Plant Growth Promoting Rhizobacteria (PGPR): Prospects for New Inoculants

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Louise M. Nelson, Vice President (Research), Okanagan University College, 3333 University Way, Kelowna BC V1V 1V7

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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