PERSPECTIVE

published: 05 April 2016

doi: 10.3389/fpls.2016.00435

A Systematic Approach to Discover

and Characterize Natural Plant
Biostimulants
Giovanni Povero *, Juan F. Mejia, Donata Di Tommaso, Alberto Piaggesi and
Prem Warrior
Global R&D Department, Valagro SpA, Atessa, Italy

The use of natural plant biostimulants is proposed as an innovative solution to address

the challenges to sustainable agriculture, to ensure optimal nutrient uptake, crop
yield, quality, and tolerance to abiotic stress. However, the process of selection and
characterization of plant biostimulant matrices is complex and involves a series of
rigorous evaluations customized to the needs of the plant. Here, we propose a highly
differentiated plant biostimulant development and production platform, which involves
a combination of technology, processes, and know-how. Chemistry, biology and omic
concepts are combined/integrated to investigate and understand the specific mode(s)
Edited by: of action of bioactive ingredients. The proposed approach allows to predict and
Michael A. Grusak, characterize the function of natural compounds as biostimulants. By managing and
Children’s Nutrition Research Center,
analyzing massive amounts of complex data, it is therefore possible to discover, evaluate
Agricultural Research Service, United
States Department of Agriculture, and validate new product candidates, thus expanding the uses of existing products to
USA meet the emerging needs of agriculture.
Reviewed by:
Ebrahim Hadavi, Keywords: abiotic stress, Ascophyllum nodosum, biostimulants, crop growth and development, plant nutrition,
seaweeds
Islamic Azad University of Karaj, Iran
Marta R. M. Lima,
University of California, Davis, USA
*Correspondence:
INTRODUCTION
Giovanni Povero
g.povero@valagro.com One of the biggest challenges for agriculture is the development of sustainable and environmentally
friendly systems to address the need to feed the growing world population. With decreasing area of
Specialty section: arable land as we approach the limits of genetic potential of staple crops, the only way to achieve this
This article was submitted to objective is by increasing the crop yield and protecting what we produce. In other words, produce
Plant Nutrition, “more with less” (Food and Agriculture Organization of the United Nations [FAO], 2012; Carvalho
a section of the journal and Vasconcelos, 2013; International Food Policy Research Institute [IFPRI], 2014). Parallel to
Frontiers in Plant Science
this, reducing energy consumption and utilizing resources more efficiently should be priorities
Received: 11 January 2016 (Gregory and George, 2011). Simultaneously, quality of crops should be enhanced, particularly
Accepted: 21 March 2016 under unfavorable growing environments. This means obtaining higher incomes for farmers,
Published: 05 April 2016
having better postharvest storage and more nutritious food for consumers (Eckardt et al., 2009).
Citation: One of the most innovative and promising solutions to address these important challenges
Povero G, Mejia JF, Di Tommaso D,
consists of the use of plant biostimulants (PBS), referred as “materials which contain substance(s)
Piaggesi A and Warrior P (2016)
A Systematic Approach to Discover
and/or microorganisms, whose function when applied to plants or the rhizosphere is to stimulate
and Characterize Natural Plant natural processes to enhance/benefit nutrient uptake, nutrient efficiency, tolerance to abiotic stress,
Biostimulants. Front. Plant Sci. 7:435. and/or crop quality, independent of its nutrient content” (European Biostimulant Industry Council
doi: 10.3389/fpls.2016.00435 [EBIC], 2016).

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Povero et al. Approach to Study Plant Biostimulants

Plant biostimulants formulations are generally proprietary and chemical characteristics of raw materials such as seaweeds,
compositions based on seaweed extracts, complex organic microorganisms and their metabolites, plant extracts, is needed
materials, plant hormone-like compounds, amino-acids, and to identify, characterize and preserve specific active ingredients
humic acids. Extensive reviews discuss the large group of PBS that can help achieve the targeted physiological responses in
derived from seaweeds, in particular Ascophyllum nodosum plants. Thus, it becomes crucial to choose the right time and
(Khan et al., 2009; Craigie, 2011; Calvo et al., 2014; Sharma et al., season to obtain -from raw materials- the most optimal yield
2014) and the beneficial effect of natural biostimulants on specific of specific biomolecules needed for researched activities (Parys
aspects of plant growth, production and fruit quality in different et al., 2009; Apostolidis et al., 2011). A typical example of this
crops (Parad̄iković et al., 2011, 2013; Bulgari et al., 2015; Saa approach is the use of Ascophyllum nodosum (L.), one of the
et al., 2015). Specific PBS activities such as increased root and most researched seaweeds (Ugarte et al., 2006), and one of
shoot growth, tolerance to abiotic stress, water uptake, reduction the raw materials utilized in the GeaPower technology. It is
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of transplant shock etc., have been also reported (Adani et al., recognized as the dominant intertidal seaweed of the North
1998; Parrado et al., 2008; Alam et al., 2014; Petrozza et al., Atlantic coastline where water temperatures do not exceed 27◦ C
2014). Biostimulants can also reduce fertilizer use and nutrient (Keser et al., 2005), although this alga is known to grow under
solution concentration in hydroponic systems (Vernieri et al., extreme temperatures, from −20◦ C in winter to air temperatures
2006). A summary of the beneficial effects of PBS is reported in of 20–25◦ C and direct sun heating in summer (Strömgren,
Figure 1. 1983).
Considering the above, we expect a critical role for Ascophyllum can be collected in Norway, where this macroalga
biostimulants in the agriculture of the future. The market is exposed to 6 months of darkness during polar night, but
of biostimulants is estimated to worth $1,402.15 million in also subjected to high solar radiation in spring, especially
2014, and is projected to reach $2,524.02 million by 2019, during low tide and high water transparency, leading to
at a Compound Annual Growth Rate (CAGR) of 12.5%. The strong oxidative stress due to the formation of reactive oxygen
expected drivers of this growth include (i) growing importance species (ROS) induced by environmental factors (Dummermuth,
for organic products in the agriculture industry (ii) increase 2003; Di Tommaso, 2012). These extreme conditions confer
of biostimulants application in developing countries (iii) more pliability, elasticity, ability to conform to the flow, and
global PBS presence and acceptance among customers, since influence the chemical composition of this seaweed, as a
the players in this market have developed a range of innovative consequence of the exposure degree (Black, 1948, 1950;
products to satisfy specific crop needs (Biostimulant Market, Biber, 2002; La Barre et al., 2004). Careful selection and
2014). harvest of algal materials with select biological attributes thus
While the knowledge on the benefits of PBS on plants is becomes extremely critical to developing select biostimulant
steadily improving – evidenced by a significant increase of preparations.
research papers focused on PBS – there has been little attention to
the critical scientific steps required for an optimal selection and
characterization of biostimulant compounds, based on chemical Extraction, Chemical/Biological
and biological analyses to develop optimal solutions for specific Characterization, and Prototyping
agronomical needs. Customized extraction processes are required to maintain a
Here we propose a robust platform that we named precise ratio of each ingredient in complex natural mixtures
GeaPower , based on different research approaches and
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thus assuring the efficacy, quality, and consistency of the final
a combination of technology, know-how and processes products.
consolidated over a decade of experience aimed to investigate Extraction procedures are calibrated in order to selectively
and develop effective PBS products. isolate categories of chemicals specific for the intended use,
utilizing appropriate solvent mixtures, pH, temperature and
eventually enzymes to drive the process. The challenges and
FROM RAW MATERIALS TO hurdles in these procedures have been very well described by
Harborne (1984). Hou et al. (1997, 2000) described methods
BIOSTIMULANTS: A STEP-BY-STEP
to isolate alginate-containing fractions, pigments, proteins, and
PROCEDURE sulphated polysaccharides, including specific processes for the
highest possible yield. Enzyme digestion procedures can also
When the Process Begins: Definition and be used in several processes to shorten the polymer length, for
Analysis of an Unmet Need example in proteins, but also in polysaccharides, resulting in
The first step that triggers the research platform for the study of enhanced biological activity and bioavailability (Jiménez-Escrig
new PBS formulations is the definition of a specific agricultural et al., 2011).
need. The study of the desired attribute along with a thorough Further, the natural extracts must be analyzed qualitatively
review of scientific literature in consultation with scientific and quantitatively for actives that they may contain. According
experts in the research field of interest, allows us to draw a list of to the specific molecule (or family of molecules) that needs to
the natural sources or active ingredients that may be included in be analyzed, one may choose liquid chromatography, such as
a future prototype. An in-depth understanding of the biological HPLC-DAD-FLD, LC-MS-MS, Q-ToF, or gas chromatography,

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Povero et al. Approach to Study Plant Biostimulants

FIGURE 1 | Reported examples of the main effects and physiological actions played by plant biostimulants (PBS).

with GC-MS. The first step is the identification of the compounds be used. Simultaneously, a primary evaluation of the formulation
of interest, using qualitative techniques, such as GC-MS, LC- for the safety profile should be performed. The technology to
MS-MS, Q-ToF. After this challenging step, the development realize prototypes in liquid, emulsion, microgranular and powder
and validation of analytical methods for each active ingredient form is required to address the market needs; often these could be
to be quantified enables the appropriate separation of the the greatest challenge in biological formulations and is generally
biomolecules of interest from other molecules present in considered proprietary information at the manufacturer level.
the background, thus allowing the realization of calibration Standard shelf-life and stability trials including the accelerated
curves and quantification, ensuring minimal batch-to-batch aging conditions, in warm (45◦ C) and cold environments
variation. (+4, −4, and −20◦ C) on each prototype in the final packaging
If, for example, we consider an alkaline extraction of are carried out. Following these evaluations, a detailed profile of
seaweeds, alginates will be partially converted to carboxylic the active ingredients evolution over time and changes in physical
acids (Niemela and Sjostrom, 1985) and their identification and parameters of each prototype under each aging condition is
quantification can be carried out by GC-MS; during the same performed to develop recommendations for appropriate shelf-life
extraction procedure, polyphenols, such as phloroethols, will be and storage conditions.
rearranged as complex dibenzofurans (Ragan and Glombitza,
1986), and HPLC-DAD will be a good analytical tool to separate, Biological Screening
qualitatively identify and quantify them against a suitable internal Chemical analyses are further substantiated by biological assays
standard. Fucoidans will undergo hydrolysis in oligomers and that characterize the composition at different levels, including
constituent sugars. After complete hydrolysis and derivatization, the physiological mechanisms activated by specific compounds.
each monosaccharide can be quantified with HPLC-DAD. This is essential to enrich the internal library, a proprietary
Betaines presence can be quantified with LC-MS-MS (Blunden database including all the information about molecules of
et al., 2010), and the structure of native phlorotannins can be interest, to link each component to a specific function and use
elucidated by NMR (Parys et al., 2007), but also by Q-ToF this information to better set prototype formulations according
(Tierney et al., 2013). to the need.
Once the desired combination of active ingredients is defined, It has become increasingly evident that understanding the
it is very important to check and match the regulatory guidelines functional links between genes/transcripts, proteins, metabolites
on the different crops and geographies where they are intended to and nutrients is one of biology’s greatest challenges, and recent

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Povero et al. Approach to Study Plant Biostimulants

technological improvements have brought major advances in

this area (Carvalho and Vasconcelos, 2013). Functional genomics
presents a powerful tool that allows us to decipher the molecular
and physiological triggers for specific responses in plant systems
(Bouchez and Höfte, 1998). DNA microarrays are clear examples
of functional genomic application: a high-throughput technology
that allows rapid and quantitative measurement of parallel
expression of thousands of genes (Aharoni and Vorst, 2002).
The transcriptomic profiling provided by microarrays datasets
can generate a picture of cellular functions under a given
experimental condition (Schena et al., 1995; Tan et al., 2009). In
the field of PBS also, the use of microarrays has allowed us to
dissect the effects of PBS at transcriptomic level, highlighting the
ability of raw materials that are used to formulate PBS to induce
the expression of various sets of genes. By using molecular tools,
it is possible to hypothesize possible modes of action of different
substances, predicting their role as biostimulants (Santaniello
et al., 2013).
Besides the so called transcriptomic fingerprint released by
a microarray analysis, a parallel and/or subsequent qPCR study
is often needed in order to validate the data, starting from a
microarray dataset or de novo selection of target genes involved
in specific physiological processes (Morey et al., 2006). For
example, to screen the effect of a set of different seaweed-
based prototypes, we selected a set of tomato genes involved
in different biochemical pathways that can be used as markers
for the qPCR screening (Figure 2A). If one or more gene
markers display a significant differential expression after the
application of a prototype in comparison with untreated control,
then it is possible to hypothesize a role of the prototype in the
physiological process(es) in which the gene(s) is/are involved.
For instance, this can suggest a possible priming effect of treated
crops against distinct stresses (Figure 2A). More generally, the list
of genes can be chosen and/or extended according to the needs
and/or desired results, and also the experimental conditions (e.g.,
normal vs. stressful conditions) can be chosen based on the
target.
In parallel with genomics, the phenomic approach permits the
study of PBS on plant growth, performance, and composition
based on multi-spectrum, high-throughput image analysis to
detect morphometric and physiological parameters (Furbank and
Tester, 2011). Such multi-spectrum analysis (infra-red, visible,
and ultraviolet light) of reflected or re-emitted light from the
plant crown, stem and leaves provides information on the
nutritional, hydrological and physio-pathological state of plants,
as well as on a plant’s ability to absorb light (Petrozza et al.,
2014, Figure 2B). One example of a phenomic facility is the high-
throughput plant phenotyping platform (LemnaTec-Scanalyzer
3D system) placed at the ALSIA Centro Ricerche Metapontum FIGURE 2 | Genomic, phenomic, and in vitro platforms to screen the
Agrobios s.r.l. (Matera, Italy; Figure 2B; Petrozza et al., 2013). effect of a set of different natural extracts-based prototypes using
tomato (Solanum lycopersicum L.) cv. Microtom as plant model.
The value of an integrated molecular/phenomic analysis (A) Relative abundance of mRNA transcripts of markers for specific
in characterizing the role of a specific PBS was recently physiological processes in response to a range of treatments with biostimulant
demonstrated in relation to the application of Megafol , a natural
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prototypes (Protot.). Transcript levels (signal intensities) are presented in the
PBS, on tomato plants in order to show the reduction of drought- form of a heat-map (using HeatMapper Plus Tool), on a color scale between
low (yellow) and high (red). (B) LemnaTec-Scanalyzer 3D System placed at
stress related damage (Petrozza et al., 2012, 2014).
ALSIA – Metapontum Agrobios Research Center (Matera, Italy).
Besides omics, in vitro assays may also be useful tools to (Continued)
speed up the process of preliminary screening. Here, plants

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Povero et al. Approach to Study Plant Biostimulants

FIGURE 2 | Continued Product Development: Phytotoxicity

The physiological and morphometric parameters that can be measured Assessment, Field Testing, REACh
using plant phenomics are UV (Ultraviolet) to fluorescence, photosynthesis
and health index; RGB (Red-Green-Blue) to plant morphology, architecture,
Compliance
digital biomass and green/yellow index; NIR (Near-Infrared) to plant water Even if certain prototypes display good efficacy in respect to the
content. (C) Comparison of UTC (untreated control) and PBS-treated (final initial need, other crucial factors need to be considered before
concentration of 0.5 mL/L) plants grown on agar-based medium. (D) Root releasing a new commercial product, in particular:
phenotype differentiation, based on imaging analysis software GiA Roots
(Galkovskyi et al., 2012), comparing UTC (untreated control) with PBS-treated
Phytotoxicity
(final concentration of 0.5 mL/L) plants. Traits displayed are: Average root
width (Width), Bushiness (Bush), Network Depth (Ndepth), Aspect ratio (AspR), In order to assess any negative/toxic effects of the selected
Network length distribution (Ldist), Major Ellipse Axis (MajA), Maximum number prototype on plants, several phytotoxicity tests are carried out on
of roots (MaxR), Network width (Nwidth), Median number of roots (MedR), different target crops, using a large range of rates of application.
Minor Ellipse Axis (MinA), Network Area (NwA), Network Convex Area (ConvA),
Network perimeter (Perim), Network solidity (NS), Specific root length (SRL),
Field Trials
Nsurf (Network surface area), Network length (Nlen), Netwok volume (Nvol) and
Network width to depth ratio (NW/D). The error bars indicate the standard error Once the agronomic performance of a certain prototype is
of the mean. determined, it is critical to validate them through a number
of trials under field conditions. In order to ensure robust
and statistically significant results, the efficacy of prototypes
should be accurately verified on target crops worldwide. Thus,
are germinated under sterile conditions in petri dishes, flasks, prototypes are tested in varying agroclimatic conditions, under
or tubes, and then grown on a liquid or a solid medium in distinct growing environments and according to local agronomic
an incubator, where light and temperature parameters can be practices.
modulated and monitored. According to the plant model, PBS REACh (Registration, Evaluation, Authorisation and
formulations are added either to the solid or the liquid medium Restriction of Chemical substances for the European Union)
at different concentrations to evaluate dose-effect response compliance is critical: in accordance with the EU guidelines
curve. Such experimental conditions allow us a fast screening for agricultural product development, the preliminary
of prototypes on plants, eliminating the influence of soil and safety evaluation is integrated with the REACh compliance
other environmental parameters, including competition with assessment that includes physicochemical, toxicological and
fungi and bacteria (Chawla, 2004). For example, it is possible ecotoxicological evaluations. Under REACh it is necessary for
to perform in vitro tests using tomato (cv. Microtom) seeds producers/importers to register chemical substances unless
on an agar-based substrate containing sucrose 1.5%, without exempt from REACh registration (European Parliament,
any additional nutrient sources. A PBS formulation may be Council of the European Union, 2006). In case of the use of
included into the medium, in order to evaluate the biostimulant microorganisms, a particular dossier should be filled out, in order
effect of selected matrices, in relation to untreated controls to specify the identity, properties (or characteristics), toxicology
(Figures 2C,D). and other attributes of the selected microbials for the intended
use as PBS (Kamilova et al., 2015).
Tests in Controlled Environment
It has been demonstrated that the use of PBS can improve The Last Step: Process Development
quantitative and qualitative parameters also if applied in and Further Scientific Validation
hydroponic or other environmentally controlled crop systems. Once a new PBS prototype is selected according to the
Some of the effects reported are the reduction of nutrient solution Geapower steps described earlier, the commercial team embarks
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concentration in floating system, besides yield and nutritional on developing a manufacturing process which is efficient,
quality increase. For this reason, screening under plant growth consistent and optimizes yields and costs. This step is carried out
chambers and greenhouses are considered robust methods to at the lab and pilot plant facilities (upstream and downstream
evaluate the agronomical validity of PBS formulations (Vernieri equipments), to simulate commercial scale up. At this point
et al., 2006; Parad̄iković et al., 2011). Plants are grown directly the prototype may be launched as a pre-commercial prototype,
on soil, pots, or liquid media (hydroponic solution), and treated which becomes available for the scientific community to perform
with prototypes (foliar and/or root applications). This approach further scientific studies. This is for example the case of a
allows us to define the best application methods, timing and biostimulant developed to overcome abiotic stress such as
rates, and provides preliminary indications on phytotoxicity. The drought, composed of specific amino acids, glycosides, vitamins,
use of the plant growth chamber is ideal for studies of specific polysaccharides, betaines, organic nitrogen and carbon derived
kinds of stress, such as temperature stress, giving quantitative and from Ascophyllum nodosum and other plant materials (Saa
qualitative evidences related to the compounds tested (Feng et al., et al., 2015). Plants pre-treated with this PBS were healthier in
2003). terms of digital biomass (image-based biomass estimation) and
In conclusion, the above described steps, provide robust chlorophyll fluorescence, and this positive effect was confirmed
scientific bases to support the development of innovative PBS at molecular level observing a lower expression of drought-
solutions for agriculture. related genes even when plants were strongly water-stressed. This

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Povero et al. Approach to Study Plant Biostimulants

suggests that treated plants were indeed experiencing a lower modulate the physiology of plants, making them more efficient
level of water stress, as a consequence of the treatment itself even under limited water and/or nutrient resources in their
(Petrozza et al., 2014). In addition to this case study, the beneficial environment (du Jardin, 2012).
attributes of PBS in recent years has resulted in an increasing Finally, in our view extensive experience with field trials,
number of research papers that validate the commercial value together with continuous research and know-how acquisition in
of these complex natural compounds. In our view, this will be terms of PBS formulation and biological effect, are and will be
bring important advances in the detection and demonstration of crucial to satisfy the needs of present and future professional
clear, measurable effects of PBS on plant production and, more agriculture. The plant biostimulants may thus represent the
generally, in agriculture. previously non-existent bridge between the biological, live
products and the prescriptive chemical products that serve the
agricultural input markets.
CONCLUSION
The increasing pressure on the land to support a fast-growing
world population has made it necessary to intensify agricultural AUTHOR CONTRIBUTIONS
production. Such pressure will inevitably lead to the development
of alternative technologies to improve the efficiency of crop GP: ideation of the manuscript; main writing and structuring of
production and food security (Beddington, 2011). The solution the paper; physiology/genomics topics inside the manuscript. JM:
we propose involves a highly differentiated PBS discovery, introduction section; topics related to microorganisms; figures.
development, characterization, and production platform, to DT: contribution in the Introduction section, especially on
which we give the name GeaPower . This approach uses the
R algae as biostimulants; chemical characterization and product
power of chemistry, biology and omics to integrate large amounts development paragraphs; AP: biological characterization section;
of complex data in order to assess and validate the inherent critical review. PW: conclusion and critical review of the entire
activities and synergies of candidate natural compound mixtures manuscript; figures choice.
and micro-organisms for commercial use in agriculture.
We believe this systematic approach, starting from customized
access to raw materials, through extraction methods to product ACKNOWLEDGMENTS
development, helps efficiently turn prospective natural active
ingredients into high quality nutrient solutions. GeaPower R
We thank Prof. Pierdomenico Perata for his critical review of the
permits us to understand what makes a PBS formulation work manuscript, and Dr. Angelo Petrozza for his critical review of
explaining the mode(s) of action of complex biomolecules, and the phenomic technology description. Megafol and GeaPower R R

discover new opportunities. With this approach it is also possible indicate trademark registered in Italy and other countries by
to predict the function of natural substances and how they Valagro. LemnaTec is a Registered Trademark of LemnaTec.
R

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Zhang, X., and Ervin, E. H. (2008). Impacts of seaweed extract-based cytokinins publication in this journal is cited, in accordance with accepted academic practice.
and zeatin riboside on creeping bentgrass heat tolerance. Crop Sci. 48, 364–370. No use, distribution or reproduction is permitted which does not comply with these
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