ISOLATION AND CHARACTERIZATION OF NITRATE REDUCTASE DEFICIENT MUTANTSOF THE ECTOMYCORRHIZAL FUNGUS HEBELOMA-CYLINDROSPORUM

To clarify the role of the fungal nitrate assimilation pathway in nitr ate reduction by mycorrhizal plants, nitrate reductase (NR)-deficient (NR-) mutants of the ectomycorrhizal basidiomycete Hebeloma cylindrosp orum Romagnesi have been selected. These mutants were produced by u.v. mutagenesis on protoplasts originating from homokaryotic mycelia belo nging to complementary mating types of this heterothallic tetrapolar s pecies. Chlorate-resistant mutants were first selected in the presence of different nitrogen (N) sources in the culture medium. Among 1495 c hlorate resistant mycelia, 30 failed to grow on nitrate and lacked a d etectable NR activity. Growth tests on different N sources suggested t hat the NR activity of all the different mutants is specifically impai red as a result of mutations in either the gene coding for NR apoprote in or genes controlling the synthesis of the molybdenum cofactor. Furt hermore, restoration of NR activity in some of the dikaryons obtained after crosses between the different mutant mycelia suggested that not all the selected mutations mapped in the same gene. Utilization of N o n a (NH4NO3)-N-15 medium was studied for two mutant strains and their corresponding wild-type homokaryons. None of the mutants could use nit rate whereas N-15 enrichment values indicated that 13-27 % of N presen t in 13-d-old wild-type mycelia originated from nitrate. Apparently, t he mutant mycelia do not compensate their inability to use nitrate by a more efficient use of ammonium. These different NR mutants still for m mycorrhizas with the habitual host plant, Pinus pinaster (Ait.), mak ing them suitable for study of the contribution of the fungal nitrate assimilation pathway to nitrate assimilation by mycorrhizal plants.