The global decrease in soil fertility leads to a new agricultural scenario where eco-friendly solutions play an important role. The plant growth promotion through the use of microbes, especially endophytes and rhizosphere microbiota, has been proposed as a useful solution. Several studies have shown that humic substances are suitable vehicles for the inoculation of plant growth promoting bacteria, and that this combination has an enhanced effect on the stimulation of plant development. In this work, cucumber plants grown hydroponically have been pre-treated with a sedimentary humic acid (SHA) with known plant growth-enhancing effects, and culturable bacterial endophytes have been isolated from these plants. The hypothesis was that this pre-treatment with SHA could lead to the isolation of certain endophytic taxa whose proliferation within the plant could have been promoted as a result of the effects of the treatment with SHA, and that could eventually reinforce a potential synergistic effect of a combined application of those endophytic bacteria and SHA. The culturable endophytes that have been isolated from humic acid-treated cucumber plants have been identified as members of four main phyla:Proteobacteria,Firmicutes,Actinobacteria, andBacteroidetes. Isolates were characterized according to the following plant growth-promoting traits: nitrogen fixation/scavenging, phosphate solubilization, siderophore production and plant hormone production. Most of the isolates were able to fix/scavenge nitrogen and to produce plant hormones (indole-3-acetic acid and several cytokinins), whereas few isolates were able to solubilize phosphate and/or produce siderophores. The most promising endophyte isolates for its use in futures investigations as plant growth-promoting bacterial inocula werePseudomonassp. strains (that showed all traits),Sphingomonassp.,Stenotrophomonassp. strains, or someArthrobactersp. andMicrobacteriumsp. isolates.

Humic substances (HS, fulvic and humic acids) are widely used as fertilizers or plant growth stimulants, although their mechanism of action still remains partially unknown. Humic substances may be applied either directly to the soil or as foliar sprays. Despite both kind of application are commonly used in agricultural practices, most of the studies regarding the elicited response in plants induced by HS are based on the root-application of these substances. The present work aimed at discriminating between the mechanisms of action of foliar application versus root application of a sedimentary humic acid (SHA) on plant development. For this purpose, six markers related to plant phenotype, plant morphology, hormonal balance and root-plasma membrane H+-ATPase were selected. Both application strategies improved the shoot and root growth. Foliar applied- and root applied-SHA shared the capacity to increase the concentration of indole-3-acetic acid in roots and cytokinins in shoots. However, foliar application did not lead to short-term increases in either abscisic acid root-concentration or root-plasma membrane H+-ATPase activity which are, however, two crucial effects triggered by SHA root-application. Both application modes increased the root concentrations of jasmonic acid and jasmonoyl-isoleucine. These hormonal changes caused by foliar application could be a stress-related symptom and connected to the loss of leaves trichomes and the diminution of chloroplasts size seen by scanning electron microscopy. These results support the hypothesis that the beneficial effects of SHA applied to roots or leaves may result from plant adaptation to a mild transient stress caused by SHA application.

Humification is a process that plant and microbiota residues experiment in natural or agronomic soils under microorganisms action and environmental conditions. Under this process natural biomolecules - such as protein, carbohydrates or lignin - experience secondary biochemical and chemical reactions yielding to the formation of new organic biomolecules normally known as soil humus or humic substances (HS). In parallel, composting of fresh organic residues may be seen as an artificial process that involves many microorganism-induced secondary biochemical reactions that are probably also included in the first steps of natural humification in soils. In this context, we have applied multivariate statistical analysis to diverse and complementary analytical techniques (UV-Visible, synchronous fluorescence, FTIR, C-13-NMR and pyrolysis GS/MS) to follow the structural evolution of three groups of organic material: (i) fresh organic matter materials, (ii) compost of the fresh organic matter materials, and (iii) humic and fulvic acids including standards and references from the International Humic Substances Society. In order to discriminate among the three groups of organic materials, the set of data obtained from each analytical technique was analyzed using complementary statistical techniques: Correlations, Kolmogorov-Smirnov Test and Principal Component Analysis (PCA). The results showed positive correlations between UV-visible and fluorescence indexes and aromatic structures ...

Availability of fresh water for crop irrigation is becoming scarce and rather expensive. In this context, the research about the potential reutilization of non-conventional water sources becomes highly relevant, principally in arid and semi-arid areas. On many occasions, these new water resources involve water with a moderate concentration of salt, making it necessary to improve plant growth under moderate saline conditions. Besides plant breeding techniques, the use of molecules able to improve plant adaptation to saline conditions has great interest. Between these molecules, humic substances (HS) have proven to be efficient as stress-protectors under specific conditions of stress intensity and moment of application. The HS are main components of the soil organic matter and dissolved organic matter resulting from the biotic and abiotic transformation of fresh organic matter in natural ecosystems. Although knowledge about their structure is still under open debate, HS contain aromatic and aliphatic domains presenting O-, N- and S- containing functional groups with high biological and chemical activities. The aim of this presentation is to summarize the main effects of humic acids (HA) applied either on the root or on the shoot, on the metabolism and hormonal balance of plants cultivated under normal and stressing conditions, from studies carried out for our group and collaborations during the last years.

Although the ability of humic (HA) and fulvic acids (FA) to improve plant growth has been demonstrated, knowledge about the mechanisms responsible for the direct effects of HA and FA on the promotion of plant growth is scarce and fragmentary. Our study investigated the causal role of both root PM H+-ATPase activity and ABA in the SHA-promoting action on both root and shoot growth. The involvement of these processes in the regulation of shoot cytokinin concentration and activity was also studied. Our aim was to integrate such plant responses for providing new insights to the current model on the mode of action of HA for promoting root and shoot growth. Experiments employing specific inhibitors and using Cucumis sativus L. plants show that both the root PM H+-ATPase activity and root ABA play a crucial role in the root growth-promoting action of SHA. With regard to the HA-promoting effects on shoot growth, two pathways of events triggered by the interaction of SHA with plant roots are essential for the increase in root PM H+-ATPase activity-which also mediates an increase in cytokinin concentration and action in the shoot-and the ABA-mediated increase in hydraulic conductivity (Lp(r)).

Iron deficiency in plants is caused by a low availability of iron in the soil, and its main visual symptom is leaf yellowing due to a decrease in chlorophyll content, along with a reduction in plant growth and fruit quality. Foliar sprays with Fe compounds are an economic alternative to the treatment with expensive synthetic Fe-chelates applied to the soil, although the efficacy of foliar treatments is rather limited. Generally, plant response to Fe-foliar treatments is monitored by measuring chlorophyll content (or related parameters as SPAD index). However, different studies have shown that foliar Fe sprays cause a local regreening and that translocation of the applied Fe within the plant is quite low. In this context, the aim of this study was to assess the effects of foliar applications of different Fe compounds [FeSO4, Fe(III)-EDTA, and Fe(III)-heptagluconate] on Fe-deficient cucumber plants, by studying the main physiological plant root responses to Fe deficiency [root Fe(III) chelate reductase (FCR) activity; acidification of the nutrient solution; and expression of the Fe deficiency responsive genes encoding FCR, CsFRO1, Fe(II) root transporter CsIRT1, and two plasma membrane H+-ATPases, CsHA1 and CsHA2], along with SPAD index, plant growth and Fe content. The results showed that the overall assessment of Fe-deficiency root responses improved the evaluation of the efficacy of the Fe-foliar treatments compared to just monitoring SPAD indexes. Thus, FCR activity and expression of Fe-deficiency response genes, especially CsFRO1 and CsHA1, preceded the trend of SPAD index and acted as indicators of whether the plant was sensing or not metabolically active Fe due to the treatments. Principal component analysis of the data also provided a graphical tool to evaluate the evolution of plant responses to foliar Fe treatments with time.

The ability of rhizospheric humic substances to improve plant growth has been well established by many studies carried out using diverse plant species cultivated under many different conditions. These beneficial effects of humic substances on plant development are expressed in both root and shoot. However, the mechanisms responsible for this action of humic substances are only partially known and poorly integrated. In fact, although the studies focused on plant root development are numerous, those dealing with plant shoot development are scarce. Likewise, studies integrating humic effects on root and shoot are also few. In this context, the main goal of this work is to summarize some of the results regarding the effects of humic substances on plant development within a hypothetical holistic framework that will allow us to interconnect these findings and disclose some features of the functional crosstalk between the effects on soil, root and shoot. Furthermore, the significance of all these mechanisms in plants growing in the field is also discussed.

The traditional method to determine humic content (humic and fulvic acids) in commercial fertilizers, biostimulants, and organic materials is based on the oxidation of the organic carbon contained in the basic-soluble but acid-insoluble fraction (humic acids) and the basic-acid soluble fraction (fulvic acids) of their alkaline water extracts. This methodology, merely operational, makes it impossible to distinguish if the quantified carbon corresponds to substances with "humic" chemical nature or to non-humic organic matter but with similar solubility properties to those of humic matter. The aim of this work is to develop a new methodology that not only quantifies the humic content in commercial products (and raw materials) but also assesses the humic quality of the quantified organic matter. To this end, humic and fulvic (-like) fractions have been isolated/purified from several humic and non-humic materials and characterized by means of elemental analysis and UV-visible, fluorescence, and infrared spectroscopies, and these data have been used to perform a discriminant analysis (DA). The model obtained from the DA is able to discriminate humic and fulvic fractions from apparently humic or fulvic ones and provides discriminant classification functions that have proven to successfully predict the "humic quality" of the fractions isolated from commercial products, after their elemental and spectroscopic characterization. Therefore, the combination of the fractionation, characterization, and evaluation by the DA is proposed as an effective methodology for quantifying and assessing the quality of the humic content claimed in the labels of commercial products.

The effect of humic acids (HAs) and their iron complexes (Fe¿HAs) on the input of the main mineral elements into wheat seedlings, as well as on the efficiency of photosynthesis and the lipid profile of plants, under iron deficiency has been studied. The input of iron from Fe¿HA complexes and its predominant accumulation in roots are demonstrated. It is found that HAs increase the efficiency of photosynthesis due to enhanced electron transport in photosystem II. It is shown that the application of HAs and Fe¿HAs is accompanied by an enhanced input of Zn into plants, which could increase the antioxidant status of plants under iron deficiency conditions. In addition, a pronounced increase in the content of lipids in plants is revealed, which is indicative of the effect of HAs on plant metabolism. The obtained results suggest that the positive effect of Fe¿HAs and HAs on plants under iron deficiency conditions is due to a combination of factors, among which the effect of HAs on the antioxidant status of plants and the plant lipid metabolism predominates.

The physiological and metabolic mechanisms behind the humic acid-mediated plant growth enhancement are discussed in detail. Experiments using cucumber (Cucumis sativus) plants show that the shoot growth enhancement caused by a structurally well-characterized humic acid with sedimentary origin is functionally associated with significant increases in abscisic acid (ABA) root concentration and root hydraulic conductivity. Complementary experiments involving a blocking agent of cell wall pores and water root transport (polyethylenglycol) show that increases in root hydraulic conductivity are essential in the shoot growth-promoting action of the model humic acid. Further experiments involving an inhibitor of ABA biosynthesis in root and shoot (fluridone) show that the humic acid-mediated enhancement of both root hydraulic conductivity and shoot growth depended on ABA signaling pathways. These experiments also show that a significant increase in the gene expression of the main root plasma membrane aquaporins is associated with the increase of root hydraulic conductivity caused by the model humic acid. Finally, experimental data suggest that all of these actions of model humic acid on root functionality, which are linked to its beneficial action on plant shoot growth, are likely related to the conformational structure of humic acid in solution and its interaction with the cell wall at the root surface.

The role of soil humus in soil fertility and crop production has been well established by many studies and practical farming experience. This role is related to the presence of a family of organic substances, known as humic substances, with the capacity to increase the pool of plant-available nutrients in soil for root uptake. The improvement in plant growth and mineral nutrition is directly linked to the physicochemical features of humic substances, and more specifically, to their capacity to form stable chemical complexes with metals. This chapter reviews the main mechanisms behind the action of humic substances in improving plant mineral nutrition. It also discusses the main signalling pathways that might be involved in the regulation of these humic substances.

A number of studies have demonstrated the key role of nitric oxide in the regulation of many fundamental physiological processes that includes plant responses to abiotic and biotic stresses. On the other hand, beneficial action of humic substances on plant growth has been well corroborated, particularly when plants are subjected to abiotic stresses. Furthermore, several recent works have reported the functional links between the plant growth promoting action of humic substances and nitric oxide production and function in plants. In this article, we try to briefly review and discuss the main results showing the relationships between nitric oxide function and humic substances action on plants, also stressing the nitric oxide-dependent involvement of other plant growth regulators, such as auxin, ethylene, abscisic acid, and cytokinins.

The main aim of this communication is to discuss the current knowledge about the potential direct mechanisms that are involved in the beneficial action of humic substances on plant development. To this end, we present and discuss here recent results obtained in our laboratory, along with other findings published by other authors. Finally, we propose a hypothetical whole mechanism for explaining the action of humic substances on plant development. In this schema, we point out those steps that remain unclear.