Subject: Nitrate non-utilizing mutants of Trichoderma harzianum

From: [email-masked] (Jerome Rigot)
Date: Thu, 08 Jun 1995 10:26:25 -0800

Hello, 

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
(916)752-5896
e-mail: [email-masked]

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From: [email-masked] (Paul Zambino)
Date: 16 Jun 1995 08:38:57 -0700

>by J.E. Puhalla on his work with Fusarium oxysporum
(using chlorate as a nitrate analog).
(more deleted)
>Please e-mail me directly.  Thanks

Jerome, It's a tough problem.  I have had success generating nitrogen
utilization mutants in Leptographium species, but when I tested growth on
chlorate and  tried to induce chlorate-resistant mutants in T. viridis and
T. harzianum and a Scopulariopsis species, I too found that with these
species, growth was only slightly reduced on chlorate, even at
concentrations up to 20 g/L of chlorate per liter of medium. I noticed that
T. harzianum and Scopulariopsis produced abundant yellow and blue pigment,
respectively, on the chlorate media, much greater than on media containing
nitrate, nitrite, or asparagine without chlorate.  At the time, I wondered
if toxic levels of nitrite and chlorite could be chemically altered by the
fungus, and/or excreted back into the medium.  Scopulariopsis is known to
occur on substrates with high protein content, and could maybe be expected
to possess an efficient mechanism of handling excess nitrogen and the
by-products of its metabolism (e.g. nitrite).  I don't know if the same
might hold true for Trichoderma.  Tolerance of chlorate raises some
additional possibilities, such as the use of chlorate in media selective
for Trichoderma, or for favoring the establishment of mycoparasitic
Trichoderma species versus pathogenic or decay fungi in tree wounds or cut
wood.

If after repeated attempts, Chlorate does not work for you, there are other
types of transport mutants that can be isolated by analogues that may be
useful for vegetative incompatibility studies, although I have not heard of
them being used for this specific purpose.  Check out The Genetic Control
of Membrane Transport by Carolyn W. Slayman, pp. 1-174 in F. Bronner and A.
Kleinzeller, eds., Current Topics in Membranes and Transport, Vol 4, 1973.
There is a nice list of substrates and analogues on pages 130-131.  My
guess is that the amino acid analogues will be useful.  Good luck!  Paul
Zambino

Paul Zambino
USDA Forest Service
Forestry Sciences Lab
5985 Highway K
Rhinelander, WI 54501
PH:(715)362-1178
FAX: (715)362-1166
EMAIL: [email-masked]
DG:p.zambino:R09F06A

Paul Zambino
USDA Forest Service
Forestry Sciences Lab
5985 Highway K
Rhinelander, WI 54501
PH:(715)362-1178
FAX: (715)362-1166
EMAIL: [email-masked]
DG:p.zambino:R09F06A