Importance of rare taxa for bacterial diversity in the rhizosphere of Bt- and conventional maize varieties

Abstract

Ribosomal 16S rRNA gene pyrosequencing was used to explore whether the genetically modified (GM) Bt-maize hybrid MON 89034 × MON 88017, expressing three insecticidal recombinant Cry proteins of Bacillus thuringiensis, would alter the rhizosphere bacterial community. Fine roots of field cultivated Bt-maize and three conventional maize varieties were analyzed together with coarse roots of the Bt-maize. A total of 547,000 sequences were obtained. Library coverage was 100% at the phylum and 99.8% at the genus rank. Although cluster analyses based on relative abundances indicated no differences at higher taxonomic ranks, genera abundances pointed to variety specific differences. Genera-based clustering depended solely on the 49 most dominant genera while the remaining 461 rare genera followed a different selection. A total of 91 genera responded significantly to the different root environments. As a benefit of pyrosequencing, 79 responsive genera were identified that might have been overlooked with conventional cloning sequencing approaches owing to their rareness. There was no indication of bacterial alterations in the rhizosphere of the Bt-maize beyond differences found between conventional varieties. B. thuringiensis-like phylotypes were present at low abundance (0.1% of Bacteria) suggesting possible occurrence of natural Cry proteins in the rhizospheres. Although some genera indicated potential phytopathogenic bacteria in the rhizosphere, their abundances were not significantly different between conventional varieties and Bt-maize. With an unprecedented sensitivity this study indicates that the rhizosphere bacterial community of a GM maize did not respond abnormally to the presence of three insecticidal proteins in the root tissue.

Figure 1

Relative sequence load of genera. Sequence load of 100% correspond to all Bacteria-assigned sequences of a sample. Percent-values specify averages of all 10 samples. The general distribution of sequence loads of all genera is displayed next to those from most abundant five phyla. Number of genera in respect to their relative sequence load (a). Sum of relative abundances of all genera assigned to the respective groups of sequence load (b).

Figure 2

Cluster analyses (UPGMA) of the rhizosphere bacterial communities from different maize varieties and microhabitats (fine roots, coarse roots) based on the relative abundances of all genera (a), the 49 most dominant genera that each represent >0.5% of all genera-assigned sequences (each genus with >0.23% of all Bacteria) (b), the remainig lower represented 461 genera (c) and the 91 genera significantly responsive to the root environments. Relative abundances were normalized for each comparison, respectively. Pearson's correlation on relative taxa abundances comprising all detected taxa of a given rank.

Figure 3

Similarities of the rhizosphere bacterial communities at different taxonomic ranks between (a) two varieties as indicated or (b) the three conventional varieties (three varieties) or the three conventional varieties and 3BT (four varieties). Significance of difference was tested by analysis of variance, P⩽0.05. Comparisons were taken at each taxonomic rank separately and only differences were labeled in the figure. Bars with no significant difference to each other are indicated by the same letter.