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Title: Alleviating salt stress in tomato inoculated with mycorrhizae: photosynthetic performance and enzymatic antioxidants
Authors: Mohsen K.H. Ebrahim, Abdel-Rahman Saleem
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S1658-3655(17)30025-0
http://dx.doi.Org/doi:10.1016/j.jtusci.2017.02.002 JTUSCI 362
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7-2-2017
12-2-2017
Please cite this article as: Mohsen K.H.Ebrahim, Abdel-Rahman Saleem, Alleviating salt stress in tomato inoculated with mycorrhizae: photosynthetic performance and enzymatic antioxidants (2010), http://dx.doi.org/10.1016/j.jtusci.2017.02.002
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Alleviating salt stress in tomato inoculated with mycorrhizae: photosynthetic performance and enzymatic antioxidants
Mohsen K.H. Ebrahim1,2 and Abdel-Rahman Saleem1,3*
1 Biology Department, Faculty of Science, Taibah University, 344 Almadinah Almunawarah, Saudi Arabia
2 Botany Department, Faculty of Science, Tanta University, 31527 Tanta, Egypt
3 Botany Department, Faculty of Science, South Valley University, 83523 Qena, Egypt
Corresponding author: Tel: +966532806243 E-mail: asaleem@hotmail.com
Fax. +966 48454770
Abstract
Tomato cultivars (Sultana-7 & Super Strain-B) were germinated under various levels (0-200 mM) of NaCl. Seed germination of Super Strain-B was promoted at 25 mM NaCl. However, the germination of both cultivars was progressively inhibited at 50 and 100 mM, and stopped at 200 mM, but the response was more pronounced in case of Sultana-7. Therefore, Super Strain-B was selected for further investigation, after growing under NaCl stress (50 & 100 mM) and inoculation with vesicular-arbuscular mycorrhizal fungus (Glomus fasciculatum, VAMF). Mineral (N, P, K, Mg) uptake by leaves and K/ Na ratio were declined by salinity, while Na uptake and N/ P ratio were increased. Salinity decreased chlorophyll (Chl) contents
coupled with an increase in Chl a/ b, diminished Hill-reaction activity, and quenched Chl a fluorescence emission. This reflect the disturbance of the structure, composition and function of the photosynthetic apparatus as well as the activity of photosystem 2. Superoxide dismutase and peroxidase activities of leaves were enhanced by salinity, whereas catalase activity was decreased. Leaf polysaccharides and proteins, and shoot biomass were depressed by salinity, but total soluble sugars and root to shoot ratio were improved. Applying VAMF to plants enhanced both photosynthesis and productivity. This confirm the role of VAMF in alleviating negative effects of salinity on the plant, by increasing its salt tolerance. Although, mycorrhizal infection showed a negative correlation with salinity, it remained relatively high (21 & 25 %) at 100 mM NaCl.
Abbreviations: CAT= catalase, cv. = cultivar, POX= peroxidase, PS2= photosystem 2, ROS= reactive oxygen species, SOD= superoxide dismutase, TSP= total soluble proteins, TSS= total soluble sugars, VAMF= vesicular-arbuscular mycorrhizal fungus.
Keywords: Mycorrhizae, tomato, salinity, minerals, photosynthetic performance and antioxidant enzymes.
1. Introduction
Water stress induced by salinity could be regarded as a major factor exerting considerable alterations in plant growth and metabolism (Aly et al., 2003). The degree of such alterations, which can be regarded as salinity resistance, mainly depends on the plant species and even
cultivar as well as the growth stage and the salt level (Levitt, 1980, Ebrahim, 2005). According to the electrical conductivity (EC in mmhos cm-1) of the irrigation water, Ebrahim and Abu-Grab (1997) have classified the soil salinity into 3 orders: (1) none "EC < 0.75", (2) moderate "0.75 < EC < 3.0 and (3) severe "EC > 3.0. The severe salinity induces detrimental effects on plant growth, yield and productivity (Aly et al., 2003).
Salt tolerance is linked through common mechanisms of salt uptake for osmotic adjustment (Ebrahim, 2005). NaCl salinity inhibits plant growth by lowering soil water potential and increasing the potentially toxic ions (Na+ and Cl-) that, in turn, can lead to water deficit and nutritional imbalance (Levitt, 1980). Such effects lead to a decrease in water and nutrient uptake by plants, and may directly or indirectly damage the other physiological processes such as germination, growth, photosynthesis, respiration and metabolite accumulation (Aly et al., 2003; Ebrahim, 2005).
Plants use molecular oxygen as a terminal oxidant in respiration. However, the presence of oxygen, as reactive oxygen species (ROS), in cellular environment poses a constant oxidative threat to cellular structures and processes (El-Shintinawy et al., 2004). Such ROS, formed during electron transport activities in plants, are linked to oxidative stress caused by most stress factors such as salinity (Meloni et al., 2003). Plant cells have evolved defense antioxidant mechanisms to combat the danger posed by the presence of ROS. These include enzymatic mechanisms involving antioxidant enzymes such as superoxide desmutases (SODs), peroxidases (POXs) and catalases (CATs) (Landberg and Greger, 2002; Meloni et al., 2003; Li, 2009).
Under natural growth conditions, plants are adapted for minimizing the damage induced by ROS. However, O2 toxicity emerges when ROS production exceeds the quenching capacity of
protective system (Liu and Wu, 2014). This occurs under various stress conditions due to the dysfunction of photosynthetic and respiratory electron flow (Meloni et al., 2003; El-Shintinawy et al., 2004). The overproduction of ROS limits the activity of several enzymes, causes protein denaturation, DNA mutation and declines the ATP generation and redox metabolites that are essential for cellular defense and repair (Landberg and Greger, 2002; Maloni et al., 2003; El-Shintinawy et al., 2004; Li, 2009; Belew et al., 2010). These detrimental effects of ROS accumulation could limit the plant tolerance to stresses, resulting in growth retardation and early senescence (Hashem et al., 2015).
Vesicular-arbuscular mycorrhizal (VAM; G. fasciculatum) fungi are ubiquitous among a wide array of soil microorganisms inhabiting the rhizosphere (Giri et al., 2003). The symbiotic association of a plant with VAM fungi allows access to mobile nutrients in nutrient-poor soils (Marschner and Dell, 1994). VAM fungi constitute an integral component of the natural ecosystem, and are known to exist in saline environments where they improve early plant growth and tolerance to salinity (Abdel Latef and Miransari, 2014; Hashem et al., 2015). Many researchers have reported that VAM fungi could enhance the ability of plants to cope with salt stress (Yano-Melo et al., 2003; Rabie, 2005; Enteshari and Hajbagheri, 2011) by improving plant nutrient uptake (Asghari et al., 2005), protecting enzyme activity (Giri and Mukerji, 2004) and facilitating water uptake (Ruiz-Lozano and Azcon, 1995). In salt-stressed soil, VAM fungi are thought to improve the supply of mineral nutrients to plants, especially the supply of P, as it tends to be precipitated phosphate salts (Al-Karaki et al., 2001). VAM fungi counter-balanced the adverse effects of salinity stress and thereby increased plant growth (Giri et al., 2003). They have also protected the host plants against the detrimental effects of stesses (Rabie, 2005) by enhancing the antioxidant responses (Garcia-
Sancheza et al., 2014) and/ or via the induction of acquired systemic tolerance (Hashem et al., 2016).
Tomato is one of the most important economic crops in many countries. It belongs to fruit vegetable of horticulture crops, and its flesh is applied not only as salad and ingredients of food, but also fresh food. It is one of the important routine fruits and vegetables. The optimal temperature for germination is ranged from 20-25°C, while the plant fruiting is largely suppressed at temperature more than 40°C and less than 10°C (Li, 2009). The plant grows well in different soil, but the best one is characterized by fertility, moderate acidity, and good drainage (Li, 2009). The plant response to salt stress was studied during germination (Li, 2009), and vegetative growth stage and fruiting stage (Cuartero et al., 2006). The above authors recorded significant effects of salinity on most morphological and physiological characters, and fruit yield, but seeds and seedlings appeared to be less stress tolerant than adults.
Despite the enormous studies of plant responses to salinity and VAM fungi (e.g., Al-karaki et al., 2001; Rabie, 2005; Belew et al., 2010; Hashem et al., 2015), relatively little is known about the underlying mechanisms, especially in case of tomato plant. Therefore, this study aimed at: (1) selecting a salt-tolerant tomato cultivar (cv.), (2) alleviating the salt stress of such cv. with VAM fungi, (3) explaining the role of VAMF on biochemical mechanisms based on the alterations in photosynthetic performance as well as in the enzymatic antioxidant system, and (4) finding a recommendation for the possibility of tomato cultivation in salinized soils.
2. Materials and Methods
Plant material and sterilization. Seeds of tomato (Solanum lycopersicon L, cvs Sultana-7 and Super Strain-B) were obtained from the Central Market (Al-Halakah) of Al-Madinah Al-Munawarah (Saudi Arabia) and surface sterilized with 2% Na-hypochlorite for 10 min., then rinsed with sterile distilled water. The sterilized seeds of each cultivar were soaked in water for 12 h, then used for both Petri- dish and pot experiments.
Petri-dish experiment. The soaked seeds were germinated in Petri-dishes (10 cm diameter, half-filled with sand which was previously washed with concentrated HCl, then with distilled water). Soaked seeds (10 per dish) were uniformly distributed throughout Petri-dishes that were randomly ranked in 5 groups supplied with tap water (for 3 days). Then with half-strength Hoagland nutrient solution (Hoagland and Arnon,1950) at 25, 50, 100 and 200 mM NaCl, respectively. They were germinated for 12 days in growth chamber (5 days in dark, and then 7 days at a 12 h photoperiod at day/ night temperature of 30/ 18°C and 65-75% relative humidity).
1. Seed germination. On the twelfth day, seed germination (%) was determined as following: Seed germination (%) = (No. of germinated seeds/ total no. of seeds) x 100 Mycorrhizal inoculum. Mycorrhizal spores of Glomus fasciculatum (Taxt.) Gerd. & Trapp, were kindly obtained from Agricultural Research Center (ARC, Giza, Egypt), then kept at 4°C until use in the pot experiment.
Pot experiment. The soaked seeds were sown in plastic pots (18 cm diameter, 14 cm height). Each pot contained 2 kg washed and sterilized sandy soil. Seeds (10 per pot) were uniformly distributed throughout the pots, irrigated with tap water (whenever needed), for 15 days, until complete germination. On day 15, the pots were grouped in two groups (I & II). Each pot of group I was inoculated with approximately 50 ml of sterile (non-mycorrhizal, n-
M) treatment, while each one of group II was supplied with 50 ml of viable (mycorrhizal, M) treatment of G. fasciculatum spores obtained as described above. On day 20, plantlets of each pot were thinned to 4, and then pots of each group (I & II) were divided into 3 subgroups (A, B, & C) that were regularly supplied with full-strength Hoagland nutrient solution (Hoagland and Arnon, 1950) at 0, 50 and 100 mM NaCl, respectively. Plants were allowed to grow for 3-months in growth chambers (Biology Department, Faculty of Science, Taibah University, KSA) at 12-h photoperiod (i.e.,12 h in the light at 120-140 (xmol/ m2 s and 12 h in the dark), at day/ night temperature of 30/ 18°C, and 65-75% relative humidity. At 2 and 3 months post sowing, leaf and shoot samples were randomly taken and prepared for analyses. 1. Mycorrhizal infection. At 2 and 3 months post sowing, fresh roots were separated, cleaned, stained (Phillips and Hayman, 1970) and examined microscopically for vesicular-arbuscular mycorrhizal (VAM) colonization. Colonization on root pieces was recorded if any mycorrhizal hyphae, vesicles, arbuscles or spores were detected.
3. Leaf minerals. At 2 months post sowing, fresh leaves were dried in an aerated oven at 70°C to constant weight, and then used for mineral (N, P, K, Mg & Na) determination. Mixed-acid-digestion method (Allen et al., 1974) was used for mineral extraction. Total-nitrogen (N) concentration was determined using the micro-Kjeldahl method (Jacobs, 1958). Phosphorus (P) concentration was spectrophotometrically determined by molybdenum-blue method (Page, 1982). Potassium (K), Magnesium (Mg) and sodium (Na) concentrations were determined according to Allen et al. (1974). Flame photometer (Corning Scientific Instruments, Model 400) was used for K and Na determinations, while Atomic-Absorption Spectrophotometer (Perkin-Elmer, 2380) was used for Mg determination.
3. Chlorophyll. At 2 months post sowing, Chl was extracted from 0.5 g fresh mass of green leaves in 10 ml of pure N, N-dimethyl formamide (Ebrahim et al., 1998). The extract was kept in dark for 2 d at 4°C, and then centrifuged for 15 min. at 66.7 rps. Chl concentration in the supernatant was spectrophotometrically determined according to the equations of Moran and Porath (1980).
4. Photosynthetic (Hill-reaction) activity. At 2 months post sowing, photosystem 2 (PS2) activity of chloroplasts isolated from green leaves expressed as electron transport rate was determined by using 2,6-dichlorophenol indophenol (DCPIP) as electron acceptor (Hashem et al., 2015). Chloroplasts were isolated in the cold as described by EL-Shintinawy (2000). The concentration of Chl a+b in the supernatant was determined according to the equation of Arnon (1949). For measuring the PS2 activity, assay sample was prepared by mixing 1.6 ml of 10 mM DCPIP (dissolved in 96% ethanol) with 50 ^g Chl, and then the volume was completed to 3 ml by the reaction buffer. The sample was irradiated (at right angles) with red actinic radiation (300 W/m2, 10 min) provided from a slide projector. The DCPIP photo-reduction was assayed spectrophotometrically according to EL-Shintinawy et al. (2004).
5. Chl a fluorescence
At 2 months post sowing, Chl a fluorescence emission spectra were measured at room temperature (26±2°C) according to Tripathy et al. (1981) with minor modifications. The blue actinic light was switched on and focused on the sample cuvette by a spectrofluorometer (model 510, Schimadzu, Japan). Chloroplast isolation and Chl determination were carried out as described above. Isolated chloroplasts were suspended in the reaction buffer, then transferred to the sample cuvette. The assay volume was 3 cm3 and the Chl concentration was
5 p,g cm-1. All samples were incubated in dark for 15 min prior to measurements. Thereafter, samples were extracted by blue actinic light (460 nm). Produced emission kinetics (signals) were photomultiplied and recorded by means of a recorder. Fluorescence data were presented as the relative fluorescence intensity in arbitrary units.
5. Antioxidant enzyme assays. At 2 months post sowing, superoxide dismutase (SOD) was assayed on the basis of its ability to inhibit the photochemical reduction of nitro blue-tetrazolium (Beauchamp and Fridovich, 1971). The reaction was initiated by switching on "White light" and allowing the reaction to run for 10 min. before being stopped by switching the light off. Thereafter, the absorbance of the reaction mixture was measured at 560 nm. Log A560 was plotted as a function of the volume of enzyme extract (0- 200 ml) in the reaction mixture. The volume of raw extract (enzyme) producing 50% inhibition of the initial rate of the reaction, in absence of the enzyme, was calculated from the resultant curve and defined as one unit of SOD activity. Peroxidase (POX) and catalase (CAT) were assayed by measuring the initial rate of disappearance of H2O2 and tetra-guaiacol, respectively (Kato and Shimizu, 1987). For POX, the decrease in tetra-guaiacol was followed as a decline in the absorbance at 470 nm and the activity was calculated using the extinction coefficient (26.6 mM/ cm at 470 nm, Meloni et al., 2003) for tetra-guaiacol. For CAT, the change in the absorbance, induced by the decrease in H2O2 was measured at 420 nm and the activity was calculated using the extinction coefficient (40 mM/ cm at 240 nm) for H2O2.
6. Leaf metabolites and shoot growth. At 3 months post sowing, leaf, root and shoot samples were randomly taken, oven-dried at 70°C until constant weight. Leaf carbohydrates and proteins were extracted in borate buffer (PH 8), then carbohydrate fractions (g glucose/ 100 g dry weight) were estimated according to Naguib (1964 and 1963, respectively), while
total soluble proteins were determined according to the method of Lowry et al. (1951). Shoot biomass (g /plant) as well as root to shoot ratio were determined on dry weight basis. Statistical analysis. Data were averaged and statistically analyzed by using two-way analysis of variance (ANOVA). The least significant difference at 0.05 level was used to compare the means indirectly by the multiple range tests of Duncan (1955) or directly according to Steel and Torrie (1980).
3. Results
The viability of tomato seedlings as well as the photosynthetic performance and the accumulation of certain chemical constituents as well as shoot productivity could be considered as integral indexes for stress-tolerance response. Hence, we concentrated on these parameters to assess the interactive effects of NaCl stress (25-200 mM) and VAMF on germination, growth and development of two tomato cultivars. However, the most important consideration for successful tomato production is the selection of a cultivar capable of maximizing the utilization of available resources under stress conditions. Therefore, this study aimed at determining the genotypic stability of two tomato cultivars (Sultana-7 and Super Strain-B) stressed by NaCl-salt. These cultivars are comparatively characterized by high yield, hence they are commonly cultivated in Saudi Arabia. A preliminary experiment showed that NaCl-salt stress (25-200 mM) had progressively reduced the percentage of seed germination of Sultana-7 (Table 1). In contrast, seed germination of Super Strain-B was stimulated at the lowest level (25 mM), but evidently inhibited by the moderate and severe levels (50 and 100 mM) and completely suppressed by the highest level (200 mM) of NaCl. Although seed germination of Sultana-7 was evidently retarded by the level of 100 mM, 46 % of the seeds of Super Strain-B were germinated. Beside the significant differences between
the germination rates of both cultivars at 25 and 50 mM, respectively, the axes grew more rapidly and their growth was more uniform in Super Strain-B compared with Sultana-7 (data not shown). These results comparatively demonstrated a higher genotypic stability and stress-tolerance for the cultivar Super Strain-B. They also allowed to select the salinity levels of 50 and 100 mM - as well as the cultivar Super Strain-B- for further studies described below. It is clear that mycorrhizal infection tended to decrease significantly with increasing salinity (Table 2), although it remained relatively high even at high salinity (21% at 100 mM NaCl). Regardless of mycorrhizal treatment; salinity caused a significant decrease in N, P, Mg and K/Na ratio, and a non-significant increase in K concentration, while the reverse was true in case of N/P ratio and Na concentration in leaves of 2-month old tomato plants (Table 3). In either moderate (50 mM) or severe (100 mM) salinity level, mycorrhizal infection induced a significant increase in leaf N, P and Mg, and a non-significant increase in case of K and Na. In contrast, the N/ P ratio decreased with mycorrhizal infection, indicating that mycorrhizae increased P rather than N.
NaCl stress caused significant and progressive reductions in Chl a+b content as well as in PS2 activity, expressed as DCPIP photoreduction of isolated chloroplasts of tomato leaves, while the reverse was shown under the effect of mycorrhizal treatment (Table 4). In contrast, Chl a/b ratio was increased by salinity as well as by mycorrhizal inoculation, suggesting that Chl b is more sensitive to salinity and mycorrhizae than Chl a. Although both moderate and severe levels (50 and 100 mM) showed similar negative effects, the magnitude of effect was comparatively higher in case of the severe level. To determine the site of the inhibitory effect of salinity on PS2 activity, Chl a fluorescence emission spectra were studied in the absence and presence of DCMU (Fig.1) which blocks the electron flow from the primary (QA) to the
secondary (QB) quinone acceptors and thus chemically isolated PS2 from PS1. On excitation at 460 nm, isolated chloroplasts showed an emission peak, at 684-685 nm, from Chl a of PS2. Whether in the absence or presence of DCMU, salinity progressively quenched the fluorescence emission with shifts in peaks observed more drastically in absence of mycorrhizae. However, in all treatments, Chl a fluorescence displayed higher intensities in the presence than absence of DCMU (Fig.1).
The intracellular alterations under salinity stress, induced by accumulating over-oxidation products, can evidently serve as triggers for appropriate protective mechanisms. In leaves of 2-month old tomato plants, activities of antioxidant enzymes (SOD, POX and CAT) were significantly (P< 5 %) influenced by salinity, mycorrhizae and their interactions (Table 5). Activities of SOD and POX were positively and progressively correlated with salinity level, while the reverse was true in case of CAT as well as under mycorrhizal treatment in case of all enzymes. The superior activities of SOD and POX were recorded in leaves of the non-mycorrhizal plants at the severe level of NaCl (100 mM), while inferior ones were shown in mycorrhizal control plants. However, a contrary trend was shown in case of catalase (Table 5).
The mycorrhizal infection showed a significant decrease with increasing salinity (Table 6). Such infections were noticed to be higher comparing with those recorded in Table 2 (2-month old tomato plants).
Regardless of mycorrhizal treatment, leaf soluble sugars (TSS) as well as root to shoot ratio have progressively increased by increasing salinity, while the reverse was true in case of all other criteria indicated in Table 7. Such contrasting responses were more pronounced at 100 mM NaCl. In addition, the mycorrhizal inoculum improved such criteria in the case of
moderate salinity level (50 mM) and alleviated the adverse effects of severe one (100 mM, Table 7). Salinity caused a progressive and significant decrease in the growth of test tomato cultivar (Super Strain-B), whereas such decrease was minimized by VAM fungus (Fig. 2).
4. Discussion
In the present investigation, tomato plant might be suffering from two problems: (1) high levels of NaCl in the soil solution, and (2) high levels of Na+ and Cl-. NaCl exclusion minimizes ion toxicity, but accelerates water deficit in plants (Gunes et al., 1996). Whereas, salt absorption facilitates osmotic adjustment, but can lead to ion toxicity and nutritional imbalance (Aly et al., 2003).
The effect of various levels of NaCl on seed germination is an important factor in predicting the possible response of tomato seedlings as well as the intact plant to stress caused by salinity. Therefore, the influence of NaCl levels on seed germination was firstly examined. Salt stress was shown to lead to cultivar and concentration-dependent changes in seed germination. These changes agree with others recorded by Al-Karaki et al. (2001). They could be also referred to the inhibition or stimulation of some early metabolic processes related to seed germination. Therefore, these processes are believed to be progressively inhibited by salinity in both cultivars. Although the mechanism of these changes is still not clear and has poorly investigated, the change in the activity of enzymes involved in the mobilization of stored compounds within seeds can be taken into account. However, the higher genotypic stability of Super Strain-B compared with Sultana-7 may be interpreted to a higher adaptive response of seedlings of Super Strain-B to alleviate the adverse effects of salt stress. Further investigation of metabolic processes, related to seed germination, as well as respiration rate, and protein and isozyme patterns may support the interpretations of this section of results.
The mycorrhizal isolate of Glomus fasciculatum (Phillips and Hayman, 1970) showed a high salt-tolerance, and could evidently grow in the presence of NaCl (up to 100 mM), but the percent of root length infected decreased by salinity. Similar results were obtained by others (Phillips and Hayman, 1970; Garcia-Sancheza et al., 2014; Hashem et al., 2015), who attributed this to the salinity problems, which could induce a spontaneous efflux of cell water and the accumulation of osmo-regulators in the cell (Table 7).
NaCl salinity increased the absorption of Na+, whereas N, P, K, and Mg uptake, and K/ Na ratio were decreased. These results are in general or partial agreement with several authors (Gunes et al., 1996; Aly et al., 2003; Giri et al., 2003) and could be due to the salinity problems described above. Increasing Na+ concentration disturbs the nutrient balance, osmotic regulation and causes specific ion toxicity (Munns, 1993). The decrease in K and N concentrations, in plant, by salinity was ascribed to the antagonism between Na+ and K+, and between Cl- and NO3-, respectively (Gunes et al., 1996). Although the VAM biofertilizer increased the accumulation of N, P, K, and Mg; but reduced Na+ concentration. This finding is in line with results reported by several authors (Marschner and Dell, 1994; Giri and Mukerji, 2004; Garcia-Sancheza et al., 2014). It could be also attributed to a decrease in Na+ concentration by mycorrhizal inoculation (Table 3).
Salinity is a stress factor experienced by plants (Levitt, 1980; Yano-Melo et al., 2003). Deleterious effects of salinity are reported to be partitioned between damage to the photosynthetic machinery and damage to the plant genome (Ebrahim et al., 1998; El-Shintinawy, 2000; Aly et al., 2003; Meloni et al., 2003). The decrease in Chl concentration by salinity has been reported (Gunes et al., 1996; Aly et al., 2003; Garcia-Sancheza et al., 2014) and might be referred to changes in the pigment biosynthesis and/ or degradation. It was also
attributed to a salt induced weakening of protein-pigment-lipid complexes (Gunes et al., 1996), increased chlorophyllase activity (Stivsev et al., 1973) and/or decreased concentrations of N and Mg in plant leaves (Table 3). However, enhancing Chl concentration by the vesicular-arbuscular mycorrhizal (VAM) fungus (Glomus fasciculatum) might be due to their role in: (1) decreasing the absorption of sodium, and (2) increasing both N and Mg concentrations of tomato leaves (see Table 3). The increase of Chl a/b ratio by salinity is mostly due to a variation in the level of light harvesting protein complex as an adaptive response to salt stress. It might be also ascribed to the grater destruction of Chl b than Chl a (data not shown). This may lead to changes in the composition and structure of Chl a/b protein complex (Meloni et al., 2003; El-Shintinawy et al., 2004) which, in turn, can influence the chloroplast development (El-Shintinawy, 2000). The comparatively decrease in PS2 activity, in in leaves of Super Strain-B under salt treatment, could be due to: (1) a declined energy transfer from the light harvesting complex to the reaction center, (2) inability of the reaction center to accept photons as a result of the altered architecture of PS2 complex and/ or (3) a dramatic decrease in the electron flow from Qa to Qb quinone acceptors of PS2 (El-Shintinawy, 2000; Ebrahim et al., 1998; Meloni et al., 2003).. To examine the above possibilities, Chl a fluorescence emission spectra were investigated in the absence and presence of DCMU (Fig.1.). In the absence of DCMU, the quenching of Chl a fluorescence reflects the inefficient energy transfer from the light harvesting complex to the reaction center of PS2 (Cao and Govindjee, 1990), probably as a result of structural alterations in the PS2 complex. This quenching was partly increased by DCMU, suggesting an inhibitory effect of salt stress on the electron transport from Qa to Qb. Therefore, these results confirm an inhibitory effect of salinity on both the donor and the acceptor sides of the isolated
chloroplasts. They also display the impairment of the photochemical efficiency of PS2 protein complex by salinity. Since this impairment was comparatively lower in mycorrhizal plants, the non- mycorrhizal plants showed more sensitivity (lower tolerance) to salt stress than mycorrhizal plants. This finding is in agreement with others reported by several workers (Al-Karaki et al., 2001; Rabie, 2005; Hashem et al., 2015).
Under oxidative stress conditions such as salt stress, plant cells have evolved defense antioxidant mechanisms to combat the danger posed by the overproduction of ROS. These include enzymatic mechanisms involving antioxidant enzymes such as superoxide dismutases (SODs), peroxidases (POXs) and catalases (CATs) (Landberg and Greger, 2002; Meloni et al., 2003). SOD is activated by increasing the level of its substrate (Meloni et al., 2003), whereas the increase of SOD activity in our work could be ascribed to an increase in its biosynthesis and/ or to an activation of its latent form or both (El-Shintinawy et al., 2004). However, SOD does not provide the complete protection of the cell against oxidative stresses, since H2O2 emerges as a product of its functioning (Meloni et al., 2003). H2O2 can be destroyed by POXs and CATs, and therefore our results in Table 5 can support this suggestion. Although the response of SOD and POX to salt stress was similar, a contrary trend was recorded in case of CAT (Table 5). This finding is in accordance with several authors (Meloni et al., 2003; El-Shintinawy et al., 2004; Hashem et al., 2015). However, the decrease of activities of antioxidant enzymes (SOD, POX and CAT) by VAM is in line with several authors (Younes et al., 2013; Garcia-Sancheza et al., 2014; Hashem et al., 2015) and could be ascribed to a decrease in their biosynthesis and/ or activation of their latent forms or both. This finding confirm the role of Glomus fasciculatum in alleviating the oxidative salt stress effects on the test plant (Liu and Wu, 2014; Hashem et al., 2015). Further investigation
of protein and isozyme patterns as well as gene expression may support the interpretation of these results.
The higher mycorrhizal infection in 3-month old tomato plants comparing with those of 2-month old ones is in agreement with results obtained by other authors (Phillips and Hayman, 1970; Garcia-Sancheza et al., 2014), and might be ascribed to adaptation of VAM fungus to stress conditions by time.
Our results of leaf metabolites, shoot biomass and root to shoot ratio are totally or partially in accordance with results reported by several workers (Gunes et al., 1996; Al-Karaki et al., 2001; Aly et al, 2003; Giri et al, 2003; Yano-Melo et al, 2003; Asghari et al, 2005; Ebrahim, 2005; Rabie, 2005; Cuartero et al., 2006; Belew et al., 2010). In our work, the correlation between TSS and Na ion concentration (Tables 3 & 7) confirms the reported role of TSS in osmoregulation which enables plants to tolerate or adapt to saline conditions (Levitt, 1980; Cuartero et al., 2006; Younes et al., 2013). Also, the reductions in polysaccharides as well as total carbohydrates and TSP concentrations could be attributed to the inhibition of their biosynthesis and/ or to the stimulation of their breakdown through affecting the activities of the respective hydrolases enzymes. The positive effect of VAM on these characters could mainly referred to the influence of VAM on osmotic adjustment and antioxidant enzyme activity (Younes et al., 2013) and/ or to the role of mycorrhizal fungi in alleviating the deleterious stress effects by enhancing water and mineral uptake (Marschner and Dell, 1994; Ruiz-Lozano and Azcon; 1995; Al-Karaki et al., 2005; Rabie, 2005; Enteshari and Hajbagheri, 2011; Abdel Latef and Miransari, 2014), but the mechanism of such role is still relatively obscure.
The inhibition of shoot growth, by salinity treatment, was reported (Aly, et al., 2003; Ebrahim, 2005; Garcia-Sancheza et al., 2014; Hashem et al., 2015). Suppression of plant growth by salinity could be attributed to an inhibitory effect on both meristematic activity and biosynthesis of auxin, whereas the alleviation of this effect by VAM was ascribed to the positive role of the fungal inoculum in water and nutrient uptakes (Marschner and Dell, 1994; Ruiz-Lozano and Azcon; 1995; Al-Karaki et al., 2005; Rabie, 2005; Abdel Latef and Miransari, 2014). In the present work, the positive correlation between the plant growth, and Chl concentration in leaves, metabolite accumulation and enhanced levels of minerals in tomato leaves (Tables 3, 4 and 7) could be taken into consideration for explaining the variation of tomato shoot growth and productivity with salinity and the mycorrhizal inoculation. Also, increasing the root to shoot ratio with salinity (Table 7) could be ascribed to an attempt of tomato plants to: 1) decrease the transpired water, and 2) increase the absorbed water. Consequently, the plant can resist water stress resulting from growing under saline conditions.
5. Conclusion
Salinity retarded tomato growth and caused a decrease in K/Na ratio, but a drastic increase in TSS content and root to shoot ratio, that have been correlated with the level of Na+ in plant leaves. Inoculating control plants with VAM fungus improved the plant growth. However, inoculation of the NaCl-stressed plants - by VAMF - alleviated the adverse effect (s) of salinity. This alleviation was enough for the plant to be able to efficiently resist the harmful effects of salinity, particularly at the moderate level. Therefore, we recommend that the test cultivar of tomato (Super Strain-B) is promising to be cultivated in salinized soils, especially
in the presence of VAMF (i.e., the present data satisfactorily support our hypothesis described in the Introduction section).
Acknowledgments
The authors gratefully acknowledge the Deanship of Scientific Research at Taibah University, Saudi Arabia, for financial support and guidance in this research project (Project No. 3015).
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Wavelength (nm)
Figure 1: Chl a fluorescence emission spectra of chloroplasts isolated from leaves of 2-month old tomato plants (cv. Super Strain-B) grown under various levels of NaCl (0, 50 & 100 mM) with respect to vesicular-arbuscular mycorrhizal fungus (VAMF). (1) Control plants, (2) 50 mM NaCl, and (3) 100 mM Na Cl.
Figure 2: Mycorrhizal (M) and non mycorrhizal (n-M) tomato plants (cultivar Super Strain-B) grown under various levels of NaCl for three month old.
Table 1: Effect of NaCl level (mM) on seed germination (%) of two tomato
cultivars
NaCl level (mM) Seed germination on day 12
Sultana-7 Super Strain-B
0 84 b 86 b
25 65 d 94 a
50 56 d 69 c
100 20 f 46 e
200 0 0
Mean values marked by the same letter do not differ significantly (P < 0.05).
Table 2: Effect of NaCl level (mM) on vesicular-arbuscular mycorrhizal fungus (VAMF) infection (%) of roots of 2-month old tomato plants (cv. Super Strain-B)
NaCl level (mM) Root length infected (%)
Mycorrhizal status
Uninoculated (n-M) Inoculated (M)
0 0.0 42 a
50 0.0 27 b
100 0.0 21 c
Mean values marked by the same letter do not differ significantly (P <
0.05).
Table 3: Interactive effects of NaCl level (mM) and vesicular-arbuscular mycorrhizal fungus (VAMF) on mineral (N, P, K, Mg & Na) content [mg/ g (dm)], and N/ P and K/ Na ratios of leaves of 2-month old tomato plants (cv. Super Strain-B).
NaCl level (mM) Mycorrhizal treatment N P K Mg Na N/ P ratio K/ Na ratio
0 n-M 20.8 11.1 11.9 5.8 4.44 1.87 2.68
M 21.6 12.0 12.2 6.1 4.27 1.80 2.86
50 n-M 18.4 8.3 11.5 5.1 5.28 2.22 2.18
M 19.9 9.4 11.9 5.7 5.03 2.12 2.21
100 n-M 13.1 4.2 11.2 3.9 6.24 3.12 1.79
M 15.0 5.7 11.7 4.2 6.01 2.63 1.95
Factor L.S.D. at 5% level
NaCl level (A) Mycorrhizae (B) Interaction (Ax B) 1.8 1.2 NS 0.64 0.38 - -
1.1 0.8 NS 0.26 NS - -
2.4 1.5 NS 0.88 0.61 - -
dm: dry matter
Table 4: Interactive effects of NaCl level (mM) and vesicular-arbuscular mycorrhizal fungus (VAMF) on Chl content (mg/ g dm), Chl a/b ratio, and PS2 activity (p,mol DCPIP reduced/ mg Chl. h) of 2-month old tomato plants (cv. Super
Strain-B).
NaCl level (mM) Mycorrhizal treatment Chl a+ b Chl a/b PS2 activity
0 n-M 5.30 2.70 70
M 5.84 2.82 73
50 n-M 5.04 2.88 63
M 5.10 2.94 68
100 n-M 4.36 3.06 50
M 4.71 3.21 59
Factor L.S.D. at 5% level
NaCl level (A) Mycorrhizae (B) Interaction (Ax B) 0.16 0.05 4.16
0.09 0.02 2.81
0.24 0.07 5.68
dm: dry m
Table 5: Interactive effects of NaCl level (mM) and vesicular-arbuscular mycorrhizal fungus (VAMF) on activities of superoxide dismutase (SOD) [unit per mg (fm) S-1], and peroxidase (POX) and catalase (CAT) [p,mol (substrate reacted) kg-1 (fm) S-1] in leaves of 2-month old tomato plants (cv. Super Strain-B).
NaCl level (mM) Mycorrhizal treatment SOD POX CAT
0 n-M 1.26 11.1 13.6
M 0.91 9.9 10.8
50 n-M 2.38 13.8 9.92
M 1.88 11.3 7.23
100 n-M 4.21 18.4 6.66
M 2.96 15.2 4.98
Factor L.S.D. at 5% level
NaCl level (A) Mycorrhizae (B) Interaction (Ax B) 0.24 1.12 1.11
0.19 0.91 0.89
0.27 1.54 1.24
fm: fresh matter
Table 6: Effect of NaCl level (mM) on vesicular-arbuscular mycorrhizal fungus (VAMF) infection (%) of roots of 3-month old tomato plants (cv. Super Strain-
NaCl level (mM) Root length infected (%)
Mycorrhizal status
Uninoculated (n-M) Inoculated (M)
0 0.0 44 a
50 0.0 33 b
100 0.0 25 c
Mean values marked by the same letter do not differ significantly (P <
0.05).
Table 7: Interactive effects of NaCl (mM) and vesicular-arbuscular mycorrhizal fungus (VAMF) on leaf metabolite content [mg/ g (dm)] and shoot biomass [g. (dm)/ plant]
yielded by 3-month old tomato plants (cv. Super Strain-B).
NaCl level (mM) Mycorrhizal treatment Total sol. Sugars (TSS) Polysacch. Total carbohyd. Total sol. Proteins (TSP) Shoot biomass Root/ Shoot (DW basis)
0 n-M 187 328 515 121 22 0.29
M 200 339 539 131 25 0.32
50 n-M 199 276 475 96 16 0.41
M 216 286 502 111 19 0.37
100 n-M 222 214 436 56 8 0.64
M 237 227 464 78 11 0.52
Factor
NaCl level (A) Mycorrhizae (B) Interaction (Ax B) 12 26 39 11.60 1.7 0.031
8 23 35 7.91 1.3 0. 022
19 32 47 18.20 2.3 0.043
dm: dry matter
