Policing the legume-Rhizobium symbiosis: a critical test of partner choice
Rhizobium are a group of Gram-negative soil bacteria that are well known for their symbiotic relationship with various leguminous (soybeans, alfalfa etc). The relationships between Rhizobium fredii and the rhizobia that nodulate Galega oflcinalis and Galega new genus of legume root nodule bacteria, Bradyrhizobium advice on phage-typing techniques, and the Applied Biochemistry. The interaction between the legume plants and rhizobial bacteria is very the host-nonhost relationship between legumes and rhizobia.
But nutrition stress especially phosphorus, potassium, zinc, iron, molybdenum, and cobalt can be corrected with fertilizers. When a nutritional stress is corrected, the legume responds directly to the nutrient, and indirectly to the increased nitrogen nutrition resulting from enhanced nitrogen fixation.
Poor nitrogen fixation in the field can be easily corrected by inoculation, fertilization, irrigation, or other management practices. Common beans are poor fixers less than 50 lbs per acre and fix less than their nitrogen needs.
Maximum economic yield for beans in New Mexico requires an additional lbs of fertilizer nitrogen per acre. However, if beans are not nodulated, yields often remain low, regardless of the amount of nitrogen applied.
Nodules apparently help the plant use fertilizer nitrogen efficiently. Other grain legumes such as peanuts, cowpeas, soybeans, and faba beans are good nitrogen fixers, and will fix all of their nitrogen needs other than that absorbed from the soil. These legumes may fix up to lbs of nitrogen per acre and are not usually fertilized. In fact, they usually don't respond to nitrogen fertilizer as long as they are capable of fixing nitrogen. Nitrogen fertilizer is applied at planting to these legumes when grown on sandy or low organic matter soils to supply nitrogen to the plant before nitrogen fixation starts.
If nitrogen is applied, the rate is low, lbs per acre. When large amounts of nitrogen are applied, the plant literally slows or shuts down the nitrogen fixation process. It is easier and less energy consuming for the plant to absorb nitrogen from the soil than to fix it from the air.
Perennial and forage legumes such as alfalfa, sweetclover, true clovers, and vetches may fix lbs of nitrogen per acre. Like the grain legumes previously discussed, they are not normally fertilized with nitrogen. They occasionally respond to nitrogen fertilizer at planting or immediately after a cutting when the photosynthate supply is too low for adequate nitrogen fixation. Almost all of the nitrogen fixed goes directly into the plant. Little leaks into the soil for a neighboring non-legume plant.
However, nitrogen eventually returns to the soil for a neighboring plant when vegetation roots, leaves, fruits of the legume die and decompose. When the grain from a grain legume crop is harvested, little nitrogen is returned for the following crop.
Most of the nitrogen fixed during the season is removed from the field. The stalks, leaves, and roots of grain legumes such as soybeans and beans contain about the same concentration of nitrogen as found in non-legume crop residue. In fact, the residue from a corn crop contains more nitrogen than the residue from a bean crop, simply because the corn crop has more residue.
Nitrogen Fixation by Legumes
A perennial or forage legume crop only adds significant nitrogen for the following crop if the entire biomass stems, leaves, roots is incorporated into the soil. If a forage is cut and removed from the field, most of the nitrogen fixed by the forage is removed. Roots and crowns add little soil nitrogen, compared to the above ground biomass.
However, a grower can make some field observations that can help indicate if nitrogen fixation is adequate in some of the common legumes. If a newly planted field is light green and slow growing, suspect insufficient nitrogen fixation. This is often seen with beans and alfalfa. In a new field, the poor fixation is often attributed to the lack of native Rhizobium to nodulate the legume, but the cause may also be poor plant nutrition or other plant stresses that inhibit nitrogen fixation.
Small nodules should be present weeks after germination. If no nodules are present, consider the following options. Replant using seed inoculated with the correct Rhizobium. Try to inoculate the plants in the field through the irrigation system or by other means.
Consider nitrogen fertilization to meet all of the plant's nitrogen needs. This may not be an option for a perennial legume such as alfalfa if the field is kept in alfalfa for several years. Also, some legumes use soil or fertilizer nitrogen more efficiently if nodules are present. If young nodules are present, sufficient soil nitrogen may not be available for the young plant before nitrogen fixation starts. The plant usually grows out of this condition, or a small amount of nitrogen can be applied.
Also, inefficient native Rhizobium may result in poor nitrogen fixation. Consider other soil stresses that may be inhibiting plant growth, especially plant nutrition and water stress.
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.
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 A given stress may also have more than one effect: 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 Populations of Rhizobium and Bradyrhizobium species vary in their tolerance to major environmental factors; consequently, screening for tolerant strains has been pursued Salt and Osmotic Stresses Salinity is a serious threat to agriculture in arid and semiarid 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 Increasing salt concentrations may have a detrimental effect on soil microbial populations as a result of direct toxicity as well as through osmotic stress 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 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 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 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 — Variability in salt tolerance among crop legumes has been reported It has been reported that some V.
Other legumes, such as ProsopisAcaciaand Medicago sativa 7are 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, 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 mM NaCl, and nodulation was completely suppressed by mM NaCl Bacterial colonization and root hair curling of V. The effects of salt stress on nodulation and nitrogen fixation of legumes have been examined in several studies 698698, The reduction of N2-fixing activity by salt stress is usually attributed to a reduction in respiration of the nodules 86, and a reduction in cytosolic protein production, specifically leghemoglobin, by nodules 85 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 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, Reduction in photosynthetic activity might also affect N2 fixation by legumes under salt stress 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 mM NaClwhile some rhizobia, e. Strains of Rhizobium leguminosarum have been reported to be tolerant to NaCl concentrations up to mM NaCl in broth culture 5 Soybean and chickpea rhizobia were tolerant to mM NaCl, with fast-growing strains being more tolerant than slow-growing strains Rhizobium strains from Vigna unguiculata were tolerant to NaCl up to 5. It has been found recently that the slow-growing peanut rhizobia are less tolerant than fast-growing rhizobia Rhizobia from woody legumes also showed substantial salt tolerance: In addition to NaCl, MgCl and chlorides are more toxic than sulfates It has been reported that the growth of R.
Nitrogen Fixation by Legumes
Many species of bacteria adapt to saline conditions by the intracellular accumulation of low-molecular-weight organic solutes called osmolytes 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 Rhizobia utilize this mechanism of osmotic adaptation 4243, An osmolyte, N-acetylglutaminyl-glutamine amide, accumulates in cells of R.
The disaccharide trehalose plays a role in osmoregulation when rhizobia are growing under salt or osmotic stress 96 Trehalose accumulates to higher levels in cells of R. Fast-growing peanut rhizobia accumulate trehalose in the presence of many carbon sources mannitol, sucrose, or lactosebut the slow growers accumulate trehalose only when cultured with mannitol as the carbon source. In a medium supplemented with mM NaCl, the content of trehalose increased intracellularly throughout the logarithmic and stationary phases of growth of peanut rhizobia The disaccharides sucrose and ectoine were used as osmoprotectants for Sinorhizobium meliloti However, these compounds, unlike other bacterial osmoprotectants, do not accumulate as cytosolic osmolytes in salt-stressed S.
One salt or osmotic stress response already identified in rhizobia is the intracellular accumulation of glycine betaine, The concentration of glycine betaine increases more in the salt-tolerant strains of R.
The addition of sodium salts to bacteroids of Medicago sativa nodules increased the uptake activity of the exogenously added glycine betaine 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. 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. 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 Therefore, at least two distinct classes of osmoprotectants exist: The content of polyamines, e.
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 fold in salt-stressed cells of Escherichia coli. The formation of osmotic shock proteins was only recently found in cells of rhizobia.
These organic osmolytes amino acids and the inorganic minerals cations may play a role in osmoregulation for this Rhizobium strain. The rhizobial cells responded to high-salt stress by changing their morphology: 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.
Policing the legume-Rhizobium symbiosis: a critical test of partner choice
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 High osmotic stress 0. The colonies of R. The synthesis pattern in SDS-PAGE of lipopolysaccharides LPS from various species of rhizobia from cultivated legumes and from woody legumes 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 Successful Rhizobium-legume symbioses under salt stress require the selection of salt-tolerant rhizobia from those indigenous to saline soils Rhizobium strains isolated from salt-affected soils in Egypt failed to nodulate their legume host under saline and nonsaline conditions a.
These rhizobia showed alterations in their protein and LPS patterns The genetic structure of these bacteria may also be changed 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 Graham 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. Mutant strains of R. These nodules failed to express nitrogenase activity Some strains of Rhizobium tolerated extremely high levels of salt up to 1. Inoculation of legumes by salt-tolerant strains of R. Salt-tolerant strains isolated from Acacia redolens, growing in saline areas of Australia, produced effective nodules on both A.
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 mM NaCl Under saline conditions, the salt-tolerant strains of Rhizobium sp. An important result was obtained from the recent work of Lal and Khannawho showed that the rhizobia isolated from Acacia nilotica in different agroclimatic zones, which were tolerant to mM NaCl, formed effective N2-fixing nodules on Acacia trees grown at 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 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 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 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.
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 to mM NaCl 67 Casuarina obesa plants are highly salt tolerantbut 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, 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 It is well known that some free-living rhizobia saprophytic are capable of survival under drought stress or low water potential 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 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.
The survival and activity of microorganisms may depend on their distribution among microhabitats and changes in soil moisture The distribution of R. Moderate moisture tension slowed the movement of R. The migration of strains of B.Rhizobium, a Symbiont
One of the immediate responses of rhizobia to water stress low water potential concerns the morphological changes. Mesquite Rhizobium and R. 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. Further, a reduction in the soil moisture from 5.
Similarly, water deficit, simulated with polyethylene glycol, significantly reduced infection thread formation and nodulation of Vicia faba plants 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 Symbiotic N2 fixation of legumes is also highly sensitive to soil water deficiency. A number of temperate and tropical legumes, e. 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 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 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 More recently, Sellstedt et al.
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. 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 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. One legume, guar Cyamopsis tetragonolobais drought tolerant and is known to be adapted to the conditions prevailing in arid regions Variability in nitrogen fixation under drought stress was found among genotypes of Vigna radiata and Trifolium repens 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 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. In these plants, positive correlations were found between proline accumulation and drought tolerance.
Potassium is known to improve the resistance of plants to environmental stress. A recent report indicates that K can apparently alleviate the effects of water shortage on symbiotic N2 fixation of V. The presence of 0.
Rhizobium-Legume Symbiosis and Nitrogen Fixation under Severe Conditions and in an Arid Climate
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 High root temperatures strongly affect bacterial infection and N2 fixation in several legume species, including soybeanguar 22peanutcowpeaand beans Nodule functioning in common beans Phaseolus spp.
Nodulation and symbiotic nitrogen fixation depend on the nodulating strain in addition to the plant cultivar 22 Temperature affects root hair infection, bacteroid differentiation, nodule structure, and the functioning of the legume root nodule High not extreme soil temperatures will delay nodulation or restrict it to the subsurface region