South African Journal of Botany 148 (2022) 1
10

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South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Effect of arbuscular mycorrhizal fungi inoculation on growth and

physiology performance of olive trees under regulated deficit irrigation
and partial rootzone drying
Badia Aganchich1,*, Said Wahbi1, Abderrahmane Yaakoubi1, Hanan El-Aououad2,
Josefina Bota2
1
Department of Biology, Biotechnology and Plant Physiology Unit, Cadi Ayyad University, Faculty of Science Semlalia, Marrakesh, Morocco
2
Research Group on Plant Biology under Mediterranean Conditions, Department of Biology-INAGEA Universitat de les Illes Balears, Carretera de Valldemossa Km
7.5, 07122 Palma de Mallorca, Spain

A R T I C L E I N F O A B S T R A C T

Article History: Agriculture water resources are limited in several Mediterranean regions. In this context, the current study
Received 13 June 2021 was conducted to examine the response of mychorrizal olive plants to two water-saving irrigation techni-
Revised 26 March 2022 ques, namely partial-root zone drying (PRD) and regulated deficit irrigation (RDI). Young olive (Olea europaea
Accepted 28 March 2022
L., cv Picholine marocaine) trees were grown with (+AMF) or without (-AMF) Rhizophagus irregularis using a
Available online 7 April 2022
split-root experiment, which consists in dividing the root system of the plant in two parts placed in separate
Edited by Dr Q. Kritzinger pots. The pots were exposed simultaneously to three different water regimes: Control, with both root com-
partments well irrigated; RDI, with both compartments irrigated (50% of the control); and PRD, with one
Keywords:
compartment irrigated (50% of the control), while the other compartment was kept dry switching every
Arbuscular mycorrhizal fungi
10 days. Stomatal conductance (gs), leaf relative water content (RWC), vegetative growth (shoot length, total
Drought resilience
leaf area and leaf number), proline and sugar contents and leaf carbon isotope discrimination (d13C) were
Olea europaea
Osmotic adjustment measured in order to evaluate the influence of arbuscular mycorrhizal (AM) symbiosis on the tolerance of
Vegetative growth the olive tree to water stress induced by RDI and PRD. The low soil moisture under this two water treatments
Water relations have a negative effect on all studied parameters. However, the olive plants inoculated with the AM fungi
(AMF) generally have significant great growth indices in comparison with the non-inoculated ones. Further-
more, at physiological (gs and RWC) and biochemical (proline, sugar and d13C) level, +AMF olive plants
exhibit better performance under drought than -AMF ones. These responses were mostly, earlier under PRD
treatment. AM olive plants can tolerate better the water deficit stress of RDI and PRD treatments saving up to
50% of the irrigation water.
© 2022 SAAB. Published by Elsevier B.V. All rights reserved.

1. INTRODUCTION and oil quality is an obligation (d'Andria et al. 1999;

The olive tree (Olea europea L.) is extensively cultivated around
the Mediterranean basin and plays an important role in the economy
of several countries of the region. In Morocco, this plant is the most
important fruit tree, covering extensive areas (1.1 M ha) (MAPM-
DREF/DSS, 2018) and providing more than 380,000 jobs annually.
Although the olive tree is a well-adapted specie against abiotic stress
(Bacelar et al. 2009), drought proves to be the essential climatic factor
responsible for the restriction of crop yield and quality
(Servili et al. 2009). Under these unpredictable and highly variable
conditions, the necessity of irrigation to increase olive production
* Corresponding author: Dr Badia Aganchich, Universite Cadi Ayyad Faculte des Sci-
ences Semlalia, Boulevard Prince My Abdellah, B.P.. . ., Tel.: +212 662835447, Marra-
kech 40000, Morocco
E-mail address: ba.aganchich@uca.ac.ma (B. Aganchich).
Aganchich et al. 2008) especially when the plant is least tolerant of
water stress.
As available water resources in Morocco become increasingly rare,
the urgent need to reduce water for irrigation and make plants more
efficient in water use is an important issue especially in the arid and
semi-arid regions.
The most effective ways to conserve water appears to be through
carefully managing deficit irrigation strategies that are supported by
an advanced irrigation system and flexible, state-of-the-art water
delivery systems. Such irrigation should emphasize on defining the
optimum water requirement that will ensure efficient water use and
avoid wastage (Evans and Salder, 2008). As a response to this objec-
tive, an important volume of applied and fundamental research has
been focused into the exploration of the capacity to optimize water
use (Wolters 1992; Burt et al. 1997; Evans and Salder, 2008).

https://doi.org/10.1016/j.sajb.2022.03.051
0254-6299/© 2022 SAAB. Published by Elsevier B.V. All rights reserved.
B. Aganchich, S. Wahbi, A. Yaakoubi et al.
The study of water use efficiency (WUE) is of particular interest in
situations where growth is affected by limited water availability
(Anyia and Herzog, 2004), and it has been demonstrated that drought
can substantially increase WUE in olive tree (Bongi and Palliotti, 1994;
Ferna ndez 2014; Abdallah et al. 2018; Trabelsi et al. 2019). There are
few studies that focus on exploring new irrigation technologies to
reduce water consumption and look for a compromise between rea-
sonable crop yield and quality and reduction of irrigation dosage.
Regulated deficit irrigation (RDI) and partial rootzone drying (PRD)
are two promising irrigation strategies to save water with a concomi-
tant WUE increase and no significant yield reduction
(Davies et al. 2002). Under RDI, the entire root zone of the plant is
irrigated with less water than required for potential evapotranspira-
tion (English et al. 1990) and, thus, maintaining soil moisture at a rel-
atively dry level. Under PRD, half of the root zone is irrigated while
the other half is allowed to dry out, with alternation of the two parts
of the root system, basically requiring two irrigation systems. The
technique based on the theoretical assumption that the irrigated
root-half would maintain the water status of the plant, whereas the
non-irrigated half would send chemical signals to the shoot via xylem
to reduce stomatal aperture and shoot growth, preventing water loss
(Gowing et al. 1990; Loveys et al. 2000; Dry et al. 2001;
Dodd et al. 2006).
It has been shown that water storage in soil and water-use effi-
ciency are positively influenced by different management practices
such as fertilizer application (Zhang et al. 2009; Grigoras et al. 2012).
Numerous researchers have demonstrated that arbuscular mycor-
rhizal fungi (AMF), which are commonly known as bio-fertilizers,
improve the quality of soil by influencing its structure and texture
(Zou et al. 2015), that increase plant tolerance to drought (Auge ,
2001; Porcel et al. 2004; Meddich et al. 2015; Ouledali et al. 2018;
Ait-El-Mokhtar et al. 2019 et al.). Over 90% of plant species including
forest trees, wild grasses, and many crops are associated with AMF
for a better exploration of soil and improvement of nutritional status
(Bonfante and Genre 2010). The symbiosis of plants roots with AMF
is one of the most ancient and widespread strategies to increase plant
tolerance to environmental stress such as drought (Wu et al. 2013).
AMF has been shown to increase plant resistance to drought
(Porcel et al. 2004) and to alleviate water stress (Sheng et al. 2008).
Arbuscular mycorrhizal plant tolerance to drought could be pri-
marily due to a large volume of soil explored by roots and the extra-
radical hyphae of the fungi (Gianinazzi et al. 2010; Zhang et al. 2015)
which can further improve the regulation of abscisic acid levels
(Aroca et al. 2008; Liu et al. 2018) and help maintain turgor pressure
by enhanced osmotic adjustment (Ruiz-Lozano and Azcon 1995;
Auge et al. 1992). Under drought conditions, AMF can also change
root hydraulic properties (Aroca et al. 2008) that facilitate water
uptake and increase net photosynthetic rates (Birhane et al. 2012).
Additionally, AMF establishes an extensive hyphal network in the
soil, mobilizing soil nutrients, mainly phosphorus (P) and nitrogen
(N) (Bompadre et al. 2013). The improvement of plant tolerance to
water deficiency by the application of AMF could be also linked to the
stimulation of the antioxidant system which up-regulates the accu-
mulation of reactive oxygen species leading to protection of major
metabolic pathways, including photosynthesis (Begum et al. 2019).
The olive tree is a particularly mycotrophic plant (Roldan Fajardo
and Barea 1986), and appears to be highly dependent on AMF under
arid conditions (Mekahlia et al. 2013). According to Vitagliano and
Citermesi (1999), the beneficial effect of AMF on olive root morphol-
ogy was the increase of lateral root frequency, giving rise to plants
with root systems consisting of a greater proportion of higher order
roots. Physiologically, the rate of photosynthesis, carboxylation effi-
ciency, transpiration and stomatal conductance in mycorrhizal olive
plants was significantly higher than that of non- mycorrhizal ones
(Mechri et al. 2014).
2
South African Journal of Botany 148 (2022) 1
10
A few studies (Neumann et al. 2009; Liu et al. 2018, Meddich et al.
2021) have reported the effect of AMF on plants under PRD and RDI
water regimes and the underlying mechanisms. In a greenhouse
experiment on potato grown with or without fertilization and sub-
jected to PRD and RDI irrigations, Liu et al. (2018) showed that inocu-
lation of Rhizophagus irregularis and Glomus proliferum maintained or
improved potato plant growth and P/N uptake when subjected to
PRD. These authors concluded that the AMF symbiosis, which facili-
tated plant P and N uptake, could fully offset the negative effect
caused by PRD and low P fertilization further increase the WUE under
low-P conditions. Recently, Meddich et al. (2021) found that the
application of PRD and RDI techniques combined with AMF signifi-
cantly improved the biomass, physiology and biochemistry of melon
plants under different levels of water stress. Our study falls under
this context, and aims to evaluate, from a morphological to a physio-
logical and biochemical level, the effect of AMF on olive plants sub-
jected to deficit irrigation with PRD and RDI strategies.
2. Materials and Methods
2.1. Plant material, experimental design and irrigation
The experiment was conducted during 2017-2018 at the Faculty
of Sciences (Marrakech, Morocco), 31° 38’ N; 8° 04’ W, 411,6m a.s.l.
One-year-old olive (Olea europaea L., cv Picholine marocaine) trees
were planted in March 2017 in pots, in a semi-controlled greenhouse
under natural light (photon flux density ranged from 500 to 750
mmol¢m
2 ¢s
1). The temperature was maintained at average 25 °C
light/dark. Relative humidity was set to 68,5 %. The root system was
split into two parts, such that each half was planted in a different
plastic pot. The two pot-compartments were tied together with the
plant crown in the middle; thus, water exchange between the two
compartments was prevented. Each pot-compartment was filled
with around 2 kg of sterilized soil; containing a 1:1:2 (v/v/v) peat/
sand/silty-clay soil mix.
At the time of dividing the root system and re-potting, half of the
plants (24 pots), were inoculated (5g, i.e. 2.5 g in each root-compart-
ment) with inoculum (Rhizophagus irregularis), which was deposited
directly below the root of the olive plantlets. AMC was multiplied by
trap culture in pots using Zea mays L. as host plant under green-
house-controlled conditions for 3 months (purified fungi strain was
obtained from the Institute of Plant Biology Research of the Univer-
sity of Quebec in Canada; Institut de Recherche en Biologie Ve ge
tale
de l’Universite Quebec. Canada). AMF inoculum consisted of a mix-
ture of rhizospheric soil from the trap culture containing spores,
hyphae and mycorrhizal root fragments.
In order to guarantee the uniformity of plants development and to
adapt to the new split-root system, all plants were kept well-watered
by irrigating both rooting compartments one year until the treat-
ments were started. Thereafter, plants of homogeneous size were
subjected to different irrigation treatments.
At the end of February 2018, six treatments with 8 replicates per
treatment were imposed in a fully randomized block design. Treat-
ments consisted of a combination of two factors: two inoculation
treatments (-AMF; non-mycorrhizal plants and +AMF; mycorrhizal
plants with Rhizophagus irregularis) and three irrigation methods
(Control, PRD and RDI). Control (irrigated with 100% of the crop
evapotranspiration, on the 2 compartments), PRD (irrigated with 50%
of the control water amount, while the other compartment was not
watered, with switching every 10 days) and RDI (both compartments
were irrigated with 50% of the control water amount, i.e. 25% in each
compartment). All plants were irrigated three times a week in the
late afternoon. The amounts of water applied were estimated based
on plant water use by the control treatment, which was measured by
weighing the pots before irrigation.
B. Aganchich, S. Wahbi, A. Yaakoubi et al.
2.2. Morphological Parameters
Shoot length was measured on one branch of current year
(which was previously tagged) per plant. The leaf number was also
counted simultaneously on the same branches. Total leaf area (LA),
of the lateral shoot was estimated from leaf dimensions i.e. leaf
length L, maximum width W and constant K (Montgomery, 1911):
LA = K * L * W. For the olive leaves K=0.67 et al.according to the
method applied by VanArkel (1978) using a sample of 100 leaves
(Aganchich et al. 2007).
2.3. Physiological parameters
2.3.1. Stomatal conductance
Stomatal conductance (gs) was measured on fully expanded
mature leaves of the same rank using a Leaf porometer (LP1989, Dec-
agonDevice, Inc., Washington, USA). Measurements were done
between 10 h and 12 h under saturating light. The measurements
were done on two leaves per plant and four replicate plants for each
water treatment.
2.3.2. Relative water content
Relative water content (RWC) was determined using two leaves
per plant and four replicates plants per treatment; they were
detached in a similar position (third leaf from top). After cutting, the
petiole was immediately immersed in distilled water inside a pre-
weighed glass tube, which was sealed. The tubes were then taken to
the laboratory and weighed; the increased weight of the tubes was
used to determine leaf fresh weight (FW). After 48 h in dim light, the
leaves were again weighed to obtain turgid weight (TW). Dry weight
(DW) was then determined after oven-drying at 80°C for 48h and
Figure 1. Evolution of shoot length of mycorrhizal (a) and non-mycorrhizal (b) olive trees subjected to three water treatments (C, RDI and PRD). Shoot length (c), Leaves number (d),
total leaf area (e) at the end of the experiment (day 96 after start of water treatment) for mycorrhizal (+AMF) and non-mycorrhizal (-AMF) olive trees subjected to three water treat-
ments (C, RDI and PRD). Values are means §SE of four replicates. Different letters indicate significant differences at P < 0.05.
3
South African Journal of Botany 148 (2022) 1
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relative water content was calculated as RWC = 100 * (FW-DW) /
(TW-DW).
2.4. Biochemical parameters
2.4.1. Plant sample collection
Four plants of each treatment were selected in each date for leaf
sampling. Each sample consisted in four healthy and fully expanded
leaves taken from newly-developed shoots. All samples were taken
and all measurements (except leaf area) were carried out regularly at
average frequency of 10 days from the beginning of April (day 30,
after the start of treatment) to the end of June (day 96, after the start
of the treatment).
2.4.2. Measurement methods
The leaf samples were transported to the laboratory and stored
immediately at
25 °C until lyophilization. Lyophilized leaves from
each treatment were ground to a fine powder which was then divided
into equal parts and stored in plastic bags. Subsequently, the leaf pow-
der samples were sent to Research Group on Plant Biology under Med-
iterranean Conditions, Department of Biology-INAGEA Universitat de
les Illes Balears, Palma de Mallorca, Spain, for biochemical analysis.
2.4.2.1. Proline content. Proline contents were determined following
the method employed by Trolls and Lindsley (1955) with some modi-
fications. A total of 0.2g of lyophilized powder was mixed with a
4.0 ml aliquot of 40% methanol in covered glass tubes and boiled in a
water bath at 100 °C during 30 min. After cooling, a 1 ml aliquot of
the extract was mixed with 2 ml glacial acetic acid, 2ml of the reagent
mixture (120 ml distilled water, 300 ml glacial acetic acid, 80 ml
orthophosphoric acid) and 1 ml ninhydrin solution (25mg/ml) and
B. Aganchich, S. Wahbi, A. Yaakoubi et al.
boiled at 100 °C for 1 h. After cooling the mixture, 4 mL of toluene
was added and it was vortexed for 15-20 s. The chromophore-con-
taining toluene was separated and absorption at 528nm was read,
using toluene as a blank. Proline concentration was calculated using
l-proline for the standard curve.
2.4.2.2. Sugar content. Soluble sugars content (expressed as
mmol¢g
1FW) was determined according to the method of Robyt and
White (1987) with some modifications. A total of 0.2g of lyophilized
leaf was mixed with a 5.0 ml of 80% methanol in covered glass tubes
and boiled in a water bath at 70 °C during 30 minutes. Sugar content
was determined with phenol-acid sulfuric. 1 ml of phenol 5% and
5 ml acid-sulfuric concentrated was added to a 1 ml extract. After agi-
tation and cooling of the mixture, absorption at 640 nm was read.
Soluble Sugars concentration was calculated using l-glucose as a
standard curve.
2.4.2.3. Carbon isotope composition in leaf dry matter. The leaf carbon
isotope (d13C) value is related to plant WUE. Lyophilized young and
completely developed leaves from different treatments were ground
into powder and subsamples of 2 mg were analyzed for isotope ratio
(d13C). Samples were combusted in an elemental analyzer (Carlo-
Erba, Rodano, Italy). CO2 was separated by chromatography and
directly injected into a continuous-flow isotope ratio mass spectrom-
eter (Thermo Finnigan Delta Plus, Bremen, Germany). Peach leaf
standards (NIST 1547, Washington) were run every eight samples.
d13C was calculated according to Eqn 1 from Farquhar and
Richards (1984):
d13 Csampleð%Þ ¼ ½ðRsample=RstandardÞ
1  1000
d13C values are referred to a Pee Dee Belemnite standard.
Figure 2. Evolution of stomatal conductance of mycorrhizal (a) and non-mycorrhizal (b) olive trees subjected to three water treatments (C, RDI and PRD). (c) Stomatal conductance
at the end of the experiment (day 96 after start of water treatment) for mycorrhizal (+AMF) and non-mycorrhizal (-AMF) olive trees subjected to three water treatments (C, RDI and
PRD). Values are means §SE of four replicates. Different letters indicate significant differences at P < 0.05.
South African Journal of Botany 148 (2022) 1
10
2.5. Statistical Analysis
Pots were randomized using completely Randomised Block
Design, with two groups of plants (inoculated +AMF and non-inocu-
lated -AMF) with four replicates and three treatments for each group
(Control, RDI and PRD). The data were subjected to analysis of vari-
ance using two-way ANOVA, followed by Tukey test to compare the
means using SPSS 26 Windows. The significance probability levels of
the results are given at the p < 0.05 level.
3. RESULTS
3.1. Plant growth
Plant growth (shoot growth, leaf number, total leaf area) was
strongly influenced by mycorrhizal colonization and water deficit
(Figures 1) Under well-watered conditions, these parameters were
higher in +AMF plants compared to -AMF ones, but shoot length was
significantly reduced in response to PRD and RDI treatments in both
-AMF plants and +AMF (Figure 1- a,b). At the end of the experiment
(day 96), for -AMF plants, PRD and RDI caused respectively 42.6% and
28.3% reduction of shoot length. However, for +AMF plants, the
effects of PRD and RDI on shoot length were similar without signifi-
cant differences, i.e. 44.4% and 37.2% reduction for PRD and RDI
respectively (Figure 1- c). In both -AMF and +AMF plants, PRD treat-
ment tends to reduce the shoot elongation more than RDI treatment.
The effect of mycorrhization is more pronounced on PRD and well-
watered plants reaching for C about 12 cm in +AMF and just 9.5 cm in
-AMF, whereas under RDI treatment, the shoot length was similar in
ð1Þ
-AMF and +AMF plants, although the effect of the mycorhrization on
shoot length was slightly stronger in comparison to the -AMF plants
(Figure 1-c).
4
B. Aganchich, S. Wahbi, A. Yaakoubi et al.
Figure 3. Evolution of leaf relative water content of mycorrhizal (a) and non-mycorrhizal (b) olive trees subjected to three water treatments (C, RDI and PRD). (c) Leaf relative water
content at the end of the experiment (day 96 after start of water treatment) for mycorrhizal (+AMF) and non-mycorrhizal (-AMF) olive trees subjected to three water treatments (C,
RDI and PRD). Values are means §SE of four replicates. Different letters indicate significant differences at P < 0.05.
The number of newly developed leaves was significantly reduced
(p˂0.05) in both groups of plants (-AMF and +AMF) in response to
deficit irrigation treatments (Figure 1- d). Although the differences
were not significant between PRD and RDI treatments, in -AMF plants
the leaves number was more reduced by RDI (37.8%) than by PRD
(26.7%). Whereas, in +AMF the leaves number reduction was more
pronounced under PRD treatment (42.9%) comparatively to the RDI
treatment (34.9%). Under well-watered conditions, +AMF plants
showed more newly developed leaves reaching a mean value around
16 leaves against only 11 leaves in -AMF plants.
Similarly, in the two groups of plants (-AMF and +AMF) the total
leaf area was significantly reduced (p˂0.05) under PRD and RDI com-
pared with the control (Figure 1- e). For both -AMF and +AMF plants,
total leaf area was much more reduced under PRD than under RDI
relative to the control. For all water treatments, AMF increased signif-
icantly (p˂0.05) leaf area especially under well-watered conditions.
3.2. Physiological changes
Stomatal conductance (gs) was affected by water deficit and
mycorrhization (Figure 2- a,b). Under well-watered conditions (C) gs
was higher in +AMF plants compared to -AMF ones. Water deficit
caused a substantial decrease of gs under PRD and RDI treatments,
but to a greater extent in -AMFplants than +AMF plants. This decrease
was rapid under PRD especially in -AMF ones; reaching gs values of
47.03 mmol m
2¢s
1 against a gs value of 55.86 mmol m
2¢s
1 under
RDI treatment. At the end of the experiment, differences among
treatments were significant either in -AMF or in +AMF plants; on the
other hand, differences between myc
and +AMF plants were signifi-
cant under the three water treatments (Figure 2- c).
5
South African Journal of Botany 148 (2022) 1
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Similarly to gs, leaf relative water content (RWC) was also affected
by water deficit and mycorrhization (Figure 3- a,b). Soil drying under
PRD and RDI irrigations lead to a significant decrease in leaf relative
water content in both +AMF and -AMF plants, being more acute in
-AMF plants (26.2% and 16.1% of RWC reduction by RDI and PRD
respectively) than +AMF ones (24.7% and 8.6% of RWC reduction by
RDI and PRD respectively) especially in day 74 after the beginning of
the experiment, which was the hottest day (39°C) during all experi-
ment’ days. Plants exposed to RDI had a constant and relatively low
RWC values than PRD, reaching a minimal value around 67.3% and
62% in +AMF and -AMF plants respectively, whereas plants subjected
to PRD treatment tend to remain closer to the control especially in
+AMF plants. At the end of the experiment, differences among water
treatments were significant, with low RWC values under RDI, inde-
pendently from the mycorrhization treatment (Figure 3-c). Under
well-watered conditions, there were no significant differences
between -AMF and +AMF plants in RWC, whereas, for plants sub-
jected to PRD and RDI treatments RWC was significantly improved by
inoculation (Figure 3- c).
3.3. Biochemical changes
3.3.1. Isotopic abundances in plant shoots
A significant increase in leaf 13C/12C isotope ratio (d13C) occurred
in response to water deficit caused by PRD and RDI irrigations
(Figure 4- a,b). This increment was more pronounced in plants sub-
jected to RDI than in those subjected to PRD. For the three water
treatments, +AMF plants have high d13C values than -AMF ones. At
the end of the experiment, differences were significant (p˂0.05)
between all water and mycorrhization treatments (Figure 4- c).
B. Aganchich, S. Wahbi, A. Yaakoubi et al.
Figure 4. Evolution of leaf carbon isotope ratio d13C (%) of mycorrhizal (a) and non-mycorrhizal (b) olive trees subjected to three water treatments (C, RDI and PRD). (c) Leaf carbon
isotope ratio d13C (%) at the end of the experiment (day 96 after start of water treatment) for mycorrhizal (+AMF) and non-mycorrhizal (-AMF) olive trees subjected to three water
treatments (C, RDI and PRD). Values are means §SE of four replicates. Different letters indicate significant differences at P < 0.05.
3.3.2. Proline and Soluble sugars contents
The evolution of leaf proline content was influenced by mycorrh-
ization and water treatments (figure 5- a,b). Under control and PRD,
proline content was slightly higher in +AMF plants than -AMF ones,
reaching maximal values (1.3; 1.9 mmole proline/gdw and 0.9; 1.4
mmole proline/g dw in +AMF and -AMF plants respectively) in day 74
after the beginning of the treatment, during which the most elevated
temperature was recorded. However, under RDI treatment proline
accumulation in the leaves of +AMF plants was more much pro-
nounced than -AMF ones, reaching maximal values around 2.7 and
1.87 mmole proline/g dw for +AMF and -AMF plants respectively
(Figure 5- a,b). Indeed, during all experiment’ days for both -AMF and
+AMF plants the accumulation of proline was significantly different
between PRD and RDI treatments. At the end of the experiment, RDI
plants accumulated 1.5-fold higher proline than PRD ones either in
-AMF or +AMF plants (Figure 5- c). Differences between -AMF and
+AMF plants were only significant under drought conditions (PRD
and RDI) (Figure 5- c).
The evolution of soluble sugars concentration in leaves was also
influenced by water deficit and mycorrhization (Figure 6- a,b). Inocu-
lation stimulated soluble sugars accumulation not only under water
deficit involved by PRD and RDI treatments but also under well-
watered conditions. In the two groups of plants (-AMF and +AMF),
PRD treatment resulted in higher sugars accumulation comparatively
to the other water treatments. On day 74 after the beginning of the
experiment (39°C), all plants had maximal soluble sugars concentra-
tions. Plants (-AMF or +AMF) subjected to PRD treatments accumu-
lated 1.5-fold higher sugars than those subjected to RDI. Differences
at the end of the experiment were significant (p˂0.05) among water
and between mycorrhization treatments (Figure 6- c).
6
South African Journal of Botany 148 (2022) 1
10
4. DISCUSSION
4.1. Effect of AMF, RDI and PRD on growth parameters
The present study shows that inoculation of young olive plants
with Rhizophagus irregularis stimulated olive growth not only under
water-deficit conditions, but also under well-watered conditions.
These observations are in agreement with several studies showing
the positive effects of AMF on the growth and the biomass of different
plant species under well-watered and water stress conditions
(Wu and Xia 2004; Meddich et al. 2015; Pozo et al. 2015;
Zou et al. 2015; Liu et al. 2018;). Also, in our study, the results
obtained from the young seedlings of ‘Picholine marocaine’ cultivated
in pot; show that plant vegetative growth was substantially reduced
under both PRD and RDI compared to the control. This reduction was
more pronounced in non-inoculated plants. The lower leaf number
caused by both the limited amount of water applied and the irriga-
tion method resulted in a significant decrease of total leaf area.
According to Medrano et al. (2002), the reduction in leaves number
and area in plants growing under drought stress influenced nega-
tively stomatal conductance and photosynthetic rate. In this study,
during all experiment’ days, the effect of PRD on shoot length was
stronger than that of RDI. This effect was also observed in leaf num-
ber and total leaf area especially at the end of the experiment. This
result confirms our previous study on mature olive trees where the
significant reduction of growth under PRD treatment resulted in an
improvement in olive and oil quality (Aganchich et al. 2008). The
association with AMF enhanced the olive growth under the three
water treatments. Recently, Khalil and El-Ansary (2020) showed in
‘Koroneiki’ and ‘Manzanillo’ olive cultivars that in both inoculated
B. Aganchich, S. Wahbi, A. Yaakoubi et al.
Figure 5. Evolution of leaf proline content of mycorrhizal (a) and non-mycorrhizal (b) olive trees subjected to three water treatments (C, RDI and PRD). (c) Leaf proline content at
the end of the experiment (day 96 after start of water treatment) for mycorrhizal (+AMF) and non-mycorrhizal (-AMF) olive trees subjected to three water treatments (C, RDI and
PRD). Values are means §SE of four replicates. Different letters indicate significant differences at P < 0.05.
and non-inoculated plants, growth rate, trunk cross-sectional area,
leaves area and total dry weights decreased with increasing deficit
irrigation levels. However, mycorrhizal inoculation significantly
improved growth performance of olive cultivars under well-watered
and water stress conditions. Moreover, the use of AMF with olive
trees has shown that mycorrhizae increases the plant development
and production (Estau  n et al. 2003). Reduced plant size could help to
conserve water, while at the same time the AMF hyphae may
increase the efficiency of roots to take nutrients and water from a
drying soil (Smith et al. 2010). In this study, the reduction of the leaf
number and total leaf area by RDI treatment was compensated by
inoculation of plants by Rhizophagus irregularis. In fact, there are no
significant differences between C -AMF and RDI +AMF for these two
parameters (Figure 1- d,e). Liu et al. (2018) observed similar results
on potato plants grown in a greenhouse with AMF+ or AMF- and sub-
jected to water deficient under PRD and deficit irrigation (DI). These
authors showed that the similar biomass production in control AMF-
plants with RDI/PRD AMF+ plants indicated that the growth limita-
tion by RDI/PRD was fully compensated by inoculation. Under PRD,
which gives the same amount of water as RDI but on the half of the
root system, differences were significant for the three growth param-
eters between C -AMF and PRD +AMF (Figure 1- c,d,e). Thus, in the
present study the limitation of olive growth observed under PRD
treatment was not compensated by inoculation. The stronger growth
inhibition caused by both the low water amount and the irrigation
method (PRD) resulted in a significant decrease of leaf area. This
reduction might result in a better balance between vegetative and
reproductive development in olive more under PRD than under RDI
treatment. In fact, Loveys et al. (2000) have found that the reduction
in canopy density was shown to result in better light penetration to
the bunch zone and therefore improvement in grape quality.
7
South African Journal of Botany 148 (2022) 1
10
4.2. Effect of AMF, RDI and PRD on physiological parameters
In both -AMF and +AMF plants, PRD and RDI treatments caused a
decrease in gs and RWC. However, these two physiological parameters
were maintained slightly higher in +AMF plants comparatively to
-AMF ones. A well-developed root system is important to provide
water uptake for plant function, development, and tolerance to water
stress (Porcel et al. 2012). According to Porras-Soriano et al. (2009),
the growth response to AMF colonization in the olive is more effective
in improving root development than shoot development (Porras-
Soriano et al. 2009). The higher leaf RWC under PRD compared with
RDI could be due, at least partially in -AMF plants, to plant’s long dis-
tance signaling system (by the dried half of roots) to control plant
growth and prevent physiological effects associated with severe water
deficits (Stoll et al. 2000; Kang an Zhang, 2004). In addition, the inocu-
lation of young olive trees with the AMF in +AMF plants would
improve their resilience to drought. Mycorrhizal association with host
plants increases the hydration status at whole-plant level, as charac-
terized by leaf relative water content (Wu and Zou, 2017;
Barros et al. 2018). It seems that the mycorrhization effect on gs is
more important under water stress treatments, which show at the end
of the experiment an increase in gs of 24.6 and 23.06% for PRD and RDI
respectively against only 9.6% for well-watered plants. Our result is in
accordance with previous studies on AMF inoculated olive plants sub-
jected to drought conditions, which report that the stomatal function
of +AMF olive plants is less affected by drought compared to -AMF
plants (Ouledali et al. 2018; Ouledali et al. 2019). AMF-inoculation of
olive plants helped to some extent to maintain transpiration and sto-
matal conductance in plants under water deficit conditions
(Ouledali et al. 2019). In potato plants, a regression of photosynthetic
rate against stomatal conductance indicated that photosynthetic rate
B. Aganchich, S. Wahbi, A. Yaakoubi et al.
Figure 6. Evolution of soluble sugars content of mycorrhizal (a) and non-mycorrhizal (b) olive trees subjected to three water treatments (C, RDI and PRD). (c) soluble sugars content
at the end of the experiment (day 96 after start of water treatment) for mycorrhizal (+AMF) and non-mycorrhizal (-AMF) olive trees subjected to three water treatments (C, RDI and
PRD). Values are means §SE of four replicates. Different letters indicate significant differences at P < 0.05.
was highly correlated (0.81) with stomatal conductance under all con-
ditions (Liu et al. 2018). In this way, AMF could increase net photosyn-
thesis by improving the plant water status because the increase of
stomatal conductance leads to a greater diffusion of CO2 within the
mesophyll (Boldt et al. 2011). The results of Zhu et al. (2018) suggest
that AM symbiosis alters stomatal morphology by changing stomatal
density and the size of the guard cells and stomatal pores, thereby
improving the stomatal conductance and water relations of wheat
leaves under abiotic stress. During our experiment, gs was more
reduced under PRD than the other water treatments, a result that is in
agreement with the findings of Dry et al. (2000), which indicate that
withholding water from half of the root system seems to induce a fast
reduction of stomatal conductance, photosynthesis and growth.
4.3. Effect of AMF, RDI and PRD on biochemical parameters
Low water availability reduces stomatal conductance and the
resulting reduction of leaf internal CO2 concentration to a more com-
plete fixation of the internal CO2 pool, increases the relative fraction
of 13CO2 incorporated, and thus elevates plants d13C
(Courty et al. 2011). The concentration of CO2 inside the leaf (Ci)
depends on the rate of photosynthesis and the opening of the stoma-
tal pores, which influences isotopic discrimination (Tomas, 2012).
The carbon isotope composition (d13C) in leaf dry matter was used as
a long-term indicator of leaf WUE (Condon et al. 2004). Furthermore,
a negative correlation was noticed between d13C values in leaves
and the soil water content for some tree species, and plants growing
in drought habitats have higher leaf d13C values (Yan et al. 1998;
Rong et al. 2008). In our study d13C was significantly increased under
water deficit (Figure 4). Similarly, El Aou-Ouad et al. (2017) showed
in Malvasia of Banyalbufar vine-cultivar that d13C has significantly
increased as a consequence of water depletion. In addition, in a study
8
South African Journal of Botany 148 (2022) 1
10
using d13C to evaluate the effect of climate change on olive trees,
Basheer salimia and Sayara (2017) have found that Roomi cultivar
exhibited less negative d13C values in dry areas and more negative
values in high rainfall areas. At leaf level, the ratio of net assimilation
(NA) to gs (NA/gs) is an estimation of WUE. It typically increases
under water stress conditions (Bota et al. 2001; Flexas et al. 2002b),
and when d13C becomes less negative, so that it correlates positively
with NA/gs (Chaves et al. 2007). In our study, plants under PRD treat-
ment were less stressed than under RDI, since their d13C was more
negative. Such a result confirms the early closure of stomata observed
under PRD (Figure 2), and consequently higher RWC values under
this treatment (Figure 3). In fact, in olive, PRD induces partial stoma-
tal closure without significantly reducing leaf water status
(Aganchich et al. 2007, 2009, 2013), and results in increased WUE
(Wahbi et al. 2005). AMF inoculation significantly increased olive
shoot d13C as compared with non-mycorrhizal plants (Figure 4) sug-
gesting that AMF improved plants water status and WUE. Our result
agrees with biomass increase after inoculation, which suggests an
enhanced photosynthesis that results in higher d13C values for inoc-
ulated plants (Country et al. 2015).
Moreover, photosynthetic rates usually correlate with lower iso-
topic fractionation and higher d13C values (Farquhar et al. 1989).
Higher d13C in mycorrhizal Olea seedlings was associated with
increased growth, which further suggests higher intrinsic photosyn-
thetic capacity in the inoculated plants during the soil dry down
period (Querejeta et al. 2003). According to these authors, the intrin-
sic WUE of the inoculated plants was higher than the controls during
drought only. Mycorrhizal olive, with its high d13C, slow growth rate
and ability to strongly increase WUE to cope with severe drought, is
clearly a representative of a conservative water use strategy
(Donovan and Ehleringer 1994). We suppose that a similar phenome-
non occurred in our experiment, with RDI and PRD treatments.
B. Aganchich, S. Wahbi, A. Yaakoubi et al.
AMF colonization can induce other changes in plants that may
increase drought tolerance. An important defense with deficit irriga-
tion is the production of non-enzymatic substances, which consist of
low-molecular-weight substances such as soluble sugars and proline
(Mansouri-Far et al. 2010). A higher concentration of osmolytes facili-
tates water diffusion in cells and maintains the turgor of plant tissues.
These substances regulate osmotic potential in plants by the law of
mass action to reduce osmotic stress and enhance plant water hold-
ing capacity (Chai et al. 2016). Our results show that under water def-
icit induced by RDI and PRD treatments, the osmotic adjustment by
proline and sugar was earlier in +AMF plants than in
AMF ones. This result agrees with previous reports showing that
most mycorrhizal plants have a better osmotic adjustment than non-
mycorrhizal plants (Porcel and Ruiz-Lozano, 2004;
Ouledali et al. 2018). According to Khalil and El-Ansary (2020), olive
plants decreased the osmotic potential by synthesizing proline that
participates in the osmotic adjustment to cope with drought stress.
In this study, mycorrhizal olives, 50% soil water-loss is reflected in
higher proline and sugar accumulation in plants subjected to RDI and
PRD respectively (Figures 5, 6). More sugar accumulation in PRD
plants occurred in the dry part of the root system, and would provide
greater capacity of osmotic adjustment to support drought stress. In
our split-root experiment, we suppose that withholding water from
half of the root system (for PRD irrigation) may have been less dam-
aging to plants with higher rates of AMF colonization in the dry part.
In fact, several researches demonstrated that PRD increases the activ-
ity of soil microorganisms and higher root nutrient uptake capacity
(Wang et al. 2013). Elhani et al. (2019) concluded from a study on
potato subjected to different water-saving irrigation techniques, that
transcript level of some key-genes involved in carbohydrate metabo-
lism was elevated at 20% water-saving in PRD, but not in RDI tubers
confirming that the mode of irrigation is sensed by the plant.
5. CONCLUSION
In conclusion, this study shows that the combination of 50% deficit
irrigation and Arbuscular mycorrhizal fungi could be a good strategy
for enhancing the resilience of olive trees to drought. Primarily deficit
irrigation, particularly PRD, controls vegetative vigor, maintains ele-
vated water content and stimulates osmotic adjustment. Secondarily,
the mycorrhizal response showed a better performance under both
PRD and RDI treatments than control. As for the other studied param-
eters, the inoculation with AMF induced a modification of olive d13C.
This parameter could be used in a future perspective to determine
WUE and carbon fixation of olive plant. These findings should con-
firm the advantage of mycorrhization as a tool that boost water-sav-
ing management through PRD and RDI strategies.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
The authors would like to thank Professor Abdelilah Meddich for
providing the mycorrhizal inoculation product. We would also like to
thank Mrs. Cyril Douthe for d13C analysis and Mss. Alicia Perera for
laboratory assistance and scientific support in antioxidant capacity
analysis (that we couldn’t put in this paper). Thanks to Research
Group on Plant Biology under Mediterranean Conditions, Department
of Biology-INAGEA Universitat de les Illes Balears, Spain. The authors
thank Professor Mohamed Ait-El-Mokhtar for assistance with statisti-
cal analysis.
9
South African Journal of Botany 148 (2022) 1
10
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