Rhizosphere organic acid efflux collection and measurement
The apple plants were treated in hydroponic nutrient solution for 4 days (pH = 5.8), and the nutrient solution was then dried to a powder in a -80℃ freeze dryer. The powder was dissolved with 5 ml of deionized water, the solution was then passed through a cation exchange resin and the filtrate was collected. After lyophilizing again, the sample was dissolved in 5 ml of deionized water and stored at -20℃. The organic acid efflux was analyzed via enzyme-linked immunosorbent assay (ELISA; Hengyuan, Shanghai, China). Organic acid was made into a solid-phase antibody, and horseradish peroxidase (HRP) was then combined with the organic acid antibody to form an antibody-antigenenzyme-labeled antibody complex. After thorough washing, the TMB substrate was added to develop the color, with the TMB converted to blue under the catalysis of HRP and finally converted to yellow under the action of acid. The shade of the color positively correlated with the organic acid concentration in the sample. Finally, the absorbance was measured using a microplate reader at a wavelength of 450 nm. The organic acid concentration was calculated from a standard curve.Effluent malate content = (337.57 ×A450 –9.0216) ×5 ×V / (134.09× M). Effluent citrate content = (506.82 ×A450 –17.148) ×5 ×V /(192.13 × M). (A: absorbance, V: volume of filtrate, M: fresh weight of apple root).
