Conversely, Douglas-fir trees are readily cross-dated and hold more promise as a candidate for future dendrochemical studies. A number of additional tree species common to salmon-bearing watersheds have been identified as suitable for dendrochemical investigations and warrant an objective assessment. Especially informative would be the analysis of tree rings from locations where once prodigious populations of anadromous salmon have been extirpated due the construction of dams. This would permit a coarse comparison of pre- and post-dam δ15N values and provide information regarding the magnitude of 15N-enrichment in wood due to SD-nitrogen inputs. Moreover, it would be instructive to examine the annual chemistry of tree rings in watersheds that contain a single dominant odd or even year population of pink salmon and few fish in the alternate years. Such conditions, if recorded with fidelity, would be expected to produce an unambiguous saw-toothed pattern of alternating high and low δ15N and [N]. However, all tree-ring based reconstructions will be essentially limited by the availability of robust escapement data for watersheds where old-growth riparian trees still exist. The regulation of plant growth and the development and alleviation of the negative effects of environmental stresses during ontogenesis, are important factors determining the productivity of cultivated plants. While it is well recognized that biotic and abiotic stress prevents essentially all crop systems from achieving their yield potential,hydroponic gutter current understanding of the mechanisms involved, and the strategies to mitigate these effects are limited.

Abiotic stresses may be prevented by optimizing plant growth conditions and through provision of water and nutrients and plant growth regulators . In addition to these traditional approaches, bio-stimulants are increasingly being integrated into production systems with the goal of modifying physiological processes in plants to optimize productivity. Plant bio-stimulants based on natural materials have received considerable attention by both the scientific community and commercial enterprises especially in the last two and a half decades . Bio-stimulants offer a potentially novel approach for the regulation/modification of physiological processes in plants to stimulate growth, to mitigate stress-induced limitations, and to increase yield. In the following review, we do not attempt to discern if the effects of bio-stimulants on plant productivity is a direct response of plants or soils to the bio-stimulant application or an indirect response of the bio-stimulant on the soil and plant microbiome with subsequent effects on plant productivity. Ultimately discerning if bio-stimulant effects are direct or microbially mediated will be critical to the development of this technology. The general goals of the current review are to provide a comprehensive analysis of the current situation in the field of bio-stimulants and to develop a science-based theoretical foundation for the conceptualization, classification, and practical application of these materials. A focus of this review is to understand and define the appropriate place of bio-stimulants among other agricultural products such as plant protection compounds and fertilizers, and to consider the unique attributes of complex, multi-component bio-stimulants. The structure of the review is based on the consideration of bio-stimulants in terms of their action on different regulatory and functional systems of plants using both conceptual and methodological approaches.

The overarching objective of the work is to highlight innovative concepts and to establish a scientific framework for future development of bio-stimulant science. To understand the development of bio-stimulant science, several seminal publications warrant discussion. To our knowledge, the first discussion of “biogenic stimulant” theory can be attributed to Prof. V.P. Filatov and was started in 1933 in the USSR . Filatov proposed that biological materials derived from various organisms, including plants, that have been exposed to stressors could affect metabolic and energetic processes in humans, animals, and plants . Blagoveshchensky further developed these ideas with specific reference to their application for plants, considering biogenic stimulants as “organic acids with stimulating effects due to their dibasic properties which can enhance the enzymatic activity in plants.” Filatov’s concept , was, however, not limited to these compounds alone . Herve’s pioneering review provides the first real conceptual approach to bio-stimulants. Herve suggests the development of novel “biorational products” should proceed on the basis of a systemic approach founded in chemical synthesis, biochemistry, and biotechnology as applied to real plant physiological, agricultural, and ecological constraints. He suggests these products should function at low doses, be ecologically benign and have reproducible benefits in agricultural plant cultivation. Zhang and Schmidt emphasized the need for comprehensive and empirical analysis of these products with particular emphasis on hormonal and antioxidant systems as the basis for many important benefits of bio-stimulants. They discuss the concept of bio-stimulants as “pre-stress conditioners,” their effects being manifested in improved photosynthetic efficiency, reduction of spread and intensity of some diseases and in better yields. Basak initiated the systematic discussion on bio-stimulants and created the conceptual preconditions for the formation of present bio-stimulant science while Du Jardin provided the first in-depth analysis of plant bio-stimulant science with an emphasis on bio-stimulant systematization and categorization on the basis of biochemical and physiological function and mode of action and origin.

Du Jardin’s analysis and categorization was influential in informing the development of subsequent legislation and regulation in the European Union. The study and development of bio-stimulants has been approached utilizing a wide range of methodological approaches including chemical and non-chemical characterization of composition , plant growth and yield studies , application of the so-called -omics strategies with variations, including microarray and physiological analysis , transcriptome , genomic , phenomic and molecular , proteomic , chemical and metabolomic . Ultimately, the integrative synthesis of results from multiple methodologies, particularly when integrated with the most relevant—omic technology, “agronomics,” will be required if the science and legitimacy of plant bio-stimulants is to advance. Several significant scientific meetings in the field of bio-stimulants have been held over the past ten years and have contributed greatly to our understanding of conceptual and methodological development of the bio-stimulant theory: “Biostimolanti in agrocoltura” , “Biostimulators in Modern Agriculture” , “bio-stimulants and Plant Growth” , among others. Of particular significance were the first and the second World Congresses on the “Use of bio-stimulants in Agriculture” which were valuable in highlighting the development of novel concepts and methodology as applied to bio-stimulants. While many of the following papers are not published in a peer-reviewed format, they do represent important advances in this field. Dumas et al. , for example, proposed a multi-part approach to study bio-stimulants based on large scale genomic approaches and high-throughput screening tests with genetically-modified reporter plants. Others suggested that bio-stimulant mode of action can be best determined using molecular microarray analysis to identify gene changes in transcript levels . This approach has the potential to reveal bio-stimulant activated signaling pathways involved in the stimulation of plant response. Microarray analysis is not, however, adequate and must be supplemented with carefully conducted field testing or high throughput plant phenotyping . The complexity of known bio-stimulant response,u planting gutter the dependency of crop environment and the diversity of bio-stimulant products demands the application of novel statistical approaches not commonly used in agronomic research . The principle espoused by Sleighter et al. is based on the identification of a subset of molecular markers that represent the active ingredients in complex bio-stimulants and then to correlate these markers with observations of plant response. Chemical genomics that utilizes small molecules to perturb target protein function is a useful strategy for bio-stimulant discovery as it overcomes constraints imposed by traditional molecular approaches that often fail due to gene redundancy and loss-of-function lethality. Botta et al. proposed probing the function of bio-stimulants using an enantiomeric analysis of active compounds in the bio-stimulant coupled with a proteomic profiling approach. In contrast, Conan et al. proposed identification of the bioactive compounds responsible for the plant growth response by means of a metabolomic profiling of bio-stimulant products and analysis of their physiological effects through transcriptomic and metabolomic strategies. Such methodology allows the determination of metabolite pathways affected by bio-stimulants as well as providing insight into gene regulation. To integrate the diversity of methodologies available Santaniello et al. emphasizes the need to use bioinformatics strategies to analyse similarities and differences in procedures of ingredient extraction and bio-stimulant formulation in terms of molecular plant responses. This integrative concept can be used to derive new technologies and novel bio-stimulant products through the identification of new target genes, enzymes and metabolites. While the development of robust, multi-faceted approach to the analysis of bio-stimulant composition and function will greatly aid in the development of this field, all advances must ultimately be interpreted in the context of plant response.

The complexity of plant response to the environment is daunting and was elegantly highlighted by Krouk who demonstrated that root response to nitrogen in the environment is mediated by combinations of signaling molecules and nitrogen sources in a manner that cannot be predicted by exposure to single compounds provided individually . Inevitably, as our understanding of the molecular networks that control plant growth improves our ability to predict plant response to bio-stimulants under specific environmental conditions, will improve. Only through a combination of methodologies will progress in bio-stimulant research be possible. The development of plant bio-stimulant science, as well as the principles governing its legislation in the context of the existing legal frameworks of plant protection products and fertilizers, requires the development of a clear definition of term “bio-stimulant.” Currently, the term “bio-stimulant” is poorly defined and includes many products that have variously been described as biogenic stimulants, metabolic enhancers, plant strengtheners, positive plant growth regulators, elicitors, allelopathic preparation, plant conditioners, phytostimulators, biofertilisers, or biofertiliser/bio-stimulant . One area of significant challenge is evoked in the question “are bio-stimulants PGRs?” Historically, bio-stimulants have been considered as a subgroup of growth regulators , as plant growth regulators , and as subgroup of bio-regulators . “From a legal point of view, bio-stimulants can contain traces of natural plant hormones, but their biological action should not be ascribed to them, otherwise they should be registered as plant growth regulators” . Likewise, bio-stimulants cannot by definition be pesticides or fertilizers . A concise and biologically meaningful definition of bio-stimulants has eluded researchers and regulators for many years. Table 1 presents a chronological evolution of concept of the term bio-stimulant. While several of bio-stimulant definitions presented are useful in their breadth, many of them have significant limitations and are overly generic, while several do not exclude possible effects of nutrients contained within any putative bio-stimulant product. In practice, bio-stimulants may deliberately include nutrients for regulatory approval as fertilizers and on occasions the included nutrients or hormones may be responsible for the perceived agronomic benefit. Given the state of public mistrust of many “bio-stimulant” products, it is necessary to provide a definition of bio-stimulants that explicitly denies the use of this term for products that do not have biological efficacy or have efficacy only by virtue of the inclusion of known plant hormones or nutrients. While the adoption of a definition of bio-stimulants for regulatory purposes is important, any definition of bio-stimulant should also be based on scientific principles.Basak , proposed that bio-stimulants could be classified depending on the mode of action and the origin of the active ingredient while Bulgari et al. , proposed that “bio-stimulants should be classified on the basis of their action in the plants or, on the physiological plant responses rather than on their composition.” Du Jardin , however, has emphasized the importance of the final impact on plant productivity when he suggests that “any definition of bio-stimulants should focus on the agricultural functions of bio-stimulants, not on the nature of their constituents nor on their modes of actions.” The term “plant productivity” is used here to describe any improvement in plant yield or quality or increased efficiency of production. These concepts reflect important differences in approaches to providing a definition of bio-stimulants as a discrete category of agricultural products. Thus, bio-stimulants could be defined by their demonstrated mode of action and origin, or solely by their demonstrated beneficial impact on plant productivity. The challenges in developing a definition are also complicated by the multi-component and largely undefined composition of many bio-stimulant products and the possibility that the activity of a bio-stimulant may not be explained by the presence of any individual constituent, but is a result of the interaction of many constituents in the product. On this basis two approaches to the definition of complex bio-stimulants emerge.