Effect of lead pollution stress on biological nitrogen fixation of alfalfa plant

Introduction Nitrogen is one of the macronutrients which alfalfa required, Upon the formation of nodules on the roots, the plant obtains a large part of its required nitrogen from the air. The rate of nitrogen fixation in alfalfa is estimated about 60 to 500 kg ha-1 per year. Symbiosis between Rhizobia and legume plants begins by exchanging molecular signals between bacteria and plant root. Plant varieties, bacterial strains, soil conditions (heavy metals, temperature, water, aeration, nutrients, salinity, nitrate, pH etc.) are factors which affect the symbiosis. Lead is one of heavy metal, pollutes environment specially agricultural lands. Biological nitrogen fixation is sensitive to low concentrations of heavy metals in soil. Also the high sensitivity of the nitrogen fixation process to heavy metals, in soils contaminated with sewage sludge have been reported. Exploitation of Lead and zinc and related operation in Zanjan province released heavy metals including lead in surrounding agricultural land, So that the agricultural land where located in the vicinity of lead and zinc processing factory, are contaminated with heavy metals The aim of this study was to examine the effects of lead contamination on alfalfa symbiotic Rhizobium indigenous to Zanjan province, and nitrogen fixation in alfalfa plants.   Materials and methods To isolate rhizobium native to heavy metals polluted soils, nodules bearing roots were collected from alfalfa farms of Zanjan province which located near Pb and Zn factories where soils were contaminated by heavy metals, then rhizobium bacteria were isolated. Resistance of isolates to Pb were examined using selective medium containing different concentration of Pb. To assess the relative resistance of strains to lead H.M medium was used. Different concentrations of lead (0, 25, 50 and 100 mg L-1) was added to the mentioned media as CdCl2. Considered isolates were cultured on media containing various concentration of lead as point spots. Five days after incubation at 25 ° C plates wereinspected, and based on the growth and Status and consistency of colonies compared to the control, isolates  were grouped as resistant (growth as control), moderately susceptible (reduced growth as compared to control) and sensitive (no growth). For each isolates was determined as the maximum resistance level (MRL). To evaluate the ability of isolates to fixing N2 in Pb polluted soil, 5 isolates were selected based on their resistance to Pb as well symbiotic effectiveness, and then were used in green house experiment. Treatments were five sinorhizobium isolates (N6, N12، N17، N41andN51) as inoculants, and five concentrations of Pb(0, 5, 25, 50, 100 mg Pb per kg) were examined on growth of alfalfa plant. Alfalfa seed after surface sterilization (1% sodium hypochlorite solution), were germinated in incubator, and then were planted in pots. Each seed was inoculated with a milliliter of a suspension which contain selected isolates (107 cfu ml-1). 90 days after planting, plants were harvested and shoot dry weight was determined. The root nodules were evaluated and classified as described by Vincent (1982). The shoot nitrogen content was measured by Kjeltec method and the absorbed nitrogen in shoot.   Results and discussion All of tested bacterial isolates could create nodules on alfalfa root systems. To evaluate the isolates resistance to lead contamination, they were cultured in media containing different concentration of lead, and then based on the growth of isolates compared with control, their resistance were evaluated. The results showed that, a few of them survived at the maximum applied concentration of lead (500 mg Pb L-1) and grew. MRL index (The maximum lead concentration where the isolates had visible growth), determined for each of isolates, then the percentage of isolates that had visible growth at different concentrations of Pb, were calculated . The Comparison interaction between isolates and lead level on shoot N concentration showed that by increasing the  lead levels, in case of all inoculated isolates, decreased shoot N concentration and this reduction was significant in most cases.The highest shoot N concentration (3.3%) belonged to control (0 mg Pb kg-1 and 70 mg N treatments) and the lowest N concentration (2.4%) belonged to the 100 mg Pb kg-1 and inoculation with N6 isolate). Means comparison showed that by increasing lead levels, reduced plant dry weight, but this reduction was different in different strains, The highest shoot dry weight (0.81 g pot-1) belonged to inoculated plants with N51 strain at the level of 25 mg Pb kg-1,while the lowest (0.15 g pot-1) was observed in non-inoculated control at 100 mg Pb kg-1.   Conclusion The tolerance of symbiotic microorganisms against environmental stresses is more than their host plants. Toxicity that comes from high concentrations of heavy metals in soil, inhibites root growth and development, as well disorderes early stages of legume- Rhizobium symbiosis and affects biochemical signals between them. Totally, 6.7% of isolates, including were very tolerant to lead, and their growth in media containing the highest lead, did not show any difference with control. While, some isolates were not able to grow on media containing more than 75 mg Pb L-1. About 64% of isolates tolerated concentrations of 25 to 150 mg per liter of lead. About 30% of the isolates survived in media containind up to 300 mg Pb  L-1 , that is a good tolerance to lead contamination. Generally application of different levels of lead decreased shoot N content of plants, but reducing the N in inoculated plants were less than the control and nitrogen treatment. Although the tolerance of alfalfa against stresses is low compared to symbiotic bacteria,  however, inoculation host plants with right rhizobia somewhat can be reduced the injury  of heavy metals.