DOI: 10.

1007/s11099-016-0243-5 PHOTOSYNTHETICA 55 (2): 308-316, 2017

Arbuscular mycorrhizal fungus Rhizophagus irregularis influences

M. TEKAYA*, +, B. MECHRI*, N. MBARKI*, H. CHEHEB**, M. HAMMAMI*, and F. ATTIA***,****

Laboratoire de Biochimie, USCR Spectrométrie de Masse, LR-NAFS/LR12ES05 Nutrition – Aliments Fonctionnels et

Abstract

In this study, we hypothesized that colonization of olive trees (Olea europaea L.) with the arbuscular mycorrhizal fungus
Rhizophagus irregularis could modify the profiles of rhizosphere microbial communities with subsequent effects on
nutrient uptake that directly affects olive tree physiology and performance. In this context, a greenhouse experiment was
carried out in order to study the effects of mycorrhizal colonization by R. irregularis on photosynthesis, pigment content,
carbohydrate profile, and nutrient uptake in olive tree. After six months of growth, photosynthetic rate in mycorrhizal (M)
plants was significantly higher than that of nonmycorrhizal plants. A sugar content analysis showed enhanced
concentrations of mannitol, fructose, sucrose, raffinose, and trehalose in M roots. We also observed a significant increase
in P, K, Ca, Mg, Zn, Fe, and Mn contents in leaves of the M plants. These results are important, since nutrient deficiency
often occurs in Mediterranean semiarid ecosystems, where olive trees occupy a major place.

Additional key words: arbuscular mycorrhizal symbiosis; carbohydrates; chlorophyll; gas exchange; lipids; mineral nutrition.

Introduction
Arbuscular mycorrhizal (AM) fungi are widespread in plants (Marschner et al. 2004, De Maria et al. 2011). It has
soils, and growth of mycorrhizal plants is often enhanced been demonstrated that some bacteria are able to
in comparison to nonmycorrhizal (NM) plants. This bene- synthesise several plant growth regulators including
ficial effect on plant growth has largely been attributed to indole-3-acetic acid and cytokinins, which can increase the
high uptake of nutrients, such as P, Zn, Cu, and Fe (Porras- root surface absorption area resulting in a better uptake of
Soriano et al. 2009). Apart from the influence of AM fungi water and nutrients (Glick et al. 1998, Wu et al. 2005).
on nutrient uptake, other positive aspects of mycor- Additionally, it has been reported that rhizosphere
rhization include an increase of plant tolerance to drought microorganisms can differently alter bioavailability of
(Ruiz-Sánchez et al. 2010), salt stress (Porras-Soriano et nutrients through the release of chelating substances,
al. 2009), as well as resistance to pathogens (Wehner et al. acidification of the microenvironment, and by changing
2010), alleviation of oxidative stress, and enhancement of the redox potential, modifying soil conditions which
antioxidant responses (García-Sánchez et al. 2014). contribute to the mobilisation and uptake of nutrient in the
AM fungi have been shown to interact with different tissues of terrestrial plants (Marschner et al. 2004).
groups of soil bacteria and to modify the rhizosphere Recently we have reported that colonisation of olive trees
microbial community (Wamberg et al. 2003, Mechri et al. with the AM fungi R. irregularis increased the number of
2014). Microbial communities can alter nutrient cycling in actinomycetes and decreased the level of Gram-negative
the rhizosphere, thus affecting nutrient availability to and Gram-positive bacteria in mycorrhizal rhizosphere soil

———
Received 21 November 2015, accepted 5 May 2016, published as online-first 19 May 2016.
+Corresponding author; phone: +216 73 462 200, fax: + 216 73 460 737, e-mail: [email protected]

Abbreviations: AM – arbuscular mycorrhizal; Car – carotenoids; Chl – chlorophyll; DM – dry mass; ICP-AES – inductively coupled
plasma atomic emission spectroscopy; FAMEs – fatty acid methyl esters; FID – flame ionization detection; M – mycorrhizal; NM –
nonmycorrhizal; Pi – inorganic phosphorus; XDH – xylitol hydrogenase; XK – xylulose kinase; XR – xylose reductase.
Acknowledgements: Special thanks go to the personnel of “Institut de l’Olivier de Sousse-Tunisia” and also to the members of “LAB-
NAFS, Faculty of Medicine of Monastir-Tunisia”.

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(Mechri et al. 2014). A higher concentration of glucose questions were addressed: how olive trees would respond
and trehalose and a lower concentration of fructose, to the specific microenvironment created after the
galactose, sucrose, raffinose, and mannitol were also colonisation of their roots by the AM fungi R. irregularis?
detected in mycorrhizal rhizosphere soil (Mechri et al. Could AM fungi contribute to the improvement of olive
2014). In view of the above background, the following tree performance?

Materials and methods

Experiment description and determination of degree of concentration was determined on dried material of M and
mycorrhization: Experimental design used in this work NM plants. Total N was determined in accordance with the
was described previously (Mechri et al. 2014). Briefly, Kjeldahl method. For the determination of other element
spores of Glomus intraradices DAOM 197198, now concentrations, about 100 mg of dry sample of leaves were
R. irregularis DAOM 197198 (Krüger et al.2012), used in ashed in a muffle furnace at 700°C for 24 h, and
this study, were inoculated in a sample of olive plantlets mineralized with HNO3. Foliar P, Ca, K, Mg, Zn, Mn, Fe,
(15 cm long and three pairs of leaves) produced in vitro. B, and Cu were analyzed by ICP-AES (Thermo Jarrell Ash
After two weeks of acclimation in a greenhouse, the olive Corp., Franklin, MA, USA).
plantlets were potted into individual 10-L pots of 20 cm in
diameter (one plant per pot) filled with a sandy P-poor soil Total lipids in the roots: Fatty acids of M and NM roots
(pH: 7.68; sand: 91.6%; silt: 1.5%, clay: 6.9%, Ctot: 4.3 g were analyzed by gas liquid chromatography after
kg–1, Olsen-P: 8.38 mg kg–1) collected directly from an conversion to the corresponding methyl esters. The
olive tree field. At the time of re-potting, 1,000 spores of procedure used was described by Schutter and Dick (2000)
R. irregularis were deposited directly below the roots of and used mild alkaline hydrolysis [0.2 M KOH in
each plantlet. The experiment was conducted in a fully methanol] to extract whole cell fatty acids and
randomized block design with two treatments and three simultaneously convert fatty acids of root lipids to methyl
replications. Treatments consisted of nonmycorrhizal esters. Briefly, 30 mg of M and NM roots plants were
plants (NM; 0 spores of R. irregularis) and mycorrhizal mixed with 15 ml of 0.2 M KOH in methanol, and the
plants (M; 1,000 spores of R. irregularis). The experiment preparation was incubated for 1 h at 37°C, during which
was carried out under controlled greenhouse conditions. ester-linked fatty acids were released and methylated.
The average air temperature in the greenhouse was Fatty acids methyl esters (FAMEs) were extracted into an
25–30°C. Plants were grown under natural light. Plants hexane organic phase, and the sample was centrifuged at
were watered every second day to maintain a soil water 480 × g for 10 min to separate the aqueous and hexane
level corresponding to 65% of the field capacity. Six phases. The hexane layer was transferred to a clean tube,
months after planting, plants were harvested and leaves and the hexane was evaporated off, after which FAMEs
and roots were collected from each treatment. were resuspended in 250 µl of hexane for analysis.
For the estimation of R. irregularis colonization, roots Samples (1 µl) of the hexane phase were separated by gas
were stained with Trypan blue (Phillips and Hayman 1970) chromatography (GC) on a HP-5MS capillary column
and the colonization of root pieces was analysed using a (30 m × 0.25 mm) and quantified using a flame ionization
stereomicroscope (Carl Zeiss, Jena GmbH, Germany). detector (6890, Agilent, USA). The following temperature
program was set: 60°C for 1 min, from 60 to 160°C at 10°C
Photosynthetic performance and pigment content: min1, from 160 to 270°C at 5°C min1, and finally
Photosynthetic rates was measured six months after remained at 270°C for 2 min in order to clean the column.
planting by using a LI-6400 gas-exchange system (Li-Cor,
Lincoln, NE, USA) on six replications per treatment. Soluble carbohydrates in the leaves and roots: Soluble
During photosynthetic measurements, the air mean tem- carbohydrates were extracted according to the method
perature was 25°C, the CO2 concentrations were 400 µmol described by Bartolozzi et al. (1997). Briefly the soluble
mol–1, and the photosynthetic photon flux density was carbohydrates from composite leaves and roots samples
maintained at 1,500 µmol m–2 s–1. Chlorophyll (Chl) and were extracted twice in 80% ethanol at 70°C. Extracts
carotenoids (Car) were extracted by grinding 0.5 g of fresh were dried and converted into trimethylsilyl ethers with a
leaves in 80% acetone. The extract was filtered and silylation mixture made up of pyridine, hexamethyl-
centrifuged at 15,000 × g for 5 min. The supernatant was disilazane, and trimethylchlorosilane. Soluble carbo-
collected and read at 663 and 647 nm for Chl a and Chl b, hydrates were analyzed using a Hewlett-Packard 5890
respectively, and at 470 nm for Car content (Perkin Elmer series II gas chromatograph equipped with a flame
Lambda 25, Boston, MA, USA). The concentrations of ionization detection (FID) system and a HP-5MS capillary
pigments were calculated according to the equations column (30 m × 0.25 mm). Injector and detector
described by Camejo et al. (2005). temperatures were 280°C and 300°C, respectively. The
following temperature program was set: 80°C for 1 min,
Nutrient concentrations in the leaves: The foliar nutrient from 80 to 170°C at 10°C min1, from 170 to 200°C at

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15°C min1, from 200 to 315°C at 25°C min1, and finally Statistical analysis: The experiment was a completely
315°C for 8 min. Using this program, 23.6 min were randomized design with three replications. The signifi-
required to elute the trimethylsilyl derivatives. Identifi- cance of differences between mean values was determined
cation of individual carbohydrates was achieved by the use by one-way analysis of variance (ANOVA). Duncan's
of the relative retention times, i.e., in comparison to that of multiple range test was used to compare the means. The
the trimethylsilyl derivates of standard carbohydrates. significance probability levels of the results are given at
the P<0.05 level.
Results

Degree of mycorrhizal colonization: The roots of NM Soluble carbohydrates and total lipids: Analyses of leaf
olive trees were observed after six months after planting, extracts from M and NM plants revealed that mannitol was
confirming the lower level of mycorrhizae (3.1%) as the predominant sugar compound of the total amount of
compared to that of the M plants (51.9%). NM olive trees soluble carbohydrates (Table 2). The amounts of glucose,
had only 0.9% of arbuscule abundance as compared to that fructose, galactose, mannose, arabinose, rhamnose,
of M plants (29.5%). xylose, inositol, mannitol sucrose, trehalose, and raffinose
in the leaves of the M plants were not significantly
Photosynthetic rate, pigment content and nutrient different from that in the leaves of NM plants (Table 2).
uptake: The rate of photosynthesis in M plants was Similarly, the most abundant sugar in M and NM roots
significantly higher than that of NM plants. In this was mannitol (Table 3). Soluble carbohydrates in roots
experiment, the percentage increase in the rate of showed a significant change under inoculation of olive
photosynthesis of M compared with NM plants was 19% trees with R. irregularis. The M roots contained
(Table 1). There was no significant difference in the Chl a, significantly higher contents of fructose, mannitol,
Chl b, and Car contents of the M plants comparing with sucrose, trehalose, and raffinose, whereas the amount of
NM plants (Table 1). In comparison with the NM plants, mannose, arabinose, rhamnose, inositol, glucose, and
leaf nutrient analysis of the M plants showed significantly galactose in the roots of the M plants were not significantly
higher concentrations of P, K, Ca, Mg, Mn, Fe, and Zn. different from that in the roots of NM plants. A significant
However, the foliar concentrations of N, Na, and Cu in the decrease of xylose was observed in M compared with NM
M plants were not significantly different from those in NM roots (Table 3). The amount of total lipids in the roots of
plants (Figs. 1, 2). The foliar B concentration decreased M plants was significantly higher than that of NM plants.
significantly in the M plants. The foliar B concentration in In this experiment, the percentage increase of total lipid
leaves of M olive trees was nearly 30% lower than that in amount was 24% in the roots of M plants compared with
NM trees (Fig. 2). NM plants.
Table 1. Photosynthetic rate, chlorophyll a, chlorophyll b, and carotenoid contents of mycorrhizal and nonmycorrhizal olive tree plants.
The effect of Rhizophagus irregularis treatment was tested with one-way ANOVA (mean value ± SE, n = 6 for photosynthesis, n = 3 for
chlorophylls, n = 3 for carotenoids), and mean values in individual line followed by the same letter(s) are not significantly different at
P<0.05 (Duncan's test).

Parameter Treatment
Mycorrhizal Nonmycorrhizal

Photosynthetic rate [µmol m2 s1] 27.5 ± 1.56A 22.2 ± 0.4B

Discussion
The present study showed a significant difference in the which corresponded well with most of the growing
percentage of root colonization between inoculated and attributes showing higher growth in Koroneiki. In this
uninoculated olive trees. This finding is consistent with experiment, noninoculated olive trees also showed some
that of Meddad-Hamza et al. (2010) who found higher degree of colonization, which may be due to contamination
percentage of olive trees root colonization using with some local AM species existing in the greenhouse
R. irregularis (70%). Seifi et al. (2014) observed a environment. Such root colonization in control plants was
significant difference in the percentage of root coloni- also observed in previous studies (Krishna et al. 2006,
zation between the cultivars. Koroneiki (66%) showed Eftekhari et al. 2012).
higher colonization as compared to Valanolia (60.4%),

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Fig. 1. The foliar nitrogen, phosphorus, potassium, calcium, magnesium, and sodium concentrations of mycorrhizal and nonmycorrhizal
olive trees. Bars represent the mean of each treatment, and error bars indicate standard deviation (n = 3). Means with different letters
are significantly different at P<0.05 (Duncan's test).

Fig. 2. The foliar copper, iron, manganese, zinc, and boron concentrations of mycorrhizal and nonmycorrhizal olive trees. Bars represent
the mean of each treatment, and error bars indicate standard deviation (n = 3). Means with different letters are significantly different at
P<0.05 (Duncan's test).
Table 2. Sugar contents in source leaves of mycorrhizal and R. irregularis increased the rate of photosynthesis and
nonmycorrhizal olive tree plants. The effect of Rhizophagus modified carbohydrate profiles in olive trees: The
irregularis treatment was tested with one-way ANOVA (mean present study showed that the rate of photosynthesis in the
value ± SE, n = 3), and mean values in individual line followed
M plants was significantly higher than that of NM plants.
by the same letter are not significantly different at P<0.05
(Duncan's test).
In the present study, Chl was not significantly different in
the M plants compared with NM plants. There are
numerous reports describing AM-induced enhancement of
Carbohydrates [µg mg1] Treatment
the photosynthetic rate (Harris et al. 1985, Boldt et al.
Mycorrhizal Nonmycorrhizal
2011). It has been hypothesized that the increased sink
Arabinose 0.34 ± 0.1A 0.27 ± 0.04A strength of mycorrhizal roots leads to faster removal of
Rhamnose 0.54 ± 22A 0.25 ± 0.04A sugars from leaves which would enable higher photo-
Xylose 0.89 ± 0.22 A 0.88 ± 0.12A synthetic rates (Wright et al. 1998, Kaschuk et al. 2009).
Fructose 2.58 ± 0.39A 4.27 ± 2.12A In this experiment, the percentage increase in the rate of
Galactose 6.98 ± 1.38A 9.21 ± 1.07A photosynthesis of M compared with NM plants was 19%.
Glucose 8.93 ± 1.09A 11.1 ± 2.6A This is in good agreement with previous studies which
Mannitol 34.8 ± 7.04A 38.2 ± 3.23A have shown that the additional amount of CO2 assimilated
Myo-inositol 3.07 ± 0.67A 3.51 ± 0.47A
by M compared with NM roots ranged from 4 to 20% of
Sucrose 4.17 ± 1.4A 4.67 ± 0.95A
Trehalose 0.40 ± 0.07 A 0.42 ± 0.09A the total net CO2 fixed by the plants (Harris et al. 1985,
Raffinose 2.53 ± 1.05A 2.43 ± 0.55A Wright et al. 1998). This suggest that the additional
assimilate gain was used to support the growth and
maintenance of the fungi.

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Table 3. Sugar contents in roots of mycorrhizal and nonmycor- carbon to the fungus.
rhizal olive tree plants. The effect of Rhizophagus irregularis The decrease of xylose in M compared with NM roots
treatment was tested with one-way ANOVA (mean value ± SE, may be explained by the need of AM fungi to acquire more
n = 3), and mean values in individual line followed by the same
carbon to support their increased metabolism. Indications
letter are not significantly different at P<0.05 (Duncan's test).
of the use of xylose as a carbon source in the AM
symbiosis are found in the work of Schliemann et al.
Carbohydrates [µg mg1] Treatment (2008) who described an increased content of xylitol in
Mycorrhizal Nonmycorrhizal M. truncatula mycorrhizal roots. Using 14C-labeled
Arabinose 0.03 ± 0.02A 0.03 ± 0.01A sugars, Helber et al. (2011) showed that the symbiotic
Rhamnose 0.06 ± 0.01A 0.06 ± 0.01A partner of plants R. irregularis DAOM 197198 expresses
Xylose 0.10 ± 0.01B 0.15 ± 0.02A a sugar transporter (MST2) which is able to transport not
Fructose 0.67 ± 0.08A 0.45 ± 0.02B only glucose but also xylose, mannose, and fructose with
Galactose 0.46 ± 0.06A 0.42 ± 0.11A decreasing affinity in that order. The authors studied the
Glucose 2.39 ± 0.73A 1.98 ± 0.43A expression of genome of R. irregularis DAOM 197198 for
Mannitol 14.2 ± 1.27A 6.85 ± 0.31B genes related to xylose catabolism during different stages
Myo-inositol 0.99 ± 0.31A 0.94 ± 0.11A of the fungal life cycle and identified two xylose
Sucrose 4.03 ± 0.27A 2.74 ± 0.45B
reductases (XR1 and XR2), one xylitol dehydrogenase
Trehalose 0.68 ± 0.16A 0.32 ± 0.05B
Raffinose 0.60 ± 0.09A 0.43 ± 0.04B (XDH) and one xylulose kinase (XK). Our data could
explain the unexpected results that artificially induced
invertase activity in roots does not alter the AM fungal
In this study, we demonstrated that pools of mannitol, colonization status (Schaarschmidt et al. 2007), suggesting
glucose, fructose, sucrose, and raffinose in the leaves of M that glucose is not the only carbon sources and/or that
and NM olive trees were similar. However, the pools of enough carbohydrates are available.
mannitol, fructose, sucrose, and raffinose were changed in Analysis of lipids revealed that total lipid content of M
M compared with NM roots. Roots of the M plants roots was significantly higher than that of NM roots. We
contained a significantly higher cncentrations of fructose, suggest that these data indicate that sugars were used for
mannitol, sucrose, trehalose, and raffinose. We suggest lipid synthesis and for the production of the large
that the pattern of carbon allocation within the M plants extrametrical mycelium. In our study, the mycelium length
was altered so that an increased proportion of assimilated in the soil as estimated using the phospholipid fatty acid
carbon was partitioned to the roots of M plants, which was 16:1ω5 was significantly higher in rhizosphere soil with
needed for energy metabolism, maintenance, and growth mycorrhizal treatment compared to rhizosphere soil with
of AM fungi. It is assumed that the increased sink strength nonmycorrhizal treatment (see Mechri et al. 2014). It has
of mycorrhizal roots leads to enhanced translocation of been shown that the mycelium length in the soil correlated
sugars from source leaves which would enable higher most closely with the content of phospholipid fatty acid
photosynthetic rates (Kaschuk et al. 2009). Consequently 16:1ω5 in the soil (Olsson et al. 1997). Previous
the increase of mannitol, sucrose, and raffinose in the experiments have shown a higher content of lipids in M
M plants may be explained by this mechanism. It is known roots (Wright et al. 1998, Bago et al. 2002). Lipids play a
that mannitol, sucrose, and raffinose are the major phloem- key role in the transport of assimilates from the plant–
translocated carbohydrates in Olea europaea (Flora and fungus interface (the arbuscule, in this case) to the extra-
Madore 1993, Conde et al. 2007, Rejšková et al. 2007). radical mycelium, where they are broken down and used
The comparison of M with NM roots revealed that the as an energy source (Pfeffer et al. 1999, Bago et al. 2002).
fructose content was higher in the M plants, while glucose
content was not affected. We suggested that a significant R. irregularis increased the contents of P, K, Ca, Mg,
proportion of sugars was used by the mycorrhizal fungus, Zn, Fe and Mn in M olive trees: The present study
because only the concentration of fructose increased, while showed that N uptake was not significantly different in the
the concentration of glucose, which is mainly transferred M plants compared with NM plants. There is evidence that
towards the fungus, was nearly constant. These results are AM fungi play a role in the uptake of nitrate and
in agreement with previous studies of Wright et al. (1998) ammonium, which are assimilated and transported within
and Boldt et al. (2011), who also suggested that glucose the mycelium as arginine (Olsson et al. 2005), but
may be the main form of carbohydrates utilized by AM compared with ectomycorrhizas, rates of N uptake by AM
fungi (Shachar-Hill et al. 1995, Bago et al. 2000). hyphae are too small to contribute substantially to plant N
The fungus-specific sugar trehalose increased by nutrition (Smith and Read 2008).
52.7% in M roots compared with the NM plants. In The results obtained from this study indicated that leaf
M roots, the glucose is incorporated by the AM fungus into P content of the M plants was higher than that of NM
trehalose and glycogen (Shachar-Hill et al.1995, Bago et plants. Several studies have demonstrated that plants
al.2000), consequently the amount of observed trehalose colonized by mycorrhizal fungi are much more efficient in
may represent a considerable allocation of the plant’s taking up soil P than noninoculated plants (Black et al.

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2000, Bücking and Shachar-Hill 2005, Abdel-Fattah et al. in leaf tissue comparing with the nonmycorrhizal control.
2014). To our knowledge, one of the reasons for the Marschner and Dell (1994) estimated that 25% of the Zn
increase of leaf P in the M plants may be due to the uptake by plants can be supplied by AM fungi. Thus, it
increase in the number of actinomycetes in mycorrhizal implies that colonisation of olive trees with R. irregularis
rhizosphere soil (see Mechri et al. 2014). It has been may improve Zn uptake from a soil characterized by high
reported that actinomycetes, by excretion of chelating pH which is generally associated with Zn deficiency (pH
substances, such as siderophores, which form stable com- of the soil used in this study was 7.68). Rillig et al. (2001)
plexes with phosphorus adsorbents, increase phosphate reported that glomalin (a glycoprotein is produced in
solubilisation (Welch et al. 2002, Hamdali et al. 2008a). copious amounts by external hyphae of all members of AM
Hamdali et al. (2008b) revealed that the presence of the genera) leads to the formation of a sticky string-bag of
actinomycete strains in the soil had the greatest stimulatory hyphae that acts as an adsorptive site for metallic cations
effect on P content of wheat tissues and plant growth in which may result in enhanced availability of Zn. Other
comparison with the control. studies mentioned that the AM fungi R. irregularis
A possible and another explanation for the increase of improved organic status, dehydrogenase and phosphatase
leaf P content of M plants could be related to the high activities, and modified the Zn fractionation pattern of
trehalose content in M roots. In this experiment, the soils that collectively contributed for the availability of Zn
percentage increase of the content of trehalose was 52.7% and may assist in alleviating Zn deficiency in crop plants
in M compared with NM plants. The higher fungal (Wamberg et al. 2003, Subramanian et al. 2009).
carbohydrate trehalose content in M roots enhances the However, the mechanisms that underlie the induction of
remobilization of polyphosphates. The remobilization of mobilisation and uptake of Zn in plants inoculated with
polyphosphates increases the intracellular inorganic AM fungi have not yet been elucidated.
phosphorus (Pi) concentration in the hyphae (Bücking and In our investigation, the R. irregularis significantly in-
Heyser 2003), and thereby promotes Pi efflux through the creased foliar Mn concentration from 21.93 mg kg1(DM)
fungal plasma membrane into the interfacial apoplast. in NM plants to 30.46 mg kg1(DM) in M plants, thus
Consequently, the increase of phosphorus in the M plants revealing the contribution of R. irregularis on improving
compared with NM plants may be explained by this Mn uptake by plants. Enhanced Mn concentrations have
mechanism. been reported for a range of AM fungal colonised plants
As our findings supported, it has been reported that (Al-Karaki and Clark 1999, Taylor and Harrier 2001). We
mycorrhizal colonization can enhance Ca (Liu et al. 2002), did not measure the population density of Mn reducers in
Mg (Jentschke et al. 2001, Liu et al. 2002), and K the rhizosphere, but it has been shown that AM
absorption by plants (Giri et al. 2007, Gholamhoseini et al. colonization increased the population density of Mn
2013). To our knowledge, the increased K, Ca, and Mg in reducers in the rhizosphere, thus increasing Mn
the M plants compared with NM plants may be explained availability to the plants and plant Mn uptake (Marschner
by the direct enhanced uptake, by enlarging the absorption and Timonen 2006).
area of root systems with extraradicular hyphae, thus Colonisation of olive trees with R. irregularis also
shortening the distance that nutrients must travel within the increased Fe concentration in plants, suggesting that
soil before they reach the roots. In our study, the hyphal R. irregularis had a positive effect on the Fe nutrition of
length in the soil, as estimated using the phospholipid lipid olive trees. This may reflect not only increased mobili-
fatty acid 16:1ω5, was significantly higher in rhizosphere sation from soil, but also enhanced transport to olive tree
soil with mycorrhizal treatment compared to rhizosphere plants. It is well known that many rhizobacteria and fungi
soil with nonmycorrhizal treatment (see Mechri et al. release iron chelators which can contribute to increased Fe
2014). Olsson et al. (1997) reported that the content of this availability to plants (Khan et al. 2006, Lemanceau et al.
phospholipid was an excellent marker for estimating 2009). Santiago et al. (2013) reported that the changes in
fungal hyphal length. Rhodes and Gerdemann (1978) microbial communities rather than the increase of
found that the external hyphae of AM fungi absorbed and microbial biomass in soil can contribute to enhanced Fe
transported 45Ca to the host plants. George et al. (1992) accumulation in plants. Within our experimental condi-
observed K depletion in a hyphal compartment colonized tions, we have reported that root colonization with the AM
by Glomus mosseae concurrently with K accumulation in fungi R. irregularis induced significant changes in the
the host plant. Jentschke et al. (2001) indicated that the microbial community structure of olive tree rhizosphere
increased Mg and K concentrations in M plants can be a compared with nonmycorrhizal plants (Mechri et al.
consequence of the increased P availability from mycor- 2014). These change in microbial community observed in
rhizal fungal activity. The authors demonstrated that the rhizosphere soil of the M plants appeared effective in
translocation to mycorrhizal plant of K and Mg was improving the content of foliar Fe.
strongly reduced, when hyphal P-fluxes were ceased.
In the present study, a significant difference in Zn Did the mycorrhizal ‘sink’ for assimilates stimulate
content was observed between M and NM olive tree plants. boron remobilization process? Boron mobility is
AM fungi inoculation increased Zn concentration by 16% species-dependent and is restricted to plant species that

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translocate photosynthates as polyols (Liakopoulos et al. circulation could promote a recycling of nutrients such as
2009). These carbohydrates readily form stable diesters boron. The historical evidence for a role of AM in sugar
with boric acid (Hu et al. 1997), thereby facilitating its transport and a possible effect of AM fungi on boron
translocation from source leaves to growing tissues. The mobility described herein are intriguing and deserving a
lower content of boron in the M plants compared with NM further study.
plants may occur due to the formation and transport of
complexes between boron and mannitol. The results Conclusion: The results of this study demonstrated that
presented here indicated that the rate of mannitol export inoculation of olive tree roots with R. irregularis induced
from leaves of the M plants, due to AM-enhanced ‘sink’ several changes in physiological parameters that influence
demand for assimilates, was enhanced, as suggested by the olive tree performance, particularly photosynthetic rate,
elevated mannitol content in M roots. Furthermore, if more nutrient uptake, and carbohydrate contents in leaves and
mannitol was continuously created in source tissues of the roots. The increased sink strength of mycorrhizal roots led
M plants compared with control, as suggested from the to faster removal of sugars from leaves which induced
elevated photosynthetic rate, and loaded into the phloem, higher photosynthetic rate and increased concentrations of
then the amounts of mannitol in the phloem of M plants some sugars (fructose, mannitol, sucrose, trehalose, and
should be increasing. This could increase the rate of raffinose) in the M plants compared with NM plants. The
mannitol–borate complex formation and facilitated the enhancement of root carbohydrate contents in turn led to
phloem mobility of boron. Hu et al. (1997) found that leaf the production of a large extrametrical mycelium which
phloem mannitol could be considered as a principal factor may contribute to the improvement of the nutrient status of
determining boron remobilisation since it forms com- olive trees.
plexes of boric acid and renders it mobile in the phloem. Hence, our results clearly illustrated that R. irregularis
Consequently the decrease of boron in the M plants is a good support for Olea europaea plants. The appli-
compared with NM plants may be explained by this cation of these microorganisms could be critical in the
mechanism. Liakopoulos et al. (2009) demonstrated that a cultivation of O. europaea under arid and semiarid
quantitative relationship exists between mannitol trans- conditions, where water and the availability of nutrients in
location and boron mobility in olive plants. Drossopoulos soil are the most important factors in determining plant
et al. (1988) have reported that mannitol can be allocated growth and yield.
to bark tissue of Olea europaea during autumn. Such

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