To clarify the role of the fungal nitrate assimilation pathway
in nitrate reduction by mycorrhizal plants, nitrate
reductase (NR)-deficient (NR−) mutants of the ectomycorrhizal
basidiomycete Hebeloma cylindrosporum
Romagnesi have been selected. These mutants were produced by u.v. mutagenesis
on protoplasts originating from
homokaryotic mycelia belonging to complementary mating types of this heterothallic
tetrapolar species. Chlorate-resistant mutants were first selected in the
presence of different nitrogen (N) sources in the culture medium.
Among 1495 chlorate resistant mycelia, 30 failed to grow on nitrate and
lacked a detectable NR activity. Growth
tests on different N sources suggested that the NR activity of all the
different mutants is specifically impaired as
a result of mutations in either the gene coding for NR apoprotein or genes
controlling the synthesis of the
molybdenum cofactor. Furthermore, 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 on a NH415NO3 medium
was studied for two mutant strains and their corresponding wild-type
homokaryons. None of the mutants could use nitrate whereas
15N enrichment values indicated that 13–27% of N
present in 13-d-old wild-type mycelia originated from nitrate. Apparently,
the mutant mycelia do not compensate
their inability to use nitrate by a more efficient use of ammonium. These
different NR mutants still form
mycorrhizas with the habitual host plant, Pinus pinaster (Ait.),
making them suitable for study of the contribution
of the fungal nitrate assimilation pathway to nitrate assimilation by mycorrhizal
plants.