Subject: For A.J. Phillips.......about Rhizoctonia

From: [email-masked]
Date: 21 AUG 96 20:02:48 GMT

Dear Prof. Phillips,
		I am sending this message through the news group as I don't
seem to be able to get through to your personal number.                
Thank you so much for your information.   This morning I found
your paper from the Canadian Journal of Micro. and I have a few
 questions that I would hope that you can help me with.
1)      The liquid media you used is referenced Weinhold 1969 in
	 Phytopathology
        I tried to grow Rhizoctonia in Peptone Yeast Glucose Media
 but I didn't
        get satisfactory results.  Is this medium too rich?

        The ingredients are as follows
Peptone         5.0g
Yeast Extract   20.0g
Glucose         10.0g
KH2PO4          1.0g
MgSO4.7H2O      0.5g
Distilled Water 1 Litre
(Biochemical techniques for Filamentous Fungi, Paterson & Bridge
 1994)

        I don't have the journal that Weinhold published in and I
 wonder if you
        could send me the ingredients for the liquid medium used
 there to
        compare with what I have been trying?

2)      After harvesting the mycelium they are washed in 3 changes
 of SDW does
        this mean centrifuging?  If so would it be along the lines
 of 10,000 X  g for 10 mins?

3)      Lastly, in your last message you suggest a rate of 0.25g of
fresh weight
        mycelia / Kg of soil.  Does this mean weigh after collection
 in the
        Buchner flask directly or do you oven - dry it before
 weighing
        Also I'm working with peat and compost...do you think that
 will make a huge difference?

Sorry for all the questions and I hope you won't regret answering my
 initial
request!

In appreciation,

Susan Rafferty :->

---

From: "A. Phillips" <[email-masked]>
Date: Fri, 23 Aug 1996 00:27:51 -0700

I'm sorry that you had problems getting through, but I collected
your message from the bionet.mycology newsgroup without any
problems.  If you have any further problems and can't get through
to my personal number, just post it up on the newsgroup - I
normally check this every day.

Since I am on vacation at the moment I don't have the recipe for
the medium for growing Rhizoctonia in liquid culture.  However,
the medium you are using does seem to be rather rich and I would
recommend that you should omit the peptone, cut the yeast extract
to about 2.0 g and maybe add 2.0 g of asparagine.  Some
micronutrients could be useful if you are using distilled water,
but this isn't always necessary if you are using tap water.  I
favoured the medium described by Weinhold et al. (1969) because
I had grand ideas to study in more detail the influence of
pathogen nutrition on virulence but never found the time to do
so.  Another suitable medium is Czapek-Dox but replace the KNO3
with asparagine and add 2g of yeast extract.  If, after trying
these media, you still have problems, please let me know and I
will send you the full details Weinhold's medium when I get back
to work about 10 days from now.

Regarding your second query.  It is not necessary to centrifuge
the mycelium.  I just poured off the liquid medium, added sterile
water, shook it briefly and then poured off the water.  Repeat
this 3 times.  Over the years I modified my procedure and finally
decided that washing was not necessary but rather strained off
the mycelium through cheese cloth and then pressed the mycelium
by hand pressure between wads of sterile paper hand towels.  This
is what I regard as the "fresh weight" of mycelium.  Details of
this method are in Annals of Applied Biology vol 118, pp 9-17
(1991).  There is no need to dry the mycelium any further (oven
drying is sure to kill it).  I once tried air-drying the mycelium
at room temperature and then ground this to a coarse powder (a
coffee grinder was perfect for the job).  This ground-up mycelium
seemed to retain its virulence even after storage for several
months in a refrigerator.  However, my preferred method was to
comminute the mycelium in water (6 g of mycelium in 50 ml of
water).  I used an Ultra Turrex tissue grinder operated at half
speed for 60 seconds, but just about any homogenizer/blender will
work provided that you don't "over blend" and destroy to much of
the mycelium.  I would then add 150 ml of water to my 6g of
mycelium in 50 ml of water to give a total volume of 200 ml.
This suspension was then added to 24 kg of soil and mixed
thoroughly.  A small concrete mixer gave a most satisfactory
mixing without to much hard labour.  In this way I had 6 g of
mycelium per 24 kg of soil which gave me my 0.25 g fresh weight
per kg of soil.

With regard to your peat and compost mixture.  I really can't
comment on this.  It could cause problems... maybe.  My
experience is that diseases caused by Rhizoctonia are most severe
in sandy soils low in organic matter.  But, on the other hand,
these disease can be a problem in high organic potting mixtures
as used in nurseries.  What is your host?  Indeed, what are you
doing with Rhizoctonia?  I really am interested to know and look
forward to hear of your successes.

Regards

Alan J.L. Phillips