High-Throughput DNA Sequence-Based Analysis of AMF Communities

Part of the Methods in Molecular Biology book series (MIMB, volume 2146)


Arbuscular mycorrhizal fungi (AMF) are obligate symbionts of most land plants. They have great ecological and economic impacts as they support plant nutrition and water supply, soil structure, and plant resistance to pathogens. Investigating AMF presence and distribution at small and large scales is critical. Therefore, research requires standard protocols to be easily implemented. In this chapter, we describe a workflow for AMF identification by high-throughput sequencing through Illumina MiSeq platform of two DNA target regions: small subunit (SSU) and internal transcribed spacer (ITS). The protocol can apply to both soil and root AMF communities.

Key words

Arbuscular mycorrhizal fungi (AMF) DNA-based species characterization High-throughput sequence Illumina MiSeq ITS primers SSU primers 



The described work was possible thanks to the financial support of the Fondazione Cassa di Risparmio di Torino (n. 2017.1966) to the project “SaffronAlp” (Italy), the FIAM bilateral project titled “MYCOTTON” (Mozambique), and the European LUCAS project.


  1. 1.
    Montagna M, Berruti A, Bianciotto V et al (2018) Differential biodiversity responses between kingdoms (plants, fungi, bacteria and metazoa) along an alpine succession gradient. Mol Ecol 27:3671–3685CrossRefGoogle Scholar
  2. 2.
    Taylor T, Remy W, Hass H, Kerp H (1995) Fossil arbuscular mycorrhizae from the early Devonian. Mycology 87:560–573CrossRefGoogle Scholar
  3. 3.
    Lichtfouse E (2017) Sustainable agriculture reviews, 1st edn. Springer International Publishing, MarseilleCrossRefGoogle Scholar
  4. 4.
    Brundrett M (2009) Mycorrhizal associations and other means of nutrition of vascular plants: understanding the global diversity of host plants by resolving conflicting information and developing reliable means of diagnosis. Plant Soil 320:37–77CrossRefGoogle Scholar
  5. 5.
    Öpik M, Davison J (2016) Uniting species and community-oriented approaches to understand arbuscular mycorrhizal fungal diversity. Fungal Ecol 24:106–113CrossRefGoogle Scholar
  6. 6.
    Kivlin SN, Hawkes CV, Treseder KK (2011) Global diversity and distribution of arbuscular mycorrhizal fungi. Soil Biol Biochem 43:2294–2303CrossRefGoogle Scholar
  7. 7.
    Öpik M, Davison J, Moora M, Zobel M (2014) DNA-based detection and identification of Glomeromycota: the virtual taxonomy of environmental sequences. Botany 92:135–147CrossRefGoogle Scholar
  8. 8.
    Öpik M, Metsis M, Daniell TJ et al (2009) Large-scale parallel 454 sequencing reveals host ecological group specificity of arbuscular mycorrhizal fungi in a boreonemoral forest. New Phytol 184:424–437Google Scholar
  9. 9.
    Stürmer SL, Bever JD, Morton JB (2018) Biogeography of arbuscular mycorrhizal fungi (Glomeromycota): a phylogenetic perspective on species distribution patterns. Mycorrhiza 28:587–603CrossRefGoogle Scholar
  10. 10.
    Davison J, Moora M, Öpik M et al (2015) Global assessment of arbuscular mycorrhizal fungus diversity reveals very low endemism. Science 349:970–973CrossRefGoogle Scholar
  11. 11.
    Hart M, Aleklett K, Chagnon P et al (2015) Navigating the labyrinth: a guide to sequence-based, community ecology of arbuscular mycorrhizal fungi. New Phytol 207:235–247CrossRefGoogle Scholar
  12. 12.
    Lekberg Y, Vasar M, Bullington LS et al (2018) More bang for the buck? Can arbuscular mycorrhizal fungal communities be characterized adequately alongside other fungi using general fungal primers? New Phytol 220:971–976CrossRefGoogle Scholar
  13. 13.
    Lindahl BD, Nilsson RH, Tedersoo L et al (2013) Fungal community analysis by high-throughput sequencing of amplified markers—a user’s guide. New Phytol 199:288–299CrossRefGoogle Scholar
  14. 14.
    Vasar M, Andreson R, Davison J et al (2017) Increased sequencing depth does not increase captured diversity of arbuscular mycorrhizal fungi. Mycorrhiza 27:761–773CrossRefGoogle Scholar
  15. 15.
    Corradi N, Brachmann A (2017) Fungal mating in the most widespread plant symbionts? Trends Plant Sci 22:175–183CrossRefGoogle Scholar
  16. 16.
    Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes - application to the identification of mycorrhizae and rusts. Mol Ecol 2:113–118CrossRefGoogle Scholar
  17. 17.
    White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis M, Gelfand D, Sninsky J, White T (eds) PCR protocols: a guide to methods and applications. Academic Press Inc, New York, p 315Google Scholar
  18. 18.
    Ihrmark K, Bodeker I, Cruz-Martinez K et al (2012) New primer to amplify the fungal ITS 2 region-evaluation by 454-sequencing of artificial and natural communities. FEMS Microbiol Ecol 82:666–677CrossRefGoogle Scholar
  19. 19.
    Berruti A, Desirò A, Visentin S et al (2017) ITS fungal barcoding primers versus 18S AMF-specific primers reveal similar AMF-based diversity patterns in roots and soils of three mountain vineyards. Environ Microbiol Rep 9:658–667CrossRefGoogle Scholar
  20. 20.
    Lee J, Lee S, Young JPW (2008) Improved PCR primers for the detection and identification of arbuscular mycorrhizal fungi. FEMS Microbiol Ecol 65:339–349CrossRefGoogle Scholar
  21. 21.
    Öpik M, Vanatoa A, Vanatoa E et al (2010) The online database MaarjAM reveals global and ecosystemic distribution patterns in arbuscular mycorrhizal fungi (Glomeromycota). New Phytol 188:223–241CrossRefGoogle Scholar
  22. 22.
    Van Geel M, Busschaert P, Honnay O, Lievensv B (2014) Evaluation of six primer pairs targeting the nuclear rRNA operon for characterization of arbuscular mycorrhizal fungal (AMF) communities using 454 pyrosequencing. J Microbiol Methods 106:93–100CrossRefGoogle Scholar
  23. 23.
    Stockinger H, Kruger M, Schußler A (2010) DNA barcoding of arbuscular mycorrhizal fungi. New Phytol 187:461–474CrossRefGoogle Scholar
  24. 24.
    Schoch C, Seifert K, Huhndorf S et al (2012) Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for fungi. Proc Natl Acad Sci 109:6241–6246CrossRefGoogle Scholar
  25. 25.
    Krüger M, Stockinger H, Krüger C, Schußler A (2009) DNA-based species-level detection of Glomeromycota: one PCR primer set for all arbuscular mycorrhizal fungi. New Phytol 183:212–223CrossRefGoogle Scholar
  26. 26.
    Tedersoo L, Bahram M, Polme S et al (2015) Response to comment on ‘global diversity and geography of soil fungi. Science 349:936CrossRefGoogle Scholar
  27. 27.
    Tedersoo L, Tooming-Klunderud A, Anslan S (2018) PacBio metabarcoding of fungi and other eukaryotes: errors, biases and perspectives. New Phytol 217:973–976CrossRefGoogle Scholar
  28. 28.
    Thiéry O, Vasar M, Jairus T et al (2016) Sequence variation in nuclear ribosomal small subunit, internal transcribed spacer and large subunit regions of Rhizophagus irregularis and Gigaspora margarita is high and isolate-dependent. Mol Ecol 25:2816–2832CrossRefGoogle Scholar
  29. 29.
    Berruti A, Bianciotto V, Lumini E (2018) Seasonal variation in winter wheat field soil arbuscular mycorrhizal fungus communities after non-mycorrhizal crop cultivation. Mycorrhiza 28:535–548CrossRefGoogle Scholar
  30. 30.
    Lumini E, Vallino M, Alguacil M et al (2011) Different farming and water regimes in Italian rice fields affect arbuscular mycorrhizal fungal soil communities. Ecol Appl 21:1696–1707CrossRefGoogle Scholar
  31. 31.
    Fernández-Ugalde O, Orgiazzi A, Jones A et al (2018) LUCAS 2018—SOIL COMPONENT: sampling instructions for surveyors, EUR 28501 EN.
  32. 32.
    Sato K, Suyama Y, Saito M, Sugawara K (2005) A new primer for discrimination of arbuscular mycorrhizal fungi with polymerase chain reaction-denature gradient gel electrophoresis. Grassl Sci 51:179–181CrossRefGoogle Scholar
  33. 33.
    Öpik M, Zobel M, Cantero J et al (2013) Global sampling of plant roots expands the described molecular diversity of arbuscular mycorrhizal fungi. Mycorrhiza 23:411–430CrossRefGoogle Scholar
  34. 34.
    Schloss PD (2009) A high-throughput DNA sequence aligner for microbial ecology studies. PLoS One 4:e8230CrossRefGoogle Scholar
  35. 35.
    Westcott S, Schloss P (2017) OptiClust, an improved method for assigning amplicon-based sequence data to operational taxonomic units. mSphere 8:2–11Google Scholar
  36. 36.
    Lekberg Y, Gibbon S, Rosendahl S (2014) Wild different OUT delineation methods change interpretation of arbuscular mycorrhizal fungal community patterns? New Phytol 202:1101–1104CrossRefGoogle Scholar
  37. 37.
    Zhang Z, Schwartz S, Wagner L, Miller W (2000) A greedy algorithm for aligning DNA sequences. J Comput Biol 7:203–214CrossRefGoogle Scholar
  38. 38.
    Yilmaz P, Parfrey L, Yarza P et al (2014) The SILVA and “all-species living tree project (LTP)” taxonomic frameworks. Nucleic Acids Res 42:D643–D648CrossRefGoogle Scholar
  39. 39.
    Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73:5261–5267CrossRefGoogle Scholar
  40. 40.
    Abarenkov K, Nilsoon H, Larson K et al (2010) The UNITE database for molecular identification of fungi recent updates and future perspectives. New Phytol 186:281–285CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Institute for Sustainable Plant ProtectionNational Research Council (CNR)TorinoItaly
  2. 2.Department of Life Sciences and Systems BiologyUniversity of TurinTorinoItaly
  3. 3.Biological Science Department, Science FacultyEduardo Mondlane University (UEM)MaputoMozambique
  4. 4.European Commission, Joint Research Centre (JRC), Directorate for Sustainable Resources, Land Resources UnitIspraItaly

Personalised recommendations