Skip to main content
SpringerLink
Log in

Multiplex qPCR assays to distinguish individual species of arbuscular mycorrhizal fungi from roots and soil

  • Original Article
  • Published:
Mycorrhiza Aims and scope Submit manuscript

Abstract

Currently, root colonization measurements of arbuscular mycorrhizal fungi (AMF) require staining and microscopy, and species-level identification of the fungi by such observations is not possible. Here, we present novel multiplex real-time PCR assays targeting the glomalin genes of 11 different species of AMF commonly found in temperate agricultural soils, which independently detect and measure the abundance of these fungi using DNA extracts from soil and or root tissue. The availability of these tools will not only increase throughput in determining levels of root colonization but can provide species-specific levels of root colonization from a single sample. This will help to establish which AMF species, or combinations of different species, provide the most benefits to crops, and will aid in the development of AMF for use as biofertilizers.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Code availability

Not applicable.

References

  • Bodenhausen N, Deslandes-Hérold G, Waelchli J, Held A, van der Heijden MG, Schlaeppi K (2021) Relative qPCR to quantify colonization of plant roots by arbuscular mycorrhizal fungi. Mycorrhiza 31(2):137–148

    Article  CAS  Google Scholar 

  • Bona E, Cantamessa S, Massa N, Manassero P, Marsano F, Copetta A, Lingua G, D’Agostino G, Gamalero E, Berta G (2017) Arbuscular mycorrhizal fungi and plant growth-promoting pseudomonads improve yield, quality and nutritional value of tomato: a field study. Mycorrhiza 27:1–11

    Article  CAS  Google Scholar 

  • Comas LH, Callahan HS, Midford PE (2014) Patterns in root traits of woody species hosting arbuscular and ectomycorrhizas: implications for the evolution of belowground strategies. Ecol Evol 4(15):2979–2990

    Article  Google Scholar 

  • Douds DD, Nagahashi G, Pfeffer PE, Reider C, Kayser WM (2006) On-farm production of AM fungus inoculum in mixtures of compost and vermiculite. Biores Technol 97(6):809–818. https://doi.org/10.1016/j.biortech.2005.04.015

    Article  CAS  Google Scholar 

  • Douds D Jr, Carr E, Shenk J, Ganser S (2017) Positive yield response of eggplant (Solanum melongena L.) to inoculation with AM fungi produced on-farm. Sci Hortic 224:48–52

    Article  Google Scholar 

  • Douds D Jr, Galvez L, Janke R, Wagoner P (1995) Effect of tillage and farming system upon populations and distribution of vesicular-arbuscular mycorrhizal fungi. Agr Ecosyst Environ 52(2–3):111–118

    Article  Google Scholar 

  • Douds D Jr, Lee J, Shenk J, Ganser S (2015) Inoculation of sweet potatoes with AM fungi produced on-farm increases yield in high P soil. J Appl Hortic 17(3):171–175

    Article  Google Scholar 

  • Douds D Jr, Reider C (2003) Inoculation with mycorrhizal fungi increases the yield of green peppers in a high P soil. Biol Agric Hortic 21(1):91–102

    Article  Google Scholar 

  • Douds DD Jr, Nagahashi G, Reider C, Hepperly PR (2007) Inoculation with arbuscular mycorrhizal fungi increases the yield of potatoes in a high P soil. Biol Agric Hortic 25(1):67–78

    Article  Google Scholar 

  • Douds DD Jr, Nagahashi G, Shenk JE, Demchak K (2008) Inoculation of strawberries with AM fungi produced on-farm increased yield. Biol Agric Hortic 26(3):209–219

    Article  Google Scholar 

  • Evans D, Miller M (1990) The role of the external mycelial network in the effect of soil disturbance upon vesicular—arbuscular mycorrhizal colonization of maize. New Phytol 114(1):65–71

    Article  CAS  Google Scholar 

  • Galvez L, Douds D, Drinkwater L, Wagoner P (2001) Effect of tillage and farming system upon VAM fungus populations and mycorrhizas and nutrient uptake of maize. Plant Soil 228(2):299–308

    Article  CAS  Google Scholar 

  • Gamper HA, Young JPW, Jones DL, Hodge A (2008) Real-time PCR and microscopy: are the two methods measuring the same unit of arbuscular mycorrhizal fungal abundance? Fungal Genet Biol 45(5):581–596

    Article  CAS  Google Scholar 

  • Giovannetti M, Mosse B (1980) An evaluation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots. New Phytol 489–500

  • Jansa J, Mozafar A, Kuhn G, Anken T, Ruh R, Sanders I, Frossard E (2003) Soil tillage affects the community structure of mycorrhizal fungi in maize roots. Ecol Appl 13(4):1164–1176

    Article  Google Scholar 

  • Jansa J, Smith FA, Smith SE (2008) Are there benefits of simultaneous root colonization by different arbuscular mycorrhizal fungi? New Phytol 177(3):779–789

    Article  CAS  Google Scholar 

  • Jansa J, Wiemken A, Frossard E (2006) The effects of agricultural practices on arbuscular mycorrhizal fungi. Geological Society, London, Special Publications 266(1):89–115

    Article  Google Scholar 

  • Jasper D, Abbott L, Robson A (1989) Soil disturbance reduces the infectivity of external hyphae of vesicular—arbuscular mycorrhizal fungi. New Phytol 112(1):93–99

    Article  Google Scholar 

  • Kabir Z, O'halloran I, Widden P, Hamel C (1998) Vertical distribution of arbuscular mycorrhizal fungi under corn (Zea mays L) in no-till and conventional tillage systems. Mycorrhiza 8(1):53–55

  • Knegt B, Jansa J, Franken O, Engelmoer DJ, Werner GD, Bücking H, Kiers ET (2016) Host plant quality mediates competition between arbuscular mycorrhizal fungi. Fungal Ecol 20:233–240

    Article  Google Scholar 

  • Kokkoris V, Stefani F, Dalpé Y, Dettman J, Corradi N (2020) Nuclear dynamics in the arbuscular mycorrhizal fungi. Trends Plant Sci 25(8):765–778

    Article  CAS  Google Scholar 

  • Krüger M, Stockinger H, Krüger C, Schüßler A (2009) DNA-based species level detection of Glomeromycota: one PCR primer set for all arbuscular mycorrhizal fungi. New Phytol 183(1):212–223

    Article  Google Scholar 

  • Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35(6):1547

    Article  CAS  Google Scholar 

  • Magurno F, Malicka M, Posta K, Wozniak G, Lumini E, Piotrowska-Seget Z (2019) Glomalin gene as molecular marker for functional diversity of arbuscular mycorrhizal fungi in soil. Biol Fertil Soils 55(4):411–417. https://doi.org/10.1007/s00374-019-01354-x

    Article  CAS  Google Scholar 

  • McGonigle T, Miller M, Evans D, Fairchild G, Swan J (1990) A new method which gives an objective measure of colonization of roots by vesicular—arbuscular mycorrhizal fungi. New Phytol 115(3):495–501

    Article  CAS  Google Scholar 

  • Morin E, Miyauchi S, San Clemente H, Chen EC, Pelin A, de la Providencia I, Ndikumana S, Beaudet D, Hainaut M, Drula E (2019) Comparative genomics of Rhizophagus irregularis, R. cerebriforme, R. diaphanus and Gigaspora rosea highlights specific genetic features in Glomeromycotina. New Phytol 222(3):1584–1598

  • Morton J, Bentivenga S, Wheeler W (1993) Germ plasm in the International Collection of Arbuscular and Vesicular-Arbuscular Mycorrhizal Fungi (INVAM) and procedures for culture development, documentation and storage. Mycotaxon 48(1):491–528

    Google Scholar 

  • Rosier CL, Piotrowski JS, Hoye AT, Rillig MC (2008) Intraradical protein and glomalin as a tool for quantifying arbuscular mycorrhizal root colonization. Pedobiologia 52(1):41–50

    Article  CAS  Google Scholar 

  • Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406–425

    CAS  Google Scholar 

  • Scholdberg TA, Norden TD, Nelson DD, Jenkins GR (2009) Evaluating precision and accuracy when quantifying different endogenous control reference genes in maize using real-time PCR. J Agric Food Chem 57(7):2903–2911

    Article  CAS  Google Scholar 

  • Schwarzott D, Schüßler A (2001) A simple and reliable method for SSU rRNA gene DNA extraction, amplification, and cloning from single AM fungal spores. Mycorrhiza 10(4):203–207

    Article  CAS  Google Scholar 

  • Six J, Bossuyt H, Degryze S, Denef K (2004) A history of research on the link between (micro) aggregates, soil biota, and soil organic matter dynamics. Soil and Tillage Research 79(1):7–31

    Article  Google Scholar 

  • Six J, Elliott E, Paustian K (2000) Soil macroaggregate turnover and microaggregate formation: a mechanism for C sequestration under no-tillage agriculture. Soil Biol Biochem 32(14):2099–2103

    Article  CAS  Google Scholar 

  • Smith SE, Read D (2008) Mycorrhizal Symbiosis. In: Smith SE, Read D (eds) Mycorrhizal Symbiosis (Third Edition). Academic Press, London, pp vii-ix. https://doi.org/10.1016/B978-012370526-6.50001-5

  • Tamura K, Nei M, Kumar S (2004) Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci 101(30):11030–11035

    Article  CAS  Google Scholar 

  • Thonar C, Erb A, Jansa J (2012) Real-time PCR to quantify composition of arbuscular mycorrhizal fungal communities—marker design, verification, calibration and field validation. Mol Ecol Resour 12(2):219–232

    Article  CAS  Google Scholar 

  • Tisserant E, Malbreil M, Kuo A, Kohler A, Symeonidi A, Balestrini R, Charron P, Duensing N, Frei Dit Frey N, Gianinazzi-Pearson V, Gilbert LB, Handa Y, Herr JR, Hijri M, Koul R, Kawaguchi M, Krajinski F, Lammers PJ, Masclaux FG, Murat C, Morin E, Ndikumana S, Pagni M, Petitpierre D, Requena N, Rosikiewicz P, Riley R, Saito K, San Clemente H, Shapiro H, Van Tuinen D, Becard G, Bonfante P, Paszkowski U, Shachar-Hill YY, Tuskan GA, Young JPW, Sanders IR, Henrissat B, Rensing SA, Grigoriev IV, Corradi N, Roux C, Martin F (2013) Genome of an arbuscular mycorrhizal fungus provides insight into the oldest plant symbiosis. Proc Natl Acad Sci 110(50):20117–20122. https://doi.org/10.1073/pnas.1313452110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Venice F, Ghignone S, Salvioli di Fossalunga A, Amselem J, Novero M, Xianan X, Sędzielewska Toro K, Morin E, Lipzen A, Grigoriev IV (2020) At the nexus of three kingdoms: the genome of the mycorrhizal fungus Gigaspora margarita provides insights into plant, endobacterial and fungal interactions. Environ Microbiol 22(1):122–141

    Article  Google Scholar 

  • Wright SF, Upadhyaya A (1996) Extraction of an abundant and unusual protein from soil and comparison with hyphal protein of arbuscular mycorrhizal fungi. Soil Sci 161(9):575–586

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful for expert technical assistance in processing root samples by Lindsay McKeever, and for the access to field sites at the Rodale Institute to conduct field experiments.

Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. The US Department of Agriculture (USDA) prohibits discrimination in all its programs and activities on the basis of race, color, national origin, age, and disability, and where applicable, sex, marital status, familial status, parental status, religion, sexual orientation, genetic information, political beliefs, reprisal, or because all or part of an individual's income is derived from any public assistance program. (Not all prohibited bases apply to all programs.) Persons with disabilities who require alternative means for communication of program information (Braille, large print, audiotape, etc.) should contact USDA’s TARGET Center at (202) 720–2600 (voice and TDD). To file a complaint of discrimination, write to USDA, Director, Office of Civil Rights, 1400 Independence Avenue, SW, Washington, DC 20250–9410, or call (800) 795–3272 (voice) or (202) 720–6382 (TDD). USDA is an equal opportunity provider and employer.

Funding

All sequence data have been deposited in GenBank and biological materials not available elsewhere may be requested from the corresponding author.

Author information

Authors and Affiliations

  1. USDA Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA, USA

    Wade P. Heller & Joseph E. Carrara

Authors
  1. Wade P. Heller
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Joseph E. Carrara
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

USDA-ARS in-house project 8072–12000-013–000-D.

Corresponding author

Correspondence to Wade P. Heller.

Ethics declarations

Ethics approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Conflict of interest

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Heller, W.P., Carrara, J.E. Multiplex qPCR assays to distinguish individual species of arbuscular mycorrhizal fungi from roots and soil. Mycorrhiza 32, 155–164 (2022). https://doi.org/10.1007/s00572-022-01069-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00572-022-01069-2

Keywords

Search

Navigation