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The Use of Biostimulants for Enhancing Nutrient Uptake
Article in Advances in Agronomy · December 2015

DOI: 10.1016/bs.agron.2014.10.001
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From Halpern, M., Bar-Tal, A., Ofek, M., Minz, D., Muller, T., Yermiyahu, U., 2015. The
Use of Biostimulants for Enhancing Nutrient Uptake. In: Sparks, D.L. (Ed.), Advances in
Agronomy, pp. 141–174.
ISBN: 9780128021378
© 2015 Elsevier Inc. All rights reserved.
Academic Press
CHAPTER TWO
The Use of Biostimulants for

Enhancing Nutrient Uptake
Moshe Halpern*, Asher Bar-Taly, 1, Maya Ofeky, Dror Minzy,
Torsten Mullerx and Uri Yermiyahu*
*Soil, Water and Environmental Sciences, Agricultural Research Organization, Gilat Research Center, Israel
y
Institute of Soil, Water and Environmental Sciences, ARO, Bet-Dagan, Israel
x
Institute of Crop Science, Fertilization and Soil Matter Dynamics, University of Hohenheim, Fruwirthstr.
Stuttgart, Germany
1
Corresponding author: E-mail: [email protected]
Contents
1. Introduction 143
2. Humic Substances 144
2.1 Introduction to HS 144
2.2 Characterization of HS 145
2.3 Effects of HS on Plant Growth 145
2.3.1 Effects of HS on Nutrient Uptake 145
2.4 Mechanisms by Which HS Affect Nutrient Uptake 147
2.4.1 HS Improve Soil Structure 147
2.4.2 HS Improve Solubility of Micronutrients and P 148
2.4.3 HS Change Root Morphology 148
2.4.4 HS Stimulate HþATPase and NO3-Assimilation Enzymes 149
3. Amino Acids 149
3.1 Introduction to AA 149
3.2 Absorption of AA by Plants 150
3.3 Effects of AA on Plants 150
3.3.1 Effects of AA on Nutrient Uptake 151
3.4 Mechanisms by Which AA Affect Nutrient Uptake 151
3.4.1 AA Increase Soil Microbial Activity 151
3.4.2 AA Chelate Micronutrients 151
3.4.3 AA Reduce Micronutrients 153
3.4.4 AA Improve Internal Translocation of Micronutrients 153
3.4.5 AA Affect Root Morphology 153
3.4.6 AA Stimulate NO3-Assimilation Enzymes 153
4. Seaweed Extract 154
4.1 Introduction to SE 154
4.2 Characterization of SE 155
4.3 Effects of SE on Plants 155
4.3.1 Effects of SE on Nutrient Uptake 155
4.4 Mechanisms by Which SE Affects Nutrient Uptake 157
Advances in Agronomy, Volume 130
© 2015 Elsevier Inc.
j
ISSN 0065-2113
http://dx.doi.org/10.1016/bs.agron.2014.10.001 All rights reserved. 141
Advances in Agronomy, First Edition, 2015, 141–174

142 Moshe Halpern et al.
4.4.1 SE Improves Soil Structure 157

4.4.2 SE Improves Micronutrient Solubility 157
4.4.3 SE Affects Root-to-Shoot Ratio 158
4.4.4 SE Promote Symbiotic Relationship Between Mycorrhizal Fungi and Roots 158
5. Plant-Growth-Promoting Bacteria 158
5.1 Introduction to PGPB 158
5.2 Mechanisms by Which PGPB Affect Plant Nutrition 160
5.2.1 Some PGPB Fix N 160
5.2.2 Some PGPB Solubilize P 160
5.2.3 Some PGPB Solubilize Fe 161
5.2.4 Some PGPB Induce Changes in Root Morphology 162
5.2.5 Some PGPB Promote a Symbiotic Relationship between Mycorrhizal Fungi 162
and Roots
6. Conclusions and Future Directions 163
Acknowledgments 167
References 167
Abstract
Fertilizer use in modern agriculture is highly inefficient; much of the applied fertilizer is
released into the environment, causing environmental degradation. One way in which
fertilizer use can be reduced without damaging plant nutrition is to enhance crop up-
take of nutrients through the use of biostimulants. A broad definition of plant bio-
stimulants, including substances sometimes categorized as biofertilizers or
biopesticides, is used throughout this review: “Plant biostimulants are substances or
materials, with the exception of nutrients and pesticides, which, when applied to plants,
seeds, or growing substrates in specific formulations, have the capacity to modify phys-
iological processes in plants in a way that provides potential benefits to growth, devel-
opment, or stress response.” This definition includes a variety of substances, four of
which will be reviewed in this article: seaweed extract, humic substances, amino acids,
and plant-growth-promoting bacteria. We will concentrate on the positive effects of
biostimulant application on plant nutrient uptake, and the underlying mechanisms,
which include positive changes in soil structure or nutrient solubility, root morphology,
plant physiology, and symbiotic relationships, will be discussed. Recommendations for
future research directions include finding the most promising substances, isolating the
active ingredients and clearly demonstrating the mechanisms by which they affect
nutrient uptake. The beneficial effects and mechanisms must be consistently demon-
strated in greenhouse and field experiments.
Abbreviations
AA Amino acids or protein hydrolysates
AspAT Aspartate aminotransferase
EDDHA Ethylenediamine-N,N0 -bis(2-hydroxyphenylacetic acid)
EDTA Ethylenediaminetetraacetic acid
DTPA Diethylene triamine pentaacetic acid

Biostimulants for Nutrient Uptake 143
GDH Glutamate dehydrogenase

GOGAT Glutamate synthase
GS Glutamate synthetase
HS Humic substances
MDH Malic dehydrogenase
NiR Nitrite reductase
NR Nitrate reductase
PGPB Plant-growth-promoting bacteria
PM HDATPase Plasma membrane HþATPase
SE Seaweed extract
1. INTRODUCTION
The application of chemical fertilizers is an inexpensive and effective
method of supplying crops with mineral nutrients (Chen, 2006). However,
fertilizers are often washed from the field in the runoff (Daverede et al.,
2004; Moe et al., 1967) or can become unavailable to the crops through
chemical, physical, or biological transformation (Sanchez et al., 2001;
Schachtman et al., 1998). To compensate for these processes, farmers
need to apply more chemical fertilizer than the plant actually needs, and
the remainder is often released into the environment, polluting the air
and water (Vance, 2001). Furthermore, the industrial production of chem-
ical fertilizers is an energy-intensive process that is known to significantly
contribute to global CO2 emissions (Vance, 2001).
Organic fertilizers, such as compost, sludge, or manure, have the advantage
of making use of nutrients that are already available in the agro-ecosystem, and
they require little input of energy to be processed. Furthermore, the mineral
nutrients that are bound in organic materials may be more stable, and
may therefore be washed away or released into the atmosphere less quickly
(Estavillo et al., 1994; King and Torbert, 2007). However, organic fertilizers
have the disadvantage of not supplying crops with nutrients in an easily
absorbable, water-soluble form when the crops need them (Chen, 2006).
One method by which it is possible overcome this disadvantage is to
grow crops with more robust root systems and higher nutrient-uptake
efficiency, to ensure that they receive the nutrients when they need them
despite their lower immediate availability when they are introduced in
organic form. Alternatively, nutrients can be made more available by pro-
moting certain types of organisms within the soil microbial community
(Vessey, 2003). Both of these approaches can be achieved by introducing
biostimulants to crop leaves, seeds, or soil as a means of stimulating root
growth (Canellas et al., 2002; Khan et al., 2009; Zandonadi et al., 2007),

efficient root uptake (Pinton et al., 1999a), and beneficial microbial popu-
lations (Chen, 2006; Vessey, 2003).
The term biostimulant is relatively new and its use in the scientific
community is still nebulous (du Jardin, 2012). One broad definition was
introduced by du Jardin (2012): “Plant bio-stimulants are substances or ma-
terials, with the exception of nutrients and pesticides, which, when applied to
plants, seeds or growing substrates in specific formulations, have the capacity
to modify physiological processes in plants in a way that provides potential
benefits to growth, development, or stress response.” Other workers differ-
entiate between biostimulants, biofertilizers, and biopesticides by their direct
hormonal effects (biostimulants) (Subler et al., 1998), indirect effects on
nutrient availability (biofertilizers) (Orhan et al., 2006), and enhanced control
of pathogens or pests (biopesticides) (Copping and Menn, 2000). Since many
different mechanisms have been proposed for the plant’s positive reactions to
the different substances discussed in this review, each can be called a bio-
stimulant or biofertilizer. Therefore, we will employ the broader definition
of biostimulants proposed by du Jardin (2012). Although this definition in-
cludes substances that help the plant overcome biotic or abiotic stresses,
in this review, we will only be considering biostimulants that have positive
effects on root growth, root efficiency, or nutrient uptake, traits that are use-
ful in shifting from a chemical to organic fertilization regime.
Four major groups of biostimulants have been shown to affect root growth
and nutrient uptake: (1) humic substances (HS), (2) protein hydrolysate and
amino acid formulations (AA), (3) seaweed extract (SE), and (4) plant-
growth-promoting microorganisms. Although mycorrhizal fungi are included
in group 4, they are not treated in this review because of the many articles and
reviews that have already been written on the topic (see for example: Bolan,
1991; Cavagnaro, 2008; Marschner and Dell, 1994). Thus only plant-growth-
promoting bacteria (PGPB) will be discussed from this group.
2. HUMIC SUBSTANCES
2.1 Introduction to HS
HS are heterogeneous organic molecules that form in the soil as
byproducts of microbial metabolism of dead organic matter (Nardi et al.,
2007). HS are one of the most common organic substances on Earth
(Sutton and Sposito, 2005), and make up 60% of the organic matter in
the world’s soils (Muscolo et al., 2007). In the past, HS were thought to
be large linked polymers of organic molecules. However, the emerging

consensus is that HS are made up of many small organic molecules that are
held together by hydrophobic interactions and hydrogen bonds (Piccolo,
2002; Simpson et al., 2002; Sutton and Sposito, 2005).
HS can be extracted from many different sources, including soils (Nardi
et al., 2000; Varanini et al., 1993; Zandonadi et al., 2007), municipal waste
(Ayuso et al., 1996), vermicomposts and earthworm casts (Canellas et al.,
2002; Russell et al., 2006), various coal deposits (Kulikova and Perminova,
2002), peat (Ayuso et al., 1996; Schmidt et al., 2007), and Leonardite
(Nikbakht et al., 2008).
HS can be applied to the plant in a number of ways, including foliar
applications (Katkat et al., 2009; Yildirim, 2007), in the irrigation water
(Salman et al., 2005), and direct application to the soil (Katkat et al., 2009).
2.2 Characterization of HS
HS are often divided into fractions according to their molecular weight
(Nardi et al., 2007; Quaggiotti et al., 2004; Russell et al., 2006; Varanini
et al., 1993). The lower molecular weight fractions tend to have greater pos-
itive biological effects on plants (Piccolo et al., 1992; Varanini et al., 1993),
but this is not always the case (Muscolo et al., 2007; Nardi et al., 2007).
Furthermore, the true molecular weight is not easy to determine, since
HS are thought to be made up of many different-sized molecules that
interact with one another on a supramolecular scale (Piccolo, 2002; Simpson
et al., 2002; Sutton and Sposito, 2005).
A number of different methods have been used to characterize HS,
including three spectroscopic ones: DRIFT (diffuse reflectance infrared
Fourier transform), 1H-NMR (H1 nuclear magnetic resonance) (Muscolo
et al., 2007), and 13C-NMR (Canellas et al., 2010). In recent studies, the
specific chemical structure of the HS rather than the sizes of their molecules
have been shown to affect the biological activity of the HS (Canellas et al.,
2010; Muscolo et al., 2007).
2.3 Effects of HS on Plant Growth
HS have a number of positive effects on plant growth, including increased
biomass (Ayuso et al., 1996; Lee and Bartlett, 1976), increased number
of fruits and flowers (Arancon et al., 2006), and improved fruit quality
(Yildirim, 2007).
2.3.1 Effects of HS on Nutrient Uptake
HS have a positive effect on nutrient uptake (see Table 1). There have been
a number of studies showing that HS increase NO3 uptake (Albuzio et al.,

146
Table 1 Examples of positive effects of humic substance (HS) application on plant nutrition
Nutrients positively
Plant affected by HS Parent material of HS References
Barley NO3 Soil Albuzio et al. (1986)

Barley NO3 Coal Piccolo et al. (1992)
Barley NO3 Soil Nardi et al. (2000)
Maize NO3 Earthworm feces Quaggiotti et al. (2004)
Barley N, P, Mn, Cu, Zn, Fe Sewage sludge, compost, leonardite, and peat Ayuso et al. (1996)
Maize P, Fe Cow manure, compost, peat, and soil Lee and Bartlett (1976)
Maize N, Zn Soil Tan and Nopamornbodi (1979)
Melon, soybean, Zn, Fe Peat and leonardite Chen et al. (2004)
and rye grass
Maize Cu, Zn, Mn Leonardite Çelik et al. (2011)
Tomato Fe Lignite (brown coal) Sanchez-Sanchez et al. (2005)
Grape Fe Not specified Sanchez-Sanchez et al. (2006)
Moshe Halpern et al.

1986; Nardi et al., 2000; Piccolo et al., 1992; Quaggiotti et al., 2004). These
studies were relatively short term, testing NO3 uptake, gene transcription,
and activities of the proteins involved in NO3 uptake and assimilation
over the course of 16–48 h in very young plants. Tan and Nopamornbodi
(1979) measured the effects of HS derived from soil on the nutrient uptake
of maize plants over the course of a growing season. Whereas N and Zn
uptake were improved at certain doses, P uptake was negatively affected
irrespective of the HS dose, while the uptake rates of other minerals were
not significantly affected. Ayuso et al. (1996) showed that HS from a number
of different parent materials can improve the uptake of total N as well as
other nutrients, such as P, Mn, Cu, Zn, and Fe in barley over the course
of an entire growing season. Plant acquisition of each of these nutrients
was affected differently by different HS doses; some doses affected certain
minerals positively and others negatively. Lee and Bartlett (1976) found
that HS greatly improve P and Fe uptake in maize when they are applied
to soils with little organic material. When applied to soils with high concen-
trations of organic material, the positive effects were small or nonexistent,
probably because the background levels of HS were already high in those
soils. Some studies found positive effects of HS on micronutrient uptake,
specifically in alkaline soils or alkaline nutrient solutions where micronu-
trients are often limiting (Çelik et al., 2011; Chen et al., 2004; Sanchez-
Sanchez et al., 2005, 2006).
2.4 Mechanisms by Which HS Affect Nutrient Uptake

HS improve plant nutrition by affecting soil processes and by directly
affecting the plant’s physiology. The mechanisms that affect the soil pro-
cesses include: (1) improvement of the soil structure, (2) improvement of
micronutrient solubility in the soil. Direct effects on the plant’s physiology
include: (3) changes in root morphology, (4) an increase in root activity of
HþATPase, and (5) an increase in the activity of NO3-assimilation enzymes.
2.4.1 HS Improve Soil Structure

HS improve plant nutrition by improving the soil structure. Piccolo et al.
(1997) found that amending the soil with HS increases aggregate stability.
They attributed this phenomenon to the HS0 ability to form clay–humic
complexes with hydrophilic components oriented toward the center of
the aggregate and hydrophobic components facing outward. This reduces
water infiltration into the aggregates, making them more stable in wetting
and drying cycles. Improved aggregate stability leads to improved soil

aeration, facilitated root penetration, greater water availability to the plant,

and less soil erosion, which indirectly contribute to enhanced nutrient up-
take (Amezketa, 1999; Bronick and Lal, 2005). However, improved aggre-
gate stability does not explain the observed improvement in plant nutrition
in hydroponic systems (Chen et al., 2004), or when the HS are applied to the
foliage rather than the soil (Katkat et al., 2009).
2.4.2 HS Improve Solubility of Micronutrients and P

Under some circumstances, micronutrients and P are highly insoluble. HS
added to the nutrient solution enhance Fe and Zn solubility by forming
metal–humic complexes (Chen et al., 2004). The Fe–humic complexes
are available to plants, regardless of whether they use strategy I (dicots and
nongraminaceous monocots) or strategy II (graminaceous monocots) for
Fe mobilization (Cesco et al., 2000; Chen et al., 2004; Pinton et al.,
1999b). In fact, the increased growth observed in plants treated with HS
may be attributed to increased Fe availability (Chen et al., 2004; Pinton
et al., 1999b). Application of the water-soluble fraction of HS increased
the solubility of Fe-hydroxides, as well as their mobility in the soil (Cesco
et al., 2000). HS have been shown to be effective replacements for artificial
chelates of Fe such as ethylenediamine-N,N0 -bis(2-hydroxyphenylacetic
acid) (EDDHA) in tomato, lemon trees, and grapevines grown in calcareous
soils (Sanchez-Sanchez et al., 2002, 2005, 2006). HS also increase the activ-
ity of plasma membrane (PM) HþATPase (Pinton et al., 1999a), which
could lead to rhizosphere acidification and hence to increased solubility of
micronutrients. HS increase the availability of P by interfering with the for-
mation of nonsoluble Ca-phosphates (Delgado et al., 2002). This explains
the increased efficiency of P use when soluble phosphate fertilizers are
applied to soils that have been amended with organic materials (Delgado
et al., 2002).
2.4.3 HS Change Root Morphology

Malik and Azam (1985) showed greater root development of wheat seed-
lings grown in distilled water supplemented with HS versus distilled water
alone. Canellas et al. (2002) and Zandonadi et al. (2007) showed that HS
derived from earthworm compost increase lateral-root proliferation and
elongation in maize. They both attributed this effect to the auxin-like activ-
ity of HS, which stimulates PM HþATPase, thereby stimulating cellular
growth. Schmidt et al. (2007) showed that water-soluble HS derived from
peat cause an increase in root-hair density in Arabidopsis, but they ruled

out the involvement of auxin-like activity by showing that HS cannot

save an auxin-deficient mutant. An increase in lateral-root and root-hair
development increases the surface area of the root, which would explain
the increased nutrient uptake induced by HS.
2.4.4 HS Stimulate HþATPase and NO3-Assimilation Enzymes

As already mentioned, HS are known to stimulate PM HþATPase (Canellas
et al., 2008; Nardi et al., 2000; Pinton et al., 1999a; Quaggiotti et al., 2004).
Zandonadi et al. (2010) showed that this stimulation involves auxin-like
activity of the HS and nitric oxide (NO) signaling, by demonstrating that
auxin inhibitors and NO-scavenging molecules inhibit PM HþATPase
stimulation by HS. By stimulating the PM HþATPase, HS acidify the rhizo-
sphere, causing the NO3 Hþ symport system to work more effectively. The
cumulative result is that the plant absorbs more NO3 (Pinton et al., 1999a;
Quaggiotti et al., 2004; Zandonadi et al., 2010).
HS also increase the rate of NO3 assimilation by causing the plant to
upregulate the enzymes involved in this process. Albuzio et al. (1986) found
that barley plants incubated with HS derived from soil and fractionated in
various ways had increased nitrate reductase (NR), glutamate dehydrogenase
(GDH), and glutamate synthetase (GS) activity. While some fractions had
more of a stimulatory effect than others, the effect did not correlate well
with fraction size. Muscolo et al. (1999) found an increase in GDH, GS,
and malic dehydrogenase (MDH) activity in carrot cells treated with HS
derived from worm castings. They attributed this stimulatory effect to the
auxin-like substances found in the castings. Vaccaro et al. (2009) found
that the hydrophilic and least structurally complex HS derived from
compost have a stimulatory effect on many NO3-assimilation enzymes,
including NR, nitrite reductase (NiR), GS, glutamate synthase (GOGAT),
and aspartate aminotransferase (AspAT).
3. AMINO ACIDS
3.1 Introduction to AA
AA are a large family of biological compounds that contain an amine
functional group and a carboxylic acid functional group. There are only 20
AA involved in protein building, but there are 250 more that are known to
have diverse functions in plants, including protection from biotic and abiotic
stresses, signaling, N storage, and chelation of metals as phytosiderophores
(Vranova et al., 2011). Commercially available AA biostimulants are mostly

mixtures of different AA and short peptides, rather than pure substances (du
Jardin, 2012). These mixtures, called protein hydrolysates, are derived from
the hydrolysis of proteins from plant (Schiavon et al., 2008), animal (Maini,
2006) and microbial (du Jardin, 2012) sources, often from industrial and agri-
cultural waste products such as crop residues (du Jardin, 2012), animal skin
(Vasileva-Tonkova et al., 2007), feathers (Grazziotin et al., 2007; Jie et al.,
2008), and blood (Polo et al., 2006). Protein hydrolysates are marketed as
plant biostimulants that can be applied as a foliar spray, soil drench, or
seed treatment (du Jardin, 2012; Maini, 2006). The scientific literature dis-
cusses both pure AA (Ghasemi et al., 2012; Rodríguez-Lucena et al., 2010;
Yuan et al., 2013; Zhou et al., 2007) and protein hydrolysates (Ertani et al.,
2009; García-Martínez et al., 2010; Maini, 2006; Schiavon et al., 2008). In
this review we will refer to both protein hydrolysates and pure AA as “AA,”
and specify the name of the pure AA when it is relevant.
3.2 Absorption of AA by Plants

Plants can absorb AA directly into the roots when they are dissolved in the
mass flow of water into the xylem (Biernath et al., 2008), through specific
transporters in the roots (N€asholm et al., 2009) or via diffusion into the leaves
(Kolomaznik et al., 2012; Pecha et al., 2011). Plants can utilize AA as a source
of N, and under some circumstances, in certain plants, AA are the main
source of N (Schimel and Chapin, 1996). Strictly speaking, when AA are
used as a source of N, they do not fit du Jardin’s, 2012 definition of bio-
stimulant, which specifies that nutrients are not considered biostimulants.
However, the dose at which AA are usually applied as biostimulants is so
low that their positive effects cannot be attributed to the increase in N avail-
ability. For example, Schiavon et al. (2008) applied 0.1 and 0.01 ppm AA
which contained 2.29% N (a total of 0.016–0.16 mmol L1N) to a nutrient
solution with 600 mmol L1 N, and Zhou et al. (2007) applied between 10
and 100 mmol L1 pure AA to a nutrient solution with 5000 mmol L1 N.
Furthermore, mechanisms other than improved N nutrition have been
implicated in the beneficial effects of AA application (Ghasemi et al.,
2012; Maini, 2006; Schiavon et al., 2008; Zhou et al., 2007).
3.3 Effects of AA on Plants

AA application has been shown to increase biomass production (Shehata
et al., 2011), help protect plants against biotic (Cohen and Gisi, 1994) and
abiotic (Maini, 2006; Polo et al., 2006) stresses, and increase the antioxidant
content of the leaves (Ardebili et al., 2012).

3.3.1 Effects of AA on Nutrient Uptake

Application of exogenous AA to plant leaves and roots has been shown to
increase nutrient uptake and nutrient-use efficiency for both macro- and
micronutrients (see Table 2). A commercial mixture of AA was shown to
increase corn yield, even when the N-fertilization rate was cut in half
(Maini, 2006). Ca can also be better utilized by the plant when it is applied
together with AA, and mixtures of AA and Ca are used to reduce Ca defi-
ciency in apples and tomatoes (Maini, 2006). However, Otero et al. (2006)
found that a mixture of AA and Ca was not effective in kiwifruit and rec-
ommended finding other methods of Ca fertilization. AA were also shown
to increase the efficiency of foliar-applied micronutrients, and mixtures of
FeSO4 and AA applied as a foliar spray were shown to be effective against
chlorosis in grapevine (Maini, 2006).
3.4 Mechanisms by Which AA Affect Nutrient Uptake

AA application can improve plant nutrition by affecting soil processes and by
affecting the plant’s physiology directly. The mechanisms affecting the soil
processes include: (1) promotion of beneficial microbial communities and
nutrient mineralization in the soil, (2) improvement of micronutrient solu-
bility in the soil through chelation and reduction of micronutrients. The
mechanisms that affect the plant’s physiology directly include: (3) improve-
ment of micronutrient mobility in the plant, (4) changes in root
morphology, (5) increased activity of NO3-assimilation enzymes.
3.4.1 AA Increase Soil Microbial Activity

AA application to the soil increases soil microbial activity, which can
improve the soil’s physical and chemical attributes (Garcia-Martinez et al.,
2010). Specifically, the increased bioactivity in the soil causes a quicker
breakdown of organic matter, which transforms organic nutrients into
plant-available mineral forms (Garcia-Martinez et al., 2010).
3.4.2 AA Chelate Micronutrients

AA can chelate metals such as Fe, Zn, Mn, Cu, making them more readily
absorbable through the roots and leaves via specific transporters, such as
lysine histidine transporter 1 (LHT1), amino acid permease 1 (AAP1) and
AAP5 (Ghasemi et al., 2012; Jie et al., 2008). In nature, plants often secrete
specific nonprotein AAs known as phytosiderophores from their roots into
the soil to improve micronutrient availability (Dakora and Phillips, 2002;
Kinnersley, 1993). For this reason, many micronutrient foliar sprays (for

152
Table 2 Examples of positive effects of amino acid (AA) application on plant nutrition
Plant affected by AA Parent material or specific AA References
Maize N Animal epithelial tissue Maini (2006)

Maize NO3 Alfalfa protein hydrolysate and meat flour Ertani et al. (2009)
(hydrolysate)
Maize NO3 Alfalfa protein hydrolysate Schiavon et al. (2008)
Tomato Fe, Zn, N Histidine, glycine, and arginine Ghasemi et al. (2012)
Rice Fe, Zn, Cu, Mn Chicken feather hydrolysate Jie et al. (2008)
Pear Fe, Zn Commercial mixture of amino acids Koksal et al. (1999)
Soybean Fe 3 commercial mixtures with the main components Rodríguez-Lucena et al. (2010)
glycine, glycine þ glutamate, and glycine þ
arginine. These mixtures also contained 3e15%
polypeptides
Maize Cu Cysteine Zhou et al. (2007)
Rice Fe, Zn Nicotianamine and other unspecified amino acids Yuan et al. (2013)

example, Koksal et al., 1999; Rodríguez-Lucena et al., 2010) and hydro-

ponic solutions (for example, Ghasemi et al., 2012) contain AA mixtures.
3.4.3 AA Reduce Micronutrients

Specific AAs may also increase the availability of micronutrients by acting as
a reductant. Zhou et al. (2007) showed that exogenous application of
cysteine to maize roots in a hydroponic solution causes an increase in Cu up-
take. They hypothesized that the cysteine acts as a reductant, changing Cu II
to Cu I, which may be more available to the roots.
3.4.4 AA Improve Internal Translocation of Micronutrients

AA chelates are also important for the translocation of micronutrients within
the plant. There have been a large number of studies showing that nicotian-
amine, a nonprotein AA, is responsible for the translocation of micronu-
trients in the phloem (Curie et al., 2009; Schmidke and Stephan, 1995;
Stephan et al., 1994). Nicotianamine has also been shown to have positive
effects on plant physiological processes, even when applied exogenously,
and it can therefore be considered a biostimulant. Specifically, exogenous
application of nicotianamine increased the translocation of Zn and Fe to
the grains of rice plants, which has important implications for human nutri-
tion (Yuan et al., 2013).
3.4.5 AA Affect Root Morphology

Exogenous application of AA has also been shown to have an effect on root
morphology. Specifically, L-glutamate application to the root inhibited
primary-root growth and stimulated root branching (Walch-Lui et al.,
2006). It also stimulated root-hair development close to the root tip
(Walch-Lui et al., 2006). This effect was specific to L-glutamate, and did
not occur in response to applications of 21 other AA, including D-glutamate
(Walch-Lui et al., 2006). Tryptophan was shown to have a different stimu-
latory effect on root growth, which the authors attributed to the fact that this
AA is a precursor for auxin production (Walch-Lui et al., 2006).
3.4.6 AA Stimulate NO3-Assimilation Enzymes

AA have also been shown to stimulate the NO3-assimilation enzymes
through hormonal action. Maini (2006) reviewed a number of studies that
showed that a commercial AA foliar spray increases NR activity. Schiavon
et al. (2008) conducted an in-depth study showing that AA derived from
a hydrolysate of alfalfa proteins applied at a rate of 0.1 mg L1 have a signif-
icant impact on the enzymes associated with NO3 assimilation in Zea mays

plants grown in full Hoagland solution. The enzymes measured in the root
and leaves included NR, NiR, GS, and GOGAT. The AA treatment led to
a statistically significant increase in all of the NO3-assimilation enzymes
measured, in both the root and the shoot. The overall N concentration
was unchanged by the treatment, but the NO3 concentration decreased,
indicating that the treatment caused the plants to assimilate the NO3
more rapidly. Enzymes involved in C metabolism were also positively
affected, indicating that C and N metabolism are regulated together, helping
the plant regulate the C:N balance. Ertani et al. (2009) studied the effects of
AA mixtures from protein hydrolysates of alfalfa or meat meal on NR and
GS; the AA mixtures had stimulatory effects, regardless of the origin of the
AA. They also measured NO3 in the roots and shoots, and found that its
concentration is significantly decreased by the application of protein hydro-
lysate compared to controls. This indicated that NO3 assimilation is stimu-
lated by the treatments (Ertani et al., 2009). Both Schiavon et al. (2008) and
Ertani et al. (2009) attributed the observed effects on the NO3-assimilation
enzymes to the auxin-like and gibberellin-like activities of the protein hy-
drolysates. In both studies, auxin-like and gibberellin-like activities were
confirmed using bioassays for the plant-based (Schiavon et al., 2008) and
animal-based (Ertani et al., 2009) protein hydrolysates.
4. SEAWEED EXTRACT
4.1 Introduction to SE
Seaweed has been applied as a fertilizer in coastal regions for centuries
(Craigie, 2011). The first method for liquefying seaweed for agricultural use
was patented in 1912 (Booth, 1969). Liquefied SE is usually manufactured
from Ascophyllum nodosum, a brown seaweed that is commonly found in
the North Atlantic, although other species, such as Durvillaea antarctica, Dur-
villaea potatorum, Macrocystis pyrifera, and Ecklonia maxima are also used (Khan
et al., 2009). The methods for liquefying seaweed are often proprietary, but
they mostly involve physical disruption of the seaweed and/or its chemical
digestion using alkalis or acids (Craigie, 2011). SE also contains some plant
nutrients, and when applied to plants grown in a nutrient-deficient medium,
the simple availability of these nutrients may improve growth and nutrient
uptake. For example, Becket and van Staden (1989) observed a positive ef-
fect on K uptake, even when the SE was “ashed” (heated at 450 C for 1 h).
The authors surmised that this effect was simply caused by the K in the SE,
which was present at a rate of 18 mg L1 of a root drench applied three times

during the growing season. Strictly speaking, when SE is used as a source

of K, it does not fit du Jardin’s (2012) definition of biostimulants, which
specifically excludes nutrients.
4.2 Characterization of SE
SE is a heterogeneous substance that can be characterized by its parent
material (Khan et al., 2009), the pH of the extraction solution (Booth,
1969), or 1H-NMR spectroscopy (Craigie et al., 2008). SE have been
shown to contain plant hormones such as auxins, cytokinins, and abscisic
acid (Khan et al., 2009). SE also contain AA (Khan et al., 2009), whose ef-
fects on plant nutrition were discussed in Section 3.
4.3 Effects of SE on Plants

SE have been shown to increase plant growth (van Staden et al., 1994), chlo-
rophyll levels (Blunden et al., 1997), flowering and yield (Crouch et al.,
1990; Eris et al., 1995), and seed germination (Demir et al., 2006; Kumar
and Sahoo, 2011). They increase the success of in vitro propagation (Vinoth
et al., 2012) and enhance plant protection against pathogens and pests
(Hankins and Hockey, 1990; Jayaraman et al., 2011; Khan et al., 2009;
Klarzynski et al., 2003; Loureiro et al., 2012; Mercier et al., 2001).
4.3.1 Effects of SE on Nutrient Uptake

SE has been shown to have a positive impact on plant nutrient uptake (see
Table 3). Turan and K€ ose (2004) tested the effects of foliar application of
three different commercial SE on nutrient uptake in a 1-year-old grapevine
planted in perlite medium. When the vines were grown with a supply of
mineral nutrients at optimal or high levels, all three SE induced significant
improvement in macronutrient and micronutrient concentrations in the
leaves, but no effect was seen when plants were grown at suboptimal mineral
nutrition. Interestingly, plant acquisition of Cu was shown to be more
strongly affected by the application of SE than by the concentration of
Cu in the growth medium.
Rathore et al. (2009) found that foliar application of SE obtained from
the red alga Kappaphycus alvarezii increases the grain concentration of N, P,
K, and S by up to 36%, 61%, 49% and 93%, respectively in soybeans grown
under rainfed conditions. The nutrient concentration in the grain showed a
clear correlation with the SE dosage, with the highest concentration found
in plants receiving the highest dosage. The nutrient levels in the straw
were affected either positively or negatively, depending on the SE dosage.

156
Table 3 Examples of positive effects of seaweed extract (SE) application on plant nutrition
Plant affected by SE Seaweed type References
Grapevine N, P, K, Ca, Mg, Fe, Ascophyllum nodosum (Maxicrop) and other commercial Turan and K€
ose (2004)
Zn, Mn, Cu extracts of unknown source (Proton and Algipower)
Soybean N, P, K, S Kappaphycus alvarezii Rathore et al. (2009)
Wheat K Ecklonia maxima (Kelpak) Beckett and van Staden (1989)
Lettuce Ca, K, Mg Ecklonia maxima (Kelpak) Crouch et al. (1990)
Cucumber P Ecklonia maxima (Kelpak) Nelson and van Staden (1984)
Strawberry Fe Ascophyllum nodosum (Actiwave) Spinelli et al. (2010)

Crouch et al. (1990) measured the effects of a commercial SE on growth,

as well as Ca, Mg, and K uptake. They found that the growth of nutrient-
stressed lettuce was not improved by root flushing with SE, but that nutrient
uptake and plant growth were positively affected when the SE was applied
to lettuce plants that were also receiving a highly concentrated nutrient
solution. Nelson and van Staden (1984) applied the same commercial SE
to cucumber plants grown in pine bark medium. The SE was applied at a
low dosage as a root drench and as a foliar treatment. The seaweed treat-
ments caused the roots to grow significantly larger. They measured the N
and P concentration in the leaves, and found that the weekly spray of SE
increased the foliar P concentration by 20%, and that the root drench caused
a statistically insignificant increase of 5% in foliar P. The root drench and the
foliar spray caused statistically significant decreases in leaf N concentration
(15% and 18%, respectively). The authors attributed the drop in foliar
N to a large N sink in the roots caused by the SE treatments, but they
did not measure the root N to confirm this hypothesis.
4.4 Mechanisms by Which SE Affects Nutrient Uptake

SE improves plant nutrition by affecting soil processes and by affecting the
plant’s physiology directly. The mechanisms that affect soil processes
include: (1) improvement of soil structure, (2) improvement of micronu-
trient solubility in the soil. The mechanisms that affect the plant’s physiology
directly include: (3) changes in root morphology, and (4) increased root
colonization by arbuscular mycorrhizal fungi.
4.4.1 SE Improves Soil Structure

SE supports plant nutrition by improving the soil structure. Brown seaweed
contains large amounts of polysaccharides such as alginates and fucoidans,
which bond with the metallic ions in the soil to produce a gel that helps
hold water and maintain an aggregate structure (Khan et al., 2009). This
helps the plant grow a robust root system, which in turn can increase
nutrient uptake. However, improved soil structure does not explain
the observed improvement in plant nutrition in hydroponic systems (e.g.,
Vernieri et al., 2006), nor the observed improvement when SE is applied
to the foliage rather than the soil (e.g., Rathore et al., 2009).
4.4.2 SE Improves Micronutrient Solubility

There have not been many studies on the chelating effects of SE on micro-
nutrients, although it is possible that the large organic molecules in the SE

can chelate micronutrients and render them more available. One recent
study involving SE and the solubility of micronutrients showed that a com-
mercial extract of A. nodosum can be used to replace sequestrene, a standard
iron chelate (Spinelli et al., 2010).
4.4.3 SE Affects Root-to-Shoot Ratio

Application of SE to either leaves or roots has been shown to lead to an in-
crease in root mass or root-to-shoot ratio in various different crops, such as
cucumber (Nelson and van Staden, 1984), barley (Steveni et al., 1992),
rocket (Vernieri et al., 2006), tomatoes (Crouch and van Staden, 1992;
Featonby-Smith and van Staden, 1983) and strawberries (Spinelli et al.,
2010), and in a mung bean bioassay (Stirk and van Staden, 1997). This effect
is believed to be a result of auxins (Crouch and van Staden, 1992; Khan
et al., 2009) or cytokinins (Steveni et al., 1992) in the SE. A larger, more
robust root system contributes to nutrient uptake (Khan et al., 2009).
4.4.4 SE Promote Symbiotic Relationship Between Mycorrhizal Fungi

and Roots
Arbuscular mycorrhizal fungi are associated with the roots of most terrestrial
plants, and they play an important role in nutrient uptake. SE has been
shown to encourage arbuscular mycorrhizal fungal growth and infection
rates (Khan et al., 2009). Kuwada et al. (2006) found that extracts from
the green and red algae Gracilaria verrucosa, Gelidium amansii, Eucheuma cotto-
nii, and Chlorella pyrenoidosa significantly stimulate the growth of the arbus-
cular mycorrhizal fungi Gigaspora margarita and Glomus caledonium both in
vitro (on petri dishes) and in vivo (on passionfruit cuttings and papaya
seedlings).
5. PLANT-GROWTH-PROMOTING BACTERIA
5.1 Introduction to PGPB
PGPB are found in the bulk soil or rhizosphere and promote plant
growth under some conditions (Bashan and de Bashan, 2005). PGPB belong
to diverse genera and promote plant growth in various different ways (see
Table 4 for examples). PGPB have been shown to have a number of positive
effects on plant growth, including pathogen control (Bashan and de Bashan,
2005), increased salt tolerance (Alavi et al., 2013), increased resistance to
heavy metals and other toxins (Lucy et al., 2004), increased growth and yield
(Alam et al., 2011; Lucy et al., 2004), and enhanced plant nutrition

Table 4 Examples of positive effects of plant-growth-promoting bacteria (PGPB) application on plant nutrition
Biostimulants for Nutrient Uptake

Crop PGPB affected by PGPB Main mechanisms References
Fingermillet, maize, Bacillus sp. P P solubilization Pal (1998)

amaranth, buckwheat,
frenchbean
Maize Rhizobium leguminosarum P P solubilization Chabot (1998)
bv. phaseoli
Ficus benjamina Bacillus coagulans, P Positively affect Srinath et al. (2003)
Trichoderma harzianum mycorrhizal fungi
Wheat Azotobacter chroococcum P P solubilization and Kumar and Narula (1999)
hormone production
Wheat Bacillus circulans and P P solubilization and Singh and Kapoor (1999)
Cladosporium herbarum interaction with
arbuscular mycorrhizal
fungi
Tomato Enterobacter agglomerans N, P P solubilization and Kim et al. (1997)
positive effect on
mycorrhizal fungi
Maize and sunflower Unknown Fe Fe solubilization Masalha et al. (2000)
Barley Pseudonomas putida Fe Fe chelation Duijff et al. (1994)
Mung bean Pseudomonas sp. Fe Fe chelation Sharma et al. (2003)
Maize Pseudomonas alcaligenes, N, P,K Hormonal effects on Egamberdiyeva (2007)
Bacillus polymyxa, root growth
Mycobacterium phlei
Peas Rhizobia N N fixation Johnston and Beringer
(1976)
Kenya clover Rhizobia N N fixation Moore and Britten (1964)
159
Chickpea Rhizobia N N fixation Yadav et al. (2011)
(Richardson et al., 2009; Vessey, 2003). In this review, we focus on the

effects of PGPB on plant nutrition.
PGPB can be inoculated onto the seed or directly into the soil (Smith,
1992), and are usually mixed with a carrier material such as peat, manure,
compost, sawdust, or vermiculite (Smith, 1992). These carriers provide a
favorable environment for the PGPB when they are initially introduced
to the often hostile soil environment (van Veen et al., 1997). Proper storage
conditions and a good understanding of the local soil ecology are essential
for successful inoculation (van Veen et al., 1997).
5.2 Mechanisms by Which PGPB Affect Plant Nutrition

There have been many studies showing the positive effects of PGPB on plant
nutrition (see Table 4). Depending on the underlying mechanisms, they can
affect the uptake of a single nutrient or a broad spectrum of nutrients. For
example, PGPB that affect mycorrhizal symbiosis will affect the uptake
of many nutrients, whereas PGPB affecting uptake through N fixation, or
P or Fe solubilization, will only affect the uptake of those specific nutrients.
5.2.1 Some PGPB Fix N

One of the earliest PGPB mechanisms discovered was N fixation, and
commercial inoculations of N-fixing Rhizobia, which form symbiotic rela-
tionships with legumes, have been available since the 1890s (Vessey, 2003).
Mixed inoculations of endophytic diazotrophic bacteria such as Gluconaceto-
bacter diazotrophicus, Burkholderia tropica, Azospirillum amazonense, Herbaspiril-
lum rubrisubalbicans, and Herbaspirillum seropedicae have also been shown
very effective at promoting N fixation in sugar cane (Oliveira et al.,
2009). However, except for sugar cane, the use of nonrhizobial N-fixing
PGPB in other nonlegumes has met with limited success (Vessey, 2003).
In fact, many free-living N-fixing PGPB which were thought to improve
plant growth because of their ability to fix N have since been shown to pro-
mote plant growth through other mechanisms. Many reviews have been
written on the topic of N-fixing PGPB, for example, Hardarson (1993),
Provorov and Tickhonovich (2003), Schubert (1995), and Vance (2001).
5.2.2 Some PGPB Solubilize P

Some PGPB have been shown to improve plant nutrition through
P solubilization (Vessey, 2003). The total concentration of P in agricultural
soils usually ranges between 400 and 1200 mg kg1. However, only
1 mg kg1 is generally present in available forms such as HPO-4 and

H2PO2 4 (Rodrı; guez and Fraga, 1999). The nonsoluble P in agricultural

soils is present in inorganic and organic forms. The nonsoluble inorganic
forms account for about 20–50% of the total soil P (Richardson, 2001), usu-
ally in the form of PO-4 ions. These ions are either adsorbed onto the posi-
tively charged constituents of the soil, or they form poorly soluble
precipitates with Fe, Al, or Ca, depending on the pH (Richardson, 2001).
The nonsoluble organic P accounts for 50–80% of the total soil P (Richard-
son, 2001), and is comprised of phosphate esters, inositol phosphates, and
large, uncharacterized organic molecules (Richardson, 2001).
Bacteria use a number of strategies to solubilize the nonsoluble inorganic
and organic P compounds. To solubilize inorganic P, bacteria often synthe-
size organic acids such as gluconic and citric acids, which chelate the insol-
uble compounds and lower the pH, both of which increase P solubility
(Gamalero and Glick, 2011). Another mechanism is to simply release pro-
tons, which lowers the pH and increases solubility without the help of che-
lates (Gamalero and Glick, 2011). Bacteria also increase P availability by
mineralizing organic P (Gamalero and Glick, 2011).
The ability to solubilize P is common in rhizospheric bacteria (Richard-
son, 2001), and many such bacteria have been isolated, including those from
the genera Pseudomonas, Bacillus, Rhizobium, Burkholderia, Enterobacter, Strep-
tomyces, Achromobacter, Agrobacterium, Micrococcus, Aereobacter, Flavobacterium,
and Erwinia (Gamalero and Glick, 2011; Rodrı; guez and Fraga, 1999).
However, not all bacteria with the ability to solubilize P benefit the plant
by increasing P uptake when inoculated into the soil (Richardson, 2001).
It may be that the mechanisms that work well in laboratory culture do
not work as well under soil conditions, or that the organisms themselves
do not thrive iń the soil (Richardson, 2001).
5.2.3 Some PGPB Solubilize Fe

Like P, Fe is also abundant in soils but mostly in the nonsoluble Fe III oxide
form, such as hematite, goethite, and ferrihydrite (Masalha et al., 2000). Fe is
particularly unavailable for plant uptake in calcareous soils because the alka-
line conditions render the Fe less soluble (Masalha et al., 2000). Certain bac-
teria produce siderophores, which chelate Fe, making it more soluble. There
is some controversy about whether plants can use Fe that has been chelated
by bacterial siderophores and whether one of the mechanisms by which
PGPB improve plant nutrition is through the release of siderophores.
Masalha et al. (2000) showed that maize and sunflower have better Fe up-
take in nonsterile calcareous soils than in their sterile counterparts, indicating

that soil microorganisms have beneficial effects on Fe uptake. Similarly,

Sharma et al. (2003) showed that a Pseudomonas sp. increases Fe uptake
and reduces chlorosis in mung bean. Both Masalha et al. (2000) and Sharma
et al. (2003) attributed the improvement in Fe nutrition to plant uptake of
bacterial siderophores. Vansuyt et al. (2007) found that siderophores pro-
duced by Pseudomonas fluorescens are easily absorbed by Arabidopsis thaliana
roots. However, Vessey (2003) called into question the efficiency with
which plants absorb bacterium–siderophore complexes, and suggested that
the main benefit to plants of siderophore-producing bacteria may be that
they compete with pathogens for scarce Fe resources. Interestingly, Duijff
et al. (1994) concluded that siderophores produced by the PGPB Pseudo-
monas putida contribute significantly to barley Fe nutrition in conditions un-
der which the natural phytosiderophores produced by the plant are
decomposed too quickly by the rhizobacteria to be useful in plant Fe uptake.
5.2.4 Some PGPB Induce Changes in Root Morphology

PGPB also enhance plant nutrition by affecting root morphology (Vessey,
2003). Sarig et al. (1992) found that inoculation with Azospirillum brasilense
increases the length and number of adventitious roots of hydroponically
grown sorghum plants. They attributed this change to auxin-like phytohor-
mones produced by A. brasilense. L opez-Bucio et al. (2007) found that inoc-
ulation with Bacillus megaterium causes an increase in root-hair number and
size in Arabidopsis; this change could not be attributed to auxins or ethylene
and they hypothesized that there is a metabolite produced by the bacteria
that may enhance root-hair growth. The same group later found evidence
that the effects of B. megaterium on Arabidopsis roots were caused by cytoki-
nins that were produced by the bacteria (Ortíz-Castro et al., 2008). Bashan
and Dubrovsky (1996) analyzed 79 different experiments reported in the
literature to determine the changes in shoot-to-root ratio caused by Azospir-
illum, and found a large number of experiments that showed a decrease in
the shoot-to-root ratio, although a similar number showed a rise in this
parameter. They suggested that Azospirillum has an effect on the partitioning
of energy and C between the different plant organs, which could explain the
nutrition enhancement by Azospirillum.
5.2.5 Some PGPB Promote a Symbiotic Relationship between

Mycorrhizal Fungi and Roots
One of the most interesting mechanisms by which PGPB enhance plant
nutrition involves promotion of the symbiotic relationship between

mycorrhizal fungi and plant roots (Frey-Klett et al., 2007). The PGPB that
promote this relationship are called mycorrhiza helper bacteria (MHB)
(Frey-Klett et al., 2007). Mycorrhizal fungi infect more than 80% of all
terrestrial plants (Giovannetti and Sbrana, 1998), and they contribute signif-
icantly to plant nutrition by increasing the absorbing surface of the roots and
excreting chelates or enzymes to mobilize insoluble nutrients (Marschner
and Dell, 1994). Many PGPB have been found to promote mycorrhizal
fungal growth, including Agrobacterium, Streptomyces, Pseudomonas, Bacillus,
Paenibacillus, Burkholderia, Arthrobacter, Azospirillum, Klebsiella, Azospirillum,
Alcaligenes, Rhizobium, Bradyrhizobium and Brevibacillus (Frey-Klett et al.,
2007). There are six major mechanisms by which MHB promote mycor-
rhizal fungal growth: (1) stimulating germination of fungal spores
(Frey-Klett et al., 2007; Garbaye, 1994; Johansson et al., 2004), (2) promot-
ing mycelial growth (Frey-Klett et al., 2007), (3) removing toxins from the
soil that inhibit mycorrhizal growth or positively changing the rhizospheric
chemistry or environment to encourage mycorrhizal growth (Frey-Klett
et al., 2007; Garbaye, 1994; Johansson et al., 2004), (4) enhancing root
receptivity to mycorrhizal infection (Frey-Klett et al., 2007; Garbaye,
1994; Johansson et al., 2004), (5) promoting root branching through hor-
monal action (Frey-Klett et al., 2007), and (6) increasing the availability of
nutrients such as N and P, thus promoting synergy between the mycorrhizal
fungi and the plant, both of which require these nutrients (Garbaye, 1994;
Johansson et al., 2004).
6. CONCLUSIONS AND FUTURE DIRECTIONS

Biostimulants have been shown to increase nutrient uptake under
certain conditions, suggesting their usefulness in reducing fertilizer use
without negatively affecting yield. Various mechanisms for these increases
have been proposed, and they are summarized in Table 5. However, despite
the fact that biostimulants are readily available in the marketplace, they have
thus far not significantly reduced fertilizer use in conventional agriculture.
Before biostimulants can be effective in reducing fertilizer use, it is important
to find the most promising ones for specific conditions, and to research how
they are best applied.
Amendment with HS seems promising for improving N uptake. There is
a large body of evidence of their effect on both N uptake and N assimilation
through stimulation of HþATPase and NO3-assimilation enzymes,
including some progress into exactly which fraction of the HS is active.

Table 5 Literature suggesting different mechanisms for biostimulants
164
Improving soil Increase Improve activity
structure or soil Chelation or Increase activity of of arbuscular
Type of microbial solubilization of H+ATPase NO3-assimilation mycorrhizal Changes in root
biostimulant activity nutrients activity Enzymes fungi morphology
Humic Piccolo et al. Chen et al. Pinton et al. Albuzio et al. Malik and Azam
substances (1997) (2004); (1999a); (1986); (1985)
Cesco et al. Canellas et al. Muscolo et al.
(2000); (2008); (1999);

Pinton et al. Zandonadi Vaccaro et al.
(1999b); et al. (2010); (2009)
Sanchez- Quaggiotti
Sanchez et al. et al. (2004)
(2006);
Sanchez-
Sanchez et al.
(2005);
Sanchez-
Sanchez et al.
(2002)
Amino acids García-Martínez Jie et al. (2008); Schiavon et al. Walch-Liu et al.
et al. (2010) Ghasemi et al. (2008); (2006)
(2012); Ertani et al.

Koksal et al. (2009);
(1999); Maini (2006)
Rodríguez-
Lucena et al.
(2010)
Biostimulants for Nutrient Uptake

Seaweed extract Khan et al. Spinelli et al. Kuwada et al. Nelson and van
(2009) (2010) (2006) Staden
(1984);
Steveni et al.
(1992);
Featonby-
Smith and van

Staden (1983)
Plant-growth- Vessey (2003); Frey-Klett et al. Vessey (2003);
promoting Richardson (2007); Sarig et al.
bacteria (2001); Garbaye (1992);
Gamalero and (1994); Ortíz-Castro
Glick (2011); Johansson et al. (2008);
Masalha et al. et al. (2004) Lopez-Bucio
(2000); et al. (2007);
Sharma et al. Bashan and
(2003); Dubrovsky
Vansuyt et al. (1996)
(2007);
Duijiff et al.
(1994)
165
One confounding factor is that HS are usually already present in soils, mak-
ing it difficult to observe positive effects of added HS. More research into the
active fraction may help determine when the addition of HS is useful, and
how and when it is best applied for positive results.
AA may also be able to decrease the use of N fertilizer by stimulating
NO3 assimilation enzymes, but they probably work most effectively as che-
lators. They could be best used for correcting micronutrient deficiencies
when applied either through foliar spray or as a soil drench with micronu-
trient fertilizer. There is evidence that AA can be used to chelate micronu-
trients, but the efficacies of different AA formulations need to be compared,
to each other, and to other common chelates such as ethylenediaminetetra-
acetic acid (EDTA), diethylene triamine pentaacetic acid (DTPA), and
EDDHA, under different types of applications (root drench or leaf spray)
and under various conditions (high or low pH levels, field, or greenhouse).
A large number of studies have shown that SE can increase root size or
root-to-shoot ratio through hormonal action. However, evidence of SE
increasing nutrient uptake is still relatively patchy. Only one field study
(Rathore et al., 2009) has shown an increase in nutrient uptake due to appli-
cation of SE, and greenhouse studies do not show consistent results. For
example, upon application of SE, an increase in leaf N was observed by
Turan and K€ ose (2004), whereas a decrease in leaf N was observed by
Nelson and van Staden (1984); it should be noted, however, that these
studies were performed with different types of SE. Other benefits of SE
application, such as increased growth, enhanced chlorophyll levels, increased
flowering and yield, increased seed germination, enhanced propagation, and
increased resistance to pests and pathogens, although not detailed in this re-
view, may offer more fruitful avenues of research. The use of SE as an alter-
native, environmentally friendly Fe chelate may also hold some promise
(Spinelli et al., 2010).
PGPB seem promising for increasing the availability of P and micronu-
trients in the soil, and enhancing the root system and the arbuscular mycor-
rihizal fungal network. However, there are technical difficulties in ensuring
the survival of the inoculum in the field, and further research is needed on
inoculation methods of the most promising PGPB.
Biostimulants hold much promise for reducing fertilizer use, but the
literature is often fragmented and unconvincing. Authors tend to look at
different nutrients and hypothesize numerous mechanisms without demon-
strating any of them consistently. If the use of biostimulants is to reduce that
of fertilizers, it is important to elucidate the specific mechanisms in the most

promising substances. Then the active ingredient in the substance should be

isolated, and examined in small-scale laboratory experiments, then larger
greenhouse experiments and finally, field experiments. The results should
consistently show the same mechanisms at work and demonstrate a reduced
need for fertilizer use while resulting in equal or greater yield. Without this
type of systematic work, it is unlikely that biostimulants will enable a reduc-
tion in global fertilizer use.
ACKNOWLEDGMENTS
This project received funding from the European Community’s Seventh Framework Pro-
gramme (FP7/2007-2013) under grant agreement n 312117 (BIOFECTOR).
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What are humic substances and what are their effects on plant growth?

What are humic substances and what are their effects on plant growth?

What are amino acids and what is their role in plant nutrition?

What are amino acids and what is their role in plant nutrition?

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The Use of Biostimulants for Enhancing Nutrient Uptake

Article in Advances in Agronomy · December 2015

Moshe Halpern Asher Bar-Tal

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Dror Minz Torsten Müller

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The Use of Biostimulants for

Advances in Agronomy, First Edition, 2015, 141–174

4.4.1 SE Improves Soil Structure 157

Advances in Agronomy, First Edition, 2015, 141–174

GDH Glutamate dehydrogenase

Advances in Agronomy, First Edition, 2015, 141–174

Advances in Agronomy, First Edition, 2015, 141–174

Advances in Agronomy, First Edition, 2015, 141–174

Plant affected by HS Parent material of HS References

Barley NO3 Soil Albuzio et al. (1986)

Moshe Halpern et al.

2.4 Mechanisms by Which HS Affect Nutrient Uptake

2.4.1 HS Improve Soil Structure

Advances in Agronomy, First Edition, 2015, 141–174

aeration, facilitated root penetration, greater water availability to the plant,

2.4.2 HS Improve Solubility of Micronutrients and P

2.4.3 HS Change Root Morphology

Advances in Agronomy, First Edition, 2015, 141–174

out the involvement of auxin-like activity by showing that HS cannot

2.4.4 HS Stimulate HþATPase and NO3-Assimilation Enzymes

Advances in Agronomy, First Edition, 2015, 141–174

3.2 Absorption of AA by Plants

3.3 Effects of AA on Plants

Advances in Agronomy, First Edition, 2015, 141–174

3.3.1 Effects of AA on Nutrient Uptake

3.4 Mechanisms by Which AA Affect Nutrient Uptake

3.4.1 AA Increase Soil Microbial Activity

3.4.2 AA Chelate Micronutrients

Advances in Agronomy, First Edition, 2015, 141–174

Plant affected by AA Parent material or speciﬁc AA References

Maize N Animal epithelial tissue Maini (2006)

Moshe Halpern et al.

example, Koksal et al., 1999; Rodríguez-Lucena et al., 2010) and hydro-

3.4.3 AA Reduce Micronutrients

3.4.4 AA Improve Internal Translocation of Micronutrients

3.4.5 AA Affect Root Morphology

3.4.6 AA Stimulate NO3-Assimilation Enzymes

Advances in Agronomy, First Edition, 2015, 141–174

Advances in Agronomy, First Edition, 2015, 141–174

during the growing season. Strictly speaking, when SE is used as a source

4.3 Effects of SE on Plants

4.3.1 Effects of SE on Nutrient Uptake

Advances in Agronomy, First Edition, 2015, 141–174

Moshe Halpern et al.

Crouch et al. (1990) measured the effects of a commercial SE on growth,