. There was a weak relationship between community-averaged NRA and leaf nitrogen
concentration (r 2 = 0.38, Figure 1a). This was also the case
within a given community (Figure 1b). For example, in the
tropical monsoon forest (Site 5), there was no relationship
between NRA and leaf nitrogen concentration, despite the
presence of species with a moderately high capacity for nitrate
reduction (about 400 pkat gfw−1).
The low rates of nitrate reduction observed in the leaves of
species in these systems could be explained if these species are
predominantly root nitrate assimilators. However, root NRA in
these types of species is also generally low (e.g., average root
NRA in mature phase rainforest trees is 39 pkat gfw−1 (Stewart
et al. 1988), 37 pkat gfw−1 in a Banksia woodland (Stewart et
al. 1993), 18 pkat gfw−1 on the coral cay and 24 pkat gfw−1 in the
coastal heathland). These results are consistent with the conclusion that the majority of species in these ecosystems utilize
sources of nitrogen other than nitrate ions.
Using data for a range of plant groups in tropical, sub-tropical and temperate ecosystems, it is possible to identify patterns
of nitrate utilization that appear to be based on the presence or
absence of mycorrhizal associations. Putatively non-mycorrhizal families (e.g., the Urticaceae, Chenopodiaceae, Amaranthaceae and Polygonaceae) had average NRA values in the

Figure 1. (a) Relationship between community-averaged nitrate reductase activity and leaf nitrogen concentration for nine north-Australian
plant communities. 1 = coastal heathland; 2 = semi-arid woodland; 3
= sub-tropical rainforest; 4 = savanna woodland; 5 = monsoon open
forest; 6 = eucalypt open forest; 7 = tropical mangrove forest; 8 = coral
cay forest; and 9 = tropical rainforest. Values represent means ± SEM
for each variable. (b) The relationship between nitrate reductase activity and leaf nitrogen concentration for plant species in a northern
Australian monsoon forest.

944

TURNBULL ET AL.

tion of nitrogen-fixers to ecosystem nitrogen status are complicated by the fact that published values for rates of N2-fixation
in the field vary widely (0.10--1.6 kg N ha −1 year −1 for four
common shrub legumes of jarrah forest (Hansen et al. 1987);
2.2 kg N ha −1 year −1 for Acacia pulchella R. Br. (Monk et al.
1981); 18.6 kg N ha −1 year −1 for Macrozamia riedlei (Fisch.
ex Gaud.--Beaup) C. Gardn., an understory component of
mixed Banksia woodland in southwest Western Australia (Halliday and Pate 1976)). Pate et al. (1993) cite δ15N values for a
range of species in a Banksia woodland that suggest that the
woody species were either assimilating a common nitrogen
source and that nitrogen fixation was of minor importance to
the nitrogen-fixing species, or that shoot nitrogen was derived
from mobilized reserves rather than current assimilation. Heterogeneity in 15N discrimination of soil precludes the use of the
15
N natural abundance technique for assessing legume N2 fixation in a jarrah forest (Hansen and Pate 1987).
Nitrogen source availability in soils

Figure 2. Average nitrate reductase activities (± SEM) for species from
a range of non-mycorrhizal (Urticaceae, Chenopodiaceae, Amaranthaceae and Polygonaceae) and mycorrhizal (including herbaceous
spp. (largely VAM), woody spp. (largely ECM or VAM--ECM), Ericaceae and Epacridaceae) groups from tropical and temperate Australian ecosystems.

of 2.6% (± 0.43 SD) compared with a community average of
1.3% (± 0.61 SD). A similar trend was observed in the subtropical eucalypt open forest (Site 6), where nitrogen fixing
species had average nitrogen concentrations of 2.92%
(± 1.36 SD) compared with 1.52% (± 0.60 SD) for non-fixing
species. At the most nitrogen-limited site (Site 1), the average
nitrogen concentration of nitrogen-fixing species was significantly higher than that of non-fixing species (1.51 ± 0.35
versus 1.0 ± 0.19%). However, the relative importance of
nitrogen fixation as a strategy and the extent to which N2-fixing
species might contribute to ecosystem nitrogen turnover may
be small. Thus, at the heathland site (Site 1), putative N2-fixing
species represent 10% of the total species, but contribute only
1.5% to the total ecosystem biomass (Bolton 1986). In a
similar community type (mixed Banksia woodland--heath) on
Stradbroke Island in southeastern Queensland, 10% of the
species are potential nitrogen fixers (Clifford and Specht
1979), as are 10% of the species in a Banksia woodland in
Western Australia (Bennett 1988) and 16% of the species in a
typical Eucalyptus open forest (Clifford and Specht 1979). Of
the greater than 400 species represented in the semi-arid Acacia aneura F.J. Muell. dominated ‘‘mulga’’ woodland (represented by Site 2), only 13% are potential nitrogen fixers
(Nelder 1984).
Because of the potentially low rates of nitrogen fixation
under natural conditions (e.g., Hansen et al. 1987), the contribution of symbiotic N2-fixation to total ecosystem nitrogen
may be relatively small. However, assessments of the contribu-

Analysis of water extracts of soil yielded similar nitrogen
profiles for the E. grandis and E. maculata forest sites (Table 1). Ammonium and nitrate were present in similar concentrations; however, the water extracts tended to underestimate
the presence of ammonium. Organic forms of nitrogen contributed significantly to total soluble soil nitrogen, and the soluble
soil components accounted for approximately 0.1% of the total
soil nitrogen in both the E. grandis and E. maculata forest
soils. In the wet season, soluble protein concentrations in soils
of the E. grandis and E. maculata forests were 32.9 and 37.0
nmol N gdw−1 soil, respectively. The concentration of soluble
proteins was lower during the drier months (6.9 and 8.1 nmol
N gdw−1 soil in July in the E. grandis and E. maculata forests,
respectively) than the wet months of the year. The highest
concentrations of amino acids were found in the E. grandis
forest soil (4.05 nmol N gdw−1) and this pool decreased dramatically in July. The major amino acids in the total pool were
serine, alanine, glycine, aspartate and leucine (data not
shown).
In the subtropical wet heathland site, ammonium, amino
acids and nitrate were present in the soil solution (Table 2). The
absolute quantities of each component varied because of environmental conditions, but ammonium dominated at all times,
and amino acids were abundant during waterlogging or immediately after fire as a result of increased rates of mobilization
of organic sources. Total available nitrogen in the soil solution
also varied considerably, and increased 100-fold in the week
following fire. Nitrate availability increased immediately after
fire, whereas the amino acid concentration in the soil solution
was significantly reduced, presumably because of increased
ammonification and nitrification.
Recent studies have stressed the importance of organic
forms of nitrogen, such as amino acids, for plant nutrition in
temperate and arctic regions (Abuarghub and Read 1988, Kielland 1994). Plants can absorb amino acids from the soil solution by an active transport mechanism (Jones and Darrah
1994), and species such as the non-mycorrhizal arctic sedge
Eriophorum vaginatum L. display a preference for amino acids

N SOURCE ACQUISITION AND SPECIALIZED ROOT ADAPTATION

945

Table 1. Mean nitrogen concentrations for inorganic and organic components of soil solution (nmol N gdw−1 soil) from forest sites dominated by
Eucalyptus grandis and E. maculata for the months of December (wet season) and July (dry season). Each value represents the mean (± SD) for
replicate soil samples at each site (n = 3 for nitrate, ammonium and amino acid determinations; n = 6 for protein determinations); nd = not detected.
Nitrogen source

Ammonium
Nitrate
Amino acids (total)
Soluble protein

E. grandis forest site

E. maculata forest site

December

July

December

July

26.2 (24.1)
2.8 (1.7)
4.1 (1.25)
32.9 (7.2)

21.9 (5.7)
46.3 (30.6)
0.01
6.9 (1.9)

37.1 (23.7)
54.4 (23.0)
1.1 (0.50)
37.0 (3.55)

26.8 (4.3)
18.9 (11.4)
nd
8.1 (2.5)

Table 2. Mean nitrogen concentrations for inorganic and organic components of soil solution (nmol N g −1 ion exchange resin) from a wet coastal
heathland in Beerwah State Forest, southeastern Queensland. Each value represents the mean for replicate samples at points during a burning cycle
and during waterlogging. The proportion of each component as a percentage of the total is indicated in parentheses.

Before burn
After burn -- 1 week
After burn -- 3 weeks
Waterlogged

NH +4

NO −3

Amino acids

Total

2.28 (46)
578 (94)
157 (91)
12.7 (50)

0.2 (4)
28 (4.5)
8 (5)
0.03 (0.1)

2.5 (50)
12 (1.5)
6.7 (4)
12.7 (50)

4.98
618
172
25.4

over inorganic forms of nitrogen (Chapin et al. 1993). The
relative importance of mineral and organic forms of nitrogen
for plant nutrition likely depends on their relative availability
in the organic and mineral horizons of the soil. The soil
nitrogen profiles for both the open Eucalyptus forests and the
coastal heathland indicate that multiple sources of nitrogen
may be available for plants in natural ecosystems and that
organic forms may contribute significantly to total soil nitrogen. Because the pool of amino acid nitrogen is highly labile
(Schmidt et al. 1960), its presence in soil solution extracts is
highly variable (Read et al. 1989). The availability of proteins
in the soil solution is also likely to be subject to significant
seasonal release and immobilization (Abuzinadah et al. 1986).
Incorporation of nitrate versus ammonium
Although it is known that the availability of nitrate and ammonium varies with the soil type, it is not known how much plant
species differ in their ability to assimilate these sources. We
found a preference for uptake and assimilation of ammonium
ions over nitrate ions in a range of species (Table 3). The rate
of incorporation of ammonium ions was nearly 3 times that of
nitrate in Eucalyptus grandis, 5--6 times that of nitrate in the
mangroves Avicennia marina (Forssk.) Vierh and Rhizophora
mangle L., 10 times that of nitrate in Hakea sp. and 25 times
that of nitrate in Leptospermum sp. In the coral cay forest
species, the preference for uptake of ammonium over nitrate
was 15 times in Pandanus sp. aff. heronensis H. St. John and
25 times in Argusia argentea (L.f.) Heine.
The nitrogen concentration and the generally low nitrate
reductase activity in the communities studied suggest that
many of the species utilize nitrogen sources other than nitrate

(Tables 1--3). This is consistent with previous findings for a
range of soil types and successional stages (Smirnoff and
Stewart 1985, Stewart et al. 1993).
Utilization of organic sources of nitrogen
Although soluble organic nitrogen represents a significant
pool in soils of natural ecosystems, few studies have been
undertaken to determine the extent to which these pools can be
accessed by plants. Non-mycorrhizal seedlings of both
E. grandis and E. maculata grew well on nitrate and ammonium, but showed little capacity to utilize organic nitrogen
(Table 4). Neither species showed growth beyond that supported by seed reserves on asparagine, glycine, histidine or
BSA. In both species, mycorrhizal infection conferred on
seedlings the ability to grow on organic sources of nitrogen
(Table 4). Both E. grandis and E. maculata seedlings infected
with Elaphomyces sp. displayed significantly greater growth
than non-mycorrhizal seedlings on asparagine, glycine, histidine and BSA (P < 0.01). The potential to utilize a broad
spectrum of organic sources has important implications for the
nutrition of Eucalyptus. In field conditions, a complex mixture
of nitrogen sources, all at potentially low concentrations, may
be available. Thus, the ability to access a diversity of nitrogen
sources may confer distinct nutritional advantages on mycorrhizal plants in forest ecosystems. When mycorrhizal seedlings
of Eucalyptus grandis were offered a mixed source of nitrogen
in the growth medium, they displayed a strong ability to utilize
organic nitrogen. Seedlings derived 48 and 55% of their nitrogen from asparagine when it was offered with nitrate and
ammonium, respectively (Table 5). The BSA protein source
provided 42 and 33% of plant nitrogen when it competed with

946

TURNBULL ET AL.

Table 3. Rates of incorporation of nitrate and ammonium (nmol 15N
gdw−1 h −1) in a range of plant species from diverse plant communities.
Each value represents the mean of replicate determinations.
Species
Eucalypt open forest
Eucalyptus grandis
Coastal heathland
Hakea sp.
Leptospermum sp.
Mangrove forest1
Avicennia marina
Rhizophora mangle
Coral cay open forest
Argusia argentea
Pandanus sp. aff. heronensis
1

Nitrate

Ammonium

246

714

180
29

1715
724

360
66

1818
408

29
98

724
1450

Table 5. Atom% excess and calculated percentage utilization of inorganic and organic sources of nitrogen in mycorrhizal seedlings of
Eucalyptus grandis grown in sterile medium at pH 5.5. Plants were
grown in medium to which was added either a single labeled source of
15
N-labeled ammonium or nitrate (10 atom% excess) or a mixture of
these labeled inorganic sources with either asparagine or BSA in the
combinations shown. Values are the means of duplicate bulked samples (n = 10--12) (± range) except for 15NO −3 / Asparagine and 15NO −3 /
BSA which are single bulked samples (n = 10). The percentage of
plant nitrogen derived from the unlabeled source was calculated according to the formula given in Materials and methods.
Source

Atom% excess
leaf material

NO −3
NO −3 / NH +4
15
NO −3 / Asparagine
15
NO −3 / BSA
15
NH +4
15
NH +4 / Asparagine
15
NH +4 / BSA

9.89 (0.47)
2.28 (0.15)
5.10
5.79
9.33 (0.11)
4.20 (0.10)
6.30 (0.05)

15
15

Data taken from Stewart et al. 1991.

nitrate and ammonium, respectively. Thus, organic sources of
nitrogen may provide a significant nitrogen source even in the
presence of readily assimilable inorganic ions, especially in
conditions where plants must scavenge for available resources
(Table 5).
Although it has been established that mycorrhizal plant
species in heathland ecosystems can utilize complex organic
nitrogen through ericoid or ecto-mycorrhizae (Bajwa et al.
1985) and have access to NH +4 through VAM (Marschner and
Dell 1994), the question arises whether predominantly nonmycorrhizal and non N2-fixing plants, such as members of the
Proteaceae, Cyperaceae and Restionaceae, also have special
means of nitrogen nutrition. In Hakea sp. seedlings offered
single nitrogen sources, a preference in the order of ammonium, glycine, nitrate was displayed in both roots and proteoid
roots (Figure 3a). Rates of incorporation of labeled glycine
were in the order of 40--60% those of ammonium and 4--5
times those of nitrate. A similar trend in preference was displayed in both root types when all three nitrogen sources were
offered simultaneously (Figure 3b). Uptake of ammonium was
reduced by 35% in proteoid roots and by 20% in roots under

Calculated %
utilization of
unlabeled
N source
77
48
42
55
33

the competitive influence of the other nitrogen sources in the
triple feeding experiment. The uptake of nitrate and glycine
was not significantly influenced by the presence of other nitrogen sources in the medium. Although incorporation of 15N-labeled substrates was consistently higher in roots than in
proteoid roots on a fresh weight basis, direct comparisons of
rates of uptake between the two root types are difficult because
of the retention of small amounts of sand in cluster roots.
Because proteoid roots are mainly found in the upper organic rich soil layer, Dinkelaker et al. (1995) have suggested
that proteoid roots have a special function in the utilization of
organic nitrogen, such as preferential uptake of amino acids.
We have demonstrated that root systems of the genus Hakea
can take up and assimilate exogenous sources of amino acids
from the soil solution, but we found no qualitative difference
between roots and proteoid roots with respect to uptake of
labeled glycine (Figure 3). We conclude, therefore, that the

Table 4. Dry weight (mg) of seedlings of Eucalyptus grandis and Eucalyptus maculata on a range of inorganic and organic sole nitrogen sources
in buffered agar culture (pH 5.5). Seedlings were grown in non-mycorrhizal form or in association with Elaphomyces sp. (isolate NQ732). Each
value represents the mean (± SEM) for replicate growth assays for seedlings (n = 12).
Nitrogen source

N free
Nitrate
Ammonium
Asparagine
Glycine
Histidine
Protein (BSA)
1

Eucalyptus grandis

Eucalyptus maculata

Non-mycorrhizal

Mycorrhizal

Non-mycorrhizal

Mycorrhizal

3 (0.3)
111 (12.5)
85 (8.0)
13 (0.5)
4 (0.5)
3 (0.5)
3 (0.5)

65 (2.0)
65 (5.0)
29 (0.5)
18 (1.0)
35 (0.5)
45 (0.5)

25 (0.5)
144 (35)
125 (13)
26 (4.5)
19 (0.5)
16 (1.5)
14 (3.0)

156 (5.0)
70 (2.5)
82 (2.0)
46 (4.0)
64 (2.0)
162 (10)

Adapted from Turnbull et al. (1995).

N SOURCE ACQUISITION AND SPECIALIZED ROOT ADAPTATION

947

tions, the ability to tap a broad spectrum of nitrogen sources
that have been acquired and concentrated by the host.

Conclusion

Figure 3. Total incorporation of 15N in roots (open bars) and proteoid
roots (filled bars) of Hakea seedlings incubated in aerated solution
culture with (a) either 1 mol m −3 of 15N-labeled nitrate, ammonium or
glycine (65 atom% excess) as sole source, or (b) 1 mol m −3 each of
nitrate, ammonium and glycine offered as a triple nitrogen source, but
with only one source labeled with 15N. Values represent determinations made on duplicate bulked root systems.

ability to utilize organic nitrogen is common to both proteoid
and normal roots in Hakea. The structure of proteoid roots and
their placement in the organic soil horizons suggests that their
functional role is intensive exploration of small soil volumes
(cf. Dinkelaker et al. 1995).
The haustoria of root hemiparasites constitute another potentially important root specialization related to the uptake of
organic nitrogen. Such specializations are found in a range of
genera (e.g., Exocarpus, Anthobolus, Santalum and Striga)
that are common in nutrient-poor ecosystems (Lamont 1984).
Not only do these structures enable the hemiparasite to tap into
host nutrient reserves, but studies with Olax phyllanthi L. have
shown that these species have the ability to transform host
organic solutes before they enter the xylem (Pate et al. 1994),
which enables this species to parasitize hosts with a wide
variety of organic transport compounds. This, in addition to the
ability to induce the production of nitrate reductase in hosts
containing high concentrations of nitrate in xylem sap, has the
potential to confer on species with such haustorial connec-

Our results confirm those obtained in previous studies (e.g.,
Stewart et al. 1993, Pate et al. 1993) suggesting complex
patterns of utilization of ecosystem nitrogen sources. We conclude that species that are predominantly nitrate assimilating
are likely to be non-mycorrhizal or weakly VAM. In contrast,
species that have specific associations with ectomycorrhizal or
ericoid mycorrhizal fungi assimilate ammonium, amino acids
or even more complex forms of nitrogen such as protein. Many
of these species have a relatively low potential to assimilate
nitrate. This is likely to have important consequences to species composition and community structure in natural ecosystems that are not highly nitrifying, and which may become
exposed to anthropogenic inputs of nitrogen. In addition, we
have demonstrated that obligately non-mycorrhizal species
may have the ability to compete with mycorrhizal species for
organic pools of nitrogen that are present in many natural
ecosystems. Other root specializations (nitrogen-fixing symbioses, hemiparasitic haustoria) extend further the spectrum of
nitrogen source utilization in natural ecosystems. There is,
thus, the possibility of both competitive and complementary
patterns of exploitation of different nitrogen sources within the
soil profile in relation to plant life from physiology, rooting
morphology and the presence of specialized feeding roots or
mycorrhizal associations (Pate et al. 1993).
Acknowledgments
This study was supported by the Australian Research Council (Grant
Nos. A19230676 and A19332711) and the Australian Flora Foundation. Research activities in Kakadu National Park were supported both
financially and logistically by the Environmental Research Institute of
the Supervising Scientist, Jabiru. The assistance of Dr N. Ashwath
during field work in the Northern Territory is gratefully acknowledged. We thank Dr Paul Reddell (CSIRO Division of Soils, Townsville) for the provision of fungal cultures.

References
Abuarghub, S.M. and D.J. Read. 1988. The biology of mycorrhiza in
the Ericaceae. XII. Quantitative analysis of individual ‘‘free’’ amino
acids in relation to time and depth in the soil profile. New Phytol.
108:433--441.
Abuzinadah, R.A. and D.J. Read. 1986. The role of proteins in the
nitrogen nutrition of ectomycorrhizal plants. III. Protein utilisation
by Betula, Picea and Pinus in mycorrhizal association with Hebeloma crustuliniforme. New Phytol. 103:507--514.
Abuzinadah, R.A., R.D. Finlay and D.J. Read. 1986. The role of
proteins in the nitrogen nutrition of ectomycorrhizal plants. II.
Utilisation of protein by mycorrhizal plants of Pinus contorta. New
Phytol. 103:495--506.
Alexander, I.J. 1983. The significance of ectomycorrhizas in the nitrogen cycle. In Nitrogen as an Ecological Factor. Eds. J.A. Lee, S.
McNeill and I.H. Rorison. Blackwell, Oxford, pp 69--94.

948

TURNBULL ET AL.

Bajwa, R., S.M. Abuarghub and D.J. Read. 1985. The biology of
mycorrhiza in the Ericaceae. X. The utilization of proteins and the
production of proteolytic enzymes by the mycorrhizal endophyte
and by mycorrhizal plants. New Phytol. 101:469--486.
Bennett, E.M. 1988. The bushland plants of King’s Park, Western
Australia. Scott Four Colour Print, Perth. 176 p.
Bolton, M.P. 1986. Community dynamics and productivity in a
subtropical wet heathland. Ph.D. Thesis, Univ. Queensland, St
Lucia, 217 p.
Bowen, G.D. 1981. Coping with low nutrients. In The Biology of
Australian Plants. Eds J.S. Pate and A.J. McComb. Univ. Western
Australia Press, Nedlands, W.A., pp 33--64.
Chapin, F.S. III, L. Moilanen and K. Kielland. 1993. Preferential use
of organic nitrogen for growth by a non-mycorrhizal arctic sedge.
Nature 361:150--152.
Clifford, H.T. and R.L. Specht. 1979. The vegetation of North Stradbroke Island. Univ. Queensland Press, St. Lucia, 141 p..
Dinkelaker, B., C. Hengeler and H. Marschner. 1995. Distribution and
function of proteoid roots and other root clusters. Bot. Acta
108:183--200.
Field, C.B. and H.A. Mooney. 1986. The photosynthesis--nitrogen
relationship in wild plants. In On the Economy of Plant Form and
Function. Ed. T.J. Givnish. Cambridge University Press, Cambridge, pp 25--55.
Finlay, R.D., Å. Frostegård and A.-M. Sonnerfeldt. 1992. Utilisation
of organic and inorganic nitrogen sources by ectomycorrhizal fungi
in pure culture and in symbiosis with Pinus contorta Dougl. ex
Loud. New Phytol. 120:105--115.
Halliday, J. and J.S. Pate. 1976. Symbiotic nitrogen fixation by coralloid roots of the cycad Macrozamia riedlei: physiological characteristics and ecological significance. Aust. J. Plant Physiol.
3:349--358.
Hansen, A.P. and J.S. Pate. 1987. Evaluation of the 15N natural abundance method and xylem sap analysis for assessing N 2 fixation of
understorey legumes in Jarrah (Eucalyptus marginata Donn ex
Sm.) forest in S.W. Australia. J. Exp. Bot. 38:1446--1458.
Hansen, A.P., J.S. Pate, A. Hansen and D.T. Bell. 1987. Nitrogen
economy of post-fire stands of shrub legumes in Jarrah (Eucalyptus
marginata Donn ex Sm.) forest of SW Australia. J. Exp. Bot.
38:26--41.
Harley, J.L. and S.E. Smith. 1983. Mycorrhizal symbiosis. Academic
Press, London, 483 p.
Jones, D.L. and P.R. Darrah. 1994. Amino-acid influx at the soil--root
interface of Zea mays L. and its implications in the rhizosphere.
Plant Soil 163: 1--12.
Kershaw, J.L. and G.R. Stewart. 1992. Metabolism of 15N-labeled
ammonium by the ectomycorrhizal fungus Pisolithus tinctorius
(Pers.) Coker and Couch. Mycorrhiza 1:71--77.
Kielland, K. 1994. Amino acids absorption by arctic plants: implications for plant nutrition and nitrogen cycling. Ecology 75: 2373-2383.
Lamont, B. 1973. Factors affecting the distribution of proteoid roots
within the root system of two Hakea species. Aust. J. Bot.
21:165--187.
Lamont, B.B. 1982. Mechanisms for enhancing nutrient uptake in
plants, with particular reference to mediterranean South Africa and
Western Australia. Bot. Rev. 48:597--689.
Lamont, B.B. 1984. Specialised modes of nutrition. In Kwongan-Plant Life of the Sandplain. Eds. J.S. Pate and J.S. Beard. Univ.
Western Australia Press, Nedlands, pp 126--145.

Lamont, B.B. 1993. Why are hairy root clusters so abundant in the most
nutrient impoverished soils of Australia? Plant Soil 155:269--272.
Marschner, H. and B. Dell. 1994. Nutrient uptake in mycorrhizal
symbiosis. Plant Soil 159:89--102.
Monk, D., J.S. Pate and W.A. Loneragan. 1981. Biology of Acacia
pulchella R.Br. with special reference to symbiotic nitrogen fixation. Aust. J. Bot. 29:579--592.
Nelder, V.J. 1984. Vegetation survey of South Central Queensland.
Queensland Botany Bulletin No. 3, Queensland Dept. Primary
Industries, Brisbane, 285 p.
Pate, J.S. and W.D. Jeschke. 1993. Mineral uptake and transport in
xylem and phloem of the proteaceous tree Banksia prionotes. In
Plant Nutrition: From Genetic Engineering to Field Practice. Ed.
N.J. Barrow. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 313--316.
Pate, J.S., G.R. Stewart and M. Unkovich. 1993. 15N Natural abundance of plant and soil components of a Banksia woodland ecosystem in relation to nitrate utilisation, life form, mycorrhizal status
and N2-fixing abilities of component species. Plant, Cell Environ.
16:365--373.
Pate, J.S., G. Woodall, W.D. Jeschke and G.R. Stewart. 1994. Root
xylem transport of amino acids in the root hemiparasitic shrub Olax
phyllanthi (Labill) R.Br. (Olacaceae) and its multiple hosts. Plant,
Cell Environ. 17:1263--1273.
Read, D.J. 1991. Mycorrhizas in ecosystems----Nature’s response to
the ‘‘Law of the Minimum’’. In Frontiers in Mycology. Ed. D.L.
Hawksworth. CAB International, pp 101--130.
Read, D.J., J.R. Leake and A.R. Langdale. 1989. The nitrogen nutrition of mycorrhizal fungi and their host plants. In Nitrogen, Phosphorus and Sulphur Utilisation by Fungi. Eds. L.L. Boddy, R.
Marchant and D.J. Read. Cambridge University Press, Cambridge,
pp 181--204.
Schmidt E.L., H.D. Putnam and E.A. Paul. 1960. Behaviour of free
amino acids in soil. Proc. Soil Soc. Am. 24:107--109.
Smirnoff, N., and G.R. Stewart. 1985. Nitrate assimilation and translocation by higher plants: comparative physiology and ecological
consequences. Physiol. Plant. 64:133--140.
Smirnoff, N., P. Todd and G.R. Stewart. 1984. The occurrence of nitrate
reduction in the leaves of woody plants. Ann. Bot. 54:363--374.
Sprent, J.I. and P. Sprent. 1990. Nitrogen fixing organisms. Chapman
and Hall, London, 256 p.
Stewart, G.R., M. Popp, I. Holzapfel, J.A. Stewart and A. DickieEskew. 1986. Localisation of nitrate reduction in ferns and its
relationship to environment and physiological characteristics. New
Phytol. 104:373--384.
Stewart, G.R., E.E. Hegarty and R.L. Specht. 1988. Inorganic nitrogen
assimilation in plants of Australian rainforest communities.
Physiol. Plant. 74:26--33.
Stewart, G.R., S.A. Robinson and A.P. Slade. 1991. Application of 15N
tracer studies in the investigation of higher plant inorganic nitrogen
metabolism. In Stable Isotopes in Plant Nutrition, Soil Fertility and
Environmental Studies. International Atomic Energy Agency, Vienna, pp 431--440.
Stewart, G.R., J.S. Pate and M. Unkovich. 1993. Characteristics of
inorganic nitrogen assimilation of plants in fire-prone Mediterranean-type vegetation. Plant, Cell Environ. 16:351--363.
Turnbull, M.H., R. Goodall and G.R. Stewart. 1995. The impact of
mycorrhization on nitrogen source utilisation in Eucalyptus grandis
and Eucalyptus maculata. Plant, Cell Environ. 18:1386--1394.