ORIGINAL RESEARCH

published: 16 July 2019

doi: 10.3389/fpls.2019.00843

Arbuscular Mycorrhizal Fungi Confer

Salt Tolerance in Giant Reed (Arundo
donax L.) Plants Grown Under Low
Phosphorus by Reducing Leaf Na+
Concentration and Improving
Phosphorus Use Efficiency
Antònia Romero-Munar 1* , Elena Baraza 2 , Javier Gulías 2 and Catalina Cabot 1
1
Research Group on Plant Biology Under Mediterranean Conditions, Department of Biology, Facultat de Ciències, University
of the Balearic Islands, Palma, Spain, 2 Institute of Agro-Environmental and Water Economy Research (INAGEA),
Palma, Spain

Salinization is one of the major causes of agricultural soil degradation worldwide. In arid
and semi-arid regions with calcareous soils, phosphorus (P) deficiency further worsens
the quality of salinized soils. Nonetheless, nutrient poor soils could be suitable of
producing second-generation energy crops. Due to its high biomass production, Arundo
Edited by:
donax L. (giant reed) is one of the most promising species for energy and second-
Pablo Cornejo, generation biofuel production. A. donax can be propagated by micropropagation, an
Universidad de La Frontera, Chile in vitro technique that produces high number of homogeneous plantlets. However, crop
Reviewed by: establishment is often compromised due to poor plantlet acclimatization to the soil
Asunción Morte,
University of Murcia, Spain environment. Arbuscular mycorrhizal fungi (AM) are components of soil-plant systems
Marcelo Pedrosa Gomes, able to increase root phosphorus uptake and to confer the plant an increase tolerance
Federal University of Paraná, Brazil
to salinity with a consequent enhancement effect of plant growth and yield. In the
*Correspondence:
Antònia Romero-Munar
present study, the relative importance of the early symbiosis establishment between AM
a.romeromunar@gmail.com fungi and A. donax micropropagated plantlets in the response to salt stress under low
phosphorus availability was determined. A commercial inoculum which contained two
Specialty section:
This article was submitted to
different AM fungi species: Rhizophagus intraradices and Funneliformis mosseae was
Plant Abiotic Stress, used. AM-symbionts (AM) and non-symbionts plants were grown at two phosphorus
a section of the journal
[2.5 µM (C) and 0.5 mM (P)] and three NaCl (1, 75 and 150 mM) concentrations
Frontiers in Plant Science
in a room chamber under controlled conditions. After 5 weeks, AM root colonization
Received: 03 February 2019
Accepted: 12 June 2019 was 60, 26 and 15% in 1, 75 and 150 mM NaCl-treated plants, respectively. At 1
Published: 16 July 2019 and 75 mM NaCl, AM plants showed increased growth. In all saline treatments, AM
Citation: plants had decreased Na+ uptake, Na+ root-to-shoot translocation, Na+ /K+ ratio and
Romero-Munar A, Baraza E,
Gulías J and Cabot C (2019)
increased P and K use efficiencies with respect to C and P plants. AM improved the
Arbuscular Mycorrhizal Fungi Confer nutritional status of A. donax plants by enhancing nutrient use efficiency rather than
Salt Tolerance in Giant Reed (Arundo
nutrient uptake. Increased phosphorus use efficiency in AM plants could have benefited
donax L.) Plants Grown Under Low
Phosphorus by Reducing Leaf Na+ ion (Na+ and K+ ) uptake and/or allocation and ultimately ameliorate the plant’s response
Concentration and Improving to saline conditions.
Phosphorus Use Efficiency.
Front. Plant Sci. 10:843. Keywords: early stage, arbuscular mycorrhiza, salinity tolerance, phosphorus scarcity, phosphorus use
doi: 10.3389/fpls.2019.00843 effieciency

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Romero-Munar et al. AM, Salinity Tolerance and PUE

INTRODUCTION response in mycorrhized salt-stressed plants was related to an AM

fungi-mediated amelioration of nutrient acquisition, especially
Salinity is one of the most damaging degradation processes phosphorous, when under saline conditions Pi absorption was
affecting soils, especially in arid and semi-arid regions, where greatly decreased (Shokri and Maadi, 2009). AM symbiosis was
salinization is considered a major cause of soil desertification. also reported to reduce Na+ uptake and translocation while
According to Dubois (2011), salinity affects 19.5% of irrigated and favored the uptake of essential cations such as K+ , Ca2 g + and
2.1% of dry agricultural lands worldwide. Mg2+ and increased the K+ /Na+ and Mg2+ /Na+ ratios in shoots
Soil salinization negatively affects plant growth and yield. (Giri et al., 2003; Giri and Mukerji, 2004; Colla et al., 2008).
The high salt concentration in the soil solution decreases the However, large variation in the effectiveness of AM symbiosis to
soil osmotic potential that may result in loss of cell turgor in salinity and phosphorous deficiency depending on plant and AM
species unable to regulate their water potential. Additionally, the fungi genotypes has been reported (Tian et al., 2004; Juniper and
excess of ions, principally Na+ and Cl− , negatively affect plant Abbott, 2006; Zou and Wu, 2011; Zribi et al., 2012).
metabolism by inducing ion toxicity or/and ion imbalance in Second-generation biofuels mainly developed in the second
plant tissues (Marschner, 2011). Nonetheless, plants have evolved half of the 2000s in response to social concerns over
multiple responses to cope with salt stress. Control of water environmental and food security issues raised by the first-
and ion homeostasis, Na+ exclusion from the shoot, Na+ tissue generation biofuels. To ensure a more sustainable used of
tolerance and the scavenging of toxic compounds are among the agricultural soils and to prevent the displacement of food crops,
principal physiological and biochemical mechanisms involved second-generation energy crops can be grown on marginal lands,
(Hasegawa et al., 2000; Munns and Tester, 2008). abandoned or unsuitable for food production (Mohr and Raman,
On the other hand, in the Mediterranean climatic regions, 2013). Nonetheless, marginal lands often compromised the crop
where calcareous and alkaline soils largely prevail (Vance et al., establishment success due to the harsh conditions that plantlets
2003), in addition to salinity, crop production is also threated face in these extreme environments (Pilu et al., 2012).
by P deficiency, especially in low-input agricultural systems. Arundo donax L. is one of the most promising species for
Changes in growth and root structure (Lambers et al., 2006) second-generation biofuel production because its high biomass
and increased synthesis and secretion of phosphatase into the production (Hidalgo and Fernandez, 2001; Shatalov and Pereira,
rhizosphere to improve soil P mobilization (Li et al., 2011) 2002; Lewandowski et al., 2003). Moreover, giant reed has also
are common plant responses to cope with P scarcity. The been reported to be an environmentally sustainable, low-cost,
exudation and increased root levels of strigolactones, compounds low-maintenance crop with very low fertilizer requirements
that trigger mechanisms involved in the plant- Arbuscular (Lewandowski et al., 2003). However, there are some bottlenecks
Mycorrhizal (AM) fungi interaction, are also promoted by P regarding giant reed physiology and cultivation. In nature, due
deficiency (Akiyama et al., 2005; Yoneyama et al., 2007). to the lack of viable seeds, giant reed principally propagates
In natural habitats, plants often simultaneously face multiple through rhizomes, while plants obtained through micro-
stresses, and it is well known, that plant responses to combined propagation of embryogenic callus are nowadays used for large-
stresses are not just the merge of the different responses triggered scale cultivation. This process suppresses the possibility of the
by individual constrains (Rizhsky et al., 2004). The combination mutualisms or symbiosis that happened between plantlets and
of salinity and phosphorous scarcity is very common in soil microorganisms in natural conditions, including AM. In
calcareous and alkaline soils of Mediterranean-type climate this line, the early inoculation of giant reed plants with AM
ecosystems (Zribi et al., 2012). The effect of salinity on P nutrition fungi has been proposed as an useful strategy to improve field
in crop plants is quite complex and depending among others, on establishment and first year crop production as well as plant
the plant genotype and environmental conditions, either positive, tolerance to marginal lands (Baraza et al., 2016; Romero-Munar
negative or no effect of salinity on the plant P status has been et al., 2018). In view of the foregoing, we hypothesized that
reported (reviewed by Grattan and Grieve, 1999). AM symbiosis could be a good tool to enhance A. donax
Arbuscular Mycorrhizal symbiosis had been defined by physiological traits and biomass production in early stages
Parniske (2008) as “the mother of plant root endosymbiosis,” under salinity and low Pi soil growing conditions, through
in natural ecosystems, AM symbiosis is also one the most changes in phosphorus use efficiency and sodium toxicity
widespread plant strategies to cope with abiotic and biotic management. The main objective of the present work was to
stresses. It has traditionally been related with improved water and study the effect of AM symbiosis on the growth and biomass
nutrient acquisition, especially P, However, research conducted allocation, water relations, nutrient use efficiency and ion
during the last decades have drawn a more complex picture and, concentration of A. donax grown at different salinity regimes and
for example, AM have been reported to be involved in nutrient phosphorous concentrations.
use efficiency, photosynthesis, respiration and plant metabolism
(Fay et al., 1996; Del-Saz et al., 2017; Romero-Munar et al., 2017).
As components of soil-plant natural ecosystems, the symbiosis MATERIALS AND METHODS
with AM fungi can ameliorate the plant’s response to salinity
and have beneficial effects on plant growth and yield, which Plant and Fungi Material
made AM fungi suitable candidates to bio-ameliorate salinized Fifty-four micropropagated bare-root plants of A. donax K12
soils (reviewed by Evelin et al., 2009). The positive growth clone were provided by Biothek Ecologic Fuel S.L. Upon arriving,

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Romero-Munar et al. AM, Salinity Tolerance and PUE

they were immediately planted in trays filled with agricultural root and the total number of root fragments examined. An
substrate previously tindalized at 120◦ C for 60 min (during three average of 300 root pieces per plant and six plants per
consecutive days to excluding other microorganisms present in treatment were examined. Percentage of mycelium, spores,
the peat) which consisted of nutrient-rich black peat (Kekkilä vesicles, arbuscules and total inoculation were determined
DSM 1 W, pH 5.9, 90% of organic matter). Principal compounds from roots to evaluate salinity effect on the average of
of black peat used: Sphagnum peat; additives: N-P2 O5 -K2 O these structures.
(16-4-17, 0.60 g l−1 ), wetting agent (0.10 g l−1 ) and dolomite
limestone (5.0 g l−1 ). Biomass Measurements
One-week-old plants were transplanted in sterilized silicic Two months after starting treatments, total number of leaves,
sand on 4L pots. Eighteen plants were inoculated in the stems and length of the highest stem were measured in six
transplanted moment with 5 mL (5 g aprox.) of commercial plants per treatment.
inoculum (AEGIS SYM ), mixture of the generalist fungi
R
To assess the biomass of the different plant tissues, each plant
Funneliformis mosseae (T.H. Nicolson & Gerd.) C. Walker & A. was divided into leaves (grouped in: young leaves, YL – two leaves
Schüßler and Rhizophagus intraradices (N.C. Schenck & G.S. Sm.) from the top of the stem; mature leaves, ML – third or fourth leaf
C. Walker & A. Schüßler (Schüßler and Walker, 2010). It was from the top of the stem; and old leaves, OL – leaves located at
chosen as its beneficial effect on the growth of A. donax was the bottom of the stem), stems and roots (grouped in thick roots,
previously reported (Baraza et al., 2016). Both fungi species are TR – diameter >5 mm; and fine roots, FR – diameter <5 mm). To
generalist with high richness in all soil types and commonly measure the dry weight (DW), plant tissue was dried in a forced-
present in commercial inocula. air oven at 70◦ C for 72 h. The roots were washed in distilled
The inocula contained 25 spores per gram of each specie. water before drying.
Inoculated plants were termed AM plants. The rest 36 non- The mycorrhizal dependency (MD) was calculated for each
inoculated plants were supplied with 5 mL (5 g approx.) of treatment according to Plenchette et al. (1983): MD (%) = (DW
autoclaved inoculum plus 3 mL of an inoculum filtrate (<20 µm) of mycorrhizal plant/DW of non-mycorrhizal plant)/DW of
to provide a general microbial population, free of AM propagules mycorrhizal plant × 100. While the mycorrhizal growth response
(Bárzana et al., 2012). was calculated according to Hoeksema et al. (2010): MGR = loge
Plants were grown for 3 months in a growth room under [DW of mycorrhizal plant/DW of non-mycorrhizal plant].
controlled conditions at 25/20◦ C day/night temperature, above Specific leaf area (SLA) was determined as the one-sided area
40% relative humidity and 12 h photoperiod (300 µmol m−2 s−1 of the fourth leaf of each plant divided by its oven-dry mass.
of photosynthetic photon flux density, PPFD). Whole-plant leaf area (LA) was calculated as the total
leaf dry weight/SLA.
Treatments Establishment
Pots were kept at field capacity by watering the plants with Leaf Physiology
25% modified Hoagland nutrient solution with 2.5 µM Pi Physiological parameters were measured in the leaves, 2-month
and 1 mM Na for 7 weeks to allow AM fungi establishment. after treatment establishment.
After AM colonization, two phosphorus and three salinity Gas exchange parameters were measured from 10:00 to
treatments where set up in a step-wise manner in sextuplicate, 12:30 h on one ML per plant leaves using an open infrared
resulting in nine treatments. Nutritional factor (N): Control, C gas exchange analyzer system (Li- 6400; Li-Cor Inc., Lincoln,
(non-inoculated plants growing with 2.5 µM P); Phosphorus NE, United States). Leaf chamber fluorometer (Li-6400-40, Li-
plants, P (non-inoculated plants growing with 0.5 mM Pi); and Cor Inc.) conditions were PPFD of 1.500 µmol m−2 s−1 ,
arbuscular mycorrhiza plants, AM (colonized plants growing with 10% of blue light, and a vapor pressure deficit of 2.0–
with 2.5 µM Pi); each N treatment was combined with three 3.0 kPa at a CO2 concentration of 400 µmol mol−1 (air).
salt concentrations (S): 1, 75 and 150 mM NaCl. Before, N The leaf temperature was set at 25◦ C, and the relative
and S treatments began, the stem length (cm) was measured humidity of the incoming air was approximately 50% throughout
to ensure the homogeneity in size among plantlets (C, P all measurements.
and AM, 82.57 ± 2.43, 84.88 ± 2.38 and 82.78 ± 2.65, To quantify chlorophyll and leaf protein concentrations,
respectively, p = 0.76). samples of the leaves used for gas exchange measurement
(ML) were frozen in liquid nitrogen and stored at −80◦ C.
Mycorrhizal Colonization Photosynthetic pigments were extracted using 96% ethanol.
The percentage of mycorrhizal root colonization was determined Chlorophyll a, chlorophyll b and total chlorophyll content were
at the end of the experiment, 2 months after starting calculated according to Lichtenthaler and Wellburn (1983). Leaf
treatments (3-month after inoculation). It was assessed by protein content was determined following the method described
visual observation of fungal colonization. Roots were digested by Bradford (1976).
with 10% KOH and stained with trypan blue (0.05% in lactic Leaf osmotic potential was measured in two mature leaves per
acid (v/v), according to Phillips and Hayman (1970). AM plant. Frozen samples of ML were thawed and grinded for 30 s.
colonization was assessed using the magnified intersections A sap volume of 10 µL was used (Gucci et al., 1991) to determine
method (Abbott and Robson, 1984), where the frequency of the leaf osmotic potential using a Wescor 5500 vapor pressure
colonization represents the ratio between fragments of colonized osmometer (Wescor Inc., Logan, UT, United States).

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Romero-Munar et al. AM, Salinity Tolerance and PUE

FIGURE 1 | AM fungi structures percentages to the total colonization of each salinity treatment.

Ion Tissue Concentration RESULTS

Ion concentration was determined in YL, ML, OL, S, TR, and FR
of each plant. Dry tissue was finely powdered using an orbital Salinity Severely Decreased AM
shaker in seal tubes with glass balls. One hundred mg of dried Symbiosis Colonization
tissue were ashed at 550◦ C. After cooling, the ash was washed The colonization of A. donax roots by R. intraradices and
into polyethylene tubes with 9.2 ml 0.08 M H2 SO4 and 0.8 ml F. mosseae reached values of 54% ± 9.98 in non-salinized
40% HF were added. The suspension was shaken for 1 h and left plants. Despite root colonization was greatly reduced by S
overnight at room temperature. One micro liter of the suspension factor (p = 0.0196, ANOVA), but differences between three salt
was transferred into polyethylene tubes with 25 ml of 0.32% treatment were partially significant with values of 23.50% ± 6.22
H3 BO3 to neutralize the excess of HF prior analysis (Xia et al., and 12.29% ± 4.33 in 75 and 150 mM NaCl-treated plants,
2000). After filtering, the Na+ , K+ , Ca2+ , Mg2+ , Pi and Si respectively. Tukey test showed significant differences between
concentrations were determined by Inductively Coupled Plasma 1 and 150 Mm, but not between 1 and 75 mM neither between
(ICP) Spectrometry (Perkin-Elmer Plasma-2000, Perkin-Elmer 75 and 150 mM. Salinity also significatively reduced the presence
Inc., Norwalk, CA, United States). of spores (p = 0.0325, ANOVA), vesicles (p = 0.0445, ANOVA)
The nutrient use efficiency was calculated dividing the and arbuscules (p = 0.0435, ANOVA) observed in roots. As well
total plant biomass by the plant nutrient concentration as in total colonization, post hoc Tukey test showed significant
(Siddiqi and Glass, 1981). differences between 1 and 150 NaCl mM treated plants, but
External and internal phosphorus use efficiency (PUE), PUEe not between them and 75 mM NaCl, in percentages of spores
and PUEi , respectively, for leaf, stem and root tissues were and vesicles. The amount of arbuscules was highly reduced
calculated as: (47%) at moderate salt stress (75 mM NaCl), while it greatly
increased (59%) at severe salt stress (150 mM NaCl) with respect
PUEe = Tissue DW/plant P concentration, according to non-salinized roots, but those differences were not significant
Zhang et al. (2009) according to Tukey test (Figure 1). C and P plants were also
PUEi = Tissue DW/tissue P concentration, following screened and no colonization was detected.
Hammond et al. (2009)

Statistical Analyses AM Symbiosis Increased Plant Growth in

All datasets were tested for a normal distribution and variance Non-salinized and Moderately Salinized
homogeneity (P<0.05), and variables were log transformed Plants Under Low P Condition
when necessary. Two-way analysis of variance (ANOVA II) Total plant biomass and its distribution were significantly
was performed to analyze the effect of the two factors (N affected by the nutritional and salinity treatments (N and S
and S) in the main studied parameters: biomass dry weight, factor, respectively), were AM plants showed the highest value,
leaf physiological parameters and ion tissue concentration. We and total biomass decreased from 1 mM >75 mM >150 mM.
performed a post hoc Tukey test to analyze differences among the The interaction of the two factors was also significant (Table 1A
nine treatments. and Figure 2A).
The analyses were performed using the JMP , Version 10 (SAS
R
In non-salinized plants, P and AM treatments increased total
Institute Inc., Cary, NC, United States 1989–2007). plant dry weight by 11 and 41%, respectively with respect to

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Romero-Munar et al. AM, Salinity Tolerance and PUE

TABLE 1A | Summary of two-way analysis of variance (ANOVA) and R saquere Regarding biomass distribution (Table 3), at 1 mM NaCl, AM
adjusted of the total model (R2 adj) for the effects of nutrient treatment (N) and salt
and P plants showed higher root-to-shoot ratio than C plants
treatment (S), with their interaction factor (N × S) on biomass, physiological and
nutrition- related traits in Arundo donax plants.
while under mild salt stress, no significant differences among
nutritional treatments were observed.
Trait R2 adj N S N×S Nutritional and salinity factors had an independent
effect on leaf area (Table 1A). In non-salinized plants,
Total Biomass 0.86 <0.0001 <0.0001 0.0007
P treatment did not increase leaf area, however, AM
Leaf Biomass 0.81 <0.0001 <0.0001 0.0992
Root Biomass 0.76 <0.0001 <0.0001 0.0005
plants showed statistically higher leaf area than P and
LA (cm2 ) 0.51 0.0053 <0.0001 0.5157
C plants (Table 3). Non-significant differences among
AN 0.56 0.1174 0.1656 0.0007
nutritional treatments were observed neither at 75 mM
gs 0.57 0.0704 0.1736 <0.0001
NaCl nor at 150 mM NaCl (Table 3). Furthermore, no
AN /gs 0.57 0.0354 0.8879 0.0007
differences in SLA, leaf area ratio (LAR) and leaf mass
9π 0.30 0.5834 <0.0001 0.2783
ratio (LMR) among nutritional or salinity treatments were
Leaf protein 0.30 0.3107 0.8212 0.0009
found (Table 3).
Chl a 0.25 0.2869 0.0576 0.0080
Na/K (leaf) 0.55 0.1221 <0.0001 0.0297
Physiological Arundo Leaf Responses to
Pi 0.69 <0.0001 0.3232 0.0165
Mg2+ 0.07 0.4631 0.0723 0.5191
Different Nutritional and NaCl Treatments
Ca2+ 0.03 0.2670 0.0489 0.8620 Leaf physiological responses of C, P and AM plants grown
Si 0.03 0.0715 0.3612 0.9733 at 1, 75 or 150 mM NaCl are shown in Figure 3. Under
KUE 0.64 0.0017 <0.0001 0.0471 non-salt stress, C plants showed statistically significant
MgUE 0.51 0.0072 <0.0001 0.2900 lower osmotic potential than P and AM plants, while
CaUE 0.22 0.1207 0.9261 0.7680 under salt stress conditions, osmotic potential declined
SiUE 0.32 0.7572 0.0045 0.9357 with increased salinity with no differences among nutritional
PUE 0.61 <0.0001 <0.0001 0.5626 treatments (Figure 3A).
PUEe Leaf 0.68 <0.0001 <0.0001 0.1906 Regarding leaf protein concentration (Figure 3B), only the
PUEe Stem 0.62 <0.0001 <0.0001 0.2791 interaction between nutritional and salinity (N × S) was
PUEe Root 0.71 <0.0001 0.0004 0.1323 significant (Table 1A). According to post hoc Tukey test, P plants
PUEi Leaf 0.69 <0.0001 <0.0001 0.1985 showed significantly lower leaf protein content than C plants
PUEi Stem 0.57 <0.0001 <0.0001 1.7833 under non-salt stress conditions. No differences were observed
PUEi Root 0.88 <0.0001 <0.0001 0.0108 in leaf protein among N levels at 75 and 150 mM.
Salinity and N factor did not affect chlorophyll a separately,
Values correspond to P values. Abbreviations as shown in the text. Significant P
values are highlighted in bold. although significant N × S interaction was found (Table 1A).
AM showed higher chlorophyll a concentration under non-
salt stress conditions, than C plants (Figure 3C). Moderate
salt stress did not affect chlorophyll a in C and P plants,
C plants, which was mainly due to higher root rather than whilst in AM plants, chlorophyll a decreased significantly and
higher leaf biomass (Figures 2B,C). This significant increase showed the lowest concentration (post hoc Tukey). No significant
in AM plants was linked with 30% of mycorrhizal dependency differences in chlorophyll a were found among nutritional
(MD) observed (Table 2). Under mild salinity (75 mM NaCl), treatments at 150 mM NaCl.
no significant differences in total biomass between P and C Photosynthesis (AN ) and stomatal conductance (g s )
treatments were observed. However, regardless the important (Figures 3D,E, respectively) were significantly affected by
decrease in AM root colonization and MD caused by salt nutritional and salinity interaction (N × S, Table 1). Under
(Table 2), AM plants showed a 14% increase in total dry biomass non-saline conditions, P plants showed significantly higher AN
due to similar positive AM effects on leaf and root growth. and g s than plants grown under low P. However, under mild
In 150 mM NaCl-treated plants, no differences on total, leaf salt stress conditions, AM plants significantly increased both
or root biomass between nutritional treatments were found parameters respect to C and P plants. No differences were found
(Figures 2A–C, respectively). at 150 mM NaCl among N levels.

TABLE 1B | Summary of three-way analysis of variance (ANOVA) and R saquere adjusted of the total model (R2 adj) for the effects of nutrient treatment (N), salt
treatment (S) and tissue (T) with their interaction (N × S, T × N, T × S and T × N × S) on sodium (Na+ , mM) and potassium concentration (K+ , mM).

R2 adj N S N×S T T×N T×S T×N×S

Na+ 0.91 <0.0001 <0.0001 0.0044 <0.0001 0.0080 <0.0001 <0.0001

K+ 0.92 0.4362 <0.0001 0.6401 <0.0001 0.1936 <0.0001 0.8449

Values correspond to P values. Significant P values are highlighted in bold.

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Romero-Munar et al. AM, Salinity Tolerance and PUE

TABLE 2 | Mycorrhizal dependency (MD) and mycorrhizal growth response (MGR)

of A. donax under three levels of NaCl concentration.

NaCl (mM) MD (%) MGR

1 29.33 0.35
75 12.56 0.13
150 5.35 0.06

It was calculated respect C plants.

concentration profile among the different tissues was dependent

on the nutritional treatment (Figures 4A–C). While in non-
salt stressed plants (Figure 4A), where Na+ was preferentially
accumulated in stems, P plants showing the highest values, at
75 and 150 mM AM plants showed the lowest ones. Under mild
and severe stress salt conditions, thick roots (TR) and stems (S)
had the highest Na+ followed by fine roots (R), mature leaves
(ML) and old leaves (OL) with the lowest concentrations found
in young leaves (YL) (Figures 4A–C). At 75 mM NaCl, AM
plants strongly reduced Na+ concentration in leaves, whilst C
and P plants only showed significant reduction in young leaves
(Figure 4B). At 150 mM, despite AM plants showed an equal
Na+ distribution among tissues, a significant reduction of Na+
concentration in mature leaves respect C and P plants was
observed (Figure 4C).
Potassium concentration was significantly affected by the
S factor and tissue distribution and their interaction, but
not by the N factor (Table 1B). The K+ distribution profile
among the different tissues was the same in all nutritional
treatments with highest values found in the aboveground tissues,
especially in stems. Salinity decreased root K+ concentrations
but its concentration was maintained in the aboveground tissues
(Figures 4D–F).
The Na+ /K+ ratio of ML increased in the order C > P > AM
(p < 0.05, ANOVA and post hoc Tukey test) at 75 mM NaCl,
while increased in the order P > C > AM (p < 0.05, ANOVA
and post hoc Tukey test) at 150 mM NaCl (Figures 4D–F).
The N factor highly affected phosphorus concentration but
its effect depended on the salt treatment (in Supplementary
Table S1A). Significantly higher P concentration was observed
in P plants, and slightly but not significantly decreases through
salinity treatments. Post hoc Tukey test showed higher P
concentration in AM plants compared with C plants under
FIGURE 2 | Total biomass (A), leaf (B) and root (C) dry weight of control (C, 1 mM NaCl treatment, but those differences disappeared
white bar), phosphorus (P, gray bar) and arbuscular mycorrhiza plants (AM, under salinity conditions. Salt stress did not significantly
black bar) grown at 1, 75 and 150 mM NaCl. Values are mean ± SE of six affect tissue Pi concentration at any nutritional treatment
replicates. Different capital letters indicate significant differences among
nutritional levels (C, P and AM) within the same salt level, and lowercase
(Table 1A). The Pi concentration response profile of the different
letters indicate significant differences among salt levels within the same plant tissues showed lower values in stems and thick roots
nutritional level, from post hoc Tukey test. respect to fine roots and all other aboveground tissues (in
Supplementary Table S1A).
Calcium concentration was dependent on the salinity
treatment while no significant differences were found in Mg2+
AM Symbiosis Modulated Salinity and or Si concentrations among N or S treatments (Table 1A). Ca2+
Low Phosphorus Effects on Ion Uptake concentrations decreased in roots and increased in leaves of P
and Distribution and AM plants with increasing salinity, while no differences
Nutritional and salinity factors had a dependent effect on were found among salt treatments in C plants. Calcium and
tissue Na+ concentrations (Table 1B). Furthermore, the Na+ Mg2+ showed the highest concentrations in OL, while Si was

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Romero-Munar et al. AM, Salinity Tolerance and PUE

0.57 ± 0.03a

0.215 ± 0.01a

Total leaf area [LA, (cm2 )], root to shoot ratio (R:S) Specific leaf area [SLA, (cm2 g−1 )]; leaf area ratio [LAR, (cm2 g−1 )] and leaf mass ratio [LMR, (g g−1 )]. Data are means ± SE of 5 to 6 replicates. Different letters
preferentially accumulated in roots and OL (in Supplementary

57 ± 2.7a
12.3 ± 0.9a
400 ± 37a
Tables S1B–D, respectively).

AM
AM Symbiosis Increased Nutrient Use
Efficiency
Potassium use efficiency (KUE) was significantly affected by N

0.219 ± 0.004a
0.56 ± 0.02a
and S factors and their interaction (Figure 5A and Table 1A).

11.2 ± 0.6a
341 ± 24a

51 ± 2a
AM plants, followed by P plants, showed higher values compared
150

to C ones at 1 mM (Figure 5A), while no differences were

observed under moderate and severe stress conditions. With the
increase of Na+ concentration, KUE was progressively decreased
in all N levels.
0.50 ± 0.02a

0.217 ± 0.01a
Statistical analysis showed Mg2+ use efficiency (MgUE) was
11.4 ± 1.2a
351 ± 40a

51.8 ± 4a

affected by S and N factors but not for their interaction

C

(Table 1A). At 1 mM NaCl, AM plants showed the highest

MgUE (post hoc Tukey test), but not differences were found
among C, P and AM plants under salinity stress conditions. Ca2+
use efficiency (CaUE) and silicon use efficiency (SiUE) showed
0.66 ± 0.06a

0.203 ± 0.01a
46.2 ± 1.5a
9.4 ± 0.4a
439 ± 20a

no statistical differences among treatments, and any statistical

effect of N and S factor and their interaction (Table 1A and
AM

Figures 5C,E, respectively).

C and AM non-salinized plants showed highest phosphor
use efficiency (PUE) than P plants. Regarding salinity stress
conditions, the post hoc Tukey showed that AM plants presented
0.49 ± 0.02a

0.222 ± 0.01a
49.3 ± 2.6a
11 ± 0.9a
421 ± 26a

higher PUE followed by C and P plants (Figure 5B) at 75 and

75

150 mM NaCl. However, in all N levels, salinity had a negative

P

indicate statisticaly significant differences among N treatments with the same S factor (p-values are shown in Table 1).

effect on PUE, decreasing at 75 mM and ranged the lowest

values at 150 mM NaCl.
Phosphorus use efficiency external and internal (PUEe and
0.63 ± 0.04b

0.191 ± 0.01a

PUEi, respectively) statistical analyses showed that both were

9.9 ± 0.7a
403 ± 23a

51.5 ± 2a

significantly affected by N and S factors and their interaction

C

(Table 1A and Figures 6A–F). Increased salinity declined PUEe

(Figures 6A,C,E) and PUEi (Figures 6B,D,F) in C and AM plants
TABLE 3 | Biomass parameters of C, P and AM plants grown at 1, 75 and 150 mM NaCl.

with little effect in P plants. PUEe showed lower values than PUEi
in all nutritional and salinity treatments. In leaves and stems, at
0.74 ± 0.06a

0.183 ± 0.01a
755 ± 120a

9.5 ± 1.2a

75 and 150 mM NaCl, AM plants showed the highest values of

51.8 ± 4a

PUEe and PUEi, followed by C and P plants (Figures 6A–D).

AM

The highest PUEi values were found in roots, where in non-

salinized conditions AM plants showed 2-fold higher values than
C plants (Figure 6F).
0.64 ± 0.03ab

0.199 ± 0.01a
10.4 ± 0.9a
574.5 ± 51b

51.7 ± 3a

DISCUSSION
P
1

In arid and semiarid regions with calcareous soils, combined

phosphorus deficiency and high salinity are often soil-borne
stress conditions that limit crop establishment and plant
0.49 ± 0.05c

0.211 ± 0.01a

production (Bargaz et al., 2018). Although unsuitable for food

11 ± 0.5a
543 ± 40b

53 ± 3a

crops, such marginal lands have potential for growing low-

C

cost, environmentally sustainable, energy crops which could also

enhance ecosystem services such as carbon sequestration and
soil structure. A. donax is a fast-growing perennial grass which
has been highlighted as a promising crop for lignocellulosic
LAR (cm2 g−1 )
SLA (cm2 g−1 )

biomass production in salinized soils (Sánchez et al., 2016

LMR (g g−1 )

and references herewith). On the other hand, AM symbiosis

N factor

LA (cm2 )
S factor

is known to ameliorate the plant response to constraining

S:R

factors in calcareous-salinized soils by enhancing Pi acquisition

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Romero-Munar et al. AM, Salinity Tolerance and PUE

FIGURE 3 | Leaf physiological traits: (A) Osmotic potential (9π), (B) Leaf protein, (C) Chlorophyll a concentration (Chl a), (D) Net Photosynthesis rate (AN ), (E)
Stomatal conductace (gs ) of C, P and AM plants (white, gray and black bars, respectively) at 1, 75 and 150 mM NaCl. Values are mean and SE of six replicates.
Different capital letters indicate significant differences among nutritional levels (C, P and AM) within the same salt level, and lowercase letters indicate significant
differences among salt levels within the same nutritional level, from post hoc Tukey test.

(Harrison and van Buuren, 1995; Wright and Upadhyaya, 1998; The effect of concurrent abiotic stresses on plant growth is
Richardson et al., 2009; Smith and Smith, 2011) and salinity related to both, the severity of each individual stress and the plant
tolerance (Mohammad et al., 2003; Evelin and Kapoor, 2014). species (Rabhi et al., 2007; Slama et al., 2008) with plant growth
Here, marginal land stress conditions were mimicked by being determined by the most growth-limiting stress factor (van
growing AM and non-AM A. donax plants at low and sufficient der Ploeg and Kirkham, 1999; Talbi Zribi et al., 2011).
phosphorous concentrations under increasing salinity. To the Regardless the marked differences between C and P plants
best of our knowledge, few studies have focused on the role of in total Pi tissue, whose values were in the high range in P
the AM symbiosis in the plant responses to combined salt stress plants (>5 mg P/g DW) and close or just below the critical
and low P (Mohammad et al., 2003; Del-Saz et al., 2017). values (3 mg P/g DW) in C plants (Veneklaas et al., 2012

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Romero-Munar et al. AM, Salinity Tolerance and PUE

FIGURE 4 | Sodium and potassium concentration: (A–C) show Na+ concentration (mM), and (D–F) show K+ concentration (mM) of different tissues (fine root – FR;
thick root – TR; stem – S; old leaf – OL; mature leaf – ML and young leaf – YL) at 1, 75 and 150mM salt levels, of C, P and AM plants (white, gray and black bars,
respectively). Values of ML Na+ /L+ ratio are show in a nested chart. Values are mean and SE of six replicates. Different capital letters indicate significant differences
among nutritional levels (C, P and AM) within the same tissue, and lowercase letters indicate significant differences among tissues within the same nutritional level,
from post hoc Tukey test.

and references herewith), the low Pi concentration supplied in could at least partly explain why plant growth, shoot/root ratio
this study did not trigger Pi scarcity-related growth responses and leaf chlorophyll in C and P plants were alike. While
in A. donax. Non-salinized C plants did not show increased similar photosynthesis values in C and P plants could be
root/shoot biomass ratio or decreased chlorophyll production explained by the highest external and internal PUE ratio in
(Hammond and White, 2011), neither a decrease in CO2 C compared with P plants since internal PUE at leaf level
assimilation. Cytokinins (CKs) have been related to Pi signaling, is a key factor to prevent a reduction in photosynthesis due
with low-P conditions decreasing plant CK concentrations to increased sucrose concentrations in response to low P
(Rouached et al., 2010). The maintenance of CK signaling (Yang et al., 2017).

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Romero-Munar et al. AM, Salinity Tolerance and PUE

FIGURE 5 | Nutrient Use Efficiency [Total Biomass, TB (g)/nutrient concentration (mg g−1 )]. (A) K+ , KUE; (B) P, PUE; (C) Mg2+ , MgUE; (D) Ca2+ , CaUE and (E) Si,
SiUE. Values are mean and SE of six replicates of of C, P and AM plants (white, gray and black bars, respectively) at the three salinity levels (1, 75 and 150 mM
NaCl). Different capital letters indicate significant differences among nutritional levels (C, P and AM) within the same salt level, and lowercase letters indicate
significant differences among salt levels within the same nutritional level, from post hoc Tukey test.

The symbiosis of A. donax with AM fungi (AM plants), reporting the positive effects of AM symbiosis in A. donax
showed the best ameliorating growth response to low P. Root and (Tauler and Baraza, 2015; Baraza et al., 2016; Romero-Munar
leaf biomass in AM plants even surpassed the P plants values. AM et al., 2017, 2018), but also negative or null effects (Pollastri
symbiosis has frequently been reported as a biological method to et al., 2018), depending on growing conditions, fungi partners
promote plant growth by increasing nutrient uptake, especially P and/or the phenological stage of the plants (Johnson et al., 1997;
(Wright and Upadhyaya, 1998; Richardson et al., 2009; Smith and Smith and Smith, 2015).
Smith, 2011). However, variation in the response from positive Despite the high resilience shown by A. donax to low P
to negative to AM symbiosis exits depending on the plant and conditions, AM symbiosis provided this species with additional
fungus genotype and the environmental/agronomic conditions benefits as a 30% degree of plant growth change was associated
(Dai et al., 2014; Johnson et al., 2015). There are several works with AM colonization.

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Romero-Munar et al. AM, Salinity Tolerance and PUE

FIGURE 6 | Phosphorus use efficiency external and internal (PUEe and PUEi , respectively), of leaves (A,B), shoots (C,D) and roots (E,F), for C, P and AM plants
(white, gray and black bars, respectively) under 1, 75 and 150 mM NaCl. Values are mean and standard error of at least four replicates per treatment. Different
capital letters indicate significant differences among nutritional levels (C, P and AM) within the same salt level, and lowercase letters indicate significant differences
among salt levels within the same nutritional level, from post hoc Tukey test.

However, in contrast with previous studies (Wright and grown under P limitation, AM colonization decreases both
Upadhyaya, 1998; Richardson et al., 2009; Smith and Smith, carboxylates exudation and respiration, and enhances biomass
2011), the increased growth response in AM plants was production (Del-Saz et al., 2017).
related to higher nutrient use efficiency rather than to Regarding to the effect of concurrent abiotic stresses on plant
tissue P concentration. Higher KUE, PUE and MgUE could growth above mentioned, in the present work, the most growth-
have benefited, amongst others, leaf water relations and limiting stress factor was salinity, since both, as single stress
photosynthesis and consequently plant growth. Moreover, (P plants) or in combination with low phosphorus (C and AM
the greatest internal root PUE in AM plants could be plants), moderate and severe salt stress conditions greatly reduce
due to an inhibitory effect of the AM-inducible root P plant biomass. Despite some studies have listed A. donax as
transporters pathway on the direct uptake pathway (Smith a salt tolerant species (Williams et al., 2008; Sánchez et al.,
et al., 2004; Campos-Soriano et al., 2012), and the activation 2015), our results showed a fast inhibition of leaf growth in
of the mycorrhizal nutrient uptake pathway, an energy saving this species caused by salt independently of P supply, which
mechanisms (Watts-Williams et al., 2015). In roots of plants compromised carbon acquisition and consequently yield, due

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Romero-Munar et al. AM, Salinity Tolerance and PUE

to its dependence on leaf production and expansion. However, affinity phosphorus transporters (Harrison et al., 2002), could
AM symbiosis ameliorated the response of A. donax to combine explain how AM plants maintain the internal and external PUE
low P and mild salinization conditions. At 75 mM NaCl, and reducing Na+ concentration in tissues, under moderate
when C and P plants growth was driven by salinity rather and severe salinity and low P availability, compared to C but
than P availability, AM plants showed 14% higher biomass also than P plants.
than C and P, despite the important reduction of AM root To summarize, the results indicate that AM symbiosis could
colonization and consequently in MD. Although AM symbiosis be a good tool to enhance A. donax physiological traits and
are present in saline soils (Landwehr et al., 2002), the osmotic biomass production under combined low phosphorus and salt
and toxic effects of salt not only affect the host plants but stress conditions during the plant’s early developmental stages.
also the fungi in a similar way (Juniper and Abbott, 2006). Despite the negative impact of high salt on AM colonization,
In fact, the effect of salinity on plant colonization by AM AM plants were able to maintain cellular homeostasis at low
depends on the fungus tolerance to salinity (Yamato et al., Pi supply by assessing higher PUE rather than increasing tissue
2008). Under our experimental conditions, root colonization by P concentrations. Thus, AM symbiosis establishment at early
R. intraradices and F. mosseae was severely decreased by salt. development stages could play a key role in the A. donax
Reduced root colonization by AM fungi in saline environments cultivation in marginal lands.
has been related to a salt effect on primary infection as more
inhibition has been reported at the early stages of AM symbiosis
(Wilson, 1984; McMillen et al., 1998). However, in this study, AUTHOR CONTRIBUTIONS
salinity treatments were started after AM colonization was
achieved and therefore the reduction in AM colonization was AR-M and CC designed and performed the research,
more likely due to a salt effect on secondary colonization. collected, analyzed and interpreted the data, and wrote the
On the other hand, the percentage of root colonization is not manuscript. JG and EB collaborated on data interpretation and
directly related to the symbiotic outcome (Giri and Mukerji, writing the manuscript.
2004). In this line, at 75 and 150 mM NaCl, AM plants
showed remarkable growth and sodium management responses
compared with C and P plants. FUNDING
Sodium specific toxic effects have been associated with the
built-up of high leaf Na+ concentrations (Munns, 2002). In This work was supported by OPTIMA project-UE FP7 (No.
barley, a salt-tolerant species, plant dry weight was found to of Contract: 289642). AR-M was funded by a fellowship from
decrease at shoot Na+ concentrations above 9.2 mg g−1 DW the Government of the Balearic Islands, University of Balearic
(Tavakkoli et al., 2011). Notably, similar and even higher Na+ Islands, and the European Social Fund.
values were found in leaves of C and P plants submitted at
the highest salt treatment, while leaf Na+ was significantly
lower in AM plants, pointing out to an enhanced effect of AM ACKNOWLEDGMENTS
fungi on the Na+ exclusion capacity of A. donax. It has been
reported that AM symbiosis increased and also orchestrated We thank Biothek Ecologic Fuel for providing the plants. We also
Na+ exclusion response (Giri and Mukerji, 2004). In AM plants thank Dr. Jonh Sibole, Jaume and Lluís Sibole Cabot and Dr. Cyril
the intraradical hyphae could have provided the plant with an Douthe for their inestimable help.
additional space for Na+ allocation and help to prevent its
translocation to the shoots (Cantrell and Linderman, 2001). This
higher Na+ exclusion capacity could be related to the ameliorated SUPPLEMENTARY MATERIAL
growth found in AM plants grown at mild salt conditions.
Furthermore, it has been propose that AM fungi excludes Na+ The Supplementary Material for this article can be found online
by discrimination in its uptake from the soil or during its transfer at: https://www.frontiersin.org/articles/10.3389/fpls.2019.00843/
to plants (Hammer et al., 2011), but also had specific and high full#supplementary-material

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pathway is not correlated with mycorrhizal responses in growth or total P Conflict of Interest Statement: The authors declare that the research was
uptake. New Phytol. 162, 511–524. doi: 10.1111/j.1469-8137.2004.01039.x conducted in the absence of any commercial or financial relationships that could
Talbi Zribi, O., Abdelly, C., and Debez, A. (2011). Interactive effects of salinity be construed as a potential conflict of interest.
and phosphorus availability on growth, water relations, nutritional status and
photosynthetic activity of barley (Hordeum vulgare L.). Plant Biol. 13, 872–880. Copyright © 2019 Romero-Munar, Baraza, Gulías and Cabot. This is an open-access
doi: 10.1111/j.1438-8677.2011.00450.x article distributed under the terms of the Creative Commons Attribution License
Tauler, M., and Baraza, E. (2015). Improving the acclimatization and establishment (CC BY). The use, distribution or reproduction in other forums is permitted, provided
of Arundo donax L. plantlets, a promising energy crop, using a mycorrhiza- the original author(s) and the copyright owner(s) are credited and that the original
based biofertilizer. Ind. Crop Prod. 66, 299–304. doi: 10.1016/j.indcrop.2014. publication in this journal is cited, in accordance with accepted academic practice. No
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Frontiers in Plant Science | www.frontiersin.org 14 July 2019 | Volume 10 | Article 843