Co-inoculations of arbuscular mycorrhizal fungi and rhizobia under salinity in alfalfa

Abstract

Alfalfa (Medicago sativa L.) is cultivated in arid and semi-arid regions where salinity is one of the main limiting factors for its production. Thus, this experiment was conducted to evaluate the efficacy of arbuscular mycorrhizal fungus (AMF), Glomus mosseae, alfalfa rhizobia Sinorhizobium meliloti (R) seed inoculation in the development of salinity tolerance of different alfalfa cultivars (Rehnani, Pioneer and Bami) under a variety of salinity levels. The results revealed that under non-stress condition, root mycorrhizal infection, nodulation (the number and weight of nodules per plant), potassium (K), calcium (Ca), phosphorus (P), zinc (Zn), copper (Cu) and magnesium (Mg) contents of the root and shoot, the value of the K/Na ratio, protein [calculated from the nitrogen (N) content] and proline contents of the shoot and the alfalfa yield were found to be the highest while Na contents of the root and shoot were seen to be the lowest when seeds were double inoculated followed by mycorrhizae, rhizobium and control treatments, respectively. Similarly, under salinity condition, the greatest amounts of mycorrhizal infection, nodulation, root and shoot P contents, the value of K/Na ratio, the shoot proline content and the root Ca content were enhanced with the least amount of leaf Na content related to the cases of seeds which were double inoculated, followed by mycorrhizae, rhizobium and control treatments respectively. The results suggested that inoculation of alfalfa seed with AMF or R, especially double inoculation, causes a considerable increase in alfalfa yield under both saline and non-saline conditions by increasing colonization, nodulation and nutrient uptake.

Key words:

• alfalfa
• mycorrhizae
• nutrition
• rhizobium
• salinity

INTRODUCTION

Salt affects more than 7% of land surface area and more than 77 million ha of cultivated land (Munns 2002). Salinity causes reduction in plant growth and yield due to the imbalance of nutrient uptake, increase in osmotic potential of the soil solution and increased toxicity of the plant as a result of excessive sodium (Na) and chlorine (Cl) absorbed by the plant in the plasma membrane (Munns 2002). Reclamation, drainage and improved irrigation practices, soil amendments, using salt-tolerant plants, conventional and modern plant breeding techniques may provide possible solutions to salinity problems in the salt-affected areas (Munns 2002). However, these methods are expensive and/or very time consuming. In contrast, the application of biological processes such as mycorrhizae may provide a relatively cost-effective and long-term solution to increase productivity in salt-affected lands. Through many researches, arbuscular mycorrhizal fungi (AMF) have been shown to promote plant growth and salinity tolerance. They promote salinity tolerance by employing various mechanisms, such as enhancing nutrient uptake, especially phosphorus (P) nutrition (Giri et al. 2002), producing plant growth hormones, improving rhizospheric condition of the soil (Linderman 1994), altering the physiological and biochemical properties of the host plants (Grant et al. 2001) and improving host physiological processes (Pfetffer and Bloss 1988). In addition, AMF colonization can reverse the effect of salinity on potassium (K) and Na⁺ nutrition by enhancing K⁺ absorption under saline conditions (Ruiz-Lozano et al. 1996; Giri et al. 2002; Sharifi et al. 2007) while preventing Na⁺ translocation to shoot tissues. These benefits of AMF have prompted it to be a suitable candidate for bio-amelioration of saline soils.

Alfalfa (Medicago sativa L.) is the most important forage legume in the world due to its high yield, nutritional quality, high protein content and adaptability to a wide range of soil and climatic conditions (McDonald et al. 1991). Alfalfa is also widely cultivated in arid and semi-arid regions where salinity is one of the main limiting factors for growing this crop (McDonald et al. 1991).

The gram-negative bacterium Sinorhizobium meliloti is able to interact with roots of alfalfa to form nitrogen (N)-fixing nodules (Biondi et al. 2003; Elboutahiri et al. 2010). Soil salinity adversely affects the nodulation and N fixation capacities of rhizobia, resulting in lower productivity of legumes (Valdenegro et al. 2001).

Alfalfa may benefit from symbiotic associations of N-fixing bacteria (Sinorhizobium meliloti) and AMF which form a tripartite symbiosis. This association can lead to enhancing the plant growth, the yield and the nutrient contents, particularly N and P contents (Valdenegro et al. 2001). In addition, research reports suggested that the plant benefits from the tripartite symbiosis are superior to those of plants inoculated with either AMF or rhizobium alone (Valdenegro et al. 2001).

It is also of great interest to note that, based on what was reported by Tsang et al. (1991), mycorrhizal wild bean plants (Strophostyles helvola) had greater vigor and enhanced growth because of increases in chlorophyll contents, shoot dry weight, root available water and the number of root nodules compared to non-mycorrhizal plants in saline conditions. The rhizosphere/mycorrhizosphere system can therefore help plants to survive under stress conditions such as salinity stress (Ianson and Linderman 1991) and drought (Goicoechea et al. 1998). However, to the best of our knowledge, there have been no reports demonstrating the negative and positive effects of combined inoculation of AMF and Sinorhizobium meliloti on nodulation parameters, root and shoot chemical contents, forage quality (protein content) and yield of alfalfa cultivars with different salt tolerance. Therefore, the objectives of this study were to investigate the effects of combined inoculation of mycorrhizal fungi Glomus mosseae and alfalfa rhizobia Sinorhizobium meliloti on yield, nutrient contents, nodulation and mycorrhizal colonization of three alfalfa cultivars under saline conditions.

MATERIALS AND METHODS

The experimental design was laid out based on a factorial (4 × 9 × 4) experiment in a completely randomized block design (CRBD) with three replicates. In this experiment, four states of inoculation (mycorrhizae, rhizobium, mycorrhizae + rhizobium and control), three types of cultivars (Rehnani, Pioneer and Bami) and four different salt levels (0, 60, 120 and 180 mM sodium chloride, NaCl) were the treatments. The four levels of salinity (0, 60, 120 and 180 mM NaCl) were produced by adding 0, 3.5, 7 and 10 g NaCl L⁻¹ in irrigation water. Pots were irrigated with water thrice weekly to maintain soil moisture at 80% field capacity.

The soil was a clay loam Typical Haplargid, with a bulk density of 1.34 g cm⁻³, a pH of 7.5 (measured in a 1:2 soil/water suspension), water-holding capacity at field capacity of 240 g kg⁻¹ (determined gravimetrically), organic carbon (C) content of 2.7 g kg⁻¹ (measured by wet-digestion analysis), total N of 300 mg kg⁻¹ (determined by the Kjeldahl digestion method), and available P and available K of 14.9 and 250 mg kg⁻¹ (measured by the Olsen method and extraction with ammonium acetate (NH₄OAc), respectively) (Soil Survey Laboratory Staff 2004).

At the first step, soil was passed through a 2-mm mesh sieve, then it was thoroughly mixed and autoclaved (110°C, 1 h, twice at 48 h intervals) to remove the effect of soil microorganisms including indigenous AM propagules and rhizobium; then, in order to provide the four above-mentioned states of inoculation, the following steps were taken: the soil was inoculated with mycorrhizae Glomus mosseae, by putting 30 g inoculum in each pot, and the seed lots were inoculated with 25 g of S. meliloti inoculum (10⁶ cfu g⁻¹ soil). Mycorrhizae Glomus mosseae and S. meliloti inoculum were purchased from the Agricultural and Biotechnology Research Institue, Karaj, Iran. In the case of double inoculation, the inoculated seeds were planted in the inoculated soil. Seeds were sown into pots of 20 cm diameter, 60 cm height, and with five plants per each pot. Each pot was filled with 8 kg of soil. This experiment was conducted on April 1 to May 30 under natural conditions at the College of Agriculture, Isfahan University of Technology, Iran (2013).

Fifty-five days after planting, plants were harvested, then thoroughly washed and, ultimately, the dry weights of shoot and root, phosphorus (P), K, calcium (Ca), magnesium (Mg), zinc (Zn), copper (Cu), Na and K/Na, protein (calculated from the N content), proline concentrations, nodulation and root mycorrhizal infection were measured. Dry weights of shoot and root were determined after drying in an oven for 48 h at 80°C. P contents of the plants were determined colorimetrically after nitric-perchloric acid digestion (Allen et al. 1984) and the K and Na contents were defined using flame photometry (Allen et al. 1984). The concentrations of Ca, Mg, Zn and Cu were specified by atomic absorption spectrometry (Allen et al. 1984), using a Perkin-Elmer model 5000 spectrophotometer.

The percentage of mycorrhizal root infection was estimated after clearing washed roots in 10% potassium hydroxide (KOH) and staining with 0.05% trypan blue in lactophenol (volume/volume), according to the method of Phillips and Hayman (1970). The weight of dry nodules was measured after drying in a forced draft oven at 70°C for 48 h.

Proline content was estimated following the method of Bates et al. (1973). Fresh leaves (0.5 g) were extracted in 3% sulphosalicylic acid and the homogenates were centrifuged at 10,000 g for 10 min. Two milliliters of the supernatant was reacted with 2 mL of acid ninhydrin reagent and 2 mL of glacial acetic acid in a test tube for 1 h at 100°C and the reaction was terminated in an ice bath. The reaction mixture was extracted with 4 mL of toluene and mixed vigorously with a vortex mixer for 15–20 s. The chromophore containing toluene was aspirated from the aqueous phase and warmed to room temperature and the absorbance was measured at 510 nm using toluene as blank. Proline concentration was calculated from a standard curve using 0–100 μg L-proline (Sigma).

N was determined using Kjeldhal apparatus and multiplied by a correction factor (6.25) to get the protein content (Jackson 1958).

The data were subjected to normal distribution. Analysis of variance and least significant difference (LSD) tests in order to compare the means were performed, using SAS (v. 9.1) software.

RESULTS

Nodulation and mycorrhizal infection

According to the results, the number of nodules per plant and nodule weight depended on the cultivar, salinity level, inoculation, cultivar × inoculation, cultivar × salinity level and inoculation × salinity level (Table 1). Without salt treatment and in the absence of mycorrhizae, the number of nodules per plant and nodule weight were 83 and 87, while with double inoculation these numbers were 124 and 129, respectively (Table 2). In the presence of salt, the number and weight of nodules decreased with increasing salt level; however, the reduction was less when the plants were double inoculated followed by rhizobia inoculation (Table 2).

Table 1 Analysis of variance for protein, proline, mycorrhiza infection, nodule number and weight and forage yield in four inoculation treatments and four salinity levels and with three cultivars of alfalfa (Medicago sativa L.)

		Means of square
Variable source	Degree of Freedom (DF)	Protein	Proline	Mycorrhiza infection	Nodule number	Nodule weight	Forage yield
Replicate	2	3.7^ns	0.7^ns	7.6^ns	0.8^ns	0.7^ns	1.7^ns
Cultivar (C)	2	10.8***	82.8***	48.7***	481.7***	558.6***	84.3***
Inoculation (I)	3	9.8***	76.1***	2325.9***	8567***	14644.8***	121.3***
Salinity (S)	3	61.5***	658.5***	117.1***	161.7***	247.5***	1094.6***
C*I	6	0.2^ns	0.2^ns	16.5***	181.7***	235.4***	0.1^ns
C*S	6	0.1^ns	2.5*	0.6^ns	6.1***	8.1***	3.9**
I*S	9	0.4^ns	1.3*	39***	67.6***	107.8***	4*
C*I*S	18	0.1^ns	0.1^ns	0.2^ns	3.6^ns	7.9^ns	0.1^ns
Error	94	5.2	1.1	10.2	1.5	4.9	2.3
Total	143
Coefficient Variance (CV)		15.5	6.2	14.4	7.7	5.7	5.1
R^2		0.73	0.96	0.98	0.99	0.99	0.97

*, ** and ***, significant at 0.05, 0.01 and 0.001 levels of probability, respectively; ^ns, non-significant.

Table 2 Interaction between salinity levels and inoculation treatments on forage yield, proline, root and leaf phosphorus (P), mycorrhiza infection, nodule number and weight, leaf sodium (Na), leaf and root potassium (K)/Na and root calcium (Ca)

Salinity	Inoculation	Forage yield (g)	Proline (µmol g^–1 FW)	Root P (mg kg^–1)	Leaf P (mg kg^–1)	Mycorrhiza infection (%)	Nodule number (No. pot^–1)	Nodule weight (mg pot^–1)	Leaf Na (mg kg^–1)	Leaf K/Na	Root K/Na	Root Ca (mg kg^–1)
0	Control	1.51^d†	0.27^i	1.62^d	0.93^e	0^h	0^h	0^h	4.8^ij	11.1^d	17.1^d	7.6^de
60	Control	1.41^e	0.42^g	1.15^i	0.46^j	0^h	0^h	0^h	5.2^gh	9.7^g	13.9^f	6.8^f
120	Control	1.03^h	0.57^e	0.91^l	0.26^k	0^h	0^h	0^h	6.1^d	7.8^k	7.4^h	4.8^h
180	Control	0.65^k	0.62^d	0.43^p	0.06^n	0^h	0^h	0^h	8.6^a	5.3^o	4.4^i	2.0^l
0	Rhizobium	1.60^bc	0.34^h	1.69^c	1.13^c	0^h	83^d	87^d	4.6^jk	11.8^c	19.3^c	8.4^c
60	Rhizobium	1.52^d	0.48^f	1.28^g	0.73^g	0^h	77^e	81^e	4.9^hi	10.3^f	15.5^e	7.6^d
120	Rhizobium	1.18^g	0.61^d	1.03^k	0.46^j	0^h	69^f	74^f	5.8^de	8.3^j	7.9^gh	5.6^g
180	Rhizobium	0.78^j	0.66^bc	0.56^o	0.13^m	0^h	62^g	67^g	7.8^b	5.9^n	4.6^i	2.6^k
0	Mycorrhiza	1.6^b	0.37^h	1.74^b	1.23^b	40^b	0^h	0^h	4.4^k	12.2^b	21.3^b	9.0^b
60	Mycorrhiza	1.58^c	0.51^f	1.41^f	0.89^f	37^c	0^h	0^h	4.8^ij	10.8^e	16.9^d	8.5^c
120	Mycorrhiza	1.25^f	0.64^cd	1.11^j	0.56^i	30^e	0^h	0^h	5.6^ef	8.7^i	8.3^gh	6.7^f
180	Mycorrhiza	0.87^i	0.68^ab	0.64^n	0.17^l	20^g	0^h	0^h	7.5^b	6.2^m	4.8^i	3.4^j
0	R + Mycorrhiza (M)	1.75^a	0.41^g	1.82^a	1.33^a	43^a	124^a	129^a	4.4^k	12.6^a	23.1^a	10.0^a
60	R + Mycorrhiza (M)	1.66^b	0.56^e	1.51^e	1.06^d	40^b	113^b	118^b	4.7^ijk	11.2^d	18.5^c	9.3^b
120	R + Mycorrhiza (M)	1.38^e	0.67^bc	1.21^h	0.66^h	33^d	94^c	96^c	5.4^fg	9.1^h	8.8^g	7.3^e
180	R + Mycorrhiza (M)	0.98^h	0.70^a	0.74^m	0.26^k	23^f	81^d	86^d	6.6^c	7.1^l	5.0^i	4.1^i
Least Significant Difference (LSD)		0.06	0.03	0.03	0.05	2	3	3	0.3	0.1	0.9	0.3

† Means within the same column with the same letters are not significantly different at 5% level.

The mycorrhizal infection rate depended on the cultivar, salinity level, inoculation, cultivar × inoculation and inoculation × salinity level (Table 1). Without salt treatment and in the absence of rhizobium, the mycorrhizal infection rate was 40% while with double inoculation this number was 43% (Table 2). In the presence of salt, the rate of mycorrhizal infection reduced with increasing salt level; however, the reduction was least when the plants were double inoculated followed by mycorrhizal inoculation (Table 2). Furthermore, mycorrhizal infection rate with or without the presence of salt was highest with double inoculation followed by mycorrhizal inoculation (Table 2).

Plant nutrients

Leaf and root K, Ca (except in the leaf) and Mg, Zn and Cu (except in the root) contents depended on the cultivar, salinity level and inoculation (Tables 3–4). Leaf and root P, K, Ca, Mg, Zn, and Cu contents and K/Na were highest in Rehnani followed by Pioneer and Bami, while Na content was highest in Bami followed by Pioneer and Rehnani, respectively (Tables 5–6). In addition, leaf and root P, K, Ca, Mg, Zn and Cu contents and K/Na were highest with double inoculation followed by mycorrhizae, rhizobium and control treatments, respectively, whereas Na content was lowest (Tables 5–6). In the presence of salt, concentrations of P, K, Ca, Mg, Zn and Cu and K/Na in the leaf and root decreased with increasing salt level (Tables 5–6). However, reduction in leaf and root P contents, root Ca content and leaf and root K/Na was least affected when plants were double inoculated followed by mycorrhizae, rhizobium and control treatments, respectively, whereas Na content was lowest.

Table 3 Analysis of variance for root and leaf phosphorus (P), zinc (Zn), copper (Cu) and magnesium (Mg) in four inoculation treatments and four salinity levels and with three cultivars of alfalfa (Medicago sativa L.)

	Degree of Freedom (DF)	Means of square
		Root (mg kg^–1)				Leaf (mg kg^–1)
Variable source		P	Zn	Cu	Mg	P	Zn	Cu	Mg
Replicate	2	3.2^ns	4.7^ns	0.6^ns	0.1^ns	3.4^ns	3.1^ns	0.9^ns	0.2^ns
Cultivar (C)	2	395.3***	10.2***	1.7^ns	255.1***	561.2***	10.3***	3.3*	279.8***
Inoculation’ (I)	3	513.7***	8.7***	4.4**	8.1***	934.1***	8.8***	3.4*	8.4***
Salinity (S)	3	7271.7***	106.2***	23.9***	70.7***	5848.7***	106.5***	21.1***	76.7***
C*I	6	1.9^ns	0.1^ns	0.1^ns	0.2^ns	1^ns	0.2^ns	0.1^ns	0.1^ns
C*S	6	20.3***	1.1^ns	0.2^ns	0.2^ns	43.3***	1.1^ns	0.3^ns	0.1^ns
I*S	9	7.3***	0.3^ns	0.4^ns	0.2^ns	41.7***	0.3^ns	0.1^ns	0.2^ns
C*I*S	18	0.9^ns	0.1^ns	0.1^ns	0.1^ns	6^ns	0.1^ns	0.1^ns	0.2^ns
Error	94	5.1	6.2	2.1	4.8	4.3	5.5	1.5	6.2
Total	143
Coefficient Variance (CV)		2.8	6.4	12.7	20.6	5.2	7.9	16.5	12.9
R^2		0.99	0.8	0.52	0.89	0.99	0.8	0.5	0.89

*, ** and ***, significant at 0.05, 0.01 and 0.001 levels of probability, respectively; ^ns, non-significant.

Table 4 Analysis of variance for root and leaf sodium (Na), potassium (K), K/Na and calcium (Ca) in four inoculation treatments and four salinity levels and with three cultivars of alfalfa (Medicago sativa L.)

	Degree of Freedom (DF)	Means of square
		Root (mg kg^–1)				Leaf (mg kg^–1)
Variable source		Na	K	K/Na	Ca	Na	K	K/Na	Ca
Replicate	2	0.1^ns	6.1^ns	0.4^ns	0.1^ns	0.1^ns	6.2^ns	0.02^ns	1.2^ns
Cultivar (C)	2	58.3***	29.9***	183.7***	234.9***	217.8***	22.9***	4812.4***	0.9^ns
Inoculation (I)	3	26.1***	3.2**	62.7***	345.5***	46.2***	2.6*	632.3***	6.0**
Salinity (S)	3	2636.6***	41.7***	1737.3***	2114.0***	614.7***	35.6***	9298.1***	37.1***
C*I	6	0.1^ns	0.1^ns	2.1^ns	0.2^ns	6.23*	0.1^ns	6.7***	0.1^ns
C*S	6	0.5^ns	0.1^ns	27.6***	1.9^ns	4.1***	0.6^ns	145.2***	0.5^ns
I*S	9	0.5^ns	0.1^ns	10.4***	1.3*	6.8***	0.1^ns	4.5***	0.1^ns
C*I*S	18	0.4^ns	0.1^ns	0.59^ns	0.2^ns	0.8^ns	0.1^ns	2.2^ns	0.1^ns
Error	94	6.23	10.5	1.0	5.2	2.1	11.1	10.2	6.4
Total	143
Coefficient Variance (CV)		6.7	7.2	8.3	5.1	5.8	6.6	1.7	19.3
R^2		0.98	0.69	0.98	0.98	0.96	0.65	0.99	0.59

*, ** and ***, significant at 0.05, 0.01 and 0.001 levels of probability, respectively; ^ns, non-significant.

Table 5 Root and leaf phosphorus (P), zinc (Zn), copper (Cu) and magnesium (Mg) as affected by salinity level, inoculation treatment and cultivar

Variable source	Root (mg kg^–1)				Leaf (mg kg^–1)
Cultivar	P	Zn	Cu	Mg	P	Zn	Cu	Mg
Rehnani	1.28^a†	53.64^a	26.8^a	2.43^a	0.77^a	43.6^a	21.0^a	3.45^a
Pioneer	1.15^b	50.62^b	26.1^a	1.41^b	0.58^b	41.5^b	20.2^ab	2.41^b
Bami	1.10^c	50.62^b	25.6^a	0.94^c	0.58^b	40.4^b	19.3^b	1.86^c
Least Significant Difference (LSD)	0.01	1.35	1.2	0.10	0.01	1.5	1.3	0.13
Inoculation
Control	1.02^d	50.0^c	24.7^c	1.42^c	0.43^d	40.0^c	18.9^c	2.40^c
Rhizobium	1.14^c	51.4^bc	25.8^bc	1.54^bc	0.61^c	41.4^bc	19.8^bc	2.51^bc
Mycorrhiza	1.23^b	52.5^ab	26.7^ab	1.63^b	0.71^b	42.5^ab	20.6^ab	2.61^b
R + Mycorrhiza (M)	1.32^a	53.8^a	27.4^a	1.79^a	0.83^a	43.8^a	21.3^a	2.78^a
Least Significant Difference (LSD)	0.01	1.5	1.5	0.15	0.03	1.5	1.6	0.16
Salinity (Mm)
0	1.72^a	56.4^a	28.5^a	1.96^a	1.15^a	46.4^a	22.4^a	2.9^a
60	1.34^b	55.3^a	27.9^a	1.87^a	0.78^b	45.3^a	21.8^a	2.8^a
120	1.06^c	52.3^b	25.8^b	1.58^b	0.48^c	42.3^b	19.7^b	2.5^b
180	0.59^d	43.7^c	22.5^c	0.95^c	0.15^d	33.7^c	16.7^c	1.8^c
Least Significant Difference (LSD)	0.01	1.6	1.5	0.2	0.01	1.5	1.6	0.1

† Means within the column for each treatment with the same letters are not significantly different at 5% level.

Table 6 Root and leaf sodium (Na), potassium (K), K/Na and calcium (Ca) as affected by salinity level, inoculation treatment and cultivar

Variable source	Root (mg kg^–1)				Leaf (mg kg^–1)
Cultivar	Na	K	K/Na	Ca	Na	K	K/Na	Ca
Rehnani	4.5^c†	49.0^a	14.2^a	7.2^a	5.0^c	51.8^a	10.8^a	13.47^a
Pioneer	4.8^b	45.7^b	12.4^b	6.4^b	5.6^b	50.2^b	9.41^b	13.01^a
Bami	5.3^a	43.8^c	10.2^c	5.7^c	6.4^a	47.3^c	7.67^c	12.77^a
Least Significant Difference (LSD)	0.2	1.7	0.4	0.1	0.1	1.3	0.06	1.02
Inoculation
Control	5.3^a	45.0^b	10.7^d	5.3^d	6.2^a	48.7^b	8.50^d	11.8^c
Rhizobium	5.0^b	45.8^ab	11.9^c	6.0^c	5.8^b	49.5^ab	9.10^c	12.6^bc
Mycorrhiza	4.6^c	46.6^ab	12.8^b	6.9^b	5.6^c	50.2^ab	9.53^b	13.5^ab
R + Mycorrhiza (M)	4.3^d	47.3^a	13.9^a	7.7^a	5.3^d	50.8^a	10.05^a	14.2^a
Least Significant Difference (LSD)	0.2	1.6	0.4	0.1	0.1	1.5	0.07	1.1
Salinity (Mm)
0	2.5^d	50.0^a	20.2^a	8.7^a	4.5^d	53.8^a	11.97^a	15.3^a
60	3.0^c	47.9^b	16.2^b	8.0^b	4.9^c	50.9^b	10.54^b	14.6^a
120	5.5^b	45.0^c	8.1^c	6.1^c	5.7^b	48.4^c	8.51^c	12.6^b
180	8.7^a	41.7^d	4.7^d	3.0^d	7.6^a	46.1^d	6.16^d	9.6^c
Least Significant Difference (LSD)	0.1	1.6	0.4	0.2	0.1	1.5	0.07	1.1

† Means within the column for each treatment with the same letters are not significantly different at 5% level.

Leaf and root P, K, Ca, Mg, Zn and Cu contents and K/Na were highest with double inoculation followed by mycorrhizae, rhizobium and control treatments, respectively, whereas Na content was the lowest. However, reduction in leaf and root P contents, root Ca content and leaf and root K/Na was the least affected when plants were double inoculated followed by mycorrhizae, rhizobium and control treatments, respectively, whereas Na content was the opposite.

Protein content

Protein content was affected by the cultivar, inoculation and salinity level (Table 1). The highest protein content was recorded in Rehnani and Pioneer, followed by Bami (Table 7). With or without salt treatment, plants treated with rhizobia and mycorrhizae (R + M) produced the highest protein content followed by mycorrhizae, rhizobia and control treatments, respectively (Table 7). As salinity increased, protein content reduced.

Table 7 Protein, proline, mycorrhiza infection, nodule number and weight, and forage yield as affected by salinity level, inoculation treatment and cultivar

Variable source	Protein (%)	Proline (µmol g^–1 FW)	Mycorrhiza infection (%)	Nodule number (No. pot^–1)	Nodule weight (mg pot^–1)	Forage yield (g)
Cultivar
Rehnani	22.82^a†	0.58^a	19.2^a	53.5^a	54.9^a	1.40^a
Pioneer	21.70^a	0.52^b	17.2^b	46.4^b	47.3^b	1.28^b
Bami	19.65^b	0.49^c	14.3^c	32.3^c	36.5^c	1.23^c
Least Significant Difference (LSD)	1.35	0.01	0.9	1.3	1.1	0.02
Inoculation
Control	19.1^c	0.47^d	0^c	0^c	0^c	1.15^d
Rhizobium	21.0^b	0.52^c	0^c	73.2^b	77.7^b	1.27^c
Mycorrhiza	22.0^ab	0.55^b	32.3^b	0^c	0^c	1.34^b
R + Mycorrhiza (M)	23.3^a	0.58^a	35.3^a	103.2^a	107.3^a	1.44^a
Least Significant Difference (LSD)	1.5	0.01	1.1	1.5	1.2	0.03
Salinity (Mm)
0	25.6^a	0.35^d	20.9^a	52^a	54.1^a	1.63^a
60	23.7^b	0.49^c	19.5^b	47^b	50.0^b	1.54^b
120	20.5^c	0.62^b	16.0^c	40^c	42.5^c	1.21^c
180	15.7^d	0.66^a	11.0^d	35^d	38.4^d	0.82^d
Least Significant Difference (LSD)	1.6	0.01	1.1	2	1.2	0.03

† Means within the column for each treatment with the same letters are not significantly different at 5% level.

Proline content

Proline content was affected by the cultivar, inoculation, salinity level, cultivar × salinity level and inoculation × salinity level (Table 1). With or without salt treatment, the highest proline content was measured in Rehnani, followed by Pioneer and Bami, respectively (Table 7). With or without salt treatment, plants treated with rhizobia and mycorrhizae (R + M) contained the highest proline content followed by mycorrhizae, rhizobia and control treatments, respectively (Table 2). In contrast to other measured parameters, proline content increased with increasing salinity level.

Forage yield

Cultivar, salinity level, inoculation, cultivar × salinity level and inoculation × salinity level affected forage yield (Table 1). Forage yield was reduced as salinity level increased, but the reduction was less when plants were treated with rhizobia or mycorrhizae, especially with double inoculation (Table 2). With or without salt treatment, the highest yield was measured in Rehnani, followed by Pioneer and Bami, respectively (Table 8). With or without salt treatment, plants treated with rhizobia and mycorrhizae (R + M) produced the highest forage yield followed by mycorrhiza, rhizobia and control treatments, respectively (Table 2).

Table 8 Forage yield, root and leaf phosphorus (P), proline, nodule number and weight, leaf sodium (Na), and leaf and root potassium (K)/Na of alfalfa (Medicago sativa L.) cultivars as affected by salinity levels

Cultivar	Salinity	Forage yield	Root P	Leaf P	Proline	Nodule number	Nodule weight	Leaf Na	Leaf K/Na	Root K/Na
Rehnani	0	1.68^a†	1.77^a	1.22^a	0.40^g	58.77^a	59.8^a	3.9^i	14.1^a	24.1^a
Pioneer	0	1.63^ab	1.71^b	1.12^b	0.33^h	53.95^bc	55.5^b	4.5^h	12.0^b	20.3^b
Bami	0	1.58^b	1.66^c	1.12^b	0.30^i	43.58^f	47.0^e	5.3^f	9.6^d	16.1^d
Rehnani	60	1.62^b	1.45^d	1.02^c	0.56^d	56.20^ab	57.7^a	4.3^h	12.1^b	18.3^c
Pioneer	60	1.53^c	1.30^e	0.67^d	0.47^e	50.33^de	51.7^c	4.8^g	10.7^c	16.7^d
Bami	60	1.48^c	1.25^f	0.67^d	0.44^f	36.58^g	40.3^g	5.6^e	8.6^e	13.5^e
Rehnani	120	1.33^d	1.15^g	0.62^e	0.66^a	51.33^cd	52.4^c	5.2^f	9.6^d	9.1^f
Pioneer	120	1.18^e	1.05^h	0.42^f	0.61^b	43.83^f	43.0^f	5.6^e	8.7^e	8.1^g
Bami	120	1.13^e	1.00^i	0.42^f	0.58^c	27.58^h	31.6^h	6.3^d	7.1^f	7.1^h
Rehnani	180	0.96^f	0.76^j	0.25^g	0.69^a	47.83^e	49.3^d	6.6^c	7.2^f	5.4^i
Pioneer	180	0.77^g	0.53^k	0.11^h	0.67^a	37.83^g	38.8^g	7.7^b	6.0^g	4.6^ij
Bami	180	0.72^g	0.48^l	0.11^h	0.64^b	21.82^i	27.0^i	8.5^a	5.1^h	4.2^j
Least Significant Difference (LSD)		0.05	0.02	0.03	0.02	2.75	2.20	0.2	0.1	0.8

† Means within rows and column for each item with the same letters are not significantly different at 5% level.

DISCUSSION

Nutrients

The results demonstrated that under non-stress conditions, the K, Ca, P, Zn, Cu and Mg contents of the root and shoot, the value of the K/Na ratio and the protein content (calculated from the N content) of the shoots were found to be the highest, while the Na contents of the roots and shoots were seen to be the lowest in double inoculation followed by mycorrhizae, rhizobium and control treatments, respectively. In line with our results, other researchers also reported that inoculation of plants with AMF increased uptake of N, P, K, Zn, Fe, Cu, Mg and Ca (Tinker and Gildon 1983; Díaz et al. 1996).

The results are also supported by the results reported by Aziz and Khan (2001), which showed that co-inoculation of alfalfa with Glomus mosseae and Sinorhizobium meliloti increased N and P contents in the plant tissues. Increases in nutrients in plant tissues could be due to increased uptake of nutrients by extra-radical mycorrhizae hyphae beyond the root hair and nutrient depletion zones, as concluded by Aziz and Khan (2001).

The results showed that in the presence of salt, with increasing salt levels, the concentrations of P, K, Ca, Mg, Zn, and Cu and K/Na in the leaf and root tissues decreased, whereas the Na content increased (Tables 5–6). However, the reductions in leaf and root P contents, root Ca content and the K/Na value of leaf and root were the least when plants were double inoculated followed by mycorrhizae, rhizobium and control treatments, respectively. The increase in the nutrient contents of plant tissues in our experiments under both stress and non-stress conditions was likely due to the increase in nodulation and mycorrhizal infection resulting from mycorrhizal and/or rhizobium inoculation, especially double inoculation, as indicated in Table 5. The improved content of P in root and shoot by AMF under salinity stress was also reported by Asghari et al. (2005). In this study, the higher root Ca content and the value of K/Na ratio in the plants inoculated with AMF, especially in the presence of rhizobia, suggest that inoculation with AMF, rhizobia and double inoculation may have the potential to increase K and Ca uptake more than Na under salt stress, resulting in salt adaptation in plants. This fact can be justified as follows: such symbioses may cause hydrogen (H) pumps that generate the driving force for increasing the value of the K/Na ratio which enhance the plant salinity tolerance (Rabie and Almadini 2005). In addition, AMF may also increase plant salinity tolerance by increasing mineral nutrition acquisition (Cordovilla et al. 1995), improving rhizospheric conditions (Linderman 1994), enhancing water potential (Hildebrandt et al. 2001; Marulanda et al. 2003), altering physiological and biochemical properties of the host plants (Smith and Read 1997), and enhancing host physiological processes such as increasing the carbon dioxide exchange rate, transpiration, stomatal conductance, root hydraulic conductivity and water use efficiency (Ruiz-Lozano et al. 1996; Smith and Read 1997; Al-Karaki et al. 2001).

Proline

With or without salt stress, proline concentrations in the leaves increased the most in the case of double inoculation followed by mycorrhizae, rhizobium and control treatments, respectively. Goicoechea et al. (1998) reported that under well-watered conditions, Rhizobium-inoculated alfalfa had the highest level of leaf proline, followed by double inoculated, AMF inoculated and control samples, respectively. In line with our results, Sharifi et al. (2007) showed that the soybean (Glycine max L.) specimens inoculated with AMF had higher proline than the control sample under saline conditions. Gloux and Le Rudulier (1989) showed that, like carbon and nitrogen, proline betaine synthesized by alfalfa can be used as an energy source for Rhizobium meliloti as well as an osmoprotectant. Our results showed that under salt stress and non-saline conditions, proline in the leaf tissues of alfalfa increased with the inoculation of rhizobia or AMF, especially with double inoculation.

Mycorrhizae

In this experiment, it was observed that with or without the presence of salt, colonization of alfalfa root occurred when seeds were inoculated with AMF. This colonization was intensified by double inoculation. In the presence of salt, the rate of mycorrhizal infection decreased with increasing salt level; however, the reduction was the least in the case of double inoculation, followed by mycorrhizal inoculation. Nourinia et al. (2007) reported that inoculation of barley (Hordeum vulgare L.) root with Glomus mosseae produced significant colonization, but colonization percentage decreased as salinity level increased, and it was concluded that this could be due to a direct effect of NaCl on the fungi. Al-Karaki et al. (2001) reported that mycorrhizal colonization was negatively affected by salinity stress and they concluded that it was possibly due to the effect of salt on initial colonization. Several other researchers have also shown that salinity reduces mycorrhizal colonization through inhibiting the germination of spores (Hirrel and Gerdemann 1980), inhibiting growth of hyphae in soil and hyphal spreading after initial infection had occurred (McMillen et al. 1998), and reducing the number of arbuscules (Tian et al. 2004).

Our results showed that AMF colonization in the root of alfalfa was affected also by cultivars. AMF infection in Rehnani was the highest followed by Pioneer and Bami, respectively; this was perhaps due to genetic differences between the cultivars. The occurrence of mycorrhizal colonization in our experiment under both stress and non-stress conditions was perhaps due to the inoculation of alfalfa seeds with AMF and especially with double inoculation, as indicated in Table 2. Similarly to our results, it was reported that AMF colonization in roots was affected by plant species (Smith and Read 1997). Scheublin and van der Heijden (2006) showed that all plant roots in several legume species inoculated with AMF were colonized by AMF; however, birdsfoot trefoil (Lotus corniculatus L.) had the highest colonization followed by white clover (Trifolium repens L.) and common restharrow (Ononis repens L.), respectively.

Nodule

The present investigation demonstrated that inoculation of alfalfa with Rhizobium, especially with AMF and Rhizobium, significantly enhanced nodulation (the number of nodules per plant and the weights of nodules). Similar results were reported by many scientists for different crops (Athar and Johnson 1997). Mizukami et al. (1991) suggested that enhancement of nodulation in the plants inoculated with AMF is possibly due to a change in the hormonal balance of the plants. This hormonal imbalance can cause improved P nutrition (Athar and Johnson 1997).

In the presence of salt, the number and weight of nodules decreased with increasing salt level. The results agreed with results reported by others (Azcón and Elatrash 1997). The reduction in nodulation under salt stress could be due to a reduction in activity of rhizobia cells (Mashhady et al. 1998). The reduction in the number and weight of nodules when the plants were inoculated with rhizobia, especially with double inoculation, was seen to be less than in the other plant samples. This indicated that the harmful effect of salinity could be reduced by dual inoculation. This was perhaps due to the presence of extra-matrical hyphae of AMF which increased the absorption of immobile elements such as P and N fixed by Rhizobium, as indicated in Table 2.

The results showed that the non-rhizobium or AMF inoculation treatments remained free from nodules. Scheublin and van der Heijden (2006) reported that inoculation of several legumes with AMF resulted in nodule colonization; however, AMF-colonized nodules never fixed N.

The Increase in the nodulation (nodules per plant and nodule weight) in this experiment under both stress and non-stress conditions was perhaps a result of the inoculation of alfalfa seeds with Rhizobium, and especially of double inoculation, as indicated in Table 2.

Yield

With or without salt treatment, plants treated with rhizobia and mycorrhizae produced the highest forage yield, followed by mycorrhizae, rhizobia and control treatments, respectively (Table 2). The results agree with Sharifi et al. (2007) who reported that the alfalfa specimens inoculated with both Rhizobium and Glomus produced the highest shoot yield, followed by mycorrhizae, rhizobium and control treatments, and they concluded that Rhizobium may affect fungal metabolism. In line with our results, Mycorrhizal wild beans (Strophostyles helvola L.) (Tsang et al. 1991) and mangrove (Ceriops tagal C.B. Rob.) (Aziz and Khan 2001) have been shown to produce higher yields in comparison to control samples under salinity conditions. In accordance with our findings, the increase in yield of the soybean specimen inoculated with Rhizobium japonicum and Glomus mosseae was also reported by Ruiz-Lozano et al. (1996). Increase in forage yield of subterranean clover inoculated with mycorrhizae under both salinity and non-salinity conditions was also reported by Asghari et al. (2005), and it was concluded that inoculation increases mycorrhizae effectiveness and enhances root colonization under salt and non-salt stresses. The results are also in line with other studies that have shown that inoculation of legumes with both rhizobium and AMF increases plant growth to a greater extent than either inoculum when added singly (Mizukami et al. 1991). An increase in N and P nutrition supply and a resulting increase in N fixation have been suggested for the benefits of the synergistic relationship (Bowen 1987).

The increase in alfalfa yield under non-stress conditions in our experiment was likely due to the increase in mycorrhizal infection and nodule weight and number, as well as increases in root and shoot K, Ca, P, Zn, Cu and Mg contents and K/Na ratio, increases in shoot protein and proline contents, and decrease in root and shoot Na contents, as indicated in Tables 5–8. On the other hand, the increase in alfalfa yield under salinity conditions was due to an increase in mycorrhizal infection and nodule weight and number as well as an increase in root and shoot P contents and K/Na ratio, shoot proline content, root Ca content and reduced leaf Na content, as indicated in Table 8 and supported by Evelin et al. (2009).

CONCLUSIONS

Based on the obtained results, under non-stress conditions, root mycorrhizal infection, nodulation (the number of nodules per plant and the weight of nodules), K, Ca, P, Zn, Cu and Mg contents of the root and shoot, the value of K/Na ratio, protein and proline content of the shoot and the alfalfa yield were found to be the highest, while Na contents of the root and shoot were seen to be the lowest when seeds were double inoculated, followed by mycorrhizae, rhizobium and control treatments, respectively. Similarly, under salinity conditions, the greatest amounts of mycorrhizal infection, nodulation, root and shoot P contents, the value of K/Na ratio, the shoot proline content and the root Ca content enhanced with the least amount of leaf Na content related to the cases of seeds were found in the double inoculated treatment, followed by mycorrhizae, rhizobium and control treatments, respectively. The results suggested that combined inoculation of alfalfa with AMF and rhizobium under both salt and non-stress conditions can enhance the growth of alfalfa. In the case of dual inoculation, the increase in alfalfa growth under salinity conditions was perhaps owing to increases in mycorrhizal infection and nodulation as well as increases in root and shoot P contents, K/Na ratio, shoot proline content and root Ca content, and decrease in leaf Na content.