Elemental contents in vacuolar granules of ectomycorrhizal fungi measured by EELS and EDXS. A comparison of different methods and preparation techniques

Abstract

The elemental composition of vacuolar granules in different ectomycorrhizal fungi, Pisolithus tinctorius, Suillus bovinus and Xerocomus badius, were determined by electron energy loss spectroscopy (EELS) and energy dispersive X-ray spectroscopy (EDXS). The investigations dealt with advantages and limits of the ECXS and EELS technique with respect to the analysis of the elemental composition of vacuolar granules and the effect of different specimen preparation techniques. Axenic cultures of these fungi as well as field mycorrhizae were used for the analysis. Results after conventional chemical fixation and dehydration of material were compared to results obtained after cryofixation followed by freeze-drying of the samples. Light microscopical studies were also carried out to control the occurrence of vacuolar granules in living hyphae. The results showed that vacuolar granules exist in living hyphae of different ectomycorrhizal fungi and are not an artifact of the fixation or other specimen preparation procedures of cells. EDXS and EELS differed in their ability to detect the elemental composition of these granules. Both analytical techniques found P in the vacuolar bodies, which indicates a deposition of polyphosphates. Polyphosphate granules are strongly negative polyanions, which contain different cations to balance the negative charge. These cations were often difficult to determine by EELS and could only be shown by EDXS, but the cations varied considerably depending on the technique used for specimen preparation. In chemically fixed and dehydrated material, especially Mg, K and Ca were detected in the granules. However, measurements of cryofixed and freeze-dried specimens showed that the most abundant cations in polyphosphate granules were K and Mg and the incorporation of Ca has to be interpreted as a result of the chemical specimen preparation. EELS revealed that N was also incorporated in the vacuolar granules independently of the specimen preparation. Based on these results, it can be assumed (1) that the N-containing granules, detected by EELS, and the P-rich granules, analysed by EDXS, were identical; (2) that these vacuolar inclusions were polyphosphate granules or metachromatic granules, which were often described in different mycorrhizal associations; and (3) that polyphosphate granules can also act as a binding site for large amounts of N.