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Biological Agriculture & Horticulture:An International Journal forSustainable Production SystemsPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/tbah20

Biostimulants and crop responses: areviewR. Bulgaria, G. Cocettaa, A. Trivellinib, P. Vernierib & A. Ferrantea

a Department of Agricultural and Environmental Sciences –Production, Landscape, Agroenergy, University of Milan, viaCeloria 2, Milano 20133, Italyb Department of Scienze Agrarie, Alimentari e Agro-ambientali,University of Pisa, Viale delle Piagge 23, Pisa 56124, ItalyPublished 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 InternationalJournal for Sustainable Production Systems, DOI: 10.1080/01448765.2014.964649

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Biostimulants and crop responses: a review

R. Bulgaria, G. Cocettaa*, A. Trivellinib, P. Vernierib and A. Ferrantea

aDepartment of Agricultural and Environmental Sciences – Production, Landscape, Agroenergy,University of Milan, via Celoria 2, Milano 20133, Italy; bDepartment of Scienze Agrarie, Alimentarie 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 orenvironmental friendly systems. The aim of modern agriculture is to reduce inputswithout reducing the yield and quality. These goals can be achieved by breedingprogrammes but would be species specific and time consuming. The identification oforganic molecules able to activate plant metabolismmay allow an improvement in plantperformance in a short period of time and in a cheaper way. Biostimulants are plantextracts 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 andenhance tolerance to biotic and abiotic stresses. In this review, the state of the art andfuture prospects for biostimulants are reported and discussed. Moreover, particularattention has been paid to intensive agricultural systems such as horticultural andfloricultural crops. In vegetables, the application of biostimulants allowed a reduction infertilizers without affecting yield and quality. In leafy vegetables susceptible to nitrateaccumulation, such as rocket, biostimulants have been able to improve the quality andkeep the nitrates under the limits imposed by EU regulations. Moreover in leafyvegetables, biostimulants increased leaf pigments (chlorophyll and carotenoids) andplant growth by stimulating root growth and enhancing the antioxidant potential ofplants. In floriculture, biostimulants used in bedding plant production stimulated thegrowth of plants, which reached the blooming and commercial stages earlier, thusoptimizing 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

q 2014 Taylor & Francis

*Corresponding author. Email: giacomo.cocetta@unimi.it

Biological Agriculture & Horticulture, 2014

http://dx.doi.org/10.1080/01448765.2014.964649

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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

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.

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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

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

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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

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 (Martınez-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-

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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

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

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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).

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 gL21); iron (Fe) chelated byethylenediaminedi(2-hydroxy-5-sulfophenylacetic) acid (EDDHSA) 0.5%(6.45 g L21); zinc (Zn) soluble in water 0.08% (1.03 gL21); zinc (Zn) chelated byEthylenediaminetetraacetic acid (EDTA) 0.08% (1.03 gL21). 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 carbon11.6%Amino acid content (% w/v): alanine 1.08, arginine 0.64, aspartic acid 0.83, cystine0.4, glutamic acid 2.02, glycine 0.65, histidine 0.41, iso-leucine 0.41, leucine 1.19,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 gL21); organic carbon (C) of biological origin: 10.0% (120 g L21). Liquidformulation

Goemar 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%

Goemar 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%; ureicnitrogen (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) chelatedby EDTA 0.1%. Liquid formulation

Seasol Nitrogen (N) 0.2% w/v, phosphorus (P) 0.02% w/v, potassium (K) 3.7%, boron (B)15mgL21, calcium (Ca) 458mgL21, cobalt (Co),0.5mgL21, copper (Cu),0.5mgL21, iron (Fe) 115mgL21, magnesium (Mg) 972mgL21, manganese (Mn)2mgL21, molybdenum (Mo),0.5mgL21, selenium (Se),0.5mgL21, silicon (Si)56mgL21, sodium (Na) 6820mgL21, sulphur (S) 2574mgL21, zinc (Zn) 5mgL21

Stimplexw Composition (w/v): cytokinin 0.01% (expressed as kinetin, corresponding 100 ppmof 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 gL21); iron (Fe) chelated byEDDHSA 0.02% (0.25 gL21). Liquid formulation

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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).

Themechanismsof action of plant secondarymetabolites dependon the kind ofmolecule,

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-Garcıa 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 (Paradikovic et al. 2011). Organic mineral fertilizers significantly

influenced the content of biologically active compounds in endive (Cichorium endivia);

the most effective preparation tested (Goemar 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

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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

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 3mLm22 (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 Goemar 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

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

SpeciesBiostimulantconcentration Plant response References

Broccoli(B. oleracea var.italica)

Seasolw

Dilutions of 1:25, 1:100,1:200, 1:500 in distilledwater (crop drenchingwith kelp extract at 25 and2.5 L ha21)

Increased leaf area, stemdiameter and biomass

Mattner et al. (2013)

Broccoli(B. oleracea var.cymosa)

Goemar BM862L ha21

Increased yield andcontent of macro- andmicronutrients

Gajc-Wolska et al.(2013)

Carrot (D. carota) Aminoplant1.5 dm3 ha21

3.0 dm3 ha21

Influenced carrotproductivity and chemicalcomposition of the roots

Grabowska et al.(2012)

Endive (C. endivia) Goemar GoteoWatering with solution0.10%AminoplantSpraying with solution0.20%

Leaves synthesized morerutoside and astragalin

Gajc-Wolska et al.(2012)

Lettuce (L. sativa) Actiwave3mLm22

Increased yield andenergy use efficiency

Amanda et al. (2009)

Lettuce (L. sativa) Radifarm125mL a.s. hL21

Stimulated root growthand induced a morefavourable root/shoot ratio

Vernieri et al. (2002)

Pepper (C. annuum) RadifarmBy watering inconcentration of 0.25% inquantity of 60mL plant21

Better root growth anddevelopment

Paradikovic et al.(2011)

MegafolBy spraying inconcentration of 0.20% inquantity of 55–60mLplant21

Effects on foliar growthand an anti-stress effect

Paradikovic et al.(2011)

VivaBy watering inconcentration of 0.25% inquantity of 120mLplant21

Improved fruit setting andreduced fruit drop

Paradikovic et al.(2011)

BenefitBy spraying inconcentration of 0.30% inquantity of 120–150mLplant21

Accelerated majormetabolic reactions andimproved and made moreuniform fruit weight andsize

Paradikovic et al.(2011)

Potato(S. tuberosum cv.Sante)

Seaweed extract‘Primo’0.5mLL21 ha21

Improvement in growth,yield and tuber quality ofpotato

Haider et al. (2012)

Rocket (E. sativa) Actiwave0.08–1.3mLL21

Increased yield, totalchlorophyll andcarotenoids. Reducednitrate accumulation inleaves

Vernieri, Borghesi,et al. (2005, 2006)and Vernieri,Ferrante, et al. (2005,2006)

(Continued)

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pepper (C. annuum) grown hydroponically and at the same time improved fruit quality

during the hot summer season (Paradikovic et al. 2011). Petrozza et al. (2013a) showed thatRadifarmw treatments on tomato (Solanum lycopersicum) plants (Table 2) stimulated a

greater root system andmore 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

Table 2 – continued

SpeciesBiostimulantconcentration Plant response References

Sacred basil(O. sanctum)

Combination of threebiostimulants T7 (spraying2% Panchakavya þ 0.2%HA þ 2% Moringa leafextract until run off)

Higher dry herbage yield Prabhu et al. (2010)

Spinach(S. oleracea)

Aminoplant1.5 dm3 ha21

3.0 dm3 ha21

Lowered dry mattercontent in leaves,positively influencednitrate reductase activity

Kunicki et al. (2010)

Strawberry(Fragariaananassa)

Actiwave10mL of productdissolved in 20mL of tapwater

Increased biomass, yield,chlorophyll content, thestomata density,photosynthesis and fruitweight

Spinelli et al. (2010)

Tomato(S. lycopersicum)

Radifarm250mL a.s. hL21

Stimulated root growthand induced a morefavourable root/shoot ratio

Vernieri et al. (2002)

Tomato(S. lycopersicum)

Radifarm3–6mLL21

Positive effects on rootsystem

Petrozza et al.(2013a)

Vivan.d.

Increasing of plant androot biomass

Petrozza et al.(2013b)

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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

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.

Actiwavewwas also tested in the nursery for improving the rooting ofCamellia 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 ofActiwave as a spray treatment to theCamellia 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) (Zeljkovic,

Paradikovic, 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 (Zeljkovic,

Paradikovic, Babic, 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

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Table

3.

Biostim

ulantconcentrationsandplantresponsesin

differentfloriculture

crops.

Species

Biostim

ulantconcentration

Plantresponse

References

Wildrose

(R.canina),

S.splendens;Begonia

(B.semperflorens)

Radifarm

concentrationof0.25%

Positiveeffect

onshootnumber

androot

weight;im

proved

growth

anddevelopmentof

root

Tkalec

etal.(2012),Zeljkovic,

Paradikovic,Babic,etal.(2010)and

Zeljkovic,P

aradikovic,T

kalec,etal.

(2010)

A.houstonianum,C.blumei,

L.maritima,I.wallerana,

S.splendens,T.patula

Actiwave,

2.5mLL21for8weeks

Increasedleaf

area,freshweight,dry

weight

VernieriandMugnai

(2003),

Vernieri,Borghesi,et

al.(2005)and

Vernieri,Ferrante,et

al.(2006)

C.grandiflora

Combinationofthreebiostim

ulants

Radifarm

andKendal

(1:1)2.5mLL21

andKendal

þViva(1:1)2.5mLL21

Stimulatedrootgrowth

andhastened

flowering

VernieriandMugnai

(2003)

C.japonicaL.

Actiwave,

0.12–0.24mLcutting21

delivered

ineightapplications

Increasedrooting,reducedthenurserystage

Ferrante

etal.(2011,2013)

Lilium

Brindisi(LA:Lilium

longiflorum£Lilium

elegans)

MAE,APH

andHS,applied

eighttimes

both

atfoliar

anddrenchinglevel

atthe

concentrationof1.5gL–1

Cropcyclecameearly,leaves

more

expanded

inthelaminaandgreener,flower

budswith

higher

diameter;rootsystem

longer,stem

and

bulb

dry

weightshigher

DeLuciaandVecchietti(2012)

Gerbera(G

.jamesoniicv.

Malibu)

HA,

500and1000mgL21

500mgL21increasedthenumber

of

harvestedflowersper

plant.

1000mgL21increasedrootgrowthandmacro

andmicronutrientcontentsofleaves

and

scapes.Thevaselife

was

extended

Nikbakhtet

al.(2008)

Gladiolus(G

ladiolus)

HA,solutionscontaining0,10,20,30and

40mmolL

21ofCfrom

HA

Accelerated

growth,hastened

andincreased

flowering

BaldottoandBaldotto(2013)

S.splendens

Combinationofthreebiostim

ulants

Radifarm

andKendal

(1:1)2.5mLL21

andKendal

þViva(1:1)2.5mLL21

Stimulatedrootgrowth,increasedbiomass,

yield,chlorophyll

VernieriandMugnai

(2003)

T.patula

Combinationofthreebiostim

ulants

Radifarm

andKendal

(1:1)2.5mLL21

andKendal

þViva(1:1)2.5mLL21

Increasedbiomass,yield,chlorophyll,

anticipated

thefloweringandincreasedthe

resistance

topathogens

VernieriandMugnai

(2003)

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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).

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.

References

Abbas SM. 2013. The influence of biostimulants on the growth and on the biochemical compositionof Vicia faba cv. Giza 3 beans. Rom Biotechnol Lett. 18:8061–8068.

Abbas SM, Akladious SA. 2013. Application of carrot root extract induced salinity tolerance incowpea (Vigna sinensis L.) seedlings. Pak J Bot. 45:795–806.

Abdalla MM. 2013. The potential of Moringa oleifera extract as a biostimulant in enhancing thegrowth, biochemical and hormonal contents in rocket (Eruca vesicaria subsp. sativa) plants. Int JPlant Physiol Biochem. 5:42–49.

Alberici A, Quattrini E, Penati M, Martinetti L, Marino Gallina P, Ferrante A. 2008. Effect of thereduction of nutrient solution concentration on leafy vegetables quality grown in floating system.Acta Hortic. 801:1167–1176.

Amanda A, Valagussa M, Piaggesi A, Ferrante A. 2009. Effect of biostimulants on quality of babyleaf lettuce grown under plastic tunnel. Acta Hortic. 807:407–412.

Apel K, Hirt H. 2004. Reactive oxygen species: metabolism, oxidative stress, and signaltransduction. Annu Rev Plant Biol. 55:373–399.

Biostimulants and crop responses 13

Dow

nloa

ded

by [

Uni

vers

ity o

f W

ater

loo]

at 0

6:10

11

Oct

ober

201

4

Aziz A, Poinssot B, Daire X, Adrian M, Bezier A, Lambert B, Joubert JM, Pugin A. 2003. Laminarinelicits defense responses in grapevine and induces protection against Botrytis cinerea andPlasmopara viticola. Mol Plant Microb Interact. 16:1118–1128.

Baldotto LEB, Baldotto MA, Giro VB, Canellas LP, Olivares FL, Bressan-Smith R. 2009.Performance of pineapple ‘Vitoria’ in response to the application of humic acids duringacclimatization. Rev Bras Ciencia Solo. 33:979–990. [In Portuguese].

Baldotto MA, Baldotto LEB. 2013. Gladiolus development in response to bulb treatment withdifferent concentrations of humic acids. Rev Ceres. 60:138–142.

Bartwal A, Mall R, Lohani P, Guru SK, Arora S. 2013. Role of secondary metabolites andbrassinosteroids in plant defense against environmental stresses. J Plant Growth Regul.32:216–232.

Beecher GR. 1999. ‘Phytonutrients’ role in metabolism: effects on resistance to degenerativeprocesses. Nutr Rev. 57:3–6.

Berlyn GP, Russo RO. 1990. The use of organic biostimulants to promote root growth. BelowgroundEcol. 2:12–13.

Billard V, Etienne P, Jannin L, Garnica M, Cruz F, Garcia-Mina JM, Ourry A. 2013. Twobiostimulants derived from algae or humic acid induce similar responses in the mineral contentand gene expression of winter oilseed rape (Brassica napus L.). J Plant Growth Regul. 33:1–12.

Cavaiuolo M, Cocetta G, Ferrante A. 2013. The antioxidants changes in ornamental flowers duringdevelopment and senescence. Antioxidants. 2:132–155.

Cavaiuolo M, Ferrante A. 2014. Nitrates and glucosinolates as strong determinants of the nutritionalquality in rocket leafy salads. Nutrients. 6:519–1538.

Chen Y, Clapp CE, Magen H. 2004. Mechanisms of plant growth stimulation by humic substances:the role of organo-iron complexes. Soil Sci Plant Nutr. 50:1089–1095.

Choudhury S, Panda P, Sahoo L, Panda SK. 2013. Reactive oxygen species signaling in plants underabiotic stress. Plant Signal Behav. 8(4):e23681.

De Lucia B, Vecchietti L. 2012. Type of biostimulant and application method effects on stem qualityand root system growth in LA Lily. Eur J Hortic Sci. 77:1–10.

Ertani A, Nardi S. 2013. Review: long-term research activity on the biostimulant properties ofnatural origin compounds. Acta Hortic. 1009:81–187.

Ertani A, Schiavon M, Altissimo A, Franceschi C, Nardi S. 2011. Phenol-containing organicsubstances stimulate phenylpropanoid metabolism in Zea mays. J Plant Nutr Soil Sci.174:496–503.

Ertani A, SchiavonM, Muscolo A, Nardi S. 2013. Alfalfa plant-derived biostimulant stimulate short-term growth of salt stressed Zea mays L. plants. Plant Soil. 364:145–158.

[FAO] Food and Agricultural Organization. 2006. Yearbook of fishery statistics. Rome: Food andAgricultural Organization of United Nations.

Ferrante A, Piaggesi A, Martinetti L. 2011. Treatments with gibberellic acid and a biostimulant toenhance the growth and development of rooted Camelia cuttings. Bullettino dell’Agricoltura,Atti della Societa Agraria di Lombardia. 3–4:87–95. [In Italian].

Ferrante A, Trivellini A, Vernieri P, Piaggesi A. 2013. Application of Actiwavew for improving therooting of Camelia cuttings. Acta Hortic. 1009:213–218.

Ferrini F, Nicese FP. 2002. Response of English oak (Quercus robur L.) trees to biostimulantsapplication in the urban environment. J Arboric. 28:70–75.

Foyer CH, Noctor G. 2005. Redox homeostasis and antioxidant signaling: a metabolic interfacebetween stress perception and physiological responses. Plant Cell. 17:1866–1875.

Fraser GA, Percival GC. 2003. The influence of biostimulants on growth and vitality of three urbantree species following transplanting. Arboric J. 27:43–57.

Gajc-Wolska J, Kowalczyk K, Nowecka M, Mazur K, Metera A. 2012. Effect of organic-mineralfertilizers on the yield and quality of endive (Cichorium endivia L.). Acta Sci Pol. HortotumCultus. 11:189–200.

Gajc-Wolska J, Spizewski T, Grabowska A. 2013. The effect of seaweed extracts on the yield andquality parameters of Broccoli (Brassica oleracea var. cymosa L.) in open field production. ActaHortic. 1009:83–89.

Gallie DR. 2013. The role of L-ascorbic acid recycling in responding to environmental stress and inpromoting plant growth. J Exp Bot. 64:433–443.

Grabowska A, Kunicki E, Sekara A, Kalisz A, Wojciechowska R. 2012. The effect of cultivar andbiostimulant treatment on the carrot yield and its quality. Veg Crop Res Bull. 77:37–48.

14 R. Bulgari et al.

Dow

nloa

ded

by [

Uni

vers

ity o

f W

ater

loo]

at 0

6:10

11

Oct

ober

201

4

Guinan KJ, Sujeeth N, Copeland RB, Jones PW, O’Brien NM, Sharma HSS, Prouteau PFJ,O’Sullivan JT. 2013. Discrete roles for extracts of Ascophyllum nodosum in enhancing plantgrowth and tolerance to abiotic and biotic stresses. Acta Hortic. 1009:127–136.

Haider MW, Ayyub CM, Pervez MA, Asad HU, Manan A, Raza SA, Ashraf I. 2012. Impact of foliarapplication of seaweed extract on growth, yield and quality of potato (Solanum tuberosum L.).Soil Environ. 31:157–162.

Hamza B, Suggars A. 2001. Biostimulants: myths and realities. Turfgrass Trends. 10:6–10.Havaux M. 2014. Carotenoid oxidation products as stress signals in plants. Plant J. 79:597–606.

doi:10.1111/tpj.12386.Heckman JR. 1994. Effect of an organic bio-stimulant on cabbage yield. J Home Consum Hortic.

1:11–113.Jannin L, ArkounM, Ourry A, Laine P, Goux D, Garnica M, Fuentes M, San Francisco S, Baigorri R,

Cruz F, et al. 2012. Microarray analysis of humic acid effects on Brassica napus growth:involvement of N, C and S metabolisms. Plant Soil. 359:297–319.

Kauffman GL, Kneivel DP, Watschke TL. 2007. Effects of a biostimulant on the heat toleranceassociated with photosynthetic capacity, membrane thermostability, and polyphenol productionof perennial ryegrass. Crop Sci. 47:261–267.

Keunen E, Peshev D, Vangronsveld J, Van den Ende W, Cuypers A. 2013. Plant sugars are crucialplayers in the oxidative challenge during abiotic stress: extending the traditional concept. PlantCell Environ. 36:1242–1255.

Kijne JW. 2006. Abiotic stress and water scarcity: identifying and resolving conflicts from plantlevel to global level. Field Crops Res. 97:3–18.

Kliebenstein DJ. 2004. Secondary metabolites and plant/environment interactions: a view throughArabidopsis thaliana tinged glasses. Plant Cell Environ. 27:675–684.

Kolomaznık K, Pecha J, Friebrova V, Janacova D, Vasek V. 2012. Diffusion of biostimulators intoplant tissues. Heat Mass Transf. 48:1505–1512.

Kumari A, Sairam RK. 2013. Moisture stress induced increases in the activity of enzymes ofosmolytes biosynthesis are associated with stress tolerance in wheat genotypes. Indian J PlantPhysiol. 18:223–230.

Kunicki E, Grabowska A, Sekara A, Wojciechowska R. 2010. The effect of cultivar type, time ofcultivation, and biostimulant treatment on the yield of spinach (Spinacia oleracea L.). FoliaHortic. 22:9–13.

Liebman M, Davis AS. 2000. Integration of soil, crop and weed management in low-external-inputfarming systems. Weed Res. 40:27–47.

Luhua S, Hegie A, Suzuki N, Shulaev E, Luo X, Cenariu D, Mittler R. 2013. Linking genes ofunknown function with abiotic stress responses by high-throughput phenotype screening.Physiol Plant. 148:322–333.

Luo J, Sun S, Jia L, Chen W, Shen Q. 2006. The mechanism of nitrate accumulation in pakchoi[Brassica campestris L. spp. chinensis (L.)]. Plant Soil. 282:291–300.

Maini P. 2006. The experience of the first biostimulant, based on amino acids and peptides: a shortretrospective review on the laboratory researches and the practical results. Fert Agrorum.1:29–43.

Mancuso S, Azzarello E, Mugnai S, Briand X. 2006. Marine bioactive substances (IPA extract)improve foliar ion uptake and water stress tolerance in potted Vitis vinifera plants. Adv HorticSci. 20:156–161.

Martin C, Zhang Y, Tonelli C, Petroni K. 2013. Plants, diet, and health. Annu Rev Plant Biol.64:19–46.

Martınez-Ballesta M, Moreno DA, Carvajal M. 2013. The physiological importance ofglucosinolates on plant response to abiotic stress in Brassica. Int J Mol Sci. 14:11607–11625.

Mattner SW, Wite D, Riches DA, Porter IJ, Arioli T. 2013. The effect of kelp extract on seedlingestablishment of broccoli on contrasting soil types in southern Victoria, Australia. Biol AgricHortic. 29:258–270.

Mena-Petite A, Lacuesta M, Munoz-Rueda A. 2006. Ammonium assimilation in Pinus radiataseedlings: effects of storage treatments, transplanting stress and water regimes after plantingunder simulated field conditions. Environ Exp Bot. 55:1–14.

Miao M, Jiang H, Jiang B, Zhang T, Cui SW, Jin Z. 2014. Phytonutrients for controlling starchdigestion: Evaluation of grape skin extract. Food Chem. 145:205–211.

Biostimulants and crop responses 15

Dow

nloa

ded

by [

Uni

vers

ity o

f W

ater

loo]

at 0

6:10

11

Oct

ober

201

4

Nardi S, Carletti P, Ertani A, Pizzeghello D. 2006. Knowledge and evaluation of plant growthstimulants. Inform Agrario. 45:41–44.

Nardi S, Carletti P, Pizzeghello D, Muscolo A. 2009. Biological activities of humic substances. In:Senesi N, Xing B, Huang PM, editors. Biophysico-chemical processes involving naturalnonliving organic matter in environmental systems. Vol 2. Hoboken, NJ: Wiley; p. 305–340.

Neilson EH, Goodger JQ, Woodrow IE, Møller BL. 2013. Plant chemical defense: at what cost?Trends Plant Sci. 18:250–258.

Nikbakht A, Kafi M, Babalar M, Xia YP, Luo A, Etemadi NA. 2008. Effect of humic acid on plantgrowth, nutrient uptake, and postharvest life of gerbera. J Plant Nutr. 31:2155–2167.

Norrie J, Keathley JP. 2006. Benefits of Ascophyllum nodosum marine-plant extract applications to‘Thompson seedless’ grape production. Acta Hortic. 727:243–247.

Paradikovic N, Vinkovic T, Vinkovic Vrcek I, Zunta I, Bojic M, MedicSaric M. 2011. Effect ofnatural biostimulants on yield and nutritional quality: an example of sweet yellow pepper(Capsicum annuum L.) plants. J Sci Food Agric. 91:2146–2152.

Pardo-Garcıa AI, Martınez-Gil AM, Cadahıa E, Pardo F, Alonso GL, Salinas MR. 2014. Oak extractapplication to grapevines as a plant biostimulant to increase wine polyphenols. Food Res Int.55:150–160.

Parks SE, Huett DO, Campbell LC, Spohr LJ. 2008. Nitrate and nitrite in Australian leafy vegetables.Aust J Agric Res. 59:632–638.

Pecha J, Furst T, Kolomaznık K, Friebrova V, Svoboda P. 2012. Protein biostimulant foliar uptakemodeling: the impact of climatic conditions. AIChE J. 58:2010–2019.

Peleg Z, Blumwald E. 2011. Hormone balance and abiotic stress tolerance in crop plants. Curr OpinPlant Biol. 14:290–295.

Petrozza A, Summerer S, Di Tommaso G, Di Tommaso D, Piaggesi A. 2013a. Evaluation of theeffect of Radifarmw treatment on the morpho-physiological characteristics of root systems viaimage analysis. Acta Hortic. 1009:149–153.

Petrozza A, Summerer S, Di Tommaso G, Di Tommaso D, Piaggesi A. 2013b. An evaluation oftomato plant root development and morpho-physiological response treated with VIVAw byimage analysis. Acta Hortic. 1009:155–159.

Petrussa E, Braidot E, Zancani M, Peresson C, Bertolini A, Patui S, Vianello A. 2013. Plantflavonoids – biosynthesis, transport and involvement in stress responses. Int J Mol Sci.14:14950–14973.

Prabhu M, Kumar AR, Rajamani K. 2010. Influence of different organic substances on growth andherb yield of sacred basil (Ocimum sanctum L.). Indian J Agric Res. 44:48–52.

Rajarathnam S, Shashirakha MN, Mallikarjuna SE. 2013. Status of bioactive compounds in foods,with focus on fruits and vegetables. Crit Rev Food Sci Nutr. doi:10.1080/10408398.2012.692736.

Sacco SM, Horcajada MN, Offord E. 2013. Phytonutrients for bone health during ageing. Brit J ClinPharmacol. 75:697–707.

Sangha JS, Ravichandran S, Prithiviraj K, Critchley AT, Prithiviraj B. 2010. Sulfated macroalgalpolysaccharides l-carrageenan and i-carrageenan differentially alter Arabidopsis thalianaresistance to Sclerotinia sclerotiorum. Physiol Mol Plant Pathol. 75:38–45.

Santaniello A, Giorgi FM, Di Tommaso D, Di Tommaso G, Piaggesi A, Perata P. 2013. Genomicapproaches to unveil the physiological pathways activated in Arabidopsis treated with plant-derived raw extracts. Acta Hortic. 1009:161–174.

SchiavonM, Ertani A, Nardi S. 2008. Effects of an alfalfa protein hydrolysate on the gene expressionand activity of enzymes of the tricarboxylic acid (TCA) cycle and nitrogen metabolism in Zeamays L. J Agric Food Chem. 56:11800–11808.

SchiavonM, Pizzeghello D, Muscolo A, Vaccaro S, Francioso O, Nardi S. 2010. High molecular sizehumic substances enhance phenylpropanoid metabolism in maize (Zea mays L.). J Chem Ecol.36:662–669.

Schreiber L. 2005. Polar paths of diffusion across plant cuticles: new evidence for an old hypothesis.Ann Bot. 95:1069–1073.

Sharif M, Khattak RA, Sarir MS. 2002. Effect of different levels of lignitic coal derived humic acidon growth of maize plants. Comm Soil Sci Plant Anal. 33:3567–3580.

Sharma HS, Fleming C, Selby C, Rao JR, Martin T. 2014. Plant biostimulants: a review on theprocessing of macroalgae and use of extracts for crop management to reduce abiotic and bioticstresses. J Appl Phycol. 26:465–490.

16 R. Bulgari et al.

Dow

nloa

ded

by [

Uni

vers

ity o

f W

ater

loo]

at 0

6:10

11

Oct

ober

201

4

Spann TM, Little HA. 2011. Applications of a commercial extract of the brown seaweedAscophyllum nodosum increases drought tolerance in container-grown ‘Hamlin’ sweet orangenursery trees. HortSci. 46:577–582.

Spinelli F, Fiori G, Noferini M, Sprocatti M, Costa G. 2010. A novel type of seaweed extract as anatural alternative to the use of iron chelates in strawberry production. Sci Hortic. 125:63–269.

Stadnik MJ, Freitas MBD. 2014. Algal polysaccharides as source of plant resistance inducers. TropPlant Pathol. 39:111–118.

Sun X, Rikkerink EH, Jones WT, Uversky VN. 2013. Multifarious roles of intrinsic disorder inproteins illustrate its broad impact on plant biology. Plant Cell Online. 25:38–55.

Tkalec M, Paradikovic N, Zeljkovic S, Vinkovic T. 2012. Influence of medium on growth anddevelopment of wild rose in vitro. In: International Conference on BioScience: Biotechnologyand Biodiversity – Step in the Future – The Forth Joint UNS–PSU Conference; 2012 Jun18–20; Novi Sad, Serbia; p. 104–108.

Vasconcelos ACFD, Zhang X, Ervin EH, Kiehl JDC. 2009. Enzymatic antioxidant responses tobiostimulants in maize and soybean subjected to drought. Sci Agric. 66:395–402.

Vernieri P, Borghesi E, Ferrante A, Magnani G. 2005. Application of biostimulants in floatingsystem for improving rocket quality. J Food Agric Environ. 3:86–88.

Vernieri P, Borghesi E, Tognoni F, Serra G, Ferrante A, Piaggesi A. 2006. Use of biostimulants forreducing nutrient solution concentration in floating system. Acta Hortic. 718:477–484.

Vernieri P, Ferrante A, Borghesi E, Magnani G. 2005. High quality flowering plants using thebiostimulants. L’Informatore Agrario. 16:57–60. [In Italian].

Vernieri P, Ferrante A, Borghesi E, Mugnai S. 2006. Biostimulants: a tool for improving quality andyield. Fertilitas Agrorum. 1:17–22. [In Italian].

Vernieri P, Malorgio F, Tognoni F. 2002. Use of biostimulants in production of vegetable seedlings.Colture Protette. 31:75–79. [In Italian].

Vernieri P, Mugnai S. 2003. Use of biostimulants for bedding plants production. L’InformatoreAgrario. 24:51–54. [In Italian].

Wang M, Zheng Q, Shen Q, Guo S. 2013. The critical role of potassium in plant stress response. Int JMol Sci. 14:7370–7390.

Wasternack C, Hause B. 2013. Jasmonates: biosynthesis, perception, signal transduction and actionin plant stress response, growth and development. An update to the 2007 review in Annals ofBotany. Ann Bot. 111:1021–1058.

Zeljkovic SB, Paradikovic NA, Babic TS, Ðuric GD, Oljaca RM, Vinkovic TM, Tkalec MB. 2010.Influence of biostimulant and substrate volume on root growth and development of scarlet sage(Salvia splendens L.) transplants. J Agric Sci. 55:29–36.

Zeljkovic S, Paradikovic N, Tkalec M, Vinkovic T, Ðuric G, Oljaca R. 2010. Nutrient content andgrowth of begonia transplants (Begonia semperflorens L.) under the influence of biostimulantapplication. Sjemenarstvo. 27:77–84.

Zeng H, Wang G, Hu X, Wang H, Du L, Zhu Y. 2014. Role of microRNAs in plant responses tonutrient stress. Plant Soil. 374:1005–1021.

Zhang X, Schmidt RE. 1999. Biostimulating turfgrasses. Ground Maint. 34:14–32.Ziosi V, Zandoli R, Di Nardo A. 2013. Biological activity of different botanical extracts as evaluated

by means of an array of in vitro and in vivo bioassays. Acta Hortic. 1009:61–66.

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