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Quantification of arbuscular mycorrhizal fungal DNA in roots: how important is material preservation?

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Abstract

Monitoring populations of arbuscular mycorrhizal fungi (AMF) in roots is a pre-requisite for improving our understanding of AMF ecology and functioning of the symbiosis in natural conditions. Among other approaches, quantification of fungal DNA in plant tissues by quantitative real-time PCR is one of the advanced techniques with a great potential to process large numbers of samples and to deliver truly quantitative information. Its application potential would greatly increase if the samples could be preserved by drying, but little is currently known about the feasibility and reliability of fungal DNA quantification from dry plant material. We addressed this question by comparing quantification results based on dry root material to those obtained from deep-frozen roots of Medicago truncatula colonized with Rhizophagus sp. The fungal DNA was well conserved in the dry root samples with overall fungal DNA levels in the extracts comparable with those determined in extracts of frozen roots. There was, however, no correlation between the quantitative data sets obtained from the two types of material, and data from dry roots were more variable. Based on these results, we recommend dry material for qualitative screenings but advocate using frozen root materials if precise quantification of fungal DNA is required.

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References

  • Alguacil MM, Lumini E, Roldan A, Salinas-García JR, Bonfante P, Bianciotto V (2008) The impact of tillage practices on arbuscular mycorrhizal fungal diversity in subtropical crops. Ecol Appl 18:527–536. doi:10.1890/07-0521.1

    Article  CAS  PubMed  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. doi:10.1111/j.1469-8137.2003.00975.x

    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 Microbiol 72:4192–4199. doi:10.1128/aem.02889-05

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Bainard LD, Klironomos JN, Hart MM (2010) Differential effect of sample preservation methods on plant and arbuscular mycorrhizal fungal DNA. J Microbiol Methods 82:124–130. doi:10.1016/j.mimet.2010.05.001

    Article  CAS  PubMed  Google Scholar 

  • Bustin SA (2004) A–Z of quantitative PCR. IUL biotechnology series, vol 5. International University Line, La Jolla

    Google Scholar 

  • 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 Microbiol 74:5776–5783. doi:10.1128/aem.00719-08

    Article  PubMed Central  CAS  PubMed  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. doi:10.1111/j.1469-8137.1995.tb03047.x

    Article  Google Scholar 

  • Davison J, Opik M, Daniell TJ, Moora M, Zobel M (2011) Arbuscular mycorrhizal fungal communities in plant roots are not random assemblages. FEMS Microbiol Ecol 78:103–115. doi:10.1111/j.1574-6941.2011.01103.x

    Article  CAS  PubMed  Google Scholar 

  • Douhan GW, Petersen C, Bledsoe CS, Rizzo DM (2005) Contrasting root associated fungi of three common oak-woodland plant species based on molecular identification: host specificity or non-specific amplification? Mycorrhiza 15:365–372. doi:10.1007/s00572-004-0341-2

    Article  CAS  PubMed  Google Scholar 

  • Farmer MJ, Li X, Feng G, Zhao B, Chatagnier O, Gianinazzi S, Gianinazzi-Pearson V, van Tuinen D (2007) Molecular monitoring of field-inoculated AMF to evaluate persistence in sweet potato crops in China. Appl Soil Ecol 35:599–609. doi:10.1016/j.apsoil.2006.09.012

    Article  Google Scholar 

  • Filion M, St-Arnaud M, Jabaji-Hare SH (2003) Direct quantification of fungal DNA from soil substrate using real-time PCR. J Microbiol Methods 53:67–76. doi:10.1016/S0167-7012(02)00225-7

    Article  CAS  PubMed  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. doi:10.1016/j.fgb.2007.09.007

    Article  CAS  PubMed  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. doi:10.1007/s00572-003-0244-7

    Article  PubMed  Google Scholar 

  • Gryndler M, Vejsadová H, Vančura V (1992) The effect of magnesium ions on the vesicular arbuscular mycorrhizal infection of maize roots. New Phytol 122:455–460. doi:10.1111/j.1469-8137.1992.tb00073.x

    Article  CAS  Google Scholar 

  • Gryndler M, Trilčová J, Hršelová H, Streiblová E, Gryndlerová H, Jansa J (2013) Tuber aestivum Vittad. mycelium quantified: advantages and limitations of a qPCR approach. Mycorrhiza 23:341–348

    Article  PubMed  Google Scholar 

  • Helgason T, Merryweather JW, Denison J, Wilson P, Young JPW, Fitter AH (2002) Selectivity and functional diversity in arbuscular mycorrhizas of co-occurring fungi and plants from a temperate deciduous woodland. J Ecol 90:371–384. doi:10.1046/j.1365-2745.2001.00674.x

    Article  Google Scholar 

  • Isayenkov S, Fester T, Hause B (2004) Rapid determination of fungal colonization and arbuscule formation in roots of Medicago truncatula using real-time (RT) PCR. J Plant Physiol 161:1379–1383. doi:10.1016/j.jplph.2004.04.012

    Article  CAS  PubMed  Google Scholar 

  • Janoušková M, Krak K, Wagg C, Štorchová H, Caklová P, Vosátka M (2013) Effects of inoculum additions in the presence of a preestablished arbuscular mycorrhizal fungal community. Appl Environ Microbiol 79:6507–6515. doi:10.1128/aem.02135-13

    Article  PubMed Central  PubMed  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. doi:10.1111/j.1469-8137.2007.02294.x

    Article  CAS  PubMed  Google Scholar 

  • Jansa J, Erb A, Oberholzer H-R, Šmilauer P, Egli S (2014) Soil and geography are more important determinants of indigenous arbuscular mycorrhizal communities than management practices in Swiss agricultural soils. Mol Ecol 23:2118–2135

    Article  CAS  PubMed  Google Scholar 

  • Kiers ET, Duhamel M, Beesetty Y, Mensah JA, Franken O, Verbruggen E, Fellbaum CR, Kowalchuk GA, Hart MM, Bago A, Palmer TM, West SA, Vandenkoornhuyse P, Jansa J, Bücking H (2011) Reciprocal rewards stabilize cooperation in the mycorrhizal symbiosis. Science 333:880–882. doi:10.1126/science.1208473

    Article  CAS  PubMed  Google Scholar 

  • Kohout P, Sudová R, Janoušková M, Čtvrtlíková M, Hejda M, Pánková H, Slavíková R, Štajerová K, Vosátka M, Sýkorová Z (2014) Comparison of commonly used primer sets for evaluating arbuscular mycorrhizal fungal communities: is there a universal solution? Soil Biol Biochem 68:482–493. doi:10.1016/j.soilbio.2013.08.027

    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. doi:10.1128/aem.02951-09

    Article  PubMed Central  PubMed  Google Scholar 

  • Koske RE, Gemma JN (1989) A modified procedure for staining roots to detect VA mycorrhizas. Mycol Res 92:486–505. doi:10.1016/S0953-7562(89)80195-9

    Article  Google Scholar 

  • Krak K, Janoušková M, Caklová P, Vosátka M, Štorchová H (2012) Intraradical dynamics of two coexisting isolates of the arbuscular mycorrhizal fungus Glomus intraradices sensu lato as estimated by real-time PCR of mitochondrial DNA. Appl Environ Microbiol 78:3630–3637. doi:10.1128/aem.00035-12

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Li S, Hartman GL, Domier LL, Boykin D (2008) Quantification of Fusarium solani f. sp. glycines isolates in soybean roots by colony-forming unit assays and real-time quantitative PCR. Theor Appl Genet 117:343–352. doi:10.1007/s00122-008-0779-2

    Article  CAS  PubMed  Google Scholar 

  • McGonigle TP, Miller MH, Evans DG, Fairchild GL, Swan JA (1990) A new method which gives an objective measure of colonization of roots by vesicular arbuscular mycorrhizal fungi. New Phytol 115:495–501. doi:10.1111/j.1469-8137.1990.tb00476.x

    Article  Google Scholar 

  • Minerdi D, Moretti M, Li Y, Gaggero L, Garibaldi A, Gullino ML (2008) Conventional PCR and real time quantitative PCR detection of Phytophthora cryptogea on Gerbera jamesonii. Eur J Plant Pathol 122:227–237. doi:10.1007/s10658-008-9276-3

    Article  CAS  Google Scholar 

  • Öpik M, Moora M, Liira J, Koljalg U, Zobel M, Sen R (2003) Divergent arbuscular mycorrhizal fungal communities colonize roots of Pulsatilla spp. in boreal Scots pine forest and grassland soils. New Phytol 160:581–593. doi:10.1046/j.1469-8137x.2003.00917.x

    Article  Google Scholar 

  • Öpik 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. doi:10.1111/j.1469-8137.2009.02920.x

    Article  PubMed  Google Scholar 

  • Öpik M, Vanatoa A, Vanatoa E, Moora M, Davison J, Kalwij JM, Reier Ü, Zobel M (2010) The online database MaarjAM reveals global and ecosystemic distribution patterns in arbuscular mycorrhizal fungi (Glomeromycota). New Phytol 188:223–241. doi:10.1111/j.1469-8137.2010.03334.x

    Article  PubMed  Google Scholar 

  • Sanders IR, Clapp JP, Wiemken A (1996) The genetic diversity of arbuscular mycorrhizal fungi in natural ecosystems—a key to understanding the ecology and functioning of the mycorrhizal symbiosis. New Phytol 133:123–134. doi:10.1111/j.1469-8137.1996.tb04348.x

    Article  Google Scholar 

  • Sanzani SM, Nicosia M, Faedda R, Cacciola SO, Schena L (2014) Use of quantitative PCR detection methods to study biocontrol agents and phytopathogenic fungi and oomycetes in environmental samples. J Phytopathol 162:1–13. doi:10.1111/jph.12147

    Article  CAS  Google Scholar 

  • Schena L, Nicosia M, Sanzani SM, Faedda R, Ippolito A, Cacciola SO (2013) Development of quantitative PCR detection methods for phytopathogenic fungi and oomycetes. J Plant Pathol 95:7–24. doi:10.4454/JPP.V95I1.016

    CAS  Google Scholar 

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

    Google Scholar 

  • Thonar C (2009) Synthetic mycorrhizal communities—establishment and functioning. Dissertation, ETH Zürich

  • 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. doi:10.1111/j.1755-0998.2011.03086.x

    Article  CAS  PubMed  Google Scholar 

  • Thonar C, Frossard E, Šmilauer P, Jansa J (2014) Competition and facilitation in synthetic communities of arbuscular mycorrhizal fungi. Mol Ecol 23:733–746. doi:10.1111/mec.12625

    Article  PubMed  Google Scholar 

  • Tisserant B, Gianinazzi-Pearson V, Gianinazzi S, Gollotte A (1993) In planta histochemical staining of fungal alkaline phosphatase activity for analysis of efficient arbuscular mycorrhizal infections. Mycol Res 97:245–250. doi:10.1016/S0953-7562(09)80248-7

    Article  CAS  Google Scholar 

  • van der Heijden MGA, Klironomos JN, Ursic M, Moutoglis P, Streitwolf-Engel R, Boller T, Wiemken A, Sanders IR (1998) Mycorrhizal fungal diversity determines plant biodiversity, ecosystem variability and productivity. Nature 396:69–72

    Article  Google Scholar 

  • Vierheilig H, Schweiger P, Brundrett M (2005) An overview of methods for the detection and observation of arbuscular mycorrhizal fungi in roots. Physiol Plant 125:393–404. doi:10.1111/j.1399-3054.2005.00564.x

    CAS  Google Scholar 

  • Wagg C, Jansa J, Stadler M, Schmid B, van der Heijden MGA (2011) Mycorrhizal fungal identity and diversity relaxes plant-plant competition. Ecology 92:1303–1313. doi:10.1890/10-1915.1

    Article  PubMed  Google Scholar 

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Acknowledgments

This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic, project no. LK11224, and by the long-term research development programs RVO 67985939 and RVO 61388971 in frame of the Joint Working Group of the Institute of Microbiology AS CR and the Institute of Botany AS CR. We are grateful to Hana Hršelová, Petra Bukovská, and Milan Gryndler for the technical advice, to Hana Gryndlerová for the technical assistance, and to two anonymous reviewers for their constructive comments.

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Authors and Affiliations

  1. Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20, Praha 4 – Krč, Czech Republic

    Martina Janoušková, David Püschel, Martina Hujslová, Renata Slavíková & Jan Jansa

  2. Institute of Botany, Academy of Sciences of the Czech Republic, Zámek 1, 252 43, Průhonice, Czech Republic

    Martina Janoušková & David Püschel

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  1. Martina Janoušková
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  2. David Püschel
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  3. Martina Hujslová
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Correspondence to Martina Janoušková.

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Janoušková, M., Püschel, D., Hujslová, M. et al. Quantification of arbuscular mycorrhizal fungal DNA in roots: how important is material preservation?. Mycorrhiza 25, 205–214 (2015). https://doi.org/10.1007/s00572-014-0602-7

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