Mineral fertilizers have sustained food security for decades, yet the long-term impacts on soil microbial communities underpinning soil health remain virtually unknown. We combine standardized field surveys with a meta-analysis to assemble a global dataset of 501 long-term agricultural experiments (≥5 years, median duration 25 years) to evaluate the impacts of sustained mineral fertilization on soil properties and microbial communities. Long-term mineral fertilization increases soil organic carbon by 14% and decreases soil pH by 0.31 units on average relative to unfertilized controls. Soil organic carbon accumulation largely explains increased microbial biomass carbon and living bacterial biomass. Mineral fertilizer-induced acidification primarily reshapes dominant bacterial taxa, with the relative abundance of Proteobacteria increasing and Firmicutes declining, whereas fungal community composition remains stable. Virulent bacteriophages increase in association with shifts in bacterial hosts. Microbial activities reveal a decoupling of more nitrogen- and phosphorus-acquisition enzymes from the unchanged production of carbon-mineralization enzymes. Microbial communities are taxonomically reorganized without reducing richness or promoting fungal pathogens. These responses are evaluated in the context of agroecosystem type, fertilization regime, and cropping regime. Our findings provide global-scale evidence for the consequences of long-term mineral fertilization on soil health, which is integral to guiding fertilizer management for sustainable agriculture.

Urease inhibitors and nitrification inhibitors delay nitrogen fertilizer transformations in soil to reduce nitrogen losses and increase nitrogen use efficiency. While new inhibitor compounds are constantly being developed, little is known about non-target effects on the soil microbiome. This is the first study to investigate non-target effects of the urease inhibitor 2-NPT and the nitrification inhibitor MPA on the soil microbiome. In addition, the more established nitrification inhibitor DMPP was investigated. Target effects and potential non-target effects on the function and composition of microbial communities in three soils from Germany were assessed. Soil microcosms were treated with practically relevant doses of inhibited and non-inhibited fertilizer products. Effects on soil nutrients and enzyme activities from nutrient cycling were analyzed. Gene abundances of bacterial and archaeal amoA as well as 16S rRNA and ITS marker genes were quantified using the QIAcuity nanoplate digital PCR. The bacterial and fungal community compositions were analyzed via amplicon sequencing of 16S rRNA and ITS marker genes. Significant reductions of target enzyme activities were found for 2-NPT and DMPP but not MPA. Effect size of inhibition was soil-dependent. Ammonium and nitrate concentrations were significantly affected by the inhibitors in one of the three soils. The non-target enzyme activities of phosphatase, beta-glucosidase, and arylsulfatase were not affected by the inhibitors. Both nitrification inhibitors primarily targeted bacterial ammonia oxidizers, as bacterial but not archaeal amoA genes were reduced. Overall bacterial and fungal communities were not clearly affected. Observed abundance shifts of soil microorganisms were linked to indirect effects driven by nutrient availability rather than direct effects of the inhibitors. Our study suggests that the newer inhibitors 2-NPT and MPA as well as DMPP do not directly affect the function and composition of the soil microbiome in the short-term. Significant target effects of the inhibitors change the availability of mineral N which causes indirect effects with minimal non-target microbial community shifts. Further tests involving other soil organisms and long-term field studies are required to further improve the environmental risk assessment of inhibitors.

Soils are among the habitats with the most species-rich communities on our planet. However, knowledge about soil biodiversity status and trends varies by organism group and this also applies to Germany. For soil microorganisms and microfauna, only a fraction of the expected number of species has been described so far. For most groups of soil mesofauna, including collembolans, oribatid mites, and enchytraeids checklists exist, but yet undescribed and cryptic species are to be expected, and data on the distribution and trends of taxa are missing. Larger animals belonging to the soil macrofauna, including earthworms, millipedes, centipedes, ants, isopods, ground beetles, and spiders, are better studied, and checklists and Red Lists exist in Germany. However, even in these rather well-studied groups, genetic information is limited and population trends for species remain largely unknown due to the lack of long-term, large-scale monitoring programs. Only few soil animal groups have been sufficiently studied to allow conclusions on their regional distribution and the degree of endemism or the distribution of possible neobiota. At the national, European, and global scale, data on soil biodiversity in space and time is very patchy and inconsistent. We therefore also lack baseline values to record and assess potential changes that are currently taking place. Moreover, soils are heterogeneous, varying spatially and temporally, and soil biodiversity distribution is influenced by the dynamics of resources and habitat characteristics. Spatial variation in the presence of soil animals depends on microhabitat structure, animal body size, animal mobility, and dispersal. At small scales, the physical structure and pore space of the soil play an essential role for soil organisms and their interactions. At larger scales, different habitat types contribute to soil biodiversity, with geological substrate playing a key role. Compared to aboveground communities, soil biota are more buffered against climatic fluctuations and climate change effects are thus likely to be less pronounced and delayed. Even in a rather well-studied country like Germany, our understanding of the status and trends of soil biodiversity is still scarce and we urgently need research initiatives of taxonomists, soil ecologists, data scientists and molecular biologists who jointly discover and monitor soil biodiversity and predict possible spatio-temporal changes and their consequences.

Wood decay fungi and bacteria play a crucial role in natural ecosystems, contributing to the decomposition of lignocellulosic materials and nutrient cycling. However, their activity poses significant challenges in timber durability, impacting industries reliant on wood as a construction material. This review examines the diversity of microorganisms damaging timber used indoors and outdoors. Additionally, traditional and advanced methods for microbial identification are discussed, with a focus on DNA-based, culture-independent sequencing methods whose importance has increased massively in recent years. It also provides an overview of the various options for wood protection, starting from wood protection by design, to chemical wood preservation and wood modification methods. This should illustrate how important it is to combine an ecological understanding of the decay organisms, precise identification and innovative wood protection methods in order to achieve a long-term and thus resource-saving use of wood. • Fungi and bacteria play a crucial role in the decomposition of timber wood. • Traditional and advanced DNA-based methods for microbial identification are discussed. • An overview of the various options for wood protection is provided.

Protists and nematodes are dominant predators of prokaryotic communities in soil. Their strong difference in size suggests different feeding strategies and preferences. Here we analyzed the relevance of size-dependent predation by distinguishing in microcosm experiments the contribution of small-sized and large-sized protists and nematodes, respectively. Sterilized soils were inoculated with soil suspensions filtrated across 250 mu m, 100 mu m, 60 mu m, 20 mu m and 5 mu m pore sized membranes and a non-filtrated control. After 60 days of incubation, only the soils inoculated with the 5 mu m-filtrate showed lower abundances of bacteria, archaea and fungi, and a differently composed protistan community. The nematode communities were always dominated by the small-sized bacterivore genus Acrobeloides. Correlation analyses indicated that small-sized protists were associated with a wider range of prokaryotic taxa than larger sized protists. In contrast to small-sized nematodes, large-sized showed no correlation with specific prokaryotic taxa. The comparison of effects on prokaryotes at DNA and RNA levels suggested a preference of the soil predators for actively growing rather than resting cells. Small-sized protists (only Cercozoa) and nematodes had a more pronounced influence on the gene abundances of microbial groups and N cycling genetic potentials, i.e., bacterial amoA, nirS, and nosZII genes. Therefore, we conclude that the common ecological size-dependent predation theory also applies to trophic interactions of protists and nematodes with soil prokaryotes. The distinct feeding preferences can alter the prevalence of different N-functional genes, which could thus potentially modify the dynamics of the N cycling in soil.

In vineyards facing soil degradation and biodiversity loss, crop diversification may improve sustainability, but its effects on the soil microbiome remain unclear. In a 3-year field study, we examined how diversifying the plant row under grapevine with aromatic plants affected topsoil properties (0-10 cm) in an organically farmed, steep-sloped vineyard. Specifically, we investigated the effects of diversification with oregano and thyme on microbial biomass, respiration, prokaryotic and fungal community compositions, enzyme activities, potential nitrification, and abiotic soil properties, including total and particulate organic carbon (TOC, POC), nutrient status, pH, and soil moisture. Grapevines alone with mechanical tillage served as control. The aromatic plants competed with grapevines by lowering soil nutrient contents and moisture. Aromatic plant litter had a small, mostly non-significant but consistent effect on POC contents, and POC stocks determined in the final year showed a slight increasing trend in the order control (10.9 +/- 2.8 t POC ha(-1)) < thyme (12.6 +/- 3.1) < oregano (13.1 +/- 4.1). Surprisingly, these changes coincided with a significant decrease in microbial biomass compared to control, indicating aromatic plant-microbe competition. Concomitant decreases in respiration and the activity of C-cycling enzymes but also the metabolic quotient, suggest lower carbon mineralisation but more efficient microbial carbon use. Multivariate statistics revealed that the prokaryotic community was primarily structured by abiotic soil properties, such as organic matter, nutrient and water availability. In contrast, the fungal community exhibited a stronger plant-specific response, with changes in composition likely driven by root-associated interactions, suggesting a more direct biotic influence. Especially, arbuscular mycorrhizal fungi and potential nitrification were promoted under both aromatic plants, which may benefit grapevine growth. Overall, we show that diversifying perennial agroecosystems such as vineyards with aromatic plants increases soil habitat heterogeneity with benefits for microbial diversity, carbon sequestration and nutrient cycling, demonstrating its positive impact on soil biodiversity and functioning.

Healthy soils and the biodiversity therein are the prerequisite for the supply of manifold ecosystem services that are essential for human well-being. Detailed knowledge, especially at the national level, is important for effective policy-making to safeguard healthy functional soils for future generations. Hence, synthesis of the state of soil biodiversity and related ecosystem functions, the driving forces affecting the soil as a habitat, avenues for sustainable soil management, and the role of stakeholders at different levels is required. Here, we present the eleven key messages of the first comprehensive soil biodiversity assessment in Germany, based on the currently available and accessible literature and expert knowledge. Among others, we highlight the high biodiversity of soils in Germany, their role for climate regulation and other ecosystem services, the impact of multiple concurrent drivers, as well as actions and schemes already in place to sustainably manage soil biodiversity and to raise awareness in different groups of the public. We conclude that national assessments of the available literature and data are an important step towards the incorporation of soil biodiversity in national policies and to provide the basis for national long-term systematic monitoring.

Abstract Current risk assessment strategies for protein safety of newly expressed proteins (NEPs) in genetically modified (GM) plants are based on chemical risk assessment principles and Codex Alimentarius guidelines for biotech‐derived foods, initially published in 2003. These guidelines were designed for proteins with multiple testing options and for GMOs expressing a low number of NEPs. However, two decades of experience in assessing GMO and biotech products, along with recent advances in the field, underscore the need to update best practices for protein safety assessment. Furthermore, new types of products challenge the application of the current international guidelines, as assessments become more complex due to NEPs that are difficult to test using existing approaches or products with numerous NEPs. This document outlines a strategy that strengthens the stepwise, weight‐of‐evidence approach, incorporating new methodologies as complementary or alternative studies. An improved strategy for protein safety assessment could include: (1) considering history of safe use (HoSU), read‐across and phylogeny defining the type of data required and remove the need for specific in vitro or in vivo studies; (2) applying advanced in silico tools, including predictive computational models and improved phylogenetic analysis to enable more accurate comparisons with known allergens, toxins or ‘safe’ proteins; (3) using standardised in vitro gastrointestinal models that replicate physiological conditions; (4) developing targeted in vivo studies; (5) evaluating the role of exposure in the safety assessment; and, where necessary, (6) considering post‐market monitoring for risk characterisation. Consensus on the definition of HoSU and the effective integration of novel methodologies into the current NEP safety assessment will be essential to meet society's demand for safer, healthier and more sustainable food/feed in a growing world. This calls for revisiting and refining the goals of protein safety risk assessment to ensure that NEPs in biotech products are evaluated appropriately, consistently and proportionately.

A sustainable use of croplands should utilize beneficial services provided by their resident soil microbiome. To identify potentially adverse environmental effects on soil microbiomes in the future, a better understanding of their natural variability is fundamental. Here, we characterized the abundance and diversity of soil microbial communities over 2 years at two-week intervals on three neighboring fields at an operational farm in Northern Germany. Field soils differed in texture (clay, loam) and tillage (soil conservation vs. conventional). PCRamplicon analyses of soil DNA revealed distinct temporal variations of bacteria, archaea, fungi, and protists (Cercozoa and Endomyxa). Annual differences and seasonal effects on all microbial groups were detected. In addition to soil pH, prokaryotic communities varied with total soil C and N, but fungi with temperature and precipitation. The C/N ratio had contrasting effects on prokaryotic phyla and protistan classes, but all fungal phyla responded positively. Irrespective of the sampling date, prokaryotic and fungal but not protistan community compositions from the three soils were distinct. Compositional turnover rates were higher for fungi and protists than for prokaryotes and, for all, lower in clay. Conventional tillage had the strongest effect on protist diversity. In co-occurrence networks, most nodes were provided by prokaryotes, but highly connected nodes by predatory protists in the first, and by saprotrophic fungi in the second year. The temporal variation established here can provide insights of what is natural and thus below the limits of concern in detecting adverse effects on the soil microbiome.

In cropland, prokaryotic microbiomes mediate the biogeochemical cycling of nitrogen (N), which is typically supplied at large amounts for plant growth promotion. While genes encoding for key enzymes of the N cycle in soil have been identified, little is known about their seasonal variation along cropping cycles. Here, we followed over a period of two years at 2-week intervals with quantitative PCR the abundance of seven N-functional genes on three neighboring fields at an operational farm in Northern Germany. The fields differed in soil texture (clay or loam) and soil management (soil conservation vs. conventional tillage). Total N (TN) was the main factor driving temporal dynamics of all seven N-functional genes, while total carbon, temperature, fertilization events and soil tillage were less important. Precipitation, and thus soil moisture, negatively affected gene abundances, and this effect was more pronounced in loam than in clay. Clear differences could be detected for the abundance of archaeal and bacterial amoA and also, though less stringent, for nirS/K but not for nosZI/nosZII. The contrasting responses of the two former suggests distinct preferences in response to different textures and tillage regimes. Overall, this study demonstrates that in cropland, the seasonal dynamics of N functional genes depends on the TN contents, which is variable in response to soil moisture, texture and tillage. The coexistence of alternative prokaryotic genes encoding for the same enzymatic reactions reflects their specific adaptations to different environmental conditions, which can result in high, but also low correlations between them.

Fungal communities in soil play important roles in decomposition processes and soil organic carbon cycling. These communities are tremendously diverse, making it challenging to assign relevant functions to individual species. Fungal communities may be differentiated at the level of functional guilds; beyond such broad classification we have little delimitation, especially in fungal taxa common in grassland and agricultural soils. To resolve the level of functional similarity in fungal communities and define traits predictive of soil carbon cycling, we characterized fungal isolates abundant in agricultural soils to test the hypotheses that (i) the majority of saprobic soil fungi have the ability to use complex carbon sources, (ii) differences in complex carbon use abilities correlate with fungal enzymatic profiles, following principles of the fungal economics spectrum and (iii) carbon use ability is a predictive trait for fungal community functions. Using specialized growth media, we isolated and characterized 105 isolates and developed a novel FungiResp approach that directly tests fungal activity on complex carbon sources. The largest amount of variance between isolates was explained by differential abilities to use cellulose and starch, with only few phylogenetically distinct fungal clades showing high respiratory activity on these biopolymers. A preference for bacterial necromass was another major distinction among taxa. These key traits correlated with soil fungal community shifts in response to carbon substrate availability. By contrast, enzymatic activity was a poor predictor of fungal carbon use ability, except for correlations in lignin use and laccase activity. The newly established functional trait of carbon use ability offers important insights into diverse fungal communities: Many taxa lack the ability to use complex carbon (on their own), while the most common enzymes analyzed in soil showed little correlation with fungal mineralization potential. The discovery of key functional traits is an important step towards predicting the significance of fungal community shifts for soil carbon cycling. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64

The natural durability of wood against basidiomycetes is an important reference value for the use of wood species as construction timber. However, only three test fungi must be used for durability testing according to European standards, although a much larger number of wood-destroying basidiomycetes are known. Here, we used a modified test protocol of the EN 113-2 standard to examine the abilities of two strains of Perenniporia meridionalis, one of them freshly isolated and the other being in culture for 23 years, to degrade wood of beech (Fagus sylavatica), English oak (Quercus robur) and black locust (Robinia pseudoacacia) by measuring the mass loss after 16 weeks of incubation at 22 degrees C, 26 degrees C and 30 degrees C, and 70 % relative humidity. The obligatory test fungus Trametes versicolor was used as a reference. We found that the degradation abilities of all fungal isolates increased with higher temperatures. Furthermore, the freshly isolated strain of P. meridionalis caused higher mass loss than the long-term cultured strain. For beech and black locust wood the highest mass loss was caused by Trametes versicolor, while for English oak it was the fresh isolate of Perenniporia meridionalis. In conclusion, our study demonstrates that Perenniporia meridionalis is a potent wood decaying fungus, which exhibited a higher degradation ability for English oak than the obligatory used white rot test fungus Trametes versicolor, and thus could become a useful indicator in the future when evaluating durability of this type of wood.

Timber wood is a building material with many positive properties. However, its susceptibility to microbial degradation is a major challenge for outdoor usage. Although many wood-degrading fungal species are known, knowledge on their prevalence and diversity causing damage to exterior structural timber is still limited. Here, we sampled 46 decaying pieces of wood from outdoor constructions in the area of Hamburg, Germany; extracted their DNA; and investigated their microbial community composition by PCR amplicon sequencing of the fungal ITS2 region and partial bacterial 16S rRNA genes. In order to establish a link between the microbial community structure and environmental factors, we analysed the influence of wood species, its C and N contents, the effect of wood-soil contact, and the importance of its immediate environment (city, forest, meadow, park, respectively). We found that fungal and bacterial community composition colonising exterior timber was similar to fungi commonly found in forest deadwood. Of all basidiomycetous sequences retrieved, some, indicative for Perenniporia meridionalis , Dacrymyces capitatus , and Dacrymyces stillatus , were more frequently associated with severe wood damage. Whilst the most important environmental factor shaping fungal and bacterial community composition was the wood species, the immediate environment was important for fungal species whilst, for the occurrence of bacterial taxa, soil contact had a high impact. No influence was tangible for variation of the C or N content. In conclusion, our study demonstrates that wood colonising fungal and bacterial communities are equally responsive in their composition to wood species, but respond differently to environmental factors. Key points • Perenniporia meridionalis and Dacrymyces are frequently associated with wood damage • Fungal community composition on timber is affected by its surrounding environment • Bacterial community composition on structural timber is affected by soil contact

The susceptibility of timber to microbial degradation is a major challenge for its long-term outdoor usage. It is important to know which microorganisms are responsible for wood degradation and which environmental factors influence the species composition in order to make adjustments for extending their service life in the future.

Genetically modified Komagataella phaffii strain MXY0541 was developed to produce soy leghemoglobin by introducing the LGB2 coding sequence encoding leghemoglobin from soybean (Glycine max). The molecular characterisation data and bioinformatic analyses do not raise any safety concerns. The safety of soy leghemoglobin as a food additive has already been assessed by the EFSA FAF Panel (EFSA-Q-2022-00031). The GMO Panel does not identify safety concerns regarding the toxicity and allergenicity of soy leghemoglobin protein as expressed in K. phaffii, and finds no evidence that the genetic modification would change its overall allergenicity. The GMO Panel concludes that the LegH Prep derived from genetically modified K. phaffii strain MXY0541 is safe for human consumption with regard to the effects of the genetic modification. No environmental impact from the use of this product is expected regarding the recombinant DNA sequences possibly remaining in the product. The GMO Panel concludes that LegH Prep from genetically modified K. phaffii strain MXY0541 is safe with respect to potential effects on human health and the environment at the proposed use and use level as far as the impact of the genetic modification is concerned. The overall conclusion is that the genetic modification does not lead to safety issues.

Diversified cropping systems and fertilization strategies were proposed to enhance the abundance and diversity of the soil microbiome, thereby stabilizing their beneficial services for maintaining soil fertility and supporting plant growth. Here, we assessed across three different long-term field experiments in Europe (Netherlands, Belgium, Northern Germany) whether diversified cropping systems and fertilization strategies also affect their functional gene abundance. Soil DNA was analyzed by quantitative PCR for quantifying bacteria, archaea and fungi as well as functional genes related to nitrogen (N) transformations; including bacterial and archaeal nitrification (amoA-bac,arch), three steps of the denitrification process (nirK, nirS and nosZ-cladeI,II) and N2 assimilation (nifH), respectively. Crop diversification and fertilization strategies generally enhanced soil total carbon (C), N and microbial abundance, but with variation between sites. Overall effects of diversified cropping systems and fertilization strategies on functional genes were much stronger than on the abundance of bacteria, archaea and fungi. The legume-based cropping systems showed great potential not only in stimulating the growth of N-fixing microorganisms but also in boosting downstream functional potentials for N cycling. The sorghum-based intercropping system suppressed soil ammonia oxidizing prokaryotes. N fertilization reduced the abundance of nitrifiers and denitrifiers except for ammonia-oxidizing bacteria, while the application of the synthetic nitrification inhibitor DMPP combined with mineral N reduced growth of both ammonia-oxidizing bacteria and archaea. In conclusion, this study demonstrates a strong impact of diversified agricultural practices on the soil microbiome and their functional potentials mediating N transformations.

Refined conversion factors for soil fungal biomarkers are proposed. ● High interspecific variability is present in all fungal biomarkers. ● A modeling approach supports the validity of biomarker estimates in diverse soils. ● ITS1 copies vary strongly, but are fungal-specific with least phylogenetic bias. ● A combination of fungal biomarkers will reveal soil fungal physiology and activity. The abundances of fungi and bacteria in soil are used as simple predictors for carbon dynamics, and represent widely available microbial traits. Soil biomarkers serve as quantitative estimates of these microbial groups, though not quantifying microbial biomass per se. The accurate conversion to microbial carbon pools, and an understanding of its comparability among soils is therefore needed. We refined conversion factors for classical fungal biomarkers, and evaluated the application of quantitative PCR (qPCR, rDNA copies) as a biomarker for soil fungi. Based on biomarker contents in pure fungal cultures of 30 isolates tested here, combined with comparable published datasets, we propose average conversion factors of 95.3 g fungal C g−1 ergosterol, 32.0 mg fungal C µmol−1 PLFA 18:2ω6,9 and 0.264 pg fungal C ITS1 DNA copy−1. As expected, interspecific variability was most pronounced in rDNA copies, though qPCR results showed the least phylogenetic bias. A modeling approach based on exemplary agricultural soils further supported the hypothesis that high diversity in soil buffers against biomarker variability, whereas also phylogenetic biases impact the accuracy of comparisons in biomarker estimates. Our analyses suggest that qPCR results cover the fungal community in soil best, though with a variability only partly offset in highly diverse soils. PLFA 18:2ω6,9 and ergosterol represent accurate biomarkers to quantify Ascomycota and Basidiomycota. To conclude, the ecological interpretation and coverage of biomarker data prior to their application in global models is important, where the combination of different biomarkers may be most insightful.

The earthworm species Lumbricus terrestris L. feeds on plant litter mixed with surrounding soil. Here, we analyzed with a mesocosm approach and soil incubations how that activity and subsequent ageing of casts (feces) affects the abundance and diversity of the soil microbiome. Earthworms were fed either with straw of sainfoin (SA, Onobrychis viciifolia; C/N ratio 22) or winter wheat (WW, Triticum aestivum, C/N ratio 101). The gut transit increased the abundances of bacteria and fungi, but reduced archaea. As indicated at the DNA and RNA level, main beneficiaries of the facilitated access to nutrients were members of Bacteroidota, especially Flavobacteriales with an estimated generation time of only 2 h. While Alphaproteobacteria were reduced, Gammaproteobacteria also increased in abundance and activity. SA was more nutritious for L. terrestris, and supported a higher bacterial abundance, probably because more N was available for growth and denitrification. During cast ageing, prokaryotic community compositions became increasingly similar to bulk soil communities. However, they remained distinguishable even after 168 d, suggesting that effects can last beyond a vegetation period. Dry-wet conditions preserved these differences better than continuous moisture. During ageing, more complex prokaryotic networks were detected with WW and dry-wet conditions. Thus, N and water limitations appeared to enhance cooperation rather than competition between the prokaryotes. Overall, this study demonstrates that earthworm soil interactions strongly affect the diversity and temporal dynamics of the soil microbiome. Legacy effects of earthworm activities should thus be kept in mind when investigating the environmental variation of soil microbiomes.

Abstract Following a request from the European Commission, the GMO Panel assessed additional information related to the application for authorisation of food and feed containing, consisting of and produced from genetically modified soybean MON × MON 87708 × MON 89788 (EFSA‐GMO‐NL‐2015‐126). The applicant conducted a 90‐day feeding study on GM soybean MON 87705 and provided a proposal for post‐market monitoring considering the altered fatty acid profile of GM soybean MON 87705 × MON 87708 × MON 89788, to fulfil the deficiencies identified by EFSA GMO Panel, addressing elements that remained inconclusive from a previous EFSA scientific opinion issued in 2020. The GMO Panel concludes that the 90‐day feeding study on GM soybean MON 87705 is in line with the requirements of Regulation (EU) No 503/2013 and that no treatment‐related adverse effects were observed in rats after feeding diets containing soybean MON 87705 meals at 30% or 15% for 90 days. The GMO Panel reiterates the recommendation for a PMM for food in accordance with Regulation (EC) No 1829/2003 and Regulation (EU) No 503/2013 and concludes that the proposal provided by the applicant is in line with the recommendations described for the PMM plan of soybean MON 87705 × MON 87708 × MON 89788 in the adopted scientific opinion. Taking into account the previous assessment and the new information, the GMO Panel concludes that soybean MON 87705 × MON 87708 × MON 89788, as assessed in the scientific opinion on application EFSA‐GMO‐NL‐2015‐126 and in the supplementary toxicity study, is as safe as its non‐GM comparator and the non‐GM reference varieties tested and does not represent a nutritional concern in humans and animals, within the scope of this application.

The analysis of 18S rRNA gene amplicons is an important tool to characterize the diversity of the eukaryotic soil microbiome. Here we analyzed two primer sets (TAReuk, EKeuk) and the impact of a newly designed antifungal peptide nucleic acid to enhance the detection of protists in silico and with soil DNA extracted from croplands and a forest. The in silico analyses showed for TAReuk pronounced specificities for protist SAR supergroup and Metazoa, while EKeuk was particularly specific for Ascomycota and Basidiomycota. In silico, the PNA matched with the majority of Ascomycota (81.3 %) and Basidiomycota (65.4 %), but with <6 % of protists. The contrasting primer specificities were confirmed with soil DNA, but the proportion of protist amplicons was similar. In contrast to in silico, effects of the PNA were not as clear with soil DNA, even though it completely inhibited the amplification of the targeted fungal sequences. PNA effects were more pronounced with TAReuk, and results with cropland and forest soil DNA were not consistent, e.g., for cropland, PNA decreased the relative abundance of fungi but for forest it was the opposite, possibly because of different fungal diversity. The divergence between PNA in silico-predictions and results with soil DNA are likely an outcome of primer binding to <100 % complementary target sequences and a still limited DNA sequence databases for soil microbial eukaryotes. With TAReuk, the presence of PNA enhanced the detection of Conosa and, thus, could be a useful tool to study this group in the future.