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Biostimulants and crop responses: a

review
a a b b a
R. Bulgari , G. Cocetta , A. Trivellini , P. Vernieri & A. Ferrante
a
Department of Agricultural and Environmental Sciences –
Production, Landscape, Agroenergy, University of Milan, via
Celoria 2, Milano 20133, Italy
b
Department of Scienze Agrarie, Alimentari e Agro-ambientali,
University of Pisa, Viale delle Piagge 23, Pisa 56124, Italy
Published online: 07 Oct 2014.

To cite this article: R. Bulgari, G. Cocetta, A. Trivellini, P. Vernieri & A. Ferrante (2014):
Biostimulants and crop responses: a review, Biological Agriculture & Horticulture: An International
Journal for Sustainable Production Systems, DOI: 10.1080/01448765.2014.964649

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 Biological Agriculture & Horticulture, 2014
http://dx.doi.org/10.1080/01448765.2014.964649

Biostimulants and crop responses: a review

R. Bulgaria, G. Cocettaa*, A. Trivellinib, P. Vernierib and A. Ferrantea
a
Department of Agricultural and Environmental Sciences – Production, Landscape, Agroenergy,
University of Milan, via Celoria 2, Milano 20133, Italy; bDepartment of Scienze Agrarie, Alimentari
e Agro-ambientali, University of Pisa, Viale delle Piagge 23, Pisa 56124, Italy
(Received 8 January 2014; accepted 9 September 2014)

Agricultural growing practices have been evolving towards organic, sustainable or

environmental friendly systems. The aim of modern agriculture is to reduce inputs
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without reducing the yield and quality. These goals can be achieved by breeding
programmes but would be species specific and time consuming. The identification of
organic molecules able to activate plant metabolism may allow an improvement in plant
performance in a short period of time and in a cheaper way. Biostimulants are plant
extracts and contain a wide range of bioactive compounds that are mostly still unknown.
These products are usually able to improve the nutrient use efficiency of the plant and
enhance tolerance to biotic and abiotic stresses. In this review, the state of the art and
future prospects for biostimulants are reported and discussed. Moreover, particular
attention has been paid to intensive agricultural systems such as horticultural and
floricultural crops. In vegetables, the application of biostimulants allowed a reduction in
fertilizers without affecting yield and quality. In leafy vegetables susceptible to nitrate
accumulation, such as rocket, biostimulants have been able to improve the quality and
keep the nitrates under the limits imposed by EU regulations. Moreover in leafy
vegetables, biostimulants increased leaf pigments (chlorophyll and carotenoids) and
plant growth by stimulating root growth and enhancing the antioxidant potential of
plants. In floriculture, biostimulants used in bedding plant production stimulated the
growth of plants, which reached the blooming and commercial stages earlier, thus
optimizing space in the greenhouse.
Keywords: bedding plants; floriculture crops; nutrient use; sustainable agriculture;
vegetables

Introduction
Research activity in the matter of agriculture systems has for years been oriented to
increase yield without considering the quality of the produce and the rational use of
resources. In contrast, attention now is mainly focused on product quality and the
sustainability of the cultivation systems. Moreover, cultivation management pays more
attention to the reduction of production costs by lowering inputs.
Protected cultivation of vegetables and floricultural crops usually requires high
amounts of fertilizers and pesticides. It is not always true that high nutrient availability
corresponds to higher quality of the products. On the contrary, excessive fertilization, and
especially high nitrogen supply, stimulates vegetative growth with a higher susceptibility
to pathogens (Liebman & Davis 2000). In leafy vegetables, the excessive availability of
nitrates often induces an accumulation in leaves with levels above the limits imposed by
EU regulation (Alberici et al. 2008; Cavaiuolo & Ferrante 2014). High rates of nitrogen

*Corresponding author. Email: [email protected]

q 2014 Taylor & Francis

 2 R. Bulgari et al.

fertilizers can have detrimental impacts on the environment, such as nitrate flows into
waterways and can increase greenhouse gas emissions of nitrous oxide (Mattner et al.
2013). The accumulation of high levels of nitrates in Brassica crops, for example, can also
impact adversely on human health (Luo et al. 2006; Parks et al. 2008; Cavaiuolo &
Ferrante 2014). Therefore, alternative methods for stimulating early growth in broccoli
and other vegetable crops are very interesting.
Floriculture crops, if highly fertilized, may have luxury consumption without benefits
for quality and, if grown in open hydroponics systems, may contaminate the environment,
because much of the nutrients used and water are lost. The cultivation of floriculture crops
is characterized by highly chemical inputs because quality is essentially defined by visual
appearance of the products. The quality of cut flowers and potted plants depends mainly on
leaf colour and flower integrity. The presence of physiological disorders related to mineral
nutrition or damage due to disease and insect attacks strongly affect quality and
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commercial value of these products. Furthermore, most floriculture species must be grown
under programmed cycles in order to be successful on the market. Bedding plants, for
example, are characterized by short growing cycles, rigid production plans and limited
growing area. Therefore, their growth must be fast in order to improve the use of labour
and distribution of work per area unit.
The wide range of fertilizers available together with growth regulators and
biostimulants frequently disorients the grower’s choice in the rational use of resources
with inefficient results or even negative effects on the quality of the products (Vernieri,
Borghesi, et al. 2005, 2006). These products can increase the efficiency of the use of
mineral nutrients reducing the leaching and guaranteeing a production more sustainable
(Vernieri, Ferrante, et al. 2005). Biostimulants have increasingly been considered as
production tools as demonstrated by the increase in scientific publications.
Biostimulants have been gaining interest in sustainable agriculture because their
application activates several physiological processes that enhance nutrient use efficiency,
stimulating plant development and allowing the reduction of fertilizers consumption
(Kunicki et al. 2010). Many biostimulants are also able to counteract the effect of biotic
and abiotic stresses, enhancing quality and crop yield by stimulating plant physiological
processes (Ziosi et al. 2013).

Biostimulant components and plant responses

Biostimulants are extracts obtained from organic raw materials containing bioactive
compounds. The most common components of the biostimulants are mineral elements,
humic substances (HSs), vitamins, amino acids, chitin, chitosan, and poly- and
oligosaccharides (Berlyn & Russo 1990; Hamza & Suggars 2001; Kauffman et al. 2007).
According to a report by FAO (2006), a substantial amount of seaweeds (15 million t y21)
are used as nutrient supplements and as biostimulants in agriculture. Seaweed extracts
have been used in agriculture as soil conditioners or as plant stimulators. They are applied
as foliar spray and enhance plant growth, freezing, drought and salt tolerance,
photosynthetic activity and resistance to fungi, bacteria and virus, improving the yield and
productivity of many crops (Norrie & Keathley 2006; Gajc-Wolska et al. 2013; Sharma
et al. 2014). Seaweeds used for biostimulant production contain cytokinins and auxins or
other hormone-like substances (Hamza & Suggars 2001). From a legal point of view, the
biostimulants can contain traces of natural plant hormones, but their biological action
should not be ascribed to them, otherwise they should be registered as plant growth
regulators.
 Biostimulants and crop responses 3

Humic acids (HAs) are naturally occurring in polymeric organic compounds and are
produced by the decay of organic materials. HAs can be found in soil, peat and lignites
(Sharif et al. 2002). HAs may stimulate plant growth by improving nutrient uptake by
exerting hormone-like effects as auxins (Baldotto & Baldotto 2013). HAs stimulate shoot
elongation and increase leaf nutrient accumulation (Chen et al. 2004) and chlorophyll
biosynthesis (Baldotto et al. 2009). Many of the active substances (a.s.) of biostimulants
can be present in very low concentrations, sometimes below the levels detectable with
commonly available technologies, but nevertheless can provide strong biological effects.
The composition of biostimulants is partly unknown; the complexity of the extracts
and the wide range of molecules contained in the solution make it very difficult to
understand which the most active compounds are. Moreover, the isolation and study of a
single component present in a biostimulant can produce unreliable results because the
effects on plants are often due to the combination and synergistic action of different
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compounds. The mechanisms activated by biostimulants are difficult to identify and still
under investigation (Ertani et al. 2011, 2013; Guinan et al. 2013). Therefore, the
biostimulants should be classified on the basis of their action in the plants or, even better,
on the physiological plant responses rather than on their composition.
The target for biostimulant activity in plants can be objectively identified using
molecular biology technologies such as transcriptome or microarray analysis, which
provide an overview of the affected pathways after biostimulants treatment (Santaniello
et al. 2013). Correlation analysis should be performed between the gene activation and
physiological responses in order to understand broadly the effects of biostimulants on
plants and the behaviour of different species. Moreover, bioinformatics analysis may
highlight the different action mechanisms in different plant species. These tools can also
be used to select different of raw materials on the basis of their effects on the transcriptome
and provide useful information on mixing different sources of organic materials. The
analysis of transcripts can also show the synergistic effects of different organic substances
and comparison studies with hormone or nutrient treatments can highlight the common
and specific genes that are up- or down-regulated by the biostimulants. The data that will
be obtained from these studies beyond the information on the biostimulant effects in plants
can be also useful for identifying markers to avoid product counterfeit.
Biostimulants can act directly on the plant physiology and metabolism or by improving
the soil conditions (Nardi et al. 2009). Biostimulants in soils affect the microflora and may
provide positive influence on plant growth. These products are usually applied in addition
to standard fertilization treatments to improve the nutrient use efficiency and products
quality (Heckman 1994). Biostimulants differ from fertilizers because they act on plant
metabolism, and their nutrient concentrations are negligible. These products are able to
modify root conformation and increase root development (Berlyn & Russo 1990; Nardi
et al. 2006; Petrozza et al. 2013a, 2013b). Biostimulants can be soil- or leaf-applied,
depending on their composition and on the desired results (Kunicki et al. 2010). They exert
their action only if they penetrate into the plant tissue. This aspect has to be considered in
comparison studies because different species may have different leaf permeability to the
biostimulants. The absorbability depends on field conditions, where plants are exposed to
different weather conditions and other extrinsic factors (Kolomaznik et al. 2012; Pecha
et al. 2012). The leaf cuticle can represent a barrier for biostimulant adsorption, and the
chemical structure of bioactive compounds can be an obstacle to their penetration in the
inner part of the leaf. The cuticle is composed by different components such as cuticular
waxes and the polymers cutin and cutan (Schreiber 2005). The layer of the cuticle and the
percentage of the different components is species specific, and these differences may affect
 4 R. Bulgari et al.

directly the efficiency of biostimulants. The ability of the biostimulants across the leaf
tissues is still to be elucidated. Biostimulants act at low concentrations (Zhang & Schmidt
1999). Unfortunately, the effect of a biostimulant can be different from species to species
and even from cultivar to cultivar and depends on environmental factors, and on the dose
and time of application (Kunicki et al. 2010). This variability of the effects often prevents
generalization and utilization of the results in other species.
The plant growth induced by biostimulants can be associated with an increase of amino
acids and enhanced protein biosynthesis. Seaweed and yeast extracts increase protein
content in plants as has been shown in Vicia faba. The higher protein content can be due to
the incorporation of amino acids used directly for protein biosynthesis. However, the
increased protein content can be also associated with an increase of carbohydrate
concentration in leaves (Abbas 2013). A higher sugar content in leaves usually speeds up
nitrogen incorporation through the nitrate assimilation pathway. The carbohydrates
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represent the carbon skeletons for the incorporation of reduced nitrate (ammonia) in amino
acids and increases protein biosynthesis. Alfalfa protein hydrolysate, used as a
biostimulant in maize, enhanced the enzymes activity involved in carbon metabolism and
N reduction and assimilation (Schiavon et al. 2008).
An increase in sugar biosynthesis in plants treated with biostimulants has been found
in several species and is associated with an increase in chlorophyll content, net
photosynthesis and quantum efficiency of photosystem II (Ferrini & Nicese 2002; Amanda
et al. 2009; Ertani & Nardi 2013). Chlorophyll a fluorescence parameters have widely
demonstrated that plants treated with biostimulants are less affected by a range of different
biotic and abiotic stresses (Fraser & Percival 2003; Amanda et al. 2009). Polysaccharides
and oligosaccharides are other important biostimulant components that affect plant
physiology. Polysaccharides in seaweed extracts applied to plants have been able to
enhance the resistance to fungal diseases. They are involved in the plant signalling
network against stresses and, in particular, biotic stresses. Arabidopsis plants treated with
l-carrageenan polysaccharides showed higher tolerance to Sclerotinia scleortiorum. The
l-carrageenan is a highly sulphated polysaccharide which acts in the plant defence
response by activation of jasmonic acid-related genes (Sangha et al. 2010).

Plant defence mechanisms

Plant response to biotic and abiotic stresses is a complex network of reactions which
involves different physiological pathways of the primary and secondary metabolism
(Kauffman et al. 2007). Reactive oxygen species (ROS) are a group of molecules that are
ubiquitous in plants. ROS derive from oxidative processes such as photosynthesis and
respiration, and, in normal conditions, they are produced in low concentration without any
negative consequences for the plants. In stressful conditions (biotic or abiotic), ROS levels
increase as an index of the oxidative burst induced by the stress agent (Foyer & Noctor
2005). A high concentration of ROS could be harmful because they can damage lipid
membranes, nucleic acids and proteins (Apel & Hirt 2004).
Plants have developed a complex series of mechanisms to counteract stress conditions
and ROS accumulation, and to control their levels. Mechanisms of plant stress responses
include the accumulation of sugars (Keunen et al. 2013), specific proteins (Sun et al. 2013)
and osmolytes (Kumari & Sairam 2013; Wang et al. 2013), an increase in the biosynthesis
or accumulation of flavonoids (Petrussa et al. 2013), glucosinolates (Martı́nez-Ballesta
et al. 2013), ascorbic acid (AsA; Gallie 2013) and carotenoids (Havaux 2014), and the
activation of hormone-mediated responsive networks that involve jasmonates (Waster-
 Biostimulants and crop responses 5

nack & Hause 2013) and other signalling molecules (Peleg & Blumwald 2011). Moreover,
recent studies have focused on the transcriptional response to stresses (Zeng et al. 2014)
and on the effects of genetic diversity (Luhua et al. 2013).
AsA is a widespread molecule which can act directly as an antioxidant or in a chain of
reactions mediated by specific antioxidant enzymes that catalyse the AsA oxidation and
recycling reactions (Choudhury et al. 2013). For this reason many studies have been
conducted with the aim of reinforcing this mechanism of defence by increasing AsA levels
and by stimulating the activity of the enzymes involved in its oxidation and recycling. In a
recent study, Vasconcelos et al. (2009) tested the effectiveness of a biostimulant based on
HSs and amino acids in combination with drought stress on the activity of superoxide
dismutases, catalase and ascorbate peroxidase. The study was conducted on two different
species, maize (Zea mays) and soybean (Glycine max), and the authors concluded that the
composition of the biostimulants was not able to enhance tolerance in plants subjected to
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water stress. On the other hand, a protein hydrolysate derived from alfalfa was able to
increase biomass in maize even under salinity stress by increasing the antioxidant systems
and speeding up the nitrogen metabolism (Ertani et al. 2013). It is thus clear that the
composition of the biostimulant determines its effect.
Among the polysaccharides, laminarin (b-1,3-glucan), a storage glucan found in the
brown alga Laminaria digitata (Stadnik & Freitas 2014), is able to induce a defence response
in plants and can be used to protect plants against pathogens such as Botrytis cinerea and
Plasmopora viticola in grapevine (Aziz et al. 2003). Laminarin acts through the activation of
defence-related enzymes [phenylalanine ammonia lyase (PAL), caffeic acid O-methyl
transferase and lipoxygenase], genes encoding various pathogenesis-related proteins with
antimicrobial properties and the accumulation of elicitor compounds such as salicylic acid.
The defence response also included a wide spectrum of events such as calcium influx,
alkalinization of the extracellular medium, an oxidative burst, activation of two mitogen-
activated protein kinases, expression of defence-related genes with increases in chitinase and
b-1,3-glucanase activities, and the production of phytoalexins (Aziz et al. 2003).
Treatment with a commercial extract of the brown seaweed (Stimplexw, Acadian
Agritech, Dartmouth, Nova Scotia, Canada, Table 1) increased drought tolerance in
Hamlin sweet orange trees (Spann & Little 2011). The effect of the product was found to
be independent of carbon fixation as photosynthesis was depressed regardless of treatment,
and the authors hypothesized that the observed response may have been due to plant
metabolite changes with consequent effects on plant – water relationships. The use of
marine bioactive substances (IPA extract, supplied by BiotechMarine, Roullier Group,
Pontrieux, France, Table 1) resulted in improved foliar ion uptake and water stress
tolerance in potted Vitis vinifera plants. The treatment acted by promoting accumulation of
mineral molecules, and this helped to maintain high leaf water potential and stomata
conductance in response to water stress (Mancuso et al. 2006). Two products derived from
seaweeds and black peat, respectively, have recently reported to promote growth of
Brassica napus (Billard et al. 2013). Both biostimulants stimulated chloroplast division
and increased Mg, Mn, Na and Cu plant concentrations, and root-to-shoot translocation of
Fe and Zn. These observations were associated with an increased expression of a Cu
transporter (COPT2) and NRAMP3, a gene involved in Fe and Zn translocation.

Phytonutrients: leaf pigments, secondary metabolites and vitamins

There are several definitions for the word phytonutrient; in brief, it can be described as a
substance derived from plants which is beneficial to human health and which is neither a
 6 R. Bulgari et al.

Table 1. Composition declared on the labels of commercial biostimulants.

Product Composition
Actiwave Composition (w/v): total nitrogen (N) 3.0% (38.7 g L21); organic nitrogen (N)
1.0% (12.9 g L21); ureic nitrogen (N) 2.0% (25.8 g L21); potassium oxide (K2O)
soluble in water 7.0% (90.3 g L21); organic carbon (C) of biological origin 12%
(154.8 g L21); iron (Fe) soluble in water 0.5% (6.45 g L21); iron (Fe) chelated by
ethylenediaminedi(2-hydroxy-5-sulfophenylacetic) acid (EDDHSA) 0.5%
(6.45 g L21); zinc (Zn) soluble in water 0.08% (1.03 g L21); zinc (Zn) chelated by
Ethylenediaminetetraacetic acid (EDTA) 0.08% (1.03 g L21). Liquid formulation
Aminoplant Contents (w/v): total nitrogen (N) 2%, organic nitrogen 2%, potassium (K2O) 2%,
K 1.66%, phosphate (P2O5) 2%, P 0.87%, total amino acids 12.5%, organic carbon
11.6%
Amino acid content (% w/v): alanine 1.08, arginine 0.64, aspartic acid 0.83, cystine
0.4, glutamic acid 2.02, glycine 0.65, histidine 0.41, iso-leucine 0.41, leucine 1.19,
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lysine 0.49, methionine 0.31, ornithine 0.25, phenylalanine 0.47, proline 1.14,
serine 0.60, threonine 0.54, tryptophane 0.03 tyrosine 0.32, valine 0.68
Benefit Composition (w/v): total nitrogen (N) 3.0% (36 g L21), organic nitrogen (N): 3.0%
(36 g L21); organic carbon (C) of biological origin: 10.0% (120 g L21). Liquid
formulation
Goëmar BM 86 Composition (w/v): total nitrogen (N) 5.0%, magnesium (Mg) 2.4%, sulphur (S)
combined 3.2%, boron (B) 2.0%, molybdenum (Mo) 0.02%, sodium (Na) 0.6%
Goëmar Goteo Composition (w/v): organic substances 1.3– 2.4%, phosphorus (P2O5) .24.8%,
potassium (K2O) .4.75%
IPA extract Composition (w/v): marine bioactive substances 0.1%
Kendal Composition (w/v): total nitrogen (N) 3.5% (45.0 g L21); organic nitrogen (N)
0.3% (4.0 g L21); ureic nitrogen (N) 3.2% (41.0 g L21); potassium oxide (K2O)
soluble in water 15.5% (200.0 g L21); organic carbon (C) of biological origin 3.0
(39.0 g L21). Liquid formulation
Megafol Composition (w/v): total nitrogen (N) 3.0% (36.6 g L21); organic nitrogen (N)
1.0% (12.2 g L21); ureic nitrogen (N) 2.0% (24.4 g L21); potassium oxide (K2O)
soluble in water 8.0% (97.6 g L21); organic carbon (C) of biological origin 9.0%
(109.8 g L21). Liquid formulation
Radifarm Composition (w/v): total nitrogen (N) 3.0%; organic nitrogen (N) 1.0%; ureic
nitrogen (N) 2.0%; potassium oxide (K2O) soluble in water 8.0%; organic carbon
(C) of biological origin 10.0%; zinc (Zn) soluble in water 0.1%; zinc (Zn) chelated
by EDTA 0.1%. Liquid formulation
Seasol Nitrogen (N) 0.2% w/v, phosphorus (P) 0.02% w/v, potassium (K) 3.7%, boron (B)
15 mg L21, calcium (Ca) 458 mg L21, cobalt (Co) ,0.5 mg L21, copper (Cu)
,0.5 mg L21, iron (Fe) 115 mg L21, magnesium (Mg) 972 mg L21, manganese (Mn)
2 mg L21, molybdenum (Mo) ,0.5 mg L21, selenium (Se) ,0.5 mg L21, silicon (Si)
56 mg L21, sodium (Na) 6820 mg L21, sulphur (S) 2574 mg L21, zinc (Zn) 5 mg L21
Stimplexw Composition (w/v): cytokinin 0.01% (expressed as kinetin, corresponding 100 ppm
of kinetin activity), other ingredients 99.99%
Viva Composition (w/v): total nitrogen (N) 3.0% (37.2 g L21); organic nitrogen (N)
1.0% (12.4 g L21); ureic nitrogen (N) 2.0% (24.8 g L21); potassium oxide (K2O)
soluble in water 8.0% (99.2 g L21); organic carbon (C) of biological origin 8.0%
(99.2 g L21); iron (Fe) soluble in water 0.02% (0.25 g L21); iron (Fe) chelated by
EDDHSA 0.02% (0.25 g L21). Liquid formulation

vitamin nor a mineral (for reviews about phytonutrients, see Beecher 1999; Martin et al.
2013). Nowadays a large number of publications report the beneficial effects of a diet rich
in phytonutrients (Martin et al. 2013; Sacco et al. 2013; Miao et al. 2014). Consumers have
become more aware about quality and health-related features of crops and growing
attention is paid to the antioxidant and health-related traits of fruit, vegetables
(Rajarathnam et al. 2013) and edible flowers (Cavaiuolo et al. 2013).
 Biostimulants and crop responses 7

From the physiological point of view, phytonutrients are often secondary metabolites
in plants. Secondary metabolites are a wide and heterogeneous group of compounds which
differ in their chemistry and are synthesized from primary metabolites. Plant secondary
metabolites perform several functions in plants. They are involved in the mechanisms of
interaction between plants and the environment and have a pivotal role in plant defence
responses to biotic or abiotic stresses by acting as phytoalexins, signal molecules and
antioxidants (Kliebenstein 2004; Bartwal et al. 2013). Some secondary metabolites, for
example anthocyanins, are also able to attract animals in order to favour seed dispersal or
flower pollination; others, however, have a repellent effect on animals. A recent
publication analysed the interactions between primary and secondary metabolisms in
stress responses and the relative costs in terms of allocation, auto toxicity, ecology, fitness
and opportunity (Neilson et al. 2013).
The mechanisms of action of plant secondary metabolites depend on the kind of molecule,
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the physiological pathways in which they are involved and their interactions with primary
metabolism. Frequently, secondary metabolites act as antioxidants, blocking the oxidative
reactions induced by stresses and enhancing the antioxidant potential of vegetables, flowers
and fruits. Some studies have been conducted on the effects of biostimulants on secondary
metabolites in crops; however, the mechanisms of action and the effects of biostimulation on
secondary metabolism are not clear yet. In a recent paper, Pardo-Garcı́a et al. (2014) showed
that oak acts as a biostimulant for grape polyphenols and determined a higher content of gallic
acid, hydroxycinnamoyl tartaric acids, acylated anthocyanins, flavanols and stilbenes.
Biostimulants derived from agroindustrial by-products were reported to be effective in
improving plant productivity, increasing the synthesis of secondary compounds involved in
several plant physiological responses, and enhancing the activity of the enzyme PAL and the
expression of ZmPAL in maize leaves (Ertani et al. 2011).
The first study showing the relationship between HSs and the phenylpropanoid
pathway was published in 2010 (Schiavon et al. 2010). This study reported that the effect
of HS on phenylpropanoids metabolism in Z. mays plants and the action of HS used were
related to its chemical composition and molecular conformation in addition to its
molecular weight. Activities of PAL and TAR as well as gene-related expression were
induced by treatment, and the levels in some phenolic compounds increased consequently.
Moreover, the authors suggested that HS may stimulate plant growth by inducing carbon
and nitrogen metabolism.
Biostimulants often increase the colour of leaves by stimulating the chlorophyll
content. This effect was observed in cowpea seeds pre-soaked in carrot extract (Abbas &
Akladious 2013). Analogous results were observed in rocket (E. sativa) treated with
Moringa oleifera extract; in this case, the chlorophyll levels increased and carotenoids
doubled (Abdalla 2013). High concentration of leaf pigments resulting from biostimulant
treatments in rocket was also observed by Vernieri, Borghesi, et al. (2005, 2006).
Biostimulants improved the antioxidant activity, vitamin and phenolic contents in fruits as
well as the pigment content in leaves of pepper (Capsicum annuum) plants grown
hydroponically (Paradiković et al. 2011). Organic mineral fertilizers significantly
influenced the content of biologically active compounds in endive (Cichorium endivia);
the most effective preparation tested (Goëmar Goteo; Table 1) caused the highest amounts
of rutoside and astragalin (kaempferol 3-O-glucoside) (Gajc-Wolska et al. 2012).
Much study is focused on measuring changes in the content of specific metabolites.
However, research activities should consider the complex network of physiological events
behind these effects by investigating the main enzymes, genes and regulatory factors
involved in the biosynthesis and turn-over of each metabolite. The recent development of
 8 R. Bulgari et al.

techniques for large-scale analysis of transcripts represents an important and significant

improvement in research. These approaches allow a wide range of information at the
transcript level to be obtained and create a great opportunity to study several plant responses
directly from sequence-level information. The use of modern techniques of analysis and
molecular biology-based approaches, that is microarrays and new generation sequencing,
could represent a useful means to reach a deeper understanding of the mechanisms of
actions by which biostimulants affect metabolism in plants and enhance the accumulation
of phytonutrients. In the past few years, the number of publications in which these
techniques have been applied has increased. The application of microarray analysis was
recently applied to evaluate the effect of biostimulants at the transcriptomic level (Table 2)
and hence on the plant physiology responses (Jannin et al. 2012; Santaniello et al. 2013).

Application of biostimulants on vegetable crops

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Biostimulants can be used in vegetable production to improve productivity and yield, and
to enhance plant tolerance to stress factors and plant health (Table 2). The biostimulant
Actiwavew (Valagro s.p.a., Atessa, Chieti, Italy, Table 1), applied as an additional
component in the nutrient solution of rocket (E. sativa) grown in a floating system,
increased yield even if the nutrient concentration was reduced (Vernieri, Borghesi, et al.
2006). In this crop, the application of Actiwavew increased the use efficiency of mineral
nutrients, and this effect was particularly significant when the nutrient solution
concentration was reduced to 10% of the standard nutrient solution. The improvement of
the nutrient use efficiency is probably obtained because in plants grown with Actiwavew
root biomass was higher as well as roots development.
The effect of Actiwavew was also confirmed in baby leaf lettuce (Lactuca sativa var.
acephala) grown in a plastic tunnel (Amanda et al. 2009). The yield was increased by the
application of 3 mL m22 (Table 2), whereas leaf nitrates were not affected because they
were already low. In strawberry (Fragaria £ ananassa), the application of Actiwavew
stimulated vegetative growth (10%), leaf chlorophyll content (11%), stomata density
(6.5%), photosynthetic activity, yield (27%) and fruit weight (Spinelli et al. 2010).
Kunicki et al. (2010) investigated the effect of a biostimulant containing amino acids
named Aminoplant (Table 1) on the yield of spinach (Spinacia oleracea), considering also
the influence of the cultivar and the time of cultivation (spring and autumn). This
biostimulant enhanced the nitrate reductase activity. On carrot (Dacus carota),
Aminoplant not only influenced productivity, but also the chemical composition of the
roots. The plant response to the biostimulant treatment depended on the cultivar more than
on environmental conditions, in particular growing seasons. Aminoplant influenced yield
of roots and leaf rosette mass, increased the soluble sugars content in carrot roots and
affected dry matter content. A significant effect of Aminoplant on nitrate content was also
observed, but the results were not repeatable in the experimental years, so different
climatic conditions may have modified carrot response (Grabowska et al. 2012).
In general, different crops treated with this biostimulant had greater yields per hectare
(Maini 2006). Aminoplant was also applied in curly endive (C. endivia var. crispum), but
no significant differences on yield were found (Gajc-Wolska et al. 2012).
The use of Goëmar BM86 (Table 1) in the cultivation of broccoli (Brassica oleracea
var cymosa) in an open field (2 L ha21) (Table 2) had a significant effect on the chemical
quality of produce. The content of macro- and micronutrients increased, as well as the
yield (Gajc-Wolska et al. 2013). Four different biostimulants, Radifarmw, Megafolw,
Vivaw and Benefitw (Valagro s.p.a., Atessa, Chieti, Italy, Table 1) increased the yield of
 Biostimulants and crop responses 9

Table 2. Biostimulant concentrations and plant responses in different vegetables.

Biostimulant
Species concentration Plant response References
w
Broccoli Seasol Increased leaf area, stem Mattner et al. (2013)
(B. oleracea var. Dilutions of 1:25, 1:100, diameter and biomass
italica) 1:200, 1:500 in distilled
water (crop drenching
with kelp extract at 25 and
2.5 L ha21)
Broccoli Goëmar BM86 Increased yield and Gajc-Wolska et al.
(B. oleracea var. 2 L ha21 content of macro- and (2013)
cymosa) micronutrients
Carrot (D. carota) Aminoplant Influenced carrot Grabowska et al.
1.5 dm3 ha21 productivity and chemical (2012)
3.0 dm3 ha21
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composition of the roots

Endive (C. endivia) Goëmar Goteo Leaves synthesized more Gajc-Wolska et al.
Watering with solution rutoside and astragalin (2012)
0.10%
Aminoplant
Spraying with solution
0.20%
Lettuce (L. sativa) Actiwave Increased yield and Amanda et al. (2009)
3 mL m22 energy use efficiency
Lettuce (L. sativa) Radifarm Stimulated root growth Vernieri et al. (2002)
125 mL a.s. hL21 and induced a more
favourable root/shoot ratio
Pepper (C. annuum) Radifarm Better root growth and Paradiković et al.
By watering in development (2011)
concentration of 0.25% in
quantity of 60 mL plant21
Megafol Effects on foliar growth Paradiković et al.
By spraying in and an anti-stress effect (2011)
concentration of 0.20% in
quantity of 55 – 60 mL
plant21
Viva Improved fruit setting and Paradiković et al.
By watering in reduced fruit drop (2011)
concentration of 0.25% in
quantity of 120 mL
plant21
Benefit Accelerated major Paradiković et al.
By spraying in metabolic reactions and (2011)
concentration of 0.30% in improved and made more
quantity of 120– 150 mL uniform fruit weight and
plant21 size
Potato Seaweed extract Improvement in growth, Haider et al. (2012)
(S. tuberosum cv. ‘Primo’0.5 mL L21 ha21 yield and tuber quality of
Sante) potato
Rocket (E. sativa) Actiwave Increased yield, total Vernieri, Borghesi,
0.08– 1.3 mL L21 chlorophyll and et al. (2005, 2006)
carotenoids. Reduced and Vernieri,
nitrate accumulation in Ferrante, et al. (2005,
leaves 2006)
(Continued)
 10 R. Bulgari et al.

Table 2 – continued

Biostimulant
Species concentration Plant response References
Sacred basil Combination of three Higher dry herbage yield Prabhu et al. (2010)
(O. sanctum) biostimulants T7 (spraying
2% Panchakavya þ 0.2%
HA þ 2% Moringa leaf
extract until run off)
Spinach Aminoplant Lowered dry matter Kunicki et al. (2010)
(S. oleracea) 1.5 dm3 ha21 content in leaves,
3.0 dm3 ha21 positively influenced
nitrate reductase activity
Strawberry Actiwave Increased biomass, yield, Spinelli et al. (2010)
(Fragaria 10 mL of product chlorophyll content, the
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ananassa) dissolved in 20 mL of tap stomata density,

water photosynthesis and fruit
weight
Tomato Radifarm Stimulated root growth Vernieri et al. (2002)
(S. lycopersicum) 250 mL a.s. hL21 and induced a more
favourable root/shoot ratio
Tomato Radifarm Positive effects on root Petrozza et al.
(S. lycopersicum) 3– 6 mL L21 system (2013a)
Viva Increasing of plant and Petrozza et al.
n.d. root biomass (2013b)

pepper (C. annuum) grown hydroponically and at the same time improved fruit quality
during the hot summer season (Paradiković et al. 2011). Petrozza et al. (2013a) showed that
Radifarmw treatments on tomato (Solanum lycopersicum) plants (Table 2) stimulated a
greater root system and more secondary roots. Therefore, the treated plants had higher water
use efficiency. The same authors demonstrated that Vivaw treatments on drought-stressed
plants of S. lycopersicum cv. Ikram increased plant biomass and enhanced root development
(Petrozza et al. 2013b). This study showed that the biostimulant was able to normalize plant
growth under abiotic stresses.
A combination of three biostimulants T7 (spraying 2% Panchakavya þ 0.2%
HA þ 2% Moringa leaf extract) used on basil (Ocimum sanctum) increased the yield
(Prabhu et al. 2010). Panchakavya is a mixture of five cow products. Three are directly
produced by cow such as dung, urine and milk, and two are derived products curd and
ghee. Haider et al. (2012) studied the effect of foliar application of seaweed extract Primo
(Table 1) as an organic biostimulant on potato (Solanum tuberosum cv. Sante) (Table 2)
and showed a significant improvement of plant growth, yield and tuber quality. Moreover,
it also improved nitrogen, total soluble solids and protein contents of the tubers.
Mattner et al. (2013) demonstrated that kelp extract (Seasolw International Pty Ltd,
Mountain Hwy, Boronia, Australia, Table 1) stimulates broccoli establishment and growth
in the glasshouse and field (Table 2) significantly increased the leaf area, stem diameter
and biomass of broccoli. Furthermore, kelp extract significantly reduced by 23% the early
incidence of white blister, caused by Albugo candida.
On lettuce (L. sativa) and tomato (S. lycopersicum), the application of Radifarm
(Table 1) at nursery level had a positive effect on plant growth by increasing the shoot and
roots development (Table 2). In lettuce, the biostimulant strongly stimulated the root
growth and showed also an increase of the leaf area. On tomato plants, the effect was
 Biostimulants and crop responses 11

stronger and all parameters measured were positively influenced. The application of
Radifarmin in both species stimulated growth and improved the root/shoot ratios (Vernieri
et al. 2002).

Application of biostimulants on floriculture crops

The quality of the most floriculture crops, such as bedding plants, is defined by the visual
appearance, plant biomass, flower number and turnover. It is well known that plants,
during transplantation, undergo several abiotic stresses causing environmental conditions
to deviate from the optimum (Kijne 2006; Mena-Petite et al. 2006). The application of
biostimulants reduces the stress in the case of adverse temperatures and increases yield,
and the consequences are fewer cases of drought, freezing, mechanical and chemical
damages as well as less viral plant infection (Maini 2006). With the use of biostimulants at
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the stage of plantlet growth and development, it is possible to create better conditions by
adding active substances such as polysaccharides, proteins, amino acids and glycosides.
The application of Actiwavew (Table 1) gave positive results on Ageratum
houstonianum, Coleus blumei, Impatiens wallerana, Lobularia maritima and Salvia
splendens by increasing fresh and dry weight of plants (Table 3) (Vernieri & Mugnai 2003;
Vernieri, Borghesi, et al. 2005; Vernieri, Ferrante, et al. 2006). The positive effect was
higher if combined with fertilizer supply. These results indicate that Actiwavew acts by
improving the use efficiency of mineral nutrients (Vernieri, Ferrante, et al. 2006).
Actiwavew also accelerated plant growth rates and flowering, improving quality of
bedding plants and reduced the growing cycle (Vernieri, Ferrante, et al. 2005). This aspect
is particularly important because it optimizes the growing area in a nursery.
Actiwavew was also tested in the nursery for improving the rooting of Camellia japonica
cuttings, because the rooting stage in this species is long and requires more than 3 months if
no rooting promoting treatments are applied (Table 3) (Ferrante et al. 2011, 2013). The
application of Actiwave as a spray treatment to the Camellia cuttings speeded up rooting and
growth. This biostimulant was more efficient than gibberellic acid. After 3 months, the
percentage of rooting was up to 70% in the treated cuttings while still zero in the control.
With Begonia semperflorens the soil application of biostimulant Radifarmw (Table 1)
positively affected the growth and development of the plants (Table 3) (Zeljković,
Paradiković, Tkalec, et al. 2010). This commercial product belongs to a group of
biostimulants containing glucosides (energy growth factors) and amino acids (arginine and
asparagine). Treatments with Radifarmw by watering on wild rose had positive effect on
the shoot number and the root weight (Tkalec et al. 2012). The biostimulant application in
Rosa canina transplant production improved growth and development of roots and above-
ground plant mass, which is important for faster plant adaptation to the environment
during transplanting. Similar results were obtained with S. splendens (Zeljković,
Paradiković, Babić, et al. 2010).
De Lucia and Vecchietti (2012) evaluated the effects and the interaction of three
different agricultural biostimulants based on hydrolysed proteins coming from algae
[Microwave Assisted Extraction (MAE)], animal epithelium [animal derived-protein
hydrolysate (APH)] and lucerne origin (HS) on longiflorum lilies £ Asiatic hybrids (LA)
lily grown in a soilless system. These biostimulants applied as foliar spray or soil drench
gave similar performances; the crop cycle of lily was shorter, the leaves more expanded in
the lamina and greener, the flower buds had a higher diameter and the root system showed
a higher length development. The effect HA on growth, macro- and micronutrient
contents, and postharvest life of gerbera (Gerbera jamesonii cv. Malibu) (Table 3) was
 Downloaded by [University of Waterloo] at 06:10 11 October 2014

12

Table 3. Biostimulant concentrations and plant responses in different floriculture crops.

Species Biostimulant concentration Plant response References

Wild rose (R. canina), Radifarm concentration of 0.25% Positive effect on shoot number and root Tkalec et al. (2012), Zeljković,
S. splendens; Begonia weight; improved growth and development of Paradiković, Babić, et al. (2010) and
(B. semperflorens) root Zeljković, Paradiković, Tkalec, et al.
(2010)
R. Bulgari et al.

A. houstonianum, C. blumei, Actiwave, 2.5 mL L21 for 8 weeks Increased leaf area, fresh weight, dry weight Vernieri and Mugnai (2003),
L. maritima, I. wallerana, Vernieri, Borghesi, et al. (2005) and
S. splendens, T. patula Vernieri, Ferrante, et al. (2006)
C. grandiflora Combination of three biostimulants Stimulated root growth and hastened Vernieri and Mugnai (2003)
Radifarm and Kendal (1:1) 2.5 mL L21 flowering
and Kendal þ Viva (1:1) 2.5 mL L21
C. japonica L. Actiwave, 0.12– 0.24 mL cutting21 Increased rooting, reduced the nursery stage Ferrante et al. (2011, 2013)
delivered in eight applications
Lilium Brindisi (LA: Lilium MAE, APH and HS, applied eight times Crop cycle came early, leaves more expanded De Lucia and Vecchietti (2012)
longiflorum £ Lilium both at foliar and drenching level at the in the lamina and greener, flower buds with
elegans) concentration of 1.5 g L – 1 higher diameter; root system longer, stem and
bulb dry weights higher
Gerbera (G. jamesonii cv. HA, 500 mg L21 increased the number of Nikbakht et al. (2008)
21
Malibu) 500 and 1000 mg L harvested flowers per plant.
1000 mg L21 increased root growth and macro
and micronutrient contents of leaves and
scapes. The vase life was extended
Gladiolus (Gladiolus) HA, solutions containing 0, 10, 20, 30 and Accelerated growth, hastened and increased Baldotto and Baldotto (2013)
40 mmol L21 of C from HA flowering
S. splendens Combination of three biostimulants Stimulated root growth, increased biomass, Vernieri and Mugnai (2003)
Radifarm and Kendal (1:1) 2.5 mL L21 yield, chlorophyll
and Kendal þ Viva (1:1) 2.5 mL L21
T. patula Combination of three biostimulants Increased biomass, yield, chlorophyll, Vernieri and Mugnai (2003)
Radifarm and Kendal (1:1) 2.5 mL L21 anticipated the flowering and increased the
and Kendal þ Viva (1:1) 2.5 mL L21 resistance to pathogens
 Biostimulants and crop responses 13

examined by Nikbakht et al. (2008). Addition of HA to the nutrient solution increased root
growth and macro- and micronutrient contents of leaves and scapes. The vase life of
harvested flowers was extended, and HA could prevent or delay the stem break incidence.
In Gladiolus, the treatment of corms, before planting, with solutions containing increasing
concentrations HAs accelerated growth and increased flowering (Baldotto & Baldotto
2013).
Three bedding plants, Calendula grandiflora, S. splendens and Tagetes patula were
treated twice at the emergence with Radifarm plus Kendal (Table 1) with an interval of
10 days; then until the end of experiments, plants were treated with Kendal plus Viva
every 10 days. The effects of treatments were evident on the total dry weight as well as on
the dry weight of the leaf area and roots. The treatments were more efficient in
C. grandiflora and S. splendens, whereas the effects were less marked in T. patula
(Vernieri & Mugnai 2003).
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Conclusions and future prospects

The application of biostimulants in vegetable and floriculture crop cultivation allows
higher levels of sustainability by the reduction of fertilizers and environmental
contamination and, at the same time, increases plant tolerance to abiotic and biotic stresses
enhancing internal and external quality. Most published papers report the effects of the
biostimulant applications on plants, but few have investigated their effects on plant
physiology and biochemistry. However, recent papers have focused their attention on the
mechanisms of action of these products. The characterization of a biostimulant should be
performed on the basis of the plant responses, indicating the physiological targets and
metabolic network involved. Moreover, the effect of the biostimulants is not always
consistent among the plant species. This may occur because in the treated plants the
sensitivity thresholds for one or more bioactive molecules of the biostimulants are
different and synergistic effects may not occur.
The use of transcriptome analysis to study a broad range of gene expression profiles
can help to understand the biostimulant targets in plants, providing information on the
physiological pathways affected and the potential receptors activated. These data will
allow a deeper knowledge of the effects and functions of the components, both known and
unknown, of biostimulants products to be obtained and can be used in the classification of
new commercial formulations and in the evaluation of their effectiveness.

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