An Assignment On PLANT GROWTH PROMOTING RHIZOBACTERIA Submitted by:Shrivastava Anukriti Ashok M.

Sc II Year

Submitted to:Dr. Mrs. S.R. Bhalsing DEPARTMENT OF BIOTECHNOLOGY NORTH MAHARASHTRA UNIVERSITY JALGAON.

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. 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. 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. 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. 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. 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 technologys reliability in the field and facilitate its commercial development.

Rhizosphere Colonization Plant growth-promoting rhizobacteria (PGPR) were first defined by Kloepper and Schroth 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. The ineffectiveness of PGPR in the field has often been attributed to their inability to colonize plant roots . A variety of bacterial traits and specific genes contribute to this process, but only a few have been identified . 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 . 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. 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 and in vitro expression technology (IVET) 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. 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. 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 , phospholipid fatty acid (PLFA), and fatty acid methyl ester (FAME) profiling. 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. 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 . These approaches can also be used to determine the impact of inoculation of plant growth-promoting rhizobacteria on the rhizosphere community.

Mechanisms of Action PGPR enhance plant growth by direct and indirect means, but the specific mechanisms involved have not all been well-characterized. Direct mechanisms of plant growth promotion by PGPR can be demonstrated in the absence of plant pathogens 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. 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 . 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 . PGPR that synthesize auxins and cytokinins or that interfere with plant ethylene synthesis have been identified. 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, 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. 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. 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 . 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 . 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. Other approaches involve selection based on traits known to be associated with PGPR such as root colonization, 1aminocyclopropane-1-carboxylate (ACC) deaminase activity, and antibiotic and siderophore production. The development of high throughput assay systems and effective bioassays will facilitate selection of superior strains .

Challenges in Field Application of PGPR The application of PGPR for control of fungal pathogens in greenhouse systems shows considerable promise, 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. 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 . 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 .

Challenges in Commercialization of PGPR Prior to registration and commercialization of PGPR products, a number of hurdles must be overcome. 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 estimated that a minimum capitalization of $1 million US is required to register a biopesticide product in North America. http://www.plantmanagementnetwork.org/pub/cm/review/2004/rhizobacteria/

Applications of plant growth promoting rhizobacteria:1.) Inoculation with two PGPR strains, Serratia sp. and Rhizobium sp., into saline soils alleviated the salinity effects on the antioxidant enzymes ascorbate peroxidase (APX) and glutathione reductase (GR), along with those on photosynthesis, mineral content and growth. As a result, an increase in salinity in the soil caused a physiological response or disorder in lettuce plants. Treatment with PGPR strains could alleviate the effect of potentially toxic ions. 2.) PGPR (Plant growth promoting rhizobacteria) have gained world wide importance and acceptance for agricultural benefits. These microorganisms are the potential tools for sustainable agriculture and the trend for the future. Scientific researches involve multidisciplinary approaches to understand adaptation of PGPR to the rhizosphere, mechanisms of root colonization, effects on plant physiology and growth, biofertilization, induced systemic resistance, biocontrol of plant pathogens, production of determinants etc. Biodiversity of PGPR and mechanisms of action for the different groups: diazotrophs, bacilli, pseudomonads, and rhizobia are shown. Effects of physical, chemical and biological factors on root colonization and the proteomics perspective on biocontrol and plant defence mechanism is discussed. Visualization of interactions of pathogens and biocontrol agents on plant roots using autofluorescent protein markers has provided more understanding of biocontrol process. Commercial formulations and field applications of PGPR are detailed. 3.) Banana requires large amounts of chemical fertilizers which are costly and can be hazardous to the environments when are used excessively. Biological N2 fixation (BNF) technology can play a vital role as substitution to commercially available N-fertilizer in crop production and reduction of environmental problem to some extent. An experiment was conducted in the shade-house of University Putra Malaysia, Malaysia under hydroponics condition using nitrogen-free

plant nutrient solution to evaluate the effect of PGPR (Plant Growth Promoting Rhizobacterial) inoculation on growth and N2 fixation of tissue-cultured banana plantlets under nitrogen (N) free hydroponics condition. The experiment was a completely randomized design with six replicates. There were three treatments viz. T1: (control; N0 -PGPR), T2: (N0 + Sp7) and T3: (N0 + UPMB10). One tissue-cultured banana plantlet (ex-laboratory, about 10-11 cm height of three-leafed stage) cv. Berangan (Musa spp. dessert type) was planted per pot (4.0 L). The results indicated that a remarkable increase in root growth, namely length (33-44%), volume (76-168%) and mass (137-141%) were recorded due to the PGPR inoculation, beside a higher shoot growth (123-202%) and N yield (94-144%). The inoculated plants showed higher formation of root hair which was visible within 7 days of inoculation. The growth attributes namely, leaf area, chlorophyll content, and consequently the total biomass were also increased due to PGPR inoculation. The overall growth performance of inoculated seedlings was higher in compare to un-inoculated control. Thus, it might be concluded that PGPR strains Sp7 and UPMB10 could be used as crop-enhancer and bio-fertilizer for vigor seedling and production of bananas. 4.) Bacillus subtilis (BSCBE4), Pseudomonas chlororaphis (PA23), endophytic P. fluorescens (ENPF1) inhibited the mycelial growth of stem blight pathogen Corynespora casiicola (Berk and Curt)Wei under in vitro. All these bacterial isolates produced both hydroxamate and carboxylate type of siderophores. But the siderophore production was maximum with the isolate ENPF1. Delivering of talc based formulation of BSCBE4 through seedling dip and foliar application effectively reduced stem blight disease incidence and increased the dry matter production under pot culture and field conditions. Application of BSCBE4, PA23 and ENPF1 increased the defense related enzymes such as peroxidase, polyphenol oxidase, chitinase and b1,3 glucanase in P. amarus up to ten days after challenge inoculation with C. cassicola. Native gel electrophoretic analysis revealed that challenge inoculation of pathogen with BSCBE4 and PA23 induced both peroxidase and polyphnol oxidase isoforms. Root Nodule Formation in Rhizobium-Legume Association

Rhizobium occurs free-living in the soil but does not fix nitrogen in this situation. It has recently been studied to fix nitrogen in the laboratory, in the absence of host when supplied with carbolic

acid,

pentose

and

small

amount

of

fixed

nitrogen.

This confirms that it is Rhizobium itself that contains all necessary genetic information for fixing nitrogen but does not imply so, when living-free in the normal soil environments; its association with leguminous roots and formation of nodules seems obligatory to fix nitrogen. The stages envolved in root nodule formation are now fairly understood and include (i) recognition and attachment, (ii) penetration and travel, (iii) bacteroid formation and development of mature nodule. (i) Recognition and Attachment

In response to the variety of organic metabolites secreted by the roots of legume plants, the rhizobia migrate towards and grow in the rhizosphere and build up to high population density. It is considered that a series of flavonoid signals are there in organic metabolites that lead to the exchange of recognition signals thus attracting specific rhizobia species to specific legume roothairs. However, all species of Rhizobium (and Bradyrhizobium) possess specific adhesion protein called rhicadhesin on their surface. Rhicadhesin is a calcium-binding protein and binds calcium complexes on the surface of root hairs. Lectins, carbohydrate containing proteins, also contribute in Rhizobium-Legume attachment. Root Nodule Formation by Rhizobium

1. Migration and growth of rhizobia in rhizosphere

2. Attachment of Rhizobia on Root Hair

3. Root Hair Curling and Penetration by Rhizobium

4. Infection Thread Growth 5. Cell to Cell Spread of Rhizobium and Rhizobial Travel

6. Bacteroids and the Differentiation of Bacteroids into Symbiosomes

7. T.S. of a Single Root Nodule

8. Mature Nodules on Legume Root

(ii) Penetration and Travel:- After attachment, the root hair curls as a result of the action of substances, excreted by the Rhizobium species called nod-factors. Some physiologists believe that curling is also affected by indole acetic acid, a plant growth hormone. After curling of the root-hair, the bacteria penetrate and enter the root-hair and induce the plant to develop a cellulosic tube, called infection thread, which extends inward to the root-hair . The Rhizobium cells then spread within the infection thread, move into the underlying root cells, and are released into cytoplasm of the host cell through the action of an organizer produced by the interaction between the rhizobial polysaccharides and component of root cells. Nod factors now stimulate root cell division eventually leading to the development of the root nodule. (iii) Bacterial Formation and Development of Mature Nodule:- When the bacteria are released from the infection thread into the host cell cytoplasm, they get transformed into swollen, irregular-shaped, branched structures called bacteroids which then become surrounded singly or

in small groups by a plant-derived membrane, called peribacteroid membrane, to form structures called symbiosomes. Symbiosomes are, in fact, the sites of nitrogen fixation. At this point, however, the symbiosomes secrete a hormone which enables the polyploid cells to divide rapidly forming the core of the nodule and the surrounding diploid cells also to divide and differentiate to cover the nodule in cortical tissue and to form vascular connections with the root. This hormone also enables the production of leghaemoglobin which protects the nitrogen fixation enzymes from oxygen. Other specific nodule components are also produced to complete the nodulation process.
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