© 2004 Plant Management Network.

Accepted for publication 14 January 2004. Published 1 March 2004.

Plant Growth Promoting Rhizobacteria (PGPR):

Prospects for New Inoculants
Symposium Homepage
Louise M. Nelson, Vice President (Research), Okanagan University
PDF version College, 3333 University Way, Kelowna BC V1V 1V7
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Corresponding author: Louise M. Nelson. lnelson@ouc.bc.ca

Nelson, L. M. 2004. Plant growth promoting rhizobacteria (PGPR): Prospects for new
inoculants. Online. Crop Management doi:10.1094/CM-2004-0301-05-RV.

Abstract
Root colonizing bacteria (rhizobacteria) that exert beneficial effects on plant
development via direct or indirect mechanisms have been defined as plant
growth promoting rhizobacteria (PGPR). Although significant control of plant
pathogens or direct enhancement of plant development has been demonstrated
by PGPR in the laboratory and in the greenhouse, results in the field have been
less consistent. Because of these and other challenges in screening,
formulation, and application, PGPR have yet to fulfill their promise and potential
as commercial inoculants. Recent progress in our understanding of their
diversity, colonization ability, mechanisms of action, formulation, and
application should facilitate their development as reliable components in the
management of sustainable agricultural systems.

Introduction
Plant growth in agricultural soils is influenced by a myriad of abiotic and
biotic factors. While growers routinely use physical and chemical approaches
to manage the soil environment to improve crop yields, the application of
microbial products for this purpose is less common. An exception to this is the
use of rhizobial inoculants for legumes to ensure efficient nitrogen fixation; a
practice that has been occurring in North America for over 100 years (39).
The region around the root, the rhizosphere, is relatively rich in nutrients, due
to the loss of as much as 40% of plant photosynthates from the roots (26).
Consequently, the rhizosphere supports large and active microbial
populations capable of exerting beneficial, neutral, or detrimental effects on
plant growth. The importance of rhizosphere microbial populations for
maintenance of root health, nutrient uptake, and tolerance of environmental
stress is now recognized (9,13). These beneficial microorganisms can be a
significant component of management practices to achieve the attainable
yield, which has been defined as crop yield limited only by the natural
physical environment of the crop and its innate genetic potential (13).
The prospect of manipulating crop rhizosphere microbial populations by
inoculation of beneficial bacteria to increase plant growth has shown
considerable promise in laboratory and greenhouse studies, but responses
have been variable in the field (9). The potential environmental benefits of
this approach, leading to a reduction in the use of agricultural chemicals and
the fit with sustainable management practices, are driving this technology.
Recent progress in our understanding of the biological interactions that occur
in the rhizosphere and of the practical requirements for inoculant formulation
and delivery should increase the technology’s reliability in the field and
facilitate its commercial development.
Rhizosphere Colonization
Plant growth-promoting rhizobacteria (PGPR) were first defined by
Kloepper and Schroth (23) to describe soil bacteria that colonize the roots of
plants following inoculation onto seed and that enhance plant growth. The
following are implicit in the colonization process: ability to survive
inoculation onto seed, to multiply in the spermosphere (region surrounding
the seed) in response to seed exudates, to attach to the root surface, and to
colonize the developing root system (22). The ineffectiveness of PGPR in the
field has often been attributed to their inability to colonize plant roots (4,8). A
variety of bacterial traits and specific genes contribute to this process, but
only a few have been identified (4,25). These include motility, chemotaxis to
seed and root exudates, production of pili or fimbriae, production of specific
cell surface components, ability to use specific components of root exudates,
protein secretion, and quorum sensing (25). The generation of mutants
altered in expression of these traits is aiding our understanding of the precise
role each one plays in the colonization process (25,31). Progress in the
identification of new, previously uncharacterized genes is being made using
nonbiased screening strategies that rely on gene fusion technologies. These
strategies employ reporter transposons (33) and in vitro expression
technology (IVET) (32) to detect genes expressed during colonization.
Using molecular markers such as green fluorescent protein or fluorescent
antibodies it is possible to monitor the location of individual rhizobacteria on
the root using confocal laser scanning microscopy (7,8,40) (Fig. 1). This
approach has also been combined with an rRNA-targeting probe to monitor
the metabolic activity of a rhizobacterial strain in the rhizosphere and showed
that bacteria located at the root tip were most active (24,40).

Fig. 1. Confocal laser scanning

micrograph of a 5-day old canola root
colonized by Pseudomonas putida
strain 6-8 labelled with green
fluorescent protein (as indicated by
the arrow). The bar is equal to 60 µm.
(From the author's laboratory, photo
by R. Pallai.)
An important aspect of colonization is the ability to compete with
indigenous microorganisms already present in the soil and rhizosphere of the
developing plant. Our understanding of the factors involved in these
interactions has been hindered by our inability to culture and characterize
diverse members of the rhizosphere community and to determine how that
community varies with plant species, plant age, location on the root, and soil
properties. Phenotypic and genotypic approaches are now available to
characterize rhizobacterial community structure. Phenotypic methods that
rely on the ability to culture microorganisms include standard plating
methods on selective media, community level physiological profiles (CLPP)
using the BIOLOG system (17), phospholipid fatty acid (PLFA) (43), and fatty
acid methyl ester (FAME) profiling (18). Culture-independent molecular
techniques are based on direct extraction of DNA from soil and 16S-rRNA
gene sequence analysis, bacterial artificial chromosome or expression cloning
systems (34). These are providing new insight into the diversity of
rhizosphere microbial communities, the heterogeneity of the root
environment, and the importance of environmental and biological factors in
determining community structure (3,5,37). These approaches can also be used
to determine the impact of inoculation of plant growth-promoting
rhizobacteria on the rhizosphere community (12,41).

Mechanisms of Action
PGPR enhance plant growth by direct and indirect means, but the specific
mechanisms involved have not all been well-characterized (20,22). Direct
mechanisms of plant growth promotion by PGPR can be demonstrated in the
absence of plant pathogens (Fig. 2) or other rhizosphere microorganisms,
while indirect mechanisms involve the ability of PGPR to reduce the
deleterious effects of plant pathogens on crop yield. PGPR have been reported
to directly enhance plant growth by a variety of mechanisms: fixation of
atmospheric nitrogen that is transferred to the plant, production of
siderophores that chelate iron and make it available to the plant root,
solubilization of minerals such as phosphorus, and synthesis of
phytohormones (20). Direct enhancement of mineral uptake due to increases
in specific ion fluxes at the root surface in the presence of PGPR has also been
reported (2,6). PGPR strains may use one or more of these mechanisms in the
rhizosphere. Molecular approaches using microbial and plant mutants altered
in their ability to synthesize or respond to specific phytohormones have
increased our understanding of the role of phytohormone synthesis as a direct
mechanism of plant growth enhancement by PGPR (20,31). PGPR that
synthesize auxins and cytokinins or that interfere with plant ethylene
synthesis have been identified (16,20,31).

Fig. 2. Example of growth promotion

of lentil following inoculation with
PGPR isolates, 2-28, 3-10, 3-31, and
3-67. Plants were grown in cone-
tainers at 80°C in a growth chamber
and sampled 11, 17, and 26 days
following inoculation. (From the
author's laboratory.)
PGPR that indirectly enhance plant growth via suppression of
phytopathogens do so by a variety of mechanisms. These include the ability to
produce siderophores that chelate iron, making it unavailable to pathogens;
the ability to synthesize anti-fungal metabolites such as antibiotics (Fig. 3),
fungal cell wall-lysing enzymes, or hydrogen cyanide, which suppress the
growth of fungal pathogens; the ability to successfully compete with
pathogens for nutrients or specific niches on the root; and the ability to
induce systemic resistance (8,20,31). Biochemical and molecular approaches
are providing new insight into the genetic basis of these traits, the
biosynthetic pathways involved, their regulation, and importance for
biological control in laboratory and field studies (8,9,20,31).

Fig. 3. Example of in vitro assay for

inhibition of fungal growth. Different
bacterial isolates were tested for their
ability to inhibit the growth of
Rhizoctonia spp., a soil-borne plant
pathogen of legumes. A zone of
inhibition can be observed around
isolate 4-31 in the upper quadrant of
the plate. (From the author’s
laboratory.)
Challenges in Selection and Characterization of PGPR
One of the challenges in developing PGPR for commercial application is
ensuring that an effective selection and screening procedure is in place, so
that the most promising organisms are identified and brought forward. In the
agricultural chemical industry, thousands of prospective compounds are
screened annually in efficient high-throughput assays to select the best one or
two compounds for further development. Similar approaches are not yet in
place for PGPR. Effective strategies for initial selection and screening of
rhizobacterial isolates are required. It may be important to consider host
plant specificity or adaptation to a particular soil, climatic conditions or
pathogen in selecting the isolation conditions, and screening assays (9,11).
The spermosphere model, an enrichment technique that relies on seed
exudates as the nutrient source, has been used for selection and isolation of
promising N2-fixing rhizosphere bacteria from rice (42). One approach for
selection of organisms with the potential to control soil-borne
phytopathogens is to isolate from soils that are suppressive to that pathogen
(44). Other approaches involve selection based on traits known to be
associated with PGPR such as root colonization (36), 1-aminocyclopropane-1-
carboxylate (ACC) deaminase activity (10,20), and antibiotic (19) and
siderophore production (10). The development of high throughput assay
systems and effective bioassays will facilitate selection of superior strains
(28,29).

Challenges in Field Application of PGPR

The application of PGPR for control of fungal pathogens in greenhouse
systems shows considerable promise (30), due in part to the consistent
environmental conditions and high incidence of fungal disease in
greenhouses. Achieving consistent performance in the field where there is
heterogeneity of abiotic and biotic factors and competition with indigenous
organisms is more difficult. Knowledge of these factors can aid in
determination of optimal concentration, timing and placement of inoculant,
and of soil and crop management strategies to enhance survival and
proliferation of the inoculant (9,29). The concept of engineering or managing
the rhizosphere to enhance PGPR function by manipulation of the host plant,
substrates for PGPR, or through agronomic practices, is gaining increasing
attention (9,27). Development of better formulations to ensure survival and
activity in the field and compatibility with chemical and biological seed
treatments is another area of focus; approaches include optimization of
growth conditions prior to formulation and development of improved carriers
and application technology (1,9,14,28,45).

Challenges in Commercialization of PGPR

Prior to registration and commercialization of PGPR products, a number
of hurdles must be overcome (15,28,29). These include scale up and
production of the organism under commercial fermentation conditions while
maintaining quality, stability, and efficacy of the product. Formulation
development must consider factors such as shelf life, compatibility with
current application practices, cost, and ease of application. Health and safety
testing may be required to address such issues as non-target effects on other
organisms including toxigenicity, allergenicity and pathogenicity, persistence
in the environment, and potential for horizontal gene transfer. The product
claim, whether as a fertilizer supplement or for biological control, will
determine to which federal agency applications for registration should be
addressed in Canada and the USA. Capitalization costs and potential markets
must be considered in the decision to commercialize. McSpadden Gardener
and Fravel (29) estimated that a minimum capitalization of $1 million US is
required to register a biopesticide product in North America.

Future Prospects
As our understanding of the complex environment of the rhizosphere, of
the mechanisms of action of PGPR, and of the practical aspects of inoculant
formulation and delivery increases, we can expect to see new PGPR products
becoming available. The success of these products will depend on our ability
to manage the rhizosphere to enhance survival and competitiveness of these
beneficial microorganisms (9). Rhizosphere management will require
consideration of soil and crop cultural practices as well as inoculant
formulation and delivery (9,29). Genetic enhancement of PGPR strains to
enhance colonization and effectiveness may involve addition of one or more
traits associated with plant growth promotion (8,20,24). Genetic
manipulation of host crops for root-associated traits to enhance establishment
and proliferation of beneficial microorganisms (27,38) is being pursued.
However, regulatory issues and public acceptance of genetically engineered
organisms may delay their commercialization. The use of multi-strain inocula
of PGPR with known functions is of interest as these formulations may
increase consistency in the field (21,35). They offer the potential to address
multiple modes of action, multiple pathogens, and temporal or spatial
variability.
PGPR offer an environmentally sustainable approach to increase crop
production and health. The application of molecular tools is enhancing our
ability to understand and manage the rhizosphere and will lead to new
products with improved effectiveness.

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