Influence of plant stage and cropping history on soil and root-associated prokaryotic community structure and composition in wheat

We conducted field studies at two locations in Germany in 2020 and 2021 to study the effect of plant developmental stage and wheat rotational position on the soil, rhizosphere and rhizoplane prokaryotic communities. Soil and root samples were taken from long-term field experiments in Hohenshulen (Kiel) and Harste (Göttingen), Germany.
Experimental design :
The crop rotational trial in Hohenschulen was established 1989 and included the factors a) cropping history, i.e. a first (W1) and a third wheat (W3) after a break crop (rapeseed), b) winter wheat cultivars (four levels), and c) N fertilization (four levels). Each field plot of 27 m² was replicated four times in a split-plot design. Treatment samples were taken in W1 and W3 plots of the cultivar “Nordkap” of the optimal N-level plots (240 kg N/ha). The crop rotational trial in Harste was established 2006 and included eight crop rotations, out of which two with winter wheat were included in this study, (1) a wheat monoculture and (2) a winter oilseed rape - winter wheat – winter wheat – grain pea – sugar beet – winter wheat rotation. Each crop rotational element is cultivated every year, which allowed us to sample three different crop rotational positions of winter wheat simultaneously per year. In Harste treatment samples were taken from the first wheat after winter oilseed rape grown as break crop (W1), from the second wheat after winter oilseed rape grown as break crop (W2), and from the long-term wheat monoculture (WM). Each plot of 110 m² was replicated three times within a split-plot design containing incomplete blocks.
For each replicate, 9 -12 soil cores (0-30 cm depth), sampled randomly across the respective total field plot area, were combined. Soil samples were sieved (4 mm mesh-size) and all samples were split among project partners for enzymology testing, fungal microbiome and prokaryotic microbiome analyses. For the root-associated samples 9-12 wheat plants were picked per treatment (wheat rotational positions) across the field plot area and combined (composite sample). Samples were taken in 2020 at the plant developmental stages flowering (BBCH 59-65) and early ripening (BBCH 69-75) and in 2021 in addition at the end of tillering (BBCH 30). Root-associated samples were split at the field sites into the rhizosphere fraction by brushing off the soil particles from roots using sterile disposable toothbrushes and wearing gloves at all times. The remaining roots were put in clean plastic bags. All samples were transported to the laboratory on ice. At the lab the rhizoplane fraction was taken by washing off the tightly adhering soil particles from the roots using 0.3 % NaCl (5 g roots/45 ml buffer) and a Stomacher machine.
The prokaryotic community in bulk soils, rhizosphere and rhizoplane of different wheat rotational positions was analyzed analyzed by high-throughput Illumina sequencing of the 16S rRNA gene fragments (V3-V4 region) amplified from total community. A total of 289 samples was investigated over two years.