Woodland mixed soil from Nanjing Laoshan Forest Farm(Nanjing, Jiangsu Province) was collected at a depth of 0-20 cm and used as the culture matrix. After 100 days of soil culture, P. massoniana seedlings receiving the same growth conditions were selected as the experimental material. The chemical components of the simulated acid rain were similar to those of natural precipitation, and the acidity was set as follows: pH 6.5 (control), pH 5.6 (micro acid treatment), pH 4.5 (mild acid treatment), pH 3.5 (moderate acid treatment), and pH 2.5 (severe acid treatment). The spray amount, time and frequency of simulated acid rain were set according to the precipitation status of Nanjing from May to August 2017. The root physiological parameters of P. massoniana seedlings were measured on treatment days 30, 60 and 100. Root activity was determined by triphenyl tetrazolium chloride-reduction spectrophotometry, the root absorbing area was determined via methylene blue adsorption, the relative permeability of roots plasma membrane was determined using the conductivity method, and ultra-trace organic acids were determined by combining accelerated solvent extraction and solid-phase extraction liquid chromatography with electrospray ionization tandem mass spectrometry. The rhizospheric soil pH was measured using a PHS-25 digital acidity meter. Data were processed by one-way analysis of variance and bivariate correlations using the SPSS 11.5 software, and tabulation and plotting were performed using the Excel software and Origin Pro 2017 software.

The root activity, total and active root absorbing area, malic acid and citric acid secretion of P. massoniana seedlings increased and then decreased as the pH of simulated acid rain decreased, reaching the maximum value under treatment with mild acid rain and sharply decreasing with moderate and severe acid rain. In addition, the longer the exposure to simulated acid rain, the greater the differences in root physiological characteristics. Compared with those for 30-day treatment, the root physiological indices of P. massoniana seedlings receiving 100-day severe acid rain treatment decreased as follows: root activity by (46.25 ± 7.23)%, total root absorbing area by (56.37 ± 8.14)%, active root absorbing area by (38.55 ± 11.58)%, malic acid secretion by (68.45 ± 5.41)% and citric acid secretion by (10.80 ± 4.93)%. On the contrary, the relative permeability of root plasma membrane decreased and then increased as the pH of simulated acid rain decreased; the permeability value was as low as (13.62 ± 1.46)% when seedlings were treated with mild acid rain for 100 days and was as high as (36.48± 2.25) % when seedlings were treated with severe acid rain for 100 days. In addition, with the increase of simulated acid rain acidity and treatment time, the root oxalic acid secretion in P. massoniana increased and reached a maximum of (1 251.93 ± 37.52) μg/L in seedlings treated with acid rain at pH 2.5 for 100 days, but the rhizospheric pH value decreased gradually. The correlation analysis showed that root activity, root absorbing area, and rhizospheric soil pH were apparently negatively correlated with the root oxalic acid secretion but significantly positively correlated with root malic acid and citric acid secretion; and root plasma membrane permeability was significantly positively correlated with the root oxalic acid secretion but markedly negatively correlated with the root malic acid and citric acid secretion.

The changes in root physiological parameters of P. massoniana seedlings treated with simulated acid rain at different acidity levels and treatment times indicate that at pH 4.5, the species shows good acid resistance: the root nutrient absorption ability of the seedlings is strong, the structure and function of the selective permeability barrier of root cells are intact, cell permeation is maintained in internal and external equilibrium, and the ability of intracellular pH and ion concentration regulation is strong. However, when the seedlings were exposed to acid rain with pH lower than 3.5 for more than 60 days, the rhizospheric soil pH and the root malic acid and citric acid secretion decreased dramatically and the root physiological indicators deteriorated significantly. These results indicate that long-term exposure of P. massoniana to acid rain at pH ≤ 3.5 can result in root membrane system damage, intracellular charge imbalance, root physiological dysfunction, and metabolic obstruction, leading to a significant decline in acid resistance.