A novel method for characterizing the microscale 3D spatial distribution of bacteria in soil A. Dechesne a , C. Pallud b , D. Debouzie c , J.P. Flandrois c , T.M. Vogel a , J.P. Gaudet b , G.L. Grundmann a, * a Ecologie Microbienne UMR CNRS 5557, Universite ´ Claude Bernard Lyon 1, 16, rue Dubois, 69622 Villeurbanne cedex, France b Laboratoire d’Etude des Transferts en Hydrologie et Environnement UMR CNRS, INPG, Universite ´ Joseph Fourrier, IRD 5564, BP 53, 38041 Grenoble cedex 9, France c Laboratoire de Biome ´trie et Biologie Evolutive UMR CNRS 5558, Universite ´ Claude Bernard Lyon 1, 16, rue Dubois, 69622 Villeurbanne cedex, France Received 14 January 2002; received in revised form 14 July 2003; accepted 21 July 2003 Abstract Knowledge of the microscale spatial distribution of bacteria in soil would improve our understanding of microbial ecology and function. A specific 3D method has been developed to characterize the spatial distribution of bacterial microhabitats in soil. The method combines a specific microsampling strategy with a statistical data analysis. The sampling strategy involves several different unit volumes (minimum sample side: length of 50 mm) subsequently tested for the presence or absence of the targeted microorganisms. The statistical analysis is based on the comparison of experimental sampling data with data expected from the limited sampling of numerous theoretical spatial distributions. Since the exact spatial pattern of the zones colonized by bacteria requires the spatial coordinates of the samples, this approach only identifies possible distributions. Distributions are characterized by descriptors relevant to soil, such as the average size of colonized patches and their average number g 21 of soil. These descriptors provide the basis for comparing spatial patterns. Computer simulations confirmed the method’s ability to distinguish between different spatial patterns. The spatial patterns in soil of two bacterial types, NH 4 þ oxidizers and 2,4-D degraders, were studied independently in repacked soil columns percolated with solutions containing either NH 4 þ or 2,4- D. The spatial analysis method distinguished between the two microbial spatial distributions ðP # 0:025Þ: Thus, this method constitutes a useful tool for identifying factors regulating bacterial spatial distributions in soil at the microscale. q 2003 Elsevier Ltd. All rights reserved. Keywords: Microscale; Spatial distribution; 2,4-D degraders; NH4 þ oxidizers; Bacteria 1. Introduction The spatial variability of soil characteristics have scales ranging from aggregate to continental size (Beckett and Webster, 1971; Burrough, 1983; Amador et al., 2000; Grundmann and Debouzie, 2000; Franklin et al., 2002). Numerous sampling strategies and statistical methods are available for the spatial analysis of soil abiotic factors at different scales (Nielsen and Alemi, 1989). The spatial heterogeneity of bacterial activity is thought to be in part responsible for the spatial variability of soil factors, at least on a small scale (Wachinger et al., 2000). Nevertheless, due to methodological difficulties (Wardle and Giller, 1996), the study of the spatial distribution of microorganisms has lagged behind studies on the spatial variability of soil physical factors. The spatial distribution of microorganisms can be considered at different scales. At the large scale (samples taken at distances greater than 100 m), bacterial diversity studies provide access to biogeographical data and, thus, to the evaluation of bacterial endemism (Cho and Tiedje, 2000). At the field scale (0.1–100 m), there are numerous data about bacterial spatial heterogeneity, such as that applied to phytopathology. This application aims at assessing the risks of microbial pathogens contaminating plants (Schuh et al., 1986; Mihail and Alcorn, 1987). The scale of sampling used for these studies is always larger than a few centimeters. Due to their size, bacteria conspicuously 0038-0717/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0038-0717(03)00243-8 Soil Biology & Biochemistry 35 (2003) 1537–1546 www.elsevier.com/locate/soilbio * Corresponding author. Tel.: þ33-4-72-43-13-78; fax: þ33-4-72-43-12-23. E-mail address: grundman@biomserv.univ-lyon1.fr (G.L. Grundmann).