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. Chlorateresistant 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 NH % "&NO $ medium was studied for two mutant strains and their corresponding wild-type homokaryons. None of the mutants could use nitrate whereas "&N 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.
S U M M A R VIntraspecific variability in the activity of nitrate reductase (NR) has been studied in the ectomycorrhizal fungus Hc'heloiun cylindrosporum Romagnesi at the interstrain and intrastrain lc\cls, the latter within a population of 1 1 wild dikaryotic strains collected in ' Les Landes' (SW France) at four locations less than 100 km a\\a\-from one another. .\n attempt was made to determine whether variability within the monokaryotic and dikaryotic progeny of the HCl fruiting strain could he used as a hasis for an improvement programme in\()hing breeding between selected monokaryons.The NR acti\ it>' of the wikl strains ranged from 201 to 700 nmol NO^ synthesized h ' mg ' fungal protein whilst that of 20 sib-monokaryons (5 per mating type) of the HCl suain varied from 51 to 510 nmol NO, s\nthesized b^' mg"' fungal protein. Fifty controlled dikaryotic mycelia were obtained from all the compatible fusions with these monokaryons. In these, variation of NR acti\it\' was of the same order of magnitude as that recorded at the interstrain Ie\el, ranging frotn 72 to 689 ntnol NO.^ synthesized h ' mg ' fungal proteiti. .Atiahsis of the componetits of the \ariatioti of NR actixity iti these controlled dikaiyotis demonstrated that the additise component of this variation accounted for less than 1 ",, of the total observed variation. The .NR acti\-itv of anv one cotitrolled dikaryon eould tiot therefore be predicted trom the actix'ity of its paretital monokarvons Howexer 14 of the 50 controlled dikaryons exhibited an NR activity higher than tliat of the HCl parental dikarvon These results indicate that breeding with sib-motiokaryons cati be used as a basis tor ati itnprovemetn programme of NR activity m this ectomyeorrhizal Basidiomycete.
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