These objectives are in line with many countries policies, like the ‘Farm to Fork’ strategy in EU, addressed to reduce the use of chemical inputs and the consumer’s demand of higher quality and safer food. The need to reduce the use of agrochemical inputs in cropping system, together with the need to preserve soil fertility and increase crop productivity, requires a better exploitation of natural resources and a more efficient use of fertilizers and other inputs. Biostimulants can contribute to the achievement of these objectives with variable levels of efficacy depending to the interactive effects among biostimulant product, genotype and environment. Therefore, the correct choice of biostimulant product, rate, timing and method of application needs to consider the objective to be pursued, the cultural practices used and the pedo-climatic conditions in which the crop cycle takes place. The agronomic and environmental benefits that can be obtained with the use of biostimulants need to be translated into positive economic results for the farmer in order to justify biostimulant use.

Identification of environmental factors limiting crop productivity

The knowledge of the factors limiting the crop productivity in the growing site represents the starting point for identifying the potential benefits of biostimulant application. The identification of the factors limiting crop productivity must be carried out in the planning phase of the crop cycle through an in-depth analysis of the pedo-climatic conditions of the site, the use of historical series of meteorological data and soil analysis. These information are usefull to identify the limiting factors of crop productivity at soil (e.g., salinity) and climate level (e.g., sub- or supra-optimal temperature), and to establish management strategies addressed to mitigate the crop stress with biostimulant applications. However, this initial assessment of crop limiting factors has many margins of error for climatic factors, and therefore it is necessary a more precise assessment of climatic conditions during the crop cycle throught real time weather forecasting and in-situ weather stations. Weather forecast models are particularly useful to predict stress conditions and to plan biostimulant application before the stress event for activating the defence mechanisms of plant against stress. Monitoring the morpho-physiological crop traits using remote sensing techniques can be helpful to highlight a stress condition on the crop especially when the stress intensity is such that it results in no visible symptoms, and to evaluate post-stress recovery of plants. Finally, it should be noted that crops under field conditions are often subjected to multiple stresses caused by several environmental factors which can act simultaneously or consecutively, amplifying the negative effects of stress factors on crop. For instance, in hot-arid climates multiple stresses caused by supra-optimal temperature and drought or by salinity and drought in non-irrigated crops are very common.

Fig. 1. High-throughput phenotyping platform of Arcadia s.r.l., Spin-off Company approved by Tuscia University, at Experimental Farm of Tuscia University, Viterbo, Italy (www.arcadia.expert).

How to chose the biostimulant

The choice of microbial and/or non-microbial biostimulant for achieving a specific objective (e.g. increase the crop resistance to one or more environmental stress factor/s, enhance the nutrient use efficiency, improve one or more crop quality trait/s) requires a proven knowledge of the biological activity of the product under similar growing conditions. This information can be available in technical reports, databases, technical and scientific articles, and specific books where the results of agronomic trials are reported. The technical reports can be provided directly by the biostimulant manufacturers while articles and book chapters can be found in web search engines some of which are accessible for free (e.g. https://scholar.google.com) while others can be consulted paying a fee (e.g. https://www.scopus.com). Studies conducted on biostimulants are more useful when they consider not only agronomic aspects (e.g. crop yield) but they also include insights to understand the mode of action of the products and the interactive effects with other biostimulants and chemical inputs (e.g. fertilizers, pesticides). When the selected biostimulant is applied together with other biostimulants or chemical inputs it is necessary to verify that the applied products interact sinergically or at least in an additive way. For istance, Rouphael et al (2017) found that application of two biostimulants (root application of an inoculum of mycorrhizal fungi and foliar applications of a vegetal-derived protein hydrolysate) on lettuce plants resulted in a synergistic interaction with an increase in the shoot fresh weight that was greater (+33%) than the sum of the effects caused by the  application of the single biostimulant alone (+16% with root application of inoculum of mycorrhizal fungi or +7% with foliar application of vegetal-derived protein hydrolyzate).

Interestingly, positive interactions have also been found between biostimulants and pesticides. For istance, the negative effects of herbicide (phytotoxicity and growth depression) on sunflower plants were reduced when the plants were foliarly spray with a combination of protein hydrolysate and  imazamox-based herbicide (Balabanova et al., 2016). Regarding mineral nutrition, various scientific works have highlighted positive interactions between biostimulants and fertilizers resulting in an improvement in uptake, translocation and assimilation of nutrients in plant. For instance, Colla et al. (2017) found an increase of leaf potassium concentration following foliar treatments with vegetal-derived protein hydrolyzate on greenhouse tomatoes while Celletti et al. (2020) reported that the same vegetal-derived protein hydrolysate increased the leaf iron concentration of tomato plants grown in hydroponics.

Negative interactions among biostimulants are typical of some microbial biostimulants;  microorganisms can compete with each other and perform reciprocal inhibition actions through antibiosis and/or mycoparasitism. The saprophytic fungus Trichoderma harzianum, for example, is well recognized for inhibiting arbuscular mycorrhizal fungi when applied to the roots (Cardarelli et al., 2016).

The above findings highlight the importance of an accurate evaluation of biostimulant activity  considering the target effects on crop. In this regard, high-throughput phenotyping platforms under controlled environment allow a precise reproduction of specific conditions of stress and/or nutrient availability and to accurately verify the effects of the products on various crops through the analysis of images acquired during the crop cycle (Figure 1).  

The biostimulant choose also depends on the available formulations which must simplify the field application using the common farm equipments. For this reason, biostimulant manufacturers have developed many formulations as powder, granular, liquid and tablet form for application to seeds, substrate in the nursery, and soil in the field, through foliar spray, irrigation system and root dipping. Many fertilizer formulations are also available in the market containing biostimulants and mineral nutrients obtained by mixing the components or complexing reactions (e.g. biochelates containing cationic nutrients complexed by peptides). These formulations allow nutrient supply and biostimulation of plants to be carried out in a single step, also favoring synergistic effects on plant nutrition between the bioactive components and the nutritive elements.

When to apply the biostimulant

The time of biostimulant application depends on the target effect, biostimulant type, crop and environmental conditions. Microbial biostimulants such as those based on inocula of mycorrhizal fungi are preferably applied once on the propagation material or in the early stages of the crop cycle while biostimulant substances are often applied repeatedly following different approaches: a) calendar application; b) application at specific crop phenological stages; c) application before, during and/or after adverse meteorological events. Calendar applications of biostimulants are preferable when the crop experiences sub-optimal conditions for most of the growing cycle, such as in the case of sub-optimal radiation and temperatures during autumn-winter-spring cropping cycles under unheated greenhouses or in the case of saline soils. However, this approach is economically feasible only for high-value crops such as vegetables or flower and ornamental species under greenhouse conditions. For long-cycle crops (e.g. wheat), where the low profitability makes economically unsustainable to carry out multiple applications of biostimulant substances, it is recomended to prioritize the biostimulant applications in the critical crop stages such as germination, flowering and fruit enlargement. Biostimulant treatments can be limited to specific phenological phases when it is necessary to promote a specific plant trait like rooting with the early applications of biostimulants or fruit set and quality with late applications of biostimulant. In the case of occasional abiotic stress (e.g sudden drop in temperatures), it is better to apply the biostimulant few days before the stress in order to activate the plant’s endogenous defenses. Recent studies carried out by Luziatelli et al (2016) have also demonstrated that foliar applications of biostimulant substances are able to stimulate natural occurring epiphytic bacteria which are able to promote plant growth and resistance to stress. After the stress event, biostimulant applications can be useful to promote a fast crop recovery. In herbaceous and vegetable crops, biostimulant substances are often applied together with pesticides for saving time  and mitigating pesticide stress on crop. However, it should be noted that before mixing different products, it is necessary to verify their compatibility and the lack of phytotoxic effects of the product mixture on crop.

Fig. 2. Ion mobility qTOF in the laboratory of lipidomics and metabolomics at oloBion S.L., Ion mobility qTOF in the laboratory of lipidomics and metabolomics at oloBion Tarragona, Spagna (www.olobion.ai) Ion mobility qTOF in the laboratory of lipidomics and metabolomics at oloBion.

Evaluation of the biostimulant performances at farm level

Biostimulant performances need to be evaluated from agronomic, economic and environmental point of view.

For evaluating the agronomic benefits of plant biostimulants under field conditions, it is necessary to measure the most interesting crop traits (e.g. germination, yield) in the biostimulant-treated crop and to compare them with the values obtained from untreated crop grown under similar environmental conditions. For microbial biostimulants, the measurements of selected crop traits should always be accompanied by laboratory analysis for verifying the establishment of the applied microorganism/s (e.g. root colonization analysis for mycorrhizal fungi). Field measurements of crop traits can be performed to quickly assess the activity of a plant biostimulant. For istance, portable instruments can allow to estimate non-destructively the chlorophyll concentration (e.g. SPAD 502) and also flavonols and anthocyanins (e.g. Multi-pigment-meter) of leaves. Leaf chlorophyll concentration is a good indicator of plant health while the ratio of chlorophyll and flavonoids (so-called nitrogen balance index) is used for assessing the nitrogen nutrition status of plants. Field measurements of leaf chlorophyll fluorescence can be performed with a portable fluorometer. The chlorophyll fluorescence data are used for determining the maximum efficiency of photosystem II (Fv/Fm); leaf Fv/Fm values of a plant in good physiological condition should range between 0.79 and 0.84 depending on the plant species. Chlorophyll concentration of leaves is a more sensitive stress marker than the Fv/Fm ratio, which varies only under severe stress conditions. For evaluating the effectiveness of a biostimulant in mitigating water stress on crop, it can be useful to monitor the degree of stomata opening in the leaves (stomatal conductance) using a portable porometer and the leaf water potential using a pressure chamber. Biostimulant effects on plant nutrient uptake can be monitored with portable instruments which allow to measure in the field nitrates, potassium, calcium or sodium on leaf petioles; the resulting values can be compared with literature data or with data obtained from untreated plants of the same field. Remote sensing can also be used for crop growth monitoring and rapid detection of plant stress on large areas. Various vegetation-related characteristics, including biochemical properties (e.g. pigments, water content) can be derived from the spectral imaging. One of the most popular vegetation indices derived from spectral imaging is the Normalized Difference Vegetation Index (NDVI) which can be used as indicator of vegetation health.

Besides field measurements of crop traits, it is usefull to carry out laboratory analysis of specific stress markers (e.g. malondialdehyde concentration as an indicator of peroxidation of cell membrane lipids; activity of enzymes of the antioxidant defense system) in plant tissues for a better evaluation of plant’s response to the biostimulant treatment under stress conditions. A better understanding of the biostimulant activity and the related mechanisms of action at the molecular level can be achieved through metabolomics analyses, which allow the characterization of the set of metabolites present in plant tissue (Figure 2). The comparison between the metabolites in plant tissue from plants treated with biostimulant and those in plant tissue from untreated plants allows to identify the metabolic pathways affected by the biostimulant treatment. For example, Bonini et al. (2020) reported that inoculation with mycorrhizal fungi (Rhizoglomus irregularis BEG72 and Funneliformis mosseae BEG234) and Trichoderma koningii TK7 increased by 24% fruit yield of greenhouse pepper in comparison with untreated control; metabolomic analysis on leaf tissues showed that the mycorrhizal-mediated fruit yield increase was associated with changes in hormonal balance (auxins, gibberellins and cytokinins) and in the secondary metabolites (carotenoids, saponins and phenols).

Metabolomic analysis on edible product also allows to determine if the biostimulant causes an improvement in the nutritional quality of the product.  

Economic analysis is essential to evaluate the convenience of applying a plant biostimulant. Biostimulant applications can increase farmers’ profitability by enhancing the marketable yield, by improving the quality traits of product affecting its selling price or by reducing the production cost due to a lower inputs’ requirement (e.g. fertilizer). Such effects can occur one by one or even jointly.

For evaluating the convenience of applying a plant biostimulant, it is necessary to consider the cost of biostimulant in terms of use (purchase and distribution) and effect (variation in the harvested yield and in the releated variable costs). The above data are used to calculate the gross margin as the difference between the production value and the costs for raw materials, services and labor. The difference between the gross margin with or without the application of biostimulant allows to evaluate the economic convenience of plant biostimulant use. Coletta (2019) reported several case studies where applications of plant biostimulants resulted in significant increases of gross margins especially for high-value vegetable crops.

The use of biostimulants in agriculture can lead to a reduction in the environmental impact of production process by reducing greenhouse gas emissions, conventionally expressed as CO₂ equivalent (carbon footprint), as well as water consumption and the land use associated with a given quantity of product. The improvement of the environmental impact indicators associated with the use of biostimulants can derive from an increase of marketable yield using the same level of inputs (e.g., irrigation water, fertilizers, energy), from a reduction of inputs for achieving the same marketable yield or from a simultaneous increase in marketable yield and reduction of inputs. The quantification of the environmental benefits induced by biostimulant application on cropping systems can be obtained with the Life Cycle Analysis (LCA) methodology applied to the primary production phase “from the cradle at the gate”. This methodology was used by Rajabi et al. (2020) in two case studies under greenhouse conditions. In the first case study, the root application of inoculum of arbuscular mycorrhizal fungi on zucchini crop reduced greenhouse gas emissions by 7.6%, expressed as CO₂ equivalent per ton of fruits, in comparison with untreated control while in the second case study the foliar applications of vegetal-derived protein hydrolysate on spinach crop significantly reduced greenhouse gas emissions by 13.5% kg of CO₂ equivalent per ton of leaves compared to the untreated control. These results were attributed to the positive effect of biostimulant applications on crop yield. The reduction of greenhouse gas emissions associated with the biostimulant-treated product can be used for green marketing initiatives aimed at promoting sustainable consumption leading to a competitive advantage for the company both in terms of income and reputation.

Conclusions

Plant biostimulants represent a good opportunity to increase crop yield especially under environmental stress, to improve product quality and to increase resource use efficiency. The use of plant biostimulants is growing worldwide and every day new biostimulants appear in the market. Nevertheless, the choice of biostimulants to be applied is often made empirically without an in-depth knowledge of the product characteristics and performances. This approach can lead to variable results that are not always optimal causing economic losses for the farmers. For maximizing the benefits of  biostimulant applications, it is therefore necessary to use a technical-scientific approach which includes the definition of the objectives to be pursued with the biostimulant application considering the factors potentially limiting crop productivity in the growing site, the identification of the best performing products on the basis of results from agronomic trials conducted in similar growing  conditions, and the definition of biostimulant application strategies considering the characteristics of the product and the needs of the crop. Monitoring the morpho-physiological and productive traits of the crop can be useful for verifying the biostimulant activity under field conditions, while laboratory analysis can highlight the biostimulant effects at molecular level and on the nutritional quality of the product. Finally, the evaluation of the convenience of using a biostimulant needs to consider a cost-benefits analysis associated with the biostimulant application and the quantification of any benefits arising from the use of biostimulant in terms of environmental impact mitigation of the production process.