# Effects of Long-Term Fertilization on Phosphorus Form and Availability in Black Soil
## 2 Materials and Methods
### Sample Collection and Testing Method  
In order to reduce errors due to a lack of repetition, the test site was divided into three equal parts along its long side and set up as three replicated plots. Samples were collected at representative points in each sample plot using the five-point sampling method, with the sampling depth set at 0 to 20 cm. The soil samples were stored in sterile boxes in a \(-\)80 \({}^{\circ}\)C refrigerator at low temperature, which helped to maintain the original state and activity of microorganisms in the soil samples and prevented the samples from degradation during preservation to ensure the accuracy and reliability of the research results. The samples were mixed, air-dried, and sieved until analysis. The soil TP and soil AP contents were determined using conventional methods.  
Soil phosphorus grading was performed via [PERSON]'s improved Hedley method [14], which involved the sequential extraction of different forms of phosphorus in the soil. Deionized water was added to determine the contents of H\({}_{2}\)O-Pi and H\({}_{2}\)O-P, 0.5 mol\(\cdot\)L\({}^{-1}\) NaHCO\({}_{3}\) was added to the residue to determine the contents of NaHCO\({}_{3}\)-Pi and NaHCO\({}_{3}\)-P, 0.1 mol\(\cdot\)L\({}^{-1}\) NaOH was added to the residue to determine the contents of NaOH-Pi and NaOH-P, and 1 mol\(\cdot\)L\({}^{-1}\) HCl was added to the residue to determine the contents of HCl-Pi and HCl-P.  
Solution \({}^{31}\)P NMR determination was performed according to the procedures of [PERSON] and [PERSON] (1996) and [PERSON] (2004) [21; 22]. The basic procedure was as follows: 0.25 mol\(\cdot\)L\({}^{-1}\) NaOH + 0.05 mol\(\cdot\)L\({}^{-1}\) EDTA mixture was added according to the liquid-soil ratio of 20:1, shaken, centrifuged, and filtered. The supernatant was obtained to determine the TP of the extract, and the other supernatant was removed for freeze-drying and then dried with 10 mol\(\cdot\)L\({}^{-1}\) NaOH. The NaOH-EDTA mixture and  
\begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline
**Plow** & **Soil** & **Organic** & **Total** & **Total** & **Holyzable** & **Available** & **Available** & **pH** \\
**Layer** & **Depth** & **Matter** & **Nitrogen** & **Photoshorus** & **Potassium** & **Nitrogen** & **Phosphorus** & **Potassium** \\
**(cm)** & **(g\(\cdot\)kg\({}^{-1}\))** & **(g\(\cdot\)kg\({}^{-1}\))** & **(g\(\cdot\)kg\({}^{-1}\))** & **(g\(\cdot\)kg\({}^{-1}\))** & **(mg\(\cdot\)kg\({}^{-1}\))** & **(mg\(\cdot\)kg\({}^{-1}\))** & **(mg\(\cdot\)kg\({}^{-1}\))** \\ \hline
**Topsol** & 0-20 & 22.8 & 1.40 & 1.39 & 22.1 & 114 & 27.0 & 190 & 7.6 \\
**Subsoil** & 20-40 & 15.2 & 1.30 & 1.35 & 22.3 & 98 & 15.5 & 181 & 7.5 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Basic properties of soil at the beginning of the experiment.  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Treatment** & \multicolumn{4}{c}{**Mineral Fertilizers**} & \multicolumn{4}{c}{**Organic Fertilizers (Pig Manure)**} \\ \cline{2-5}  & **N** & **P\({}_{2}\)O\({}_{5}\)** & **K\({}_{2}\)O** & **N** & **P\({}_{2}\)O\({}_{5}\)** & **K\({}_{2}\)O** \\ \hline CK & 0 & 0 & 0 & 0 & 0 & 0 \\ NPK & 165 & 83 & 83 & 0 & 0 & 0 \\ M1 NPK & 50 & 83 & 83 & 115 & 92 & 113 \\ M2 NPK & 165 & 83 & 83 & 150 & 120 & 147 \\
1.5M1 NPK & 75 & 124 & 124 & 173 & 138 & 170 \\ \hline \hline \end{tabular}
\end{table}
Table 2: Annual application rate of mineral and organic fertilizer by treatment (kg\(\cdot\)hm\({}^{-2}\)).  
D\({}_{2}\)O were redissolved, shaken, and centrifuged at low temperature and high speed, and the supernatant was taken for NMR spectroscopy. NMR spectra were collected from an AVANCE III HD 500 spectrometer ([PERSON], Biospin, Fallanden, Switzerland), and only one composite sample per treatment was used for NMR testing.