Skip to main content
SpringerLink
Log in

Detection of arbuscular mycorrhizal fungi (Glomales) in roots by nested PCR and SSCP (Single Stranded Conformation Polymorphism)

  • Published:
Plant and Soil Aims and scope Submit manuscript

Abstract

PCR amplification of a region of the large subunit ribosomal DNA sequence with Glomus specific primers was used to detect arbuscular mycorrhizal fungi in root tissue of four plant species. The primers were specific to Glomus mosseae, Glomus caledonium, Glomus geosporum, Glomus coronatum, Glomus fragilistratum and Glomus constrictum, and did not recognise sequences from Glomus claroideum. Sequence differences between isolates were detected by Single Stranded Conformation Polymorphisms (SSCPs) in polyacrylamide gels under non-denaturing conditions. Isolates of G. mosseae, G. caledonium and G. coronatum could be separated by their SSCP patterns, while three isolates of G. geosporumshowed no variation. Specific SSCP patterns from isolates of G. mosseae and G. caledonium allowed detection of both fungi in the same root segment. Sequence differences leading to variations in SSCP patterns were confirmed by direct sequencing.

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

Similar content being viewed by others

References

  • Abbas J D, Hetrick B A D and Jurgenson J E 1996 Isolate specific detection of mycorrhizal fungi using genome specific primer pairs. Mycologia 88, 939–946.

    CAS  Google Scholar 

  • Abbott L K 1982 Comparative anatomy of vesicular-arbuscular mycorrhizas formed on subterranean clover. Aust. J. Bot. 30, 485–499.

    Article  Google Scholar 

  • Abbott L K and Robson A D 1981 Infectivity and effectiveness of five endomycorrhizal fungi: competition with indigenous fungi in field soils. Aus. J. Agric. Res. 32, 621–630.

    Article  Google Scholar 

  • Bärtschi H, Gianinazzi-Pearson V and Vegh I 1981 Vesiculararbuscular mycorrhiza formation and root rot disease (Phytophthora cinnamomi) development in Chamaecyparis lawsoniana. Phytopathol. Zeit. 102, 213–218.

    Google Scholar 

  • Bentivenga S P and Morton J 1994 Stability and heritability of fatty acid methyl ester profiles of glomalian endomycorrhizal fungi. Mycol. Res. 98, 1419–1426.

    CAS  Google Scholar 

  • Clapp J P, Young J W P, Merryweather J W and Fitter A H 1995 Diversity of fungal symbionts in arbuscular mycorrhizas from a natural community. New Phytol. 130, 259–265.

    Article  Google Scholar 

  • Cordier C, Gianinazzi-Pearson V and Gianinazzi S 1996 An immunological approach for the study of spatial relationships between arbuscular mycorrhizal fungi in planta. In Mycorrhizas in Integrated Systems: from Genes to Plant Development. Eds. C Azcon-Aguilar and J M Barea. pp 25–30. European Commision, EUR 16 728, Luxembourg.

    Google Scholar 

  • Di Bonito R, Elliott M L and Des Jardin E A 1995 Detection of an arbuscular mycorrhizal fungus in roots of different plant species with the PCR. Appl. Environ. Microbiol. 61, 2809–2810.

    PubMed  CAS  Google Scholar 

  • Gardes M and Bruns T 1993 ITS primers with enhanced specificity for basidiomycetes - application of mycorrhizae and rusts. Mol. Ecol. 2, 113–118.

    PubMed  CAS  Google Scholar 

  • Gargas A and DePriest P T 1996 A nomenclature for fungal PCR primers with examples from intron-containing SSU rDNA. Mycologia 88: 745–748.

    CAS  Google Scholar 

  • Gerdemann J W and Nicolson T H 1963 Spores of mycorrhizal Endogone species extracted from soil by wet sieving and decanting. Trans. Br. Mycol. Soc. 46, 235–244.

    Article  Google Scholar 

  • Giovannetti M and Gianinazzi-Pearson V 1994 Biodiversity in arbuscular mycorrhizal fungi. Mycol. Res. 98: 705–715.

    Article  Google Scholar 

  • Graham J H, Linderman R G and Menge J A 1982 Development of external hyphae by different isolates of mycorrhizal Glomus spp. in relation to root colonization and growth of Troyer citrange. New Phytol. 9, 183–189.

    Article  Google Scholar 

  • Helgason T, Daniell T J, Husband R, Fitter A and Young P 1998 Ploughing up the wood-wide web? Nature 394, 431.

    Article  PubMed  CAS  Google Scholar 

  • Hepper C M, Azcon-Aguilar C, Rosendahl S and Sen R 1988 Competition of three species of Glomus used as spatially separated introduced and indigenous mycorrhizal inocula for leek (Allium purrum L.). New Phytol. 110, 207–215.

    Article  Google Scholar 

  • Jakobsen I, Abbot L K and Robson A D 1992 External hyphae of vesicular-arbuscular mycorrhizal fungi associated with Trifolium subterraneum L. 1. Spread of hyphae and phosphorus inflow into roots. New Phytol. 120, 371–380.

    Article  CAS  Google Scholar 

  • John M E 1992 An efficient method for isolation of RNA and DNA from plants containing polyphenolics. Nucleic Acids Res. 20, 2381.

    PubMed  CAS  Google Scholar 

  • Millner P D, Mulbry W W, Reynolds S L and Patterson C A 1998 A taxon-specific oligonucleotide probe for temperate zone soil isolates of Glomus mosseae. Mycorrhiza 8, 19–27.

    Article  CAS  Google Scholar 

  • Orita M, Iwahana H, Kanazawa H, Hayashi K and Sekiya T 1989 Detection of polymorphisms of human DNA by gel electrophoresis as single-stranded conformation polymorphisms. Proc. Nat. Acad. Sci. 86, 2766–2770.

    Article  PubMed  CAS  Google Scholar 

  • Rosendahl S, Sen R, Hepper C M and Azcon-Aguilar C 1989a Quantification of three vesicular-arbuscular mycorrhizal fungi (Glomus spp) in roots of leek (Allium purrum) on the basis of activity of diagnostic enzymes after polyacrylamide gel electrophoresis. Soil Biol. Biochem. 21, 519–522.

    Article  Google Scholar 

  • Rosendahl S and Sen R 1992 Isozyme analysis of mycorrhizal fungi and their mycorrhiza. In Methods of Microbiology.Vol 24. Eds. A K Varma, D J Read and J R Norris. pp 169–194. Academic Press, London.

    Google Scholar 

  • Rosendahl S, Rosendahl C N and Sochting U 1989b Distribution of VA mycorrhizal endophytes amongst plants from a Danish grassland community. Agric. Ecosys. Environ. 29: 329–335.

    Article  Google Scholar 

  • Rosendahl S, Dodd J C and Walker C 1994 Taxonomy and phylogeny of the Glomales. In Impact of Arbuscular Mycorrhizas on Sustainable Agriculture and Natural Ecosystems. Eds. S Gianinazzi and H Shuepp. pp. 1–12. Birkhauser Verlag, Basel, Switzerland.

    Google Scholar 

  • Sanders I R, Alt M, Groppe K, Boller T and Wiemken A 1995 Identification of ribosomal DNA polymorphisms among and within spores of the Glomales: application to studies on the genetic diversity of arbuscular mycorrhizal fungal communities. New Phytol. 130, 419–427.

    Article  CAS  Google Scholar 

  • Simon L, Levesque R C and Lalonde M 1993 Identification of endomycorrhizal fungi colonizing roots by fluorescent singlestrand conformation polymorphism-polymerase chain reaction. App. Environ. Microbiol. 59, 4211–4215.

    CAS  Google Scholar 

  • Smith S E and Read D J 1997 Mycorrhizal symbiosis. 2nd edn. Academic Press, London.

    Google Scholar 

  • Tisserant B, Brenac V, Requena N, Jeffries P and Dodd J C 1998 The detection of Glomus spp. (arbuscular mycorrhizal fungi) forming mycorrhizas in three plants, at different stages of seedling development, using mycorrhiza-specific isozymes. New Phytol. 138, 225–239.

    Article  CAS  Google Scholar 

  • Van Tuinen D, Jacquot E, Zhao B, Gollotte A and Gianinazzi-Pearson V 1998 Characterization of root colonization profiles by a microcosm community of arbuscular mycorrhizal fungi using 25S rDNA-targeted nested PCR. Mol. Ecol. 7, 879–887.

    Article  PubMed  CAS  Google Scholar 

  • White T J, Bruns T, Lee S and Taylor J 1990 Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols. A Guide to Methods and Applications. Eds. M Innis, D Gelfand, J Sninsky, T White and FL Orlando. pp. 315–322. Academic Press, London.

    Google Scholar 

  • Wright S F and Morton J B 1989 Detection of vesicular-arbuscular mycorrhizal fungus colonization of roots by using a dotimmunoblot assay. Appl. Environ. Microbiol. 55, 761–763.

    PubMed  Google Scholar 

  • Yap E P H and McGee J O 1993 Nonisotopic discontinuous phase single strand conformation polymorphism (DP- SSCP): genetic profiling of D-loop of human mitochondrial (mt) DNA. Nucleic Acids Res. 21, 4155.

    PubMed  CAS  Google Scholar 

  • Zézé A, Dulieu H and Gianinazzi-Pearson V 1994 DNA cloning and screening of a partial genomic library from an arbuscular mycorrhizal fungus, Scutellospora castanea. Mycorrhiza 4, 251–254.

    Article  Google Scholar 

Download references

Author information

Author notes

  1. Rasmus Kjøller

    Present address: Department of Plant and Microbial Biology, University of California, 111 Koshlan Hall, Berkeley, CA, 94720, USA

Authors and Affiliations

  1. Department of Mycology, Botanical Institute, University of Copenhagen, Øster Farimagsgade 2D, DK-1353, Copenhagen K, Denmark

    Rasmus Kjøller

  2. Department of Mycology, Botanical Institute, University of Copenhagen, Øster Farimagsgade 2D, DK-1353, Copenhagen K, Denmark

    Søren Rosendahl

Authors
  1. Rasmus Kjøller
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Søren Rosendahl
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kjøller, R., Rosendahl, S. Detection of arbuscular mycorrhizal fungi (Glomales) in roots by nested PCR and SSCP (Single Stranded Conformation Polymorphism). Plant and Soil 226, 189–196 (2000). https://doi.org/10.1023/A:1026499923717

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1026499923717

Search

Navigation