Genomic DNA Extraction and Barcoding of Endophytic Fungi

  • Patricia L. Diaz
  • James R. Hennell
  • Nikolaus J. Sucher
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 862)

Abstract

Endophytes live inter- and/or intracellularly inside healthy aboveground tissues of plants without causing disease. Endophytic fungi are found in virtually every vascular plant species examined. The origins of this symbiotic relationship between endophytes go back to the emergence of vascular plants. Endophytic fungi receive nutrition and protection from their hosts while the plants benefit from the production of fungal secondary metabolites, which enhance the host plants’ resistance to herbivores, pathogens, and various abiotic stresses. Endophytic fungi have attracted increased interest as potential sources of secondary metabolites with agricultural, industrial, and medicinal use. This chapter provides detailed protocols for isolation of genomic DNA from fungal endophytes and its use in polymerase chain reaction-based amplification of the internal transcribed spacer region between the conserved flanking regions of the small and large subunit of ribosomal RNA for barcoding purposes.

Key words

Fungal endophytes Internal transcribed spacer Fusarium oxysporum Polymerase chain reaction PCR Molecular identification techniques 

References

  1. 1.
    Tan RX and Zou WX (2001) Endophytes: a rich source of functional metabolites. Natural Product Reports 18(4):448–459PubMedCrossRefGoogle Scholar
  2. 2.
    Rodriguez RJ, White JF, Arnold AE, et al (2009) Fungal endophytes: diversity and ­functional roles. New Phytologist 182(2): 314–330PubMedCrossRefGoogle Scholar
  3. 3.
    Zhang HW, Song YC, and Tan RX (2006) Biology and chemistry of endophytes. Nat Prod Rep 23(5):753–771PubMedCrossRefGoogle Scholar
  4. 4.
    Seifert KA (2009) Progress towards DNA barcoding of fungi. Molecular Ecology Resources 9:83–89PubMedCrossRefGoogle Scholar
  5. 5.
    McLaughlin DJ, Hibbett DS, Lutzoni F, et al (2009) The search for the fungal tree of life. Trends Microbiol 17(11):488–497PubMedCrossRefGoogle Scholar
  6. 6.
    Stajich JE, Berbee ML, Blackwell M, et al (2009) The fungi. Curr Biol 19(18): R840–845PubMedCrossRefGoogle Scholar
  7. 7.
    Saikkonen K, Faeth SH, Helander M, et al (1998) Fungal endophytes: A continuum of interactions with host plants. Annual Review of Ecology and Systematics 29:319–343CrossRefGoogle Scholar
  8. 8.
    Belesky DP and Bacon CW (2009) Tall fescue and associated mutualistic toxic fungal endophytes in agroecosystems. Toxin Reviews 28(2–3):102–117CrossRefGoogle Scholar
  9. 9.
    Schardl CL, Grossman RB, Nagabhyru P, et al (2007) Loline alkaloids: Currencies of mutualism. Phytochemistry 68(7):980–996PubMedCrossRefGoogle Scholar
  10. 10.
    Schulz B, Boyle C, Draeger S, et al (2002) Endophytic fungi: a source of novel biologically active secondary metabolites. Mycological Research 106:996–1004CrossRefGoogle Scholar
  11. 11.
    Strobel G (2006) Harnessing endophytes for industrial microbiology. Current opinion in microbiology 9(3):240–244PubMedCrossRefGoogle Scholar
  12. 12.
    Strobel G and Daisy B (2003) Bioprospecting for microbial endophytes and their natural products. Microbiol Mol Biol Rev 67(4):491–502PubMedCrossRefGoogle Scholar
  13. 13.
    Strobel G, Daisy B, Castillo U, et al (2004) Natural products from endophytic microorganisms. Journal of natural products 67(2):257–268PubMedCrossRefGoogle Scholar
  14. 14.
    Sucher NJ and Carles MC (2008) Genome-based approaches to the authentication of medicinal plants. Planta Med 74(6):603–623PubMedCrossRefGoogle Scholar
  15. 15.
    Hebert PD, Cywinska A, Ball SL, et al (2003) Biological identifications through DNA barcodes. Proc Biol Sci 270(1512):313–321PubMedCrossRefGoogle Scholar
  16. 16.
    Stockinger H, Kruger M, and Schussler A (2010) DNA barcoding of arbuscular mycorrhizal fungi. New Phytol 187(2):461–474PubMedCrossRefGoogle Scholar
  17. 17.
    Hebert PD and Gregory TR (2005) The promise of DNA barcoding for taxonomy. Syst Biol 54(5):852–859PubMedCrossRefGoogle Scholar
  18. 18.
    Chase MW and Fay MF (2009) Barcoding of Plants and Fungi. Science 325(5941):682–683PubMedCrossRefGoogle Scholar
  19. 19.
    Martin KJ and Rygiewicz PT (2005) Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts. BMC Microbiol 5:28PubMedCrossRefGoogle Scholar
  20. 20.
    White T, Bruns T, Lee S, et al (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis M, Gelfand D, Sninsky J, & White T (ed) PCR Protocols: A Guide to Methods and Applications. Academic Press Inc, New YorkGoogle Scholar
  21. 21.
    Wang B, Priest MJ, Davidson A, et al (2007) Fungal endophytes of native Gossypium species in Australia. Mycol Res 111(3):347–354PubMedCrossRefGoogle Scholar
  22. 22.
    Egger KN (1995) Molecular Analysis of Ectomycorrhizal Fungal Communities. Can J Bot 73:S1415–S1422CrossRefGoogle Scholar
  23. 23.
    Gardes M and Bruns TD (1993) Its Primers with Enhanced Specificity for Basidiomycetes – Application to the Identification of Mycorrhizae and Rusts. Mol Ecol 2(2): 113–118PubMedCrossRefGoogle Scholar
  24. 24.
    Gargas A and DePriest PT (1996) A nomenclature for fungal PCR primers with examples from intron-containing SSU rDNA. Mycologia 88(5):745–748CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Patricia L. Diaz
    • 1
  • James R. Hennell
    • 1
  • Nikolaus J. Sucher
    • 2
  1. 1.Centre for Complementary Medicine Research, School of Science and HealthUniversity of Western SydneyPenrithAustralia
  2. 2.Department of Science, Technology, Engineering & Math (S.T.E.M.)Roxbury Community CollegeBostonUSA

Personalised recommendations