Progress toward DNA barcoding the vast diversity of fungi

1. 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

2. Fungi are everywhere! Mushrooms Polypores Mycorrhizae Morels Truffles Yeasts Parasites Saprobes Molds

3. 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

4. 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

5. All Fungi Barcode of Life Planning Workshop Smithsonian Conservation and Research Center Front Royal, Virginia 13-15 May 2007

6. 2nd Workshop on DNA Barcoding of Fungi17 September 2007, Taipei, Taiwan

7. FBI – Fungal Barcode Initiative Connecting above ground with below ground fungi Ectomycorrhizae Bolete fruiting Conifer forest in Northern California photographed Feb. 15, 2003 by Eric Guinther. Healthy forests

8. 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

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

10. Environmental samples of fungi in alpine soils. Are these unknown or just unsequenced lineages? Schadt et al in Science 301:1359-1361. 2003

11. What region to use for DNA barcode for fungi? COI ITS of rDNA ITS plus LSU ITS plus tef, beta tubulin, RPB1

12. COI in Penicillium and Aspergillus http://www.botany.utoronto.ca/ResearchLabs/MallochLab/Malloch/Moulds 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 . . .

13. Seifert et al. 2007

14. Figure 3 Taylor and Berbee 2006 Mycologia 98:838-849

15. Figure 3

16. Figure 3

17. All Fungi Barcode of Life Planning Workshop Decision on DNA Barcode for Fungi ITS 1- 5.8S - ITS 2, ~525-700 bp

18. 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.

19. 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

20. 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

21. 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)

22. 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.

25. 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.

26. 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 AUSTRIA Irina Druzhinina & Barbara Paulus

27. …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

28. opportunistic pathogens of mammals mycopathogens 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

29. promoters of plant growth roots of faba bean (Vicia faba) with Trichoderma without Trichoderma T. Belayneh, Ethiopia, 2005 …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)

30. 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

32. …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

33. …an oligonucleotide BarCode for Trichoderma >1500 ITS1 and 2 sequences ~180 ITS1 and 2 alleles ~100 species Second gene region, tef, often needed forspecies identification

34. 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

35. Rapid identification of indoor air fungi Aeroallergens, black mold (Stachybotrys chartarum)

36. Correlate fungi with environmental parameters Rapid identification of indoor and outdoor air fungi

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

38. 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.

39. 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. http://images.google.com/imgres?imgurl=http://cropwatch.unl.edu/photos/ 2004: Discovered following Hurricane Hugo, urediniospores in upper atmosphere

40. Equipment available for effective sampling at high altitudes

41. 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!