k.a. seifert - algae, protists & fungi plenary

… 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 keith.seifert@agr.gc.ca

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