Plant Soil
https://doi.org/10.1007/s11104-023-06342-7
RESEARCH ARTICLE
Natural nodulation and nitrogen fixation of Acacia
Auriculiformis grown in technosol eco-engineered from Fe
ore tailings
Zhen Li · Songlin Wu · Yunjia Liu · Fang You ·
Merinda Hall · Longbin Huang
Received: 28 July 2023 / Accepted: 9 October 2023
© The Author(s) 2023
Abstract
Aims Nitrogen deficiency in eco-engineered techno-
sol from iron (Fe) ore tailings limits the productivity
of colonising soil microbes and pioneer plants, which
are critical to further development of the technosol.
Symbiotic biological N ­ 2 fixation may be a strategy
to supply N in the moderately alkaline early techno-
sols since native legumes such as Acacia auriculi-
formis are tolerant of saline and alkaline soil condi-
tions as those in the technosol. It is hypothesized that
Responsible Editor: Michael Komárek.
Supplementary Information The online version
contains supplementary material available at https://​doi.​
org/​10.​1007/​s11104-​023-​06342-7.
Z. Li · S. Wu (*) · Y. Liu · F. You · M. Hall · L. Huang
Centre for Mined Land Rehabilitation, Sustainable
Minerals Institute, The University of Queensland,
Brisbane, QLD 4072, Australia
e-mail: songlin.wu@uq.edu.au
Z. Li
School of Environmental Science and Engineering,
Southern University of Science and Technology,
Shenzhen 518055, China
S. Wu
State Key Laboratory of Urban and Regional Ecology,
Research Center for Eco‑Environmental Sciences, Chinese
Academy of Sciences, Beijing 100085, China
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tolerant native legume A. auriculiformis could form
functional nodules to fix ­N2 when grown in early eco-
engineered technosols.
Methods A. auriculiformis growth and root nodula-
tion in the early tailing technosols were investigated
using a glasshouse experiment, and plant ­N2 fixation
was evaluated using the 15 N natural abundance iso-
tope method. Key factors influencing root nodulation
and ­N2 fixation have also been evaluated, including
water supply and phosphorous nutrition.
Results The results indicated that A. auriculiformis
grew well in the tailing technosols and naturally
formed nodules with rhizobia. The nodules were
functional in ­N2 fixation, leading to improved plant
N nutrition. The nodulation and ­ N2 fixation were
severely limited by water deficiency stress. Improved
phosphorous supply favoured nodulation and ­ N2
fixation by A. auriculiformis plants under water defi-
ciency stress.
Conclusions These findings suggested that A.
auriculiformis could grow in early tailings technosols
and fixed N­ 2, and proper water and phosphorous fer-
tilizer management could improve Acacia plant’s per-
formance and N ­ 2 fixation functions. It is possible to
introduce tolerant native legumes such as A. auricu-
liformis to improve N supply in the early technosols.
Keywords Acacia auriculiformis · Alkaline Fe ore
tailings · Nodulation · N2 fixation · Water deficiency ·
Phosphorous nutrition
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Introduction
The extraction and processing of Fe ores have been
generating large volumes of Fe ore tailings, which
have been stored in many purposely engineered tail-
ings storage facilities worldwide, destroying large
areas of landscapes and posing environmental health
risks (Franks et al. 2020). Eco-engineering Fe ore
tailings into soil-like technoosls through harnessing
biological driving forces in pedogenesis, such as pio-
neer plants and microbes, has been demonstrated to
be a promising and sustainable way for in-situ mine
tailings land rehabilitation (Huang et al. 2012; Wu
et al. 2019, 2021a). Specific tolerant microbial com-
munity and pioneer plant colonization are key steps
for early eco-engineered tailing technosols forma-
tion (Wu et al. 2023). However, the productivity of
soil microbes and pioneer plants requires sustainable
N supply, which is deficient in tailings and resultant
technosols. Mineral N fertiliser strategy is not feasi-
ble and cost-effective due to the high loss of N in the
forms of ­NH3, NO, N ­ 2O and/or N
­ 2 under moderately
alkaline pH conditions (Bai et al. 2014; Huang et al.
2002; Su et al. 2021). Besides, irrigation in highly
compacted tailings during eco-engineered pedo-
genesis could also cause N loss through enhancing
denitrification (Bai et al. 2014). As a result, tolerant
symbiotic biological ­N2 fixation in native leguminous
species (e.g., Acacia spp.) would be a feasible option
to drive soil-like technosols formation from tailings
and the development of a primary succession of soil-
plant systems (Jach et al. 2022; Yu et al. 2021). How-
ever, it is still unknown whether native Acacia plants
could survive in Fe ore tailings technosols and carry
out ­N2 fixation by forming functional nodules with
indigenous ­N2 fixing bacteria.
Our previous study showed that the original phys-
icochemical conditions in the tailings and inade-
quately improved conditions in early technosols were
unfavourable for forming functional nodules to fix N ­ 2
in roots of Acacia holosericea, that is a tolerant native
Acacia species (Yu et al. 2022). It is recognised that
root affinity towards rhizobia and symbiosis forma-
tion with specific rhizobia strains may vary among
Acacia spp., depending on rhizobia and root interac-
tions (Assefa and Kleiner 1998; Craig et al. 1991).
Compared to A. holosericea, A. auriculiformis, a
commonly used rehabilitation species, may have
stronger tolerance to the physicochemical conditions
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Plant Soil
in the early technosols and then form well symbiosis
with rhizobia strains in the technosol, as it is adap-
tive to various adverse soil conditions (from acidic to
alkaline) and capable of fast-growing and fixing ­N2
(Boland et al. 1990; Reza et al. 2019).
Moreover, we found that field-sampled Fe ore tail-
ings contained tolerant ­ N2 fixing bacteria such as
Frankia sp., Rhizobium sp., and Aureimonas altami-
rensis (belongs to the order Rhizobiales) (Wu et al.
2021c). After early eco-engineered pedogenesis with
exogenous organic matter (OM) amendment and pio-
neer plant colonization, the microbial community was
boosted with the improved physical structure, neu-
tralized pH conditions, elevated nutrient level, and
active plant root activities (Wu et al. 2019, 2021b,
2023). These diverse microbial communities, espe-
cially ­N2-fixing bacteria, could form a symbiosis with
A. auriculiformis to form nodules and possess ­ N2
fixation capacity. However, it is unclear if A. auriculi-
formis could form nodules naturally through associa-
tion with N ­ 2-fixing bacteria in eco-engineered tailings
technosols, and whether the symbiosis could fix N ­2
from the atmosphere to avoid exogenous N fertilizer
application for sustainable ecosystem build-up in Fe
ore tailings.
Water deficiency is generally regarded as the main
restriction for successful revegetation, as many mine
sites are located in arid or semi-arid areas. Previous
studies showed that Acacia spp. are drought-tolerant
and nitrogen-fixing trees, which are widely distrib-
uted in arid or semi-arid tropics (Sheokand et al.
2012). The strong root penetration and supplied N
nutrition from the atmosphere facilitate their accli-
mation or avoidance of drought stress (Minucci et al.
2017). Whereas a large number of studies reported
that drought stress suppressed nodulation and further
­N2 fixation (Serraj et al. 1999). In addition, the eco-
engineered tailings and native soils around the remote
mine sites are infertile, especially lacking available
phosphorous (P) supply (Li et al. 2022a, b). The posi-
tive influence of improved P supply on plant growth
and Acacia spp. ­ N2 fixing has been widely docu-
mented even under drought conditions (Crews 1993;
Zheng et al. 2016). Moreover, phosphorous is essen-
tial for plant metabolism, rhizobia bacteria growth,
and nitrogenase activity (Alberty 2005). As a result,
this study aims to investigate whether water defi-
ciency affects Acacia spp. growth and its N ­ 2 fixation
in eco-engineered tailings technosols, and increasing
Plant Soil

P addition improves their growth and N ­ 2 fixation
function under water deficit conditions.
The present study aimed to investigate whether A.
auriculiformis could grow and form functional nod-
ules to fix N
­ 2 when growing in eco-engineered Fe ore
tailings technosols in a glasshouse experiment. The
15
N natural abundance isotope method was employed
to estimate N ­ 2 fixed in plant biomass. The responses
of plant growth and N ­ 2 fixation were investigated in
plants subject to water deficit and P-deficiency treat-
ments. We hypothesized that (1) A. auriculiformis
would be tolerant of the physicochemical conditions
in the early technosols and form functional nodules
on roots; (2) water deficiency adversely affects plant
growth and N ­ 2 fixation, but which could be amelio-
rated by increasing P addition. The expected findings
would provide an important methodology to improve
N supply in the infertile technosols for sustainable
growth of colonising plant communities, without
resorting to mineral N fertilisers.
Materials and methods
Materials and experimental design
­MnSO4, 1 µM ­ZnSO4, 0.05 µM ­Na2MoO4,100 µM
The eco-engineered Fe ore tailings technosol (‘ET’)
was used in the present study as the original Fe ore
tailings are difficult to support phytostabilization
based on our preliminary test. Briefly, the origi-
nal tailings were firstly amended through incubat-
ing with Lucerne hay (2%, w/w) and native top-
soil (10%, w/w) under field water capacity for one
month, and followed by 3.5 months pioneer plant
colonization (Wu et al. 2019). After that, the eco-
engineered tailing technosol, namely ‘ET’, was
employed for the current experiment. The basic
physiochemical properties of ‘ET’ were: pH of
8.15, electronic conductivity of 0.35 mS c­ m−1, total
organic carbon (OC) of 2.12 g ­kg−1, total nitrogen
of 0.38 g ­kg−1, ­NO3− of 48.8 mg ­kg−1, ­NH4+ of
42.9 mg ­kg−1, and Olsen-P of 5.02 mg ­kg−1. Also,
the ‘ET’ had a better physical structure owing to
the water-stable aggregate formation during amend-
ment compared with the original tailings (Wu et al.
2023).
The A. auriculiformis was selected as the test
plant as it easily forms a symbiosis with ­N2-fixing
bacteria. The seeds were purchased from the native
plant seeds supplier Austrahorte (https://​www.​austr​
ahort.​com.​au). Firstly, the seeds were soaked with
hot water (80 oC) for half an hour, and then germi-
nated in a moist filter paper in a growth chamber
with 16/8 of day/night and at 28 oC. After germi-
nation, the seedings were transferred into clean
and sterilized river sand, and cultured in a glass-
house with 14/10 h (day/night) and 28/21℃ (day/
night). The Hoagland nutrition was applied once
a week, and then, the A. auriculiformis seedlings
were transplanted into the ‘ET’. During cultivation
in ‘ET’, no exogeneous nutrition was applied. There
were nearly no nodules identified upon transplanta-
tion. The composition of Hoagland nutrition was: 2
mM ­KNO3, 2 mM Ca(NO3)2, 0.5 mM ­KH2PO4, 1
mM ­MgSO4, 0.2 µM C ­ uSO4, 10 µM H­ 3BO4, 2 µM
NaFe(III)EDTA, and the pH was around 6.5.
Two experimental factors, including phospho-
rus (P) level and water condition (D), were consid-
ered in the current experimental design. Two levels
of P addition (0 ppm and 40 ppm, in the form of
­NaH2PO4), and two levels of water content (16%
and 8%, w/w, indicating well-watered and water-
deficit conditions, respectively) resulted in four
treatments (Table 1), and each treatment had four
replicates, resulting in 16 pots in total. Three A.
auriculiformis in river sand with similar plant bio-
mass were transplanted into a pot with 500 g ‘ET’
inside. After transplantation, all treatments were
maintained under well-watered conditions for two

Table 1  The abbreviation of different treatments and corresponding representation

Abbreviation Representation

A + P-D The A. auriculiformis grown in tailings with P addition under well-watered conditions
A-P-D The A. auriculiformis grown in tailings without P addition under well-watered conditions
A+P+D The A. auriculiformis grown in tailings with P addition under water deficiency conditions
A-P + D The A. auriculiformis grown in tailings without P addition under water deficiency conditions

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weeks to support plant adaption, after which half
pots received water deficiency treatment with 8%
watering every day through weighting.
Plant harvest
After 14 weeks of cultivation, the number of leaves
increased from 5 to 6 at transplantation to 12–14 in
the ‘A-P + D’ treatment, and in the ‘A + P-D’ treat-
ment, the number of leaves reached above 30.
According to our previous experience, there may be
some plant growth constraints caused by the volume
of the pots, which should be considered. Besides, in
this study, we mainly focused on the plant growth
and ­N2 fixation in growth period rather than propa-
gation stage. Therefore, we harvested the plants after
14 weeks. The shoots and roots were separately har-
vested after washing them with DI water. The nod-
ules on the root were removed using a tweezer, and
the number of nodules was counted. In addition, the
activity of nodules was checked by colouration with
pink indicating active nodule. Then, about 5 g fresh
shoots were stored at -80℃ freezer for chlorophyll
analysis, and the remaining shoot and root tissues
were oven-dried at 70℃ for 48 h. Then the dry shoot
and root tissues were ground finely for TOC and TN
analysis using an automatic C/N analyser (Elementar
Vario MAX, Germany). The N content of the plant
shoot and root was calculated by multiplying the TOC
or TN concentration and the corresponding plant tis-
sue weight. At last, around 20 g of fresh tailings tech-
nosol samples were stored in a cold room (4℃) for
­NO3− and N ­ H4+ analysis. In addition, a little number
of nodules were left in the tailings during harvest,
thus, around 20 g of tailings technosol was selected
randomly after shaking thoroughly to infer the num-
ber of nodules in the tailings. The total number of
nodules in each pot was calculated by summing the
nodules of the root and tailings. The remaining tail-
ings after treatments were collected and air-dried for
basic physicochemical characterization.
Biological ­N2 fixation
Subsamples of homogenized dry plant shoot tis-
sues were submitted for δ15N composition analysis
using an Elemental PrecisION Isotope ratio mass
spectrometry (IRMS) (Elementar, German) at the
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Plant Soil
Stable Isotope Geochemistry Laboratory, the Uni-
versity of Queensland. The analysis precision was
0.2 per mil at one sigma for an internal standard
(USGS40, L-glutamic acid) at δ15N of -4.52‰.
The δ15N, i.e., 15 N abundance (the per mil 15 N
excess over atmospheric ­N2) was calculated using
the standard ­N2 gas and following Eq. (1):
Rsample
[ ]
15
𝛿 N= − 1 × 1000‰ (1)
Rstandard
Where, Rsample and Rstandard mean the 15 N/14 N
ratio of samples and standard ­N2 gas, respectively.
The proportion of nitrogen in plant shoot or root
derived from the atmosphere (Ndfa%) was calcu-
lated based on the Eq. (2):
[ ]
𝛿 15 Nref − 𝛿 15 Nsample
Ndfa% = × 100% (2)
𝛿 15 Nref − B
Where, δ15Nref and δ15Nsample indicate the 15 N
abundance (δ15N) in the shoot (or root) of the ref-
erence plant and samples being measured, respec-
tively. The B value represents the 15 N abundance of
Acacia spp. growing in an N-free nutrient condition.
The reference plant is important for Ndfa% cal-
culation and normally required to reflect the 15 N
abundance of the plant growth matrix. In the pre-
sent study, we selected one Acacia plant grown in
tailings without any nodulation from the ‘A-P + D’
treatment as a reference plant, which probably could
reflect the 15 N abundance of ‘ET’. As for the B
value, on one hand, the B value should be less than
the δ15N of plant tissues with the highest nodula-
tion and ­N2 fixation (Tye and Drake 2011); on the
other hand, it is reasonable that the Ndfa% should
be less than 90% based on the B value (Nebiyu et al.
2013). Thus, in this study, the B value (-2.9) of Aca-
cia spp. from previous studies (Boddey et al. 2000;
Raddad et al. 2005; Tye and Drake 2011; Vincent
et al. 2018), were used to reflect the 15 N abundance
of Acacia spp. shoot growing in an N-free nutrient
condition. In addition, the 15 N abundance of Acacia
spp. roots from ‘A + P-D’ treatment with abundant
nodules was selected as the B value to determine
the ­N2 fixation by roots from the atmosphere since
few studies have determined the %Ndfa of roots
(Mercado et al. 2010; Unkovich et al. 2000).
Plant Soil
Plant mineral nutrition
The dry plant shoots and roots were first cut into short
segments and then finely ground using a ball mill.
Aliquots of homogenized ground tissues were firstly
soaked into concentrated ­HNO3 for 48 h at room tem-
perature and then digested at 70 ℃ for 1 h and 125
℃ for another 1 h using a hot block (Thermo Scienti-
ficTM 88,870,004; USA). Then the concentration of
the element, including Fe, K, Ca, Mg, and P, in the
digested solution was detected by inductively coupled
plasma optical emission spectrometry (ICP-OES,
Varian Vista Pro II). The Apple Leaves (NIST-SRM
1515) were used as the standard reference material to
control the digestion quality.
Available N in tailings
The mineral nitrogen, including N ­ O3− and N
­ H4+, of
fresh tailings were firstly extracted by 2 M KCl solu-
tion and then determined colourimetrically using a
segmented flow analyser (AQ2 + discrete analyser,
SEAL Analytical Company, Germany). In brief, after
thoroughly mixing the tailings in each pot, 1 g tail-
ings sample was collected and mixed with 10 ml 2 M
KCl solution and shaken for one hour using an end-
over-end shaker at room temperature. Then, the mix-
ture was centrifuged for 4 min (4000 g) and filtered
through the Whatman #40 filter, and the supernatant
was used for N ­ O3− and ­NH4+ concentration analysis.
−
The ­NO3 and ­ NH4+ concentration was expressed
based on the dry weight of tailings by calculating
the water content of fresh tailings. The Olsen-P con-
centration of air-dried tailings was determined col-
ourimetrically based on the molybdate blue method
using a spectrophotometer (Spectramax 250, USA)
after being extracted by 0.5 M ­NaHCO3 solution (pH
of 8.5). In addition, the pH and EC of tailings were
measured by using a pH meter (TPS 2100) and an EC
(TPS 900-P) meter at a solution with a ratio of tailing
to deionized water of 1:5, respectively.
Statistical analysis
The Two-way ANOVA was employed to assess the
interactive effects of P addition and water condition
on plant growth, biological ­ N2 fixation and other
physiochemical properties of tailings. Tukey’s multi-
ple range test with the assumption of a P value less
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than 0.05 was used to compare the mean difference
of indices among treatments. The Pearson correlation
analysis with a two-tailed significance test (P < 0.05)
was applied to detect the correlation of plant growth,
such as the plant N uptake and biological N ­ 2 fixation,
with plant nodules. The normal distribution of data
was tested by Shapiro-Wilk analysis, and Levene’s
test was used to assess the equality of variances anal-
ysis. For the data that cannot meet the requirement
of normal distribution and variance equality, log-
transformed data were used for analysis but shown as
the original data. All statistical tests were conducted
using SPSS software (Ver 20, IBM, Armonk, NY,
USA).
Results
Plant growth, physiology and mineral nutrition
The statistical analysis showed that there was no
interaction between P addition and water condition
on the plant’s dry weight and chlorophyll concentra-
tion (Table S1). Water deficiency decreased the plant
shoot dry weight under P addition (P < 0.05, Fig. 1a).
It also reduced the biomass of plant roots regardless
of P addition (P < 0.05, Fig. 1b). In addition, drought
stress under water-deficit conditions reduced the chlo-
rophyll concentration of fresh leaves regardless of P
addition (P < 0.05, Fig. 1c). P addition did not signifi-
cantly alter the plant biomass and chlorophyll concen-
tration no matter of tailings’ water condition.
Water deficiency increased the Fe concentration
but decreased the K concentration in plant shoots
under no P addition conditions (P < 0.05, Table 2).
Moreover, it also decreased the Fe, K, Mg, and P
concentration in plant roots no matter of P addition
(P < 0.05, Table 2). P addition had no effects on the
shoot P concentration but increased the root P con-
centration regardless of tailings’ water conditions
(P < 0.05, Table 2).
Plant root nodulation and total N/C content
The A. auriculiformis survived in most of the treat-
ments and formed an association with ­N2 fixing bac-
teria as revealed by the plentiful and pink nodules
on the root tissues (Fig. 2). However, there are few
black nodules on the root in ‘A-P + D’ treatments,
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 Plant Soil

Fig. 1  The plant shoot dry weight (a), root dry weight (b), and treatments explained in Table 1. The different letters above the
chlorophyll concentration in plant leaves (c) among treatments. columns represent significant differences among treatments by
Note, the ‘A + P-D’, ‘A-P-D’, ‘A + P + D’, and ‘A-P + D’ are Tukey’s test (N = 4, P < 0.05)

Table 2  Mineral
elemental concentration Treatment Nutritional elements in plant shoot (mg ­g−1)
in plant shoots and roots, Fe K Ca Mg P
as influenced by water A + P-D 0.13(0.01)b 12.67(0.52)b 11.6(0.65)a 1.69(0.05)a 0.72(0.08)a
conditions and phosphorous A-P-D 0.12(0.01) b
15.66(0.91) a
11.2(1.42)a 1.69(0.19)a 0.67(0.10)a
level ab b
A+P+D 0.12(0.01) 11.83(0.71) 12.1(1.65)a 1.43(0.33)a 0.51(0.15)a
a b
A-P + D 0.18(0.01) 13.51(0.59) 12.5(0.35)a 1.79(0.05)a 0.47(0.07)a
significance
D ** *** ns ns ns
P ns *** ns ns ns
Note, the ‘A + P-D’, D×P ns ** ns ns ns
‘A-P-D’, ‘A + P + D’, and Treatment Nutritional elements in plant root (mg g­ −1)
‘A-P + D’ are treatments Fe K Ca Mg P
explained in Table 1. D:
experimental factor of A + P-D 21.12(1.82)a 23.42(0.80)a 9.18(0.46)a 3.35(0.15)a 1.18(0.03)a
b a
tailings’ water condition; A-P-D 15.69(2.41) 27.13(1.53) 10.2(0.31)a 3.39(0.18)a 0.91(0.03)b
P: experimental factor of A+P+D 10.99(1.11) bc
17.34(2.48) b
8.86(0.59)a 2.53(0.14)b 0.92(0.06)b
Phosphorous addition. A-P + D 8.46(1.18) c
9.39(1.30) c
7.79(1.47)a 1.91(0.32)b 0.53(0.06)c
Different letters show
significant differences significance
among treatments by D *** *** ns *** ***
Tukey’s test (N = 4, P * ns ns ns ***
P < 0.05). * P < 0.05; ** D×P ns ** ns ns ns
P < 0.01; *** P < 0.001

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Plant Soil
indicating that A. auriculiformis − rhizobia symbiosis
cannot develop under water and P deficit conditions.
It is essential to point out that the new and fresh nod-
ules should be white and/or pink (based on our pre-
experiment), and the black nodules collected from
roots upon harvest may be those senescent or dead,
or probably covered by clay-sized minerals from
tailings. Moreover, it is evident that the number of
nodules declined in response to water-deficit treat-
ment (Fig. 3a). No interaction between P addition
and water condition on the nodules’ conditions was
revealed by the two-way ANOVA analysis (Table S1).
Water deficiency reduced the biomass of nodules
regardless of P addition (P < 0.05, Fig. 3b, c). It also
decreased the number of nodules under no P-addition
conditions (P < 0.05, Fig. 3a). P addition increased
the fresh weight of nodules regardless of the water
conditions of ‘ET’, and the number of nodules under
water-deficit conditions (P < 0.05, Fig. 3a, b).
Water condition and P addition did not affect the C
and N concentration in plant shoot or root but showed
interaction on the C: N ratio of plant root (P < 0.05,
Table S2). Water deficiency reduced the N concen-
tration of plant shoot and enhanced the C: N ratio
of plant shoot no matter of P additions (P < 0.05,
Fig. 4a, c). Whereas it increased the plant root N con-
centration and decreased its C: N ratio under P addi-
tions (P < 0.05, Fig. 4d, f). P addition had no effects
on the C and N concentration and C: N ratio of plant
shoot but increased the C concentration in plant roots
under well-watered conditions and N concentration in
plant roots under water-deficit conditions (P < 0.05,
Fig. 4d, e).
Overall, the ‘A + P-D’ treatment produced the
highest shoot N content of 67.8 (6.63) mg p­ ot−1, fol-
lowed by the ‘A-P-D’ treatment. The ‘A + P + D’ and
‘A-P + D’ treatments produced lower shoot N con-
tent (Fig. S1). Similarly, the ‘A + P-D’ treatment also
yielded the highest root N content of 74.90 (17.8) mg
­pot−1 among all treatments (Fig. S1).
Biological ­N2 fixation
The 15 N abundance in plant shoots or roots was
much lower in treatments with well-watered condi-
tions than in treatments with water-deficit conditions
regardless of P addition (P < 0.05, Fig. 5a, b). The N
content in plant shoots or roots derived from biologi-
cal ­N2 fixation (Ndfa%) ranged from 18 to 54% and
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23–91%, respectively (Fig. 5c, d). Water deficiency
reduced the N in shoots or roots derived from bio-
logical ­N2 fixation regardless of P addition (P < 0.05,
Fig. 5c, d). P addition increased the Ndfa% in roots
but did not influence the Ndfa% in shoots under both
well-watered and water-deficit conditions (P < 0.05,
Fig. 5c, d). Overall, the A. auriculiformis − rhizobia
symbiosis exerted N ­ 2 fixation functions in the ‘ET’.
However, the Ndfa% by A. auriculiformis − rhizo-
bia symbiosis was reduced by water deficiency, and
P addition increased the Ndfa% of roots (P < 0.05,
Fig. 5d).
Physicochemical properties of tailings technosol
The water condition and P addition treatment had
­ O3− and ­NH4+ concentra-
interactive effects on the N
tion rather than pH and EC values, as well as Olsen-
P concentration (P < 0.05, Table 3). Water deficiency
led to lower pH but higher EC values of ‘ET’ com-
pared with well-watered conditions regardless of P
additions. In addition, P addition resulted in lower
pH and higher EC values regardless of water condi-
tions (P < 0.05, Table 3). The P addition and water
deficiency (‘A + P + D’ treatment) resulted in the
lowest pH value and highest EC. Tailings had higher
­NO3− concentration under water-deficit conditions
than that under well-watered conditions. P addition
caused a lower ­NO3− concentration under water-def-
icit conditions (P < 0.05, Table 3). Water deficiency
decreased the ­NH4+ concentration, particularly under
P addition conditions (P < 0.05, Table 3). P addition
led to a higher Olsen-P concentration which was not
affected by water conditions (P < 0.05, Table 3).
Discussion
The present study has investigated the establishment,
nodulation and biological ­ N2 fixation functions of
Acacia auriculiformis in eco-engineered Fe ore tailings
technosol through the 15 N natural abundance method.
The results indicated that A. auriculiformis could
survive well in eco-engineered tailings technosol and
form nodules with N ­ 2 fixation functions. The study
also indicated that water condition and phosphorous
nutrient level influence plant nodulation and ­ N2
fixation in tailings technosol.
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 Plant Soil
◂Fig. 2  Pictures of plant roots and nodules among treatments.
Note: the ‘A + P-D’, ‘A-P-D’, ‘A + P + D’, and ‘A-P + D’ are
treatments explained in Table 1
A. auriculiformis grew and formed nodules in
eco‑engineered Fe ore tailings technosol
It is revealed in the present study that A. auricu-
liformis survived well in the tailings technosol. The
abundant pink nodules on plant roots demonstrated
that A. auriculiformis – rhizobia symbiosis success-
fully developed in the eco-engineered Fe ore tailings
(Fig. 2). Moreover, more than 18% N in plant shoots
derived from biological N ­ 2 fixation (Fig. 5) indicat-
ing that the rhizobia exerted an N ­ 2 fixation function
in eco-engineered tailings technosol. The neutralized
pH, improved organic matter, nutrition, and physical
structure during eco-engineering processes (Rob-
ertson et al. 2020; Wu et al. 2019) probably collec-
tively contributed to the survival of A. auriculiformis,
since our preliminary test showed that A. auriculi-
formis seedings could not survive in Fe ore tailings if
without eco-engineering processes. High alkalinity
in tailings usually hinders plant nutrient uptake, ion
balance, and further plant root physiological func-
tions (Javid et al. 2012), which may inhibit nodula-
tion (Zahran 1999). Yu et al. (2022) also showed that
soil alkalinity resulting from Fe ore tailings amend-
ment reduced the nodule dry weight from 0.8 g ­pot−1
to 0.02 g p­ ot−1 in legume plant Acacia holosericea. In
addition, the eco-engineered tailings had high micro-
bial abundance compared with the original tailings.
For example, after OM amendment and pioneer plant
colonization, the sequence depth for both bacteria and
fungi increased from less than 100 in original tailings
to more than 40,000 in eco-engineered tailings, and
Proteobacteria (38–45%), Planctomycetes (7–10%),
Chloroflexi (5–9%), Bacteroidetes (6–10%), Actino-
bacteria (7–10%), and Acidobacteria (2–6%) were
found to constitute the microbial community in ‘ET’
(Wu et al. 2021b). This facilitates plant root infec-
tion by rhizobia and nodulation. Moreover, ­N2 fixa-
tion in nodules is a high energy-demanding process,
which requires O ­ 2 supply to ensure a high respira-
tion rate and then provide sufficient ATP for rhizobia
(Minchin 1997). The eco-engineered tailings tech-
nosol with water-stable aggregates formed possibly
provides porosity space and ­O2 for root and microbi-
ome respiration (Li et al. 2023), favouring nodulation
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
processes. Moreover, the study found that A. auricu-
liformis could survive and fix ­N2 in eco-engineered
tailings technosol, which was not the case for Acacia
holosericea as shown in our previous study (Yu et al.
2022). This may be due to the stronger adaptability of
A. auriculiformis compared with A. holosericea. For
instance, Immanuel and Ganapathy (2007) reported
that A. auriculiformis is more tolerant to high salinity
compared with A. holosericea. Moreover, A. auriculi-
formis, rather than A. holosericea was recommended
to be one of the five priority species for intensive
development in the humid tropical lowlands by the
International Union of Forest Research Organiza-
tions (Boland et al. 1990). In addition, although both
of them were widely introduced to initiate new eco-
systems in virgin land, A. auriculiformi, had a higher
risk score than A. holosericea as invasive species in
the Pacific Island Ecosystems at Risk, due to its wide
adaptability (Reza et al. 2019).
Water deficiency inhibits plant growth and biological
­N2 fixation
Water and mineral nutrients are necessary resources
for plant growth. In the current study, either plant
shoot dry weight or root dry weight declined more
than 44% by water shortage stress (Fig. 1). These
findings agree with a recent investigation, which
found that the leaf dry weights of mungbean (Vigna
radiata (L.) R. Wilczek) declined 43% under drought
stress (Meena et al. 2021). Similar results that
drought suppressed plant growth have been widely
reported (Mahieu et al. 2009; Ohashi et al. 1999;
Qiao et al. 2022). The low mobility of mineral nutri-
ents under water-deficit conditions, such as N and P,
would restrict photosynthesis and reduce cell division
(Farooq et al. 2009; Goh and Bruce 2005), restricting
plant production. The lowered foliar chlorophyll con-
centration in treatment under water-deficit conditions
compared to treatments with well-watered conditions
confirmed this assumption (Fig. 1). The suppressed
plant growth under water deficiency further impacted
nodulation and corresponding ­N2 fixation.
The restrained nodulation of plants under water-
deficit conditions was confirmed by the significantly
declined fresh weight of nodules (Fig. 3), as well as
the black nodules (unhealthy nodules) present in plant
roots from ‘A-P + D’ treatment (Fig. 3). The negative
effects of water-deficient stress on nodulation have
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Fig. 3  The number (a), fresh weight (b), and dry weight (c)
of nodules among treatments. Note: the ‘A + P-D’, ‘A-P-D’,
‘A + P + D’, and ‘A-P + D’ are treatments explained in Table 1.
long been reported for all other legume plants (Hun-
gria and Vargas 2000; Mahieu et al. 2009; Sheokand
et al. 2012). The direct mechanism may lie in that
the lower moisture under drought could restrict the
mobility of bacteria and the infection process of root
hair (Graham 1992; Zahran et al. 1994). In addition,
the indirect mechanisms may be that the reduced pho-
tosynthesis rate and plant N demand under water-def-
icit conditions could reduce investment in biological
­N2 fixation, which normally costs more energy than
soil N uptake (Vitousek and Field 1999). The higher
­NO3− concentration of the growth matrix under water
deficiency conditions (Table 3) could also restrain
nodulation and ­N2 fixation due to that plant relies
more on ­NO3− rather than biological N­ 2 fixation (But-
tery and Dirks 1987).
The construction of nodules underpins biological
­N2 fixation (Gutschick 1981), which was confirmed
by the positive correlation between %Ndfa and nod-
ule number (or fresh weight of nodules) (P < 0.05,
Table S3). In the present study, the declined nodule
number or fresh weight probably decelerated the N ­2
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Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Plant Soil
The different letters above the columns present significant dif-
ferences among treatments by Tukey’s test (N = 4, P < 0.05)
fixation from the atmosphere and the N accumulation
in plant tissues, as revealed by the declined %Ndfa
in shoots or roots (Fig. 5). Various field surveys and
glasshouse experiments demonstrated the high sen-
sitivity of biological ­N2 fixation to drought stress
(Minucci et al. 2019; Serraj et al. 1999). For one
thing, water shortage influenced plant photosynthe-
sis, which further decreased C allocation to root and
nitrogenase activity (Serraj et al. 1999). For another
thing, water deficiency may alter the nodule O ­ 2 per-
meability in the nodule (Minchin 1997) and restrict
the export of products of ­N2 fixation from nodules,
which could also inhibit the nitrogenase activities
(Serraj et al. 1999). Moreover, some investigations
showed that lowered leaf water potential under water-
deficit conditions might suppress nodule physiology
as the transport of water and compounds to the nod-
ules is mostly through the phloem (Walsh 1995). In
the current study, the negative or close to zero δ15N
values in shoots (Fig. 5a) are consistent with previ-
ous studies reporting that legume tree had negative
foliar δ15N value in tropic or subtropic climate zone
Plant Soil
Fig. 4  The concentration of C, N, and C: N ratio in plant
shoots and roots. Note: the ‘A + P-D’, ‘A-P-D’, ‘A + P + D’, and
‘A-P + D’ are treatments explained in Table 1. The different let-
(Fang et al. 2012; Mordelet et al. 1996). The lower
δ15N values in shoots should result from the isotopic
fractionation during N transport from roots to shoots.
Generally, the lower δ15N values indicate that more
N is obtained from atmosphere ­ N2 via biological
­N2 fixation (Fang et al. 2011; Wang et al. 2014). It
was noted that the δ15N value of the shoots or roots
was higher under water-deficit conditions than under
well-watered conditions. This may result from the
restricted ­N2 fixation in plant roots grown in water
deficit eco-engineered tailings technosols. Under
water-deficit conditions, the declined nodules, nitro-
genase and microbial activities possibly reduced
biological ­N2 fixation from the atmosphere and/or
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
ters above the columns represent significant differences among
treatments by Tukey’s test (N = 4, P < 0.05)
isotope fractionation by nitrifier and nitrogen-fixing
bacteria, leading to the elevated δ15N value. Conse-
quently, the proportion of N derived from ­N2 fixation
(%Ndfa) in shoots and roots grown in tailings tech-
nosol under water-deficit conditions was much lower
than that under well-watered conditions.
Phosphorus addition enhanced plant drought
tolerance but did not influence ­N2 fixation
Phosphorous is required for ATP formation, nitroge-
nase synthesis, and cell growth of N-fixer (Alberty
2005), and thus, it is expected to favour the nodula-
tion process. In the present study, P addition did
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 Plant Soil

Fig. 5  The δ15N values of plant shoots (a) and roots (b) ments explained in Table 1. The different letters above the
among treatments (a), and the proportion of N in plant shoot columns represent significant differences among treatments by
(c) and root (d) tissues derived from ­ N2 fixation (%Ndfa). Tukey’s test (N = 4, P < 0.05)
Note: ‘A + P-D’, ‘A-P-D’, ‘A + P + D’, and ‘A-P + D’ are treat-

Table 3  The pH, EC, mineral nitrogen, and Olsen-P concentration in tailings technosol with various treatments
Treatment pH EC NO3− NH4+ Olsen-P
(mS ­cm−1) (mg ­kg−1) (mg ­kg−1) (mg ­kg−1)

A + P-D 8.74(0.03)b 0.23(0.01)b 1.23(0.16)c 41.1(0.56)a 7.84(0.96)a

A-P-D 8.86(0.04)a 0.17(0.00)c 1.44(0.12)c 41.7(0.83)a 2.67(0.07)b
A+P+D 8.50(0.04)c 0.28(0.01)a 3.59(0.25)b 39.1(0.46)b 9.19(0.37)a
A-P + D 8.73(0.02)b 0.25(0.01)b 5.62(0.64)a 40.2(0.30)ab 2.52(0.10)b
Significance
P *** *** ** ns ns
D *** *** *** ns ns
PхD ns ns * * ns
‘A + P-D’, ‘A-P-D’, ‘A + P + D’, and ‘A-P + D’ are treatments explained in Table 1; the different letters indicate significant differences
among treatments by Tukey’s test (N = 4, P < 0.05); P represents phosphorus addition; D represents tailings’ water condition factor; *
P < 0.05; ** P < 0.01; *** P < 0.001

elevate the nodules’ number or weight even under
water-deficit conditions (Fig. 3). This was consistent
with previous research that nitrogen-fixing bacteria
biomass was controlled by soil P conditions (Binkley
et al. 2003; Crews 1993), and P addition generally ele-
vated the nodules number or biomass, and N ­ 2 fixation
(Míguez-Montero et al. 2019). Generally, P addition
could increase the diversity and abundance of rhizo-
bia strains (Galiana et al. 1990; Reed et al. 2010).
Compared with plants, the rhizobia bacteria are more
resistant to soil desiccation and can survive in water
films around soil particles (Williams and Sicardi de
Mallorca 1984), favouring root infection and nodu-
lation. Moreover, P addition may increase plant

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Plant Soil
photosynthesis rate and allocate more C to plant roots
(Crews 1993), which facilitates N mineral nutrients
acquisition from tailings to maintain C: N stoichi-
ometry (Elser et al. 2010). Then, the decreased avail-
able N to plants can trigger an increased investment
in nodulation (Wurzburger and Miniat 2014). Fur-
ther, the improved P nutrition status in legume plants
could facilitate plants to generate more energy for N
fixation, therefore favouring nodule formation. This is
consistent with our findings that there was a higher
root N concentration in the ‘A + P + D’ treatment
than that in the ‘A-P + D’ treatment (Fig. 4; Table 3).
The elevated root C concentration and decreased C:
N ratio by P addition under well-watered conditions
also supported the more C allocation into plant roots
(Fig. 4). In addition, some experiments demonstrated
that phosphorous fertilizer application could improve
plant water use efficiency, which favours to plant
growth and further nodules development and N ­ 2 fixa-
tion (Alkhader and Rayyan 2013; Olatunji et al. 2018;
Tariq et al. 2017). Overall, coinciding with previous
conclusions, P addition stimulated nodulation possi-
bly through improving host plant growth and N nutri-
tion demand.
However, the increased nodule number or weight
by P addition did not elevate the Ndfa% in plant
shoots (Fig. 5), which did not corroborate previous
findings of a significant rise in Ndfa% with improved
soil P availability (Isaac et al. 2011a; Zheng et al.
2016). Probably, the A. auriculiformis − rhizobia
symbiosis adopted a facultative fixation strategy, that
the ­N2 fixation was controlled by a balance between
soil N supply and plant N demand (Batterman et al.
2013; Minucci et al. 2017). When soil nitrogen is
insufficient, the symbiosis activates ­ N2 fixation to
meet the N demands of biomass growth, but excising
fixation once the nitrogen supply is sufficient (Bar-
ron et al. 2011). Under well-watered conditions, the
soil available N is too low to meet plant N demand
no matter of P addition, as revealed by the approxi-
mate ­NO3− concentration of tailings from ‘A + P-D’
and ‘A-P-D’ treatments (Table 3). Therefore, no mat-
ter of P addition, the N deficiency in tailings could
trigger biological ­N2 fixation to meet plant N demand.
Additionally, P addition did not induce significant
shoot biomass and shoot N concentration increment
in the current study (Figs. 1 and 4). As a result, the N
in plant shoots derived from the atmosphere was not
impacted by exogenous P addition. In contrast, water
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
deficiency may reduce plant N demand (Minucci
et al. 2017), resulting in the N supply being rela-
tively unlimited. This can be reflected by the higher
­NO3− concentration compared with well-watered
conditions (Table 3). Likewise, the approximate
plant shoot and root biomass, regardless of P addition
under water-deficit conditions possibly also account
for the unchanged shoot N derived from the atmos-
phere (Fig. 5c). Isaac et al. (2011b) also reported that
the N acquisition strategies of A. Senegal changed
with P supply and reduced the reliance on N ­ 2-fixation
under non-limiting N conditions.
However, it should be noted that the unchanged
%Ndfa of plant shoots by P addition could not
exclude that plant fixed more ­N2 under elevated P
conditions. This is because that the fixed ­N2 may be
used for various physiological activities other than
storing in leaf tissues. The P addition increased the
nodule number and weight (Fig. 3), probably leading
to more N ­ 2 fixation from the atmosphere. The fixed
­N2 driven by P addition was probably partially stored
in plant roots as the Ndfa% of roots were elevated
by P addition regardless of tailings’ water condi-
tions (Fig. 5d). The fixed ­N2 may firstly be used to
support root development for stronger nodulation
and adaptation to the tailings’ environment. In addi-
tion, in response to water-deficit stress, there may
be an investment of fixed N driven by P addition in
photosynthetic enzymes, which improves carboxyla-
tion rate and water use efficiency (Kitao et al. 2007;
Wright et al. 2001), strengthening plants’ drought tol-
erance (Freitas et al. 2022; Minucci et al. 2017; Sheo-
kand et al. 2012).
Conclusion
The present study found that Acacia auriculiformis
could grow in eco-engineered Fe ore tailings techno-
sol, form nodules, and exhibit biological ­N2-fixation
functions. However, water-deficit stress restrained
nodulation and N ­ 2 fixation. Exogeneous phosphorous
application facilitated the nodulation process, and
increased ­N2 fixation by plant roots but did not affect
­N2 fixation by plant shoots. This study gives impor-
tant implications for using Acacia spp. in ecological
restoration of mine tailing sites, especially in arid and
semi-arid areas.
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13

Acknowledgements Kim Baublys, in the stable isotope geo-
chemistry laboratory, earth and environmental science, the
University of Queensland, was acknowledged for her help in
15
N analysis.
Author contributions Zhen Li: Experiment conduction,
conceptualization, methodology, data analysis, writing-original
draft. Songlin Wu: Conceptualization, experimental design,
supervision, data analysis, methodology, writing-review &
editing. Yunjia Liu: Methodology. Fang You: Methodology.
Merinda Hall: Methodology. Longbin Huang: Supervision,
project administration, funding acquisition, writing-review &
editing.
Funding Open Access funding enabled and organized
by CAUL and its Member Institutions This research was
funded by the Australian Research Council Linkage Project
(LP160100598), Zhen Li was funded by the China Scholarship
Council scholarship (File No. 201906350122), and Songlin
Wu is funded by the Hundred Talents Program of the Chinese
Academy of Sciences.
Data Availability Available on request.
Declarations
Competing interests We declare that the authors have no
competing financial interests.
Open Access This article is licensed under a Creative Com-
mons Attribution 4.0 International License, which permits
use, sharing, adaptation, distribution and reproduction in any
medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Crea-
tive Commons licence, and indicate if changes were made. The
images or other third party material in this article are included
in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not
included in the article’s Creative Commons licence and your
intended use is not permitted by statutory regulation or exceeds
the permitted use, you will need to obtain permission directly
from the copyright holder. To view a copy of this licence, visit
http://creativecommons.org/licenses/by/4.0/.
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