# Presentation: 467593

## 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 **
**Saprobes**
**Molds**

## 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

## 2nd Workshop on DNA Barcoding of Fungi17 September 2007, Taipei, Taiwan

## Ectomycorrhizae

**FBI – Fungal Barcode Initiative**
**Connecting above ground with below ground fungi**

**Bolete fruiting**

**Healthy forests**

- Conifer forest in Northern California
- photographed Feb. 15, 2003 by Eric Guinther.

## Aecidial state of black stem rust on unrelated host - barberry

**FBI – Fungal Barcode Initiative**
**Connecting alternate states of plant pathogenic fungi **

**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

**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?

**COI**
**ITS of rDNA**
**ITS plus LSU**
**ITS plus tef, beta tubulin, RPB1**

## COI in Penicillium and Aspergillus

- COI distinguished most species in *Penicillum* sect. *Penicillium*.
- And, the authors note a problem with COI introns . . .

- http://www.botany.utoronto.ca/ResearchLabs/MallochLab/Malloch/Moulds

- COI in *Penicillium *and* Aspergillus*

## Seifert et al. 2007

## Figure 3

- Taylor and Berbee 2006
- Mycologia 98:838-849

## Figure 3

## Figure 3

## 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
*Positives:*
- 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.
*Negatives*:
- 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)**
- http://unite.ut.ee

**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**

## 5) Sequences must be associated with specimen data

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

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

## 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.**

## Slide 23

## Slide 24

## Tomentella & Thelephora

***Tomentella***** & Thelephora**
**695 ITS sequences**
**fruit body 229**
**ectomycorrhiza 341**
**orchid mycorrhiza 72**
**etc.**
**Europe 402**
**N-America 202**
**S-America 33**
**Australasia 28**
**etc.**

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

**Group of Fungal Evolution and Biodiversity**
- Research Area of Gene Technology and Applied Biochemistry
- Institute of Chemical Engineering, Vienna University of Technology
**AUSTRIA**

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

**Irina Druzhinina & **
**Barbara Paulus**

## ...become acquainted with Trichoderma

- ...become acquainted with *Trichoderma*

**mitosporic genus of *****Hypocrea***
**Hypocreales, Ascomycota **

**the third most important genus of industrial fungi **

**present in nearly all soils and other diverse habitats**
**in soil, they frequently are the most prevalent culturable fungi**

**photo: W. Jaklitsch, 2005**

## ...become acquainted with Trichoderma

## ...become acquainted with Trichoderma

**endophytes**

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

## efficient producers of many extracellular enzymes 

**efficient producers of many extracellular enzymes **

## Slide 31

## DNA BarCode

**molecular identification**

- sequence similarity search (for example, BLAST)
**	****	****   *****Tricho*****BLAST, Kopchinskiy et al., 2005**

- phylogenetic analysis (single locus or multilocii)

**DNA BarCode**

## >1500

**>1500**

**ITS1 and 2 sequences**

**~180**

**ITS1 and 2 alleles**

**~100**

**species**

- ...an oligonucleotide BarCode for *Trichoderma*

**Second gene region, tef, often needed for**** ****species identification**

## ...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

**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 Gorgonia ventalina. Infected sea fans are charaterized by lesions of necrotic tissue surrounded by a dark purple halo. Photo by Kiho Kim. 

**Symptoms and effects of *****Aspergillus***** infection on the sea fan *****Gorgonia***** *****ventalina*****. 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

- _[http://images.google.com/imgres?imgurl=http://cropwatch.unl.edu/photos/](http://images.google.com/imgres?imgurl=http://cropwatch.unl.edu/photos/)_
**2004: Discovered following Hurricane Hugo, urediniospores in upper atmosphere**

**Tracking movement of plant pathogenic fungi**

## Equipment available for effective sampling at high altitudes

**Equipment available for effective sampling at high altitudes**

## No Fungi

**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!**