Annals of Botany 87: 303±311, 2001
doi:10.1006/anbo.2000.1305, available online at http://www.idealibrary.com on
Reduced 15N-nitrogen Transport Through Arbuscular Mycorrhizal Hyphae to Triticum
aestivum L. Supplied with Ammonium vs. Nitrate Nutrition
H E I D I - J AY N E H AW K I N S *{ and E C K H A R D G E O R G E {
{Department of Botany, University of Cape Town, Private Bag, Rondebosch, 7700, Cape Town, South Africa and
{Institute of Plant Nutrition (330), Hohenheim University, Stuttgart, 70599, Germany
Received: 23 February 2000 Returned for revision: 21 August 2000
Accepted: 21 September 2000
Published electronically: 26 January 2001
This study compared the in¯uence of NH ‡4 or NO ÿ3 nutrition on the contribution of extraradical hyphae of the
arbuscular mycorrhizal fungus Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe (BEG 107) to NH ‡4 or NO ÿ3
uptake by Triticum aestivum L. `Hano' (summer wheat) with sucient or insucient N supply in semi-hydroponic
culture. Roots and root-distant hyphae were spatially separated in compartmentalized pots. Although NH ‡4 -fed
plants supplied with sucient N had higher N concentrations than their NO ÿ3 -fed counterparts, this did not
favourably a€ect colonization rates, hyphal length densities or 15N amounts transported via hyphae to the plants.
Ammonium supply did not result in higher P or reduced carbohydrate concentrations in the plants, so these factors
could not explain the reduced hyphal lengths. It was concluded that the e€ect of NH ‡4 supply on hyphal length may
be related to the reduced root growth and/or a direct e€ect of NH ‡4 on hyphal growth. Plant N de®ciency reduced the
percentage root length colonized, hyphal length, total 15N uptake by hyphae and dry weight of both NO ÿ3 - and NH ‡4 fed mycorrhizal plants. This was more obvious for NO ÿ3 -fed plants because plant biomass and hyphal lengths of
NH ‡4 -fed plants were relatively low in mycorrhizal plants irrespective of the N concentration supplied.
# 2001 Annals of Botany Company
Key words: Ammonium, arbuscular mycorrhiza, Glomus mosseae, nitrate, nitrogen uptake,
aestivum, wheat.
I N T RO D U C T I O N
NO ÿ3
NH ‡4
or
nutrition
This study compares the e€ect of
on arbuscular mycorrhizal (AM) colonization and N
uptake through hyphae under controlled nutrient conditions in an inert substrate. Results of Bago et al. (1996)
con®rm that extraradical hyphae of AM actively take up
NO ÿ3 ( probably via an NO ÿ3 : H ‡ symport), but the actual
contribution of this fungal-mediated nitrate uptake is
unknown. AM hyphae have also been shown to deplete
15
NH ‡4 -labelled soil and transport this 15N to the plant (e.g.
Frey and SchuÈepp, 1993; Johansen et al., 1993).
For practical purposes, most studies of N uptake through
AM hyphae have used NH ‡4 as the N source (Johansen
et al., 1992, 1994; Tobar et al., 1994) although NO ÿ3 is the
predominant N form in most agricultural soils (Marschner,
1995). As a result, very few studies have compared the e€ect
of NH ‡4 and NO ÿ3 ions as N sources for mycorrhizal plants
while also excluding the e€ect of these ions on rhizosphere
pH. For instance, NH ‡4 has been shown to have a
deleterious e€ect on mycorrhizal colonization of plants in
soil compared to NO ÿ3 ions, due to lowered rhizosphere
pH with NH ‡4 nutrition (Chambers et al., 1980a). Lowered
rhizosphere pH can a€ect spore germination (Green et al.,
1976), root colonization and growth of AM fungi
(Chambers et al., 1980a). E€ects related to the NH ‡4 ion
but not pH may include a direct salt e€ect (Chambers et al.,
* For correspondence. Fax ‡27 21 650 4041, e-mail hhawkins@
botzoo.uct.ac.za
0305-7364/01/030303+09 $35.00/00
15
N studies, Triticum
1980a) or a lower carbohydrate status of the root, since
NH ‡4 uptake requires immediate incorporation of the N
into amino acids in the root, which in turn requires Cskeletons from the TCA cycle (Cramer and Lewis, 1993a).
Other e€ects may be increased shoot : root ratio or increased
tissue P concentration (Johnson et al., 1984), where the
latter may decrease membrane permeability and therefore
root exudates that are thought to sustain the germination
and growth of the fungus (Graham et al., 1981).
Ammonium absorption occurs `downhill' and results in
high N contents and allows increased P absorption in some
plants. The increased P absorption may be a result of faster
synthesis of a nitrogenous P carrier and/or absence of anion
competition with NO ÿ3 for the P uptake site. Therefore, it
was expected that NH ‡4 nutrition would, due to one or
more of the above factors and independently of rhizosphere
pH, decrease mycorrhizal colonization and possibly hyphal
length, which might a€ect the amount of N transported
through the hyphae to the plant roots.
The compartmentalized culture system used in this study
(Hawkins and George, 1999) allows the direct comparison
of NO ÿ3 and NH ‡4 nutrition of non-mycorrhizal and
mycorrhizal plants under pH-bu€ered and controlled
nutrient conditions, thus eliminating any pH e€ects of the
N forms. It was the aim of this study to test (1) the
dependence of colonization, hyphal growth and hyphal 15N
transport by G. mosseae to T. aestivum on N form without
the e€ect of pH, and increase understanding of the mechanism responsible for possible di€erences in the colonization
of NH ‡4 - and NO ÿ3 -fed AM plants and (2) the dependence
# 2001 Annals of Botany Company
304
Hawkins and GeorgeÐE€ect of N Form on Hyphal N Uptake
of colonization, hyphal growth and hyphal 15N transport
by G. mosseae to T. aestivum on N plant status when plants
were supplied with NH ‡4 or NO ÿ3 .
M AT E R I A L S A N D M E T H O D S
Experimental culture containers
Containers with a central root compartment and two outer
hyphal compartments were used in a Perlite (2 mm) and
drip-irrigation system as described by Hawkins and George
(1999).
Culture and harvest of plants and fungus
Triticum aestivum `Hano' (summer wheat) was cultivated
with or without the mycorrhizal fungus Glomus mosseae
(BEG 107). Two days before harvesting, 15N was supplied
to the hyphal compartments as NO ÿ3 or NH ‡4 (see below).
Shoots and roots were harvested into liquid N2 , freezedried and analysed for plant dry weight, N and P
concentrations, 15N abundance and carbohydrate concentration. Roots were analysed for mycorrhizal colonization
and the Perlite in the hyphal compartments was analysed
for hyphal length density.
Nutrient media
All nutrient media were based on Long Ashton nutrient
solution (LANS; Hewitt, 1966), which was modi®ed as
described by Hawkins and George (1999) where N was
provided as NO ÿ3 (2 or 0.2 mM) or NH ‡4 (2 or 0.2 mM).
Three di€erent levels of N and P supply, represented as
‡N ‡ P (2 mM N, 0.1 mM P), ‡N ÿ P (2 mM N, 10 mM P)
and ÿN ÿ P (0.2 mM N, 10 mM P) were used. The ®rst
treatment, containing higher N and P, was used to grow
adequately fed control plants. These were compared with
‡AM and ÿAM plants that were supplied with either low
P, or low N and P. The plants receiving the ‡N ‡ P treatment are also referred to as controls in tables, ®gures and
text.
Hyphal compartments were irrigated with complete,
modi®ed LANS (Hawkins and George, 1999) to allow
the hyphae access to a more concentrated nutrient
solution than was available to the roots directly. It was
calculated that the amounts of N and P supplied to the
hyphal compartments were sucient to ameliorate the
expected N or P de®ciency, should the total amount of
these nutrients be taken up by the hyphae and transported
to the plants. Nutrient de®ciency and suciency were based
on expected plant growth rates and adequate levels of N
and P in T. aestivum (Reuter and Robinson, 1988).
A nitri®cation inhibitor (e.g. N-serve) was not used since
toxic e€ects below 10 mg kg ÿ1 soil or above 15 mg kg ÿ1
soil N-serve have been known to occur in soil microorganisms (Laskowski et al., 1975) and AM fungi
(Chambers et al., 1980b) respectively.
Nitrogen,
15
N and P analysis
Nitrogen concentration was determined from dried,
pulverized shoot and root material by dry oxidation
(Macro N, Heraeus Holding Ltd, Hanau, Germany) using
asparagine as a standard. The percentage atom enrichment
of 15N in dried, pulverized samples was determined by dry
oxidation and reduction (Roboprep CN Biological Sample
Converter, Europa Scienti®c Ltd, Crewe, UK) and subsequent mass spectrometry (Tracermass Sample Isotope
Analyser, Europa Scienti®c Ltd) using pulverized pea shoot
(2.5 % N, 0.45 % 15N) as a standard. Plant P concentration
was determined using the molybdenum blue assay (Murphy
and Riley, 1962) on dried, pulverized shoot and root
material that had been dry ashed at 5008C for 4 h,
moistened with H2O and 1 : 3 (v/v) HNO3 , which was
evaporated o€ to split SiO2 from other compounds.
Samples were then dissolved in 1 : 30 (v/v) HCl and boiled
for 2 min (to convert metaphosphates and pyrophosphates
to orthophosphates) before being made up to volume.
Carbohydrate analysis
Reducing sugars and sucrose were determined according
to a modi®ed method of Blakeney and Mutton (1980).
About 30 mg of freeze-dried plant material was extracted
three times in 2.5 ml 70 % (v/v) ethanol (end volume
7.5 ml) by consecutive mixing and centrifugation at 2500 g
and 48C (Minifuge, Hereaus GmbH, Stuttgart, Germany).
Chlorophyll in leaf extracts was removed by addition of
activated charcoal (about 10 mg ml ÿ1), mixing and
centrifugation at 1800 g (Mikroliter, Hettich Zentrifugen,
Tuttlingen, Germany). The clear supernatant was used for
reducing sugar and sucrose determination. Reducing sugars
were determined by mixing 0.2 ml of the clear supernatant
with 0.8 mM Na-acetate ( pH 4.8) and sucrose was
determined by mixing 0.1 ml supernatant with 0.1 ml
invertase solution (50 U ml ÿ1 bu€ered in 0.2 M Na-acetate)
and 0.8 ml 0.1 M Na-acetate ( pH 4.8). The mixtures were
incubated for 2 h in a 308C water bath to allow sucrose to
be converted to glucose equivalents. After incubation, 5 ml
of a colour reagent (0.03 M hydroxybenzoic acid hydrazide,
0.05 M Na-citrate, 0.01 M CaCl2 and 0.5 M NaOH) was
added to the sample solution and boiled for 4 min in a
water bath. The cooled yellow solution was measured
spectrophotometrically at 415 nm.
Starch was determined in the residual pellet according to
Blakeney and Matheson (1984). The pellet was dissolved in
2 ml dimethyl sulfoximine by boiling for 10 min in a water
bath. The sample was centrifuged at 2500 g (Minifuge,
Hereaus GmbH, Stuttgart, Germany) and washed with
8 ml 0.1 mM Na-acetate ( pH 4.8) solution. One millilitre of
the sample solution was mixed with 2 ml amyloglucosidase
solution (1.2 U ml-1 bu€ered in 0.2 M Na-acetate) and
incubated for at least 12 h at 378C in a water bath. After
incubation, 5 ml of the enzyme colour reagent (4000 U
glucose oxidase, 1000 U peroxidase, 0.07 M Na2HPO4 ,
0.4 M NaH2PO4 , 0.016 M benzoic acid, 0.5 mM 4-amino
antipyrin, 0.01 M p-hydroxy benzoic acid) was added to
each 1 ml sample solution and incubated in a 408C water
Hawkins and GeorgeÐE€ect of N Form on Hyphal N Uptake
305
bath for 15 min. The cooled pink solution was measured
spectrophotometrically at 510 nm.
of NO ÿ3 -fed plants due to a relatively smaller root growth
(P 5 0.05, t-tests, Table 1).
Mycorrhizal root colonization and hyphal length density
Nutrient contents and concentrations
The percentage of root length colonized by mycorrhizal
fungi was determined on roots stained in trypan blue (Koske
and Gemma, 1989) using the gridline-intersect method
(Giovannetti and Mosse, 1980). For the determination of
hyphal length density, samples were taken from hyphal
compartments using a 10 mm wide cork borer. The Perlite
was thoroughly mixed before sampling. These samples were
air-dried, weighed and used to determine hyphal length
density according to the modi®ed agar ®lm technique
described by Li et al. (1991). Hyphal lengths were expressed
on a length per unit volume basis to enable comparison with
previously reported hyphal lengths from soil extracts.
With sucient P (controls), there was, as expected, a
higher P concentration in the NH ‡4 -fed compared to NO ÿ3 fed plants (Fig. 1). However, with insucient P (‡N ÿ P
and ÿN ÿ P treatments), this was not the case and P
concentrations were even higher in NO ÿ3 -fed plants than in
NH ‡4 -fed plants (P 5 0.05, Student t-tests), regardless of
the mycorrhizal or N treatments (Fig. 1). As expected,
mycorrhizal colonization resulted in an increased P
concentration of the shoots of both NO ÿ3 and NH ‡4 -fed
plants, regardless of the N treatment (Fig. 1). Mycorrhizal
colonization signi®cantly increased P concentrations in the
root of NO ÿ3 -fed plants only (Fig. 1).
Nitrate-fed ‡AM plants had a higher N content
compared to ÿAM plants, irrespective of the level of N
supplied while this e€ect could be seen only for the shoots
of NH ‡4 -fed, ÿN ÿ P plants (Fig. 2A). Although the N
concentration of shoots of NO ÿ3 -fed ‡N ÿ P plants was
also greater for mycorrhizal plants (31 % increase), as was
the case for the roots of NH ‡4 -fed ÿN ÿ P plants (19 %
increase, Fig. 2B), the e€ect of mycorrhizal colonization on
the N concentration of either NO ÿ3 or NH ‡4 -fed plants was
not large. In general, a greater mycorrhizal e€ect was visible
for NO ÿ3 -fed as opposed to NH ‡4 -fed plants with respect to
both N content and N concentration. In ‡N ÿ P plants,
roots of NH ‡4 -fed plants had higher N concentrations than
those of NO ÿ3 -fed plants (Fig. 2B, P 5 0.05, Student's
t-test).
15
Nitrogen feeding
Nitrogen (20 ml of 5 mM 95 % atom enriched 15NO ÿ3 or
NH ‡4 ; Chemotrade GmbH, Leipzig, Germany) was
supplied to each hyphal compartment of both ‡AM and
ÿAM treatments 48 h before harvesting. The shoots were
harvested after 48 h and, at the same time, the hyphal
connection between the root and hyphal compartments was
severed using a scalpel. Speci®c uptake rates of 15N by the
plant via hyphae were expressed as mmol 15N g ÿ1 plant
d.wt h ÿ1 and speci®c uptake rates per unit hyphae were
expressed as nmol 15N m ÿ1 hyphae h ÿ1.
15
Statistics
Four replicate containers per treatment with ®ve
plants per container were used. For most comparisons,
Student's t-tests were applied to determine di€erences
due to presence or absence of mycorrhizal fungi within
one nutrient treatment or between NO ÿ3 - and NH ‡4 -fed
plants with the same N supply (concentration supplied).
Root colonization data and other data presented as percentages were normalized by arcsine square root transformation
before performing Student's t-tests. Di€erences within
all ®ve treatments (‡N ‡ P ÿ AM, ‡N ÿ P ÿ AM,
‡N ÿ P ‡ AM, ÿN ÿ P ÿ AM, ÿN ÿ P ‡ AM) were
calculated by mean separation (P 5 0.05, pair-wise multiple
range Tukey test) following a one-way analysis of variance.
Results in the tables, text and ®gures are given as
means + s.e.
R E S U LT S
Dry weight
Nitrate-fed plants were larger that NH ‡4 -fed plants,
regardless of mycorrhizal or N treatment (P 5 0.05, pairwise multiple range Tukey test, Table 1). The largest
di€erences in dry weight between ‡AM and ÿAM plants
were evident when the plants were provided with sucient
N (‡N ÿ P) and NO ÿ3 as the N source. The shoot : root
ratio of NH ‡4 -fed plants was signi®cantly higher than that
Carbohydrate concentrations
The most obvious di€erence between ÿAM and ‡AM
plants was seen in the sucrose concentrations of the root
and shoot of NO ÿ3 -fed plants: There was signi®cantly less
sucrose in both shoot and root (P 5 0.001, Student's
t-tests) in ‡AM compared to ÿAM plants, which resulted
in the total sugar concentration being lower (P 5 0.001,
Student's t tests, Fig. 3). There was no di€erence between
the starch concentration of the shoot while the starch
concentration in the root was higher (P ˆ 0.026, Student's
t-test) in ‡AM compared to ÿAM ‡ N ÿ P plants
(Fig. 3). There were no signi®cant di€erences between
ÿAM and ‡AM plants in the ÿN ÿ P treatment (Fig. 3).
In contrast to NO ÿ3 -fed plants, the ‡AM ‡N ÿ P NH ‡4 fed plants had higher concentrations of reducing sugars
(P ˆ 0.010, P 5 0.001, Student's t-test for shoot and root,
respectively) and higher concentrations of sucrose
(P 5 0.001, P 5 0.001, Student's t-test for shoot and
root, respectively) while the starch concentrations were
signi®cantly lower (P ˆ 0.027, P 5 0.001, Student's t-test
for shoot and root, respectively, Fig. 3) than in the
corresponding ÿAM plants.
Therefore, the pattern and allocation of C into carbohydrates in ÿAM and ‡AM plants was similar in NO ÿ3
and NH ‡4 -fed plants in the ÿN ÿ P treatment, while the
absolute concentrations of starch and sucrose were higher
306
Hawkins and GeorgeÐE€ect of N Form on Hyphal N Uptake
T A B L E 1. Dry weight and shoot : root ratio (S : R) of non-mycorrhizal (ÿAM) and mycorrhizal (‡AM) T. aestivum shoots
and roots grown with low or high P supply (ÿP; ‡P) and low or high NO ÿ3 or NH ‡4 supply (ÿN; ‡N)
Dry weight (g)
‡N ‡ P (control)
‡N ÿ P
ÿN ÿ P
ÿAM
ÿAM
‡AM
ÿAM
‡AM
Nitrate-fed plants
Shoot
Root
S:R
36.25 + 0.64
32.59 + 1.12
1.12 + 0.06
7.57 + 0.52a
5.60 + 0.64a
1.41 + 0.18a
10.17 + 0.59b
13.21 + 1.15b
0.86 + 0.17a
3.63 + 0.11a
3.63 + 0.20a
1.01 + 0.04a
4.46 + 0.15b
4.37 + 0.52a
1.06 + 0.12a
Ammonium-fed plants
Shoot
Root
S:R
14.84 + 0.50
2.93 + 0.30
5.17 + 0.47
4.84 + 0.26a
2.23 + 0.11a
2.20 + 0.21a
7.42 + 0.45b
2.45 + 0.13a
3.04 + 0.14b
4.10 + 0.25a
2.05 + 0.11a
2.00 + 0.03a
4.48 + 0.23a
2.15 + 0.14a
2.12 + 0.20a
Di€erent superscripts indicate a statistically signi®cant di€erence between ÿAM and ‡AM treatments (P 5 0.05, Student's t-test) at the same
level of nutrient supply. Values are means + s.e. of four replicates.
8
Nitrate-fed plants
Ammonium-fed plants
P concentration (mg g–1 d. wt)
6
4
b
2
0
–
a
–
a
+
a
2
–
a
b
+
–
a
b
b
a
–
b
a
b
+
–
+
a
a
a
4
6
8
+N+P
+N–P
–N–P
+N+P
+N–P
–N–P
N treatment
F I G . 1. Phosphorus concentrations of non-mycorrhizal and mycorrhizal T. aestivum shoots and roots supplied with either nitrate or ammonium
and grown with low or high P supply (ÿP; ‡P) and low or high N supply (ÿN; ‡N). Di€erent letters indicate a statistically signi®cant di€erence
between the roots or shoots of ÿAM and ‡AM treatments (P 5 0.05), Student's t-test) at the same level of nutrient supply. Values are means of
four replicates. (h), Shoot; (D), root; (ÿ/‡), without/with AM.
in NH ‡4 -fed plants (P 5 0.05, Student's t-tests, Fig. 3). For
the ‡N ÿ P treatment, the pattern and allocation of C into
carbohydrates in ÿAM and ‡AM was signi®cantly
di€erent (P 5 0.05, Student's t-tests) and reversed (Fig. 3).
higher hyphal length (P 5 0.001), Student's t-test, Table 2)
while in NH ‡4 -fed plants, hyphal lengths were low,
regardless of N supply to the plant.
15
Mycorrhizal root colonization and hyphal length density
Plants not inoculated with the fungus were not colonized.
There was no signi®cant di€erence in the colonization rates
of NO ÿ3 and NH ‡4 -fed plants, regardless of N treatment
(Table 2) while hyphal lengths were signi®cantly reduced in
NH ‡4 -fed plants, regardless of N treatment (Table 2). In
NO3-fed plants, a higher N supply resulted in a signi®cantly
Nitrogen uptake
There was no signi®cant 15N enrichment of the NO ÿ3 -fed
ÿAM plants above the natural abundance level of 0.366 %,
i.e. uptake rates were essentially zero (Table 3) because no
mass ¯ow of 15N occurred from the hyphal to the root
compartments in non-mycorrhizal variants. There was
some small uptake by the ÿAM, NH ‡4 -fed plants
(Table 3). For both the NO ÿ3 - and NH ‡4 -fed plants, the
Hawkins and GeorgeÐE€ect of N Form on Hyphal N Uptake
800
Nitrate-fed plants
Ammonium-fed plants
307
A
600
Total N (mg)
400
b
200
0
a
–
–
+
a
–
a
a
a
b
+
–
b
a
–
+
a
a
a
–
a
b
+
a
200
b
400
+N+P
+N–P
–N–P
+N+P
+N–P
–N–P
N treatment
40
Nitrate-fed plants
Ammonium-fed plants
B
N concentration (mg N g–1 d. wt)
30
b
20
a
a
10
0
a
–
–
+
–
a
10
20
a
a
+
–
–
a
a
–
+
a
b
a
a
+N+P
+
b
30
40
a
+N–P
–N–P
+N+P
a
+N–P
–N–P
N treatment
F I G . 2. Nitrogen contents per pot (A) and concentrations (B) of non-mycorrhizal and mycorrhizal T. aestivum shoots and roots supplied with
either nitrate or ammonium and grown with low or high P supply (ÿP; ‡P) and low or high N supply (ÿN; ‡N). Di€erent letters indicate a
statistically signi®cant di€erence between the roots or shoots of ÿAM and ‡AM treatments (P 5 0.05, Student's t-test) at the same level of
nutrient supply. Values are means of four replicates. (h), Shoot; (D), root; (ÿ/‡), without/with AM.
uptake rate of the ‡AM plants was higher than for
the ÿAM plants, although this was not signi®cant for the
NH ‡4 -fed plants (Table 3). There was no signi®cant
di€erence between the uptake rate of ‡N ÿ P and
ÿN ÿ P plants but a higher percentage of the total 15N
supplied was taken up into the NO ÿ3 -fed, ‡N ÿ P plants
compared to the ÿN ÿ P plants (P ˆ 0.043, Table 3).
There was no signi®cant di€erence between the speci®c
uptake rates per unit hyphae of ‡N ÿ P and ÿN ÿ P
variants with the same N form supplied (Fig. 4). This
implies that the di€erences in 15N uptake into the plants
were due to di€erences in hyphal length and not di€erences
in speci®c hyphal uptake rates. There was also no
signi®cant di€erence between the speci®c uptake rates per
unit hyphae in the NO ÿ3 and NH ‡4 -fed variants with the
same N concentration supplied (P 4 0.05, Student's t-test),
which also implies that the di€erence in uptake rates and
percentage uptake between the NO ÿ3 and NH ‡4 -fed variants
was due to hyphal length densities and not the speci®c
uptake capacity of the hyphae.
308
Hawkins and GeorgeÐE€ect of N Form on Hyphal N Uptake
1200
Glucose equivalents (µmol glucose g–1 d. wt)
1000
800
Nitrate-fed plants
Ammonium-fed plants
Shoot
600
400
200
0
–
–
+
–
+
–
–
+
–
+
200
400
600
800
1000
Root
+N+P
+N–P
–N–P
+N+P
+N–P
–N–P
N treatment
F I G . 3. Concentration of reducing sugars, sucrose and starch measured as glucose equivalents in non-mycorrhizal and mycorrhizal T. aestivum
shoots and roots supplied with either nitrate or ammonium and grown with low or high P supply (ÿP; ‡P) and low or high N supply (ÿN; ‡N).
Values are means of four replicates. (h), Reducing sugars; (D), sucrose; (F
G), starch; (ÿ/‡), without/with AM.
T A B L E 2. Root colonization and extraradical hyphal length
densities of nitrate or ammonium-fed mycorrhizal T.
aestivum plants grown with low or high P supply (ÿP;
‡P) and low or high N supply (ÿN; ‡N)
T A B L E 3. Uptake rates of 15N and total percentage of 15N
taken up over the 48 h feeding period in nitrate or
ammonium-fed T. aestivum plants grown with low or high
P supply (ÿP; ‡P) and low or high N supply (ÿN; ‡N)
N treatment
‡N ÿ P
ÿN ÿ P
Nitrate-fed plants
Colonization rate ( %)
Hyphal length (m cm ÿ3)
85 + 2.5a
8.86 + 0.98a
70 + 5.6a
1.61 + 0.10b
Ammonium-fed plants
Colonization rate ( %)
Hyphal length (m cm ÿ3)
90 + 0.8a
0.89 + 0.45a
67 + 3.5b
0.49 + 0.24a
Di€erent superscripts indicate a statistically signi®cant di€erence
between the ‡N ÿ P and ÿN ÿ P treatments (P 5 0.05, Student's
t-test) at the same N form. Values are means of four replicates + s.e.
DISCUSSION
When N was supplied as NH ‡4 , plant biomass, hyphal
length densities and also 15N transport via the hyphae to
the AM plant were lower than when NO ÿ3 was supplied to
the AM plant, irrespective of plant N status. The N
concentrations of plants supplied with NH ‡4 were greater
than for the corresponding NO ÿ3 -fed plants and this may
have been a concentration e€ect due to the lowered biomass
of these plants. Short-term assimilation experiments by
Johansen et al. (1996) found that cucumber colonized by
G. intraradices assimilated more NH ‡4 into amino acids
N uptake rate per unit root mass
(nmol 15N g-1 root d.wt h ÿ1)
ÿAM
‡AM
Nitrate-fed plants
‡N ‡ P (control)
‡N ÿ P
ÿN ÿ P
0.0 + 0.4
0.0 + 1.4a
0.0 + 0.6a
Ð
56.2 + 10.1b
66.4 + 13.5b
Ammonium-fed plants
‡N ‡ P (control)
‡N ÿ P
ÿN ÿ P
2.9 + 6.9
0.0 + 5.0a
4.4 + 5.0a
Ð
57.5 + 26.3a
85.7 + 49.6a
Percentage of supplied 15N taken up over
48 h ( %)
ÿAM
‡AM
Nitrate-fed plants
‡N ‡ P (control)
‡N ÿ P
ÿN ÿ P
0.00 + 0.1
0.00 + 0.05a
0.00 + 0.01a
Ð
4.8 + 1.1b
1.8 + 0.4b
Ammonium-fed plants
‡N ‡ P (control)
‡N ÿ P
ÿN ÿ P
0.05 + 0.13
0.00 + 0.07a
0.06 + 0.07a
Ð
1.0 + 0.5a
1.1 + 0.6a
Di€erent superscripts indicate a statistically signi®cant di€erence
between the ÿAM and ‡AM treatments (P 5 0.05, Student's t-test) at
the same N supply. Values are means of four replicates + s.e.
N uptake rate (nmol 15N m–1 hyphae h–1)
Hawkins and GeorgeÐE€ect of N Form on Hyphal N Uptake
18
a
16
14
12
10
a
a
8
6
4
a
2
0
Nitrate
Ammonium
N form supplied
F I G . 4. Nitrogen uptake into nitrate or ammonium-fed T. aestivum
plants expressed per unit hyphal length of G. mosseae in those variants.
Plants were grown with low or high N supply [ÿN (D); ‡N (h)] and
low P supply. Similar letters indicate that there was no statistically
signi®cant di€erence between the ‡N ÿ P and ÿN ÿ P treatments
(P 5 0.05, Student's t-test) with the same N form. Values are means of
four replicates + s.e.
compared to when NO ÿ3 was supplied, indicating that the
former might be more easily assimilated. This may
represent an alternative explanation to a concentration
e€ect observed in this study since ‡N ÿ P NH ‡4 -fed plants
had higher N concentrations compared to NO ÿ3 -fed plants
but this was evidently not bene®cial for the symbiosis.
Lower biomass accumulation (Haynes and Goh, 1978;
Falkengren-Grerup, 1995), higher shoot : root ratios
(Cramer and Lewis, 1993a) and higher N concentrations
of T. aestivum (Cramer and Lewis, 1993b) and other plants
(Falkengren-Grerup, 1995) under NH ‡4 compared to sole
NO ÿ3 nutrition in a pH-bu€ered nutrient solution have
been reported previously. In a study using various
herbaceous and graminoid northern forest species, it was
found that no species, even if they were acid tolerant, grew
preferentially on NH ‡4 compared to NO ÿ3 plus NH ‡4
(Falkengren-Grerup, 1995). This growth e€ect may also
be the result of so-called NO ÿ3 -de®ciency, since NO ÿ3 has
been shown to play a likely role in maintaining biosynthesis
and/or root to shoot transfer of cytokinins (Walch-Liu
et al., 2000). Also, NO ÿ3 regulates the gene expression of
not only nitrate transporters, nitrate reductase and nitrite
reductase but also of glutamine synthetase and glutamate
synthase in plants and Neurospora and Aspergillus (Crawford, 1995). Therefore, an absence of NO ÿ3 might have
consequences not only for NO ÿ3 assimilation but also for
expression of glutamine synthetase and glutamate synthase,
enzymes that are also required for NH ‡4 assimilation.
The higher N concentrations did not result in increased
hyphal length densities compared to NO ÿ3 -fed plants;
rather the opposite was true, as mentioned. This is in
contrast to the trend reported previously (Hawkins and
George, 1999), namely that plants with sucient N supply
had higher hyphal length densities and 15N uptake rates.
This may indicate that, although the ‡N ÿ P, NH ‡4 -fed
plants had relatively high N concentrations, this was a
309
consequence of reduced root growth, and the NH ‡4 ion is a
relatively less favourable N source compared to NO ÿ3 with
respect to hyphal growth and 15N uptake via hyphae,
regardless of N status. In this and a previous study
(Hawkins and George, 1999), greater hyphal lengths were
generally related to larger amounts of 15N transported to
the plant via hyphae. Various factors (increased P
concentration, reduced carbohydrate concentration or a
direct NH ‡4 ion e€ect) could be responsible for the reduced
hyphal (and root) growth of mycorrhizal NH ‡4 -fed plants.
Ortas et al. (1996) found that NH ‡4 nutrition led to
increased P concentration in ‡AM plants independent of
rhizosphere pH. However, the NH ‡4 -fed plants in this
experiment did not have higher P concentrations than the
NO ÿ3 -fed plants. Higher P concentration in NH ‡4 -fed
compared to NO ÿ3 -fed plants was therefore not responsible
for the reduced hyphal lengths in NH ‡4 -fed plants.
It was also hypothesized that under NH ‡4 nutrition there
would be relatively less carbohydrate available for the
fungus (due to the requirement of NH ‡4 for immediate
assimilation into amino acids with the consumption of Cskeletons) than with NO ÿ3 nutrition. According to Cramer
and Lewis (1993c), the allocation of carbon to the amino-N
fraction occurred at the expense of carbohydrate fractions,
especially in the root of T. aestivum supplied with NH ‡4 as
compared to NO ÿ3 . This was the case for the ÿAM
‡N ÿ P treatment. However, the corresponding ‡AM
plants supplied with NH ‡4 did not have less carbohydrates
in the roots compared to those supplied with NO ÿ3 . The
higher carbohydrate concentrations in ‡AM NH ‡4 -fed
plants may re¯ect the relatively smaller sink of the lesser
hyphal lengths in NH ‡4 -supplied plants compared to NO ÿ3 fed plants.
Therefore, the negative e€ect of NH ‡4 supply on hyphal
growth was neither directly related to increased P concentrations nor to decreased carbohydrate concentrations in
the root. The reduced colonization, hyphal length and 15N
transport to the plant may be related to the observed
reduced root growth and/or, NH ‡4 may possibly have had a
direct inhibitory e€ect on hyphal growth. The fact that
NH ‡4 supply to the plant/fungus a€ected the extraradical
hyphal length but not the colonization rate supports the
idea that the NH ‡4 ion may be deleterious to extraradical
fungal growth but has less e€ect on colonizing structures
protected inside the bu€ered milieu of the root.
In this and a previous study (Hawkins and George,
1999), greater hyphal lengths were generally related to
larger amounts of 15N being transported to the plant.
However, in this study, this trend was only signi®cant when
NO ÿ3 was supplied to the plant. In this case the hyphae
transported 1.8 % (ÿN ÿ P) or 4.8 % (‡N ÿ P) of the 15N
supplied. For NH ‡4 -fed plants, the ®gure was about 1 %,
regardless of N treatment. The largest mycorrhizal e€ect
with respect to biomass accumulation, hyphal length and
15
N uptakeÐand even N concentrationÐwas evident for
‡N ÿ P NO ÿ3 -fed plants. The results of these two studies
imply that an adequate N status of the plant is indeed
important for mycelial development but that supplying
NH ‡4 as the sole N source has a detrimental e€ect on
hyphal and root growth, despite an adequate root N status.
310
Hawkins and GeorgeÐE€ect of N Form on Hyphal N Uptake
This is evidently not the case for the ectomycorrhizal
symbiosis which usually grows and preferentially takes up
NH ‡4 compared to NO ÿ3 (Finlay et al., 1988, 1989; Martin
et al., 1994) although inter- and intraspeci®c di€erences
occur. This is understandable in evolutionary terms since
ectomycorrhizal fungi have largely evolved in environments
where NH ‡4 is the predominant N form due to reduced
nitri®cation at higher altitudes or latitudes where ectomycorrhiza are prevalent.
Those plants with the largest mycorrhizal e€ect in terms
of biomass accumulation and 15N transport were also those
plants with a reduced sucrose concentration compared to
the ÿAM plants in the ‡N ÿ P treatment. The reduced
sucrose concentration observed in these ‡AM plants is in
agreement with the idea from ectomycorrhizal research that
a gradient of sucrose is maintained between plant and
fungus by the rapid conversion of plant sugar to fungal
sugar by the fungus. In this way, the fungus maintains a
strong sink (Wallander, 1992). The actual carbohydrate
status of the root (generally low in mycorrhizal plant roots)
therefore does not necessarily re¯ect the degree to which
carbohydrate is available to the fungus.
In conclusion, the results indicate that the smaller hyphal
lengths of ‡AM, NH ‡4 -fed plants were due to a direct ion
e€ect of NH ‡4 that reduced root and/or extraradical hyphal
biomass. In addition, it was con®rmed that sucient N
supply was important for the development of the fungal
mycelium and this development was related to the amount
of N acquired by the fungus and transported to the plant.
Since development of the fungus was generally reduced
under NH ‡4 nutrition, this latter trend was more obvious
for NO ÿ3 -fed plants.
AC K N OW L E D G E M E N T S
We thank the Hochschulsonderprogramm III, Hohenheim
UniversitaÈt, Germany for the award of a bursary during this
study.
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