While many articles have attempted to describe the composition of complex bio-stimulants, these descriptions are frequently incomplete since the vast majority of biological molecules that would be present in crude extracts of complex origin, have not yet been characterized and the mere presence of a specific compound does not a priori demonstrate that compound is functional. The composition of most biologically derived bio-stimulant feed stock will also vary with the season of production, species, physiological state of the source organism and growth conditions. Indeed, there is an implication in the marketing of many bio-stimulants that stress conditions experienced by the plant or microbe utilized to produce the bio-stimulant, results in the production of stress metabolites and amino acids with consequent beneficial effects on plant response. In the absence of knowledge of the functional component of a bio-stimulant, changes in composition of a bio-stimulant over time and between batches and commercial sources cannot be interpreted. In the most rigorously prepared bio-stimulants from leading companies, high-throughput analytical methods have been employed to ensure consistent product quality . Methods such as chromatography, mass spectrometry, NMR spectroscopy, elemental analysis, ELISA, spectrophotometry, etc. are typically used for this purpose . The complexity of this challenge is illustrated in the analysis of a four-year algae composition sequence using a profile or finger print technique employing NMR .bio-stimulants have been used at all stages of agricultural production including as seed treatments,strawberry gutter system as foliar sprays during growth and on harvested products. The mode/mechanisms action of “bio-stimulants” is equally diverse and may include the activation of nitrogen metabolism or phosphorus release from soils, generic stimulation of soil microbial activity or stimulation of root growth and enhanced plant establishment.
Various bio-stimulants have been reported to stimulate plant growth by increasing plant metabolism, stimulating germination, enhancing photosynthesis, and increasing the absorption of nutrients from the soil thereby increasing plant productivity . bio-stimulants may also mitigate the negative effects of abiotic stress factors on plants and marked effects of bio-stimulants on the control of drought, heat, salinity, chilling, frost, oxidative, mechanical, and chemical stress, have been observed . Alleviation of abiotic stress is perhaps the most frequently cited benefit of bio-stimulant formulations. The following text describes the primary modes/mechanisms of action that have been demonstrated or claimed for bio-stimulants in the primary scientific literature.An example of a “mechanism of action” would be a stimulation of photosynthesis or the down regulation of a plant stress signaling pathway without an understanding of the explicit biochemical or molecular “mode of action.” For many bio-stimulant products, however, neither a specified mode of action, nor a known mechanism of action, has been identified. The presence of some spurious products in the marketplace compromises the market for all players resulting in the assumption by many, that bio-stimulants as a whole, are “snake oils” , a pejorative term implying the product is of no value. Multi-component bio-stimulants are particularly difficult to reconcile since they may have constituents for which the mode of action is known and components of no known functional benefit. Furthermore, multi-component bio-stimulants will frequently contain measureable but biologically irrelevant concentrations of known essential elements, amino acids, and plant hormones etc., for which the mode of action is known but the concentrations are irrelevant when used at recommended rates. Thus, for many of the multi-component bio-stimulant in the marketplace today, we propose that a demonstration of a clear “mechanism of action” is a more rationale and attainable regulatory goal than requiring an unequivocal demonstration of the “mode of action.”
Insight into the use of the terms “mode and mechanism” of action can be drawn from the pesticide science and pesticide development. In pesticide science, the “mechanism of action” describes the integral of all the biochemical events following application while the “mode of action” characterizes the main features of a bio-active molecule and its specific biochemical action leading to its effect in treated plants . In reference to plant bio-regulators, Halmann suggests that ideally an understanding of the mode of action of plant bio-regulators on the molecular level requires the identification of the receptor site for each regulator, as well as the elucidation of the subsequent reactions. In reality this standard is often not met in bio-pesticide or bio-stimulant products where the identification of the molecular targets of all bio-available compounds within a given extract cannot be easily achieved. The identification of the target binding sites of the natural bio-molecules has, however, proven to be helpful in the design of new insecticidal molecules with novel modes of action . At the present time, given the difficulty in determining a “mode of action” for a complex multi-component product such as a bio-stimulant, and recognizing the need for the market in bio-stimulants to attain legitimacy, we suggest that the focus of bio-stimulant research and validation should be upon determining the mechanism of action, without a requirement for the determination of a mode of action. This is the standard of practice for many pharmacological products. With the development of advanced analytical equipment, bio-informatics, systems biology and other fundamentally new methodologies a more complete understanding of the mechanisms and even possible modes of action of these materials may be achieved in the future. While this proposal suggests that the development and marketing of a bio-stimulant may not require a demonstration of the mode of action, it is still in the interest of the manufacturers of these products to pursue an understanding of the mode of action so that the product can be improved and the use can be optimized for various environments and cropping systems.This is primarily due to the heterogeneous nature of raw materials used for production and the complex mixtures of components contained in bio-stimulant products which makes it almost impossible to identify exactly the component responsible for biological activity and to determine the involved mode of action .
Therefore, focus should be upon the identification of the “mechanisms of action” of bio-stimulants as indicated by general positive impacts on plant productivity through enhancement in processes such as photosynthesis, senescence, modulation of phytohormones, uptake of nutrients and water, and activation of genes responsible for resistance to abiotic stresses and altered plant architecture and phenology . An example of this process is the advances in use of protein-based bio-stimulants for which recent studies have identified the target metabolic pathways and some of the mechanisms through which they exert their effects on plants . To further our understanding of modes/mechanisms of bio-stimulant action we have systematized the stages of bio-stimulants action on plants after their application: penetration into tissues, translocation and transformation in plants, gene expression, plant signaling and the regulation of hormonal status, metabolic processes and integrated whole plant effects. The penetration of amino acids and peptide based bio-stimulants into plant tissues has been investigated using radiolabeled amino acids and mathematical modeling . The components of a bio-stimulant preparation of animal origin,grow strawberry in containers labeled with 14C proline and glycine, were shown to penetrate rapidly into treated leaves and where subsequently distributed to other leaves . The mathematical model based on the “mechanism of diffusion” allows the estimation of the time required for the absorption of a minimal amount of the active component of a bio-stimulant. Furthermore, it describes the process of its transport from the moment of penetration into the leaf until the arrival at more distant tissues . The penetration of protein hydrolysates into a plant tissues occurs via diffusion of protein molecules through membrane pores and is energy-dependent . bio-stimulants must have a good solubility in water or other suitable solvents. This is a precondition for most types of application and for sufficient penetration of active ingredients into internal structures of treated plants. Surfactants and other additives may be required to overcome solubility and uptake limitations including lipophilicity and molecular size of active components . Ultimately a full understanding of the biological activity of complex bio-stimulant preparations will require a detailed understanding of the mechanism of action and effects on plant productivity and the identification of the biologically active molecules and their molecular mode of action . A wide array of molecular methods has been used to attempt to discern the active compounds found in bio-stimulants including microarrays, metabolomics, proteomic, and transcriptomics methods. These technologies have been applied to bio-stimulants to probe changes in gene expression following the application of bio-stimulants . Further research on the effects of complex bio-stimulants and their components on the complete genome/transcriptome of plants will be required to understand the mechanisms of action involved in growth responses and stress mitigation . The search for the mode of action of bio-stimulants is complicated by the observation that many bio-stimulants have been shown to induce genes and benefit productivity only when plants are challenged by abiotic and biotic stress. Experimental methods must therefore be developed to produce relevant and reproducible stress conditions so that the application of any molecular tool to probe gene function produces results that are relevant to the purported effects on plant productivity. The role of signaling molecules in plant response to environmental cues has been an area of active research in plant biology. The process of signal transmission involves the synthesis of signaling molecules , their translocation, their binding to receptors, the resulting cellular responses, and, finally, the degradation of the signaling molecules . When the signaling molecule binds to its receptor, the initial cellular response is the activation of secondary messengers, or intracellular signaling mediators, which cause a further series of cellular responses. Among the substances that may act as secondary messengers are: lipids, sugars, ions, nucleotides, gases, Ca2+, cAMP, cGMP, cyclic ADP-ribose, small GTPase, 1,2-diacylglycerol, inositol-1,4,5- triphosphate, nitric oxide, phosphoinosides, and others . Generally, a membranemediated action is typical for water-soluble compounds, while cytosol-mediated activity is primarily triggered by lipophilic compounds.
Whereas, enzymes interact with their substrates in a geometrical way , signaling molecules are thought to have a topochemical affinity to their receptors. It is assumed that the interaction of such components at the receptor site is cooperative and quantized . The bioactive compounds in some bio-stimulants are also proposed to display signaling activity in plants or induce signaling pathways. Various amino acids , and peptides function as signaling molecules in the regulation of plant growth and development . Peptide signaling is important in various aspects of plant development and growth regulation including meristem organization, leaf morphogenesis, and defense responses to biotic and abiotic stress . specific signaling peptides contained in a plant-derived protein hydrolysate have been shown to affect plant growth and development, defense responses, callus growth, meristem organization, root growth, leaf-shape regulation, and nodule development . Protein hydrolysates from soybean and casein have been shown to act as elicitors to enhance grapevine immunity against Plasmopara viticola . Proteins may also contain hidden peptide sites, “cryptides” or “crypteins” in their amino acid sequence, which may have their own biological activities, distinct from its precursor . Evidence that cryptides can trigger plants defense reactions have recently been demonstrated and there are reports of the isolation of cryptides by hydrolysis of proteins from marine organisms, including seaweeds, and cryptides may be present naturally in a variety of biological derived products .Exogenous amino acids may affect biological processes by acting directly as signal molecules or by influencing hormone action via amino acid conjugation . It has been suggested that amino acid based bio-stimulants are readily absorbed and translocated by plant tissues and once absorbed, they have the capacity to function as compatible osmolytes, transport regulators, signaling molecules, modulators of stomatal opening, and may detoxify heavy metals among other benefits . Sugars and fatty acids and plant lipids are also known to act as signaling molecules and mitigators of stress response in plants . Animal based lipid soluble fractions, have also been observed to produce an auxin-like response , while sugars, sucrose, and its cleavage products , are also known to act as signaling molecules through regulation of gene expression and by interaction with other hormone signals including auxins. In a sunflower meal hydrolyzate, amino acids, humic substances, microelements, and sugars present in the bio-stimulant appeared to coordinate, with auxin-like compounds in complex signaling cross-talk promoting plant growth, enhancing plant transplanting success and increasing final crop yield .