The establishment of the term ‘biostimulant’
The word 'biostimulant' appeared when it became evident that some products applied to plants were able to stimulate growth at low doses, which could neither be explained by the supply of nutrients nor by some plant protection against pests and pathogens. The pioneering work of two research teams should be mentioned here.
In the 1980s to early 1990s, at the School of Forestry and Environmental Studies of the Yale University, Professor G. P. Berlyn and his team studied the response of woody and grass species to bioactive substances - seaweed extracts, humic acids and vitamins - combined in a proprietary mixture named Roots™. Improvements in root and shoot growth, drought resistance and nitrogen use efficiency were reported (Russo and Berlyn, 1991). There are two remarkable things to be pointed out in this paper. First its title, 'The Use of Organic Biostimulants to Help Low Input Sustainable Agriculture', which, to the best of our knowledge, is the first to use the word 'biostimulant' in a peer- reviewed article. The scope of using biostimulants in agriculture is also far- reaching: low-input agriculture. The second thing is how the authors describe the action of their biostimulant product. After listing the bioactive ingredients of Roots, they propose that 'the innovation of mixing them and capitalizing on their synergistic effects is a real contribution in terms of agricultural production'. Whether the unique properties of biostimulant products rely on synergistic and/or emerging properties of blended bioactive compounds is an issue which we will cover later in this chapter. In a later article describing the effects of Roots on beans (Russo and Berlyn, 1992), the authors define biostimulants as 'nonnutritional products that may reduce fertilizer use and increase yield and resistance to water and temperature stresses', also saying that they 'stimulate plant growth in relatively small amounts'.
In the same period, another team led by Professor R. E. Schmidt - at Department of Crop and Soil Environmental Sciences, Virginia Polytechnic Institute and State University - performed similar studies on growth and stress responses of turfgrass treated by seaweed- and humic acids-based biostimulants. Although the word 'biostimulant' was used and defined by these authors in a popular web magazine in 1997 (du Jardin, 2015), they preferred using the wording 'metabolic enhancers' and 'hormone-containing products' in their peer-reviewed articles (Zhang and Schmidt, 2000). Regarding the action mechanisms of biostimulants, these authors concluded that PBs modify hormone homeostasis and stimulate antioxidant activities which play a major role in environmental stress responses (Zhang and Schmidt, 2000).
In a review paper dated 1993, Kinnersley introduces biostimulants as a category of phytochelates, which are organic compounds and substances promoting plant growth by chelating micro- and macronutrients (Kinnersley, 1993). Humic acids, components of many biostimulant products since that time and still today, enhance metal uptake - for example, magnesium as a constituent of chlorophyll, increasing photosynthetic capacity - and also protect the plant against metal toxicity, which are typical behaviors of phytochelates. The carboxyl groups of humic acids are proposed to be responsible fortheir chelating activity by Kinnersley (Kinnersley, 1993). The hypothesis of an iron-chelating activity of seaweed extracts was also one the reasons for the interest of Professor Schmidt and colleagues toward biostimulants, in the context of their work on iron nutrition in creeping bentgrass (R. Schmidt, personal communication).
In conclusion, this early literature on biostimulants paved the way for the later research and development, by highlighting some of their main characteristics: their action on plant homeostasis at low doses, their modulation of the plant stress response, their role in plant nutrition and growth promotion by acting on nutrients availability and uptake and the synergistic effects resulting from the combination of bioactive compounds from the same or different substances. These topics are still the agenda of many research teams studying and developing biostimulants (Calvo et al., 2014; du Jardin, 2015; Brown and Saa, 2015; Yakhin et al„ 2016).
Plant biostimulants as functional ingredients of fertilizing products
Today, PBs are applied to a wide range of crops, and in different ways, for the purpose of improving yield and quality, saving nutrients and water and making the crop more resilient to environmental stress (Fig. 1).
Plant biostimulants: a new paradigm for the sustainable intensification of crops
Figure 1 Application of biostimulant products to crop plants and claimed agricultural functions.
Biostimulants introduce a new paradigm in plant production. They appear as a relatively new category of agricultural inputs, which aims at acting on the plant's physiology in ways which are distinct from supplying nutrients or protecting the plants against potentially harmful organisms. Changes in plant physiology and development are targeted by breeding techniques and farming practices, and it is now realized that many plant traits are also amenable to modification through the external application of bioactive substances. In this respect, biostimulant products resemble existing technologies based on plant growth regulators, marketed as pesticides, but most biostimulants are biosourced materials with complex compositions, and their activity is deemed to be supported by many interacting bioactive constituents.
There is some analogy in the way plant nutrition and human nutrition are considered today. Human diets not only provide the body with the nutrients needed for growth and maintenance, but they also supply bioactive constituents which target specific physiological mechanisms, enhancing cell and organ homeostasis. This is achieved via protection against environmental damage and sources of, for example, antioxidants (including vitamins C and E, but also many phenolics), which are examples of such 'functional ingredients' of foods ameliorating human health and welfare. Once the activities of specific food constituent are identified, new products can be developed and marketed which combine relatively high amounts of the bioactive substances and are taken by human consumers as food supplements.
In a way, biostimulant products are food supplements of cultivated plants. But as with food supplements consumed by humans, the 'fertilizer supplements', or biostimulants, will be used by growers if the expected benefits are clear and supported by fair marketing practices, including adequate labeling, and by a sound regulatory framework. This emphasizes the importance of the claims as defined by the regulation in order to inform growers about the intended uses of biostimulant products. Let us now describe in more detail the claims included in the regulatory definition of biostimulants in Europe.
Improving nutrient use efficiency
Nutrient use efficiency (NUE) has been defined in many ways, depending on the crop and the scope of the study, but the word 'efficiency' always refers to a ratio between outputs and inputs. Both the outputs and inputs can be expressed using different variables: outputs can be grain yield or the amount of exported nitrogen, while the inputs can be the total nutrient content of the soil, its bioavailable fraction or the applied fertilizer. A review of available definitions and calculation methods of NUE can be found in Ussiri and Lai (2013).
In order to analyze the effectiveness of biostimulants in improving NUE, the following function is useful, as it dissects NUE into two components which are controlled by different plant traits and can be influenced by biostimulants according to distinct mechanisms:
Nutrient Use Efficiency = Uptake Efficiency * Utilization Efficiency where:
- • NUE is the dry mass (or yield) produced per nutrient input (gram total plant biomass or harvested biomass per gram nutrient input).
- • Uptake efficiency is the amount of nutrient taken up by the plant per nutrient input (gram nutrient uptake per gram nutrient input).
- • Utilization efficiency is the dry mass produced per nutrient taken up by the plant (gram total plant biomass or harvested biomass per gram nutrient uptake).
Taking phosphorus (P) as an example, three strategies for high P use efficiency have been distinguished (Richardson et al., 2011): 'root foraging strategy', that is, efficient capture of P by developing roots even in low-P soils; 'root mining strategy', that is, enhancing the desorption, solubilization or mineralization of P from sparingly available pools; and improved 'internal P-utilization efficiency' (higher yield per unit of P uptake). Each of the described strategies is related to specific plant traits, amenable to modification using biostimulants: upregulation of genes encoding membrane transporters for the uptake of nutrients (Ertani et al., 2017), promotion of root growth (Barone et al., 2018) and of root hairs development (Canellas et al., 2010) and control of the partitioning of nutrients between plant parts via cell membrane transporters (Billard et al., 2014).
Despite the evidence that biostimulants can modify several plant traits involved in NUE, difficulties persist. First, we still largely ignore which phenotypes best serve NUE, at both the developmental and biochemical levels, not to forget the progress made in some crop/nutrient models (Richardson et al., 2011; Lynch, 2013). In this research field, 'phenes' are defined as discrete units of the phenome, making up the phenotype, like genes are the discrete units of the genome making up the genotype. Advances in plant phenome research will benefit the development of biostimulant products, as they will improve the way in which biostimulants can be screened in the laboratory and moved to practical applications on the basis of known mechanisms of action. Second, it is likely more important from an NUE perspective to promote the responsiveness of plants and their roots to changing concentrations of nutrients in their environments, in time and space, than to promote any 'ideal' root architecture. This is more difficult to address in the laboratory, but the interplay of biostimulants with the regulatory gene networks well characterized in model plants is worth studying.
The third and last point is that few field experiments on biostimulants measuring NUE are described in the public literature (Storer et al., 2016). Indeed, most field studies only report on yield and other characteristics directly valued by the market (like grain protein contents). Yet the positive effect of some biostimulants on plant growth and tissue composition when applying a reduced amount of fertilizers (Carillo et al., 2019; Mola et al., 2019; Nguyen et al. 2019a,b) encourages their consideration for a 'high-output-low-input' agriculture.
Improving tolerance to abiotic stress
Tolerance to abiotic stress is a major concern when aiming at high crop productivity on a warming planet. In all cropping systems of the world, reducing the yield gap between the achievable and actual yields will depend on how farming practices and the genetic properties of plants can be improved to cope with the environmental stresses, like drought, salinity and heat. The magnitude of this yield gap is much different from one farmland to another across the world (Van Ittersum et al., 2013), but, whatever it is, improving tolerance to abiotic stress will be key to increasing crop productivity.
When the yield is threatened by pathogens and pests, farmers know how to deal with them by applying plant protection products, whetherthey are synthetic chemicals, natural substances or living organisms. The action of these products can be by direct toxicity toward the pathogen or its biological vectors, or by triggering the plant's immunity. Before the advent of PBs, there were few, if at all, solutions to reduce the abiotic stress suffered by the plant, at least in non- controlled growing conditions. Drought mitigation by irrigation is an important exception, but it cannot always be putin practice at affordable costs. Biostimulants appear as a breakthrough in crop protection against abiotic stress, and analyzing their potential in this respect seems to be of paramount importance.
Increased tolerance to abiotic stress is a claim of biostimulant products in all regulatory norms so far. However, quantifying their actual effects on abiotic stress is challenging. When dealing with stress response of an organism, three phases need to be considered; before, during and after the stress episode. Of special interest is the capacity of the organism to be 'primed' against abiotic stressors, which means that some physical, chemical or biological treatment 'before' stress improves the responsiveness of the organism toward subsequent stress application. Although such priming effects have been described since long in the context of both environmental and pathogen stresses, with concepts like hardening and immunity (Hilker et al., 2016), it is only recently that it was realized that the 'priming stimulus' and the 'trigger stimulus' (i.e. the stressor) could be the same or different (Jakab et al., 2005). Interestingly, some chemical treatments are able to prime the organism against a pathogen or abiotic stress, despite any resemblance of the priming molecules to known physiological effectors of the plant (Sawides et al., 2016). When such compounds, substances or microorganisms are developed into commercial products ameliorating plant tolerance to abiotic stress, they may be referred to as PBs. In fact, this appears as a most promising marketing opportunity for PBs, as it complements all other technologies aiming at increasing crop productivity under stress and provides growers with a tool to reduce yield penalties whenever stressful events can be anticipated, like heat and drought episodes.
As an interesting case study, the priming activity of three types of biostimulants in the protection of Arabidopsis thaliana against drought stress was studied by Fleming et al. (2019), using phenotypic, biochemical and gene expression analyses. This study emphasizes the importance of applying the biostimulant at the right time with respect to the stress episode and at right doses, showing enhancement of drought tolerance in specific conditions only. Most notably, seaweed extracts proved to be effective as priming stimulus, when applied one week before stress, and ineffective when the stressor and the biostimulant were applied together or when the biostimulant was applied after the onset of stress. In contrast, an amino acid-based biostimulant tested by the same study showed a less stringent timing requirement for efficacy and did help the plants even when applied together with or during the stress (Fleming et al., 2019).
The timing of application of the biostimulants is critical, and their roles in stress tolerance might be described as 'defense primers' (acting before stress), 'rescuers' (acting during stress) or 'restorers' (acting after stress). As an example of enhanced restoring activity, Dalai et al. (2019) have shown the contrasting responses of two biostimulants derived from seaweed extracts and amino acids in pepper and have shown that the latter did improve the resilience of the plants subjected to drought, indicated by the faster water reabsorption activity of roots after stress withdrawal.
Although such studies are scarce, they can explain the 'inconsistent' efficacy of biostimulants observed in the field by some growers. 'Inconsistency' actually reflects insufficient knowledge of the biostimulation mechanisms as well as of the stress experienced by the recipient plants. This shortcoming prevents from delivering sound advice to growers on the application conditions of biostimulants, raising skepticism among them.
Furthermore, molecular studies have shown that plants respond to the combination of stress factors, like drought and heat, in a unique way, different from the addition of the responses to each individual stress (Mittler and Blumwald, 2010). Since plants experience multiple stressors simultaneously in the field, the extrapolation of laboratory experiments applying single stress to the performance of crops in their actual growing conditions can be problematic.
In conclusion, biostimulation is a promising technology to increase yield in adverse environments, but, as with gene technologies, our limited understanding of plant stress physiology, due to the complexity of the plant's response and the resulting methodological challenges, seems to be a major bottleneck to implement efficient strategies in crops. Our capacity to monitor abiotic stress in the field needs to be improved as well in order to determine the suitable timings of application and to quantify the benefits of biostimulant products.
Improving crop quality
Crop quality refers to specific characteristics of agricultural and horticultural products, which impact their market value and are targeted by breeding and growing practices. Some of the quality traits are closely related to the nutritional characteristics of the plant, such as the protein content in grains under the control of N nutrition, or to abiotic stress tolerance, such as post-harvest chilling injury in fruits controlled by antioxidant metabolism. Hence, there is some overlap between the first three claims introduced by the European regulation on FPs: improvements in NUE, tolerance to abiotic stress and crop quality.
Considering the many cellular targets of PBs and the cross talk between biostimulants, phytohormones and other plant gene regulators, a wide range of effects in a wide range of crops can be anticipated. For example, the effects of the foliar application of biostimulants on fruit tree physiology have been reviewed by Tanou et al. (2017). In vegetable and fruit crops, contents in secondary metabolites are important quality traits. Applications of A. nodosum seaweed extracts have been shown to increase the tissue contents of phenolics and flavonoids in woody and non-woody crops (Frioni et al., 2018; Lola-Luz et al., 2013,2014a,b).
Although post-harvest biology is an important aspect of the market value of vegetable and fruit crops, little attention has been paid so far to the capacity of biostimulants to improve the shelf-life of crop products. An exception to this is chitosan, a deacetylated derivative of chitin, which has been shown to protect fruits from post-harvest damage, resulting from both biotic and abiotic stress, from ageing caused by the oxidative attack and from deterioration due to water loss (Romanazzi et al., 2017; Sharma et al., 2016). However, for chitosan, like for other biopolymers used as biostimulants, the molecular mass profile of the breakdown products of the polymer has a strong influence on their bioactivity, and film-forming properties of high molecular mass chitosan need to be distinguished from the cellular effects of oligochitosans (Malerba and Cerana, 2016; Kerch, 2015; Dzung et al„ 2017). Processing of the biopolymer needs to be tailored to its marketing claim.
Improving the availability of confined nutrients in the soil or rhizosphere
A fourth claim was added to the definition of biostimulants by the European regulation on FPs during the parliamentary process, which is an improvement in the 'availability of confined nutrients in the soil or rhizosphere'. It is so far unclear which mechanisms will be referred to by this claim, and clarification is expected from the future European standards supporting the implementation of the regulation. Anyway, there seems to be a clear overlap with the first claim of the definition bearing on NUE, as increased bioavailability of nutrients in the soil and rhizosphere can be regarded as one of the components of the uptake efficiency of nutrients, which is itself a component of NUE, as explained before.
As a matter of fact, both microbial and non-microbial biostimulants are known to influence the bioavailability of nutrients, via their chelating/ complexing activities and via the biochemical activities of microbes releasing sequestered soil nutrients. Phosphate-solubilizing bacteria and fungi are important examples of such microorganisms, which liberate soluble phosphates from insoluble organic and inorganic forms of phosphate, accounting for most of the total P content of the soil (Richardson et al., 2011; Owen et al., 2015).