Fig 1.
Example of split-root setup showing one representative replicate of treatment Control/+ARD 35 days after planting.
Reddish-brown discolouration of roots is visible in both compartments and is typical for apple roots. Colour is related to the high concentrations of phloridzin and quercetin in apple roots.
Fig 2.
Root length development of apple roots in the two compartments of the split-root boxes for the four treatment combinations: -ARD/-ARD, +ARD/+ARD, -ARD/+ARD, Control/+ARD.
Root length development was derived from surface scanning of the split-root boxes at 2–4 days intervals during the 34-day growth period. Asterisks indicate significant differences between the two compartments of one treatment at the respective point in time (p<0.05). Means of four replicates. Error bars show standard errors (SE).
Fig 3.
Root length of apple roots in the two compartments of the split-root boxes for the four treatments -ARD/-ARD, +ARD/+ARD, -ARD/+ARD, Control/+ARD 34 days after planting.
Root length was determined with WinRhizo after destructive sampling. The substrate in the respective compartment is indicated by colour/pattern code (see legend). Different letters indicate significant differences at p<0.05. Means of four replicates. Error bars show SE.
Fig 4.
Shoot dry weight (a) and shoot N-uptake (b) 34 days after planting for apple seedling growing on -ARD or +ARD soil only, and treatments with part of their root system growing in +ARD soil vs -ARD soil and control soil, respectively. Different letters indicate significant differences at p<0.05. Means of four replicates. Error bars show SE.
Fig 5.
15N abundance in different soil and plant fractions after 34-day growing period of apple trees in split-root boxes.
Split-root boxes were filled with -ARD soil in one compartment and +ARD soil in the second compartment. Normal nitrogen (14N) fertilizer was crosswise replaced with 15N labelled nitrogen fertilizer, i.e. in one treatment -ARD soil was labelled with 15N (white columns), in the other treatment +ARD soil was labelled with 15N (black columns). Grey-shaded background indicates that these samples have been retrieved from the labelled side of the split-root boxes. Asterisk indicates significant differences (p<0.05) between treatments for the respective fraction. Means of four replicates. Error bars show SE.
Fig 6.
Quantification of 16S rRNA gene and ITS fragment copy numbers in the two compartments of the split-root boxes for the four treatments -ARD/-ARD; +ARD/+ARD; -ARD/+ARD; Control/+ARD based on soil dry weight (bulk and rhizosphere soil) or centrifuged soil sample (rhizoplane), respectively.
Data are provided separately for three microhabitats (rhizoplane, rhizosphere and bulk soil) at the end of the 34-day growth period. +ARD soil (black), -ARD soil (white), Control soil (cross-hatched). Different letters indicate significant differences at p<0.05 (only shown for bulk soil). Means of four replicates. Error bars show SE.
Fig 7.
Bacterial response to ARD depending on the microhabitat studied.
Bacterial DGGE fingerprints based on 16S rRNA gene fragments amplified from total genomic DNA extracted from different microhabitats (Rhizoplane, Rhizosphere and Bulk soil) from plants grown in Control and +ARD soils, at the same time. On the right side the respective UPGMA cluster analysis of the samples is shown. Red arrows indicate differentiating bands (S3 Fig) detected only in the bacterial fingerprint of ARD soils. The bands were excised, reamplified, cloned and sequenced. BS: Bacterial standard of 16S rRNA gene fragments from 11 bacterial strains included according to Heuer et al. [38]. Samples are enumerated with the same number in DGGE and the UPGMA tree.
Fig 8.
Fungal response to ARD depending on the microhabitat studied.
Fungal DGGE fingerprints based on ITS-fragments amplified from total genomic DNA extracted from different microhabitats (Rhizoplane/RP, Rhizosphere/RS and Bulk soil/BK) from plants grown in Control and +ARD soils at the same time. FS: Fungal standard of ITS gene fragments from 16 fungal strains. On the right side the respective UPGMA cluster analysis of the samples is shown. Red arrows indicate fungal populations that are enriched in the respective substrate. Samples are enumerated with the same number in DGGE and UPGMA tree.
Fig 9.
Rhizoplane bacterial response to different substrates.
Bacterial DGGE fingerprints based on 16S rRNA gene fragments amplified from total genomic DNA extracted from plants grown in the different substrates (+ARD, -ARD and Control), coming from different combinations of substrates: +ARD, +ARD; -ARD, -ARD; -ARD, +ARD and +ARD, Control. On the right side the respective UPGMA cluster analysis of the samples. Red arrows indicate differentiating bands detected only in the bacterial fingerprint of ARD soils. The bands were excised, reamplified, cloned and sequenced (S2 Fig). BS: Bacterial standard of 16S rRNA gene fragments from 11 bacterial strains included according to Heuer et al. [38]. Samples are enumerated with the same number in DGGE and UPGMA tree.
Table 1.
Substrate effect on the microbial community structure in the rhizosphere and rhizoplane.
Differences (dissimilarity-values) between the DGGE fingerprints from the substrates within each variant (-ARD, -ARD), (+ARD, +ARD), (+ARD, -ARD) (+ARD, Control) in two microhabitats (Rhizoplane, rhizosphere). D-values were calculated based on pairwise Pearson’s similarity coefficients. Significant differences are indicated by asterisk (p<0.05) and n.s. = not significant difference.
Table 2.
Substrate effect on different taxonomic groups in rhizoplane, rhizosphere and bulk soil.
Differences (dissimilarity-values) between the DGGE fingerprints (Fungi: Fig 8, Bacteria: Fig 7, other DGGEs not shown) from the substrates within the treatment (+ARD/Control). N.d = not determined. D-values were calculated based on pairwise Pearson similarity coefficients. Significant differences are indicated by asterisk (p<0.05).
Table 3.
Putative phylogenetic affiliation of 16S rRNA partial gene sequences (V6-V8 region) from the rhizoplane of the bands excised from DGGE (Figs 7 and 9).