The role of arbuscular mycorrhizal fungi in alleviating ...

The role of arbuscular mycorrhizal fungi in alleviating salt stress in Medicago sativa L. var. icon Angela Campanelli, Claudia Ruta, Giuseppe De Mastro & Irene MoroneFortunato Symbiosis ISSN 0334-5114 Volume 59 Number 2 Symbiosis (2013) 59:65-76 DOI 10.1007/s13199-012-0191-1

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Author's personal copy Symbiosis (2013) 59:65–76 DOI 10.1007/s13199-012-0191-1

The role of arbuscular mycorrhizal fungi in alleviating salt stress in Medicago sativa L. var. icon Angela Campanelli & Claudia Ruta & Giuseppe De Mastro & Irene Morone-Fortunato

Received: 23 January 2012 / Accepted: 28 September 2012 / Published online: 16 October 2012 # Springer Science+Business Media Dordrecht 2012

Abstract Medicago sativa L. is the most important forage crop in arid and semi-arid areas, where increased salinity is a major factor limiting plant growth and crop productivity. The role of arbuscular mycorrhizal (AM) fungus Glomus viscosum H.T. Nicolson strain A6 in protecting alfalfa plants from salt stress, induced by sodium chloride (NaCl), was studied in two ways. Firstly, the root systems of 3-month old M. sativa plants, both mycorrhizal (AM+) and nonmycorrhizal (non-AM) (M. sativa L. var. icon), were placed in solutions of increasing salt concentrations (0, 50, 100, 150, 200 mM NaCl) to study the wilting response. G. viscosum improved the tolerance to salinity stress and the benefit was expressed in terms of the time required to reach the T4 stage in the wilting experiment. Secondly, to evaluate the ability of the Glomus-alfalfa symbiosis to tolerate salt, a pot experiment was set up in a glasshouse in which 3-month old alfalfa plants (M. sativa var. icon) were grown in a peat substratum at three salinity levels (0, 100, 150 mM NaCl). The AM symbiosis stimulated plant height, leaf area, root density, fresh and dry plant weight under saline conditions. Furthermore, proline accumulation was higher in mycorrhizal M. sativa plants than in non-mycorrhizal plants under conditions of salt stress. These and other results indicated that the micropropagated selected clone of M. sativa var. icon, when in symbiosis with G. viscosum H.T. Nicolson strain A6, exhibited better growth and physiological activities under saline conditions than non-AM plants. The AM+ A. Campanelli : C. Ruta (*) : G. De Mastro : I. Morone-Fortunato Department of Agricultural and Environmental Sciences, “A. Moro” University of Bari, Via Amendola 165/a, Bari, Italy e-mail: [email protected]

plants also had lower sodium and chloride concentrations in tissues than non-AM plants. Keywords Glomus viscosum . Alfalfa . Symbiosis . NaCl . Morphological parameters . Physiological parameters . Salt tolerance

1 Introduction Medicago sativa L., alfalfa, is a forage species that is grown worldwide as a result of its ability to produce biomass, its longevity and the fact that it can improve the soil where it is cultivated as a result of nitrogen fixation by the root nodules. Alfalfa is moderately salt sensitive (Maas and Hoffman 1977) although this varies with cultivar and climatic factors. Salinity affects plants via osmotic, nutritional and ionic stress. The need to produce crops with enhanced tolerance to salt stress has been the stimulus for research. Several studies have demonstrated that inoculation with mycorrhizal fungi improves growth and productivity when plants are exposed to salt stress (Evelin et al. 2009; Kumar et al. 2010; Khalil et al. 2011; Sheng et al. 2011; Porcel et al. 2012). This may be due to an increased uptake of water and nutrients and/or to a decrease in the sodium and chloride uptake (Talaat and Shawky 2011). Studies have shown higher chlorophyll contents in the leaves of mycorrhizal plants, confirming that the symbiosis plays a key role in the modifying photosynthetic and metabolic activities (Huang et al. 2011). Arbuscular mycorrhizal (AM) fungi also influence stomatal conductance and transpiration in the host plant (Augé et al. 2008), but this varies widely with plant species. The mycorrhizal symbiosis may improve osmotic balance by inducing the host plant to

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synthesis osmolytes, such as proline which accumulates in the plant tissues (Ruiz-Lozano et al. 1995; Hatimi 1999; Sharifi et al. 2007; Borde et al. 2010; Kumar et al. 2010; Dudhane et al. 2011; Talaat and Shawky 2011). Many studies have demonstrated the effectiveness of arbuscular mycorrhiza in various Medicago sativa varieties (Saif 1987; Dissing-Nielsen 1990; Srivastava and Mukerji 1995; Goicoechea et al. 1997, 1998; Larose et al. 2002; Zhang et al. 2011). However, little research has been done on the influence of this symbiotic relationships on tolerance to increasing salt stress (Azcón and El-Atrash 1997). The present study sets out to investigate the effect of the AM mycorrhizal fungus Glomus viscosum on the growth and physiology of a selected clone of Medicago sativa var. icon treated with different NaCl concentrations.

2 Materials and methods The starting inoculum of Glomus viscosum H.T. Nicolson strain A6 was provided by the Department of Crop Plant Biology (University of Pisa, Italy). It was cultured in a growth chamber at the Department of Agricultural and Environmental Sciences of the “A. Moro” University of Bari (Italy) using strawberry plantlets as the mycorrhizal trap crop, and the steamed sand was employed as the substratum in order to avoid culture contamination according to Dalpé and Monreal (2004). A micropropagated clone of Medicago sativa var. icon, selected as being salt tolerant in an in vitro screening on saltenriched culture medium (200 mM NaCl), was used. This NaCl-tolerant clone was multiplied in vitro and was maintained in a growth chamber (23°C, 16/8-hour photoperiod) at the Department of Agricultural and Environmental Sciences of the University of Bari (Italy). The ex-vitro experiments were performed in the glasshouse (Bari, Apulia, Italy) from June to October 2010. Acclimatization of the micropropagated clone was done in a glasshouse at 20°C and a relative humidity of 40%, in natural daylight. The plantlets were grown in a sterilized commercial peat/ soil mixture (organic carbon 46%, organic nitrogen 1-2%, organic matter 80%) mixed with perlite at a 2:1 (v/v) ratio which was used to fill the 0.2 dm3 pots. At transplanting, one batch of the plantlets were inoculated with the AM fungus G. viscosum. Ten grams of crude AM inoculum were added to each pot at the base of the rooted microplants. The AM inocula consisted of soil which contained spores (about 50–60 spores in 5 g of inoculum), external mycelium and infected strawberry root fragments from the strawberry pot cultures inoculated with G. viscosum H.T. Nicolson strain A6. After 30 days, the acclimatized plantlets of the selected clone of M. sativa var. icon were transplanted into 3dm3

A. Campanelli et al.

pots using the same sterile peat/soil/perlite mix. The plants were watered daily with distilled water. After 90 days, both the mycorrhizal and non-mycorrhizal control plantlets were evaluated for their salt tolerance; firstly by a quick test in saline solutions, and secondly by a pot trial in the glasshouse, using irrigation water with different levels of salinity. 2.1 Quick test in saline solutions Ninety days after mycorrhizal inoculation, the root systems of the M. sativa var. icon plants, either inoculated or not, were carefully washed to remove the substratum, and each plantlet was placed in polypropylene boxes (9 cm diameter and 14 cm height, Microbox Round High, Duchefa) containing 0.75 dm3 distilled water with increasing salt concentrations. The 5 × 2 experimental design included two treatments (with or without G. viscosum) and five salinity levels (0, 50, 100, 150, 200 mM NaCl). Ten replicates were used for each treatment and for each salinity level. To study wilting, wilting indexes were defined based on visual characteristics: T1 leaves flaccid, T2 leaves and stem flaccid, T3 stem bent, T4 total wilting (Rosendhal and Rosendhal 1991). The time taken to reach each wilting indexes was recorded for both the mycorrhizal and non-mycorrhizal plants. The data were subjected to an analysis of variance. In addition, the Student-Newman-Keul (SNK) test was used to compare the mean of wilting times between mycorrhizal or non-mycorrhizal treatments for each salt concentration following Anova (P≤0.01) (Miller 1981). 2.2 Glasshouse salinity tolerance assessment After 90 days, the above soil portions of the M. sativa var. icon plants were cut at the collar level and used for the potbased salinity trial. The experiment was randomized in complete blocks with three levels salinity used in the irrigation water (0, 100, 150 mM NaCl) and two treatments (with or without G. viscosum) to give a 3×2 factorial, with 15 replicates per NaCl concentration. The salt was added to the irrigation water. The salt concentration was increased gradually to avoid osmotic shock, adding 50 mM NaCl at a time in the irrigation water up to reach the levels of 100 mM or 150 mM NaCl. During the first 2 weeks, the plantlets were irrigated on alternative days with saline or distilled water. After the first 2 weeks the plants were irrigated daily with the appropriate salt solution. The volume of each irrigation was 150 ml per pot. At the beginning and at the end of the glasshouse experiments, the salinity level in each pot was assessed by measuring the electrical conductivity (ECe) of the peat/soil mix in each pot using a portable conductivity meter, WTW model LF 330 (Sonneveld et al. 1974). The salinity trial in pots lasted 60 days. To assess the salt tolerance of mycorrhizal and non-mycorrhizal plants, the

Author's personal copy The role of arbuscular mycorrhizal fungi

following morphological and physiological parameters were used to evaluate nutritional, osmotic and toxic stresses. 2.2.1 Mycorrhizal colonization and plant growth responses The analysis of M. sativa var. icon roots was carried out following Phillips and Hayman (1970) and the samples were then observed using an optical microscope (Leica DMLB100). AM colonization (%) 0 100* root length infected/root length observed (Biermann and Linderman 1981; Graham and Syvertsen 1985; Trouvelot et al. 1986). Mycorrhizal colonization was estimated on M. sativa roots 1 month after mycorrhizal inoculation to verify the establishment of symbiosis. An assessment of the roots to check for AM fungi colonization was repeated before the quick test and at the beginning and the end of the salinity trial in pots. At the end of the salinity trial in pots, ten plants were processed per treatment. Shoots and roots were weighed to determine the fresh weight, and were then oven-dried (48h, 60°C) and reweighed to determine the dry weight. The modified line intersect method (Tennant 1975) was used to measure the root density. Leaf area was measured using a leaf area meter (Delta-T Area Meter MK2 Devices Ltd, England). Mycorrhizal dependency (M.D.) was calculated using the individual total dry weight (DW) of mycorrhizal plants (AM) and the mean dry weight of non-mycorrhizal plants (Non-AM) at each salinity level, according to Plenchette et al. (1983) as: M:D: ¼ ðDW AMDW Non AMÞ=DW AM 100 Plant tolerance (Pt) was calculated according to Hatimi (1999) as: Ptð%Þ ¼ 100 ðDWSP=DWNSPÞ where DWSP is the dry weight of the stressed plant and DWNSP is the dry weight of the non-stressed plant. A relatively high Pt would be indicative of a relatively high tolerance level (Hatimi 1999). 2.2.2 Gas exchange measurements Stomatal conductance was measured using a leaf porometer (Model SC-1, Decagon Device, Washington, DC, USA). These measurements were made every 10 days, and were carried out on the completely expanded, but not senescent, leaves, with two repeated readings for five sample plants (both mycorrhizal and non-mycorrhizal). The measurements were made between 11 a.m. and 1 p.m. on the abaxial leaves. A total of 420 measurements were taken during the experiments [2 (mycorrhizal treatments) x 3 (levels of salinity) x 2 (replications) x 5 (plants per replication) x 7 (measurement periods)].

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2.2.3 Clorophyll Content The chlorophyll content was measured in two ways. It was determined indirectly with a Chlorophyll meter SPAD502 (Minolta Camera Co. Ltd, Japan). The SPAD meter is a simple, portable diagnostic tool that measures the greenness or chlorophyll content of leaves. Compared with the traditional destructive methods of chlorophyll extraction, the use of this equipment saves time, space and resources allowing to monitor over time the plant physiological status because there are high correlations among SPAD readings, total leaf chlorophyll and foliar N content (Percival et al. 2008). The SPAD values were monitored every 10 days at same time (between 9 a.m. and 11 a.m.). The SPAD measurements were carried out on the first fully expanded leaves with three readings repeated for five sample plants (both mycorrhizal and non-mycorrhizal). A total of 630 measurements were taken during the experiments [2 (mycorrhizal treatments) x 3 (levels of salinity) x 3 (replications) x 5 (plants per replication) x 7 (measurement periods)]. At harvest, chlorophyll determinations were made by solvent extraction on three sample plants for each mycorrhizal treatment and salinity level. Prior to extraction, fresh leaf samples were cleaned with deionized water to remove any surface contamination. Chlorophyll extraction was carried out on fresh fully expanded leaf material; the leaf sample (1g) was ground in 90% acetone using a pestle and mortar. Absorbance was measured with a UV/visible spectrophotometer (Lambda 3B - Perkin Elmer) and chlorophyll concentrations were calculated using the following equation proposed by Strain and Svec (1966): Chl a mg ml1 ¼ 11:64 ðA663Þ 2:16 ðA645Þ Chl b mg ml1 ¼ 20:97 ðA645Þ 3:94 ðA663Þ A663 and A645 represent the absorbance values read at 663 and 645 nm wavelengths, respectively. 2.2.4 Water relations Relative leaf water was determined on the basis of the method developed by Kramer and Boyer (1995). Ten uniform leaves from three randomly chosen plants were removed at the stem and their fresh mass (FW) was measured immediately. Then the leaves were floated on distilled water in a closed Petri dish in the laboratory with an ambient room temperature of about 25°C to obtain the turgid weight (TW). The dry weight (DW) was determined after over-drying at 80°C for 24°C. RWC ¼ ½ðFW DWÞ=ðTW DWÞ 100 The osmotic potential (ψs) of the xylem sap was measured at harvest using the Scholander pressure chamber


(Model 3000, ICT International, Australia). The measurements were taken at around 1 p.m., at the time of day associated with the maximum light intensity, when the plants have the lowest water content and the maximum leaf water potential. 2.2.5 Stomata density Stomata density was determined on the abaxial epidermis of leaves of mycorrhizal and non-mycorrhizal plants treated in a glasshouse with saline water at different concentrations. Ten leaves (fully-expanded terminal leaflets) were chosen from five plants in each treatment. The lower epidermis was peeled off, observed and photographed under a light microscope (Leica DMLB100) at 400x magnification: a total of 300 impressions were sampled. Stomata density was calculated from photomicrographs made with X-PRO, an advanced software designed to optimize image capture with a Nikon DXM 1200 Digital Camera. Stomata were counted on five microscope fields (0.035 mm2 each) taken at random from each sample. Leaf edges and the areas next to the middle rib were avoided.


was measured by an ion chromatograph (761 Compact IC Metrohm Ltd, Herisau, Switzerland). Instead, the concentrations of sodium (Na), potassium (K), calcium (Ca) and magnesium (Mg) were determined by an atomic absorption spectrophotometer (932 Plus, GBC Scientific Equipment, Melbourne, Australia) because the high concentration of sodium in the vegetal tissues interfered with the determination of low concentrations of the other analyte cations with similar retention times, causing the peak shape problems which compromised chromatographic analysis. 2.2.8 Statistical analyses The data collected were subjected to analysis of variance. The SNK test (P≤0.01 - P≤0.05) was used to compare the means of morphological or physiological parameters in mycorrhizal or non-mycorrhizal treatments for each salt concentration following Anova (Miller 1981). Since the data were expressed as percentages, to overcome the difficulty of irregularities the measured values were transformed according to the angular transformation.

2.2.6 Proline determination

3 Results

The proline content of fresh vegetative tissue was determined by means of the method described by Bates et al. (1973). The proline content was estimated on three plants for each treatment and for each saline level. Approximately 1 g of M. sativa tissue was weighed and homogenized in 2 ml of 3% sulfosalicilic acid solution. The homogenate was centrifuged at 13000g for 10 min and 1 ml of supernatant was placed in a test tube. Glacial acetic acid (1ml) and acid ninhydrin (1 ml) were added to each tube, which was closed and heated in a 100°C water bath for 1 hour. After the sample had cooled in an ice bath for 15 min, 2 ml of toluene were added to every sample and mixed on a vortex for 20 s under a fume hood. The test tubes were left undisturbed for at least 10 min to allow for the separation of the toluene and aqueous phases. The toluene phase was carefully pipetted out into a glass test tube and the absorbance was measured at 520 nm, using pure toluene as a blank. The standard curve was prepared using proline in a 3% sulfosalicylic acid solution. Proline content was expressed as micromoles per gram of fresh weight of plant material.

3.1 Quick test in saline solutions

2.2.7 Mineral analysis

3.2.1 Mycorrhizal colonization and plant growth responses

Ions were extracted from leaves and roots (Baker et al. 1964; Watson and Isaac 1990). Three randomly selected plants per replicate were dry-ashed at 550°C for 4 h, mixed with hot 1M HCl, filtered, and then brought to a final volume of 50 ml with distilled water. The chloride content

The initial conductivity of the substratum (0.5 dS/m) increased with salt exposure. The level of salinity reached in the substratum for each treatment was 1.2 dS/m (0 mM NaCl), 12.2 dS/m (100 mM NaCl) and 18.2 dS/m (150 mM NaCl), respectively. Anova analysis (Table 1) showed

Before exposure to saline conditions, the evaluation of mycorrhizal colonization in the root system showed an extensive colonization with mycorrhizal hyphae and vesicles in the 50% of the observed roots. The effects of salt stress induced by sodium chloride on inoculated and control plants of Medicago sativa var. icon are shown in Figure 1. Glomus viscosum improved the tolerance to salinity stress and the benefit was expressed in terms of the time required to reach the T4 stage in the wilting experiment. The rate of increase in the wilting index was correlated with the concentration of sodium chloride in the solution. Mycorrhizal plants reached higher wilting index values later than the non-mycorrhizal plants. At lower concentrations of NaCl (50 mM or 100 mM), the mycorrhizal plants never reached the T4 stage. Thus the AM mycorrhizal symbiosis appeared to mitigate the adverse effects of salt on plant wilting. 3.2 Glasshouse salinity tolerance assessment

Author's personal copy

Time (hours)

Fig. 1 Wilting trend and time necessary for maximum wilting from salt stress caused by NaCl (mM) in M. sativa var. icon, three months old, inoculated (AM+) or not inoculated (nonAM) with G. viscosum. T1 (leaves flaccid), T2 (leaves and stem flaccid), T3 (stem bent), T4 (total wilting) are the wilting indexes evaluated after exposure to NaCl. The values represent means of 10 replicates ± standard error. Test SNK, **Significant difference between treatments (P≤0.01)

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Time (hours)

The role of arbuscular mycorrhizal fungi

160 140 120 100 80 60 40 20 0

160 140 120 100 80 60 40 20 0

**

**

50 mM NaCl AM+ 50 mM NaCl Non-AM T1

T2 T3 Wilting index

**

**

T4

**

100 mM NaCl AM+ 100 mM NaCl Non-AM

T1

T2

T3

T4

Time (hours)

Wilting index 160 140 120 100 80 60 40 20 0

**

**

**

**

150 mM NaCl AM+ 150mM NaCl Non-AM T1

T2

T3

T4

Time (hours)

Wilting index 160 140 120 100 80 60 40 20 0

**

**

**

**

200 mM NaCl AM+ 200 mM NaCl Non-AM T1

T2

T3

T4

Wilting index

that both the levels of salinity in the substrata, and the mycorrhizal treatment, affected growth. The AM symbiosis

stimulated plant height, leaf area, root density, fresh and dry plant weight under saline conditions. The increased salinity



Table 1 Height, leaf area, fresh weight (FW), dry weight (DW), root density and plant tolerance (Pt) of M. sativa var. icon inoculated with G. viscosum (AM+) or non inoculated (Non-AM) under three saline conditions. Shoot AM treatment

Non-AM

AM+

Root

Plant

Salinity

Height cm plant-1

Leaf area cm2 plant-1

FW g plant-1

DW g plant-1

FW g plant-1

DW g plant-1

Density cm cm-3 plant-1

Total DW g plant-1

0 mM NaCl 100 mM NaCl 150 mM NaCl 0 mM NaCl 100 mM NaCl 150 mM NaCl

43.7 42.9 30.7 56.4 53.4 48.6

296.7 c 246.4 d 231.5 d 488.4 a 411.0 ab 350.9 b

17.0 b 13.4 c 11.8 c 25.6 a 20.1 b 17.3 b

3.6 2.9 2.5 5.1 4.6 4.0

24.3 d 20.5 e 18.0 e 47.1 a 41.8 b 29.8 c

4.1 3.6 2.6 7.7 7.1 6.1

0.35 c 0.27 d 0.24 d 0.48 a 0.41 b 0.34 c

7.7 c 6.5 cd 5.1 d 12.8 a 11.7 ab 10.1 b

** ** NS

** ** NS

** ** NS

** ** *

** ** NS

** ** NS

** ** NS

Significance AM treatment (M) Salinity (S) MxS

** ** NS

c c d a ab b

bc cd d a a ab

c cd d a ab b

Pt %

84.4 66.2 91.4 78.9

The values represent means of 10 replicates. Different letters within each column indicate significant differences between treatments. Test SNK, **Significant P≤0.01, *significant P≤0.05, NS non significant

reduced the growth of the plants; the interactions between the treatments also had a significant effect on root fresh weight. On the basis of total dry weight, plant tolerance (Pt), was greater in mycorrhizal plants (91.4% and 78.9% in plants irrigated with 100 mM or 150 mM NaCl, respectively) than in non-mycorrhizal plants (84.4% and 66.2%, respectively). The mycorrhizal symbiosis decreased growth inhibition due to salt stress at all levels. Table 2 shows that for plant dry weight, mycorrhizal dependency increased at higher sodium chloride levels. This suggests that the symbiotic association between the mycorrhizal fungus and the Medicago sativa plants was strengthened in the saline environment once the association was established. However, the level of mycorrhizal colonization decreased with increasing sodium chloride concentrations in the substratum (Table 2). Thus, salinity is also a stress for G. viscosum, limiting its ability to colonize root systems, but this reduction did not influence the effectiveness of the symbiosis.

Table 2 Root arbuscular mycorrhizal (AM) colonization and mycorrhizal dependency of M. sativa var. icon inoculated with G. viscosum under three saline conditions Salinity

AM colonisation (%)

AM dependency (%)

0 mM NaCl 100 mM NaCl

69.7 a 57.1 b

41.0 a 44.8 ab

150 mM NaCl

47.7 c

50.2 b

The values represent means of 10 replicates. Different letters indicate significant differences between treatments. Test SNK, P≤0.01.

3.2.2 Gas exchange measurements Stomatal conductance was affected by AM colonization and by salt stress conditions (Table 3). Mycorrhizal plants had higher values of stomatal conductance and these were be consistent with the observed higher rates of gas exchange. A physiological adaptation may be necessary to supply the carbon needs of the fungal symbiont. Promotion of stomatal conductance by saline irrigation water (especially at moderate salinity, i.e. 100 mM NaCl) was higher during the first 30 days of the trial than during the last 30 days when conductance values gradually decreased. Gradual stomatal closure allowed for the maintenance of plant water status and physiological activity while the plant salt stress persisted. 3.2.3 Chlorophyll determinations The SPAD value indirectly quantifies chlorophyll content and provides an indirect measure of the plant nitrogen status (Chang and Robison 2003; Gáborčík 2003; Wu et al. 2007; Esfahani et al. 2008). In this experiment, the mycorrhizal treatment exhibited higher SPAD values irrespective of the level of salinity (Table 4). This indicates a higher chlorophyll content and, hence, a higher photosynthetic potential. Saline treatments did not influence SPAD readings significantly. The mycorrhizal symbiosis appeared to sustain the activities and synthesis of the mesophyll (Table 5). Mycorrhizal plants had a higher chlorophyll content than nonmycorrhizal plants, particularly with respect to chlorophyll a levels.

Author's personal copy The role of arbuscular mycorrhizal fungi Table 3 Stomatal conductance of M. sativa var. icon inoculated with G. viscosum (AM+) or not inoculated (Non-AM) under three saline conditions.

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AM treatment

Salinity

Stomatal conductance (mmol m-2 s-1) Days after inoculation 105


Non-AM

AM+ The values represent means of 10 replicates. Different letters within each column indicate significant differences between treatments. Test SNK, **Significant P≤0.01, *significant P≤0.05, NS non significant

397 595 585 433 645 630

115 c b b c a a

448 640 590 491 676 657

125 d a b c a a

546 691 604 623 738 673

135 d ab c c a b

505 564 490 576 619 550

145 c b c b a b

458 404 320 494 461 360

155 ab b c a ab c

441 387 305 487 435 373

165 b c d a b c

388 ab 290 c 192 e 411 a 350 b 244 d


** ** NS

Salinity decreased significantly the total chlorophyll content; particularly it reduced leaf content of chlorophyll a (Chl a) but had no significant effect on chlorophyll b (Chl b). 3.2.4 Leaf relative water content, Osmotic potential, Proline determination and Stomatal density The salt treatment induced the reduction of relative leaf water content (Table 5). This decrease indicated a loss of turgor. Mycorrhizal inoculation improved the water status of the M. sativa plants; mycorrhizal plants maintained a higher leaf water content under saline conditions. The osmotic potential of salt-treated plants decreased with increasing NaCl concentration (Table 5), but there was no difference

** ** NS

** ** NS

** ** NS

** ** NS

** ** NS

** ** NS

between mycorrhizal and non-mycorrhizal plants at each salinity level. Foliar proline content increased in response to an increase in NaCl (Table 5). One of the mechanisms of salt tolerance in plants is osmotic adjustment. Proline accumulation in response to osmotic stress is widely reported and can play an important role in stress adaptation (Porcel and Ruiz-Lozano 2004; Karimi et al. 2005; Sharifi et al. 2007; Borde et al. 2010; Kumar et al. 2010; Dudhane et al. 2011; Talaat and Shawky 2011). Proline accumulation was higher in mycorrhizal M. sativa plants than in nonmycorrhizal plants and the values were significant as shown by the interactions (SxM) between salinity (S) and AM treatments (M) (Table 5). It is possible that mycorrhizal fungi are able to promote adaptation in the host under salt stress conditions.

Table 4 SPAD values of M. sativa var. icon inoculated with G. viscosum (AM+) or not inoculated (Non-AM) under three saline conditions AM treatment

Salinity

SPAD values Days after inoculation

Non-AM

AM+ Significance AM treatment (M) Salinity (S) MxS


105

115

125

32.0 b 32.6 b 32.6 b 42.0 a 38.9 a 38.5 a

34.8 b 35.2 b 34.7 b 41.8 a 40.2 a 40.5 a

36.1 35.9 36.1 43.5 43.1 42.4

** NS NS

** NS NS

** NS NS

b b b a a a

135

145

155

38.5 b 38.0 b 37.8 b 46.6 a 44.2 a 44.1 a

39.2 b 38.0 b 36.3 b 45.0 a 44.1 a 43.5 a

37.3 37.2 36.5 44.5 43.7 41.6

** NS NS

** NS NS

** NS NS

165 b b b a a a

36.7 b 36.5 b 35.7 b 44.0 a 41.2 a 41.1 a ** NS NS

The values represent means of 15 replicates. Different letters within each column indicate significant differences between treatments. Test SNK, **Significant P≤0.01, *significant P≤0.05, NS non significant



Table 5 Chlorophyll content (Chl a, Chl b, Chl tot), relative water content (RWC), osmotic potential, proline content, stomatal density of M. sativa var. icon inoculated with G. viscosum (AM+) or not inoculated (Non-AM) under three saline conditions AM treatment

Non-AM

AM+

Salinity

Chl a Chl b mg g-1 FW

Chl tot

RWC %

Osmotic potential Mpa

Proline content μmol g-1 FW

Stomatal density units mm-2


2.4 1.9 1.9 2.8 2.3 2.3

3.3 b 2.8 c 2.7 c 3.9 a 3.4 b 3.4 b

85.8 80.4 73.3 90.5 85.4 78.8

−0.9 a −1.2 b −1.7 c −0.9 a −1.2 b −1.6 c

0.4 f 1.3 d 2.1 c 0.6 e 2.5 b 6.0 a

240.0 222.9 217.1 291.4 271.4 254.3

** ** NS

** ** NS

NS ** NS

** ** **

** * NS


** ** NS

b c c a b b

0.9 0.9 0.8 1.1 1.1 1.1 * NS NS

b b b a a a

b c d a b c

ab b b a ab ab

The chlorophyll content (Chl a, Chl b, Chl tot) values represent means of 3 replicates. The relative water content(RWC) values represent means of 30 replicates. The osmotic potential and the proline content values represent means of 5 replicates. The stomatal density values represent means of 50 replicates. Different letters within each column indicate significant differences between treatments. Test SNK, **Significant P≤0.01, *significant P≤0.05, NS non significant

Stomata numbers (per unit of leaf area in abaxial epidermis) tended to decrease with increasing NaCl levels in control non-mycorrhizal plants (Table 5), and this could be an adaptation to alleviate salt stress (Kaya et al. 2007). In contrast, mycorrhizal plants showed a rise in stomatal density (Table 5). 3.2.5 Mineral analysis All element concentrations in shoots and roots of M. sativa var. icon are displayed in Table 6. Sodium (Na) and chloride (Cl) contents in leaf tissue and in roots were significantly raised when the salinity of the substrata was increased. Mycorrhizal plants showed lower levels of Na and Cl in all organs. The levels of potassium (K), an antagonist of Na, were reduced by saline treatments, although mycorrhizal plants had higher K concentrations than non-mycorrhizal plants. Thus, symbiosis improved K uptake by M. sativa plants at all salinity levels in comparison with controls. The concentrations of Na, K and Cl in leaf tissues and in roots were significantly influenced by the SxM interaction (Table 6). Salinity reduced calcium (Ca) and magnesium (Mg) contents, but there were no differences between mycorrhizal and non mycorrhizal plants.

4 Discussion The results of the quick test show that mycorrhizal symbiosis has a beneficial effect in alleviating the negative osmotic stress caused by high saline concentrations (Rosendhal and Rosendhal 1991). The growth and physiological results obtained in the pot trial provide more evidence on the role of

the arbuscular mycorrhizal symbiosis in alleviating salt stress of Medicago sativa var. icon. As found in the present and previous studies, salinity affects the performance of M. sativa plants negatively (Ashraf et al. 1986; Khan et al. 1997; Anand et al. 2000; Maggio et al. 2009). However as found with various plant species, arbuscular mycorrhizae help to mitigate the effects of salinity treatments (Cantrell and Linderman 2001; Zuccarini 2007; Sharifi et al. 2007; Sheng et al. 2008; Borde et al. 2011). Although shoot and root dry biomass production decreased in M. sativa with increasing salinity in both mycorrhizal and non-mycorrhizal plants, the mycorrhizal plants had a greater ability to induce tolerate salinity and exhibited the smallest decreases in growth under salt stress conditions. AM-mediated growth could be the result of an improvement in nitrogen status, as suggested by the SPAD values monitored during the glasshouse trial. The SPAD values are closely linked to leaf chlorophyll content (Smith et al. 2004; Netto et al. 2005) and closely linked to leaf N content (Chang and Robison 2003; Gáborčík 2003; Netto et al. 2005; Wu et al. 2007; Esfahani et al. 2008). The chlorophyll analysis confirmed that the photosynthetic rate and chlorophyll content were higher in mycorrhizal plants than in non-mycorrhizal plants as found in previous studies (Busquets et al. 2010; Abdel Latef and Chaoxing 2011; Borde et al. 2011; Zhu et al. 2011). In the mycorrhizal plants there was a greater quantity of photosynthetic pigments compared with controls, even under salt stress conditions. The reduced chlorophyll pigments under salinity treatments could be attributed to the increased activity of chlorophyllase enzyme (Reddy and Vora 1985), or disruption of fine structure of the chloroplast and instability of pigment

Author's personal copy The role of arbuscular mycorrhizal fungi

73

Table 6 Shoot and root mineral content in M. sativa var. icon inoculated with G. viscosum (AM+) or not inoculated (Non-AM) under three saline conditions. AM treatment

Non-AM

AM+ Significance AM treatment (M) Salinity (S) MxS

Salinity


Leaf ion content (mg g-1 DW)

Root ion content (mg g-1 DW)

Na

K

Ca

Mg

Cl

1.3 e 30.4 c 36.8 a 1.3 e 25.2 d 33.3 b

27.6 b 21.9 c 20.8 c 30.7 a 26.4 b 22.8 c

23.3 a 21.3 ab 19.0 b 23.1 a 21.0 ab 19.4 b

5.1 a 4.8 a 4.0 b 5.3 a 5.0 a 4.2 b

13.8 29.8 39.8 12.0 26.7 29.0

** ** **

** ** **

** NS NS

** NS NS

** ** **

d b a d c b

Na

K

Ca

1.4 d 21.7 b 25.9 a 1.4 d 20.0 c 22.7 b

18.5 a 12.2 c 10.1 d 20.4 a 18.7 a 13.9 b

8.7 7.7 6.2 8.4 7.2 6.1

** ** **

** ** **

** NS NS

Mg a b c a b c

5.8 4.9 4.4 6.0 5.1 4.5 ** NS NS

Cl a ab b a ab b

6.8 e 14.6 c 18.5 a 4.4 f 9.7 d 12.5 b ** ** **

The values represent means of 3 replicates. Test SNK, **Significant P≤0.01, *significant P≤0.05, NS non significant

and protein complexes by ions (Djanaguiraman et al. 2006; Jaleel et al. 2008). The role of the AM-symbiosis in sustaining leaf photosynthesis could be due to a better water supply as a result of the increased density of roots in the mycorrhizal plants. This would also help to maintain the leaf water content (Krishna et al. 2005; Colla et al. 2008; Huang et al. 2011) and account for the significant differences in the relative water content of leaves in the salt treatments. Salinity reduced the osmotic potential in M. sativa plants but this decline could be mitigated by solute accumulation within the plant cells. The accumulation of proline in plants following salt stress is related to the osmoregulatory role of this compound (Heuer 1999). Many studies have highlighted the role of proline accumulation as a reliable index of salt tolerance (Giridarakumar et al. 2003; Meloni et al. 2004; Karimi et al. 2005). In the present study, salinity stress resulted in proline accumulation, especially in mycorrhizal plants. Previous studies on proline accumulation in plant tissues of mycorrhizal plants subjected to salt stress have yielded conflicting results. Ruiz-Lozano et al. (1996), Rabie and Almadini (2005), Wu and Xia (2006), Al-Khaliel (2010), Sheng et al. (2011), all found a decrease in the proline content in plant tissues of mycorrhizal plants subjected to osmotic stress. However, other studies (Ruiz-Lozano et al. 1996; Hatimi 1999; Sharifi et al. 2007; Borde et al. 2010; Kumar et al. 2010; Dudhane et al. 2011) found an increase of proline concentration in the plant tissues of mycorrhizal plants as was found in the present study. It seems likely that in M. sativa var. icon, the accumulation of proline is a physiological response to the plant-fungus interaction which activates the endogenous production of proline. As a result the mycorrhizal plants displayed a

greater degree of osmotic adjustment than the nonmycorrhizal plants under saline stress conditions. M. sativa var. icon plants showed the typical morphological and physiological adaptations to increasing salt concentrations, such as the regulation of water use through a gradual decrease of stomatal conductance and a lowering of leaf area to reduce the rate of water loss (Anand et al. 2000; Kao et al. 2006). The stomatal conductance of the leaves can be modified by the presence of AM fungi in the roots as found by Augè et al. (2001; 2004; 2008). Our results confirmed that Glomus viscosum symbiosis in M. sativa was able to increase stomatal conductance with subsequent greater carbon dioxide capture in the leaves, a higher photosynthetic rate, and hence, a greater plant biomass accumulation. The higher rates of gas exchange associated with mycorrhizal symbiosis are presumed to be necessary to supply the carbon needs of the fungal symbiont (Augé et al. 2008). Indeed, the higher stomatal conductance would be consistent with a slight increase of stomata number per mm2 in mycorrhizal plants. Environmental conditions in which plants grow can also influence stomatal frequency (Cole and Dobrenz 1970; Kang et al. 2011). Saline conditions modify the uptake of mineral nutrients and nutrient balance (Giri et al. 2007). Na and Cl concentrations were lower in mycorrhizal plants than in nonmycorrhizal plants. Some researchers have hypothesized that lower Na and Cl concentrations in plant tissues may be due to the capability of the fungus to retain these ions in intraradical fungal hyphae or to compartmentalize them in the root cell vacuoles (Cantrell and Linderman 2001; AlKaraki 2006). Others propose that AM symbiosis improves plant growth and this results in a subsequent dilution of the ions in the tissues (Jarrell and Beverly 1981; Al-Karaki 2000). In the present study, the salt treatment decreased K


concentrations in the plant tissues. This reduction in K uptake, caused by Na, is likely to be the result of the competitive intracellular influx of both ions (Cerda et al. 1995; Colla et al. 2008). Mycorrhizae had a positive influence in helping to maintain the potassium content at all salinity exposure levels. Potassium (K) is important for water regulation, stomatal behavior, cell expansion, etc. (Kaya et al. 2007); it also activates a range of enzymes, and Na cannot substitute it in this role (Giri et al. 2007). A high level of Na or a high Na:K ratio can disrupt various enzymatic processes in the cytoplasm; it seems that higher K accumulation by mycorrhizal plants under salt stress conditions may help in reducing the Na:K ratio in the plant, thus limiting the metabolic toxicity of Na. In conclusion, the arbuscular mycorrhiza is a key component in helping M. sativa var. icon plants to cope with adverse environmental conditions, particularly salinity. This is consistent with previous reports on the beneficial effects of mycorrhizal fungi in promoting plant growth and alleviating the negative effects of salt stress (Ruiz-Lozano et al. 1996; Yano-Melo et al. 2003; Zuccarini 2007; Colla et al. 2008; Sheng et al. 2008; Borde et al. 2011). By understanding more about the arbuscular mycorrhizal symbiosis between G. viscosum H.T. Nicolson strain A6 and the selected clone M. sativa var. icon, it is hoped that new cultivars can be developed that will be more productive in the semi-arid regions of the world where salt stress is a common feature. Acknowledgments The authors are grateful to Ms. Sara Donahue, English Language Professor in the Faculty of Foreign Languages and Literatures, University of Bari, for her invaluable language assistance in the preparation of this manuscript. Thanks are due to Antonio Corleto, Professor in the Department of Plant Production Sciences, University of Bari, for his precious advice and for providing Medicago sativa seeds. The authors also wish to thank Prof. Manuela Giovannetti of the Department of Crop Plants, University of Pisa (Italy) for providing the arbuscular-mycorrhizal inoculum.

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