Alvarez M., Godoy R., Gieseke A., & Härtel S.
Biology & Fertility of Soils 42(6), 561-568,

For the quantification of surface bound phosphomonoesterase activity (SBPA) of fungi, roots, or mycorrhiza, a colorimetric method based on p-nitrophenyl-phosphate (pNPP) is widely used. Unfortunately, this method does not reveal information about the localisation of the surface bound phosphomonoesterase (SBP). We introduce a method which localises and quantifies SBPA in living hyphae of ectomycorrhizal (EM) fungi using confocal laser scanning microscopy (LSM) of the hydrophilic substrate ELF-97 (enzyme-labeled fluorescence) and compare it to the pNPP assay. ELF-97 turns into a strongly-fluorescent precipitate upon activation by SBPA and forms bright fluorescent centres on the outer cell wall of the hyphae. Our data shows that the enzymatic reaction is not substrate limited during an incubation period of 15 min in fungal hyphae of Pisolithus tinctorius, Cenococcum geophilum, and Paxillus involutus. Image processing routines determined the total intensity and the average number of the fluorescent ELF-97 centres / µm fungal hyphae of C. geophilum and P. involutus. ELF-97 and pNPP detected similar variations of the SBPA at different pH values (3-7) during the measurement and different phosphorus (P) concentrations during the growth period of the fungi. The ELF-97 method revealed that C. geophilum and P. involutus adapt in different ways to the variation of the P concentrations during the growth period by varying the number, the activity, or both properties of the SBP centres. The phosphatases show peak activities at different pH values, so the response of the fungal mycelium to varying P concentrations in soils is pH selective. In conclusion, ELF-97 is a promising substrate to reveal SBPA and adaptation strategies on a structural-physiological level.

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