Frequency and Diversity of Nitrate Reductase Genes among Nitrate-Dissimilating <i>Pseudomonas</i> in the Rhizosphere of Perennial Grasses Grown in Field Conditions

A total of 1246 <i>Pseudomonas</i> strains were isolated from the rhizosphere of two perennial grasses (<i>Lolium perenne</i> and <i>Molinia coerulea</i>) with different nitrogen requirements. The plants were grown in their native soil under ambient and elevated atmospheric CO₂ content (pCO₂) at the Swiss FACE (Free Air CO₂ Enrichment) facility. Root-, rhizosphere-, and non-rhizospheric soil–associated strains were characterized in terms of their ability to reduce nitrate during an <i>in vitro</i> assay and with respect to the genes encoding the membrane-bound (named NAR) and periplasmic (NAP) nitrate reductases so far described in the genus <i>Pseudomonas</i>. The diversity of corresponding genes was assessed by PCR-RFLP on <i>narG</i> and <i>napA</i> genes, which encode the catalytic subunit of nitrate reductases. The frequency of nitrate-dissimilating strains decreased with root proximity for both plants and was enhanced under elevated pCO₂ in the rhizosphere of <i>L. perenne</i>. NAR (54% of strains) as well as NAP (49%) forms were present in nitrate-reducing strains, 15.5% of the 439 strains tested harbouring both genes. The relative proportions of <i>narG</i> and <i>napA</i> detected in <i>Pseudomonas</i> strains were different according to root proximity and for both pCO₂ treatments: the NAR form was more abundant close to the root surface and for plants grown under elevated pCO₂. Putative denitrifiers harbored mainly the membrane-bound (NAR) form of nitrate reductase. Finally, both <i>narG</i> and <i>napA</i> sequences displayed a high level of diversity. Anyway, this diversity was correlated neither with the root proximity nor with the pCO₂ treatment.