Root nodulation of alfalfa by Ensifer meliloti in petroleum contaminated soil

One of the limiting factors of soil fertility is contamination with petroleum hydrocarbons, which has affected soil fertility's effective biological activity, such as the symbiosis of nitrogen fixing bacteria with legumes. In this study, some petroleum hydrocarbons' effect on the nodulation of Rhizobium sp. in alfalfa roots under greenhouse conditions was evaluated. For this purpose, 25 bacterial isolates were isolated from alfalfa surface-sterilized root nodules, 5 of which could grow in yeast mannitol media containing crude oil up to 5%. These strains also had the ability to coexist with alfalfa roots and form nodules under in-vitro condition. Isolate B20 formed the highest number of nodules on alfalfa roots at concentrations of 1, 3, and 5% crude oil, which according to the 16S rRNA gene sequencing result, belonged to Ensifer meliloti. Then the effect of different concentration of crude oil (1-3-5% v/w), paraffin (1-3-5% v/w), toluene (50-100-200 ppm), naphthalene (50-100-200 ppm), and phenanthrene (50-100-200 ppm) was investigated on alfalfa growth in the presence of Ensifer meliloti. According to the results, the highest number of nodules (53.33) was observed in the control treatment, and the lowest number of nodules (14.66) was observed at the concentration of 200 mg/kg of phenanthrene. As the number of benzene rings increased, or the pollutant concentration increased, plant growth decreased as assayed by root and shoot biomass, plant nitrogen, number of root nodules, and soil microbial respiration. Contaminants or metabolites produced by the activity of soil microorganisms may inhibit the nodulation activity of Ensifer meliloti. •Ensifer meliloti B20 is a novel passenger bacteria endophyte isolated from alfalfa nodules.•E. meliloti B20 enhances alfalfa growth in hydrocarbon contaminated soil.•Nodule formation in the presence of E. meliloti B20 was reduced by petroleum hydrocarbons.