## 2 Materials and Methods
### Sampling and Analysis  
In the spring before fertilization and in the fall after tobacco harvest, soil samples consisting of five subsamples of 1 kg were taken from the layer from 0 to 20 cm of each experimental plot, and composite samples of treatment were prepared for each plot by mixing the subsamples. The composite samples were air-dried for 7 days, removed of plant residue and stones, crushed, and passed through a 2 mm sieve for available metals analyses and through a 0.15 mm sieve for total metals analyses. The soil samples were used for determinations of the pH (soil to carbon dioxide-free water = 1:2.5, w/v, UB-10, Denver Instrument, U.S.), available N (1.0 mol L\({}^{-1}\) NaOH and H\({}_{3}\)BO\({}_{3}\)), available P (0.5 mol L\({}^{-1}\) NaHCO\({}_{3}\)), and available K (1.0 mol L\({}^{-1}\) NH\({}_{4}\)OAc) [33]. For total metals analyses, 0.2000 g of soil \begin{table}
\begin{tabular}{c c c c c c c c c c c c c} \hline \multirow{2}{*}{**Item**} & \multirow{2}{*}{**pH**} & **Organic** & \multirow{2}{*}{**Available**} & **Available** & **Available** & \multicolumn{4}{c}{**Total Content**} & \multicolumn{4}{c}{**DTPA Extracted**} \\  & & **Matter** & & **N** & **P** & **K** & **Cd** & **Pb** & **Cu** & **Zn** & **Cd** & **Pb** & **Cu** & **Zn** \\ \hline Soil & 7.8 & 21 & 109 & 12 & 141 & 3.82 & 21.31 & 36.02 & 146.69 & 0.73 & 1.42 & 3.38 & 4.46 \\ Chinese standard & \(>\)7.5 & – & – & – & – & 0.6 & 170 & 100 & 300 & – & – & – & – \\ \hline \multicolumn{11}{c}{\(-\), the value was not defined in Chinese soil environmental quality standards (no. GB15618-2018).} \\ \end{tabular}
\end{table} 
Table 1: Soil characteristics and Chinese soil environmental quality standards (total concentration in mg\(\cdot\)kg\({}^{-1}\)). was digested by 8 mL of HNO\({}_{3}\)/HCl (1/3, v/v). The available metals contents were extracted from 5.00 g of soil with 20 mL of an aqueous solution of diethylene triamine pentaacetate acid (DTPA) (Sinopharm Chemical Reagent Co., Shanghai, China) [34]. The soil samples were air-dried and ground to pass through a 60-mesh sieve, and then the modified BCR sequential extraction method (European Communities Bureau of Reference) was applied to measure different fractions of Cd, Pb, Cu, and Zn in the soil as follows [25; 35]:  
**Exchangeable fraction of metals** The air-dried soil samples (2 g dry weight) were extracted with 20 mL of 1 mol L\({}^{-1}\) MgCl\({}_{2}\) (pH = 7.0) in Teflon centrifuge tubes for 1 h at 25 \(\pm\) 1 \({}^{\circ}\)C with continuous agitation. The suspension was then centrifuged (5000 g for 5 min). The supernatant liquid was filtered through 0.45 \(\upmu\)m filter paper and stored at 4 \({}^{\circ}\)C prior to analysis. **Reducible fraction** The residue from the exchangeable fraction was extracted with 20 mL of 0.04 mol\(\cdot\)L\({}^{-1}\) NH\({}_{2}\)OH\(\cdot\)HCl (solvent is HAc solution, Hac: water = 1:4) with a pH of 1.5 for 6 h at 25 \(\pm\) 1 \({}^{\circ}\)C with continuous agitation. The suspension was then centrifuged and filtered as in the first step. **Oxidizable fraction** The residue from the reducible fraction was extracted with 20 mL of 30% H\({}_{2}\)O\({}_{2}\) (pH = 2.0, with 1M HNO\({}_{3}\)) for 2 h at 85 \(\pm\) 2 \({}^{\circ}\)C with occasional agitation and an additional 20 mL of 3.2 mol L\({}^{-1}\) NH\({}_{4}\)Ac for 16 h at 85 \(\pm\) 2 \({}^{\circ}\)C with continuous agitation. The suspension was centrifuged and filtered as described in the first step. **Residual fraction of metals** The residue from the oxidizable fraction was removed and microwave-digested with 8 mL of HNO\({}_{3}\), 2 mL of HClO\({}_{4}\), and 2 mL of HF. The flowers were removed when the tobacco was mature. The leaves were divided into upper, middle and lower leaves according to the different developmental stages of each plant from top to bottom at maturity. The lower leaves were the 1 st to 6 th leaves, the middle leaves were the 7 th to 12 th leaves, and the upper leaves were the 13 th to 19 th leaves.