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Untreated roots (T1) featured high OTU richness (86 �� 12). However, as evident from the high standard errors of the mean, the OTU richness differed highly between replicates, suggesting that observed ��-diversity between root communities is not always the result of the treatment effect. Thus, the inversed Simpson concentration index (2D) was calculated for each community, which puts more emphasis on changes in the abundances of the dominant OTUs. For root samples, a steady decrease in 2D values was found between untreated roots and NaOCl-treated roots with washed and sonicated root samples ranking in between, except T6 Carboplatin (2DT1-R: 7.3 �� 1.6 SE; 2DT2-R: 4.3 �� 0.5; 2DT3-R: 3.2 �� 0.3; 2DT4-R: 3.9 �� 0.5; 2DT5-R: 4.4 �� 0.6; 2DT6-R: BI-6727 7.3 �� 0.6; 2DT7-R: 2.1 �� 0.2). For the root samples, 2D values were higher for the sonication bath treatments than for the probe sonicated roots. For microbial assemblies detached from roots, much higher values for 2D were observed in general, and no clear trend was visible due to high within-sample heterogeneity (2DT2-D: 11.0 �� 3.1 SE; 2DT3-D: 12.1 �� 1.8 2DT4-D: 8.8 �� 0.5; 2DT5-D: 7.6 �� 0.7; 2DT6-D: 10.0 �� 3.7). Non-metric multi-dimensional scaling based on Bray�CCurtis-distances revealed a clear separation of populations sampled from remaining roots and treatment suspensions along the first axis (Figure ?Figure44). The highest distance was observed between communities in the suspensions with detached cells obtained from the washing step (samples T2-D) and root-associated communities after NaOCl-treatment most likely representing the endophytic community (samples T7-R). The NMDS analysis was complemented with anosim under 999 permutations, which showed a significant (p DZNeP research buy scaling (NMDS) of T-RFLP fingerprints (Hellinger-transformed abundance data). Labeling represents roots (#-R) and root-detached microorganisms (#-D) for different treatments (T1�CT7), and replicates (n = 3 each, a�Cc). ... Discussion Our understanding of the dynamics of root-associated microbial communities rapidly improves by utilizing high-throughput sequencing techniques (Knief, 2014). However, our current definitions of the different root-associated communities are constrained by technical limitations (i.e., incomplete microbiome separation). Implications of these limitations on root microbiome research need to be assessed, especially when molecular tools are used that are able to detect very small subpopulations (i.e., rare biosphere).