Nitrogen is the most limiting nutritional factor for the growth of plants. Since plants cannot reduce atmospheric N2, they require exogenously fixed nitrogen for growth and development. Atmospheric N2 must be first reduced to ammonia to be used by plants. In practice, chemical N fertilizers are used to provide nitrogen nutrition to plants. However, manufacture and use of N fertilizers are associated with environmental hazards that include release of greenhouse gases at the time of manufacture, as well as contamination of underground and surface water due to leaching out of nitrates. Moreover, manufacture of chemical fertilizers requires non-renewable resources like coal and petroleum products. Excess and continuous use of chemical fertilizers to improve the yield of commercial crops has negative effect on soil fertility and reduces their agricultural sustainability. All these concerns necessitate the search for an alternative strategy that can provide nitrogen nutrition to the plants in an efficient and sustainable manner. Here biological nitrogen fixation has immense potential and can be used as an alternate to chemical fertilizers. Biological nitrogen fixation has been reported to be exclusively carried out by few members of the prokaryotic organisms. Biological nitrogen fixation is a process where atmospheric N2 is reduced to NH3. This process is catalyzed by microbial enzyme nitrogenase. Microorganisms having the capacity to fix atmospheric N2 can be used as efficient biofertilizer.In this chapter, we review application, properties, ecology, and advances in biology of nitrogen fixing bacteria with reference to endophytic bacteria that colonize the interior of plant without exerting any substantive harm to their host plant. Nitrogen-fixing endophytic bacteria have edge over its rhizospheric counterparts because, being sheltered inside plant tissues, they face less competition and can make available the fixed nitrogen directly to plants. Moreover, the partial pressure of oxygen inside the plant tissue is more acquiescent for efficient nitrogen fixation. Nitrogen fixing endophytic bacteria have been isolated from several plant species and found to contribute upto 47% of nitrogen derived from air, which in turn enhance plant growth. Nitrogen fixing ability of bacteria can be evaluated by total nitrogen difference method, acetylene reduction assay, analysis of nitrogen solutes in xylem and other plant parts and N-Labeling Methods. Furthermore, molecular approaches such as amplification, analysis of nitrogen-fixing genes (nif genes), and qualitative and quantitative estimation of their products can be used for evaluation of nitrogen fixing ability of the bacteria.In addition to nitrogen-fixation ability, these bacteria can influence plant growth through one or more properties. These include production of phytohormones, siderophores, induced systemic tolerance through production of 1-aminocyclopropane-1-carboxylase deaminase, induced systemic resistance and antagonistic activities. The make-up of endophytic bacterial communities depends on various factors such as soil type, soil composition, soil environment, plant genotype and physiological status, bacterial colonization traits, and agricultural management regimes. Colonization and abundance of different bacterial species varies widely with host plants. Endophytic bacterial community can be analyzed employing stable isotope probing as well as various modern molecular approaches which are based on analysis of 16S ribosomal deoxyribonucleic acid (DNA), gene encoding products for nitrogen fixation and repetitive DNAs. Moreover, metagenomic approaches allow estimation and analysis of unculturable bacteria at genomic as well as functional genomic level. Colonization process of an endophytic bacterium involves various steps which include migration towards root surface, attachment and microcolony formation on plant surface, distribution along root and growth and survival of the population inside plant tissue. Ongoing progress towards in-depth analysis of genomic and whole protein profile of some of the potential endophytic bacteria such as Azoarcus sp., Gluconoacetobacter diazotrophicus, Herbaspirillum seropedicae, Serratia marcesens can help understand mechanism involved in plant-endophyte interaction which in turn will be deterministic in use of suitable formulations of endophytic bacteria to be used as biofertilizer for sustainable agriculture.

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