Effects of bacterial inoculants on the indigenous microbiome and secondary metabolites of chamomile plants

Abstract

Plant-associated bacteria fulfill important functions for plant growth and health. However, our knowledge about the impact of bacterial treatments on the host's microbiome and physiology is limited. The present study was conducted to assess the impact of bacterial inoculants on the microbiome of chamomile plants Chamomilla recutita (L.) Rauschert grown in a field under organic management in Egypt. Chamomile seedlings were inoculated with three indigenous Gram-positive strains (Streptomyces subrutilus Wbn2-11, Bacillus subtilis Co1-6, Paenibacillus polymyxa Mc5Re-14) from Egypt and three European Gram-negative strains (Pseudomonas fluorescens L13-6-12, Stenotrophomonas rhizophila P69, Serratia plymuthica 3Re4-18) already known for their beneficial plant-microbe interaction. Molecular fingerprints of 16S rRNA gene as well as real-time PCR analyses did not show statistically significant differences for all applied bacterial antagonists compared to the control. In contrast, a pyrosequencing analysis of the 16S rRNA gene libraries revealed significant differences in the community structure of bacteria between the treatments. These differences could be clearly shown by a shift within the community structure and corresponding beta-diversity indices. Moreover, B. subtilis Co1-6 and P. polymyxa Mc5Re-14 showed an enhancement of the bioactive secondary metabolite apigenin-7-O-glucoside. This indicates a possible new function of bacterial inoculants: to interact with the plant microbiome as well as to influence the plant metabolome.

Keywords: bioactive secondary metabolites; biological control agents; chamomile; microbial communities; soil-borne pathogens.

Figure 1

Content (%) of apigenin-7-O-glucoside (A) and apigenin (B) in Chamomilla recutita (L.) Rauschert samples. Averages of individual HPLC-MS measurements and confidences are shown. Significant differences (p < 0.05) of bacterial treatments (Streptomyces subrutilus Wb2n-11, Bacillus subtilis Co1-6, Paenibacillus polymyxa Mc5Re-14, Pseudomonas fluorescens L13-6-12, Stenotrophomonas rhizophila P69, Serratia plymuthica 3Re4-18) to the control are indicated by asterisks.

Figure 2

Cluster analysis of eubacterial community fingerprints. Similarities between SSCP fingerprints were calculated using the curve-based Pearson correlation coefficient and grouped according to their similarity using the hierarchical UPGMA. Treatments (Streptomyces subrutilus Wb2n-11, Bacillus subtilis Co1-6, Paenibacillus polymyxa Mc5Re-14, Pseudomonas fluorescens L13-6-12, Stenotrophomonas rhizophila P69, Serratia plymuthica 3Re4-18, and water control) and sampling times (1st) after 4 weeks and (2nd) after 8 weeks are indicated. Numbers 1–5 mark the five independent replicates per treatment.

Figure 3

Principal component analysis (PCA) of eubacterial (A), Pseudomonas (B) and Firmicutes (C) community fingerprints. Treatments (Streptomyces subrutilus Wb2n-11, Bacillus subtilis Co1-6, Paenibacillus polymyxa Mc5Re-14, Pseudomonas fluorescens L13-6-12, Stenotrophomonas rhizophila P69, Serratia plymuthica 3Re4-18, and water control) are indicated by colors. Sampling time (1st) after 4 weeks is shown on the left side and (2nd) after 8 weeks is shown on the right side. PCA was calculated based on relative positions and intensity of DNA bands.

Figure 4

Classification of bacterial communities associated with Chamomilla recutita (L.) Rauschert. Pyrosequencing reads were classified at phylum (A) and genus (B) level against RDP core set within QIIME pipeline with an 80% confidence threshold. Taxa below 1% of relative abundance are included in “Other.” Multi-colored charts at the legend are shown for each sample correspondingly.

Figure 5

Comparison of the microbial communities of Chamomilla recutita (L.) Rauschert rhizosphere by jackknifed principal coordinate analysis. The 2D-plot illustrates the compositional similarity between samples based on weighted UniFrac. The positions of the points are the averages for the jackknifed replicates generated by QIIME and are shown with ellipses representing the interquartile range (IQR) in each axis. Larger ellipses represent more diverse communities. Colors correspond to the different treatments.

Figure 6

Comparison of the microbial communities of Chamomilla recutita (L.) Rauschert rhizosphere by jackknifed principal coordinate analysis. The biplot illustrates the compositional similarity between samples based on weighted UniFrac. Taxa coordinates are indicated by gray orbs with size, as a function of relative abundance. To confine the biplot, the number of the displayed taxa was restricted to 5. The positions of the points are the averages for the jackknifed replicates generated by QIIME and are shown with ellipses representing the interquartile range (IQR) in each axis. Larger ellipses represent more diverse communities. Colors correspond to the different treatments.

Figure 7

Root epidermis of a chamomile seedling 14 days after inoculation. Visualization of DsRed marked bacteria with confocal laser scanning microscopy. (A) xy, xz, and yz maximum projections showing a colonizing of small colonies and single cells of Serratia plymuthica 3Re4-18 of the surrounding area of the root. Red: bacterial cells, blue: root, scale bar: 30 μm. (B) Surface model of (A) shows the root-surface localization of Serratia plymuthica 3Re4-18 in the three-dimensional space. Red: bacterial cells, brown: root, scale bar: 10 μm.