Background Analysis of species count data in ecology often requires normalization to an identical sample size. Rarefying (random subsampling without replacement), which is the current standard method for normalization, has been widely criticized for its poor reproducibility and potential distortion of the community structure. In the context of microbiome count data, researchers explicitly advised against the use of rarefying. Here we introduce a normalization method for species count data called scaling with ranked subsampling (SRS) and demonstrate its suitability for the analysis of microbial communities. Methods SRS consists of two steps. In the scaling step, the counts for all species or operational taxonomic units (OTUs) are divided by a scaling factor chosen in such a way that the sum of scaled counts equals the selected total number of counts Cmin. The relative frequencies of all OTUs remain unchanged. In the subsequent ranked subsampling step, non-integer count values are converted into integers by an algorithm that minimizes subsampling error with regard to the population structure (relative frequencies of species or OTUs) while keeping the total number of counts equal Cmin. SRS and rarefying were compared by normalizing a test library representing a soil bacterial community. Common parameters of biodiversity and population structure (Shannon index H’, species richness, species composition, and relative abundances of OTUs) were determined for libraries normalized to different size by rarefying as well as SRS with 10,000 replications each. An implementation of SRS in R is available for download (https://doi.org/10.20387/BONARES-2657-1NP3). Results SRS showed greater reproducibility and preserved OTU frequencies and alpha diversity better than rarefying. The variance in Shannon diversity increased with the reduction of the library size after rarefying but remained zero for SRS. Relative abundances of OTUs strongly varied among libraries generated by rarefying, whereas libraries normalized by SRS showed only negligible variation. Bray–Curtis index of dissimilarity among replicates of the same library normalized by rarefying revealed a large variation in species composition, which reached complete dissimilarity (not a single OTU shared) among some libraries rarefied to a small size. The dissimilarity among replicated libraries normalized by SRS remained negligibly low at each library size. The variance in dissimilarity increased with the decreasing library size after rarefying, whereas it remained either zero or negligibly low after SRS. Conclusions Normalization of OTU or species counts by scaling with ranked subsampling preserves the original community structure by minimizing subsampling errors. We therefore propose SRS for the normalization of biological count data.

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.

Integration of trees in agroforestry systems can increase the system sustainability compared to monocultures. The resulting increase in system complexity is likely to affect soil-N cycling by altering soil microbial community structure and functions. Our study aimed to assess the abundance of genes encoding enzymes involved in soil-N cycling in paired monoculture and agroforestry cropland in a Phaeozem soil, and paired open grassland and agroforestry grassland in Histosol and Anthrosol soils. The soil fungi-to-bacteria ratio was greater in the tree row than in the crop or grass rows of the monoculture cropland and open grassland in all soil types, possibly due to increased input of tree residues and the absence of tillage in the Phaeozem (cropland) soil. In the Phaeozem (cropland) soil, gene abundances of amoA indicated a niche differentiation between archaeal and bacterial ammonia oxidizers that distinctly separated the influence of the tree row from the crop row and monoculture system. Abundances of nitrate ( napA and narG ), nitrite ( nirK and nirS ) and nitrous oxide reductase genes ( nosZ clade I) were largely influenced by soil type rather than management system. The soil types’ effects were associated with their differences in soil organic C, total N and pH. Our findings show that in temperate regions, conversion of monoculture cropland and open grassland to agroforestry systems can alter the abundance of soil bacteria and fungi and soil-N-cycling genes, particularly genes involved in ammonium oxidation.

Background Tree-based intercropping (agroforestry) has been advocated to reduce adverse environmental impacts of conventional arable cropping. Modern agroforestry systems in the temperate zone are alley-cropping systems that combine rows of fast-growing trees with rows of arable crops. Soil microbial communities in these systems have been investigated intensively; however, molecular studies with high taxonomical resolution are scarce. Methods Here, we assessed the effect of temperate agroforestry on the abundance, diversity and composition of soil bacterial communities at three paired poplar-based alley cropping and conventional monoculture cropland systems using real-time PCR and Illumina sequencing of bacterial 16S rRNA genes. Two of the three systems grew summer barley (Hordeum vulgare); one system grew maize (Zea mays) in the sampling year. To capture the spatial heterogeneity induced by the tree rows, soil samples in the agroforestry systems were collected along transects spanning from the centre of the tree rows to the centre of the agroforestry crop rows. Results Tree rows of temperate agroforestry systems increased the abundance of soil bacteria while their alpha diversity remained largely unaffected. The composition of the bacterial communities in tree rows differed from those in arable land (crop rows of the agroforestry systems and conventional monoculture croplands). Several bacterial groups in soil showed strong association with either tree rows or arable land, revealing that the introduction of trees into arable land through agroforestry is accompanied by the introduction of a tree row-associated microbiome. Conclusion The presence of tree row-associated bacteria in agroforestry increases the overall microbial diversity of the system. We speculate that the increase in biodiversity is accompanied by functional diversification. Differences in plant-derived nutrients (root exudates and tree litter) and management practices (fertilization and tillage) likely account for the differences between bacterial communities of tree rows and arable land 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.