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Biologia, Bratislava, 61/Suppl. 19: S324—S328, 2006
Effects of arbuscular mycorrhizal fungi and Bradyrhizobium japonicum on drought stress of soybean Nasser Aliasgharzad, Mohammad Reza Neyshabouri & Ghobad Salimi Department of Soil Science, Faculty of Agriculture, University of Tabriz, Tabriz, I.R. of Iran; fax: ++98-411-3345332, e-mail:
[email protected]
Abstract: Mycorrhizal symbiosis can potentially improve water uptake by plants. In a controlled pot culture experiment, soybean plants were inoculated with two species of arbuscular mycorrhizal fungi, Glomus mosseae (Gm) or Glomus etunicatum (Ge), or left non-inoculated (NM) as control in a sterile soil. Four levels of soil moisture (Field capacity, 0.85 FC, 0.7 FC, 0.6 FC) in the presence or absence of the Bradyrhizobium japonicum, were applied to the pots. Relative water content (RWC) of leaf at both plant growth stages (flowering and seed maturation) decreased with the dryness of soil; RWC was higher in all mycorrhizal than non-mycorrhizal plants irrespective of soil moisture level. At the lowest moisture level (0.6 FC) Ge was more efficient than Gm in maintaining high leaf RWC. Leaf water potential (LWP) had the same trend as RWC in flowering stage but it was not significantly influenced by decrease in soil moisture to 0.7 FC during seed maturation stage. Seed and shoot dry weights were affected negatively by drought stress. Mycorrhizal plants, however had significantly higher seed and shoot dry weights than non-mycorrhizal plants at all moisture levels except for seed weight at 0.6 FC. Root mycorrhizal colonization was positively correlated with RWC, LWP, shoot N and K, and seed weight, implying improvement of plant water and nutritional status as a result of colonization. Regardless of moisture treatments, bacterial inoculation caused a significant enhancement in N content and the highest N occurred in rhizobial inoculated plants at 0.85 FC and 0.7 FC. Shoot K was enhanced considerably by both bacterial and fungal inoculations, particularly in plants with dual inoculations where the highest shoot K levels were found. The relatively higher shoot and seed dry weights in plants inoculated with both G. etunicatum and B. japonicum could be ascribed to their higher RWC and LWP, suggesting that drought avoidance is main mechanism of this plant-microbe association in alleviation of water stress in soybean. Key words: soybean, arbuscular mycorrhizal fungi, drought stress, B. japonicum
Introduction Water deficit is an important abiotic factor limiting plant growth and yield in many areas on the earth that is increasingly topical because of climate change and water shortages (Ruiz-Lozano et al., 2001; Jafarzadeh & Abbasi, 2006). Several eco-physiological studies have demonstrated that arbuscular mycorrhizal (AM) symbiosis often results in altered rates of water movement into, through and out of the host plant, with consequent effects on tissue hydration and plant physiology. It is now accepted that the contribution of AM symbiosis to plant drought tolerance is the result of accumulative physical, nutritional, physiological and cellular effects (Ague, 2001). The pioneer studies of Allen (1982) and Hardie (1985) indicated a possible role of AM fungal in water uptake and transfer to the host plant. Allen (1991) estimated that the rate of water transport by extraradical hyphae to the root was 0.28 ng s−1 per entry point, a level sufficient to modify plant water relations. Another mechanism for increasing drought resistance in plants is osmotic ad-
justment which allows cells to maintain turgor and the processes that depend on it, such as cellular expansion and growth, stomatal opening and photosynthesis, as well as keeping a gradient of water potential favorable to water entrance into the plant (Ruiz-Lozano, 2003). The solutes that participate in osmotic adjustment are inorganic ions (mainly K+ ) or uncharged organic compounds proline or glycine betaine as well as carbohydrates like sucrose, pinitol or mannitol (Aliasgharzad et al., 2005; Ruiz-Lozano, 2003). In addition, other studies have shown that the AM symbiosis can alleviate drought-induced nodule senescence in legume plants. The most remarkable observation was the substantial reduction in oxidative damage to lipids and proteins in nodules of mycorrhizal plants subjected to drought as compared to the nodules of non-mycorrhizal plants (Ruiz-Lozano et al., 2001). Drought resistance in plants can occur via drought avoidance (maintenance of high internal water potential) or drought tolerance (survival of low internal water potential) (Ludlow, 1989). In this respect, relative water content (RWC) and water potential (LWP) of leaf are suitable param-
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Table 1. Analysis of variance (mean squares) of the effects of mycorrhizal fungi, Bradyrhizobium and soil moisture levels on RWC, LWP, seed and shoot dry weights and shoot N and K. RWCa Source of variance
Block Bacteria(B) Fungi (F) BxF Moisture(M) BxM FxM BxFxM Error
LWP
Dry weight
Shoot
df
3 1 2 2 3 3 6 6 69
Fl
Sm
Fl
Sm
seed
shoot
N
K
0.000 0.000 0.023** 0.000 0.400** 0.000 0.001* 0.000 0.000
0.001* 0.000 0.033** 0.000 0.046** 0.000 0.001** 0.000 0.000
0.535 0.042 9.661** 0.023 21.845** 0.097 0.564 0.132 0.404
0.772 0.388 12.441** 0.096 16.927** 0.187 0.502 0.120 0.693
0.023 0.002 5.808** 0.004 4.947** 0.008 1.009** 0.009 0.009
0.253* 0.408* 15.709** 0.009 15.681** 0.038 2.295** 0.036 0.068
0.017 5.241** 1.581** 0.366** 0.823** 0.157 0.084 0.054 0.043
0.008* 0.057** 0.186** 0.007* 0.002 0.000 0.001 0.001 0.001
*, ** significant at α levels of 0.05 and 0.01, respectively a RWC, relative water content; LWP, leaf water potential; Fl, flowering stage; Sm, seed maturation stage.
Material and methods In a controlled pot culture experiment, soybean (Glycine max L. cv. Harkor) plants were inoculated with two species of arbuscular mycorrhizal fungi, Glomus mosseae (NICOL and GERD) GERDEMANN and TRAPPE (Gm) or Glomus etunicatum BECKER and GERDEMANN (Ge), or left nonmycorrhizal (NM) as control in a sterile soil. The soil used in this study was a sandy loam in texture, salinity measure (1.8 dS m−1 ), pH 7.2, and low organic carbon (3900 mg kg−1 ) and available NaHCO3 – extractable P of 4.4 mg kg−1 and medium available K of 200 mg kg−1 . Four levels of soil moisture (Field Capacity, 0.85 FC, 0.7 FC and 0.6 FC) in the presence or absence of Bradyrhizobium japonicum KIRCHNER (JORDAN) were applied to the pots. Soil moisture at FC was 15.7% (w/w). Pots were weighed every other day and water was added when their soil water content dropped to about 10% below the desired level (ALIASGHARZAD et al., 2005). Based on soil chemical analysis, potassium sulfate at a rate of 44 mg kg−1 was added evenly to the pots to supply sufficient K levels. The experiment was conducted in a greenhouse with average daytime and nighttime temperatures of 28 ± 2 ◦C and 18 ± 2 ◦C respectively, and average day length of 14 hours from June to October 2002. The pots were arranged in a factorial randomized block design with three factors (fungi, bacteria and soil moisture levels) and four replications. Relative water content (RWC) and leaf water potential (LWP) were measured at two plant growth stages: flowering and seed maturation (BENNET et al., 1987). At harvest, seed and shoot dry weights, and concentrations of N and K in shoots were measured (COTTENIE, 1980). Roots
were cleared and stained for determination of mycorrhizal root colonization percentage (DALPE, 1993).
Results and discussion Relative water content The main effects of AM fungi (F) and moisture levels (M) and the interaction of FxM were significant (p < 0.01) on leaf RWC at both plant growth stages (Tab. 1). Leaf RWC at both plant growth stages decreased with increase in water stress and RWC was higher in all mycorrhizal than non-mycorrhizal plants irrespective of soil moisture level. At the lowest moisture level (0.6 FC) the fungus Ge was more efficient than Gm in maintaining high leaf RWC (Figs 1, 2). Hardie (1985) reported that AM fungal hyphae with a diameter of 2–5 µm can penetrate soil pores inaccessible to root hairs (10–20 µm diameter) and so absorb water that is not available to non-mycorrhizal plants. Faber et al. (1991) measured rates of water transport by AM fungal hyphae ranging from 375 to 760 nL H2 O h−1 . Ruiz-Lozano & Azcon (1995) in their experiment with lettuce plants designed an AM hyphal compartment in the root zone using a 50-µm nylon screen that allowed penetration by AM hyphae but not by roots; water was applied by injection to this compartment. They found an increase in plant fresh weight of nearly 150% in mycorrhizal as compared
100
b RWC (%)
eters for evaluation of plant water status which have been gained less attention in mycorrhizal relationships. Also, the nutritional status of plants influences their water balance and stomatal behaviour. Leaf potassium and nitrogen concentrations, for example, are integrally involved in the physiology of stomatal opening and closing (Mansfield et al., 1990). This study was undertaken to determine the effects of both mycorrhizal and rhizobial symbiosis on leaf RWC and LWP, plant growth, seed production as well as K and N status of soybean plants in water-stressed condition.
a a
90
NM
b b d
Gm
c c
Ge
d c
e f
80 70 60 FC
0.85 FC
0.7 FC
0.6 FC
Fig. 1. Effect of mycorrhizal fungi and soil moisture levels on leaf RWC of flowering stage. Abbreviations: FC – field capacity; RWC – relative water content of leaf; NM – non-mycorrhizal; Gm – Glomus mosseae; Ge – Glomus etunicatum.
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N. Aliasgharzad et al.
RWC (%)
100 90
NM
a a
b
cd
b b de
c
Gm
Ge
c
d c e
80 70 60 FC
0.85 FC
0.7 FC
0.6 FC
Fig. 2. Effect of mycorrhizal fungi and soil moisture levels on leaf RWC of seed maturation stage (see Fig. 1 for abbreviations).
flowering FC
0.85 FC
maturation
0.7 FC
0.6 FC
LWP (KPa)
0 -200 -400 -600
a
b c
c
c
d
cd
-800
e
Fig. 3. Main effect of soil moisture levels on leaf water potential (LWP) at two plant growth stages.
flowering NM
maturation
Gm
Ge
LWP (KPa)
0 -200 -400 b
-600 a -800
that drought avoidance is the main mechanism of AM fungi in the alleviation of water stress in plants. The most accepted mechanism for increasing drought resistance by AM in plants is osmotic adjustment which allows cells to maintain turgor and turgor dependent processes, such as cellular expansion and growth, stomatal opening and photosynthesis, as well as keeping a gradient of water potential favorable to water entrance into the plant (Auge, 2001). Safir et al. (1972) have reported that the presence of mycorrhiza decreased root resistance to water flow in soybeans which could lead to improved water balance and high RWC and turgor in leaves. Mycorrhizal and non-mycorrhizal plants also often show different critical points or thresholds of stomatal behaviour under water stress condition. For example, leaf water potential was about 0.2 MPa lower in mycorrhizal wheat plants than in similar-sized nonmycorrhizal plants when the stomata began to close (Allen & Boosalis, 1983). Similarly, leaf water potential at stomatal closure was 0.7 MPa lower in AMF rose plants than in non-mycorrhizal plants of the same size (Auge et al., 1986). Seed and shoot dry weights The main effects of AM fungi and moisture levels and the interaction of FxM were significant (p < 0.01) on seed and shoot dry weights, but the main effect of bacteria was only significant (p < 0.05) on shoot dry weight (Tab. 1). Seed and shoot dry weights were negatively affected by water stress. Mycorrhizal plants, however had significantly higher seed and shoot dry weights than non-mycorrhizal plants at all moisture levels except for seed weight at 0.6 FC (Tab. 2).
c c
cd
c
Fig. 4. Main effect of mycorrhizal fungi on leaf water potential at two plant growth stages (see Fig. 1 for abbreviations).
to the non-mycorrhizal plants. Similarly, leaf water content increased in mycorrhizal plants with water applied to the hyphal compartment. Leaf water potential The main effects of AM fungi and moisture levels were significant (p < 0.01) on LWP at both plant growth stages (Tab. 1). Leaf water potential had the same trend as RWC in the flowering stage, but it was not significantly influenced by a decrease in soil moisture to 0.7 FC during the seed maturation stage. In similar moisture levels and mycorrhizal treatments, LWP was higher (less negative) at flowering than seed maturation stage (Figs 3, 4). Both mycorrhizal plants had higher LWP at two growth stages, than nonmycorrhizal plants. However, Ge was more efficient than Gm in maintaining a higher LWP (Fig. 4). It seems
Shoot N and K The main effects of bacteria (B) and fungi (F) and the interaction of BxF were significant (p < 0.01) on shoot N and K concentrations, but the main effect of moisture levels was only significant (p < 0.05) on shoot N (Tab. 1). Regardless of the moisture treatments, bacterial inoculation caused a significant enhancement in shoot N. B. japonicum may improve soybean growth in a water-stressed condition by supplying nitrogen. Radin (1990) pointed out that leaf N concentration can affect stomatal behaviour. Although mycorrhizal fungi had no significant effect on shoot N, but in nonbacterial plants it increased slightly in the presence of either fungi (Tab. 3). Root nodule number and fresh weight were not affected by moisture levels until 0.7 FC, but they were significantly diminished at 0.6 FC. Reduction in oxidative damage to lipids and proteins in nodules of mycorrhizal plants subjected to drought, as mentioned above, can alleviate drought induced nodule senescence in legume plants. Moreover, AM symbiosis can increase the glutathione reductase (GR) activity considerably both in roots and nodules of soybean plants subjected to drought stress (Porcel et
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Table 2. Interaction of moisture levels and mycorrhizal fungi on seed and shoot dry weights. Moisture Levels
FC
0.85 FC
0.7 FC
0.6 FC
Fungi
NM*
Gm
Ge
NM
Gm
Ge
Nm
Gm
Ge
NM
Gm
Ge
Seed (g plant−1 ) Shoot (g plant−1 )
0.6d 2.8de
3.52a 7.0a
3.54a 7.4a
0.6d 2.4e
2.8b 5.8b
2.6b 5.2c
0.4d 1.8e
1.3c 3.7d
1.1c 3.6d
0.4d 1.4f
0.65d 1.8ef
0.7d 2.6e
Means in each row followed by same letter are not significantly different (p < 0.05) (* see Fig. 1 for abbreviations). Table 3. Interaction of mycorrhizal fungi and bacterial inoculations on shoot K and N concentrations. Bacteria
Without Bradyrhizobium
With Bradyrhizobium
Fungi
NM*
Gm
Ge
NM
Gm
Ge
K (mg g−1 ) N (%)
0.35d 2.80b
0.42b 3.40ab
0.41b 3.50ab
0.34c 3.60a
0.45a 3.70a
0.44a 3.70a
Means in each row followed by same letter are not significantly different (p < 0.05) * see Fig. 1 for abbreviations
al., 2003). Shoot K was enhanced considerably by both bacterial and fungal inoculations and the highest was achieved in plants with dual inoculations (Tab. 3). Contribution of K+ in osmotic adjustment and stomatal conductance of plants has already been reported (RuizLozano, 2003). Root mycorrhizal colonization Root mycorrhizal colonization was positively correlated with RWC, LWP, shoot N and K, and seed weight, implying improvement of plant water and nutritional status as a result of mycorrhizal colonization. Given the higher RWC and LWP in mycorrhizal than non-mycorrhizal plants in this study, it can be concluded that mycorrhizal fungi may contribute to increase drought avoidance ability of plants, thus leading to alleviation of water stress in soybean. Mycorrhizal and bacterial inoculated plants had relatively higher K and N in shoots. Potassium and nitrogen are two essential nutrients that play key role in osmotic adjustment and stomatal behaviour. The relatively higher shoot and seed dry weights in plants inoculated with both G. etunicatum and B. japonicum could be ascribed to their higher RWC and LWP, suggesting that plant water status is effectively improved by this plant-microbe association. References ALIASGHARZAD, N., BARIN, M. & SAMADI, A. 2005. Effects of NaCl-induced and mixture salinities on leaf proline and growth of tomato in symbiosis with arbuscular mycorrhizal fungi, pp. 203–207. In: Proc. of the Int. Conf. on Environ. Management, 28–30 October 2005, Hyderabad, India. ALLEN, M.F. 1982. Influence of vesicular-arbuscular mycorrhiza on water movement through Buteloua gracilis LAG ex STEUD. New Phytol. 91: 191–196. ALLEN, M.F. & BOOSALIS. M.G. 1983. Effects of two species of VA mycorrhizal fungi on drought tolerance of winter wheat. New Phytol. 93: 67–76.
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