FW: [Mycology] trichoderma primers

Rossman, Amy via mycology%40net.bio.net (by Amy.Rossman from ARS.USDA.GOV)
Mon Nov 30 09:24:55 EST 2009


Here is the response from our person who does molecular work on
Trichoderma.  I hope this is helpful.

Amy

Amy Rossman, Research Leader
Systematic Mycology & Microbiology Laboratory
USDA-ARS, Rm. 304, B011A
10300 Baltimore Ave.
Beltsville, MD 20705
301-504-5364
Fax 301-504-5810
http://ars.usda.gov/ba/psi/smml
 


-----Original Message-----
From: Ismaiel, Ed 
Sent: Monday, November 30, 2009 8:12 AM
To: Rossman, Amy
Cc: Samuels, Gary
Subject: RE: [Mycology] trichoderma primers

Dr Amy,

Here is my answer about Trichoderma primers- we can provide further info
if he needs.

Primers for identification of trichoderma species are available in many
of Dr Gary Samuel's publication such as "Samuels et al. 2006.  The
Trichoderma koningii aggregate species.  Studies in Mycology 56:67-133"

We use a section of the gene "translation elongation factor 1 alpha"
sequence for identification of all Trichoderma species.  I am sending
you the sequences of the primers used for amplification of this gene.
Below, Primer 1 (forward) and primer 2 (reverse) are used in PCR and
will give a product of about 600-700 bp.  Use 30 PCR cycles of
denaturation at 94C 30 sec, annealing 48C 30 sec and amplification 72C 1
min.  Once you get the sequence, BLAST it at NCBI and you should get
perfect identification of your culture unless your culture is a new
species.
Primers 3 and 4 are forward and reverse primers for amplification of
rRNA gene ITS (ITS1, 5.8S rRNA, ITS2).  These two primers are used for
amplification of ITS of all fungi.  The primers work very well for most
fungi but for Trichoderma it will not work for closely related species.
In other words, the ITS sequence for some closely related species of
Trichoderma are identical.  Therefore, you may not be able to identify
your culture correctly. Still, you get good idea about the position of
your culture within the phylogeny of the genus Trichoderma. 

1)	EF-728M		5'-CAT YGA GAA GTT CGA GAA GG
2)	TEF1-R		5'-GCC ATC CTT GGA GAT ACC AGC 
3)	ITS5			5'-GGA AGT AAA AGT CGT AAC AAG G
4)	ITS4			5'-TCC TCC GCT TAT TGA TAT GC

Ed	 
-----Original Message-----
From: Rossman, Amy 
Sent: Friday, November 27, 2009 9:53 AM
To: Ismaiel, Ed
Cc: Samuels, Gary
Subject: FW: [Mycology] trichoderma primers

Ed,

Can you respond to this request?  Please copy me on your answer.
Thanks.

Amy

Amy Rossman, Research Leader
Systematic Mycology & Microbiology Laboratory USDA-ARS, Rm. 304, B011A
10300 Baltimore Ave.
Beltsville, MD 20705
301-504-5364
Fax 301-504-5810
http://ars.usda.gov/ba/psi/smml
 

-----Original Message-----
From: mycology-bounces from oat.bio.indiana.edu
[mailto:mycology-bounces from oat.bio.indiana.edu] On Behalf Of Thomas
Delaney
Sent: Thursday, November 26, 2009 12:30 PM
To: mycology from magpie.bio.indiana.edu
Subject: [Mycology] trichoderma primers

Hi,

I was researching on the internet regarding Trichoderma where i came
across your email address. I am doing a research project in Ireland with
trichoderma to see if has a effect on straw decomposition. i was just
woundering did you have any luck in finding any primers for the
trichoderma fungus?

Any help or Information regarding Trichoderma or Trichoderma primers
would be of great help.

Many Thanks
Thomas

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