Mycorrhiza

, Volume 21, Issue 7, pp 589–600 | Cite as

Optimized assay and storage conditions for enzyme activity profiling of ectomycorrhizae

  • Karin Pritsch
  • Pierre Emanuel Courty
  • Jean-Louis Churin
  • Benoit Cloutier-Hurteau
  • Muhammad Arif Ali
  • Coralie Damon
  • Myriam Duchemin
  • Simon Egli
  • Jana Ernst
  • Laurence Fraissinet-Tachet
  • Francisco Kuhar
  • Elvira Legname
  • Roland Marmeisse
  • Alex Müller
  • Petia Nikolova
  • Martina Peter
  • Claude Plassard
  • Franck Richard
  • Michael Schloter
  • Marc-André Selosse
  • Alain Franc
  • Jean Garbaye
Original Paper

Abstract

The aim of a joint effort by different research teams was to provide an improved procedure for enzyme activity profiling of field-sampled ectomycorrhizae, including recommendations on the best conditions and maximum duration for storage of ectomycorrhizal samples. A more simplified and efficient protocol compared to formerly published procedures was achieved by using manufactured 96-filter plates in combination with a vacuum manifold and by optimizing incubation times. Major improvements were achieved by performing the series of eight enzyme assays with a single series of root samples instead of two series, reducing the time needed for sample preparation, minimizing error-prone steps such as pipetting and morphotyping, and facilitating subsequent DNA analyses due to the reduced sequencing effort. The best preservation of samples proved to be storage in soil at 4–6°C in the form of undisturbed soil cores containing roots. Enzyme activities were maintained for up to 4 weeks under these conditions. Short-term storage of washed roots and ectomycorrhizal tips overnight in water did not cause substantial changes in enzyme activity profiles. No optimal means for longer-term storage by freezing at −20°C or storage in 100% ethanol were recommended.

Keywords

Enzyme activity profiling Sample storage Ectomycorrhizae Method optimization 

References

  1. Abuzinadah RA, Read DJ (1989) The role of proteins in the nitrogen nutrition of ectomycorrhizal plants. V. Nitrogen transfer in birch (Betula pendula) grown in association with mycorrhizal and non-mycorrhizal fungi. New Phytol 112:61–68CrossRefGoogle Scholar
  2. Agerer R (1991) Characterization of ectomycorrhiza. Methods Microbiol 23:25–73CrossRefGoogle Scholar
  3. Agerer R (2001) Exploration types of ectomycorrhizae. Mycorrhiza 11:107–114CrossRefGoogle Scholar
  4. Bernard M, Mouyna I, Dubreucq G, Debeaupuis J-P, Fontaine T, Vorgias C, Fuglsang C, Latgé J-P (2002) Characterization of a cell-wall acid phosphatase (PhoAp) in Aspergillus fumigatus. Microbiology 148:2819–2829PubMedGoogle Scholar
  5. Buée M, Vairelles D, Garbaye J (2005) Year-round monitoring of diversity and potential metabolic activity of the ectomycorrhizal community in a beech (fagus silvatica) forest subjected to two thinning regimes. Mycorrhiza 15:235–245Google Scholar
  6. Buee M, Courty PE, Mignot D, Garbaye J (2007) Soil niche effect on species diversity and catabolic activities in an ectomycorrhizal fungal community. Soil Biol Biochem 39:1947–1955CrossRefGoogle Scholar
  7. Chróst RJ, Velimirov B (1991) Measurement of enzyme kinetics in water samples: effect of freezing and soluble stabilizer. Mar Ecol Prog Ser 70:93–100CrossRefGoogle Scholar
  8. Cosby CN, Troiano NW, Kacena MA (2008) The effects of storage conditions on the preservation of enzymatic activity in bone. J Histotechnol 31:169–173PubMedGoogle Scholar
  9. Courty P-E, Pritsch K, Schloter M, Hartmann A, Garbaye J (2005) Activity profiling of ectomycorrhiza communities in two forest soils using multiple enzymatic tests. New Phytol 167:309–319PubMedCrossRefGoogle Scholar
  10. Courty P-E, Breda N, Garbaye J (2007) Relation between oak tree phenology and the secretion of organic matter degrading enzymes by Lactarius quietus ectomycorrhizas before and during bud break. Soil Biol Biochem 39:1655–1663CrossRefGoogle Scholar
  11. Courty PE, Franc A, Garbaye J (2010) Temporal and functional pattern of secreted enzyme activities in an ectomycorrhizal community. Soil Biol Biochem 42:2022–2025Google Scholar
  12. Courty PE, Labbe J, Kohler A, Marçais B, Bastien C, Churin JL, Garbaye J, Le Tacon F (2011) Effect of poplar genotypes on mycorrhizal infection and secreted enzyme activities in mycorrhizal and non-mycorrhizal roots. J Exp Bot 62:249–260Google Scholar
  13. De Bolle X, Vinals C, Fastrez J, Feytmans E (1997) Bivalent cations stabilize yeast alcohol dehydrogenase I. Biochem J 323:409–413PubMedGoogle Scholar
  14. Duponnois R, Garbaye J (1991) Techniques for controlled synthesis of the Douglas-fir–Laccaria laccata ectomycorrhizal symbiosis. Ann For Sci 48:641–650CrossRefGoogle Scholar
  15. Hedh J, Wallander H, Erland S (2008) Ectomycorrhizal mycelial species composition in apatite amended and non-amended mesh bags buried in a phosphorus-poor spruce forest. Mycol Res 112:681–688PubMedCrossRefGoogle Scholar
  16. Hoppe H-G (1983) Significance of exoenzymatic activities in the ecology of brackish water: measurements by means of methylumbelliferyl-substrates. Mar Ecol Prog Ser 11:299–308CrossRefGoogle Scholar
  17. Horton TR, Bruns TD (2001) The molecular revolution in ectomycorrhizal ecology: peeking into the black-box. Mol Ecol 10:1855–1871PubMedCrossRefGoogle Scholar
  18. Kacena MA, Troiano NW, Coady CE, Horowitz MC (2004) Effects of ethanol post-fixation on the histological, histochemical, and immunohistochemical analysis of murine bone specimens. J Histotechnol 27:15–20Google Scholar
  19. Latgé J-P (2007) The cell wall: a carbohydrate armour for the fungal cell. Mol Microbiol 66:279–290PubMedCrossRefGoogle Scholar
  20. Lehto T, Brosinsky A, Heinonen-Tanski H, Repo T (2008) Freezing tolerance of ectomycorrhizal fungi in pure culture. Mycorrhiza 18:385–392PubMedCrossRefGoogle Scholar
  21. Martin F, Selosse M-A (2008) The Laccaria genome: a symbiont blueprint decoded. New Phytol 180:296–310PubMedCrossRefGoogle Scholar
  22. Martin F, Kohler A, Murat C, Balestrini R, Coutinho PM, Jaillon O, Montanini B, Morin E, Noel B, Percudani R, Porcel B, Rubini A, Amicucci A, Amselem J, Anthouard V, Arcioni S, Artiguenave F, Aury J-M, Ballario P, Bolchi A, Brenna A, Brun A, Buee M, Cantarel B, Chevalier G, Couloux A, Da Silva C, Denoeud F, Duplessis S, Ghignone S, Hilselberger B, Iotti M, Marcais B, Mello A, Miranda M, Pacioni G, Quesneville H, Riccioni C, Ruotolo R, Splivallo R, Stocchi V, Tisserant E, Viscomi AR, Zambonelli A, Zampieri E, Henrissat B, Lebrun M-H, Paolocci F, Bonfante P, Ottonello S, Wincker P (2010) Perigord black truffle genome uncovers evolutionary origins and mechanisms of symbiosis. Nature 464:1033–1038PubMedCrossRefGoogle Scholar
  23. Mogge B, Loferer C, Agerer R, Hutzler P, Hartmann A (2000) Bacterial community structure and colonization patterns of Fagus sylvatica l. Ectomycorrhizospheres as determined by fluorescence in situ hybridization and confocal laser scanning microscopy. Mycorrhiza 9:271–278CrossRefGoogle Scholar
  24. Mosca E, Montecchio L, Scattolin L, Garbaye J (2007) Enzymatic activities of three ectomycorrhizal types of Quercus robur l. In relation to tree decline and thinning. Soil Biol Biochem 39:2897–2904CrossRefGoogle Scholar
  25. Niederer M, Pankow W, Wiemken A (1989) Trehalose synthesis in mycorrhiza of Norway spruce: an indicator of vitality. Eur J For Pathol 19:14–20CrossRefGoogle Scholar
  26. Perez-Moreno J, Read DJ (2000) Mobilization and transfer of nutrients from litter to tree seedlings via the vegetative mycelium of ectomycorrhizal plants. New Phytol 145:301–309CrossRefGoogle Scholar
  27. Pitarch A, Sanchez M, Nombela C, Gil C (2002) Sequential fractionation and two-dimensional gel analysis unravels the complexity of the dimorphic fungus Candida albicans cell wall proteome. Mol Cell Proteomics 1(12):967–982PubMedCrossRefGoogle Scholar
  28. Plassard C, Guérin-Laguette A, Véry A-A, Casarin V, Thibaud J-B (2002) Local measurements of nitrate and potassium fluxes along roots of maritime pine. Effects of ectomycorrhizal symbiosis. Plant Cell Environ 25:75–84CrossRefGoogle Scholar
  29. Pretzsch H, Kahn M, Grote R (1998) The mixed spruce-beech forest stands of the “Sonderforschungsbereich” “growth or parasite defence?” in the forest district Kranzberger Forst. J For Res 117:241–257Google Scholar
  30. Pritsch K, Garbaye J (2011) Enzyme secretion by ECM-fungi and exploitation of mineral nutrients from soil organic matter. Ann Forest Sci 68(1). doi:10.1007/s13595-010-0004-8
  31. Pritsch K, Raidl S, Marksteiner E, Blaschke H, Agerer R, Schloter M, Hartmann A (2004) A rapid and highly sensitive method for measuring enzyme activities in single mycorrhizal tips using 4-methylumbelliferone labelled fluorogenic substrates in a microplate system. J Microbiol Meth 58:233–241CrossRefGoogle Scholar
  32. R Development Core Team (2005) R: a language and environment for statistical computing. R Development Core Team, ViennaGoogle Scholar
  33. Rast DM, Baumgartner D, Mayer C, Hollenstein GO (2003) Cell wall-associated enzymes in fungi. Phytochemistry 64:339–366PubMedCrossRefGoogle Scholar
  34. Richard F, Millot S, Gardes M, Selosse MA (2005) Diversity and specificity of ectomycorrhizal fungi retrieved from an old-growth Mediterranean forest dominated by Quercus ilex. New Phytol 166:1011–1023PubMedCrossRefGoogle Scholar
  35. Rineau F, Garbaye J (2009) Does forest liming impact the enzymatic profiles of ectomycorrhizal communities through specialized fungal symbionts? Mycorrhiza 19:493–500PubMedCrossRefGoogle Scholar
  36. Slayman CL (1965) Electrical properties of Neurospora crassa: effects of external cations on the intracellular potential. J Gen Physiol 49:69–92PubMedCrossRefGoogle Scholar
  37. Staddon PL, Fitter AH (2001) The differential vitality of intraradical mycorrhizal structures and its implications. Soil Biol Biochem 33:129–132CrossRefGoogle Scholar
  38. Tedersoo L, Köljalg U, Hallenberg N, Larsson K-H (2003) Fine scale distribution of ectomycorrhizal fungi and roots across substrate layers including coarse woody debris in a mixed forest. New Phytol 159:153–165CrossRefGoogle Scholar
  39. Tedersoo L, Partel K, Jairus T, Gates G, Poldmaa K, Tamm H (2009) Ascomycetes associated with ectomycorrhizas: molecular diversity and ecology with particular reference to the Helotiales. Environ Microbiol 11(12):3166–3178PubMedCrossRefGoogle Scholar
  40. Tibbett M, Sanders FE (2002) Ectomycorrhizal symbiosis can enhance plant nutrition through improved access to discrete organic nutrient patches of high resource quality. Ann Bot 89:783–789PubMedCrossRefGoogle Scholar
  41. Wallenius K, Rita H, Simpanen S, Mikkonen A, Niemi RM (2010) Sample storage for soil enzyme activity and bacterial community profiles. J Microbiol Meth 81:48–55CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Karin Pritsch
    • 1
  • Pierre Emanuel Courty
    • 3
    • 4
  • Jean-Louis Churin
    • 3
  • Benoit Cloutier-Hurteau
    • 7
  • Muhammad Arif Ali
    • 7
  • Coralie Damon
    • 6
  • Myriam Duchemin
    • 7
  • Simon Egli
    • 5
  • Jana Ernst
    • 10
  • Laurence Fraissinet-Tachet
    • 6
  • Francisco Kuhar
    • 11
    • 2
  • Elvira Legname
    • 7
  • Roland Marmeisse
    • 6
  • Alex Müller
    • 5
  • Petia Nikolova
    • 1
  • Martina Peter
    • 5
  • Claude Plassard
    • 7
  • Franck Richard
    • 8
  • Michael Schloter
    • 10
  • Marc-André Selosse
    • 8
  • Alain Franc
    • 9
  • Jean Garbaye
    • 3
  1. 1.Institute of Soil EcologyGerman Research Center for Environmental Health (GmbH)NeuherbergGermany
  2. 2.Biodiversidad y Biología Experimental, CABAUniversidad de Buenos AiresBuenos AiresArgentina
  3. 3.UMR 1136, Interactions Arbres Micro-organismesCentre INRA de NancyChampenouxFrance
  4. 4.Botanical InstituteUniversity of BaselBaselSwitzerland
  5. 5.Swiss Federal Research Institute WSLBirmensdorfSwitzerland
  6. 6.UMR CNRS 5557 d’Ecologie MicrobienneUniversité de Lyon, Université Lyon 1Villeurbanne CedexFrance
  7. 7.UMR 1222, Ecologie Fonctionelle et Biogéochimie des SolsINRA/IRD/SupAgroMontpellier Cedex 01France
  8. 8.UMR 5175Centre d’Ecologie Fonctionnelle et EvolutiveMontpellier 5France
  9. 9.UMR Biodivers Genes & CommunautesINRA PierrotonCestasFrance
  10. 10.Institute of Soil Ecology, Terrestrial EcogeneticsGerman Research Center for Environmental Health (GmbH)NeuherbergGermany
  11. 11.Institute of Soil Ecology, Helmholtz Zentrum MünchenGerman Research Center for Environmental Health (GmbH)NeuherbergGermany

Personalised recommendations