Nitrate non-utilizing mutants of Trichoderma harzianum

Jerome Rigot jfrigot at
Thu Jun 8 13:26:25 EST 1995


I am trying to obtain nitrate non-utilizing mutants of Trichoderma
harzianum.  I have been using the procedure developed by Cove as modified
by J.E. Puhalla on his work with Fusarium oxysporum (Classification of
strains of Fusarium oxysporum on the basis of vegetative compatibility.
.1985. Can. J. Bot.  63: 179-183), in which he sucessfully isolated
several nitrate non-utilizing mutants of Fusarium oxysporium.  The
procedure is fairly simple, the fungus is plated on mineral media (MM)
containing sucrose or glucose as carbon source, 0.2% NaNO3, 0.16%
L-asparagine, and 1.5% or 3% KClO3 (potassium chlorate), and agar.  The
plates are inoculated with agar blocks from a fungus culture grown on a
complete medium (CM), and incubated at the desired temperature.  The
plates are examined periodically for the appearance of fast-growing
sectors from the initially restricted colony.  Growth of wild-type strains
is restricted on chlorate , presumably because chlorate is reduced by
nitrate reductase to highly toxic chlorite.  Nitrate non-utilizing mutants
are unable to reduce chlorate to chlorite and hence are chlorate
resistants.  The mutants obtained are plated on MM with NaNO3 as the only
Nitrogen source, and will grow as thin and expansive colonies with no
aerial mycelium (Correll, J.C. et al, 1987.  Nitrate non-utilizing mutants
of Fusarium oxysporum and their use in vegetative compoatibility tests. 
Phytopathology 77:1640-1646).
So far I have been unsucessful with Trichoderma harzianum, I don't see any
fast-growing sectors, and when I replate on MM with NaNO3 only, I always
get good growth with aerial mycelium and fruiting bodies, no thin and
expansive growth a mutant is suppose to display.  
I would appreciate any help or advice, and I will give you more details. 
Please e-mail me directly.  Thanks

Jerome Rigot

Jerome Rigot
Dept. Environmental Toxicology
University of California
Davis, CA 95616
e-mail: jfrigot at

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