Skip to main content
SpringerLink
Log in
Book cover

Mycotoxigenic Fungi pp 201–214Cite as

Targeting Fumonisin Biosynthetic Genes

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1542))

Abstract

The fungus Fusarium is an agricultural problem because it can cause disease on most crop plants and can contaminate crops with mycotoxins. There is considerable variation in the presence/absence and genomic location of gene clusters responsible for synthesis of mycotoxins and other secondary metabolites among species of Fusarium. Here, we describe a quantitative real-time PCR (qPCR) method for distinguishing between and estimating the biomass of two closely related species, F. proliferatum and F. verticillioides, that are pathogens of maize. The qPCR assay is based on differences in the two species with respect to the genomic location of the gene cluster responsible for synthesis of fumonisins, a family of carcinogenic mycotoxins. Species-specific qPCR primers were designed from unique sequences that flank one end of the cluster in each species. The primers were used in qPCR to estimate the biomass of each Fusarium species using DNA isolated from pure cultures and from maize seedlings resulting from seeds inoculated with F. proliferatum alone, F. verticillioides alone, or a 1:1 mixture of the two species. Biomass estimations from seedlings were expressed as the amount of DNA of each Fusarium species per amount of maize DNA, as determined using maize-specific qPCR primers designed from the ribosomal gene L17. Analyses of qPCR experiments using the primers indicated that the assay could distinguish between and quantify the biomass of the two Fusarium species. This finding indicates that genetic diversity resulting from variation in the presence/absence and genomic location of SM biosynthetic gene clusters can be a valuable resource for development of qPCR assays for distinguishing between and quantifying fungi in plants.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   79.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   99.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   129.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Leslie JF, Summerell BA (2006) The Fusarium laboratory manual. Blackwell Publishing, Ames

    Book  Google Scholar 

  2. Desjardins AE (2006) Fusarium mycotoxins chemistry, genetics and biology. APS Press, St. Paul

    Google Scholar 

  3. Siou D, Gelisse S, Laval V et al (2015) Interactions between head blight pathogens: consequences for disease development and toxin production in wheat spikes. Appl Environ Microbiol 81:957–965

    Article  PubMed  PubMed Central  Google Scholar 

  4. Picot A, Hourcade-Marcolla D, Barreau C et al (2012) Interactions between Fusarium verticillioides and Fusarium graminearum in maize ears and consequences for fungal development and mycotoxin accumulation. Plant Pathol 61:140–151

    Article  CAS  Google Scholar 

  5. Cole RJ, Jarvis BB, Schweikert MA (2003) Handbook of secondary fungal metabolites. Academic, San Diego

    Google Scholar 

  6. Keller NP (2015) Translating biosynthetic gene clusters into fungal armor and weaponry. Nat Chem Biol 11:671–677

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Keller NP, Turner G, Bennett JW (2005) Fungal secondary metabolism—from biochemistry to genomics. Nat Rev Microbiol 3:937–947

    Article  CAS  PubMed  Google Scholar 

  8. Frandsen RJN, Nielsen NJ, Maolanon N et al (2006) The biosynthetic pathway for aurofusarin in Fusarium graminearum reveals a close link between the napthoquinones and naphthopyrones. Mol Microbiol 61:1069–1080

    Article  CAS  PubMed  Google Scholar 

  9. Wiemann P, Willmann A, Straeten M et al (2009) Biosynthesis of the red pigment bikaverin in Fusarium fujikuroi: genes, their function and regulation. Mol Microbiol 72:931–946

    Article  CAS  PubMed  Google Scholar 

  10. Studt L, Wiemann P, Kleigrewe K et al (2012) Biosynthesis of fusarubins accounts for pigmentation of Fusarium fujikuroi perithecia. Appl Environ Microbiol 78:4468–4480

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Bömke C, Tudzynski B (2009) Diversity, regulation, and evolution of the gibberellin biosynthetic pathway in fungi compared to plants and bacteria. Phytochemistry 70:1876–1893

    Article  PubMed  Google Scholar 

  12. Alexander NJ, Proctor RH, McCormick SP (2009) Genes, gene clusters, and biosynthesis of trichothecenes and fumonisins in Fusarium. Toxin Rev 28:198–215

    Article  CAS  Google Scholar 

  13. Brown DW, Lee SH, Kim LH et al (2015) Identification of a 12-gene fusaric acid biosynthetic gene cluster in Fusarium species through comparative and functional genomics. Mol Plant Microbe Interact 28:319–332

    Article  CAS  PubMed  Google Scholar 

  14. Kim YT, Lee Y-R, Jin J et al (2005) Two different polyketide synthase genes are required for synthesis of zearalenone in Gibberella zeae. Mol Microbiol 58:1102–1113

    Article  CAS  PubMed  Google Scholar 

  15. Niehaus EM, Kleigrewe K, Wiemann P et al (2013) Genetic manipulation of the Fusarium fujikuroi fusarin gene cluster yields insight into the complex regulation and fusarin biosynthetic pathway. Chem Biol 20:1055–1066

    Article  CAS  PubMed  Google Scholar 

  16. Sørensen JL, Sondergaard TE, Covarelli L et al (2014) Identification of the biosynthetic gene clusters for the lipopeptides fusaristatin A and W493 B in Fusarium graminearum and F. pseudograminearum. J Nat Prod 77:2619–2625

    Article  PubMed  Google Scholar 

  17. Sørensen JL, Hansen FT, Sondergaard TE et al (2012) Production of novel fusarielins by ectopic activation of the polyketide synthase 9 cluster in Fusarium graminearum. Environ Microbiol 14:1159–1170

    Article  PubMed  Google Scholar 

  18. Kakule TB, Sardar D, Lin Z et al (2013) Two related pyrrolidinedione synthetase loci in Fusarium heterosporum ATCC 74349 produce divergent metabolites. ACS Chem Biol 8:1549–1557

    Article  CAS  PubMed  Google Scholar 

  19. Vesonder RF, Golinski P (1989) Metabolites of Fusarium. Fusarium Mycotoxins, Taxonomy and Pathogenicity. In: Chelkowski J (ed) Topics in secondary metabolism, vol 2. Elsevier, Amsterdam, pp 1–39

    Google Scholar 

  20. Proctor RH, Plattner RD, Brown DW et al (2004) Discontinuous distribution of fumonisin biosynthetic genes in the Gibberella fujikuroi species complex. Mycol Res 108:815–822

    Article  CAS  PubMed  Google Scholar 

  21. Proctor RH, Van Hove F, Susca A et al (2013) Birth, death and horizontal transfer of the fumonisin biosynthetic gene cluster during the evolutionary diversification of Fusarium. Mol Microbiol 90:290–306

    CAS  PubMed  Google Scholar 

  22. Stepien L, Koczyk C, Waskiewicz A (2010) FUM cluster divergence in fumonisins-producing Fusarium species. Fungal Biol 115:112–123

    Article  PubMed  Google Scholar 

  23. Wiemann P, Sieber CMK, von Bargen KW et al (2013) Deciphering the cryptic genome: genome-wide analyses of the rice pathogen Fusarium fujikuroi reveal complex regulation of secondary metabolism and novel metabolites. PLoS Pathog 9, e1003475

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Malonek S, Bömke C, Bornberg-Bauer E et al (2005) Distribution of gibberellin biosynthetic genes and gibberellin production in the Gibberella fujikuroi species complex. Phytochemistry 66:1296–1311

    Article  CAS  PubMed  Google Scholar 

  25. Glenn AE, Zitomer NC, Zimeri AM et al (2008) Transformation-mediated complementation of a FUM gene cluster deletion in Fusarium verticillioides restores both fumonisin production and pathogenicity on maize seedlings. Mol Plant Microbe Interact 21:87–97

    Article  CAS  PubMed  Google Scholar 

  26. Van Hove F, Waalwijk C, Logrieco A et al (2011) Gibberela musae (Fusarium musae) sp. nov.: a new species from banana is sister to F. verticillioides. Mycologia 103:570–585

    Article  PubMed  Google Scholar 

  27. Brown DW, Proctor RH (2016) Insights into natural products biosynthesis from analysis of 490 polyketide synthases from Fusarium. Fungal Genet Biol 89:37–51

    Article  CAS  PubMed  Google Scholar 

  28. Malonek S, Tudzynski B (2003) Evolutionary aspects of gibberellin biosynthesis in the Gibberella fujikuroi species complex. Fungal Genet Newslett 50:140

    Google Scholar 

  29. Lysoe E, Harris LJ, Walkowiak S et al (2014) The genome of the generalist plant pathogen Fusarium avenaceum is enriched with genes involved in redox, signaling and secondary metabolism. PLoS One 9, e112703

    Article  PubMed  PubMed Central  Google Scholar 

  30. O’Donnell K, Rooney AP, Proctor RH et al (2013) Phylogenetic analyses of RPB1 and RPB2 support a middle Cretaceous origin for a clade comprising all agriculturally and medically important fusaria. Fungal Genet Biol 52:20–31

    Article  PubMed  Google Scholar 

  31. Wingfield BD, Ades PK, Al-Naemi FA et al (2015) IMA genome-F 4: draft genome sequences of Chrysoporthe austroafricana, Diplodia scrobiculata, Fusarium nygamai, Leptographium lundbergii, Limonomyces culmigenus, Stagonosporopsis tanaceti, and Thielaviopsis punctulata. IMA Fungus 6:233–248

    Article  PubMed  PubMed Central  Google Scholar 

  32. Ma LJ, van der Does HC, Borkovich KA et al (2010) Comparative genomics reveal mobile pathogenicity chromosomes in Fusarium. Nature 464:367–373

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Srivastava SK, Huang X, Brar HK et al (2014) The genome sequence of the fungal pathogen Fusarium virguliforme that causes sudden death syndrome in soybean. PLoS One 9, e81832

    Article  PubMed  PubMed Central  Google Scholar 

  34. Coleman JJ, Rounsley SD, Rodriguez-Carres M et al (2009) The genome of Nectria haematococca: contribution of supernumerary chromosomes to gene expansion. PLoS Genet 5, e1000618

    Article  PubMed  PubMed Central  Google Scholar 

  35. Mulè G, Susca A, Stea G et al (2004) A species-specific PCR assay based on the calmodulin partial gene for identification of Fusarium verticillioides, F. proliferatum and F. subglutinans. Eur J Plant Pathol 110:495–502

    Article  Google Scholar 

  36. Edwards SG, Pirgozliev SR, Hare MC et al (2001) Quantification of trichothecene-producing Fusarium species in harvested grain by competitive PCR to determine efficacies of fungicides against Fusarium head blight of winter wheat. Appl Environ Microbiol 67:1575–1580

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Fernandez-Ortuno D, Waalwijk C, Van der Lee T et al (2013) Simultaneous real-time PCR detection of Fusarium asiaticum, F. ussurianum and F. vorosii, representing the Asian clade of the F. graminearum species complex. Int J Food Microbiol 166:148–154

    Article  CAS  PubMed  Google Scholar 

  38. Proctor RH, Brown DW, Plattner RD et al (2003) Co-expression of 15 contiguous genes delineates a fumonisin biosynthetic gene cluster in Gibberella moniliformis. Fungal Genet Biol 38:237–249

    Article  CAS  PubMed  Google Scholar 

  39. Taylor S, Wakem M, Dijkman G et al (2010) A practical approach to RT-qPCR-Publishing data that conform to the MIQE guidelines. Methods 50:S1–S5

    Article  CAS  PubMed  Google Scholar 

  40. Bustin SA, Benes V, Garson JA et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622

    Article  CAS  PubMed  Google Scholar 

  41. Nicolaisen M, Suproniene S, Nielsen LK et al (2009) Real-time PCR for quantification of eleven individual Fusarium species in cereals. J Microbiol Methods 76:234–240

    Article  CAS  PubMed  Google Scholar 

  42. Vaughan MM, Huffaker A, Schmelz EA et al (2014) Effects of elevated [CO2] on maize defence against mycotoxigenic Fusarium verticillioides. Plant Cell Environ 37:2691–2706

    Google Scholar 

  43. Proctor RH, McCormick SP, Alexander NJ et al (2009) Evidence that a secondary metabolic biosynthetic gene cluster has grown by gene relocation during evolution of the filamentous fungus Fusarium. Mol Microbiol 74:1128–1142

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. USDA ARS NCAUR, 1815 N. University St., Peoria, IL, 61604, USA

    Robert H. Proctor

  2. United States Department of Agriculture, National Center for Agricultural Utilization Research, Peoria, IL, USA

    Robert H. Proctor & Martha M. Vaughan

Authors
  1. Robert H. Proctor
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Martha M. Vaughan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Robert H. Proctor .

Editor information

Editors and Affiliations

  1. Institute of Sciences of Food Production National Research Council, Bari, Italy

    Antonio Moretti

  2. Institute of Sciences of Food Production National Research Council, Bari, Italy

    Antonia Susca

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Proctor, R.H., Vaughan, M.M. (2017). Targeting Fumonisin Biosynthetic Genes. In: Moretti, A., Susca, A. (eds) Mycotoxigenic Fungi. Methods in Molecular Biology, vol 1542. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6707-0_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6707-0_13

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6705-6

  • Online ISBN: 978-1-4939-6707-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation