A rapid and highly sensitive method for measuring enzyme activities in single mycorrhizal tips using 4-methylumbelliferone-labelled fluorogenic substrates in a microplate system

Journal of Microbiological Methods
K PritschA Hartmann

Abstract

A microplate fluorimetric assay was developed for measuring potential activities of extracellular enzymes of individual ectomycorrhizal (EM) roots using methylumbelliferone (MU)-labelled fluorescent substrate analogues and microsieves to minimise damage due to manipulation of excised mycorrhizal roots. Control experiments revealed that enzyme activities remained stable over the whole time of the experiment suggesting a strong affinity of the studied enzymes to the fungal cell walls. The same mycorrhizal tips thus could be used repeatedly for enzyme detection and subsequently analysed for the projection area by automated image analysis. The developed system was evaluated on four different EM species measuring pH optimum and substrate saturation of phosphatase, chitinase and beta-glucosidase. The four EM species studied were Lactarius subdulcis, Russula ochroleuca, Cortinarius obtusus and Xerocomus cf. chrysenteron. Depending upon the enzyme, each species exhibited different levels of enzymatic activities as well as enzyme kinetics and showed also differences in pH optima.

References

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Aug 29, 2003·Phytochemistry·Dora M RastG O Hollenstein

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Citations

Aug 28, 2012·Current Microbiology·Maria Swiontek Brzezinska, Urszula Jankiewicz
Nov 1, 2013·The ISME Journal·Lori A PhillipsMelanie D Jones
Mar 19, 2013·Journal of Microbiological Methods·Doreen FischerMichael Schloter
Feb 20, 2007·Mycorrhiza·Magali di PietroJean Garbaye
Jun 14, 2005·The New Phytologist·Pierre-Emmanuel CourtyJean Garbaye
Sep 24, 2014·Journal of the American Chemical Society·Yuanli Liu, Marco Bonizzoni
Mar 27, 2010·Journal of Evaluation in Clinical Practice·Maria D Perez-CarcelesAurelio Luna
Aug 31, 2010·Mycorrhiza·Marleen IjdoStéphane Declerck
Nov 18, 2015·Virchows Archiv : an International Journal of Pathology·J Han J M Van KriekenAndreas Jung
Apr 4, 2021·International Journal of Molecular Sciences·Gheorghita MenghiuRaluca Ostafe
Dec 23, 2016·Journal of Microbiological Methods·Shiping DengMichael N Weintraub

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