Soil Biology & Biochemistry 37 (2005) 395–412

www.elsevier.com/locate/soilbio

Intracellular and extracellular PGPR: commonalities and distinctions

in the plant–bacterium signaling processes
E.J. Gray, D.L. Smith*
Department of Plant Science, McGill University, Macdonald Campus, 21,111 Lakeshore Road, St Anne-de-Bellevue, Que., Canada H9X 3V9
Received 24 January 2004; received in revised form 13 May 2004; accepted 27 August 2004

Abstract
Plant growth promoting bacteria (PGPR) associations range in degree of bacterial proximity to the root and intimacy of association.
In general, these can be separated into extracellular PGPR (ePGPR), existing in the rhizosphere, on the rhizoplane or in the spaces between
cells of the root cortex, and intracellular PGPR (iPGPR), which exist inside root cells, generally in specialized nodular structures. The latter
includes rhizobia and Frankia species, both of which fix nitrogen in symbiosis with higher plants. There has been considerable development
in understanding signaling mechanisms of rhizobia (iPGPR) during the establishment of the rhizobia–legume symbiosis, and this may serve
as a model of knowledge regarding cross-talk and plant growth promoting mechanisms. We provide a detailed review of this process,
including plant-to-bacteria signal molecules, followed by bacterial perception and consequent production of bacteria-to-plant signals.
A history of PGPR discovery is also provided, indicating progress in understanding each of the PGPR groups. Recent advances in
understanding plant growth responses to microbial signals are reviewed, along with the research areas that require attention. Based on new
understandings of signaling mechanisms in the iPGPR (rhizobia) and recent findings with ePGPR we are able to speculate regarding general
patterns of signaling in the ePGPR.
q 2004 Elsevier Ltd. All rights reserved.

Keywords: Plant growth promoting rhizobacteria (PGPR); Rhizobia; Nod factors; Lipo-oligosaccharides; Interorganismal signaling; Plant growth

1. Introduction of plant growth promotion, PGPR must colonize the

Bacteria able to colonize plant root systems and promote
plant growth are referred to as plant growth promoting
rhizobacteria (PGPR) (Kloepper and Schroth, 1978). PGPR
can affect plant growth either indirectly or directly; indirect
promotion of plant growth occurs when PGPR lessen or
prevent the deleterious effects of one or more phytopatho-
genic organisms; while direct promotion of plant growth by
PGPR involves either providing plants with a compound
synthesized by the bacterium or facilitating the uptake of
certain nutrients from the environment (Glick, 1995). Thus,
in the broadest sense, PGPR include the N2-fixing
rhizobacteria that colonize the rhizosphere, providing N to
plants, including the rhizobia of the well-characterized
legume–rhizobia symbiosis. Regardless of the mechanisms
* Corresponding author. Tel.: C1 514 398 7851; fax: C1 514 398 7897.
E-mail address: donald.smith@mcgill.ca (D.L. Smith).
rhizosphere around the roots, the rhizoplane (root surface)
or the root itself (within root tissues) (Glick, 1995).
PGPR are associated with plant roots, and either directly
or indirectly stimulate plant growth. Among rhizobacteria
there is a gradient of root proximity and intimacy as follows:
(i) bacteria living in the soil near roots, utilizing metabolites
leaked from roots as C and N sources, (ii) bacteria
colonizing the rhizoplane (root surface), (iii) bacteria
residing in root tissue, inhabiting spaces between cortical
cells and lastly (iv) bacteria living inside cells in specialized
root structures, or nodules, which generally fall into two
groups, the legume associated rhizobia and the woody plant
associated Frankia species. These are discussed in order of
intimacy with the associated plant, from almost casual, to
extremely regulated and housed in specialized structures.
Within this classification, various mechanisms of plant
growth promoting effects have been established; with the
greatest understanding being of the rhizobia–legume

0038-0717/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.soilbio.2004.08.030
396 E.J. Gray, D.L. Smith / Soil Biology & Biochemistry 37 (2005) 395–412
symbiosis. As the quest for definitive knowledge presses on,
research into processes underlying plant growth effects of
PGPR, at each of the intimacy levels just described, is
underway.
The rhizosphere can be defined as any volume of soil
specifically influenced by plant roots and/or in association
with roots and hairs, and plant-produced material (Andrade
et al., 1997; Mahaffee and Kloepper, 1997; Bringhurst et al.,
2001). This space includes soil bound by plant roots, often
extending a few mm from the root surface (Bringhurst et al.,
2001) and can include the plant root epidermal layer
(Mahaffee and Kloepper, 1997). Plant exudates in the
rhizosphere, such as amino acids and sugars, provide a rich
source of energy and nutrients for bacteria, resulting in
bacterial populations greater in this area than outside the
rhizosphere. Most rhizosphere organisms occur within
50 mm of root surface and populations within 10 mm of
root surface may reach 1.2!108 cells cmK3 or 109–1012
microbial cells gK1 soil. Despite large numbers of bacteria
in the rhizosphere, only 7–15% of the total root surface is
generally occupied by microbial cells (Foster et al., 1983;
Pinton et al., 2001).
Rhizobacteria that establish inside plant roots, forming
more intimate associations, are endophytes. These include
a wide range of soil bacteria forming less formal
associations than the rhizobia–legume symbiosis; endo-
phytes may stimulate plant growth, directly or indirectly
(Kobayashi et al., 1995) and include the rhizobia. Given
the semantic overlap, the following definition for endo-
phytic bacteria has been proposed: ‘those bacteria that can
be isolated from surface-disinfected plant tissue or
extracted from within the plant, and that do not visibly
harm the plant’ (Hallmann et al., 1997). In general, a
greater proportion of endophytes are PGPR than is the case
for bacteria inhabiting the rhizoplane or rhizosphere
(Nowak, 1998). Nodulating (rhizobia) and other N2-fixing
rhizobacteria are also endophytes (reviewed in Lodewyckx
et al. (2002)), living in specially developed root organs;
given their ability to promote plant growth through N
fixation these bacteria [primarily rhizobia and the woody
plant associated Frankia (Gardner et al., 1984; Reddell and
Spain, 1991; Rosbrook and Reddell, 1995; Nickel et al.,
2001; Rojas et al., 2001), although the cyanobacterial N2-
fixing symbionts of the cycads (Rai et al., 2000) could also
be included] are also PGPR. To aid in this conceptualiz-
ation, two simple terms have been adopted: intracellular
PGPR—(iPGPR) bacteria residing inside plant cells,
producing nodules and being localized inside those
specialized structures, and extracellular PGPR (ePGPR)—
those bacteria living outside plant cells and not producing
nodules, but enhancing plant growth through production of
signal compounds that directly stimulate plant growth,
improve plant disease resistance, or improve mobilization
of soil nutrients. ePGPR can be subdivided into three types,
based on degree of association with plant roots: those
living near, but not in contact with, the roots; those
Fig. 1. Degree of bacterial associations among plant roots: iPGPR
producing nodules and inhabiting the plant root (in this case (i) on the
surface of a root hair during infection and (ii) in the plant cytoplasm); and
ePGPR ((i) living near, but not in contact with roots; (ii) colonizing the root
surface; (iii) living in spaces between cells of the root cortex).
colonizing the root surface; and those living in the spaces
between cells of the root cortex (Fig. 1). This concept was
briefly touched upon in a recent well-written review
(Vessey, 2003); discussing rhizospheric and endophytic
PGPR in terms of their use as biofertilizers. To further
expand on this we have introduced the novel terms: iPGPR
and ePGPR. We feel that this distinction will play an
important role in the development of concepts and
understanding related to processes underlying the mech-
anisms of plant growth promotion and that such a
classification will highlight differences in the way bacteria
induce plant growth. There is a need for conceptual
distinction between rhizobacteria enhance plant growth
through very near organelle status relationships, while
residing intracellularly within specialized structures, and
those that colonize extracellular tissues and exist in or near
the plant in an unmodified state. The reasons for these
differences are fundamental and should be addressed as
components of understanding the mechanisms of plant
growth promotion. This is clearly illustrated in the
Burkholderia spp. relationships where the same group of
closely related organisms can be either iPGPR or ePGPR.
Burkholderia is a unique genus containing members that
are soil bacteria, human pathogens and PGPR. For
instance, Burkholderia cepacia has been shown to colonize
roots of Sorghum bicolor and promote plant growth
(Chiarini et al., 1998), while other members of Burkhol-
deria have nitrogen fixation properties and have been
isolated from sugarcane (Boddey et al., 2003).
It is important to note that the iPGPR–ePGPR grouping
represents a convenient division of what is really a
continuum of intimacy; we will comment of the similarities
and differences between ePGPR and iPGPR throughout this
review. As signaling in the iPGPR is best understood for
rhizobia, we will focus on this group; little is understood
regarding signaling in Frankia and cyanobacterial-cycad
associations (Rasmussen and Svenning, 1998; Rai et al.,
2000; Parsons and Sunley, 2001).
Soil bacteria in the genera Rhizobium, Bradyrhizobium,
Sinorhizobium, Azorhizobium, Mesorhizobium and
Allorhizobium, belonging to the family Rhizobiaceae,
 E.J. Gray, D.L. Smith / Soil Biology & Biochemistry 37 (2005) 395–412
invade plant root systems and form root nodules
(Martinez-Romero and Wang, 2000). Collectively they
are often referred to as rhizobia. These iPGPR are mostly
Gram-negative and rod-shaped, with a lower proportion
being Gram-positive rods, cocci and pleomorphic forms.
The primary mechanism by which rhizobia increase plant
growth is N2 fixation. ePGPR do not form nodules, but
increase plant growth through a variety of mechanisms;
these include genera such as Bacillus, Pseudomonas,
Erwinia, Caulobacter, Serratia, Arthrobacter, Micrococ-
cus, Flavobacterium, Chromobacterum, Agrobacterium,
Hyphomycrobium and free-living nitrogen-fixing bacteria
(Foster et al., 1983; Prithiviraj et al., 2003).
Over the course of the 20th century, PGPR, including
rhizobia and other root endophytic bacteria, have been
increasingly exploited in agricultural production. It is well
known that plant bacterial symbioses, other than the N2-
fixing systems (with ePGPR), can have pronounced effects
on plant growth. These may be manifested through
accelerated growth and development due to: production of
growth stimulating phytohormones, enhanced availability
of minerals, for example, Fe, through production of
bacterial siderophores, or phosphorus through enhanced
solubilization (Kloepper et al., 1980b); or increased
resistance to pathogens and/or abiotic stresses such as
frost damage (Sturz et al., 2000). More recently, Glick et al.
(1998) have shown that some PGPR can promote growth by
modulating the levels of plant signals (ethylene). In some
cases there can be favorable interactions between iPGPR
and ePGPR; many ePGPR strains, as well as some activators
produced by them, promote legume nodulation and nitrogen
fixation in association with rhizobia (Dashti et al., 1997;
Zhang and Smith, 1996, 1997). They have been added to
rhizobial inoculants, promoting legume nodulation and
nitrogen fixation.
The use of rhizobia to replace nitrogen fertilizers is,
once again, being investigated and discussed (Vessey,
2003), as demand for food has increased due to increasing
global human population while, at the same time, there is a
realized need to reduce the negative environmental impacts
of crop production. The expanded use of nitrogen
fertilizers has allowed increased crop yield. Unfortunately,
it has also had deleterious effects on ecosystems: NO3
pollution of ground and surface waters, soil acidification,
and production of the greenhouse gas N2O through
denitrification (Biswas et al., 2000). Therefore, manipulat-
ing signaling between legumes and rhizobia, including
both flavanoid (from the plant) and lipo-chitoologisac-
chride (LCO—from the bacteria) components, in order to
reduce N fertilizer utilization, provides an alternative to
commercial mineral N fertilizers. Improved understanding
of the way iPGPR promote plant growth can lead to
expanded exploitation of these ‘biofertilizers’ and a
reduction in the potential negative environmental effects
associated with food and fiber production.
397
1.1. PGPR discovery
Early reports of iPGPR date back to the late 1880s, about
the time Beijerinck (1988) isolated the first rhizobia from
nodules. At about the same time Hellriegel and Wilfarth
(1888) demonstrated the ability of bacteria, residing in
‘swellings’ on root tissue, to convert atmospheric N2 into
plant usable forms. Since these initial discoveries, research
on iPGPR has advanced to the detailed investigations of
signaling mechanisms seen today. Our understanding of
iPGPR is now advancing at genomic, proteomic, cellular,
whole plant and environmental levels. Examples of plants
able to form symbioses with rhizobia include soybean
(Glycine max), alfalfa (Medicago sativa), bean (Phaseolus
vulgaris), pea (Pisum sativum), clover sp. (Trifolium sp.),
peanut (Arachis hypogaea), acacia (Acacia sp.) lentil (Lens
culinaris), vetch sp. (Coronilla sp.), birdsfoot trefoil (Lotus
corniculatus), chickpea (Cicer arietinum), pigeonpea
(Cajanus cajan) and red bud (Cercis canadensis). Among
the rhizobial species involved examples are Bradyrhizobium
japonicum (Guerinot and Chelm, 1984), Rhizobium giardi-
nii (Amarger et al., 1997), Rhizobium ciceri (Nour et al.,
1994), Rhizobium etli (Segovia et al., 1993), Rhizobium
galegae (Lindstrom, 1989), Rhizobium gallicum (Amarger
et al., 1997), Rhizobium fredii (Scholla and Elkan, 1984),
Sinorhizobium fredii (Chen et al., 1988), Sinorhizobium
medicae (Rome et al., 1996), Sinorhizobium arboris (Nick
et al., 1999), Mesorhisobium chacoense (Velazquez et al.,
2001), Mesorhizobium pluriforium (de Lajudie et al.,
1998a), Azorhizobium caulinodans (Dreyfus et al., 1988)
and Allorhizobium undicola (de Lajudie et al., 1998b).
Understanding of the Frankia sp. symbioses expanded
rapidly in the 1970s and 1980s, and their ability to produce
nodules on sea buckthorn (Hippophae rhamnoides) (Ore-
mus, 1980) solidified a new line of dynamic research.
Concomitantly, techniques for isolation of Frankia strains
were being reported (Burggraaf et al., 1981). The conclusive
identification of Frankia isolates from tree species
expanded to organisms such as alder sp. [Alnus glutinosa
(Akkermans et al., 1983), Alnus rubre, Alnus sinuate
(Carpenter et al., 1984), and Alnus rubra (Crannell et al.,
1994)], poplar (Populas trichocarpa) (Du Cros et al., 1984),
hemp (Datisca cannabina) (Akkermans et al., 1983),
mountain mahogany (Cerocarpus ledifolius) (Wood et al.,
1989), silverberry (Elaeagnus commutata) and buffaloberry
(Shepherdia canadensis) (Visser et al., 1991), Casuarina (a
Mediterranean tree genus) (Rosbrook and Reddell, 1995),
common bayberry (Myrica pensylvanica) (Clawson and
Benson, 1999) and anchor plant (Discaria trinervis)
(Valverde and Wall, 2002) among others. Examples of
isolated Frankia strains include ACC13 (Jiang et al., 1984),
ORS021011 (Diem and Dommergues, 1985), HFPCc13
(Lancelle et al., 1985), HFPCg14 (Mansour et al., 1990) and
Frankia alni (Cournoyer and Normand, 1994).
Research on ePGPR initially focused on Bacillus and
Anthrobacter spp. (Brown, 1974). Applications of these
398 E.J. Gray, D.L. Smith / Soil Biology & Biochemistry 37 (2005) 395–412
symbioses have been investigated in corn (Zea mays), wheat
(Triticum aesitivum), oat (Avena sativa), barley (Hordeum
vulgare), pea (P. sativum), canola (Brassica napus),
soybean (G. max), potato (Solanum tuberosum), tomato
(Lycopersicum esculentum), lettuce (Lactuca serriola),
radish (Raphanus sativus) and cucumber (Cucumis sativa).
The ePGPR are reported to include species such as
Actinobacter sp. (Tanii et al., 1990), Aeromonas caviae
(Inbar and Chet, 1991), Agrobacterium radiobacter (Ryder
and Jones, 1990), Alcaligenes sp. (Yuen et al., 1985),
Bacillus cereus (Handelsman et al., 1990; Ryder et al.,
1999), Bacillus circulans (Berge et al., 1990), Bacillus
firmus, Bacillus licheniformis (Chen et al., 1996), Bacillus
subtilis (Turner and Blackman, 1991; Zhang and Smith,
1996), Enterobacter agglomerans (Tanii et al., 1990),
Enterobacter cloacae, Erwinia herbicola (Nelson, 1998),
Flavobacterium spp. (Tanii et al., 1990), Phyllobacterium
sp. (Lambert et al., 1990), Pseudomonas aureofaciens
(Kluepfel and McInnis, 1991), Pseudomonas cepacia (De
Freitas and Germida, 1991), Pseudomonas fluorescens
(Voisard et al., 1989), Pseudomonas putida (De Freitas
and Germida, 1991; Mayak et al., 1999), Serratia fonticola
(Chanway et al., 1991), Serratia liquefaciens (Zhang and
Smith, 1996), Serratia macescens (Ordentlich et al., 1988),
Serratia proteamaculans (Chanway et al., 1991; Zhang
and Smith, 1996) and Bacillus thuringiensis (Bai et al.,
2002a,b).
2. Perceptions on communication: starting
with the microbes
Many bacterial species utilize a signaling mechanism
that regulates gene expression in a population density-
dependent manner, a phenomenon referred to as quorum
sensing, discovered over 25 years ago in marine bacteria
(Nealson and Hastings, 1979). Bacteria will produce unique
signal compounds that, upon reaching a critical threshold,
will induce gene expression encoding for a variety of
responses, both phenotypical and physiological. Responses
include bioluminescence, pathogenicity and pigment induc-
tion, in addition to cell conjugation, growth regulation,
nodulation and cell motility (Fray, 2002). Arguably the most
characterized quorum-sensing molecule is the N-acyl-
homoserine lactone (AHL), employed by a range of
bacterial species, notably Gram-negative species.
Since its discovery several quorum-sensing models have
been described in other bacteria, involved in plant–bacterial
symbioses (reviewed in Loh et al. (2002)), most notably the
rhizobia–legume symbiosis. Rhizobium leguminosarum
produces several AHLs (Cha et al., 1998; Lithgow et al.,
2000) and it has been postulated that these compounds
regulate nodule development, though additional research is
needed. However, recent evidence from B. japonicum has
shown that induction of nodulation repression genes (nodD2
and nolA) is regulated by a cell density factor (CDF).
In cultures, this factor represses nod gene expression
directly through reductions in the activity of NodD2 and
NolA (Loh et al., 2001). Though little is known about the
CDF, it does not appear to be related to AHLs. Another
PGPR utilizing quorum sensing is P. putida. P. putida strain
IsoF contains a ppu quorum-sensing locus involved in
production of AHLs in response to population density
(Steidle et al., 2002). Interestingly, Gram negative P. putida
WCS358 produces and secretes cyclic-peptides involved in
cross-talk with the quorum-sensing luxI and luxR homologs
(Degrassi et al., 2002). Current research is showing the
potential of Gram-negatives in producing modified
peptides that can interact with AHLs.
It is well understood that Gram positive bacteria produce
and secrete oligo-peptides, illustrated in Staphylococcus
aureus competence (Korem et al., 2003) and B. subtilis
competence/sporulation (Dubnau, 1991; Grossman, 1995).
B. subtilis has a two-component system exploiting a sensor
kinase and a response regulator that directs this signaling. A
third component of this system, acting as an intermediatary
fine-tune control, is a set of aspartyl phosphatases (Raps),
which can de-phosphorylate compounds, reversing the
effects of the sensor kinases (Gray, 1997). In addition to
producing regulatory peptides Bacillus produces enzymes to
degrade the AHL moieties produced by Gram negatives.
Genes encoding for AHL degrading enzymes, aiiA have
been found in B. thuringiensis and various subspecies
(Lee et al., 2002). The presence of such proteins allows
Bacillus spp. to cleave the lactone bond of AHLs via
hydrolysis, suggesting a mechanism for autoinducer-
degrading activity, allowing these bacteria to compete
with other Gram negatives.
Recent evidence shows that plant host species have
evolved responses to AHLs. When exposed to a wide
concentration range (1 nM–50 mM) of AHLs, Medicago
trunculata responded with an initial decrease in various
protein concentration at 24 h, followed by a later increase of
the same proteins after 48 h (Mathesius et al., 2003). Some
of these proteins included members of defense/stress
response, cytoskeleton structure/function, isoflavone pro-
duction and metabolic enzyme families. This new and
dynamic area of research presents an exciting line of
understanding as to how bacteria communicate among
themselves and how plants have evolved mechanisms to
respond to these signal compounds.
Since the initial PGPR discoveries understanding of
growth stimulating compounds involving the N2-fixing
symbiosis has dramatically expanded. Today, we are
embarking on another line of research involving signaling
in the iPGPR and ePGPR relationships. As these signal
exchanges are understood, one can begin to manipulate the
parameters of communication to further increase plant
growth. Rhizobial signaling is now understood reasonably
well from perspectives such as chemical ecology, biochem-
istry, and genomics. It is through studies of signaling in the
iPGPR–legume symbiosis that a detailed understanding of
 E.J. Gray, D.L. Smith / Soil Biology & Biochemistry 37 (2005) 395–412
signal effects on plant growth has begun to take shape
(reviewed in Miklashevichs et al. (2001)) and we can exploit
this as a model for PGPR-plant systems. It should be noted
that rhizobia of the legume–rhizobia symbiosis can also be
ePGPR (Vessey, 2003). By recognizing that rhizobia can be
extracellular or intracellular, depending on the symbiosis,
one can begin to ask the questions regarding mechanisms
and motivations for the different relationships. Are the
signaling processes involved in these very different
relationships similar? Recognizing the potential differences
associated with the two types of relationships highlights
how much is left to understand about plant–bacteria
symbioses. As extensive knowledge has come from
iPGPR signaling with rhizobia, a brief summary of this
process is provided in Section 2.1.
2.1. Signaling in the iPGPR symbiosis: a platform
of understanding
Stimulation of plant growth by rhizobacteria can involve
a wide range of signal exchanges between plant roots and
bacteria. In the case of rhizobia, phenolic signaling
molecules launching this communication are secreted by
plant roots and represent the first step in a signaling cascade.
Flavanoids, isoflavanoids and phenolic signaling molecules
excreted by the plant root induce expression of rhizobial nod
genes. For a detailed review on additional root exudates and
other signaling mechanisms refer to Bais et al. (2004). In
response to these compounds rhizobia produce a series of
host-specific signaling molecules, LCOs also referred to as
Nod factors (NFs). Genistein, a phenolic compound secreted
by soybean and other plants, activates nod gene expression
in B. japonicum (Ip et al., 2001). Along with genistein,
potential signal compounds secreted from soybean include
diadzein, O-acetyldiadzein, 6 0 -O-malonylgenistin, 6 0 -O-
malonyldiadzin, glycitin and 6 0 -O-malonylglycin, which
are all able to induce nod gene expression in B. japonicum
(Smit et al., 1992). B. japonicum, when exposed to 5 mM
genistein, produces a characteristic set of Nod factors,
including Nod Bj V (C18:1, MeFuc) (Souleimanov et al.,
2002b), which acts as the primary signaling molecule
inducing soybean plant growth (Souleimanov et al., 2002a).
Although gene regulation of LCO production may vary,
the initial transcriptional regulator responsible for these
signaling compounds includes nodD (Stougard, 2000).
NodD proteins, part of the LysR-analagous family of
transcriptional factors, bind to 47 bp DNA motifs (Fisher
et al., 1988). The nodD gene is constitutively expressed, so
that some amount NodD is present even in the absence of
plant-to-rhizobia signal compounds. Bradyrhizobium spp.
contain all nod genes on their chromosome and have more
sophisticated regulation mechanisms for nod gene
expression than other rhizobia (Knight et al., 1986; Schla-
man et al., 1992; Stacey et al., 1995). Nod genes have been
characterized as either ‘common’, such as nodDABCIJ,
which show significant homology among Rhizobium
399
and Bradyrhizobium species, or ‘host-specific’, such as
nodZ of B. japonicum, nodEFLMN of R. leguminosarum
and nodEFGHQ of Rhizobium meliloti (Horvath et al.,
1986; Stacey et al., 1995). Most of the nod genes are not
expressed until bacterial inducers from a compatible host
plant are present. The nodD protein is a common regulator
of NF production and is responsible for activating various
structural nod genes; it is ubiquitous in all rhizobia and is
localized to the cytoplasmic membrane (Denarie and
Debelle, 1996). Specific genes involved in the diversity of
LCO structure and nodulation vary among bacterial species
and are host specific (Denarie and Debelle, 1996). Rhizobia
can contain more than one nodD protein, as indicated by the
presence of nodD1 and nodD2 in B. japonicum. Further,
nodD proteins show distinct responses, based on exposure to
specific inducing signals. For instance, in R. meliloti, nodD1
is activated by the plant flavanoid, luteolin, while nodD3 in
the same bacterium regulates nod gene expression in
absence of inducers (Mulligan and Long, 1989). Inhibition
of nodulation is regulated by nodD2 and nolA and, as
described earlier, new evidence from B. japonicum shows
that repression through NodD2 and NolA is also regulated
by CDFs. This line of research represents yet another piece
of the signaling puzzle that is beginning to be understood.
2.1.1. LCO synthesis
The nod genes common to all rhizobia include nod A, B
and C. These are responsible for the biosynthesis of the
basic LCO structure (Wais et al., 2002; Perret et al., 2000).
It has been postulated that LCO synthesis takes place in
enzyme complexes located at the interface between the
cytoplasmic membrane and cytosol (Denarie and Debelle,
1996). LCO synthesis begins with chitin oligomer
elongation at the non-reducing terminus via a nodC UDP-
N-acetyl glucosaminyltransferase (Perret et al., 2000;
Denarie and Debelle, 1996). The gene nodC, localized to
the cytoplasmic membrane, also encodes for b-glucososyl
transferases which incorporate N-acetyl glucosamine into
cell wall polysaccharides (Hirsh et al., 2001). The nodB
gene encodes for deacetylase activity, and its product
removes the N-acetyl moiety from the non-reducing
terminus of the N-acetylglucosamine oligosaccharide
(Spaink et al., 1991). This is followed by nodA N-
acyltransferase activity linking the acyl chain to the
acetyl-free C-2 carbon of the non-reducing end of the
oligosaccharide (Perret et al., 2000). The resulting general
LCO structure consists of a m-1,4-linked N-acetyl-D-
glucosamine backbone containing 4–5 residues of which
the non-reducing terminal residue is substituted at the C2
position (Geurts and Bisseling, 2002). Attached to this core
is an acyl chain which varies in length and degree of
unsaturation, depending on the rhizobial species.
2.1.1.1. Acyl chain structure and function. There are two
basic types of fatty acyl chains, the common saturated or
monounsaturated fatty acids (stearic and cis-vaccenic)
400 E.J. Gray, D.L. Smith / Soil Biology & Biochemistry 37 (2005) 395–412
and the unsaturated compounds containing 2–4 double
bonds found in R. leguminosarum and R. meliloti. Products
of nodEF are responsible for the unsaturated components of
the acyl chain. The nodF gene shows homology to acyl
carrier proteins, while nodE is a b-acetoacetylsynthase.
Following induction of nodEF in Rhizobium viciae, in
absence of the nod box, the fatty acyl chain C18:4 was
synthesized (Geiger et al., 1998), highlighting that the fatty
acyl chains are synthesized independent of the LCO chitin
back-bone.
Nodulation efficiency has been linked to the chitin
backbone and acyl chain structures. Mutations in nodE of R.
leguminosarum bv. viciae result in lack of nodulation of
Trifolium species, while nodulation will occur in pea (Perret
et al., 2000). Modifications in nodEF in R. meliloti result in
delayed nodulation of alfalfa (M. sativa) highlighting the
importance of nodEF and their role in host specificity of
nodulation (Swanson et al., 1987). Comparison of chain
structures shows that the most physiologically active LCOs
are those containing 2 double bonds and 16 carbon atoms as
illustrated by the most abundant N-acyl chain found in
Sinorhizobium meliloti—C16:2. This LCO structure was
shown to have the most efficient effect on nodulation with
alfalfa roots (Demont-Caulet et al., 1999). In addition to
nodulation efficiency associated with acyl chain length,
various mitogenic properties have been associated with acyl
chain features. For instance, LCOs contributing to nodula-
tion of Vicia sativa subsp. nigra, are mitogenic and process
a C18:4 chain (Spaink et al., 1991).
2.1.1.2. Additional features on LCOs—role in host speci-
ficity. Specific nod genes, required for the ‘decorative’
features found on the chitin backbone of LCOs play a role in
regulating the specificity of the symbiosis; these species-
specific features of LCOs contribute to host specificity of
bacterium-to-plant signaling and response mechanisms.
Acetylation, involves addition of acetyl groups on either
end of the chain; nodL, nodX, and nolL are involved in
acylation (Perret et al., 2000). Fucosylation, encoded by
nodZ, nolK, involves addition of a fucose sugar moiety to
the chitin backbone. Presence of a fucose plays a role in
nodulation for some legumes; for instance, nodulation of M.
atropurpureum by B. japonicum. In this case, the nodZ gene
of B. japonicum is essential for nodulation (Stacey et al.,
1994). Sulfation, encoded by nodH and noeE, involves
sulfation at the reducing terminus of the chitin backbone.
Host specificity via this structure is seen in nodH sulfation
required for successful nodulation by R. meliloti (Lerouge et
al., 1990). N methylation and carbamoylation are encoded
by nodS, nodU, and nolO (Mylona et al., 1995). Inactivation
of nodS in NGR234 inhibits nodulation of P. vulgaris
(Waelkens et al., 1995). Lastly, 2-0 methylation is encoded
by noeI. By encoding for decorative features, nod genes and
their products play an important role in aspects of host
specificity and recognition.
2.1.2. Manipulations of LCO signaling in agricultural
systems
The demand for increased crop yield continues to be one
of the major driving forces in agriculture. Thus, the quest to
find a mechanism to increase grain yield is fundamental.
The possible role of genistein, a known plant-signaling
molecule secreted by plant root systems, represents a
possibility. Extensive research has explored the possibility
of increasing LCO levels in iPGPR and results are
promising. Commonly secreted by soybean, genistein
regulates transcription of nod genes in B. japonicum
(Pan et al., 2002), by binding to the two-component
regulatory system NodVW and/or the NodD1 transcrip-
tional activator (Loh et al., 1997). Most B. japonicum strains
produce maximum amounts of LCO in the presence of 5 mM
genistein, however, mutant strains have been developed that
respond well to as little as 0.1 mM genistein (Zhang et al.,
2002). Pre-incubation of wild-type B. japonicum with
genistein increases overall soybean yield and protein
content (Belkheir et al., 2001).
As a subtropical legume, the optimum temperature range
for soybean nodulation and nitrogen fixation is 25–30 8C
(Lynch and Smith, 1993). In cooler Canadian climates, soil
temperature conditions are often a limiting factor in soybean
yield, as nodule formation and symbiotic nitrogen fixation
are limited in cooler climates. Successful attempts to
overcome this involved pre-incubation of B. japonicum
cells used in inoculants with genistein, or even genistein
treatment of soil where sufficient indigenous populations of
B. japonicum are present (Zhang and Smith, 1995; Belkheir
et al., 2001; Pan et al., 2002). For instance, pre-incubation of
B. japonicum with genistein will increase nodulation at
suboptimal root zone temperatures (17.5–15 8C—Zhang
and Smith, 1995). Further, the onset of nitrogen fixation
occurs earlier in plants inoculated with genistein pre-
incubated bacteria (Zhang and Smith, 1995). Applying both
Serratia and genistein to soybean increases nitrogen fixation
more than PGPR or genistein alone (Dashti et al., 2001).
Additional research on incubating non-rhizobial PGPR
strains, Serratia spp. and Bacillus spp. with genistein and
related compounds is underway. Perhaps, in the future,
plant-to-bacteria signals will be shown to enhance the
production of growth stimulating materials produced by
non-rhizobial PGPR.
The successful application of genistein to increase
soybean crop yield and protein content resulted in a US
patent highlighting this technology—SoyaSignal (Smith
and Zhang, 1999). Furthermore, soybean genotypes having
higher yield potentials responded with yield increases of up
to 21% when treated with SoyaSignal technology. As world
populations increase there will be a need to find technol-
ogies that increase crop yield with a minimum amount of
environmental effect. Although much research has been
conducted on iPGPR, very little work has been done with
ePGPR. This field constitutes a largely unexplored method
to increase crop plant productivity; iPGPR that produced
 E.J. Gray, D.L. Smith / Soil Biology & Biochemistry 37 (2005) 395–412
elevated levels of growth stimulating materials should
cause greater increases in crop yield. Manipulating such
compounds will have applications in both basic science
laboratory research and field-crop research and production.
2.1.3. Rhizobial infection/nodulation: general overview
The best characterized for PGPR relationship is the
nitrogen-fixing rhizobia–legume symbiosis. Understanding
what is known about this plant–microbe interaction will
facilitate investigations of similar, perhaps less intimate,
associations. Nodule formation begins with the mutual
exchange of diffusible signal molecules (flavonoids and
LCOs as described above), followed by attachment of
bacteria to plant root hairs. Through Nod factor–receptor
reactions, bacteria loosely attach to root hairs as single cell
moieties. This process is mediated by a bacterial surface the
Ca2C-binding protein, rhicadhesin. Rhizobia anchor to the
root hair tip leading to ‘cap formation’. Plant lectins and
bacterial appendages such as cellulose fimbriae and a
rhizobial lectins are essential (Hirsh, 1999). Although
several factors are used in bacterial attachment, Nod factor
binding in the microbial cell membrane, along with host
plant lectin is the most apparent selective mechanism
(Vlassak and Vanderleyden, 1997). Esseling et al. (2003)
recently have shown that nod factors, in absence of bacteria,
are sufficient to induce root hair curling.
Once established on the root hair tip root hair curling
and growth brings the bacteria to one side of the root hair
and surrounded by the curled root hair. From here and via
infection threads, bacteria invade the root and, eventually,
specific plant cells. Various genes involved in LCO
production are active during infection thread formation.
Mutations in nodL remove the ability for infection thread
formation, thereby preventing bacteria from entering into
root hairs. After the infection threads penetrate into cortical
cells, bacteria enter into the host cell cytoplasm. Nodule
primordia, induced by the bacteria-to-plant signals,
develop into mature nodules, while bacteria differentiate
into an endosymbiotic form–bacteroids. Bacteroids syn-
thesize dinitrogenase, responsible for catalyzing the
reduction of nitrogen into ammonia (reviewed in Mylona
et al. (1995)). Cortical cell division is anticlinal initially
and is first seen in the hypodermis. As nodulation develops,
cell division is seen in the pericycal and inner cortex.
Derivatives of hypodermal cells form the central tissue of
the nodule, which is composed of cytoplasm-rich cells.
Cell derivatives of the inner cortex and pericycle develop
into the nodule parenchyma and vascular strands differen-
tiate in the nodule parenchyma (Stougard, 2000). In the
central tissue of the nodule, only some cells are infected.
Small-uninfected cells are dispersed among the infected
cells and in soybean uninfected cells outnumber the
infected cells by 3:2 (Vandenbosch and Newcomb,
1986). Recent evidence is showing the use of plant
breeding to produce plant strains that are more susceptible
to nitrogen-fixing bacteria (Hungria and Stacey, 1997;
401
Provorov and Tikhonovich, 2003). This line of research
proposes innovative ideas allowing the benefits of nitrogen
fixation to be further exploited.
2.1.4. Rhizobial infection/nodulation: molecular overview
Given their amphipathic nature, LCOs accumulate in
plant cell walls and bind to cell wall components. LCOs also
bind to various receptors in plant cell membranes located in
root epidermal cells. Upon binding host signaling pathways
are activated, stimulating the development of root hair
deformation and infection thread formation-ultimately
leading to microbial entry into the plant root system (Perret
et al., 2000). Early signs of infection involve root hair
deformation characterized by a re-organization of the
cellular cytoskeleton, rearrangement of actin filaments and
cytoplasmic streaming. Morphological symptoms include
swelling at the root tip (Denarie and Debelle, 1996). Root
tip growth is reinitiated and there is a change in growth
direction (Geurts and Bisseling, 2002). Besides the
flavonoids and LCOs exchanged between legume plants
and rhizobial cells, it is now realized that a multitude of
additional signals from both symbiotic parties are likely
involved in the nodulation process. These additional
molecules include the following structural features: extra-
cellular polysaccharides also referred to as exo-polysac-
charides (EPS), lipo-polysaccharides (LPS), capsular
polysaccharides (CPS) and cyclic b-glucans (Perret et al.,
2000). A fifth component, secreted from the bacteria
accumulates at the plant–bacterium interface and is known
as surface polysaccharides (SPS) (Perret et al., 2000).
Experimental evidence shows that bacterial mutants in
exo- or LPS are unable to from functional nodules. In R.
meliloti, two classes, EPSI and EPSII, are now recognized.
It appears that EPSI mutant strains can still cause nodule
formation, however these nodules are devoid of bacteria and
are therefore ineffective. Additional research shows that
mutants of EPSI can be rescued by products of EPSII and
nodulation will proceed (Leigh and Lee, 1988). Perhaps,
EPSI has a more significant role in nodulation than EPSII.
Furthermore, low molecular weight EPSII can restore
nodule invasion in mutants of EPSI.
2.1.4.1. Rhizobium secreted proteins. Additional bacterial
components required for nodulation are proteins secreted by
rhizobia. One example is the product of the nodO gene,
secreted by strains of R. leguminosarum bv viciae
(Stougard, 2000). The products of nodO are required by
V. hisuta for nodulation. This gene encodes for a Ca2C-
binding protein that forms a Ca2C transport ion channel in
the cell membranes of leguminous plants. It is thought that
nodO products may also be involved in LCO transport
across membranes and in depolarization of membranes via
Ca2C spiking.
The bacterial strain NGR234, has a flavanoid-inducible
gene coding for a type III protein secretion system (TTSS)
on its symplasmid. The TTSS is common to pathogenic
402 E.J. Gray, D.L. Smith / Soil Biology & Biochemistry 37 (2005) 395–412
bacteria and functions in delivery of virulent pathogenicity
factors directly into the cytosol (Lee, 1997). It is thought
that activity of TTSS commences upon contact between
bacteria and plant root hairs. The TTSS of NGR234
involves a number of genes (nolX, rhcC1, nolB, rhcJ,
nolU and nolV), which regulate infection of soybean. As
mentioned, TTSS secretes gene products responsible for
flavanoid induction, one being products of y4xI. Mutants in
NGR234 with limited secretion of y4xI and nolX products
are not able to nodulate an appropriate host. The connection
between a flavanoid signaling cascade and nodule formation
remains vague at this time. Future developments in this area
will provide a basis in the signaling pathways of early stages
of nodulation.
2.1.5. The plant–microbe interface: beginning of plant
responses
LCOs are amphiphylic with a hydrophilic sugar back-
bone and hydrophobic lipid tail, and are localized to the
plasma membrane via insertion of the acyl chain (Goedhart
et al., 1999). Transport of LCOs out of the cell is dependent
on a set of genes common to all rhizobia, nodIJ. The nodI
gene encodes an ATP-binding cassette protein located in the
inner membrane and nodJ encodes for a hydrophobic trans-
membrane protein (Spaink et al., 1995). Together, nodIJ are
responsible for LCO export out of the cell; LCOs do not
accumulate in the growth media of Rhizobium trifolii nodIJ
mutants (McKay and Djordjevic, 1993). Once at the plant
root interface, Nod factors in the rhizosphere are cleaved by
various chitinases excreted by the root (Perret et al., 2000;
Prithiviraj et al., 2000). Chitinases cut LCOs at the b-1,4-
linkage in the carbohydrate moiety into either di-, tri- or
tetrameric acylated byproducts. In doing so, chitinases
regulate the levels of LCOs in the rhizosphere which helps
to suppress defense responses in the host plant, stimulated
by high levels of LCOs (Perret et al., 2000).
Presently, extensive knowledge of the mechanisms by
which LCOs bind to plant receptors is lacking. However,
two Nod factor binding sites (NFBS1 and NFBS2) have
been isolated from alfalfa (Niebel et al., 1997). Both sites
require the oligo-chitooligosaccharide backbone attached to
the acyl chain for binding and show a higher affinity for
intact LCOs than chitin fragments (Cullimore et al., 2001).
The NFBS1 site has a lower affinity for LCOs and does not
discriminate between sulfated and non-sulfated types
(Gressent et al., 1999) on the other hand, NFBS2 shows a
much higher affinity for LCOs (Kd: 4 nM). Binding proteins
of a lectin type have been isolated from Dolichos biflorus,
which have the ability to bind LCOs with high affinity
(Etzler et al., 1999). Such proteins, for example, lectin-
nucleotide phosphorylase (LNP), show nucleotide phos-
phorylase activity and are part of the ATPase superfamily
responsible for catalyzing the hydrolysis of adenosine
diphosphate (ADP). It has been postulated that LNPs
may serve as signals between bacteria and plants
(Stougard, 2000).
2.1.6. Signal transduction: inside the host cell
Upon LCO binding to receptors, membrane depolariz-
ation takes place (Cardenas et al., 2000). This process
initiates the first steps in a signal transduction cascade
within the plant and is detectable approximately 1 min after
LCO addition (Geurts and Bisseling, 2002). A rise in
cytosolic alkalinization, due to a rapid Ca2C influx into the
cell, occurs and nod genes are essential for Ca2C spiking
(Ehrhardt et al., 1996). In response to elevated cytosolic
Ca2C levels, membrane depolarization is induced by an
efflux of ClK ions (Felle et al., 1998). Membrane
repolarization occurs through KC efflux and HC influx
into the cytosol to reestablish membrane polarity.
Mutations in S. meliloti nodF and nodL genes disrupt the
ability of the bacteria to initiate nodule development and
infection thread formation, however, the Ca2C flux response
is not lost (Wais et al., 2002). These findings illustrate that
Ca2C spiking alone is not sufficient to trigger nodulation.
Further, S. meliloti nodH mutants failed to elicit Ca2C
spiking in vetch, indicating the necessity for nodH in
S. meliloti to induce calcium spiking and nodulation
(Wais et al., 2002). Additional genes involved in Ca2C
spiking have been isolated in Medicago truncatula. LCO
induced calcium spiking does not take place in sym8, sym10
or sym19 mutants of pea. Interestingly, these genes are also
known to play a role in controlling infection thread
formation in pea (Geurts and Bisseling, 2002).
Further analysis of the signal transduction pathway has
demonstrated importance of a heterotrimeric GTP protein.
Research using the pharmacological compound mastoparan,
shows that activation of trimeric G-proteins is involved
(Law and Northrop, 1994). As mastoparan induces activity
of G proteins in plant systems, it is postulated that Nod
factors induce activity similar to these compounds. Given
that plants contain remarkably less G proteins than that in
mammalian systems, the degree to which the plant relies on
G proteins as part of the Nod factor induced signaling
pathways remains to be clearly determined. Mastoparan, as
well as LCOs, have also been shown to elicit phospholipid
signaling molecule responses (Geurts and Bisseling, 2002).
Both compounds induce an increase in phosphatidic acid
(PA) and diacylglycerol pyrophosphate (DGPP). LCO
causes increased PA concentrations due to the activity of
a phospholipase C and phospholipase D, both compounds
essential for inducing root hair formation. Phospholipid
moieties are presumed to be signaling molecules in plants.
Feedback mechanisms regulating these responses involve a
range of compounds. Dmi3, a mutant incapable of
successful infections, has proven hypersensitivity to LCOs
and will detect levels 10-fold less than other mutants. Due to
its hypersensitivity dmi3 is involved in negative upstream
regulation of calcium spiking (Oldroyd et al., 2001a).
Another compound known for its regulatory ability is
ethylene; its effect on nodule formation is through
regulation of Nod factor signaling (Caba et al., 1998).
Ethylene, when applied to plant cells, will stop calcium
 E.J. Gray, D.L. Smith / Soil Biology & Biochemistry 37 (2005) 395–412
oscillations in the cytosol and is also involved in regulating
the number of nodule primordial established at the onset
of rhizobial infection of legume roots (Penmetsa and
Cook, 1997).
Various genes recognized in the process of nodulation
include the early nodulation (ENOD) genes, enod12,
enod40, enod5 and rip1. Enod5 and enod12 encode a
proline rich protein thought to be part of the cell-wall
(Denarie and Debelle, 1996). Expression of enod12 in the
root epidermis occurs within minutes to hours after LCO
application, depending on species involved. However, the
gene products are not seen in nodule meristems (Scheres et
al., 1990). Along with enod12, protein products of rhizobia-
induced peroxidase, rip1 are seen in the root epidermis
(Cullimore et al., 2001). Products of enod40 are small 1–13
amino acid peptides and enod40 activity is up-regulated in
root pericycle cells. Expression of enod40 in pericycle cells
of the root cortex causes these cells to loose their identity
and undergo a series of cell divisions. Expression of both
enod12 and enod40 takes place in the inner root cortex
causing induction of the nodule primordium (Geurts and
Bisseling, 2002).
3. Plant reactions to bacterial signals: a hormonal-type
response
The relatively detailed understanding of signaling
between rhizobia and legumes, can be viewed as a model
for hormone-type signaling between PGPR and plants. It is
interesting to note that the signaling occurring at the
beginning of the N2 fixation symbiosis involves the
exchange of flavonoids and chitin based compounds. Plants
have a well characterized ability to detect chitin fragments,
elicitors of plant defense reactions and constituents of
fungal cell walls, and to produce phytoalexins in response
(Ebel and Mithofer, 1998). In many cases, these phytoa-
lexins are flavonoids and isoflavonoids, such as genistein.
Thus, it may be that during the course of evolution a hostile
signaling system, established to detect and respond to the
presence of pathogenic fungi, has been converted to a
friendly signaling system, used in the establishment of an
extremely important symbiosis. It may also be that vestiges
of the original protective function remain in the symbiotic
signaling system.
Plants will respond to compounds produced by PGPR
through systemic acquired resistance (SAR) in which a
cascade of defense mechanisms are employed (Metraux,
2001). For instance, SAR involves signal transduction,
generation of phytoalexins, oxidative stress protection, and
lignification (Reymond and Farmer, 1998). In all of these
defense mechanisms salicylic acid (SA), jasmonic acid
(JA) and ethylene play roles. These hormones may also
have a part in plant growth stimulation effects. However,
less is known about the role of these compounds in
response to infection by ePGPR. Given that some ePGPR
403
are reported to stimulate plant growth through production
of plant hormones, it may be that ethylene, SA and JA
are active here as well. However, additional research on
this is badly needed, and demonstration of parallel roles
for these plant-internal signals could determine common-
alities between plant–microbe signaling in iPGPR and
ePGPR.
Some hormonal effects are known to mirror those of
LCOs. For instance, stem injection of SA into corn produces
an increase in photosynthetic rates and also yield (Zhou
et al., 1999), while a spray application to soybean increased
photosynthetic rates and plant dry weight (Khan et al.,
2003). These effects show a marked similarity to effects
seen upon application of LCOs. Application of LCOs to
tobacco (John et al., 1997) or to tomato (Staehelin et al.,
1994) induces a signal transduction response, largely
controlled by SA, JA and ethylene (Reymond and Farmer,
1998; Metraux, 2001). This represents a dynamic and
expanding area of PGPR research.
3.1. Ethylene
Ethylene plays a role in various developmental pro-
cesses, such as leaf senescence, leaf abscission, epinasty and
fruit ripening (Vogel et al., 1998). As previously mentioned
ethylene also regulates nod factor signaling and nodule
formation and has primary functions in plant defense
systems. Molecular studies have revealed various genes
encoding biosynthetic enzymes responsible for ethylene
synthesis (Yang and Hoffman, 1986). Aminocyclopropane-
1-carboxylate synthase (ACC synthase) catalyzes the first
irreversible step in ethylene synthesis, the conversion of S-
adenosyl methionine to 1-aminocyclopropane-1-carboxy-
late. The last step in ethylene synthesis involves the
conversion of ACC, to ethylene via ACC oxidase. It has
been postulated that different subsets of genes encoding
ACC synthase are expressed in response to different
developmental, environmental and hormonal signals
(Vogel et al., 1998). At this time the role of genes encoding
ACC synthase, in response to PGPR invasion, remains
unclear.
Ethylene production increases as a result of plant
infection by Rhizobium and Bradyrhizobium (Schmidt et
al., 1999). One PGPR strain, P. putida GR12–2 is also
known to cause increased ethylene levels. A proposed
mechanism for this response includes bacterial synthesis of
indole-3 acetic acid, which stimulates ethylene production
(Mayak et al., 1999). In M. trunculata nodulation can be
restored in the presence of ethylene by application of
ethylene inhibitors such as AgC (Caba et al., 1998).
Explanations specify that bacteria sequester the ethylene
precursor 1-aminocyclopropane-1-carboxylate (ACC)
for hydrolyzation via ACC deaminase (Glick, 2004).
Ethylene inhibits nodule formation for most nitrogen-fixing
legumes. However, recent evidence shows that this effect is
not seen in all species. For instance, in the presence of
404 E.J. Gray, D.L. Smith / Soil Biology & Biochemistry 37 (2005) 395–412
enhanced ethylene levels soybean does not show a decrease
in nodule number (Schmidt et al., 1999). Further, a proposed
signaling pathway has been suggested, highlighting the
feedback role ethylene plays in regulating the number of
nodules on a plant (Chanway et al., 1989; Oldroyd et al.,
2001a), suggesting that ethylene acts upstream of Ca2C
spiking (Oldroyd et al., 2001b) and as a link between the
nitrogen status of the plant and the nodulation process
(Wood, 2001). Ethylene insensitivity has been attributed to
the ethylene-insensitive mutant Sickle, skl gene. Expression
of this gene results in increased sensitivity to LCOs as well
as increased insensitivity to ethylene. Expression of skl in
M. trunculata results in the formation of more nodule
primordia (Penmetsa and Cook, 1997; Geurts and Bisseling,
2002).
Understanding the various mechanisms of ethylene
action and its direct role in nodulation needs further
investigation. These questions remain unanswered and
answering them will provide fundamental knowledge to
the picture of Nod factor induced signal transduction and
host plant molecular responses.
3.2. Jasmonic and methyl jasmonic acids
JA has many roles in plant systems, such as activation of
plant defense genes, onset of senescence, root formation and
ethylene synthesis. JA also inhibits seed germination, callus
growth and chlorophyll production (Mueller and
Brodschelm, 1994). JA synthesis relies on the activation
of genes encoding its biosynthesis: 13-lipoxygenase
(13-LOX), allene oxide synthase (AOS), allene oxide
cyclase (AOC) and oxophytodienoic acid reductase (OPR)
(Hause et al., 2002). JA is produced through the octadeca-
noid pathway; linolenic acid, 18:3, is converted to oxylipin
signal molecules such as JA (van Spronsen et al., 2003). JA
levels rise in response to plant invasion by pathogens. JA
activates plant defense genes: defensins, thionins and
proteinase inhibitors (Hause et al., 2002).
Presently little is known about the interactions between
ePGPR and JA synthesis. However, experimental results
show that genes responsible for JA synthesis are activated in
barley leaves and root tips when the plants are infected by
the mycorrhizal fungus G. intraradicies, suggesting a role in
symbiotic infections. The gene JIP23 is expressed in root
tips, leaves and leaf bases and its expression in these tissues
correlates with enhanced JA levels (Hause et al., 2002).
When treated with G. intradices, elevated levels of JIP23
were detected in the inner root cortex, yet they were not
found in the central root cylinder or in the rhizodermis.
Expression of the JIP23 mRNA was not detected in non-
mycorrhizal roots. Perhaps expression of these genes would
be seen upon infection by rhizobia or even ePGPR;
experimental trials need to be conducted to assess for this
type of gene expression.
3.3. Salicylic acid
SA has many roles in plant defense systems and is
involved in signaling related to SAR response. Further, two
PGPR strains, P. fluorescens Pf4 and P by inducing SA
production, can increase plant growth (Singh et al., 2003).
SA can inhibit the synthesis of JA by disrupting the
octadecanoid pathway (van Spronsen et al., 2003). It has
been suggested that Nod factors are involved in the
inhibition of SA-mediated defense mechanisms in legumes;
this could explain why rhizobia can successfully infect
legume plants without triggering a host defense response
(Pinton et al., 2001). SA is known to inhibit indeterminate
nodulation, but not determinate nodulation. Seven days after
inoculation of V. sativa with R. leguminosarium, nodulation
was completely inhibited (van Spronsen et al., 2003).
Moreover, 10K4 M of SA did not inhibit determinate
nodulation in P. vulgaris colonized with R. etli CE3 or
Glycine soja with S. firedii. It has been hypothesized that
SA disrupts nodulation via LCO signaling in one of three
ways: (i) inhibition of induction of rhizobial nod genes,
(ii) inhibition of LCO production, (iii) inhibition of LCO
perception and signal transduction in plant tissues. Research
in this area is currently juvenile and additional experimen-
tation is needed to reveal the complete story of SA effects on
indeterminate nodulation.
4. ePGPR signaling: the future of PGPR signaling
research
Signaling between plants and ePGPR is not well
understood and, in general, this type of effect remains to
be investigated. Research into the isolation of bacterial-to-
plant signal molecules has expanded to ePGPR strains that
are known to induce plant growth already. For instance,
Bacillus spp. is an ePGPR species promoting growth of
several crop species. B. cereus UW85 can enhance soybean
nodulation under both field and growth chamber conditions
(Halverson and Handelsman, 1991). Srinivasan et al. (1996)
isolated 22 Bacillus spp. from the rhizosphere of P. vulgaris
that can produce significant amounts of the phytohormone
indole acetic acid (IAA). B. circulans GY92 produces a
plant growth promoting chemical compound when cultured
with 5 mM of genistein (Lian et al., 2001) that induces root
hair deformation in soybean. However, the identity of this
compound remains unknown. Rice exudates have been
shown to increase the chemotaxis response to Bascillus
pumilus to root systems (Bacilio-Jimenez et al., 2003). This
perhaps provides a link that signaling mechanisms are
occurring between plant root systems and ePGPR and that
colonization can be enhanced with signaling compounds.
Another ePGPR known for its growth enhancing effects
is S. proteamaculans 1–102 tested on lentil and pea
(Chanway et al., 1989) and on soybean (Dashti et al.,
1997, Bai et al., 2002a,b). Past research has speculated that
 E.J. Gray, D.L. Smith / Soil Biology & Biochemistry 37 (2005) 395–412
S. proteamaculans 1–102 strain might promote plant growth
by producing a diffusible substance (Zhang et al., 1996). In
fact, ePGPR species S. proteamaculance 1–102 and
B. thuringiensis NEB17 produce biological activators
which promote plant growth. Recent experiments (Bai et
al., 2002a,b) demonstrate that an activator is produced by
S. proteamaculance 1–102.
4.1. Bacillus and Serratia: model species
4.1.1. Bacillus spp.
Bacillus spp., a Gram positive ePGPR, may be used
separately or co-inoculatied with, for instance, B. japoni-
cum, to increase plant growth. B. polymaxa (H5), a
phosphate-solubilizing bacterium, increases available soil
phosphorus levels when co-inoculated with Rhizobium on
chickpea (Alagawadi and Gaur, 1988), resulting in
increased phosphorus uptake, and thus to increased crop
yield and nitrogen uptake. Colonization and nodulation by
B. japonicum on soybean (Li and Alexander, 1988) can be
enhanced with Bacillus spp. thereby increasing plant dry
weight and seed yield. Bacillus spp., isolated from the
rhizosphere of P. vulgaris produce significant amounts of
the phytohormone IAA (Srinivasan et al., 1996). IAA
promotes root growth and/or nodulation when added with R.
etli on P. vulgaris, resulting in increased nodule number,
nodule fresh weight, nitrogenase activity and leghemoglo-
bin content (Srinivasan et al., 1996). Application of Bacillus
spp., alone, also has growth promoting effects on various
plants and this may vary depending on soil type, illustrated
with Alder and B. licheniformis (Ramos et al., 2003).
Bacillus has also been shown to increase growth of tomato
when applied to a soilless medium (Yan et al., 2003)
highlighting its diverse application. B. subtilis promotes the
growth of peanut seedlings, resulting in yield increases of K
3.5 to 37% (Turner and Blackman, 1991), as well as
improved germination, seedling emergence, plant nutrition
and increased root growth. Inoculation with B. licheniformis
CECT 5106 and B. pumilus CECT5105 enhances growth of
Pinus seedlings. B. cereus UW85 enhances soybean
nodulation under both field and growth chamber conditions
(Halverson and Handelsman, 1991). Interestingly, in field
experiments, differences in nodulation were seen 25–35
days after planting, however, as the trial progressed the
differences decreased and by 49 days, differences in
nodulation were no longer observed. These results show
initial growth promoting effects, but are not effective in the
long-term. However, initial growth effects could be
beneficial in enhancing growth of juvenile plants, position-
ing them to better deal with environmental stresses they
might encounter later in development.
4.1.2. Serratia spp.
Research on the ability of Serratia spp. to improve plant
growth under low root zone temperatures (RZTs) (Zhang
and Smith, 1996), revealed two Serratia spp. particularly
405
beneficial for increasing nodulation. In this study, S.
proteamaculans 1–102 applied at RZTs of 17.5 and 15 8C
and S. liquefaciens 2–68 applied at 25 8C resulted in the
largest increases in soybean growth. At 15 8C RZT,
inoculation of soybean seedlings with 1–102 increased
nitrogen fixation approximately 46%; at 25 8C RZT, plants
inoculated with 2–68 had high levels of fixed nitrogen. At
25 8C RZT, inoculation with 2–68 increased plant total dry
weight, leaf number and area, and the photosynthetic rate by
24–25%. At 15 8C RZT, inoculation with 1–102 increased
plant leaf area and plant dry weight, as well as plant
photosynthetic rate, by 30–37% (Zhang and Smith, 1997).
Inoculation effects of S. proteamaculans 1–102 and S.
fonticola 2–114 were analyzed on lentil and pea (Chanway
et al., 1989). Strain 1–102 increased lentil nodule number by
50% although there were no effects on emergence, vigor,
acetylene reduction, root weight and shoot weight. Inocu-
lation with 2–114 increased acetylene reduction ability by
approximately 150%, but other variables were not affected.
Serratia spp. can have additive affects when co-inoculated
with rhizobia. Co-inoculation of soybean with B. japonicum
and 2–68 or 1–102 in the field, increased soybean grain
yield by 23 and 29%, respectively, and protein yield by 60
and 50%, respectively (Dashti et al., 1997). Further,
application of culture supernatants to soybean roots
produced effects similar to inoculation with cells, although
not under sub-optimal conditions (Dashti et al., 1997).
4.2. Direct and indirect benefits of ePGPR
Some ePGPR can produce and secrete low molecular
weight (400–1000 Da) iron-binding molecules (sidero-
phores) with a high affinity (KdZ10K20–10K50) for Fe3C
(Castignetti and Smarrelli, 1986). Siderophores bind
available Fe3C in the rhizosphere, preventing pathogens in
the vicinity of the root from proliferating, due to a lack of
iron (O’Sullivan and O’Gara, 1992). Examples of ePGPR
with this activity include P. putida and P. aerruginosa. The
former inhibits Fusarium oxysporum, a tomato pathogen
(Vandendergh and Gonzalez, 1984); while the latter inhibits
Pythium, also a tomato pathogen (Buysens et al., 1994).
Unlike microbial phytopathogens, plants are not generally
harmed by the localized depletion of iron caused by ePGPR.
Most plants can grow at much lower (about 1000-fold) iron
concentrations than microorganisms (O’Sullivan and
O’Gara, 1992). Some plants can bind the bacterial iron–
siderophore complex, transporting it through the plant; at
which time the iron is released from the siderophore and is
available to the plant (Crowley et al., 1988).
Many ePGPR benefit crop production through antibiosis
or biocontrol mechanisms; they are able to suppress
numerous plant pathogenic bacteria, fungi, nematodes, and
viruses. A wild-type strain of P. fluorescence protects
cucumber against Pythium ultimum. Following genetic
manipulation to overproduce the antibiotics pyoluteorin
and 2,4-diacetylphloroglucinol, the resulting strain can
406 E.J. Gray, D.L. Smith / Soil Biology & Biochemistry 37 (2005) 395–412
protect cucumber more effectively (Maurhofer et al., 1992;
Schnider et al., 1994); while non-antibiotic-producing
mutants of suppressive bacterial strains were no longer
able to prevent phytopathogen-caused damage to plants
(Guterson et al., 1986; Keel et al., 1992). Finally, ePGPR
produce various enzymes beneficial to plants. Pseudomonas
spp. can produce hydrogen cyanide which inhibits some
phytopathogenic fungi (Voisard et al., 1989). Other PGPR
strains can produce enzymes hydrolyzing fungal cell walls.
P. stutzeri produces extracellular chitinase and laminarinase
which digest fungal mycelia (Lim et al., 1991).
4.3. ePGPR phytohormones: another signaling mechanism
The production of phytohormones, namely auxin,
cytokinin and gibberellin are the most commonly invoked
mechanisms of plant growth promotion by ePGPR; with
auxin being of primary interest (Garcia de Salamone et al.,
2001). The auxins are well characterized class of plant
hormones, and this is particulary so of indole-3-acetic acid
(IAA), known to stimulate both short and long term
responses in plants (Cleland, 1990; Hagen, 1990). Some
ePGPR strains can produce IAA. Early reports of IAA
production by ePGPR date back to the late 1970s. Tien et al.
(1979) showed that Azospirillum brasilense will produce
IAA as well as indole lactic acid when exposed to
tryptophan. Further, IAA production was shown to increase
with the age of the culture. Bacillus spp., isolated from the
rhizosphere of P. vulgaris produce significant amounts IAA
(Srinivasan et al., 1996). IAA producing Azospirillum
brasilense can stimulate formation of wheat seedling lateral
roots, while a mutant strain, synthesizing low levels of IAA,
does not (Barbieri et al., 1986; Barbieri and Galli, 1993).
Further, 72 h after tryptophan addition, Azospirillum
brasilense strain Sp 245 produces 15 mg/l of IAA
(Zakharova et al., 1999). Acetobacter diazotrophicus
produced 32 and 21 ng IAA mlK1 when cultured with 10
and 15% sucrose (Bastian et al., 1998). In this same study,
Herbaspirillum seropediacae produced less IAA,
7 ng IAA mlK1, when cultured in NFb medium containing
no sucrose. P. fluorescens BSP53a also produces IAA and a
mutant overproducing IAA stimulates root development of
blackcurrant softwood cuttings and inhibits cherry (Dubei-
kovesky et al., 1993). These results indicate that the
response of plants treated with an IAA-secreting PGPR is
affected by both the amount of IAA produced by the
bacteria and the characteristics of the plants.
Cytokinins influence plant growth and development such
as cell division, seed germination, root elongation (Stenlid,
1982), chlorophyll accumulation (Weidhase et al., 1987),
leaf expansion and delay senescence (Hackett, 1985).
Cytokinins are N6-substituted aminopurines and it has
been postulated that cytokinins produced by rhizobacteria
influence plant growth and development. Some P. fluor-
escens strains producing cytokinins promote seedling
emergence and increase root length of several crop species
under controlled conditions (Gacia de Salamone et al.,
2001). Recent evidence suggests that some bacteria produce
volatile compounds that influence plant growth (Ryu et al.,
2003). Ryu et al. (2003) propose that a blend of airborne
chemicals is responsible for increased growth in Arabidop-
sis. Further, the potent growth promoting strains B. subtilis
GB03 and Bacillus amyloliquefaciens IN937a produced
volatiles of 3-hydroxy-2-butanone (acetoin) and 2,3 buta-
nediol (Ramos et al., 2000).
Bacillus spp. produces high levels of gibberellins: A1,
A3, A4 and A20, inducing positive effects on stem and shoot
elongation in A. glutinosa (Gutierrez Manero et al., 1996;
Gutierrez Munero et al., 2001). Acetobacter diazotrophicus
and Herbaspirillum seropediacae produce indole-3-acetic
acid and gibberellins, A1 and A3, enhancing growth of
members of the Graminaceae (Bastian et al., 1998). There is
some information on signaling between ePGPR and plants,
however, compared to the rhizobia–legume association, far
less is known regarding the molecular genetics of such
signaling. This presents a fundamental challenge in under-
standing PGPR signaling and molecular effects on plant
growth.
5. Applications of iPGPR and ePGPR
The possible role of ePGPR signals in plant growth
stimulation presents exciting possibilities and research
opportunities. As already outlined, ePGPR increase plant
growth and the mechanisms underlying this are poorly
understood. However, where they are understood they could
be exploited to increase plant growth. Agronomically,
iPGPR and ePGPR effects are of particular interest and play
roles in crop production (reviewed in Broughton et al.
(2003)). The research required to fully understand them will
require work at all levels, from ecology to proteomics and
metabolomics. In a time where greenhouse gas emissions
are increasing and climate change is rapidly occurring,
mechanisms to combat these detrimental occurrences are
fundamental (Rosa, 2001). Manipulations of source-sink
demand may increase plant sequestration of CO2 into tissues
and then into soil (Rodriguez and Oijen, 1999). Further as
the demand for food increases, with increasing human and
livestock populations, grain and forage yields are of interest.
Both grain and straw yields of wheat infected with
R. leguminosarium bv. Trifolii and G. max infected with
B. japonicum are greater than uninfected controls. Yields of
both plants are even higher when commercial fertilizer is
applied (Biswas et al., 2000). Rice infected with R.
leguminosarium yields more grain, although only in the
presence of fertilizer N. The authors proposed interdepen-
dence between fertilizer-N inputs and inoculation, resulting
in higher rice grain yields. Lateral root number and root
size of Pennisetum americanum L. increase when inocu-
lated with Azospirillum brasilense (Tien et al., 1979).
B. japonicum LCOs stimulate root branching in soybean,
 E.J. Gray, D.L. Smith / Soil Biology & Biochemistry 37 (2005) 395–412
producing an increase in root surface area and nodule size
(Souleimanov et al., 2002a). Injection of 10K7–10K10 M
LCO into stems of soybean plants increased growth,
suggesting hormone-like effects. A similar increase in
potato growth occurs following infection with Pseudomo-
nas (Kloepper et al., 1980a), increasing total plant weight up
to 100% when compared to the control. Bacillus spp.,
producing various isomers of gibberellins (GA1, GA3, GA4
and GA20) were found to increase stem elongation in
alder (Gutierrez Munero et al., 2001). Growth of Pinus
pinea L. seedlings increases when exposed separately to
B. licheniformis CECT 5106 and B. pumilus CECT 5105
(Probanza, 2002). Inoculation with either B. japonicum
strain USDA 30 or USDA 31 increased soybean grain
protein and total protein levels (Zhang et al., 2002). These
bacteria grow well under cooler soil conditions, highlighting
the importance of selecting bacteria for inoculation that are
suitable for specific environmental conditions.
6. Conclusions
As we have outlined in this paper, much is known about
signaling in the iPGPR–legume symbiosis and a certain
amount of ‘reasoning by analogy’ will help understanding
signal exchange between ePGPR and plants. Within the
various ePGPR associations there is a much greater
diversity between proximity to the host plant, mode of
infection and, most importantly, mode of growth effects
than in the iPGPR. Signaling molecules from ePGPR can be,
as recently shown, volatile compounds. At this point,
production of volatile compounds from iPGPR is unknown.
A clear distinction between the two types of associations has
been made and with this understanding one can begin to fit
pieces of this puzzle together to enhance the plant growth
promoting effects. Research is being conducted on ePGPR
to determine the identities of signaling compounds pro-
duced and of course, the detailed signaling response
mechanisms that follow. Additional research exploiting
the use of activator inducers on ePGPR activator production
would provide a means to further increase plant growth
promoting potential; as well as expand this concept to
signaling of other ePGPR and iPGPR species. An improved
understanding of this aspect of plant–microbe relationships
will allow for manipulation of these compounds to enhance
plant growth, increasing crop yield and allowing for
efficient and less environmentally damaging production of
plant biomass for food and feed production as well as
increasing uptake of CO2 into plant biomass for climate
change management.
During the last two decades, we have achieved a
remarkable understanding of the subtleties of signaling
between rhizobia and their legume hosts. While there are
still things to learn about this, it is clear that the signaling
process in this relationship is extremely sophisticated. It is
also clear that there are less intimate plant-rhizobacterial
407
associations, providing a wide range of growth enhancing
materials to plants. Will we find signaling systems spanning
the gradient from the rhizobial–legume system, to a mere
chance association? This seems likely. Plants growing in
field soil cannot be viewed as single organisms. They are a
source of solar-derived reduced carbon and, as such, are
colonized, for good or ill, by a wide range of microbes and
other organisms—these plants are communities of organ-
isms, and we do not understand the nature of the
communities to any great extent. We can expect many
more discoveries and many more communication systems.
The road ahead will be frustrating at times, but exciting
when each milestone is achieved.
References
Akkermans, A.D.L., Roelofsen, W., Blom, J., Huss-Danell, K., Harkink, R.,
1983. Utilization of carbon and nitrogen compounds by Frankia in
synthetic media and in root nodules of Alnus glutinosa, Hippophae
rhamnoides, and Datisca cannabina. Can. J. Bot. 61, 2793–2800.
Alagawadi, A.R., Gaur, A.C., 1988. Associative effect of Rhizobium and
phosphate-solubilizing bacteria on the yield and nutrient uptake of
chickpea. Plant Soil 105, 241–246.
Amarger, N., Macheret, V., Laguerre, G., 1997. Rhizobium gallicum sp.
nov. and Rhizobium giardinii sp. nov., from Phaseolus vulgaris
nodules. Int. J. Syst. Bacteriol. 47, 996–1006.
Andrade, K.L., Mihara, R.G., Linderman, G., Bethlenfalvay, G.J., 1997.
Bacteria from rhizosphere and hyphosphere soils of different arbus-
cular-mycorrhizal fungi. Plant Soil 192, 71–79.
Bacilio-Jimenez, M., Aguilar-Flores, S., Ventura-Zapata, E., Perez-
Campos, E., Bouquelet, S., Zenteno, E., 2003. Chemical characteriz-
ation of root exudates from rice (Oryza sativa) and their effects on the
chemotactic response of endophytic bacteria. Plant Soil 249, 271–277.
Bai, Y., Souleimanov, A., Smith, D.L., 2002a. An inducible activator
produced by Serratia proteamaculans strain and its soybean growth-
promoting activity under greenhouse conditions. J. Exp. Bot. 53,
1495–1502.
Bai, Y., D’Aoust, F., Smith, D.L., Driscoll, B.T., 2002b. Isolation of plant-
growth-promoting Bacillus strains from soybean root nodules. Can.
J. Microbiol. 48, 230–238.
Bais, H.P., Park, S.W., Wier, T.L., Callaway, R.M., Vivanco, J.M., 2004.
How plants communicate using the underground information super-
highway. Trends Plant Sci. 9, 26–32.
Barbieri, P., Zanelli, T., Galle, E., Zanetti, G., 1993. Effect on wheat
root development of innoculation with an Azospirillum brasilense
mutant with altered indole-3-acetic acid production. Res. Microbiol.
144, 69–75.
Bastian, F., Cohen, A., Piccoli, P., Luna, V., Baraldi, R., Bottini, R., 1998.
Production of indole-3-acetic acid and gibberellins A1 and A3 by
Acetobacter diazotrophicus and Herbaspirillum seropedicae in chemi-
cally-defined culture media. Plant Growth Reg. 24, 7–11.
Beijerinck, M., 1988. Die Bakterien de Papilionaceenknollchen. Botanische
Zeitung 46, 725–804. In Milestones in Microbiology: 1556 to 1940,
translated and edited by Thomas D. Brock, ASM Press, 1998, p. 220.
Belkheir, A.M., Zhou, X., Smith, D.L., 2001. Variability in yield and yield
component responses to genistein pre-incubated Bradyrhizobium
japonicum by soybean (Glycine max L. Merr) cultivars. Plant Soil
229, 41–46.
Berge, O., Fages, J., Mulard, D., Balandreau, J., 1990. Effects of inoculation
with Bacillus circulans and Azospirillum lipoferum on crop-yield in
field grown maize. Symbiosis 9, 259–266.
408 E.J. Gray, D.L. Smith / Soil Biology & Biochemistry 37 (2005) 395–412
Biswas, J.C., Ladha, J.K., Dazzo, F.B., Yanni, Y.G., Rolfe, B.G., 2000.
Rhizobial inoculation influences seedling vigor and yield of rice.
Agron. J. 92, 880–886.
Boddey, R.M., Urquiaga, S., Alves, B.J.R., Reis, V., 2003. Endophytic
nitrogen fixation in sugarcare: present knowledge and future appli-
cations. Plant Soil 252, 139–149.
Bringhurst, R.M., Cardon, Z.G., Gage, D.J., 2001. Galactosides in the
rhizosphere: utilization by Sinorhizobium meliloti and development of a
biosensor. PNAS 98, 4540–4545.
Broughton, W.J., Zhang, F., Perret, X., Staehelin, C., 2003. Signals
exchanged between legumes and Rhizobium: agricultural uses and
perspectives. Plant Soil 252, 129–137.
Brown, M.E., 1974. Seed and root bacterization. Annu. Rev. Phytopath. 12,
181–197.
Burggraaf, A.J.P., Quispel, A., Tak, T., Valstar, J., 1981. Methods of
isolation and cultivation of Frankia species from actinorhizas. Plant
Soil 61, 157–168.
Buysens, S., Poppe, J., Hofte, M., 1994. Role of siderophores in plant
growth simulation and antagonism by Pseudomonas aeruinosa 7NSK2,
in: Ryder, M.H., Stephens, P.M, Bowen, D.G. (Eds.), Improving Plant
Productivity with Rhizosphere Baceria. Commonwealth Science and
Industrial Research Organization, Adelaide, Australia, pp. 139–141.
Caba, J.M., Recalde, L., Ligero, F., 1998. Nitrate-induced ethylene
biosynthesis and the control of nodulation in alfalfa. Plant Cell
Environ. 21, 87–93.
Cardenas, L., Holdaway-Clarke, T.L., Sanchez, F., Qunito, C., Feijo, J.A.,
Kunkel, J.G., Hepler, P.K., 2000. Ion changes in legume root hairs
responding to nod factors. Plant Phys. 123, 443–452.
Carpenter, C.V., Robertson, L.R., Gordon, J.C., Perry, D.A., 1984. The
effect of four new Frankia isolates on growth and nitrogenase activity in
clones of Alnus rubra and Alnus sinuata. Can. J. Forest Res. 14,
701–706.
Castignetti, D., Smarrelli, J.J., 1986. First year field performance of spruce
seedlings inoculated with plant growth promoting rhizobacteria. Can.
J. Microbiol. 39, 1084–1088.
Cha, C., Gao, P., Chen, Y.C., Shaaw, P.D., Farrand, S.K., 1998. Production
of acyl-homoserine lactone quorum-sensing signals by Gram-negative
plant-associated bacteria. Mol. Plant Microbe Inter. 11, 1119–1129.
Chanway, C.P., Hynes, R.K., Nelson, L.M., 1989. Plant growth-promoting
rhizobacteria: effects on growth and nitrogen fixation of lentil (Lens
escuienta Moench) and pea (Pisum sativum L.). Soil Biol. Biochem. 21,
511–517.
Chanway, C.P., Radley, R.A., Holl, F.B., 1991. Inoculation of conifer seed
with plant growth promoting Bacillus strains causes increased seedling
emergence and biomass. Soil Biol. Biochem. 23, 575–580.
Chen, W.X., Yan, G.H., Li, J.L., 1988. Numerical taxonomic study of fast-
growing soybean rhizobia and a proposal that Rhizobium fredii be
assigned to Sinorhizobium gen. nov. Int. J. Syst. Bacteriol. 38, 392–397.
Chen, Y.X., Mei, R.H., Lu, S., Liu, L., Kloepper, J.W., 1996. The use of
yield increasing bacteria (YIB) as plant growth-promoting rhizobacteria
in Chinese agriculture, in: Utkehede, R.S., Gupta, V.K. (Eds.),
Management of Soil-Borne Disease. Kalyani, New Delhi, pp. 165–184.
Chiarini, L., Bevivino, A., Tabacchioni, S., Dalmastri, C., 1998. Inoculation
of Burkholderia cepacia, Pseudomonas fluorescens and Enterbacter sp.
on Sorghum biclor: root colonization and plant growth promotion of
dual strain inocula. Soil Biol. Biochem. 30, 81–87.
Clawson, M.L., Benson, D.R., 1999. Natural diversity of Frankia strains in
actinorhizal root nodules from promiscuous hosts in the family
Myricaceae. Appl. Environ. Microbiol. 65, 4521–4527.
Cleland, R.E., 1990. Auxin and cell elongation, in: Davies, P.J. (Ed.), Plant
Hormones and their Role in Plant Growth and Development. Kluwer
Academic Publishers, Dordrecht, The Netherlands, pp. 132–148.
Cournoyer, B., Normand, P., 1994. Characterization of a spontaneous
thiostrepton-resistant Frankia alni infective isolate using PCR-RFLP of
nif and glnII genes. Soil Biol. Biochem. 26, 553–559.
Crannell, W.K., Tanaka, Y., Myrold, D.D., 1994. Calcium and pH
interaction on root nodulation of nursery-grown red alder (Alnus
rubra Bong.) seedlings by Frankia. Soil Biol. Biochem. 26, 607–614.
Crowley, D.E., Reid, C.P.P., Szaniszlo, P.J., 1988. Utilization of microbial
siderophores in iron acquisition by oat. Plant Phys. 87, 680–685.
Cullimore, J.V., Ranjeva, R., Bono, J.J., 2001. Perception of lipo-
chitooligosaccharidic Nod factors in legumes. Trends Plant Sci. 6,
24–30.
Dashti, N., Zhang, F., Hynes, R., Smith, D.L., 1997. Application of plant
growth-promoting rhizobacteria to soybean (Glycine max (L.) Merr.)
increases protein and dry matter yield under short-season conditions.
Plant Soil 188, 33–41.
Dashti, N., Prithiviraj, B., Hynes, R.K., Smith, D.L., 2001. Root and
rhizosphere colonization of soybean (Glycine max (L.) Merr.) by plant-
growth-promoting rhizobacteria at low root zone temperatures and
under short-season conditions. J. Agron. Crop Sci. 185, 15–22.
De Freitas, J.R., Germida, J.J., 1991. Pseudomonas cepacia and
Pseudomonas putida as winter wheat inoculants for biocontrol of
Rhizobium solani. Can. J. Microbiol. 37, 780–789.
Degrassi, G., Aguilar, C., Bosco, M., Zahariev, S., Pongor, S., Venturi, V.,
2002. Plant growth-promoting Pseudomonas putida WCS358 produces
and secretes four cyclic dipeptides: cross-talk with quorum sensing
bacterial sensors. Curr. Microbiol. 45, 250–254.
de Lajudie, P., Willems, A., Nick, G., Moreira, F., Molouba, F., Hoste, B.,
Torck, U., Neyra, M., Collins, M.D., Lindstrom, K., Dreyfus, B.,
Gillis, M., 1998a. Characterization of tropical tree rhizobia and
description of Mesorhizobium plurifarium sp. nov. Int. J. Syst.
Bacteriol. 48, 369–382.
de Lajudie, P., Laurent-Fulele, E., Willems, A., Torck, U., Coopman, R.,
Collins, M.D., Kersters, K., Dreyfus, B., Gillis, M., 1998b. Allorhizo-
bium undicola gen. nov., sp. nov., nitrogen-fixing bacteria that
efficiently nodulate Neptunia natans in Senegal. Int. J. Syst. Bacteriol.
48, 1277–1290.
Demont-Caulet, N., Maillet, F., Tailer, D., Jacquinet, J.C., Prome, J.C.,
Nicolaou, K.C., Truchet, G., Beau, J.M., Denarie, J., 1999. Nodule-
inducing activity of synthetic Sinorhizobium meliloti nodulation factors
and related lipo-chitooligosaccharides on alfalfa. Importance of the acyl
chain structure. Plant Phys. 120, 83–92.
Denarie, J., Debelle, F., 1996. Rhizobium lipo-chitooligosaccharide
nodulation factors: signaling molecules mediating recognition and
morphogenesis. Ann. Rev. Biochem. 65, 503–535.
Diem, H.G., Dommergues, Y.R., 1985. In vitro production of specialized
reproductive torulose hyphae by Frankia strain ORS 021001 isolated
from Casuarina junghuhniana root nodules. Plant Soil 81, 17–29.
Dreyfus, B., Garcia, J.L., Gillis, M., 1988. Characterization of Azorhizo-
bium caulinodans gen. nov., sp. nov., a stem-nodulating nitrogen-fixing
bacterium isolated from Sesbania rostrata. Int. J. Syst. Bacteriol. 38,
89–98.
Dubeikovesky, A.N., Modokhova, E.A., Kocheskov, V.V.,
Polikarpova, F.P., Boronin, A.M., 1993. Growth promotion of black-
currant softwood cuttings by recombinant strain Pseudomonas fluor-
escens BSP53a synthesizing an increased amount of indole-3-acetic
acid. Soil Biol. Biochem. 25, 1211–1221.
Dubnau, D., 1991. Genetic competence in B. subtilis. Microbiol. Rev. 55,
395–424.
Du Cros, E.T., Jung, G., Bariteau, M., 1984. Alder-Frankia interaction and
alder–poplar association for biomass production. Plant Soil 78,
235–243.
Ebel, J., Mithofer, A., 1998. Early events in the elicitation of plant defense.
Planta 206, 335–348.
Ehrhardt, D.W., Wais, R., Long, S.R., 1996. Calcium spiking in plant root
hairs responding to Rhizobium nodulation signals. Cell 85, 673–681.
Esseling, J.J., Lhuissier, F.G.P., Emons, A.M.C., 2003. Nod factor-induced
root hair curling: continuous polar growth towards the point of nod
factor application. Plant Physiol. 132, 1982–1988.
 E.J. Gray, D.L. Smith / Soil Biology & Biochemistry 37 (2005) 395–412
Etzler, M.E., Kalsi, G., Ewing, N.N., Roberts, N.J., Day, R.B.,
Murphy, J.B., 1999. A Nod factor binding lectin with apyrase activity
from legume roots. PNAS 96, 5856–5861.
Felle, H.H., Kondorosi, E., Kondorosi, A., Schultze, M., 1998. The role of
ion fluxes in Nod factor signaling in Medicago sativa. Plant J. 13,
455–463.
Fisher, R.F., Egelhoff, T.T., Mulligan, J.T., Long, S.R., 1988. Specific
binding of proteins from Rhizobium meliloti cell-free extracts contain-
ing NodD to DNA sequences upstream of inducible nodulation genes.
Genes Dev. 2, 282–293.
Foster, R.C., Rovira, A.D., Cock, T.W., 1983. Ultrastructure of the Root–
Soil Interface. The American Phytopathological Society, St Paul, MN
p. 157.
Fray, R.G., 2002. Altering plant–microbe interaction through artificially
manipulating bacterial quorum sensing. Ann. Bot. 89, 245–253.
Garcia de Salamone, I.E., Hynes, R.K., Nelson, L.M, 2001. Cytokinin
production by plant growth promoting rhizobacteria and selected
mutants. Can. J. Microbiol. 47, 404–411.
Gardner, I.C., Clelland, D.M., Scott, A., 1984. Mycorrhizal improvement in
non-leguminous nitrogen-fixing associations with particular reference
to Hippophae rhamnoides L. Plant Soil 78, 189–199.
Geiger, O., Glushka, J., Lugtenberg, B.J.J., Spaink, H.P., Thomas-
Oates, J.E., 1998. NodFE-dependent fatty acids that lack an alpha–
beta unsaturation are subject to differential transfer, leading to novel
phospholipids. Mol. Plant Microbe Inter. 11, 33–44.
Geurts, R., Bisseling, T., 2002. Rhizobium Nod factor perception and
signaling. Plant Cell 14, S239–S249.
Glick, B.R., 1995. The enhancement of plant growth by free-living bacteria.
Can. J. Microbiol. 41, 109–117.
Glick, B.R., Penrose, D.M., Li, J., 1998. A model for the lowering of plant
ethylene concentrations by plant growth promoting bacteria. J. Theor.
Biol. 190, 63–68.
Glick, B.R., 2004. Bacterial ACC deaminase and the alleviation of plant
stress. Adv. Appl. Microbiol. 56, 291–312.
Goedhart, J., Rohrig, H., Hink, M.A., Van Hoek, A., Visser, A.J.,
Bisseling, T., Gadella, T.W., 1999. Nod factors integrate spontaneously
in biomembranes and transfer rapidly between membranes and to
root hairs, but transbilayer flip-flop does not occur. Biochemistry 28,
10898–10907.
Gray, K.M., 1997. Intercellular communication and group behavior in
bacteria. Trends Microbiol. 5, 184–188.
Gressent, F., Drouillard, S., Mantegazza, N., Samain, E., Geremia, R.A.,
Canut, H., Niebel, A., Driguez, H., Ranjeva, R., Cullimore, J.,
Bono, J.J., 1999. Ligand specificity of a high-affinity binding site for
lipo-chitooligosaccharidic Nod factors in Medicago cell suspension
cultures. PNAS 96, 4704–4709.
Grossman, A.D., 1995. Genetic networks controlling the initiation of
sporulation and the development of competence in Bacillus subtilis.
Ann. Rev. Genet. 29, 477–508.
Guerinot, M.L., Chelm, B.K., 1984. Isolation and expression of the
Bradyrhizobium japonicum adenylate cyclase gene (cya) in Escherichia
coli. J. Bacteriol. 159, 1068–1071.
Guterson, N.L., Layton, T.J., Ziegle, J.S., Warren, G.J., 1986. Molecular
cloning of genetic determinants for inhibition of fungal growth by a
fluorescent pseudomonas. J. Bacteriol. 165, 696–703.
Gutierrez Manero, F.J., Acero, N., Lucas, J.A., Probanza, A., 1996. The
influence of native rhizobacteria on European alder (Alnus glutinosa L.
Gaertn.) growth. II. Characterization and biological assays of
metabolites from growth promoting and growth inhibiting bacteria.
Plant Soil 182, 67–74.
Gutierrez Munero, F.J., Ramos-Solano, B., Probanza, A., Mehouachi, J.,
Tadeo, F.R., Talon, M., 2001. The plant-growth-promoting rhizobac-
teria Bacillus pumilus and Bacillus licheniformis produce high amounts
of physiologically active gibberellins. Physiol. Plant 111, 206–211.
Hackett, B.P., 1985. Hormonal regulation of leaf senescence in Rumex:
ethylene and cytokinins. Dissertation Abstracts International B
Sciences & Engineering 46 (4) 1018B.
409
Hagen, G., 1990. The control of gene expression by auxin, in: Davies, P.J.
(Ed.), Plant Hormones and their Role in Plant Growth and Develop-
ment. Kluwer Academic Publishers, Dordrecht, The Netherlands,
pp. 149–163.
Hallmann, J., Quadt-Hallmann, A., Mahaffee, W.F., Kloepper, W., 1997.
Bacterial endophytes in agricultural crops. Can. J. Microbiol. 43,
895–914.
Halverson, L.J., Handelsman, J., 1991. Enhancement of soybean nodulation
by Bacillus cereus UW85 in the field and in a growth chamber. Appl.
Environ. Microbiol. 57, 2767–2770.
Handelsman, J., Raffel, S., Mester, E.H., Wunderlich, L., Crau, C.R., 1990.
Biological control of damping-off of alfalfa seedlings with Bacillus
cereus UW85. Appl. Environ. Microbiol. 56, 713–718.
Hause, B., Maier, W., Miersch, O., Kramell, R., Strack, D., 2002. Induction
of jasmonate biosynthesis in arbuscular mycorrhizal barley roots. Plant
Physiol. 130, 1213–1220.
Hellriegel, H., Wilfarth, H., 1888. Untersuchungen uber die Stickstoffnah-
rung der Gramineen und Leguminosen. Beilageheft zu der Zeitschrift
des Vereins fur Rubenzucker-Industrie Deutschen Reichs 1888;, 234.
Hirsh, A., 1999. Role of lectins (and rhizobial exopolysarrharides) in
legume nodulation. Curr. Opin. Plant Biol. 2, 320–326.
Hirsh, A.M., Lum, M.R., Downie, J.A., 2001. What makes the rhizobia–
legume symbiosis so special?. Plant Physiol. 127, 1484–1492.
Horvath, B., Kondorosi, E., John, M., Schmidt, J., Torok, I., Gyorgypal, Z.,
Barabas, L., Weineke, U., Schell, J., Kondorosi, A., 1986. Organization
structure and symbiotic function of Rhizobium meliloti nodulation
genes determining host specificity for alfalfa. Cell 46, 335–343.
Hungria, M., Stacey, G., 1997. Molecular signals exchanged between host
plants and rhizobia: basic aspects and potential application in
agriculture. Soil Biol. Biochem. 29, 819–830.
Inbar, J., Chet, I., 1991. Evidence that chitinase produced by Aeromonas
caviae is involved in the biological control of soil-borne plant
pathogens by bacterium. Soil Biol. Biochem. 23, 974–978.
Ip, H., D’Aoust, F., Begum, A.A., Zhang, H., Smith, D.L., Driscoll, B.T.,
Charles, T.C., 2001. Bradyrhizobium japonicum mutant with enhanced
sensitivity to genistein resulting in altered nod gene regulation. Mol.
Plant–Microbe Inter. 14, 1404–1410.
Jiang, J., Yang, H., Huang, J., Zhu, B., Liu, H., Zhong, Y., Bifang, Y., 1984.
Isolation and cultivation of Frankia strain ACC13 from root nodules of
Alnus cremastogyne. Wei Sheng Wu Hsueh Pao 24, 37–40.
John, M., Schmidt, J., Walden, R., Czaja, I., Dulz, M., Schell, J., Rohrig, H.,
1997. Lipochitooligosaccharide-induced tobacco cells release a peptide
as mediator of the glycolipid signal. PNAS 94, 10178–10182.
Keel, C., Schnider, U., Maurhofer, M., Voisard, C., Laville, J., Burger, U.,
Wirthner, P., Haas, D., Defago, G., 1992. Suppression of root diseases
by Pseudomonas fluorescens CHAO: importance of the bacterial
secondary metabolite 2, 4-diacetylphloroglucinol. Mol. Plant Microbe
Inter. 5, 4–13.
Khan, W., Prithiviraj, B., Smith, D.L., 2003. Photosynthetic responses of
corn and soybean to foliar application of salicylates. J. Plant Physiol.
160, 485–492.
Kloepper, J.W., Schroth, M.N., 1978. Plant growth-promoting rhizobac-
teria on radishes, Fourth International Conference on Plant Pathogen
Bacteria, Angers, France, vol. 2 1978 pp. 879–882.
Kloepper, J.W., Schroth, M.N., Miller, W., 1980a. Effects of rhizosphere
colonization by plant growth-promoting rhizobacteria on potato plant
development and yield. Ecol. Epidemiol. 70, 1078–1082.
Kloepper, J.W., Leong, J., Teintze, M., Schroth, M.N., 1980b. Enhanced
plant growth by siderophores produced by plant growth-promoting
rhizobacteria. Nature 286, 885–886.
Kluepfel, D.A., McInnis, T.M., 1991. Biological control of ring nematodes
by Pseudomonas aureofaciens. Phytopathology 81, 1178–1186.
Knight, C.D., Rossen, L., Robertson, J.G., Wells, B., Downie, J.A., 1986.
Nodulation inhibition by Rhizobium leguminosarum multi copy nod
ABC genes and analysis of early stages of plant infection. J. Bacteriol.
166, 552–558.
410 E.J. Gray, D.L. Smith / Soil Biology & Biochemistry 37 (2005) 395–412
Kobayashi, M., Suzuki, T., Fujita, T., Masuda, M., Shimizu, S., 1995.
Occurrence of enzymes involved in biosynthesis of indole-3-acetic acid
from indole-3-acetonitrile in plant-associated bacteria, Agrobacterium
and Rhizobium. PNAS 92, 714–718.
Korem, M., Sheoran, A.S., Gov, Y., Tzipori, S., Borovok, I., Balaban, N.,
2003. Characterization of RAP, a quorum sensing activator of
Staphylococus aureus. FEMS Microbiol. Lett. 223, 167–175.
Lambert, B., Joos, H., Dierick, S., Vantomme, R., Swings, J., Kerters, K.,
Van Montagu, M., 1990. Identification and plant interaction of
Phyllobacterium sp., a predominant rhizobacterium of young sugar
beat. Appl. Environ. Microboil. 56, 1093–1102.
Lancelle, S.A., Torrey, J.G., Hepler, P.K., Callaham, D.A., 1985.
Ultrastructure of freeze-substituted Frankia strain HFPCcI3, the
actinomycete isolated from root nodules of Casuarina cunning-
hamiana. Protoplasma 127, 64–72.
Law, G.J., Northrop, A.J., 1994. Synthetic peptides to mimic the role of
GTP binding proteins in membrane traffic and fusion. Ann. NY Acad.
Sci. 710, 196–208.
Lee, C.A., 1997. Type III secretion systems: machines to deliver bacterial
proteins into eukaryotic cells?. Trends Microbiol. 5, 149–156.
Lee, S.J., Park, S.Y., Lee, J.J., Yum, D.Y., Koo, B.T., Lee, J.K., 2002.
Genes encoding the N-acyl homoserine lactone-degrading enzyme are
widespread in many subspecies of Bacillus thuringiensis. Appl.
Environ. Microbiol. 68, 3919–3924.
Leigh, J.A., Lee, C.C., 1988. Characterization of polysaccharides of
Rhizobium meliloti exo mutants that form ineffective nodules.
J. Bacteriol. 170, 3327–3332.
Lerouge, P., Roche, P., Faucher, C., Maillet, F., Truchet, G., Prome, J.C.,
Denarie, J., 1990. Symbiotic host-specificity of Rhizobium meliloti is
determined by a sulfated and acylated glucosamine oligosaccharide
signal. Nature 344, 781–784.
Li, D., Alexander, M., 1988. Co-inoculation with antibiotic-producing
bacteria to increase colonization and nodulation by rhizobia. Plant Soil
108, 211–219.
Lian, B., Prithiviraj, B., Souleimanov, A., Smith, D.L., 2001. Evidence for
the production of chemical compounds analogous to Nod factor by the
silicate bacterium Bacillus circulans GY92. Micro Res. 156, 289–292.
Lim, H., Kim, Y., Kim, S., 1991. Pseudomonas stutzeri YPL-1 genetic
transformation and antifungal mechanisms against Fusarium solani, an
agent of plant root rot. Appl. Environ. Microbiol. 57, 510–516.
Lindstrom, K., 1989. Rhizobium galegae, a new species of legume root
nodule bacteria. Int. J. Syst. Bacteriol. 39, 365–367.
Lithgow, J.K., Wilkinson, A., Hardman, A., Rodelas, B., Wisniewski-
Dye, F., Williams, P., Downie, J.A., 2000. The regulatory locus cinR1
in Rhizobium leguminosarum controls a network of quorum-sensing
loci. Mol. Microbiol. 37, 81–97.
Lodewyckx, C., Vangronsveld, J., Porteous, F., Moore, E.R.B., Taghavi, S.,
Mezgeay, M., Van der Lelie, D., 2002. Endophytic bacteria and their
potential applications. Crit. Rev. Plant Sci. 21, 583–606.
Loh, J., Garcia, M., Stacey, G., 1997. NodV and NodW, a second flavanoid
recognition system regulating nod gene expression in Bradyrhizobium
japonicum. J. Bacteriol. 179, 3013–3020.
Loh, J., Yuen-Tsai, J.P.Y., Welborn, A., Stacey, G., 2001. Population
density-dependent regulation of the Bradyrhizobium japonicum nodu-
lation genes. Mol. Microbiol. 42, 3746.
Loh, J., Pierson, E.A., Pierson, L.S., Stacey, G., Chatterjee, A., 2002.
Quorum sensing in plant-associated bacteria. Curr. Opin. Plant Biol. 5,
1–6.
Lynch, D.H., Smith, D.L., 1993. Soybean (Glycine max (L.) Merr.)
nodulation and N2 fixation as affected by period of exposure to a low
root zone temperature. Physiol. Plant 88, 212–223.
Mahaffee, W.F., Kloepper, J.W., 1997. Temporal changes in the bacterial
communities of soil, rhizosphere, and endorhiza associated with field-
grown cucumber (Cucumis sativus L.). Microb. Ecol. 34, 210–223.
Mansour, S.R., Dewedar, A., Torrey, J.G., 1990. Isolation, culture, and
behavior of Frankia strain HFPCgI4 from root nodules of Casuarina
glauca. Bot. Gaz. 151, 490–496.
Martinez-Romero, E., Wang, E.T., 2000. Sesbania herbacea–Rhizobium
huautlense nodulation in flooded soils and comparative characterization
of S. herbaces nodulating rhizobia in different environments. Microbe
Ecol. 41, 25–32.
Mathesius, U., Mulders, S., Gao, M., Teplitski, M., Caetano-Anolles, G.,
Rolfe, B.G., Bauer, W.D., 2003. Extensive and specific responses of a
eukaryote to bacterial quorum-sensing signals. PNAS 100, 1444–1449.
Maurhofer, M., Keel, C., Schnider, U., Voisard, C., Hass, D., Defago, G.,
1992. Influence of enhanced antibiotic production in Pseudomonas
fluorescens strain CHAO on its disease suppressive capacity.
Phytopathology 82, 190–195.
Mayak, S., Tirosh, T., Glick, B.R., 1999. Effect of wild-type and mutant
plant growth-promoting rhizobacteria on the rooting of mung bean
cuttings. J. Plant Growth Reg. 18, 49–53.
McKay, I.A., Djordjevic, M.A., 1993. Production and excretion of Nod
metabolites by Rhizobium legminosarum bv. trifolii are disrupted by the
same environmental factors that reduce nodulation in the field. Environ.
Microbiol. 59, 3385–3392.
Metraux, J.P., 2001. Systemic acquired resistance and salicylic acid:
current state of knowledge. Eur. J. Plant Pathol. 107, 13–18.
Miklashevichs, E., Rohig, H., Schell, J., Schmidt, J., 2001. Perception and
signal transduction of rhizobial NOD factors. Crit. Rev. Plant Sci. 20,
373–394.
Mueller, M.J., Brodschelm, W., 1994. Quantification of jasmonic acid by
capillary gas chromatography—negative chemical ionization—mass
spectrometry. Analyt. Biochem. 218, 425–435.
Mulligan, J.T., Long, S.R., 1989. A family of activator genes regulates
expression of Rhizobium meliloti nodulation genes. Genetics 122, 7–18.
Mylona, P., Pawlowski, K., Bisseling, T., 1995. Symbiotic nitrogen
fixation. Plant Cell 7, 869–885.
Nealson, K.H., Hastings, J.W., 1979. Bacterial bioluminescence: its control
and ecological significance. Microbiol. Rev. 43, 496–518.
Nelson, E.B., 1998. Biological control of pythium seed rot and
preemergence damping-off of cotton with Enterobacter cloacae and
Ervinis herbicola applied as seed treatments. Plant Dis. 72, 140–142.
Nick, G., de Lajudie, P., Eardly, B.D., Soumalainen, S., Paulin, L.,
Zhang, X., Gillis, M., Lindstrom, K., 1999. Sinorhizobium arboris sp.
nov. and Sinorhizobium kostiense sp. nov., isolated from leguminous
trees in Sudan and Kenya. Int. J. Syst. Bacteriol. 49, 1359–1368.
Nickel, A., Pelz, O., Hahn, D., Saurer, M., Siegwolf, R., Zeyer, J., 2001.
Effect of inoculation and leaf litter amendment on establishment of
nodule-forming Frankia populations in soil. Appl. Environ. Microbiol.
67, 2603–2609.
Niebel, A., Bono, J.J., Ranjeva, R., Cullimore, J.V., 1997. Identification of a
high affinity binding site for lipo-oligosaccharidic NodRm factors in the
microsomal fraction of Medicago cell suspension cultures. Mol. Plant
Microbe Inter. 10, 132–134.
Nour, S.M., Fernandez, M.P., Normand, P., Cleyet-Marel, J.C., 1994.
Rhizobium ciceri sp. nov., consisting of strains that nodulate chickpeas
(Cicer arietinum L.). Int. J. Syst. Bacteriol. 44, 511–522.
Nowak, J., 1998. Benefits of in vitro ‘biotization’ of plant tissue cultures
with microbial inoculants. In Vitro Cell. Dev. Biol. Plant 34, 122–130.
Oldroyd, G.E., Engstrom, E.M., Long, S.R., 2001a. Ethylene Inhibits the
Nod factor signal transduction pathyway of Medicago truncatula. Plant
Cell 13, 1835–1849.
Oldroyd, G.E., Mitra, R.M., Wais, R.J., Long, S.R., 2001b. Evidence for
structurally specific negative feedback in the Nod factor signal
transduction pathway. Plant J. 28, 191–199.
Ordentlich, A., Elad, Y., Chet, I., 1988. The role of chitinase of Serratia
marcescens for control of Sclerotium rolfsii. Soil Boil. Biochem. 19,
747–751.
Oremus, P.A.I., 1980. Occurrence and infective potential of the endophyte
Frankia of Hippophae rhamnoides L. ssp. rhamnoides in coastal sand-
dune areas. Plant Soil 56, 123–139.
O’Sullivan, D.J., O’Gara, F., 1992. Traits of fluorescent Pseudomonas spp.
involved in suppression of plant root pathogens. Microbiol. Rev. 56,
662–676.
 E.J. Gray, D.L. Smith / Soil Biology & Biochemistry 37 (2005) 395–412
Pan, B., Vessey, J.K., Smith, D.L., 2002. Response of field-grown soybean
to co-inoculation with the plant growth promoting rhizobacteria
Serratia proteamaculans or Serratia liquefaciens, and Bradyrhizobium
japonicum pre-incubated with genistein. Eur. J. Agron. 17, 143–153.
Parsons, R., Sunley, R., 2001. Nitrogen nutrition and the role of root–shoot
nitrogen signalling particularly in symbiotic systems. J. Exp. Bot. 52,
435–443.
Penmetsa, R.V., Cook, D.R., 1997. A legume ethylene-insensitive mutant
hyperinfected by its rhizobial symbiont. Science 275, 527–530.
Perret, X., Staehelin, C., Broughton, W.J., 2000. Molecular basis of
symbiotic promiscuity. Microbiol. Mol. Cell Biol. Rev. 64, 180–201.
Pinton, R., Varanini, Z., Nannipieri, P., 2001. The rhizosphere as a site of
biochemical interactions among soil components, plants, and microor-
ganisms, in: Pinton, R., Varanini, Z., Nannipieri, P. (Eds.), The
Rhizosphere. Mercel Dekker, Inc, New York, USA, pp. 1–18.
Prithiviraj, B., Souleimanov, A., Zhou, X., Smith, D.L., 2000. Differential
response of soybean (Glycine max (L.) Merr) genotypes to lipo-chito-
oligosaccharide Nod Bj V (C18:1 MeFuc). J. Exp. Bot. 51, 2045–2051.
Prithiviraj, B., Zhou, X., Souleimanov, A., Kahn, W., Smith, D.L., 2003. A
host-specific bacteria-to-plant signal molecule (Nod factor) enhances
germination and early growth of diverse crop plants. Planta 216, 437–
445.
Probanza, A., Lucas Garcia, J.A., Ruiz Palomino, M.R., Ramos, B.,
Gutierrez Manero, F.J., 2002. Pinus pinea L. seedling growth and
bacterial rhizosphere structure after inoculation with PGPR Bacillus
(B. licheniformis CET 5106 and B. pumilus CECT 5105). Appl. Soil
Ecol. 20, 75–84.
Provorov, N.A., Tikhonovich, I.A., 2003. Genetic resources for improving
nitrogen fixation in legume–rhizobia symbiosis. Genet. Resour. Crop
Evol. 50, 89–99.
Rai, A.N., Soderback, E., Bergman, E., 2000. Cyanobacterium-plant
symbiosis. New Phytol. 147, 449–481.
Ramos, H.C., Hoffmann, T., Nedjari, H., Presecan-Siedel, E., Dreesen, O.,
Glaser, P., Jahn, D., 2000. Fermentative metabolism of Bacillus subtilis:
physiology and regulation of gene expression. J. Bacteriol. 182,
3072–3080.
Ramos, B., Lucas Garcia, J.A., Probanza, A., Barrientos, M.L., Gutierrez
Manero, F.J., 2003. Alterations in the rhizobacterial community
associated with European alder growth when inoculated with PGPR
strain Bacillus licheniformis. Environ. Exp. Bot. 2003, 61–68.
Rasmussen, U., Svenning, M.M., 1998. Fingerprinting of cyanobacteria
based on PCR with primers derived from short and long tandemly
repeated repetitive sequences. Appl. Environ. Microbiol. 64, 265–272.
Reddell, P., Spain, A.V., 1991. Transmission of infective Frankia
(actinomycetales) propagules in casts of the endogeic earthworm
Pontoscolex corethrurus (Oligochaeta: Glossoscolecidae). Soil Biol.
Biochem. 23, 775–778.
Reymond, P., Farmer, E.E., 1998. Jasmonate and salicylate as global
signals for defense gene expression. Curr. Opin. Plant Biol. 5, 404–411.
Rodriguez, D., Oijen, M., van Schapendonk, A.H.M.C, Lingra, C.C., 1999.
A sink-source model to simulate the impact of climate change and
management on grassland productivity. New Phytol. 144, 359–368.
Rojas, N.S., Li, C.Y., Perry, D.A., Ganio, L.M., 2001. Frankia and
nodulation of red alder and snowbrush grown on soils from Douglas-fir
forests in the H.J. Andrews experimental forest of Oregon. Appl. Soil
Ecol.: Sect. Agric. Ecosys. Environ. 17, 141–149.
Rome, S., Fernandez, M.P., Brunel, B., Normand, P., Cleyet-Marel, J.C.,
1996. Sinorhizobium medicae sp. nov., isolated from annual Medicago
spp. Int. J. Syst. Bacteriol. 46, 972–980.
Rosa, E.A., 2001. Global climate change: background and sociological
contributions. Soc. Nat. Res. 14, 491–499.
Rosbrook, P.A., Reddell, P., 1995. Isolation of Frankia from root nodules
of three species of Casuarina. Soil Biol. Biochem. 27, 427–429.
Ryder, M.H., Jones, D.A., 1990. Biological control of crown gall, in:
Hornby, D., Cook, R.J., Henis, Y. (Eds.), Biological Control of Soil-
Borne Plant Pathogens. CAB International, Oxford, UK, pp. 45–63.
411
Ryder, M.H., Yan, Z., Terrace, T.E., Rovira, A.D., Tang, W., Correll, R.L.,
1999. Use of strains of Bacillus isolated in China to suppress take-all
and rhizoctonia root rot, and promote seedling growth of glasshouse-
grown wheat in Australian soils. Soil Biol. Biochem. 31, 19–29.
Ryu, C.M., Farag, M.A., Hu, C.H., Reddy, M.S., Wei, H.X., Pare, P.W.,
Kloepper, J.W., 2003. Bacterial volatiles promote growth in Arabi-
dopsis. PNAS 100, 4927–4932.
Scheres, B., Van de Wiel, C., Zalensky, A., Horvath, B., Spaink, H., Van
Eck, H., Zwartkruis, F., Wolters, A.M., Gloudemans, T., Van
Kammen, A., Bisseling, T., 1990. The ENOD12 gene product is
involved in the infection process during the pea-Rhizobium interaction.
Cell 60, 281–294.
Schlaman, H.R.M., Okker, R.J.H., Lugtenberg, B.J.J., 1992. Regulation of
nodulation gene expression by NodD in rhizobia. L.. Bacteriology 174,
5177–5182.
Schmidt, J.S., Harper, J.E., Hoffman, T.K., Bent, A.F., 1999. Regulation of
soybean nodulation independent of ethylene signaling. Plant Physiol.
119, 951–960.
Schnider, U., Blumer, C., Troxler, J., Defago, G., Haas, D., 1994.
Overproduction of the antibiotics 2,4-diacetylphloroglucinol and pyo-
luteorin in Pseudomonas fluorescens strain CHAO, in: Ryder, M.H.,
Stephens, P.M., Bowen, G.D. (Eds.), Improving Plant Productivity with
Rhizosphere Bacteria. Commonwealth Science and Industrial Research
Organization, Adelaide, Australia, pp. 120–121.
Scholla, M.H., Elkan, G.H., 1984. Rhizobium fredii sp. nov., a fast-growing
species that effectively nodulates soybeans. Int. J. Syst. Bacteriol. 34,
484–486.
Segovia, L., Young, J.P.W., Matinez-Romero, E., 1993. Reclassification of
American Rhizobium leguminosarum biovar Phaseoli type I strains as
Rhizobium etli sp. nov. Int. J. Syst. Bacteriol. 43, 374–377.
Singh, U.P., Sarma, B.K., Singh, D.P., 2003. Effect of plant growth-
promoting rhizobacteria and culture filtrate of Sclerotium rolfsii on
phenolic and salicylic acid contents in chickpea (Cicer arietinum). Curr.
Microbiol. 46, 131–140.
Smit, G., Puvanesarajah, V., Carlson, R.W., Barbour, W.M., Stacey, G.,
1992. Bradyrhizobium japonicum nod D1 can be specifically induced
by soybean flavanoids that do not induce the nodYABCSUIJ operon.
J. Biol. Chem. 267, 310–318.
Smith, D.L., Zhang, F., 1999. Composition for Enhancing Grain Yield and
Protein Yield of Legumes Grown under Environmental Conditions that
Inhibit or Delay Nodulation Thereof. US Patent 5922316.
Souleimanov, A., Prithiviraj, B., Carlson, R.W., Jeyaretnam, B.,
Smith, D.L., 2002a. Isolation and characterization of the major Nod
factor of Bradyrhizobium japonicum strain 532C. Microbiol. Res. 157,
25–28.
Souleimanov, A., Prithiviraj, B., Smith, D.L., 2002b. The major Nod factor
Bradyrhizobium japonicum promotes early growth of soybean and corn.
J. Exp. Bot. 53, 1929–1934.
Spaink, H.P., Weinman, J., Djorjevic, M.A., Wijffelman, C.A.,
Okker, R.J.H., Lugtenberg, B.J.J., 1991. A novel highly unsaturated
fatty acid moiety of lipo-oligosaccharide signals determines host
specificity of Rhizobium. Nature 354, 125–130.
Spaink, H.P., Wijfjes, A.H.M., Lugtenberg, B.J.J., 1995. Rhizobium NodI
and NodJ proteins play a role in the efficiency of secretion of lipochitin
oligosaccharides. J. Bacteriol. 177, 6276–6281.
Srinivasan, M., Peterson, D.J., Holl, F.B., 1996. Influence of IAA producing
Bacillus isolates on the nodulation of Phaseolus vulgaris by Rhizobium
etli. Can. J. Microbiol. 42, 1006–1014.
Stacey, G., Luka, S., Sanjuan, J., Banfalvi, Z., Nieuwkoop, A.J., Chun, J.Y.,
Forsberg, L., Carlson, R.W., 1994. nodZ, a unique host-specific
nodulation gene, is involved in the fucosylation of the lipopolysacchar-
ide nodulation signal of Bradyrhizobium japonicum. J. Bacteriol. 176,
620–633.
Stacey, G., Sanjuan, J., Lka, S., Dockendorff, T., Carlson, R.W., 1995.
Signal exchange in the Bradyrhizobium-soybean symbiosis. Soil Biol.
Biochem. 27, 473–483.
412 E.J. Gray, D.L. Smith / Soil Biology & Biochemistry 37 (2005) 395–412
Staehelin, C., Granado, J., Muller, J., Wiemken, A., Mellor, R.B., Felix, G.,
Regenass, M., Broughton, W.J., Boller, T., 1994. Perception of
Rhizobium nodulation factors by tomato cells and inactivation by root
chitinases. PNAS 91, 2196–2200.
Steidle, A., Allesen-Holm, M., Riedel, K., Berg, G., Givskov, M., Molin, S.,
Eberl, L., 2002. Identification and characterization of an N-acylhomo-
serine lactone-dependent quorum-sensing system in Pseudomonas
putida strain IsoF. Appl. Environ. Microbiol. 68, 6371–6382.
Stenlid, G., 1982. Cytokinins as inhibitors of root growth. Physiol. Plant 56,
500–506.
Stougard, J., 2000. Regulators and regulation of legume root nodule
development. Plant Physiol. 124, 531–540.
Sturz, A.V., Christie, B.R., Nowak, J., 2000. Bacterial endophytes:
Potential role in developing sustainable systems of crop production.
Crit. Rev. Plant Sci. 19, 1–30.
Swanson, J.A., Tu, J.K., Ogawa, J.M., Sanga, R., Fisher, R., Long, S.R.,
1987. Extended region of nodulation genes in Rhizobium meliloti 1021:
I. Phenotypes of Tn5 insertion mutants. Genetics 117, 181–189.
Tannii, A., Takeuchi, T., Horita, H., 1990. Biological control of scab, black
scruff and soft rot of potato by seed bacterization, in: Hornby, D.,
Cook, R.J., Henis, Y. (Eds.), Biological Control of Soil-Borne Plant
Pathogens. CAB International, Oxford, UK, pp. 143–146.
Tien, T.M., Gaskins, M.H., Hubbell, D.H., 1979. Plant growth substances
produced by Azospirillum brasilense and their effect on the growth of
pearl millet (Pennisetum americanum L.). Appl. Environ. Microbiol.
37, 1016–1023.
Turner, J.T., Blackman, P.A., 1991. Factors related to peanut yield
increases following Bacillus subtilis seed treatment. Plant Dis. 75,
347–353.
Valverde, C., Wall, L.G., 2002. Nodule distribution on the roots of
actinorhizal Discaria trinervis (Rhamnaceae) growing in pots. Environ.
Exp. Bot. 47, 95–100.
Vanden Bosch, K.A., Newcomb, E.H., 1986. Immunogold localization of
nodule-specific uricase in developing soybean nodules. Planta 167,
198–201.
Vandendergh, P.A., Gonzalez, C.F., 1984. Methods for protecting the
growth of plants employing mutant siderophore producing strains of
Pseudomonas putida. US Patent No. US4 479936.
Van Spronsen, P.C., Tak, T., Rood, A.M.M., Van Brussel, A.A.N.,
Kijne, J.W., Boot, K.J.M., 2003. Salicylic acid inhibits indeterminate-
type nodulation but not determinate-type nodulation. Mol. Plant
Microbe Inter. 16, 83–91.
Velazquez, E., Igual, J.M., Willems, A., Fernandex, M.P., Munoz, E.,
Mateos, P.F., Abril, A., Toro, N., Normand, P., Cervantes, E., Gillis, M.,
Martinez-Molina, E., 2001. Mesorhizobium chacoense sp. nov., a novel
species that nodulates Prosopis alba in the Chaco Arido region
(Argentina). Int. J. Syst. Evol. Microbiol. 51, 1011–1021.
Vessey, K.V., 2003. Plant growth promoting rhizobacteria as biofertlizers.
Plant Soil 255, 571–586.
Visser, S., Danielson, R.M., Parkinson, D., 1991. Field performance of
Elaeagnus commutata and Shepherdia canadensis (Elaeagnaceae)
inoculated with soil containing Frankia and vesicular–arbuscular
mycorrhizal fungi. Can. J. Bot. 69, 1321–1328.
Vlassak, K.M., Vanderleyden, J., 1997. Factors influencing nodule
occupancy by inoculant rhizobia. Crit. Rev. Plant Sci. 16, 163–229.
Vogel, J.P., Woeste, K.E., Theologis, A., Kieber, J.J., 1998. Recessive and
dominant mutations in the ethylene biosynthetic gene ACS5 of
Arabidopsis confer cytokinin insensitivity and ethylene overproduction,
respectively. Plant Biol. 95, 4766–4771.
Voisard, C., Keel, C., Haas, D., Defago, G., 1989. Cyanide production by
Pseudomonas fluorescens helps suppress Black Root Rot of tobacco
under gnotobiotic conditions. EMBO J. 8, 351–358.
Waelkens, F., Voets, T., Vlassak, K., Vanderleyden, J., Van Rhijn, P., 1995.
The nodS gene of Rhizobium tropici CIAT899 is necessary for
nodulation of Phaseolus vulgaris and Leucaena leucocephala. Mol.
Plant Microbe Inter. 8, 147–154.
Wais, R.J., Keating, D.H., Long, S.R., 2002. Structure-function analysis of
Nod factor-induced root hair calcium spiking in Rhizobium–legume
symbiosis. Plant Physiol. 129, 211–224.
Weidhase, R.A., Lehmann, J., Kramell, H., Sembdner, G., Parthier, B., 1987.
Degradation of ribulose-1,5-bisphosphate carboxylase and chlorophyll
in senescing barley leaf segments triggered by jasmonic acid
methylester, and counteraction by cytokinin. Physiol. Plant 69, 161–166.
Wood, N., 2001. Nodulation by numbers: the role of ethylene in symbiotic
nitrogen fixation. Trends Plant Sci. 6, 501–502.
Wood, S.M., Newcomb, W., Nelson, D., 1989. Fine structure of the
microsymbiont of the actinorhizal root nodules of mountain mahogany
(Cercocarpus ledifolius, family Rosaceae). Can. J. Bot. 67, 116–120.
Yan, Z., Reddy, M.S., Kloepper, J.W., 2003. Survival and colonization of
rhizobacteria in a tomato transplant system. Can. J. Microbiol. 49,
383–389.
Yang, S.F., Hoffman, N.E., 1986. Ethylene biosynthesis and its regulation
in higher plants. Ann. Rev. Plant Physiol. 35, 155–189.
Yuen, G.Y., Schroth, M.N., McCain, A.H., 1985. Reduction of Fusarium
wilt of carnation with suppressive soils and antagonistic bacteria. Plant
Dis. 69, 1071–1075.
Zakharova, E., Shcherbakov, A., Brudnik, V., Skripko, N., Sh. Bulkhin, N.,
Ignatov, V., 1999. Biosynthesis of indole-3-acetic acid in Azospirillum
brasilense: insights from quantum chemistry. Eur. J. Biochem. 259,
572–576.
Zhang, F., Smith, D.L., 1995. Preincubation of Bradyrhizobium japonicum
with genistein accelerates nodule development of soybean at sub-
optimal root zone temperatures. Plant Physiol. 108, 961–968.
Zhang, F., Smith, D.L., 1996. Genistein accumulation in soybean (Glycine
max (L.) Merr.) root systems under suboptimal root zone temperatures.
J. Exp. Bot. 47, 785–792.
Zhang, F., Dhasti, N., Hynes, R., Smith, D.L., 1996. Plant growth
promoting rhizobacteria and soybean [Glycine max (L.) Merr.]
nodulation and nitrogen fixation at suboptimal root zone temperatures.
Ann. Bot. 77, 453–459.
Zhang, R., Smith, D.L., 1997. Application of genistein to inocula and soil to
overcome low spring soil temperature inhibition of soybean nodulation
and nitrogen fixation. Plant Soil 192, 141–151.
Zhang, H., Prithiviraj, B., Souleimanov, A., D’Aoust, F., Charles, T.C.,
Driscoll, B.T., Smith, D.L., 2002. The effect of temperature and
genistein concentration on lipo-chitooligosaccharide (LCO) production
by wild-type and mutant strains of Bradyrhizobium japonicum. Soil
Biol. Biochem. 34, 1175–1180.
Zhou, X.M., Mackenzie, A.F., Madramootoo, C.A., Smith, D.L., 1999.
Effects of stem-injected plant growth regulators, with or without
sucrose, on grain production, biomass and photosynthetic activity of
field-grown corn plants. J. Agron. Crop Sci. 183, 103–110.