ELEMENTAL CONTENTS IN VACUOLAR GRANULES OF ECTOMYCORRHIZAL FUNGI MEASURED BY EELS AND EDXS - A COMPARISON OF DIFFERENT METHODS AND PREPARATION TECHNIQUES

The elemental composition of vacuolar granules in different ectomycorr hizal fungi, Pisolithus tinctorius, Suillus bovinus and Xerocomus badi us, were determined by electron energy loss spectroscopy (EELS) and en ergy dispersive X-ray spectroscopy (EDXS). The investigations dealt wi th advantages and limits of the EDXS and EELS technique with respect t o the analysis of the elemental composition of vacuolar granules and t he effect of different specimen preparation techniques. Axenic culture s of these fungi as well as field mycorrhizae were used for the analys is. Results after conventional chemical fixation and dehydration of ma terial were compared to results obtained after cryofixation followed b y freeze-drying of the samples. Light microscopical studies were also carried out to control the occurrence of vacuolar granules in living h yphae. The results showed that vacuolar granules exist in living hypha e of different ectomycorrhizal fungi and are not an artifact of the fi xation or other specimen preparation procedures of cells. EDXS and EEL S differed in their ability to detect the elemental composition of the se granules. Both analytical techniques found P in the vacuolar bodies , which indicates a deposition of polyphosphates. Polyphosphate granul es are strongly negative polyanions, which contain different cations t o balance the negative charge. These cations were often difficult to d etermine by EELS and could only be shown by EDXS, but the cations vari ed considerably depending on the technique used for specimen preparati on. In chemically fixed and dehydrated material, especially Mg, K and Ca were detected in the granules. However, measurements of cryofixed a nd Freeze-dried specimens showed that the most abundant cations in pol yphosphate granules were K and Mg and the incorporation of Ca has to b e interpreted as a result of the chemical specimen preparation. EELS r evealed that N was also incorporated in the vacuolar granules independ ently of the specimen preparation. Based on these results. it can be a ssumed(1) that the N-containing granules, detected by EELS, and the P- rich granules, analysed by EDXS, were identical; (2) that these vacuol ar inclusions were polyphosphate granules or metachromatic granules, w hich were often described in different mycorrhizal associations; and ( 3) that polyphosphate granules can also act as a binding site for larg e amounts of N. (C) 1998 Elsevier Science Ltd. All rights reserved.