Journal of Experimental Botany, Vol. 75, No. 13 pp.

3797–3817, 2024
https://doi.org/10.1093/jxb/erae156 Advance Access Publication 17 April 2024

REVIEW PAPER

A practical guide to the discovery of biomolecules with

biostimulant activity

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Jing Li1, , Robin Lardon1, , Sven Mangelinckx2, , and Danny Geelen1,*,
1
HortiCell, Department Plants and Crops, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000 Ghent,
Belgium
2
SynBioC, Department of Green Chemistry and Technology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653,
9000 Ghent, Belgium
* Correspondence: Danny.Geelen@UGent.be

Received 13 November 2023; Editorial decision 4 April 2024; Accepted 16 April 2024

Editor: Karl-Josef Dietz, Bielefeld University, Germany

Abstract
The growing demand for sustainable solutions in agriculture, which are critical for crop productivity and food quality
in the face of climate change and the need to reduce agrochemical usage, has brought biostimulants into the spot-
light as valuable tools for regenerative agriculture. With their diverse biological activities, biostimulants can con-
tribute to crop growth, nutrient use efficiency, and abiotic stress resilience, as well as to the restoration of soil health.
Biomolecules include humic substances, protein lysates, phenolics, and carbohydrates have undergone thorough
investigation because of their demonstrated biostimulant activities. Here, we review the process of the discovery
and development of extract-based biostimulants, and propose a practical step-by-step pipeline that starts with ini-
tial identification of biomolecules, followed by extraction and isolation, determination of bioactivity, identification of
active compound(s), elucidation of mechanisms, formulation, and assessment of effectiveness. The different steps
generate a roadmap that aims to expedite the transfer of interdisciplinary knowledge from laboratory-scale studies to
pilot-scale production in practical scenarios that are aligned with the prevailing regulatory frameworks.

Keywords: Bioactivity, bioassay, biomolecule, biostimulant discovery and development, effectiveness, efficacy, extraction,
formulation.

Introduction
The extensive and prolonged use of synthetic agrochemicals
carries enduring long-term health risks, despite strict regula-
tory measures governing maximum residual levels (Marchand,
2019). In addition, there is a pressing need to curtail the usage
of mined and synthetic fertilizers (Horton et al., 2021). In
Europe, the ‘Farm to Fork Strategy’ advocates for a 20% re-
duction in chemical fertilizer application by 2030, instigating
an agricultural transformation that demands a diverse array of
alternatives for crop production and management (European
Council, 2022). One such alternative lies in the adoption
of plant biostimulants, which are either microbial or non-
microbial products that, applied in modest quantities, enhance
crop yield and quality (Zhang and Schmidt, 1999; du Jardin,
2015; Rouphael and Colla, 2020). Biostimulants are defined
by their agronomic functionalities. When applied to either the
plant or the rhizosphere, they stimulate the plant development

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3798 | Li et al.

process, leading to improvements in nutrient use efficiency, re-
silience to abiotic stress, and crop quality. These functions are
independent of the biostimulants’ nutrient composition (du
Jardin et al., 2020). Extract-based biostimulants, derived from a
diverse variety of plant, microbial, or animal biomass (Harvey
et al., 2015), commonly consist of a wide spectrum of pri-
mary and/or secondary metabolites, such as monomers, oligo-
mers, polymers, and various small molecules that participate
in a diverse array of metabolic processes. These biomolecules
influence gene regulation and protein activity to inducing
property protection and commercialization (a process typi-
cally spanning 2–5 years), and a relatively low percentage of
patentable end-products (are characterized by high turnover
rates (<10%; EBIC, 2023a). A particularly noteworthy chal-
lenge lies in the absence of clearly defined standards that guar-
antee the effectiveness of biostimulant products, a factor that
contributes to public scepticism surrounding their use (EBIC,
2021). The European Committee for Standardization (CEN)
has proposed an obligation to provide both evidence about the
claimed effects and relevant instructions for use and this will

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phenotypic responses that culminate in improved productivity
(Rinschen et al., 2019). Secondary metabolites are evolutionary
optimized for specific biological functions and cover a broad
chemical landscape (Atanasov et al., 2021). The chemodiversity
of these molecules can further increase during extraction and
processing, generating novel molecules with potentially dif-
ferent levels—or even novel—bioactivity.
In 2022, the global biostimulant market was projected to
attain a value of 1.5–2 billion euros, with an anticipated com-
pound annual growth rate of 10–12% (EBIC, 2023a). Seaweed
extracts, with a usage history spanning over 80 years (Craigie,
2011), presently constitute the largest share (~33%) of the
global biostimulant market (Bio4Safe, 2022). Various botanical
extracts employed as biostimulants have demonstrated notable
efficacy in eliciting robust responses in plant growth and crop
yield (Herrmann et al., 2022; Li et al., 2022). However, only a
small proportion of commercial biostimulants originate from
terrestrial plants, accounting for up to just 3% of the market
(Van Iperen International, 2022a). Protein hydrolysates and
humic substances represent two additional categories of bio-
stimulants and are derived from organic matter sourced from
plant residues and animal by-products (Canellas et al., 2015;
Colla et al., 2015). Natural polymers such as chitin and chitosan,
the structural components in insects, crustacean skeletons, and
fungi, also exhibit broad-spectrum plant g­rowth-promoting
activities (Haneef et al., 2017; Li et al., 2020). This diverse
array of biostimulants requires specific expertise in intellectual
greatly improve the quality of biostimulants (CEN, 2022), al-
though the detailed guidance is still awaiting approval. The use
of mixtures presents a significant barrier to uniform produc-
tion (Yakhin et al., 2017). There is currently abundant unreal-
ized potential in terms of yet-to-be-discovered biomolecules
and diverse bioactivities, and this offers considerable opportu-
nities for both academic and industrial researchers within the
biostimulant sector (Yan et al., 2018).
The road to discovering and developing a biostimulant is
very bumpy and presents many challenges and difficulties.
Here, we review recent technological advances in biomolecule
research and plant science in order to provide a platform for
facilitating the process of product discovery and development.
We present a systematic and step-by-step strategy designed to
tackle the primary obstacles encountered in the development
of biostimulants, and emphasize the importance of implement-
ing quality-control measures in pilot-scale manufacturing to
help ensure their effectiveness. A glossary of commonly used
terminology is presented in Box 1.
A roadmap of biomolecule-based
biostimulant discovery and development
A roadmap designed to facilitate the discovery and develop-
ment of biomolecule-based biostimulants is presented in Fig. 1.
In this review, we aim to provide a thorough exploration of a

Box 1. Glossary of terms (in the context of this review)

Bioactive compound or substance: a compound or substance with a biological effect, which could be registered as
an active ingredient or substance in a product.
Biofertilizer: nutrient-rich substances of natural origin used to fertilize plants.
Biomolecule: also called biological molecule, a chemical compound present in organisms.
Biostimulant: a natural substance or microorganism, excluding nutrient elements, that promotes plant growth in small
amounts either directly or indirectly.
Efficacy: the quantifiable ability to produce the desired effect under ideal conditions.
Effectiveness: the ability to produce the desired effect in normal circumstances.
Metabolite: also called small molecule, a metabolic intermediary or end-product in the metabolic process including
primary and secondary metabolites.
Plant growth regulator: a chemical used to promote or inhibit plant growth that is regulated under the Fertilizing
Products Regulation or the Plant Protection Products in Europe.
 Guide to the discovery of biomolecules with biostimulant activity | 3799

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Fig. 1. A roadmap for the discovery and development of biomolecule-based biostimulants. Step 1. If the biomass is considered to be of potential
interest, then biomolecule mining in natural extracts is used to predict the bioactivity. If not, then find an alternative one based on the suggestions given
in the text. Step 2. If the biomolecule(s) are known to be of interest and harbor potential bioactivity, then a dedicated extraction is recommended for
characterization. If not, a crude extract could be obtained to search for relevant compounds as an alternative. Step 3. If there is a known agricultural
function of interest, then a dedicated bioassay is recommended for the identification of the constituent biomolecule(s). If not, then a broad bioactivity
screen is needed in order to determine the relevant functions. If the biomolecule(s) responsible for the bioactivity is identified, then its efficacy should
be ascertained. If not, it is recommended to conduct bioassay-guided fractionation. Step 4. If the formulation based on the chosen compound(s) is
proved to be safe and bioactive, then the efficacy should be verified by the dedicated bioassay in Step 3. If not, a formulation assay would be helpful to
reformulate the biomolecule(s). The effectiveness of the formulation is evaluated in practice when its efficacy has been verified. If it does not appear to be
effective, then the optimal conditions for the application need to be tested. Step 5. The product registration process can be initiated if the performance
of the prototype biostimulant meets the regulation criteria of the region in which it will be marketed. If not, then work back step-by-step through the
roadmap to identify and solve the problem(s).

wide diversity of biomolecule sources, encompassing plants, an-
imal by-products, microbial cell-free supernatants, and culture
filtrates. In addition, we examine recent technological advances
in other botanical drug industries that expedite pre-market de-
velopment of agri-health biological materials. The develop-
ment of extract-based biostimulants encompasses the extraction
of biomolecules from target organisms, the detection of bioac-
tivity, the determination of efficacy, formulation, and the assess-
ment of effectiveness either in controlled greenhouse settings
or field conditions (Fig. 1). These steps form a decision-making
tree comprising six pivotal considerations.
Step 1: mining of biomolecules in natural extracts
Living organisms synthesize a vast array of metabolites, result-
ing in a wide spectrum of chemicals that potentially offer
promising avenues for bioactivity screening (Rinschen et al.,
2019). The fundamental building blocks of large biomolecules
(ranging from 10−5 to 10−3 mm in length) are oxygen, carbon,
nitrogen, hydrogen, phosphorus, and sulfur (Marth, 2008) (Fig.
2A). Within living organisms, these macromolecules serve pur-
poses such as structural support, energy storage (carbohydrates,
lipids), metabolic catalysis, signaling (proteins), and conveyance
3800 | Li et al.
Fig. 2. The relative elemental compositions of large biomolecules and the distribution of abundance of small biomolecule compounds in microbes,
animals, and plants. (A) The constituents of carbon (C), nitrogen (N), phosphorus (P), hydrogen (H), oxygen (O), and sulfur (S) comprise the primary
components of macromolecules, including nucleic acids, proteins, carbohydrates, and lipids (adapted from Anon, 2023). (B) The distribution of annotated
small biomolecules (<3000 Da) across microbial domains (i.e. Archaebacteria, Protista, bacteria, and fungi), animals, and plants (adapted from Ntie-Kang
and Svozil, 2020).
of genetic information (nucleic acids) (Dhara and Nayak, 2022).
Plant-derived small molecules (< 1000 Da) constitute ~67.3%
of the compounds cataloged in the ‘Dictionary of Natural
Products’, followed by animal-derived (12.6%), and microbes
(18.8%) (Ntie-Kang and Svozil, 2020). and the diverse distri-
bution of biomolecular metabolites by class produced in these
three groups is illustrated in Fig. 2B. Notably, plants represent
one of the most adaptable life forms and account for up to
82.5% of the Earth’s total biomass, equivalent to 450 Gt of
carbon (Bar-On et al., 2018). Remarkably, an estimated 15%
of land plants remain to be discovered (Brummitt et al., 2021).
Secondary metabolites typically have a specialized function in
plant development and play a role in the responses to biotic
and abiotic stresses (Kessler and Kalske, 2018). The selection of
individual plants with important agronomic traits has included
those with an altered secondary metabolite profile, and has
contributed to crop domestication (Alseekh et al., 2021). The
secondary metabolites play diverse signaling and regulatory
roles and represent valuable sources of biostimulants (ben Mrid
et al., 2021; Maeda and Fernie, 2021). Their production is
frequently induced by environmental cues or at specific de-
velopmental stages (Kliebenstein and Lunn, 2023). Molecules
produced by microorganisms have become the second-largest
source of crop protection agents (Sparks et al., 2023). Natural
resources from higher plants, animal by-products, algae (e.g.
seaweed, microalgae), and cell-free supernatants or filtrates
from microorganisms are employed to generate novel biostim-
ulants (Matthews et al., 2022) (Fig. 3A).
For the large-scale production of biostimulants from biomass
sources, several key principles should be considered. These in-
clude effective management of quality control and consist-
ency of supply, minimizing costs for collection and storage,
and striving to use homogeneous resources (Xu and Geelen,
2018). An economical and bio-accessible raw material resource
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is one that allows the utilization of organic waste (e.g. crop
residues) or by-products collected along the agri-food supply
chain. To reduce variation of metabolic content, raw resources
should be selected with minimal geographical, temporal, and
organ-specific divergence, in other words plant material from
genetically related families and cultivated in specific geological
regions under similar climates (Kessler and Kalske, 2018). This
can be facilitated by using the KNApSAcK metabolite–plant
species database, which has systematically integrated geograph-
ical zone–species relationships with searchable metabolite in-
formation (Afendi et al., 2012).
Having identified a suitable potential resource, the next step
involves the anticipation of it providing biological function-
ality in facilitating plant growth predicated upon the estab-
lished composition of biomolecules. Examples of biomolecules
that have previously been identified as harboring biostimu-
lant activity together with their modes of action are listed in
Table 1. Recent advances in omics methodologies have led
to a proliferation of annotated biomolecule repositories, cat-
egorized by species (Sorokina and Steinbeck, 2020). Notably,
the Plant Metabolic Network database (https://plantcyc.org)
encompasses metabolic profiles from a diverse array of sources,
including algae, model organisms, crops, and medicinal plants
(Hawkins et al., 2021) Biomolecular networks are progres-
sively being elucidated through the integration of multi-omics
modeling with augmented metagenomic data (Dorado et al.,
2021). In one particular breakthrough, Skinnider et al. (2020)
predicted the previously unelucidated structure of secondary
metabolites from microbial genome sequences and biosynthetic
pathways. This approach offers a novel pathway for exploring
previously untested natural products and identifying organisms
that produce similar biomolecules. Compounds exhibiting
structural similarities tend to also share similar physiochem-
ical attributes and biological functionalities (Lo et al., 2018).
 Guide to the discovery of biomolecules with biostimulant activity | 3801

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Fig. 3. A workflow of biostimulant identification and characterization from bioactive compounds in natural resources. (A) The biological scale of particle-
size distribution is depicted in terms of length. The initial phase in the retrieval of target biomolecules involves reducing the particle size of the natural
resource to the millimeter scale, thereby increasing the diffusion rate during extraction. A diverse array of biomass sources can harbor biostimulants,
encompassing plants (including seaweeds and microalgae) and animal residues, as well as cell-free supernatants or filtrates derived from microalgae and
microbes (i.e. bacteria and fungi). (B) To acquire both extra- and intracellular molecules from source organisms (illustrated here for plant cells) requires
mechanical and non-mechanical pretreatment and extraction methodologies that disrupt the cell wall and membranes. (C) After extraction, separation of
the biomolecules is carried out according to their physicochemical characteristics. (D) The purified biomolecules are then characterized through diverse
techniques such as UV absorption, mass spectrometry, and nuclear magnetic resonance spectroscopy. Created with Biorender.com.

The potential biological roles of biomolecules can be surmised
based on their chemical configurations and data derived from
publicly accessible bioactivity databases, such as ChEMBL
(Mendez et al., 2019) and PubChem (Kim et al., 2023), al-
though there is a lack of specialized sections for plant bioas-
says. The prevalence and/or specialization of specific classes of
biomolecules can thus be identified through the aggregation
of data about the chemical constitution and bioactivities of a
given biomass source, and this information can influence sub-
sequent extraction methodologies and the design of appro-
priate bioassays.
Step 2: extraction and characterization of biomolecules
The next challenge involves extracting the identified biomole-
cules from the natural source in a way that achieves a high yield
while preserving their bioactivity. Biomass is notable for its
complexity and heterogeneity, stemming from the significant
spatial and temporal variations present within living organ-
isms (Resch and Ladisch, 2020). In many instances, a crude
extraction is conducted for screening of bioactivity, particularly
when there are no specific classes of biomolecules that have
been identified that would necessitate a specialized extraction
process (Fig. 1). Alternatively, where individual biomolecules of
interest are known to be present, targeted extractions can be car-
ried out using established or custom-made protocols. The rate-
limiting factors in biomolecule extraction are diffusivity and
solubility, both of which are interconnected with the size and
physiochemical properties of the biomolecules in question
and the choice of extraction solvent (Zhang et al., 2018). The
extraction of metabolites from plant material requires ef-
fective cell lysis through mechanical or chemical disruption
Table 1. Biomolecule classes harboring biostimulant activity and their modes of action
Biomolecule
class (subclass)
Flavonoids, simple aromatic natural prod-
ucts, and lignans (flavonoids, phenolic acids,
coumarins, lignin)
Terpenoids (diterpenes, triterpenes, tetrater-
penoids, sesquiterpenes, isoprenoids)
Carbohydrates (chitosan, oligosaccharides,
fructan, pectin)
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Biostimulant activity and potential mode of action
Crop yield and quality
• Flavonoids function as indirect growth regulators
through interaction with phytohormone signaling
(Laoué et al., 2022).
• Quercetin derivatives (flavonoids) antagonize ABA
and induce stomatal closure to regulate water loss
and gas exchange (Watkins et al., 2017)
• Ortho-dihydroxy B-ring-substituted flavonoids
modulate auxin signaling and transport by regulat-
ing PINOID (PID) proteins (Brunetti et al., 2018).
• Phenolic acids serve as structural units in lignin bi-
osynthesis and cell wall formation (Antonova et al.,
2012).
• Salicylic acid (phenolic acid) is a growth regu-
lator involved in root development, plant growth,
and crop yield (Hassoon and Abduljabbar, 2019;
Bagautdinova et al., 2022)
• cis-Cinnamic acid (phenolic acid) is a natural plant
growth-promoting compound that stimulates cell
division and expansion (Steenackers et al., 2019)
• Gibberellins (diterpenes), abscisic acid (tetraterpe- •
noids, derived from carotenoids), strigolactones
(sesquiterpenes, derived from carotenoids), and
brassinosteroids (triterpenes) are phytohormones
with primary roles in plant growth (De Bruyne et al.,
2014)
• Carotenoids (isoprenoids) are photosynthetic
complex-bound and function in light-harvesting and
photoprotection (Sun et al., 2022)
• The apocarotenoid signal derived from cis-carotene
activates chloroplast development and promotes
leaf virescence (Cazzonelli et al., 2020).
• Chitosan and related oligosaccharides promote • Soluble sugars scavenge reactive oxygen
plant growth by regulating photosynthesis, C/N
metabolism, and plant stress (Mukhtar Ahmed et
al., 2020)
• Fructan regulates plant growth via phytohormone
signaling (Márquez-López et al., 2022)
• Pectin affects plant signaling by regulating stem
cells, cell morphogenesis, and cell–cell communi-
cations (Shin et al., 2021)
Abiotic stress tolerance
• Flavonoids and phenolic acids are antioxi-
dants that stabilize cellular redox status by
scavenging reactive oxygen species (e.g.
through their hydroxyl groups on B-ring) (Agati •
et al., 2012).
• Lignin biosynthesis and deposition enhance
plant tolerance to environmental stresses in-
cluding heavy metals (Liu et al., 2018)
Sterols (triterpenes) are cell membrane
components that regulate cell wall develop-
ment and hormone signaling (Rogowska and
Szakiel, 2020)
species and are osmoprotectants that stabi-
lize membranes (Keunen et al., 2013)
Nutrient use efficiency (via rhizo-
sphere and/or soil)
• Flavonoids regulate nodulation and sym-
3802 | Li et al.
biotic nitrogen fixation (Okazaki et al.,
2013)
Flavonoids stimulate fungal growth in
arbuscular mycorrhizal symbiosis and
phosphorus acquisition (Tian et al.,
2019)
• Metal chelating with flavonoids
increases the bioavailability of micro-
nutrients and soil quality (Cesco et al.,
2010)
• Coumarins are involved in the
­microbiome-to-root-to-shoot communica-
tions that shape microbiome composition
and iron mobilization (Stassen et al., 2021)
• Humic and fulvic acids interact with
plant growth-promoting bacteria leading
to root biomass increase, nutrient ab-
sorption, and stress damage control (da
Silva et al., 2021)
• Strigolactones (sesquiterpene) signal
the interaction with beneficial arbuscular
mycorrhiza and nodulation (López-Ráez
et al., 2017)
• The root system serves as a carbon sink
in response to sugar transport in plant–
rhizobacteria interactions (Hennion et
al., 2019)
 Guide to the discovery of biomolecules with biostimulant activity | 3803

(Islam et al., 2017) (Fig. 3B). In view of environmental con-

naling, transport, and metabolism during

metabolites are involved in sensing, sig-

plant–microbe interactions (Moormann

cerns, the extraction process should be energy-efficient and
Nutrient use efficiency (via rhizo-

Amino acids and their specialized sustainable (Rahman et al., 2022); Table 2 shows comparisons
of various eco-friendly, cost-effective, and scalable pretreat-
ment techniques for biostimulant production. Briefly, non-
thermal techniques, such as steam/CO2 explosion and pulsed
electric field, are more effective and sustainable. The extraction
sphere and/or soil)

efficiency is optimized by adjusting parameters such as temper-

et al., 2022)

ature, duration of extraction, particle size, solvent type, liquid-

to-solid ratio, and pH (Zhang et al., 2018).

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Following the initial crude extraction, various separation
techniques utilizing differential partition coefficients, molec-
•

ular sizes, and ionic strengths can be employed to enrich the

proteins as chemical chaperones, and avoid-

recovery of specific molecules (Zhang et al., 2018) (Fig. 3C).

quenching hydroxyl radicals (˙OH), stabilizing
peptides are osmoprotectants that accumu-
late during abiotic stress (Batista-Silva et al.,

Proline enhances abiotic stress tolerance by

ing freezing as an osmolyte (Patriarca et al.,

Purification of the bioactive compounds is technically very

Glutamate, proline betaines, and derived

challenging (García-García et al., 2020), which explains why

the majority of biostimulants that are currently marketed are
complex mixtures (Ricci et al., 2019). Simplifying the chem-
ical composition is strongly recommended to mitigate po-
Abiotic stress tolerance

2019; Kim et al., 2021)

tential unwanted side effects (detailed in the section below).

Given the substantial environmental impacts associated with
conventional separation techniques such as liquid separation,
distillation, adsorption, and absorption, alternative methods
such as membrane separation can be considered (Calabrase
2021)

et al., 2020). Biomolecules are identified by employing liquid

and gas chromatography coupled with mass spectrometry or
Biostimulant activity and potential mode of action

nuclear magnetic resonance spectroscopy to align their mass

Phosphorothioate oligonucleotides (S-DNA) induce

spectra with those of previously characterized chemicals (Fig.

by overexpressing CLAVATA3/ESR-related (CLEs)
trolling root and shoot development (Steenackers

C-terminally encoded peptide (CEP) improves ni-

Bioactive peptides modulate plant signaling con-

pollen tubes as well as lateral root and root hairs

Bovine serum albumin promotes root growth by
enhancing nitrogen (Paungfoo-Lonhienne et al.,

3D). However, many natural products remain uncharacterized,

genes (Paungfoo-Lonhienne et al., 2010a, b)
trate uptake rate in roots (Roy et al., 2022)

due to their high complexity and the occurrence of molecules

with high structural similarity. Recent advances have tackled
this issue by employing molecular networking topology and
structural similarity fingerprinting from fragmented spectra
databases (Borelli et al., 2023; Morehouse et al., 2023).
Initial extraction from plant biomass is commonly performed
Crop yield and quality

on small-scale samples, and scaling-up is therefore required for

pilot-scale biorefinery processing. Belwal et al. (2020) investi-
gated the scaling-up of extraction methods to optimize solvent
et al., 2019)

consumption and processing time with capacity and yield. The

2008)

transition from the initial research scale (~1 l) to the indus-

trial level (~100–10000 l) presents challenges that are typically
•

related to equipment design, the scaling-up of mass transfer,

processing kinetics, optimization, and evaluations of techno-
economic and environmental costs. Pilot-scale production of
biostimultants aims to adapt the extraction and separation pro-
cesses developed at the research stage to a commercial scale
Proteins (amino acids, peptides)

whilst taking into account practical considerations such as

feedstock supply and operating costs, as well as market demand
Table 1. Continued

(Gong et al., 2016).

class (subclass)

As shown in the example in Box 2, humic substances from

Nucleic acids
Biomolecule

unused biomass can be successfully processed for bioactive

extracts using customized methods that have been optimized.
Crude extracts can be refined to produce active compounds, as
detailed in the following step.
•
 3804 | Li et al.

Step 3. Identification of agricultural functions and

Ziegler-Devin et al. (2021)

bioactive compound(s)
Bagade and Patil (2019)

Bocker and Silva (2022)

Herrero et al. (2013)

Shukla et al. (2023)

Gullón et al. (2020)
Zheng et al. (2022)
Throughout the entire process of discovery, the central focus is

Khan et al. (2019)

Rao et al. (2021)
on defining the biological functions and identifying the cor-
Reference

responding active biomolecules (Fig. 4). The functions are in-

tricately tied to specific plant traits (Bertoni et al., 2021). The
spatial and temporal assessment of bioactivities involves a broad
array of phenotype bioassays that include in vitro assays and
Chemical con-

trials at the greenhouse, field, and climate zone scales (De

Diego and Spíchal, 2022). Once a bioactivity has been defined,

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sumption
Moderate
Moderate

specialized biotests are conducted (Fig. 1); a list of bioassays that

Sustainability benefit

None

None

High

can be employed to evaluate biostimulant activity is provided

Low
Low

Low
Low

in Table 3. It is recommended to choose bioassays for examin-

Energy con-

ing agricultural functions based on established standards and/

sumption

or peer-reviewed scientific literature (CEN, 2022).

Moderate
Moderate

Moderate

Bioassays need to be customized and standardized for effi-

High
Low

Low
Low

Low
Low

cient screening and rapid validation of quality control.A diverse

range of bioassays should be considered in order to broaden
the range of active substances that are identified (Hughes et al.,
Table 2. Comparisons of recently developed, environmentally friendly extraction methods for biomolecules

Moderate
Moderate

Moderate

2011). The primary requirements for these initial bioassays

Sub- and supercritical extraction methods, for example pressurized liquid extraction and supercritical fluid extraction.
Yield

are speed, stability, sensitivity, specificity, technical repeatability,

High

High
High
Low

Low
Low

and biological reproducibility (Ghisalberti, 2007). In summary,

Efficiency benefit

the selection of bioassays that are tailored for biostimulants is

Process time

critical in determining their potential at the start of a screen

(Povero, 2020).
Moderate

Moderate
Moderate

When aiming to replicate the bioactivity observed the in

Short
Short
Short

Short

Long
Long

original biomass, the chemical composition and abundance

of the bioactive substances serve as the fundamental basis for
proprietary products. Generally, more intense separation and
Feasibility

Moderate
Moderate
Moderate

Moderate

purification operations are required for products that contain

simpler compositions. Identification of the active chemical in a
High

High

High
Low

Low

biostimulant requires multiple rounds of fractionation that are

guided by the results of the bioassays (Nothias et al., 2018). The
Operational benefit

process consists of three crucial steps: fractionating the crude

extract; characterizing the chemical composition of the ac-
tive fraction; and assessing the relative abundance of candidate
Scalability

Moderate

metabolites. The identity of the candidate active biomolecule(s)

is subsequently validated (Ory et al., 2019). Even though bio-
High

High
High

High
High

High
Low

Low

stimulants with pure active compounds offer more advantages

due to their superior standardization of quality and our better
(Natural) deep eutectic solvents

understanding of the mechanisms of their action, extract-based

Enzymes, bacterial, and fungi

mixtures dominate the current market due to their reduced

High-pressure processing
Hydrodynamic cavitation

costs in terms of less-precise active ingredient identification

Steam/CO2 explosion
Compressed fluids1

Pulsed electric field

and purification (García-García et al., 2020).

Once the composition of active molecule(s) has been de-
Ultrasonication
Evaluation
technique

termined, dose–response experiments are used to assess the

Microwave

optimal concentration for maximal activity at minimal eco-

toxicity (Jalal et al., 2021) (Fig. 5A). In terms of efficacy data,
the potency of the active compound is represented as stan-
dardized measures (Dougall and Unitt, 2015). A lower half-
Non-thermal

maximal effective concentration (EC50) typically demonstrates

Biological
Chemical
Method

Thermal

more potent activity with greater efficacy (Holland-Letz and

Kopp-Schneider, 2015). When analysing a mixture of active
1
 Guide to the discovery of biomolecules with biostimulant activity | 3805

Box 2. Biostimulants based on humic substances generated from biowaste

Humic-rich substances are historically known to contribute to soil fertility (Susic, 2018). They originate from the aerobic
composting of lignocellulosic-rich organic waste, including sewage sludge, wastewater, food residuals, and agricultural
by-products (Guo et al., 2019). The humification process initiates with the hydrolysis of macromolecular and low-molecular-
weight compounds from the source materials, followed by their degradation and decomposition that ultimately culminates
in the formation of the humic substance end-products (Sutradhar and Fatehi, 2023). Humic substances represent a
supramolecular association of small, heterogeneous molecules characterized by an undefined chemical structure (Piccolo,
2002). They are pigmented compounds that are categorized into humins, humic acids, and fulvic acids based on their
molecular weight and distinctive characteristics (Gautam et al., 2021). These biomolecular groups manifest as complex

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compositions that depend on the nature of the source material and the extraction methodology employed (Mahler et al.,
2021). Their physiochemical attributes and relative abundance are discerned and characterized via elemental and
functional group analyses, Fourier-transform infrared spectroscopy, nuclear magnetic resonance spectroscopy (NMR),
and excitation–emission matrix spectroscopy, with the recommended methodologies being outlined by the International
Humic Substances Society (Hu et al., 2022).
Humic acid fractions have been extensively studied due to their notable chemical reactivity that confers resistance to
microbes (Billingham, 2013). The functions of humic acid fractions depend on the structures associated with the source
materials. Assessments of the structure–activity relationships show that the molecular the conformations of humic acids
in aqueous solution are a pivotal factor in influencing their plant-promoting properties (Aranaz et al., 2023). Savy et al.
(2020) identified a positive correlation between the methoxyl and aromatic moieties of humic substances and root growth
in maize; however, it remains difficult to determine the ultimate efficacy, as the most potent single molecule is hard
to identify. Moreover, studies into the mechanisms of action require the use of standardized and well-characterized
compounds, whereas extracts derived from humic substances are inherently complex (Klučáková, 2018).
As an example, humic acids obtained from Leonardite (sedimentary humic acids, SHA) are commonly marketed
as biostimultants (JISA Advanced Agro, 2015). Petrov et al. (2017) used the Vienna Soil Organic-Matter Modeler tool
to construct models of the SHAs to investigate their structure and dynamics across a range of hydration levels, and
combining this computational technique with experimental studies yielded insights into their modes of action. Qian et al.
(2015) had previously characterized aqueous solutions of SHAs with varying molecular weights using NMR, and tested
their effects on snap beans. The results growth-promoting effects on both roots and shoots, particularly with the solutions
containing low-molecular-weight compounds. Several studies have shown that SHAs boost root and shoot growth by
augmenting H+-ATPase activity in the root plasma membranes, in conjunction with elevating the contents of abscisic acid
and jasmonic acid (Olaetxea et al., 2019; De Hita et al., 2020).

Fig. 4. The agricultural functions of biostimulants and corresponding plant traits, bioactivities, and underlying mechanisms. Biostimulants are
characterized by three distinct categories of agricultural functions that highlight their impact on plant traits, their bioactivities, and the underlying
mechanisms that are driven by the active biomolecules. Illustrative examples of each of these categories are listed. Created with BIorender.com.
 3806 | Li et al.

Table 3. Examples of bioassays used to evaluate biostimulant activity.

Agricultural function Biostimulant activity/plant trait1 Example evaluation indices References for example descriptions of bioas-
says
Crop yield and quality Agronomical trait Yield, height, stem diameter, leaf number, leaf area, fresh Gallegos-Cedillo et al. (2021); Li et al. (2021)
weight, root system quality parameters
Marketable trait Nutritional properties Proteins, lipids, trace elements, vitamins, amino acids Capstaff and Miller 2018); Yu and Tian (2018)
Organoleptic properties Appearance, related metabolites (e.g. sugars, organic acids, Pott et al. (2019); Bashir et al. (2023)
phenolics, terpenoids, volatiles)
Techno-functional properties2 Related metabolites for food, feed, energy, cosmetics, phar- Kai et al. (2018); Rashwan et al. (2024)
macology, building materials (e.g. proteins, polysaccharides,
lignin, pectins)
Abiotic stress tolerance Thermal Visual damage, biomembrane functions, chlorophyll fluores- Ramel et al., (2012); Potocka and Szymanowska-Pulka
Light cence, water potential, heat-shock proteins, phytohormones, (2018); Ding et al. (2019); Sun et al. (2020); Van Zelm et
Mechanical anthocyanin, reactive oxygen species, antioxidants al. (2020); Geange et al. (2021); Shi et al. (2022)
Water
Chemical
Xenobiotic
Nutrient use efficiency Nutrient availability Chemical soil analysis of nutrient status based on extractable Marschner and Rengel (2012)
quantity, mobility, spatial availability (i.e. rhizosphere)
Uptake efficiency Root ion uptake kinetics Griffiths and York (2020)
Utilization efficiency Plant biomass or organic compounds produced per unit of Santa-María et al.( 2015)
nutrient over a given period
1
Examples are adapted from CEN (2022).
2
Physio-chemical attribute that impacts the transformation process or end use.

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 Guide to the discovery of biomolecules with biostimulant activity | 3807

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Fig. 5. A roadmap for identifying bioactive compounds and determining the effectiveness of biostimulants. (A) The methodological connection between
the biomolecule and its agricultural impact is established through the identification of active biomolecules and the determination of their efficacy.
Bioassay-guided fractionation aims to isolate the bioactive fraction from the crude extract. As an example, molecular size-exclusion chromatography
(1) leads to a heatmap of the bioactivity in each of the fractions obtained (2). Successive rounds of bioassays are used to pinpoint the bioactive fraction
containing the individual biomolecule(s) responsible for the observed bioactivity (3). The efficacy of individual active biomolecule(s) is determined by dose–
response relationships (4) and interactive effects between biomolecules are examined (5). (B) The objective of formulation is to enhance the uptake rate of
the active biomolecules that have been identified, ultimately leading to the creation of a potential commercial product. This has to take into account the
basic form of the material, its characteristics, and the application method. (C) The final stage in the process of translating laboratory findings to practical
field performance involves assessing effectiveness. The identified potential product(s) should undergo rigorous testing in a variety of trials encompassing
greenhouses, open fields, and diverse crop cultivation and growth conditions. The results gained from these trials will be crucial in gaining registration of
the product for commercial use and in providing recommendations for its application.

biomolecules, the biological interactions, including synergistic,
additive, potentiation, and antagonism effects, and their corre-
lation with the biological EC50 values within the context of
composition–structure–activity relationships should be deter-
mined (Yadav et al., 2015).
In the pursuit of consistent extracts in mixed forms such
as biostimulants, the practice of traditional Chinese medicine
(TCM) serves as a pertinent paradigm for popular botanical
products that incorporate complex mixtures; however, despite
increasing demand for public health applications, quality con-
cerns related to TCM have emerged as a significant barrier
to global market entry (Song et al., 2013). Regional regula-
tory authorities have employed ‘Pharmacopoeia’ guidelines to
apply standardized and comprehensive instructions for various
aspects of TCM production, including plant collection, ex-
traction, storage, quality assays, and processing (Leong et al.,
2020). The combination of bioactive molecules within the ex-
tract gives it a ‘metabolic fingerprint’, and together with rapid
bioassay tests it can serve as quality assessment tools for batch
evaluation, whether for the industrial-scale production of pure
compounds or for mixed forms of prospective biomolecules
(Li et al., 2019). Well-documented and quality-validated pro-
cedures are imperative to ensure the integrity of the entire
product supply chain during the extraction of biostimulants.
For industrial-scale production, the metabolic fingerprint of
bioactive components in mixtures can function as an indi-
cator of quality, leading to a more reproducible and predictable
product. This approach can help mitigate against variations
arising from batch effects in plant materials, extracts, and
fractions.
At the molecular level, biostimulant activity is determined
by analysing the biochemical changes in transcript, protein,
Table 4. Biosensors used to monitor signaling pathways for in vivo plant bioassays

Signaling molecule Functional group Biosensors for in vivo detection in plant bioassay
3808 | Li et al.

Chemical probe Genetically encoded fluorescent protein1

Reactive oxygen species Redox actor Non-fluorescent probes: 3,3´-diaminobenzidine (DAB), nitro-blue tetrazo- Hydrogen peroxide: HyPer (Ugalde et al., 2021),
lium (NBT) roGFP2-Orp1(Nietzel et al., 2019), roGFP2-
Tsa2ΔCR (Niemeyer et al., 2021)
Fluorescent probes: dihydroethidium (DHE), 2´,7´-dichlorodihydrofluorescein
diacetate (H2DCFDA), N-acetyl-3,7-dihydroxyphenoxazine (Amplex red),
boronate-based probes such as peroxy orange-1 (PO1), singlet oxygen
sensor green (SOSG), bis-N-methylacridinium dinitrate (LC2+) (Ortega-
Villasante et al., 2018).
Glutathione redox potential and state Antioxidant Fluorescent probes: monochlorobimane (mchlB), monobromobimane Grx1-roGFP2 (Wagner et al., 2019), FROG/B
(mbrB) (Gasperl et al., 2022) (Sugiura et al., 2020).
NADH/NAD+/NADPH Antioxidant, energy NA1 iNAP, SoNar (Lim et al., 2020), Peredox-mCherry
(Steinbeck et al., 2020)
Thioredoxins Redox protein NA CROST1, CROST2 (Sugiura et al., 2019)
Protons (pH) Ion Non-fluorescent probes: phenol red, bromocresol purple (Steinegger et al., phGFP, Pt-GFP, apo-pHusion, pHluorin
2020) (Moreau et al., 2021)
Fluorescent probes: dextran-conjugated forms of fluorescein isothiocya-
nate (FITC), 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS),
BCECF (2´,7´-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein), and SNARF-1
(seminaphthorhodafluor-1) (Tsai and Schmidt, 2021).
Ca2+ Secondary messenger Fluorescent probes: Calcium Green-11, Fura-2, and Indo-1 (Kanchiswamy Multiple markers (reviewed by Grenzi et al., 2021).
et al., 2014)
ATP Energy NA ATeam, MgATP2- (De Col et al., 2017)
Auxin Phytohormone3 Fluorescent labeling probes: fluorescein isothiocyanate and rhodamine iso- DII-VENUS, R2D2, AuxSen (Herud-Sikimić et al.,
thiocyanate, nitrobenzoxadiazole (NBD) fluorophore (Pařízková et al., 2021) 2021)
Abscisic acid Fluorescent probes: fluorescent chromophores (AIEgens) (Wu et al., 2022), ABACUS1–2μ, ABAleon2.1, SNACS (Zhang et al.,
flubactin (Yang et al., 2022) 2020)
Gibberellin Fluorescent labeling probes: fluorescein GPS1 (Rizza et al., 2017)
Glucose and sucrose Primary metabolites Fluorescent labeling probe: O-BODIPY (Kanyan et al., 2022) FlipSuc90 µ, FLIPglu-Δ13, FLII12Pglu-700μ∂6 (Zhu
et al., 2017)
Amino acid Reviewed by Xiong et al. (2021) Glutamate: FLIPW (Castro-Rodríguez et al., 2022)
Inorganic phosphate Nutrient NA cpFLIPPi (Mukherjee et al., 2015)
Zn2+ Reviewed by Pratt et al. (2021). eCALWY-1 (Lanquar et al., 2014)
Nitrate and ammonium NA mcpGFP (De Michele et al., 2013), mCitrine-NLP7
(Liu et al., 2022)
1
NA, no available applications were found in the literature.
For reviews on genetically encoded biosensors see Gjetting et al. (2013), Walia et al. (2018), Isoda et al. (2021), and Müller-Schüssele et al. (2021). For a review on phytohormone-related
biosensors see Balcerowicz et al. (2021).

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 Guide to the discovery of biomolecules with biostimulant activity | 3809
and metabolite levels (Xu et al., 2020). Biostimulants are well-
recognized for their ability to modulate plant signaling path-
ways, including hormones and secondary messengers (Yakhin
et al., 2017). These signaling pathways can be observed using
biosensors (Akter et al., 2021), allowing investigation in vivo
(Table 4). In vivo biomarker imaging, incorporating multi-
parameter fluorescent indicators in plants, facilitates detailed
monitoring of molecular dynamics and simultaneous bio-
logical processes (Waadt et al., 2021). Identifying the active
molecule(s) and comprehending the molecular mechanism of
activity represent major challenges in the discovery process, but
it is highly recommended in order to develop evidence-based
formulation and application methods.
Various types of bioactive compounds can be fractionated
from humic substances and investigated for their specific bios-
timulant activities (Box 2). The optimal configurations of the
active compounds often remain unclear, and an in-depth un-
derstanding of the mode of actions would serve as a functional
tool for employing structure–activity relationship studies.
Step 4. Formulation and effectiveness
Formulating biostimulants is a crucial step in achieving op-
timal uptake by the plants to which they are applied and in
prolonging the shelf life of the bioactive ingredients (Myung
et al., 2014) (Fig. 1). The formulation method is determined by
the physiochemical properties of the active molecules and the
intended delivery method. The composition and susceptibility
to degradation pose significant challenges in formulating nat-
ural organic compounds (Shaw, 2021). Most final biostimulant
products exist in either liquid or solid form (Fig. 5B), and their
efficacy in plants highly depends on the adhesiveness, wettabil-
ity, penetration, and stability of the formulated product, which
can vary across crop species (Dima et al., 2020). The com-
plexity of active components and application conditions can
make it challenging to find the ideal formulation.
Application of biostimulants can be through various meth-
ods including seed treatment, foliar spraying, drenching, and
soil and root irrigation (Nairn et al., 2016; Zhao et al., 2018).
Among these, foliar application is the most common, ac-
counting for ~80% of global market use (Grand View Research,
2021). Agrochemical companies have developed proprietary
formulations, such as adjuvants, to enhance the uptake of
their products. Water-dispersible granules and adjuvants have
been specifically engineered to formulate water-based extracts
(Dipak Kumar et al. 2020). Encapsulation, lyophilization, in-
clusion of liposomes, and water-compatible excipients are
also suitable for botanical water extracts (Borges et al. 2018).
In addition, nanomaterials-based formulation and encapsula-
tion is employed to improve aqueous solubility, chemical sta-
bility, and bioavailability (Zhao et al., 2018;Vega-Vásquez et al.,
2020). The choice of formulation strategy is closely aligned
with the intended application methods for biostimulants.
Selecting the appropriate application method and formulation
technique is crucial in product development. This decision is
made on a case-by-case basis, taking into account specific plant
developmental stages, to ensure optimal efficacy.
Drawing from over 30 years of experience of biophar-
maceutical development, the technique of high-throughput
formulation (HTF) has been adapted, coupled with high-
throughput analysis, in the orthogonal design of experiments
to rapidly screen for optimal formulation stability and activity
(Arvinte et al., 2018). Capelle et al. (2007) have described the
practical considerations of applying HTF for stabilizing ther-
apeutic proteins, and the associated physical and chemical
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analyses (Capelle and Arvinte, 2008). The transport and up-
take dynamics of molecules in plants are also important factors
influencing in vivo efficacy. This has recently been measured by
compound-specific stable isotope analysis using 13C and 15N
labeled amino acids (Svennerstam and Jämtgård, 2022). The
absorption of essential nutrients and trace elements can be ac-
curately monitored in intact living plant tissues (Mijovilovich
et al., 2020). The labeling of single molecules enables real-time
imaging and tracking within live cells (Ha et al., 2022).
Commercially available biostimulants should have dem-
onstrated increased crop performance in greenhouse and/or
field experiments (Ricci et al., 2019) (Fig. 5C). The scien-
tific evidence should follow various standards such as those
approved by the European Committee for Standardization
(CEN), including the claims on which the objective of the
trial has been based, experimental design, crop groups, growth
conditions, treatment methods, assessment modes, statistical
analysis, and a final report (CEN, 2022). It is crucial to note
that the biostimulant effect must be validated independently
from the nutrient content, by incorporating positive and neg-
ative controls in the experimental design. Using both indoor
and field phenotyping, multiple greenhouse and field trials are
carried out to assess the efficacy of the biostimulant under var-
ious crop cultivation conditions, with different plant varieties
and application methods, in diverse climate zones, and across
different growing seasons (Araus et al., 2018; Jägermeyr and
Frieler, 2018). Determining the optimal concentrations for
greenhouse and/or field applications is a crucial aspect (Traon
et al., 2014). To ensure objective results, it is advisable to have
a third party conducting the experiments, adhering to estab-
lished industry standards and competition criteria (European
Seed, 2021). National and regional agricultural experiments,
as well as research stations overseen by governments, universi-
ties, and research institutions, serve as external certifiers for
demonstrating the effectiveness of biostimulant treatments in
greenhouse and field settings (Pearson and Atucha, 2015).
The first commercial plant extract-based biostimulants de-
rived from cultivated crops have recently been registered
under new EU regulation (European Union, 2019; van Iperen
International, 2022b). These products were assessed in 120
field trials across 12 crops, spanning six climate zones, over
three consecutive production years, in collaboration with var-
ious agricultural cooperatives in European countries. This rig-
orous evaluation revealed a 60% positive effect on increases of
water and nutrient use efficiencies.
 3810 | Li et al.

Table 5. The current state of global regulations on biostimulants

Region Country Recognition of biostimulants? Latest regulation (year issued) Legislation status1 Reference
Europe European Union countries Recognizes biostimulants as a distinct Regulation (EU) 2019/1009 (2019) Adopted European Union (2019)
category
North America United States Provides a federal definition of biostimu- Plant Biostimulant Act (2023) Proposal US Congress (2023)
lants and excludes them from the Federal
Insecticide, Fungicide, and Rodenticide Act
(FIFRA)
Canada No definition of biostimulants and they are Fertilizers Regulations (2020) Adopted Government of Canada
regulated as supplements (2020)
South America Brazil Regulates biostimulants separately as Decree No. 10.375 (2020) Adopted Diário Oficial da União (2023)
biofertilizers
Argentina No definition of biostimulants Resolution number 1004/2023 (2003) Adopted Gottems (2023)
Asia China No definition of biostimulants and they are Measures for the Administration of Ferti- Adopted Lagre (Beijing) Agriculture
regulated as other fertilizer products lizer Registration (Measures) (2022) Technology (2022)
India Defines biostimulants that do not include Fertilizer (Inorganic, Organic or Mixed) Adopted Government of India (2021)
pesticides or plant growth regulators (Control) Amendment Order (2021)
Oceania Australia No definition of biostimulants and they are The Agricultural and Veterinary Chemi- Adopted Australian Pesticides and Vet-
regulated as biological agricultural products cals Code Act (Agvet Code) (1994), The erinary Medicines Authority
Biosecurity Act (2015), and The Gene (2022)
Technology Act (2020)
Africa South Africa Regulated as a Group 3 fertilizer. A product Fertilizers, Farm Feeds, Seeds and Rem- Adopted South African Government
can be both a biostimulant and a biopes- edies Act 36 of 1947 (2019) (2019)
ticide
1
The legislation status is simplified to proposal and adopted, based on European Parliament (1999).

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 Guide to the discovery of biomolecules with biostimulant activity | 3811
An unbiased approach to consolidating reproducible evi-
dence from individual studies is through meta-analysis (Anon.,
2016). Ultimately, optimal formulation and assessment of effec-
tiveness will mean that biostimulant applications can be seam-
lessly integrated into sustainable farming practices, providing
consistent benefits.
Step 5: regulation and commercialization
In terms of product registration, commercial biostimulants
must adhere to the regulatory framework of the relevant nation
or region in which they are sold. The current state of global
regulations for biostimulants is shown in Table 5. These reg-
ulatory measures aim to ensure the safety and efficacy of bio-
stimulants, together with their proper labeling to ensure they
are used appropriately.
As an example in Europe, the ‘Component Material
Categories’ outline 10 types of natural resources (excluding
microorganisms) permitted for use in biostimulant production
in agreement with EU regulations (European Union, 2019),
and it is required that the chemical composition of biostimu-
lants for compounds that exceed 1% abundance is character-
ized. Strict safety requirements are in place to govern the reuse
of materials and to prevent any contamination that might be
harmful to public health or the ecosystem. The Registration,
Evaluation, Authorisation, and Restriction of Chemicals
(REACH+) requirements are reinforced in the ‘Fertilizing
Products Regulation’, which necessitates the provision of a
chemical safety report to all users of chemicals. This report
includes exposure scenarios covering both the processes of
manufacturing and usage (EBIC, 2023b). Furthermore, it is
crucial that non-microbial biostimulants adhere to strict stan-
dards to avoid contamination by heavy metals and pathogens,
ensuring compliance with specified limit values (European
Union, 2019). Biostimulants derived from animal b­ y-products
are subject to additional European regulations (European
Commission, 2009; European Union, 2019). Alongside the
safety data sheet, which details aspects such as physical char-
acteristics, production methodology, and expiration dates, bio-
stimulant registration requires the inclusion of information
about the claimed agricultural effect on the target plant spe-
cies, together with guidelines regarding efficacy and applica-
tion methods (European Union, 2019).
Ensuring product quality and delivering positive outcomes
are essential for building long-term customer trust. Though
EU regulations do not specifically require information on the
active ingredient(s) responsible for bioactivity, understanding
these components can greatly enhance product performance
and reduce operational costs associated with inconsistent ac-
tivity. Therefore, it is recommended that the biostimulant in-
dustry invests in the identification of bioactive molecules and
their underlying mechanisms to gain a deeper understanding
of their products. This will provide information to improve
product performance and may lead to more reliable and effec-
tive biostimulant applications.
Conclusions and outlook
Plant biostimulants contribute to crop growth, nutrient use
efficiency, and abiotic resilience, as well as the restoration of
soil health, benefiting both organic and conventional agricul-
ture. With new regulatory frameworks and growing evidence
of their efficacy ensuring their safety, security, transparency, and
sustainability, biostimulants are gaining in credibility. The de-
velopment and commercialization of new biostimulants will
be improved by analysing their bioactive components and by
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application of reliable bioassays. The step-by-step approach
presented here will help in avoiding potential pitfalls and guide
the transition from lab-scale research to pilot-scale biostimu-
lant production.
While the future of biostimulants appears promising from a
policy perspective, the path forward is marked by uncertainties
and formidable challenges. Two significant challenges impede
the discovery of biostimulants from natural biomass enriched
with biomolecules. The first arises from the complicated com-
positional mixtures in the extracts, posing technical challenges in
isolating individual or small groups of bioactive molecules. The
extraction of the required fractions becomes problematic, com-
pounded by the need for a series of separations from crude
extracts. The active compound(s) should be in a state allowing
optimal bioactivity, aligning with the appropriate range of field
doses. The second challenge lies in defining the desired bio-
activity as a clear target for screening. Biostimulant activity is
inherently broad, encompassing various traits due to the com-
plex chemical composition, creating an obstacle in pinpointing
a specific target. These challenges are interrelated, as complex
mixtures are likely to exhibit a wide range of bioactivities.
The key lies in understanding the composition–structure–
activity relationships within well-studied modes of action.
Subsequent investigations into doses and response times are es-
sential for providing valuable insights to guide practical applica-
tions. It is important to acknowledge that a single biostimulant
cannot universally address the needs of every crop under all con-
ditions. Therefore, the development of biostimulants with spe-
cific functions is required and requires a nuanced understanding
of the product and its application scenarios. To fully unlock the
potential of bioactivities carried by biomolecules, a comprehen-
sive strategy of advertising their value must be adopted to po-
sition biostimulants in the market. The practical objective is to
achieve consistent and robust bioactivity in biostimulants. This
holistic approach aims to pave the way for the successful inte-
gration of biostimulants into sustainable agricultural practices.
Acknowledgements
We thank the examining committee of JL’s doctorate, Prof. Kris
Audenaert, Prof. Katleen Raes, Prof. Cinzia Bertea, and Dr Juan Carlos
Cabrera, for their critical comments and suggestions throughout the de-
velopment of this review. We also would like to thank the biostimulant
team from HortiCell Lab and CropFit, a multidisciplinary consortium, at
Ghent University for their support.
3812 | Li et al.
Author contributions
JL: investigation, visualization, writing—original draft; RL, SM, and DG:
writing—review and editing, supervision; JL and DG: conceptualization.
Conflict of interest
The authors declare that they have no conflicts of interest in relation to
this work.
Funding
The research associated with this review was supported by Fonds
Wetenschappelijk Onderzoek–Vlaanderen (FWO) under project
Bio2Bio (S006017N), by Flanders Innovation & Entrepreneurship
(VLAIO) (HBC.2019.2244) and the European Union’s Horizon 2020
research and innovation program under the grant agreement of sustain-
able and resilient agriculture for food and non-food systems (FACCE
SURPLUS) (No. 652615) under project BioSUNmulant, by Flanders
Innovation & Entrepreneurship (VLAIO) (HBC.2019.2797), and joint
CORE Organic & ERA-Net Co-fund on Sustainable Food produc-
tion and consumption (SUSFOOD) (Project ID:2019.39) under project
Bio4Food. JL was supported by the China Scholarship Council (CSC)
Grant No. 201706350259 and by the Special Research Fund of Ghent
University (BOF) No. 01CD5821.
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