**8. Factors that effective on symbiotic nitrogen fixation in soybean**

Nitrogen fixation is one of the important soil microbial activity which was affected by all ongoing processes in soil as well as other soil microorganisms. The biological nitrogen fixing process depends on the occurrence and survival of Rhizobium in soils and also on their efficiency (Adamovich, Klasens, 2001).

The rate of the nitrogen fixation was affected by many different physiological and environmental factors in soil, such as temperature, water holding capacity, water stress, salinity, nitrogen level, pH and other nutrients. Many of these factors, including temperature, affect many aspects of nitrogen fixation and assimilation, as well as factors such as respiratory activity, gaseous diffusion and the solubility of dissolved gasses, which ultimately affect plant growth (Dogan et al 2010; Keerio et al., 2001).

High amount of mineral nitrogen in soil has negative effect on nodulation. Wide or narrow C:N ratio decreases nodule formation, therefore nitrogen fixation. If the C:N ratio is in expected ratio (15-30) nodulation and N2-fixation regularly realizes. Inhibitory effect of nitrate causes the reduction of capillary roots development as well as preventing particular infection's strands. This effect is very similar to herbicides' effect. Many researches have shown that adequate nitrate, nitrite, ammonium and urea concentrations in soil causes to decrease the number of infections, to delay to the first formation of nodules, to decrease to the nodule number and weight. Temperature is the main factor affecting N2-fixation; however, optimum temperature for N2-fixation is depending on various soil properties. Optimum N2-fixation temperature value is between 20-40 °C. Nodulation and nitrogen fixation in soybean is composed of between 20-30 °C. High soil temperature diminishes root growth as well as nodule formation. Furthermore, temperature changes affects to the competitive ability of Rhizobium/Bradyrhizobium species. Low temperatures decreased to nodule formation and N2-fixation. However, N2-fixation in natural legumes is not influenced extreme cold conditions (Bordeleau and Prevost, 1994).

Soil reaction (pH) is one of the most important factor influencing legume and Rhizobium symbiosis. A higher concentration of H+ ions increases the solubility of Al, Mn and Fe, and higher amount of these elements may become toxic for rhizobium. *Sinorhizobium meliloti* and *Rhizobium galegae* are highly sensitive to acid pH and soluble Al when the critical soil pH is 4.8–5.0 (Bordeleau ve Prevost, 1994). *Rhizobium leguminosarum bv. trifolii* and *Rhizobium leguminosarum bv. Viciae* in comparison with alfalfa rhizobia are more tolerant to soil acidity. However, pH less than 4.6 inhibits their activity. Legumes and Rhizobium have form an efficient symbiosis and fix high amounts of biological nitrogen when soil pH is no less than 5.6–6.1. Soil acidification inhibited the root-hair infection process and nodulation. Optimum soil pH for nodulation and yield for soybean is between 6.2 and 6.8 (Lapinskas, 1998).

The results of a study indicate that *Rhizobium leguminosarum* bv. trifolii is widely distributed in slightly acid soils with pHKCl 5.6–6.0. The average content of rhizobia was 540.0 • 103 cfu g–1 of soil. Less *Rhizobium leguminosarum* bv. viciae and significantly less *Sinorhizobium meliloti* and *Rhizobium galegae* were found. Rhizobium significantly declined in acid soils (pHKCl 4.1–5.0). Most of biological nitrogen was fixed at soil pHKCl 6.1–7.0. In this case, *Rhizobium galegae* accumulated 196 to 289 kg N ha–1 of nitrogen, whereas rhizobia of alfalfa and clover were less, and it depended on strain efficiency and soil pH. Soil liming had a positive effect on nitogenase activity in red clover. The soil liming (CaCO3 rate 6.2 t ha–1) in combination with inoculation have increased biological nitrogen fixation by red clover at 106 kg N ha–1. Associative diazotrophes in non–legume rhizoplane have fixing the biological nitrogen too. The effective strains of *Rhizobium spp.*, *Agrobacter radiobacter* and *Arthrobacter mycorens* have made up an active association with barley, timothy and spring rape and accumulated 11.0 to 20.4 kg N ha–1 of biological nitrogen (Lapinskas, 2008).

Soil moisture can affect to nitrogen fixation both directly and indirectly. In low moisture condition in soil, nodule respiration decreases and nitrogen in nodule moves out slowly. This case is direct effect of low soil moisture. However in the same condition, nitrogen fixation decreased due to deterioration of generating photosynthesis units assimilate and in this case, N2-fixation was affected indirectly.

Iron (Fe) and molybdenum (Mo) are located in structure of the Nitrogenase enzyme which is working with legumes for symbiotic nitrogen fixation (Fig. 6). Therefore, the amount of these nutrients in the soil and plant uptake affects the symbiotic N2-fixation of legumes directly (Werner, 1987; Durrant, 2001).

Fig. 6. Structure of nitrogenase enzyme (Durrant, 2001)

Nitrogen fixation in soybean is negatively affected by increasing salt contents of the soil. N2 fixation of Rhizobium bacteria and their activities decreased in accordance with increasing soluble salt contents. Thus, increasing salt concentration in irrigation water was found to reduce a significant amount of grain and nodule weight in soybean (FAO, 1982).

According to many research it was determined to development of soybean was decreased in soil condition of 0.08% CaCl2 and 1.5% ZnSO4 (Anonymous, 1982). According to the results of many similar studies show that salt tolerance of rhizobium bacteria, optimum pH, antibiotic resistance and so on has revealed important differences (Gok and Martin, 1993).

#### **9. References**

178 Soybean Physiology and Biochemistry

tolerant to soil acidity. However, pH less than 4.6 inhibits their activity. Legumes and Rhizobium have form an efficient symbiosis and fix high amounts of biological nitrogen when soil pH is no less than 5.6–6.1. Soil acidification inhibited the root-hair infection process and nodulation. Optimum soil pH for nodulation and yield for soybean is

The results of a study indicate that *Rhizobium leguminosarum* bv. trifolii is widely distributed in slightly acid soils with pHKCl 5.6–6.0. The average content of rhizobia was 540.0 • 103 cfu g–1 of soil. Less *Rhizobium leguminosarum* bv. viciae and significantly less *Sinorhizobium meliloti* and *Rhizobium galegae* were found. Rhizobium significantly declined in acid soils (pHKCl 4.1–5.0). Most of biological nitrogen was fixed at soil pHKCl 6.1–7.0. In this case, *Rhizobium galegae* accumulated 196 to 289 kg N ha–1 of nitrogen, whereas rhizobia of alfalfa and clover were less, and it depended on strain efficiency and soil pH. Soil liming had a positive effect on nitogenase activity in red clover. The soil liming (CaCO3 rate 6.2 t ha–1) in combination with inoculation have increased biological nitrogen fixation by red clover at 106 kg N ha–1. Associative diazotrophes in non–legume rhizoplane have fixing the biological nitrogen too. The effective strains of *Rhizobium spp.*, *Agrobacter radiobacter* and *Arthrobacter mycorens* have made up an active association with barley, timothy and spring rape and accumulated 11.0 to 20.4 kg N ha–1 of biological

Soil moisture can affect to nitrogen fixation both directly and indirectly. In low moisture condition in soil, nodule respiration decreases and nitrogen in nodule moves out slowly. This case is direct effect of low soil moisture. However in the same condition, nitrogen fixation decreased due to deterioration of generating photosynthesis units assimilate and in

Iron (Fe) and molybdenum (Mo) are located in structure of the Nitrogenase enzyme which is working with legumes for symbiotic nitrogen fixation (Fig. 6). Therefore, the amount of these nutrients in the soil and plant uptake affects the symbiotic N2-fixation of legumes

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Fig. 6. Structure of nitrogenase enzyme (Durrant, 2001)

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