Quantification of species composition in grass-clover swards using RGB and multispectral UAV imagery and machine learning.

Introduction: Growing grass-legume mixtures for forage production improves both yield productivity and nutritional quality, while also benefiting the environment by promoting species biodiversity and enhancing soil fertility (through nitrogen fixation). Consequently, assessing legume proportions in grass-legume mixed swards is essential for breeding and cultivation. This study introduces an approach for automated classification and mapping of species in mixed grass-clover swards using object-based image analysis (OBIA). Methods: The OBIA procedure was established for both RGB and ten band multispectral (MS) images capturedby an unmanned aerial vehicle (UAV). The workflow integrated structural (canopy heights) and spectral variables (bands, vegetation indices) along with a machine learning algorithm (Random Forest) to perform image segmentation and classification. Spatial k-fold cross-validation was employed to assess accuracy. Results and discussion: Results demonstrated good performance, achieving an overall accuracy of approximately 70%, for both RGB and MS-based imagery, with grass and clover classes yielding similar F1 scores, exceeding 0.7 values. The effectiveness of the OBIA procedure and classification was examined by analyzing correlations between predicted clover fractions and dry matter yield (DMY) proportions. This quantification revealed a positive and strong relationship, with R2 values exceeding 0.8 for RGB and MS-based classification outcomes. This indicates the potential of estimating (relative) clover coverage, which could assist breeders but also farmers in a precision agriculture context.

The impact of antibiotic residues on resistance patterns in leek at harvest.

When crops are cultivated on fields fertilized with animal manure, the risk exists that plants may take up antibiotic residues and may be exposed to antibiotic resistance genes and antibiotic resistant bacteria. During cultivation in a greenhouse pot experiment, leek (Allium porrum) was fertilized with either pig slurry or mineral fertilizer and exposed to either no antibiotics, doxycycline (10,000 µg/kg manure), sulfadiazine (1000 µg/kg manure), or lincomycin (1000 µg/kg manure). At harvest, 4.5 months later, lincomycin, sulfadiazine or doxycycline were not detected in any of the leek samples nor in their corresponding soil samples. Further, antimicrobial susceptibility testing was performed on 181 Bacillus cereus group isolates and 52 Pseudomonas aeruginosa isolates from the grown leek. For the B. cereus group isolates, only a small shift in MIC50 for lincomycin was observed among isolates from the lincomycin and control treatment. For P. aeruginosa, only in the setup with doxycycline treatment a higher MIC50 for doxycycline was observed compared to the control, specifically the isolates selected from growth media supplemented with 8 mg/L doxycycline. Nine antibiotic resistance genes (tet(B), tet(L), tet(M), tet(O), tet(Q), tet(W), erm(B), erm(F) and sul2) were investigated at harvest in the leek and soil samples. In the leek samples, none of the antibiotic resistance genes were detected. In the soil samples fertilized with pig slurry, the genes erm(B), erm(F), tet(M), sul2, tet(W) and tet(O) were detected in significantly higher copy numbers in the lincomycin treatment as compared to the other antibiotic treatments. This could be due to a shift in soil microbiota induced by the addition of lincomycin. The results of this study indicate that consumption of leek carries a low risk of exposure to antibiotic residues or antibiotic resistance to doxycycline, sulfadiazine or lincomycin.

Response of a Diverse European Soybean Collection to "Short Duration" and "Long Duration" Drought Stress.

Drought causes significant damage to a high value crop of soybean. Europe has an increasing demand for soybean and its own production is insufficient. Selection and breeding of cultivars adapted to European growth conditions is therefore urgently needed. These new cultivars must have a shorter growing cycle (specifically for adaptation to North-West Europe), high yield potential under European growing conditions, and sufficient drought resistance. We have evaluated the performance of a diverse collection of 359 soybean accessions under drought stress using rain-out shelters for 2 years. The contrasting weather conditions between years and correspondingly the varying plant responses demonstrated that the consequences of drought for an individual accession can vary strongly depending on the characteristics (e.g., duration and intensity) of the drought period. Short duration drought stress, for a period of four to 7 weeks, caused an average reduction of 11% in maximum canopy height (CH), a reduction of 17% in seed number per plant (SN) and a reduction of 16% in seed weight per plant (SW). Long duration drought stress caused an average reduction of 29% in CH, a reduction of 38% in SN and a reduction of 43% in SW. Drought accelerated plant development and caused an earlier cessation of flowering and pod formation. This seemed to help some accessions to better protect the seed yield, under short duration drought stress. Drought resistance for yield-related traits was associated with the maintenance of growth under long duration drought stress. The collection displayed a broad range of variation for canopy wilting and leaf senescence but a very narrow range of variation for crop water stress index (CWSI; derived from canopy temperature data). To the best of our knowledge this is the first study reporting a detailed investigation of the response to drought within a diverse soybean collection relevant for breeding in Europe.

Impact of fertilization with pig or calf slurry on antibiotic residues and resistance genes in the soil.

Antibiotic residues and antibiotic resistance genes can enter the environment via fertilization with calf and pig manure. In a longitudinal study, nine antibiotic resistance genes (tet(B), tet(L), tet(M), tet(O), tet(Q), tet(W), erm(B), erm(F) and sul2) and 56 antibiotic residues were investigated in 288 soil samples and 8 corresponding slurry samples from 6 pig farms and 2 veal farms using qPCR and LC-MS/MS, respectively. A significant increase in gene copy number of tet(M), erm(B), erm(F) and sul2 was observed in all the soil layers between sampling times prior to (T1) and 2-3 weeks after fertilization (T3). Tet(B), tet(Q) and tet(L) were least abundant in the soil among the genes tested. From 7 classes of antibiotics, 20 residues were detected in soil and slurry using an optimized and validated extraction method. Flumequine was detected in all soil samples in concentrations below 100 µg/kg despite being detected in only half of the corresponding slurry samples. Doxycycline, oxytetracycline, lincomycin and sulfadiazine were also frequently detected in concentrations ranging from 0.1 µg/kg to 500 µg/kg and from 2 µg/kg and 9480 µg/kg in soil and slurry, respectively. Furthermore a positive association between the presence of antibiotic residues (total antibiotic load) and antibiotic resistance genes in soil was found. One possible explanation for this is a simultaneous introduction of antibiotic residues and resistance genes upon application of animal slurry.

Physicochemical Characterization of Thirteen Quinoa (Chenopodium quinoa Willd.) Varieties Grown in North-West Europe-Part II.

Quinoa cultivation has gained increasing interest in Europe but more research on the characteristics of European varieties is required to help determine their end use applications. A comparative study was performed on 13 quinoa varieties cultivated under North-West European field conditions during three consecutive growing seasons (2017-2019). The seeds were milled to wholemeal flour (WMF) to evaluate the physicochemical properties. The WMFs of 2019 were characterized by the highest water absorption capacity (1.46-2.06 g/g), while the water absorption index (WAI) between 55 °C (2.04-3.80 g/g) and 85 °C (4.04-7.82 g/g) increased over the years. The WMFs of 2018 had the highest WAI at 95 °C (6.48-9.48 g/g). The pasting profiles were characterized by a high viscosity peak (1696-2560 mPa.s) and strong breakdown (-78-643 mPa.s) in 2017. The peak viscosity decreased in 2018 and 2019 (823-2492 mPa.s), while breakdown (-364-555 mPa.s) and setback (19-1037 mPa.s) increased. Jessie, Summer Red, Rouge Marie, Vikinga, and Zwarte WMFs were characterized by low WAIs and high shear resistance. Bastille WMF developed high viscosities and, along with Faro WMF, showed a high breakdown. The wide variation in physicochemical properties suggests that the potential food applications of WMFs depend on the variety and growing conditions.

Yield and Nutritional Characterization of Thirteen Quinoa (Chenopodium quinoa Willd.) Varieties Grown in North-West Europe-Part I.

The cultivation of quinoa has gained increasing interest in Europe. Different European varieties exist, but more research is required to understand the individual variety characteristics for end-use applications. The objective of this study is to evaluate the agronomic performance of 13 quinoa varieties under North-West European field conditions during three growing seasons (2017-2019). Furthermore, seeds were qualitatively characterized based on characteristics and composition. Yield differed among varieties and growing seasons (0.47-3.42 ton/ha), with lower yields obtained for late-maturing varieties. The saponin content varied from sweet to very bitter. The seeds contained high protein levels (12.1-18.8 g/100 g dry matter), whereas varieties had a similar essential amino acid profile. The main fatty acids were linoleic (53.0-59.8%), α-linolenic (4.7-8.2%), and oleic acid (15.5-22.7%), indicating a high degree of unsaturation. The clustering of varieties/years revealed subtle differences between growing seasons but also reflected the significant interaction effects of variety and year. Most varieties perform well under North-West European conditions, and their nutritional content is well within the values previously described for other cultivation areas. However, optimal yield and quality traits were not combined in one variety, illustrating the importance of breeding for adapted quinoa varieties.

Segregation of rol Genes in Two Generations of Sinningia speciosa Engineered Through Wild Type Rhizobium rhizogenes.

Rhizobium rhizogenes infects and transforms a wide range of plant species. It thereby introduces new genes located on transfer-DNA of the root inducing plasmid (pRi) into the plant genome and one of its abilities is to alter the host root system. Explants from pRi transformed roots from Sinningia speciosa were regenerated to create naturally transgenic Ri lines. The presence of rol and aux genes in the Ri lines was linked with altered growth characteristics: shorter peduncles, wrinkled leaves, delayed flowering and enhanced root growth. The potential of Ri lines for breeding was evaluated through consecutive backcrossing with the original host genotype. The progeny of reciprocal crosses showed non-Mendelian inheritance suggesting partial transmission of the of the aux and rol genes. The typical Ri phenotype observed in the primary Ri line was partially inherited. These results revealed that the Ri phenotype is a complex trait influenced by the genetic background of the Ri line.

Temporal changes in genetic diversity and forage yield of perennial ryegrass in monoculture and in combination with red clover in swards.

Agricultural grasslands are often cultivated as mixtures of grasses and legumes, and an extensive body of literature is available regarding interspecific interactions, and how these relate to yield and agronomic performance. However, knowledge of the impact of intraspecific diversity on grassland functioning is scarce. We investigated these effects during a 4-year field trial established with perennial ryegrass (Lolium perenne) and red clover (Trifolium pratense). We simulated different levels of intraspecific functional diversity by sowing single cultivars or by combining cultivars with contrasting growth habits, in monospecific or bispecific settings (i.e. perennial ryegrass whether or not in combination with red clover). Replicate field plots were established for seven seed compositions. We determined yield parameters and monitored differences in genetic diversity in the ryegrass component among seed compositions, and temporal changes in the genetic composition and genetic diversity at the within plot level. The composition of cultivars of both species affected the yield and species abundance. In general, the presence of clover had a positive effect on the yield. The cultivar composition of the ryegrass component had a significant effect on the yield, both in monoculture, and in combination with clover. For the genetic analyses, we validated empirically that genotyping-by-sequencing of pooled samples (pool-GBS) is a suitable method for accurate measurement of population allele frequencies, and obtained a dataset of 22,324 SNPs with complete data. We present a method to investigate the temporal dynamics of cultivars in seed mixtures grown under field conditions, and show how cultivar abundances vary during subsequent years. We screened the SNP panel for outlier loci, putatively under selection during the cultivation period, but none were detected.

A zone-specific fish-based biotic index as a management tool for the Zeeschelde estuary (Belgium).

Fish-based indices monitor changes in surface waters and are a valuable aid in communication by summarising complex information about the environment (Harrison and Whitfield, 2004). A zone-specific fish-based multimetric estuarine index of biotic integrity (Z-EBI) was developed based on a 13 year time series of fish surveys from the Zeeschelde estuary (Belgium). Sites were pre-classified using indicators of anthropogenic impact. Metrics showing a monotone response with pressure classes were selected for further analysis. Thresholds for the good ecological potential (GEP) were defined from references. A modified trisection was applied for the other thresholds. The Z-EBI is defined by the average of the metric scores calculated over a one year period and translated into an ecological quality ratio (EQR). The indices integrate structural and functional qualities of the estuarine fish communities. The Z-EBI performances were successfully validated for habitat degradation in the various habitat zones.

Effects of willow stands on heavy metal concentrations and top soil properties of infrastructure spoil landfills and dredged sediment-derived sites.

The effects of willow stand development on top soil properties of uncontaminated infrastructure spoil landfills (ISL) and contaminated dredged sediment landfills (DSL) were assessed. For the ISL, significant increases in Cd, Zn and organic C levels in the top soil (0-10 cm) were detected more than 20 years after disposal. The increases in Cd and Zn concentrations in the top soil were attributed to leaf-associated metal transfer and leaf fall: the relatively high Cd and Zn concentrations in willow leaves resulted in top soil enrichment for these elements. Higher absolute amounts of Cd, Zn and Mn were taken up and recycled during leaf fall on DSL than on ISL, but did not result in significant differences between top soil and deeper soil (10-30 cm) for the DSL. Direct comparison of top soil development between both types of sites is not possible due to differences in stand age and time since disposal. The DSL were characterised by a higher short-range variance for the Cd, Cr, Cu, Pb and Zn concentrations in the top soil than the ISL. During the first years of ripening and dewatering, significant sulphate leaching occurred in the top soil of the DSL.

Uptake of Cd, Zn and Mn by willow increases during terrestrialisation of initially ponded polluted sediments.

Metal concentration of plants growing on contaminated soils among other factors may depend on changes in the hydrological regime of the soil. Foliar and stem metal concentrations in Salix cinerea (grey sallow) were measured in 2 consecutive growing seasons on a submerged sediment-derived soil that underwent gradual terrestrialisation. Foliar and stem cutting concentrations for Cd, Zn and Mn increased on plots that were submerged during the first year, but emerged in the second year of monitoring. The litter layer was sampled under the shrubs of a plot with a recent abrupt change in hydrological regime and on the reference plot. It was separated in three size fractions through sieving. Analysis of the litter fractions suggested that Cd and Zn concentrations remained constant during fragmentation. However, Cr, Cu, Ni and Pb concentrations increased, which was attributed to adhesion of mineral soil particles on the fine fraction. After correction for the metal content in the mineral fraction, an increase in Cd, Mn and Cu concentration during fragmentation of the organic part of the litter layer was observed for the polluted plot. Net litter layer decomposition rate was low, which may indicate low colonisation by the decomposing community. Terrestrialisation resulted in higher Cd, Mn and Zn uptake by willows. The deviant litter layer metal concentrations for Cd, Zn and Mn and low decomposition rate must be further monitored. Feasibility of measures aiming at re-establishing wetland conditions for the dredged sediment landfill must be considered.

Differences in Cd and Zn bioaccumulation for the flood-tolerant Salix cinerea rooting in seasonally flooded contaminated sediments.

Several authors suggest that a hydrological regime aiming at wetland creation is a potential management option that favours reducing bioavailability for metal-contaminated sites. The hydrological conditions on a site constitute one of the many factors that may affect the availability of potentially toxic trace metals for uptake by plants. Bioavailability of Cd, Mn and Zn on a contaminated dredged sediment landfill (DSL) with variable duration of submersion was evaluated by measuring metal concentrations in the wetland plant species Salix cinerea in field conditions. Longer submersion periods in the field caused lower Cd and Zn concentrations in the leaves in the first weeks of the growing season. Foliar Cd and Zn concentrations at the end of the growing season were highest on the initially flooded plot that emerged early in the growing season. Foliar Zn concentrations were also high at a sandy-textured oxic plot with low soil metal concentrations. Zn uptake in the leaves was markedly slower than Cd uptake for trees growing on soils with prolonged waterlogging during the growing season, pointing at a different availability. Zn availability was lowest when soil was submerged, but metal transfer from stems and twigs to leaves may mask the lower availability of Cd in submerged soils. Especially for Cd, a transfer effect from one growing season to the next season was observed: oxic conditions at the end of the previous growing season seem to determine at least partly the foliar concentrations for S. cinerea through this metal transfer mechanism. Duration of the submersion period is a key factor for bioavailability inasmuch as initially submerged soils emerging only in the second half of the growing season resulted in elevated Cd and Zn foliar concentrations at that time.

The effect of hydrological regime on the metal bioavailability for the wetland plant species Salix cinerea.

The hydrological conditions on a site constitute one of the many factors that may affect the availability of potentially toxic trace metals for uptake by plants. Bioavailability of Cd, Mn and Zn in a contaminated dredged sediment-derived soil under different hydrological regimes was determined by measuring metal uptake by the wetland plant species Salix cinerea, both in field circumstances and in a greenhouse experiment. Longer submersion periods in the field caused lower Cd concentrations in leaves and bark. The wetland hydrological regime in the greenhouse experiment resulted in normal Cd and Zn concentrations in the leaves, while the upland hydrological regime resulted in elevated Cd and Zn concentrations in the leaves. Field observations and the greenhouse experiment suggest that a hydrological regime that creates or sustains a wetland is a potential management option that reduces metal bioavailability to willows. This would constitute a safe management option of metal-polluted, willow-dominated wetlands provided that wetland conditions can be maintained throughout the full growing season.

Growth and trace metal accumulation of two Salix clones on sediment-derived soils with increasing contamination levels.

The growth and metal uptake of two willow clones (Salix fragilis 'Belgisch Rood' and Salix viminalis 'Aage') was evaluated in a greenhouse pot experiment with six sediment-derived soils with increasing field Cd levels (0.9-41.4 mg kg-1). Metal concentrations of eight elements were measured in roots, stems and leaves and correlated to total and soil water metal concentrations. Dry weight root biomass, number of leaves and shoot length were measured to identify eventual negative responses of the trees. No growth inhibition was observed for both clones for any of the treatments (max. 41.4 mg kg-1 Cd, 1914 mg kg-1 Cr, 2422 mg kg-1 Zn, 655 mg kg-1 Pb), allowing their use for phytoextraction on a broad range of contaminated sediments. However, dry weight root biomass and total shoot length were significantly lower for S. viminalis compared to S. fragilis for all treatments. Willow foliar Cd concentrations were strongly correlated with soil and soil water Cd concentrations. Both clones exhibited high accumulation levels of Cd and Zn in aboveground plant parts, making them suitable subjects for phytoextraction research. Cu, Cr, Pb, Fe, Mn and Ni were found mainly in the roots. Bioconcentration factors of Cd and Zn in the leaves were highest for the treatments with the lowest soil Cd and Zn concentration.

Assessment of the pollution status of alluvial plains: a case study for the dredged sediment-derived soils along the Leie River.

The concept of an integrated multifunctional river management is gaining importance. For major rivers, restoring the contact between rivers and alluvial plains is an important goal, as riparian areas have a specific role for several riverine processes. However, former and current human activities are an obstruction or a limitation for river restoration. We studied the influence of former dredging activities along the river Leie on the alluvial plain quality. A soil survey and an archive query for reconstructing the history of dredging operations were conducted simultaneously. The geographical impact expressed as topographical changes and covering of the original soil profile and related processes and biota was large. The pollution status of dredged sediment-derived soils was found to be far from negligible: concentrations of Cd, Cr, and Zn were, in 10% of the cases, higher than 20, 480, and 2800 mg kg(-1) DM, respectively. Both agriculture and nature rehabilitation on dredged sediment-derived soils can only be accepted after profound risk assessment, and management should focus on ecological risk reduction. Results indicate the importance of soil quality assessment in alluvial plains for an integrated river management, rather than a priori assuming pristine soil conditions. The collected "off-line" sediment data can be used as a reconstruction of past sediment pollution, especially when long-term sediment monitoring programs are not available.

Foliar concentrations of volunteer willows growing on polluted sediment-derived sites versus sites with baseline contamination levels.

Many alluvial soils along navigable waterways are affected by disposal of dredged sediments or overbank sedimentation and contain metal concentrations that are elevated compared to baseline levels. Uptake patterns for metals and other elements by several volunteer Salix species growing on these sites were determined during a growing season in field plots and compared with the same species growing on soils with baseline contamination levels. For Cd and Zn, foliar concentrations were clearly higher on dredged sediment landfills. Uptake patterns differed significantly between species. A high uptake of Mn and low uptake of Cu, K and S in S. cinerea was attributed to wetland soil chemistry. Site effects on metal uptake were evaluated in more detail for Salix cinerea and S. alba growing on different sediment-derived sites under field conditions. Foliar Cd concentrations were higher in S. cinerea than in S. alba. This appeared to be a genetic feature not influenced by soil chemical properties, as it was observed both on clean sites and polluted sediment-derived sites. For S. cinerea, soil chemistry was reflected in foliar concentrations, while foliar Cd concentrations and bioavailability were found to be independent of the thickness of the polluted horizon. Dredged sediment landfills and freshwater tidal marshes with comparable Cd soil pollution had significantly different foliar Cd concentrations.

Earthworm biomass as additional information for risk assessment of heavy metal biomagnification: a case study for dredged sediment-derived soils and polluted floodplain soils.

The important role of earthworms in the biomagnification of heavy metals in terrestrial ecosystems is widely recognised. Differences in earthworm biomass between sites is mostly not accounted for in ecological risk assessment. These differences may be large depending on soil properties and pollution status. A survey of earthworm biomass and colonisation rate was carried out on dredged sediment-derived soils (DSDS). Results were compared with observations for the surrounding alluvial plains. Mainly grain size distribution and time since disposal determined earthworm biomass on DSDS, while soil pollution status of the DSDS was of lesser importance. Highest earthworm biomass was observed on sandy loam DSDS disposed at least 40 years ago.