Selected mercury-tolerant rhizobia facilitate phytoremediation capability of shrub legumes

Abstract

The Rhizobium-legume symbiosis is being tested as an alternative tool for phytoremediation of Hg-contaminated soils in Almadén (Ciudad Real, Spain). Previously, rhizobia from these soils were isolated and characterized to obtain a collection of rhizobia, adapted to the soil characteristics of the Almadén Mining District, that could be used as inoculants of legumes for phytoremediation purposes (1). The two main objectives of the present work were to continue analyzing the diversity of rhizobia from different areas of Almadén and to evaluate the phytoremediation and revegetation capacity of shrub legumes inoculated with the previously-selected Hg-tolerant strains, first in greenhouse and then in field experiments. 16S rRNA gene sequencing was used for the species affiliation of 14 strains of rhizobia isolated from 7 species of legumes belonging to the genera Medicago, Trifolium, Vicia, Lupinus, Retama, Phaseolus and Colutea. The greenhouse experiment consisted on the inoculation of four shrub legumes (Adenocarpus hispanicus, Spartium junceum, Retama monosperma and Cytisus striatus) with selected rhizobial strains. The plants were grown in pots, with vermiculite as a substrate, under controlled conditions. Control plants were watered with Hg-free nutrient solution while the rest were watered with Hg-containing nutrient solution. The results showed that, for most legume-rhizobium pairs, Hg negatively affected different traits such as aerial part and root weights, number and weight of nodules or nitrogenase activity. However, Retama plants inoculated with either of two Hg-tolerant strains of Rhizobium radiobacter and Bradyrhizobium canariense, were able to maintain similar values of the above-mentioned traits in the absence as well as the presence of Hg. Mercury accumulation was very high in nodules and roots, but not in leaves, suggesting that phytoremediation of Hg-contaminated soils using legumes inoculated with Hg-tolerant rhizobia may be achieved through rhizostabilization. The field experiment consisted of two plantations of shrub legumes inoculated with selected Hg-tolerant rhizobia (the same as in the greenhouse experiment) established in two areas which soils had different levels of Hg contamination. The results showed that the survival and accumulation capacity of the plants varied depending on the legume species and the inoculated strain. A high number of gaps were detected, although a higher persistence of legumes inoculated with the more Hg-tolerant strain was observed. Monitoring of the nodulation occupancy (analysis of the 16S-23S rRNA intergenic spacers (IGS)) has confirmed the presence of the inoculated strains in nodules of Cytisus and Spartium in the Almadén soils.