… The plague is on the walls of the housewith ingrained streaks, greenish orreddish … and the dust that they scrapeoff the...
microbial culturing                    (conventional & d2e)field biology                          DNA detection  (in a hou...
Penicillium italicum      Kent Loeffler / Kathie Hodge          Cornell Mushroom Blog
Stachybotrys
Classical indoor moulds in the Eumycota  Common mycobiota: 100-200 species                         Eurotiomycetes         ...
Wallemia sebi
IM-BOL Global house dust survey• What is the indoor fungal diversity on a world scale?• What is the correlation in the pro...
Next generation DNA sequencing  • Millions of sequences directly from a sample      – Culture independent  • In fungi, usu...
Global dust survey: Pyrosequencing results                                    ITS (1 and 2)               LSU             ...
Hoekstra et al. 1994. In Health implications of fungi inindoor environments. ed. Samson et al. pp. 169-177.
Wallemiomycetes                  Sordariomycetes                                                                          ...
454 sequences … Cladosporium                                                 Alignment minus 170 bases                    ...
454 sequences… AlternariaReference alignment from: Pryor, B. M., and Gilbertson, R. L.2000. Mycological Research 104: 1312...
454 sequences … Epicoccum                            In house ITS data plus GenBank data
… a problem with unidentified sequencesFX2FH6V03C9FN4 – ID = DikaryaATCCCTACCTGATCCGAGGTCAACCGTAGAAATGGGGGTTTCTGGAGGCGGGCG...
Dilution to Extinction (d2e)
‘High throughput’ isolation from global dust samples
Cryptocoryneum rilstonei
Triadelphia uniseptata
Bartheletia paradoxa
Keith’s house. Pyro-fungi.LCA analysis600 unique DNA sequences53 unassigned to taxonomic order (<10%)
Keith’s house. Pyro-fungi.
Fusarium sambucinum dry rot of potato      (teleomorph: Gibberella pulicaris)
Fusarium oxysporum wilt of basil
Keith’s house. Pyro-fungi.
Friday Afternoon Mycologist / Kathie Hodge / Kent Loeffler                                  Cornell Mushroom Blog
Keith’s house. Pyro-fungi.                                          Conifer endophytes600 unique sequences53 unassigned to...
www.cbs.knaw.nl/indoor   • now includes classical isolations only   • d2e isolations to be added   • to be linked with MoB...
IM-Bol conclusions• Vast increase in indoor fungal species diversity on the  global scale• Enhanced importance of the clas...
Concluding thoughts• Identifications in environmental DNA studies   – Very sensitive to data gaps and taxonomic imprecisio...
microbial culturing                    (conventional & d2e)field biology                          DNA detection  (in a hou...
pyrosequencing                 direct observation     culturing
pyrosequencing                                      direct observation  alternative culturing   culturing
Thanks to                       IM-BOL:        The Indoor Alfred P. Sloan Foundation of Life                   Mycota Barc...
K.A. Seifert - Algae, Protists & Fungi Plenary
K.A. Seifert - Algae, Protists & Fungi Plenary
K.A. Seifert - Algae, Protists & Fungi Plenary
K.A. Seifert - Algae, Protists & Fungi Plenary
K.A. Seifert - Algae, Protists & Fungi Plenary
K.A. Seifert - Algae, Protists & Fungi Plenary
K.A. Seifert - Algae, Protists & Fungi Plenary
K.A. Seifert - Algae, Protists & Fungi Plenary
K.A. Seifert - Algae, Protists & Fungi Plenary
K.A. Seifert - Algae, Protists & Fungi Plenary
K.A. Seifert - Algae, Protists & Fungi Plenary
K.A. Seifert - Algae, Protists & Fungi Plenary
K.A. Seifert - Algae, Protists & Fungi Plenary
K.A. Seifert - Algae, Protists & Fungi Plenary
K.A. Seifert - Algae, Protists & Fungi Plenary
K.A. Seifert - Algae, Protists & Fungi Plenary
K.A. Seifert - Algae, Protists & Fungi Plenary
K.A. Seifert - Algae, Protists & Fungi Plenary
K.A. Seifert - Algae, Protists & Fungi Plenary
K.A. Seifert - Algae, Protists & Fungi Plenary
K.A. Seifert - Algae, Protists & Fungi Plenary
K.A. Seifert - Algae, Protists & Fungi Plenary
K.A. Seifert - Algae, Protists & Fungi Plenary
K.A. Seifert - Algae, Protists & Fungi Plenary
K.A. Seifert - Algae, Protists & Fungi Plenary
K.A. Seifert - Algae, Protists & Fungi Plenary
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K.A. Seifert - Algae, Protists & Fungi Plenary

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K.A. Seifert - Algae, Protists & Fungi Plenary

  1. 1. … The plague is on the walls of the housewith ingrained streaks, greenish orreddish … and the dust that they scrapeoff they shall pour out in an unclean placeoutside the city. Leviticus 14
  2. 2. microbial culturing (conventional & d2e)field biology DNA detection (in a house) (pyrosequencing) comprehensive DNA barcode databases
  3. 3. Penicillium italicum Kent Loeffler / Kathie Hodge Cornell Mushroom Blog
  4. 4. Stachybotrys
  5. 5. Classical indoor moulds in the Eumycota Common mycobiota: 100-200 species Eurotiomycetes • Penicillium and Aspergillus Dothideomycetes • Alternaria • Cladosporium Sordariomycetes • Fusarium • Stachybotrys Wallemiomycetes • Wallemia
  6. 6. Wallemia sebi
  7. 7. IM-BOL Global house dust survey• What is the indoor fungal diversity on a world scale?• What is the correlation in the profile of species detected bydifferent methods?• Are we talking about unculturable or uncultured organisms?• What is the biological significance of the species detected?
  8. 8. Next generation DNA sequencing • Millions of sequences directly from a sample – Culture independent • In fungi, usually ITS +/- 28S • Many platforms and technologies – 454 pyrosequencing – Illumina sequencing – Ion Torrent 18S 5.8S 5.8S 8 25- 25-28SIGS ITS1 S ITS1 ITS2 ITS2 S IGS
  9. 9. Global dust survey: Pyrosequencing results ITS (1 and 2) LSU • 7,032 OTUs Sequence Length 397 bp 445 bp • 56% represented once Mean Total Reads 288,361 282,883 (204,000) (183,000)Amend et al., 2010. Proc. Nat. Acad. Sci. 107, 13748-13753.
  10. 10. Hoekstra et al. 1994. In Health implications of fungi inindoor environments. ed. Samson et al. pp. 169-177.
  11. 11. Wallemiomycetes Sordariomycetes LeotiomycetesDothideomycetes Eurotiomycetes Tremellomycetes Classical isolation study - Netherlands454 pyrosequencing study – Northern hemisphere dy Nort y ? Data reformatted from: Hoekstra et al. 1994. In Health implications of fungi in indoor environments. ed. Samson et al. pp. 169-177. Amend et al., 2010. Proc. Nat. Acad. Sci. 107, 13748- 13753.
  12. 12. 454 sequences … Cladosporium Alignment minus 170 bases Reference alignment from TreeBase: Schubert et al. 2009. Persoonia, 22: 111-122.Full alignment
  13. 13. 454 sequences… AlternariaReference alignment from: Pryor, B. M., and Gilbertson, R. L.2000. Mycological Research 104: 1312-1321.
  14. 14. 454 sequences … Epicoccum In house ITS data plus GenBank data
  15. 15. … a problem with unidentified sequencesFX2FH6V03C9FN4 – ID = DikaryaATCCCTACCTGATCCGAGGTCAACCGTAGAAATGGGGGTTTCTGGAGGCGGGCGGCGCCGAACCTGGAAAGCTGGAGAATTTACTACGCTTGAGGTTCAACACCACCGCCGAGGCCTTTAGGGAGCGTCCGCGACAGGGACGGCACCCAATACCAAGCAGGGCTTGAAGGTTGATAATGACGCTCGAACAGGCATGCTCCCCGGAATACCAGGGAGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCAG
  16. 16. Dilution to Extinction (d2e)
  17. 17. ‘High throughput’ isolation from global dust samples
  18. 18. Cryptocoryneum rilstonei
  19. 19. Triadelphia uniseptata
  20. 20. Bartheletia paradoxa
  21. 21. Keith’s house. Pyro-fungi.LCA analysis600 unique DNA sequences53 unassigned to taxonomic order (<10%)
  22. 22. Keith’s house. Pyro-fungi.
  23. 23. Fusarium sambucinum dry rot of potato (teleomorph: Gibberella pulicaris)
  24. 24. Fusarium oxysporum wilt of basil
  25. 25. Keith’s house. Pyro-fungi.
  26. 26. Friday Afternoon Mycologist / Kathie Hodge / Kent Loeffler Cornell Mushroom Blog
  27. 27. Keith’s house. Pyro-fungi. Conifer endophytes600 unique sequences53 unassigned to taxonomic order (<10%)
  28. 28. www.cbs.knaw.nl/indoor • now includes classical isolations only • d2e isolations to be added • to be linked with MoBEDAC
  29. 29. IM-Bol conclusions• Vast increase in indoor fungal species diversity on the global scale• Enhanced importance of the class Dothideomycetes – especially Epicoccum, Phoma and Alternaria• Confirmed importance of the class Eurotiomycetes – especially Penicillium and Aspergillus• Confirmed significance of the genus Wallemia – undoubtedly several species rather than one
  30. 30. Concluding thoughts• Identifications in environmental DNA studies – Very sensitive to data gaps and taxonomic imprecision – Especially Last Common Ancestor analysis – Results can vary day by day!• Dilution to extinction – Very labour intensive – Isolates common as well as rare, unusual fungi – Including species not detected by pyrosequencing• Determining biological significance requires multifaceted approach – Living growing moulds vs transients or dead cells – Are all fungi really detected by 454 sequencing?
  31. 31. microbial culturing (conventional & d2e)field biology DNA detection (in a house) (pyrosequencing) comprehensive DNA barcode databases
  32. 32. pyrosequencing direct observation culturing
  33. 33. pyrosequencing direct observation alternative culturing culturing
  34. 34. Thanks to IM-BOL: The Indoor Alfred P. Sloan Foundation of Life Mycota Barcode Real moulds and virtual moulds in your house Genome Canada, NSERC, Canadian Network for DNA Barcoding, Consortium for the Barcode of Life Keith A. Seifert, Joey Tanney, Kalima Mwange, Hai Nguyen, Ed Whitfield Biodiversity, Agriculture & Agri-Food Canada, Ottawa, Canada ECORC colleagues: Tom Graefenhan, Wen Chen, Chris Lewis, Robert A. Samson, Martin Meijer, Vincent Robert André Lévesque CBS Fungal Biodiversity Centre, Utrecht, the Netherlands Thomas D. Bruns, Anthony Amend Plant & Microbial Biology, University of California at Berkeley, USAKeith Seifert keith.seifert@agr.gc.ca

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