ORIGINAL PAPER

Role of native and exotic mycorrhizal symbiosis to developmorphological, physiological and biochemical responses copingwith water drought of date palm, Phoenix dactylifera

Marouane Baslam • Ahmed Qaddoury •

Nieves Goicoechea

Received: 18 June 2013 / Revised: 11 September 2013 / Accepted: 12 September 2013

� Springer-Verlag Berlin Heidelberg 2013

Abstract

Key Message Arbuscular mycorrhizal (AM) symbiosis

can improve date palm growth and alleviate drought-

related impacts than non-mycorrhizal plants due to the

ability of AMF for modifying plant metabolism and

physiology.

Abstract Date palm (Phoenix dactylifera L.) is an

important agricultural and commercial crop in the North of

Africa and Middle Eastern countries. During the last dec-

ade, date palm plantations were subjected to degradation

due to an extensive exploitation and to drastic environ-

mental conditions such as drought. Currently, there is a

growing interest in the valorization of water due to envi-

ronmental problems and economic aspects. The use of ar-

buscular mycorrhizal fungi (AMF) can offer a possibility to

overcome these problems. The objective of this study was

to study the influence of different Glomus species—Glo-

mus intraradices, G. mosseae and Complex Aoufous

(native AMF)—on the development of date palm grown

under two water regimes (optimal irrigation, 75 % of field

capacity or water deficit, 25 % of field capacity). Our

results revealed that the beneficial effect of mycorrhizal

symbiosis on plant growth depended on the fungal species

and the water regime applied to the palm date seedling.

While the native Complex Aoufous was the most effective

in increasing the shoot height and biomass under well-

watered conditions, G. intraradices was the most beneficial

fungus for improving growth of plants that undergo

restricted water supply. This positive effect of G. intrara-

dices under drought conditions was not related to an

enhancement of the antioxidant enzymatic activities in

leaves; the association of palm date with G. intradices

caused an increase in the elasticity of cell walls in leaves

and allowed maintaining high water content in leaves

without lowering leaf water potential under stressful con-

ditions. The adequate selection of the AMF species is

crucial for improving growth of palm date seedlings, and it

must be in accordance with the water regime that will be

applied to plants.

Keywords Antioxidant � Arbuscular mycorrhizal

fungi (AMF) � Date palm � Drought tolerance �Oxidative damage � Plant water status

Abbreviations

n Cell elastic modulus

hsymp Symplastic water

Ww Leaf water potential

Wp0 Osmotic potential at turgor loss

Wp100 Osmotic potential at full turgor

APX Ascorbate peroxidase

BSA Bovine serum albumin

CA Complex Aoufous

CAT Catalase

DM Dry matter

FC Field capacity

FW Fresh weight

Gi Glomus intraradices

Communicated by R. Hampp.

M. Baslam (&) � N. Goicoechea

Grupo de Fisiologıa del Estres en Plantas (Unidad Asociada al

CSIC, EEAD, Zaragoza e ICVV, Logrono), Departamento de

Biologıa Ambiental, Facultades de Ciencias y Farmacia,

Universidad de Navarra, C/Irunlarrea, 1, 31008 Pamplona, Spain

e-mail: bmarouane@alumni.unav.es

M. Baslam � A. Qaddoury

Team of Plant Biotechnology and Agrophysiology of Symbiosis,

Faculty of Sciences and Techniques (FSTg), PO Box 549,

40000 Marrakech, Morocco

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Trees

DOI 10.1007/s00468-013-0939-0

Gm Glomus mosseae

GPOX Guaiacol peroxidase

M Mycorrhizal

MEI Mycorrhizal efficiency index

NM Non-mycorrhizal

P–V curves Pressure–volume curves

PVPP Polyvinylpolypyrrolidone

RDM Root dry matter

ROS Reactive oxygen species

RWC Relative water content

SDM Shoot dry matter

SOD Superoxide dismutase

SPC Soluble phenolic compounds

TBA Thiobarbituric acid

TBARS Thiobarbituric acid reacting substances

TCA Trichloroacetic acid

TSP Total soluble proteins

TSS Total soluble sugars

TW Turgid weight

WD Water drought

WW Well-watered

Introduction

Date palm (Phoenix dactylifera L.), a dioecious species, is

a major fruit crop in North Africa, several tropical coun-

tries located in the Near East, United States of America and

Australia. According to FAOSTAT (FAO Statistics Divi-

sion) 2012, the world production of dates in 2010 reached

7,626,448 tons on about 1,104,331 ha. Over the history,

date palm tree has played a major role in the life of human

beings as a major agricultural crop. It could be used for

generations to come due to its remarkable nutritional,

health and economic value in addition to its esthetic and

environmental benefits. Every part of the date palm is

useful and the importance of the date in human nutrition

comes from the acceptable taste and its rich composition of

carbohydrates, salts and minerals (selenium, potassium,

calcium, magnesium, manganese, and iron), dietary fiber,

vitamins, carotenoids, fatty acids, amino acids and protein

(Al-Farsi and Lee 2008; Biglari et al. 2009). Indeed, date

palm fruit possess anti-tumor activity (Ishurd and Kennedy

2005), antioxidant and antimutagenic properties in vitro

(Vayalil 2002; Mansouri et al. 2005) and this is due, partly,

to its high content of polyphenolic compounds and vita-

mins as important dietary constituents (Mansouri et al.

2005; Awad et al. 2011). Pits of date palm fruit constitute

about 10–15 % of the fruit weight and contain fats, car-

bohydrates, minerals, proteins, steroids, vitamins, phenols,

and crude fibers. Although of potentially useful nutritive

content, pits generally may be used as organic fertilizers, as

feed supplement for livestock and activated charcoal.

Recently, Gad et al. (2010) and Trigueros et al. (2012)

successfully used date extract and date blanching water to

reconstitute skim milk powder in processing fortified

yogurt with highest antioxidant activities compared with

those of control yogurt. Furthermore, the remnants of palm

trees (unripe dates, date seeds and palm leaves) may be

used to feed some animals (sheep, goats and rabbits),

which represents an alternative to manufactured fodders

(Al-Banna et al. 2010).

The role of the tree has increased when growers realized

that the tree is drought and salt tolerant, in addition to its

impact in combating desertification. Moreover, date palm

assumes importance because production of other fruit trees

is limited in the harsh environment. Cultural areas of date

palm depend on groundwater, the main source of water for

agriculture in these regions. The limited precipitation and

the huge increase in the area of agricultural land have put

pressure on groundwater usage, since the agricultural

demand for fresh water in this region is in growing (Ab-

dullah and Hasbini 2004). Therefore, drought stress can

cause significant yield losses in date palm and can affect

their productivity negatively. Providing water around the

country for agricultural uses, mapping the vegetation

through change detection via satellite image techniques and

applying biotechnology approaches utilizing new breeding

programs are useful tools that could be used to improve

resistance to important constraints affecting the date palm.

However, the high costs of these projects (Al Hammadi

2006) induce to look for other alternatives in order to

improve growth and alleviate drought-related impacts. In

this context, specific associative bacteria or symbiotic

eukaryotes could have the potential to mitigate drought

stress of plants. Among all these, arbuscular mycorrhizal

fungi (AMF) can protect host plants against detrimental

effects of water restriction. Mycorrhizal fungi colonize the

roots of over 90 % of plant species to the mutual benefit of

both plant host and fungus (Harley and Smith 1983). The

most common are the arbuscular mycorrhizas (AM), which

are formed by the majority of crop, horticultural plants, and

trees including date palm. AMF play a critical role in plants

mineral nutrition and terrestrial ecosystem functioning.

AMF association benefits host plants not only by improv-

ing mineral nutrition, but also by increasing plant resis-

tance to drought (Goicoechea et al. 2004), soil salinity (Giri

et al. 2003) and pathogens (Garmendia et al. 2004).

The main objectives of this study were: (1) to evaluate

the effect of mycorrhizal symbiosis on growth and biomass

production of date palm seedlings cultivated at either

optimal or restricted irrigation; (2) to assess if the appli-

cation of AMF can improve water status and/or activate

antioxidant mechanisms in data palm plants subjected to

drought and (3) to compare the effectiveness of native

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123

AMF (collected from field soil where date palm usually

grow) with that of non-native or ‘exotic’ AMF in protect-

ing date palm plants against harmful effects of water

deficit.

Materials and methods

Biological material and experimental design

Phoenix dactylifera cv. Khalt was the cultivar of date palm

chosen for this study. The experiment was conducted as a

factorial design with two major factors and eightfold rep-

lication. The first factor had two levels: non-mycorrhizal

plants (NM) and plants inoculated (M) with AMF. The

second factor included two irrigation regimes: optimal

irrigation (WW) (75 % of field capacity, FC) and water

deficit (WD) (25 % FC). Seeds of P. dactylifera cv. Khalt

were surface sterilized by 10 % bleach for 10 min and

sown in sand and maintained at 38 �C in the dark. Two

weeks later, seedlings were transferred to room at 32/24 �C

day/night. When seedlings had two fully developed leaves

(2 months), the most similar seedlings were selected and

transferred to 10-L plastic pots filled with a mixture of soil

diluted with compost (2/3:1/3, soil:compost, v/v). Loamy

soil was collected from the date palm groves in Tafilalet

(Meknes Province) in the South of Morocco (33�54’900N5�3205200W) and sieved (5 mm). In this study, the compost

used was prepared from date palm by-products and was

provided by ‘‘Pepeniere Communale’’, Marrakech, Mor-

occo. The compost was obtained according to the method

described by Chakroune et al. (2005). Soil and compost

were previously sterilized at 100 �C for 1 h on 3 consec-

utive days.

At transplanting, 16 pots of date palm were inoculated

with the native mycorrhizal fungus Complex Aoufous

(CA), another 16 pots were inoculated with Glomus in-

traradices (Schenck and Smith) (actually Rhizophagus

intraradices (Schenck and Smith) Walker & Schubler

comb. nov.), another 16 pots were inoculated with Glomus

mosseae (Nicol. and Gerd.) Gerd. and Trappe [actually

Funneliformis mosseae (Nicol. and Gerd.) Walker &

Schubler comb. nov.], and another 16 pots were not

inoculated and kept as non-mycorrhizal controls, thus

making a total of 64 pots. G. intraradices and G. mosseae

came from a pot culture with alfalfa (Medicago sativa L.)

as host plant, and inoculum consisted of 10.0 g of soil with

alfalfa root fragments, spores, and hyphae per 10 L of total

substrate. The same amount of autoclaved inoculum was

added to non-mycorrhizal plants. Complex Aoufous (CA)

is a complex of native AMF coming from the Aoufous date

palm grove in the south of Morocco. CA was identified

using morphological and molecular techniques. The spe-

cies was clearly identified as a composition of many G.

mosseae strains (Zeze et al. 2007), which has been recently

reassigned to F. mosseae (Nicol. and Gerd.) Walker &

Schubler comb. nov.). The G. mosseae strains of CA can

act synergically, which may contribute to increase the

effectiveness of these inocula for enhancing the resistance

of host plants to drought (Zeze et al. 2007).

The experimental pots were placed in a greenhouse

under natural light, where no temperature controlling

equipment was available. The photon flux density ranged

from 600 to 1,400 lmol m-2 s-1. The average day/night

temperature was 32/22 �C; the relative humidity (RH) was

50/85 %. Greenhouse was placed at the Faculty of Sciences

and Techniques campus (31�3705100N, 8�0100200W; Mar-

rakesh, Morocco). The plants were watered regularly to

75 % FC until 7 months after sowing (5 months after

inoculation). At this stage, half of plants from every

treatment (eight non-inoculated with AMF and eight

inoculated with AMF) was subjected to water deficit by

reducing the volume of water supplied to 25 % FC as

described by Meddich et al. (2000).

Growth parameters

At harvest, plant growth was estimated by calculating shoot

height and biomass, leaf area and number of leaves per

plant. Shoot height was measured from the steam base to

the crossing point of the last leaf (the oldest leaf). Leaf area

per plant was determined by a non-destructive method

based on the weight of paper cut-out of the leaf tracing

compared to the weight of known areas of the same paper.

Dry matter (DM) of shoots (SDM) and roots (RDM) was

determined after drying plant material in the oven at 80 �C

until weight was constant.

Mycorrhizal analysis

Root samples of date palm plants were cleared and stained

as described by Phillips and Hayman (1970) and the per-

centage of mycorrhizal root colonization was determined

by visual observation of fungal colonization. Percentage of

AMF colonization was assessed on each root sample by

using the gridline intersects method (Giovanetti and Mosse

1980). The mycorrhizal efficiency index (MEI) was esti-

mated according to Bagyaraj (1994): MEI = (DM of

inoculated plant - DM of non-inoculated plant) 9 100/

DM of inoculated plant. Determination of MEI allows

assessment of the growth improvement brought about by

inoculation of plants with a mycorrhizal fungus.

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Leaf water status

Relative water content (RWC) was estimated by a modi-

fication of the method of Weatherley (1950) and calculated

as RWC = 100 9 (FW - DM)/(TW - DM). FW and

DM denote, respectively, fresh weight and dry matter.

Turgid weight (TW) was calculated after fully hydrating

fresh leaves in darkness at 4 �C for 24 h. Results were

expressed as percentages. Leaf water potential (Ww) was

measured using a pressure chamber (Scholander et al.

1965). Osmotic potential at both full turgor (Wp100) and

turgor loss (Wp0), symplastic water (hSymp) and cell elas-

ticity modulus (n) were inferred from pressure–volume

curves following the free-transpiration method (Brodribb

and Holbrook 2003).

Non-structural carbohydrates, proline and total soluble

phenolics in leaves

Starch and total soluble sugars (TSS) were quantified in

potassium phosphate buffer (KPB; 50 mM, pH = 7.5)

extracts of fresh leaves (1 g). These extracts were filtered

through four cheese cloth layers and centrifuged at

38,720g for 10 min at 4 �C. The supernatant was collected

and stored at 4 �C for TSS. The pellet was used for starch

determination (Jarvis and Walker 1993). The content of

TSS in leaves was analyzed with the anthrone reagent in a

Spectronic 2000 (Bausch and Lomb, Rochester, NY, USA)

(Yemm and Willis 1954).

Free proline determination of the supernatant was esti-

mated by reacting 5 mL of ninhydrin (3.125 g ninhydrin

dissolved in 50 mL of phosphoric acid 6 M and 75 mL of

glacial acetic acid) and placed in boiling water for 45 min.

The absorbance was read in a spectrophotometer at 515 nm

(Paquin and Lechasseur 1979). Free proline concentration

was calculated from a calibration curve using proline as a

standard.

Total phenolic compounds were extracted according to

Chapuis-Lardy et al. (2002) with some modifications. 0.5 g

FW of leaves were pulverized in liquid nitrogen, mixed

with 20 mL of 80 % methanol, and homogenized at room

temperature for 1 min. After filtration, 0.5 mL of each

sample was mixed with 10 mL of distilled water. Total

phenolic content was determined from aqueous solutions

by spectrophotometric analysis at 760 nm with Folin–Ci-

ocalteu reagent (Waterman and Mole 1994) (one mea-

surement per extract). Although it is not completely

specific for phenolic compounds (e.g., it is affected by

other constituents) and not all phenolic compounds exhibit

the same level of activity in the assay (Kang and Saltveit

2002), the Folin–Ciocalteu method is commonly used to

measure phenolic content. Results were expressed as mg of

gallic acid per g of DM.

Hydrogen peroxide and lipid peroxidation in leaves

The H2O2 measurement was based on that described by

Patterson et al. (1984) adapted from Aranjuelo et al. (2008)

with some modifications. Frozen leaf samples (0.5 g) were

homogenized in a cold mortar with 10 mL of 5 % tri-

chloroacetic acid (TCA) containing 0.2 g activated char-

coal and 0.1 g polyvinylpolypyrrolidone (PVPP). The

homogenate was centrifuged at 18,000g for 10 min and the

supernatant was filtered through a Millipore filter

(0.45 lm) and then used for the assay. The reaction mix-

ture consisted in 0.1 mL of extract, 1.9 mL of 0.1 M

potassium phosphate buffer (pH 7.0) and 1 mL of a col-

orimetric reagent [1:1, v/v of 0.6 mM potassium titanium

oxalate and 0.6 mM 4-2 (2-pyridylazo) resorcinol (diso-

dium salt)] prepared daily. The mixture (2 mL) was placed

into a hot bath (60 �C) for 45 min and finally was mea-

sured at 508 nm. Blanks were made by replacing the leaf

extract with 5 % TCA. Lipid peroxidation was estimated

by measuring the concentration of TBARS (thiobarbituric

acid reacting substances), as described by Heath and

Packer (1965). Frozen leaf samples (0.5 g) were homoge-

nized in 10 mL of 0.1 % TCA, filtered and centrifuged at

18,000g for 10 min. The reaction mixture consisted in

2 mL of extract and 2 mL of 0.5 % TBA (thiobarbituric

acid) in 20 % TCA. The mixture was heated in a bath at

95 �C for 30 min. After cooling, samples were measured at

532 nm and corrected for nonspecific turbidity by sub-

tracting the absorbance at 600 nm. TBARS concentration

was calculated using the following formulae:

(A532 9 1,000) - (A600 9 1,000)/155, ‘155’ being the

extinction coefficient in mM-1 cm-1 (Heath and Packer

1965).

Antioxidant enzymes and total soluble proteins

in leaves

For determining antioxidant enzymatic activities, frozen

leaf samples (0.5 g) were homogenized in 10 mL of 0.1 M

potassium phosphate buffer (pH 7.0) with 0.1 g PVPP with

a pestle and cold mortar. The homogenate was filtered and

centrifuged at 18,000g and 4 �C for 10 min. The super-

natant was separated in aliquots (1 mL) and stored at -

80 �C for the determinations of antioxidant enzymes’

activities. All enzymatic activities were expressed per mg

of protein (specific activity). Superoxide dismutase (SOD,

EC 1.15.1.1) was determined as described by Becana et al.

(1986) with some modifications, by the nitro blue tetra-

zolium (NBT) method. The reaction mixture contained

4 mL of 20 mM riboflavin, 140 mM methionine, 80 mM

NBT, 0.1 M potassium phosphate buffer and 0.1 mL of

extract. The mixtures were illuminated in a small home-

made-designed illuminated chamber (25 �C) with

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123

fluorescent lamps. The reduction of NBT was determined

at 560 nm. Blanks and controls were performed without

illumination and enzyme, respectively. Results are

expressed in units of activity. One unit of SOD activity

was defined as the amount of enzyme necessary to inhibit

by 50 % the NBT photoreduction under the assay condi-

tions. Catalase (CAT, EC 1.11.1.6) activity was deter-

mined according to Aebi (1984) with some modifications

by measuring the rate of H2O2 disappearance at 260 nm.

The incubation mixture contained 0.2 mL of extract and

1.9 mL of 10 mM H2O2 diluted in 0.1 M potassium

phosphate buffer. A calibration curve was set with H2O2

concentrations. Ascorbate peroxidase (APX, EC

1.11.1.11) was assayed by the ascorbate oxidation method

at 260 nm. The activity was determined as described by

Nakano and Asada (1981) with some modifications. A

reaction mixture containing 0.1 mL of extract, 1 mL

0.1 M ascorbate and 1 mL 0.5 mM H2O2 was prepared,

and then the absorbance was determined. A calibration

curve was set with ascorbate concentrations. Guaiacol

peroxidase (GPOX, EC 1.11.1.7) activity was estimated

according to Putter (1974) with some modifications.

Activity was assayed in a reaction mixture containing 50

lL guaiacol (20 mM) as substrate, 50 lL H2O2

(12.3 mM), 2.4 mL phosphate buffer (100 mM, pH 7.0)

and 100 lL of enzyme extract. The rate of formation of

oxidized guaiacol was followed spectrophotometrically at

436 nm. All enzymatic activities were measured at 25 �C.

Total soluble proteins (TSP) were measured by the protein

dye-binding method of Bradford (1976) using bovine

serum albumin (BSA) as a standard.

Statistical analysis

Data were analyzed with two-way ANOVA (SPSS v. 15.0)

to partition the variance into the main effects. AMF

(A) and water status (B) were used as first and second

factor, respectively. Significant differences between factors

were calculated at 5 %. Means ± standard errors (SE)

were calculated, and when F ratio was significant accord-

ing to the ANOVA analysis, least significant differences

were evaluated using least significant difference post hoc

test (LSD) (P B 0.05). All these statistical analyses were

carried out with the SPSS statistical package version 15.0

programs for windows XP (SPSS inc. Chicago, IL). All

values shown in the figures are mean ± SE.

Results

Date palm plants non-inoculated with any AMF never

showed fungal structures in roots (Table 1). Therefore, we

will refer indistinctly to these plants as ‘non-inoculated’ or

‘non-mycorrhizal’. Percentages of mycorrhizal coloniza-

tion reached values ranging between 50 and 55 % after the

application of either native or ‘exotic’ AMF (Table 1).

Reduced irrigation disfavored the colonization of date palm

roots by AMF (Table 1) (AMF 9 water status,

P B 0.001). MEI increased in date palm seedlings inocu-

lated with Gi and cultivated under water regimes equiva-

lent to 25 % FC compared with their respective WW

controls (AMF 9 water status, P B 0.001). The imposition

of WD had negative effect on MEI in plants inoculated

Table 1 Mycorrhizal colonization and growth parameters of date

palm seedlings non-mycorrhizal (NM) or mycorrhizal (M) inoculated

with Glomus intraradices (Gi), Glomus mosseae (Gm) or Complex

Aoufous (CA) and subjected to well-watered (WW) or water deficit

(WD) conditions

Water

status

AMF AM

colonization (%)

MEI

(%)

SDM

(g plant-1)

RDM

(g plant-1)

Leaf number

(per plant)

Plant height

(cm)

Root length

(cm)

Leaf area

(cm2 plant-1)

WW NM nd ND 3.49 c 1.62 bc 4.50 b 28.38 bcd 35.38 bc 43.44 c

Gi 53.11 a 35.85 c 4.90 b 2.68 a 4.75 b 31.88 b 60.00 a 52.44 b

Gm 50.45 b 21.70 d 4.32 b 1.29 cd 4.25 bc 26.50 cde 41.88 bc 33.33 d

CA 54.81 a 52.89 b 7.41 a 1.79 b 6.00 a 42.75 a 66.50 a 86.14 a

WD NM nd ND 1.37 f 0.63 e 2.67 d 23.33 e 39.67 bc 10.20 f

Gi 46.22 c 60.28 a 2.81 d 1.14 d 3.75 c 29.38 bc 43.50 b 20.63 e

Gm 22.33 e 12.38 e 1.28 f 0.34 e 2.63 d 23.69 e 34.00 bc 13.52 ef

CA 25.09 d 48.84 b 2.14 e 0.52 e 3.00 d 24.36 de 32.57 c 16.49 ef

AMF (A) *** *** *** *** *** *** *** ***

Water status (B) *** ** *** *** *** *** *** ***

A 9 B *** *** *** ns *** *** *** ***

Values are means (n = 3–6). Within each parameter data followed by the same letter indicate that values are similar (P B 0.05)

MEI mycorrhizal efficiency index, nd not detected, ND not determined, SDM shoot dry matter, RDM root dry matter

ANOVA: ns not significant; significant at * P B 0.05; ** P B 0.01 and *** P B 0.001

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123

with Gm and had no affect in plants that received the native

CA (Table 1).

Shoot and root DM were very sensitive to WD (water

status, P B 0.001) and decreased significantly in date palm

seedlings subjected to drought regardless they were or not

inoculated with AMF (Table 1). Number of leaves, height

and leaf area of seedlings were also significantly reduced

under water stress (Table 1). Mycorrhizal symbiosis

always benefited growth of plants, independently of water

regime (AMF 9 water status, P B 0.001), the positive

effect being especially evident when the plants were

inoculated with either Gi or CA.

Parameters related to water status are shown in Table 2.

When cultivated under optimal irrigation, leaf RWC

always reached values above 98 %. With the exception of

plants associated with Gi, the imposition of WD caused

significant decreases in leaf RWC, the reduction being

stronger in non-mycorrhizal than in mycorrhizal seedlings.

Non-mycorrhizal controls showed more negative values of

water potential (Ww) than mycorrhizal plants regardless of

the water regime (AMF, P B 0.001) (Table 2). When

subjected to drought, leaf Ww only remained unchanged in

plants colonized by Gi. Leaf osmotic potential varied sig-

nificantly among the different strains of AMF used in this

experiment: while WD reduced leaf osmotic potential at

full turgor (Wp100) in plants associated with Gi, Wp

100

remained unchanged in plants inoculated with CA and

increased in seedlings colonized by Gm (Table 2). Water

stress also reduced the amount of symplastic water (hsymp)

in leaves of non-mycorrhizal plants and those associated

with Gm or Gi; however, hsymp was similar in leaves of all

seedlings inoculated with CA regardless of the water

regime applied to them (Table 2). Our results showed that

cell elastic modulus (n) can vary significantly depending on

the water regime (water status, P B 0.001), the presence or

absence of mycorrhizal symbiosis (AMF, P B 0.001) and

the species of AMF associated to roots (Table 2). The

imposition of WD always caused a significant drop in n(water status, P B 0.001). The lowest values of n corre-

sponded to seedlings colonized by Gm under both WW and

WD conditions.

Analyses of hydrogen peroxide (Fig. 1a) revealed that

the levels of H2O2 were affected by drought in non-

mycorrhizal plants. In contrast, concentrations of H2O2

were similar in leaves of mycorrhizal seedlings under both

WW and WD conditions. The imposition of WD enhanced

lipid peroxidation in leaves of non-mycorrhizal date palm

seedlings as well as in leaves of plants associated with

either Gi or Gm (Fig. 1b). However, WD did not induce the

accumulation of TBARS in leaves of plants inoculated with

the native CA (Fig. 1b).

Date palm seedlings did not accumulate detectable

contents of starch in their leaves regardless of whether they

were or not associated with AMF and independently of

water regime (data not shown). Plants associated with

AMF (with the exception of those associated with Gm and

subjected to WD) had greater amounts of TSS than their

respective non-mycorrhizal controls under both WW and

WD conditions (Fig. 2a) (AMF, P B 0.001). The highest

concentrations of soluble proteins TSP were found under

optimal irrigation in non-mycorrhizal plants (6.1 mg g-1

DM) and in plants associated with Gi (5.6 mg g-1 DM)

(Fig. 2b). Water stress only induced an accumulation of

proteins in plants inoculated with CA (Fig. 2b). Interaction

Table 2 Relative water content (RWC), leaf water potential (Ww),

symplastic water (hsymp), osmotic potential at full turgor (Wp100),

osmotic potential at turgor loss (Wp0) and cell elastic modulus (n) of

date palm seedlings non-mycorrhizal (NM) or mycorrhizal (M) with

Glomus intraradices (Gi), Glomus mosseae (Gm) or Complex

Aoufous (CA) and subjected to well-watered (WW) or water deficit

(WD) conditions

Water status AMF RWC (%) Ww (-MPa) hSymp Wp100 (-MPa) Wp

0 (-MPa) n

WW NM 98.62 a 30.50 e 5.53 a 4.80 a 15.32 c 1.77 c

Gi 99.11 a 27.20 c 5.22 a 6.90 b 16.65 c 3.52 a

Gm 98.62 a 23.47 a 5.52 a 10.00 c 8.33 a 0.57 e

CA 99.41 a 27.50 c 4.00 b 15.40 e 15.60 c 3.73 a

WD NM 92.42 c 33.53 f 2.50 c 20.09 f 28.53 f 1.32 d

Gi 96.96 ab 27.95 cd 1.65 c 13.33 d 25.05 e 0.54 e

Gm 93.48 bc 25.52 b 1.90 c 6.62 b 20.02 d 0.12 f

CA 94.57 b 29.30 de 3.80 b 15.44 e 13.72 b 2.34 b

AMF (A) *** *** ns *** *** ***

Water status (B) *** *** *** *** *** ***

A 9 B ** ns *** *** *** ***

Values are means (n = 3). Within each parameter data followed by the same letter indicate that values are similar (P B 0.05)

ANOVA: ns not significant; significant at * P B 0.05; ** P B 0.01; *** P B 0.001

Trees

123

between AMF and water status was highly significant

(P B 0.001) for both TSS and TSP in leaves of date palm

seedlings.

In general terms, water regime did not affect the con-

centration of proline in leaves, independently of the pre-

sence or absence of mycorrhizal symbiosis (Fig. 3a). The

only exception was the reduced proline content in leaves of

plants inoculated with Gm when subjected to WD. Under

WW conditions and except for plants inoculated with Gi,

the concentrations of soluble phenolics compounds (SPC)

were similar in non-mycorrhizal and mycorrhizal date palm

(Fig. 3b). Drought caused an accumulation of SPC only in

leaves of non-mycorrhizal plants (Fig. 3b).

The application of WD increased SOD activity only in

leaves of mycorrhizal plants (AMF 9 water status,

P B 0.001) (Fig. 4a). Contrariwise, non-mycorrhizal

plants had similar SOD activity in leaves under WW and

WD conditions. The activity of CAT (Fig. 4b) in leaves

of date palm depended on water regime, the presence of

absence of mycorrhizal symbiosis and the species of

AMF inoculated to seedlings. The highest CAT activity

was measured in non-mycorrhizal plants subjected to

WD. In contrast, WD reduced CAT activity in leaves of

plants inoculated with Gi or Gm (Fig. 4b). APX activity

in leaves (Fig. 4c) always increased under drought (water

status, P B 0.001), the greatest activity being found

in non-mycorrhizal plants and in those inoculated with

CA. The highest GPOX activity was found in leaves of

non-mycorrhizal plants regardless water regime (Fig. 4d).

In mycorrhizal plants, GPOX activity significantly

increased under WD (AMF 9 water status, P B 0.001)

(Fig. 4d).

TB

AR

S(n

mol

g-1 p

rote

in)

0

10

20

30

40

50

60

H2O

2

(µm

ol m

g-1 p

rote

in)

5

10

15

20

25

30

35WW WS a bcab

bcbcbc bcc

ab b

cc

c cd

d

NM Gi Gm CA

0

ANOVAAMF (A) Water status (B) A x B

H2O2 * * ns

TBARS *** *** **

a

b

Fig. 1 Concentration (lmol mg-1 protein) of H2O2 (a) and lipid

peroxidation, determined by the TBARS concentration (nmol g-1

protein) (b) in the leaves of date palm seedlings non-mycorrhizal

(NM) or mycorrhizal (M) inoculated Glomus intraradices (Gi),

Glomus mosseae (Gm) or the Complex Aoufous (CA) and subjected to

well-watered (WW, white histograms) or water deficit (WD, black

histograms) conditions. Values are means (n = 3–6). Within each

parameter data followed by the same letter indicate that values are

similar (P B 0.05). ANOVA: ns not significant; *, ** and *** sig-

nificant at P B 0.05, P B 0.01 and P B 0.001, respectively

Tot

al S

olub

le S

ugar

s (T

SS)

(mg

g-1 D

M)

20

40

60

80

100

WW WS

Tot

al S

olub

le P

rote

ins

(TSP

)(m

g g

-1 D

M)

0

1

2

3

4

5

6

NM

a

Gi Gm CA

ab

bcbcd

cde

eff

f

aa

b

cccc

d

ANOVA

AMF (A) Water status (B) A x BTSS *** ns ***

TSP ** *** ***

0

a

b

Fig. 2 Concentrations (mg g-1 DM) of total soluble sugars (TSS)

(a) and total soluble proteins (TSP) (b) in leaves of date palm

seedlings non-mycorrhizal (NM) or mycorrhizal (M) inoculated with

Glomus intraradices (Gi), Glomus mosseae (Gm) or the Complex

Aoufous (CA) and subjected to well-watered (WW, white histograms)

or water deficit (WD, black histograms) conditions. Values are means

(n = 3–6). Within each parameter data followed by the same letter

indicate that values are similar (P B 0.05). ANOVA: ns not signif-

icant; *, ** and *** significant at P B 0.05, P B 0.01 and P B 0.001,

respectively

Trees

123

Discussion

Percentages of AMF colonization in roots of inoculated

plants were higher under optimal irrigation than when

plants were subjected to WD. These data agree with several

studies in which mycorrhizal symbiosis decreased when

applied water stress to host plants (Goicoechea et al. 1997,

2004; Wu and Xia 2006; Lee et al. 2012), although some

authors (Davies et al. 1992) have found enhanced devel-

opment of external hyphal network under drought. The

highest level of root colonization under low soil water

content corresponded to the exotic Gi, which suggests that

this fungal species was the best adapted and/or the most

aggressive colonizer under WD. The application of AMF

always benefited plant growth (mainly shoot biomass

production), but the greatest improvements for the date

palm plants occurred with Gi and CA. Water restriction

had no effect, decreased or benefited the efficiency of

mycorrhizal symbiosis (MEI). The nature of the water

stress can affect the mycorrhizal effect on the physiology

and growth of plants (Ruiz-Lozano et al. 1995). The ben-

eficial effect of mycorrhizal association on growth of date

palm plants, under either WW or WD, may be at least

partially explained by greater uptake of nutrients with low

mobility arising from the substrate. In fact, a previous

study of our group demonstrated that date palm plants

inoculated with AMF accumulated more K?, Ca2? Mg2?

and P in leaves than non-mycorrhizal plants when sub-

jected to drought (Faghire et al. 2010). However, non-

mycorrhizal plants and those inoculated with Gm achieved

similar height, root biomass and number of leaves

regardless water regime. This observation could be due to

carbon drain effect by the mycorrhizal fungus and/or dys-

function of plant metabolism (Liu et al. 2004). Similar

results were reported by Porcel and Ruiz-Lozano (2004)

and Zhu et al. (2012).

Mycorrhizal and non-mycorrhizal plants often display

different physiological characters. It is well documented

that AM symbiosis can improve the water status of host

plants. Porcel and Ruiz-Lozano (2004) and Baslam and

Goicoechea (2012) reported higher leaf water contents in

mycorrhizal than in non-mycorrhizal plants subjected to

drought. In general terms, our study has shown that AM

symbiosis enhanced RWC in date palm plant leaves under

WD. This improved water status of mycorrhizal plants may

be a consequence of: (1) external hyphal extraction of soil

water (Ruiz-Lozano et al. 1995); (2) stomatal regulation

through hormonal signals (Aroca et al. 2008); (3) higher

stomatal conductance and transpiration fluxes (Zhu et al.

2012); (4) indirect effect of improved phosphate and other

nutrient uptake (Goicoechea et al. 1997); (5) greater

osmotic adjustment (Wu and Xia 2006) and/or (6) higher

root hydraulic conductivity (Auge 2001) than non-mycor-

rhizal plants. However, the leaf RWC in mycorrhizal and

non-mycorrhizal plants were similar under WW conditions,

which reinforces the idea that mycorrhizal symbiosis may

be more beneficial for their host plants under adverse

conditions (Goicoechea et al. 2004).

In general terms, leaf osmotic potentials (Wp) became

more negative when date palm seedlings were subjected to

WD, but such decreases of Wp were not correlated with

increased contents of soluble sugars and/or proline.

Therefore, reductions of Wp do not indicate an osmotic

adjustment in leaves of date palms under water stress;

decreased Wp would be a consequence of passive water

loss as indicated by decreased RWC. Rapid loss of turgor

usually stops growth and other physiological processes

(Hsiao et al. 1976). Except for plants inoculated with the

Pro

line

(pm

ol g

-1 D

M)

2

4

6

8

10

WW WS

Solu

ble

Phe

nolic

Com

poun

ds (

SPC

)(m

g g

-1 D

M)

0

2

4

6

8

10

a

ab

bcbc bcc cc

a

bbcbcbc

ccc

NM Gi Gm CA

0

ANOVAAMF (A) Water status (B) A x B

Proline * ** *

SPC *** ns ns

a

b

Fig. 3 Concentrations (pmol g-1 DM) of proline (a) and total

soluble phenolic compounds (SPC) (mg g-1 DM) (b) in leaves of

date palm seedlings non-mycorrhizal (NM) or mycorrhizal (M) inoc-

ulated with Glomus intraradices (Gi), Glomus mosseae (Gm) or the

Complex Aoufous (CA) and subjected to well-watered (WW white

histograms) or water deficit (WD black histograms) conditions.

Values are means (n = 3–6). Within each parameter data followed by

the same letter indicate that values are similar (P B 0.05). ANOVA:

ns not significant; *, ** and *** significant at P B 0.05, P B 0.01

and P B 0.001, respectively

Trees

123

native AMF (CA), imposition of drought reduced the

amount of symplastic water (hSymp) in leaves of plants

colonized by Gi or Gm, which is in agreement with results

of Goicoechea et al. (1997) working with mycorrhizal

alfalfa. Only under WW conditions and except for plants

associated with Gm, mycorrhizal seedlings had stiffer walls

(higher n values). Water restriction decreased n values in

all plants. Low n values (flexible cell walls) have been

correlated with drought-adaptation of mycorrhizal plants

(Goicoechea et al. 1997) and may provide cells with a high

resistance to short-term water stress. Increased flexibility of

cell walls in leaves of mycorrhizal plants subjected to WD

can be due to a reduced lignification process (Lee et al.

2012). However, increased tissue rigidity may also occur in

response to WD and can result in physiological and eco-

logical advantages (Chaves et al. 2003).

Low water availability induces reduction in stomatal

conductance and photosynthesis, and when radiation is

high, the combined effects may stimulate the formation of

reactive oxygen species (ROS) (Munne-Bosch and Penu-

elas 2004). ROS accumulation may inhibit several key

plants processes such as photosynthesis and respiration and

may affect membrane permeability, gene expression and

protein functions (Erice et al. 2007). Our results proved

that under WD there were lower levels of MDA and H2O2

in leaves of mycorrhizal date palm in comparison with

those found in the non-mycorrhizal controls, which may

indicate enhanced drought tolerance in plants associated

with AMF. The higher oxidation of membrane lipids

(TBARS) in non-mycorrhizal plants than in those inocu-

lated with AMF when grown under WD is a reliable

indication of uncontrolled free-radical production and

hence of oxidative stress in palm seedlings not associated

with AMF (He et al. 2007). Abbaspour et al. (2012) sug-

gested two possibilities to explain the low oxidative dam-

age found in plants colonized by AMF: either mycorrhizal

plants suffered less drought stress due to a primary drought

avoidance effect by the symbiosis (e.g., direct water uptake

from soil by fungal hyphae and transfer to the host plant) or

mycorrhizal colonization induced the activation of a set of

defense enzymes involved in the elimination of ROS. The

osmolytes proline, soluble sugars (TSS) and phenols are

also considered as indexes of drought avoidance and anti-

oxidative plant defense responses. In our study, when

applied WD, the highest concentrations of TSS were found

in leaves of plants inoculated with either Gi or CA, may be

as a consequence of enhanced photosynthetic rates (San-

chez-Dıaz et al. 1990). Photosynthesis in mycorrhizal

CA

T s

peci

fic

acti

vity

(µm

ol m

g-1 p

rote

in m

in-1)

0.00

0.05

0.10

0.15

0.20

0.25

200

400

600

800

1000

GP

OX

specific activity(µ

mol m

g-1 protein m

in-1)

0

1

2

3

4

AP

X specific activity

(µm

ol mg

-1 protein min

-1)

2

4

6

8

10

a

b

c

d de

ff

a aaa

a

bbb

aa

abbb

ccc

a

ab bc

c

d d dd

NM Gi Gm CA NM Gi Gm CA

WW WS

WW WS

ANOVAAMF (A) Water status (B) A x B

SOD ns *** ***

CAT *** *** ***

ANOVAAMF (A) Water status (B) A x B

APX ns *** *

GPOX *** *** ***

a

b

c

d0 0

SOD

spe

cifi

c ac

tivi

ty

(SO

D u

nits

mg

-1 p

rote

in m

in-1)

Fig. 4 Activities of antioxidant

enzymes: superoxide dismutase

(SOD; units mg-1 protein

min-1; a), catalase (CAT;

lmol mg-1 protein min-1; b),

ascorbate peroxidase (APX;

lmol mg-1 protein min-1;

c) and guaiacol peroxidase

(GPOX; lmol mg-1 protein

min-1; d) in leaves of date palm

seedlings non-mycorrhizal (NM)

or mycorrhizal (M) with Glomus

intraradices (Gi), Glomus

mosseae (Gm) or the Complex

Aoufous (CA) and subjected to

well-watered (WW, white

histograms) or water deficit

(WD, black histograms)

conditions. Values are means

(n = 3–6). Within each

parameter data followed by the

same letter indicate that values

are similar (P B 0.05).

ANOVA: ns not significant; *,

** and *** significant at

P B 0.05, P B 0.01 and

P B 0.001, respectively

Trees

123

plants can be improved by the sink effect of the AMF

demanding sugars from leaves to roots (Porcel and Ruiz-

Lozano 2004). Under certain conditions, some plants pro-

duce large amounts of proline to enhance osmosis and

prevent dehydration (Lu et al. 2009). As explained before,

in this study, proline was not accumulated in leaves of

palm plants subjected to WD, regardless they were or not

associated with AMF. Our results are consistent with the

inability to downregulate genes under stress, thereby

causing continued proline catabolism and reduced proline

accumulation (Verslues and Sharma 2010). The imposition

of WD only induced an accumulation of soluble phenolic

compounds in leaves of non-mycorrhizal date palm plants.

Oh et al. (2009) found that the activity of the enzyme

phenylalanine ammonia-lyase (PAL), involved in the bio-

synthesis of many phenolic compounds, increased in plants

exposed to water stress. Lee et al. (2012) found higher

concentration of PAL and polyphenol oxidase (PPO) in

non-mycorrhizal than in mycorrhizal ryegrass subjected to

drought. The enhanced activity in most enzymes impli-

cated in phenols and lignin metabolism in non-mycorrhizal

plants was significantly associated with decreased Ww as

the duration of drought treatment was prolonged.

Taking into account that CAT, APX and GPOX activi-

ties are induced by H2O2 (Droillard et al. 1987) and that

H2O2 is a product of SOD activity, the similar SOD activity

together with the higher activity of CAT in leaves of non-

mycorrhizal plants in comparison with that found in leaves

of mycorrhizal seedlings under WD leads us to hypothesize

that non-mycorrhizal plants were more sensitive than

mycorrhizal ones to water stress and enhanced SOD

activity earlier than mycorrhizal plants did. Lower CAT

activity together with reduced H2O2 concentration in leaves

of mycorrhizal plants than in those of non-mycorrhizal

plants under WD provides evidence that mycorrhizal

symbiosis alleviated oxidative stress. Similarly, Roldan

et al. (2008) found lower SOD activity in mycorrhizal

Juniperus oxycedrus than in non-mycorrhizal seedlings

under drought, which was attributed to primary drought

avoidance mechanisms (Abbaspour et al. 2012).

Conclusions

According to our results, the association of date palm with

AMF benefited growth under well-watered conditions,

being the native CA the most effective in increasing shoot

height and biomass. However, when plants were under-

going restriction of water, the exotic Gi appeared as the

most beneficial fungus for improving plant growth. This

positive effect was not due to enhanced activities implied

in the antioxidant metabolism in leaves of seedlings asso-

ciated with Gi and subjected to drought. These plants

increased cell wall elasticity and were able to maintain

high water content in leaves without lowering leaf water

potential under stressful conditions. Our results demon-

strate the great importance that the adequate selection of

AMF has for improving growth of palm date plants culti-

vated under different water regimes.

Acknowledgments The authors highly acknowledge Moroccan-

Spanish; AECI-MAEC; bilateral cooperation (A/5367/06), Moroccan

ministerial fellowship for Marouane Baslam grant, Dr. A. Meddich

(nursery for date palm production, Marrakech; Morocco) for helping

to prepare and provide the native mycorrhizal inoculum (Complex

Aoufous), soil and compost and Dr. J.J. Irigoyen and Dr. I. Pascual for

excellent technical support.

Conflict of interest The authors declare that they have no conflict

of interest and there has been no significant financial support for this

work that could have influenced its outcome.

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