Microbiology and Molecular Biology Reviews, December 1999, p. 968-989, Vol. 63, No. 4
Department of Botany, Faculty of Science,
Beni-Suef, 62511 Egypt
1092-2172/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Rhizobium-Legume Symbiosis and Nitrogen
Fixation under Severe Conditions and in an Arid Climate
SUMMARY
THE NITROGEN FIXATION PROCESS
NITROGEN-FIXING ORGANISMS
SIGNIFICANCE OF BIOLOGICAL N2
FIXATION TO SOIL FERTILITY
EFFECTS OF SEVERE CONDITIONS ON NITROGEN FIXATION
Environmental Conditions
Salt and Osmotic Stresses
Soil Moisture Deficiency
High Temperature and Heat Stress
Soil Acidity and Alkalinity
Nutrient Deficiency Stress
Soil Amendments and Ameliorations
Sewage sludge treatment and organic fertilizers.
Fertilizer application.
Pesticide application.
NITROGEN FIXATION IN ARID REGIONS
Arid Regions and Arid Climates
Improving the Fertility of Arid Regions
Biological N2 Fixation in Arid Regions
Rhizobium-Legume Symbioses and
Rehabilitation of Arid Regions
Drought-tolerant Rhizobium-legume symbiosis.
Salt-tolerant Rhizobium-legume symbiosis.
Significance of woody (tree)-legume-Rhizobium
symbioses to the rehabilitation of arid regions.
CONCLUSIONS
REFERENCES
SUMMARY
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Biological N2 fixation represents the major source of N input in agricultural soils including those in arid regions. The major N2-fixing systems are the symbiotic systems, which can play a significant role in improving the fertility and productivity of low-N soils. The Rhizobium-legume symbioses have received most attention and have been examined extensively. The behavior of some N2-fixing systems under severe environmental conditions such as salt stress, drought stress, acidity, alkalinity, nutrient deficiency, fertilizers, heavy metals, and pesticides is reviewed. These major stress factors suppress the growth and symbiotic characteristics of most rhizobia; however, several strains, distributed among various species of rhizobia, are tolerant to stress effects. Some strains of rhizobia form effective (N2-fixing) symbioses with their host legumes under salt, heat, and acid stresses, and can sometimes do so under the effect of heavy metals. Reclamation and improvement of the fertility of arid lands by application of organic (manure and sewage sludge) and inorganic (synthetic) fertilizers are expensive and can be a source of pollution. The Rhizobium-legume (herb or tree) symbiosis is suggested to be the ideal solution to the improvement of soil fertility and the rehabilitation of arid lands and is an important direction for future research.
THE NITROGEN FIXATION PROCESS
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The element nitrogen, or
"azote," meaning "without life," as Antonie Lavoisier called it
about 200 years ago, has proved to be anything but lifeless, since it
is a component of food, poisons, fertilizers, and explosives
(277). The atmosphere contains about 1015 tonnes
of N2 gas, and the nitrogen cycle involves the
transformation of some 3 × 109 tonnes of
N2 per year on a global basis (244). However,
transformations (e.g., N2 fixation) are not exclusively
biological. Lightning probably accounts for about 10% of the world's
supply of fixed nitrogen (301). The fertilizer industry also
provides very important quantities of chemically fixed nitrogen. World
production of fixed nitrogen from dinitrogen for chemical fertilizer
accounts for about 25% of the Earth's newly fixed N2, and
biological processes account for about 60%. Globally the consumption
of fertilizer-N increased from 8 to 17 kg ha
1 of
agricultural land in the 15-year period from 1973 to 1988 (107). Significant growth in fertilizer-N usage has occurred in both developed and developing countries (238). The
requirements for fertilizer-N are predicted to increase further in the
future (306); however, with the current technology for
fertilizer production and the inefficient methods employed for
fertilizer application, both the economic and ecological costs of
fertilizer usage will eventually become prohibitive.
For more than 100 years, biological nitrogen fixation (BNF) has commanded the attention of scientists concerned with plant mineral nutrition, and it has been exploited extensively in agricultural practice (50, 91). However, its importance as a primary source of N for agriculture has diminished in recent decades as increasing amounts of fertilizer-N have been used for the production of food and cash crops (238). However, international emphasis on environmentally sustainable development with the use of renewable resources is likely to focus attention on the potential role of BNF in supplying N for agriculture (91, 238). The expanded interest in ecology has drawn attention to the fact that BNF is ecologically benign and that its greater exploitation can reduce the use of fossil fuels and can be helpful in reforestation and in restoration of misused lands to productivity (50, 301).
Currently, the subject of BNF is of great practical importance because the use of nitrogenous fertilizers has resulted in unacceptable levels of water pollution (increasing concentrations of toxic nitrates in drinking water supplies) and the eutrophication of lakes and rivers (19, 91, 301). Further, while BNF may be tailored to the needs of the organism, fertilizer is usually applied in a few large doses, up to 50% of which may be leached (301). This not only wastes energy and money but also leads to serious pollution problems, particularly in water supplies.
NITROGEN-FIXING ORGANISMS
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Organisms that can fix nitrogen, i.e., convert the stable nitrogen gas in the atmosphere into a biologically useful form, all belong to a biological group known as prokaryotes. All organisms which reduce dinitrogen to ammonia do so with the aid of an enzyme complex, nitrogenase. The nitrogenase enzymes are irreversibly inactivated by oxygen, and the process of nitrogen fixation uses a large amount of energy (91, 244). Nitrogenase activity is usually measured by the acetylene reduction assay, which is cheap and sensitive (91, 141, 301). The 15N isotopic method, which is also used to measure N2 fixation, is accurate but expensive.
A wide range of organisms have the ability to fix nitrogen. However, only a very small proportion of species are able to do so; about 87 species in 2 genera of archaea, 38 genera of bacteria, and 20 genera of cyanobacteria have been identified as diazotrophs or organisms that can fix nitrogen (91, 301, 361). This wide variety of diazotrophs ensures that most ecological niches will contain one or two representatives and that lost nitrogen can be replenished.
SIGNIFICANCE OF BIOLOGICAL N2
FIXATION TO SOIL FERTILITY
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BNF is an efficient source of nitrogen (238). The total annual terrestrial inputs of N from BNF as given by Burns and Hardy (49) and Paul (235) range from 139 million to 175 million tonnes of N, with symbiotic associations growing in arable land accounting for 25 to 30% (35 million to 44 million tons of N) and permanent pasture accounting for another 30% (45 million tons of N). While the accuracy of these figures may be open to question (301), they do help illustrate the relative importance of BNF in cropping and pasture systems and the magnitude of the task necessary if BNF is to be improved to replace a proportion of the 80 to 90 million tonnes of fertilizer-N expected to be applied annually to agricultural land by the end of the decade (238, 239). Much land has been degraded worldwide, and it is time to stop the destructive uses of land and to institute a serious reversal of land degradation (50). BNF can play a key role in land remediation.
An examination of the history of BNF shows that interest generally has focused on the symbiotic system of leguminous plants and rhizobia, because these associations have the greatest quantitative impact on the nitrogen cycle. A tremendous potential for contribution of fixed nitrogen to soil ecosystems exists among the legumes (46, 238, 313). There are approximately 700 genera and about 13,000 species of legumes, only a portion of which (about 20% [301]) have been examined for nodulation and shown to have the ability to fix N2. Estimates are that the rhizobial symbioses with the somewhat greater than 100 agriculturally important legumes contribute nearly half the annual quantity of BNF entering soil ecosystems (313). Legumes are very important both ecologically and agriculturally because they are responsible for a substantial part of the global flux of nitrogen from atmospheric N2 to fixed forms such as ammonia, nitrate, and organic nitrogen. Whatever the true figure, legume symbioses contribute at least 70 million tonnes of N per year, approximately half deriving from the cool and warm temperature zones and the remainder deriving from the tropics (46). Increased plant protein levels and reduced depletion of soil N reserves are obvious consequences of legume N2 fixation. Deficiency in mineral nitrogen often limits plant growth, and so symbiotic relationships have evolved between plants and a variety of nitrogen-fixing organisms (116).
Most of the attention in this review is directed toward N2 fixation inputs by legumes because of their proven ability to fix N2 and their contribution to integral agricultural production systems in both tropical and temperate climates (238). Successful Rhizobium-legume symbioses will definitely increase the incorporation of BNF into soil ecosystems. Rhizobium-legume symbioses are the primary source of fixed nitrogen in land-based systems (313) and can provide well over half of the biological source of fixed nitrogen (313).
Atmospheric N2 fixed symbiotically by the association
between Rhizobium species and legumes represents a renewable
source of N for agriculture (239). Values estimated for
various legume crops and pasture species are often impressive, commonly
falling in the range of 200 to 300 kg of N ha1
year1 (238). Yield increases of crops planted
after harvesting of legumes are often equivalent to those expected from
application of 30 to 80 kg of fertilizer-N ha1. Inputs of
fixed N for alfalfa, red clover, pea, soybean, cowpea, and vetch were
estimated to be about 65 to 335 kg of N ha1
year1 (313) or 23 to 300 kg of N
ha1 year1 (339). However, the
measured amounts of N fixed by symbiotic systems may differ according
to the method used to study N2 fixation (279).
Inputs into terrestrial ecosystems of BNF from the symbiotic relationship between legumes and their rhizobia amount to at least 70 million tons of N per year (46); this enormous quantity will have to be augmented as the world's population increases and as the
natural resources that supply fertilizer-N diminish. This objective
will be achieved through the development of superior legume varieties,
improvements in agronomic practice, and increased efficiency of the
nitrogen-fixing process itself by better management of the symbiotic
relationship between plants and bacteria.
The symbioses between Rhizobium or Bradyrhizobium
and legumes are a cheaper and usually more effective agronomic practice for ensuring an adequate supply of N for legume-based crop and pasture
production than the application of fertilizer-N. The introduction of
legumes into these pastures is seen as the best strategy to improve
nitrogen nutrition of the grasses. Large contributions (between 75 and
97 kg of N ha1 in 97 days of growth) by
Stylosanthes guianensis were found (333). 15N data suggested that over 30% of the N accumulated by
the grass in mixed swards could be derived from nitrogen fixed by the
associated legume (333). Other recent studies
(199) revealed that the nitrogen contribution of
Arachis hypogaea to the growth of Zea mays in intercropping systems is equivalent to the application of 96 kg of
fertilizer-N ha1 at a ratio of plant population densities
of one maize plant to four groundnut plants.
Actinorhizal interactions (Frankia-nonlegume symbioses) are
major contributors to nitrogen inputs in forests, wetlands, fields, and
disturbed sites of temperate and tropical regions (313). These associations involve more than 160 species of angiosperms classified among six or seven orders. The contributions of fixed nitrogen to native as well as managed ecosystems by the actinorhizal symbioses are comparable to those of the more extensively studied Rhizobium-legume interactions. Typical contributions by
Alnus associations are 12 to 200 kg of N ha1
year1, and those by Hippophae associations are
27 to 179 kg of N ha1 year1
(27).
The above overview clearly indicates the significance of Rhizobium-legume symbioses as a major contributors to natural or biological N2 fixation. Therefore, the following discussion centers on the behavior of these symbioses under severe environmental conditions and also for applications in arid regions.
EFFECTS OF SEVERE CONDITIONS ON NITROGEN FIXATION
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Environmental Conditions
Several environmental conditions are limiting factors to the growth and activity of the N2-fixing plants. A principle of limiting factors states that "the level of crop production can be no higher than that allowed by the maximum limiting factor" (46). In the Rhizobium-legume symbiosis, which is a N2-fixing system, the process of N2 fixation is strongly related to the physiological state of the host plant. Therefore, a competitive and persistent rhizobial strain is not expected to express its full capacity for nitrogen fixation if limiting factors (e.g., salinity, unfavorable soil pH, nutrient deficiency, mineral toxicity, temperature extremes, insufficient or excessive soil moisture, inadequate photosynthesis, plant diseases, and grazing) impose limitations on the vigor of the host legume (46, 239, 315).
Typical environmental stresses faced by the legume nodules and their symbiotic partner (Rhizobium) may include photosynthate deprivation, water stress, salinity, soil nitrate, temperature, heavy metals, and biocides (337). A given stress may also have more than one effect: e.g., salinity may act as a water stress, which affects the photosynthetic rate, or may affect nodule metabolism directly. The most problematic environments for rhizobia are marginal lands with low rainfall, extremes of temperature, acidic soils of low nutrient status, and poor water-holding capacity (44). Populations of Rhizobium and Bradyrhizobium species vary in their tolerance to major environmental factors; consequently, screening for tolerant strains has been pursued (176). Biological processes (e.g., N2 fixation) capable of improving agricultural productivity while minimizing soil loss and ameliorating adverse edaphic conditions are essential.
Salt and Osmotic Stresses
Salinity is a serious threat to agriculture in arid and semiarid regions (252). Nearly 40% of the world's land surface can be categorized as having potential salinity problems (69); most of these areas are confined to the tropics and Mediterranean regions. Increases in the salinity of soils or water supplies used for irrigation result in decreased productivity of most crop plants and lead to marked changes in the growth pattern of plants (69). Increasing salt concentrations may have a detrimental effect on soil microbial populations as a result of direct toxicity as well as through osmotic stress (313). Soil infertility in arid zones is often due to the presence of large quantities of salt, and the introduction of plants capable of surviving under these conditions (salt-tolerant plants) is worth investigating (86). There is currently a need to develop highly salt-tolerant crops to recycle agricultural drainage waters, which are literally rivers of contaminated water that are generated in arid-zone irrigation districts (129). Salt tolerance in plants is a complex phenomenon that involves morphological and developmental changes as well as physiological and biochemical processes. Salinity decreases plant growth and yield, depending upon the plant species, salinity levels, and ionic composition of the salts (86).
As with most cultivated crops, the salinity response of legumes varies greatly and depends on such factors as climatic conditions, soil properties, and the stage of growth (70-72). Variability in salt tolerance among crop legumes has been reported (353, 354). Some legumes, e.g., Vicia faba, Phaseolus vulgaris, and Glycine max, are more salt tolerant than others, e.g., Pisum sativum. It has been reported that some V. faba tolerant lines sustained nitrogen fixation under saline conditions (6, 72). Other legumes, such as Prosopis (105), Acacia (367), and Medicago sativa (7), are salt tolerant, but these legume hosts are less tolerant to salt than are their rhizobia.
The legume-Rhizobium symbioses and nodule formation on legumes are more sensitive to salt or osmotic stress than are the rhizobia (98, 330, 354, 365). Salt stress inhibits the initial steps of Rhizobium-legume symbioses. Soybean root hairs showed little curling or deformation when inoculated with Bradyrhizobium japonicum in the presence of 170 mM NaCl, and nodulation was completely suppressed by 210 mM NaCl (323). Bacterial colonization and root hair curling of V. faba were reduced in the presence of 50 to 100 mM NaCl or 100 to 200 mM polyethylene glycol as osmoticum (352, 365); the proportion of root hairs containing infection threads was reduced by 30 and 52% in the presence of NaCl and polyethylene glycol, respectively. The effects of salt stress on nodulation and nitrogen fixation of legumes have been examined in several studies (6, 9, 86, 98, 159, 223, 330, 352). The reduction of N2-fixing activity by salt stress is usually attributed to a reduction in respiration of the nodules (86, 159, 337) and a reduction in cytosolic protein production, specifically leghemoglobin, by nodules (85, 86). The depressive effect of salt stress on N2 fixation by legumes is directly related to the salt-induced decline in dry weight and N content in the shoot (72). The salt-induced distortions in nodule structure could also be reasons for the decline in the N2 fixation rate by legumes subject to salt stress (302, 352, 360). Reduction in photosynthetic activity might also affect N2 fixation by legumes under salt stress (122).
Although the root nodule-colonizing bacteria of the genera Rhizobium and Bradyrhizobium are more salt tolerant than their legume hosts, they show marked variation in salt tolerance. Growth of a number of rhizobia was inhibited by 100 mM NaCl (350), while some rhizobia, e.g., Rhizobium meliloti, were tolerant to 300 to 700 mM NaCl (99, 146, 214, 272). Strains of Rhizobium leguminosarum have been reported to be tolerant to NaCl concentrations up to 350 mM NaCl in broth culture (5, 45). Soybean and chickpea rhizobia were tolerant to 340 mM NaCl, with fast-growing strains being more tolerant than slow-growing strains (96). Rhizobium strains from Vigna unguiculata were tolerant to NaCl up to 5.5%, which is equivalent to about 450 mM NaCl (216). It has been found recently that the slow-growing peanut rhizobia are less tolerant than fast-growing rhizobia (124). Rhizobia from woody legumes also showed substantial salt tolerance: strains from Acacia, Prosopis, and Leucaena are tolerant to 500 to 850 mM NaCl (188, 364, 367). In addition to NaCl, MgCl and chlorides are more toxic than sulfates (96). It has been reported (167) that the growth of R. meliloti was severely inhibited by Mg2+ ions, whereas Na+ and K+ ions had little inhibitory effect.
Many species of bacteria adapt to saline conditions by the intracellular accumulation of low-molecular-weight organic solutes called osmolytes (77). The accumulation of osmolytes is thought to counteract the dehydration effect of low water activity in the medium but not to interfere with macromolecular structure or function (292). Rhizobia utilize this mechanism of osmotic adaptation (42, 43, 292, 295, 362). In the presence of high levels of salt (up to 300 to 400 mM NaCl), the levels of intracellular free glutamate and/or K+ were greatly increased (sometimes up to sixfold in a few minutes) in cells of R. meliloti (43, 167, 189), R. fredii (118, 119, 350), Sinorhizobium fredii (307), and rhizobia from the woody legume Leucaena leucocephala (349), K+ strictly controls Mg2+ flux during osmotic shock. An osmolyte, N-acetylglutaminyl-glutamine amide, accumulates in cells of R. meliloti (292, 294, 295); the accumulation of these osmolytes is dependent on the level of osmotic stress, the growth phase of the culture, the carbon source, and the presence of osmolytes in the growth medium.
The disaccharide trehalose plays a role in osmoregulation when rhizobia are growing under salt or osmotic stress (96, 151). Trehalose accumulates to higher levels in cells of R. leguminosarum (45) and peanut rhizobia (124) under the increasing osmotic pressure of hypersalinity. Fast-growing peanut rhizobia accumulate trehalose in the presence of many carbon sources (mannitol, sucrose, or lactose), but the slow growers accumulate trehalose only when cultured with mannitol as the carbon source. In a medium supplemented with 400 mM NaCl, the content of trehalose increased intracellularly throughout the logarithmic and stationary phases of growth of peanut rhizobia (123). The disaccharides sucrose and ectoine were used as osmoprotectants for Sinorhizobium meliloti (132). However, these compounds, unlike other bacterial osmoprotectants, do not accumulate as cytosolic osmolytes in salt-stressed S. meliloti cells.
One salt or osmotic stress response already identified in rhizobia is the intracellular accumulation of glycine betaine (189, 272, 293). The concentration of glycine betaine increases more in the salt-tolerant strains of R. meliloti than in sensitive strains (189, 293). The addition of sodium salts to bacteroids of Medicago sativa nodules increased the uptake activity of the exogenously added glycine betaine (113). These osmoprotective substances may play a significant role in the maintenance of nitrogenase activity in bacteroids under salt stress. When externally provided, glycine betaine and choline enhance the growth of Rhizobium tropici, S. meliloti, S. fredii, R. galegae, Mesorhizobium loti, M. huakuii, and Agrobacterium tumefaciens (40). However, the main physiological role of glycine betaine in the family Rhizobiaceae seems to be as an energy source, while its contribution to osmoprotection is restricted to certain strains. Another osmoprotectant, ectoine, was as effective as glycine betaine in improving the growth of R. meliloti under adverse (0.5 M NaCl) osmotic conditions (308). Ectoine does not accumulate intracellularly and therefore would not repress the synthesis of endogenous compatible solutes such as glutamate and trehalose; it may play a key role in triggering the synthesis of endogenous osmolytes (308). Therefore, at least two distinct classes of osmoprotectants exist: those such as glycine betaine or glutamate, which act as genuine osmolytes, and those such as ectoine, which act as chemical mediators.
The content of polyamines, e.g., homospermidine, increases in
salt-tolerant cells and acid-tolerant strains of R. fredii
(118). This polyamine may function to maintain the
intracellular pH and repair the ionic imbalance caused by osmotic
stress. Osmotic stress (shock) results in the formation of specific
proteins in bacteria. Botsford (42) reported that the
production of 41 proteins was increased at least 10-fold in
salt-stressed cells of Escherichia coli. The formation of
osmotic shock proteins was only recently found in cells of rhizobia.
Zahran et al. (364) reported the appearance of new protein
bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) profiles of rhizobia from woody legumes grown under salt
stress. The synthesis pattern of proteins and amino acids (free or
total) changes in cowpea rhizobia after high-salt (10% NaCl = 1.64 M NaCl) stress treatment (362, 365a). The
Na+, K+, and Mg2+ concentrations
are increased in cells of cowpea Rhizobium under salt
stress. These organic osmolytes (amino acids) and the inorganic minerals (cations) may play a role in osmoregulation for this Rhizobium strain. Zahran et al. (362) extended
their work on this halotolerant strain of cowpea Rhizobium
and examined its cell morphology and ultrastructure under salt stress
(1.64 M NaCl). The rhizobial cells responded to high-salt stress by
changing their morphology: the cells appeared as spiral or
filament-like structures, and the cell size greatly expanded. The cell
ultrastructure was severely affected, the cell envelope was distorted,
and the homogeneous cytoplasm was disrupted. It has been reported
(51) that cells of a strain of R. meliloti
appeared with irregular morphology at potentials below 
0.5 MPa.
Strains of rhizobia from different species modified their morphology
under salt stress, and rhizobia with altered morphology have been
isolated from salt-affected soils in Egypt (363). High
osmotic stress (0.2 to 1.44 MPa) modified the synthesis pattern of
extracellular and capsular polysaccharides of R. leguminosarum bv. trifolii (45). The colonies of
R. meliloti EFB1 grown in the presence of 0.3 M NaCl show a
decrease in mucoidy, and in salt-supplemented liquid medium this
organism produces a 40% lower level of exopolysaccharides
(193). The synthesis pattern in SDS-PAGE of
lipopolysaccharides (LPS) from various species of rhizobia from
cultivated legumes (355) and from woody legumes
(364) was modified by salt, in the presence of which the
length of side chains increased. Changing the surface antigenic polysaccharide and LPS, by salt stress, might impair the
Rhizobium-legume interaction. LPS are very important
for the development of root nodules (38, 312).
Successful Rhizobium-legume symbioses under salt stress require the selection of salt-tolerant rhizobia from those indigenous to saline soils (354). Rhizobium strains isolated from salt-affected soils in Egypt failed to nodulate their legume host under saline and nonsaline conditions (359a). These rhizobia showed alterations in their protein and LPS patterns (355). The genetic structure of these bacteria may also be changed (356) since they showed little DNA-DNA hybridization to reference rhizobia. The Rhizobium strains that are best able to form effective symbiosis with their host legumes at high salinity levels are not necessarily derived from saline soils (305). Graham (133) reported that salt-tolerant strains of rhizobia represent only a small percentage of all strains isolated and identified; therefore, further research in selecting salt-tolerant and effective strains of rhizobia is strongly recommended. In fact, and as indicated in recent reports, some strains of salt-tolerant rhizobia are able to establish effective symbiosis, while others formed ineffective symbiosis. Isolates of R. leguminosarum from the lentil-growing regions of the Southern Nile Valley of Egypt were salt tolerant but were not effective in N2 fixation (212). Mutant strains of R. leguminosarum bv. viciae, which grow at 200 mM NaCl, formed ineffective nodules on roots of V. faba. These nodules failed to express nitrogenase activity (63). Some strains of Rhizobium tolerated extremely high levels of salt (up to 1.88 M NaCl) but showed significantly decreased symbiotic efficiency under salt stress (223).
Inoculation of legumes by salt-tolerant strains of R. leguminosarum bv. trifolii and R. meliloti enhanced nodulation and N content under salt stress up to 1% NaCl (95). Salt-tolerant strains isolated from Acacia redolens, growing in saline areas of Australia, produced effective nodules on both A. redolens and A. cyclops grown in sand at salinity levels up to 80 mM NaCl (75). The growth, nodulation, and N2 fixation (N content) of Acacia ampliceps, inoculated with salt-tolerant Rhizobium strains in sand culture, were resistant to salt levels up to 200 mM NaCl (370). Under saline conditions, the salt-tolerant strains of Rhizobium sp. formed more effective N2-fixing symbiosis with soybean than did the salt-sensitive strains (97). An important result was obtained from the recent work of Lal and Khanna (188), who showed that the rhizobia isolated from Acacia nilotica in different agroclimatic zones, which were tolerant to 850 mM NaCl, formed effective N2-fixing nodules on Acacia trees grown at 150 mM NaCl. It was concluded from these results that salt-tolerant strains of Rhizobium can nodulate legumes and form effective N2-fixing symbioses in soils with moderate salinity. Therefore, inoculation of various legumes with salt-tolerant strains of rhizobia will improve N2 fixation in saline environments (370). However, tolerance of the legume host to salt is the most important factor in determining the success of compatible Rhizobium strains to form successful symbiosis under conditions of high soil salinity (75). Evidence presented in the literature suggests a need to select plant genotypes that are tolerant to salt stress and then match them with the salt-tolerant and effective strain of rhizobia (70, 329). In fact, the best results for symbiotic N2 fixation under salt stress are obtained if both symbiotic partners and all the different steps in their interaction (nodule formation, activity, etc.) resist such stress (34, 122, 364).
The use of actinorhizal associations to improve N2 fixation in saline environments was also studied but not as extensively as Rhizobium-legume associations. One of these actinorhizal associations (Frankia-Casuarina) is known to operate in dry climates and saline lands and was reported to be tolerant to salt up to 250 to 500 mM NaCl (67, 94). Casuarina obesa plants are highly salt tolerant (254), but growth under saline conditions depends on the effectiveness of symbiotic N2 fixation. Successful plantings of Casuarina in saline environments require the selection of salt-tolerant Frankia strains to form effective N2-fixing association.
Soil Moisture Deficiency
The occurrence of rhizobial populations in desert soils and the effective nodulation of legumes growing therein (164, 165, 336) emphasize the fact that rhizobia can exist in soils with limiting moisture levels; however, population densities tend to be lowest under the most desiccated conditions and to increase as the moisture stress is relieved (313). It is well known that some free-living rhizobia (saprophytic) are capable of survival under drought stress or low water potential (117). A strain of Prosopis (mesquite) rhizobia isolated from the desert soil survived in desert soil for 1 month, whereas a commercial strain was unable to survive under these conditions (284). The survival of a strain of Bradyrhizobium from Cajanus in a sandy loam soil was very poor; this strain did not persist to the next cropping season, when the moisture content was about 2.0 to 15.5%. The survival and activity of microorganisms may depend on their distribution among microhabitats and changes in soil moisture (231). The distribution of R. leguminosarum in a loamy sand and silt loam soil was influenced by the initial moisture content (245). Moderate moisture tension slowed the movement of R. trifolii (139); the migration of bacteria ceased when water-filled pores in soil became discontinuous as a result of water stress. The migration of strains of B. japonicum from the initial point of placement was found to be very limited (335); the effective strains migrated into the soil to a greater extent than the ineffective strains did.
One of the immediate responses of rhizobia to water stress (low water potential) concerns the morphological changes. Mesquite Rhizobium (284) and R. meliloti (51) showed irregular morphology at low water potential. The modification of rhizobial cells by water stress will eventually lead to a reduction in infection and nodulation of legumes. Low water content in soil was suggested to be involved in the lack of success of soybean inoculation in soils with a high indigenous population of R. japonicum (156). Further, a reduction in the soil moisture from 5.5 to 3.5% significantly decreased the number of infection threads formed inside root hairs and completely inhibited the nodulation of T. subterraneum (345). Similarly, water deficit, simulated with polyethylene glycol, significantly reduced infection thread formation and nodulation of Vicia faba plants (352, 365). A favorable rhizosphere environment is vital to legume-Rhizobium interaction; however, the magnitude of the stress effects and the rate of inhibition of the symbiosis usually depend on the phase of growth and development, as well as the severity of the stress. For example, mild water stress reduces only the number of nodules formed on roots of soybean, while moderate and severe water stress reduces both the number and size of nodules (342).
Symbiotic N2 fixation of legumes is also highly sensitive to soil water deficiency. A number of temperate and tropical legumes, e.g., Medicago sativa (7, 21), Pisum sativum (5), Arachis hypogaea (286), Vicia faba (5, 138, 365), Glycine max (89, 179, 251), Vigna sp. (233, 331), Aeschynomene (15), and the shrub legume Adenocarpus decorticand (215) exhibit a reduction in nitrogen fixation when subject to soil moisture deficit. Soil moisture deficiency has a pronounced effect on N2 fixation because nodule initiation, growth, and activity are all more sensitive to water stress than are general root and shoot metabolism (14, 365). The response of nodulation and N2 fixation to water stress depends on the growth stage of the plants. It was found that water stress imposed during vegetative growth was more detrimental to nodulation and nitrogen fixation than that imposed during the reproduction stage (236). There was little chance for recovery from water stress in the reproductive stage. Nodule P concentrations and P use efficiency declined linearly with soil and root water content during the harvest period of soybean-Bradyrhizobium symbiosis (115). More recently, Sellstedt et al. (279) found that N derived from N2 fixation was decreased by about 26% as a result of water deficiency when measured by the acetylene reduction assay.
The wide range of moisture levels characteristic of ecosystems where legumes have been shown to fix nitrogen suggests that rhizobial strains with different sensitivity to soil moisture can be selected. Laboratory studies have shown that sensitivity to moisture stress varies for a variety of rhizobial strains, e.g., R. leguminosarum bv. trifolii (117), R. meliloti (51), cowpea rhizobia (232), and B. japonicum (196). Thus, we can reasonably assume that rhizobial strains can be selected with moisture stress tolerance within the range of their legume host. Optimization of soil moisture for growth of the host plant, which is generally more sensitive to moisture stress than bacteria, results in maximal development of fixed-nitrogen inputs into the soil system by the Rhizobium-legume symbiosis (313).
Drought-tolerant, N2-fixing legumes can be selected, although the majority of legumes are sensitive to drought stress. Moisture stress had little or no effect on N2 fixation by some forage crop legumes, e.g., M. sativa (175), grain legumes, e.g., groundnut (Arachis hypogaea) (331), and some tropical legumes, e.g., Desmodium intortum (13). One legume, guar (Cyamopsis tetragonoloba), is drought tolerant and is known to be adapted to the conditions prevailing in arid regions (332). Variability in nitrogen fixation under drought stress was found among genotypes of Vigna radiata (248) and Trifolium repens (262). These results assume a significant role of N2-fixing Rhizobium-legume symbioses in the improvement of soil fertility in arid and semiarid habitats.
Several mechanisms have been suggested to explain the varied physiological responses of several legumes to water stress. The legumes with a high tolerance to water stress usually exhibit osmotic adjustment; this adjustment is partly accounted for by changing cell turgor and by accumulation of some osmotically active solutes (112). The accumulation of specific organic solutes (osmotica) is a characteristic response of plants subject to prolonged severe water stress. One of these solutes is proline, which accumulates in different legumes, e.g., Glycine max (120) and Phaseolus vulgaris (172). In these plants, positive correlations were found between proline accumulation and drought tolerance. Other compounds, e.g., the free amino acids and low-molecular-weight solutes such as pinitol (o-methylinositol), accumulate in several tropical legumes under drought stress (112, 185). Potassium is known to improve the resistance of plants to environmental stress. A recent report (269) indicates that K can apparently alleviate the effects of water shortage on symbiotic N2 fixation of V. faba and P. vulgaris. The presence of 0.8 or 0.3 mM K+ allowed nodulation and subsequent nitrogen fixation of V. faba and P. vulgaris under a high-water regimen (field capacity to 25% depletion). It was also shown that the symbiotic system in these legumes is less tolerant to limiting K supply than are the plants themselves. Species of legumes vary in the type and quantity of the organic solutes which accumulate intracellularly in leguminous plants under water stress. This could be a criterion for selecting drought-tolerant legume-Rhizobium symbioses that are able to adapt to arid climates.
High Temperature and Heat Stress
High soil temperatures in tropical and subtropical areas are a major problem for biological nitrogen fixation of legume crops (210). High root temperatures strongly affect bacterial infection and N2 fixation in several legume species, including soybean (218), guar (22), peanut (180), cowpea (249), and beans (154, 241). Critical temperatures for N2 fixation are 30°C for clover and pea and range between 35 and 40°C for soybean, guar, peanut, and cowpea (210). Nodule functioning in common beans (Phaseolus spp.) is optimal between 25 and 30°C and is hampered by root temperatures between 30 and 33°C (241). Nodulation and symbiotic nitrogen fixation depend on the nodulating strain in addition to the plant cultivar (22, 218). Temperature affects root hair infection, bacteroid differentiation, nodule structure, and the functioning of the legume root nodule (265, 266).
High (not extreme) soil temperatures will delay nodulation or restrict it to the subsurface region (133). Munns et al. (221) found that alfalfa plants grown in desert environments in California maintained few nodules in the top 5 cm of soil but were extensively nodulated below this depth. Nodulation of soybean was markedly inhibited at 42 and 45°C during 12-h and 9-h days, respectively (186), with no correlation between the ability of plant strains to grow at high temperature and to induce nodulation under temperature stress. Piha and Munns (241) reported that bean nodules formed at 35°C were small and had low specific nitrogenase activity, and Hernandez-Armenta et al. (148) found that transferring nodulated bean plants from a daily temperature of 26 to 35°C markedly inhibited nitrogen fixation. Some soybean varieties appear somewhat more heat tolerant, with nitrogen fixation being severely inhibited only by daytime temperatures higher than 41°C (187). The acetylene reduction activity of nodulated roots excised from unstressed bean plants (Phaseolus) was strongly diminished at 35 or 40°C when plants were nodulated by heat-sensitive or heat-tolerant strains (210).
For most rhizobia, the optimum temperature range for growth in culture is 28 to 31°C, and many are unable to grow at 37°C (133). However, 90% of cowpea Rhizobium strains obtained from the hot, dry environment of the Sahel Savannah grew well at 40°C (93). Strain adaptation to high temperature has also been reported by Hartel and Alexander (144) and Karanja and Wood (173). The latter authors found that a high percentage of the strains that persisted at 45°C lost their infectiveness. They attributed these losses in infectiveness to plasmid curing. Heat treatment of R. phaseoli at 35 and 37°C resulted in mutant strains lacking a plasmid DNA implicated in the synthesis of melanin and is related to the loss of symbiotic properties of these bacteria (36). Screening of R. leguminosarum bv. phaseoli showed that some strains were able to nodulate Phaseolus vulgaris at high temperatures (35 and 38°C) but that the nodules formed at high temperatures were ineffective and plants did not accumulate N in shoots (154).
Rhizobial survival in soil exposed to high temperature is greater in soil aggregates than in nonaggregated soil and is favored by dry rather than moist conditions (133). Ten inoculant strains of Rhizobium spp. examined by Somasegaran et al. (297) showed a gradual decline in population during 8 weeks of incubation at 37°C, while exposure to 46°C was lethal to all strains in less than 2 weeks. A decrease in the infectivity of cowpea rhizobia following storage at 35°C has also been documented (343). High soil temperature could contribute to the frequency of noninfective isolates in soil; Segovia et al. (278) noted that such noninfective isolates actually outnumbered those that were infective in the rhizosphere of bean. R. leguminosarum isolates from lentil plants in the Southern Nile Valley of Egypt were tolerant to 35 to 40°C; however, these heat-tolerant rhizobia formed less effective symbiosis with their legume hosts (212). Several heat-tolerant N2-fixing bean-nodulating Rhizobium strains (which grow at 40°C) have been described recently (155, 210).
Heat shock proteins have been found in Rhizobium (1) but have not been studied in detail (133). The synthesis of heat shock proteins was detected in both heat-tolerant and heat-sensitive bean-nodulating Rhizobium strains (210) at different temperatures. An increased synthesis of 14 heat shock proteins in heat-sensitive strains and of 6 heat shock proteins in heat-tolerant strains was observed at 40 and 45°C, respectively (210). Heat-tolerant rhizobia are likely to be found in environments affected by temperature stress. Rhizobia isolated from the root nodules of Acacia senegal and Prosopis chilensis, growing in hot, dry regions of Sudan, had high maximum growth temperatures (44.2°C) (364, 367). Heat stress (35 and 40°C) changed the pattern of LPS mobility of some strains of tree rhizobia, as shown by SDS-PAGE (364). The same authors found that temperature stress consistently promoted the production of a protein with a relative mobility of 65 kDa in four strains of tree legume rhizobia. The 65-kDa protein that was detected under heat stress was heavily overproduced. This protein was not overproduced during salt or osmotic stress (364), which indicates that it is a specific response to heat stress.
Soil Acidity and Alkalinity
Soil acidity is a significant problem facing agricultural production in many areas of the world and limits legume productivity (41, 65, 73, 133). Most leguminous plants require a neutral or slightly acidic soil for growth, especially when they depend on symbiotic N2 fixation (41, 47). It has been recently reported (207, 309) that pasture and grain legumes acidify soil to a greater extent and that the legume species differ in their capacity to produce acids. Legumes and their rhizobia exhibit varied responses to acidity. Some species, like lucerne (M. sativa), are extremely sensitive to acidity, while others, such as Lotus tenuis, tolerate relatively low soil pH (73). Soil acidity constrains symbiotic N2 fixation in both tropical and temperate soils (220), limiting Rhizobium survival and persistence in soils and reducing nodulation (47, 136, 157). Rhizobia with a higher tolerance to acidity have been identified (136). These strains usually but not always perform better under acidic soil conditions in the field (134). It has been found that R. loti multiplied at pH 4.5 but Bradyrhizobium strains failed to multiply (68); the acid-tolerant strains of R. loti demonstrate a comparative advantage over acid-sensitive strains in the ability to nodulate their host legume at pH 4.5. R. tropici and R. loti are moderately acid tolerant (344), while R. meliloti is very sensitive to acid stress (47, 318). However, R. meliloti WSM 419 has recently been shown (65) to perform satisfactorily in the field in acidic soils (pH 5.0 to 5.5). Strains of a given species vary widely in certain cases in their pH tolerance. The fast-growing strains of rhizobia have generally been considered less tolerant to acid pH than have slowly growing strains of Bradyrhizobium (134), although some strains of the fast-growing rhizobia, e.g., R. loti and R. tropici, are highly acid tolerant (68, 78, 134, 344). Recent reports, however, support the existence of acid-tolerant fast-growing strains, since both fast- and slow-growing strains of Vigna unguiculata which are tolerant to pH values as low as 4.0 have been isolated (216). The basis for differences in pH tolerance among strains of Rhizobium and Bradyrhizobium is still not clear (73, 134), although several workers have shown that the cytoplasmic pH of acid-tolerant strains is less strongly affected by external acidity (60, 62, 131, 230). Aarons and Graham (1) reported high cytoplasmic potassium and glutamate levels in acid-stressed cells of R. leguminosarum bv. phaseoli, a response which is similar to that found in osmotically stressed cells. Differences in LPS composition, proton exclusion and extrusion (60, 62), accumulation of cellular polyamines (118), and synthesis of acid shock proteins (150) have been associated with the growth of cells at acid pH. The composition and structure of the outer membrane could also be a factor in pH tolerance (134). Studies on the genetic basis of tolerance to low pH suggest that at least two loci of either megaplasmid or chromosomal location for pH genes are necessary for the growth of rhizobia at low pH (60-62). Acid tolerance in R. loti (73) was related to the composition and structure of the membrane, and acid-tolerant strains showed one membrane protein of 49.5 kDa and three soluble proteins of 66.0, 85.0, and 44.0 kDa. The expression of these proteins increased when the cells were grown at pH 4.0. The same authors (73) suggested that acid tolerance in R. loti involves constitutive mechanisms, such as permeability of the outer membrane, together with adaptive responses, including the state of bacterial growth and concomitant changes in protein expression.
The failure of legumes to nodulate under acid-soil conditions is common, especially in soils of pH less than 5.0. The inability of some rhizobia to persist under such conditions is one cause of nodulation failure (30, 55, 136), but poor nodulation can occur even where a viable Rhizobium population can be demonstrated (133, 134). Evans et al. (104) found that nodulation of P. sativum was 10 times more susceptible to acidity than was either rhizobial multiplication or plant growth. Some legumes, e.g., Trifolium subterranean, T. balansae, Medicago murex, and M. truncatula, showed tolerance to soil acidity as indicated by dry-matter yield; however, the establishment of nodules was more sensitive to soil acidity in most of these plants than was indicated by the relative yields of dry matter (102). Despite this, elevated inoculation levels have enhanced the nodulation response under acidic conditions in some studies (243). The growth, nodulation, and yield of V. faba were improved after inoculation with strains of R. leguminosarum bv. viciae in acid soils (55). It appears that the pH-sensitive stage in nodulation occurs early in the infection process and that Rhizobium attachment to root hairs is one of the stages affected by acidic conditions in soils (54, 326). Taylor et al. (314) reported that acidity had more severe effects on rhizobial multiplication than did Al stress and low P conditions. They suggested that colonization of soils and soybean roots by B. japonicum may be adversely affected by acidity, an effect which will result in reduced nodulation.
The host cultivar-rhizobial strain interaction at acid pH has also been investigated. Munns et al. (221) noted that nodulation and nitrogen fixation by some strains of Bradyrhizobium at acidic pH differ with the cultivar of mung bean used. Vargas and Graham (327) examined the cultivar and pH effects on competition for nodule sites between isolates of Rhizobium in beans (P. vulgaris) under acidic conditions. They found a significant effect of host cultivar, ratio of inoculation, and pH on the percentage of nodule occupancy by each strain. However, it has been suggested (326) that only one of the symbionts needed to be acid tolerant for good nodulation to be achieved at pH 4.5. Inoculation of Medicago polymorpha by an acid-tolerant R. meliloti strain has extended the area of acidic soils in Western Australia that can be sown with annual legumes to some 350,000 ha (153). The performance of the R. trifolii-Trifolium pratense symbiosis under acidic conditions is best when the rhizobial strains were isolated from the most acidic soils, i.e., acid-tolerant strains (191). Rhizobia appear to be vary in their symbiotic efficiency under acidic conditions. Van Rossum et al. (325) compared 12 strains of Bradyrhizobium for their symbiotic performance with groundnut in acidic soils and found that some strains were totally ineffective under acidic stress (pH 5.0 to 6.5) while others performed well under these conditions. Acid-tolerant alfalfa-nodulating strains of rhizobia, isolated from acidic soils, were able to grow at pH 5.0 and formed nodules in alfalfa with a low rate of nitrogen fixation (87). The results also demonstrate the complexity of the rhizobial populations present in the acidic soils, represented by a major group of nitrogen-fixing rhizobia and a second group of ineffective and less predominant isolates.
The host legume appears to be the limiting factor for establishing Rhizobium-legume symbiosis under acidic conditions. Legume species differ greatly in their response to low pH with regard to growth and nodulation (311). Recently, it has been found that the amount of N2 fixed by forage legumes on low-fertility acidic soil is dependent on legume growth and persistence (316). However, selection of acid-tolerant rhizobia to inoculate legume hosts under acidic conditions will ensure the establishment of the symbiosis and also successful performance (73, 128, 229).
Recent reports indicated the destructive effects of acidic soils on Rhizobium-legume symbiosis and N2 fixation. Low pH reduced the number of R. leguminosarum bv. trifolii cells in soils, which resulted in no or ineffective nodulation by clover plants (157). The number of nodules, the nitrogenase activity, the nodule ultrastructure, and the fresh and dry weights of nodules were affected to a greater extent at a low medium pH (<4.5) (328).
In acidic soils with pH of >5.0, where heavy-metal activity is relevant, the presence of available aluminum inhibits nodulation (35, 41). Rhizobia showed varied responses to aluminum toxicity in acidic soils and cultures. Strains of Rhizobium (326, 344) and Bradyrhizobium (133) that were resistant to aluminum (50 µM) at low pH (>5.0) were identified; however, rhizobia from clover were sensitive to these conditions (344). Johnson and Wood (168) reported that Al was taken up and bound to the DNA of both sensitive and tolerant strains but that DNA synthesis by the tolerant strains of R. loti was not affected. However, Richardson et al. (261) found that 7.5 µM Al depressed nod gene expression at low pH (4.8).
Legume species vary markedly in their tolerance to Al3+ and Mn2+, with some plants being significantly more strongly affected by these ions than are the rhizobia (133). Therefore, for acid soils with high Al content, improvement is achieved by manipulating the plant rather than the rhizobia (314). Nodulation of legumes appears more sensitive to Al than does plant growth (133); at pH 4.5 and with 0.5 mM Ca2+, nodulation in cowpea was delayed by 12.7 µM Al and nodule number and dry weight were severely depressed (20). Availability of Ca2+ in acidic soils with a high Al content appears very important for nodulation; a low Ca2+ concentration (0.13 mM) at pH 4.5 greatly affected nodule number, nitrogenase activity, and nodule ultrastructure of the common bean, Phaseolus (328).
Two strategies have been adopted to solve the problem of soil acidity: (i) selecting tolerant plants, and (ii) liming the acidic soil to ameliorate the effects of acidic conditions. Few cultivated legumes are adapted to low pH levels. The primary protective mechanism of acid tolerance in certain cultivars of lentil (Lens culinaris) is excess production of citric, malic, aspartic, glucenic, and succinic acids in root exudate under acidic conditions (247). It has been recently reported that some pasture legumes acidified soils to a greater extent and that the amount of acid produced per gram of shoot dry matter (specific acid production) varied between species and with growth stages, ranging from 44 to 128 cmol/kg of dry shoot matter (309). Similarly, some grain legumes produced large amounts of acids (207), with the production of H+ ranging between 77 and 136 cmol/kg of dry matter. It has been suggested that Al-tolerant (acid-tolerant) plant species contain and exude more organic acid and other ligands that form stable chelates with Al and thereby reduce its chemical activity and toxicity (114).
In recent studies, trials were performed to study the effects of
treating soil acidity by applying lime (at rate of 2,500 kg
ha1) and superphosphate (at rates up to 20 kg
ha1) to acidic soils (239). The amelioration
increased the soil pH from 4.5 to 4.9, decreased the concentration of
extractable Al and Mn, and improved growth and N2 fixation
of T. subterranean. Amelioration of subsoil acidity was also
done by applying coal-derived calcium fulvate (324), and
this treatment increased the pH more than did amelioration by gypsum,
Ca-EDTA, Ca (OH)2, or CaCO3. Previous reports
also indicate the importance of liming for improvement of growth and
nodulation of legumes in acidic soils, since they indicated that liming
raised the pH from 5 to 6.5 and increased the percentage of nodule
occupancy of T. subterraneum (17). However,
amelioration by lime and other substances, e.g., carbonate, must be
optimized to avoid increasing the pH to a level which would be
inhibitory to growth and symbiotic performance of legumes. Applied
carbonate was found to react with Na and raise the pH (41).
Addition of bicarbonate decreased nodulation, growth, and shoot
nitrogen in some grain legumes (311). Nodulation inhibition in Lupinus angustifolius grown in a limed sand at a pH of
>7.0 has also been reported. Nodulation of groundnut (Arachis
hypogaea) was also inhibited when plants grew in nutrient solution
containing carbonate (309). High pH (>6.0 and up to 10.0)
totally inhibited the nodulation of some lupins (310), and
the authors suggested that pH values above 6.0 have a specific effect
in the impairment of nodulation of lupins. However, rhizobia appear to
be more tolerant to alkalinity than do their legume hosts. The number
of R. leguminosarum bv. trifolii cells was greater in
carbonate-treated soil (103); increasing the soil pH
increased both the rate at which rhizobia colonized the soil and the
frequency of nodule formation. It has also been reported
(253) that while germination of pigeon pea was decreased at
pH values of >8.8, growth of rhizobia was unaffected up to pH 11.5. These authors also found that uninoculated pigeon pea plants had as
good a nodulation as did those grown from plant seeds inoculated with
Rhizobium in reclaimed alkaline soils in a greenhouse study.
The tolerance of actinorhizal plants to soil acidity and acidic conditions was also reported. Solution culture studies have shown reduced nodulation of black alder (Alnus glutinosa) and other actinorhizal plants at low pH. The effect of soil acidity on nodulation of A. glutinosa grown in mine soils, limed to various pH values, was also studied (137). The authors found that soil pH was a significant factor affecting nodulation in the mine soil, with the highest level of nodulation occurring between soil pH values of 5.5 and 7.2 and the level being reduced below pH 5.5. There was also evidence of decreased viability of the endophyte (Frankia) below pH 4.5 (137). In a recent study, Igual et al. (158) reported a decrease in nodulation of Casuarina cunninghamiana at high levels of Al, with the nitrogen-fixing efficiency being decreased from 0.20 to 0.10 mg of N fixed per mg (dry weight) of nodule at 880 µM Al3+. They found that the mean N concentration of nodules was significantly lower at pH 4.0 (1.83%) than at pH 6.0 (2.01%).
Nutrient Deficiency Stress
Soil salinity and acidity are usually accompanied by mineral
toxicity (specific ion toxicity), nutrient deficiency, and nutrient disorder. Salt damage to nonhalophytic plants grown in nutrient solution is often due to the effect of ion imbalance (disorder) rather
than the osmotic potential (347). This disorder might occur
by specific toxicity of ions such as Na+ and
Cl and might be balanced by increasing the concentration
of counterions, like K+ and Ca2+, against
Cl (127). It has been suggested that
K+ and NO3 inhibited
Na+ and Cl translocation from the roots to
the shoots of Arachis hypogaea, so that leaf growth was
protected against salt damage (285). The dominant ions in
saline waters and saline soils which are available in arid zones are
Na+ and Cl. Excess Na+ often
harms nonhalophytes by displacing Ca2+ from root membranes
and thus changing their integrity and their normal functioning
(76). Also, acidic stress markedly affects ion absorption by
and growth of roots (320); the membrane structure and
function of the roots suffer fatal changes under these stress conditions. The requirement of some essential elements, e.g., Ca2+ and P, is increased under severe stress conditions.
The requirement of Ca2+ for growth of R. meliloti was increased under osmotic stress (51). The
Ca-depleted cells of R. leguminosarum are swollen, lack
rigidity, and express an additional somatic antigen normally blocked by
side chains of the LPS O antigen (88). High levels of
salinity (up to 10% NaCl) decreased the Ca2+ content of
Rhizobium cells (362), and the outer membrane
structure of the Rhizobium cells was greatly distorted. In
the same way, calcium appears significantly more important in cells
exposed to low pH (133). O'Hara et al. (230)
found that in acid-sensitive strains of R. meliloti, 1.2 mM
Ca2+ was needed for cytoplasmic pH maintenance, and Beck
and Munns (32) noted that phosphorus-limited cells or cells
grown at low pH needed Ca2+ for phosphorus mobilization in
the cell. Lack of Ca2+ produced some changes in ion
transport, which are caused mostly by changes in membrane properties
(66), and Ca2+ plays an essential role in cell
division, elongation, and membrane structure and function
(320). At low pH, addition of Ca2+ to the
incubation medium improves both growth and ion uptake by roots
(320); it was also suggested that Ca2+ offset
the harmful effects of ions such as K+ and H+
and of stress. Ca2+ seems to have two effects on K
transport, (i) control of K+ permeability and (ii)
activation of K+ uptake through the acidification of the
cytoplasm. Calcium-dependent cell surface components affect the
attachment of Rhizobium to root hair cells (54,
293). It has been found (365) that salt stress (100 mM
NaCl) reduced the attachment to and colonization of root hairs of
V. faba plants by R. leguminosarum; this was attributed to the effect of salt on calcium availability. The effects
of salt stress or acidity on calcium availability and on the initial
stages of nodule formation will affect the net nodulating capacity of
legumes. Both pH (4.5) and aluminum (100 µM) caused delays in
nodulation of Vigna unguiculata, particularly at low
Ca2+ levels (0.3 mM), while at a high calcium concentration
(3.0 mM), nodulation was improved (152). The critical
Ca2+ level for nodule formation in pigeon pea and guar is
more than 50 µM, whereas peanut and cowpea nodulated very well in
solution culture with 2 µM calcium (35). Nodulation and
nodule development in cowpea were strongly depressed at low pH (4.5 to
5.5) and low calcium concentration (0.05 to 2.5 mM) (20).
Stress conditions may inhibit nodulation of legumes through the
inhibition of genetic activity. It has been reported that
Ca2+ (10 mM) increased nod gene induction and
expression activities of clover plants 5- to 10-fold at pH 4.5 to 5.2 (261).
Phosphorus is one of several elements which affects N2 fixation, and, along with N, it is a principal yield-limiting nutrient in many regions of the world (240). Strains of rhizobia differ markedly in tolerance to phosphorus deficiency (33). Rhizobial P deficiency when there is a P deficiency in the soil and rhizosphere is a real possibility, especially under acidic conditions, where dissolved phosphorus salts may be precipitated in the presence of aluminum (133). Slow-growing strains of rhizobia appear more tolerant to low P levels than do fast-growing R. meliloti, in particular (33); this bacterium failed to grow at 0.06 µM P, regardless of the Ca2+ concentration, and some strains needed high Ca2+ levels to grow at 0.5 and 5.0 µM P. Phosphate-limited cultures of both fast- and slow-growing rhizobia do take up phosphate 10- to 180-fold faster than cells grown with adequate P (291), and inducible alkaline phosphatase activity was detected in P-limited cells of fast-growing R. trifolii strains (290, 291). Recently, it has been reported (18) that free-living R. tropici and bacteroids respond to P stress by increasing their P transport capacity and inducing both acid and alkali phosphatases. This P stress response occurred when the medium P concentration decreased below 1 µM. Leguminous species differ in their phosphorus requirement for growth from 0.8 to 3 µM (110).
Phosphorus appears essential for both nodulation and N2 fixation (240, 303). Nodules are strong sinks for P and range in P content from 0.72 to 1.2% (142, 143); as a consequence, N2 fixation-dependent plants will require more of this element than those supplied with combined nitrogen (56, 57). Nodulation, N2 fixation, and specific nodule activity are directly related to the P supply (163, 190, 288). Application of KH2PO4 (25 mg of P per kg of soil) to acidic soils significantly increased the percent nodule occupancy of Trifolium subterranean by R. leguminosarum bv. trifolii (17). The nodulation and N2 fixation (nitrogenase activity) of T. vesiculosum increased significantly after the addition of P (100 mg per kg of soil) and K (300 mg per kg of soil); nitrogenase activity was doubled when the P concentration increased to 400 mg per kg of soil (194). The interaction of P and Zn and their effects on nodulation of legumes under salt stress were studied. Saxena and Rewari (273) found that application of phosphate (20 and 40 ppm) improved the growth and nodulation of chickpea (C. arietinum) in the presence of Zn2+ (5 ppm) at two levels (4.34 and 8.3 dS/m) of salinity. They suggested that augmentation with Zn2+ provided protection to the plant under saline conditions by reducing the Na+/K+ ratio in the shoot; the shoot N content after augmentation with Zn2+ and in the presence of phosphate was equal to that of nonsaline control. Differences between cultivars of some legume species with regard to phosphorus requirements have been reported (48). Variability of N2 fixation under low P availability existed between lines of P. vulgaris; high N2-fixing and high-yielding progeny lines were detected (240).
Mycorrhizal infection of roots of legumes has been reported to stimulate both nodulation and N2 fixation, especially in soils low in available P (121, 257). The role of mycorrhizal fungi in the protection of the Medicago sativa-R. meliloti symbiosis against salt stress was recently studied (26), and it was found that the interaction between soluble P in the soil mycorrhizal inoculum and the degree of salinity in relation to concentration and nodule formation increased with the amount of plant-available P or mycorrhizal inoculum in the soil and generally declined as the salinity in the solution culture increased. Azcon and Elatrash (26) found that the mycorrhizal inoculation protected plants from salt stress more efficiently than did any amount of plant-available P in the soil, particularly at the highest salinity level applied (43.5 dS/m).
Nitrogen fixation by the Frankia-actinorhizal symbiosis may be limited by low available P in soils. Sangina et al. (270) observed increased N2 fixation by Casuarina equisetifolia by adding phosphate to P-deficient soil, and Reddell et al. (255) found a greater increase in the yield (wood volume) of Frankia-inoculated Casuarina cunninghamiana by adding phosphate to soil. Low P status is a frequent limitation to nodulation of actinorhizal plants. It has been reported that symbiotic N2 fixation of the Frankia-Casuarina association requires higher P levels than those required for plant growth, when the P concentration in soil is low (270). Genetic variations among species of Allocasuarina in relation to P requirement were identified; species showed different nodulation abilities in soils with low available P (270).
Soil Amendments and Ameliorations
Sewage sludge treatment and organic fertilizers. Sewage sludge application to agricultural soils is an economical way of disposal (109, 202, 228). It improves the physical characteristics of the soil (340) and increases organic matter content and essential plant nutrients, in particular N and P (109). Sewage sludge contains numerous components required for microbial growth and may increase the activity of soil microorganisms (299), including rhizobial growth (177). Contaminants associated with certain fertilizers such as sewage sludge may also negatively affect the survival of various soil microorganisms (202). Concern about the use of sewage sludge contaminated by heavy metals has increased. Heavy metals are known to persist in the soil over long periods and have ecotoxicological effects on plants and soil microorganisms (100). There is increasing evidence of adverse effects on microbial processes related to nutrient cycling in these types of soils (228).
Sewage sludge may contain a variety of materials potentially toxic to rhizobia, such as soluble salts (195) and heavy metals (126, 206). Despite the presence of metal-impacted agricultural soils, there have been few studies of metal resistance in rhizobia. A decline in rhizobial populations (e.g., R. japonicum) in higher-sludge soils (5 parts soils to 1 part sludge) may be due to the presence of heavy metals which are available during the mineralization of sludge in soils (256). Kinkel et al. (177) examined two genera of soybean-nodulating rhizobia to determine the level of resistance to eight different metals. Marked variations were found with several heavy metals, even for rhizobial strains belonging to the same species. Relatively large numbers of R. leguminosarum bv. trifolii were found in soils treated with organic (e.g., sewage sludge) and inorganic fertilizers; however, these numbers were related to soil pH, and all isolates were equally effective (202). Tong and Sadowsky (319) reported that Bradyrhizobium strains were more resistant to heavy metals than were Rhizobium strains. However, this observation is not always correct, since it has been found that resistance to tellurite, selenite, and selenate was observed only in R. meliloti and R. fredii strains but not Bradyrhizobium strains (178). More recently, it has been reported (16) that the growth of some bacteria, e.g., R. leguminosarum and Agrobacter tumefaciens, was affected by copper treatment. The cells of these bacteria remain viable but nonculturable. However, some heavy metals, e.g., nickel, are essential elements in several biological processes, including H2 oxidation in some bacteria and urea hydrolysis by plants. Klucas et al. (181) found that the addition of Ni (5 to 8 µM) to both the nitrate-grown and symbiotically grown soybean plants resulted in a 7- to 10-fold increase in urease activity in leaves and significantly increased the hydrogenase activity in isolated nodule bacteroids. They also found that free-living R. japonicum, cultured under chemolithotrophic conditions, required Ni for growth and for the expression of hydrogenase activity. Therefore, Ni is an important micronutrient element in the biology of the soybean plant and R. japonicum. The survival of B. japonicum in sludge-amended soils was also studied (195) and the soluble salts of the sludge (not the heavy metals) were shown to be primarily responsible for a short-term reduction in bradyrhizobial populations following sludge application to soil. Adverse effects of heavy metals on nodulation and N2 fixation of legumes have been reported for clover (206, 264) and chickpea (348). Giller et al. (126) suggested two possibilities to explain the mechanism by which the elevated metal concentrations eliminated N2 fixation: (i) one or more of the metals present might have prevented the formation of N2-fixing nodules by effective Rhizobium strains present in the soil or (ii) the metal contamination might have resulted in elimination of the effective Rhizobium strains from the soil. Inoculation of white clover plants grown in metal-contaminated soil with an effective strain of R. leguminosarum bv. trifolii promoted N2 fixation, but this did not occur when inoculation was carried out 2 months before sowing, unless a very large inoculation (1010 cells per g of dry soil) was used (126). It was also found that the plasmid profiles of these isolates were all very similar, indicating a lack of genetic diversity in the population surviving at high concentrations of heavy metals. These strains were all ineffective in N2 fixation. These authors concluded that white clover rhizobia are unable to survive (or at least unable to remain infective) in the presence of concentrations of heavy metals close to the current Commission of the European Communities guidelines for environmental protection. The survival and the number of effective strains of R. leguminosarum bv. trifolii in soils amended with anaerobically digested and undigested sewage sludge (at rates up to 300 m3 ha1
year1) were studied (228).
Rhizobium was found in most of the contaminated soils, apart
from the most contaminated treatment in the soil of lower pH, despite
the absence of the host plant from the field sward. Lack of nodulation
and N2 fixation for Trifolium repens grown in
these soils was indicated. Obbard et al. (228) suggested that important effects on the sizes of effective rhizobial populations were determined by soil pH, sludge type and addition rates, and concentration of heavy metals present. The Rhizobium
symbiosis with T. subterranean was recently studied in soil
fertilized with sewage sludge, lime, and standard mineral (PK)
fertilizer (109). Nodulation was decreased only with the
highest rate (60 tons ha1) of sludge amendments and was
greater after standard mineral fertilization than after sewage sludge
and lime amendments. After 1 year, nodulation of plants grown in soils
treated with large quantities of sludge was greater than nodulation of
plants grown in soils amended with mineral fertilizer. Improved
nodulation after sludge treatment in the second year was attributed to
an increase of the rhizobial population and breakdown of soil organic matter. Ferreira and Castro (109) suggested that sewage
sludge be used as organic fertilizer, although the nutrient content and the pH of different types of sludge are different. The effects of heavy
metals from biosolids on the population and N2-fixing potential of R. leguminosarum bv. trifolii, under two pH
regimens, were studied by Ibekwe et al. (157). They found
few significant effects of biosolid-borne heavy metals on plants,
N2 fixation, and number of rhizobia at the concentrations
of metals studied, as long as the soil pH was maintained near 6.0. Where reduction in rhizobial number and plant parameters was observed,
the decrease was attributed primarily to low soil pH and, to a lesser
extent, to heavy metal toxicity from biosolids. Smith (296)
reported that strains of R. leguminosarum bv. trifolii,
which are effective in N2 fixation with Trifolium
repens, were present in different soils from long-term sewage
sludge-treated sites. The rhizobia survived in soil and formed an
effective symbiosis with the host plant where the metal concentration
in soil increased to 300 mg of Cu kg of soil1 and 2,000 mg of Zn kg of soil1. This work demonstrated that
nodulation and N2 fixation by white clover occur in
sludge-treated soils containing more than the current United Kingdom
maximum permissible concentrations of heavy metals. It appears that
some Rhizobium-legume associations are able to cope with a
high content of heavy metals in soils treated with sludge or organic
fertilizers; however, knowledge of the chemical and physical properties
of organic fertilizers, e.g., sludge, prior to their application to
soil is of the utmost importance (106). This ensures the
quality and quantity of the required components and that heavy metals
are not above the toxic limit for plants. The characteristics of some
local organic fertilizers (e.g., dried sludge and limed sludge) in
Egypt were recently studied (106). The tested composts
showed high levels of some nutrients (i.e., K, P, B, Fe, Zn, Mn, and
Cu) but these fell within the permissible levels. However, the Ni and
Cd levels were higher than the permissible levels and should be
reduced. Application of organic fertilizers with low concentrations
(within permissible levels) of heavy metals will improve soil fertility
in reclaimed soils. Furthermore, the legume-root nodule symbiosis can
be used as an effective parameter for ecotoxicological evaluation of
contaminated soils (341). Substances affecting the macro-
and/or microsymbiont vitality, such as certain heavy metals or
polycyclic aromatic hydrocarbons (PAHs), reduce nodulation before
visible damage of the plant occurs. Wetzel and Werner (341)
found a dose-responsive decrease in nodulation of alfalfa after
application of CdCl2, NaAsO2, fluoranthene, and
other PAHs, although PAH-contaminated soil (75 mg/kg) caused only a
slight reduction in nodulation of alfalfa.
Fertilizer application.
Most combined N available to
crop legumes is in the form of NO3, formed by
oxidation of NH4 from residual fertilizer and
mineralization of organic N. Nitrate has been recorded in soils at
levels up to 20 mM or 280 ppm (31). Both
NO3 assimilation and N2 fixation
of legumes are strongly dependent on the plant cultivar, bacterial
strain, ontogeny, and environmental factors (e.g., soil
NO3 concentration, carbon and water
availability, and temperature). Invariably, N2-fixing
activity is confined to areas with low NO3
availability (31). NO3 may allow
the plant to conserve its energy, since in overall terms more energy is
required to fix N2 than to utilize
NO3. It is therefore necessary that the plant
be able to detect the presence and level of
NO3 in the rooting medium and to adjust its
N2 fixation accordingly. Symbiotic N2 fixation
in field legumes takes place against a changing background of mineral N
availability as a consequence of mineralization, leaching, and, often,
fertilizer application (81). Uptake of fertilizer N by
plants depends on soil moisture and is higher in normal than in wet
soils (with uptakes of 32 and 27%, respectively) as a result of
different N-leaching losses (224).
ions
(147). It has been established for many years that soil NO3 inhibits root infection (8),
nodule development (25, 160), and nitrogenase activity
(23, 246, 271). Maximum N2 fixation in a legume
requires that the legume be adequately nodulated. Scanty and poorly
distributed nodules on the root system do not usually satisfy the
nitrogen needs of the plant, resulting in a greater reliance on soil N
for growth. Supplemental inoculation to correct initial suboptimal
nodulation has been recommended (64, 80). Nodulation and
N2 fixation of soybean have been improved by using this
approach. Furthermore, Danso et al. (80) found that the
inhibition of soybean N2 fixation at higher N levels (83 mg
of N kg of soil1) was significantly reduced by a second
inoculation; this treatment resulted in at least a doubling of both the
percentage and total amount of N2 fixed by soybean plants
after the single slurry inoculation.
Variation among strains of Rhizobium and
Bradyrhizobium in their tolerance to the inhibitory effects
of combined nitrogen on their population in soil and growth media has
been reported (208, 353). Semu et al. (280)
showed that nitrogen application of up to 200 kg of N ha1
did not change the population size of B. japonicum in a
soybean field. In Rhizobium-legume symbioses, the formation
of nodules is the result of a complex multistep process in which
rhizobial attachment to legume root hairs is an early step and may be
involved in the control of specificity. Combined N is one of the many
environmental factors which limit the development and success of the
Rhizobium-legume symbiosis in nature and can regulate
rhizobial adsorption to host root hairs and root hair infection
(82, 90, 219). It has been found that addition of
NO3 (5 or 16 mM) to the seedling growth
medium significantly decreased the number of rhizobial cells adhering
to lucerne seedling roots. The attachment of R. leguminosarum bv. trifolii and the level of trifoliin A (a lectin)
on the root surface of white-clover seedlings grown with high nitrate
were also decreased (82, 83). It has been found
(283) that excess nitrate did not repress the synthesis of
the lectin (trifoliin A) in the root but did affect the distribution
and activity of this lectin in a way which reduced its ability to
interact with cells of rhizobia. Since it has been suggested
(84) that legume lectins confer specificity in the Rhizobium-legume symbiosis by interacting with the bacterial
symbiont, factors, such as excess nitrate, which affect lectin activity and interaction with rhizobia might inhibit the symbiotic process.
The inhibitory effect of N on nodulation is probably plant mediated;
however, differences in tolerance to nitrate and ammonium have also
been found between rhizobial isolates when investigated in nodulated
systems (225). Gibson and Harper (125) have shown that different strains of B. japonicum have varied tolerance
to external nitrogen application in their nodulation and nitrogen fixation characteristics. Rhizobia showed varied responses to combined
N with regard to competition for nodule occupancy. Variations in the
competitive abilities of three strains of B. japonicum for
nodulation of soybean (G. max) at increasing fertilizer-N concentrations (up to 10 g of N m2) have been
reported (201). It has also been reported that combined N
altered the nodule occupancy of two strains of rhizobia in soybean (208), while nitrogen treatment had no significant effect on nodule occupancy by three strains each of B. japonicum
(nodulating soybean) and R. leguminosarum bv. phaseoli
(2), although nitrogen application reduced the nodule number
and mass of both legumes. Recently, it has been found (282)
that increased soil fertility (soil N) had no effect on nodule
occupancy of chickpea (Cicer arietinum).
Nitrogen fertilization is sometimes needed to achieve a substantial
yield of legumes (e.g., soybean) when the symbiotic N2 fixation is unable to provide enough nitrogen (52). However, fertilizer rates exceeding those exerting a "starter nitrogen" effect generally reduce nodulation and N2 fixation
(11). The response of the Rhizobium-legume
symbiosis to added nitrogen fertilizer is definitely determined by time
of application (growth stage), level and form of N, and the legume
species (8, 160, 182). Nitrates are more inhibitory to
nodulation than is ammonia, especially if added shortly after planting.
Application of fertilizer-N (25 mg of N kg of soil1)
during sowing was less detrimental to N2 fixation by
P. vulgaris than during vegetative growth (217).
Experiments have demonstrated that NO3
inhibits nodule formation on legumes primarily as a root-localized
effect rather than as a function of whole-plant N nutrition (8,
92). When NO3 levels were sufficiently
high to completely suppress nodulation on the original root (primary
root), there were profuse nodulation and significant nitrogenase
activity (C2H2 reduction) on the adventitious roots of soybean (92) and nodules were formed on the lateral roots of V. faba and P. sativum (8).
Application of urea (90 kg of N ha1) to soybean plants
suppressed nodulation by curtailing the enrichment of
Bradyrhizobium spp. on the host plant (315). The
root system of pigeon pea (Cajanus cajan) was poorly
developed after application of fertilizer-N (up to 60 kg of N
ha1), and this also affected other N2-fixing
parameters, e.g., nodule number, nitrogenase activity, nodule dry
weight, shoot weight, and root and shoot nitrogen (174).
However, recent reports suggest a role for the whole process of
metabolism of host legumes and their rhizobia in response to applied
fertilizer-N. The host legume, but not rhizobia, controls the
efficiency of respiration in nodules under normal or stressed
conditions (59). Nitrogenase-linked respiration was markedly
raised by the addition of nitrate, but the host affected mainly the
changes of resistance to O2 diffusion in nodules, and the
presence of combined N depleted the reaction capability of nodules to
adjust to changing O2 levels in the rhizosphere. Salt
stress (150 mM NaCl), nitrate stress (10 mM
NO3), and drought inhibited N2
fixation by soybean through the inhibition of nodule enzymatic activity
(130). These authors proposed that N2 fixation
in soybean nodules is mediated by both the oxygen-diffusion barrier and
the potential to metabolize sucrose via sucrose synthase. The response
to the environmental perturbation may involve down-regulation of the
nodule sucrose synthase gene.
The inhibitory effect of exogenous nitrate on N2 fixation
has variously been attributed to a direct competition between nitrate reductase and nitrogenase for reducing power (304) or to the fact that nitrite (a by-product of nitrate reductase) inhibits the
function of nitrogenase and leghemoglobin (31). The high levels of nitrite and amino acids in nodules of alfalfa plants treated
with nitrate (10 mM KNO3) correlated with reduced
leghemoglobin content and nitrogenase activity (74), but
this does not explain directly the mechanism of suppression of nitrogen
fixation in alfalfa exposed to nitrate. Low levels of nitrite
accumulate in nodules of V. faba plants compared to those in
roots (53); the root enzymes nitrate reductase (NRA) and
nitrite reductase (NiRA) were significantly stimulated by exogenous
nitrate, while negative (NRA) or little effect (NiRA) was found for
nodules. In a more recent study, treatment of soybean plants with
nitrite (10 mM) and salt (150 mM NaCl) did not significantly affect
leghemoglobin levels and other enzyme activities (130). In
addition, it has been found that the decline in total nitrogenase
activity, measured as H2 production in 20%
O2-80% Ar, upon exposure to nitrate was independent of
the N2-fixing efficiency (i.e., the amount of
N2 reduced by nitrogenase) of the symbiosis
(39). In fact, the mechanism of inhibition of N2
fixation (nitrogenase activity) by nitrate and ammonia implies various
factors. A comparison of experiments is hampered by the use of
different concentrations and durations of fertilizer-N treatment and by
the fact that the legumes differ in their sensitivity to
NO3 and NH4 (31).
Increased N input via fixation is self-limiting, since high soil
nitrogen levels inhibit fixation. This limitation could be overcome by
using a legume which will continue to fix N2 in the presence of combined nitrogen. The potential for breeding genotypes of
legumes with improved nodulation and N2 fixation when grown with combined nitrogen was examined. Mytton and Rys (222)
and Rys and Mytton (268) demonstrated the existence of
heritable genetic factors controlling nodulation and N2
fixation under sodium nitrate treatment (up to 22.8 mM). Herridge and
Betts (149) selected 4 genotypes of soybean from 32 previously tested for tolerance of 2.5 mM
NO3. The four genotypes showed the highest
levels of symbiotic activity when inoculated with B. japonicum and sown into a soil which contained a high level of
nitrate (260 kg of N ha1 at depths of 0 to 120 cm) and
which was free of soybean rhizobia. Abdel-Wahab and Abd-Alla
(4) provided evidence for nodulation and growth variability
of soybean cultivars fertilized with different levels of N (up to about
120 kg of N ha1). Several soybean mutants
(101) and a supernodulating genotype of soybean
(298) displayed a nitrate-tolerant symbiosis in the presence
of fertilizer-N at 40 and 180 kg of N ha1. Genotypes of
P. vulgaris, which form a successful symbiosis with
Rhizobium in the presence of fertilizer-N (at 12.5 mg of N
kg of soil1) in saline-sodic soil, were identified
(234). Supernodulating genotypes of pea (227) and
cowpea (287) with better nodulation and N2
fixation efficiency under fertilizer-N treatment (20 mM NO3 and 120 kg of N ha1,
respectively) were also identified. Different species of legumes showed
different responses to the same treatment of fertilizer-N; application
of 200 kg of N ha1 decreased N2 fixation only
by 18% in groundnut but by 54% in cowpea (351).
In general, high soil N levels, applied or residual, reduce nodulation
and N2 fixation. To improve BNF by the legumes under such
circumstances, the soil N concentration must be managed through inclusion of appropriate nitrate-tolerant high-N2-fixing
legume crops or the genotype of a given crop (339). Analysis
of the different contributions to BNF by legumes discussed in this
review suggests that there is a potential to select appropriate legume crops or cultivars of a given legume for specific areas with high soil
N contents without decreasing their BNF contribution to the system.
Pesticide application.
The use of a vast array of
pesticides to overcome the economic losses in agriculture exerts varied
environmental stresses on nontarget organisms present in the soil. The
maximum benefits of BNF may not be achieved if other constraints are
placed on the system. One of the most important and potentially
limiting factors to BNF is the use of herbicides, fungicides, and other pesticides. Herbicides applied to leguminous crops constitute a
potential hazard to the establishment and performance of the N2-fixing root nodules. It has been reported that foliar
application of the herbicides bentazone and MCPA
(4-chloro-2-methylphenoxyacetic acid) to red clover (Trifolium
pratense) at the recommended rates altered the morphology of root
hairs and reduced nodule numbers and nitrogenase activity
(192). Application of the herbicide dinoseb
(2-sec-butyl-4,6-dintrophenol) to red clover in the field reduced the levels of nitrogenase activity of the plants
(191). Rhizobium strains isolated from root
nodules of red clover plants treated with dinoseb showed resistance to
the herbicide when grown in laboratory media containing dinoseb;
however, these strains did not improve the symbiotic performance of red
clover treated with dinoseb at 200 µg ml1 (the
recommended rate). Lindström et al. (191) suggested
that foliar application of dinoseb did not act directly on the nodules but affected nitrogen fixation by damaging the photosynthetic system of
the plant. Reduction in nodulation and N2 fixation of a
legume, e.g., V. faba, was found only in cases where
herbicide injury to the plant was evident (37). Herbicides
have been reported to affect B. japonicum growth in vitro
and to reduce the nodulation of soybeans under greenhouse conditions
(197, 198). Five herbicides tested under Canadian field
conditions showed no effect on soybean nodulation and nitrogen fixation
(258). The herbicides sethoxydim, alachlor, fluazifop butyl,
and metalachlor did not have detrimental effects on N2
fixation or seed yields when added at the recommended rates for weed
control in soybean plantations (183). However, paraquat
significantly reduced the amount of N2 fixed by soybean as
measured by the 15N dilution method. Similarly, herbicides
were reported to induce reduction in nodulation and N2
fixation of soybean (162) and bean (276). In most
published studies, herbicides varied in the extent of their effect on
N2 fixation. Consequently, the effect of herbicides on
N2 fixation in soybeans must eventually be determined under
field conditions, and if the results are to be relevant, the effect
must be studied in the area where they are to be used (184).
Fungicides are also dangerous to the Rhizobium-legume symbiosis. Field rates and higher rates of captan reduced nodulation of
and N2 fixation by T. repens (111).
Thiram and captan are harmful to nodulation and N2 fixation
of several grain and forage legumes (135, 145, 259).
NITROGEN FIXATION IN ARID REGIONS
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Arid Regions and Arid Climates
About one-third of the land area of the world comprises arid and semiarid climates (169). Arid desert soils were previously considered economically unimportant; however, during the past three decades, the economic and agricultural utilization of arid lands has emerged as a critical element in maintaining and improving the world's food supply (289). Arid lands in Egypt represent about 97% of the total area of the country. Egypt occupies the northeastern part of the African continent, and its total area is a little more than 106 km2. The whole country forms part of the great desert belt that stretches from the Atlantic across the whole of North Africa through Arabia (366). An extremely arid climate prevails in Egypt; the high temperature, low relative humidity, high evaporation, and scanty rainfall (1.4 to 5.3 mm/year) all contribute to the fact that the greater part of Egypt is barren and desolate desert (3, 366). The desert lands also include saline areas; saline lands represent about 15% of the arid and semiarid lands of the world (281, 358). In saline areas, evaporation greatly exceeds precipitation, and there may be salination to a sufficient degree to eliminate most plants from these habitats (29, 358). Saline lands, like arid lands, have been largely ignored and are usually considered to be abandoned, nonproductive lands. Desert ecosystems are characterized by a lack of moisture and nitrogen, but drought and salt stresses are probably the most important environmental factors that inhibit the growth of organisms in arid and semiarid regions.
Improving the Fertility of Arid Regions
Plant productivity in many arid regions is often limited by low soil fertility; therefore, the nutrient content of the soil should be considered in conjunction with the amount of moisture when selecting plants for deserts (3, 300). One of the suggested treatments for building up the fertility of desert soils in Egypt is by application of a mixture of Nile sediments (clay) and organic manure; however, this suggestion was considered a nonpractical solution to improve the fertility of desert soils (3), since the huge amount of sediments required for the amelioration of large areas is not available.
Amelioration of low-fertility soils by application of organic fertilizers (e.g., sewage sludge and animal organic manures) is a strategy that has been adopted in many countries in recent years. Recent reports have emphasized the significance of this treatment in improving the physical characteristics of soil and supplying soil with the nutrients required for growth of different organisms. It is also a less expensive means of soil amendment than fertilizer-N (106, 109, 202, 228). Organic fertilizers are rich in several valuable nutrients, but they also contain high levels of toxic heavy metals. Recent reports, however, indicate that heavy metals are probably not the only factors which harm the activity of soil organisms (e.g., plants and microorganisms) but that the presence of soluble salts and the reduction in pH as a result of high levels of ammonia could also suppress the activity of soil organisms (157, 195, 296). In contrast to previous reports, recent reports demonstrate the tolerance of some N2-fixing bacteria to conditions created in soils after the application of sewage sludge. Some strains of R. leguminosarum bv. trifolii survived and formed an effective symbiosis with clover cultivated in soil treated with sewage sludge containing levels of heavy metals above the maximum permissible concentrations (296). Therefore, we suggest that organic fertilizers be used to ameliorate the newly reclaimed arid lands. Organic fertilizers with low concentrations of heavy metals, containing essential nutrients, and slightly acidic (pH less than 6.0) are recommended. Rhizobium-legume symbioses, which are tolerant to moderate levels of acidity, can be grown in these ameliorated soils.
Application of mineral fertilizers, e.g., fertilizer-N, is a common
practice to improve soil fertility in developed and developing countries; however, there is increasing concern about serious pollution
of drinking water by fertilizers such as nitrates. However, we suggest
that fertilizer-N be used to increase fertility in N-poor soils or
desert lands in order to achieve substantial yield of legumes when
symbiotic N2 fixation is unable to provide enough N for
maximum yield. The application of fertilizer-N, however, requires the
selection of N-tolerant legumes. The negative effects of nitrate and
other combined nitrogen sources on the symbiosis of
Rhizobium and legumes are well documented and are discussed above in detail. Variations between strains of rhizobia from the same
species in the presence of high concentrations of nitrate were
demonstrated, and high concentrations (up to 200 kg of N ha1) of fertilizer-N had no significant effects on the
population size of rhizobia. Recent reports discussed in this review
demonstrate the potential of breeding genotypes of legumes, e.g. pea,
bean, and soybean, with improved nodulation and N2 fixation
in the presence of combined nitrogen (4, 227, 298, 339). The
application of fertilizer-N in arid regions, however, should be
optimized to reduce its marginal effects. The inclusion of appropriate
nitrate-tolerant N2-fixing legumes will maximize the yield
of crops and also reduce N losses through leaching.
Biological N2 Fixation in Arid Regions
BNF is the major way to introduce N into desert ecosystems. BNF in deserts is mediated mainly by some heterotrophic bacteria, associative bacteria, cyanobacteria, actinorhizal plants, and legumes. The N2 fixed by heterotrophic free-living bacteria is of minor importance as a mechanism for N input in arid soils (3, 361). Associative dinitrogen-fixing bacteria may be potentially important in supplying relatively small amounts of N to associated grasses in arid lands (346). Bacteria that associate with the roots of C4 grasses perhaps are the most potentially important in arid climates (300) since such grasses are known to have high levels of water use efficiency. The total amount of N fixed by cyanobacteria may be small; nevertheless, they could contribute to the fertility of arid lands, which are N-limited environments (300). The actinorhizal plants, e.g., the Frankia-Casuarina symbiosis, grow very well in hot, dry, and saline environments and fix appreciable amount of N. There are recent reports of desiccation tolerance in Frankia spp.; these species also survive and form effective symbiosis at high temperature and low soil moisture (274, 275). A new Frankia-Atriplex symbiosis was recently discovered (58) (Atriplex is a perennial forage shrub in dry and salt-affected soil in Argentina); this symbiosis is an N2-fixing mechanism. Further research is needed for an exact quantification of the contribution of actinorhizal plants to BNF in arid regions. Rhizobium-legume symbioses represent the major mechanism of BNF in arid lands. This point is discussed in detail in the following sections; however, emphasis is placed on the naturally occurring herb or woody legumes in deserts.
Rhizobium-Legume Symbioses and Rehabilitation of Arid Regions
Drought-tolerant Rhizobium-legume symbiosis. Improved cultivars of plants for arid lands must have drought resistance mechanisms to enable them to grow and survive in areas with low moisture availability. In fact, Rhizobium-legume symbioses are currently the most important nitrogen-fixing systems, which may have the potential to increase N input in arid lands. The leguminous plants include species or varieties which are extremely well adapted to the drastic conditions of arid lands. Examples are Medicago sativa, Arachis hypogaea, Cyamposis tetragonoloba, and Melilotus spp.; these legumes are known to be adapted to conditions prevailing in arid regions. In addition, a drought-tolerant cultivar of Phaseolus vulgaris has recently been identified (250). The dry weight of this legume was not affected by water stress (50 and 30% of field capacity), although the number and weight of nodules as well as N2 fixation (acetylene reduction) were obviously reduced. However, these legume species require drought-tolerant rhizobia to form effective symbiosis under arid climates. Rhizobia with survival ability, which showed effective symbiotic characteristics with their host legumes (e.g., Prosopis rhizobia) in desert soils and arid regions, were identified (117, 164, 165). Effective rhizobia are competitive and able to migrate under conditions of scarce moisture (335). In a recent work, Athar and Johnson (24) reported that two mutant strains of R. meliloti were competitive with naturalized alfalfa rhizobia and were symbiotically effective under drought stress. These results suggest that nodulation, growth, and N2 fixation in alfalfa can be improved by inoculating plants with competitive and drought-tolerant rhizobia. This could be an economically feasible way to increase alfalfa (M. sativa) production in water-limited environments.
The N2-fixing legume-Rhizobium symbioses that would be selected are those that grow rapidly when temperature and moisture conditions are favorable (169). These associations could be either annuals or perennials. Peoples et al. (237) reported that perennial pastures containing lucerne (M. sativa) provide consistently greater annual herbage production and fix an average of 90 to 150% as much N2 as do neighboring subterranean clover-based pastures. In the 1994 drought (in Australia), when annual pastures failed, lucerne still managed to fix >70 kg of N ha1. It is proposed that lucerne-based pastures could
represent a more reliable means of improving soil fertility than annual
pastures. A positive correlation was found between proline accumulation and drought tolerance of legumes (112, 120, 172). In a
recent study, Straub reported that under mild water stress, soybean
plants inoculated with bacteria that were unable to catabolize proline suffered twice the percent decrease in seed yield as did plants inoculated with bacteria that were able to catabolize proline. These
results suggest that increasing the oxidative flux of proline in
bacteroids might provide an agronomically significant yield advantage
when the stress is modest. Various criteria which influence the
competition and saprophytic competence of rhizobia under environmental factors such as moisture deficiency should be considered. These criteria were recently reviewed (334) and include rhizobial
movement in the soil, inoculum placement, legume root exudates
precluding the early stages of nodulation, and competition for nodule induction.
Naturally occurring forage legumes (annuals and perennials) are well
nodulated, and their root nodules are active in fixing N2
(3, 359). These legumes may be found in desert or in
cultivated lands as wild plants. Recently, the suitability of
rhizobium-inoculated wild herb legumes for providing vegetation cover
and improving soil fertility in unreclaimed lands was suggested
(166). Isolation of effective rhizobia from wild legumes to
inoculate other legume crops is a new strategy to improve the
efficiency of the Rhizobium-legume symbiosis. The rhizobia
of wild legumes may have better traits than the homologous rhizobia.
Rhizobium strains from Astragalus cicer
successfully nodulate M. sativa and P. vulgaris
(368). Rhizobia isolated from the wild plants of northern
deserts and cultivated lands of Egypt (365a) formed
effective nodules on V. faba and P. sativum.
One of the adaptations of legumes to arid lands (poor in N and P) and
those with low moisture availability is their infection by mycorrhizal
fungi in addition to Rhizobium. Mycorrhizal inoculation has
alleviated the effects of drought stress on Acacia and
Leucaena under arid conditions (263). Recent
reports support the ability of mycorrhizal infection to improve
N2 fixation by legumes under drought stress (257,
260). Rhizobium bacteria, arbuscular mycorrhiza, and
plant-growth-promoting bacteria were isolated from a representative area of a desertified semiarid ecosystem in the south-east of Spain
(260). Phosphate-solubilizing bacteria could release
phosphate ions from soil in association with mycorrhiza
(321). The biodegradation of phosphonomycin (an antibiotic)
by Rhizobium huakuii was also reported (205).
This bacterium was able to degrade up to 10 mM phosphonomycin as a
carbon, energy, and phosphorus source and to release inorganic
phosphate (Pi). High temperatures may prevail in arid
regions; therefore, the drought-tolerant symbiotic systems that are
selected should also be heat tolerant. High soil temperature (35 to
40°C) usually results in the formation of ineffective nodules; however, several strains of rhizobia, e.g., R. leguminosarum
bv. phaseoli, were recently reported to be heat tolerant and to form an
effective symbiosis with their host legumes (155, 210).
These associations will be relevant for cultivation in arid climates.
Salt-tolerant Rhizobium-legume symbiosis. An allied concern with regard to soil moisture in the reclamation of environmentally impacted sites, e.g., arid lands, can be the effect of salinity on the survival of rhizobia in soil systems. Salinity affects the survival and distribution of rhizobia in soil and the rhizospheres of plants (165, 313); however, salt-tolerant rhizobia were isolated from various crop and wild legumes (see above). Some of these rhizobia are tolerant to higher levels of salts, up to 1.8 M NaCl. These salt-tolerant rhizobia underwent morphological and metabolic changes, as well as structural modifications, to cope with and adapt to salt stress. However, effective salt-tolerant rhizobia were isolated from nonsaline as well as saline environments. Recent reports support the finding that some rhizobia have the potential to form a successful symbiosis with legumes under salt stress. Most of the work done emphasizes the previous understanding about the sensitivity of the host legumes to salt stress. Therefore, salt-tolerant legume genotypes should be selected. In the last few years, rhizobia which form successful N2-fixing Rhizobium-legume symbioses under salt stress (up to 120 to 150 mM NaCl) have been selected. Examples are strains of R. meliloti and R. leguminosarum bv. trifolii from forage legumes (95), soybean (97) and woody legumes (75, 188, 370). In a recent study reported by Mashhady et al. (203), R. meliloti formed a successful symbiosis with Medicago sativa under saline conditions (100 mM NaCl). These rhizobia are local strains isolated from Saudi Arabian soil in arid lands. Also, recent reports point out that rhizobia from naturally growing tree legumes in the deserts are prominent and effective salt-tolerant rhizobia.
Significance of woody (tree)-legume-Rhizobium symbioses to the rehabilitation of arid regions. The use of leguminous trees for a variety of food, feed, and fuel wood purposes in semiarid regions has been reviewed (105, 108). Trees of the genera Acacia and Prosopis are of central importance in the rural economy of many of the world's arid and semiarid areas. Species of both genera provide high-quality animal fodder, timber, fuel wood, charcoal, gums, and other products, as well as contributing to soil stabilization and improvement through N2 fixation (105). Their particular value in arid zones lies in their extreme resistance to heat, drought, salinity, and alkalinity; they are better able to establish growth in disturbed areas of arid regions than are herb legumes (242). Tree legumes are widely distributed (Acacia, in particular, is native to every continent), they are widespread in Africa and the Middle East (209), and many are valuable for fixing atmospheric N2. It has been reported that woody legumes, e.g., Prosopis, are well nodulated under drought stress; however, the value of many shrubby and woody legumes in arid areas probably lies in their extensive, deep root systems, in addition to their potential to fix N2 (164, 302, 357). Prosopis forms a unique system of deep roots with significant tolerance to water stress (226).
Evaluation of the nodulating ability, N2 fixation, and agroforestry potential of woody legumes has been the subject of many recent reports (170, 204, 317, 369). These recent studies prove that a high percentage of the trees examined form effective nodules. The N2 fixed (measured as a percentage of the total nitrogen) in the tree legumes Leucaena, Albizia and Cliricidia ranged from about 20 to 74, 28 to 72, and 44 to 84%, respectively (170). In one of the most recent and interesting studies, Dakora and Keya (79) determined the contribution of some tree legumes to soil fertility. Tree legumes fix about 43 to 581 kg of N ha1, compared to about 15 to 210 kg of N
ha1 for grain legumes (79). This high
N2-fixing potential makes leaf pruning of these tree
legumes an important component of sustainability in agroforestry and
soil fertility. It was estimated that in a single year, the pruning of
a tree or shrub like Sesbania sesban can provide up to 1 ha
of cereal crop, up to 448 kg of N, 31.4 kg of P, 125 kg of K, 114 kg of
Ca, and 27.3 kg of Mg, thus making the foliage of this legume the
"ideal" fertilizer. In arid regions, where soil moisture and low
fertility often limit yields, research on neglected symbiotic native
tree legumes with nitrate and drought-tolerant traits would constitute
a sound basis for increased sustainable production in arid regions.
Tree legumes are nodulated by a diverse group of rhizobia
(Rhizobium and Bradyrhizobium). Some isolates of
these rhizobia have been phenotypically and genotypically characterized
(358, 364, 367); however, the potential of these root nodule
bacteria as symbionts with tree legumes has not yet been explored.
Woody-legume rhizobia, e.g., Prosopis rhizobia, were
isolated from the surface layers of desert soils which are usually
exposed to fluctuating environmental conditions, e.g., soil moisture,
temperature, and salinity (164, 165). Under these
conditions, fast-growing rhizobia are more relevant than slow-growing
ones. One interesting approach is the usage of rhizobia from woody
legumes as an inoculum for crop legumes. Wange (338)
succeeded in obtaining effective symbioses between woody rhizobia from
Acacia and other trees with peanut and cowpea. This
symbiosis was more effective than the symbiosis between the trees and
their homologous rhizobia. This new approach is the focus of interest
of several nitrogen fixation laboratories, including ours.
CONCLUSIONS
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This review recognizes the role of BNF as a nonpolluting and more cost-effective way to improve soil fertility compared to other ways, such as fertilizer-N and sewage sludge, with their high levels of toxic metals. The Rhizobium-legume symbiosis is superior to other N2-fixing systems with respect to N2 fixing potential and adaptation to severe conditions. Several symbiotic systems of legumes which are tolerant to extreme conditions of salinity, alkalinity, acidity, drought, fertilizer, metal toxicity, etc., were identified. These associations might have sufficient traits necessary to establish successful growth and N2 fixation under the conditions prevailing in arid regions. In fact, the existence of Rhizobium-tree legume symbioses, which are able to fix appreciable amount of N2 under arid conditions, is fascinating. These symbioses represent the best source of the "ideal" fertilizer in arid regions and therefore command great interest as the subject of future research.
FOOTNOTES
* Mailing address: Department of Botany, Faculty of Science, Beni-Suef, 62511 Egypt. Phone: 02082-324897/316933. Fax: 02082-324897/314384.
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