Bacterial Distribution in the Rhizosphere of Wild Barley under Contrasting Microclimates

Background: All plants in nature harbor a diverse community of rhizosphere bacteria which can affect the plant growth. Our samples are isolated from the rhizosphere of wild barley Hordeum spontaneum at the Evolution Canyon (‘EC’), Israel. The bacteria which have been living in close relationship with the plant root under the stressful conditions over millennia are likely to have developed strategies to alleviate plant stress. Methodology/Principal Findings: We studied distribution of culturable bacteria in the rhizosphere of H. spontaneum and characterized the bacterial 1-aminocyclopropane-1-carboxylate deaminase (ACCd) production, biofilm production, phosphorus solubilization and halophilic behavior. We have shown that the H. spontaneum rhizosphere at the stressful South Facing Slope (SFS) harbors significantly higher population of ACCd producing biofilm forming phosphorus solubilizing osmotic stress tolerant bacteria. Conclusions/Significance: The long-lived natural laboratory ‘EC ’ facilitates the generation of theoretical testable and predictable models of biodiversity and genome evolution on the area of plant microbe interactions. It is likely that the bacteria isolated at the stressful SFS offer new opportunities for the biotechnological applications in our agro-ecologica

Cross section of the ‘Evolution Canyon’ indicating the collection sites on South Facing Slope (SFS) 1 and 2 and North Facing Slope (NFS) 5 and 7.

<p>Cross section of the ‘Evolution Canyon’ indicating the collection sites on South Facing Slope (SFS) 1 and 2 and North Facing Slope (NFS) 5 and 7.</p

Endospore forming bacterial distribution on the ‘EC’ SFS and NFS slopes.

1<p>The endospore forming bacterial fraction was isolated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017968#s2" target="_blank">Material and Methods</a>.</p>2<p>ACC (1-Aminocyclopropane-1-carboxylate) medium was composed of salts for <i>P. polymyxa</i> minimal medium there the nitrogen source is replaced with ACC.</p>3<p>Biofilm formation was estimated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017968#s2" target="_blank">Material and Methods</a>. The isolates with OD >1 were considered as biofilm formers.</p>4<p>Halophilic growth was determined in the medium containing 2 M NaCl (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017968#s2" target="_blank">Material and Methods</a>).</p>5<p>P solubilization was estimated using NBRIP medium (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017968#s2" target="_blank">Material and Methods</a>). The colonies with index >2 were considered as P solubilizers.</p

Solid surface assay of the South Facing Slope (SFS) and North Facing Slope (NFS) bacterial biofilm formation.

<p>The crystal violet assay was used to measure solid surface biofilm formation at 30C. Preparation and analysis were as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017968#s2" target="_blank">Material and Methods</a>.</p

Bacterial distribution on the ‘EC’SFS and NFS slopes.

1<p>The data are expressed per gram of fresh weight. Wild barley roots were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017968#s2" target="_blank">Material and Methods</a>.</p><p>Each data point represents three independent experiments. The culturable aerobic fraction of the total bacterial CFUs was determined on TSA plates as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017968#s2" target="_blank">Material and Methods</a> s-sun area; sh-shaded area.</p>2<p>Spores were counted using Shaeffer-Fulton staining.</p>3<p>ACC (1-Aminocyclopropane-1-carboxylate) medium was composed of salts for <i>P. polymyxa</i> minimal medium there the nitrogen source is replaced with ACC.</p>4<p>Biofilm formation was estimated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017968#s2" target="_blank">Material and Methods</a>. The isolates with OD >1 were considered as biofilm formers.</p>5<p>Halophilic growth was determined in the medium containing 2 M NaCl (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017968#s2" target="_blank">Material and Methods</a>).</p>6<p>P solubilization was estimated using NPRJP medium (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017968#s2" target="_blank">Material and Methods</a>). The colonies with index >2 were considered as P solubilizers.</p

Endospore forming bacterial phosphorus solubilization.

1<p>Endospore forming bacterial isolates as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017968#pone-0017968-t002" target="_blank">Table 2</a>. Solubilization index and pH change was studied of each isolate in three independent experiments.</p>2<p>Bacterial phosphorus solubilization index was determined after bacterial growth in NBRIP agar plates for 7 days at 30C (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017968#s2" target="_blank">Material and Methods</a>).</p>3<p>Change of pH by the bacteria was determined after 7 days of incubation in NBRIP broth (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017968#s2" target="_blank">Material and Methods</a>).</p><p>*<i>P. polymyxa, B. megaterium, B. cereus</i> and <i>B. pumilus</i> SFS and NFS isolate values (mean value±SE) were compared. Numbers followed by different letter are significantly different (P<0.05) according to Fisher's least significance difference test.</p