Microarray Analysis of Bacterial Diversity and Distribution in Aggregates from a Desert Agricultural Soil

Biol Fertil Soils (2008) 44:1003–1011 DOI 10.1007/s00374-008-0291-5 ORIGINAL PAPER Microarray analysis of bacterial diversity and distribution in aggregates from a desert agricultural soil Jong-Shik Kim & Robert S. Dungan & David Crowley Received: 1 February 2008 /Revised: 2 April 2008 /Accepted: 8 April 2008 /Published online: 7 May 2008 # Springer-Verlag 2008 Abstract Previous research has shown that soil structure distribution and organization of bacterial populations at can influence the distribution of bacteria in aggregates and, small scales (van Gestel et al. 1996; Ranjard et al. 2000; thereby, influence microbiological processes and diversity Mummey and Stahl 2004; Schutter and Dick 2002). Early at small spatial scales. Here, we studied the microbial research using cultivation-based methods demonstrated that community structure of inner and outer fractions of micro- Gram-positive bacteria were predominant in the outer aggregates of a desert agricultural soil from the Imperial fractions of microaggregates, whereas Gram-negative bac- Valley of Southern California. To study the distribution of teria occupied the inner fractions (Hattori and Hattori soil bacteria, 1,536 clones were identified using phyloge- 1976). Differences in the distribution of bacterial species netic taxon probes to classify arrays of 16S rRNA genes. in different aggregate size classes have also been revealed Among the predominant taxonomic groups were the α- using biochemical profiling and molecular methods (Kabir Proteobacteria, Planctomycetes, and Acidobacteria. When et al. 1994; Ranjard et al. 2000; Schutter and Dick 2002; compared across all phyla, the taxonomic compositions and Blackwood et al. 2006;Mummeyetal.2006). Low- distributions of bacterial taxa associated with the inner and resolution molecular methods include community profiling outer fractions were nearly identical. Our results suggest by denaturing gradient gel electrophoresis of 16S rRNA that the ephemeral nature of soil aggregates in desert genes (Kandeler et al. 2000) and terminal restriction agricultural soils may reduce differences in the spatial fragment length polymorphism analyses (Sessitsch et al. distribution of bacterial populations as compared to that 2001). The most recent work has used cloning and which occur in soils with more stable aggregates. sequence analysis of 16S rRNA genes to elucidate the relationship between inner- and outer-aggregate fractions Keywords 16S rRNA . Microaggregates . Macroarray . and microbial community structure (Mummey et al. 2006). Soil aggregate . Microbial community While many studies, so far, have revealed differences in the species richness of inner- and outer-aggregate fractions with inner-aggregates generally harboring greater species diver- Introduction sity, other studies using terminal restriction fragment length polymorphism have shown that differences in bacterial The size, stability, and chemical properties of soil aggre- community composition between the exterior and interior gates are important factors that influence the spatial fractions in some soils are marginal or not significant (Blackwood et al. 2006). This suggests that the influence of microsite location is dependent on the chemical and J.-S. Kim : D. Crowley (*) Department of Environmental Sciences, University of California, physical properties of the soils from which the aggregates Riverside, CA 92521, USA are formed. e-mail: [email protected] Here, we were interested in characterizing the difference in bacterial community structure and species composition R. S. Dungan Northwest Irrigation and Soils Research Laboratory, USDA-ARS, for inner- and outer-aggregate fractions from a desert Kimberly, ID 83341, USA agricultural soil in California’s Imperial Valley. Soils of 1004 Biol Fertil Soils (2008) 44:1003–1011 the Southwestern US are particularly difficult to manage On a mass basis, the inner-aggregate fraction accounted for due to the low amounts of organic matter and rapid 96.1% of the total aggregate mass. decomposition of organic materials that lead to low soil aggregation and poor soil structure (Martens et al. 2005). Soil DNA extraction This area receives less than 10 cm of rain per year and relies entirely on irrigation. Many farmers in the Imperial DNA was extracted from the inner- and outer-aggregate and Coachella Valley are using organic amendments and fractions using the Fast DNA Spin Kit for soil (Bio 101, green manures to improve soil structure. Hence, there is QBiogene, Vista, CA, USA). The DNA extraction proce- interest in whether organic amendments are also improving dure was conducted according to the manufacturer’s or affecting soil microbial diversity, presumably by im- protocol. The DNA was then concentrated using a Speed- proving the diversity of microhabitats that are provided by vac (AES 1000, Savant, Holbrook, NY, USA), gel eluted, improved soil aggregation using organic amendments. and purified with a QIAquick gel extraction kit (Qiagen, Nonetheless, the ephemeral nature of aggregates that are Chatsworth, CA, USA). formed in these loamy sand soils with low of amounts of clay may preclude development of distinct microbial 16S rRNA clone library construction communities since the organic matter in these fractions is relatively unprotected and short lived. To address this A clone library of the 16S rRNA genes from the inner- and question, we examined the distribution of different phylo- outer-aggregate fractions was generated by polymerase genetic groups of the inner- and outer-aggregate fractions of chain reaction (PCR) of the purified DNA templates. The the Imperial soil using a DNA approach, combined with PCR reactions were performed using the 27f (5′-GAG CTC sequencing of 16S rRNA gene clones. AGA GTT TGA TCM TGG CTC AG-3′) and 1492r (3′- CAC GYT ACC TTG TTA CGA CTT-5′) primers (Lane 1991). Reaction mixtures contained 20 pmol of each primer, 5 U of Ampli Taq DNA polymerase (Applied Materials and methods Biosystems, Foster, CA, USA), 2.5 μl of the thermophilic DNA polymerase 10× reaction buffer, 200 μM of each Soil sampling, characterization, and fractionation deoxynucleoside triphosphate, 10 μL of soil DNA com- posed of equal volumes of DNA from each soil replicate, Nine 10-cm soil cores were collected from within a 100-m2 and sterile deionized water to a final volume of 25 μl. The quadrant of a field planted with lettuce in the Imperial PCR temperature conditions were as follows: 94°C for Valley, California and were immediately transported to the 2 min, followed by 35 cycles of 94°C for 5 s, 48°C for 20 s, laboratory where they were stored at 4°C for 72 h. The and 72°C for 40 s, and a final primer extension at 72°C for Imperial soil was classified as a loamy sand (fine, smectitic, 2 min. The PCR products were visualized on 1% agarose calcareous, hyperthermic Vertic Torrifluvents). The soil has gels by UV transillumination with ethidium bromide a pH of 7.2, electrical conductivity of 20.9 dS m-1, organic staining. Bands were removed and purified with a QIA- carbon content of 1.7%, total nitrogen content of 0.1%, and quick gel extraction kit (Qiagen). particle size of 79% sand, 7% silt, and 14% clay. Prior to The PCR products were ligated into pGEM-T Easy fractionation, the soil samples were passed through a 2-mm Vector, transformed into competent Escherichia coli JM109 sieve using a Tyler Ro-Tap Sieve Shaker (Mentor, OH, (Promega, Madison, WI, USA), and plated on Luria- USA) and then pooled. Bertani (LB) agar plates for selection of transformants. The soil was treated to obtain the inner and outer fraction Construction of 16S rRNA clone library arrays was of soil aggregates as described by Ranjard et al. (1997). The accomplished by spotting near full length 16S rRNA gene inner fraction of the aggregate contains micropores with a sequences onto nylon membranes as described by Valinsky diameter of 2 to 6 μm, while the outer fraction is et al. (2002). Microdroplets (0.5 μl) of freshly grown represented by the macropores with a diameter >6 μm overnight cultures of the rRNA gene clones were added to (Hattori 1988). The entire fractionation procedure was the PCR reagents using a 384-pin solid pin replicator (V & performed twice on 30 g soil (dry weight) and involved P Scientific, San Diego, CA, USA). The PCR was 15 successive soil washes in sterile 0.8% NaCl solution performed using a PTC-200 thermal cycler (Bio-Rad). with shaking at 100 rpm on a rotary shaker for 1 min. The The PCR conditions were as follows: 94°C for 10 min; supernatants, containing bacteria that are easily washed 35 cycles of 94°C for 1 min, 65°C for 1 min and 72oC for from the outer fraction of the aggregates, were pooled and 2 min, and 72°C for 10 min. The products were printed centrifuged at 9,800×g for 20 min. The remaining soil onto dry Hybond N + membranes (GE Healthcare, Piscat- represented bacteria adhered to the inner-aggregate fraction. away, NJ, USA) with a QPix robot (Genetix, Hampshire, Biol Fertil Soils (2008) 44:1003–1011 1005 UK) using a 384-pin gridding head and a 23 nl slot pin. A signal intensities was performed with control probes. The total of 1,536 clones were spotted onto the 11×14 cm array. results were entered in an Excel file as binary data with the One membrane was made for each oligonucleotide probe criteria of that zero values had values below the maximum tested in this study. of the negative controls and 1 values having a minimum of Hybridization of the oligonucleotide probes was con- the value for the positive control sequences (Valinsky et al. ducted as described by Valinsky et al.( 2002). The DNA 2002). oligonucleotide probes (Table 1) were synthesized by Operon Biotechnologies (Huntsville, Al, USA) and were Phylogenetic analysis by 16S rRNA clone sequencing 33P end-labeled with T4 polynucleotide kinase (T4 PNK; New England Biolabs, Ipswich, MA, USA). The hybrid- To determine the phylogenetic composition of the microbial ization solutions containing 1 nM DNA oligonucleotide communities from the aggregate fractions and confirm the probe were applied to the membranes and incubated specificity of the array approach, clones were randomly overnight at 11°C.

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