Bacterial origin and community composition in the barley phytosphere as a function of habitat and presowing conditions

Abstract

An understanding of the factors influencing colonization of the rhizosphere is essential for improved establishment of biocontrol agents. The aim of this study was to determine the origin and composition of bacterial communities in the developing barley (Hordeum vulgare) phytosphere, using denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes amplified from extracted DNA. Discrete community compositions were identified in the endorhizosphere, rhizoplane, and rhizosphere soil of plants grown in an agricultural soil for up to 36 days. Cluster analysis revealed that DGGE profiles of the rhizoplane more closely resembled those in the soil than the profiles found in the root tissue or on the seed, suggesting that rhizoplane bacteria primarily originated from the surrounding soil. No change in bacterial community composition was observed in relation to plant age. Pregermination of the seeds for up to 6 days improved the survival of seed-associated bacteria on roots grown in soil, but only in the upper, nongrowing part of the rhizoplane. The potential occurrence of skewed PCR amplification was examined, and only minor cases of PCR bias for mixtures of two different DNA samples were observed, even when one of the samples contained plant DNA. The results demonstrate the application of culture-independent, molecular techniques in assessment of rhizosphere bacterial populations and the importance of the indigenous soil population in colonization of the rhizosphere.

FIG. 1

(A) DGGE analysis of 16S rDNA fragments from soil and barley samples obtained 6 days after sowing. The gel shown is the original photograph before straightening of lanes and alignment of bands. P1 to P3 refer to bands with barley rDNA (see Table 1). (B) Dendrogram showing the relatedness of the DGGE banding patterns. Bands with barley DNA and the phylloplane samples, which contained no bacterial bands, were not considered in the cluster analysis. Abbreviations: RP, rhizoplane; ER, endorhizosphere; PP, phylloplane; EP, endophyllosphere; RS, rhizosphere soil; BS, bulk soil; CS, control soil. The number after each abbreviation indicates replicate number.

FIG. 2

(A) DGGE analysis of 16S rDNA fragments from soil and barley samples obtained 6, 12, 18, and 36 days after sowing. The gel shown is the original photograph before straightening of lanes and alignment of bands. PCR products of triplicate samples were pooled prior to the DGGE analysis. Sequenced bands are labeled; 1 to 8 refer to bacterial bands, and P1 to P6 refer to bands with barley rDNA (see Table 1). U1 to U6 refer to bands that could not be sequenced. (B) Dendrogram showing the relatedness of the DGGE banding patterns. Bands with barley DNA were not considered in the cluster analysis. Abbreviations: SP, spermoplane; ES, endospermosphere; RP, rhizoplane; ER, endorhizosphere; RS, rhizosphere soil; BS, bulk soil. The number after each abbreviation indicates sampling day. For the SP and ES samples, the RP and ER of the 5- to 10-mm-long primary roots are included.

FIG. 3

Dendrogram for DGGE banding patterns of barley samples showing the impact of seed pregermination. Abbreviations: WT, water source; SP, spermoplane; ES, endospermosphere; RP, rhizoplane; ER, endorhizosphere; RS, rhizosphere soil; BS, bulk soil; CS, control soil; UP, upper part of root; LP, lower part of root; 2+6 and 6+6, number of days of seed pregermination + number of days of growth in soil. For the SP and ES samples, the RP and ER of the 5- to 10-mm-long primary roots are included.