Tree Physiology 33, 464–474
doi:10.1093/treephys/tpt024
Research paper
Two-way transfer of nitrogen between Dalbergia odorifera and its
hemiparasite Santalum album is enhanced when the host is
effectively nodulated and fixing nitrogen
J.K. Lu1, L.H. Kang1, J.I. Sprent2, D.P. Xu1,6 and X.H. He3,4,5,6
1Research
Institute of Tropical Forestry, Chinese Academy of Forestry, Guangdong 510520, China; 2Division of Plant Sciences, University of Dundee at James Hutton Institute,
Dundee DD2 5DA, UK; 3School of Life Sciences, Yunnan Normal University, Kunming, Yunnan 650500, China; 4Ministry of Agriculture Key Laboratory of Crop Nutrition and
Fertilization, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing 100081, China; 5School of Plant Biology, University of
Western Australia, Crawley, WA 6009, Australia; 6Corresponding author: [email protected] and ([email protected])
Received October 29, 2012; accepted March 10, 2013; published online April 19, 2013; handling Editor Heinz Rennenberg
Nutrient translocation from a host plant is vital to the growth and survival of its root parasitic plant, but few studies have
investigated whether a parasitic plant is also able to transfer nutrients to its host. The role of N2-fixation in nitrogen (N) transfer between 7-month-old Dalbergia odorifera T. Chen nodulated with Bradyrhizobium elkanii DG and its hemiparasite
Santalum album Linn. was examined by external 15N labeling in a pot study. Four paired treatments were used, with 15N given
to either host or hemiparasite and the host either nodulated or grown on combined N. N2-fixation supplied 41–44% of total
N in D. odorifera. Biomass, N and 15N contents were significantly greater in both nodulated D. odorifera and S. album grown
with paired nodulated D. odorifera. Significantly higher total plant 15N recovery was in N donor D. odorifera (68–72%) than
in N donor S. album (42–44%), regardless of the nodulation status in D. odorifera. Nitrogen transfer to S. album was significantly greater (27.8–67.8 mg plant−1) than to D. odorifera (2.0–8.9 mg plant−1) and 2.4–4.5 times greater in the nodulated
pair than in the non-nodulated pair. Irrespective of the nodulation status, S. album was always the N-sink plant. The amount
of two-way N transfer was increased by the presence of effective nodules, resulting in a greater net N transfer (22.6 mg
plant−1) from host D. odorifera to hemiparasite S. album. Our results may provide N management strategies for D. odorifera/
S. album mixed plantations in the field.
Keywords: Bradyrhizobium, Dalbergia odorifera, haustorium, hemiparasite, nitrogen fixation, Santalum album, two-way N
transfer.
Introduction
Nitrogen (N) transfer between plants is of fundamental importance in agricultural and natural ecosystems (Forrester et al.
2006, Moyer-Henry et al. 2006, He et al. 2009, Richards et al.
2010, Pirhofer-Walzl et al. 2012). Numerous studies over the
past two decades have demonstrated that N transfer from an N
donor to an N receiver is through both soil mass flow and diffusion (Paynel et al. 2001, Høgh-Jensen 2006, Sierra and
Nygren 2006, Sierra and Desfontaines 2009, Kurppa et al.
2010, Pirhofer-Walzl et al. 2012) and common mycorrhizal network (CMN) linkages (Johansen and Jensen 1996, He et al.
2004, 2005, 2009, Moyer-Henry et al. 2006, Li et al. 2009).
Parallel with this, studies have also shown that N2 fixation could
enhance two-way N transfer between N2-fixing and non-­N2-­fixing
plants under both non-mycorrhizal (Shen and Chu 2004,
Høgh-Jensen 2006) and mycorrhizal colonization (Newman
1988, Johansen and Jensen 1996, He et al. 2004, 2005,
2009, Moyer-Henry et al. 2006, Li et al. 2009).
© The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
Two-way transfer of nitrogen between D. odorifera and its hemiparasite S. album 465
Parasitic plants are parasites on other plants and cover a
wide range of morphologies, life strategies and growth forms
(~19 families, 227 genera and 4100 species). Parasitic plants
are functionally or morphologically classified to hemiparasites
(containing chlorophyll) and holoparasites (lacking chlorophyll),
xylem and phloem feeders or stem and root parasites (see Bell
and Adams 2011). Root parasitic plants are linked to their hosts
by haustoria for taking up water, mineral nutrients or organic
compounds (Calladine et al. 2000, Hibberd and Jeschke 2001,
Pate 2001, Press and Phoenix 2005, Irving and Cameron 2009,
Bell and Adams 2011). For instance, 15N labeling, 15N natural
abundance and mass flow modeling methods have demonstrated that up to 70% of N in the parasites is obtained from the
hosts (Tennakoon et al. 1997a, 1997b, Pate 2001, Pageau et al.
2003, Jiang et al. 2004, Cameron and Seel 2007), indicating
that such N translocations may be vital for the growth and survival of the parasitic plants. However, no studies have investigated whether a parasitic plant is also able to transfer N to its
host. Pate et al. (1994) did suggest that the hemiparasite Olax
phyllanthi (Labill.) R.Br. might allow the backflow of nitrogenous
compounds to its multiple hosts.
Indian sandalwood (Santalum album Linn.), an evergreen and
xylem-feeding hemiparasitic tree, has been over-exploited for
its aromatic heartwood, which has cosmetic, religious and
medicinal significance (Dhanya et al. 2010). Large-scale plantations of it have been established in Australia, China, India and
Indonesia over the last two decades (Dhanya et al. 2010, Lu
2011). Both pot and field plantations have shown that sandalwood growth is greatly enhanced after its successful establishment on suitable hosts, particularly on N2-fixing species
(Radomiljac et al. 1998, 1999b). Compared with a singlespecies plantation, a mixture of non-N2-fixing and N2-fixing plants
is important to the socio-economy of local growers (Press and
Phoenix 2005, Forrester et al. 2006, 2007, Irving and Cameron
2009, Dhanya et al. 2010, Bell and Adams 2011, Lu 2011).
A mixed plantation of S. album with Dalbergia odorifera
T. Chen (another valuable indigenous tree in China) was successfully established in the Jianfengling Arboretum of Hainan
Island, China (18°42′N, 108°49′E) in 1989. There, S. album
has successfully parasitized D. odorifera nodulated with
Bradyrhizobium and newly identified Burkholderia strains (Lu
et al. 2011, 2012). The establishment of these two relationships indicates potential relocation of resources including
­carbon, N and water from one to the other plant species (Lu
2011). Unfortunately, to date, an appropriate understanding of
the N2-fixation capacity, parasitic relationship and either oneway or two-way N transfer has not been studied between
these two precious tropical forest trees.
In a pot study, using (15NH4)2SO4 as 15N tracer to address the
possible interplant N movement and role of N2-fixation in N
transfer between 7-month-old hemiparasite S. album and its
N2-fixing host D. odorifera, we asked four questions: (i) Does N
transfer occur between hemiparasitic S. album and host
D. odorifera? (ii) If N transfer occurs, how much is transferred?
(iii) Is N transfer affected by N2-fixation? (iv) In which direction
does net N transfer occur? Our objectives are not only to reveal
N movement between the hemiparasite S. album and its
N2-fixing host D. odorifera but also to provide useful information
about N management for these two high-value tropical trees.
Materials and methods
Plant growth
To stimulate germination, seeds of S. album were soaked in
0.1% gibberellic acid for 12 h (Radomiljac, 1998). Seeds of
both D. odorifera and S. album were then surface-sterilized
with 3% sodium hypochlorite for 5 min, washed with distilled
water and then germinated on sterilized sand soaked with distilled water at 28–30 °C for 3–4 weeks.
One S. album seedling was transplanted 10 cm away from
one D. odorifera seedling in a plastic pot (15 × 11 × 14 cm), filled
with 250 g potting mix (vermiculite : perlite, 2 : 1, v/v, pH 6.8).
Plants were grown for 7 months (April to October 2010) in a
greenhouse, located in the Research Institute of Tropical
Forestry, Guangzhou, China (23°11′N, 113°23′E), under
33/25 °C and 65/75% relative humidity (day/night). Each pot
was given 300 ml of N-free nutrient solution before seedlings
were transplanted. Plants were watered twice a week with distilled water depending on vigor, and weekly with 5 ml Jensen’s
N-free nutrient solution and 2.5 ml 0.5% (14NH4)2SO4 (2.65 mg
N pot−1 week−1) for the first 6 months (127.14 mg N pot−1 year−1).
The 15N signature of (14NH4)2SO4 was 0.366783 ± 0.000032
(15N, atom %) or 1.32 ± 0.10 (δ15N, ‰).
Rhizobium inoculum
Bradyrhizobium elkanii DG, originally isolated from the active
nodules of Pterocarpus macrocarpus Kurz and known to be
effective in nodulating D. odorifera (Lu 2011, Lu et al. 2011,
2012), was used. It was maintained on a solid yeast extract–
mannitol (YM) medium (Vincent 1970), and incubated in
250 ml triangular flasks at 28 °C for 5 days. The resultant culture was then centrifuged at 4000 rpm for 5 min and suspended in sterilized 0.75% NaCl solution (107–109 colony ml−1
at 600 nm optical density).
Geminated Dalbergia seedlings with a 2–3 cm radicle were
soaked in B. elkanii slurry for 10 min and then transplanted into
the plastic pots, and given a further 1 ml pot−1 of inoculum
around the tap root area 3 days after transplanting. All noninoculated seedlings were given the same amount of autoclaved (121 °C for 30 min) B. elkanii inoculum.
Experimental design and
15N
labeling
Either D. odorifera or S. album served as the 15N donor, and
thus net N transfer could be calculated from the difference of
Tree Physiology Online at http://www.treephys.oxfordjournals.org
466 Lu et al.
15N
translocation from D. odorifera to S. album and from
S. album to D. odorifera. In doing so, there were four pairs or
treatments with four replicates of each: (i) non-nodulated
D. odorifera/S. album (Dnod− → S−), (ii) S. album/non-nodulated
D. odorifera (S− → Dnod−), (iii) nodulated D. odorifera/S. album
(Dnod+ → S +) and (iv) S. album/nodulated D. odorifera
(S + → Dnod+) (Table 1). The two non-nodulated pairs were
used to measure N transfer through the growth medium (soil
pathway), while the two nodulated pairs were to study the
effect of N2 fixation on N transfer. One leaf from an N donor
seedling was inserted into a 1.5 ml sterilized centrifuge tube
containing 1.0 ml 0.15% (15NH4)2SO4 (N dissolved in sterilized
Milli-Q water, 99.36 at % 15N) solution according to He et al.
(2006). For each N donor seedling, a total of 3.0 ml
(15NH4)2SO4 (1.006 mg N) was given as 1.0 ml each to the
third, fourth and fifth fully expanded leaves. The centrifuge
tubes were vertically attached to the iron wire and immediately
sealed using Parafilm (Menasha, WI, USA) after the branch
wrapping to avoid spillage and evaporation (Figure 1). All 15N
solution had been imbibed after 4 weeks and the 15N labeling
tubes were then removed without any obvious sign of solute
residue in the tube. No 14N was supplied to the donor and
receiver plants during this 4 week period.
Plant harvest and analysis
Fresh leaves of D. odorifera before 15N treatment were collected to
estimate N2 fixation using the 15N natural abundance method
(Knowles and Blackburn 1993). Plants of D. odorifera and S. album
were separately harvested after 4 weeks of 15N addition to the N
donor plant. Oven-dried (70 °C for 72 h) plant t­issues including
shoots, roots, nodules and haustoria (see Figure 1) were ground
in a ball mill (Retsch GmbH & Co. KG, Haan, Germany) for the
analysis of total N and 15N (Isotope Ratio Mass Spectrometer,
Thermo Fisher Scientific Inc., Waltham, MA, USA).
The calculation of plant N derived from fixation (%Nfixed) was
according to Knowles and Blackburn (1993):
%Nfixed = (δ15 Nnon− fixing plant – δ15 Nfixing plant ) /
(δ15 Nnon− fixing plant – B) × 100 (1)
where B is the δ15N value of nodulated N2-fixing plants grown
without N supplement. Three indigenous woody plants
(Bischofia polycarpa (Levl.) Airy Shaw, Heteropanax fragrans
(Roxb.) Seem. and Murraya exotica L. Mant.), which have similar root and phenological characteristics to the N2-fixing plant
of D. odorifera growing in the same natural habitat in southern
China, were chosen as non-N2-fixing reference plants. The
averaged foliar δ15N values of these three reference plants
were then used to calculate the N2-fixation.
Nitrogen transfer was expressed as the percentage N transfer
(Eq. (2)), the amount of N transferred (mg plant−1, Eq. (4)) and
percentage NDFT (N in the receiver derived from transfer, Eq. (5))
using the formulas (Johansen and Jensen, 1996, He et al. 2004):
%Ntransfer =
Ncontent receiver × 100
Ncontent receiver + 15 Ncontent donor 15
15
(2)
Table 1. ​Experimental design for two-way N transfer between D. odorifera and S. album seedlings grown in pots
Pair name
Non-nodulated pairs
(Dnod−/S−
pair)
Nodulated pairs (Dnod+/S + pair)
N donor
N receiver
Nodulation status
Code
Dalbergia odorifera
Santalum album
Dalbergia odorifera
Santalum album
Santalum album
Dalbergia odorifera
Santalum album
Dalbergia odorifera
–
–
+
+
Dnod− → S−
S− → Dnod−
Dnod+ → S +
S + → Dnod+
14N
supplement to both the N donor and N receiver plant was ceased when plants were 6 months old and then only 15N was supplied to the leaves
of donor plant for another 4 weeks. D, D. odorifera; S, S. album; nod+ or nod−, inoculated or non-inoculated with a Bradyrhizobium strain; S + or S−,
S. album paired with inoculated or non-inoculated D. odorifera.
Figure 1. ​Setups of 15N (99.36 at % 15N) labeling with plant leaves in greenhouse. (A) Dalbergia odorifera and (B) S. album. DoL, D. odorifera leaf;
SaL, S. album leaf; IW, iron wire; NS, 0.15% N solution.
Tree Physiology Volume 33, 2013
Two-way transfer of nitrogen between D. odorifera and its hemiparasite S. album 467
where
15
s­ ignificant difference post hoc test at P ≤ 0.05 using SPSS
11.5 (SPSS Inc., Chicago, IL, USA).
Ncontent plant =
Atom %15 N excessplant × total Nplant
(3)
Atom %15 N excesslabeled N
Formation of haustoria and nodules and N2-fixation
capacity of D. odorifera
Amount of N transferred (mg plant−1):
Ntransfer =
% Ntransfer × total Ndonor
100 − %Ntransfer
(4)
% of N in receiver derived from transfer (% NDFT):
% NDFT =
Ntransfer × 100
Total Nreceiver (5)
Partial 15N recovery (%) by plants is calculated as follows since
no growth media samples were taken:
%15 N recovery =
15
15
Nplant
Nfertilizer × 100 Results
(6)
Data analysis
Each treatment had four replicates for a total of 16 pots or 32
paired plants. Data (means ± SE, n = 4) were subjected to a
one-way analysis of variance and significant differences
between the treatments were compared with the least
Bell-shaped S. album haustoria attached to D. odorifera roots
(Figure 2A) and penetrated into D. odorifera nodules
(Figure 2B–D). The numbers of haustoria averaged 7.5 ± 3 per
pair and were similar between all four paired treatments.
Nodules from both the nodulated D. odorifera donor and
receiver were reddish-brown with pink internal tissues, indicating that they were active in N2 fixation.
Compared with the 6-month-old nodulated D. odorifera,
foliar δ15N values were lower in the no-N-supplied D. odorifera,
while higher in the three non-N2-fixing reference plants (see
captions in Table 2). There were no significant differences in
N2-fixation capacity between the donor and receiver Dalbergia
seedlings (Table 2). Foliar δ15N before external 15N labeling,
nodule number and biomass, percentage of N2-fixation and
specific nodule activity were similar between the nodulated
D. odorifera donor and receiver. N2 fixation supplied ~43.00%
of the total N requirement, and the specific nodule activity was
~45.0 µg N mg−1 day−1 wt nodule. In addition, after 4 weeks
15N labeling, foliar δ15N values were the highest in nodulated
D. odorifera donors (672.42 ± 38.89‰), much less in the
Figure 2. ​Haustoria of S. album attaching on D. odorifera roots (A) and nodules (B, C, D) in 7-month-old plants grown in pots. An immature haustorium (C) and a mature haustorium (D) attaching on functional nodule. Scale bars, 2 mm (A), 1 mm (B, D) and 0.5 mm (C). DoR, D. odorifera root;
E, endophyte of haustorium; Hstr, haustorium; Nod, nodule; SaR, S. album root.
Tree Physiology Online at http://www.treephys.oxfordjournals.org
468 Lu et al.
Table 2. ​N2-fixation capacity of 6-month-old nodulated D. odorifera seedlings grown in pots
Plants
Foliar δ15N
(‰)
Nodule
numbers
Nodule
biomass (mg)
Nodule N
concentration (%)
Nfixed
(%)
Specific nodule activity
(µg N mg−1 day−1 wt nodule)
D. odorifera (donor)
D. odorifera (receiver)
1.39 ± 0.10a
1.35 ± 0.19a
51 ± 14a
58 ± 10a
88.19 ± 11.11a
95.13 ± 6.72a
1.61 ± 0.53a
1.31 ± 0.24a
44 ± 4a
41 ± 2a
44.85 ± 0.81a
44.97 ± 1.50a
Data (means ± SE, n = 4) followed by the same letter for each parameter are not significantly different (P = 0.05). For the plants used for the
N2-fixation measurements, foliar δ15N values were −1.76 ± 0.08‰ for non-N-fertilized D. odorifera, 3.70 ± 0.25‰, 3.87 ± 0.30‰ and 3.20 ± 0.44‰
for non-N2-fixing B. polycarpa, H. fragrans and M. exotica, respectively. N2-fixation was hence calculated by Eq. (1) using the averaged foliar δ15N
values of these three non-N2-fixing plants. For the 7-month-old D. odorifera seedlings, foliar δ15N values were 672.42 ± 38.89‰ and 2.61 ± 0.45‰
for the D. odorifera 15N donor and receiver, respectively. The soil in the Jianfengling Arboretum of Hainan Island, China (18°42′N, 108°49′E), where
the plants are naturally grown, is a red soil (Humic Acrisols) with a δ15N 7.13 ± 0.60‰ (n = 12).
three non-N2-fixing plants (3.20–3.87‰) and the least in the
nodulated receivers (2.61 ± 0.45‰) (Table 2).
Plant biomass production
Plant biomass production was similar whether D. odorifera and
S. album served as the N donor or N receiver (Figure 3A), so
the data from the donor and receiver were combined.
Significantly more (1.4–1.5 times) biomass was produced for
both D. odorifera and S. album in the nodulated pair than in the
non-nodulated pair. D. odorifera produced about 2.5 times as
much biomass as S. album irrespective of whether it was nodulated or not (Figure 3A).
Plant nitrogen concentration and content
Plant %N or N content were similar whether D. odorifera and
S. album served as the N donor or N receiver (Figure 3B and
C), so the data from the donor and receiver were combined.
Both D. odorifera and S. album had significantly higher %N in
the nodulated pair than in the non-nodulated pair (Figure 3B).
In contrast, plant %N was similar between D. odorifera and
S. album regardless of the nodulation status of the former.
Plant N content in both D. odorifera and S. album was 1.9–
3.2 times greater in the nodulated pair than in the non-­
nodulated pair, and D. odorifera had significantly more N than
S. album whether D. odorifera was nodulated or not (Figure 3C).
In both species, the plant biomass production was positively
correlated with plant N content (Figure 4, r2 ranged from 0.94
to 0.99). Both greater biomass and N content were found in
the nodulated pair.
15N
at% excess,
15N
content and plant
15N
15N
recovery
Values of
at % excess between nodulation treatments for a
given species were similar no matter whether a plant species
served as the N donor or N receiver (Table 3). Values of 15N at
% excess between plant species for a given nodulation
­treatment were significantly greater in D. odorifera than in
S. album when D. odorifera was the donor or in S. album than
in D. odorifera when S. album was the donor (Table 3).
Both D. odorifera and S. album had significantly more 15N
in the non-nodulated pair than in the nodulated pair when
Tree Physiology Volume 33, 2013
Figure 3. ​Effects of nodulation on biomass (A), nitrogen concentration
(B) and content (C) of 7-month-old D. odorifera (solid bars) and
S. album (open bars) seedlings grown in pots. Plants were fertilized
with (14NH4)2SO4 for 6 months and then labeled with (15NH4)2SO4 (N
donor only) for 4 weeks before harvest. Since the identity of the donor
plant did not affect biomass, N% and N content, data (means ± SE,
n = 8) were combined. Values followed by different letters designate
the significant differences (P = 0.05) between treatments for the same
plant species (a, b), and between species for a given treatment (x, y).
D, D. odorifera; S, S. album; nod+ or nod−, inoculated or non-­inoculated
with a Bradyrhizobium strain; S + or S−, S. album paired with inoculated
or non-inoculated D. odorifera.
Two-way transfer of nitrogen between D. odorifera and its hemiparasite S. album 469
D. odorifera served as the N donor, while the opposite was true
when S. album served as the N donor (Table 3). Plant 15N contents were significantly greater in D. odorifera than in S. album
when D. odorifera was the donor or in S. album than in D. odorifera when S. album was the donor (Table 3).
Values of 15N recovery for a given treatment were significantly greater in the N donor than in the N receiver no matter
whether D. odorifera nodulated or not (Table 4). Values of 15N
recovery between treatments were similar no matter whether
D. odorifera or S. album served as the N donor, whereas they
were significantly greater in the N donor than in the N receiver
no matter whether D. odorifera or S. album served as the N
receiver (Table 4). Percentage of total plant 15N recovery was
significantly higher when D. odorifera served as the N donor
(68–72%) than when S. album served as the N donor (42–
44%), regardless of the nodulation status in D. odorifera
(Table 4).
Nitrogen transfer between D. odorifera and S. album
seedlings
Data on N transfer were calculated from 15N at % excess and
15N content of donor and receiver plants (Table 3). N transfer
Figure 4. ​Relationships between biomass and N content of 7-month-old Dalbergia odorifera (circles) and Santalum album (triangles) seedlings
grown in pots. Data are means ± SE (n = 4) and the 15N donor are indicated by solid and 15N receiver by open symbols. Regressions are shown for
S. album (dashed lines) and for D. odorifera (solid lines). D, D. odorifera; S, S. album; nod+ or nod−, inoculated or non-inoculated with a Bradyrhizobium
strain; S + or S−, S. album paired with inoculated or non-inoculated D. odorifera.
Table 3. ​15N at % excess and total 15N content of D. odorifera and S. album seedlings grown in pots
Codes
N donor
Non-nodulated pairs
Dalbergia
Dnod− → S−
Santalum
S− → Dnod−
Nodulated pairs
Dalbergia
Dnod+ → S +
Santalum
S + → Dnod+
15N
N receiver
At % excess
mg plant−1
0.559 ± 0.059a,x
0.481 ± 0.100a,x
0.193 ± 0.028a,x
0.080 ± 0.010b,x
0.460 ± 0.050a,x
0.480 ± 0.042a,x
0.128 ± 0.025b,x
0.119 ± 0.012a,x
15N
At % excess
mg plant−1
Santalum
Dalbergia
0.385 ± 0.080a,y
0.034 ± 0.005a,y
0.124 ± 0.006a,y
0.007 ± 0.001b,y
Santalum
Dalbergia
0.374 ± 0.036a,y
0.050 ± 0.016a,y
0.107 ± 0.002b,y
0.015 ± 0.003a,y
Plants were fertilized with (14NH4)2SO4 for 6 months and then with (15NH4)2SO4 (N donor only) for 4 weeks before harvest. Data (means ± SE,
n = 4) followed by different letters designate the significant differences (P = 0.05) between treatments for a given species within columns (a, b),
and between species for a given treatment within rows (x, y).
D, D. odorifera; S, S. album; nod+ or nod−, inoculated or non-inoculated with a Bradyrhizobium strain; S + or S−, S. album paired with inoculated or
non-inoculated D. odorifera.
Tree Physiology Online at http://www.treephys.oxfordjournals.org
470 Lu et al.
Table 4. ​15N recovery of Dalbergia odorifera and Santalum album seedlings grown in pots
Codes
Non-nodulated pairs
Dnod− → S−
S− → Dnod−
Nodulated pairs
Dnod+ → S +
S + → Dnod+
N donor
species
15N
Dalbergia
Santalum
Dalbergia
Santalum
recovery (%)
N receiver
species
15N
44.94 ± 4.76ax
39.07 ± 6.88a,x
Santalum
Dalbergia
27.32 ± 3.21a,y
2.84 ± 0.47b,y
72.26 ± 7.66a
41.91 ± 6.31b
37.28 ± 4.01a,x
38.93 ± 3.41a,x
Santalum
Dalbergia
30.36 ± 2.08a,y
4.93 ± 0.92a,y
67.63 ± 5.91a
43.86 ± 3.39b
N donor
recovery (%)
N receiver
15N
recovery
(%) Total
Plants were fertilized with (14NH4)2SO4 for 6 months and then with (15NH4)2SO4 (N donor only) for 4 weeks before harvest. Data (means ± SE,
n = 4) were calculated from the total amount of 15N supplied in each pot and 15N in plants in Table 3 and followed by different letters designate
significant differences (P = 0.05) between the treatments for a given species within columns (a, b), and between species for a given treatment
within rows (x, y). The total amount of 15N in each pot during the whole 7-month experimental period = 0.2332 mg 15N (63.57 mg supplied N pot−1
during 6-months growth as 2.5 ml 0.5% (14NH4)2SO4 week−1 × 0.366783 atom 15N %) + 1.0001 mg 15N (1.0065 mg labeled N pot−1 during
1-month 15N labeling as 3 ml 0.15% (15NH4)2SO4 × 99.36 atom 15N %) = 1.2333 mg 15N. D, D. odorifera; S, S. album; nod+ or nod−, inoculated or
non-inoculated with a Bradyrhizobium strain; S + or S−, S. album paired with inoculated or non-inoculated D. odorifera.
was expressed in three parameters: percentage N transfer
(Figure 5A); percentage N in the receiver derived from transfer
(%NDFT) (Figure 5B); and the total amount of N transferred
(mg plant−1) (Figure 5C). Two-way N transfer occurred in all
four D. odorifera/S. album pairs, regardless of the nodulation
status. In both nodulated and non-nodulated treatments, %N
transfer, %NDFT and the amount of N transferred were significantly higher from D. odorifera to S. album (37.5 and 43.8%,
60.8 and 81.6%, and 27.8 and 67.8 mg plant−1, respectively)
than from S. album to D. odorifera (10.2 and 16.6%, 7.4 and
12.8%, and 2.0 and 8.9 mg plant−1, respectively) (Figure 5A–c).
Our study was able to calculate net N transfer in paired
D. odorifera/S. album seedlings (Figure 5D). The amounts of
net N transfer were significantly higher in the nodulated
(22.6 ± 4.7 mg plant−1) than in the non-nodulated treatment
(16.9 ± 0.8 mg plant−1). Regardless of the nodulation status,
S. album was the sink plant for N acquisition from D. odorifera.
Discussion
Formation of haustoria roots
Santalum album is a xylem-feeding parasitic plant which
obtains water and solutes from its host plants via haustoria
(Tennakoon and Cameron 2006). Such solute transfers are
driven by the elevated transpiration rate and reduced water
potential of the parasite compared with the host (Radomiljac
et al. 1999a, Ackroyd and Graves 1997). A successful haustorial attachment to a superior host species can greatly promote
S. album growth under both pot and field conditions (Radomiljac
et al. 1998, 1999a, 1999b).
In the present study, there was no significant difference in
numbers of haustoria between treatments, suggesting that
haustorial formation is independent of plant N status. We also
observed direct parasitism of the nodules of D. odorifera
(Figure 2B), as previously described for S. album and another
Tree Physiology Volume 33, 2013
hemiparasite, O. phyllanthi, attaching to legume hosts
(Tennakoon et al. 1997a, Radomiljac et al. 1999b). Although
only a small proportion of the host nodules had haustoria, it
suggests that the parasite can tap the rich source of fixed
nitrogen exported from nodules. It would be interesting to
know whether, given the choice, a parasite would choose to
parasitize a N2 fixing rather than a non-N2-fixing host.
Biological nitrogen fixation
The 15N natural abundance method has been increasingly used
to estimate N2 fixation based on natural ecosystems (see for
example Boddey et al. 2000, Unkovich et al. 2008). Its major
disadvantage is the selection of suitable non-fixing reference
plants having similar root and phenological to the N2 fixating
plant. To minimize such limitations in our study, we used three
indigenous woody species, B. polycarpa, H. fragrans and
M. exotica, from the same natural habitat in southern China as
our plantation (Lu 2011). These three non-N2-fixing reference
plants had significantly different, yet greater foliar δ15N values
(3.20–3.87‰) than D. odorifera (1.35–1.39‰) under greenhouse N-fertilized conditions (Table 2). Further, the respective
foliar δ15N values of D. odorifera were −1.76 and 1.37‰ under
non-N-fertilized and N-fertilized conditions (Table 2), which
are within the range of other similar plants (Unkovich et al.
2008).
We used the rhizobium strain B. elkanii which is highly effective in nodulation on D. odorifera roots (Lu et al. 2011, 2012). In
our study, the percentage of N derived from N2-fixation was from
41 ± 2 to 44 ± 4% for 6-month-old nodulated D. odorifera
grown under greenhouse condition, and the rest of the plant’s N
was derived from the growth media or nutrition, seed and root N
exudation, etc. (Unkovich et al. 2008). These 41–44% N2-fixation
rates were comparable with 38–73% in 2-year-old Calliandra
calothyrsus Meissn. and Gliricidia sepium (Jacq.) Walp. (Peoples
et al. 1996) and 38% in 11-month-old Casuarina ­cunninghamiana
Two-way transfer of nitrogen between D. odorifera and its hemiparasite S. album 471
Figure 5. ​Percentage of nitrogen transfer (A), N in the receiver derived from transfer (NDFT, B), amount of N transfer (C) and net N transfer (D) in
7-month-old D. odorifera/S. album pairs as affected by nodulation (nod) and species of N donor or N receiver. Data are means ± SE (n = 4). For
each parameter, values followed by different letters designate significantly the differences (P = 0.05) between treatments for the given directions
of transfer (a, b) and between the directions of transfer in pairs for a given treatment (x, y). D, D. odorifera; S, S. album; nod+ or nod−, inoculated
or non-inoculated with a Bradyrhizobium strain; S + or S−, S. album paired with inoculated or non-inoculated D. odorifera.
Miq. (He et al. 2005), but were different from 29% in 5-monthold C. cunninghamiana (He et al. 2004), 50–80% in 2- to
4-year-old Cyclopia sp. (Spriggs and Dakora 2009) and 68–79%
in 1- to 2-year-old Elaeagnus angustifolia L. (Khamzina et al.
2009). Nevertheless, to our knowledge, this is the first study to
adopt the 15N natural abundance method to evaluate the
N2-fixation capacity of D. odorifera. Even then, further estimation
of N2-fixation capacity by D. odorifera under field or natural habit
would provide better management strategy of N economy for D.
odorifera plantation and its neighboring plants including hemiparasitic S. album if they are growing together.
Effect of nodulation on plant growth
Nodulation in D. odorifera enhanced biomass production and N
accumulation in both D. odorifera and S. album (Figure 3). In
our study, seedlings, not only D. odorifera but also S. album,
were taller, healthier and had larger root systems in the
7-month-old nodulated pair than in the same age non-­
nodulated pair. This is important to the hemiparasite S. album
for their early establishment in the field, but may affect the
growth of the host D. odorifera over a longer period.
N2 fixation enhances N transfer between plants
Nitrogen transfer between plants can take place by indirect or
direct routes (Høgh-Jensen 2006). Indirect N transfer via soil or
medium comes from dead and decayed plant tissues (e.g. roots,
leaves and nodules) and exudates such as ammonium, amino
acids and amides from plant roots (Paynel et al. 2001, Walker
et al. 2003, Høgh-Jensen 2006, Sierra and Nygren 2006, Sierra
and Desfontaines 2009, Kurppa et al. 2010, Pirhofer-Walzl et al.
2012). These compounds are deposited in soil or medium and
can be absorbed by adjacent plants. Leaf absorption of volatilized N-containing compounds is another way of indirect N transfer (Frank et al. 2004). Direct N transfer may be mediated by
mycorrhizal hyphae or haustoria, which connect plants (He et al.
2009, Bell and Adams 2011). One-way and two-way N transfer
via CMNs between plants has been determined (Johansen and
Jensen 1996, He et al. 2004, 2005, 2006). Numerous studies
Tree Physiology Online at http://www.treephys.oxfordjournals.org
472 Lu et al.
have also shown that fixed N can enhance N transfer between
plants under non-mycorrhizal (Shen and Chu 2004, HøghJensen 2006, Kurppa et al. 2010) and mycorrhizal plants (He
et al. 2004, 2005, 2009, Li et al. 2009).
Schulze and Ehleringer (1984) suggested that high transpiration rates of mistletoes enable them to take up sufficient
xylem N from its hosts, suggesting that the N transfer from a
host to a parasite plays a vital role in parasite growth and survival. Few studies have quantified one-way N transfer from
host plants to parasitic plants (see review by Bell and Adams
2011), let alone two-way N transfer. As far as we know, the
holoparasite obtained almost all its N from a N2-fixing or nonN2-fixing host (Hibberd et al. 1998, Hibberd and Jeschke
2001). For hemiparasites, the proportion of N transferred from
the host ranged widely, from low (0.2–18%) via non-N2-fixing
hosts to high (56–70%) via N2-fixing plants (Tennakoon et al.
1997b, 1997c, Jiang et al. 2004, Cameron and Seel 2007).
Hence, N2-fixing legumes have been assumed to be superior
hosts by supplying N to parasites (Tennakoon et al. 1997a,
1997b, Radomiljac et al. 1998, Cameron and Seel 2007).
However, N2-fixation in legumes might not strengthen their
own performance when parasitized by Rhinanthus minor Linn.
(Jiang et al. 2008).
Most importantly, our results demonstrated two-way N
transfers generated from the 15N signature of both donor and
receiver plants, between 7-month-old paired D. odorifera and
S. album seedlings supplied with (15NH4)2SO4. Transfer of N
under the respective non-nodulated or nodulated condition
was as low as 10.2 or 16.6% of N transfer and 7.4 or 12.8% in
NDFT to D. odorifera, while as high as 37.5 or 43.8% of N
transfer and 60.8 and 81.6% in NDFT to S. album. The much
higher N transfer in the nodulated treatment indicated that D.
odorifera with effective nodules enhanced N fluxes between
host D. odorifera and hemiparasite S. album, suggesting that
effective N2-fixation could supply more nitrogenous substances
in the xylem sap to the S. album haustoria. This might be one
of the primary factors why legumes make good hosts (Jiang
et al. 2008).
Nitrogen was transferred from host D. odorifera to S. album.
A similar phenomenon was reported for host Acacia rostellifera
Benth. to hemiparasite Santalum acuminatum (R. Br.) A. DC.,
resulting in 70% or more N of Santalum depending on fixing
atmospheric N from the leguminous hosts (Tennakoon et al.
1997c). From an evolutionary perspective, in order to acquire
nutrients of xylem sap from associated hosts, parasites may
have evolved with reduced water potential and increased transpiration relative to hosts (Ackroyd and Graves 1997, Schulze
and Ehleringer 1984, Radomiljac et al. 1999a). Indeed, where
the N level is low in a field, it may be economically and sustainably advantageous to meet the N demand of the S. album, if an
accessible N source is available from symbiotically fixed N
rather than from external N fertilizations.
Tree Physiology Volume 33, 2013
Backflow of amino compounds such as S-ethenyl cysteine
was demonstrated in the natural habitat and in pot cultures
involving O. phyllanthi parasitizing its hosts (Pate et al. 1994,
Tennakoon et al. 1997b, Pate 2001), indicating that the hemiparasite was able to transfer its own nitrogenous compound to
its associated host via haustoria. Our data are the first to report
that S. album is able to transfer N to its host D. odorifera.
However, this study did not prove whether the labeled N in the
host D. odorifera had actually passed via haustoria, or just
merely been taken up by D. odorifera roots from root exudates
or deposition of S. album. To demonstrate direct transfer and/
or eliminate transfer through indirect pathways, further
research is required, including designing a pot system to
exclude root contact and water movement, but allowing haustoria attachment and/or mycorrhizal linkage on host roots in
order to accurately determine the N transfer from S. album to
D. odorifera via haustorial connections.
We calculated net N transfer to locate the N flux direction
between D. odorifera and S. album regardless of the nodulation
status. Significantly greater net N transfer was from D. odorifera to S. album in the nodulated than in the non-nodulated
treatment, indicating that effective N2-fixation could improve N
transfer between S. album and its host. Moreover, S. album
was always the N-sink plant, whether nodulated or not. As a
consequence, field N fertilization strategy should pay more
attention to the S. album management if it was in a mixed stand
with a N2-fixation host.
In summary, our results showed that almost half of N in
D. odorifera was from N2-fixation, and N2-fixation enhanced
N accumulation, biomass production and N transfer
between host D. odorifera and hemiparasite S. album. When
nodulated D. odorifera as the N donor, and when S. album
as the N donor and partnered with nodulated D. odorifera,
plant biomass production and N content were maximized in
both plant species. Our data support previous reports that
N2-fixation plays an important role in N translocation to
non-N2-fixation partner. This would have practical consequences and may provide useful information about N management for mixed plantations of D. odorifera and S. album
in the field.
Acknowledgments
We thank Professor Bernard Dell and Dr Treena Burgess of
Murdoch University, Australia, and Chris Done, Don Spriggins,
Paul Brennan and Ron Wilson of the Institute of Foresters of
Australia for their valuable comments on an early version of the
manuscript.
Conflict of interest
None declared.
Two-way transfer of nitrogen between D. odorifera and its hemiparasite S. album 473
Funding
This study was financially supported by the National Natural
Science Foundation of China (31170582) and the Guangdong
Forestry Science and Technology for Creativity and Special
Fund, China (2009KJCX002-01).
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