Plant and Soil (2006) 287:69–75 DOI 10.1007/s11104-006-9061-z
Ó Springer 2006
Phosphate-solubilizing microorganisms isolated from rhizospheric and bulk soils of colonizer plants at an abandoned rock phosphate mine I. Reyes1, A. Valery & Z. Valduz Laboratorio de Biofertilizantes, Decanato de Investigacio´n, Universidad Nacional Experimental del Ta´chira, Paramillo, San Cristo´bal, Ta´chira, Repu´blica Bolivariana de Venezuela. 1Corresponding author* Received: 31 May 2006
Key words: rock phosphate, hydroxyapatite solubilization, biodiversity, rhizosphere, Penicillium sp., Azotobacter sp.
Abstract The abandoned ‘‘Monte-Fresco’’ rock phosphate mine in Ta´chira, Venezuela, was sampled to study the biodiversity of phosphate-solubilizing microorganisms (PSM). Rhizosphere and bulk soils were sampled from colonizer plant species growing at a mined site where pH and soluble P were higher than the values found at a near by unmined and shrubby soil. Counting and isolating of PSM choosing strains showing high solubilization halos in a solid minimal medium with hydroxyapatite as phosphate source were evaluated using ammonia or nitrate as nitrogen sources and dextrose, sucrose, and mannitol as carbohydrate sources. A larger number of PSM were found in the rhizospheric than in the bulk soil. Six fungal strains belonging to the genus Penicillium and with high hydroxyapatite dissolution capacities were isolated from bulk soil of colonizer plants. Five of these strains had similar phenotypes to Penicillium rugulosum IR94MF1 but they solubilized hydroxyapatite at different degrees with both nitrogen sources. From 15 strains of Gram-negative bacteria isolated from the rhizosphere of colonizer plants, 5 were identified as diazotrophic free-living encapsulated Azotobacter species able to use ammonium and/or nitrate to dissolve hydroxyapatite with glucose, sucrose and/or mannitol. Different nitrogen and carbohydrate sources are parameters to be considered to further characterize the diversity of PSM.
Introduction Interactions between microorganisms that release organic acids and other products onto the surfaces of minerals may liberate ions from their surface layers. In this sense, rock phosphate dissolution by microorganisms directly affects fertility of soils (Reyes et al. 2002). The rhizosphere is a dynamic changing environment that differs from bulk soil both in physical and chemical properties (Bowen and Rovira 1999). In this sense, plant root exudates selectively influence the growth of microorganisms that colonize the rhizosphere when altering the chemistry of soil * E-mail:
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aggregates and concurrently, rhizospheric microbial populations change the composition and quantity of root exudates through their effect on plant nutrition (Bowen and Rovira 1999; Glick 1995). In mineral soils, bacterial and fungal populations increase in abundance and diversity as minerals are weathered and transformed to soil (Banfied et al. 1999). During the initial stages of weathering, apatite rock phosphate is replaced by chemically- or microbially-precipitated secondary phosphate minerals (e.g., strengite and variscite) and these are completely solubilized in the soil after microbial colonization (Banfied et al. 1999). It has been demonstrated that fungi (Reyes et al. 2001; Vassilev et al. 1996) and bacteria (Goldstein 1995) release organic acids such
70 as citric, gluconic, and keto-gluconic, to dissolve phosphates and other complexing compounds such as siderophores (Watteau and Berthelin 1994), and reducing mechanisms of cations also liberate phosphates into soil (Altomare et al. 1999). Colonizer plants growing in soil disturbed by strip mining after exploitation of rock phosphates may reflect in their rhizospheres the processes of mineral transformations of such soils. Spoil banks, left by strip mining, are composed of debris of the rock phosphate, skeletal minerals, and other mixed materials from the disturbed soil horizons. Colonizer plant species could bring some insights about strategies that nature uses for mineral cycling and revegetation of a disturbed land. The purpose of the present work is to determine the phosphate-solubilizing microorganisms (PSM) diversity existing in a rock phosphate mine soil. In the mined site of the mine, diverse strains of Penicillium, Azotobacter and other unidentified bacteria and fungi were found associated to the colonizer plant species’ bulk and rhizosphere soils, respectively. In order to characterize the diversity among their phosphate solubilizing (PS) metabolism Penicillium sp. and Azotobacter sp. strains were assessed using different carbohydrate and nitrogen sources.
Materials and methods Site characterization and sampling Monte Fresco rock phosphate mine is a low-solubility fluorapatite situated in the Andean piedmont at the southwest region of Venezuela (1000 m a.s.l.). Two sites were sampled: an area mined and abandoned since 1994 characterized by a low vegetation stratum distributed in a random-clustered pattern, and a second site, used as a control for the bulk soil, located in an unmined area around 150 m of the first one composed of a shrubby pasture. Sampling of the rhizosphere and bulk soils was done after the end of the rainy season. The unmined and mined sites were sampled for their bulk soils to determine total and solubilizing cultivable fungi and bacteria populations. The mined soil was sampled for the rhizosphere soils of different colonizer plants, which were not present in the shrubby pasture
site, in order to isolate Monte Fresco phosphate solubilizers. For the bulk soils composite samples were obtained from the first 10 cm depth and for the rhizosphere soils the whole plants were transported in a cool container with a square of soil around the roots. Some relevant chemical and physical properties of the sampled bulk soils were determined: P, Ca2+, Mg2+, K+, % organic matter, pH and soil texture. Adsorbed forms of phosphate were extracted following the method of Bray and Kurtz (1945), where neutral ammonium fluoride is used as the extraction reagent for soils with a pH
