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

A microplate fluorimetric assay was developed for measuring potential activities of extracellular enzymes of individual ectomycorrhizal (EM) roots using metbylumbelliferone (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. chtysenteron. Depending upon the enzyme, each species exhibited different levels of enzymatic activities as well as enzyme kinetics and showed also differences in pH optima. (C) 2004 Elsevier B.V. All rights reserved.