Biostimulants in Plant Science: A Global Perspective — TEI-tagged view

O. I. YakhinpersName, A. A. LubyanovpersName, I. A. YakhinpersName, Patrick H. BrownpersName (2017date). “Biostimulants in Plant Science: A Global Perspective.” Frontiers in Plant Sciencetitle·venue.

Object of study — the term biostimulantterm·dom itself - its competing definitions, synonyms and regulatory categories

Research question — what a biostimulantterm·dom is, such that the category can be distinguished from fertilisers, hormones and pesticides

Contribution — a proposed definition turning on emergent propertiesterm·dom, and a table of the competing definitions it replaces

Full text — tagged

REVIEWterm·mth published: 26 January 2017 doi: 10.3389/fpls.2016.02049 Biostimulantsterm·dom in Plant Science: A Global Perspective Oleg I. YakhinpersName 1, 2*, Aleksandr A. LubyanovpersName 2, Ildus A. YakhinpersName 2 and Patrick H. BrownpersName 3 1 Institute of Biochemistry and Genetics, Ufa Scientific Center, Russian Academy of Sciences, Ufa, Russia, 2 R&D Company Eco Priroda, Ulkundy, Russia, 3 Department of Plant Sciences, University of California, Davis, Davis, CA, USA This reviewterm·mth presents a comprehensive and systematic study of the field of plant biostimulantsterm·dom and considers the fundamental and innovative principles underlying this technology. The elucidation of the biological basis of biostimulantterm·dom function is a prerequisite for the development of science-based biostimulantterm·dom industry and sound regulations governing these compounds. The task of defining the biological basis of biostimulantsterm·dom as a class of compounds, however, is made more complex by the diverse sources of biostimulantsterm·dom present in the market, which include bacteria, fungi, seaweeds, higher plants, animals and humate-containing raw materials, and the wide diversity of industrial processes utilized in their preparation. To distinguish biostimulantsterm·dom from the existing legislative product categories we propose the following definition of a biostimulantterm·dom as “a formulated product of biological origin that improves plant productivityterm as a consequence of the novel or emergent propertiesterm·dom of the complex of constituents, and not as a sole consequence of the presence of known essential plant nutrientsterm·dom, plant growth regulatorsterm·dom, or plant protective compoundsterm·dom.” The definition provided here is important as it emphasizes the principle that biological function can be positively modulated through application of molecules, or mixtures of molecules, for which an explicit mode of actionterm·dom has not been defined. Given the difficulty in determining a “mode of actionterm·dom” for a biostimulantterm·dom, and recognizing the need for the market in biostimulantsterm·dom to attain legitimacyterm·dom, we suggest that the focus of biostimulantterm·dom research and validation should be upon proof of efficacyterm·dom and safety and the determination of a broad mechanism of actionterm·dom, without a requirement for the determination of a specific mode of actionterm·dom. While there is a clear commercial imperative to rationalize biostimulantsterm·dom as a discrete class of products, there is also a compelling biological case for the science-based development of, and experimentation with biostimulantsterm·dom in the expectation that this may lead to the identification of novel biological molecules and phenomenon, pathways and processes, that would not have been discovered if the category of biostimulantsterm·dom did not exist, or was not considered legitimate.

Keywords: biostimulantsterm·dom, mode of actionterm·dom, definition, classification, regulation, concepts, methodology, emergent propertiesterm·dom INTRODUCTION 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, current understanding of the mechanisms Edited by: Stefania De Pascale, University of Naples Federico II, Italy Reviewed by: Yuksel Tuzel, Ege University Faculty of Agriculture, Turkey Patrick Du Jardin, Gembloux Agro-Bio Tech - University of Liège, Belgium *Correspondence: Oleg I. YakhinpersName yakhinpersName@anrb.ru Specialty section: This article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science Received: 27 October 2016 Accepted: 21 December 2016 Published: 26 January 2017 Citation: YakhinpersName OI, LubyanovpersName AA, YakhinpersName IA and BrownpersName PH (2017) Biostimulantsterm·dom in Plant Science: A Global Perspective. Front. Plant Sci. 7:2049. doi: 10.3389/fpls.2016.02049 Frontiers in Plant Science | www.frontiersin.org January 2017 | Volume 7 | Article 2049 YakhinpersName et al.

Biostimulantsterm·dom in Plant Science: A Global Perspective 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 regulatorsterm·dom (PGRs—auxins, cytokinins, gibberellins, strigolactones, brassinosteroids). In addition to these traditional approaches, biostimulantsterm·dom are increasingly being integrated into production systems with the goal of modifying physiological processes in plants to optimize productivity. Plant biostimulantsterm·dom 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 (Crouch and van Staden, 1993a; Herve, 1994; Zhang and Schmidt, 1999; Maini, 2006; Khan et al., 2009; Apone et al., 2010; Craigie, 2011; Sharma et al., 2014; BrownpersName and Saa, 2015; Du Jardin, 2015; YakhinpersName et al., 2016a). Biostimulantsterm·dom 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 reviewterm·mth, we do not attempt to discern if the effects of biostimulantsterm·dom on plant productivityterm is a direct response of plants or soils to the biostimulantterm·dom application or an indirect response of the biostimulantterm·dom on the soil and plant microbiome with subsequent effects on plant productivityterm. Ultimately discerning if biostimulantterm·dom effects are direct or microbially mediated will be critical to the development of this technology. The general goals of the current reviewterm·mth are to provide a comprehensive analysis of the current situation in the field of biostimulantsterm·dom and to develop a science-based theoretical foundation for the conceptualization, classification, and practical application of these materials. A focus of this reviewterm·mth is to understand and define the appropriate place of biostimulantsterm·dom among other agricultural products such as plant protection compounds and fertilizers, and to consider the unique attributes of complex, multi-component biostimulantsterm·dom. The structure of the reviewterm·mth is based on the consideration of biostimulantsterm·dom in terms of their action on different regulatory and functional systems of plants (signaling, metabolism, uptake, and transport mechanisms, etc.) 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 biostimulantterm·dom science.

GENERAL CONCEPTS AND METHODOLOGY To understand the development of biostimulantterm·dom 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, 1944, 1951a,b; Gordon, 1947; Sukhoverkhov, 1967). 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 (Table 1). Blagoveshchensky (1945, 1955, 1956) 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 (1951b), was, however, not limited to these compounds alone (Filatov, 1951b). Herve’s (1994) pioneering reviewterm·mth provides the first real conceptual approach to biostimulantsterm·dom. 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 low doses, be ecologically benign and have function at reproducible benefits in agricultural plant cultivation. Zhang and Schmidt (1999) 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 biostimulantsterm·dom. They discuss the concept of biostimulantsterm·dom 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 (2008) initiated the systematic discussion on biostimulantsterm·dom and created the conceptual preconditions for the formation of present biostimulantterm·dom science while Du Jardin (2012, 2015) provided the first in-depth analysis of plant biostimulantterm·dom science with an emphasis on biostimulantterm·dom systematization and categorization on the basis of biochemical and physiological function and mode of actionterm·dom and origin. Du Jardin’s (2015) analysis and categorization was influential in informing the development of subsequent legislation and regulation in the European Union.

The study and development of biostimulantsterm·dom has been approached utilizing a wide range of methodological approaches including chemical and non-chemical characterization of composition (Crouch and van Staden, 1993b; YakhinpersName et al., 2005; Parrado et al., 2008; Sharma et al., 2012a,b; Ertani et al., 2013a,b; Aremu et al., 2015a,b), plant growth and yield studies (Khan et al., 2009; Kunicki et al., 2010; Paraąikovi´c et al., 2011; Zodape et al., 2011; YakhinpersName et al., 2012, 2016b; Chbani et al., 2013; Kurepin et al., 2014; Colla et al., 2015; Saa et al., 2015; Tandon and Dubey, 2015; Tian et al., 2015), application of the so-called -omics strategies with variations, including microarray and physiological analysis (Jannin et al., 2012, 2013), transcriptome (Wilson et al., 2015; Goñi et al., 2016), genomic (Santaniello et al., 2013), phenomic and molecular (Petrozza et al., 2014), proteomic (Martínez-Esteso et al., 2016), chemical and metabolomic (Ertani et al., 2014). 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 legitimacyterm·dom of plant biostimulantsterm·dom is to advance. in the field of biostimulantsterm·dom have been held over the past ten years and have contributed greatly to our understanding of conceptual and methodological development of the biostimulantterm·dom theory: “Biostimolanti in agrocoltura” (Italy, 2006), “Biostimulatorsterm·dom in Modern Agriculture” (Poland, 2008), “Biostimulantsterm·dom and Plant Growth” (Belgium, 2014), among others. Of particular significance were the first (France, 2012) and the second (Italy, 2015) World Congresses on the “Use of Biostimulantsterm·dom in Agriculture” which were valuable in highlighting the development of novel concepts and methodology as applied scientific meetings significant Several Frontiers in Plant Science | www.frontiersin.org January 2017 | Volume 7 | Article 2049 YakhinpersName et al.

Biostimulantsterm·dom in Plant Science: A Global Perspective TABLE 1 | Terminology in the biostimulantterm·dom field: Evolution and diversity of concepts*.

Terms, variants, and synonyms of the term of “biostimulantterm·dom”

Biogenic stimulators Biogenic stimulants Biogenic stimulants Organic Biostimulantterm·dom Biostimulatorsterm·dom Biostimulantsterm·dom Allelopathic Preparation Biostimulantsterm·dom Biostimulantsterm·dom Biostimulantterm·dom Biostimulantterm·dom Plant Strengtheners Biostimulantterm·dom (Positive Plant Growth Regulatorterm·dom), (Metabolic Enhancer) Biostimulantsterm·dom Original definitions and hypotheses Translation from Russian publications into English is verbatim (word for word, literatim) References “Every living tissue (human, animal and plants), when exposed to unfavorable, but non-lethal conditions, undergoes biochemical restructuring with the formation in it of special substances which are biogenic stimulators of non-specific nature, stimulating the life reactions of the organism, in which they introduced in, one way or another.” “1. Organisms, either animal or plant, when exposed to such environmental factors that complicate their lives, are subjected to biochemical restructuring. Consequently, there are formed substances that stimulate biochemical processes in these tissues. These substances which help the tissues to preserve life under adverse conditions, are named stimulants of biological origin (biogenic stimulators).” “2. Biogenic stimulators, injected one way or another in any organism activates vital processes in it. By strengthening metabolism, thus they increase physiological functions of the organism. In this manner biogenic stimulants increase the organism’s resistance to pathogenic factors and enhance its regenerative and absorbable properties, which facilitates recovery.” “3. Biogenic stimulators emerge as a result of biochemical restructuring, and in whole living organisms subjected to non-lethal but unfavorable external or internal environmental conditions.” “4. Environmental factors that cause the emerging of biogenic stimulants in the organism or in tissues separated from it, can be diverse.” “5. The emergence of biogenic stimulants under the influence of unfavourable factors of the environment is a common law for all wildlife. Biogenic stimulators are formed wherever there is a adaptation to new conditions of existence and the struggle for life.” “6. Biogenic stimulators accumulate in tissues and organisms when exposed to such external and internal factors that lead to the disruption of their normal metabolism, and are chemically products of disturbed metabolism.” “7. Biogenic stimulants act on the whole organism. This explains the breadth of the range of their action on the organism.” “8. The action of biogenic stimulators is expressed in changing of metabolic and energetic processes of organism.” “Substances which are produced in living tissues when using the method of Filatov following a series of disturbances of normal metabolism for the organism (according to Filatov - resistance factors), that have a stimulating effect on various processes in the organism.” [sic] “Biogenic stimulators can not substitute for fertilizer.” “These compounds increase plant growth and vigor through increased efficiency of nutrient and water uptake. Definitions for biostimulantsterm·dom vary greatly and there are still some arguments surrounding these compounds. However, they are defined as non-fertilizer products which have a beneficial effect on plant growth. Many of these biostimulantterm·dom materials are natural products that contain no added chemicals or synthetic plant growth regulatorsterm·dom.”

Filatov, 1951a Filatov, 1951b Blagoveshchensky, 1956 Russo and Berlyn, 1991 “Materials of little or no fertilizer value that accelerate plant growth, usually when used at low concentrations.” Goatley and Schmidt, 1991 “Plant hormone-containing substances that can stimulate growth when exogenously applied.”

Schmidt, 1992 “Multi-component balanced systems of biologically active substances of metabolic origin on the basis of plant raw materials with a broad spectrum of biological activity.”

Naumov et al., 1993 A subgroup of plant growth regulatorsterm·dom but are quite different from nutritional additives. … It is proposed to limit the use of the term biostimulantterm·dom to products aimed at improving yield through various metabolistics pathways.

Herve, 1994 “Products that are nonnutritive promoters of growth. Growth can be promoted by stimulating nutrient uptake, chelating nutrients, providing plant growth hormones, or enhancing plant hormonal activity. Biostimulantsterm·dom that contain plant growth hormones can be produced synthetically or obtained from natural plant extracts.”

Elliott and Prevatte, 1996 “Materials that, in minute quantities, promote plant growth.”

“An ambiguous term used to encompass non-nutritional growth-promoting substances such as microbes, plant growth hormones, soil conditioners and microbe energy sources.”

Zhang and Schmidt, 1999 McCarty, 2001 “Products intended to protect plants against harmful organisms by stimulating defence mechanisms in the plant or by competing with harmful organisms for space and nutrients in the phyllosphereterm·dom or rhizosphereterm·dom.”

(Anonymous 2001) quoted by Sharma K. et al., 2012 An organic material that, when applied in small quantities, enhances plant growth and development such that the response cannot be attributed to application of traditional plant nutrients. … If applied before stress occurs, biostimulantsterm·dom can help plants tolerate stress.

James Beard from Schmidt et al., 2003 “Natural or synthetic products of either mineral or organic composition that by their mode of actionterm·dom positively contribute to crop nutrition and the development of healthy plants.”

(S.D. Hankins, personal communication) Dixon and Walsh, 2004 (Continued) Frontiers in Plant Science | www.frontiersin.org January 2017 | Volume 7 | Article 2049 YakhinpersName et al.

Biostimulantsterm·dom in Plant Science: A Global Perspective TABLE 1 | Continued Terms, variants, and synonyms of the term of “biostimulantterm·dom”

Original definitions and hypotheses Translation from Russian publications into English is verbatim (word for word, literatim) References Metabolic Enhancer “Non-mineral substances that, when exogenously applied in very small quantities, stimulate the metabolic activities in plants.”

Doak et al., 2005 Biostimolanti - Biostimulantsterm·dom “Products that brings to other fertilizer and/or to the soil and/or to the plant, substances that favour or Ciavatta and Cavani, 2006 Biostimulantsterm·dom Organic Biostimulantterm·dom Biostimulantsterm·dom Biostimulantsterm·dom Biostimulantterm·dom Plant Growth Promoters Growth-Promoting PGRs Biostimulatorsterm·dom Biostimulatorsterm·dom Biostimulatorsterm·dom Biofertiliser/Biostimulantterm·dom regulate the absorption of the nutrients or correct some physiological anomalies.” “It must be remind that the biostimulantterm·dom activity does not have to derive from the addition of phytohormones (Attached 6, codicil 4.1). In other words, a fertilizer with “biostimulantterm·dom activity” must contribute positively to the improvement of the nutrition and the development of the plants, independently from the presence of the nutrients, with the exclusion of the phytohormones, whose presence is prohibited, and of the products with declared and specific phytosanitary function.”

“This term commonly identifies formulations supporting the plant in the improvement of its performances without using synthesis hormones.”

Tagliavini and Kubiskin, 2006 “Other than the direct influence on the nitrogen balance in plants, Siapton acts also indirectly improving the activity of some enzyme systems and regulating some plant growth regulatorsterm·dom (PGR) functions and biochemical processes. Moreover, Siapton makes easier the absorption and the transport of the nutritional macro- and micro-elements. These statements allow to define the product as a real “organic biostimulantterm·dom” and natural nutritional equilibrator for plants, more than a simple foliar organic fertilizer.”

Maini, 2006 “Product generally of organic nature which increase the soil microbial activity and/or plant growth.”

Nardi et al., 2006 “Single compounds or mixtures of compounds which mitigate the effects caused by abiotic stress agents, through the induction of innate plant defense responses.”

Apone et al., 2006 “Organic or hormone-containing compounds derived from natural products which can stimulate plant growth and development without causing known harm to the environment.”

Huang, 2007 “Compounds of biological origin and should act by increasing natural capabilities of plants to cope with stresses. Biostimulatorsterm·dom do not act neither as nutrients nor affect directly the stress factors making them less harmful for plants.”

Gawro ´nska, 2008 “Agents which at very low concentrations improve the basic biochemical processes in plants and soil, and thereby improve the growth and development of plants, and increase their resistance to stress. Biostimulatorsterm·dom are not a substitute for fertilizers, manure or other sources of mineral nutrients.”

Anonymous, cited by Basak, 2008 “Innovative natural stimulators of plant growth and development, obtained from biological raw materials, and having a hormone- or fertilizer-like mode of actionterm·dom.”

Anonymous, cited by Basak, 2008 “Are organic products composed of peptides, amino acids, polysaccharides, peptides, humic acids, and/or phytohormones, etc. for immediate uptake and availability within the plant. Their absorption does not depend on the photosynthetic activity as they are directly absorbed by the plant, resulting in lower energy consumption. The aim of these products is not to supply nutrition, but rather to favour and stimulate the metabolism of the plant, decrease plant stress, etc.”

Parrado et al., 2008 Organic Biostimulantsterm·dom “Non-nutrient containining products which have beneficial effect on plant growth. Such products consist of humic acids, marine algae (sea weed) extracts, non-hormonal plant metabolites and vitamins.”

Kumar and Shivay, 2008 Phytostimulatorterm·def “Microorganism with the ability to produce or change the concentration of growth regulators such as indole acetic acid, gibberellic acid, cytokinins and ethylene.”

Martínez-Viveros et al., 2010 Agricultural Biostimulantsterm·dom Biostimulatorsterm·dom Biostimulantsterm·dom Plant Biostimulantsterm·dom Antitranspirant “Include diverse formulations of compounds, substances and other products that are applied to plants or soils to regulate and enhance the crop’s physiological processes, thus making them more efficient. Biostimulantsterm·dom act on plant physiology through different pathways than nutrients to improve crop vigour, yields, quality and post-harvest shelf life/conservation.”

European Biostimulantsterm·dom Industry Council EBIC, 2011b “They mean inorganic and organic substances or its mixtures positively affecting plant development or other physiological processes in plants. One of the requirements for biostimulatorsterm·dom is that they pose no risk for human, animal, or natural environment due to its application.”

Smole ´n S, 2012 “Materials that are neither a fertilizer nor a pesticide, but when applied to a plant will enhance their health, growth, and protection.”

Banks and Percival, 2012 “Substances and materials, with the exception of nutrients and pesticides, which, when applied to plants, seeds or growing substrates in specific formulations, have the capacity to modify physiological processes of plants in a way that provides potential benefits to growth, development and/or stress response.”

Du Jardin, 2012 “Indicates the overall effect on the plant, the chemical compounds and underlying mechanisms are very diverse. Some of the compounds have physical effects at the surface and/or within the plant organs, others are regulators of the leaves openings diffusing water vapor, called stomata.”

Du Jardin, 2012 Metabolic Antitranspirants “Stomatal regulators, acting on the complex hormonal control of the highly specialized cells bordering the stomatal pore (guard cells).”

Du Jardin, 2012 (Continued) Frontiers in Plant Science | www.frontiersin.org January 2017 | Volume 7 | Article 2049 YakhinpersName et al.

Biostimulantsterm·dom in Plant Science: A Global Perspective TABLE 1 | Continued Terms, variants, and synonyms of the term of “biostimulantterm·dom”

Plant Biostimulantsterm·dom Biostimulantterm·dom Biostimulantsterm·dom Plant Strengthener (Biostimulantterm·dom) Biostimulantterm·dom Bio-Stimulatory Bio-stimulatory Agent Biostimulantterm·dom Biostimulantterm·dom Microorganisms Phytostimulatorsterm·def Biostimulatorsterm·dom Plant Biostimulantterm·dom Biostimulantsterm·dom Biostimulantsterm·dom Biostimulantterm·dom Biostimulantsterm·dom Biostimulantterm·dom Biostimulantsterm·dom Original definitions and hypotheses Translation from Russian publications into English is verbatim (word for word, literatim) References “Contain substance(s) and/or micro-organisms whose function when applied to plants or the rhizosphereterm·dom is to stimulate natural processes to enhance/benefit nutrient uptake, nutrient efficiency, tolerance to abiotic stress, and crop quality. Biostimulantsterm·dom have no direct action against pests, and therefore do not fall within the regulatory framework of pesticides.”

European Biostimulantsterm·dom Industry Council EBIC, 2012 “Material that, when applied to a plant, seed, soil or growing media - in conjunction with established fertilization plans, enhances the plant’s nutrient use efficiency, or provides other direct or indirect benefits to plant development or stress response.”

Beaudreau, 2013, Biostimulantterm·dom Coalition “Compounds that produce non-nutritional plant growth responses and reduce stress by enhancing stress tolerance. This is in contrast to fertilizers, which produce a nutritional response. Many important benefits of biostimulantsterm·dom are based on their ability to influence hormonal activity.” “Compounds in biostimulantsterm·dom can alter the hormonal status of a plant and exert large influences over its growth and health.”

Daniels, 2013 A class of “borderline” products used in agriculture in some member states … which act only on plant strength without direct effects against pests and have no main nutritional activity, … enhance the resistance of plants to harmful organisms and protect plants against non-parasitic impairments.”

Torre et al., 2013 “Products which, alone or mixed with other fertilizers, contribute to improve plant growth by exploiting different physiological processes.”

“The term “bio-stimulatory” means according to the invention, if not otherwise specified, an activity or efficacyterm·dom which stimulates, increases or improves many different processes in the plant or plant parts, such as improved generation of growth promoting substances like sugars and amino acids, improved adequate supply of cells with available nutrients and growth regulators, enhanced cell metabolism, improved cell decontamination, enhanced immune defense, promotion of growth and yield, induction of systemic acquired resistance (SAR), inhibition of growth and yield of competing plants (allelopathy). The bio-stimulatory activityterm·def can be caused by agents, plant extracts and compositions including metabolic compounds synthesized by the plant to be protected after induction of their synthesis by said bio-stimulatory agent. A “bio-stimulatory agent” according to the invention is a biological plant protecting agent as specified above, which shows the above-specified bio-stimulatory properties in a plant treated with this agent in vitro and/or in vivo.”

Migliore et al., 2013 Pretorius, 2013 “Is an organic material that, when applied in small quantities, enhances plant growth and development such that the response cannot be attributed to the application of traditional plant nutrients.”

Sharma et al., 2014 “Both biocontrol microorganisms (BCMs) and plant growth-promoting microorganisms (PGPMs) can be defined as “biostimulantterm·dom microorganisms,” able to foster plant growth and defence against pathogens throughout the crop life cycle, from seed germination to plant maturity.”

“This category includes microorganisms that promote plant growth usually by hormonal action.”

“Any substance or microorganism, in the form in which it is supplied to the user, applied to plants, seeds or the root environment with the intention to stimulate natural processes of plants benefiting nutrient use efficiency and/or tolerance to abiotic stress, regardless of its nutrients content, or any combination of such substances and/or microorganisms intended for this use.”

“Are extracts obtained from organic raw materials containing bioactive compounds.”

“Are materials, other than fertilizers, that promote plant growth when applied in small quantities. These environmental friendly and natural substances promote vegetative growth, mineral nutrient uptake and tolerance of plants to abiotic stresses.”

Sofo et al., 2014 Aguado-Santacruz et al., 2014 Traon et al., 2014 Bulgari et al., 2015 Chojnacka et al., 2015 “Any substance or microorganism applied to plants with the aim to enhance nutrition efficiency, abiotic stress tolerance and/or crop quality traits, regardless of its nutrients content. By extension, plant biostimulantsterm·dom also designate commercial products containing mixtures of such substances and/or microorganisms.”

Du Jardin, 2015 “Are substances or materials, with the exception of nutrients and pesticides, which, when applied to plants, seeds, or growing substrates in specific formulations, have the capacity to modify physiological processes in plants in a way that provides potential benefits to growth, development, or stress response.”

Halpern et al., 2015 “Refers to a compound or composition that is neither a fertilizer nor pesticide, but which when applied to a plant will enhance the health and growth of a plant.”

Lovatt, 2015 “Products mostly based on natural raw materials, used in the ultra-small and small doses for modification of physiological and biochemical plant processes with the aim of more complete realization of genetic potential of their productivity due to changes in hormonal status, activation of metabolic processes, increase of efficiency of nutrition, stimulation of growth, development and strengthening the ability to withstand to the negative effects of various stress factors.”

YakhinpersName et al., 2016a *The definitions are provided as exact quotes from the primary sources without correction of spelling or grammar.

Frontiers in Plant Science | www.frontiersin.org January 2017 | Volume 7 | Article 2049 YakhinpersName et al.

Biostimulantsterm·dom in Plant Science: A Global Perspective to biostimulantsterm·dom. 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. (2012), for example, proposed a multi-part approach to study biostimulantsterm·dom based on largescale genomic approaches and high-throughput screening tests with genetically-modified reporter plants. Others suggested that biostimulantterm·dom mode of actionterm·dom can be best determined using molecular microarray analysis to identify gene changes in transcript levels (Gates et al., 2012). This approach has the potential to reveal biostimulantterm·dom 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 (Summerer et al., 2013). The complexity of known biostimulantterm·dom response, the dependency of crop environment and the diversity of biostimulantterm·dom products demands the application of novel statistical approaches not commonly used in agronomic research (Sleighter et al., 2015). The principle espoused by Sleighter et al. (2015) is based on the identification of a subset of molecular markers that represent the active ingredients in complex biostimulantsterm·dom 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 biostimulantterm·dom 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. (2015) proposed probing the function of biostimulantsterm·dom using an enantiomeric analysis of active compounds in the biostimulantterm·dom coupled with a proteomic profiling approach. In contrast, Conan et al. (2015) proposed identification of the bioactive compounds responsible for the plant growth response by means of a metabolomic profiling of biostimulantterm·dom products and analysis of their physiological effects through transcriptomic and metabolomic strategies. Such methodology allows the determination of metabolite pathways affected by biostimulantsterm·dom as well as providing insight into gene regulation. To integrate the diversity of methodologies available Santaniello et al. (2015) emphasizes the need to use bioinformatics strategies to analyse similarities and differences in procedures of ingredient extraction and biostimulantterm·dom formulation in terms of molecular plant responses. This integrative concept can be used to derive new technologies and novel biostimulantterm·dom products through the identification of new target genes, enzymes and metabolites.

While the development of robust, multi-faceted approach to the analysis of biostimulantterm·dom 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 (2015) 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 (Krouk, 2016; Krouk et al., 2009, 2010, 2011). Inevitably, as our understanding of the molecular networks that control plant growth improves our ability to predict plant response to biostimulantsterm·dom under specific environmental conditions, will improve. Only through a combination of methodologies will progress in biostimulantterm·dom research be possible.

TERMINOLOGY AND DEFINITIONS The development of plant biostimulantterm·dom 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 the term “biostimulantterm·dom” is term “biostimulantterm·dom.” Currently, poorly defined and includes many products that have variously been described as biogenic stimulants, metabolic enhancers, plant strengtheners, positive plant growth regulatorsterm·dom, elicitors, allelopathic preparation, plant conditioners, phytostimulatorsterm·def, biofertilisers, or biofertiliser/biostimulantterm·dom (Table 1). One area of significant challenge is evoked in the question “are biostimulantsterm·dom PGRs?” Historically, biostimulantsterm·dom have been considered as a subgroup of growth regulators (Herve, 1994), as plant growth regulatorsterm·dom (Huang, 2007), and as subgroup of bioregulators (Basak, 2008). “From a legal point of view, biostimulantsterm·dom 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 regulatorsterm·dom” (Bulgari et al., 2015). Likewise, biostimulantsterm·dom cannot by definition be pesticides or fertilizers (Russo and Berlyn, 1991; Karnok, 2000; Hamza and Suggars, 2001; Banks and Percival, 2012; Du Jardin, 2012; Torre et al., 2013, 2016). and biologically meaningful definition of A concise biostimulantsterm·dom has for eluded researchers and regulators many years. Table 1 presents a chronological evolution of concept of the term biostimulantterm·dom. While several of biostimulantterm·dom 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 biostimulantterm·dom product. In practice, biostimulantsterm·dom 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 “biostimulantterm·dom” products, it is necessary to provide a definition of biostimulantsterm·dom that explicitly denies the use of this term for products that do not have biological efficacyterm·dom or have efficacyterm·dom only by virtue of the inclusion of known plant hormones or nutrients.

While the adoption of a definition of biostimulantsterm·dom for regulatory purposes is important, any definition of biostimulantterm·dom should also be based on scientific principles. Several concepts have been proposed to define plant biostimulantsterm·dom. Basak (2008), proposed that biostimulantsterm·dom could be classified depending on the mode of actionterm·dom and the origin of the active ingredient while Bulgari et al. (2015), proposed that “biostimulantsterm·dom 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 (2015), however, has emphasized the importance of the final impact on plant productivityterm when he suggests that “any definition of biostimulantsterm·dom should focus on the agricultural functions of biostimulantsterm·dom, not on the nature of their constituents nor on their modes of actions.” The term Frontiers in Plant Science | www.frontiersin.org January 2017 | Volume 7 | Article 2049 YakhinpersName et al.

Biostimulantsterm·dom in Plant Science: A Global Perspective “plant productivityterm” 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 biostimulantsterm·dom as a discrete category of agricultural products. Thus, biostimulantsterm·dom could be defined by their demonstrated mode of actionterm·dom and origin, or solely by their demonstrated beneficial impact on plant productivityterm. The challenges in developing a definition are also complicated by the multi-component and largely undefined composition of many biostimulantterm·dom products and the possibility that the activity of a biostimulantterm·dom 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 biostimulantsterm·dom emerge. The first is based on the possibility that the biostimulantterm·dom contains within it, previously unrecognized molecules that are the sole and discrete cause of the observed improvement in plant productivityterm. This concept emphasizes both the need for clear demonstration of plant productivityterm benefits and the unknown nature of the mode of actionterm·dom. Thus, a biostimulantterm·dom could be defined as “a formulated product that improves plant productivityterm by a mechanism of actionterm·dom that is not the sole consequence of the presence of known essential plant nutrientsterm·dom, plant hormones, plant growth regulatorsterm·dom or plant protective compoundsterm·dom.” By this definition, once the primary biological mechanism of biostimulantterm·dom function has been identified it should henceforth, be subject to classification on the basis of that functional component.

The majority of biostimulantsterm·dom in use today are complex mixtures of chemicals derived from a biological process or extraction of biological materials. The complexity of these mixtures is often considered to be essential to the performance of the biostimulantterm·dom, and biostimulantsterm·dom may have properties of the whole, that cannot be fully elucidated by knowing the characteristics of the separate components or their combinations. This theory of complexity or “emergence” was described by Mayr (1982), who argued that in many biological systems “the properties of the whole cannot be fully elucidated by knowing the characteristics of the separate components or their combinations.” “The term emergence describes the onset of novel properties that arise when a certain level of structural complexity is formed from components of lower complexity. In the last few decades, emergence has been discussed in a number of different research fields, such as cybernetics, theory of complexity, artificial intelligence, non-linear dynamics, information theory, and social systems organization” (Luisi, 2002). “Emergence” and “emergent propertiesterm·dom” are thus closely related with the notion of the “systems biology” (Luisi, 2002; Johnson, 2006; Korosov, 2012; Lüttge, 2012; Bertolli et al., 2014). Emergence was described by Johnson (2006) as “unexpected behaviors that stem from interaction between the components of an application and their environment,” “there is, however, considerable disagreement about ‘emergent propertiesterm·dom.’ Some include almost any unexpected properties exhibited by a complex system. Others refer to emergent propertiesterm·dom when an application exhibits behaviors that cannot be identified through functional decomposition. In other words, the the nature of system is more than the sum of its component parts” (Johnson, 2006).

Thus, a biostimulantterm·dom could also be defined as “a formulated product of biological origin that improves plant productivityterm as a consequence of the emergent propertiesterm·dom of its constituents.”

To our knowledge, however, there have been no clear demonstrations that any biostimulantterm·dom exhibits truly emergent propertiesterm·dom. This is not however a unique challenge and all “biological systems are extremely complex and have emergent propertiesterm·dom that cannot explained, or even predicted, by studying their individual parts” (Van Regenmortel, 2004). Emergent propertiesterm·dom have been demonstrated in the networks of biological signaling pathways (Bhalla and Iyengar, 1999); in system-level study of traditional Chinese medicine (Chen et al., 2014), and in microbial communities (Wintermute and Silver, 2010; Chiu et al., 2014). To adequately explain the biological complexity present in plants and their interactions with the environment, Lüttge (2012) and Bertolli et al. (2014) emphasize that classic reductionist biology/chemistry is indeed insufficient.

While the two theoretical definitions provided in this section share a requirement that the mode of actionterm·dom is unknown, they differ in the core assumption that biostimulantterm·dom function is a consequence of the discrete components in the biostimulantterm·dom or as a consequence of the “emergent” properties of the biostimulantterm·dom as a whole. Each of these definitions is also incomplete in that it is certainly possible that a biostimulantterm·dom may contain several molecules that act synergistically while not being truly “emergent,” and it is indeed possible and indeed likely, that even if a biostimulantterm·dom is demonstrated to have emergent propertiesterm·dom, that not all components of that biostimulantterm·dom are required for that property to be expressed.

We propose, therefore, a definition of a biostimulantterm·dom that integrates these two concepts. Thus, a biostimulantterm·dom is defined here as: “a formulated product of biological origin that improves plant productivityterm as a consequence of the novel, or emergent propertiesterm·dom of the complex of constituents, and not as a sole consequence of the presence of known essential plant nutrientsterm·dom, plant growth regulatorsterm·dom, or plant protective compoundsterm·dom.”

Consistent with this definition, the ultimate identification of a novel molecule within a biostimulantterm·dom that is found to be wholly responsible for the biological function of that biostimulantterm·dom, would necessitate the classification of the biostimulantterm·dom according to the discovered function.

CLASSIFICATION A reviewterm·mth of the history of biostimulantsterm·dom and related products provides insight into the diversity of these products and the development of this field of study. The evolution of biostimulantterm·dom classifications as described by various authors is presented in the Table 2. To the best of our knowledge, one of the first attempts to categorize biostimulantsterm·dom was provided by Filatov (1951b) when 4 groupings of biogenic stimulants were suggested. Karnok (2000) compiled a list of 59 materials presenting in Frontiers in Plant Science | www.frontiersin.org January 2017 | Volume 7 | Article 2049 YakhinpersName et al.

Biostimulantsterm·dom in Plant Science: A Global Perspective [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fi [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fi [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fi [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fi [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] Frontiers in Plant Science | www.frontiersin.org January 2017 | Volume 7 | Article 2049 YakhinpersName et al.

Biostimulantsterm·dom in Plant Science: A Global Perspective 15 biostimulantsterm·dom; Ikrina and Kolbin (2004) systematized patent literature and specified 9 categories of natural raw materials used to derive biostimulantsterm·dom; Basak (2008) suggested that biostimulantsterm·dom could be grouped on the basis of single or multicomponent formulations and classified on the origin of the active ingredient, and the mode of actionterm·dom of the active ingredient. Du Jardin (2012) developed a scientific rationale of classification considering 8 categories of biostimulantsterm·dom and subsequently reduced this list to 7 categories (Du Jardin, 2015). Du Jardin (2012) was explicit in his exclusion of microorganisms from his categorization primarily to avoid conflict with existing categorization of microorganisms as biopesticides and sources of plant hormones. Later Bulgari et al. (2015) proposed a biostimulantterm·dom classification on the basis of their mode of actionterm·dom rather than on their composition.

Many biostimulantterm·dom products have been classified into completely divergent groups and categories of function, use, and type of activity (Tables 3, 4). For example, humatebased products are often described as soil health amendments while plant growth promoting rhizobacteria (PGPRs) could be categorized as biofertilizers, phytostimulatorsterm·def, and biopesticides (Martínez-Viveros et al., 2010; Bhattacharyya and Jha, 2012). Du Jardin (2015) has proposed that biofertilisers are a subcategory of biostimulantsterm·dom. Seaweed extracts have been considered as biofertilizers (Zodape, 2001) and microorganisms have also been described as biofertilizers (Vessey, 2003; Fuentes-Ramirez and Caballero-Mellado, 2006; Roy et al., 2006; Malusá et al., 2012; Bhardwaj et al., 2014; Malusá and Vassilev, 2014). Some inorganic elements or small molecules that are not known to be essential may also be classified as biostimulantsterm·dom if evidence of plant growth promotion is available (Michalski, 2008; Kleiber and Markiewicz, 2013; Radkowski and Radkowska, 2013). Thao and Yamakawa (2009), for example, consider phosphites to be biostimulantsterm·dom since plant response to phosphites frequently cannot be explained as a consequence of the known anti-fungal function of these molecules. While the categorization of biostimulantsterm·dom by their origin does not, a priori, provide information on their mode of actionterm·dom this categorization may still be a useful tool to aid in the process of discovery and facilitate comparison between similar products.

LEGISLATION AND LEGAL FRAMEWORK Registration of products used in agriculture is crucial to ensure their practical, safe and legitimate application. In the absence of a sound definition of biostimulantsterm·dom as a discrete group of products (Basak, 2008), the registration procedure and subsequent classification regime is untenable and this inevitably creates a barrier to trade and development. Various countries, states, and administrative regions have developed different categories for registration of potential biostimulantsterm·dom including terminology such as plant conditioners, “other fertilizers,” supplements, soil improvers, plant strengtheners, fitofortificants, etc. (Basak, 2008; Torre et al., 2013; Traon et al., 2014). In many jurisdictions regulatory practices require an itemized description and identification of substances in all commercial product classifications while in others the registration of non-fully identified substances is allowed if those products are considered of complex composition. There is even a proposal for complex biostimulantsterm·dom to not specify the chemical name (IUPAC) and note as “None” with the definition that “this product is a complex mixture of chemical substances” (Traon et al., 2014). If we accept the concept that a biostimulantterm·dom is a product of clear benefit but unknown mode of actionterm·dom, then it can only be regulated by its safety and proof of efficacyterm·dom. For example, in pharmacology it has been suggested that “the demand to demonstrate the mode of actionterm·dom of each single component in a phytopharmaceutical may not be obligatory any more” (Ulrich-Merzenich et al., 2009).

The complex multicomponent nature of many biostimulantsterm·dom clearly complicates discovery of their modes/mechanisms of action, production, registration and use. What is clearly needed however, is a regulatory mechanism to ensure that the products are “generally recognized as safe,” have “a positive benefit on crop productivity” and are discrete from exisiting categories of products. The task of identifying function and agronomic utility can then be pursued independently and will be driven by the marketplace imperative for product quality and consistency. Coordinating national legislation within this framework will become critical for the optimization of biostimulantsterm·dom and trade between different countries. The possible place of biostimulantsterm·dom in the regulatory system of pesticides and agrochemicals is illustrated in Figure 1.

PRIMARY SOURCES OF RAW MATERIALS We have conducted an exhaustive analysis of the literature and categorized the majority of the reported biostimulantsterm·dom by origin (Table 4). Microorganisms are widely used for the production of biostimulantsterm·dom and may be derived from bacteria, yeasts, and fungi. These preparations may include living and/or nonliving microorganisms and their metabolites. The concept of microorganism-based preparations as biostimulantsterm·dom is described by Xavier and Boyetchko (2002), Sofo et al. (2014), Colla et al. (2015), Matyjaszczyk (2015), and Ravensberg (2015). Different species of algae, mostly seaweeds, are also commonly used for producing biostimulantsterm·dom. Seaweed-based preparations as biostimulantsterm·dom are described in reviewsterm·mth by Crouch and van Staden (1993a), Khan et al. (2009), Craigie (2011), Sharma et al. (2014); and experimental papers by Goatley and Schmidt (1991), Jannin et al. (2013), Billard et al. (2014), Aremu et al. (2015b). Raw materials for biostimulantsterm·dom are also commonly based on higher plant parts including seeds, leaves, and roots and exudates from families Amaryllidaceae, Brassicacae, Ericaceae, Fabaceae, Fagaceae, Moringaceae, Plantaginaceae, Poaceae, Rosaceae, Solanaceae, Theaceae, Vitaceae, among others (Naumov et al., 1993; YakhinpersName et al., 1998, 2011a, 2012, 2014; Pretorius, 2007, 2013; Parrado et al., 2008; Apone et al., 2010; Ertani et al., 2011a, 2013a, 2014; Colla et al., 2014; Yasmeen et al., 2014; Lucini et al., 2015; Ugolini et al., 2015). Biostimulantsterm·dom may also be based on protein hydrolysates and amino acids of animal origin including wastes and by-products (Mladenova et al., 1998; Maini, 2006; Kolomazník et al., 2012; Ertani et al., 2013b; Rodríguez-Morgado et al., 2014), and insect derived chitin and chitosan derivatives (Sharp, 2013). Humate-based raw materials are widely used to derive biostimulantsterm·dom and have been reviewed by Sanders et al. (1990), Kelting et al. (1998), Ertani Frontiers in Plant Science | www.frontiersin.org January 2017 | Volume 7 | Article 2049 YakhinpersName et al.

Biostimulantsterm·dom in Plant Science: A Global Perspective TABLE 3 | Examples of different terminology used for commercial biostimulantsterm·dom.

Preparations Source, composition Found in the literature related terms References Actiwave® Ascophillum nodosum Agrispon® Natural plant extract Aminoplant (Siapton®) Epithelial tissues (natural substances animal origin) Asahi SL (Atonik) Sodium para-nitrophenolate, sodium ortho-nitrophenolate, sodium 5-nitroguaiacolate Metabolic enhancer Biostimulantterm·dom Biostimulantterm·dom Bioregulator, Biostimulantterm·dom Biostimulatorterm·dom Organic biostimulantterm·dom, Soil fertilizer Biostimulantterm·dom Fertilizer Biostimulantterm·dom Bioregulator Spinelli et al., 2010 Vernieri et al., 2006; Ferrante et al., 2013 Rouse, 1984 Dubravec et al., 1995 Michalski, 2008 Maini, 2006 Betti et al., 1992; Mladenova et al., 1998; Apone et al., 2006; Cambri et al., 2008; Kunicki et al., 2010 Mladenova, 1978 Basak, 2008; Przybysz et al., 2014 Michalski, 2008 Bio-Algen® Phaeophyceae Biostimulatorterm·dom, Bioregulator Basak, 2008 Biozyme® ComCat® Ergostim® Ascophyllum nodosum (GA3+IAA+zeatin+ chelated micronutrients) Biostimulantterm·dom Bioregulator Tandon and Dubey, 2015 Belakbir et al., 1998; Ruiz et al., 2000 Lychnis viscaria Plant growth regulatorterm·dom, biostimulantterm·dom, Van der Watt and Pretorius, 2013 L-cysteine and folic acid derivative Plant growth regulatorterm·dom, Biostimulantterm·dom Biostimulantterm·dom Bioregulator, Biostimulantterm·dom Cutler and Cutler, 2004 Gupta and MacLeod, 1982; Sanders et al., 1990; Kinnersley, 1993 Dubravec et al., 1995 Fantac (Quantum) Mixture of 5% N-Acetyl thiazolidine carboxylic acid (N-ATCA) and 0.1% folic acid Biostimulantterm·dom, growth promoter Srivastava et al., 2008, 2010 FOLIAR (Macro-Sorb Foliar) A complex water soluble solution derived from the enzymatic hydrolysis of animal membranes Biofertilizer Biostimulantterm·dom Goëmar BM 86® Ascophyllum nodosum Kelpak® Ecklonia maxima Fertilizer, Biostimulatorterm·dom, Bioregulator Fertilizer Biostimulantterm·dom Aylward, 2005 Kauffman et al., 2007 Basak, 2008 Craigie, 2011 Arthur et al., 2013; Stirk et al., 2014 Biostimulatorterm·dom, Bioregulator Basak, 2008 Plant growth regulatorterm·dom, bioregulator Michalski, 2008 Fertilizer Plant growth stimulant Dhargalkar and Pereira, 2005 Khan et al., 2009 Maxicrop® Ascophyllum nodosum Biostimulatorterm·dom, Bioregulator Basak, 2008 Seasol (Agrikelp) Durvillea potatorum Stifun* The complex of biologically active substances of natural origin Fertilizer Plant growth stimulant Plant growth stimulant Liquid organic fertiliser Biostimulantterm·dom Bioregulator Plant growth regulatorterm·dom Biostimulantterm·dom Dhargalkar and Pereira, 2005 Khan et al., 2009 Khan et al., 2009 Tay et al., 1987; Kurepin et al., 2014 Sharma et al., 2014 YakhinpersName et al., 2006, 2007, 2011a YakhinpersName et al., 2011b, 2012, 2013 YakhinpersName et al., 2014, 2016a,b (Continued) Frontiers in Plant Science | www.frontiersin.org January 2017 | Volume 7 | Article 2049 YakhinpersName et al.

TABLE 3 | Continued Biostimulantsterm·dom in Plant Science: A Global Perspective Preparations Source, composition Found in the literature related terms References SM3 (Sea Magic 3) Laminariaceae and Fucaceae species Biostimulatorterm·dom, Bioregulator Basak, 2008 Tytanit® Titanium Biostimulantterm·dom Fertilizer Basak, 2008 Kleiber and Markiewicz, 2013 Wuxal®-Ascofol Ascophyllum nodosum Biostimulatorterm·dom, Bioregulator Basak, 2008 moringa leaf extract Plant growth stimulant Biostimulantterm·dom Yasmeen et al., 2013 Abdalla, 2013; Yasmeen et al., 2014 *By the results of state registration tests Stifun was recommended for registration but does not registered yet.

et al. (2011b), and Jannin et al. (2012). A final category of biostimulantsterm·dom includes those derived from extracts of food waste or industrial waste streams, composts and compost extracts, manures, vermicompost, aquaculture residues and waste streams, and sewage treatments among others. Because of the diversity of source materials and extraction technologies, the mode of actionterm·dom of these products is not easily determined.

TECHNOLOGIES OF PRODUCTION The technologies used in the production and preparation of biostimulantsterm·dom are highly diverse and include cultivation, extraction, fermentation, processing and purification, hydrolysis, and high-pressure cell rupture treatment (Table 4). In some instances, a biostimulantterm·dom product may also contain mixes of components derived from different sources and production methods. Frequently the rationale for utilizing extracts rather than raw biomass is a consequence of the need for a standardized manufacturing process to produce a uniform commercial product (Michalak and Chojnacka, 2014). For many products, the production processes are driven by process and marketing the result of a targeted strategy to demands and are not optimize the biological efficacyterm·dom of the commercial product. While the ultimate composition and possible function of commercial biostimulantterm·dom products may be partially determined by both the source of raw material and the process by which it is prepared (Traon et al., 2014), there may be manufacturing processes and product treatments utilized that result in compounds that are not present in the initial (primary raw) material. An example of this is the multitude of commercial seaweed extracts, often derived from the same species, that are rarely equivalent (Craigie, 2011). Commercial biostimulantterm·dom manufactured from similar sources are usually marketed as equivalent products, but may differ considerably in composition and thereby in efficiency (Lötze and Hoffman, 2016). Many manufacturers do not reveal the technology of biostimulantterm·dom production since that is a commercial secret (Traon et al., 2014).

BIOACTIVE COMPONENTS AND METHODS OF QUALITY CONTROL A diversity of substances contained in raw materials is used for the production of biostimulantsterm·dom. Whereas, primary metabolites the presence are contained in most preparations de facto, sugars compounds sulfur-containing nitrogen-containing including glycolysis, of secondary metabolites is more specific and depends to a large extent on the raw material used (species, tissue, growing conditions). Primary metabolites include amino acids, sugars, nucleotides, and lipids (Aharoni and Galili, 2011). Secondary metabolites are formed from different primary metabolic pathways, the tricarboxylic acid cycle (TCA), aliphatic amino acids (AA), the pentosephosphate and shikimate pathways which are primarily the source of aromatic AA and phenolic compounds (PC), terpenoids/isoprenoids, compounds (alkaloids), (glucosinolates); (Aharoni and Galili, 2011). Frequently, biostimulantsterm·dom are shown to have a multicomponent composition and may include plant hormones or hormone-like substances, amino acids, (carbohydrates, oligo-, betaines, peptides, proteins, lipids, vitamins, and polysaccharides), aminopolysaccharides, nucleotides or nucleosides, humic substances, beneficial elements, phenolic compounds, furostanol glycosides, sterols, etc. (Table 4). While many articles have attempted to describe the composition of complex biostimulantsterm·dom, 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 biostimulantterm·dom 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 biostimulantsterm·dom that stress conditions experienced by the plant or microbe utilized to produce the biostimulantterm·dom, 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 biostimulantterm·dom, changes in composition of a biostimulantterm·dom over time and between batches and commercial sources cannot be interpreted. In the most rigorously prepared biostimulantsterm·dom from leading companies, high-throughput analytical methods have been employed to ensure consistent product quality (Sharma et al., 2012b). Methods such as chromatography, mass spectrometry, NMR spectroscopy, elemental analysis, ELISA, spectrophotometry, etc. are typically used for this purpose (Table 4). The complexity of this challenge is illustrated in the analysis of a four-year algae composition sequence using a profile or fingerprint technique employing NMR (Craigie et al., 2009).

Frontiers in Plant Science | www.frontiersin.org January 2017 | Volume 7 | Article 2049 YakhinpersName et al.

Biostimulantsterm·dom in Plant Science: A Global Perspective [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fi [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fi [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fi [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fi [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fl [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fl [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fl [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fl [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fl [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fi [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fi [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fi [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] Frontiers in Plant Science | www.frontiersin.org January 2017 | Volume 7 | Article 2049 YakhinpersName et al.

Biostimulantsterm·dom in Plant Science: A Global Perspective d n a t i u r f ; t i u r f e z s d n a t n a p ; g M d n a K , a C , u C e k a t p u ; [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] ; y c n e c fi [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fi [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fl [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fi [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] P, [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] −3 O C H , l , e d x o , [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] , l o k c e : [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] ; [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] - 2 2 : [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] , [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] , [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] . [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] , [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] ; [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] - 4 2 ; l o n a t r a o c y c e n e y h t e M - 4 2 , ) R T o e d s o b i r i l o p o t - o h t r o d n a . c t e , s e d s o c u g n n k o t y c , ) T o i l o p o t o h t r o , ) R T m e d s o b i r i l o p o t - a t e m , ) T m i l o p o t - a t e m , ) R A B e d s o b i r e n n e d a y z n e b . y c n e c fi [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fl [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fi [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fl [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fi [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] Frontiers in Plant Science | www.frontiersin.org January 2017 | Volume 7 | Article 2049 YakhinpersName et al.

Biostimulantsterm·dom in Plant Science: A Global Perspective [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fl y x o r d y h i r t - ′ 4 , 7 , 5 [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] . [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] . y c n e c fi f e , [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fl [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fi [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fl [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fl [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] P, [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] P, [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] P, [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fl [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fi [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fi [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fl [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fi f o . s y a m Frontiers in Plant Science | www.frontiersin.org January 2017 | Volume 7 | Article 2049 YakhinpersName et al.

Biostimulantsterm·dom in Plant Science: A Global Perspective [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] P, P , c o n a t c o , c o n a x e h s d c a , l o n a x e h - 1 s o h o c a , ) c o n a c e d , l o e n p r e t – , l o o a n i l , l o n a n o n - 1 , l o - 3 - n e t c o - 1 , l o - 1 - n e x e h - 3 - 2 , l o h o c a y z n e b , l o c a a u g s e d y h e d a , ) l o n a h t e y n e h p , ) n i l l i n a v , l a n a n o n , e d y h e d a z n e b d n a 6 B , 3 B , 2 B , 1 B s n m a t i v , [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] ; e s a t u m s d e d x o r e p u s , [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] ; [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] , [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] . [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] , [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] , ) A R T o n a t n o c a i r t , ) [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] - [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] , s e d s o c y g ; 7 A + 4 A n i l l e r e b b G d c a , s e i t e o m e s o u l l e c m e h d n a n n g i l t h g e w r a u c e o m w o L , s d p L ; e d y h e d a r e f i n o c ; e d y h e d a g n i r y s ; n i l l i n a v : s o n e h p y o p d c a i l l i n a v ; e d y h e d a p a n s c g a l l d c a i l l a g ; d c a c g n i r y s : s e d s o e c u n n n o t a e M d c a , s e d i t o e c u n , e n d m i r y p , [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] , e s a d x o r e P , s e d i t p e p n o b r a c d c a c e f f a c - s n a r t : s d c a i l o n e h p d c a i l u r e d c a c i r a m u o c - p - s n a r t d c a c g a l l d c a c i r a t u o c - p - s n a r t d c a c i r a t f a c - s n a r t d c a c u h c e t a c o t o r p d c a i l l a g , ) d c a i l l i n a v d c a c g n i r y s i l l e r e b b g : s n i l l e r e b b g s e d s o c y g e s r e v d a d e c u d n i - [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] ; [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] ; [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] . [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] . [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] - O - i r T - ′ 4 , 2 , 4 - [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] / [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] / [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fi [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fl [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] Frontiers in Plant Science | www.frontiersin.org January 2017 | Volume 7 | Article 2049 YakhinpersName et al.

Biostimulantsterm·dom in Plant Science: A Global Perspective s t c e f f e a c g o o B n o i t c a f o m s n a h c e m / [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] / n o i t a c fi [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fl [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] P, [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] ´c v o k ą a r a P 0 1 0 2 , . l a t e a r e v R 0 1 0 2 , . l a t e e n o p A ; 9 0 0 2 , . l a t e t e u a C ; 4 1 0 2 , . l a t e i r e i l g a B ; 4 1 0 2 , z u R - z e d n á n r e H d n a o a n r A ; 3 1 0 2 , . l a t e s o Z ; 4 1 0 2 , 3 1 0 2 , . l a t e n e e m s a Y ; 3 1 0 2 , [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] ; 3 1 0 2 , . l a t e ´c v o k ą a r a P 3 1 0 2 , k y m s o P d n a s a n a J 3 1 0 2 , s e n a D 3 1 0 2 , . l a t e i t t e o o t s i r h C . 5 1 0 2 , . l a t e i l o g U ; 4 1 0 2 , . l a t e a c r a G - o d r a P ; 5 1 0 2 , . l a t e n c u L ; 4 1 0 2 , . l a t e z e m ó G - z e h c n á S 4 1 0 2 , . l a t e b a h h c a L 4 1 0 2 , . l a t e l l o C 4 1 0 2 , [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] ; 4 1 0 2 Frontiers in Plant Science | www.frontiersin.org [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fi [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] ; 7 0 0 2 , . [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] ; 6 0 0 2 , i n a M ; 6 0 0 2 , . l a t e e n o p A ; 3 0 0 2 , . l a t e n a h K ; 2 0 0 2 , a n e x a S d n a h c r u M ; 8 9 9 1 , . l a t e a v o n e d a M ; 2 9 9 1 , . l a t e i t t e B ; 8 7 9 1 , a v o n e d a M 9 5 9 1 , . l a t e h c a b a s s e W 5 5 9 1 , . l a t e r e l l i : s e c n e r e f e R d n a s a n a J ; 2 1 0 2 , . l a t e a h c e P ; 2 1 0 2 , . l a t e k n z a m o o K ; 2 1 0 2 , i t t e h c c e V d n a a c u L e D ; 1 1 0 2 , . l a t e ´c v o k ą a r a P ; 1 1 0 2 , . l a t e a k c e s L ; 9 0 0 2 , h c i r d e i r F d n a á e s e V ; a 3 1 0 2 9 0 0 2 , . l a t e n a t r E 8 0 0 2 , . l a t e o d a r r a P ; 8 0 0 2 , . l a t e . 4 1 0 2 , . l a t e a d a e T ; 4 1 0 2 , . l a t e o d a g r o M - z e u g í r d o R ; 4 1 0 2 , . l a t e b a h h c a L ; 4 1 0 2 , . l a t e e t r o C ; 4 1 0 2 , z u R - z e d n á n r e H d n a o a n r A ; 3 1 0 2 , . l a t e a v o k s a V ; 3 1 0 2 p r a h S 3 1 0 2 , . l a t e ´c [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. 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The tags cut from this paper are unaffected. ] P, , a N S A M / P C ; s y a s s a o b ; ) S M - I C P A . s t o o r d n a s t o o h s ) t h g e w e n e g e t a u g e R . s u t a t s a n o m r o h e t a u g e R c m u h r a u c e o m - h g h ; s d o n o v a fl e g n a c g a m n o i t a z i r a o p - s s o r c h t w o r g e s a e r c n . h t w o r g a e n o i t a c o s n a r t t o o h s - o t - t o o r e s a e r c n . s u t a t s , e F , u C , a C : s t n e m e e y r t e m o r t c e p s s s a m - n o i t a z n o e t s a w , l i o s c n a c o v , t s o p m o c m r e v , l i o s , t a e p . s a i r e t a m January 2017 | Volume 7 | Article 2049 [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fl t a e m YakhinpersName et al.

Biostimulantsterm·dom in Plant Science: A Global Perspective P, M R Z H D G O R Z c G O Z c P, M R Z c R Z c Z c G O R Z t G O Z t P, M R Z t , 4 A G , 3 A G , 2 A G , 1 A G s n i l l e r e b b g , 3 1 A G , 9 A G , 8 A G , 7 A G , 6 A G , 5 A G , 9 2 A G , 4 2 A G , 0 2 A G , 9 1 A G , 5 1 A G ; ) 3 5 A G , 1 5 A G , 4 4 A G , 4 3 A G ; ) P M R P R P G O R Z H D . s n e t o r p FUNCTION AND EFFECTS ON WHOLE PLANTS Biostimulantsterm·dom have been used at all stages of agricultural production including as seed treatments, as foliar sprays during growth and on harvested products. The mode/mechanisms action of “biostimulantsterm·dom” 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 biostimulantsterm·dom 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 productivityterm (Table 4). Biostimulantsterm·dom may also mitigate the negative effects of abiotic stress factors on plants and marked effects of biostimulantsterm·dom on the control of drought, heat, salinity, chilling, frost, oxidative, mechanical, and chemical stress, have been observed (Table 4). Alleviation of abiotic stress is perhaps the most frequently cited benefit of biostimulantterm·dom formulations. The following text describes the primary modes/mechanisms of action that have been demonstrated or claimed for biostimulantsterm·dom in the primary scientific literature.

MODES OF ACTION/MECHANISMS OF ACTION Understanding the modes of action of an agricultural chemical has been a fundamental requirement for effective marketing and frequently a regulatory requirement for manufactured products used in agriculture. Mode of actionterm·dom is used here to mean “a specific effect on a discrete biochemical or regulatory process,” thus the “mode of actionterm·dom” of Glyphosate is to inhibit the activity of the enzyme enolpyruvylshikimate-3-phosphate synthase (EPSPS). Biostimulantsterm·dom frequently do not meet this standard of specificity and indeed there are few biostimulantterm·dom products for which a specific biochemical target site and known mode of actionterm·dom has been identified. For a small subset of biostimulantsterm·dom, however, a demonstrated impact on general biochemical or molecular pathways or physiological processes, termed here as a “mechanism of actionterm·dom,” has been identified even though the explicit “mode of actionterm·dom” may not be known. An example of a “mechanism of actionterm·dom” 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 actionterm·dom.”

For many biostimulantterm·dom products, however, neither a specified mode of actionterm·dom, nor a known mechanism of actionterm·dom, 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 biostimulantsterm·dom as a whole, are “snake oils” (Basak, 2008), a pejorative term implying the product is of no value. Multicomponent biostimulantsterm·dom are particularly difficult to reconcile since they may have constituents for which the mode of actionterm·dom is known and components of no known functional benefit. Furthermore, multicomponent biostimulantsterm·dom , . l a t e n a t r E ; 1 1 0 2 , . l a t e o r i e d r o C ; 0 1 0 2 , . l a t e n a s v e r T ; 0 1 0 2 , . l a t e n o v a h c S ; 0 1 0 2 , . l a t e a r o M ; 0 1 0 2 , . l a t e s s b b o D ; 9 0 0 2 , . l a t e s o e c n o c s a V ; 9 0 0 2 , . l a t e e r r i u g A 0 1 0 2 7 0 0 2 , . l a t e d a n o d n a Z 7 0 0 2 6 0 0 2 , 5 0 0 2 , . l a t e d r a N ; 5 0 0 2 , e l l o b m a h C ; 4 0 0 2 , . l a t e n e h C ; 0 1 0 2 , 2 0 0 2 , . l a t e s a l l e n a C ; 0 0 0 2 , t d m h c S d n a g n a h Z ; 8 9 9 1 , . l a t e g n i t l e K ; 8 9 9 1 , . l a t e n a d A ; 1 9 9 1 , [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] 0 9 9 1 , . 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The tags cut from this paper are unaffected. ] / [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] fi [ Passage withheld — in our stored copy of this paper these characters are set one per token and run backwards, which is what a PDF extractor returns for a table or a caption printed sideways. It is not what the page of the article looks like, so we do not reproduce it. The tags cut from this paper are unaffected. ] Frontiers in Plant Science | www.frontiersin.org January 2017 | Volume 7 | Article 2049 YakhinpersName et al.

Biostimulantsterm·dom in Plant Science: A Global Perspective FIGURE 1 | The distribution of various categories of products among the plant protection products and fertilizers.

will frequently contain measureable but biologically irrelevant concentrations of known essential elements, amino acids, and plant hormones etc., for which the mode of actionterm·dom is known but the concentrations are irrelevant when used at recommended rates. Thus, for many of the multicomponent biostimulantterm·dom in the marketplace today, we propose that a demonstration of a clear “mechanism of actionterm·dom” is a more rationale and attainable regulatory goal than requiring an unequivocal demonstration of the “mode of actionterm·dom.”

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 actionterm·dom” describes the integral of all the biochemical events following application while the “mode of actionterm·dom” characterizes the main features of a bioactive molecule and its specific biochemical action leading to its effect in treated plants (Aliferis and Jabaji, 2011). In reference to plant bioregulators, Halmann (1990) suggests that ideally an understanding of the mode of actionterm·dom of plant bioregulators on the molecular level requires the receptor site for each regulator, the identification of the subsequent reactions. In as well as the elucidation of reality this standard is often not met in biopesticide or biostimulantterm·dom products where the identification of the molecular targets of all bioavailable (and frequently uncharacterized) compounds within a given extract cannot be easily achieved. The identification of the target binding sites of the natural biomolecules has, however, proven to be helpful in the design of new insecticidal molecules with novel modes of action (Rattan, 2010).

At the present time, given the difficulty in determining a “mode of actionterm·dom” for a complex multicomponent product such as a biostimulantterm·dom, and recognizing the need for the market in biostimulantsterm·dom to attain legitimacyterm·dom, we suggest that the focus of biostimulantterm·dom research and validation should be upon determining the mechanism of actionterm·dom, without a requirement for the determination of a mode of actionterm·dom. This is the standard of practice for many pharmacological products. With the development of advanced analytical equipment, bioinformatics, systems biology and other fundamentally new methodologies a more complete understanding of the mechanisms and even Frontiers in Plant Science | www.frontiersin.org January 2017 | Volume 7 | Article 2049 YakhinpersName et al.

Biostimulantsterm·dom in Plant Science: A Global Perspective possible modes of action of these materials may be achieved in the future. While this proposal suggests that the development and marketing of a biostimulantterm·dom may not require a demonstration of the mode of actionterm·dom, it is still in the interest of the manufacturers of these products to pursue an understanding of the mode of actionterm·dom so that the product can be improved and the use can be optimized for various environments and cropping systems.

The mechanisms of action of all but a few biostimulantsterm·dom remain largely unknown (Rayorath et al., 2008; Khan et al., 2009; Rathore et al., 2009). This is primarily due to the heterogeneous nature of raw materials used for production and the complex mixtures of components contained in biostimulantterm·dom products which makes it almost impossible to identify exactly the component(s) responsible for biological activity and to determine the involved mode(s) of action (Paraąikovi´c et al., 2011). Therefore, focus should be upon the identification of the “mechanisms of action” of biostimulantsterm·dom as indicated by general positive impacts on plant productivityterm 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 (Khan et al., 2009; Sharma et al., 2012b). An example of this process is the advances in use of protein-based biostimulantsterm·dom for which recent studies have identified the target metabolic pathways and some of the mechanisms through which they exert their effects on plants (Nardi et al., 2016).

To further our understanding of modes/mechanisms of biostimulantterm·dom action we have systematized the stages of biostimulantsterm·dom action on plants after their application: (1) penetration into tissues, translocation and transformation in plants, (2) gene expression, plant signaling and the regulation of hormonal status, (3) metabolic processes and integrated whole plant effects.

PENETRATION INTO TISSUES, TRANSLOCATION, TRANSFORMATION IN PLANTS The penetration of amino acids and peptide based biostimulantsterm·dom into plant tissues has been investigated using radiolabeled amino acids (Maini, 2006) and mathematical modeling (Kolomazník et al., 2012; Pecha et al., 2012). The components of a biostimulantterm·dom labeled with 14C proline and preparation of animal origin, glycine, were shown to penetrate rapidly into treated leaves and where subsequently distributed to other leaves (Maini, 2006). 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 biostimulantterm·dom. Furthermore, it describes the process of its transport from the moment of penetration into the leaf until the arrival at more distant tissues (Kolomazník et al., 2012; Pecha et al., 2012). The penetration of protein hydrolysates into a plant tissue occurs via diffusion of protein molecules through membrane pores (Kolomazník et al., 2012) and is energy-dependent (Parrado et al., 2008). Biostimulantsterm·dom 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 (Kolomazník et al., 2012; Pecha et al., 2012).

GENE EXPRESSION, SIGNALING, AND HORMONE INTERACTIONS Ultimately a full understanding of the biological activity of complex biostimulantterm·dom preparations will require a detailed understanding of the mechanism of actionterm·dom and effects on plant productivityterm and the identification of the biologically active molecules and their molecular mode of actionterm·dom (Henda and Bordenave-Juchereau, 2014). A wide array of molecular methods has been used to attempt to discern the active compounds found in biostimulantsterm·dom including microarrays, metabolomics, proteomic, and transcriptomics methods. These technologies have been applied to biostimulantsterm·dom to probe changes in gene expression following the application of biostimulantsterm·dom (Jannin et al., 2012, 2013; Santaniello et al., 2013). Further research on the effects of complex biostimulantsterm·dom 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 (Khan et al., 2009). The search for the mode of actionterm·dom of biostimulantsterm·dom is complicated by the observation that many biostimulantsterm·dom 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 productivityterm.

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 (ligands), their translocation, their binding to receptors, the resulting cellular responses, and, finally, the degradation of the signaling molecules (Zhao et al., 2005; Wang and Irving, 2011). 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, ions, nucleotides, gases, Ca2+, cAMP, cGMP, cyclic sugars, inositol-1,4,5- ADP-ribose, small GTPase, 1,2-diacylglycerol, triphosphate, nitric oxide, phosphoinosides, and others (Zhao et al., 2005; Wang and Irving, 2011). Generally, a membranemediated action is typical for water-soluble compounds, while cytosol-mediated activity is primarily triggered by lipophilic compounds.

Whereas, enzymes in a geometrical way (“lock and key”), signaling molecules are thought to have a topochemical affinity to their receptors.

interact with their substrates Frontiers in Plant Science | www.frontiersin.org January 2017 | Volume 7 | Article 2049 YakhinpersName et al.

Biostimulantsterm·dom in Plant Science: A Global Perspective is assumed that the interaction of such components at It the receptor site is cooperative and quantized (Gafurov and Zefirov, 2007). The bioactive compounds in some biostimulantsterm·dom are also proposed to display signaling activity in plants or induce signaling pathways. Various amino acids (Forde and Lea, 2007; Arbona et al., 2013), and peptides (Ivanov, 2010) function as signaling molecules in the regulation of plant growth and development (Ertani et al., 2009; Mochida and Shinozaki, 2011). 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 (Schiavon et al., 2008). 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 (Matsubayashi and Sakagami, 2006; Colla et al., 2013). Protein hydrolysates from soybean and casein have been shown to act as elicitors to enhance grapevine immunity against Plasmopara viticola (Lachhab et al., 2014).

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 (Ivanov, 2010; Samir and Link, 2011). Evidence that cryptides can trigger plants defense reactions have recently been demonstrated (Yamaguchi and Huffaker, 2011) 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 (Henda and Bordenave-Juchereau, 2014; Hayes et al., 2015).

Many small molecular weight substances are known to participate in signaling cascades in vivo. Exogenous amino acids may affect biological processes by acting directly as signal molecules or by influencing hormone action via amino acid conjugation (Tegeder, 2012). It has been suggested that amino acid based biostimulantsterm·dom 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 (Kauffman et al., 2007). Sugars (Smeekens, 2000; Eveland and Jackson, 2012) and fatty acids and plant lipids (Kachroo and Kachroo, 2009) are also known to act as signaling molecules and mitigators of stress response in plants (Okazaki and Saito, 2014). Animal based lipid soluble fractions, have also been observed to produce an auxin-like response (Kauffman et al., 2007), while sugars, sucrose, and its cleavage products (hexoses), 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 biostimulantterm·dom appeared to coordinate, with auxin-like compounds in complex signaling cross-talk promoting plant growth, enhancing plant transplanting success and increasing final crop yield (Ugolini et al., 2015).

Hormones are of central importance for the regulation of metabolic processes and plant development in a complex system of interacting hormones and cofactors, the functions of which status are closely intertwined and mutually dependent (Wang and Irving, 2011). Biostimulantsterm·dom developed from humic substances, complex organic materials, seaweeds, antitranspirants, free amino acids (Du Jardin, 2012), and crude extracts of lower (Rathore et al., 2009) and higher plants (YakhinpersName et al., 2012) have been frequently demonstrated to have an effect on plant hormonal (Kurepin et al., 2014). While hormone-like compounds may be present in biostimulantsterm·dom, it is also possible that de novo synthesis of hormones may be induced by such preparations in treated plants (Jannin et al., 2012) and amino acids, glycosides, polysaccharides and organic acids are contained in many biostimulantsterm·dom and may act as precursors or activators of endogenous plant hormones (Paraąikovi´c et al., 2011). Hormones or hormone-like effects could therefore be responsible for the action of natural biostimulantsterm·dom derived from microorganisms, algae, higher plants, animal, and humate based raw material (Table 4).

METABOLIC EFFECTS Information on currently available biostimulantsterm·dom gives some insight into the possible biochemical and molecular genetic effects of biostimulantsterm·dom derived from different natural raw (Table 4). Many published reports are available materials suggesting various biostimulantsterm·dom improve plant productivityterm through increased assimilation of N, C, and S (Jannin et al., 2012, 2013), improved photosynthesis, improved stress responses, altered senescence, and enhanced ion transport (Gajic, 1989; Khan et al., 2009; Paraąikovi´c et al., 2011). Biostimulantsterm·dom are also reported to increase free amino acids, protein, carbohydrates, phenolic compounds, pigment levels, and various enzymes (Table 4). The protective effect of many biostimulantsterm·dom against biotic and abiotic stresses has been associated with a reduction of stress-induced reactive oxygen species, activation of the antioxidant defense system of plants, or increased levels of phenolic compounds (Ertani et al., 2011a, 2013a).

While it is clear that many biologically derived biostimulantsterm·dom contain small molecular weight compounds that are involved in signaling events and may directly influence plant metabolic processes, it remains unclear how an exogenous soil or foliar application of an uncharacterized product can have predictable and beneficial responses in plants. It is well-known, for example, that application of exogenous plant hormones or compounds that disrupt hormone function (PGR’s) can have markedly negative effects on plants and that optimization of PGR materials and their applicaitons requires precise information on dosage and timing. Application of biostimulantsterm·dom for which the dosage and efficacyterm·dom of the functional compounds is unknown, cannot, therefore, be expected to result in predictable plant responses and identification of molecules with effects on plant metabolic processes is not, in of itself, a sufficient explanation for the function of a biostimulantterm·dom. It is also uncertain why the application of a biostimulantterm·dom with purported function as a PGR, signaling molecule or other discrete compound would be superior to, or Frontiers in Plant Science | www.frontiersin.org January 2017 | Volume 7 | Article 2049 YakhinpersName et al.

Biostimulantsterm·dom in Plant Science: A Global Perspective more easily controlled, than a direct application of the purified product itself.

TOXICOLOGICAL AND ECOLOGICAL ASPECTS Modern crop production requires a balance of high and consistent productivity with maximum safety for consumers, agricultural workers, and the environment (Rathore et al., 2009; Jannin et al., 2012; Pecha et al., 2012). While some biostimulantsterm·dom have been analyzed with regard to unwanted side effects including negative impact on the natural environment (Janas and Posmyk, 2013) most biostimulantsterm·dom have not been fully characterized but have been regarded as generally recognized as safe (GRAS in the US) on the basis of the biological origin of their constituents (Thomas et al., 2013). Generally, biostimulantsterm·dom are assumed to be biodegradable, non-toxic, nonpolluting and non-hazardous to various organisms. While this may be a rational conclusion for many formulations derived from biological materials such as seaweed extracts and their components (Turan and Köse, 2004; Dhargalkar and Pereira, 2005; Rathore et al., 2009; Michalak and Chojnacka, 2014; Stadnik and de Freitas, 2014), higher plants (Onatsky et al., 2001; Abdalla, 2013; YakhinpersName et al., 2013), chitin and chitosan (Bautista-Baños et al., 2006; Cabrera et al., 2013) it is not clear that this is a valid assumption for microbial products or products that would not normally be present in agricultural fields.

Biostimulantsterm·dom have been utilized as bioremediants and have been shown to improve ATP levels and phosphatase and urease activity (Tejada et al., 2011a), and hence increase the rate of degradation of xenobiotics in the soil (Tejada et al., 2010, 2011b) and to enhance beneficial soil microbial communities under semi-arid climates (Tejada et al., 2011b). Biostimulantsterm·dom may also help reduce the amount of potentially risky agrochemicals (Kolomazník et al., 2012) including reducing the use of fertilizers and pesticides (Hamza and Suggars, 2001). Most compounds contained in biostimulantsterm·dom are natural constituents of terrestrial and aquatic ecosystems (Jannin et al., 2012) and metabolites of plant and microbial origin and as such most are generally regarded as safe, particularly at the low rates at which they are typically applied. Thus, it has been proposed that biostimulantsterm·dom can be positioned as eco-friendly products for sustainable agriculture (Mladenova et al., 1998; Ertani et al., 2011a; Ghannam et al., 2013; Vijayanand et al., 2014). In many countries, however, biostimulantsterm·dom are not subject to rigorous toxicological screening (Traon et al., 2014) and there remains the potential for the persistence of human pathogens in materials of animal origin and for the synthesis of novel compounds of unknown function or toxicology during the manufacturing process.

ECONOMIC ASPECTS Even though there have been relatively demonstrations of few rigorous the benefit of biostimulantsterm·dom, and to a “market”

the market the validity of large extent the mode of actionterm·dom of these products remains uncertain, the industry for biostimulantsterm·dom is substantial and rapidly growing. Though many recent studies for these products is growing at a show that remarkable rate, these analyses must be considered with care as they frequently do not provide an explicit definition of term “biostimulantsterm·dom.” The value of the European biostimulantsterm·dom market ranged from e200 to e400 million in 2011, e500 million in 2013 and may grow to more than e800 million in 2018 with annual growth potential in 10% and more (EBIC, 2011a, 2013; Traon et al., 2014). France, Italy, Spain are the leading EU countries in the production of biostimulantsterm·dom (Traon et al., 2014). In North America, the biostimulantterm·dom market was valued at $0.27 billion in 20131, and is expected to grow at a growth rate of 12.4% annually, to reach $0.69 billion by 2018, the USA is the largest producer and consumer of biostimulantsterm·dom in the region (http://www.micromarketmonitor.com/). In 2014, the USA market was assessed at $313.0 million and is projected to reach $605.1 million by 20192, at a CAGR of 14.1% (http://news.agropages.com/). The biostimulantsterm·dom market in the Asia-Pacific was valued at $0.25 billion in 2013, and is expected to grow at a CAGR of 12.9% annually, to reach $0.47 billion by 2018 (Asia Biostimulantsterm·dom Market, 2015)3. China and India are key countries playing a significant role. The Southeast Asian & Australasian biostimulantsterm·dom market was valued at $233.8 million in 2015, and is projected to reach $451.8 million by 2021 (http://news.agropages.com/)4. The market in Latin America was valued at $0.16 billion in 20135, and is expected to grow at a CAGR of 14.4% annually, to reach $0.32 billion by 2018 (http://www.micromarketmonitor.com/). This market is mostly concentrated in Brazil and Argentina. The regional market shares of the global biostimulantsterm·dom market6 are: EU—41.7%, North America—21.5%, the Asia-Pacific region—20%, Latin America—12.9%. Globally, it biostimulantsterm·dom were valued at $1402.15 million in 2014 and are projected to have aCAGR of 12.5% reaching $2524.02 million by 20197, largely as a consequence of growing interest in organic products. Wu (2016) summised that “the global biostimulantsterm·dom market is projected to reach $2.91 billion by 2021, with a CAGR (compound annual growth rate) of 10.4% from 2016 to 2021. In terms of area of application, the biostimulantsterm·dom market is projected to reach 24.9 million hectares by 2021 and is 1North America biostimulantsterm·dom market (2015). Available online at http://www. micromarketmonitor.com/ (Accessed February 27, 2015). 2North America biostimulantsterm·dom market to reach $605.1 million by 2019 (2015). Available online at: http://news.agropages.com/ (accessed August 18, 2015). 3Available online at: http://www.micromarketmonitor.com/ (Accessed February 27, 2015). 4Southeast Asian and Australasian Biostimulantsterm·dom Market Trends and Forecasts to 2021 Available online at: http://news.agropages.com/ (Accessed August 15, 2016). 5Latin America biostimulantsterm·dom market (2015). Available online at: http://www. micromarketmonitor.com/ (Accessed February 27, 2015). 6Global biostimulantsterm·dom product market to reach $2241.0 million by 2018. Available online at: http://news.agropages.com/ (Accessed December 30, 2015). 7Global biostimulantsterm·dom market to reach $2.52 bn by 2019. Available online at http://news.agropages.com/ (Accessed December 30, 2015).

Frontiers in Plant Science | www.frontiersin.org January 2017 | Volume 7 | Article 2049 YakhinpersName et al.

Biostimulantsterm·dom in Plant Science: A Global Perspective projected to grow at a CAGR of 11.7% from 2016 to 2021” (Wu, 2016).”

PROBLEMS AND PROSPECTS The biostimulantterm·dom industry faces many problems and challenges. Until recently biostimulantterm·dom products based on natural raw materials and particularly waste stream has mainly been developed based on observational and less commonly, empirical data. While many contemporary biostimulantsterm·dom have been shown to be effective in practice, very few biostimulantsterm·dom can claim to understand the mechanisms or modes of action (Khan et al., 2009). Furthermore, while biostimulantsterm·dom can be categorized by source of origin, this is frequently inadequate as very substantial differences can exist between products even within a common feed stock origin. The challenge to biostimulantterm·dom science is further exacerbated since composition and content of active substances in the original plant raw material can be affected by many factors including the location and growing conditions, season, species, variety, organ, and the phase of growth (Naumov et al., 1993; Dragovoz et al., 2009; Sharma et al., 2012b). Similarly, the response of the target crop can be expected to vary across crops and environments. One solution to this problem is to derive the raw materials for the biostimulantterm·dom under highly regulated conditions. This approach has been successfully implemented by leading seaweed producers and fermentation based products that have developed harvesting and manufacturing processes that ensure uniformity of product performance through time. The development of a product with uniformity of response is not, however, a guarantee that the product is optimized for biological efficacyterm·dom.

To address these issues, developments in -omics approaches will be critical in accelerating the discovery of mode of actionterm·dom of bioactive compounds (Aliferis and Jabaji, 2011; Craigie, 2011; Jannin et al., 2012) and optimizing their use. Metabolomics, phenomics and agronomics represent the integration of gene expression, protein interactions, and other regulatory processes as they impact on plant productivityterm and thus are more appropriate tools for discovery in this field than mRNA, transcripts, or proteins analyzed in isolation (Arbona et al., 2013). Integrative, multidisciplinary approaches using tools from transcriptomics in conjunction with metabolomics and biochemical analysis are necessary to establish the mechanism of actionterm·dom and to identify the active components in the extracts (Lee et al., 2012). The difficulty in identifying modes of action and subsequent standardization of composition of multicomponent biostimulantsterm·dom based on natural raw materials will continue to hamper the use, certification and registration of biostimulantsterm·dom. The solution to this problem will require the collaborative efforts of specialists from different fields: chemists, biologists, plant physiologists, industrial manufacture, sales and distribution and those with expertise in practical agricultural production (Raldugin, 2004; Craigie, 2011; Jannin et al., 2012; Lee et al., 2012).

Products with a single active represent a in which the physiological effects and substance simpler construct readily determined mechanism of actionterm·dom can be more and hence certification and registration is simpler. The multicomponent composition of many preparations, however, are much more difficult to characterize (Bozhkov et al., 1996), though they may offer novel insight into biological synergy (Bulgari et al., 2015), multifunctionality and emergence which may be crucial to product efficacyterm·dom (Gerhardson, 2002). In the absence of a functional rationale for every constituent is likely in a multicomponent biostimulantterm·dom, it that may positively that or negatively influence plant productivityterm. Currently, it is chemical-synthetic, and genetic engineering approaches to reproduce the full suite of molecules and complexes of biologically active substances (Kershengolts et al., 2008) that are present in most biostimulantsterm·dom.

there will be molecules present impossible using available almost PRO’S AND CON’S OF BIOSTIMULANTSterm·dom SCIENCE AND PRACTICE Many have noted the state confusion in the field of biostimulantsterm·dom (Torre et al., 2013; Traon et al., 2014) and this has resulted in the opinion that much of the biostimulantterm·dom market is not based on science or efficacyterm·dom and that many products are little more than recycled waste products sold on the basis of pseudoscience and marketing. Indeed, research on several biostimulantterm·dom products has shown them to be ineffective or to contain inactive, unstable or inconsistent properties with several showing negative effects compared when contrasted with well-designed controls (Csizinszky, 1984, 1986; Albregts et al., 1988; Di Marco and Osti, 2009; Vasconcelos et al., 2009; Banks and Percival, 2012; Cerdan et al., 2013; de Oliveira et al., 2013; Carvalho et al., 2014). For example, foliar and root application of a product containing amino acids from animal origin have been reported to cause severe plant-growth depression and negative effects on Fe nutrition while a second product containing amino acids from plant origin stimulated plant growth (Cerdan et al., 2013). In another report that tested several biostimulantterm·dom products it was concluded that “none of the biostimulantterm·dom products tested achieved a sufficient degree of pathogen control to warrant replacement of or supplementation with conventional synthetic fungicides” (Banks and Percival, 2012), and there have been demonstrated positive and negative impacts and overall questions of the economic feasibility of the use of humic substances for increasing crop yields (Rose et al., 2014). Since biological systems are inherently complex, and given that most biostimulantterm·dom products have not been characterized and have received relatively little replicated and rigorous independent validation, it is perhaps not surprising that many products are ineffective or highly variable in response. Nevertheless, there are a significant number of rigorous independent reports of benefits from some biostimulantterm·dom formulations and market growth data demonstrates that there is a good deal of support for these products within agricultural producer communities. That such market growth has occured, even in the absence of a known “mechanism of function” suggests that there are aspects of plant Frontiers in Plant Science | www.frontiersin.org January 2017 | Volume 7 | Article 2049 YakhinpersName et al.

Biostimulantsterm·dom in Plant Science: A Global Perspective metabolism and productivity constraints that are not understood but are potentially important if we are to achieve the goal of increased global food production.

The market euphoria that is taking place in the biostimulantterm·dom industry recognizes these unknowns and biostimulantsterm·dom are viewed by many innovators and investors as a mechanism to conduct broadscale, if unfocussed, discovery of novel biologically derived molecules. Much as the exploration of marine organisms, and plants and microbes from diverse ecosystems has led to the discovery of novel pharmaceuticals, so too the development of biostimulantsterm·dom from the broad range of source materials, holds significant promise of discovery. Recent years have seen rapid growth in the number of published studies, increased numbers of scientific conferences and development of legal framework and legislation. These trends will inevitably improve the image of this industry and the efficacyterm·dom of products. Two significant problems still exist within the industry broadly: (1) preparations of products with highly complex multicomponent and incompletely identified composition make the identification of a primary mode of actionterm·dom extremely difficult and (2) the current classification and legislation/legal framework for regulation of biostimulantsterm·dom is based primarily on source material and not on biological mode of actionterm·dom. Hence there is insufficient capacity to differentiate products, and there is the potential for the successful demonstration of a single product within a biostimulantterm·dom category, to falsely indicate the efficacyterm·dom of the group as whole.

Several topical questions need consideration in the future: 1. Can living cultures of microorganisms, which have the ability to stimulate the growth of plants be referred to biostimulantsterm·dom? 2. Are non-essential elements that result in improved plant productivityterm, biostimulantsterm·dom?

3. How should biostimulantsterm·dom with a complex completely the components and unidentified structure where all modes/mechanisms involved have not been established be registered and regulated in national and international legislation?

4. What standard of proof of efficacyterm·dom is appropriate that both stimulates development and discourages the sale of materials of no benefit?

5. On what principles, should the final classification of biostimulantsterm·dom be based and what categories should it contain?

CONCLUSIONS Modern biostimulantsterm·dom are complex mixtures derived from raw materials of highly diverse origin utilizing highly diverse manufacturing processes and as such can be expected to have a broad spectrum of possible biological activity and safety. To distinguish biostimulantsterm·dom from the existing legislative product categories including essential nutrients, pesticides, or plant hormones a biostimulantterm·dom should not solely function by virtue of the presence of elements or compounds of known function. We propose, therefore, a definition of a biostimulantterm·dom as “a formulated product of biological origin that improves plant productivityterm as a consequence of the novel or emergent propertiesterm·dom of the complex of constituents and not as a sole consequence of the presence of known essential plant nutrientsterm·dom, plant growth regulatorsterm·dom, or plant protective compoundsterm·dom.” Consistent with this definition, the ultimate identification of a novel molecule within a biostimulantterm·dom that is found to be wholly responsible for the biological function of that biostimulantterm·dom, would necessitate the classification of the biostimulantterm·dom according to the discovered function.

This novel definition is inspired by three observations: (1) that the development of the biostimulantterm·dom industry will inevitably result in the discovery of novel biologically active molecules and that the identification and classification of these molecules will benefit biological discovery more greatly if these molecules are explicitly described than if they were merely labeled as “biostimulantsterm·dom,” (2) that there is a need for the nascent biostimulantterm·dom industry to explicitly discourage the inclusion of nutrient elements and known biologically active molecules under the guise of a “biostimulantterm·dom” and (3) that there is a need to recognize that classic reductionist biology/chemistry may indeed be insufficient to explain biological complexity (Luisi, 2002; Lüttge, 2012; Bertolli et al., 2014).

The definition provided here is important as it emphasizes the principle that biological function can be modulated through application of complex mixtures of molecules for which an explicit mode of actionterm·dom has not been defined. The definition also requires a demonstration of beneficial impacts of the biostimulantterm·dom on plant productivityterm. Given the difficulty in determining a “mode of actionterm·dom” for a biostimulantterm·dom, and recognizing the need for the market in biostimulantsterm·dom to attain legitimacyterm·dom, we suggest that the focus of biostimulantterm·dom research and validation should be upon determining the mechanism of actionterm·dom, without a requirement for the determination of a mode of actionterm·dom. This can be achieved through careful agronomic experimentation, molecular or biochemical demonstration of positive impact on biological processes or the use of advanced analytical equipment to identify functional constituents. Given the prerequisite multi-component and emergent characteristics of biostimulantsterm·dom, the discovery of the mode of actionterm·dom is likely to require application of new techniques in bioinformatics and systems biology. While the definition proposed here suggests that the development and marketing of a biostimulantterm·dom does not require a demonstration of the mode of actionterm·dom, it is still in the interest of the commercial producers of these products to pursue an understanding of these products so that the product can be improved and optimized for use in various environments and cropping systems.

While there is a clear commercial imperative to rationalize biostimulantsterm·dom as a discrete class of products, there is also a compelling biological case for the science-based development of the biostimulantterm·dom science that is grounded in the observation that the application of biological materials derived from various organisms, including plants, that have been exposed to stressors can affect metabolic and energetic processes in humans, animals, and plants (Filatov, 1951a,b). This hypothesis is based upon the premise that functional chemical communication occurs between individuals or organs that favorably modulate metabolic pathways and networks at different plant hierarchical levels. Inter and intra organism communication and consequent molecular Frontiers in Plant Science | www.frontiersin.org January 2017 | Volume 7 | Article 2049 YakhinpersName et al.

Biostimulantsterm·dom in Plant Science: A Global Perspective and metabolic regulation are at the heart of the science of systems biology and the tools of systems biology will inevitably be critical to the realization of mode of actionterm·dom of many biostimulantsterm·dom. Continued investments by commercial entities in biostimulantterm·dom research and product development will serve as a critical driver of discovery in this realm and will inevitably lead to the identification of novel biological phenomenon, pathways and processes that would not have been discovered if the category of biostimulantsterm·dom did not exist, or was not considered legitimate.

AUTHOR CONTRIBUTIONS All authors OY, AL, IY, PB, contributed equally to this reviewterm·mth.

ACKNOWLEDGMENTS We thank Russian Foundation for Basic Research (RFBR- Agidel No 02-04-97917), Russian Science Support Foundation, Foundation for Assistance to Small Innovative Enterprises in Science and Technology (“Start Program,” FASIE, Russia).