Abstract: Relationship among nitrogen application rate, biochemical substrate content, such as flavonoid, phenol, soluble sugar, free amino acid, as well as leaf nitrogen at topping, and leaf brown spot occurrence after topping were studied in two different climatic counties, Shilin and Xundian, respectively. Results showed that the relationship mentioned above was similar in leaves on different positions of the stalk at two experimental sites. Higher nitrogen application rate caused higher accumulation of N and free amino acid content, but less flavonoid, phenol and soluble sugar. In addition, increased severity of leaf brown spot was observed with increasing nitrogen rate treatment.Significant negative correlations existed between the content of flavonoid, phenol, soluble sugar and the occurrence of the brown spot disease, while positive correlation was observed between the content of free amino acid and N in leaf and the occurrence of the brown spot. Result suggested that the different N treatment may cause flavonoid, phenol, soluble sugar, free amino acid content difference at topping, and the brown spot severity difference after topping. Result also recommended that the moderate application of nitrogen rate should be considered for the control of brown spot disease under experimental conditions.