International Commission for the Taxonomy of Fungi (ICTF)
International Union of Microbiological Societies (IUMS, Mycology Division)
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How to identify Trichoderma

flowchart.gifHow to identify Trichoderma

in a nut shell

In general, the following order of steps may be suggested for those researchers who study Hypocrea/Trichoderma biodiversity or perform a global screening for industrially important Trichoderma strains.


1) Prepare monospore (single spore) cultures for every strain to be identified.

This step is of crucial importance since several Trichoderma spp. may occupy one ecological niche. Moreover, it is well known that due to homoplasy of morphological characters it is often impossible to discriminate species.

2) Extract genomic DNA from young cultures preferably before sporulation begins

We suggest to cultivate Trichoderma strains on agar plates covered by cellophane no longer than 2 - 4 days in order to control possible cross contamination by other Trichoderma cultures.

It is also advisable to check DNA quality on agarose gels. If possible, measure DNA concentration

3) make PCR amplification and sequencing of the following marker(s):

 

Case Phylogenetic marker (locus) Tools to be used

Large scale biodiversity assays with a big number of Trichoderma strains isolated from several locations with certain gradient of conditions;

Identification of individual strains

Species identity control for industrial isolates

Screening for certain Hypocrea/Trichoderma species distinguished by ITS1 and 2

ITS1 and 2

internal transcribed spacers�1�and�2�of rRNA gene cluster

a universal fungal BarCode marker

TrichOKEY

DNA oligonucleotide BarCode program

Advantages:

  • gives absolute identification result
  • recognises multiple sequences in FASTA format
  • identifies 95% of known Hypocrea/Trichoderma species
  • easy PCR amplification of ITS1 and 2
  • distinguishes potentially new species or allels

Disadvantages:

  • depends on sequence quality and compleatness (ITS1 - 5.8S rRNA - ITS2)
  • does not discriminate species within Koningii  and Rufa complexes and does not distinguish T. longibrachiatum - H. orientalis and T. tomentosum - T. cerinum species pairs

Identification of strains which were not identified by TrichOKEY

Identification of species from Trichoderma Section Trichoderma

Detection of new species

tef1

translation elongation factor 1-alpha encoding gene

Sequence similarity search tools

TrichoMARK and TrichoBLAST

TrichoMARK is highly recommended as an important tool for pre-BLAST sequence diagnosis. The program retrieves the most diagnostic fragments for the subsequent similarity search what significantly increases the accuracy of the similarity search in TrichoBLAST or NCBI BLAST

TrichoBLAST a multiloci local alignment each tool against verified database of Hypocrea/Trichoderma sequences

it includes three phylogenetic loci within tef1 gene (see here for details), ITS1 and 2 and rpb2

Detection of a new species
  • rpb2

RNA polymerase

  • tef1
  • cal1

calmoduline

  • chi18-5

endochitinase 18 - 5 (former ech42)

Multiloci phylogenetic analysis with sequences of the most related species.

Detect next neighbours using TrichoBLAST (Note: cal1 and chi18-5 are not yet included)

Retrieve sequences of corresponding sequences using ISTH Multiloci Database of Phylogenetic Markers

This database contains only verified type sequences of all molecularly characterized species of Hypocrea/Trichoderma. Database may be searched by species or locus name, sequences may be retrieved in the multiple FASTA format suitable for subsequent alignment and phylogenetic analysis

 

Copyright: Irina Druzhinina & Alexey Kopchinskiy 2004 - 2008