The sustainability of different management practices to enhance soil organic carbon (SOC) sequestration and reduce nitrogen (N) losses remains debated due to site-specific variability. Investigating soil physicochemical and biological factors is crucial, and soil functional genes can serve as key indicators of nutrients cycling and soil health. The present 4-year on-farm experiment in northeastern Italy, developed in collaboration with local farmers and a land reclamation authority, investigated the effects of two organic fertilizers (compost or digestate), a triticale (X triticosecale) cover crop treatment (CC) under rainfed vs. irrigated conditions on SOC, total N (nitrates - NO3- and Total Kjeldahl Nitrogen -TKN), and soil N and C functional genes in a biennial maize-soybean rotation. Compost application led to higher short-term SOC accumulation and greater GH48 cellulolytic enzyme activity than digestate supposedly due to its more recalcitrant molecules. In compost treatments, higher SOC levels enhanced microbial denitrification (nosZ). While triticale CC did not impact SOC, it reduced soil NO3content. Digestate exhibited greater competition with microbial N fixation (lower nifH gene copies) than compost. Elevated NO3- levels in the soil stimulated nitrification activity (AOA) across all management practices. A general increase in N, SOC, and microbial-mediated activities throughout the four experimental years suggested an overall enhancement of soil biological fertility. Agronomic management strategies influenced N and SOC dynamics, and functional genes linked to C and N cycling showing their potential as proxies for tracking these changes. However, while gene abundance was sensitive to management practices, environmental conditions also influenced the results, making it challenging to isolate single key factors.

European grasslands are vital carbon (C) sinks, contributing to climate change mitigation. Grazing intensity significantly influences soil C and nitrogen (N) cycles through effects on soil conditions and microbial communities. While heavy grazing is linked to soil C loss and altered N processes, existing studies show conflicting outcomes. This study examines the impact of cattle grazing on soil C and N cycles in a historical alpine pasture in the eastern Italian Alps (1868 m a.s.l.). The following three grazing intensities were analyzed: heavy (8.19 LU ha−1), moderate (0.59 LU ha−1), and light (0.06 LU ha−1). Soil was sampled from two depth layers (0–5 cm, 5–10 cm) and analyzed for bulk density, C and N content, C/N ratio, exchangeable N, δ15N, and microbial genes targeting general abundance (16S), N fixation (nifH), nitrification (amoA), and denitrification (nirK, nosZ) using real-time PCR. The results revealed decreased C and N concentrations with increasing grazing intensity, exclusively in the 0–5 cm soil layer. Higher δ15N and enhanced nitrification and denitrification suggest a more open N cycle under heavy grazing. These findings highlight the potential of microbial gene markers and δ15N isotopic ratios to monitor N cycle dynamics in alpine pastures, informing sustainable grazing management.

The Arctic region is characterized by severe temperatures and a unique ecosystem with largely unexplored microbiomes. Whereas soil microbiomes in temperate regions play key roles in nutrient cycling, organic matter decomposition, greenhouse gas fluxes, and overall ecosystem functioning, Arctic microbiomes remain poorly understood, highlighting the need for a thorough characterization to better predict and manage soil health and resilience. In this study, we compared the microbial profiles of three raised beaches on Devon Island (Nunavut, Canadian Arctic Archipelago), which emerged sequentially between eight and two thousand years ago, to assess their similarities and differences. Samples were collected by genetic horizons along excavations from the top layer to the permafrost. For each horizon, total soil DNA, 16S gene copies dPCR quantification, 16S metabarcoding, and functional prediction were carried out. Total DNA quantification revealed a consistently comparable concentration of genetic material across the three soil beaches (AB2 μ = 2.28 ± 5.44 μg ∙ g -1 , AB1 μ = 4.71 ± 2.35 μg ∙ g -1 , AB3 μ = 5.44 ± 2.91 μg ∙ g -1 ), regardless of site age (AB2 = 2,360 YBP, AB1 = 6,726 YBP, AB3 = 8,410 YBP). Conversely, clear differences emerged by comparing the different horizons at each site. The hierarchical cluster analysis based on the Bray-Curtis dissimilarity matrix revealed a clear separation between surface and deep horizons. The core microbiome analysis highlighted Actinobacteria, Proteobacteria, and Firmicutes as the three predominant phyla accounting for relative abundances of 42%, 22%, and 18%, respectively. Remarkable evidence was the unexpectedly high taxonomic diversity that was recorded in these sites and that surprisingly matched with the commonly observed values in soils of temperate regions. Since these stony shores developed under cold, life-limiting conditions, their apparent microbial richness raises doubts about the potential biases in inferring physiological contexts and active biodiversity directly inferred from culture-independent DNA-based studies. The reason is that such inventories can be possibly inflated, in all environments, by chronically accumulated cells from passive immigration events through atmospheric discharge.

Context Alpine pastures are seminatural grasslands which play a crucial role in biodiversity conservation, service provisioning, and mountain livestock systems. The soil microbial communities of pasture are fundamental in ecosystem nutrient cycles, but they are relatively underexplored in European Alpine pastures. Aims We explored the many soil microbial genes encoding key functions in the nitrogen cycle in three historical alpine pastures grazed by dairy cattle, considering different soils, temporal dynamics, and exclusion of cattle grazing for one summer. Methods 216 samples were collected across four sampling times. The abundance of genetic determinants involved in nitrogen fixation (nifH), nitrification (amoA bacterial and archaeal), and denitrification (nirK and nosZ) were quantified using real-time polymerase chain reaction. Key results The terminal denitrification nosZ gene was the most sensitive indicator and responded significantly to soil chemical composition and animal grazing. Sampling time affected nitrogen fixation nifH and intermediate denitrification nirK in relation to rainfall cumulation dynamics. The amoA nitrification genes showed high variability but no significant effects from the tested factors. Conclusions In spite of a general homeostatic trend occurring in these habitats and of the short term analysis, some genes acted as sensitive reporters of soil compositional differences, intraseasonal climatic variations, and grazing disturbance. Implications A stocking rate of >0.6 livestock units per hectare can be recommended, to combine animal production with conditions that favour complete denitrification, thus potentially reducing the nitrous oxide greenhouse gas emissions. Higher livestock grazing intensity can be withstood by the ecosystem without denitrification-related drawbacks when the preceding 10 days display a cumulated rainfall lower than 22 mm.

In the last years, plant organelles have emerged as central coordinators of responses to internal and external stimuli, which can induce stress. Mitochondria play a fundamental role as stress sensors being part of a complex communication network between the organelles and the nucleus. Among the different environmental stresses, salt stress poses a significant challenge and requires efficient signaling and protective mechanisms. By using the why2 T-DNA insertion mutant and a novel knock-out mutant prepared by CRISPR/Cas9-mediated genome editing, this study revealed that WHIRLY2 is crucial for protecting mitochondrial DNA (mtDNA) integrity during salt stress. Loss-of-function mutants show an enhanced sensitivity to salt stress. The disruption of WHIRLY2 causes the impairment of mtDNA repair that results in the accumulation of aberrant recombination products, coinciding with severe alterations in nucleoid integrity and overall mitochondria morphology besides a compromised redox-dependent response and misregulation of antioxidant enzymes. The results of this study revealed that WHIRLY2-mediated structural features in mitochondria (nucleoid compactness and cristae) are important for an effective response to salt stress.

Cover crops (CCs) strongly interact with soil water content (SWC) and nitrogen (N) cycling. However, their impact on these two resources may vary throughout the crop succession seasons. This seasonal variability is often missed by research studies where a single sampling time point is considered. The study aims to investigate how the initial introduction of diverse CCs affects the seasonal variability of the soil chemical (NO3-) and biological (soil N functional genes – NFGs) constituents of the N cycle, the SWC, and the cash crop yield in a three-year maize-soybean succession in northeastern Italy. Three CC managements were compared: a fixed treatment with triticale; a 3-year succession of rye, crimson clover, and mustard; and a control with no CC. No CC-dependent difference in the cash crop yields was observed. All the CCs didn’t exhibit water competition with ensuing cash crops compared to the control. At the CC termination time, grasses reduced soil NO3- (catch crops) and stimulated potential microbial N-fixing activity (nifH), whereas clover induced the highest residual NO3- and potential N nitrification (AOA). Agronomic operations probably disrupted differences in NFG abundances after CC residue incorporation. During the cash crop season, higher N release was estimated by the CC-NCALC model for clover, mustard, and weeds (including wild legumes) than for rye and triticale (immobilization). Nevertheless, consistent N nitrification and denitrification potentials were observed under all treatments except weed-free bare soil, with greater NFG abundance when soybean was cultivated instead of maize. This underscores the role of cash crop species in shaping the N transformation dynamics. The observed high seasonal variability highlights that the effective utilization of NFGs and chemical indicators to evaluate the impact of CCs on the N dynamics and the SWC requires careful consideration of the timing of sample collection within a crop succession (e.g. termination time for CCs).

The mymarid wasp Platystethynium triclavatum (Hymenoptera: Mymaridae) has been recently recorded as an egg parasitoid of the bush-cricket Barbitistes vicetinus (Orthoptera: Tettigoniidae). This species is endemic to northeastern Italy and has become an economically significant agricultural and forest pest. Biological attributes of P. triclavatum in relation to its host were still to be defined. Field parasitization rates and the number of individuals emerging from a single host egg were assessed by extracting eggs from soil at five outbreak sites in the Euganean Hills, while a laboratory choice experiment was conducted to evaluate the parasitoid’s preference for eggs of different ages. The natural emergence period was determined by periodically inspecting parasitized eggs buried in the ground. Additionally, genetic characterization of subpopulations from the Euganean Hills was conducted by analyzing both nuclear and mitochondrial DNA fragments. We found low parasitization rates across all sites. However, parasitoids successfully oviposited in B. vicetinus eggs of different ages, exhibiting a higher parasitism rate in one-year-old eggs compared to newly laid eggs. The average number of individuals that can hatch from a single B. vicetinus egg (95) can better reflect the potential impact of this parasitoid on regulating host populations. The hatching period was observed from the end of July to August. Subpopulations showed low values of haplotype diversity, with two main haplotypes diffused across almost all the sites. The absence of a geographical structure and a wide distribution within the landscape suggest a natural dispersal ability of P. triclavatum in the local area.

Context: Cover crops (CCs) exert significant influences on both soil water content (SWC) and nitrogen (N) cycling, with their effects often varying across different stages of crop succession. Many research endeavors overlook this seasonal variability by focusing solely on single sampling time points. Objective: This study seeks to explore how the introduction of diverse CCs influences the seasonal fluctuations of soil nitrate nitrogen (NO3-N) and biological (soil N functional genes - NFGs) components of the N cycle, SWC, and cash crop yield over a three-year maize-soybean succession in northeastern Italy. Methods: Three CC management systems were compared: a fixed treatment with triticale; a 3-year succession of rye, crimson clover, and mustard; and a weedy fallow as control. Soil N cycling was assessed using real-time PCR and Ion Chromatography, SWC through Sentek's Diviner2000. Results: CCs had no impact on cash crop yields and did not display water competition with subsequent cash crops compared to the weedy fallow. At CCs termination, grasses decreased soil NO3-N content (as catch crops) while enhancing potential microbial N-fixing activity (nifH), whereas clover led to the highest residual NO3-N and potential N nitrification (AOA). Agronomic operations likely mitigated differences in NFG abundances following CC residue incorporation. During the cash crop season, clover, mustard, and weeds (including wild legumes) were estimated to release higher amounts of N according to the CC-NCALC model, compared to rye and triticale which exhibited immobilization. Nonetheless, consistent N nitrification and denitrification potentials were observed across all treatments except for weedy fallow, with higher NFG abundance when soybean was cultivated instead of maize, underscoring the influence of cash crop species on N transformation dynamics. Conclusions: CC and cash crop species, seasonality of crops sequence, and management operations represent pivotal factors shaping the soil N cycling dynamics intricately governed by N-cycling microbial communities and the temporal variability of the SWC. Upon CC termination, grass CCs decrease soil NO3-N content, whereas clover CC sustain high NO3-N content, enhancing microbial nitrification. Agronomic operations disrupt differences in N processes subsequent to the incorporation of different CCs. However, all CC residues enhance microbe-mediated nitrification and denitrification by cash crop harvest time, potentially more pronounced in the presence of soybean cash crop compared to maize. Significance: The substantial seasonal variability observed emphasizes the necessity of carefully timing sample collection within a crop succession (e.g., at CC termination) for effectively utilizing NFGs and chemical indicators to assess the impacts of CCs on N dynamics.

Background The threatening impact of conventional agriculture (CA) on soils could be due to the detrimental effects on soil microbial communities. Conversely, organic agriculture (OA) is envisaged as potentially enhancing helpful microbial communities and is proposed as environmentally sustainable. The soil microbiome influences soil health and quality, hence, it requires deeper investigation and understanding. In this study, applying 16S metabarcoding and qPCR techniques, we compared the microbial patterns of long-term organically and conventionally managed soils to explore their similarities and differences. Results Total DNA quantification showed an over 20-fold higher amount of DNA in OA soils (mean = 22.1 ± 3.92 μg g −1 ), compared to CA soils (mean = 0.95 ± 0.17 μg g −1 ). While 16S metabarcoding evidenced the absence of significant differences among communities of the two farming systems in terms of ecological indices, the qPCR analyses targeting functional genes reported a significantly higher abundance of all considered targets in OA sites spanning up to four-fold log increases. While OA and CA did not appear to affect overall bacterial diversity or evenness per se, qPCR-based functional analysis in OA showed a consistently higher abundance of all the salient microbial genes tested, when compared to CA, underlying a potentially beneficial impact on soil fertility and sustainability. Conclusions In essence, the sequencing-based analysis of absolute bacterial diversity could not differentiate the farming systems based on the amount of diversity but identified a unique set of taxa defining each. Hence, pairing this evaluation with the qPCR-based functional gene analyses can be a suitable approach to distinguish the exerted effects of CA or OA on soils. Graphical Abstract

Highly stabilized digestate from sewage sludge and digestate-derived ammonium sulphate (RFs), were used in a comparison with synthetic mineral fertilizers (SF) to crop maize in a three-year plot trial in open fields. RFs and SF were dosed to ensure the same amount of mineral N (ammonia-N). In doing so, plots fertilized with digestate received much more N (+185 kg ha-1 of organic N) because digestate also contained organic N. The fate of nitrogen was studied by measuring mineral and organic N in soil at different depths, ammonia and N2O emissions, and N uptake in crops. Soil analyses indicated that at one-meter depth there was no significant difference in nitrate content between RF, SF and Unfertilized plots during crop season indicating that more N dosed with digestate did not lead to extra nitrate leaching. Ammonia emissions and N content in plants and grains measured were also similar for both RF and SF. Measuring denitrification activity by using gene makers resulted in a higher denitrification activity for RF than SF. Nevertheless, N2O measurements showed that SF emitted more N2O than RF (although it was not statistically different) (7.59 ± 3.2 kgN ha-1 for RF and 10.3 ± 6.8 kgN ha-1 for SF), suggesting that probably the addition of organic matter with digestate to RF, increased the denitrification efficiency so that N2 production was favoured. Soil analyses, although were not able detecting N differences between SF and Rf after three years of cropping, revealed a statistical increasing of total carbon, suggesting that dosing digestate lead to carbon (and maybe N) accumulation in soil. Data seem to suggest that N2O/N2 emission and organic N accumulation in soil can explain the fate of the extra N dosed (organic-N) in RF plots.

The microbial diversity is, among soil key factors, responsible for soil fertility and nutrient biogeochemical cycles, and can be modified upon changes in main soil physicochemical properties and soil pollution. Over the years, many restoration techniques have been applied to restore degraded soils. However, the effect of these approaches on soil microbial diversity is less understood and thus requires more investigation. In this study, we analyzed the impact, on soil microbial diversity of a patented novel technology, used to restore degraded soils. Soil samples were collected from three nearby sites located in Borgotrebbia, Piacenza, Italy, and categorized as reconstituted, degraded, and agricultural soils. After total soil DNA extraction, 16S rDNA multi-amplicon sequencing was carried out using an Ion GeneStudio S5 System to compare soils’ bacterial community profiles. Sequenced reads were processed to assign taxonomy and then key microbial community differences were identified across the sampling sites. Species diversity featured significant abatement at all rank levels in the degraded soil when compared to the agricultural control. The 5 year restoration technique showed full recovery of this index at the genus level but not at the phylum level, displaying a rank-dependent gradient of restored richness. In parallel, the abundance of genes involved in the nitrogen (N) biogeochemical cycle was assessed using quantitative Real-Time PCR (qPCR). Total DNA content was significantly higher (p < 0.05) in degraded (μ = 12.69 ± 2.58 μg g−1) and reconstituted (μ = 11.73 ± 1.65 μg g−1) soil samples when compared to the agricultural soil samples (μ = 2.39 ± 0.50 μg g−1). The taxonomic diversity of each soil site was significantly different, with some instances unique of the agricultural soil even at the phylum level. The analysis of N functional genes showed that the relative abundance of bacterial amoA (p < 0.05) and nosZ (p < 0.01) genes were significantly lower in the agricultural than in the reconstituted and degraded soils. We concluded that the application of the soil reconstitution technique appears to enhance the active microbial community, with distinct diversity and functionality towards genes involved in N biogeochemical cycle, as compared to both the degraded and the agricultural soil.

The fungus Cercospora beticola causes Cercospora Leaf Spot (CLS) of sugar beet ( Beta vulgaris L.). Despite the global importance of this disease, durable resistance to CLS has still not been obtained. Therefore, the breeding of tolerant hybrids is a major goal for the sugar beet sector. Although recent studies have suggested that the leaf microbiome composition can offer useful predictors to assist plant breeders, this is an untapped resource in sugar beet breeding efforts. Using Ion GeneStudio S5 technology to sequence amplicons from seven 16S rRNA hypervariable regions, the most recurring endophytes discriminating CLS-symptomatic and symptomless sea beets ( Beta vulgaris L.ssp. maritima ) were identified. This allowed the design of taxon-specific primer pairs to quantify the abundance of the most representative endophytic species in large naturally occurring populations of sea beet and subsequently in sugar beet breeding genotypes under either CLS symptomless or infection stages using qPCR. Among the screened bacterial genera, Methylobacterium and Mucilaginibacter were found to be significantly (p < 0.05) more abundant in symptomatic sea beets with respect to symptomless. In cultivated sugar beet material under CLS infection, the comparison between resistant and susceptible genotypes confirmed that the susceptible genotypes hosted higher contents of the above-mentioned bacterial genera. These results suggest that the abundance of these species can be correlated with increased sensitivity to CLS disease. This evidence can further prompt novel protocols to assist plant breeding of sugar beet in the pursuit of improved pathogen resistance.

The identification of efficient molecular markers related to low bolting tendency is a priority in sugar beet (Beta vulgaris L.) breeding. This study aimed to identify SNP markers associated with low bolting tendency by establishing a genome-wide association study. An elaborate 3-year field trial comprising 13 sugar beet lines identified L14 as the one exhibiting the lowest bolting tendency along with an increased survival rate after autumnal sowing. For SNP discovery following phenotyping, contrasting phenotypes of 24 non-bolting and 15 bolting plants of the L14 line were sequenced by restriction site-associated DNA sequencing (RAD-seq). An association model was established with a set of 10,924 RAD-based single nucleotide polymorphism (SNP) markers. The allelic status of the most significantly associated SNPs ranked based on their differential allelic status between contrasting phenotypes (p < 0.01) was confirmed on three different validation datasets comprising diverse sugar beet lines and varieties adopting a range of SNP detection technologies. This study has led to the identification of SNP_36780842 and SNP_48607347 linked to low bolting tendency and can be used for marker-assisted breeding and selection in sugar beet.

The present study aimed to explore the effects of foliar application of a leonardite-based product on sugar beet (Beta vulgaris L.) plants grown in the field. The approach concerned the evaluation of the community compositional structure of plant endophytic bacteria through a metabarcoding approach, the expression level of a gene panel related to hormonal metabolism and signaling, and the main sugar beet productivity traits. Results indicated that plants treated with leonardite (dosage of 2,000 ml ha(-1), dilution 1:125, 4 mg C l(-1)) compared with untreated ones had a significant increase (p < 0.05) in (i) the abundance of Oxalicibacterium spp., recognized to be an endophyte bacterial genus with plant growth-promoting activity; (ii) the expression level of LAX2 gene, coding for auxin transport proteins; and (iii) sugar yield. This study represents a step forward to advance our understanding of the changes induced by leonardite-based biostimulant in sugar beet.

Mineral phosphorus (P) fertilization in calcareous soils is not efficient enough to ensure optimal plant growth. Therefore, a higher P input is generally needed. Polymer-coated fertilizers are a promising fertilizer category that seems to affect soil extractable P, thus permitting a reduction in fertilizer rates. We tested this hypothesis in a short-term (45 days) field trial by evaluating both the agronomic and the environmental implications. In this study, two conventional fertilizers (single superphosphate, SSP; mono-ammonium phosphate, MAP) and a slow P-release fertilizer (polymer-coated MAP, PCMAP) were tested for their effects on soil P pools by combining different P rates and degrees of coating. The P soil test was determined with either Olsen or Mehlich-3 solution (available P), whereas the P soil release was estimated through water extraction. The efficiency of fertilizers was evaluated by assessing the growth of Hypericum x moserianum (L.) plants. As expected, both SSP and MAP influenced the soil Mehlich-3-P, Olsen-P, and water-P, as concentrations increased with the fertilizer rate. Conversely, PCMAP decreased the soil extractable P with increasing coating. The plant dry weight and P uptake linearly correlated with the fertilizer rate for SSP and MAP, whilst they achieved the maximum yield with PCMAP. This result indicates the underdosing for conventional fertilizers. With reference to the soil test P with water-P, the presence of change points showed low water-P release concentrations for PCMAP and SSP, and high water-P release for MAP. In conclusion, in the short-term period both soil extractable P and water-P depend on the type of fertilizer, whereas the amount of added P has rather a secondary role. PCMAP, in particular, ensures high plant P use efficiency with minimum environmental impacts.