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Humic substances (HS) are powerful natural plant biostimulants. However, there is still a lack of knowledge about the relationship between their structure and bioactivity in plants. We extracted HS (THE1-2) from two forest soils covered with Pinus mugo (1) or Pinus sylvestris (2). The extracts were subjected to weak acid treatment to produce size-fractionated HS (high molecular size, HMS1-2; low molecular size, LMS1-2). HS were characterized for total acidity, functional groups, element and auxin (IAA) contents, and hormone-like activity. HS concentrations ranging from 0 to 5 mg C L−1 were applied to garlic (Allium sativum L.) plantlets in hydroponics to ascertain differences between unfractionated and size-fractionated HS in the capacity to promote mineral nutrition, root growth and cell differentiation, activity of enzymes related to plant development (invertase, peroxidase, and esterase), and N (nitrate reductase, glutamine synthetase) and S (O-acetylserine sulphydrylase) assimilation into amino acids. A positive linear dose-response relationship was determined for all HS in the range 0–1 mg C L−1, while higher HS doses were less effective or ineffective in promoting physiological-biochemical attributes of garlic. Bioactivity was higher for size-fractionated HS according to the trend LMS1-2>HMS1-2>THE1-2, with LMS2 and HMS2 being overall more bioactive than LMS1 and HMS1, respectively. LMS1-2 contained more N, oxygenated functional groups and IAA compared to THE1-2 and HMS1-2. Also, they exhibited higher hormone-like activities. Such chemical properties likely accounted for the greater biostimulant action of LMS1-2. Beside plant growth, nutrition and N metabolism, HS stimulated S assimilation by promoting the enrichment of garlic plantlets with the S amino acid alliin, which has recognized beneficial properties in human health. Concluding, this study endorses that i) treating THE with a weak acid produced sized-fractionated HS with higher bioactivity and differing in properties, perhaps because of novel molecular arrangements of HS components that better interacted with garlic roots; ii) LMS from forest soils covered with P. mugo or P. sylvestris were the most bioactive; iii) the cover vegetation affected HS bioactivity iv); HS stimulated N and S metabolism with relevant benefits to crop nutritional quality. [ABSTRACT FROM AUTHOR]

Background and aim: Humic substances (HS) are known to influence plant physiological processes, enhancing crop yield, plant growth and nutrient uptake. The present study sought to gain a better understanding of the specific effects of HS application on the abundance of metabolites in plant tissues, using mass spectrometry analyses.Methods: Arabidopsis thaliana plants, grown in hydroponic conditions, were treated for 8 h with indole-3-acetic acid (IAA), HS from International Humic Substances Society (IHSS) and HS from earthworm faeces (EF), respectively. Humic substances structural characteristics were analyzed by 1H NMR an FT-IR spectroscopies. Root and leaf free amino acids, sugar alcohols and carbohydrate contents, and leaf amino acids from protein hydrolysis were identified and quantified by gas chromatography-mass spectrometry (GC/MS), and liquid chromatography-mass spectrometry (LC/MS). Canonical discriminant analysis (CDA) was used to evaluate the influence of the treatments on the studied parameters.Results: EF treatment had the highest influence on metabolite profiles compared to the control, IAA and IHSS. CDA analysis highlighted a clear distinction between EF and IHSS plant physiological responses, depending on the different chemical and structural properties of the HS. IAA-treated plants showed no significant difference from the control.Conclusions: A better understanding of the specific effects of different HS, also related to their chemical characteristics, might serve as a basis for the identification of marker compounds for HS bioactivity. [ABSTRACT FROM AUTHOR]

Background and aims: Biostimulants are natural compounds that enhance plant growth and plant nutrient use efficiency. In this study, biostimulant effects of humic substances (HS) extracted from leonardites were analysed on the metabolism of maize plants grown in hydroponic conditions. Methods: HS extracted from four leonardites were tested for their auxin-like and gibberellin-like activities. Then, 11 day old maize seedlings were treated for 48 h with five concentrations (0, 0.1, 0.5, 1, and 10 mg C L) of HS. After sampling, root growth and morphology, glutamine synthetase (GS) activity, glutamate synthase (GOGAT) activity, total protein content, soluble sugars content, phenylalanine ammonia-lyase (PAL) activity, soluble phenols, and free phenolic acids were analysed. Results: HS from leonardites had similar spectroscopic pattern, with small differences. The HS from the South Dakota lignite (HS_USA) had more carboxylic groups, whereas the three from Turkish mines had more aromatic and aliphatic structures. HS_USA best enhanced total root growth, root surface area, and proliferation of secondary roots. Plant nutrient use efficiency was enhanced by HS_4, HS_USA and HS_B, with increment of GS and GOGAT enzymes activity and total protein production. HS stimulated also PAL enzyme activity, followed by a higher production of total soluble phenols, p-hydroxybenzoic acid, p-coumarilic acid, and chlorogenic acid. Conclusion: This study found that, although the activity of the HS depended on the origin of the leonardite, these compounds can be attributed to the biostimulant products, eliciting plant growth, nitrogen metabolism, and accumulation of phenolic substances. [ABSTRACT FROM AUTHOR]

Soil carbon sequestration is strongly affected by soil properties, climate, and anthropogenic activities. Assessing these drivers is key to understanding the effect of land use on soil organic matter stabilization. We evaluated land use and soil depth influencing patterns of soil organic matter stabilization in three types of soil profiles located under the same pedogenetic matrix and alpine conditions but with different vegetation cover. The stock in soil organic carbon in the mean 0-20 cm layer increased from prairie (31.9 t ha-1) to prairie in natural reforestation (42 t ha-1) to forest (120 t ha-1), corresponding to increments of 1.3-fold prairie, for prairie in natural reforestation, and of 3.8-fold prairie for forest. The forest showed the highest humic carbon (21.7 g kg-1), which was 2.8 times greater than the prairie in natural reforestation and 4 times higher than the prairie. 13C-NMR spectroscopic measurements suggested a different C pattern. The prairie in natural reforestation and the prairie were characterized by a higher content in O,N-alkyl C with respect to the forest. Alkyl C and aromatic C in the prairie in natural reforestation and prairie did not show relevant differences while they decreased with respect to the forest. Carboxyl and phenolic C groups were markedly higher in forest and prairie than prairie in natural reforestation. Alkyl C, carboxyl C, and phenolic C prevailed in the Ah horizons whereas aromatic C and O,N-alkyl C were dominant in the B horizons. Overall, the marked distribution of O,N-alkyl C and alkyl C in humic substances (HS) indicates a low degree of humification. Nevertheless, in forest, the relatively high presence of aromatic C designated HS endowed with a relatively high humification degree. Thus, our results might suggest that in the alpine environment of NE Italy differences in soil organic matter (SOM) stocks and characteristics are affected by land use and anthropic activities. [ABSTRACT FROM AUTHOR]

The biological activity of humic substances (HS) has been elucidated in the last 40 years. Growth enhancement from HS has been demonstrated in several plants in the laboratory and the field. Morphogenesis effects have also been investigated and include induction of lateral root formation and root hair initiation in intact plants and stimulation of root and shoot development in treated cell calluses. HS enhance nutrient use efficiency, aiding assimilation of both macro and microelements and promoting plant growth by the induction of carbon, nitrogen, and secondary metabolism. The review aims are to: (1) shed light on the mechanism by which plants 'talk' with soil through humic substances, (2) elucidate the plant responses to the stimulatory effects of HS, the regulatory circuits that allow plants to cope with humus, and the feedback between plant community structures, and (3) show (in light of recent debate about the alkaline extraction of soil humic substances) the plant capability to acquire biologically active substances from soil. It will be shown that plants modify soils, creating and maintaining favorable habitats for growth and survival. Therefore, organic substances exuded by roots are not a wasteful loss of carbon and energy. They represent an evolved strategy by which plants 'talk' to the soil. The mobilization of bioactive organic/humic substances from bulk soil or bulk humus is critical to plant and soil health. [ABSTRACT FROM AUTHOR]

In alpine environments, colonies of snow vole ( Chionomys nivalis Martins) cause strong pedoturbation, which may affect humification process and soil organic matter ( SOM) cycling, with repercussions on the hormone-like activity of organics. We investigated the effect of snow vole pedoturbation on the chemical and spectroscopic features of soil organic fractions, and the potential hormone-like activity of humic and fulvic acids ( HA, FA). The study site was located on the high-mountain environment of the Majella massif (central Italy). Pedoturbated and regular soils were morphologically described and characterized for pH and content of total organic carbon, total extractable carbon, HA, and FA. Both HA and FA were extracted and investigated using attenuated total reflectance/Fourier transform infrared ( ATR/ FTIR), nuclear magnetic resonance with high-resolution magic angle spinning ( HRMAS- NMR), and 1H-13C heteronuclear single quantum coherence ( HSQC). HA and FA were also tested for their auxin-like and gibberellin-like activities. Results provide evidences that bioturbated and regular soils contain a poorly decomposed SOM, but HA and FA with a well-defined molecular structure. The HA and FA from both bioturbated and regular soils show a hormone-like activity with a different allocation along the soil profile. In the regular soil, the highest auxin-like activity was shown by HA and FA from Oe1 horizon, while gibberellin-like activity was expressed by FA from Oe2 horizon. Burrowing activity determines a redistribution of organics throughout the profile with a relatively high auxin-like activity in the FA from straw tunnel wall ( STW) and gibberellin-like activity in the HA from vole feces ( VF). The relative high presence of carboxylic acids, amides, proteins, and amino acids in the FA from STW and the aromatic moieties in the HA from VF put evidences for their different behavior. The fact that snow vole activity has modified the chemical and biological properties of SOM in these soils otherwise considered governed only by low temperature has important ecological implications such as the preservation of soil fertility and vegetal biodiversity. [ABSTRACT FROM AUTHOR]

Abstract: Organic matter is the soil component most sensitive to fire, being the fuel on the ground and below. It is affected by fire in terms of both abundance and composition. Incomplete combustion yields condensed carbon forms characterized by higher recalcitrance to biological and chemical attack compared to the parent material. In this study, thermogravimetry (TG-DTG), differential scanning calorimetry (DSC), attenuated total reflectance/Fourier transform infrared spectroscopy (ATR/FT-IR), and Raman spectroscopy were applied to (1) charcoal particles accumulated on the ground, (2) the top 10cm of soil, and (3) the organic matter extracted using an alkaline solution, to investigate the wildfire effect on the organic pool of the loamy and sub-acid soil of a maritime pine (Pinus pinaster Ait.) forest. The same investigations were performed on the litter layer and the mineral soil of an adjacent unburnt area, similar to the burnt one before fire occurrence. TG-DTG and DSC measurements highlighted an accumulation of soil organic matter (SOM) in the burnt soil, which suggests an incorporation of partly charred necromass into soil and the formation of pyrogenic highly refractory organic matter, i.e. black carbon. The ATR/FT-IR showed changes in the fire affected extractable SOM compatible with the formation of stable N containing compounds, which was well confirmed also by the presence of CN group vibration in the Raman spectrum. The combined application of thermal and spectroscopic techniques resulted to be highly useful to rapidly estimate many of the effects of fire on soil organic matter. [Copyright &y& Elsevier]

Abstract: Increasing attention has been devoted in the last few years to the mechanisms by which humic substances (HS) influence plant growth. Nevertheless, amongst the biologically active compounds that might be present in HS, only indoleacetic acid has been identified. An enzyme-linked immunosorbent assay (ELISA) was applied to detect the presence of isopentenyladenosine (IPA), a cytokinin, in two lignosulphonate-humates, leonardite humic acid and in a high molecular weight humic fraction extracted from earthworm faeces. The IPA concentration ranged from 34 to 145pmolmg−1 humic C and was estimated by using a linear dose–response curve between the carbon (C) concentration and percentage of binding of an anti-IPA monoclonal antibody. The cytokinin-like activity of HS was evaluated by weighing small radish (Raphanus sativus L.) cotyledon leaves and measuring chlorophyll and protein contents, the activities of ATP sulfurylase and O-acetylserine sulfhydrylase on maize plants. It is noteworthy that, amongst the four HS, those from earthworm faeces showed the highest amount of IPA and the strongest cytokinin-like activity in both plants. The presence of IPA confirmed that HS influence plant metabolism at different levels. Our results open new perspectives in the plant–HS relationship and shed new light on the active components of HS. [Copyright &y& Elsevier]

Abstract: Charcoal is mainly composed by aromatic C but is characterized by several degrees of aromaticity, which complicate its identification and quantification in natural environments by conventional analyses. Charcoal is an almost ubiquitous component of soil, although often occurring in minor amounts. Hence, there is a great interest in studying the charcoal structure and understanding the behavior of charcoal in soils. In soil, it has been demonstrated that the chemical structure of charcoal is also dependant on its particle-size. In this paper we have applied infrared, normal Raman, SERS and SEF spectroscopies to study four size-fractions of charcoal (>2mm, 2–1mm, 1–0.5mm and <0.5mm) and their alkaline extracts from a burnt Pinus pinea forest. Second derivative FT-IR in agreement with the normal Raman spectroscopy have given information about the size of aromatic rings and the nature of substituted groups in size-fractions of charcoal. Carbon cluster size progressively decreased in the smallest fractions suggesting that higher graphitization and crystallization occurred in the coarsest fraction (>2mm). The presence of organic N was well evidenced by the vibration of iso-cyanate and cyanamide groups in the fractions. This result is of great environmental importance because N may be immobilized in form of stable molecules like N-heterocyclic poliaromatic structures, and so persists in soils. The soluble organic C (SOC) extracted from charcoal size-fractions did not differ from the bulk charcoal in terms of IR and Raman spectra. Instead SERS and SEF provided further information regarding the ratio between the aromatic component and the aliphatic cross-linking one in studied fractions. [Copyright &y& Elsevier]

To understand if the biological activity of humic substances may be related to their molecular weight or chemical structure, two humic substances, derived from an Ah horizon of uncultivated couch grass [Elytrigia repens (L.) Desv. ex Nevski] and an Ah horizon of forest soil, were extensively characterized by means of different spectroscopic techniques (diffuse reflectance infrared Fourier transform [DRIFT] and ¹H nuclear magnetic resonance [NMR]). The two humic substances, each separated in fractions with low (<3500 Da) and high (>3500 Da) relative molecular mass were compared for their effects on Pinus nigra J.F. Arnold callus. Growth of callus, the soluble sugar content, free amino acid pool, and the activities of the key enzymes involved in C and N metabolism were investigated. Callus was grown for a subculture period (28 d) on basal Murashige and Skoog medium plus humic matters with or without different hormones: indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid, or 6-benzylaminopurine. The results of ¹H-NMR spectra and the DRIFT spectroscopy showed significant differences in the chemical composition between forest and grass humic substances. A large amount of aliphatic and H-sugarlike component and an intense chemical shift of the β-CH3 region in both grass humic fractions were observed, while high contents of betaine, organic acid, and COOH groups in both forest humic fractions were detected. A different biological activity between the grass and forest humic fractions was also observed. Thus, the different activity of the two humic substances used seems related to the diverse chemical composition rather than to different molecular weights. [ABSTRACT FROM AUTHOR]

In this work, we have developed a simple method to carry out the quantitative analysis of deethylhydroxyatrazine (DEHA), the most abundant metabolite of atrazine herbicide (ATZ), based on the surface-enhanced Raman scattering technique. Since this ATZ product can undergo pH-dependent tautomerization, a previous characterization of the DEHA vibrational spectrum was accomplished. This study consisted of the Raman scattering study, both experimental and theoretical, of the enolic and ketonic tautomers of this molecule. SERS spectra were recorded at different pH in order to assess the effect of the metal surface in nanoparticles along with the pH on the structure of DEHA and to find the optimal experimental conditions of the quantitative detection of DEHA. Additionally, the interaction of DEHA with two types of humic acid reference standards, the Elliot humic and leonardite humic ones, was also performed by SERS. This interaction was conducted with two different objectives: to evaluate the interaction mechanism of the ATZ degradation product with humic substances and to check if this interaction can modify the sensitivity of the SERS detection of DEHA. The results presented in this study have clearly demonstrated that SERS spectroscopy is a very powerful technique for characterizing DEHA and other triazine sub-products at a very low concentration in water and also for analyzing the interaction of these important pollutants with humic substances. [ABSTRACT FROM AUTHOR]

Humic substances (HS) are dominant components of soil organic matter and are recognized as natural, effective growth promoters to be used in sustainable agriculture. In recent years, many efforts have been made to get insights on the relationship between HS chemical structure and their biological activity in plants using combinatory approaches. Relevant results highlight the existence of key functional groups in HS that might trigger positive local and systemic physiological responses via a complex network of hormone-like signaling pathways. The biological activity of HS finely relies on their dosage, origin, molecular size, degree of hydrophobicity and aromaticity, and spatial distribution of hydrophilic and hydrophobic domains. The molecular size of HS also impacts their mode of action in plants, as low molecular size HS can enter the root cells and directly elicit intracellular signals, while high molecular size HS bind to external cell receptors to induce molecular responses. Main targets of HS in plants are nutrient transporters, plasma membrane H+-ATPases, hormone routes, genes/enzymes involved in nitrogen assimilation, cell division, and development. This review aims to give a detailed survey of the mechanisms associated to the growth regulatory functions of HS in view of their use in sustainable technologies. [ABSTRACT FROM AUTHOR]

Significant benefit of soil organic matter (SOM) to crop productivity is scientifically well documented. The main constituents and active fractions of SOM are humic substances (HS) and phenolic compounds. Since both these two components strongly impact plant–soil relationship, it is importantly from an ecological point of view to discriminate their biological effects and relating them to their composition. In this study we compared the biological effects of HS, and the soil water soluble phenols (SWSP) on growth, antioxidant activities, carbohydrates, proteins, phenols, and vitamins of Pinus laricio callus. Each extract was assessed for the content of low molecular weight organic acids, soluble carbohydrates, fatty acids, and phenolic acids. Moreover, Fourier transform infrared (FT-IR) and surface-enhanced Raman scattering (SERS) spectroscopies were applied to study their molecular structure. The results showed that HS produced better callus growth compared to the control and SWSP. Carbohydrates decreased in presence of HS while proteins, vitamin C and E increased. In contrast, in callus treated with SWSP the amount of glucose and fructose increased as well as all the antioxidant activities. The data evidenced that HS rich in tartaric and fatty acids had beneficial effects on callus growth contrary to soil water-soluble phenols rich in aldehydes, and syringic, ferulic, and benzoic acids. [ABSTRACT FROM AUTHOR]

Humic substances (HS) are important soil components playing pivotal roles in guaranteeing long-term soil fertility. In this study, the chemical and biological properties of HS extracted from earthworm coprolites collected in soils subjected to different fertilization inputs (no fertilization, NF; fertilization with farmyard manure, FM; mineral input, M; mixed inputs, FMM, half farmyard manure plus half mineral input) were investigated. Results indicated a relationship between fertilization input and composition, molecular complexity and apparent molecular weight distribution of HS produced by earthworms. Coprolites from FM and FMM soils were the most enriched in organic carbon (OC), and HS from coprolites of FM soil were the highest in humic carbon (HC). Also, soil amendment with manure increased carboxylate and aromatic groups in HS, and the fraction with a high degree of polycondensation, thus indicating a positive impact of manure on plant residues' degradation processes. These HS were the only to display hormone-like activity, which likely accounted for their most pronounced positive effects on plant growth and metabolism, including accumulation of chlorophylls, mineral nutrition, and activity of nitrogen assimilation enzymes, in oat (Avena sativa L.) plants growing in a soil-less system. We conclude that manure input favored the turnover of OC towards the humification process that led to the production of high-quality coprolites and HS with superior biological activity, and suggests that OC in coprolites and HC in HS from earthworms might be used as reliable indicators of soil fertility. [ABSTRACT FROM AUTHOR]

Abstract This study investigated the effects of hydroperiod on soil organic matter quality in three different salt marshes in the Baiona lagoon (N Italy) representing terrestrial, intertidal and subaqueous ecosystems in the area. The study specifically aimed to gain some insight into how soil waterlogging (hydroperiod) affects the chemical and biological properties of soils as well as the quality and structure of the soil organic matter (SOM). Total contents of selected nutrients, total organic carbon and carbon stable isotope (δ13C) were measured in all soil profiles. The results of these analyses enabled us to define the different origin of the SOM by discriminating between terrestrial and aquatic SOM sources. The findings also show that accumulation of nutrients and SOM is significantly magnified in intertidal systems, in which pedoturbation effects induced by water movements are particularly strong. In addition, DRIFT spectra of humic acids revealed the changes in the main functional groups in relation to increased waterlogging, highlighting the lower aromaticity and complexity in subaqueous soils (SASs), which is possibly due to the effect of the soil water saturation on the chemical and biological SOM transformation processes. Microbial biomass carbon (MBC), microbial quotient (Qmic) and the activities of some soil enzymes were measured to estimate soil metabolic activity in the systems and to evaluate how the microbial pool contributes to transforming the SOM. In all systems, the enzymatic activities were generally higher in subsurface horizons than in the surface horizon. This unexpected behaviour can be explained by the combined effect of water movement, erosion processes and preservation of SOM under anaerobic conditions. This study represents an attempt to investigate and understand the ongoing degradation processes in salt marsh ecosystems. The findings emphasize the strong influence of water flow and erosional processes associated with soil waterlogging on chemical and biological reactions in intertidal and subaqueous systems. Highlights • Soil waterlogging affects both physicochemical and biochemical soil properties. • Isotopic C signature enabled identifying the origin of organic matter in saltmarshes. • Erosion processes and water flow pedoturbation affect soil biochemical reactions. • The functional groups of humic substances change with increasing soil waterlogging. [ABSTRACT FROM AUTHOR]

Abstract: Charcoal added to soil by wildfires is usually considered to be part of the most stable pool of soil organic matter (SOM). It consists of a continuum of slightly burned plant residues to completely charred material. We sampled the macroscopic charcoal pool produced by a moderate intensity wildfire in a pine coastal forest in Tuscany (Central Italy) with the aim of assessing the relationship between its particle size and chemical composition as well as its reactivity. Our conceptual approach included particle size separation by dry sieving and analysis of four size fractions (>2mm, 2–1mm, 1–0.5mm and <0.5mm) for elemental composition. The composition of the charcoal fractions was studied using solid-state 13C nuclear magnetic resonance (NMR) spectroscopy, diffuse reflectance Fourier transform infrared (DRIFT) spectroscopy, analytical pyrolysis–gas chromatography/mass spectrometry (Py–GC/MS) and CuO oxidation. The reactivity of the fractions was assessed using acid dichromate oxidation, while the morphology was observed using scanning electron microscopy (SEM). There were clear differences in chemistry and reactivity between the fractions. The different fractions are probably the result of both type of original plant material and the fire conditions. In fact, wood-derived charcoal largely prevalent in the largest fraction, while pine needles and herbs were most probably precursors of charcoal in the smallest fractions. The degree of charring was greatest for the coarse fraction, which represented 56% of the total charcoal, and decreased gradually to the finest fraction. The composition was completely aromatic for the coarsest fraction, whereas the finest fraction showed a greater contribution from aliphatic C- and N-containing components. The greatest reactivity was found for the smallest fraction, which represented 24% of the total charcoal. It is particularly rich in N and potentially susceptible to microbial decomposition. The study demonstrates that properties of charcoal added to soil during wildfires may be reliably discriminated on the basis of particle size. [Copyright &y& Elsevier]