The effect of the plant growth stimulant bactozole on Rhizobium leguminosarum bv. viciae 250a and its nitrogen-tolerant mutant M-71 under varied nitrogen supply

The effect of the plant growth stimulant bactozole on the growth of Rhizobium leguminosarum bv. viciae 250a and its nitrogen-tolerant mutant M-71 and the synthesis of extracellular carbohydrates was studied. At a low content of nitrate (6 mM) in the medium, all three bactozole concentrations tested (0.001, 0.01, and 0.1%) exerted similar stimulating effects on the growth of the parent strain 250a (about 1.5-fold) and the synthesis of extracellular carbohydrates (about 2-fold). At a high content of nitrate (20 mM) in the medium, when the growth of the parent strain and the synthesis of extracellular carbohydrates were inhibited, bactozole at all three concentrations exerted only a growth-stimulating effect. At the same time, mutant M-71 showed better growth at higher concentrations of bactozole, whereas the ability of the mutant to synthesize extracellular carbohydrates decreased with increasing bactozole concentration. The cell biomass of the mutant accumulated at 20 mM nitrate was 1.8-2.5 times greater than it was at 6 mM nitrate. Bactozole enhanced the symbiosis of legume plants with both parent and mutant strains, raising the mass of plants and enhancing nodulation and the nitrogen-fixing activity of root nodules. The symbiotic parameters of mutant M-71 were better (irrespective of whether bactozole was present or not) when its inoculum was grown at a high nitrogen content (20 mM nitrate), whereas the respective parameters of the parent strain were better when it was grown at 6 mM nitrate. The inference is made that the better physiological characteristics of the mutant in the high-nitrate medium is due to its higher nitrate reductase activity (as compared with the parent strain) in both the free-living state and in legume nodules.     

Functional activity of exoglycans from Rhizobium leguminosarum bv. viciae 250a and its nitrogen-resistant mutant M-71 during the formation of legume-rhizobia symbiosis against a high-nitrogen background

The functional activity of the exoglycan complex (EGC) polysaccharides from Rhizobium leguminosarum bv. viciae 250a and its nitrogen-resistant mutant M-71 capable of inducing the formation of nitrogen-fixing nodules on pea roots against a high-nitrogen background (4.8 mM NO3-) was studied in vegetation tests. For this purpose, the bacterial inoculum washed free of its own exoglycans was supplemented with EGC of this or another strain grown in the presence of 6 or 20 mM nitrate. The best symbiotic characteristics (nodule number and nitrogenase activity, mass of the roots and aerial parts of plants) were recorded when the inoculum cells and exoglycans were obtained from strain M-71 grown in the presence of 20 mM nitrate. When the plants were inoculated with the cells (grown at 6 mM nitrate) + EGC (obtained at 6 mM nitrate) of this strain, the nodulation characteristics and the effectiveness of symbiosis decreased 1.5-2-fold. Partial recovery of the symbiotic potential of strain M-71 was observed when EGC (obtained at 20 mM nitrate) was substituted for its exoglycans (obtained at 6 mM nitrate). In the presence of exoglycans of the parent strain 250a (obtained at 6 or 20 mM nitrate), the mutant formed a substantially lesser number of nodules with a very low nitrogen-fixing activity. In turn, the mutant exoglycans synthesized in medium with either high or low nitrate nitrogen concentration did not recover the fix+ phenotype of strain 250a capable of forming symbiosis with pea plants only against a low-nitrogen background. When studying the relative content of high-molecular-weight exopolysaccharide components and low-molecular-weight glycans in the exoglycan complex, it was established that, in strain 250a (grown at 6 and 20 mM nitrate), as well as in its mutant M-71 (grown at 6 mM nitrate), exopolysaccharides prevailed, accounting for 72-75% of the sum of both types of glycopolymers, while low-molecular-weight glycans accounted for 25-28%. In contrast, in the EGC of strain M-71 obtained at 20 mM nitrate, which was the most active inducer of the formation of the symbiotrophic system by strain M-71 in the presence of a high mineral nitrogen concentration, low-molecular-weight glycans were the main component, accounting for 61% of total glycopolymers, while the polysaccharide content was 39%. Low-molecular-weight exoglycans are supposed to be involved in maintaining the physiological activity and the symbiotic status of rhizobia under unfavorable environmental conditions.     

Functional Activity of Exoglycans from Rhizobium leguminosarum bv. viciae 250a and Its Nitrogen-Resistant Mutant M-71 during the Formation of Legume–Rhizobia Symbiosis against a High-Nitrogen Background

The functional activity of the exoglycan complex (EGC) polysaccharides from Rhizobium leguminosarum bv. viciae 250a and its nitrogen-resistant mutant M-71, capable of inducing the formation of nitrogen-fixing nodules on pea roots against a high-nitrogen background (4.8 mM NO₃ ^–), was studied in vegetation tests. For this purpose, the bacterial inoculum washed free of its own exoglycans was supplemented with EGC of the same or another strain grown in the presence of 6 or 20 mM nitrate. The best symbiotic characteristics (nodule number and nitrogenase activity, mass of the roots and aerial parts of plants) were recorded when the inoculum cells and exoglycans were obtained from strain M-71 grown in the presence of 20 mM nitrate. When the plants were inoculated with the cells (grown at 6 mM nitrate) + EGC (obtained at 6 mM nitrate) of this strain, the nodulation characteristics and the effectiveness of symbiosis decreased 1.5- to 2-fold. Partial recovery of the symbiotic potential of strain M-71 was observed when EGC (obtained at 20 mM nitrate) was substituted for its exoglycans (obtained at 6 mM nitrate). In the presence of exoglycans of the parent strain 250a (obtained at 6 or 20 mM nitrate), the mutant formed a substantially lesser number of nodules with a very low nitrogen-fixing activity. In turn, the mutant exoglycans synthesized in medium with either high or low nitrate nitrogen concentration did not recover the fix⁺ phenotype of strain 250a, capable of forming symbiosis with pea plants only against a low-nitrogen background. In study of the relative content of high-molecular-weight exopolysaccharide components and low-molecular-weight glycans in the exoglycan complex, it was established that, in strain 250a (grown at 6 and 20 mM nitrate), as well as in its mutant M-71 (grown at 6 mM nitrate), exopolysaccharides prevailed, accounting for 72–75% of the sum of both types of glycopolymers, while low-molecular-weight glycans accounted for 25–28%. In contrast, in the EGC of strain M-71 obtained at 20 mM nitrate, which was the most active inducer of the formation of the symbiotrophic system by strain M-71 in the presence of a high mineral nitrogen concentration, low-molecular-weight glycans were the main component, accounting for 61% of total glycopolymers, while the polysaccharide content was 39%. Low-molecular-weight exoglycans are supposed to be involved in maintaining the physiological activity and the symbiotic status of rhizobia under unfavorable environmental conditions.     

Effect of the synthetic polysaccharide MOD-19 on the formation and function of symbiosis between <Emphasis Type="Italic">Pisum sativum</Emphasis> L. and <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">viciae</Emphasis>

The physiological action of the MOD-19 polysaccharide (PS), synthesized similarly to bacterial glucans, on the nodule bacteria Rhizobium leguminosarum bv. viciae and pea seeds was studied. It was found that MOD-19 stimulated nodule bacterium growth and bacterial biomass accumulation. It also altered metabolism in rhizobia grown in solid and liquid media containing this polymer. Treatment of pea seeds with MOD-19 before sowing increased the intensity of root formation, plant tissue peroxidase activity, and general symbiosis efficiency owing to secondary nodule formation on lateral roots and prolongation of their intense nitrogen fixation.     

The Effect of the Plant Growth Stimulant Bactozole on Rhizobium leguminosarum bv. viciae 250a and Its Nitrogen-Tolerant Mutant M-71 under Different Nitrogen Supply Conditions

The effect of the plant growth stimulant bactozole on the growth of Rhizobium leguminosarum bv. viciae 250a and its nitrogen-tolerant mutant M-71 and the synthesis of extracellular carbohydrates was studied. At a low content of nitrate (6 mM) in the medium, all three bactozole concentrations tested (0.001, 0.01, and 0.1%) exerted similar stimulating effects on the growth of the parent strain 250a (about 1.5-fold) and the synthesis of extracellular carbohydrates (about 2-fold). At a high content of nitrate (20 mM) in the medium, when the growth of the parent strain and the synthesis of extracellular carbohydrates were inhibited, bactozole at all three concentrations exerted only a growth-stimulating effect. At the same time, mutant M-71 showed better growth at higher concentrations of bactozole, whereas the ability of the mutant to synthesize extracellular carbohydrates decreased with increasing bactozole concentration. The cell biomass of the mutant accumulated at 20 mM nitrate was 1.8–2.5 times greater than it was at 6 mM nitrate. Bactozole enhanced the symbiosis of legume plants with both parent and mutant strains, raising the mass of plants and enhancing nodulation and the nitrogen-fixing activity of root nodules. The symbiotic parameters of mutant M-71 were better (irrespective of whether bactozole was present or not) when its inoculum was grown at a high nitrogen content (20 mM nitrate), whereas the respective parameters of the parent strain were better when it was grown at 6 mM nitrate. The inference is made that the better physiological characteristics of the mutant in the high-nitrate medium are due to its higher nitrate reductase activity (as compared with the parent strain) in both the free-living state and in legume nodules.     

The biological activity of the Sinorhizobium meliloti glucan

The study of the effect of the periplasmic glucan isolated from the root-nodule bacterium S. meliloti CXM1-188 on the symbiosis of another strain (441) of the same root-nodule bacterium with alfalfa plants showed that this effect depends on the treatment procedure. The pretreatment of alfalfa seedlings with the glucan followed by their bacterization with S. meliloti 441 insignificantly influenced the nodulation parameters of symbiosis (the number of root nodules and their nitrogen-fixing activity) but induced a statistically significant increase in the efficiency of symbiosis (expressed as the masses of the alfalfa overground parts and roots). At the same time, the pretreatment of S. meliloti 441 cells with the glucan brought about a considerable decrease in the nodulation parameters of symbiosis (the number of the root nodules and their nitrogen-fixing activity decreased by 2.5-11 and 7 times, respectively). These data suggest that the stimulating effect of rhizobia on host plants may be due not only to symbiotrophic nitrogen fixation but also to other factors. Depending on the experimental conditions, the treatment of alfalfa plants with the glucan and their bacterization with rhizobial cells enhanced the activity of peroxidase in the alfalfa roots and leaves by 10-39 and 12-27%, respectively.     

The Biological Activity of the Sinorhizobium meliloti Glucan

The study of the effect of periplasmic glucan isolated from the root-nodule bacterium Sinorhizobium meliloti CXM1-188 on the symbiosis of another strain (441) of the same root-nodule bacterium with alfalfa plants showed that this effect depends on the treatment procedure. The pretreatment of alfalfa seedlings with glucan followed by their bacterization with S. meliloti 441 insignificantly influenced the nodulation parameters of symbiosis (the number of root nodules and their nitrogen-fixing activity) but induced a statistically significant increase in the efficiency of symbiosis (expressed as the masses of the alfalfa overground parts and roots). At the same time, the pretreatment of S. meliloti 441 cells with glucan brought about a considerable decrease in the nodulation parameters of symbiosis (the number of root nodules and their nitrogen-fixing activity decreased by 2.5–11 and 7 times, respectively). These data suggest that the stimulating effect of rhizobia on host plants may be due not only to symbiotrophic nitrogen fixation but also to other factors. Depending on the experimental conditions, the treatment of alfalfa plants with glucan and their bacterization with rhizobial cells enhanced the activity of peroxidase in the alfalfa roots and leaves by 10–39 and 12–27%, respectively.