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A real-time polymerase chain reaction (PCR) method was applied to quantify the nifH gene pool in cucumber shoot and root and to evaluate how nitrogen (N) supply and plant age affect the nifH gene pool. In shoots, the relative abundance of the nifH gene was affected neither by different stages of plant growth nor by N supply. In roots, higher numbers of diazotrophic bacteria were found compared with that in the shoot. The nifH gene pool in roots significantly increased with plant age, and unexpectedly, the pool size was positively correlated with N supply. The relative abundance of nifH gene copy numbers in roots was also positively correlated (r = 0.96) with total N uptake of the plant. The data suggest that real-time PCR-based nifH gene quantification in combination with N-content analysis can be used as an efficient way to perform further studies to evaluate the direct contribution of the N2-fixing plant-colonizing plant growth promoting bacteria to plant N nutrition.