### The Use of Biostimulants for Enhancing Nutrient Uptake

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), 1

H-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.,

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 (C ̧ elik 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).

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 L1

N) 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

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-
i cant 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.

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

(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 HPO4
- and

H2PO4-2 (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 PO4

- 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 in 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. Lopez-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).