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Progress toward DNA barcoding the vast diversity of fungi . Dr. Amy Y. Rossman USDA Agricultural Research Service Systematic Mycology & Microbiology Laboratory U.S. National Fungus Collections. Fungi are everywhere!. Mushrooms Polypores Mycorrhizae Morels Truffles Yeasts Parasites

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progress toward dna barcoding the vast diversity of fungi

Progress toward DNA barcoding the vast diversity of fungi

Dr. Amy Y. Rossman

USDA Agricultural Research Service

Systematic Mycology & Microbiology Laboratory

U.S. National Fungus Collections

fungi are everywhere
Fungi are everywhere!











Fungi are polyphyletic. True fungi are more closely related to animals than to any other major group of organisms. The Oomycetes including the cause of Sudden Oak Death (SOD) and late blight of potatoes are more closely related to heterokont algae than other fungi. Myxomycetes or slime molds are relatively closely related to protozoans.

Rhododendron with Sudden Oak Death

Oospores of Phytophthora ramorum, cause of SOD

Based on ratio of fungi:plant of 5-6:1 in a few well-studied places. Only 5-10% are known, thus 90-95% have yet to be discovered.

Fungi are extremely diverse,

second only to the insects in number of species.

1.5 million species estimated to exist


All Fungi Barcode of Life Planning Workshop

Smithsonian Conservation and Research Center

Front Royal, Virginia

13-15 May 2007


FBI – Fungal Barcode Initiative

Connecting above ground with below ground fungi


Bolete fruiting

Conifer forest in Northern California

photographed Feb. 15, 2003 by Eric Guinther.

Healthy forests

aecidial state of black stem rust on unrelated host barberry

FBI – Fungal Barcode Initiative

Connecting alternate states of plant pathogenic fungi

Aecidial state of black stem rust on unrelated host - barberry

Black stem rust

of wheat


Many, many undescribed species of arbuscular mycorrhizal fungi in one deciduous forest in Japan.Yamoto, M. & K. Iwase. 2005. Mycoscience 46: 334-342.


Environmental samples of fungi in alpine soils.

Are these unknown or just unsequenced lineages?

Schadt et al in Science 301:1359-1361. 2003

what region to use for dna barcode for fungi
What region to use for DNA barcode for fungi?



ITS plus LSU

ITS plus tef, beta tubulin, RPB1


COI in Penicillium and Aspergillus

Seifert et al. (2007) PNAS. COI primers were designed from Penicillium marneffei and from two Aspergillus species.

COI distinguished most species in Penicillum sect. Penicillium.

And, the authors note a problem with COI introns . . .


Figure 3

Taylor and Berbee 2006

Mycologia 98:838-849


All Fungi Barcode of Life Planning Workshop

Decision on DNA Barcode for Fungi

ITS 1- 5.8S - ITS 2, ~525-700 bp


All Fungi Barcode of Life Planning Workshop

Decision on DNA Barcode for Fungi

ITS 1- 5.8S - ITS 2, ~525-700 bp


Stable priming regions, thus few primers can cover the fungi.

Variable regions provide specificity.

Size of one sequencing run, ~ 525-700bp.

Many ITS sequences already exist.


Not a good region for phylogenetics

ITS variation does not necessarily match phylogenetic species.


UNITE - reliable identification of ectomycorrhizal fungi - DNA barcoding in action

Urmas Kõljalg

(University of Tartu, Estonia)

Joe Bishoff, Francois Lutzoni, & Urmas Kõljalg


1)Fungal fruitbodies from which DNA are extracted must be maintained in public herbaria.

2) Only specimens that are well developed and show all the structures necessary for an accurate identification should be used as sources of DNA

3) All sequenced specimens must be identified by an experts and the name of the expert referenced in the database


4) DNA from sequenced sporocarps should be saved and maintained along with the sporocarps

5) Sequences must be associated with specimen data

6) Whenever possible, specimens from which DNA is derived should be illustrated (e.g. photos)


Database structure and data

At present, the database holds only sequences from the ITS regions.

As of Sep 3 2007, the UNITE database includes 2511 ITS sequences of 1046 species from 118 genera of basidio- and ascomycete fungi.


Tomentella & Thelephora

695 ITS sequences

fruit body 229

ectomycorrhiza 341

orchid mycorrhiza 72


Europe 402

N-America 202

S-America 33

Australasia 28



Integrated multiloci BarCode system for Hypocrea/Trichoderma species identification powered by sequence diagnosis, oligonucleotide BarCode and similarity search tools

Irina S. Druzhinina, Alexey G. Kopchinskiy, Christian P. Kubicek

Group of Fungal Evolution and Biodiversity

Research Area of Gene Technology and Applied Biochemistry

Institute of Chemical Engineering, Vienna University of Technology


Irina Druzhinina &

Barbara Paulus


…become acquainted with Trichoderma

mitosporic genus of Hypocrea

Hypocreales, Ascomycota

present in nearly all soils and other diverse habitats

in soil, they frequently are the most prevalent culturable fungi

the third most important genus of industrial fungi

photo: W. Jaklitsch, 2005


opportunistic pathogens of mammals


green mould disease of cultivated Agaricus and

Pleurotus in North America,

Europe and Asia

Skin lesion on the medial aspect of the wrist of a pediatric patient with aplastic anemia

Munoz et al. (1997): J. Clin. Microbiol. 35, 499-503

Hatvani, Kreditcs, 2006

…become acquainted with Trichoderma


promoters of plant growth

roots of faba bean (Vicia faba)

with Trichoderma

without Trichoderma

T. Belayneh, Ethiopia, 2005

…become acquainted with Trichoderma


Trichoderma theobromicola reisolated from the upper portion of cacao stem 1 m after inoculation of cacao shoots with conidial suspension (Samuels et al., 2006)


textile industry

food industry and agriculture

used in poultry feed to increase the digestibility of hemicelluloses from barley or other crops

"biostoning" of denim fabrics to give rise to the soft, whitened fabric-stone-washed denim

…become acquainted with Trichoderma

efficient producers of many extracellular enzymes


…identification of Trichoderma species

molecular identification

sequence similarity search (for example, BLAST)

TrichoBLAST, Kopchinskiy et al., 2005

phylogenetic analysis (single locus or multilocii)

DNA BarCode


…an oligonucleotide BarCode for Trichoderma


ITS1 and 2 sequences


ITS1 and 2 alleles



Second gene region, tef, often needed forspecies identification


Druzhinina et al., 2005

Jaklitsch et al., 2006b

…ITS1 and 2 phylogeny is concordant with other loci

Bayesian radial tree showing Hypocrea/Trichoderma phylogeny based on ITS1 and 2

Bayesian phylogenetic tree as inferred from the analysis of partial rpb2 sequences


Rapid identification of indoor air fungi

Aeroallergens, black mold (Stachybotrys chartarum)


Correlate fungi with environmental parameters

Rapid identification of indoor and outdoor air fungi


Dirty dust. The global transport of dust carries microbes across oceans and continents.Image credit: NASA


Symptoms and effects of Aspergillus infection on the sea fan Gorgoniaventalina. Infected sea fans are charaterized by lesions of necrotic tissue surrounded by a dark purple halo. Photo by Kiho Kim.


Tracking movement of plant pathogenic fungi

2003: While soybean rust has not yet been identified in the United States, researchers say it's just a matter of time until it enters. The yellow areas indicate countries where soybean rust has been identified.

2004: Discovered following Hurricane Hugo, urediniospores in upper atmosphere


Mycologists are excited about DNA barcoding and have a number of projects in process.

For fungi, ITS is used almost universally and

was selected as the standard DNA barcode region.

In some groups ITS is not unique for individual species and must be combined with second gene.

For many groups of fungi, the basic systematic knowledge does not yet exist to develop DNA barcodes.

Mycologists are poised to tackle one or more big international collaborative projects.

No Fungi

No Future!