DIVISION S-3-SOIL BIOLOGY & BIOCHEMISTRY A Simplified Method for Extraction of Ergosterol from Soil N. S. Hash,* P. D. Stahl, T. B. Parkin, and D. L. Karlen ABSTRACT Ergosterol is a sterol common to many fungi and may be useful for estimating fungal biomass in soil. Our objective was to compare three different methods for extracting ergosterol from soil. These included: (i) a published method that involves extraction with metha- nol, saponification with KOH, and separation with hexane before analysis using high-pressure liquid chromatography (HPLC); (ii) a recently published supercritical fluid extraction (SFE) method; and (iii) a simplified extraction method developed in our laboratory that is similar to method (i) but requires 80 to 90% less reagents. Similar quantities of ergosterol were extracted from both prairie and cropland soil with method (i) and with simplified method (iii). In contrast, the SFE method (ii) did not recover naturally occurring ergosterol from soil. Extraction efficiencies of ergosterol standards added to soil were between 75 and 88% for the three procedures, with coefficients of variation of <15% for all methods. Our simplified method substantially reduced cost, extraction time, and chemical waste per sample. Results of this study indicate that the simplified method was the most efficient technique for extraction of ergosterol from soil. F UNGI play an important role in ecosystem function (Christensen, 1989) and contribute significantly to soil quality (Eash et al., 1994). They are a major compo- nent of the soil microbial community and represent a dynamic pool of nutrients in the form of living and dead microorganisms (Doran and Linn, 1994). Fungi are the primary decomposers of plant residue, mineralizing and immobili/ing important nutrients that support successive crops following lysis, death, and decomposition (Baath and Soderstrom, 1979; Paul and Clark, 1989). Fungi serve as a food source for soil fauna, are an important mediator in soil aggregate formation (Eash, 1993; Jas- trow and Miller, 1991; Molope, 1987; Tisdall and Oades, 1982), and cause disease in plants. Until accurate and reliable methods for evaluating fungal biomass in soil are determined, the role of fungi as "conservers" and "cyclers" of nutrients cannot be accurately quantified (Franklandetal., 1990; Newell, 1992; Parkinson, 1994). Current methods for assessing fungal presence in soil include direct microscopy, ergosterol extraction, immu- noassays, enzyme activities, and selective inhibition assays (Newell, 1992). The usefulness of these methods, however, is disputed due to problems intrinsic to each. Direct microscopy is the most common technique to N.S. Eash, Plant and Soil Science Dep., Univ. of Tennessee, Knoxville, TN 37901; P.O. Stahl, T.B. Parkin, and D.L. Karlen, USDA-ARS, National Soil Tilth Lab., 2150 Pammel Drive, Ames, IA 50011. Mention of trademark, proprietary product, or vendor does not constitute a guarantee or warranty of this product by the USDA and does not imply its approval to the exclusion of other products or vendors that may also be suitable. Received 24 Oct. 1994. *Corresponding author (eash@utkvx.utk.edu). Published in Soil Sci. Soc. Am. J. 60:468-471 (1996). estimate soil fungal biomass, but results are often influ- enced by observer subjectivity (Stahl et al., 1995; New- ell, 1992). Estimation of fungal biomass from soil ergo- sterol concentration is gaining in popularity but requires the use of conversion factors that are not yet well estab- lished. Results of previous studies (Stahl et al., 1995; West et al., 1987) suggest that using direct counts in combination with ergosterol measurements can increase the reliability and accuracy of estimating fungal biomass. Use of both methods can also aid in the comparison of estimates from independent laboratories. Ergosterol is an indicator of live fungal biomass (West et al., 1987) because it is quickly degraded. Davis and LaMar (1992) found >95% reduction in ergosterol con- tent within 2 wk following fumigation and death of the fungal cells. Ergosterol correlates with fungal surface area (West et al., 1987) and hyphal length (Matcham et al., 1985), and is more sensitive than chitin or extracellu- lar laccase assays (Matcham et al., 1985; Seitz et al., 1979). Given that ergosterol is the most frequently en- countered fungal sterol (Davis and LaMar, 1992; Grant and West, 1986; Mercer, 1984; Peacock and Goosey, 1989; Pierce et al., 1979; Weete, 1973), ergosterol may be a good indicator of fungal biomass in soil. The most commonly used method for extracting ergo- sterol from soil (Grant and West, 1986) is resource intensive because it requires large volumes of solvent and a time-consuming reflux step. A recently published method that potentially overcomes these limitations is SFE (Young and Games, 1993). This method accom- plishes extraction using liquid CC*2 as the solvent; thus, no hazardous wastes are generated. However, the SFE method was developed for grain samples and has not been tested on soil. As an alternative to the Grant and West method, we developed a simplified procedure for ergosterol extraction from soil. The objective of this study was to compare these three methods for extraction of ergosterol from soil. To accomplish this goal, we performed two experiments, one in which the three meth- ods were compared with regard to extraction of naturally occurring ergosterol from each of two soils, and another where we evaluated the efficiency of the methods by measuring recovery of ergosterol standards added to soil. MATERIALS AND METHODS Three methods for ergosterol extraction from soil were evaluated with respect to extraction efficiency and variability. The methods compared were: (i) the technique of Grant and West (1986), (ii) the SFE as described by Young and Games Abbreviations: HPLC, high pressure liquid chromatography; SFE, super- critical fluid extraction; CV, coefficient of variation; ANOVA, analysis of variance. 468