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… The plague is on the walls of the house with ingrained streaks, greenish or reddish … and the dust that they scrape off they shall pour out in an unclean place outside the city.
Leviticus 14
microbial culturing (conventional & d2e)
DNA detection (pyrosequencing)
field biology (in a house)
comprehensive DNA barcode databases
Penicillium italicum Kent Loeffler / Kathie Hodge
Cornell Mushroom Blog
Stachybotrys
Classical indoor moulds in the Eumycota
Eurotiomycetes
• Penicillium and Aspergillus
Dothideomycetes
• Alternaria
• Cladosporium
Sordariomycetes
• Fusarium
• Stachybotrys
Wallemiomycetes
• Wallemia
Common mycobiota: 100-200 species
Wallemia sebi
• What is the indoor fungal diversity on a world scale?
• What is the correlation in the profile of species detected by different methods?
• Are we talking about unculturable or uncultured organisms?
• What is the biological significance of the species detected?
IM-BOL Global house dust survey
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
5.8S 25-28S
ITS1 ITS2 IGS
18S
IGS
5.88S 2255-
ITSS1 ITSS2
Global dust survey: Pyrosequencing results
Amend et al., 2010. Proc. Nat. Acad. Sci. 107, 13748-13753.
ITS (1 and 2) LSU
Sequence Length Mean
397 bp
445 bp
Total Reads 288,361 (204,000)
282,883 (183,000)
• 7,032 OTUs • 56% represented once
Hoekstra et al. 1994. In Health implications of fungi in indoor environments. ed. Samson et al. pp. 169-177.
Classical isolation study - Netherlands
454 pyrosequencing study – Northern hemisphere
Dothideomycetes
Sord
ario
myc
etes
Leot
iom
ycet
es
Tremellomycetes
Wal
lem
iom
ycet
es
Eurotiomycetes
? 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.
dyy –– ortNN
454 sequences … Cladosporium
Reference alignment from TreeBase: Schubert et al. 2009. Persoonia, 22: 111-122.
Full alignment
Alignment minus 170 bases
454 sequences… Alternaria
Reference alignment from: Pryor, B. M., and Gilbertson, R. L. 2000. Mycological Research 104: 1312-1321.
454 sequences … Epicoccum
In house ITS data plus GenBank data
FX2FH6V03C9FN4 – ID = Dikarya ATCCCTACCTGATCCGAGGTCAACCGTAGAAATGGGGGTTTCTGGAGGCGGGCGGCGCCGAACCTGGAAAGCTGGAGAATTTACTACGCTTGAGGTTCAACACCACCGCCGAGGCCTTTAGGGAGCGTCCGCGACAGGGACGGCACCCAATACCAAGCAGGGCTTGAAGGTTGATAATGACGCTCGAACAGGCATGCTCCCCGGAATACCAGGGAGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCAG
… a problem with unidentified sequences
Dilution to Extinction (d2e)
‘High throughput’ isolation from global dust samples
Cryptocoryneum rilstonei
Triadelphia uniseptata
Bartheletia paradoxa
600 unique DNA sequences 53 unassigned to taxonomic order (<10%)
Keith’s house. Pyro-fungi.LCA analysis
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
Conifer endophytes
600 unique sequences 53 unassigned to taxonomic order (<10%)
Keith’s house. Pyro-fungi.
www.cbs.knaw.nl/indoor
• now includes classical isolations only
• d2e isolations to be added
• to be linked with MoBEDAC
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
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?
microbial culturing (conventional & d2e)
DNA detection (pyrosequencing)
field biology (in a house)
comprehensive DNA barcode databases
pyrosequencing
culturing
direct observation
pyrosequencing
culturing
direct observation
alternative culturing
Keith A. Seifert, Joey Tanney, Kalima Mwange, Hai Nguyen, Ed Whitfield
Biodiversity, Agriculture & Agri-Food Canada, Ottawa, Canada
Robert A. Samson, Martin Meijer, Vincent Robert CBS Fungal Biodiversity Centre, Utrecht, the Netherlands
Thomas D. Bruns, Anthony Amend Plant & Microbial Biology, University of California at Berkeley, USA
IM-BOL: The Indoor Mycota Barcode of Life Real moulds and virtual moulds in your house
Keith Seifert [email protected]
Thanks to
Alfred P. Sloan Foundation
Genome Canada, NSERC, Canadian Network for DNA Barcoding, Consortium for the Barcode of Life
ECORC colleagues: Tom Graefenhan, Wen Chen, Chris Lewis,
André Lévesque
