Agroforestry, which is the integration of trees into monoculture cropland, can alter soil properties and nutrient cycling. Temperate agroforestry practices have been shown to affect soil microbial communities as indicated by changes in enzyme activities, substrate-induced respiration, and microbial biomass. Research exploring soil microbial communities in temperate agroforestry with the help of molecular tools which allow for the quantification of microbial taxa and selected genes is scarce. Here, we quantified 13 taxonomic groups of microorganisms and nine genes involved in N cycling (N 2 fixation, nitrification, and denitrification) in soils of three paired temperate agroforestry and conventional monoculture croplands using real-time PCR. The agroforestry croplands were poplar-based alley-cropping systems in which samples were collected in the tree rows as well as within the crop rows at three distances from the tree rows. The abundance of Acidobacteria, Actinobacteria, Alpha-and Gammaproteobacteria, Firmicutes, and Verrucomicrobia increased in the vicinity of poplar trees, which may be accounted for by the presence of persistent poplar roots as well as by the input of tree litter. The strongest population increase was observed for Basidiomycota, which was likely related to high soil moisture, the accumulation of tree litter, and the absence of tillage in the tree rows. Soil microorganisms carrying denitrification genes were more abundant in the tree rows than in the crop rows and monoculture systems, suggesting a greater potential for nitrate removal through denitrification, which may reduce nitrate leaching. Since microbial communities are involved in critical soil processes, we expect that the combination of real-time PCR with soil process measurements will greatly enhance insights into the microbial control of important soil functions in agroforestry systems.

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.

Background: Temperate agroforestry is regarded as a sustainable alternative to monoculture agriculture due to enhanced provisioning of ecosystem services. Plant health and food safety are crucial requirements for sustainable agriculture; however, studies of fungal diseases and mycotoxin contamination of crops grown under temperate agroforestry are lacking. This study therefore aimed to compare fungal colonization and mycotoxin contamination of crops grown in temperate agroforestry against conventional monoculture. Methods: The biomass of plant pathogenic fungi in oilseed rape plants and barley and wheat grain harvested in 2016 to 2018 at four paired agroforestry and monoculture sites was quantified using species-specific real-time PCR. Mycotoxin content of barley and wheat grain was determined by HPLC-MS/MS. Results: The colonization of oilseed rape plants with the vascular pathogen Verticillium longisporum and wheat grain with the head blight pathogen Fusarium tricinctum was lower in agroforestry than in conventional monoculture. Mycotoxin content of barley and wheat grain did not differ between agroforestry and monoculture systems and did not exceed the legal limits of the EU. Remarkably, fumonisin B1 was detected in wheat grains at two sites in two years, yet the low levels found do not raise food safety concerns. No differences were found between the two production systems with regard to infection of wheat and barley grain with five Fusarium species (F. avenaceum, F. culmorum, F. graminearum, F. poae, and F. proliferatum) and oilseed rape with fungal pathogens Leptosphaeria biglobosa, Leptosphaeria maculans, and Sclerotinia sclerotiorum. Conclusions: Temperate agroforestry does not negatively affect the infection of wheat, barley and oilseed rape with major fungal pathogens though it may suppress the infection of oilseed rape with V. longisporum and wheat grain with F. tricinctum. Furthermore, temperate agroforestry does not increase mycotoxin contamination of barley and wheat. Therefore, temperate agroforestry does not negatively affect food safety.

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.