Skip to main content
SpringerLink
Log in

Comparison of morphological and molecular genetic quantification of relative abundance of arbuscular mycorrhizal fungi within roots

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

Abstract

Nested PCR amplicons of ribosomal RNA genes have been used to identify individuals within assemblages of arbuscular mycorrhizal (AM) fungi in roots and to estimate their relative abundance. Microscopy has also been used to identify their relative abundance in roots, but only at low resolution, usually the genus level. We evaluated the robustness of using nested PCR amplicons of ribosomal RNA genes to estimate the relative abundance of undefined AM fungi in uniformly aged roots in comparison to visual estimates. The relative abundance of AM fungi was assessed as per cent root length colonised by morphotypes and relative sequence type abundance in clone libraries. Plants were grown in coastal soil to obtain assemblages of unknown AM fungi at two times (spring and autumn). Relative abundance of dominant genera of AM fungi in roots (Archaeospora and Glomus) based on an analysis of ribosomal RNA genes did not consistently correspond with relative abundance of morphotypes. This microscopic vs. molecular genetic comparison supports previous conclusions that there can be limitations in using nested PCR amplicons for quantifying the relative abundance of AM fungi in roots, with a sampling bias likely to be of significance. Both molecular genetic and morphological methods are used to estimate relative abundance of AM fungi as a precursor to understanding mycorrhizal function in field soils, but they are rarely verified using alternative approaches although this may be necessary.

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

References

  • Abbott LK (1982) Comparative anatomy of vesicular arbuscular mycorrhizas formed on subterranean clover. Aust J Bot 30:485–499

    Article  Google Scholar 

  • Abbott LK, Gazey C (1994) An ecological view of the formation of VA mycorrhizas. Plant Soil 159:69–78

    Google Scholar 

  • Abbott LK, Robson AD (1978) Growth of subterranean clover in relation to the formation of endomycorrhizas by introduced and indigenous fungi in a field soil. New Phytol 81:575–587

    Article  Google Scholar 

  • Abbott LK, Robson AD (1981) Infectivity and effectiveness of 5 endomycorrhizal fungi—competition with indigenous fungi in field soils. Aust J Agr Res 32:621–630

    Article  Google Scholar 

  • Abbott LK, Robson AD (1984) Colonization of the root system of subterranean clover by three species of vesicular–arbuscular mycorrhizal fungi. New Phytol 96:275–281

    Article  Google Scholar 

  • Alguacil MM, Torres MP, Torrecillas E, Díazb G, Roldána A (2011) Plant type differently promote the arbuscular mycorrhizal fungi biodiversity in the rhizosphere after revegetation of a degraded, semiarid land. Soil Biol Biochem 43:167–173

    Article  CAS  Google Scholar 

  • Alkan N, Gadkar V, Coburn J, Yarden O, Kapulnik Y (2004) Quantification of the arbuscular mycorrhizal fungus Glomus intraradices in host tissue using real-time polymerase chain reaction. New Phytol 161:877–885

    Article  CAS  Google Scholar 

  • Alkan N, Gadkar V, Yarden O, Kapulnik Y (2006) Analysis of quantitative interactions between two species of arbuscular mycorrhizal fungi, Glomus mosseae and G. intraradices, by real-time PCR. Appl Environ Microb 72:4192–4199

    Article  CAS  Google Scholar 

  • Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    Article  PubMed  CAS  Google Scholar 

  • Beck A, Haug I, Oberwinkler F, Kottke I (2007) Structural characterization and molecular identification of arbuscular mycorrhiza morphotypes of Alzatea verticillata (Alzateaceae), a prominent tree in the tropical mountain rain forest of South Ecuador. Mycorrhiza 17:607–625

    Article  PubMed  Google Scholar 

  • Bell J, Wells S, Jasper DA, Abbott LK (2003) Field inoculation with arbuscular mycorrhizal fungi in rehabilitation of mine sites with native vegetation, including Acacia spp. Aust Syst Bot 16:131–138

    Article  Google Scholar 

  • Brundrett MC, Abbott LK (1995) Mycorrhizal fungus propagules in the jarrah forest.2. Spatial variability in inoculum levels. New Phytol 131:461–469

    Article  Google Scholar 

  • Brundrett M, Bougher N, Dell B, Grove T, Malajczuk N (1996) Working with mycorrhizas in forestry and agriculture. Australian Centre for International Agricultural Research, Canberra, A.C.T.

  • Cesaro P, van Tuinen D, Copetta A, Chatagnier O, Berta G, Gianinazzi S, Lingua G (2008) Preferential colonization of Solanum tuberosum L. roots by the fungus Glomus intraradices in arable soil of a potato farming area. Appl Environ Microb 74:5776–5783

    Article  CAS  Google Scholar 

  • Clapp JP, Young JPW, Merryweather JW, Fitter AH (1995) Diversity of fungal symbionts in arbuscular mycorrhizas from a natural community. New Phytol 130:259–265

    Article  Google Scholar 

  • Corradi N, Croll D, Colard A, Kuhn G, Ehinger M, Sanders IR (2007) Gene copy number polymorphisms in an arbuscular mycorrhizal fungal population. Appl Environ Microb 73:366–369

    Article  CAS  Google Scholar 

  • Djuuna IAF, Abbott LK, Solaiman ZM (2009) Use of mycorrhiza bioassays in ecological studies. In: Varma A (ed) Symbiotic fungi, vol. 18. Soil biology. Springer, Berlin, pp 41–50

    Chapter  Google Scholar 

  • Dumbrell AJ, Ashton PD, Aziz N, Feng G, Nelson M, Dytham C, Fitter AH, Helgason T (2011) Distinct seasonal assemblages of arbuscular mycorrhizal fungi revealed by massively parallel pyrosequencing. New Phytol 190:794–804

    Article  PubMed  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 45:581–596

    Article  PubMed  CAS  Google Scholar 

  • Gamper HA, Walker C, Schüßler A (2009) Diversispora celata sp. nov: molecular ecology and phylotaxonomy of an inconspicuous arbuscular mycorrhizal fungus. New Phytol 182:495–506

    Article  PubMed  CAS  Google Scholar 

  • Gamper HA, van der Heijden MGA, Kowalchuk GA (2010) Molecular trait indicators: moving beyond phylogeny in arbuscular mycorrhizal ecology. New Phytol 185:67–82

    Article  PubMed  CAS  Google Scholar 

  • Gazey C, Abbott LK, Robson AD (1992) The rate of development of mycorrhizas affects the onset of sporulation and production of external hyphae by 2 species of Acaulospora. Mycol Res 96:643–650

    Article  Google Scholar 

  • Gollotte A, van Tuinen D, Atkinson D (2004) Diversity of arbuscular mycorrhizal fungi colonising roots of the grass species Agrostis capillaris and Lolium perenne in a field experiment. Mycorrhiza 14:111–117

    Article  PubMed  Google Scholar 

  • Helgason T, Daniell TJ, Husband R, Fitter AH, Young JP (1998) Ploughing up the wood-wide web? Nature 394:431

    Article  PubMed  CAS  Google Scholar 

  • Helgason T, Fitter AH, Young JPW (1999) Molecular diverisity of arbuscular mycorrhizal fungi colonising Hyacinthoides non-scripta (bluebell) in a seminatural woodland. Mol Ecol 8:659–666

    Article  CAS  Google Scholar 

  • Hempel S, Renker C, Buscot F (2007) Differences in the species composition of arbuscular mycorrhizal fungi in spore, root and soil communities in a grassland ecosystem. Environ Microbiol 9:1930–1938

    Article  PubMed  CAS  Google Scholar 

  • Horton TR, Bruns TD (2001) The molecular revolution in ectomycorrhizal ecology: peeking into the black-box. Mol Ecol 10:1855–1871

    Article  PubMed  CAS  Google Scholar 

  • Huber JA, Morrison HG, Huse SM, Neal PR, Sogin ML, Welch DBM (2009) Effect of PCR amplicon size on assessments of clone library microbial diversity and community structure. Environ Microbiol 11:1292–1302

    Article  PubMed  CAS  Google Scholar 

  • Isayenkov S, Mrosk C, Stenzel I, Strack D, Hause B (2005) Suppression of allene oxide cyclase in hairy roots of Medicago truncatula reduces jasmonate levels and the degree of mycorrhization with Glomus intraradices. J Plant Physiol 139:1401–1410

    Article  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Junier P, Kim OS, Hadas O, Imhoff JF, Witzel KP (2008) Evaluation of PCR primer selectivity and phylogenetic specificity by using amplification of 16S rRNA genes from betaproteobacterial ammonia-oxidizing bacteria in environmental samples. Appl Environ Microb 74:5231–5236

    Article  CAS  Google Scholar 

  • König S, Wubet T, Dormann CF, Hempel S, Renker C, Buscot F (2010) TaqMan real-time PCR assays to assess arbuscular mycorrhizal responses to field manipulation of grassland biodiversity: effects of soil characteristics, plant species richness, and functional traits. Appl Environ Microbiol 76:3765–3775

    Article  PubMed  Google Scholar 

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

    Article  PubMed  Google Scholar 

  • Liu Y, Mao L, He X, Cheng G, Ma X, An L, Feng H (2011) Rapid change of AM fungal community in a rain-fed wheat field with short-term plastic film mulching practice. Mycorrhiza. doi:10.1007/s00572-011-0378-y

  • Lueders T, Friedrich MW (2003) Evaluation of PCR amplification bias by terminal restriction fragment length polymorphism analysis of small-subunit rRNA and mcrA genes by using defined template mixtures of methanogenic pure cultures and soil DNA extracts. Appl Environ Microb 69:320–326

    Article  CAS  Google Scholar 

  • Mandyam K, Jumpponen A (2008) Seasonal and temporal dynamics of arbuscular mycorrhizal and dark septate endophytic fungi in a tallgrass prairie ecosystem are minimally affected by nitrogen enrichment. Mycorrhiza 18:145–155

    Article  PubMed  Google Scholar 

  • Merryweather JW, Fitter AH (1991) A modified method for elucidating the structure of the fungal partner in a vesicular–arbuscular mycorrhiza. Mycol Res 95:1435–1437

    Article  Google Scholar 

  • Merryweather J, Fitter A (1998a) The arbuscular mycorrhizal fungi of Hyacinthoides non-scripta I. Diversity of fungal taxa. New Phytol 138:117–129

    Article  Google Scholar 

  • Merryweather J, Fitter A (1998b) The arbuscular mycorrhizal fungi of Hyacinthoides non-scripta II. Seasonal and spatial patterns of fungal populations. New Phytol 138:131–142

    Article  Google Scholar 

  • Morton JB, Redecker D (2001) Two new families of Glomales, Archaeosporaceae and Paraglomaceae, with two new genera Archaeospora and Paraglomus, based on concordant molecular and morphological characters. Mycologia 93:181–195

    Article  Google Scholar 

  • Mummey DL, Rillig MC (2007) Evaluation of LSU rRNA-gene PCR primers for analysis of arbuscular mycorrhizal fungal communities via terminal restriction fragment length polymorphism analysis. J Microbiol Meth 70:200–204

    Article  CAS  Google Scholar 

  • Newman EI (1966) A method of estimating total length of root in a sample. J Appl Ecol 3:139–145

    Article  Google Scholar 

  • Opik M, Metsis M, Daniell TJ, Zobel M, Moora M (2009) Large-scale parallel 454 sequencing reveals host ecological group specificity of arbuscular mycorrhizal fungi in a boreonemoral forest. New Phytol 184:424–437

    Article  PubMed  CAS  Google Scholar 

  • Palatinszky M, Nikolausz M, Svába D, Márialigeti K (2011) Preferential ligation during TA-cloning of multitemplate PCR products—a factor causing bias in microbial community structure analysis. J Microbiol Meth 85:131–136

    Article  CAS  Google Scholar 

  • Pearson JN, Abbott LK, Jasper DA (1993) Mediation of competition between two colonizing VA mycorrhizal fungi by the host plant. New Phytol 123:93–98

    Article  Google Scholar 

  • Plenchette C, Morel C (1996) External phosphorus requirement of mycorrhizal and non-mycorrhizal barley and soybean plants. Biol Fert Soils 21:303–308

    Article  Google Scholar 

  • Polz MF, Cavanaugh CM (1998) Bias in template-to-product ratios in multitemplate PCR. Appl Environ Microb 64:3724–3730

    CAS  Google Scholar 

  • Redecker D, Kodner R, Graham LE (2000) Glomalean fungi from the Ordovician. Science 289:1920–1921

    Article  PubMed  CAS  Google Scholar 

  • Redecker D, Hijri I, Wiemken A (2003) Molecular identification of arbuscular mycorrhizal fungi in roots: perspectives and problems. Folia Geobot 38:113–124

    Article  Google Scholar 

  • Renker C, Weißhuhn K, Kellner H, Buscot F (2006) Rationalizing molecular analysis of field-collected roots for assessing diversity of arbuscular mycorrhizal fungi: to pool, or not to pool, that is the question. Mycorrhiza 16:525–531

    Article  PubMed  CAS  Google Scholar 

  • Robinson-Boyer L, Grzyb I, Jeffries P (2009) Shifting the balance from qualitative to quantitative analysis of arbuscular mycorrhizal communities in field soils. Fungal Ecol 2:1–9

    Article  Google Scholar 

  • Rosendahl S (2008) Communities, populations and individuals of arbuscular mycorrhizal fungi. New Phytol 178:253–266

    Article  PubMed  Google Scholar 

  • Rosendahl S, Matzen HB (2008) Genetic structure of arbuscular mycorrhizal populations in fallow and cultivated soils. New Phytol 179:1154–1161

    Article  PubMed  Google Scholar 

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

    PubMed  CAS  Google Scholar 

  • Santos-Gonzalez JC, Finlay RD, Tehler A (2007) Seasonal dynamics of arbuscular mycorrhizal fungal communities in roots in a seminatural grassland. Appl Environ Microb 73:5613–5623

    Article  CAS  Google Scholar 

  • Schechter SP, Bruns TD (2008) Serpentine and non-serpentine ecotypes of Collinsia sparsiflora associate with distinct arbuscular mycorrhizal fungal assemblages. Mol Ecol 17:3198–3210

    Article  PubMed  CAS  Google Scholar 

  • Scheltema MA, Abbott LK, Robson AD (1987) Seasonal variation in infectivity of VA mycorrhizal fungi in annual pastures in a Mediterranean environment. Aust J Agr Res 38:707–715

    Article  Google Scholar 

  • Smith SE, Read DJ (2008) Mycorrhizal symbiosis, 3rd edn. Academic, Cambridge

    Google Scholar 

  • Stockinger H, Kruger M, Schüßler A (2010) DNA barcoding of arbuscular mycorrhizal fungi. New Phytol 187:461–474

    Article  PubMed  CAS  Google Scholar 

  • Sýkorová Z, Ineichen K, Wiemken A, Redecker D (2007) The cultivation bias: different communities of arbuscular mycorrhizal fungi detected in roots from the field, from bait plants transplanted to the field, and from a greenhouse trap experiment. Mycorrhiza 18:1–14

    Article  PubMed  Google Scholar 

  • Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599

    Article  PubMed  CAS  Google Scholar 

  • Tawaraya K, Imai T, Wagatsuma T (1999) Importance of root length in mycorrhizal colonization of welsh onion. J Plant Nutr 22:589–596

    Article  CAS  Google Scholar 

  • Thompson JD, Gibson TJ, Higgins DG (2002) Multiple sequence alignment using ClustalW and ClustalX. Current Protoc Bioinformatics. John Wiley & Sons, Inc.

  • Van Tuinen D, Jacquot E, Zhao B, Gollotte A, 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  Google Scholar 

  • Verbruggen E, Röling WFM, Gamper HA, Kowalchuk GA, Verhoef HA, van der Heijden MGA (2010) Positive effects of organic farming on below-ground mutualists: large-scale comparison of mycorrhizal fungal communities in agricultural soils. New Phytol 186:968–979

    Article  PubMed  CAS  Google Scholar 

  • White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic, San Diego, pp 315-322

  • Wilde P, Manal A, Stodden M, Sieverding E, Hildebrandt U, Bothe H (2009) Biodiversity of arbuscular mycorrhizal fungi in roots and soils of two salt marshes. Environ Microbiol 11:1548–1561

    Article  PubMed  Google Scholar 

  • Wu BY, Hogetsu T, Isobe K, Ishii R (2007) Community structure of arbuscular mycorrhizal fungi in a primary successional volcanic desert on the southeast slope of Mount Fuji. Mycorrhiza 17:495–506

    Article  PubMed  CAS  Google Scholar 

  • Zarei M, Hempel S, Wubet T, Schäferb T, Savaghebi G, Jouzani GS, Nekouei MK, Buscot F (2010) Molecular diversity of arbuscular mycorrhizal fungi in relation to soil chemical properties and heavy metal contamination. Environ Pollut 158:2757–2765

    Article  PubMed  CAS  Google Scholar 

  • Zhao B, Trouvelot A, Gianinazzi S, Gianinazzi-Pearson V (1997) Influence of two legume species on hyphal production and activity of two arbuscular mycorrhizal fungi. Mycorrhiza 7:179–185

    Article  Google Scholar 

Download references

Acknowledgements

This research was enabled by the support of the Australian Research Council and Linkage grant partner Alcoa of Australia under project LP07765963 lead by Associate Professor Daniel Murphy and the China Scholarship Council under the State Scholarship Fund. We thank Drs. Yoshi Sawada and Mark Dobrowlski for assistance during the sampling, Dr. Zakaria Solaiman for technical advice on mycorrhizal morphology, Dr. Kevin Murray for statistical analysis and Dr. Ian Phillips for project support. A sampling permit was provided by the Department of Environment and Conservation, Western Australia. We appreciate the valuable comments on our manuscript made by Drs. Hayley Ridgeway, Deirdre Gleeson, Ian Abbott and anonymous reviewers.

Author information

Authors and Affiliations

  1. State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, 430070, People’s Republic of China

    P. Shi & B. Zhao

  2. Soil Biology Group, School of Earth and Environment, UWA Institute of Agriculture, Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley, 6009, WA, Australia

    P. Shi, L. K. Abbott & N. C. Banning

Authors
  1. P. Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. L. K. Abbott
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. N. C. Banning
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. B. Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to L. K. Abbott or B. Zhao.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Tables

(DOC 88.5 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Shi, P., Abbott, L.K., Banning, N.C. et al. Comparison of morphological and molecular genetic quantification of relative abundance of arbuscular mycorrhizal fungi within roots. Mycorrhiza 22, 501–513 (2012). https://doi.org/10.1007/s00572-011-0425-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00572-011-0425-8

Keywords

Search

Navigation