479 Mycologia, 96(3), 2004, pp. 479–487.  2004 by The Mycological Society of America, Lawrence, KS 66044-8897 Surface-bound phosphatase activity in living hyphae of ectomycorrhizal fungi of Nothofagus obliqua Maricel Alvarez 1 Roberto Godoy Universidad Austral de Chile, Instituto de Bota ´ nica, Campus Isla Teja, Casilla 567, Valdivia, Chile Wolfgang Heyser University of Bremen, Institute of Environmental Science and Technology (UFT), Plant Physiology and Plant Anatomy, Leobenerstrasse, D-28359 Bremen, Germany Steffen Ha ¨rtel Centro de Estudios Cientı ´ficos (CECS), Arturo Prat 514, Valdivia, Chile y Facultad de Ciencias Quı ´micas, Departamento de Quı ´mica Biolo ´gica, Pabello ´n Argentina, Universidad Nacional de Co ´rdoba, Ciudad Universitaria, 5000 Co ´rdoba, Argentina Abstract: We determined the location and the activ- ity of surface-bound phosphomonoesterase (SBP) of five ectomycorrhizal (EM) fungi of Nothofagus oblique. EM fungal mycelium of Paxillus involutus, Austropaxillus boletinoides, Descolea antartica, Cenococ- cum geophilum and Pisolithus tinctorius was grown in media with varying concentrations of dissolved phos- phorus. SBP activity was detected at different pH val- ues (3–7) under each growth regimen. SBP activity was assessed using a colorimetric method based on the hydrolysis of p-nitrophenyl phosphate (pNPP) to p-nitrophenol phosphate (pNP) + P. A new tech- nique involving confocal laser-scanning microscopy (LSM) was used to locate and quantify SBP activity on the hyphal surface. EM fungi showed two funda- mentally different patterns of SBP activity in relation to varying environmental conditions (P-concentra- tions and pH). In the cases of D. antartica, A. bole- tinoides and C. geophilum, changes in SBP activity were induced primarily by changes in the number of SBP-active centers on the hyphae. In the cases of P. tinctorius and P. involutus, the number of SBP-active centers per m hyphal length changed much less than the intensity of the SBP-active centers on the hyphae. Our findings not only contribute to the dis- cussion about the role of SBP-active centers in EM fungi but also introduce LSM as a valuable method for studying EM fungi. Accepted for publication November 22, 2003. 1 Corresponding author. Telephone: +56-63-234513. Fax: +56-63- 234517. E-mail: malvarez@uni-bremen.de Key words: ectomycorrhiza, ELF-97, image pro- cessed confocal fluorescence microscopy, P-uptake INTRODUCTION Phosphorus is one of the most important minerals for plant nutrition. At the same time, it is the most inaccessible soil nutrient (Holford 1997, Narang et al 2000). For this reason, plants have developed nu- merous morphological, physiological, biochemical and molecular adaptation strategies for its acquisition (Raghothama 1999). Among these strategies, the ex- pression of P-cleaving enzymes (mainly phospho- monoesterases and phosphodiesterases) by mycorrhi- zas and EM fungi represents an important mecha- nism to increase P availability for plant symbionts ( Jayachandran et al 1992, Smith and Read 1997, van Aarle et al 2001). Despite the numerous studies on phosphatase activity in EM fungal mycelium, little is known about adaptive changes in SBP activity in re- sponse to different soil conditions. In this study, we mapped the distribution of SBP activity in mycelium of five EM partners of Nothofagus obliqua (Mirb.) Oerst: Paxillus involutus (Batsch : Fr.) Sing., Austropaxillus boletinoides (Sing.) Brsky & Ja- rosh, Descolea antartica Sing., Cenococcum geophilum Fr. and Pisolithus tinctorius (Pers.) Coker & Couch. SBP activity was determined with a colorimetric assay, based on the hydrolysis of pNPP to pNP + P, which is a well established method for quantification of phosphomonoesterase activity in EM fungi (Antibus et al 1992, Tibbett et al 1998). In addition, SBP ac- tivity was determined using a new approach; we com- bined confocal LSM, a simple staining procedure with the UV-sensitive fluorogenic substrate, enzyme- labeled fluorescence (ELF-97) and image processing routines. This technique enabled us to localize and quantify the SBP activity of the fungal mycelium at defined pH and phosphorus levels. MATERIALS AND METHODS Cultivation of EM fungi.—Cultures of the EM fungi Paxillus involutus, Austropaxillus boletinoides and Descolea antartica were obtained from fruiting bodies that were collected in temperate forests of Nothofagus obliqua, near Quita Calzo ´n, 39°78'S, 73°02'W, Valdivia, X Region, Chile. Ectomycorrhi-