As our understanding of soil biology deepens, there is a growing demand for investigations addressing microbial processes in the earth beneath the topsoil layer, called subsoil. High clay content in subsoils often hinders the recovery of sufficient quantities of DNA as clay particles bind nucleic acids. Here, an efficient and reproducible DNA extraction method for 200 mg dried soil based on sodium dodecyl sulfate (SDS) lysis in the presence of phosphate buffer has been developed. The extraction protocol was optimized by quantifying bacterial 16S and fungal 18S rRNA genes amplified from extracts obtained by different combinations of lysis methods and phosphate buffer washes. The combination of one minute of bead beating, followed by ten min incubation at 65°C in the presence of 1 M phosphate buffer with 0.5% SDS, was found to produce the best results. The optimized protocol was compared with a commonly used cetyltrimethylammonium bromide (CTAB) method, using Phaeozem soil collected from 60 cm depth at a conventional agricultural field and validated on five subsoils. The reproducibility and robustness of the protocol was corroborated by an interlaboratory comparison. The DNA extraction protocol offers a reproducible and cost-effective tool for DNA-based studies of subsoil biology.

Intensively managed open croplands are highly productive but often have deleterious environmental impacts. Temperate agroforestry potentially improves ecosystem functions, although comprehensive analysis is lacking. Here, we measured primary data on 47 indicators of seven ecosystem functions in croplands and 16 indicators of four ecosystem functions in grasslands to assess how alley-cropping agroforestry performs compared to open cropland and grassland. Carbon sequestration, habitat for soil biological activity, and wind erosion resistance improved for cropland agroforestry (P ≤ 0.03) whereas only carbon sequestration improved for grassland agroforestry (P < 0.01). In cropland agroforestry, soil nutrient cycling, soil greenhouse gas abatement, and water regulation did not improve, due to customary high fertilization rates. Alley-cropping agroforestry increased multifunctionality, compared to open croplands. To ameliorate the environmental benefits of agroforestry, more efficient use of nutrients is required. Financial incentives should focus on conversion of open croplands to alley-cropping agroforestry and incorporate fertilizer management.

Aims Temperate alley-cropping agroforestry systems maintain agricultural production while offering several environmental benefits. Central benefits of agroforestry systems such as the ‘safety-net’-role of the trees for leached nutrients are mainly due to processes occurring below the soil surface: the subsoil. Microorganisms in the subsoil may play a key role in the ‘safety-net’-function as they can improve the capturing and uptake of nutrients by the trees. Systematic investigations of microbial communities in temperate agroforestry systems, however, are restricted to topsoil. Methods We quantified bacteria, fungi, and functional groups of microorganisms in the topsoil and subsoil of two alley-cropping systems using real-time PCR. Topsoil and subsoil samples were collected in the tree rows and at multiple distances from the trees within the crop rows of the agroforestry systems as well as at an adjacent monoculture cropland. Results Microbial population size decreased with soil depth likely due to limited resource availability in subsoil. Tree rows in agroforestry systems not only promote soil microbial populations in both the topsoil and subsoil but the promotion also extends gradually into the crop rows of the systems. The promotion of microorganisms through trees is stronger in subsoil than topsoil, pointing at more intense resource scarcity in the subsoil than topsoil. Conclusions We propose that tree root-derived resources and root litter, which are scarce in agricultural subsoils, triggered the strong positive response of the subsoil community to the trees. Finally, we provide initial evidence that subsoil microorganisms contribute to the ‘safety-net’-role of the trees in agroforestry systems.