Arbuscular mycorrhizal fungi improve growth and mineral uptake of olive tree under gypsum substrate

Ecological Engineering 73 (2014) 290–296

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Arbuscular mycorrhizal fungi improve growth and mineral uptake of olive tree under gypsum substrate Wahid Khabou a, *, Basma Hajji b , Mohamed Zouari b , Hafedh Rigane c , Ferjani Ben Abdallah b a b c

Laboratory of Improvement and Protection of Olive tree Germplasm, Olive tree Institute, B.P. 1087, 3029, University of Sfax, Sfax, Tunisia Laboratory of Plant Biodiversity and Dynamics of Ecosystems in Arid Area, Faculty of Sciences of Sfax, B.P. 802, 3018, University of Sfax, Sfax, Tunisia Department of Earth Sciences, Faculty of Sciences of Sfax, B.P. 802, 3018, University of Sfax, Sfax, Tunisia

A R T I C L E I N F O

A B S T R A C T

Article history: Received 16 May 2014 Received in revised form 18 August 2014 Accepted 13 September 2014 Available online xxx

In Tunisian dry land soils, the gypsum is ubiquitous with water scarcity and represents a major limitation for olive and several other fruit tree cultures. However, the effect of gypsum on this species is little understood. One-year old olive plants cv. “Chemlali”, which were obtained from semi hardwood cuttings, were inoculated in a nursery with an endomycorrhizae (Glomus intraradices) and they were grown in a substrate containing different proportions of gypsum. The mycorrhizae inoculation played an important role in the attenuation of the effect of sulphates contained in gypsum substrate. In fact, the obtained results showed that growth, shoot elongation and leaf area were affected by the presence of mycorrhizae. A significant growth reduction of plants was obtained with increasing gypsum rate, but this reduction was less pronounced in mycorrhizal plants. Besides, the leaves relative water content decreased as the gypsum content increased. Interestingly, G. intraradices produced an important water supply by increasing leaves relative water content in mycorrhizal plants. Calcium and phosphorus contents were significantly higher in mycorrhizal plants, which were mainly accumulated in leaves. Sulphate was accumulated in the plant roots and G. intraradices was able to attenuate such effect in mycorrhizal plants. ã 2014 Elsevier B.V. All rights reserved.

Keywords: Olive tree Gypsum Glomus intraradices Growth Leaf relative water content Mineral uptake Mycorrhizal dependence

1. Introduction In Tunisia, like in circum-Mediterranean, olives are considered as the most popularly grown fruit tree species. Olive has traditionally been cultivated under rain-fed conditions, since it is a well adapted crop to the semi-arid and arid Mediterranean region. Actually, more importance has been given for this drought and salinity tolerant species. Therefore, in arid areas the extension of the olive tree like other species is limited by water scarcity as well as ubiquitous gypsum in the soil. The extent of soils containing gypsum around the world is difficult to establish, but it has been estimated that there are 207 million ha of soils with gypsic or petrogypsic horizons. Most of these soils occur in aridic and xeric soil moisture regimes and where low precipitation prevents gypsum from being leached. Both the osmotic stress and the ion-specific (SO4) toxicity for plants are caused by gypsum (Parsons, 1976). Palacio et al. (2007a,b) and Montserrat-Martí et al. (2011) have recently noted that many

* Corresponding author. Tel.: +216 74 241 240; fax: +216 74 241 033. E-mail addresses: [email protected], [email protected] (W. Khabou). http://dx.doi.org/10.1016/j.ecoleng.2014.09.054 0925-8574/ ã 2014 Elsevier B.V. All rights reserved.

gypsophile sub-shrubs show small, hard xerophytic leaves and marked oscillations of their photosynthetic biomass throughout the year. Escudero et al. (2014) suggest that gypsophily is linked to two types of limitations (chemical and physical) that are operating simultaneously and that plants growing in gypsum environments display a complex array of plant responses to cope with these limitations. For the above reasons, there is a need for a better understanding of soils containing high proportions of gypsum and there are a set of concepts and terms as a basis for the exchange of knowledge. Together with the arid conditions, gypsum soils have particularly stressful physical and chemical properties for plant life including the presence of hard soil crusts, high mechanical instability, low soil porosity, extreme nutritional deficits and a high concentration of sulphates (Guerrero-Campo et al., 1999). Some studies have indicated that arbuscular mycorrhizal fungi (AMF) can increase plant growth, uptake of nutrients and decrease yield losses for many species under saline conditions (Ruiz-Lozana et al., 1996; Al-Karaki, 2000). It has been widely accepted that mycorrhizal fungi are able to adapt to edaphic conditions (Abbaspoura et al., 2012). In a recent investigation, developments on the effectiveness of plant growth promoting rhizobacteria and mycorrhizal fungi enhance plant growth under stressful

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environments (Nadeem et al., 2014). Root colonization by AMF involves a series of morpho-physiological and biochemical events that are regulated by the interaction of plant and fungus, as well as by environmental factors. To some extent, these fungi have been considered as bio-ameliorators of saline soils (Azcón-Aguilar et al., 1997; Rao, 1998; Zhang et al., 2014). Therefore, knowledge of the relationship between plants and the fungi is of prime importance for the successful utilization of AMF under particular conditions (Tian et al., 2013). For the same reason, Oliveira et al. (2012) showed that the ectomycorrhizal fungi (ECM) are an integrant part of forest ecosystems, vital for tree survival and development which should be considered in forestry. The mechanism by which AMF improves salt resistance remains unclear. A number of studies have revealed that mycorrhizal way is important for improving plant growth and nutrient uptake under saline conditions, especially of immobile soil nutrients such as P, Cu and Zn (Porras et al., 2009; Zhang et al., 2011; Talaat and Shawky, 2014). Many of the studies on mycorrhizal infection have been conducted under heavy-metal contaminated and drought soils (Azcón et al.,1996; Kaya et al., 2003; Yu et al., 2012). The majority of plant species form arbuscular mycorrhizae that improve the ability of plants to uptake nutrients has been suggested as important factors for plant edaphic adaptation (Schechter and Bruns, 2008). Plants growing on gypsum soils inoculated with AMF showed an enhanced nutrient uptake (Rao and Tak, 2001; Fillion et al., 2011). Little is known about AMF symbiosis in the roots of plants with different specificity to gypsum soils. The extent to which plant adaptation to gypsum substrates could be mediated by differential AMF colonisation also remains unknown. Gypsophile species can have symbiosis with fungal types specific to gypsum and hence presumably more chances for successful root colonisation than gypsovags and the high nutrient concentrations observed in most gypsophiles to be related to increase mycorrhizal infection (Alguacil et al., 2009a,b). The effects of Glomus species on olive tree production and growth have been studied in the Mediterranean zone (Beligh et al., 2014). Thus, the use of new biological methodologies is a necessary and practical way to improve agricultural plant tolerance under salinity. Studies have found that AMF symbiosis can alleviate the stress of salinity on plant growth (Allen, 2007). In this investigation, we think that transplanting inoculated olive trees in the fields can mitigate and reduce the gypsum effect soils with appropriate AMF immediately prior to transplanting a horticultural crop or to adopt cultural practices that encourage native populations of AMF in the field soil. This model of plant production has been widely adopted by nursery growers owing to the advantages it offers. The present study, therefore, examines the effects of root colonization by a strain of G. intraradices on olive trees grown in substrate that contains different proportions of gypsum. Both agronomical and physiological responses have been evaluated. 2. Materials and methods 2.1. Plant material and growth conditions Trials were conducted at the Olive Tree Institute of Sfax, Tunisia (34
43 N; 10
41 E). Uniform one-year-old self-rooted olive trees (Olea europaea L.) cv. “Chemlali”, were transplanted into 10 l pots filled with six sterilized substrate (Table 1) with physico-chemical characteristics summarized in Table 2. The pots were kept under ambient environmental conditions with natural sunlight and temperature (semi-arid climate). The design is a two-factorial experiment with six kinds of substrate and mycorrhizal treatment. The mycorrhizal treatment is divided into two plots: inoculated plants (M) by 5 g of an improved strain of endomycorrhizae G. intraradices (Shink and Smith), consisted of spores and

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Table 1 Composition of substrates. Substrate

Gypsum soil

Reference soil

G0 G1 G2 G3 G4 G5

0 1/4 1/3 1/2 2/3 1

1 3/4 2/3 1/2 1/3 0

fragments of hyphae of G. intraradices isolated from axenic carrot. The arbuscular mycorrhizal fungi (AMF) cultures were mixed with sterilized substrate and non-inoculated plants (NM). The trial was conducted during 7 months. During the experiment, all plants were irrigated weekly with the same water volume where the field capacity is reached. After 7 months of transplanting, the 60 plants were examined and the following variables were recorded. 2.2. Plant growth measurements The shoot length was measured using a regulate punt. The leaf area (LA) was determined using an Area Meter AM300 (ADC Bioscientific Ltd.) scanner. The leaf thickness was measured at the end of a seven-month study of treatment using a digital slide caliper. For each plant, the thickness of five leaves was determined. At the end of the experiment, the thickness of the totality of the leaves of plant was given. Total shoot length, leaf area and thickness were measured to characterize plant growth. 2.3. Phosphorus and calcium determination Minerals were extracted from samples (leaves, stems and roots) by the dry-ashing according to Chapman and Pratt (1982) method. The determination of the phosphorus was performed with the presence of nitrovanadomolybdate reagent. The sample digestion by phosphoric acid gives a yellow complex with phospho-molybdic (Chapman and Pratt, 1961). The calcium was determined according to the method previously described by Bedbabis et al. (2010). The ash was dissolved in 10 ml of nitric acid, heated at 70
C and then filtered. After that the volume was adjusted to 100 ml of the filtrate. Elemental analysis of phosphorus and calcium was conducted by atomic absorption spectrophotometry (PerkinElmer Analyst 300, PerkinElmer Inc., Willesley, MA, USA). 2.4. Sulphates analysis Sulphates were determined by gravimetric analysis as barium sulfate as previously described by Kenkel (2003). Four grams of dry matter sample were mineralised with concentrated HCl in the presence of MgNO3 to transfer the sulfur in sulfate. The extracts were assayed with 10 ml of BaCl2. Then, a filtration and a rinse with hot distilled water were realized. Filter paper and their contents were placed in crucibles for calcinations at 500
C. The sulphate rate was calculated from the mass of barium sulphate using the following formula: 1 SO2 4 ðmgg DMÞ ¼ ðP  TÞ  0:1029

where P: BaSO4 gross mass (mg) and T: crucible tare mass (mg). 2.5. Leaf relative water content Leaf relative water content (LRWC) was calculated based on the method of Yamasaki and Dillenburg (1999). Leaves were collected from the mid-part of the plants in order to minimize age effects. Individual leaves were first removed from the stem and then

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Table 2 Physico-chemical characterization of substrates (values are mean of 5 replicates  SD).

Sandy (%) Loam (%) Clay (%) CEs (meq/100 g) Ca (%) P (ppm) SO4 (ppm)

G0

G1

G2

G3

G4

G5

90.05a 9.89e 0.04b 5.81  0.02d 0.61  0.03c 2.20  0.38b 148  35.7e

80.68b 19.29d 0.02b 6.21  0.01c 2.28  0.3b 1.82  0.07 c 298  75.2d

72.28c 27.69c 0.03b 3.085  0.02e 3.33  0.25b 1.73  0.13c 322  54.2c

80.78b 19d 0.2a 7.13  0.05b 3.88  0.12b 2.2  0.21b 329  41c

68.3d 31.7b 0 7.61  0.02b 6.56  0.77a 2.34  0.02a 358.3  11.5b

23.13e 77a 0 7.95  0.03a 7.03  0.37a 1.56  0.16d 403.3  31a

Values in lines followed by different letters are significantly different at the 5% level according to the Duncan test.

weighed to obtain fresh mass (FM). In order to determine the turgid mass (TM), leaves were floated on distilled water inside a closed Petri dish. Maximum turgidity was determined by weighing leaves (after gently wiping the water from the leaf surface with tissue paper until no further mass increase occurred). At the end of the imbibitions period, leaf samples were placed in a pre-heated oven at 80
C for 48 h, in order to obtain dry mass (DM). All mass measurements were made using an analytical scale, with a precision of 0.0001 g. Values of FM, TM and DM were used to calculate LRWC using the equation below: LRWCð%Þ ¼

FW  DW  100 TW  DW

2.6. Mycorrhizal dependence determination Mycorrhizal dependence (MD) (based on dry matter yield) was calculated using the following equation: ½DWofMplants  DWofMplant  100 DWofMplant where DW: dry weight; FW: fresh weight; M: mycorrhizal plants and NM: non mycorrhizal plants (Porras et al., 2009). 2.7. Experimental design and statistical analysis The experimental design was a test with two factors crossed with two treatments (substrates and mycorrhizae). Each treatment was divided into two groups with five repetitions for each group. The first group consisted of mycorrhizal plants (M), the other with the non mycorrhizal plants (NM). The results were expressed as mean  standard deviation (SD) of five repetitions for each treatment. Significant differences between the values of all parameters were determined at P < 0.05 according to the one-way ANOVA. Duncan

Fig. 1. Variation of shoots elongation (cm) of mycorrhizal (&) and non mycorrhizal (&) olive plants grown in G0 sandy substrate (control), G1, G2, G3, G4 and G5 gypsum substrates. Data are expressed as mean  SD (n = 5). Different letters above the bars indicate significant differences at the 5% level according to Duncan test.

test (0.05%) was used for the means separation using SPSS Statistics 17.0 for Windows (SPSS Inc., 2008). 3. Results 3.1. Shoot elongation, leaf area and thickness The presence of mycorrhizae G. intraradices in the culture medium induced a slight improvement of growth in height and number of vegetative shoots of plants as compared to controls. The positive effect of mycorrhizal fungi on the growth in plant height was observed in the different concentrations of gypsum (Fig. 1). Indeed, the extension of vegetative shoots in mycorrhizal plants was more important than non-mycorrhizal ones. In the substrate containing a high proportion of gypsum (G5), longer vegetative shoots were up to 8 cm which was 1.5 times larger (5 cm) than non-inoculated plants grown in the same substrate. At the end of experiment, the leaf area (LA) was measured. The results reported in Fig. 2 showed a decrease in the surface of the leaves of all plants (mycorrhizal or non-mycorrhizal plants) with the increase of gypsum proportion. However, the comparison between the two types of plants showed that the LA of inoculated plants was more important than non-inoculated plants. For plants grown in the G5 substrate, the average LA was 555.42 mm2 in mycorrhizal plants, but it was only 212.91 mm2 in non-mycorrhizal plants (Fig. 2). Fig. 3 showed a slight difference between the leaf thicknesses (LT) of mycorrhizal plant leaves than those of non-mycorrhizal ones. In fact, a slight LT reduction in M-plants was observed (from 0.461 to 0.411 mm), whereas, an increase of LT for NM-plants (from 0.475 to 0.551 mm) was noticed. In response to the gypsum stress, the plant increased the level of protection structures by a thickening of the epidermis and producing higher elongation

Fig. 2. Leaf area (mm2) variation in mycorrhizal (&) and non-mycorrhizal (&) olive plants grown in G0 (sandy substrate) (control), G1, G2, G3, G4 and G5 gypsum substrates. (Each bar indicate the mean  SD, (n = 10). Data are expressed as mean  SD (n = 5). Means with different letters above the bars were significantly different at the 5% level as determined by Duncan test.

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Fig. 3. Variation in leaf thickness (mm) according to mycorrhizal (&) and non-mycorrhizal (&) plants grown in G0 (control), G1, G2, G3, G4 and G5 gypsum substrates. Data are expressed as mean  SD (n = 10). Same lowercase letter above the columns indicate non significant differences between treatments at the 5% level as determined by Duncan test.

parenchyma cells. With a low gypsum level (G1 and G2), leaf thickness of M plants was slightly higher or equal to that of NM plants grown in the same substrate. In G3, G4 and G5 substrate with higher amounts of gypsum, the leaves are significantly thicker in plants NM (Fig. 3). These results suggested that G. intraradices helped the plant to improve the morphological and structural changes in leaves. 3.2. Leaf relative water content (LRWC) The results presented in Fig. 4 showed a decrease in LRWC plants when gypsum rate increased in the substrate. The LRWC decrease was more pronounced among non-mycorrhizal plants. Indeed, in non-mycorrhizal plants, LRWC reduction is 15%, while in mycorrhizal plants this reduction did not exceed 2%. 3.3. Nutrients uptake For nutrients uptake, the incorporation of the gypsum in the substrates involved an increase in the calcium content at all the studied plants since it is an essential component of this substrate (CaSO42H2O). The concentration of calcium in the different parts was always slightly higher in the mycorrhizal plants. This increase of Ca2+ was noticed in the leaves and it was more marked when the concentration of the gypsum in the substrate increased. With a high concentration of the gypsum in the substrate (G5), the content of the leaf calcium was 1.42% DM and 1.26% DM for mycorrhizal and non-mycorrhizal plants, respectively. Whereas, for the G0 substrate (without gypsum) Ca2+ contents of the same organs was 1.3% in mycorrhizal and 0.81% in non-mycorrhizal plants. However, it was observed that the

Fig. 4. LRWC (%) variation in mycorrhizal (4) and non mycorrhizal (~) plants and substrates G0 (control), G1, G2, G3, G4, G5. (values are the mean of 5 replicates). Different letters under and above the curves indicate significant differences at the 5% level according Duncan test.

293

roots and the stem presented the same Ca2+ concentrations in different substrates (G1 and G5) for all the mycorrhizal and non-inoculated plants (Table 3). Results of this study showed that gypsum has slowed the movement of phosphorus, known for it is poorly absorped by plants. This slow down was more pronounced when the rate of gypsum increased (Table 3). Despite its low content in the substrate, phosphorus decreased with the increase of the gypsum in the substrate and M plants have accumulated more P than non-inoculated plants. By comparing the distribution of this element in the analyzed organs (leaves, stem and roots) and despite its small amount (from 0.1 to 0.6% of all confused substrate), the roots were the favorite places for phosphorus accumulation for both non- inoculated and mycorrhizal plants. The root extension by the mycelial hyphae of G. intraradices facilitated P uptake from substrate (despite its small amount) and ensure its translocation to the root cortex. The sulfate ion, which is an essential element of gypsum, has negatively affected both vegetative growth and metabolism of the olive plants used. Statistical analyses showed strong correlations between the parameters studied and sulfate content of substrates. Chemical analyses of the substrates have revealed relatively high levels of SO4 in the substrate, which vary from 148 to 400 ppm before inoculation with G0 and G5, respectively. At the end of the trial, these rates ranged from 91 (G0) to 253 ppm (G5) in the substrate inoculum and 102 (G0) to 353 ppm (G5) in the substrate free of fungus. Indeed, the analysis of results (Table 3) showed that the highest rates of sulphates were stored in the roots and those of mycorrhizal plants hold the highest levels (excluding the substrate used). Although they were very small, the quantities transported and stored in the wood and leaves (from 0 to 0.6% MS) have played an important role in reducing the growth and partial deterioration of physiological processes and ecophysiological biochemical plan. The contribution of mycorrhizae has partially mitigated the toxic effects of sulphate ions. Mycorrhizal plants accumulated fewer sulfates in organs than in non-mycorrhizal ones. 3.4. Mycorrhizal dependence The results recorded in Fig. 5 showed that mycorrhizal dependence (MD) increased significantly with increasing gypsum in the substrate. Indeed, the highest percentage (54%) of MD observed in substrate G5, which contains the highest proportion of gypsum. These results suggested that olive plants depend increasingly on mycorrhizae to survive and ensure their optimum growth and development and to resist to the harmful effects of sulphates contained in the substrate. 4. Discussion Facing on an abiotic stress, the plants grown in gypsum substrate undergo agronomical and physiological changes. In our case, the results showed an increase in the leaf thickness by increasing the cuticle thickness, which is a barrier to the external severe environmental conditions. This is one of the strategies used to maintain the balance of water status within the sheet and to protect against membrane damage and water loss. Similarly, Bacelar et al. (2007, 2009), Boughalleb and Hajlaoui (2011) and Pierantozzi et al. (2013) reported that the upper epidermis palisade parenchyma of olive leaf is thicker to ensure the growth and survival of the olive tree in water deficit conditions. This provides a better protection of interior fabrics. Symbiosis between plants and AMF is one of the plant strategies for growing under a variety of stressful conditions (Entry et al., 2002). It is well-known that AMF symbiosis protects host plants against the harmful effects of biotic and abiotic stress. The ecological impact of AMF is particularly relevant for arid and semi-arid ecosystems where they would

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Table 3 Variation of mineral element P (phosphorus), Ca (calcium) and Sulphate (SO4) in percentage of dry matter of the leaves, shoots and roots of mycorrhizal (M) and non-mycorrhizal plants (NM) grown in G0 sandy substrate (control), G1, G2, G3, G4 and G5 gypsum substrate (values are mean of 5 replicates  SD). G0 P

Leaves Stems Roots

Ca

Leaves Stems Roots

SO4

Leaves Stems Roots

M NM M NM M NM M NM M NM M NM M NM M NM M NM

0.43 0.26 0.45 0.36 0.6 0.5 1.3 0.81 1.01 0.8 0.8 0.6 0.03 0.03 0.03 0.04 0.07 0.08

G1 0.00c 0.00d 0.02c 0.02d 0.04a 0.01b 0.01a 0.11c 0.02b 0.01c 0.04c 0.11d 0.00b 0.00b 0.00b 0.00b 0.00a 0.00a

0.36 0.19 0.38 0.31 0.52 0.46 1.21 0.91 1.02 0.82 1.05 0.71 0.06 0.08 0.05 0.07 0.1 0.09

G2 0.01c 0.02d 0.01c 0.01c 0.01a 0.01b 0.06a 0.04c 0.01b 0.02c 0.02b 0.02d 0.01b 0.00a 0.00b 0.00a 0.01a 0.00a

0.32 0.16 0.35 0.25 0.49 0.43 1.22 1.18 1.2 1.17 1.19 1.17 0.1 0.13 0.08 0.18 0.46 1

G3 0.01b 0.00d 0.01b 0.01c 0.01a 0.00a 0.06a 0.01a 0.05a 0.01b 0.01a 0.03b 0.00c 0.00c 0.00d 0.00c 0.05b 0.04a

0.28 0.14 0.3 0.21 0.44 0.37 1.34 1.2 1.23 1.19 1.22 1.18 0.27 0.38 0.25 0.35 0.88 1.28

G4 0.00b 0.01c 0.02b 0.02b 0.02a 0.01b 0.02a 0.01b 0.01b 0.01b 0.03b 0.04b 0.00d 0.00c 0.02d 0.01c 0.09b 0.06a

0.25 0.11 0.28 0.17 0.4 0.32 1.4 1.24 1.25 1.22 1.25 1.2 0.47 0.68 0.35 0.45 1.28 1.62

G5 0.07b 0.00c 0.01b 0.01c 0.01a 0.02a 0.02a 0.00b 0.00b 0.01b 0.01b 0.00c 0.04d 0.02c 0.04e 0.04b 0.01b 0.14a

0.2 0.08 0.22 0.11 0.37 0.27 1.42 1.29 1.3 1.22 1.25 1.19 0.65 0.75 0.48 0.61 1.56 1.81

0.01b 0.00c 0.01b 0.02c 0.01a 0.01b 0.00a 0.00b 0.02b 0.02c 0.01c 0.00c 0.06d 0.05c 0.02e 0.02d  0.05b 0.04a

Values in columns followed by different letters are significantly different at the 5% level as determined by Duncan test.

enable greater plant tolerance to environmental stresses, which characterize these ecosystems (Allen, 2011). Assessment of the native species composition is an important issue as AMF plays important roles in the vigor of plant communities and the restoration of disturbed ecosystems (Duponnois et al., 2007; Fillion et al., 2011). Moreover, the use of AMF adapted to salinity could be a critical issue for success in recovering saline areas either in natural environments or in agricultural lands that have become salinised due to inappropriate land use (Miransari, 2010; Estrada et al., 2013). Rao and Tak (2001) reported the effect of inoculation with G. fasciculatum and gypsum on the extension of different plants Acacia ampliceps. They noted that mycorrhizal inoculation improved the longer vegetative shoots of the studied plants. In the present study, the largest leaf areas were recorded in mycorrhizal plants grown in G0 (782.4 mm2). For the same substrate, the leaf area of non-mycorrhizal plants was found to be 588.5 mm2. The obtained results confirmed those reported by Rinaldelli and Manuso (1996) on olive plants (Olea eurpopaea L.) inoculated with Glomus mosseae and treated with NaCl. The fall of the LRWC was probably a direct consequence of the increase in the concentration of sulfates in the gypsum content. In fact, gypsum sulphate ions exerted on plants an osmotic pressure, similar to that exerted by NaCl, resulted in difficulty in the water absorption by plants. These results were consistent with those obtained by Giorio et al. (1999) who reported a LRWC inversely proportional to water stress. This is an adaptive response of non- mycorrhizal trees, corresponding to a reduction of the internal water potential. Thus, one can notice that in the absence or in the presence of sulphate in the substrate, G. intraradices improved water status in the host plant through the development of the ability to maintain a given state of hydration. Indeed, Brazana et al. (2012) suggests that mycorrhizal hyphae improve the hydraulic root conductivity through apoplasmic and symlasmic transport and decrease the resistance flow of water inside the plant. This can be explained by the mycelium hypha by enabling the plant to collect larger amounts of phosphorus and ensuring translocation to the root cortex via specific fungal carriers (Sawers et al., 2008). During the cultivation, an increase in sulphate concentration in the gypsum substrate caused a significant increase in the contents of SO4 in all analyzed plant parts. The increase of SO4 in the leaves, stems and roots (Table 3) was a consequence of higher sulphate intake. According to Randle et al. (1999), plants grown in the condition of higher sulphate intake in relation to the needs accumulate the

excess of uptaken sulphates in vacuoles. A high content of total SO4 in both M and NM plants indicated a surplus intake of sulphates. However, simultaneous accumulation of sulphates in the root zone of the plants in all units indicated the activity of mechanisms blocking sulphate intake in the amounts being toxic for plants. External symptoms of toxicity related to surplus sulphate content in the nutrient solution were not observed. The effect of different sulphate contents on crop yield and quality was not proved either, which was documented by others (Kowalska and Sady, 2003). According to Herschbach and Ronnenberg (1994), when sulphate concentration in the root zone is excessive, their uptake by roots increases the synthesis of glutathione in the leaves and a certain amount of it is transported to the roots, which signals the necessity to decrease sulphates intake. The concentration of the analyzed components also depended on the stage of the plant growth. With plant aging, total SO4 concentration in the leaves increased. In younger plants, SO4 leaves content increased with the increase of gypsum in substrate. Similar tendencies were also observed by Lopez et al. (2002). Detailed information concerning changes in sulphate content in particular units and at particular stages of growth phases were published in previous work (Kowalska, 2004). Others studies showed that sulphate content exceeding 45 dm3 in nutrient solution decreased tomato leaf area (Cerda and Martinez, 1988). The same authors indicate that sulphates do not have significant effect on the vegetative development of tomato plants, which is probably a

Fig. 5. Effect of Glomus intraradices (Shenk and Smith) and the substrates G0, G1, G2, G3, G4 and G5 compared with G0 sandy substrate (control) on mycorrhizal dependence (MD) on olive plants. Data are expressed as mean  SD (n = 5). Same lowercase letters above the bars indicate non significant differences between treatments at the 5% level as determined by Duncan test.

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consequence of adequate plant nutrition. In our study, G. intraradices promoted the growth of olive plants under low level of gypsum in the substrate by increasing P and Ca uptake (Table 3). At high level of gypsum G4 and G5, a significant increase of plant dry mass and mineral uptake was also noted in M plants than non-mycorrhizal plants. This suggested that G. intraradices played a different mechanism to improve the sulphate tolerance of olive plants at higher gypsum levels. The same results have been reported by Ruiz-Lozana et al. (1996) who has concluded that the mechanisms underlying AM plant growth improvement in Lactuca sativa under saline conditions were based on physiological processes (increased carbon dioxide exchange rate, transpiration, stomatal conductance and water use efficiency) rather than on nutrient uptake (N or P). The role of mycorrhizae in improving the balance sheet mineral has been also noted by (Kaya, 2009). Regarding the content of SO4 substrates, the results showed that mycorrhizae have minimized the amount of sulphates despite the increase of gypsum compared to non-inoculated media (Table 3). The obtained results showed that the assisted roots with mycelial hyphae mitigated the ascent of this toxic element to the shoots. These findings confirmed those of Rao and Tak (2001). Similar results have been obtained by Allen and Shashar-Hill (2009) who reported the role of some arbuscular mycorrhizae in reducing the harmful effects of sulfates in the soil. Proportionally with the rate of gypsum, the average of calcium content in the stem increased by 0.42% DM and 0.37% DM in the mycorrhizal and in non-mycorrhizal plants, respectively. The level of DM in the roots varied between 0.45% and 0.6% in mycorrhizal and in non-mycorrhizal plants, respectively (Fig. 4). In the control, the increase of DM was about 0.2% in mycorrhizal plants. Similarly to our results, Rao and Tak (2001) showed an increase in the content of calcium in the organs of A. ampliceps inoculated with G. fasciculatum. Statistically, the effects of the mycorrhizae and the substrate on the accumulation of calcium in the different organs are significant with the threshold of 5%. Statistical analysis showed a significant effect of mycorrhizae and the substrate on the distribution of phosphorus and calcium between the organs (Table 3). In the same way, Porras et al. (2009) observed an improvement in mineral absorption including potassium mycorrhizal trees and irrigated with different concentrations of NaCl. Palacio et al. (2012) indicate that AM colonization could be a mechanism allowing non-specialist plants (gypsovags) to cope with the restrictive conditions of gypsum. The mycorrhizal dependence (MD), which was another parameter, was also analyzed. The results showed a positive correlation between gypsum rate and MD in M plants. In similar and under salinity conditions, Porras et al. (2009) revealed the increase of mycorrhizal dependency of olive plants with increasing salinity. This may be a sign showing the ecological importance of AMF association for plant survival. 5. Conclusions The results showed clearly the role played by the endomycorrhizae G. intraradices in the attenuation to the depressive effect of the gypsum. Indeed, the experiment showed a significant improvement in growth of the mycorrhizal plants. The variation of the concentration of the principal biogenic salts (P, Ca and SO4) in the different parts (leaves stem and roots) indicated that the increase of sulphate content in the substrate decreased the growth and the mineral uptake in both M and NM plants. A negative correlation between all parameters and the gypsum content in the substrate was observed. A mechanism played by roots and the AMF to reduce the effect of sulfates contained in the gypsum on the plant. This mechanism was built by the preferential accumulation of sulphates ions in the roots as compared to the other parts (leaves

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and stems) of the plant. The mineral improvement of the plant was a consequence of the extension of mycorrhizal hyphae. In view of these results, it is possible to recommend mycorrhizal inoculation to attain reasonable growth and mineral uptake like P under gypsum conditions. Obtained results also showed that AM fungal inocula should be used to enhance the growth of olive plant species in gypsum substrate (containing sulphate) by improving nutrient uptake and the capacity to survive due to the ability of mycorrhizal fungi in improving tolerance in plants. These fungi could certainly play an important role in the arid zone where the gypsum in the soil is ubiquitous, and it might be expected that nurseries production of inoculated olive plants would have a higher capacity to tolerate the edaphic contrast in the Mediterranean arid zone. Acknowledgements Authors wish to thank Pr. Fortin researcher in Premier Tech Québec, Canada, for providing the G. intraradices strain and Dr. Hafedh Nasr for supporting this study. References Abbaspoura, H., Saeidi-Sarb, S., Afsharia, H., Abdel-Wahhab, M.A., 2012. Tolerance of Mycorrhiza infected Pistachio (Pistacia vera L.) seedling to drought stress under glasshouse conditions. J. Plant Physiol. 169, 704–709. Alguacil, M.M., Roldan, A., Torres, M.P., 2009a. Assessing the diversity of AM fungi in arid gypsophilous plant communities. Environ. Microbiol. 11 (2), 649–2659. Alguacil, M.M., Roldan, A., Torres, M.P., 2009b. Complexity of semiarid gypsophilous shrub communities mediates the AMF biodiversity at the plant species level. Microb. Ecol. 57, 718–727. Al-Karaki, G.N., 2000. Growth of mycorrhizal tomato and mineral acquisition under salt stress. Mycorrhiza 10, 51–54. Allen, M.F., 2007. Mycorrhizal fungi: highways for water and nutrients in arid soils. Vadose Zone J. 6, 291–297. Allen, M.F., 2011. Linking water and nutrients through the vadose zone: a fungal interface between the soil and plant systems. J. Arid Land 3 (3), 155–163. Azcón, R., Gomez, M., Tobar, R., 1996. Physiological and nutritional responses by Lactuc sativa L. to nitrogen sources and mycorrhizal fungi under drought conditions. Biol. Fertil. Soils 22, 156–161. Azcón-Aguilar, C., Cantos, M., Troncoso, A., Barea, J.M., 1997. Beneficial effect of arbuscular mycorrhizas on acclimatization of micropropagated cassava plantlets. Sci. Hortic. 72, 63–71. Bacelar, E.A., Dario, L.S., Peirera, L.S., Berta, L., Correira, C.M., Conçalves, B.C., 2007. Physiological behaviour, oxydative damage and oxydative protection of olive trees grown under different irrigation regimes. Plant Soil 292 (1–2), 1–12. Bacelar, E.A., Moutinho-Periera, C.M., Gonc alves, J.M., Lopez, J.I., Correira, C.M., 2009. Physiological responses to different olive genotypes to drought conditions. Acta Physiol. Plant. 31, 611–621. Bedbabis, S., Ferrara, G., Ben Rouina, B., Boukhris, M., 2010. Effects of irrigation with treated wastewater on olive tree growth: yield and leaf mineral elements at short term. Sci. Hortic. 126, 345–350. Beligh, M., Anicet, G.B.M., Meriem, T., Faouzi, A., Hechmi, C., Fethi, B.M., Mohamed, B., Dalenda, B., Mohamed, H., 2014. Changes in microbial communities and carbohydrate profiles induced by the mycorrhizal fungu (Glomus intraradices) in rhizosphere of olive trees (Olea europaea L.). Appl. Soil Ecol. 75 (14), 124–133. Boughalleb, F., Hajlaoui, H., 2011. Physiological and anatomical changes induced by drought in two olive cultivars (cv. Zalmati and Chemlali). Acta Physiol. Plant 33, 53–65. Brazana, G., Aroca, R., Paz, J.A., Chaumont, F., Martinez-Ballesta, M.C., Carvajal, M., Ruiz-Lozano, J.M., 2012. Arbuscular mycorrhizal symbiosis increases relative water apoplastic water flow in roots of the host plant under both well-watered and drought stress conditions. Ann. Bot. 109, 1007–1009. Chapman, H.D., Pratt, F.P., 1961. Ammonium Vandate-molybdate Method for Determination of Phosphorus: Methods of Analysis for Soils, Plants and Water, 1st ed. Agriculture Division, California University, USA, pp. 184–203. Chapman, H.D., Pratt, F.P., 1982. Determination of Minerals by Titration Method: Methods of Analysis for Soils, Plants and Water, 2nd ed. Agriculture Division, California University, USA, pp. 169–170. Cerda, A., Martinez, V., 1988. Nitrogen fertilization under saline conditions in tomato and cucumber plants. J. Hortic. Sci. 63, 451–458. Duponnois, R., Plenchette, C., Prin, Y., Ducousso, M., Kisa, M., Galiana, B.A.M., 2007. Use of mycorrhizal inoculation to improve reafforestation process with Australian Acacia in Sahelian ecozones. Ecol. Eng. 29 (1), 105–112. Entry, J.A., Rygiewicz, P.T., Watrud, L.S., Donnelly, P.K., 2002. Influence of adverse soil conditions on the formation and function of arbuscular mycorrhizas. Adv. Environ. Res. 7 (1), 123–138. Escudero, A., Palacio, S., Maestre, F.T., Luzuriaga’, A.L., 2014. Plant life on gypsum: a review of its multiple facets. Biol. Rev Willey ed..

296

W. Khabou et al. / Ecological Engineering 73 (2014) 290–296

Estrada, B., Aroca, R., Barea, J.M., Ruiz-Lozano, J.M., 2013. Native arbuscular mycorrhizal fungi isolated from a saline habitat improved maize antioxidant systems and plant tolerance to salinity. Plant Sci. 201–202, 42–51. Fillion, M., Brisson, J., Guidi, W., Labrecque, M., 2011. Increasing phosphorus removal in willow and poplar vegetation filters using arbuscular mycorrhizal fungi. Ecol. Eng. 37 (2), 199–205. Giorio, P., Sorrentino, G., D’andria, R., 1999. Stomatal behaviour, leaf water status and photosynthetic response in field-grown olive trees under water deficit. Environ. Exp. Bot. 42, 95–104. Guerrero-Campo, J., Francisco, A.M., John, H., Gabriel, M.M., 1999. Plant community patterns in a gypsum area of NE Spain: II. Effects of ion washing topographic distribution of vegetation. J. Arid Environ. 41 (4), 41–419. Herschbach, C., Ronnenberg, H., 1994. Influence of glutathione (GSH) on net uptake of sulphate and sulphate transport in tobacco plants. J. Exp. Bot. 45, 1069–1076. Allen, J.W., Shashar-Hill, Y., 2009. Sulfur transfer through an arbuscular mycorrhiza. Plant Physiol. 149, 549–560. Kaya, C., Bekir, Ak., Higgs, D., 2003. Response of salt stressed strawberry plants to supplementary calcium nitrate and/or potassium nitrate. J. Nutr. 26 (3), 543–560. Kaya, A., 2009. Macrofungal diversity of Nemrut Mount National Park and its environs (Adıyaman–Turkey). Afr. J. Biotechnol. 8, 2978–2983. Kenkel, J., 2003. Analytical Chemistry for Technicians, 3rd ed. CRC Press, LLC, USA, pp. 130–142. Kowalska, I., Sady, W., 2003. Effects of different sulphate levels at t he root zone on the concentration of mineral compounds in the leaves of greenhouse tomato grown on NFT. Acta Hortic. 60 (2), 499–504. Kowalska, I., 2004. The effect of sulphate levels in the nutrient solution on mineral composition of leaves and sulphate accumulation in the root zone of tomato plants. Folia Hort. 16 (1), 3–14. Lopez, J., Parent, L.E., Trembay, N., Gosselin, A., 2002. Sulfate accumulation and calcium balance in hydroponic tomato culture. J. Plant Nutr. 25 (7), 1585–1597. Miransari, M., 2010. In: Fulton, S.M. (Ed.), Champignons mycorhiziens et des écosystèmes efficacité, Dans: champignons mycorhiziens: sols, de l’agriculture et de l’environnement Implications. Publié par Nova Publishers, USA. Montserrat-Martí, G., Palacio, S., Milla, R., Gimenez, L., 2011. Meristem growth, phenology, and architecture in chamaephytes of the Iberian Peninsula: insights into a largely neglected life form. Folia Geobot. 46, 117–136. Nadeem, S.M., Ahmad, M., Zahir, A.Z., Javaid, A., Ashraf, M., 2014. The role of mycorrhizae and plant growth promoting rhizobacteria (PGPR) in improving crop productivity under stressful environments. Biotechnol. Adv. 32, 429–448. Oliveira, R.S., Franco, A.R., Castro, P.M.L., 2012. Combined use of Pinus pinaster plus andinoculation with selected ectomycorrhizal fungi as an ecotechnology to improve plant performance. Ecol. Eng. 43, 95–103. Palacio, S., Escudero, A., Montserrat-Martí, G., Maestro, M., Milla, R., Albert, A., 2007a. Plants living on gypsum: beyond the specialist model. Ann. Bot. 99, 333–343. Palacio, S., Maestro, M., Montserrat-Martí, G., 2007b. Relationship between shoot-rooting and root-sprouting abilities and the carbohydrate and nitrogen reserves of Mediterranean dwarf shrubs. Ann. Bot. 100, 865–874. Palacio, S., Johnson, D., Escudero, A., Montserrat-Martí, G., 2012. Root colonisation by AM fungi differs between gypsum specialist and non-specialist plants: links to the gypsophile behavior. J. Arid Environ. 76, 128–132. Parsons, R.F., 1976. Gypsophily in plants. A review. Am. Midland Nat. 96, 1–20.

Pierantozzi, P., Torres, M., Bodoira, R., Maestri, D., 2013. Water relations: biochemical – physiological and yield responses of olive trees (Olea europaea L. cvs. Arbequina and Manzanilla) under drought stress during the pre-flowering and flowering period. Agric. Water Manage. 125, 13–25. Porras, Maria, L., Andrés, P., Rosario, A., 2009. Arbuscular mycorrhizal fungi increased growth, nutrient uptake and tolerance to salinity in olive trees under nursery conditions. J. Plant Physiol. 166, 1350–1359. Randle, W.M., Kopsel, D.E., Kopsell, D.A., Snyder, R.L., 1999. Total sulfur and sulfate accumulation in onion is affected by sulfur fertility. J. Plant Nutr. 22 (1), 45–51. Rao, D.L., 1998. Biological amelioration of salt-affected soils. Microbial Interactions in Agriculture and Forestry, vol. 1. Science Publishers, Enfield, USA, pp. 21–238. Rao, D.L., Tak, R., 2001. Influence of mycorrhizal fungi on the growth of different tree species and their nutrient uptake in gypsum mine spoil in India. Appl. Soil Ecol. 17, 279–284. Rinaldelli, E., Manuso, S., 1996. Response of young mycorrhizal and non mycorrhizal plants of olive tree (Olea europaea L.) to saline conditions. I. Short-term electrophysiological and long term vegetative salt effects. Adv. Hort. Sci. 10, 126– 134. Ruiz-Lozano, J.M., Azcón, R., Gómez, M., 1996. Alleviation of salt stress by arbuscular-mycorrhizal Glomus species in Lactuca sativa plants. Physiol. Plant 98, 767–772. Sawers, R.J.H., Gutjahr, C., Paszkowski, U., 2008. Cereal mycorrhiza: an ancient symbiosis in modern agriculture. Trends Plant Sci. 13 (2), 93–97. Schechter, S.P., Bruns, T.D., 2008. Serpentine and non- serpentine ecotypes of Collinsia sparsiflora associate with distinct arbuscular mycorrhizal fungal assemblages. Mol. Ecol. 17 (13), 3198–3210. SPSS Inc. 2008. SPSS Statistics 17 .1. Talaat, N.B., Shawky, B.T., 2014. Protective effects of arbuscular mycorrhizal fungi on wheat (Triticum aestivum L.) plants exposed to salinity. Environ. Exp. Bot. 98, 20–31. Tian, H., Drijberc, R.A., Zhang, J.L., Li, X.L., 2013. Impact of long-term nitrogen fertilization and rotation with soybean on the diversity and phosphorus metabolism of indigenous arbuscular mycorrhizal fungi within the roots of maize (Zea mays L.). Agric. Ecosyst. Environ. 164, 53–61. Yamasaki, S., Dillenburg, L.R., 1999. Measurements of leaf relative water content in Araucaria angustifolia. Rev. Bras. Fisiol. Veg. 11 (2), 69–75. Yu, Z.X., Zhang, Q., Yang, H.S., Tang, J.J., Weiner, J., Chen, X., 2012. The effects of salt stress and arbuscular mycorrhiza on plant neighbor effects and self-thinning. Basic Appl. Ecol. 13, 673–680. Zhang, Y.F., Wang, F., Yang, Y.F., Bi, Q., Tian, S.Y., Shi, X.W., 2011. 2011. Arbuscular mycorrhizal fungi improve reestablishment of Leymus chinensis in bare salinealkaline soil: implication on vegetation restoration of extremely degraded land. J. Arid Environ. 75 (9), 773–778. Zhang, H.S., Zai, X.M., Wu, X.H., Qin, P., Zhang, W.M., 2014. An ecological technology of coastal saline soil amelioration. Ecol. Eng. 67, 80–88.