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Endophytic bacteria represents a unique class of bacteria that can colonize interior tissues of plant and provide a range of benefits to the plant similar to those provided by the rhizospheric bacteria. Certain endophytic bacteria can provide nitrogen to the plants through biological nitrogen fixation, which is an important source of nitrogen input in agriculture and represents a promising substitute for chemical fertilizers, and are known as endophytic diazotrophic bacteria. Besides fixing nitrogen, endophytic bacteria can produce plant growth hormones like auxin and gibberellin, help in nutrient uptake, and increase the plant's tolerance to biotic and abiotic stresses. Various direct and indirect methods have been used to quantify the amount of nitrogen fixed by these bacteria, including the acetylene reduction assay, which is a quick but indirect method, and the 15 N isotopic dilution assay, which is a robust and accurate method. Research on endophytic diazotrophic bacteria has come a long way, and in this chapter, we have briefly discussed the mechanisms of biological nitrogen fixation and methods to quantify the fixed nitrogen along with reviewing recent studies focused on evaluating the role of endophytic diazotrophic bacteria in promoting plant growth in both native and nonnative crop hosts.

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Green fluorescent protein (GFP), a renowned marker protein, is typically believed to be inert in affecting the physiology of host bacteria. We analyzed the effects of GFP-tagging on the ability of an endophytic diazotroph, Paenibacillus polymyxa P2b-2R, to fix nitrogen and promote overall growth of corn plants. The growth response and the amount of nitrogen fixed by P2b-2Rgfp-inoculated plants were compared with uninoculated controls and P2b-2R-inoculated plants at three harvests. P2b-2Rgfp inoculation significantly increased the biomass of corn plants as compared to non-inoculated controls and P2b-2R-treated plants. In vitro tests revealed that strains P2b-2R and P2b-2Rgfp possess various plant-growth-promoting characteristics, namely phosphate solubilization, production of siderophores, IAA, ammonia, and enzymes like cellulase, protease, and catalase. P2b-2Rgfp-inoculated plants fixed 18% atmospheric nitrogen, significantly higher than P2b-2R-inoculated plants (15%). This difference led us to compare the expression of structural nif genes (nifH, nifD, nifK) of strains P2b-2R and P2b-2Rgfp. It was observed that expression levels of structural nif genes of strain P2b-2Rgfp were 1.5-fold higher than those of strain P2b-2R. These results indicate that GFP-tagging positively affects the efficacy of strain P2b-2R to promote plant growth and fix nitrogen, perhaps by increasing the expression levels of structural nif genes.

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