Aqueous biphasic system to extract arbuscular mycorrhizal spores from soils

The aqueous biphasic system (ABS) method using PEG and ammonium sulphate improved the recovery of spores from tropical soils as compared with the sucrose gradient method. The ABS was not affected by low temperatures. The optimal recovery of spores was achieved when 100% PEG and 50% ammonium sulphate concentration and PEG8000 instead of PEG4000 were used. Under these conditions an optimal interface formation which facilitates the localization and recovery of spores was obtained. Both phases of the ABS are aqueous and did not affect either the viability of spores or the level of root colonization reached by these spores. Soil composition did not affect the number of spores obtained by the ABS method either. 2008 Elsevier Ltd. All rights reserved. Spores are the main propagules of the arbuscular mycorrhizal (AM) fungi. Despite the fundamental importance of extraction of spores from soils, there have been few reports of new methods for the isolation of these propagules. The method of wet sieving and decanting has been the most common to extract spores from soil (Gerdemann and Nicolson, 1963). However, from tropical soils with high clay and organic matter content the extraction of spores has been very difficult and only the sucrose gradient method developed by Jenkins (1964) seems to be effective (Sieverding, 1983; Brundett et al., 1999; Martin et al., 2001). Aqueous biphasic system (ABS) has been widely used for protein, virus and cell recovery (Diamond and Hsu, 1990). In ABS, two separate phases are formed when polymers such as polyethylene glycol (PEG) are mixed with salts such as ammonium sulphate in particular concentrations. The equilibrium distribution (partitioning) of a substance in ABS depends not only on its own surface properties such as charge and hydrophobicity but also on the physicochemical properties of the two phases which can be manipulated by adjusting factors such as the polymer molecular weight and concentration, and salt concentration (Baskir et al., 1989). To study the efficiency of ABS the method, spores were isolated from four soils located in the Ejido Raymundo Enriquez (ERE) field, Tapachula, Mexico. The texture, pH and organic matter were determined (Walkley and Black, 1934; Bouyoucos, 1951) (Table 1). Three replicate root-soil samples were collected from each soil. Each sample consisted of five bulked subsamples taken from the top 10 cm of soil. In order to calibrate the optimal ABS conditions, the sandy-loam soil cultivated with Theobroma cacaowas used. AM spores were isolated from 100 g of each soil sample by the wet sieving and decanting technique (Gerdemann and Nicolson, 1963), counted in a Doncaster dish (Doncaster, 1962) and considered as 100% of spore recovery. The ABS was made by mixing the same quantities (V/V) of PEG8000 or PEG4000 aqueous solutions (from 10 to 100% of saturation) with ammonium sulphate aqueous solutions (from 10 to 100% of saturation). The isolates from the sieves were suspended in 15 ml of ammonium sulphate solutions and, after 1 min agitation, they were mixed with and equal volume of PEG. The mixture was centrifuged for 1 min at 500 rpm and the spores were placed in the interphase and were separated with a pipette, rinsed with distilled water and counted in a Doncaster dish. The ABS with 100% PEG8000 and 50% ammonium sulphate concentrationwas tested at temperatures of 28 and 4 C. The sucrose gradient method (Jenkins, 1964) was carried out with the isolates from the sieves which were centrifuged at 3500 rpm for 2 min in a 33, 50 or 69% (W/V) of sucrose. The spores obtained in the interphase of the gradient were separated with a pipette, rinsed with distilled water and counted in a Doncaster dish. For each of the ABS and sucrose * Corresponding author. Tel.: þ34 58 121011; fax: þ34 958 129600. E-mail addresses: [email protected] (M. Salvador-Figueroa), jocampo@ eez.csic.es (J.A. Ocampo).