马尾松 (Pinus massoniana) 是我国人工林的重要组成树种，对酸沉降高度敏感。随着我国大气酸沉降的日趋严重，马尾松林出现针尖坏死，树枝枯萎，甚至死亡等受害症状。木本植物响应酸沉降根系生理变化过程的研究相对较少，酸沉降对马尾松根系生理特性的影响仍不清楚。笔者研究马尾松幼苗根系生理指标在不同酸沉降强度和时间下的变化，探究马尾松根系对大气酸沉降的适应能力。

以南京老山林场林地0~20 cm混合土壤为培养基质，土培100 d长势一致的马尾松幼苗为试验材料，调节pH 6.5模拟雨水为对照，配制化学组分类似于自然降水的模拟酸雨，其pH设置为5.6(微酸化处理)、4.5(轻度酸处理)、3.5(中度酸处理)和2.5(重度酸处理)，按2017年5—8月南京市实际降水量、时间和频率对栽培马尾松幼苗喷淋处理。在栽培30、60和100 d时测定幼苗根系各生理指标，采用TTC还原分光光度法测定根系活力，甲烯蓝吸附法测定吸收面积，电导率法测定质膜透性，加速溶剂萃取(accelerated solvent extraction ASE)-固相萃取(solid?phase extraction, SPE)-高效液相色谱(HPLC)-电喷雾电离(electrosprag ionization, ESI)-串联质谱(MS/MS)法测定根系超痕量有机酸，复合电极法测定根际土壤pH。用SPSS 20.0软件处理数据，用One-Way ANOVA进行单因素方差分析，用Bivariate correlations进行相关性分析。

随模拟酸雨pH的下降，马尾松幼苗根系活力、根系总吸收面积、活跃吸收面积、苹果酸与柠檬酸分泌量均呈现先升高后降低的趋势，在轻度酸处理下达最高值，中、重度酸处理下各指标显著降低；随酸雨喷淋时间推移，差异越明显，重度酸处理100 d的上述根系各生理指标相比于栽培初期(30 d)分别下降了(46.25 ± 7.23)%、(56.37 ± 8.14)%、(38.55 ± 11.58)%、(68.45 ± 5.41)%和(10.80 ± 4.93)%。而马尾松幼苗根系质膜透性的变化趋势则相反，随模拟酸雨pH下降，质膜透性先减弱后增强，在轻度酸处理100 d时达最小值，为(13.62 ± 1.46)%；在重度酸处理100 d时达最大值，为(36.48 ± 2.25)%。此外，随模拟酸雨酸度的增加和酸处理时间的延长，马尾松根系草酸分泌量不断增加，在重度酸处理100 d时高达（1 251.93 ± 37.52） μg/L，而幼苗根际土壤pH则逐步下降。从相关性分析来看，草酸分泌量与植物根系活力、吸收面积、根际土壤pH呈极显著负相关，与质膜透性呈极显著正相关；苹果酸、柠檬酸分泌量与植物根系活力、吸收面积、根际土壤pH呈极显著正相关，与质膜透性呈极显著负相关。

对马尾松幼苗根系生理指标在不同酸沉降强度和时间下变化的研究结果反映了在轻度模拟酸雨(pH 4.5)处理下，马尾松幼苗根系养分吸收能力强，根细胞选择透过性屏障结构和功能完好，细胞内外渗透维持在平衡状态，胞内pH和离子浓度调节能力强，马尾松表现出较好的耐酸性。而当马尾松根系接触酸雨的pH ≤ 3.5，时间超过60 d时，根际土壤pH大幅降低，幼苗根系苹果酸和柠檬酸分泌量减少，各生理指标显著恶化，这表明酸雨(pH ≤ 3.5)的长期处理会导致马尾松根系膜系统受损，细胞内电荷失衡，生理功能失调，代谢受阻，最终引起马尾松抗酸能力明显下降。

Pinus massoniana is an important plantation species in China， it is highly sensitive to acid deposition. As atmospheric acid deposition has increased, symptoms of injury, such as needle tip necrosis, branch withering, and even death, have been observed in P. massoniana forests. However, there are relatively few studies assessing changes in root physiological processes in forest woody plants in response to acid deposition. Moreover, the effects of acid deposition on the physiological characteristics of P. massoniana roots are still unknown. We focused on the changes in root physiological parameters of P. massoniana seedlings exposed to simulated acid rain (with different acidity levels and treatment times) and studied the adaptability of the speciesto atmospheric acid deposition.

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.