Subject: Manipulations with Filamentous Fungi

From: [email-masked] (John J Weiland)
Date: 15 Oct 1994 08:41:53 -0700

I'm kind of new to these beasts, and am looking for some input into the 
following questions.  The fungus I'm working with is a pathogen of barley 
called Pyrenophora teres.

1)	We currently store long-term cultures of the fungus on silica 
gel.  Is this a standard long term storage media?  Can spores be stored 
in glycerol at -80 degrees C.  Can they be stored as stabs in agar (like 
many bacterial isolates)?

2)	Conidia of P. teres can be collected from mycelia grown in 
culture.  Ascospores, however, result from the mating of two opposite 
mating types of the fungus on barley plants (but not in culture).  Has 
anyone seen mention of mutations in the fungus, or culture conditions, 
that may induce productive mating of such a fungus in culture, rather 
than on its normal host plant?

3)	Any papers out there on the "bulk segregation analysis" of traits 
in haploid fungi using PCR?   Did Michelmore's group do it with the 
fungus, or just with the plant traits?

Thanks in advance!

John Weiland		[email-masked]

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From: [email-masked] (Brett Tyler)
Date: 17 Oct 1994 08:02:28 -0700

>John Weiland            [email-masked] 

Regarding 1)  In our experience with Neurospora, silica gel is in fact the
best long term storage method.  Slants or glycerol can be used, depending
on the fungus, but can be unreliable.  Liquid nitrogen storage is another
excellent long term storage method.

Regarding 3) We are using bulked segregant analysis for Phytophthora sojae.
 There is no reason at all why this method can't be used on any genetic
progeny showing random segregation of genes.  In haploids, BSA should work
even better than diploids because dominance and recessiveness (especially
of the RAPDs themselves) is not a problem.  The most important
considerations for BSA are (i) that the trait of interest is determined by
a single locus, (ii) that you have at least  8-10 progeny for each bulk
(you should have much more than this if the genetics have been done well),
(iii) that you are absolutely sure that none of the chosen progeny have
been mis-scored and (iv) that there is a sufficient level of DNA sequence
polymorphism between the parents (at least 5% of all RAPD bands should
differ between the two parents, otherwise you will have a real struggle).

Can't help with 2).

****************
Brett Tyler
Professor
[email-masked]
****************

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From: [email-masked] ("David Brayford ", IMI)
Date: 18 Oct 1994 04:23:43 -0700

1. Silica gel is fine for, say, 5 years if you keep it refrigerated.  Liquid 
nitrogen or lyophilisation are better, but obviously more expensive.  It is 
not recommended to keep them growing on agar since they are likely to 
mutate.

2.  I don't know a synthetic medium for crossing this fungus.  However, a 
good ploy is to mimic the natural conditions by pairing isolates on bits of 
autoclaved barley straw (say 2-3 cm lengths) placed on plain agar.  Allow 
the isolates to grow for 2 weeks to allow colonisation of the straw, then 
transfer to cool conditions (10 C) for 2-3 weeks, then transfer to 20-25 C 
in alternating dark and daylight.  The fungus usually forms its sexual state 
in spring after a cold winter snap - so the cool incubation period may help 
promote perithecial formation - it certainly does in other fungi.

Dave Brayford
International Mycological Institute
[email-masked]

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From: BIOSCI-REQUEST
To: mycology
Subject: Manipulations with Filamentous Fungi
Date: 15 October 1994 8:41

Thanks in advance!