The difference method is not necessarily reliable for comparing the nitrogen use efficiency of hybrid rice cultivars with different tolerance to low nitrogen conditions

The difference method is one of the most commonly used methods to measure crop nitrogen (N) use efficiency. This study aimed to test the reliability of using this method to evaluate the N use efficiency of low-N tolerant hybrid rice. Two years of N fertilization experiments were conducted to compare yield attributes and N use efficiency measured by the difference method between two hybrid rice cultivars with contrasting tolerance to low N conditions, i.e. Deyou 4727 (D4727) with higher tolerance and Y-liangyou 900 (Y900) with relatively lower tolerance. Results showed that D4727 produced 17-24% higher grain yield than Y900 under zero-N fertilization conditions, confirming the higher tolerance of D4727 to low N conditions. Higher grain weight, harvest index, and internal N use efficiency were responsible for the higher grain yield in D4727 than in Y900. However, recovery efficiency of N determined by the difference method was not consistently different between D4727 and Y900, and physiological N use efficiency (PEN) and agronomic N use efficiency (AEN) determined by the difference method were 31-68% lower in D4727 compared to Y900. The lower PEN and AEN in D4727 than in Y900 were mainly due to a higher grain yield in D4727 than in Y900 under unfertilized conditions. This study suggests that the difference method is not necessarily reliable for comparing the N use efficiency of rice cultivars with different tolerance to low-N conditions, and both crop performance under unfertilized conditions and N responses under fertilized conditions should be considered when using this method to evaluate the N use efficiency of rice.