A simple method for quantitative estimation of rhizosphere pH along root axes through visualization

Abstract

A pH indicator agar gel is widely used in rhizosphere pH studies, but its use was mostly confined to visualization of pH changes. A few complex methods are available to measure pH in agar gels. We improved such methods to enable non-destructive quantification of pH dynamics along a root axis by using an image scanner and image analysis software. A thin agar gel containing Bromocresol Purple was used for 2-dimensional image analysis. A taproot of cowpea was embedded in the agar gel containing 1 mM nitrate, and incubated in the dark at 30°C. Every 2 hours, the agar gel was scanned to capture a full color image that reflected rhizosphere pH. In image analysis, optical properties of the pH indicator showed a linear (R2 = 0.99) relationship between pH and optical density in the pH range of 4.4 to 7.2. This analysis allowed us to map the pH gradient in the rhizosphere at a resolution of 0.2 pH. An apparent proton flux was calculated to integrate rhizosphere pH gradients, which quantifies alkalizing/acidifying abilities of the root at various portions. Using this protocol, we examined the effect of pre-cultural N-levels on alkalizing (proton influx) ability of root at various portions under subsequent uniformly nitrate-fed conditions. Results showed that the estimated cumulative proton production was about two times higher in high-N- than in low-N pre-cultured roots. The ratio of proton flux in high-N- to low-N pre-cultured roots was the greatest at the middle (2.94), followed by the basal (2.08) and the apical (1.23) portions of the root, suggesting that nitrate uptake is partitioned along the root axis.