An Integrated Multilevel Approach Unveils Complex Seed-Nanoparticle Interactions and Their Implications for Seed Priming.

Nanotechnology has the potential to revolutionize agriculture with the introduction of engineered nanomaterials. However, their use is hindered by high cost, marginal knowledge of their interactions with plants, and unpredictable effects related to massive use in crop cultivation. Nanopriming is an innovative seed priming technology able to match economic, agronomic, and environmental needs in agriculture. The present study was focused on unveiling, by a multilevel integrated approach, undisclosed aspects of seed priming mediated by iron oxide magnetic nanoparticles in pepper seeds (Capsicum annuum), one of the most economically important crops worldwide. Inductively coupled plasma atomic emission mass spectrometry and scanning electron microscopy were used to quantify the MNP uptake and assess seed surface changes. Magnetic resonance imaging mapped the distribution of MNPs prevalently in the seed coat. The application of MNPs significantly enhanced the root and vegetative growth of pepper plants, whereas seed priming with equivalent Fe concentrations supplied as FeCl3 did not yield these positive effects. Finally, global gene expression by RNA-sequencing identified more than 2,200 differentially expressed genes, most of them involved in plant developmental processes and defense mechanisms. Collectively, these data provide evidence on the link between structural seed changes and an extensive transcriptional reprogramming, which boosts the plant growth and primes the embryo to cope with environmental challenges that might occur during the subsequent developmental and growth stages.

Quantitative Evaluation of Noncovalent Interactions between 3,4-Dimethyl-1H-pyrazole and Dissolved Humic Substances by NMR Spectroscopy.

Nitrification inhibitors (NI) represent a valid chemical strategy to retard nitrogen oxidation in soil and limit nitrate leaching or nitrogen oxide emission. We hypothesized that humic substances can complex NI, thus affecting their activity, mobility, and persistence in soil. Therefore, we focused on 3,4-dimethylpyrazole phosphate (DMPP) by placing it in contact with increasing concentrations of model fulvic (FA) and humic (HA) acids. The complex formation was assessed through advanced and composite NMR techniques (chemical shift drift, line-broadening effect, relaxation times, saturation transfer difference (STD), and diffusion ordered spectroscopy (DOSY)). Our results showed that both humic substances interacted with DMPP, with HA exhibiting a significantly greater affinity than FA. STD emphasized the pivotal role of the aromatic signal, for HA-DMPP association, and both alkyl methyl groups, for FA-DMPP association. The fractions of complexed DMPP were determined on the basis of self-diffusion coefficients, which were then exploited to calculate both the humo-complex affinity constants and the free Gibbs energy (Kd and ΔG for HA were 0.5169 M and -1636 kJ mol-1, respectively). We concluded that DMPP-based NI efficiency may be altered by soil organic matter, characterized by a pronounced hydrophobic nature. This is relevant to improve nitrogen management and lower its environmental impact.

Advanced printable hydrogels from pre-crosslinked alginate as a new tool in semi solid extrusion 3D printing process.

With the aim to overcome alginate shape fidelity issue during the semisolid extrusion 3D printing and matrix collapsing after drying, we speculated that a pre-crosslinking step of the alginate ink-gel with low amount of Ca+2 could improve the hydrogel performance. To verify this, the influence of pre-crosslinker concentration (10-25 mM) on the ink gel rheological properties were studied and flow behaviour and viscoelastic properties were determined. The developed ink gels were fully characterised by DSC and Magnetic Resonance Imaging (MRI). Moreover, extrudability and the shape retention of extruded forms after printing and after drying were studied. The rheological and MRI data, combined with the morphological analysis of printed forms allowed us to identify the relationship between printability, shape retention and shear thinning behaviour of gels, showing good extrudability for all the pre-crosslinked gels with a calcium concentration between 0.15 and 0.25, corresponding to both egg-box dimers and multimers interactions.

Hydrochar obtained with by-products from the sugarcane industry: Molecular features and effects of extracts on maize seed germination.

Sugarcane bagasse, vinasse and a mixture of sugarcane bagasse and vinasse were hydrothermally carbonized (HTC), with and without the addition of phosphoric acid, in order to propose new applications of sucroenergetic industry by-products on soil. Detailed information on the composition and properties of hydrochars has been obtained through elemental composition, thermogravimetric analysis, nuclear magnetic resonance and, thermochemolysis GC-MS. The soluble acidic fraction from the hydrochar samples were applied to maize seeds to evaluate the agronomic potential as biostimulants and relate the molecular features with maize seed germination. The HTC treatment converted polysaccharide-based biomasses into hydrochars with hydrophobic characteristics (C-Aryl and C-Akyl). Furthermore, the addition of phosphoric acid further increased the overall hydrophobicity and shifted the thermal degradation of the hydrochars to higher temperatures. Biomass influenced the hydrochars that formed, in which the molecular features of sugarcane bagasse determined the formation of more polar hydrochar, due to the preservation of lignin and phenolic components. Meanwhile, the HTC of vinasse resulted in a more hydrophobic product with an enrichment of condensed and recalcitrant organic fractions. The germination assay showed that polar structures of bagasse may play a role in improving the maize seeds germination rate (increase of ~11%), while the hydrophobic domains showed negative effects. The responses obtained in germination seems to be related to the molecular characteristics that organic extracts can present in solution.

Bio-Based Hydrogels Composed of Humic Matter and Pectins of Different Degree of Methyl-Esterification.

We prepared humo-pectic hydrogels through ionotropic gelation by crosslinking natural pectins of different degree of methyl-esterification with either humic substances (HS) extracted from cow manure compost or humic-like substances (HULIS) from depolymerized lignocellulose biorefinery waste. The hydrogels were characterized by solid-state 13C-NMR spectroscopy, scanning electron microscopy, spectroscopic magnetic resonance imaging and rheological analyses. Their ability to work as controlled release systems was tested by following the release kinetics of a previously incorporated model phenolic compound, like phloroglucinol. Our results indicated that the release properties of hydrogels were influenced by the molecular composition of HS and HULIS and by the different degrees of methyl-esterification of pectins. The hydrogel made by the high methoxyl pectin and HS showed the fastest rate of phloroglucinol release, and this was attributed not only to its morphological structure and crosslinking density but also to the least formation of ionic interactions between phloroglucinol and the polysaccharidic chains. Our study suggests that the efficiency of novel humo-pectic hydrogels as sustainable carriers of agroproducts to crops is related to a careful choice of the characteristics of their components.

A community-built calibration system: The case study of quantification of metabolites in grape juice by qNMR spectroscopy.

Nuclear Magnetic Resonance (NMR) is an analytical technique extensively used in almost every chemical laboratory for structural identification. This technique provides statistically equivalent signals in spite of using spectrometer with different hardware features and is successfully used for the traceability and quantification of analytes in food samples. Nevertheless, to date only a few internationally agreed guidelines have been reported on the use of NMR for quantitative analysis. The main goal of the present study is to provide a methodological pipeline to assess the reproducibility of NMR data produced for a given matrix by spectrometers from different manufacturers, with different magnetic field strengths, age and hardware configurations. The results have been analyzed through a sequence of chemometric tests to generate a community-built calibration system which was used to verify the performance of the spectrometers and the reproducibility of the predicted sample concentrations.

The fate of cigarette butts in different environments: Decay rate, chemical changes and ecotoxicity revealed by a 5-years decomposition experiment.

Cigarette butts (CBs) are the most common litter item on Earth but no long-term studies evaluate their fate and ecological effects. Here, the role of nitrogen (N) availability and microbiome composition on CBs decomposition were investigated by a 5-years experiment carried out without soil, in park grassland and sand dune. During decomposition, CBs chemical changes was assessed by both 13C CPMAS NMR and LC-MS, physical structure by scanning electron microscope and ecotoxicity by Aliivibrio fischeri and Raphidocelis subcapitata. Microbiota was investigated by high-throughput sequencing of bacterial and eukaryotic rRNA gene markers. CBs followed a three-step decomposition process: at the early stage (∼30 days) CBs lost ∼15.2% of their mass. During the subsequent two years CBs decomposed very slowly, taking thereafter different trajectories depending on N availability and microbiome composition. Without soil CBs showed minor chemical and morphological changes. Over grassland soil a consistent N transfer occurs that, after de-acetylation, promote CBs transformation into an amorphous material rich in aliphatic compounds. In sand dune we found a rich fungal microbiota able to decompose CBs, even before the occurrence of de-acetylation. CBs ecotoxicity was highest immediately after smoking. However, for R. subcapitata toxicity remained high after two and five years of decomposition.

Molecular characterization of organic matter in two calcareous soils: the effects of an acid decarbonation treatment.

The molecular composition of soil organic matter (SOM) of two calcareous soils highly rich in carbonates was assessed before and after decarbonation by acid washing with HCl through 13C-CPMAS-NMR spectroscopy and off-line thermochemolysis coupled with gas chromatography and mass spectrometry (THM-GC-MS). The acidic treatment promoted a considerable concentration of organic matter in both soils, thus improving the identification of molecules otherwise not easily detectable. Decarbonation induced only a slight loss of soil organic carbon (SOC), corresponding to 1.4 and 2.7% for A and B soils respectively. The acidic treatment also led to an increase in the organic carbon/total nitrogen (OC/N) ratio in soil A, while an opposite variation was found for the second soil. Moreover, variations in the concentration and molecular distribution of specific compound classes present in SOM were caused by the acid washing of soils. As confirmed by both 13C-CPMAS-NMR and thermochemolysis results, the molecules most susceptible to the acid treatment were the carbohydrates, lignin monomers (G14 and G15), fatty acids (C18 saturated and unsaturated), fatty acids of microbial origin (C15, C17, and C19), hydroxy acids (C16, C18), and dioic acids (C18) which represent the components weakly bound to the organic matrix. Our findings not only showed the efficacy of the decarbonation treatment of calcareous soils with 3 N HCl, but also indicated how the acidic washing can improve the differentiation of soils on the basis of SOM molecular characteristics. Graphical abstract.

Chlamyphilone, a Novel Pochonia chlamydosporia Metabolite with Insecticidal Activity.

Metabolites from a collection of selected fungal isolates have been screened for insecticidal activity against the aphid Acyrthosiphon pisum. Crude organic extracts of culture filtrates from six fungal isolates (Paecilomyces lilacinus, Pochonia chlamydosporia, Penicillium griseofulvum, Beauveria bassiana, Metarhizium anisopliae and Talaromyces pinophilus) caused mortality of aphids within 72 h after treatment. In this work, bioassay-guided fractionation has been used to characterize the main bioactive metabolites accumulated in fungal extracts. Leucinostatins A, B and D represent the bioactive compounds produced by P. lilacinus. From P. griseofulvum and B. bassiana extracts, griseofulvin and beauvericin have been isolated, respectively; 3-O-Methylfunicone and a mixture of destruxins have been found in the active fractions of T. pinophilum and M. anisopliae, respectively. A novel azaphilone compound, we named chlamyphilone, with significant insecticidal activity, has been isolated from the culture filtrate of P. chlamydosporia. Its structure has been determined using extensive spectroscopic methods and chemical derivatization.

HRMAS-NMR metabolomics of Aglianicone grapes pulp to evaluate terroir and vintage effects, and, as assessed by the electromagnetic induction (EMI) technique, spatial variability of vineyard soils.

Aglianicone is an autochthonous variety of black berried wine grape cultivated exclusively in southern Italy, but with limited information on the molecular composition of its grapes. HRMAS NMR spectroscopy was used to identify the primary metabolome of Aglianicone grape pulp from two different vineyards and the observed metabolomic changes reflected the local terroir conditions or harvesting years. Moreover, the berries pulp metabolome was related to Apparent Electrical Conductivity (ECa) maps deriving from on-site near-surface geophysical measurements of vineyard soils obtained by an electromagnetic induction (EMI) technique. Most of primary metabolites of Aglianicone grape varied systematically as a function of the spatial variability of soil properties as well as vineyards terroir and harvesting years. Therefore, the proposed approach that couples HRMAS NMR spectroscopy with ECa maps of vineyard soils represents an innovative support for wine producers who can select the best vine/soil combination to ensure the maximum wine quality.

An alternative to mineral phosphorus fertilizers: The combined effects of Trichoderma harzianum and compost on Zea mays, as revealed by 1H NMR and GC-MS metabolomics.

The ability of Trichoderma harzianum (strain OMG-08) as plant growth promoting fungus (PGPF), was tested on Zea mays plants grown in soil pots added with different inorganic (triple superphosphate and rock phosphate) and organic (cow and horse manure composts) P fertilizers. The effect of treatments was evaluated by following the variations of plants dry biomass and nutrient content, as well as the metabolic changes in plant leaves by both GC-MS and NMR spectroscopy. A synergic effect was observed in treatments with both composts and fungus inoculation, in which not only plant growth and P uptake were enhanced, but also the expression of different metabolites related to an improved photosynthetic activity. Conversely, the combination of Trichoderma with inorganic fertilizers was less effective and even showed a reduction of plants shoot biomass and N content. The corresponding plant metabolome revealed metabolic compounds typical of biotic or abiotic stresses, which may be attributed to a reduced capacity of inorganic fertilizers to provide a sufficient P availability during plant growth. Our findings also indicate that the molecular composition of compost differentiated the Trichoderma activity in sustaining plant growth. The positive effects of the combined Trichoderma and compost treatment suggest that it may become an alternative to the phosphorus mineral fertilization.

Replacing calcium with ammonium counterion in lignosulfonates from paper mills affects their molecular properties and bioactivity.

Lignosulfonates are important by-products of the paper industry and may be transformed into different commodities. We studied the molecular properties of ammonium (LS-AM) and calcium Lignosulfonates (LS-C) and evaluated their bioactivity towards the early development of maize plantlets. The FT-IR, 13C NMR and 1H-13C-HSQC-NMR spectra showed that the two lignosulfonates varied in hydroxyl, sulfonate and phenolic content, while DOSY-NMR spectroscopy suggested a similar diffusivity. High Performance Size Exclusion Chromatography (HPSEC) was used to simulate the effects of root-exuded acids and describe the conformational dynamics of both LS substrates in acidic aqueous solutions. This technique showed that LS-C was stabilized by the divalent Ca2+ counterion, thus showing a greater conformational stability than LS-AM, whose components could not be as efficiently aggregated by the monovalent NH4+ counter-ion. The plant bioassays revealed that LS-AM enhanced the elongation of the root system, whereas LS-C significantly increased both total and shoot plant weights. We concluded that the lignosulfonate bioactivity on plant growth depended on the applied concentrations, their molecular properties and conformational stability.

Biochars from olive mill waste have contrasting effects on plants, fungi and phytoparasitic nematodes.

Olive mill waste (OMW), a byproduct from the extraction of olive oil, causes serious environmental problems for its disposal, and extensive efforts have been made to find cost-effective solutions for its management. Biochars produced from OMW were applied as soil amendment and found in many cases to successfully increase plant productivity and suppress diseases. This work aims to characterize biochars obtained by pyrolysis of OMW at 300 °C to 1000 °C using 13C NMR spectroscopy, LC-ESI-Q-TOF-MS and SEM (Scanning Electron Microscopy). Chemical characterization revealed that biochar composition varied according to the increase of pyrolysis temperature (PT). Thermal treated materials showed a progressive reduction of alkyl C fractions coupled to the enrichment in aromatic C products. In addition, numerous compounds present in the organic feedstock (fatty acids, phenolic compounds, triterpene acids) reduced (PT = 300 °C) or completely disappeared (PT ≥ 500 °C) in biochars as compared to untreated OMW. PT also affected surface morphology of biochars by increasing porosity and heterogeneity of pore size. The effects of biochars extracts on the growth of different organisms (two plants, one nematode and four fungal species) were also evaluated. When tested on different living organisms, biochars and OMW showed opposite effects. The root growth of Lepidium sativum and Brassica rapa, as well as the survival of the nematode Meloidogyne incognita, were inhibited by the untreated material or biochar produced at 300 °C, but toxicity decreased at higher PTs. Conversely, growth of Aspergillus, Fusarium, Rhizoctonia and Trichoderma fungi was stimulated by organic feedstock, while being inhibited by thermally treated biochars. Our findings showed a pattern of association between specific biochar chemical traits and its biological effects that, once mechanistically explained and tested in field conditions, may lead to effective applications in agriculture.

High-Resolution Magic-Angle-Spinning NMR and Magnetic Resonance Imaging Spectroscopies Distinguish Metabolome and Structural Properties of Maize Seeds from Plants Treated with Different Fertilizers and Arbuscular mycorrhizal fungi.

Both high-resolution magic-angle-spinning (HRMAS) and magnetic resonance imaging (MRI) NMR spectroscopies were applied here to identify the changes of metabolome, morphology, and structural properties induced in seeds (caryopses) of maize plants grown at field level under either mineral or compost fertilization in combination with the inoculation by arbuscular mycorrhizal fungi (AMF). The metabolome of intact caryopses was examined by HRMAS-NMR, while the morphological aspects, endosperm properties and seed water distribution were investigated by MRI. Principal component analysis (PCA) was applied to evaluate 1H CPMG (Carr-Purcel-Meiboom-Gill) HRMAS spectra as well as several MRI-derived parameters ( T1, T2, and self-diffusion coefficients) of intact maize caryopses. PCA score-plots from spectral results indicated that both seeds metabolome and structural properties depended on the specific field treatment undergone by maize plants. Our findings show that a combination of multivariate statistical analyses with advanced and nondestructive NMR techniques, such as HRMAS and MRI, enables the evaluation of the effects induced on maize caryopses by different fertilization and management practices at field level. The spectroscopic approach adopted here may become useful for the objective appraisal of the quality of seeds produced under a sustainable agriculture.

The mechanisms of humic substances self-assembly with biological molecules: The case study of the prion protein.

Humic substances (HS) are the largest constituent of soil organic matter and are considered as a key component of the terrestrial ecosystem. HS may facilitate the transport of organic and inorganic molecules, as well as the sorption interactions with environmentally relevant proteins such as prions. Prions enter the environment through shedding from live hosts, facilitating a sustained incidence of animal prion diseases such as Chronic Wasting Disease and scrapie in cervid and ovine populations, respectively. Changes in prion structure upon environmental exposure may be significant as they can affect prion infectivity and disease pathology. Despite its relevance, the mechanisms of prion interaction with HS are still not completely understood. The goal of this work is to advance a structural-level picture of the encapsulation of recombinant, non-infectious, prion protein (PrP) into different natural HS. We observed that PrP precipitation upon addition of HS is mainly driven by a mechanism of "salting-out" whereby PrP molecules are rapidly removed from the solution and aggregate in insoluble adducts with humic molecules. Importantly, this process does not alter the protein folding since insoluble PrP retains its α-helical content when in complex with HS. The observed ability of HS to promote PrP insolubilization without altering its secondary structure may have potential relevance in the context of "prion ecology". These results suggest that soil organic matter interacts with prions possibly without altering the protein structures. This may facilitate prions preservation from biotic and abiotic degradation leading to their accumulation in the environment.

A molecular zoom into soil Humeome by a direct sequential chemical fractionation of soil.

A Humeomics sequential chemical fractionation coupled to advanced analytical identification was applied directly to soil for the first time. Humeomics extracted ~235% more soil organic carbon (SOC) than by the total alkaline extraction traditionally employed to solubilise soil humic molecules (soil Humeome). Seven fractions of either hydro- or organo-soluble components and a final unextractable humic residue were separated from soil. These materials enabled an unprecedented structural identification of solubilised heterogeneous humic molecules by combining NMR, GC-MS, and ESI-Orbitrap-MS. Identified molecules and their relative abundance were used to build up structure-based van Krevelen plots to show the specific contribution of each fraction to SOC. The stepwise isolation of mostly hydrophobic and unsaturated molecules of progressive structural complexity suggests that humic suprastructures in soil are arranged in multi-molecular layers. These comprised molecules either hydrophobically adsorbed on soil aluminosilicate surfaces in less stable fractions, or covalently bound in amorphous organo-iron complexes in more recalcitrant fractions. Moreover, most lipid molecules of the soil Humeome appeared to derive from plant polyesters rather than bacterial metabolism. An advanced understanding of soil humic molecular composition by Humeomics may enable control of the bio-organic dynamics and reactivity in soil.

Isolation and Characterization of Gramineae and Fabaceae Soda Lignins.

Some agricultural residues such as wheat or barley straw, as well as certain fast-growing plants like Leucaena leucocephala and Chamaecytisus proliferus, could be used as raw materials for the paper industry as an alternative to traditional plants (eucalyptus, pine, etc.). In the present study, four types of lignin obtained from the spent liquors produced by the pulping processes using the abovementioned feedstocks were isolated and characterized. Lignin samples were acquired through an acid precipitation from these spent liquors. The characterization of the precipitated lignin samples were performed using a Fourier transform infrared spectroscopy (FT-IR) and both liquid- and solid-state nuclear magnetic resonance spectroscopy (NMR) to analyse the chemical structure, and thermogravimetric analysis (TGA) for determining the thermal properties. Additionally, chemical composition of lignin fractions was also measured. Even though they were of different botanical origin, all the studied samples except for wheat straw lignin had a similar chemical composition and thermal behaviour, and identical chemical structure. Wheat straw lignin showed a greater amount of Klason lignin and lower carbohydrate content. Furthermore, this lignin sample showed a higher thermal stability and significantly different cross-peak patterns in the 2D-NMR experiments. The molecular structures corresponding to p-coumarate (PCA), ferulate (FA) and cinnamyl aldehyde end-groups (J) were only detected in wheat isolated lignin.

The molecular properties of biochar carbon released in dilute acidic solution and its effects on maize seed germination.

It is not yet clear whether the carbon released from biochar in the soil solution stimulates biological activities. Soluble fractions (AQU) from wheat and maize biochars, whose molecular content was thoroughly characterized by FTIR, 13C and 1H NMR, and high-resolution ESI-IT-TOF-MS, were separated in dilute acidic solution to simulate soil rhizospheric conditions and their effects evaluated on maize seeds germination activity. Elongation of maize-seeds coleoptile was significantly promoted by maize biochar AQU, whereas it was inhibited by wheat biochar AQU. Both AQU fractions contained relatively small heterocyclic nitrogen compounds, whose structures were accounted by their spectroscopic properties. Point-of-Zero-Charge (PZC) values and van Krevelen plots of identified masses of soluble components suggested that the dissolved carbon from maize biochar behaved as humic-like supramolecular material capable to adhere to seedlings and deliver bioactive molecules. These findings contribute to understand the biostimulation potential of biochars from crop biomasses when applied in agricultural production.

Metabolomics by Proton High-Resolution Magic-Angle-Spinning Nuclear Magnetic Resonance of Tomato Plants Treated with Two Secondary Metabolites Isolated from Trichoderma.

Trichoderma fungi release 6-pentyl-2H-pyran-2-one (1) and harzianic acid (2) secondary metabolites to improve plant growth and health protection. We isolated metabolites 1 and 2 from Trichoderma strains, whose different concentrations were used to treat seeds of Solanum lycopersicum. The metabolic profile in the resulting 15 day old tomato leaves was studied by high-resolution magic-angle-spinning nuclear magnetic resonance (HRMAS NMR) spectroscopy directly on the whole samples without any preliminary extraction. Principal component analysis (PCA) of HRMAS NMR showed significantly enhanced acetylcholine and γ-aminobutyric acid (GABA) content accompanied by variable amount of amino acids in samples treated with both Trichoderma secondary metabolites. Seed germination rates, seedling fresh weight, and the metabolome of tomato leaves were also dependent upon doses of metabolites 1 and 2 treatments. HRMAS NMR spectroscopy was proven to represent a rapid and reliable technique for evaluating specific changes in the metabolome of plant leaves and calibrating the best concentration of bioactive compounds required to stimulate plant growth.

Cremenolide, a new antifungal, 10-member lactone from Trichoderma cremeum with plant growth promotion activity.

Trichoderma based products are considered an alternative to synthetic pesticides and fertilizers. These Trichoderma spp. are among the most studied and applied fungal BCAs in industry and agriculture and are known to secrete several secondary metabolites with different biological activities. The analysis of metabolic profiles (the 'metabolome') of Trichoderma species is complex because of the wide range of compounds produced and the molecular activities identified, including the recently determined role in the activation of plant resistance to biotic and abiotic stresses and growth promotion. A new 10-member lactone, but-2-enoic acid 7-acetoxy-6-hydroxy-2-methyl-10-oxo-5,6,7,8,9,10-hexahydro-2H-oxecin-5-yl ester, named cremenolide (1), has been isolated from culture filtrates of Trichoderma cremeum. The structure of cremenolide was determined by spectroscopic methods, including UV, MS, and 1D and 2D NMR analyses. In vitro tests showed that the purified compound inhibited the radial mycelium growth of Fusarium oxysporum, Botrytis cinerea and Rhizoctonia solani, and exerted a significant promotion of growth of tomato seedlings.