Effect of Azospirillum brasilense inoculation on rhizobacterial ...

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A. Lerner et al. / Soil Biology & Biochemistry 38 (2006) 1212–1218 1215 Fig. 1. Cluster analysis <strong>of</strong> DGGE pr<strong>of</strong>iles <strong>of</strong> bacterial populations in the rhizosphere, collected in a Chromoxererts sandy loam soil (A) and from root tips samples collected in a Haploxeralfs sandy soil (B), amplified with 968f/1401r primers. C, control; Cd, <strong>Azospirillum</strong> <strong>brasilense</strong> Cd; 245, Azospirilum <strong>brasilense</strong> Sp 245. (A) Sample number 1–3, day 10; 4–6, day 20; 7–9, day 30; 10–12, day 40 post-<strong>inoculation</strong>. (B) Sample number 4–6, week 1; 7–9, week 3 post-<strong>inoculation</strong>. detected in all experiments in the two soils. They occurred in the rhizosphere and in all rhizoplane compartments under both growth conditions, as shown in two representative analyses in Fig. 1. In each <strong>of</strong> the two soils, the pr<strong>of</strong>iles <strong>of</strong> the rhizosphere bacterial populations obtained with the two primer sets were highly similar. However, in the endorhizosphere-rhizoplane <strong>of</strong> roots growing in the sandy soil, primers 968f/1401r always yielded a larger bacterial diversity than primers Gm5f/907r (P!0.05). A similar, non-significant effect was detected when plants were grown in the sandy loam Chromoxererts soil (data not shown). Different primer sets may lead to different results, because <strong>of</strong> differences in oligonucleotide design strategies, target sequences and amplification conditions (Heuer et al., 1997; Wintzingerode et al., 1997; Marchesi et al., 1998; Schmalenberger et al., 2001). For example, preferential amplification <strong>of</strong> abundant plastid DNA from the root may cause biased PCR amplification by hindering amplification <strong>of</strong> less dominant populations. Various strategies have been designed, and some recent development may help reduce this kind <strong>of</strong> bias (Green, 2004). In the sandy soil, diversity significantly decreased between the rhizosphere and the rhizoplane-endorhizosphere (P!0.05). No such effect was found in plants grown in the sandy loam soil. However, community pr<strong>of</strong>iles always differed between the compartments (data not shown). Rhizosphere communities may vary spatially along a radial axis from the endorhizosphere, towards the rhizoplane and the rhizosphere, as well as in specific root locations along the root axis (Marilley et al., 1998; Yang and Crowley, 2000). Species selection occurring at various sites may involve a number <strong>of</strong> characters, such as bacterial growth rate (Nacamulli et al., 1997; Ciccillo et al., 2002) and copiotrophy (Semenov et al., 1999) which may lead to population expansion, site colonization and the exclusion <strong>of</strong> competitors. These effects may be stronger nearer to the nutrient source (the root) than further from it (the rhizosphere). These processes may reduce diversity but, as the results <strong>of</strong> the experiments with the Chromoxererts sandy loam soil used in this study show, growth conditions and environmental factors may strongly blur this effect. In all experiments, replicated samples exhibited some variability both in the DGGE and ARISA analyses (Figs. 1 and 2). This variability was repeatable in recurrent PCR amplifications. This may reflect stochastic events, which occur during root colonization while the root tips grow past soil and organic matter particles, which are colonized by various bacteria, or by method-inherent inconsistencies in extraction and amplifications <strong>of</strong> DNA (Yang and Crowley, 2000; Johnsen et al., 2001). Alternatively, nutrient depletion by bacterial colonizers along the root may lead to cell death. This, in turn may induce oscillations <strong>of</strong> rhizomicrobial communities along the root as the root tip progresses (Semenov et al., 1999), explaining some <strong>of</strong> this variability between replicates. 3.2. Rhizosphere genetic fingerprints <strong>of</strong> DGGE versus ARISA Automated ribosomal intergenic spacer analysis was carried out using the DNA samples collected in the two experiments performed in the Haploxeralfs sandy soil. The overall complexity in the rhizosphere was higher using ARISA than DGGE <strong>of</strong> 16S rDNA amplicons (Fig. 2(A) versus (B)). Analyses based on intergenic spacers (between the 16S and
1216 A. Lerner et al. / Soil Biology & Biochemistry 38 (2006) 1212–1218 Fig. 2. ARISA (A) and DGGE (B) pr<strong>of</strong>iles <strong>of</strong> the rhizobacterial populations in the rhizosphere, experiment 2, carried out in a Haploxeralfs sandy soil. C, control; Cd, <strong>Azospirillum</strong> <strong>brasilense</strong> Cd; 245, <strong>Azospirillum</strong> <strong>brasilense</strong> Sp 245. Samples C, Cd and 245 numbers 10–12, week 1; 13–15 week 3 post-<strong>inoculation</strong>. 23S rRNA genes) <strong>of</strong>fer higher resolution at the taxonomic level <strong>of</strong> species (and below) than 16S rDNA intragenic analysis, because <strong>of</strong> the higher divergence accumulated in these sequences (Garcia-Martinez et al., 1999). However, band identification is usually impossible since available sequence information on these loci is very scarce. PCA analysis on the patterns acquired with ARISA showed that in each experiment the principal, significant effect affecting the bacterial
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