Investigation into the role of carboxylic acid and phenolic hydroxyl groups in the plant biostimulant activity of a humic acid purified from an oxidized sub-bituminous coal.

Introduction: Humic substances (HS) are increasingly being applied as crop plant biostimulants because they have been shown to increase plant productivity, especially under environmentally stressful conditions. There has been intense interest in elucidating the HS molecular structures responsible for eliciting the plant biostimulant response (PBR). The polar and weakly acidic carboxylic (COOH) and phenolic hydroxyl (ArOH) functional groups play major roles in the acid nature, pH dependent solubilities, conformation, and metal- and salt-binding capabilities of HS. Reports on the role played by these groups in the PBR of HS found growth parameters being both positively and negatively correlated with COOH and ArOH functionalities. Materials and methods: To investigate the role of COOH and ArOH in HS biostimulant activity we used a humic acid (HA), purified from an oxidized sub bituminous coal to prepare HAs with COOH groups methylated (AHA), ArOH groups acetylated (OHA), and with both COOH and ArOH groups methylated (FHA). The original HA was designated (NHA). The four HAs were subjected to elemental, 13C-NMR, FTIR, and EPR analyses and their antioxidant properties were assessed using the trolox equivalents antioxidant capacity assay (TEAC). 13C-NMR and FTIR analysis revealed significant alkylation/acetylation. To determine the effects of alkylating/acetylating these functional groups on the HA elicited PBR, the HAs were evaluated in a plant bioassay on corn (Zea mays L.) seedling under nutrient and non-nutrient stressed conditions. Treatments consisted of the four HAs applied to the soil surface at a concentration of 80 mg C L-1, in 50 ml DI H2O with the control plants receiving 50ml DI H2O. Results: The HA-treated plants, at both fertilization rates, were almost always significantly larger than their respective control plants. However, the differences produced under nutrient stress were always much greater than those produced under nutrient sufficiency, supporting previous reports that HA can reduce the effects of stress on plant growth. In addition, for the most part, the HAs with the alkylated/acetylated groups produced plants equal to or larger than plants treated with NHA. Conclusion: These results suggests that COOH and ArOH groups play a limited or no role in the HA elicited PBR. Alternatively, the HA pro-oxidant to antioxidant ratio may play a role in the magnitude of the biostimulant response.

Quantification of Humic and Fulvic Acids in Humate Ores, DOC, Humified Materials and Humic Substance-Containing Commercial Products.

The purpose of this method is to provide an accurate and precise concentration of humic (HA) and/or fulvic acids (FA) in soft coals, humic ores and shales, peats, composts and humic substance-containing commercial products. The method is based on the alkaline extraction of test materials, using 0.1 N NaOH as an extractant, and separation of the alkaline soluble humic substances (HS) from nonsoluble products by centrifugation. The pH of the centrifuged alkaline extract is then adjusted to pH 1 with conc. HCl, which results in precipitation of the HA. The precipitated HA are separated from the fulvic fraction (FF) (the fraction of HS that remains in solution,) by centrifugation. The HA is then oven or freeze dried and the ash content of the dried HA determined. The weight of the pure (i.e., ash-free) HA is then divided by the weight of the sample and the resulting fraction multiplied by 100 to determine the % HA in the sample. To determine the FA content, the FF is loaded onto a hydrophobic DAX-8 resin, which adsorbs the FA fraction also referred to as the hydrophobic fulvic acid (HFA). The remaining non-fulvic acid fraction, also called the hydrophilic fulvic fraction (HyFF) is then removed by washing the resin with deionized H2O until all nonabsorbed material is completely removed. The FA is then desorbed with 0.1 N NaOH. The resulting Na-fulvate is then protonated by passing it over a strong H+-exchange resin. The resulting FA is oven or freeze dried, the ash content determined and the concentration in the sample calculated as described above for HA.

Use of Ore-Derived Humic Acids With Diverse Chemistries to Elucidate Structure-Activity Relationships (SAR) of Humic Acids in Plant Phenotypic Expression.

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Bioactivity of Humic Acids Extracted From Shale Ore: Molecular Characterization and Structure-Activity Relationship With Tomato Plant Yield Under Nutritional Stress.

The increasing demands for biostimulants in the agricultural market over the last years have posed the problem of regulating this product category by requiring the industry to make available the information about efficacy and safety, including the explanation of mode of action and the definition of bioactive constituents. In the present study, we tested the biostimulant proprieties of a sedimentary shale ore-extracted humic acid (HA) on Micro Tom tomato plants under increasing nutritional stress and investigated the correlation with the chemical features of HA by means of ultra-high resolution FT-ICR MS, FT-ATR, and 13C-NMR. Humic acid application proved effective in alleviating the nutritional stress by improving nutrient use efficiency, with results comparable to the control treatment supplied with higher NPK nutrition. Increased yield (up to +19%) and fruit quality (in the range +10-24%), higher ascorbic acid content and a better root growth were the main parameters affected by HA application. Molecular-level characterization identified the possible chemical drivers of bioactivity, and included flavonoids, quinones, and alkaloids among the most represented molecules, some of which exhibiting antioxidant, pro-oxidant, and antimicrobial activity. The redox effect was discussed as a determinant of the delicate homeostasis balance, capable of triggering plant defense response and eventually inducing a protective priming effect on the plants.

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.

Molecular characteristics of water-extractable organic matter from different composted biomasses and their effects on seed germination and early growth of maize.

Four water extractable organic matter (WEOM) were obtained from composts made out of residues of: 1. artichoke (C-CYN), 2. artichoke/fennel (C-CYNF), 3. tomato/woodchips (C-TOM), 4. Municipal solid waste (C-MSW), and their bioactivity was tested for maize seed germination and maize seedling growth. The molecular properties of both original composts and their WEOM were characterized by spectroscopic (13C-CPMAS- and 1H NMR, FTIR-ATR), thermochemolysis-GC/MS, and thermal methods (TGA, DSC). While all WEOM had significant effects on plant growth, CYN-WEOM was the only material that concomitantly increased germination rate and primary and lateral root length of maize seedlings. The lignin-rich WEOM from green composts were generally more effective than those obtained from equally hydrophobic, but mainly alkyl-rich municipal organic wastes. A flexible conformational structure, due to the balanced content of aromatic compounds and carbohydrates, appeared to facilitate the release of bioactive molecules from WEOM suprastructures and stimulate plant growth.