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Journal of Stress Physiology & Biochemistry, Vol. 10 No. 3 2014, pp. 134-152 ISSN 1997-0838 Original Text Copyright © 2014 by Kadian, Yadav and Aggarwal
ORIGINAL ARTICLE
Application of AM Fungi with Bradyrhizobium japonicum in improving growth, nutrient uptake and yield of Vigna radiata L. under saline soil
Nisha Kadian, Kuldeep Yadav and Ashok Aggarwal*
Department of Botany, Kurukshetra University, Kurukshetra-136119, Haryana, India Tel.: +91 1744 238410; fax: +91 1744 238277 *E-Mail: aggarwal [email protected]
Received April 2, 2014
A pot experiment was conducted under polyhouse conditions, to evaluate the effect of two different arbuscular mycorrhizal fungi (G. mosseae and A. laevis) in combination with Bradyrhizobium japonicum on growth and nutrition of mungbean plant grown under different salt stress levels (4 dS m-1, 8dS m-1 and 12 dS m-1). It was found that under saline conditions, mycorrhizal fungi protect the host plant against the detrimental effect of salinity. The AM inoculated plants showed positive effects on plant growth, dry biomass production, chlorophyll content, mineral uptake, electrolyte leakage, proline, protein content and yield of mungbean plants in comparison to non-mycorrhizal ones but the extent of response varied with the increasing level of salinity. In general, the reduction in Na uptake along with associated increase in P, N, K, electrolyte leakage and high proline content were also found to be better in inoculated ones. The overall results demonstrate that the co-inoculation of microbes with AM fungi promotes salinity tolerance by enhancing nutrient acquisition especially phosphorus (P), producing plant growth hormones, improving rhizospheric and condition of soil by altering the physiological and biochemical properties of the mungbean plant.
Key words: Vigna radiata, Arbuscular mycorrhizal fungi, Bradyrhizobium japonicum, Soil salinity, mineral uptake, proline
ORIGINAL ARTICLE
Application of AM Fungi with Bradyrhizobium japonicum in improving growth, nutrient uptake and yield of Vigna radiata L. under saline soil
Nisha Kadian, Kuldeep Yadav and Ashok Aggarwal*
Department of Botany, Kurukshetra University, Kurukshetra-136119, Haryana, India Tel.: +91 1744 238410; fax: +91 1744 238277 *E-Mail: assarwal [email protected]
Received April 2, 2014
A pot experiment was conducted under polyhouse conditions, to evaluate the effect of two different arbuscular mycorrhizal fungi (G. mosseae and A. laevis) in combination with Bradyrhizobium japonicum on growth and nutrition of mungbean plant grown under different salt stress levels (4 dS m-1, 8dS m-1 and 12 dS m-1). It was found that under saline conditions, mycorrhizal fungi protect the host plant against the detrimental effect of salinity. The AM inoculated plants showed positive effects on plant growth, dry biomass production, chlorophyll content, mineral uptake, electrolyte leakage, proline, protein content and yield of mungbean plants in comparison to non-mycorrhizal ones but the extent of response varied with the increasing level of salinity. In general, the reduction in Na uptake along with associated increase in P, N, K, electrolyte leakage and high proline content were also found to be better in inoculated ones. The overall results demonstrate that the co-inoculation of microbes with AM fungi promotes salinity tolerance by enhancing nutrient acquisition especially phosphorus (P), producing plant growth hormones, improving rhizospheric and condition of soil by altering the physiological and biochemical properties of the mungbean plant.
Key words: Vigna radiata, Arbuscular mycorrhizal fungi, Bradyrhizobium japonicum, Soil salinity, mineral uptake, proline
Soil salinity is worldwide problem of grave concern because it negatively affects plant productivity and yield of plants particularly in arid and semi-arid and tropical regions of the world. Excessive salts, decline
soil water availability for plants, inhibit plants metabolism, nutrients uptake and is also responsible for osmotic imbalance (Evelin et a., 2009). In the recent years, the consumption of chemical fertilizers
has increased exponentially throughout the world, causing serious environmental problems. Thus, exploitation of soil microorganisms in soil amendment is of considerable importance (Yadav et al., 2013). Among the various biological approaches to enhance the plant growth in saline conditions, the role of biofertilizers such as Rhizobia and Arbuscular Mycorrhizal (AM) fungi in tolerating environmental stress has been well established and steadily receiving increased recognition from scientists (Kadian et al., 2013a). This could be attributed to the fact that they pose no ecological threats having a long lasting effect and considered as bio ameliorators of saline soils (Kadian et al., 2013).
AM fungi are ubiquitous soil microorganisms inhabiting the rhizosphere and establish a symbiotic relationship with more than 90% of plant species of natural ecosystems and are also known to occur in saline soils. Symbiotic association of a plant with AMF results in higher ability for taking up the immobile nutrients in nutrient-poor soils as well as improvement of tolerance to salinity (Dixon et al., 1993).
Vigna radiata (L.) Wilczek commonly known as mung bean is an important grain legume crop in South East Asia and Africa, and a source food that has a high nutritive value (Kumar et al., 2002; Salunke et al., 2005). It is not only a rich and economical source of protein, phosphorus, carbohydrate, minerals and provitamin A, but also commonly used as fodder and green manure. Mung bean contains bioactive components with antioxidant, antimicrobial and insecticidal properties (Bounce 2002; Kaprelynts et al., 2003; Madhujith et al., 2004; Ahmad et al., 2008).
Researches in the past few decades on various aspects of root symbionts have shown that dual interaction of AM fungi and Rhizobium has improved the growth, nodulation and yield and also nutrient status in legumes. In the light of the above, the present study was carried out to study the efficiency of dual inoculation of AM fungus (Glomus mosseae and Acaulospora laevis) with Bradyrhizobium japonicum in alleviating the adverse effect of salinity stress of mungbean.
MATERIALS AND METHODS
Growth conditions
The experiment was carried out under poly house
at Botany Department, Kurukshetra University, India
at a temperature (300C ± 50C) and humidity (50%
-70%). Light was provided by cool white fluorescent
lamps (8000 lux) under a 16-hour photoperiod. The
glasshouse also received sunlight. The soil
characteristics are as follows: sand-64.2%, silt-
21.81%, clay-3.90%, pH-6.890, EC- 1.00 dS/m,
organic carbon-0.40%, total N-0.042%, P-0.0018
Kg/m2, K-0.022 Kg/ m2, and S-14.80 ppm.
Mass multiplication of bio-inoculants
Mass multiplication of arbuscular mycorrhizal fungi (AMF)
The native predominant AM fungi Glomus mosseae (T.H. Nicolson and Gerd.) Walker and Schüßler and Acaulospora laevis (Gerdemann and Trappe) were isolated from the rhizosphere of mungbean plants. Both AM fungi were mass multiplied in sterilized soil and sand (3:1) substrate using maize as a suitable host in polyhouse conditions. The starter inoculum or pure culture of
each selected dominant AM fungus (G. mosseae and A. laevis) was raised by the funnel technique of Menge and Timmer using maize as a host.
Mass multiplication of Rhizobium sp.
The culture of Bradyrhizobium japonicum was procured from Department of Microbiology, CCS Haryana Agricultural University, Hisar, India and was used as a basal dose.
Plant material
The seeds of mungbean were surface sterilized with 0.5% (v/v) sodium hypochlorite for 10 minutes, subsequently washed with sterilized deionized water. Before sowing seeds 10 ml of a liquid suspension of Bradyrhizobium sp. with a density 108 cells/ml, was applied to each pot. After 10 days, emergence seedlings were thinned to 6 plants per pot. Experimental setup
The experiment was laid out in a randomized complete block design, with five replicates of each treatment. Soil from experimental site was collected and mixed with sand in a proportion of 3:1 (soil: sand). This mixture was then sieved through 2-mm sieve and autoclaved at 1210C for two hours for two consecutive days. Earthen pots (24.5 x 25 cm) were selected and filled with 2.5 kg soil. Initially, the pots were saturated with three different levels of saline solution, i.e. 4, 8, and 12 dSm-1(sodium chloride, calcium chloride and sodium sulphate, 7:2:1 w/v as per Richards (1954). Then, chopped AM colonized root pieces of maize having 80%-85% of colonization along with the soil having AM spores (620-650 per100 g inoculum) were used as AM inoculum. To each pot 10% (w/w), i.e. 200g/pot inoculum of AM fungi alone and in
combinations were added into the soil before plantation. Pots were watered regularly with saline solution to maintain the required salinity level and fertilized with a nutrient solution after 15 days (Weaver and Fredrick 1982), which contained half the recommended level of phosphorus and no nitrogen. The experiment had 4 treatments with a single inoculum, a combined inoculum or no inoculums as outlined below:
1. Uninoculated (autoclaved sterile sand: soil without AM inoculum but having Bradyrhizobium sp.)
2. Glomus mosseae (G) having Bradyrhizobium sp.
3. Acaulospora laevis (A) having Bradyrhizobium sp.
4. G + A having Bradyrhizobium sp.
Plant harvest, growth and nutrient analysis
Plants were harvested after 100 days by uprooting them from the soil and various morphological and physiological parameters were measured. For determining root and shoot fresh and dry weight, roots and shoots were harvested after 100 days, weighed and then, oven dried at 70 °C and weighed again. Amount of chlorophyll a, chlorophyll b and total chlorophyll was estimated using the method of Arnon (1949). Phosphorus concentration were determined using the ‘Vanado-molybdo-phosphoric yellow colour method' (Jackson, 1973) and nitrogen (N) was calculated by Kjeldahl method (Kelplus nitrogen estimation system, supra-LX, Pelican Equipments, Chennai, India). Analysis of Sodium and Potassium was done by inductively coupled plasma analyzer-Mass spectrometry (ICP-MS). Phosphatase activity was assayed using p-nitrophenyl phosphate (PNPP)
as a substrate, which is hydrolyzed by the enzyme to
p-nitrophenol (Tabatabi and Bremner, 1969).
Identification and quantification of the number and colonization by AM spores
AM spores (G. mosseae and A. laevis) were identified by using the identification manual used by Walker (1983), Scheneck and Perez (1990), Morton and Benny (1990) and Mukerji (1996). Quantification of the number of AM spores was done using the Adholeya and Gaur ‘Grid Line Intersect Method' (1994). Mycorrhizal colonization of roots was determined using the ‘Rapid Clearing and Staining Method' of Phillips and Hayman (1970). Percentage AM colonization of roots was: (Number of root segments colonized / number of root segments studied) x 100.
Electrolyte leakage
To resolve electrolyte leakage, fresh leaf samples (200 mg) were cut into small discs (i.e. 5mm in diameter) and placed in test tubes containing 10 ml distilled, deionized water. The tubes covered with cotton plugs were placed in a water bath at a constant temperature of 32±8oC. After 2 h the initial electrical conductivity of the medium (EC1) was measured using electrical conductivity meter. The samples were autoclaved afterwards at 121±8oC for 20 minutes to kill the tissues completely and release all electrolytes. The samples were then cooled to 25 ±8oC and final electrical conductivity (EC2) was measured. The electrolyte leakage (EL) was estimated using the formula of Dionisio-Sese and Tobita:
EL= EC1/EC2 X 100
Proline Determination
Proline was determined by the Bates et al. (1973). The proline content was estimated by using the formula:
Proline content = 34.11 x A520 x 10/ 2x 0.5 Protein content
Total protein was estimated by method of Bradford (1976).
Statistical analysis
Data were subjected to analysis of variance and means separated using the least significant difference test in the Statistical Package for Social Sciences (ver.11.5, Chicago, IL, USA).
RESULTS Plant height
All the treatments resulted in increment in plant
height over control at different salinity levels (Table 1).
Maximum change in plant height was recorded in dual
inoculation i.e. G. mosseae + A. laevis at 4 dS m-1
followed by single inoculation of F. mosseae.
Plant biomass
Biomass of all the inoculated plants of mungbean
increased significantly in terms of fresh and dry shoot
& root weight. Maximum increment in shoot biomass
(fresh and dry) was recorded in single inoculation of
G. mosseae followed by dual inoculation of F.
mosseae + A. laevis at 4 dS m-1 and 8dS m-1 salinity
levels (Table 1). According to the results root biomass
was also found to be increased significantly
irrespective of treatments over control. After 100 days,
the increase in root biomass (both fresh and dry) was
observed maximum in dual inoculation of G. mosseae
+ A. laevis followed by single inoculation of G.
mosseae at all salinity levels.
Root length
The study revealed a prominent increment in root
length in all the treated plants at different salinity
levels. However, the best results were observed in
plants inoculated with G. mosseae followed by the
single inoculation of A. laevis (Table1). It is clearly
evident that increased level of salinity from 4 dS m-1 to
12 dS m-1 resulted in a reduction of root length.
AM association (AM spore number and % root colonization)
All the treated plants were found to harbor more AM association in comparison to control at all the salinity levels but the AM association also decreases as the salinity level increases (Table1). The results were found to be most significant in dual inoculation of G. mosseae + A. laevis having highest number of spores and percent root colonization at all the three different level of salinity followed by single inoculation of G. mosseae.
Chlorophyll content
Chlorophyll content was found to be increased in all treated plants over control (Table 2). The highest increase in total chlorophyll content was observed in single inoculation of G. mosseae followed by dual inoculation of F. mosseae + A. laevis at all the three different levels of salinity.
Electrolyte leakage
Salt stress caused a significant increase in electrolyte leakage compared to that in the nonstressed plants (Table 2). However, mycorrhizal inoculation significantly reduced the electrolyte leakage in the salt-stressed plants of mungbean. The
dual inoculation of G+A was found to be effective in all the three levels of salt stress in lowering the uptake of electrolytes.
Proline content
In general, proline content in leaves of mycorrhizal mungbean plants was significantly higher than that of non-mycorrhizal plants grown in saline soil. Such increase in proline content was linked to the degree of mycorrhizal infection. Among all the treated plants, dual inoculation of G+A was the most efficient for their ability to improve proline content (Table 2).
Protein content
The protein content of mungbean plants was significantly reduced under salinity stress conditions in all the treated plants. It can also be seen that protein level in AM plants was higher than that of non-AM plants (Table 2). In all the treated plants, G+A was more effective for their ability to improve protein content.
Phosphatase activity
In saline soil, acid and alkaline phosphatases activities were significantly higher in mycorrhizal than in non-mycorrhizal mungbean plants. In all the treated plants alkaline phosphatase activity was found to be more than the acidic phosphatase activity and the effect were more pronounced in the plants treated with dual inoculation of G+A under different levels of salinity stress.
Mineral uptake
AM fungi have been shown a positive influence on the composition of mineral nutrients of plants grown in salt stressed conditions by enhancing selective uptake of nutrients. The concentration of N, K and P
JOURNAL OF STRESS PHYSIOLOGY & BIOCHEMISTRY Vol. 10 No. 3 2014
Table 1: Effect of AM fungi along with Rhizobium on growth parameters and mycorrhization of Mung bean plant under salinity stress
Salinity Treatments Plant Shoot weight (g) Root length Root weight (g) (%) Root AM spore
level height (cm] Fresh Dry (cm} Fresh Dry colonization number/1 Og of soil
4DS/m Control G A G+A 09.611 14s 24.0± 1.58a 19.4± 1.94a 27.8± 1.92* 1.0310.035* 4.3610.032* 2.95±0.041a 4.03±0.039“ 0.3510.023" 0.9510.022' 0.6810.025'1 0.7210.019e 6.411.14* 14.411.81* 9.6±1.14B 9211.64“ 0.3910.023'1 0.6210.044“ 0.5410.025“ 0.8710.025“ 0.0810.004® 0.1610.025“ 0.1310.021“ 0.4010.025* 2.010.70' 45.213.03“ 33.614.03* 57.6+3.04* 3.411.67* 52.41207° 45-2+2.86“ 65.0+2.54*
3 D S/m Control G A G+A 07.2+0.83" 19.6l1.8111 14.0+1.58* 21.8± 1.78“ 0-8610.0401 3.0510.030' 2.1310.0341 2.7710.037* 0.2810.028' 0.7810.028“ 0.4210.031* 0.6110.039* 4.6+1.51" 8.810.83“ 7.2+164““ 6.210.37- 0.3010.026" 0.5010.052“ 0.4510.039“ 0.7110.054“ 0.0710.002*’ 0.1110.017* 0.0910.002*' 0.2510.033“ 0.610.89' 34.414.61 ” 29.411.941 40.812.68“ 20+158" 43.013.46“ 38.212.04'’ 51.212.28“
12DS/m Control G A G+A 22+0.83“ 9.0±1.58* 4.412.70*' 12.0+1 87' 0.7310.0241 1.7610.026’' 0.9810.025' 1.9910.019s 0.0710.007' 0.4910.034' 0.3210.035* 0.5910.018* 3.710.33" 8.411.81* 6.811.09““ 6.1 + 0.08*1 02210.039' 0.4310.030* 0.3610.042' 0.6810.026“ 0.0510.003* 0.0810.003*'’ 0.0610.003* 0.1810.021c 0.0010.00' 24.613.84* 17.013.46" 32.012.12“ 0.010.00' 32.611.94* 26211.78' 42.41207e
L.S.D (PS0.05) ANOVA F(,u„ 1 9768 135.067 0042 6942.964 0.0352 399.340 1.6027 24.249 0.0475 124.760 0.4423 159.448 36229 209.236 2.7568 490.972
F values Salinity (s) Parameter (p) s x p 449.049 331.756 6.868 14356.265 11952.495 1718.149 799.714 1844.220 76273 58371 52.625 2.3B6 340.072 827.780 1.349 135.971 1231.684 24.890 81.246 1026.997 26.032 344263 1492.044 41.326
G: Glomus mosseae, A: Acaulospora laevis; each value is a mean ot five replicates.
±: standard deviation AM: Arbuscular mycorrhizal.
Values in columns followed by the same letter are not significantly different, P < 0.05, least significant difference test.
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JOURNAL OF STRESS PHYSIOLOGY & BIOCHEMISTRY Vol. 10 No. 3 2014
Table 2: Effect of AM fungi along with Rhizobium on physiological parameters of Mung bean plant under salinity stress
Salinity level Treatments Chlorophyll content Chi a Chi b Total Chi Proline content Root Shoot Electrolyte leakage Protein content
Control 0.93+0.004' 0.63+0.01 V 1.57+0.009' 0.10+ 0.002“ 2.04 +0.003" 26.7 ± 1 429 11.3 + 0.27"
4DS,m G 1.51 ±0.003“ 1,22±0.007“ 2.74±0.008* 0.34 ± 0.003h 5.11± 0.003c 20.8 ± 0.97h 21.5 ±0.94°
A 1.17+0.006' 0.93+0.009* 2.10±0.003* 0.23 ± 0.003' 4.09± 0.003* 21.3 ± 126h 16.0± 0.37*
G+A 1.41 ±0.002° 1.11+0.008° 2.53±0.010° 0.40 + 0.0029 6.19± 0.003c 19.8 ±1.49° 24.4± 0.31“
Control 0.87+0.003' 0.59+0.0071 1.47+0.01 C 0.17 + 0.0031 2.72 +0.0049 40.2 + 0.99c 7.6 + 0.24'
3D 5/m G 1.34±0.004c 1.02+0.006c 2.36±0.005c 0.61 +0.005* 4.77± 0.00311 32.4 + 1.01f 17.7±0.40d
A 1.11 ±0.004' 0.84+0.008' 1.96±0.006' 0.44 ± 0.004' 4.19 ±0.003* 34.5 ± 2.00*' 15.0 ±0.36'
G+A 1.29±0.003d 0.99±0.005d 2.29±0.009d 0.64 ± 0.003° 5.79 ± 0.003c 32.9 ± 1.87*' 19.1 ±0.71e
12DS/m Control 0.85±0.003" 0.51+0.00/ 1.36±0.010* 0.30 ± 0.004' 3.41 ± 0.002' 48.8 ± 1.28“ 3.1 ±0.00l
G 1.06±0.0029 0.80+0.0079 1.86+0.0099 1.02 ± 0.003° 7.16 ±0.003° 36.9 ± 2.89d 13.6 ± 0.039
A 0.95±0.003' 0.66+0.005' 1.61 ±0.008' 0.68 ± 0.003c 6.82 ± 0.004° 42.7 ±1.72" 11.7 ±0.46°
G+A 1.01 ±0.002h 0.77+0.006h 1.79±0.004h 1.33 ± 0.002“ 7.84 ± 0.003* 34.8 ± 1.43* 14.0 ±0.2/
L.S.D (P<0.05) 0.0053 0.0142 1.3715 0.012 0.4915 20597 0 5564
anovaf,,,^ 14123.280 1919.234 13548.763 7315.701 110.411 158.721 873.160
F values Salinity (s) Parameter (p) Salinity X P 49339.958 16359.285 2565.520 8135.940 3195.020 171.621 41069.469 23227.069 1578.953 16409.891 15186.612 1902.297 182.900 267.200 7.895 794.612 93.651 9891 2153.312 1793.629 65.569
G: Glomus mosseae, A: Acaulospora laevis; each value is a mean of five replicates.
±: standard deviation AM: Arbuscular mycorrhizal.
Values in columns followed by the same letter are not significantly different, P < 0.05, least significant difference test.
141 Application of AM Fungi..
JOURNAL OF STRESS PHYSIOLOGY & BIOCHEMISTRY Vol. 10 No. 3 2014
Table 3: Effect of AM fungi along with Rhizobium on nutrient uptake of Mung bean plant under salinity stress
Salinity level T reatments Nitrogen content (%) Root Shoot Phosphorus content (%) Root Shoot Potassium content (%) Root Shoot Sodium content (%) Root Shoot
4DSm Control 0.38±0.024' 0.5510.025" 0.50Í0.0381 0.3710.039* 1.0010035' 0.7710.038" 1.4510.047* 1.5910.040'
Û 0.7710.036“ 1.9410.033“ 1.3410.027“ 1.0310.034“ 2.1910.039* 2.0210.039* 12810.026“ 1.3110.040'
A 0.70±0.030“ 1.75+0.033' 1.00+0.027* 0.7910.028a 1.3310.043' 1.1810.025“ 0.63+0.027* 0.7010.032'
<j+A 1.0010.053* 2.10+0.038“ 1.6910.027* 1.1710.030* 1.6710.032a 1.35+0.049d 1.08+0.052' 12210.046’
flDSi'm Control 02510.031* 0.3710.033' 0.2410.0361 02210.038Í 0.9010.033’ 0.6810.031 i 1.7910.022* 1.8210.057*
G 0.7010.016“ 0.9010.043* 1.0810.046a 0.8110.020“ 2.0610.040“ 1.7710.034" U910.026' 15710.035'
A Q-SOtOM? 0.7110.038' 0.8610.040' 0.6110.0381 1.1710.034’ 1.0010.033' 0.7510.041* 0.9510.018*
û+A 0.7510.041“ 1.0810.02811 1.25l0.042c 0.8510.033' 1.3910.037* 1.1710.036* 12710.024“ 1.4010.040*
12DSrm Control Ü 0.1310.027* 0.5310.056* 0.17l0.029i 0.6610.024* 0.1910.021 ‘ 0.7410.027* 0.1210.018j 0.4910.042* 0.8310.066* 1.8910.039' 0.5910.036' 1.6710.032e 15010.038* 12910.048“ 1.6810.054* 1.4710.027a
A 0.4310.0311 0.6510.041* 0.6810.050* 0.40i0.038* 1.0810.041" 0.9210.037* 0.7210.041* 0.9210.059*
Û+A 0.65i0.030a 0.8510.040* 0.8810.0271 0.6810.054* 1.3110.043' 1.1410.036* 12010.038* 12710.039*
L.S.D(P<0.05) 0.2679 0.0442 0.0452 0.0463 0.0524 0.0464 0.0473 0.0542
AN0VA(F11,24) 203.046 1610.916 774.780 387.885 612.873 749.173 427.687 308.827
F values Salinity (s) 288.096 6945.698 334.48 602.424 855.117 1146.350 2163.875 165.958
Parameter (p) 700.530 3212.258 1089.046 3356.748 14045.681 7060287 1392.734 479.158
Salinity x p 11.360 278.981 36.596 134.197 19.095 11.764 4.906 3.238
G: Glomus mosseae, A: Acaulospora laexis; each value is a mean of five replicates.
±: standard deviation AM: Arbuscular mycorrhizal.
Values in columns followed by the same letter are not significantly different. P < 0.05, least significant difference test.
Kadian et al.
JOURNAL OF STRESS PHYSIOLOGY & BIOCHEMISTRY Vol. 10 No. 3 2014
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Table 4: Effect of AM fungi along with Rhizobium on phosphatase activity, yield and nodulation of Mung bean plant under salinity stress
Salinity level Treatments No. of pods Yield (per pot) Wt of pods (g) Number of nodules/pot Phosphatase Activity Acidic Alkaline
4D5/m Control 2.4 ± 1.14r 1.02±0.019g 3.4 ± 1.14* 0.038 ± O-OOT* 0.082 ± 0.006 '
G 9.6 ± 1.67* 2.82±0.019b 18.0 ± 2.54° 0.19510.007e 0.328 ± 0.008c
A 6.2 ± 0.83d 2.07±0.033e 13.4 ± 1.14c 0.125 ± 0.006* 0.258 ± 0.00/
G+A 12.8 ± 130“ 3.23±0.030a 24.6 ± 3.57* 0240 ± 0.008* 0.387 ± 0.006*
8D5/m Control G 0.0± 0.00* 6.2 + 1.30d 0.00 ± 0.00g 2.24±0.027d 20±0.7tyl 15.0 ± 1.87° 0.032 ± 0.007" 0.140 ± 0.008° 0.061 ± 0.006k 0.245 ± 0.007*
A 03.8±1.30e 1.48±0.027t 9.8 ± 1.48° 0.097 ± 0.008' 0.218 ± 0.007'
G+A 8.0 ± 0.70" 2.50±0.023c 18.4±2.70t 0223 ± 0.007° 0.346 ± 0.005°
12D Sim Control 0.0 ± 0.00’ 0.00 ± 0.00' 1.0 ± 0.7tf 0.024 ± 0.005* 0.045 ± 0.0061
G 0.00 ± 0.00g 0.00 ± O.OO1 7.2 ±2.16d 0.075 ± 0.006’ 0.1 16 ±0.000
A 0.00 ± 0.00g 0.00 ± 0.001 4.6 ± 2.07* 0.064 ± 0.008" 0.154 ± 0.006’
G+A 2.2 ± 0.83' 0.94±0.016h 9.2+ 2.28" 0.044 ± 0.009' 0.144 ± 0.008”
L.S.D (p< 0.05) 1.223 0.0259 2.5775 0.0097 0.0092
ANOVA F 99.984 17568.266 67.795 496.970 1285.755
F values Salinity (s) 418.819 21538.190 107.684 680.865 6490.335
Parameter (p) 92.917 44056.000 113.574 913.120 3194.482
Salinity X p 20.300 4280.779 14.302 251.308 264.851
G: Glomus mosseae, A: Acaulospora laevis: each value is a mean of five replicates.
±: standard deviation AM: Arbuscular mycorrhizal.
Values in columns followed by the same letter are not significantly different, P < 0.05. least significant difference test.
^ >
ST
CD Co"
143 Application of AM Fungi..
pods and consequently higher yields under different salinity levels (Table 4). Treatment with a mixture of G. mosseae and A. laevis at 4ds/m resulted in the greatest increase in yield in terms of the number of pods followed by those plants treated with G. mosseae alone. However, the yield was found to be decreased as the salinity level increases from 4ds/m to 8ds/m and 12ds/m. That is, the greatest yield was of plants inoculated with both G. mosseae and A. laevis which operated together more effectively in supplying their host plants with their nutrient requirements than when either operated on its own. Colonization of a legume by AM fungi also increased the number of nodules as evident from Table 4.
DISCUSSION
Salt stress adversely affects the morphology as well as physiology of plants grown under saline soil by increasing the osmotic stress, ion toxicity and nutrient deficiency. However, colonization of mungbean plant with AM fungi significantly increased growth response. The G+A treatments gave better results at all the three different salinity levels than other treatments because AM fungi counteract the toxic effects of salts by the better acquisition of nutrition especially phosphorus (Sharifi et ai., 2007) and other elements by extraradical mycorrhizal hyphae, and transferring them to the root tissues (Wu et al., 2010; Giri and Mukerji 2004). The results of present investigation are in close conformity with those of Colla et al. (2008) who reported improved growth of Cucurbita pepo colonized with Glomus intraradices under salinity stress.
Giri and Mukerji (2003) also reported a significant
increase in root and shoot dry weights of Acacia auriculiformis inoculated with mycorrhizal treated plants than non mycorrhizal plants. Similar results are also reported by Al karaki (2000) for tomato plant when inoculated with mycorrhizal fungi. This increment may be due to more absorption of nutrients especially P via an increase in root surface area through AM fungi (Prakash et al., 2011) and ability of plants for replacement of K by Na (Haijiboland and Joudmand, 2009). Under saline soil, greater CO2 assimilation could adequately provide carbohydrates for the fungal partner and results in more benefits to plants from AM association. These AM isolates from saline soils have a better ability to improve the survival, growth and ultimately biomass of host plants (Tain et al., 2004).
The better results in root length was obtained when only the plants were inoculated with F. mosseae alone at different salinity levels may be due to space and nutrition for its multiplication and survival in sterilized soil resulting in absorption of more nutrients from the soil. Similarly, Quilambo (2000) and Shekoofeh and Sepideh (2011) also observed significant increment in root length with an indigenous AM fungi at various salinity levels. Correlation of root length with mycorrhizal inoculation amount of root is probably related to suitable ventilation of soil, that is the result of hypha network of mycorrhizal fungi that connects particles of soil and as result the root spreads into deep soil (Turk et al., 2006).
AM inoculated plants showed higher percentage of colonized roots as compared to control. On the basis of present investigation, it was found that root
colonization and AM spore number were greatly influenced by increasing soil salinity level. Similarly, Al-Khaliel (2010) reported that mycorrhizal colonization and spore density decreases under highest salinization level in peanut treated with G. mosseae. The suppressed spore number and colonization of arbuscular mycorrhizal under different salinity level may be attributed to the reduced spore germination and hyphal extension of AMF that were inhibited by salt (Belew et a/., 2010).
The single inoculation of G. mosseae results in maximum increase in chlorophyll content at 4DS/m due to less interference of salt with chlorophyll synthesis in mycorrhizal than in non-mycorrhizal plants (Giri and Mukerji, 2004). Under saline conditions, mycorrhization helps in better absorption of Mg in plants and the antagonisitic effect of Na+ on Mg+ uptake is counter balanced and suppressed resulting in increased chlorophyll synthesis (Giri and Mukerji, 2003). In Glomus etunicatum inoculated maize plant, increase in photosynthesis speed, transpiration and chlorophyll a, b density was reported under stress (Zhu et a/., 2010).
Mycorrhizal plants had significantly higher root P concentration than shoot. The phosphorus concentration in plant tissuses rapidly lowered under salt stress because phosphate ion precipitates with Ca, Mg and Zn, then being unavailable to plants (Evelin et a/., 2009; Park et a/., 2009). Mycorrhizal inoculation can increase P concentration in plants by enhancing its uptake facilitated by the extensive hyphae of the fungus which allows them to explore more soil volume than the non-mycorrhizal plants
(Ruiz-Lozano and Azcon, 2000). Our results are in accordance with those of Shokri and Maadi (2009) who reported that the concentration of phosphorus in Trifolium alexandrium plants was found to be higher relative to non-inoculated ones but it decreases with the increasing level of salinity. Similar results were also obtained by Giri and Mukerji (2004) who reported maximum uptake of phosphorus in roots when the plants were inoculated with Glomus macrocarpum. Higher P uptake by mycorrhizal plants under salt stress increases the plant ability of reducing of negative effects of Na+ and Cl" ions (Feng et al., 2002) by maintaining vacuolar membrane integrity, which facilitates compartmentalization within vacuoles and selective ion intake, thereby preventing ions from interfering in metabolic pathways of growth (Cantrell and Lindermann, 2001).
AM fungi can function as a facilitator for N uptake through activation of a plant ammonium transporter (Guether et al., 2009) and salts interferes less with nitrogen acquisition and utilization by influencing different stages of N metabolism, such as NO"3 uptake and reduction and protein synthesis (Frechill et al., 2001). Thus, improved uptake of N in mycorrhizal plants under salt stress may be due to better nutrient uptake and maintenance of ionic balance and better acquisition of N (both nitrate and ammonium ions) from the soil. Garg and Manchanda (2008) also recorded highest accumulation of N in shoots of mycorrhizal Cajanus cajan than non mycorrhizal plants at all salinity levels.
AM induce a buffering effect on the uptake of Na+ when the content of Na+ is within the permissible limit
(Allen and Cunningham, 1983). This also indicates the possibility of a regulatory mechanism operating in the plant contain Na* ions. The accumulation of Na is strongly influenced by the form of N available (NO3 and NH4) and it may also be influenced by the synthesis and storage of polyphosphate (Orlovich and Ahford, 1993) as well as by other cations, particularly K (Giri et al., 2003). The results of present investigation are in consonance with Giri et al., 2007 in Acacia nilotica when inoculated with Glomus fasciculatum. Similar results are also obtained by Tian et al (2004) when the plants of Gossypium arboretum were inoculated with G. mosseae under salt stress.
Sharifi et al. (2007) and Zuccarini and Okurowska (2008) also observed increase in K+ content when inoculated with Glomus etunicatum under different levels of salt stress. Higher K+ accumulation in mycorrhizal plant under salt stress conditions may help in maintaining a high K/Na ratio, thus preventing the disruption of various enzymatic processes and inhibition of protein synthesis. This capacity of plants to maintain a high cytosolic K+: Na + is one of the important factor of plant salt tolerance (Maathuis and Amtmann, 1999).Our results are in accordance with the findings of Shokri and Maadi (2009), Porras-Soriano et al. (2009) who reported efficacy of G. intraradices in maintaining favourable K+: Na+ ratio.
In saline soil, acid and alkaline Phosphatase activities were significantly higher in mycorrhizal than non-mycorrhizal ones. Such increases in those activities were related to the degree of active mycorrhizal infection of each fungal species. Gianinazzi-Pearson and Gianinazzi (1978) and Ezawa
and Yoshida (1994) detected mycorrhizal-specific Phosphatase (MSPase) only in the mycorrhizal root extract, and it was of fungal origin. The close relation between mycorrhizal growth responses and the active arbuscular phase of the infection supports the hypothesis that the Phosphatase enzyme is somehow involved in assimilation of phosphorus by arbuscular mycorrhizal fungi (Abdel-Fattah, 2001).
Under saline conditions, many plants accumulate proline as a non-toxic and protective osmolyte to maintain osmotic balance under low water potentials (Ashraf and Foolad, 2007; Parida et al., 2002). It also acts as a reservoir of energy and nitrogen for utilization during salt stress conditions (Goas et al., 1982). Proline levels were found to be increased significantly with salinity stress in mycorrhizal plants when compared to non-mycorrhizal plants. Sharifi et al. (2007) also reported a higher proline concentration in AM soybean than non-AM plants at different salinity level. This increment in proline could be due to the induction of proline biosynthesis enzymes and/or to the reduction of oxidation to glutamate (Stewart, 1981). Several roles have been attributed to this supraoptimal level of proline; for instance, osmoregulation and detoxification of free radicals (Kaul et al., 2008).
Salt stress caused a significant increase in electrolyte leakage compared to that in the nonstressed plants. Mycorrhizal treated plants have lower electrolyte leakage as compared to non-mycorrhizal plants by maintaining improved integrity and stability of membrane (Zhongoun et al., 2007; Manchanda, 2008). Mycorrhizal plants had much lower root plasma
membrane electrolyte permeability than the non-mycorrhizal plants (Kaya et al., 2009). The increased membrane stability has been attributed to mycorrhizal mediated enhanced P uptake and increased antioxidant production (Feng et al., 2002).
Higher protein concentration could be due to higher efficiency of the osmotic regulation mechanism in mungbean plants which in turn prevents protein reduction under salt stress (Flowers and Yeo, 1995; Kumar et al., 2010) and induces the synthesis of osmotin like protein structure. This protein increment lead to membrane stabilization and helps plants to grow and develop under saline conditions (Goudarzi and Pakniyat, 2009). Mycorrhizal and nodule symbioses often act synergistically on infection rate, mineral nutrition and plant growth (Patreze and Cordeiro, 2004; Rabie, 2005) which support the need for both N and P and increased tolerance of plants to salinity stress (Rabie and Almadini, 2005).
CONCLUSION
In conclusion, the results of the present investigation showed that the tripartite symbiosis of bacterial-AM-legume significantly alleviated the harmful effects of salt stress in legumes plant. However, many practical problems remain, such as the selection of better strain dosages, salinity tolerance of symbionts, choice of good symbionts to plant and appropriate time for inoculation that needs further studies.
ACKNOWLEDGMENT
The authors are grateful to Kurukshetra University, Kurukshetra, for providing laboratory facilities to carry
out the research work.
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