*5.3. Photosynthetic Pigment Content and Leaf Nitrogen Content*

After 14 d of treatment, the third trifoliate leaf on alfalfa seedlings was collected for photosynthetic pigment and leaf nitrogen content measurement. Chlorophyll a (Chl a), Chlorophyll b (Chl b) and carotenoids (Car) were extracted from all leaf samples. Six leaf discs (0.6 cm in diameter) were cut from the middle part of each middle leaflet with a puncher, and they were placed in 25 mL of 95% acetone in the dark for 24 h, at which time the sample was colorless. Concentration of Chl a, Chl b and Car was measured at wavelengths of A663, A<sup>645</sup> and A<sup>470</sup> nm, respectively, using a UV spectrophotometer (UV-2700, Shimadzu, Kyoto, Japan), and calculated according to Pan et al. (2020) [18].

The dried leaf tissues were ground to obtain homogenous samples. A subsample of about 0.1 g was then digested with 5 mL of concentrated H2SO<sup>4</sup> for 2 h in 420 ◦C, and K2SO<sup>4</sup> and CuSO4·5H2O (K2SO4:CuSO4·5H2O = 10:1) as the catalyzer. Finally, the leaf nitrogen concentration was measured by an automatic flow injection analyzer (AA3, SEAL).
