Plant and Soil 255: 571–586, 2003.

© 2003 Kluwer Academic Publishers. Printed in the Netherlands.

571

Plant growth promoting rhizobacteria as biofertilizers

J. Kevin Vessey∗
Department of Plant Science, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada

Received 14 August 2002. Accepted in revised form 27 March 2003

Key words: Diazotrophs, endophytes, growth-promotion, phytohormones, rhizosphere, root morphology

Abstract
Numerous species of soil bacteria which flourish in the rhizosphere of plants, but which may grow in, on, or
around plant tissues, stimulate plant growth by a plethora of mechanisms. These bacteria are collectively known as
PGPR (plant growth promoting rhizobacteria). The search for PGPR and investigation of their modes of action are
increasing at a rapid pace as efforts are made to exploit them commercially as biofertilizers. After an initial clari-
fication of the term biofertilizers and the nature of associations between PGPR and plants (i.e., endophytic versus
rhizospheric), this review focuses on the known, the putative, and the speculative modes-of-action of PGPR. These
modes of action include fixing N2 , increasing the availability of nutrients in the rhizosphere, positively influencing
root growth and morphology, and promoting other beneficial plant–microbe symbioses. The combination of these
modes of actions in PGPR is also addressed, as well as the challenges facing the more widespread utilization of
PGPR as biofertilizers.

Introduction ation to stimulate plant growth in agriculture has been

Plant growth promoting rhizobacteria (PGPR) repres-
ent a wide variety of soil bacteria which, when grown
in association with a host plant, result in stimulation of
growth of their host. Biofertilizer is a recently coined
term whose exact definition is still unclear, but which
most commonly refers to the use of soil microorgan-
isms to increase the availability and uptake of mineral
nutrients for plants. The focus of this review is the
mode of action of PGPR which act as biofertilizers,
either directly by helping to provide nutrient to the
host plant, or indirectly by positively influencing root
growth and morphology or by aiding other beneficial
symbiotic relationships. Not all PGPR are biofertil-
izers. Many PGPR stimulate the growth of plants by
helping to control pathogenic organism (for reviews
of PGPR as biocontrol agents see, Whipps, 2001;
Zehnder et al., 2001).
Although bacteria were not proven to exist un-
til von Leeuwenhoek in 1683 discovered microscopic
‘animals’ under the lens of his microscope, their utiliz-
∗ FAX No: +1-204-474-7528.
E-mail: k_vessey@umanitoba.ca
exploited since ancient times. Theophrastus (372–287
BC) suggested the mixing of different soils as a means
of ‘remedying defects and adding heart to the soil’
(Tisdale and Nelson, 1975). Such mixing of soils may
have had various positive effects, but undoubtedly
the introduction of beneficial microflora, particularly
rhizobia for legume production, would have been one
of the positive effects. In his poetic work, Georgics,
Virgil identified that the beneficial effects of legume
crops grown in rotation were well established circa 30
BC (Chew, 2002), and no doubt the establishment of
legumes on newly cultivated land required some sort
of soil inoculation process.
Research on PGPR has been increasing at an ever
increasing rate since the term was first used by Kloep-
per and coworkers in the late 1970s (Kloepper and
Schroth, 1978; Suslow et al., 1979). For example, the
term PGPR appears in 11 citations from the 1980s in
the USDA’s Agricola Electronic Database (National
Agricultural Library, Washington, DC); during the
first half of the 1990s the term appears in 34 citations,
and during the second half of the decade, 72 cita-
tions. Likewise, the term biofertilizer appears to have
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come into common use in the scientific literature in the
late 1970s. The Agricola Electronic Database indic-
ates approximately 60 citations per decade which use
the term biofertilizer during the 1980s and 1990s. This
survey demonstrates the trend of an increasing rate
of research on PGPR, but is not an absolute measure
of the activity in the area. Many papers investigating
bacterial-facilitated promotions of plant growth, but
which did not use the terms PGPR or biofertilizer, are
not reflected in the survey.
The objectives of this review article are to ini-
tially define the term biofertilizer, to characterize the
two types of relationships between host plants and
their PGPR (rhizospheric and endophytic), and two
investigate the modes of action of PGPR which act
as biofertilizers. Although there is evidence of PGPR
having biofertilizing effects on forest tree species (e.g.
Elo et al., 2000; Shishido et al., 1999), the focus in this
review will be on crop species.
Definition of biofertilizers
Before initiating a discussion of PGPR as biofertil-
izers, it is necessary to define the term, biofertilizer.
Modern dictionaries do not contain a definition of the
term. A search of the world-wide-web with the word
‘biofertilizer’ results in a great number of internet sites
with a myriad of interpretations of the term. These
sites identify biofertilizers as anything from seaweed
extracts, to composted urban wastes, to various mi-
crobial mixes with unidentified constituent, to chem-
ical fertilizer formulations supplemented with organic
compounds (e.g., proteins and vitamins). Likewise,
the scientific literature has a very open interpreta-
tion of the term biofertilizer, representing everything
from green manures (Rao and Gill, 1995; Ventura and
Ladha, 1997), to animal manures (Abdel-Magid et al.,
1995), to plant extracts (Zodape, 2001).
It is proposed here that biofertilizer be defined
as a substance which contains living microorganisms
which, when applied to seed, plant surfaces, or soil,
colonizes the rhizosphere or the interior of the plant
and promotes growth by increasing the supply or avail-
ability of primary nutrients to the host plant. This
definition is based on the logic that the term biofertil-
izer is a contraction of the term biological fertilizer. As
biology is the study of living organisms, biofertilizer
should contain living organisms which increase the
nutrient status of the host plant through their on-going
existence in association with the plant. This definition
separates biofertilizer from organic fertilizer (fertil-
izer containing organic compounds which directly, or
by their decay, increase soil fertility). Likewise, the
term biofertilizer should not be used interchangeably
with the terms, green manure, manure, intercrop,
or organic-supplemented chemical fertilizer. The pro-
posed definition of biofertilizer is not in agreement
with the position of Okon and Labandera-Gonzalez
(1994) who argue that rhizospheric organisms which
improve utilization of soil nutrients, but do not re-
place soil nutrients (like chemical fertilizers) should
not be called biofertilizers. Whether the existence of
a microorganism increases the growth of plants by
replacing soil nutrients (e.g., by biological N2 fixa-
tion (BNF)) or making nutrients more available (e.g.,
by solubilization of phosphates) or increasing plant
access to nutrients (e.g., by increasing root surface
area), as long as the nutrient status of the plant has
been enhanced by the microorganism, the substance
that was applied to the plant or soil containing the
microorganisms, is referred to here as a biofertilizer.
Not all PGPR can be considered biofertilizers.
Bacteria that promote plant growth by control of de-
leterious organism are biopesticides, but not biofer-
tilizers. Interestingly some PGPR appear to promote
growth by acting as both biofertilizer and biopesti-
cides. For example, strains of Burkholderia cepacia
have been shown to have biocontrol characteristics to
Fusarium spp., but also can stimulate growth of maize
under iron-poor conditions via siderophore production
(Bevivino et al., 1998).
As defined, biofertilizers must contain living mi-
croorganisms which promote plant growth by improv-
ing the nutrient status of the plant. However, not all
biofertilizers will contain PGPR. Rhizospheric fungi
such as arbuscular mycorrhizae (for a review, see
Bethlenfalvay, 1993) and Penicillium bilaii (Vessey
and Heisinger, 2001) are long known to have plant
growth promoting effects via increasing nutrient status
of host plants, however, as our focus is PGPR, we will
not be discussing fungi as biofertilizers here.
Relationships between biofertilizing-PGPR and
their hosts: Rhizospheric and endophytic
For PGPR to have a beneficial effect on plant growth
via an enhancement of the nutrient status of their host,
there obviously needs to be a intimate relationship
between the PGPR and the host plant. However, the
degree of intimacy between the PGPR and the host

plant can vary depending on where and how the PGPR
colonizes the host plant. Relationships between PGPR
and their hosts can be categorized into two levels of
complexity: (1) rhizospheric and (2) endophytic.
In rhizospheric relationships, PGPR may colonize
the rhizosphere, the surface of the root, or even super-
ficial intercellular spaces (although this latter situation
may often involve dead cell layers; McCulley, 2001).
Not any soil bacterium can colonize these areas. By
definition, the plant changes the physical and chemical
composition of the soil in the rhizosphere compared to
the bulk soil which can affect the ability of a PGPR to
colonize the rhizosphere. These differences are mani-
fested by changes in soil pH, water potential, partial
pressure of O2 , and a myriad of other physical and
chemical characteristics due to plant exudations (Grif-
fiths et al., 1999; Revsbech et al., 1999; Tavaria and
Zuberer, 1998; Uren and Reisenauer, 1988; Xu, 2000).
In many rhizospheric relationships, the PGPR will
actually attach to the surface of the plant (Andrews
and Harris, 2000). Scanning electron micrographs of
bacteria on the surface of plants roots are common
in the scientific literature. However, the means of at-
tachment is much less often known. Azospirillum sp.
is one group of PGPR where mechanisms involved in
attachment have been well characterised (Michiels et
al., 1991; Steenhoudt and Vanderleyden, 2000). Col-
onization of root surfaces by PGPR is not uniform.
For example, Kluyvera ascorbata colonized the up-
per two-thirds of the surface of canola roots, but no
bacteria were detected around root tips (Ma et al.,
2001).
In endophytic relationships, PGPR actually reside
within apoplastic spaces inside the host plant. Al-
though there is some evidence of endophytes occupy-
ing intracellular spaces (e.g., An et al., 2001), these
reports are rare. The best characterized symbioses in-
volving colonization of hosts by endophytes are the
legume–rhizobia symbioses. The chemo-attraction, at-
tachment, and infection of the microsymbiont and the
development of the root nodules that house the bac-
teria is a highly complex and highly regulated process
that has been well described in a number of recent re-
views (Gualtieri and Bisseling, 2000; Spaink, 2000).
Likewise the infection process and development of
N2 -fixing specialized structures in the non-legume
symbioses of Parasponia–rhizobia (Gresshoff et al.,
1984), Alnus–Frankia (Huss-Danell, 1997), Azolla–
Anabaena (Peters and Meeks, 1989), Gunnera–Nostoc
(Bergman et al., 1992) and cycads–cyanobacteria
573
(Korzhenevskaya et al., 1999) have also been well
characterized.
In many endophytic relationships where no spe-
cialize structures such a root nodules are formed, the
means of infection of the PGPR and the location
and environmental milieu in which the microsymbiont
is found is not as clearly understood as in legume–
rhizobia symbioses. Firstly, the means of infection is
not clear. Although many studies exist which point to
entry of PGPR into apoplastic spaces of the host at
the junction of lateral roots from primary roots (e.g.,
O’Callaghan et al., 2001; Spencer et al., 1994), Mc-
Cully (2001) presents elegant arguments that much of
the evidence for this form of entry is artefactual. She
argues that fractures introduced during the removal
of plants from soil or artificial rooting media are re-
sponsible for the appearance of these entry portals
around lateral root junctions, and that in non-disturbed
roots, such entry points do not exist because swell-
ing of root cortical cells seals these junction areas
(McCully, 1987; Peterson and Moon, 1993). Some
PGPR endophytic species are known to have cellu-
lase and pectinase activities (e.g., Kovtunovych et al.,
1999; Verma et al., 2001) and these activities could
not doubt aid in infection, however heavily suberized
and/or lignified cell layers would still present a barrier
to such bacteria (McCulley, 2001). Some endophytic
PGPR may utilize other organisms as vectors to gain
access to apoplastic spaces in their host. For example
both the pink sugarcane mealybug (Saccharicoccus
sacchari) (Franke et al., 2000) and arbuscular my-
corrhizae (Isopi et al., 1995) have been implicated in
the infection of host plants by the endophytic diazo-
troph, Gluconacetobacter diazotrophicus (formerly
Acetobacter diazotrophiucs).
Depending on the host plant and the endophyte,
biofertilizing PGPR may be found in all parts of plants
(seed, roots, stems, leaves, fruit, etc). Within these
organs, there is evidence of endophytes living in apo-
plastic intercellular space within parenchyma tissue
(e.g., Dong et al., 1994, 1997) and xylem vessel apo-
plast (e.g., Fuentes-Ramirez et al., 1999; James et
al., 2001). In fact, while some researchers consider
xylem apoplast an ideal place for the location of endo-
phytes (e.g., constant supply of nutrients; circulation
systems for beneficial products from the microsym-
biont) (James and Olivares, 1998), others feel that this
is a most unsuitable habitat for endophytes, and note
that many plant pathogens bring about plant death by
colonization of the xylem apoplast (Dong et al., 1997;
McCully, 2001).
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Mode of action of PGPR as biofertilizers
The means by which PGPR enhance the nutrient status
of host plants can be categorized into five areas: (1)
biological N2 fixation, (2) increasing the availability of
nutrients in the rhizosphere, (3) inducing increases in
root surface area, (4) enhancing other beneficial sym-
bioses of the host, and (5) combination of modes of
action. Each of these areas will be examined in this
section of the review.
Unfortunately in many studies reported in the lit-
erature, the mode of action of PGPR as biofertilizers
are not addressed. We are referring to the plethora of
papers which can be found in the literature where stim-
ulation of growth in plants is reported by inoculation
with a putative PGPR, but no mode of action of the
PGPR is identified. Often these sorts of papers may
only report stimulation of growth by measurements of
total above ground biomass or yield, and not enough
morphometric data (e.g., root weight, root length) is
collected to even suggest a possible mode of action of
the PGPR. We researchers in this field should strive to
design and execute experimentation which can not just
identify putative PGPR, but implicate their underlying
modes of action as well. Ultimately, understanding of
mode of action at the physiological and genetic level
is most useful. The work of Vanderleyden and co-
workers (Costacurta et al., 1994; Dobbelaere et al.,
1999; Vande Broek et al., 1999) on the role of indole-
3-acetic acid (IAA) by Azospirillum brasilense in the
promotion of plant growth, and the work of Glick and
co-workers (Holguin and Glick, 2001; Li et al., 2000;
Mayak et al., 1999; Saleh and Glick, 2001; Shah et al.,
1998; Wang et al., 2000) on 1-aminocyclopropane-1-
carboxylate deaminase production by some PGPR, are
two rare examples of demonstration of the mode of
action of a PGPR to the genetic level.
PGPR that fix N2 for their host
The most studied and longest exploited PGPR are the
rhizobia (including the Allorhizobium, Azorhizobium,
Bradyrhizobium, Mesorhizobium, Rhizobium, and
Sinorhizobium) for their ability to fix N2 in their
legume hosts. Commercial rhizobia inoculants for use
on legume crops were first introduced in the 1890s
(Fred et al., 1932). Because the field of legume–
rhizobia symbioses is so vast, and many excellent
review articles and monographs cover the topic well
(e.g., Hansen, 1994; Gualtieri and Bisseling, 2000;
Schultze and Kondorosi, 1998; Sessitsch et al., 2002),
it is not reviewed here. The focus of this review is on
associative N2 fixers.
It is interesting that so many PGPR have the ability
to fix N2 , yet rarely is their mode of action for the
stimulation of plant growth credited to BNF. PGPR
that have the ability to fix N2 , but for which there is
little evidence, or even counter evidence, that their
stimulation of growth of a specific host plant is due
to nitrogenase activity include Azoarcus sp. (Hurek
et al., 1994), Beijerinckia sp. (Baldani et al., 1997),
Klebsiella pneumoniae (Riggs et al., 2001), Pantoea
agglomerans (Riggs et al., 2001), and Rhizobium sp.
(Antoun et al., 1998; Yanni et al., 2001).
It is also interesting that so many PGPR are diazo-
trophs, yet the mechanism underlying their growth-
promoting effects is not the supply of fixed N to the
host. This suggests that possibly (1) there is something
about the ability to fix N2 that makes the organisms
well adapted to living in the rhizosphere (e.g., lower
mineral N levels due to plant absorption), (2) what
may be considered insignificant levels of N2 fixation
(i.e., in agricultural terms) may actually be beneficial
to host plants in nature, or (3) researchers commonly
select for nitrogenase activity or use culture media
well suited to diazotrophs (e.g., minimal N media)
when attempting to isolate PGPR. Of these possible
explanations, the author believes that the latter reason
may be more important.
Despite much excitement and research on associ-
ative N2 fixation in a vast array of non-legume crop
plants in the late 1970s and early 1980s (e.g., see
Vose and Ruschel, 1981; Wani, 1986), there is still
little evidence of inoculation of non-legumes lead-
ing to agronomically significant levels of BNF from
PGPR in most crops. For example, it was once widely
believed the beneficial effects of Azospirillum brasi-
lense on non-legumes was via BNF, but it is now
well recognized that its growth promoting effects ori-
ginate predominantly from other mechanisms (i.e.,
phytothormone production, effects on root morpho-
logy, etc.). A list of PGPR for which evidence exists
that their promotion of plant growth is based on their
ability to fix N2 in situ are provided in Table 1.
An exception to the generally low influence of
associative BNF are the symbioses between sugar-
cane and endophytic diazotrophs, in which the host
routinely obtains 20–60% of its N requirements from
the microsymbiont (Boddey et al., 2001). In particular,
Gluconacetobacter diazotrophicus has been shown to
contribute significantly to the N-nutrition of sugar-
cane under controlled conditions (Sevilla et al., 2001).
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Table 1. Plant growth promoting rhizobacteria (PGPR) for which evidence exists that their
stimulation of plant growth is related to their ability to fix N2

PGPR Relationship to host Host crops Sample References

Azospirillum sp. Rhizospheric Maize de Salamone et al., 1996

Rice Malik et al., 1997
Wheat Boddey et al., 1986

Azoarcus sp. Endophytic Kallar grass Hurek et al., 2002

Sorghum Stein et al., 1997
Rice Egener et al., 1999

Azotobacter sp. Rhizospheric Maize Pandey et al., 1998

Wheat Mrkovacki and Milic, 2001

Bacillus polymyxa Rhizospheric Wheat Omar et al., 1996

Burkholderia sp. Endophytic Rice Baldani et al., 2001

Cyanobacteria∗ Rhizospheric Rice Hashem, 2001

Wheat Obreht et al., 1993

Gluconacetobacter Endophytic Sorghum Isopi et al., 1995

diazotrophicus Sugarcane Boddey et al., 2001
Sevilla et al., 2001

Herbaspirillum sp. Endophytic Rice James et al., 2002

Sorghum James et al., 1997
Sugarcane Pimentel et al., 1991

∗ Numerous species; predominantly of the genera Anabaena and Nostoc.

Another N2 -fixing endophytic species of sugarcane,
Herbaspirillum seroepdicae, has been shown to infect
rice and result in increased 15 N2 incorporation (James
et al., 2002).
Some researchers have been pursing the formation
of ‘para-nodules’ on non-leguminous plants as poten-
tial sites to host diazotrophs. Treatment of the roots of
some plants with natural, synthetic, or precursors of
the phytohormone auxin will result in the formation
of nodular-looking tumours with of varying degrees
of tissue organization, a para-nodule. These tumours
likely originate from lateral root meristems and are
more correctly viewed as modified root tissue, rather
than an organ with ontogenesis similar to legume nod-
ules (Sprent, 1990). Inoculation of such treated roots
with rhizospheric or endophytic diazotrophs some-
times results in colonization of the para-nodule and
some degree of nitrogenase activity. Such experiments
have involved the colonization of para-nodules on as-
sociations involving wheat and various diazotrophs
(Chen et al., 1993; Gantar and Elhai, 1999; Tchan
et al., 1991; Youssef et al., 1998), Azorhizobium
caulinodans and rice (Christiansen-Weniger, 1996),
Azotobacter nigricans and rape (Koval’skaya et al.,
2001) and Azospirillum sp. and maize (Christiansen-
Weniger and Vanderleyden, 1994). Although this is an
interesting line of research, it is questionable whether
the formation of para-nodules offers any advantage
in promoting associations between diazotrophs and
non-legume hosts compared to research pursuing the
promotion of the symbioses with normal root tissues.
PGPR that increase the availability of nutrients in the
rhizosphere
There is ample evidence that the mode of action of
many PGPR is by increasing the availability of nu-
trients for the plant in the rhizosphere (Glick, 1995;
Rodriguez and Fraga, 1999). The method by which
these increases take place involve solubilization of
576
unavailable forms of nutrients and/or siderophore pro-
duction which helps facilitate the transport of certain
nutrients (notably ferric iron).
Solubilization of phosphates
Phosphorus is second only to nitrogen in mineral nutri-
ents most commonly limiting the growth of terrestrial
plants. Ironically, soils may have large reserves of
total P, but the amounts available to plants is usually
a tiny proportion of this total (Stevenson and Cole,
1999). The low availability of P to plants is because
the vast majority of soil P is found in insoluble forms,
and plants can only absorb P in two soluble forms,
the monobasic (H2 PO4 − ) and the diabasic (HPO4 2− )
ions (Glass, 1989). Phosphate solubilizing bacteria
are common in the rhizosphere and secretion of or-
ganic acids and phosphatases are common method of
facilitating the conversion of insoluble forms of P to
plant-available forms (Kim et al., 1998).
The solubilization of P in the rhizosphere is the
most common mode of action implicated in PGPR that
increase nutrient availability to host plants (Richard-
son, 2001). Examples of recently studied associations
include Azotobacter chroococcum and wheat (Ku-
mar and Narula, 1999), Bacillus circulans and Cla-
dosporium herbarum and wheat (Singh and Kapoor,
1999), Bacillus sp. and five crop species (Pal, 1998),
Enterobacter agglomerans and tomato (Kim et al.,
1998), Pseudomonas chlororaphis and P. putida and
soybean (Cattelan et al., 1999), Rhizobium sp. and
Bradyrhizobium japonicum and radish (Antoun et al.,
1998), and Rhizobium leguminosarum bv. phaseoli
and maize (Chabot et al., 1998).
Phosphate-solubilizing bacteria are common in
rhizospheres (e.g., Nautiyal et al., 2000; Vazquez
et al., 2000b). However, the ability to solubilize P
by no means indicates that a rhizospheric bacterium
will constitute a PGPR. For example, Cattelan et al.
(1999) found only two of five rhizospheric isolates
positive for P solubilization actually had a positive
effect on soybean seedling growth. Likewise, not all P-
solubilizing PGPR increase plant growth by increasing
P availability to the hosts. For example, de Freitas et
al. (1997) found a number of P-solubilizing Bacillus
sp. isolates and a Xanthomonas maltophilia isolate
from canola (Brassica napus L.) rhizosphere which
had positive effects on plant growth, but no effects on
P content of the host plants.
Facilitated absorption of iron
Iron is an essential nutrient of plants, but it is rel-
atively insoluble in soil solutions. Plant roots prefer
to absorb iron as the more reduced ferrous (Fe2+ )
ion, but the ferric (Fe3+ ) ion is more common in
well aerated soil although it is easily precipitated in
iron-oxide forms (Salisbury and Ross, 1992). Plants
commonly excrete soluble organic compounds (che-
lators and phytosiderophores) which binds Fe3+ and
helps to maintain it in solution. Chelators ‘deliver’ the
Fe3+ to the root surface where it is reduced to Fe2+
and immediately absorbed (i.e., ‘Strategy I’ plants).
Phytosiderophores, excreted by grasses (i.e., ‘Strategy
II’ plants), are absorbed with the Fe3+ across the
plasmalemma (e.g., von Wiren et al., 2000).
Some rhizospheric bacteria also produce sidero-
phores and there is evidence that a number of plant
species can absorb bacterial Fe3+ -siderophore com-
plexes (Bar-Ness et al., 1991; Wang et al., 1993).
However, there is a controversy as the significance of
bacterial Fe3+ -siderophore uptake to the iron nutrition
of plants. While some believe that there contribu-
tion of these siderophores to the overall iron require-
ments of plants is small (Glick, 1995), other suggest
an important role (Duijff et al., 1994), even a vital
role, especially in calcareous soils (Masalha et al.,
2000). Bar-Ness et al. (1992), who had earlier sup-
ported the concept of bacterial siderophore uptake by
plants (Bar-Ness et al., 1991), concluded that two
bacterial siderophores (pseudobactin and ferrioxam-
ine B) were inefficient as iron sources for plants and
that rhizospheric siderophore-producing bacteria can
be in competition with the plant for iron. In fact, the
vast majority of research on microbial siderophores
in the rhizosphere is associated with their biocontrol
activities due to their competitive effects with plant
pathogens (e.g., Hiifte et al., 1994).
Positive effects on root growth and morphology
The Barber and Cushman model (Barber and Cush-
man, 1981) is a mechanistic mathematical model
used to simulate and predict nutrient uptake in plants.
The model includes ion uptake kinetic parameters,
soil nutrient supply parameters, and root morphology
parameters. Sensitivity analysis of model simulations
based on empirical studies consistently point to the
importance of root morphology parameters in the up-
take of a variety of nutrients (NO3 − , P, and K)(Barber
and Silverbush, 1984). This indicates that despite wide

ranges in the solubilities and availabilities of soil nu-
trient species, PGPR that affect root morphology, and
more specifically, increase root surface area, can have
a huge influence on nutrient uptake potentials.
By far the most evidence for the positive effects
of biofertilizing-PGPR points to bacteria-mediated
changes in root growth and morphology. Bacterial-
mediated increases in root weight are commonly re-
ported responses to PGPR inoculations (e.g., Bashan
and Dubrovsky, 1996; Bertrand et al., 2001; Frommel
et al., 1991; Vessey and Buss, 2002). More import-
antly, increases in root length and root surface area
are sometimes reported (e.g., Galleguillos et al., 2000;
German et al., 2000; Holguin and Glick, 2001; Jac-
oud et al., 1999; Volkmar and Bremer, 1998). Fallik
et al. (1994) reported that inoculation of maize with
Azospirillum brasilense resulted in a proliferation of
root hairs which could have dramatic effects on in-
creasing root surface area. The reporting of root length
and root surface area are important because increase in
these parameters are more reflective of an increase the
volume of soil explored, than that which would be in-
dicated by just increases in root weight. For example,
treatment of clipped soybean roots with A. brasilense
Sp7 caused a 63% increase in root dry weight, but
more than a 6-fold increase in specific root length
(root length per unit root dry weight), and more than
a 10-fold increase in total root length (Molla et al.,
2001).
Many actual and putative biofertilizing-PGPR pro-
duce phytohormones that are believed to be related to
their ability to stimulate plant growth. In most cases
these phytohormones are believed to be changing as-
similate partitioning patterns in plants and affecting
growth patterns in roots to result in bigger roots, more
branched roots, and/or roots with greater surface area.
Indole-3-acetic acid is a phytohormone which is
known to be involved in root initiation, cell division,
and cell enlargement (Salisbury, 1994). This hormone
is very commonly produced by PGPR (Barazani and
Friedman, 1999). Table 2 lists recent papers where the
production of this hormone has been implicated in the
growth promotion by biofertilizing-PGPR. Most com-
monly, IAA-producing PGPR are believed to increase
root growth and root length, resulting in greater root
surface area which enables the plant to access more
nutrients from soil.
Production of other phytohormones by biofertilizing-
PGPR has been identified, but not nearly to the same
extent as bacteria which produce IAA. Cytokinins
are a class of phytohormones which are known to
577
promote cell divisions, cell enlargement, and tissue
expansion in certain plant parts (Salisbury, 1994). Re-
searchers have recently begun to identify cytokinin
production by PGPR (Table 2). Gibberellins (gibber-
ellic acid; GA) are a class of phytohormones most
commonly associated with modifying plant morpho-
logy by the extension of plant tissue, particularly stem
tissue (Salisbury, 1994). Evidence of GA production
by PGPR is rare, however Gutierrez-Manero et al.
(2001) provide evidence that four different forms of
GA are produced by Bacillus pumilus and Bacillus
licheniformis. Inoculation of alder (Alnus glutinosa)
with these PGPR could effectively reverse a chemic-
ally induced inhibition of stem growth. Although this
is not strong evidence of GA production being a com-
mon method of growth promotion by PGPR, it does
suggest that it may have a role, and indicates that more
research is this area is warranted.
Ethylene is the only gaseous phytohormone. It is
also known as the ‘wounding hormone’ because its
production in the plant can be induced by physical
or chemical perturbation of plant tissues (Salisbury,
1994). Among its myriad of effects on plant growth
and development, ethylene production can cause an
inhibition of root growth. Glick et al. (1998) put
forward the theory that the mode of action of some
PGPR was the production of 1-aminocyclopropane-
1-carboxylate (ACC) deaminase, an enzyme which
could cleave ACC, the immediate precursor to ethyl-
ene in the biosynthetic pathway for ethylene in plants.
They submitted that ACC deaminase activity would
decrease ethylene production in the roots of host plants
and result in root lengthening. Subsequently, Glick’s
research program has provided numerous empirical
studies with wild-type and genetically modified PGPR
to support their theory (Holguin and Glick, 2001; Li
et al., 2000; Mayak et al., 1999; Saleh and Glick,
2001; Shah et al., 1998; Wang et al., 2000). In
some cases, the growth promotion effects of ACC-
deaminase-producting PGPR appear to be best ex-
pressed in stressful situations such as flooded (Grichko
and Glick, 2001) or heavy metal-contaminated soils
(Burd et al., 1998).
As stated above, IAA production in PGPR had
been identified for a long time as a mode of action
on the promotion of the growth of host plants. How-
ever, the more recent discoveries of the involvement
of cytokinins, ACC deaminase, and possibly GA pro-
ducing PGPR opens the possibility that even more
plant growth-regulating substances may be involved in
the promotion of plant growth by some PGPR. Given
578
Table 2. Plant growth promoting rhizobacteria (PGPR) for which evidence exists that their promotion
of plant growth is due to influences on or by phytohormones

Factor PGPR Host Reference

produced

IAA Aeromonas veronii Rice Mehnaz et al., 2001

Agrobacterium sp. Lettuce Barazani and Friedman, 1999
Alcaligenes piechaudii Lettuce Barazani and Friedman, 1999
Azospirillum brasilense Wheat Kaushik et al., 2000
Bradyrhizobium sp. Radish Antoun et al., 1998
Comamonas acidovorans Lettuce Barazani and Friedman, 1999
Enterobacter cloacae Rice Mehnaz et al., 2001
Enterobacter sp. Sugarcane Mirza et al., 2001
Rhizobium leguminosarum Radish Antoun et al., 1998

Cytokinin Paenibacillus polymyxa Wheat Timmusk et al., 1999

Pseudomonas fluorescens Soybean de Salamone et al., 2001
Pine Bent et al., 2001
Rhizobium leguminosarum Rape & lettuce Noel et al., 1996

Gibberellin Bacillus sp. Alder Gutierrez-Manero et al., 2001

ACC deaminase Alcaligenes sp. Rape Belimov et al., 2001

Bacillus pumilus Rape Belimov et al., 2001
Enterobacter cloacae Rape Saleh and Glick, 2001
Pseudomonas cepacia Soybean Cattelan et al., 1999
Pseudomonas putida Mung bean Mayak et al., 1999
Pseudomonas sp. Rape Belimov et al., 2001
Variovorax paradoxus Rape Belimov et al., 2001

that many ‘newer’ plant growth-regulating substances
have been identified in plants (e.g., brassinosteroids
and triacontanol; Salisbury, 1994), undoubtedly more
plant growth-regulating substances have yet to be dis-
covered. It is likely that the mode of action of currently
identified and yet to be discovered PGPR will involve
production of substances which will mimic or influ-
ence the action of these newer plant growth-regulating
substances.
Aside from the effects of bacterial-facilitated ef-
fects of phytohormones on root growth, there is evid-
ence in some systems that PGPR may be directly
affecting root respiration which in turn leads to in-
creases in root growth. Inoculation of various plant
species with Azospirillum has shown to increase root
respiration rates (Sarig et al., 1992; Vedder-Weiss et
al., 1999). Phillips et al. (1999) discovered that the
release of lumichrome, a breakdown product of ri-
boflavin, from Sinorhizobium meliloti resulted in an
increase in root respiration by alfalfa. This increase
in root respiration caused a compensatory increase in
carbon assimilation in the shoot, leading to an 8%
increase in plant growth.
‘Helper’ bacteria
It is not uncommon to see the effects of biofertilizing-
PGPR be via a synergism or promotion of the bene-
ficial effects of a third-party rhizospheric microorgan-
ism. In these cases, the PGPR aiding the other host–
symbiont relationship is often referred to as ‘helper’
bacteria. The vast majority of studies investigating
PGPR as aids of other host-symbiont relationships
involve either legume–rhizobia symbioses or plant–
fungi symbioses.
Stimulation of legume–rhizobia symbioses
PGPR aiding aspects of the legume–rhizobia symbi-
osis is the most common of these types of reports. As
early as 1979, Singh and Subba Rao reported positive
 579
Table 3. Plant growth promoting rhizobacteria (PGPR) that stimulate the rhizobia–legume symbioses and percentage change on
several characteristics of the symbioses due to inoculation1

Crop PGPR Nodule Nodule Nitrogen Reference

Number Weight Benefit2

Alfalfa Pseudomonas +122 to +141 123 +48 to +194 Knight and Langston-Unkefer, 1988
syringae

Common Azospirillum 17 NM3 NM Burdman et al., 1996

bean brasilense
Bacillus sp. +37 to +87 +33 to +83 +76 to +115 Srinivasan et al., 1996

Lentil P. putida 0 to +46 NM- 0 to +228 Chanway et al. 1989

Pea Pseudomonas sp. 0 0 0 Chanway et al. 1989

P. fluorescens 298 NM NM Andrade et al., 1998

Peanut Azospirillum +47 +31 to +70 NM Raverkar and Konde, 1988

lipoferum

Red clover Pseudomonas sp. +210 NM +124 Marek-Kozaczuk and Skorupska, 2001

Soybean Bacillus sp. and +55 to +57 NM NM Li and Alexander, 1988

Pseudomonas sp.
Aeromonas 0 to +73 0 to +300 0 to +140 Zhang et al., 1996, 1997
hydrophila
Serratia 0 to +191 0 to +1351 0 to +92 Dashti et al., 1997, 1998
proteamaculans
S. liqufaciens 0 to +231 0 to +1190 0 to +111 Dashti et al., 1997, 1998
Isolates GW3205 0 to +189 +166 to +200 NM Cattelan et al., 1999
& LN32124
P. chlororaphis 0 0 to +200 NM Cattelan et al., 1999
A. brasilense +730 +681 NM Molla et al., 2001
B. cereus +16 None 12 Vessey and Buss, 2002
1 Partially compiled by Buss (1998). 2 Measured as nitrogenase activity or N accumulation. 3 NM – Not measured. 4 Species not
identified.

effects of A. brasilense inoculation on nodule num-
ber, nodule dry weight and shoot growth of soybean.
Table 3 lists many of the PGPR which have been im-
plicated as helper bacteria in various legume–rhizobia
symbioses.
There is evidence for a number of modes of ac-
tion for PGPR stimulation of legume–rhizobia sym-
bioses, but the most commonly implicated mode is
phytohormone-induced (usually IAA) stimulations of
root growth (e.g., Molla et al., 2001; Srinivasan et
al., 1996; Vessey and Buss, 2002). In this way, the
stimulation of nodulation is most commonly an indir-
ect effect; the PGPR stimulates root growth, which
provides more sites for infection and nodulation. How-
ever, this is not always the case. Cattelan et al. (1999)
found that a number of putative PGPR had positive
effects on shoot and/or root growth in soybean and
were positive for production of IAA or ACC deam-
inase, but these putative PGPR had no positive effects
on nodulation. In fact, Cattelan et al. (1999) found
several rhizospheric isolates which stimulated aspects
of the soybean–bradyrhizobia symbiosis and which
had β-glucoanase or cyanide production (if these sub-
stances were involved or how they might stimulate the
soybean–bradyrhizobia symbiosis is unclear).
Some PGPR that stimulate legume–rhizobia sym-
bioses appear to more directly influence the develop-
ment of the symbioses. Burdman et al. (1996) related
Azospirillum brasilense-mediated stimulation in nod-
ulation of common bean to an increased production
580
of flavonoids by the legume host. These flavonoids
are the initial chemical signals secreted by the legume
host to induce nod genes in rhizobia and thereby
initiate the legume–rhizobia symbiosis (Schultze and
Kondorosi, 1998). Andrade et al. (1998) speculated
that an increase in nodulation in pea mediated by
inoculation with Pseudomonas fluorescens was due
to an increase in flavonoid exudation by the host
plant. Proposed alternative modes of action include
toxin (i.e., tabtoxinine-β-lactam) release by Pseudo-
monas syringae stimulating the alfalfa–rhizobia sym-
biosis (Knight and Langston-Unkefer, 1988) and B
vitamins secretion by Pseudomonas sp. enhancing the
red clover-rhizobia symbiosis (Marek-Kozaczuk and
Skorupska, 2001).
Stimulation of plant-fungal symbioses
Although the action of biopesticidal PGPR is often
an antogonistic effect on fungal pathogens of plants
(Whipps, 2001), biofertilizing PGPR sometimes en-
hance plant growth indirectly by stimulating the rela-
tionship between the host plant and beneficial rhizo-
spheric fungi such as arbuscular mycorrhizae (AM).
Although AM by themselves are well known to en-
hance the uptake of various soil nutrients (especially
phosphorus) (e.g., Bethlenfalvay, 1993; Marschener,
1998; Ness and Vlek, 2000; Tobar, 1994), studies are
proving that co-inoculation with some PGPR can en-
hance the relationship between plant and fungal sym-
bionts. Although research in agriculturally important
plants is common (see below), tripartite relationships
among PGPR, fungal symbionts (both AM and ecto-
mycorrhizal fungi), and forest tree species are coming
to the fore (e.g., Frey-Klett et al., 1999; Garbaye,
1994; Geric et al., 2000; Shishido and Chanway,
1998).
Ratti et al. (2001) found that the combination
of the arbuscular mycorrhizal fungus Glomus ag-
gregatum, and the PGPR Bacillus polymyxa and Azos-
pirillum brasilense maximized biomass and P con-
tent of the aromatic grass palmarosa (Cymbopogon
martinii) when grown with an insoluble source of
inorganic phosphate. Likewise Toro et al. (1997)
found that both Enterobacter sp. and Bacillus sub-
tilis promoted the establishment of the AM, Glomus
intraradices, and increased plant biomass and tissue
N and P contents. Kim et al. (1998) found that P
content was increased with inoculation with either the
AM, Glomus etunicatum, or the phosphate solubiliz-
ing PGPR, Enterobacter agglomerans; however, the
highest N and P uptake was observed when tomatoes
were inoculated with both organism. It is interesting
that in each of the above reports, one or more of the
helper bacteria are known to have P solubilizing cap-
abilities and clearly suggest that the bacteria are acting
in concert with the AM to improve P acquisition of the
host plant. However, no doubt other mechanisms, such
as phytohormone production (possibly affecting the
potential sites and success of infection processes) are
at work. Working out the mode of action in the these
tripartite relationships can prove to be complicated
(Azcon, 1993).
The relationship between PGPR and AM are not
always positive. Studies of spring wheat (Germida
and Walley, 1996; Walley and Germida, 1997) found
variable results of co-inoculation and AM fungi and
PGPR strains of Pseudomonas cepacia, P. aeruginosa,
P. fluorescens or P. putida. The results indicated that
some PGPR inoculants may have negative effects on
the mutualistic association between host plants and
AM. Requena et al. (1997) working with semi-arid
pioneering legume, Anthyllis cytisoides, and exotic
and native genotypes of AM and PGPR, identified the
importance of physiological and genetic adaptation in
the relationship among host, PGPR and AM. They
found that the greatest benefit to the host arose from
the use of native, and not introduced strains, of AM
and PGPR.
Combinations of modes of action
Although to this point this review has presented single
modes of actions of biofertilizing PGPR, in fact in
many cases (possibly most cases) a single PGPR will
display several of these modes of action. Likewise,
the isolation of bacteria from rhizospheres will often
reveal multiple modes of action from the population
of putative PGPR inhabiting the rhizosphere. For ex-
ample, Cattelan et al. (1999) identified 22 isolates
from soybean rhizosphere positive for PGPR traits.
Not all bacteria that had PGPR traits stimulated soy-
bean growth, but six isolates positive for ACC deam-
inase production, four isolates positive for siderophore
production, three isolates positive for β-1,3-glucanase
production, and two isolates positive for P solubiliz-
ation increased at least one aspect of early soybean
growth. Antoun et al. (1998) surveyed 266 strains of
rhizobia and found 83% produced siderophores, 58%
produced IAA, and 54% could solubilize phosphorus.
Inoculation of radish with these strains revealed 25%
of the stains to be PGPR, but 64% to have no ef-

fects, and 11% to actually have detrimental effects on
plant growth. Belimov et al. (2001) investigated 15
strains of bacterial isolated from the rhizoplane of pea
and Indian mustard representing a variety of bacterial
species. They found that of five Pseudomonas isol-
ates which stimulated some aspect of growth of rape
grown in uncontaminated or Cd-contaminated soils,
three were positive for ACC-deaminase and phosphate
solubilization activity, and two were positive for ACC-
deaminase activity, phosphate solubilization activity,
and IAA production. These studies indicate that PGPR
will often have multiple modes of actions. We can
also infer from these studies that PGPR co-existing in
the rhizosphere that have single modes of action may
act synergistically to stimulate the growth of the host
plant, such as that indicated in Rojas et al. (2001).
Conclusions and syntheses
This review has shown that there is huge potential
for the use of PGPR as biofertilizing agents for a
wide variety of crop plants in a wide range of cli-
matic and edaphic conditions. It is interesting that so
much research in this area has focussed on associative
diazotrophs, when, aside from the Gluconacetobac-
ter diazotrophicus–sugarcane symbiosis, there is little
evidence for consistent, significant levels of fixed-N
supply by rhizospheric or endophytic bacteria to their
host plants. As stated earlier, the focus on diazotrophs
may be reflective of the selection criteria which re-
searcher use in looking for biofertilizing PGPR, or
indicative that there is something about the nature of
diazotrophic bacteria which make them well suited for
life in the rhizosphere.
The vast majority of studies that investigate the
mode of action of biofertilizing PGPR indicate or sug-
gest bacterial influences on plant growth regulating
substance (e.g., phytohormone production, production
of enzymes which decrease phytohormone produc-
tion by the host, or induction of the host to produce
signally substances to other symbionts (i.e., flavon-
oids)). These factors often affect root development
and morphology resulting in greater root surface area
for the absorption of nutrients or enhance other host–
symbiont relationships (i.e., the ‘helper’ bacteria ef-
fect). It is interesting that so much of the beneficial ef-
fects of biofertilizing PGPR can come from influences
on plant growth regulation, because what is essentially
happening in these case is simply a modification of
plant processes, i.e., changes in root architecture and
581
assimilate partitioning patterns, and not that the bac-
teria are providing an increased supply to nutrient to
the host plant. If bacterial actions can result in im-
proved root morphology for nutrient uptake, this begs
the question, ‘Why do plants have less than ideal root
morphology for nutrient uptake in the first place?’ No
doubt what we are observing is a modification of plant
processes for better plant performance as humans see
fit (i.e., increased growth and yield in agricultural set-
tings), but not which the plant might associate with
increased fitness. If this is the case, then we must
be aware that modification of plant processes to im-
prove plant growth may increase the risk of adverse
effects for the plant (e.g., do longer, more slender roots
make the plant more susceptible to the infection by
certain pathogens?). Second to effects on plant growth
regulation, solubilization of phosphorus is the most
commonly suggested mode of action of biofertilizing
PGPR. This effect, as well as the less commonly re-
ported effect of siderophore production to aid in the
uptake of iron, may be more easily seen as a direct
biofertilizing action (i.e., physiological activity of the
PGPR results in increased supply of a nutrient to the
plant).
Aside from the use of rhizobial inoculants on
legumes which has existed for over a century (Fred et
al., 1932), there has been little extensive commercial-
ization of biofertilizing PGPR. An exception to this is
Azospirillum inoculant which is available for a vari-
ety of crops in Europe and Africa (Dobbelaere et al.,
2001). No doubt the lack of consistent responses in
different host cultivars (e.g., Dashti et al., 1998) and
different field sites (e.g., Bullied et al., 2002; Hilali
et al., 2001) are reasons limiting the more widespread
commercialization of biofertilizing PGPR. Our under-
standing of rhizosphere ecology (for reviews on the
topic see, Andrews and Harris, 2000; O’Donnell et
al., 2000; Schloter et al., 2000; Toal et al., 2000), and
in particular, the influence of rhizospheric organisms
on each other (e.g., Rojas et al., 2001; Vazquez et
al., 2000a), must increase before we can be assured
that biofertilizing PGPR in inoculants will success-
fully colonize host rhizosphere (de Freitas, 2000) and
consistently promote the growth of host plants.
Acknowledgements
The author’s research program on PGPR is largely
supported by the Natural Sciences and Engineering
Research Council (NSERC) of Canada, Agriculture
582
and Agri-Food Canada, Cargill Ltd. and Philom Bios
Inc.
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