Scientia Horticulturae 196 (2015) 28–38

Contents lists available at ScienceDirect

Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Review

Protein hydrolysates as biostimulants in horticulture

Giuseppe Colla a,∗ , Serenella Nardi b , Mariateresa Cardarelli c ,
Andrea Ertani b , Luigi Lucini d , Renaud Canaguier e , Youssef Rouphael f
a
Department of Agriculture, Forestry, Nature and Energy, University of Tuscia, via San Camillo De Lellis snc, 01100 Viterbo, Italy
b
Department of Agronomy, Food, Natural Resources, Animals and the Environment. University of Padova, Agripolis, 35020 Legnaro, Italy
c
Consiglio per la Ricerca in Agricoltura e l’analisi dell’economia agraria, Centro di ricerca per lo studio delle Relazioni tra Pianta e Suolo, Via della Navicella
2-4, Roma, Italy
d
Institute of Environmental and Agricultural Chemistry, Università Cattolica del Sacro Cuore, 29122 Piacenza, Italy
e
Nixe Laboratory, 06905 Sophia Antipolis Cedex, France
f
Department of Agricultural Sciences, University of Naples Federico II, Via Università 100, 80055 Portici, Italy

a r t i c l e i n f o a b s t r a c t

Article history: In recent years, new strategies have been proposed in order to improve the sustainability of production
Received 21 June 2015 systems for horticultural crops. A promising tool would be the use of substances and/or microorganisms
Received in revised form 21 August 2015 defined also as ‘biostimulants’ able to enhance crop quality parameters, nutrient efficiency and abiotic
Accepted 24 August 2015
stress tolerance. Protein hydrolysates (PHs) are an important group of plant biostimulants based on a
Available online 4 September 2015
mixture of peptides and amino acids that have received increasing attention in the recent years due
to their positive effects on crop performances. PHs are mainly produced by enzymatic and/or chemical
Keywords:
hydrolysis of proteins from animal- or plant-derived raw materials. The current review gives an overview
Amino acids
Biostimulants
of the biostimulant properties of PHs on productivity and product quality of horticultural crops, in par-
Enzymatic hydrolysis ticular fruit trees, vegetables, flower crops and ornamentals. After a brief introduction on PHs as plant
Horticultural crops biostimulants, this review focuses on the classification and chemical composition of PHs according to the
Peptides source of proteins and method of protein hydrolysis. The plant uptake and transport of amino acids and
Product quality peptides and the effects of PHs on primary and secondary metabolism as well as the biochemical and
physiological processes conferring tolerance to abiotic stress are also covered. The review concludes by
proposing several perspectives for future research aiming to understand the mode of action of PHs based
on their composition and also to define the suitable time and dose of application.
© 2015 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2. Classification and chemical characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3. Effects of protein hydrolysates on plant metabolism and physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.1. Plant uptake and transport of amino acids and peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.2. Effects on primary plant metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.3. Effects on secondary plant metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
4. Agronomic response of horticultural crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
4.1. Growth, yield and flowering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
4.2. Product quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
5. Protein hydrolysates and abiotic stress tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
6. Conclusions and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

∗ Corresponding author. Fax: +39 761 357558.

E-mail address: giucolla@unitus.it (G. Colla).

http://dx.doi.org/10.1016/j.scienta.2015.08.037
0304-4238/© 2015 Elsevier B.V. All rights reserved.
 G. Colla et al. / Scientia Horticulturae 196 (2015) 28–38
1. Introduction
The world’s horticultural systems face a great balancing act
between two needs: (1) raise the supply of food produced on the
available farmland, since the global population will increase to
more than 9.3 billion by 2050, and (2) reduce agriculture’s impact
on the environment and human health (Searchinger, 2013). Meet-
ing these two targets presents a major sustainability challenge to
scientists and producers, which might be fostered by using natural
products known as plant biostimulants (Calvo et al., 2014). Protein
hydrolysates (PHs) are a category of plant biostimulants defined
as ‘mixtures of polypeptides, oligopeptides and amino acids that
are manufactured from protein sources using partial hydrolysis’
(Schaafsma, 2009). There has been growing interest in PHs dur-
ing recent years due to their positive effects on crop performances,
especially under environmental stress conditions (du Jardin, 2012).
PHs are mainly produced by chemical (with strong acids or
alkalis) and/or enzymatic hydrolysis of proteins contained in agro-
industrial by-products from animal (i.e., leather, viscera, feathers,
blood) or plant origin (i.e., vegetable by-products) and in biomass
of dedicated legume crops (i.e., seeds, hay) (Maini, 2006; Schiavon
et al., 2008; du Jardin, 2012; Halpern et al., 2015 and references
cited therein). PHs obtained by agro-industrial by-products repre-
sent a sustainable solution to the problem of waste disposal, making
their production interesting from environmental and economical
points of view (Kasparkova et al., 2009; Pecha et al., 2012; Baglieri
et al., 2014). Actually, more than 90% of the PH market in horticul-
ture is based on products obtained through chemical hydrolysis of
proteins from animal origin (e.g., collagen from leather by-products
in Europe, India and China; fish by-products in United States),
while the enzymatically produced PHs from plant biomass are less
common because they have been recently introduced to the bios-
timulant market.
PHs have been identified to improve the performance of several
horticultural crops, including increased shoot, and root biomass
and productivity (Kunicki et al., 2010; Lisiecka et al., 2011;
Paradikovic et al., 2011; Colla et al., 2014; Ertani et al., 2014).
Application of PHs to plant leaves and roots has been shown to
increase Fe and N metabolism, nutrient uptake, and water and
nutrient use efficiencies for both macro and microelements (Cerdán
et al., 2009; Ertani et al., 2009; Halpern et al., 2015). The higher
nutrient uptake in PH-treated plants has been attributed to (1) an
increase in soil microbial activity and soil enzymatic activities, (2)
improvement of micronutrient mobility and solubility, in partic-
ular Fe, Zn, Mn and Cu, (3) modifications in the root architecture
of plants, in particular root length, density and number of lateral
roots and, (4) an increase in nitrate reductase, glutamine synthetase
and Fe(III)-chelate reductase activities (Cerdán et al., 2009; Ertani
et al., 2009; García-Martínez et al., 2010; Colla et al., 2014; Lucini
et al., 2015). PHs could also interfere with the phytohormone bal-
ance of the plant, thereby influencing plant development due to
the presence of specific peptides and precursors of phytohormone
biosynthesis, such as tryptophan (Colla et al., 2014). Several bioac-
tive peptides produced in a variety of plants have been identified
to have hormone-like activities (Ito et al., 2006; Kondo et al., 2006).
Moreover, many scientific papers reported that the application of
plant-derived PHs elicited auxin- and gibberellin-like activities and
thus promoted crop performances (Schiavon et al., 2008; Ertani
et al., 2009; Matsumiya and Kubo, 2011; Colla et al., 2014). PHs
have been shown not only to improve plant nutrition but also
the quality of fruits and vegetables in terms of phytochemicals
(i.e., carotenoids, flavonoids, polyphenols) (Parrado et al., 2007;
Paradikovic et al., 2011; Gurav and Jadhav, 2013; Ertani et al., 2014),
and they can reduce undesired compounds, such as nitrates (Liu
et al., 2008). In addition, PH application has been also been shown
to avoid or reduce losses in production caused by unfavourable
29
soil conditions and environmental stresses. These include thermal
stress, salinity, drought, alkalinity, and nutrient deficiency (Botta,
2013; Cerdán et al., 2013; Ertani et al., 2013; Colla et al., 2014;
Petrozza et al., 2014; Lucini et al., 2015; Visconti et al., 2015). How-
ever, the maximum benefits from PHs are obtained at very low
dosages (Ertani et al., 2014) and are dependent on species/cultivar,
environmental conditions, phenological stages, time and mode of
applications (foliar vs. root) and leaf permeability to the biostimu-
lant (Kauffman et al., 2007; Kunicki et al., 2010; Ertani et al., 2014).
The later factor is crucial, since biostimulants are usually foliarly
applied; hence, their penetration into plant tissue is a necessary
condition for a reliable efficiency (Pecha et al., 2012).
Phytotoxic effects and growth depression of fruiting crops have
been also reported after repeated applications of animal-derived
PH products (Cerdán et al., 2009; Lisiecka et al., 2011), whereas
no phytotoxicity and growth reduction was recorded in tomato
plants (Solanum lycopersicum L.) after applications of a plant-
derived PH (Cerdán et al., 2009). Similarly, several trials carried
out at the University of Tuscia-Italy on basil demonstrated that
foliar spray of some commercial animal-derived PHs caused leaf
chlorosis, whereas no injury symptoms were recorded in basil after
foliar spraying with the commercial plant-derived PH ‘Trainer’,
up to 10 times the recommended dose. The detrimental effect of
some animal derived-PHs on plant growth can be attributed to an
unbalanced amino acid composition (Oaks et al., 1977), higher con-
centration of free amino acids (Moe, 2013) and high salinity (Colla
et al., 2014). Besides phytotoxicity effects, there is an increased
concern on the use of animal-derived PHs in terms of food safety,
as demonstrated by the European Regulation No. 354/2014, which
prohibited the application of these products on the edible parts of
organic crops. However, Corte et al. (2014), evaluating safety and
fertiliser efficacy of animal-derived PHs, concluded that PHs did not
negatively affect eukaryotic cells and soil ecosystems, and PHs can
be used in conventional and organic farming without posing harm
to human health and the environment.
In this review, we focussed on recent advances in the biostim-
ulant properties of PHs on growth, yield and product quality of
horticultural crops (fruit trees, vegetables, flower and ornamen-
tal crops). The effects of PH on the primary and secondary plant
metabolism and physiology as well as the tolerance to unfavourable
chemical soil conditions and environmental stresses are covered.
2. Classification and chemical characteristics
PHs can be classified on the basis of protein source, and method
of protein hydrolysis (Fig. 1). Production process and protein
source strongly affect the chemical characteristics of PHs. Chemical
hydrolysis of proteins under acid or alkaline conditions is usu-
ally preferred for producing animal-based PHs. Acid hydrolysis is a
very aggressive process carried out at high temperature (>121 ◦ C)
and pressure (>220.6 kPa). In acid hydrolysis, hydrochloric and
sulphuric acid are mainly used to hydrolyse proteins, the most com-
mon being hydrochloric acid (Pasupuleti and Braun, 2010). Alkaline
hydrolysis is a fairly simple and straightforward process where pro-
teins are solubilised by heating followed by the addition of alkaline
agents, such as Ca, Na or potassium hydroxide, and maintaining the
temperature to a desired set point (Pasupuleti and Braun, 2010).
Chemical hydrolysis attacks all peptide bonds of proteins, leading
to a high degree of protein hydrolysis (high content of free amino
acids in total) and destruction of several amino acids (e.g., trypto-
phan is usually totally destroyed with acid hydrolysis; cysteine,
serine and threonine are partially lost; and asparagine and glu-
tamine are converted to their acidic forms with acid hydrolysis).
Moreover, other useful thermolabile compounds (e.g., vitamins) are
also mostly destroyed during chemical hydrolysis. One other crit-
30 G. Colla et al. / Scientia Horticulturae 196 (2015) 28–38
Fig. 1. Classification criteria of protein hydrolysates on the basis of protein source
and the method of protein hydrolysis used in the production process.
ical aspect of chemical hydrolysis is the conversion of free amino
acids from the L-form to D-form (racemisation). Since the amino
acids in proteins of living organisms are only in the L-form, plants
cannot use directly D-amino acids in their metabolism, making the
PH less effective or even potentially toxic for plants (Cavani et al.,
2003; Lisiecka et al., 2011). Finally, the large use of acids/alkalis
during chemical hydrolysis lead to an increase in salinity of PHs.
Enzymatic hydrolysis is usually adopted for producing plant-
based PHs. The protein hydrolysis is carried out by proteolytic
enzymes originating from animal organs (e.g., pancreatin, pepsin),
plants (e.g., papain, ficin, bromelain) or microorganisms (e.g.,
alcalase, flavourzyme). Proteolytic enzymes hydrolyse proteins
more gently than acids/alkalis and do not require high tempera-
ture (<60 ◦ C), and they usually target specific peptide bonds (e.g.,
the enzyme pepsin cuts an amino acid chain where there is a pheny-
lalanine or leucine bond; papain cuts the chain adjacent to arginine,
lysine and phenylalanine; pancreatin cuts the chain where there are
arginine, lysine, tyrosine, tryptophan, phenylalanine and leucine
bonds). PHs resulting from enzymatic hydrolysis are a mixture of
amino acids and peptides of varying length with a low salinity and
a constant composition over time.
PH production processes based on enzymatic hydrolysis are
also more environmentally friendly then those based on chemical
hydrolysis due to the low energy requirement and the carbon diox-
ide emissions (Bernabei, 2015). Combined chemical and enzymatic
hydrolytic processes have been also proposed to save energy and
to preserve the structure of amino acids (Niculescu et al., 2009).
Protein/peptides and free amino acid contents in PHs widely
differ, usually ranging from 1 to 85% (w/w) and 2–18% (w/w),
respectively (Calvo et al., 2014). Animal-derived PHs usually con-
tain a higher amount of total amino acids than plant-derived PHs
(Ertani et al., 2009). The major amino acids in PHs differ according
to the source of proteins (Table 1). Collagen-based PHs contain a
high proportion of glycine and proline, while in legume-based PHs
aspartic acid and glutamic acid dominate. Similarly, fish-based PHs
have a high proportion of aspartic acid and glutamic acid (Ertani
et al., 2009; Chalamaiah et al., 2012; Ertani et al., 2013). Casein-
derived PHs contain a high proportion of glutamic acid and pro-
line. Moreover, collagen-derived PHs contain two non-standard
amino acids, namely hydroxyproline and hydroxylysine, which are
present at negligible levels in plant-derived PHs.
Peptide concentration and molecular weight distribution can
be extremely variable depending on the production process. Pep-
tide concentrations are usually higher in enzymatically derived PHs
than in chemically derived ones. Molecular weight distribution of
peptides can vary from several hundred to several thousand Dal-
tons, with the highest plant biostimulant action of peptides having
low molecular weight (Quartieri et al., 2002). Moreover, specific
peptides from plant origin have been shown to act as signalling
molecules for plant defence, growth and development (Ryan et al.,
2002). Enzymatic hydrolysis, being more selective compared to
chemical hydrolysis, enables industry to maximise yield of these
bioactive peptides in PHs. For instance, an alkaline protease pro-
duced from Bacillus circulans HA12 was successfully used to obtain
a soybean-based PH enriched with a bioactive peptide called ‘root
hair promoting peptide’ (Matsumiya and Kubo, 2011). Finally, the
peptide molecular weight of animal-derived PHs must be lower
than 10 kDa to prevent any risk of BSE/TSE prion transmission to
humans (Supattapone et al., 1999).
Besides amino acids and peptides, PHs contain other compounds
that can contribute to the biostimulant action. These compounds
include fats, carbohydrates, phenols, mineral elements, phyto-
hormones and other organic compounds (e.g., polyamines). For
instance, two phytohormones (i.e., indoleacetic acid, and isopen-
tenyladenosine) were found in an alfalfa-based PH (Ertani et al.,
2014). Triacontanol, a fatty alcohol that stimulates plant growth,
has also been identified in an alfalfa-derived PH (Ertani et al.,
2013). Moreover, plant-based PHs contain soluble carbohydrates
and phenols, which play an important role in energy metabolism
and oxidative stress defences. In contrast, animal-derived PHs lack
carbohydrates, phenols and phytohormones. Mineral content is
also affected by protein source, usually being higher in the prod-
ucts from plant-derived PHs. Indeed, Ertani et al. (2009) reported
that a high amount of Na could be measured in a PH from meat
flour, whereas high levels of nutrients including Mg, Mn, Fe and Zn
were found in the plant-derived one. Additionally, in the case of
PHs prepared from raw materials derived from leather production,
a concern regarding the contamination by Cr forms might be raised
(Mu et al., 2003).
3. Effects of protein hydrolysates on plant metabolism and
physiology
3.1. Plant uptake and transport of amino acids and peptides
Amino acids and small peptides are absorbed by both roots
and leaves and then translocated into the plant, as demonstrated
by Watson and Fowden (1975), Soldal and Nissen (1978) and
Matsumiya and Kubo (2011). However, the root availability of
amino acids and peptides can be strongly reduced by soil micro-
bial activity (Wilson et al., 2013). Studies measuring consumption
of amino acids by soil microorganisms estimated a half-life of 1–6 h
for amino acids in soils (Moe, 2013), although a longer half-life for
amino acids (up to 32 d) has been reported in a taiga forest soil dur-
ing incubation at 10 ◦ C (Jones and Kielland, 2012). Several studies
indicated that soil microorganisms used 30–40% of amino acid-C
for respiration and the remaining amino acid-C for cell biomass
production and maintenance (Jones et al., 2005, 2009). Moreover,
30–40% of the N associated with the respired amino acid-C was
consistently excreted into the soil as ammonium-N, which can be
taken up by plants and microbes or further oxidised to produce
nitrate.
Using dual-labelled 13 C-15 N-amino acids in tomato, wheat and
grass experiments, it has been demonstrated that plant roots are
poor competitors for externally applied amino acids, taking up
only 6–25% of the added amino acids, with the remainder captured
by soil microorganisms. Moreover, the relative amounts of amino
acids taken up by plant roots can vary with plant species/variety,
abiotic soil conditions, amino acid applied and concentration at
 G. Colla et al. / Scientia Horticulturae 196 (2015) 28–38 31

Table 1
Amino acid composition of protein sources from animal and plant origin (g per 100 g of proteins).

Amino acid Bovine collagena Blood mealb Chicken feathersc Fish meald Caseine Alfalfa hayf Soybean mealg

Alanine 8.2 9.1 4.9 6.7 2.9 5.4 3.9

Arginine 7.4 2.8 6.8 6.3 3.7 4.3 6.4
Aspartic acid 5.5 12.8 7.0 9.9 6.6 15.5 11.1
Cysteine 0.0 1.7 5.1 1.0 0.3 2.2 1.4
Glutamic acid 9.8 10.4 10.7 14.4 21.5 8.2 18.3
Glycine 23.5 5.2 7.5 6.4 2.1 5.1 4.5
Histidine 0.7 3.7 1.3 2.9 3.1 3.9 2.6
Hydroxyproline 10.6 – – – – – –
Hydroxylysine 0.9 – – – – – –
Isoleucine 1.7 0.9 4.8 5.4 5.1 4.6 4.2
Leucine 3.2 12.2 8.6 8.3 9.0 8.1 8.0
Lysine 3.9 8.3 2.9 8.8 3.8 5.9 6.6
Methionine 0.7 1.2 0.8 2.9 2.7 1.8 1.4
Phenylalanine 2.2 6.0 5.1 4.3 5.1 4.5 5.5
Proline 13.1 5.5 9.0 4.8 10.7 9.4 5.2
Serine 2.9 5.6 9.6 4.0 5.6 4.6 5.9
Threonine 2.0 4.5 4.8 4.7 4.3 4.5 3.8
Tryptophan 0.0 1.2 0.6 0.0 1.3 1.7 1.4
Tyrosine 0.8 2.7 2.9 3.4 5.6 4.3 4.3
Valine 2.9 6.2 7.6 5.8 6.6 6.0 5.5
a
http://albumen.conservation-us.org/library/c20/vonendt1991.html.
b
http://www.johnrothandson.com/bloodspecs.htm.
c
http://www.uspoultry.org/ppfc/docs/FMFORPOULTRYARTICLE.pdf.
d
http://rnd.edpsciences.org/articles/rnd/pdf/1963/05/ABABB 0003-388X 1963 3 hs1 ART0002.pdf.
e
http://www.scientificpsychic.com/fitness/aminoacids1.html.
f
Purwin et al. (2014).
g
Nixe Laboratory analysis.

which each is applied (Moe, 2013). Foliar application of PHs can
increase amino acid and peptide availability for plant uptake by
reducing the competition with microorganisms. Stiegler et al.
(2013) measured in a creeping bentgrass crop leaf N uptakes of
52, 51 and 48% when labelled N (15 N) was sprayed 8 h before
analysis with proline, glutamic acid and glycine, respectively. In
another study, peach leaf uptakes of amino acids after foliar appli-
cation were 14, 10, 25 and 26% for alanine, glutamic acid, glycine
and lysine, respectively (Furuya and Umemiya, 2002). The rate of
foliar N absorption increased as the molecular weights of amino
acids decreased. However, such a relationship was not always
found, and the foliar absorption rates of N from arginine and lysine
were significantly higher than other amino acids having the same
molecular weights. Following root/foliar uptake, amino acids and
peptides are transported from cell to cell and over long distances
through the plant vascular system (xylem and phloem) in support
of plant metabolism and development. Several classes of integral
membrane proteins are involved in amino acid and peptide trans-
port through cell membranes in plants. For instance, members of
the lysine–histidine-like transporters family, amino acid permease
family and proline transporter family play a direct role in amino
acid uptake through the roots (Tegeder, 2012).
Amino acids and amides represent in most plants the princi-
pal transport form for organic N, and they can be used directly
for protein synthesis and other essential N compounds or may
be metabolised (Rentsch et al., 2007). Moreover, amino acids and
peptides can play an important role as signalling compounds. Spe-
cific receptors on cell membranes interact with peptides (elicitors)
for signal transduction, leading to morpho-physiological and bio-
chemical changes in plants (Ryan et al., 2002). Microorganisms
living in the phyllosphere and rhizosphere can also affect crop
response to PH application not only because they are competing
with plants for amino acids and peptides but also because they
secret enzymes that can hydrolyse peptides into small fragments
that may act as signalling compounds, modulating crop response.
A similar scenario has been proposed for sugars sprayed on leaves
(Trouvelot et al., 2014). Moreover, recent studies at Tuscia Uni-
versity showed that application of a plant-derived PH on lettuce
stimulated desirable, naturally occurring microorganisms, such as
N2 -fixing, P-solubilising and indoleacetic acid-producing bacteria.
The above findings suggest that PHs may also act as plant biostim-
ulants through a microorganism-mediated enhancement of plant
growth.
3.2. Effects on primary plant metabolism
Protein hydrolysates have been shown to stimulate N
metabolism and assimilation (Ertani et al., 2009, 2013; Baglieri
et al., 2014; Calvo et al., 2014). In plants, all inorganic N is first
reduced to ammonia before it is incorporated into organic form
(Crawford et al., 1993; Hoff et al., 1994). The N assimilation is
a key process controlling plant growth and development. Inor-
ganic N derived from fertilisers is assimilated into amino acids
glutamine, glutamate, asparagine and aspartate, which serve as
important N carriers in plants. Maini (2006) summarised the early
literature showing enhanced activity of NAD-dependent glutamate
dehydrogenase, nitrate reductase and malate dehydrogenase in
maize following application of a biostimulant based on amino acids.
Enzymatic and molecular approaches have recently demonstrated
that PHs supplied to maize plants influence different metabolic
pathways, such as glycolysis and the Krebs cycle (Ertani et al.,
2009, 2013). Schiavon et al. (2008) demonstrated that the activ-
ity of enzymes involved in C metabolism (malate dehydrogenase,
isocitrate dehydrogenase, citrate synthase) and N reduction and
assimilation (nitrate reductase, nitrite reductase, glutamine syn-
thetase, glutamate synthase, aspartate aminotransferase) were
stimulated by the PH application. To confirm these data, the effect of
an alfalfa PH on the transcriptional pattern of genes coding for the
enzymes functioning in the tricarboxylic acid cycle was analysed
via reverse transcription polymerase chain reaction. The results
showed that the transcript accumulation of the genes encoding
for the studied enzymes was up-regulated by the treatment and
that the gene expression of these enzymes was in line with their
enzyme activities. The same approach was used to verify the bios-
timulant effects of a product derived from hydrolysis of tanning
residues. Similarly, the PH was effective in improving the enzymes
32 G. Colla et al. / Scientia Horticulturae 196 (2015) 28–38
involved in N and C metabolism, from the enzymatic and molecular
biology points of view (Ertani et al., 2013). Similarly, application
of a plant-derived PH (Trainer) enhanced N assimilation in corn
seedlings grown under controlled environmental conditions (Colla
et al., 2013). Due to the rise in photosynthesis and energy sup-
ply for cell metabolism generated by PH applications, it may be
speculated that the increase of N assimilation in plants may be a
result of the positive effects of PHs on production of C skeletons
and energy supply, which are needed for amino acid biosynthesis.
Pace et al. (1990) measured nitrate uptake and reduction of maize
seedlings under carbon dioxide-limited photosynthesis, and they
found that nitrate uptake and reduction were regulated by the sup-
ply of energy and C skeletons required to support these processes,
rather than by the potential enzymatic capacity to catalyse nitrate
reduction, as measured by in vitro nitrate reductase activity.
Inhibition effects of PHs on root nitrate uptake and nitrate reduc-
tase activity have also been observed, especially in plants supplied
with a low rate of nitrates. Ruiz et al. (2000) reported that foliar
application of an animal-derived PH containing micronutrients
(Fosfonutren) reduced root nitrate uptake and N assimilation in
pepper. Applications of PHs containing high concentrations of free
amino acids lead to a strong phloem loading with amino acids,
which repress root nitrate uptake and, consequently, reduce the
rate of nitrate reduction. The inhibition effect depends on nitrate
concentration and the type and concentration of amino acids in the
PH; for instance, exogenously applied glutamine decreased nitrate
and ammonium influx and transporter transcript abundance in bar-
ley roots (Fan et al., 2006; Miller et al., 2007). The above findings
confirm that amino acids (especially glutamine and arginine) play
a signalling role in regulating N uptake by roots (Fan et al., 2006;
Miller et al., 2007).
3.3. Effects on secondary plant metabolism
Several studies (Kauffman et al., 2007; Apone et al., 2010; Ertani
et al., 2013) reported the positive effects exerted by PHs and amino
acids, including proline and betaine, in inducing secondary plant
metabolism and increasing plant defence responses and tolerance
to stresses, including salinity, drought and temperature and oxida-
tive conditions. Apone et al. (2010) found that a mix of amino acids,
sugars and peptides induced the expression of genes involved in
the oxidative stress response in Arabidopsis thaliana plants. Ertani
et al. (2013), studying the effects of alfalfa PH on maize grown
hydroponically under salt conditions, observed an accumulation of
flavonoids in sodium chloride-treated plants after PH application
as a result of stimulation of flavonoid biosynthesis. The increase
of flavonoids was consistent with the variation in activity of a key
enzyme (phenylalanine ammonia lyase; PAL) involved in phenyl-
propanoid biosynthesis. It has been reported that the gene encoding
for this protein is responsive to a number of abiotic and biotic
stresses in many plant species (Huang et al., 2011) and that it can
be induced by biostimulants (Schiavon et al., 2010 Ertani et al.,
2011). The results proved that PAL is responsive to salt stress and
that its activity and gene expression are both up-regulated by PHs
under salinity conditions. The stimulation of the phenylpropanoid
pathway by PHs could be due to the improvement of N assim-
ilation, although an alternative metabolic pathway that couples
the proline biosynthesis to the oxidative pentose phosphate path-
way has been proposed (Shetty et al., 2003; Shetty and Wahlqvist,
2004). According to this hypothesis, the increase of proline under
stress would be partially responsible for the triggering of the phe-
nolic compound build-up. In a metabolomics study, Lucini et al.
(2015) observed an increase of several secondary metabolites (e.g.,
terpenes, glucosinolates) after application of a plant-derived PH
(Trainer) on lettuce plants grown under saline conditions. These
secondary metabolites were involved in improving salinity toler-
ance of lettuce by modulating the signalling that activates defence
pathways. In grapevine, Lachhab et al. (2014) reported that foliar
application of PHs from casein and soybean up-regulated genes
encoding the stilbene synthase enzyme responsible for the biosyn-
thesis of resveratrol. As expected, the two PHs, in particular the
soybean-derived PH, induced a high and rapid accumulation of
resveratrol in leaves. Both hydrolysates proved to act as elicitors to
enhance grapevine immunity against Plasmopara viticola, the causal
agent of grapevine downy mildew. The authors concluded that the
soybean and casein hydrolysates could be considered good can-
didates to reduce fungicide applications in a modern sustainable
viticulture (Lachhab et al., 2014).
4. Agronomic response of horticultural crops
4.1. Growth, yield and flowering
Applications of PHs have been shown to promote the vegetative
growth and macro- and micronutrient uptake in several horti-
cultural crops, resulting in increased crop productivity (Halpern
et al., 2015). Morales-Pajan and Stall (2003) observed in papaya
that monthly foliar applications with an animal-derived PH (Siap-
ton) increased the marketable yield by 22% in comparison to
untreated plants. The improvement in papaya yield was attributed
to increased fruit numbers, as PHs may have increased fruit set
(Morales-Pajan and Stall, 2003). In banana, the soil-based or foliar
application of hydrolysed feather waste, a by-product from poultry
processing, shortened the harvesting by 28 days and enhanced the
number of hands per bunch, fingers per hand and bunch weight
by 10, 24 and 16%, respectively, in comparison to the control. The
higher crop performance in PH-treated banana was attributed to
the highest chlorophyll and reducing sugar contents. The authors
concluded that the application of feather degradation products can
play a dual role as a bioenhancer to reduce use of fertilisers in hor-
ticulture and to eliminate poultry waste (Morales-Pajan and Stall,
2003).
Quartieri et al. (2002) tested the foliar applications of differ-
ent rates and molecular weight fractions of animal-derived PHs on
growth and leaf N content of potted kiwifruit plants. The authors
reported that the fractions with low molecular weights (1–3 kDa)
stimulated, especially at low rates, shoot and root growth, while
shoot growth alone was enhanced by the fractions with high molec-
ular weights (3–10, and >10 kDa) only at the highest application
rates.
Greenhouse application with a carob germ hydrolysate or with
an animal enzymatic PH increased tomato plant height and number
of flowers per plant compared to untreated plants, while only those
treated with plant-derived PHs reported enhanced numbers of fruit
per plant (Parrado et al., 2008). Overall, these authors confirmed the
advantage of applying PHs obtained from plant origin rather than
animal origin. Similarly, Koukounararas et al. (2013) suggested that
the application (through roots or foliage) of a PH commercial prod-
uct (Amino 16) could increase greenhouse tomato yield and yield
components (fruit mean weight and number) under varying fertili-
sation doses. It is rather evident that the application of amino acids
and peptides in the nutrient solution has a beneficial effect on the
leaf mineral status, in particular cations such as K, Ca, Mg, Fe, Cu
and Zn (Garcia et al., 2011), consequently resulting in better growth
of the crop. In a recent study, Colla et al. (2014) demonstrated
that increasing the concentration of a commercial plant-derived PH
(Trainer) from 0 to 10 mL L−1 improved the tomato shoot and root
biomass, chlorophyll (SPAD index), and N content of leaves by 20,
27, 15 and 21%, respectively. The authors indicated that the greater
plant biomass of plants treated with PH containing amino acids and
small peptides could be attributed to an increase in leaf N content,
 G. Colla et al. / Scientia Horticulturae 196 (2015) 28–38
which may account for improved photosynthesis and enhanced
translocation of synthates to the sinks. It is well known that plant N
assimilation involves the synthesis and conversion of amino acids
through the reduction of nitrate by several key enzymes (Ertani
et al., 2009; Colla et al., 2014). This was demonstrated by Ertani
et al. (2009), where the root application of two PHs enhanced N
assimilation in maize seedlings through an increase of glutamine
synthetase and nitrate reductase activities. Colla et al. (2014) con-
cluded that the vigorous root apparatus may also have contributed
to increased N uptake and thus N use efficiency in tomato plants
treated with the plant-derived PH ‘Trainer’.
The increase in leaf chlorophyll concentration and N assim-
ilation have been also attributed to an auxin-like effect (Nardi
et al., 2009). In line with the previous statement, the application
of plant-derived PH ‘Trainer’ increases rooting of tomato cuttings,
and the shoot lengths of gibberellin-deficient dwarf pea plants
provide evidence of an auxin-like activity and a gibberellin-like
activity, respectively (Colla et al., 2014). Also Ertani et al. (2009)
indicated that the application of PHs improved the shoot lengths
of lettuce compared to those under the application of gibberellin,
demonstrating a strong gibberellin-like activity. These data suggest
that low-molecular-size peptides and free amino acids are easily
absorbed and can act as signalling compounds, promoting endoge-
nous phytohormone biosynthesis (Cavani and Ciavatta, 2007). This
was also confirmed by Matsumiya and Kubo (2011), who demon-
strated that root application of degraded soybean meal products,
containing a root hair-promoting peptide, exhibited auxin-like
activity, stimulating root hair length and numbers in tomato, mus-
tard greens, and lettuce.
In greenhouse lettuce, crop uniformity was significantly
increased when plants received an animal-derived PH (Amino
16), without negative effects on yield and nutritional components
(Tsouvaltzis et al., 2014). Liu and Lee (2012) indicated that the appli-
cation of mixed amino acids led to positive effects on radish, and
red pepper, specifically regarding N utilisation and concentrations
of proteins and amino acids in shoot tissue. However, the positive
effect of PH biostimulants is not a general phenomenon, since no
significant yield increase between treatments (with and without
application of Siapton) was recorded in either spinach or endive
(Kunicki et al., 2010; Gajc-Wolska et al., 2012).
Concerning flower crops, De Lucia and Vecchietti (2012) inves-
tigated the effects of two PHs (from animal epithelia and alfalfa) on
the flower quality of lily. The authors showed that both PHs reduced
the length of crop cycle and increased the leaf area, the diameter
of flower buds, the root system and the stem quality in comparison
to untreated plants.
4.2. Product quality
In the past decade, phytochemical compounds found in
fruits and vegetables have generated significant interest among
scientists, food nutritionists and producers due to their health-
benefitting properties, and they likely could become the driving
force in the future (Gruda, 2009; Khanam et al., 2012). Phytochem-
icals are natural antioxidant compounds able to reduce or prevent
chronic diseases, such as cardiovascular problems, ischemic stroke,
arthritis and inflammatory bowel, as well as some cancers (Oude
Griep et al., 2011; Slavin and Lloyd, 2012). Two recent reviews
(Rouphael et al., 2010, 2012) revealed that the quality in fresh fruits
and vegetables may depend on several factors, such as plant geno-
type, grafting, growing conditions, cultural practices and farming
management (organic vs. conventional). Furthermore, several stud-
ies (Parrado et al., 2007; Gurav and Jadhav, 2013; Ertani et al., 2014)
showed that application of PHs are able to modify the primary
and secondary metabolism, stimulating the production and accu-
mulation of antioxidant compounds (carotenoids, polyphenols,
33
flavonoids, etc.). For instance, banana plants supplemented with
feather degradation products rich in peptides, amino acids and min-
erals showed an increase in contents of total phenolics, flavonoids
and protein and in antioxidant activity (Gurav and Jadhav, 2013).
This is in line with previous studies reporting an increase in phenols
in different species amended with organic wastes (McGrath et al.,
1994).
Similarly, Parrado et al. (2007) investigated the effect of an
enzymatic vegetable extract (EVE) coming from agricultural wastes
on the expression of quality parameters, such as colour and
anthocyanin induction, in red grapes. The application of the EVE
containing peptides, free amino acids and phytohormones (aux-
ins, gibberellins, cytokinins) had higher total phenolic and total
anthocyanin values (22 and 70% higher, respectively) in compari-
son to untreated plants. The authors attributed the production and
accumulation of anthocyanins using EVE to the presence of the phy-
tohormones and nutrients (Ban et al., 2003; Jeong et al., 2004). In
chili pepper, the application of two PHs, one derived from alfalfa
and the other obtained from red grapes, significantly increased
capsaicin content in red fruits, while green pepper fruits exhib-
ited higher concentrations of chlorogenic acid, p-hydroxybenzoic
acid and p-coumaric acid (Ertani et al., 2014). The beneficial effects
of plant-derived PHs on nutraceutical properties were related to
the activation of secondary metabolism, in particular the increase
of gene expression of the phenylalanine (tyrosine) ammonia-lyase
enzyme involved in the phenylpropanoid pathway (Schiavon et al.,
2010 Ertani et al., 2011).
PH application can also reduce nitrates in leafy vegetables
belonging to the families of Brassicaceae (rocket, radish), Chenopo-
diaceae (Swiss chard, spinach) and Amaranthaceae as well as
Asteraceae (lettuce) and Apiaceae (celery, parsley) (Liu and Lee, 2012
and references cited therein). Ruiz et al. (2000) reported that foliar
applications of an animal-derived PH reduced root nitrate uptake
and N assimilation in pepper leaves. Similarly, the application of
a PH solution reduced the nitrate concentration in greenhouse
lettuce by 29% in comparison to those coming from inorganic fer-
tilisation treatment (Tsouvaltzis et al., 2014).
The reduction of nitrate content in plants has been reported in
lettuce, Chinese cabbage, onion, pak choi, radish and other leafy
crops (Gunes et al., 1994, 1996; Chen and Gao, 2002; Wang et al.,
2004; Liu and Lee, 2012). Wang et al. (2007) carried out a trial to
study the effects of single amino acids on growth and nitrate accu-
mulations in shoots of hydroponically grown pak choi. The authors
reported that, with the exceptions of cysteine, glycine, histidine
and arginine, the replacement of 20% of nitrate-N in the solution
with single amino acids significantly reduced nitrate accumula-
tion in shoots in comparison with plants supplied with 100% of
nitrate-N. However, with the exceptions of arginine, asparagine
and glutamine, growth depression was also observed when 20%
of nitrate-N was replaced with single amino acids. The authors
concluded that asparagine and glutamine may be used to partially
replace nitrate-N, leading to a reduction of nitrate content in pak
choi shoots and an increase of macronutrient content in plants
without detrimental effects on growth. Overall, the main role of
amino acids is the regulation of several processes and metabolic
pathways of plant N metabolism, in particular nitrate and nitrite
reductase, glutamine synthetase, glutamate synthase and aspar-
tate aminotransferase activities (Liu et al., 2008; Liu and Lee, 2012;
Calvo et al., 2014).
5. Protein hydrolysates and abiotic stress tolerance
Horticultural crops are frequently cultivated under
unfavourable environmental and chemical soil conditions, includ-
 34
Table 2
Effects of protein hydrolysates (PHs) on horticultural crops.

Crop Type of PH PH application mode Experimental conditions Effects References

Banana Chicken feather derived PH Root and foliar Field trial Early flowering; increased nutrient, Gurav and Jadhav (2013)
chlorophyll content, and proline in leaves;
reduced sugars, proteins, amino acids,
phenolics and flavonoids in fruits
Corn PHs from meat flour or alfalfa Root Hydroponic system under growth chamber Increased root and leaf growth, and nitrate Ertani et al. (2009)
reductase and glutamine synthetase activities
Corn Alfalfa derived PH Root Hydroponic system under growth chamber Increased crop salinity tolerance, nitrogen Ertani et al. (2013)
assimilation and activity of antioxidant
systems
Grapevine PH of distiller’s dried grains and carob germ flour Root Field trial Increased total phenolics, and anthocyanin Parrado et al. (2007)
content in grape juice
Grapevine PHs from soybean or casein Foliar Field trial Up-regulated defense genes encoding Lachhab et al. (2014)
pathogenesis-related proteins and the stilbene
synthase enzyme; increased resistance to
Plasmopara viticola

G. Colla et al. / Scientia Horticulturae 196 (2015) 28–38

Grapevine Plant derived PH Foliar Field trial Increased tolerance to drought, soluble solids, Boselli et al. (2015)
total phenols and anthocyanins in fruits
Kiwifruit Animal derived PHs with different molecular weights Foliar Pot trial Shoot and root biomass were increased by PH Quartieri et al. (2002)
fractions with the lowest molecular weight
especially at low rates
Lettuce Plant derived PH (Trainer) Root and foliar Pot culture under greenhouse using saline and Increased crop tolerance to salinity, Lucini et al. (2015)
non-saline solution chlorophyll fluorescence, nitrogen and
phosphorus content of leaves
Lettuce Plant derived PH (Trainer) Root Hydroponic system with two concentration of Increased yield, SPAD index, and nitrogen Colla et al. (2013)
nutrient solution content of leaves
Lettuce Plant derived PH (Aminol 16) Root and foliar Greenhouse crop during winter season; foliar Increased crop uniformity, and antioxidant Tsouvaltzis et al. (2014)
and soil application of PH activity; reduced nitrates in leaves
Lettuce Animal derived PH (Terra-Sorb Foliar) Foliar Pot culture in growth chamber under cold Increased plant fresh weight and stomatal Botta (2013)
stress conditions conductance
Lily Animal derived PH and alfalfa derived PH Foliar Pot culture under greenhouse conditions Reduced the length of crop cycle; increased De Lucia and Vecchietti (2012)
leaf area, diameter of flower buds, and stem
and bulb dry weight
Olive Animal derived-PH (Siapton) Foliar In vivo and in vitro trials Increased pollen tube elongation Viti et al. (1990)
Papaya Animal derived-PH (Siapton) Foliar Field trial Increased yield Morales-Pajan and Stall (2003)
Passionfruit Animal derived PH Foliar Nursery Increased seedling growth Morales-Pajan and Stall (2004)
Pepper Alfalfa derived PH Foliar Pot culture under greenhouse conditions Increased fresh weight and number of fruits, Ertani et al. (2014)
and secondary metabolites in fruits
Pepper Animal derived PH plus micronutrients (Fosfonutren) Foliar Pot culture under greenhouse conditions Decreased growth, yield and efficiency and Ruiz et al. (2000)
during fall-winter season utilization of nitrates
Persimmon Animal derived PH containing Ca (Stressal) Root Field trial under saline conditions Decreased Cl uptake, leaf necrosis, and leaf Visconti et al. (2015)
water potential
Spinach Animal derived PH (Siapton) Foliar Field trials in spring and autumn seasons using No effect on yield; positive or no effect on dry Kunicki et al. (2010)
two cultivars matter and nitrate content of leaves
Strawberry Animal derived PH (Aminoflor) Foliar Bag culture under greenhouse conditions Decreased weight of daughter plants Lisiecka et al. (2011)
Tomato Carob germ derived PH Root Pot culture under greenhouse condition Increased plant height, number of flowers, and Parrado et al. (2008)
number of fruits
Tomato Animal and plant derived PHs Root and foliar Hydroponic system with plants grown in Growth depression with animal derived PH Cerdán et al. (2013)
Fe-sufficient nutrient solution or in while plant derived PH enhanced root
lime-induced Fe deficiency Fe(III)-chelate reductase activity, chlorophyll
concentration, and leaf Fe concentration under
lime conditions
Tomato Plant derived PH (Trainer) Root Soilless culture in growth chamber Increased rooting and shoot growth Colla et al. (2014)
 G. Colla et al. / Scientia Horticulturae 196 (2015) 28–38
ing salinity, drought, thermal stress, adverse soil pH and nutrient
deficiency, leading to stunted growth and loss in productivity.
The application of PHs and specific amino acids can induce
plant defence responses and increase plant tolerance to a vari-
ety of abiotic stresses and oxidative conditions (Chen and Murata,
2008; Kauffman et al., 2007; Apone et al., 2010; Ertani et al., 2013;
Calvo et al., 2014). Small peptides and other amino acid deriva-
tives have been extensively studied for their effects on inducing
plant defence responses, thus increasing plant tolerance to a wide
range of stresses (Tuteja, 2007). Exogenous application of glycine
betaine, for example, improves growth and survival of a wide vari-
ety of plants under stress (Park et al., 2006; Cuin and Shabala, 2005).
Application of glutamate and/or ornithine, precursors of proline,
can also enhance tolerance to salt stress (Chang et al., 2010; Da
Rocha et al., 2012), whereas arginine, which plays an important
role in N storage and transport in plants, has been shown to accu-
mulate under abiotic and biotic stress (Lea et al., 2006). Also, the
beneficial effects of proline against abiotic stresses and heavy metal
toxicity have been well documented (Sharma and Dietz, 2009).
Dramatic accumulation of proline due to increased synthesis
and decreased degradation under a variety of stress conditions,
such as salt, drought and excess metal ions, has been documented
in many plants after biostimulant addition (Lucini et al., 2015).
Similarly, a decrease in the level of accumulated proline in the
rehydrated plants is due to both down-regulation of proline biosyn-
thetic pathway enzymes and up-regulation of proline-degrading
enzymes. On this account, Ertani et al. (2013) investigated the
capacity of a PH derived from alfalfa plants to stimulate short-
term growth in the presence of sodium chloride in maize plants. PH
treatment significantly stimulated the growth of sodium chloride-
treated plants, likely due to its contents of plant growth regulators,
such as indoleacetic acid and triacontanol. Exogenous indoleacetic
acid has been shown to improve the root and shoot growth of
wheat seedlings under saline conditions (Egamberdieva, 2009), and
triacontanol ameliorated salt stress-induced damage when exoge-
nously applied to wheat and radish plants (Çavuşoğlu and Kabar,
2008; Perveen et al., 2011). Moreover, salinity increased the activity
of catalase (CAT), guaiacol peroxidase (GPX) and ascorbate perox-
idase (APX), which are considered crucial enzymes in regulating
intracellular hydrogen peroxide levels in plants. The increase of
antioxidant enzyme activity indicates both protection against dam-
age and control of redox signalling and supports the evidence that
the antioxidative defence system plays a pivotal role in overall plant
salt tolerance (De Azevedo Neto et al., 2005; Abogadallah, 2010).
The increase of antioxidant enzyme activity was probably due to the
higher content of proline measured in plants grown under sodium
chloride stress. Indeed, in several studies proline has been reported
to accumulate in response to salinity and induce the activity of
antioxidant enzymes (Khedr et al., 2003; Hoque et al., 2007).
Visconti et al. (2015) addressed the question of whether the
application of an animal-derived PH containing Ca (Stressal) is able
to counteract salt stress in fruit trees. The authors reported that
application of PH on persimmon trees grafted on Diospyros lotus
alleviated the detrimental effects of salinity by lowering Cl− uptake.
The lower Cl− uptake in treated trees was accompanied by lower
leaf potential and less leaf necrosis, even though the marketable
yields of treated and untreated fruit trees were not significantly
different (Visconti et al., 2015). The improvement in tolerance was
attributed to the composition of the PH, characterised by high
amounts of proline, glycine and glycine betaine, which stimu-
lated the plant’s metabolism to build up compatible solutes and
enhanced the ability to exclude Cl− ions. Moreover, Lucini et al.
(2015) demonstrated that root and especially leaf-root applica-
tions of a plant-derived PH (Trainer) increased marketable yield,
dry biomass and root dry weight of greenhouse lettuce under salin-
ity conditions (25 mM sodium chloride). The improvement of crop
35
performance using PHs was accompanied with an enhancement of
plant N metabolism and an increase in the maximum quantum use
efficiency of Photosystem II in the dark-adapted state. Lucini et al.
(2015) also reported that PH-treated lettuce plants exhibited oxida-
tive stress alleviation, osmotic adjustment and changes in hormone
networks as well as sterols, terpenes and glucosinolates.
Drought is one of the most important environmental factors
inducing physiological changes in plants, such as a decrease in
the water potential of the cells, stomatal closure and develop-
ment of oxidative processes mediated by reactive oxygen species
(ROS). A common response to stress is the generation of ROS
that trigger oxidative stress (Blokhina et al., 2003; De Azevedo
Neto et al., 2005). Plants control the concentrations of ROS by an
array of non-enzymatic and enzymatic antioxidants, the former of
which include superoxide dismutase (SOD), CAT, APX, GPX and glu-
tathione reductase. Feitosa de Vasconcelos et al. (2009) studied
the antioxidant enzymes SOD, CAT and APX in maize and soy-
bean plants under well-watered or drought stress conditions. These
authors showed that the application of biostimulants based on
amino acids increased the activity of SOD, CAT and APX, helping
plants overcome stress conditions.
Another common environmental stress inflicting heavy eco-
nomic yield losses in agricultural and horticultural crops is thermal
stress. In a recent study, Botta (2013) carried out a cold stress
experiment on lettuce under controlled environmental conditions
using an animal-based PH obtained by enzymatic hydrolysis (Terra-
Sorb Foliar). The author reported that the application of PH on
plants grown under cold stress induced higher shoot and root
fresh weights and stomatal conductance compared to untreated
plants. In the same article, Botta (2013) stated an improvement
of heat stress tolerance on ryegrass plants, as demonstrated by
the higher photosynthetic efficiency, and levels of chlorophylls and
carotenoids in leaves of plants treated with PH in comparison with
untreated plants (control) grown at 36 ◦ C.
The effectiveness of root and foliar applications of two com-
mercial products containing amino acids to improve alkalinity
tolerance in tomato has been also demonstrated by Cerdán et al.
(2013). The product containing amino acids from animal origin
showed toxicity symptoms with no positive effects of Fe nutri-
tion under alkaline conditions (pH 8.0). In contrast, the application
of plant-derived PH increased tomato seedling growth due to
an enhancement in chlorophyll concentration, root and leaf Fe
chelate reductase activity and leaf Fe concentration, especially
when applied to the roots. Finally, Colla et al. (2014) indicated
that foliar application of a plant-derived PH could be considered an
effective tool in reducing fertiliser application and environmental
pollution. The aim of the former study was to determine whether
the use of a commercial plant-derived PH (Trainer) could enhance
the growth and N uptake of lettuce grown in a raft floating system
using full and reduced nutrient solution concentrations (100 and
10%, respectively). Weekly foliar applications of ‘Trainer’ increased
the fresh biomass, SPAD index and N uptake by 50, 11 and 11%,
respectively, indicating the importance of PH applications for cop-
ing with nutrient deficiency conditions.
6. Conclusions and future perspectives
Protein hydrolysate-based biostimulants are effective tools for
making horticulture more sustainable. Many research studies have
documented the benefits of PH applications on growth, yield, prod-
uct quality, resource use efficiency and tolerance to environmental
and chemical soil stresses of several horticultural crops (Table 2).
However, knowledge about the potential benefits derived from
applications of PHs is far from complete. Therefore, more research is
needed to understand the modes of action of PHs in relation to their
36 G. Colla et al. / Scientia Horticulturae 196 (2015) 28–38
composition. A deep investigation is also necessary for defining the
application time and dose of PHs for each crop and environmental
conditions. Seed coating with PHs is also a promising application
technique that deserves further investigation in order to enhance
germination and early growth of vegetable and flower crops, foliage
plants, and turf grasses. Limited knowledge is also available on the
effects of PHs on microbial communities living in the rhizosphere or
phyllosphere. Because PH-based products have been often reported
to exhibit variation in their compositions, it is important to stan-
dardise the production process in terms of raw material, enzyme
source and hydrolysis conditions. The regulatory situation of bios-
timulants in general and PHs in particular is still complex today,
in the absence of any harmonised and specific regulatory frame-
work in the United States and also in the European Union (EU).
For instance in the EU, PHs are sold as fertilisers, whereas in some
European countries, such as Italy, national legislation allows sell-
ing PHs as plant biostimulants. The main reasons for this situation
include lack of a clear definition and acceptance of the concept
by regulatory bodies; thus, a supranational regulatory framework
is an urgent need to promote the use of PHs as biostimulants in
horticulture.
References
Abogadallah, G.M., 2010. Antioxidative defense under salt stress. Plant Signal.
Behav. 5, 369–374.
Apone, F., Tito, A., Carola, A., Arciello, S., Tortora, A., Filippini, L., Monoli, I.,
Cucchiara, M., Gibertoni, S., Chrispeels, M.J., Colucci, G., 2010. A mixture of
peptides and sugars derived from plant cell walls increases plant defense
responses to stress and attenuates ageingassociated molecular changes in
cultured skin cells. J. Biotechnol. 145, 367–376.
Baglieri, A., Cadili, V., Mozzetti Monterumici, C., Gennari, M., Tabasso, S.,
Montoneri, E., Nardi, S., Negre, M., 2014. Fertilization of bean plants with
tomato plants hydrolysates. Effect on biomass production, chlorophyll content
and N assimilation. Sci. Hortic. 176, 194–199.
Ban, T., Ishimaru, M., Kobayashi, S., Shiozaki, S., Goto-Yamamoto, N., Horiuchi, S.,
2003. Abscisic acidand 2,4-dichlorohenoxyacetic acid affect the expression of
anthocyanin biosynthetic pathway genes in ‘Kyoho’ grape berries. J. Hortic. Sci.
Biotechnol. 78, 586–589.
Bernabei, G., 2015. Costo energetico ed impatto ambientale relativo ai processi
produttivi degli idrolizzati proteici. Master thesis in Agricultural and
Environmental Sciences at Tuscia University, Viterbo, Italy.
Blokhina, O., Violainen, E., Fagerstedt, K.V., 2003. Antioxidants, oxidative damage
and oxygen deprivation stress: a review. Ann. Bot. 91, 179–194.
Boselli, M., Bahouaoui, M., Lachhab, N., Sanzani, S.M., Ippolito, A., 2015. Vite:
idrolizzati proteici contro lo stress idrico. L’Informatore Agrario 22, 39–42.
Botta, A., 2013. Enhancing plant tolerance to temperature stress with amino acids:
an approach to their mode of action. Acta Hortic. 1009, 29–35.
Calvo, P., Nelson, L., Kloepper, J.W., 2014. Agricultural uses of plant biostimulants.
Plant Soil 383, 3–41.
Cavani, L., Ciavatta, C., Gessa, C., 2003. Determination of free l- and d-alanine in
hydrolysed protein fertilisers by capillary electrophoresis. J. Chromatogr. A
985, 463–469.
Cavani, L., Ciavatta, C., 2007. Attività biostimolante degli idrolizzati proteici.
L’informatore Agrario 44, 46–52.
Çavuşoğlu, K., Kabar, K., 2008. Effects of some plant growth regulators on leaf
anatomy of radish seedlings grown under saline conditions. J. App. Biol. Sci. 2,
47–50.
Cerdán, M., Sánchez-Sánchez, A., Oliver, M., Juárez, M., Sánchez-Andreu, J.J., 2009.
Effect of foliar and root applications of amino acids on iron uptake by tomato
plants. Acta Hortic. 830, 481–488.
Cerdán, M., Sánchez-Sánchez, A., Jordá, D.J., Juárez, M., Andreu, J.S., 2013. Effect of
commercial amino acids on iron nutrition of tomato plants grown under
lime-induced iron deficiency. J. Plant Nutr. Soil Sci. 176, 1–8.
Chalamaiah, M., Dinesh Kumar, B., Hemalatha, R., Jyothirmayi, T., 2012. Fish
protein hydrolysates: proximate composition, amino acid composition,
antioxidant activities and applications: a review. Food Chem. 135, 3020–3038.
Chang, C., Wang, B., Shi, L., Li, Y., Duo, L., Zhang, W., 2010. Alleviation of salt
stress-induced inhibition of seed germination in cucumber (Cucumis sativus L.)
by ethylene and glutamate. J. Plant Physiol. 167, 1152–1156.
Chen, G., Gao, X., 2002. Effect of partial replacement of nitrate by amino acid and
urea on nitrate content of nonheading Chinese cabbage and lettuce in
hydroponics. Sci. Agr. Sin. 35, 187–191 (Chinese).
Chen, T.H.H., Murata, N., 2008. Glycinebetaine: an effective protectant against
abiotic stress in plants. Trends Plant Sci. 13, 499–505.
Corte, L., Dell’Abate, M.T., Magini, A., 2014. Assessment of safety and efficiency of
nitrogen organic fertilizers from animal-based protein hydrolysates—a
laboratory multidisciplinary approach. J. Sci. Food Agric. 94, 235–245.
Colla, G., Svecova, E., Rouphael, Y., Cardarelli, M., Reynaud, H., Canaguier, R.,
Planques, B., 2013. Effectiveness of a plant –derived protein hydrolysate to
improve crop performances under different growing conditions. Acta Hortic.
1009, 175–179.
Colla, G., Rouphael, Y., Canaguier, R., Svecova, E., Cardarelli, M., 2014. Biostimulant
action of a plant-derived protein hydrolysate produced through enzymatic
hydrolysis. Front. Plant Sci. 5, 448.
Crawford, D.L., Lynch, J.M., Whipps, J.M., Ousley, M.A., 1993. Isolation and
characterization of actinomycete antagonists of a fungal root pathogen. Appl.
Environ. Microbiol. 59, 3899–3905.
Cuin, T.A., Shabala, S., 2005. Exogenously supplied compatible solutes rapidly
ameliorate NaCl-induced potassium efflux from barley roots. Plant Cell Physiol.
46, 1924–1933.
du Jardin, P., 2012. The science of plant biostimulants-a bibliographic analysis.
Contract 30-CE0455515/00-96, ad hoc Study on bio-stimulants products.
http://ec.europa.eu/enterprise/sectors/chemicals/files/fertilizers/final report
bio 2012 en.pdf.
Da Rocha, I.M., Vitorello, V.A., Silva, J.S., Ferreira-Silva, S.L., Viegas, R.A., Silva, E.N.,
2012. Exogenous ornithine is an effective precursor and the delta-ornithine
amino transferase pathway contributes to proline accumulation under high N
recycling in salt-stressed cashew leaves. J. Plant Physiol. 169, 41–49.
De Azevedo Neto, A.D., Prisco, J.T., Eneı́as-Filho, J., Medeiros, J.V., Gomes-Filho, E.,
2005. Effect of salt stress on antioxidative enzymes and lipid peroxidation in
leaves and roots of salt-tolerant and salt-sensitive maize genotypes. Environ.
Exp. Bot. 56, 87–94.
De Lucia, B., Vecchietti, L., 2012. Type of biostimulant and application method
effects on stem quality and root system growth in LA Lily. Eur. J. Hortic. Sci. 77,
1–10.
Egamberdieva, D., 2009. Alleviation of salt stress by plant growth regulators and
IAA producing bacteria in wheat. Acta Physiol. Plant 31, 861–864.
Ertani, A., Cavani, L., Pizzeghello, D., Brandellero, E., Altissimo, A., Ciavatta, C., Nardi,
S., 2009. Biostimulant activities of two protein hydrolysates on the growth and
nitrogen metabolism in maize seedlings. J. Plant. Nutr. Soil. Sci. 172, 237–244.
Ertani, A., Francioso, O., Tugnoli, V., Righi, V., Nardi, S., 2011. Effect of commercial
Lignosulfonate-Humate on Zea mays L. metabolism. J. Agric. Food Chem. 59,
11940–11948.
Ertani, A., Schiavon, M., 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.
Ertani, A., Pizzeghello, D., Francioso, O., Sambo, P., Sanchez-Cortes, S., Nardi, S.,
2014. Capsicum chinensis L. growth and nutraceutical properties are enhanced
by biostimulants in a long-term period: chemical and metabolomic
approaches. Front. Plant Sci. 5, 1–12.
Fan, X., Gordon-Weeks, R., Shen, Q.R., Miller, A.J., 2006. Glutamine transport and
feedback regulation of nitrate reductase activity in barley roots leads to
changes in cytosolic nitrate pools. J. Exp. Bot. 57, 1333–1340.
Feitosa de Vasconcelos, A.C., Zhang, X., Ervin, E.H., de Castro Kieh, J., 2009.
Enzymatic antioxidant responses to biostimulants in maize and soybean
subjected to drought. Sci. Agric. (Piracicaba, Braz.) 66, 395–402.
Furuya, S., Umemiya, Y., 2002. The influence of chemical forms on foliar-applied
nitrogen absorption for peach trees. Acta Horticulturae 594, 97–103.
Gajc-Wolska, J., Kowalczyk, K., Nowecka, M., Mazur, K., Metera, A., 2012. Effect of
organic-mineral fertilizers on the yield and quality of Endive (Cichorium
endivia L.). Acta Sci. Pol 11, 189–200.
Garcia, A.L., Madrid, R., Gimeno, V., Rodriguez-Ortega, W.M., Nicolas, N.,
Garcia-Sanchez, F., 2011. The effects of amino acids fertilization incorporated
to the nutrient solution on mineral composition and growth in tomato
seedlings. Span. J. Agric. Res. 9, 852–861.
García-Martínez, A.M., Díaz, A., Tejada, M., Bautista, J., Rodríguez, B., María, C.S.,
Revilla, E., Parrado, J., 2010. Enzymatic production of an organic soil
biostimulant from wheat condensed distiller solubles: effects on soil
biochemistry and biodiversity. Process Biochem. 45, 1127–1133.
Gruda, N., 2009. Do soilless culture systems have an influence on product quality
of vegetables? J. Appl. Bot. Food Qual. 82, 141–147.
Gunes, A., Post, W.H.K., Kirkby, E.A., Akas, M., 1994. Influence of partial replacement
of nitrate by amino acid nitrogen or urea in the nutrient medium on nitrate
accumulation in NFT grown winter lettuce. J. Plant Nutr. 17, 1929–1938.
Gunes, A., Inal, A., Aktas, M., 1996. Reducing nitrate content of NFT grown winter
onion plants (Allium cepa L.) by partial replacement of NO3 with amino acid in
nutrient solution. Sci. Hortic. 65, 203–208.
Gurav, R.G., Jadhav, J.P., 2013. A novel source of biofertilizer from feather biomass
for banana cultivation. Environ. Sci. Pollut. Res. Int. 20, 4532–4539.
Halpern, M., Bar-Tal, A., Ofek, M., Minz, D., Muller, T., Yermiyahu, U., 2015. The use
of biostimulants for enhancing nutrient uptake. Adv. Agron. 130, 141–174.
Hoff, T., Truong, H.N., Caboche, M., 1994. The use of mutants and transgenic plants
to study nitrate assimilation. Plant Cell Environ. 17, 489–506.
Hoque, M.A., Okuma, E., Banu, M.N.A., Nakamura, Y., Shimoishi, Y., Murata, Y.,
2007. Exogenous proline mitigates the detrimental effects of salt stress more
thanthebetaine by increasing antioxidant enzyme activities. J. Plant Physiol.
164, 553–561.
Huang, T., Jander, G., de Vos, M., 2011. Non-protein amino acids in plant defense
against insect herbivores: representative cases and opportunities for further
functional analysis. Phytochemistry 72, 1531–1537.
Ito, Y., Nakanomyo, I., Motose, H., Iwamoto, K., Sawa, S., Dohmae, N., 2006.
Dodeca-CLE peptides as suppressors of plant stem cell differentiation. Science
313, 842–845.
 G. Colla et al. / Scientia Horticulturae 196 (2015) 28–38
Jeong, S.T., Goto-Yamamoto, N., Kobayashi, S., Esaka, M., 2004. Effects of plant
hormones and shading on the accumulation of anthocyanins and the
expression of anthocyanin biosynthetic genes in grape berry skins. Plant Sci.
167, 247–252.
Jones, D.L., Kemmitt, S.J., Wright, D., Cuttle, S.P., Bol, R., Edwards, A.C., 2005. Rapid
intrinsic rates of amino acid biodegradation in soils are unaffected by
agricultural management strategy. Soil Biol. Biochem. 37, 1267–1275.
Jones, D.L., Kielland, K., Sinclair, F.L., Dahlgren, R.A., Newsham, K.K., Farrar, J.F.,
Murphy, D.V., 2009. Soil organic nitrogen mineralization across a global
latitudinal gradient. Glob. Biogeochem. Cycles 23, GB1016.
Jones, D.L., Kielland, K., 2012. Amino acid, peptide and protein mineralization
dynamics in a taiga forest soil. Soil Biol. Biochem. 55, 60–69.
Kasparkova, V., Kolomaznik, K., Burketova, L., Sasek, V., Simek, L., 2009.
Characterization of low-molecular weight collagen hydrolysates prepared by
combination of enzymatic and acid hydrolysis. J. Am. Leather Chem. Assoc.
104, 46–51.
Kauffman, G.L., Kneival, D.P., Watschke, T.L., 2007. Effects of biostimulant on the
heat tolerance associated with photosynthetic capacity, membrane
thermostability, and polyphenol production of perennial ryegrass. Crop Sci. 47,
261–267.
Khanam, U.K.S., Oba, S., Yanase, E., Murakami, Y., 2012. Phenolic acids, flavonoids
and total antioxidant capacity of selected leafy vegetables. J. Funct. Foods 4,
979–987.
Khedr, A.H.A., Abbas, M.A., Wahid, A.A.A., Quick, W.P., Abogadallah, G.M., 2003.
Proline induces the expression of salt-stress responsive proteins and may
improve the adaptation of Pancratium maritimum L. to salt-stress. J. Exp. Bot.
54, 2553–2562.
Kondo, T., Sawa, S., Kinoshita, A., Mizuno, S., Kakimoto, T., Fukuda, H., 2006. A plant
peptide encoded by CLV3 identified by in situ MALDI-TOF MS analysis. Science
313, 845–848.
Koukounararas, A., Tsouvaltzis, P., Siomos, A.S., 2013. Effect of root and foliar
application of amino acids on the growth and yield of greenhouse tomato in
different fertilization levels. J. Food Agric. Environ. 11, 644–648.
˛
Kunicki, E., Grabowska, A., Sekara, A., Wojciechowska, R., 2010. The effect of
cultivar type, time of cultivation, and biostimulant treatment on the yield of
spinach (Spinacia oleracea L.). Folia Hortic. 22, 9–13.
Lachhab, N., Sanzani, S.M., Adrian, M., Chiltz, A., Balacey, S., Boselli, M., Ippolito, A.,
Benoit, P., 2014. Soybean and casein hydrolysates induce grapevine immune
responses and resistance against Plasmopara viticola. Front. Plant Sci. 5, 716.
Lea, P.J., Sodek, L., Parry, M.A.J., Shewry, P.R., Halford, N.G., 2006. Asparagine in
plants. Ann. Appl. Biol. 150, 1–26.
Lisiecka, J., Knaflewski, M., Spizewski, T., Fraszczak, B., Kaluzewicz, A., Krzesinski,
W., 2011. The effect of animal protein hydrolysate on quantity and quality of
strawberry daughter plants cv. ‘Elsanta’. Acta Sci. Pol. Hortorum Cultus 10,
31–40.
Liu, X.Q., Ko, K.Y., Kim, S.H., Lee, K.S., 2008. Effect of amino acid fertilization on
nitrate assimilation of leafy radish and soil chemical properties in high nitrate
soil. Commun. Soil Sci. Plant Anal. 39, 269–281.
Liu, X.Q., Lee, K.S., 2012. Effect of mixed amino acids on crop growth. In: Aflakpui,
G. (Ed.), Agricultural Science. InTech Europe Publisher, Rijeka, pp. 119–158.
Lucini, L., Rouphael, Y., Cardarelli, M., Canguier, R., Kumar, P., Colla, G., 2015. The
effect of a plant-derived biostimulant on metabolic profiling and crop
performance of lettuce grown under saline conditions. Sci. Hortic. 182,
124–133.
Maini, P., 2006. The experience of the first biostimulant, based on amino acids and
peptides: a short retrospective review on the laboratory researches and the
practical results. Fertilitas Agrorum 1, 29–43.
Matsumiya, Y., Kubo, M., 2011. Soybean peptide: novel plant growth promoting
peptide from soybean. In: El-Shemy, H. (Ed.), Soybean and Nutrition. In Tech
Europe Publisher, Rijeka, pp. 215–230.
McGrath, S.P., Chang, A.C., Page, A.L., Witter, E., 1994. Land application of sewage
sludge: scientific perspectives of heavy metal loading limits in Europe and the
United States. Environ. Rev. 2, 108–118.
Miller, A.J., Fan, X., Shen, Q., Smith, S.J., 2007. Amino acids and nitrate as signals for
the regulation of nitrogen acquisition. J. Exp. Bot. 59, 111–119.
Moe, L.A., 2013. Amino acids in the rhizosphere: from plants to microbes. Am. J.
Bot. 100, 1692–1705.
Morales-Pajan, J.P., Stall, W.M., 2003. Papaya (Carica papaya) response to foliar
treatments with organic complexes of peptides and amino acids. Proc. Fla.
State Hortic. Soc. 116, 30–32.
Morales-Pajan, J.P., Stall, W., 2004. Passion fruit (Passiflora edulis) transplant
production i affected by selected biostimulants. Proc. Fla. State Hortic. Soc. 117,
224–227.
Mu, C., Lin, W., Zhang, M., Zhu, Q., 2003. Towards zero discharge of
chromium-containing leather waste through improved alkali hydrolysis.
Waste Manage. 23, 835–843.
Nardi, S., Carletti, P., Pizzeghello, D., Muscolo, A., 2009. Biological activities of
humic substances. In: Senesi, N., Xing, B., Huang, P.M. (Eds.),
Biophysico-chemical Processes Involving Natural Nonliving Organic Matter in
Environmental Systems. Wiley, Hoboken, pp. 305–339.
Niculescu, M., Bajenaru, S., Gaidau, C., Simion, D., Felipescu, L., 2009. Extraction of
the protein components as amino-acids hydrolysates from chrome leather
wastes through hydrolytic processes. Rev. Chim. 60, 1070–1078.
Oaks, A., Aslam, M., Boesel, I., 1977. Ammonium and amino acids as regulators of
nitrate reductase in corn roots. Plant Physiol. 59, 391–394.
37
Oude Griep, L.M., Verschuren, W.M., Kromhout, D., Ocke, M.C., Geleijnse, J.M., 2011.
Colors of fruit and vegetables and 10-year incidence of stroke. Stroke 42,
3190–3195.
Pace, G.M., Volk, R.J., Jackson, W.A., 1990. Nitrate reduction in response to
CO2 -limited photosynthesis: relationship to carbohydrate supply and nitrate
reductase activity in maize seedlings. Plant Physiol. 92, 286–292.
Paradikovic, N., Vinkovic, T., Vrcek, I.V., Zuntar, I., Bojic, M., Medic-Saric, M., 2011.
Effect of natural biostimulants on yield and nutritional quality: an example of
sweet yellow pepper (Capsicum annuum L.) plants. J. Sci. Food Agric. 91,
2146–2152.
Park, E.J., Jeknic, Z., Chen, T.H.H., 2006. Exogenous application of glycinebetaine
increases chilling tolerance in tomato plants. Plant Cell Physiol. 47, 706–714.
Parrado, J., Escudero-Gilete, M.L., Friaza, V., Garcia-Martinez, A., González-Miret,
M.L., Bautista, J.D., Heredia, F.J., 2007. Enzymatic vegetable extract with
bioactive components: influence of fertilizer on the colour and anthocyanins of
red grapes. J. Sci. Food Agric. 87, 2310–2318.
Parrado, J., Bautista, J., Romero, E.J., Garcia-Martinez, A.M., Friaza, V., Tejada, M.,
2008. Production of a carob enzymatic extract: potential use as a biofertilizer.
Bioresour. Technol. 99, 2312–2318.
Pasupuleti, V.K., Braun, S., 2010. State of the art manufacturing of protein
hydrolysates. In: Pasupuleti, V.K., Demain, A.L. (Eds.), Protein Hydrolysates in
Biotechnology. Springer, Dordrecht Heidelberg, New York, NY, USA, pp. 11–32.
Pecha, J., Fürst, T., Kolomaznik, K., Friebrova, V., Svoboda, P., 2012. Protein
biostimulant foliar uptake modeling: the impact of climatic conditions. AIChE
J. 58, 2010–2019.
Perveen, S., Shahbaz, M., Ashraf, M., 2011. Modulation in activities of antioxidant
enzymes in salt stressed and non-stressed wheat (Triticum aestivum L.) plants
raised from seed treated with triacontanol. Pak J. Bot. 43, 2463–2468.
Petrozza, A., Santaniello, A., Summerer, S., Di Tommaso, G., Di Tommaso, D.,
Paparelli, E., 2014. Physiological responses to Megafol® treatments in tomato
plants under drought stress: a phenomic and molecular approach. Sci. Hortic.
174, 185–192.
Purwin, C., Fijałkowska, M., Pysera, B., Lipiński, K., Sienkiewicz, S., Piwczyński, D.,
Puzio, N., 2014. Nitrogen fractions and amino acid content in alfalfa and red
clover immediately after cutting and after wilting in the field. J. Elementol. 3,
723–734.
Quartieri, M., Cavani, L., Lucchi, A., Marangoni, B., Tagliavini, M., 2002. Effects of the
rate of protein hydrolysis spray concentration on growth of potted kiwifruit
(Actinidia deliciosa) plants. Acta Hortic. 594, 341–347.
Rentsch, D., Schmidt, S., Tegeder, M., 2007. Transporters for uptake and allocation
of organic nitrogen compounds in plants. FEBS Lett. 581, 2281–2289.
Rouphael, Y., Schwarz, D., Krumbein, A., Colla, G., 2010. Impact of grafting on
product quality of fruit vegetables. Sci. Hortic. 127, 172–179.
Rouphael, Y., Cardarelli, M., Bassal, A., Leonardi, C., Giuffrida, F., Colla, G., 2012.
Vegetable quality as affected by genetic, agronomic and environmental factors.
J. Food Agric. Environ. 10, 680–688.
Ruiz, J.M., Castilla, N., Romero, L., 2000. Nitrogen metabolism in pepper plants
applied with different bioregulators. J. Agric. Food Chem. 48, 2925–2929.
Ryan, C.A., Pearce, G., Scheer, J., Moura, D.S., 2002. Polypeptide hormones. Plant
Cell, Supplement S251–S264.
Schaafsma, G., 2009. Safety of protein hydrolysates, fractions thereof and bioactive
peptides in human nutrition. Eur. J. Clin. Nutr. 63, 1161–1168.
Schiavon, M., Ertani, A., Nardi, S., 2008. Effects of an alfaalfa protein hydrolysate on
the gene expression and activity of enzymes of TCA cycle and N metabolism in
Zea mays L. J. Agric. Food Chem. 56, 11800–11808.
Schiavon, M., Pizzeghello, D., Muscolo, A., Vaccaro, S., Francioso, O., Nardi, S., 2010.
High molecular size humic substances enhance phenylpropanoid metabolism
in maize (Zea mays L.). J. Chem. Ecol. 36, 662–669.
Searchinger, T., 2013. The Great Balancing Act: Installment 1 of Creating a
Sustainable Food Future. WRI, Washington, D.C.
Sharma, S.S., Dietz, K.J., 2009. The relationship between metal toxicity and cellular
redox imbalance. Trends Plant Sci. 14, 43–50.
Shetty, P., Atallah, M.T., Shetty, K., 2003. Stimulation of total phenolics, l-DOPA and
antioxidant activity through proline-linked pentose phosphate pathway in
response to proline and its analog in germinating fava beans (Vicia faba). Proc.
Biochem. 38, 1707–1717.
Shetty, K., Wahlqvist, M.L., 2004. A model for the role of the proline-linked
pentose-phosphate pathway in phenolic phytochemical bio-synthesis and
mechanism of action for human health and environmental applications. Asia
Pac. J. Clin. Nutr. 13, 1–24.
Slavin, J.L., Lloyd, B., 2012. Health benefits of fruits and vegetables. Adv. Nutr. 3,
506–516.
Soldal, T., Nissen, P., 1978. Multiphasic uptake of amino acids by barley roots.
Physiol. Plant. 43, 181–188.
Stiegler, J.C., Richardson, M.D., Karcher, D.E., Roberts, T.L., Norman, R.J., 2013. Foliar
absorption of various inorganic and organic nitrogen sources by creeping
bentgrass. Crop Sci. 52, 1148–1152.
Supattapone, S., Bosque, P., Muramoto, T., Wille, H., Aagaard, C., Reretz, D., Nguyen,
H.O., Heinrich, C., Torchia, C., Safar, J., Cohen, F.E., DeArmond, S.J., Prusiner, S.B.,
Scott, M., 1999. Prion protein of 106 residues creates an artificial transmission
barrier from prion replication in transgenic mice. Cell 96, 869–878.
Tegeder, M., 2012. Transporters for amino acids in plant cells: some functions and
many unknowns. Curr. Opin. Plant Biol. 15, 315–321.
Trouvelot, S., Héloir, M.C., Poinssot, B., Gauthier, A., Paris, F., Guillier, C., Combier,
M., Trdá, L., Daire, X., Adrian, M., 2014. Carbohydrates in plant immunity and
38 G. Colla et al. / Scientia Horticulturae 196 (2015) 28–38
plant protection: roles and potential application as foliar sprays. Front. Plant
Sci. 5 (592).
Tsouvaltzis, P., Koukounaras, A., Siomos, S.A., 2014. Application of amino acids
improves lettuce crop uniformity and inhibits nitrate accumulation induced by
the supplemental inorganic nitrogen fertilization. Int. J. Agric. Biol. 16,
951–955.
Tuteja, N., 2007. Mechanisms of high salinity tolerance in plants. Methods
Enzymol. 428, 419–438.
Visconti, F., de Paz, J.M., Bonet, L., Jordà, M., Quinones, A., Intrigliolo, D.S., 2015.
Effects of a commercial calcium protein hydrolysate on the salt tolerance of
Diospyros kaki L. cv. Rojo Brillante grafted on Diospyros lotus L. Sci. Hortic. 185,
129–138.
Viti, R., Bartolini, S., Vitagliano, C., 1990. Growth regulators on pollen germination
in olive. Acta Hortic 286, 227–230.
Wang, H., Wu, L., Tao, Q., 2004. Nitrate accumulation and variation of nutrient
quality in pakchoi after application of several amino acids in summer and
autumn. J. Agro-Environ. Sci. 23, 224–227 (Chinese).
Wang, H.J., Wu, L.H., Wang, M.Y., Zhu, Y.H., Tao, Q.N., Zhang, F.S., 2007. Effects of
amino acids replacing nitrate on growth, nitrate accumulation, and
macroelement concentrations in Pak-choi (Brassica chinensis L.). Pedosphere
17, 595–600.
Watson, R., Fowden, L., 1975. The uptake of phenylalanine and tyrosine by seedling
root tips. Phytochemistry 14, 1181–1186.
Wilson, A.R., Nzokou, P., Guney, D., Kulac, S., 2013. Growth response and nitrogen
use physiology of Fraser fir (Abies fraseri), red pine (Pinus resinosa), and hybrid
poplar under amino acid nutrition. New For. 44, 281–295.