Plant growth, yield, and soil characteristics
In the present study, the soil pH decreased was decreased in all treatments, except for F0. Treatment NF had the lowest pH, and with the application of manure fertilizer, the pH decreased slowly (Table 1). This is consistent with other reports suggesting that the application of mineral fertilizer significantly reduces soil pH (Du et al., 2010) while organic fertilizer increases it (Butterly et al., 2016). The results show that fertilization increased the yield, total C, N, and P, and test P in soil. Moreover, applying manure fertilizer combined with N and P fertilizers gave the best effect, which is consistent with other studies (Xin et al., 2019). Compared with the NF and MN treatments, the extraction efficiencies and concentrations were higher in the P-fertilized treatments (NP and MNP) at different depths, which is consistent with the literature (Table 1; Xin et al., 2019). Our results are also consistent with the findings of Cadot (2018) who reported that fertilization can significantly increase the number of results per corn plant and the weight of stems and leaves (Table 2). In the current study, higher plant height and thicker stem diameter were found after the manure fertilizer treatments (MN, MNP), but there was no significant difference between the unfertilized and mineral fertilizer (NF, NP) soils, which is inconsistent with previous research suggesting that fertilizer application increases the height and stem of plants (Jiang et al., 2019). The higher nutrient content in soil caused by the application of manure, and a significant reduction in the soil pH after application of mineral fertilizer (Table 1; Kmet’ova and Kovacik, 2013). Our results show that yield was significantly increased by the application of P fertilizer or manure (NP, MN, and MNP), but there was no significant change in crop yield with the single application of nitrogen fertilizer (CK or NF) or under the no-fertilizer treatment. This result could be due to the lower pH value caused by the application of nitrogen fertilizer (Aula et al., 2016), making the soil unsuitable for crops to bear fruit. Additionally, the application of manure causes an increase in microbial biomass content; some studies have shown that a large amount of microbial biomass content leads to a higher content of hormones or humus in the soil, which is beneficial for increasing crop yields (Arancon et al., 2003).
The forms of inorganic P affect long-term fertilization at different soil depths and plant characteristics
Our results show that the concentrations and distributions of pyrophosphate were generally low in all treatments (0–60 cm soil depths, or years; Table 2–3), which is contrary to many other field experiments (Annaheim et al., 2015; Deiss et al., 2016). In addition, the concentrations of inorganic P and orthophosphate were significantly positively correlated with stem diameter, plant height, number of corn cobs, and yield (Plenet et al., 2000), but there were no significant relationships with stem and leaf weight (Table 6). Because inorganic P (the main component of inorganic P is orthophosphate) is present primarily in the corn fruit, but there is only a small amount in the stems and leaves. However, pyrophosphate was significantly correlated with the stem diameter. Because pyrophosphate had a significant correlation with total N, and a larger N may be associated with an increased plant stem diameter (Puntel, 2012).
Orthophosphate in soils
There were significant differences in the concentrations of orthophosphate between fertilization treatments, soil depths, and fertilization years (Fig. 3–4). The trends observed in this study are related to the direct addition of orthophosphate to the soil as a result of fertilization. One interesting finding is that over the years of fertilization, the proportion of orthophosphate decreased gradually, while the concentration increased gradually (Table 3). Schneider et al. (2016) confirmed that chemical fertilizers increase the content of orthophosphate. The higher the concentration of orthophosphate, the higher the Olsen-P concentration and yield (Table 1–2). In addition, the content of orthophosphate is positively correlated with the concentration of soil P that plants can absorb and utilize, and with grain yield (Schneider et al., 2016; Xin et al., 2019). Thus, we conclude that the content of orthophosphate was greatly increased by the use of N fertilizer, and the addition of P may enhance this effect (Table 3). The primary cause is a shift in the dominance of the major P compounds in the soil due to P addition. Specifically, added P changes the dominant P form from phosphate monoester to orthophosphate. This is significant because it can be provided to plants in relatively small amounts for a long time. This is because inositol compounds are the main components of phosphate monoesters (Ahlgren et al., 2013), while orthophosphate is readily and immediately available. The effect of organic fertilizer was better than that of mineral fertilizer because there was a large amount of orthophosphate in the mineral fertilizer, and the content of orthophosphate is indirectly increased by the presence of a large number of microorganisms (Ahlgren et al., 2013). Therefore, orthophosphate concentrations in soil may also increase with high P-fertilization rates, whereas forms of organic P can increase at low P-fertilization rates (Barbara et al., 2017).
Pyrophosphate in soils
In the present study, the P compound with the third-highest content was pyrophosphate, which can be absorbed and utilized by plants and is also a reactive species (Barbara et al., 2017). In general, the concentration of pyrophosphate was low in all treatments, soil depths, and years (Fig. 3–4), as previously reported (Deiss et al., 2016; Xin et al., 2019). Interestingly, we found that the content of pyrophosphate was significantly higher in the 0–40 cm soil layer than in the other layers, and that manure combined with P fertilizer had a significant effect on the pyrophosphate content (Table 4 and Fig. 3). The addition of P (organic or chemical P fertilizer) is an efficient way to increase the amount of P that is absorbed and utilized by plants. There were no significant differences in the composition of organic P in the soil according to the use of combined organic plus mineral fertilizer or mineral fertilizer alone (Ahlgren et al., 2013). Other studies have also found that pyrophosphate does not seem to be affected by P addition to soil. Additionally, there are many microorganisms in manure, and pyrophosphate is generally associated with microbes. The present study found that turning maize stubble from the previous year into the soil could increase oxygen and water retention and microbial activity; thus, rapid decomposition could explain the low overall level of pyrophosphate in the investigated soil. Several other studies have confirmed that to be readily used by plants, the composition of extractable P should be changed, such as by adding P to the soil (Watson et al., 1998).
Effects of long-term fertilization on the forms of organic P at different depths and on plant characteristics
The present study revealed that the main form of organic P in the soil was phosphate monoester. The low amounts of phosphate diester in the soil are consistent with the findings of other studies (Table 2–3; Fig. 2; Yang et al., 2019). In addition, the content of total organic P increased each year, and the proportion of total organic P increased gradually at the 20–40 cm soil depth but decreased at the 40–60 cm soil depth (Table 3). It could be that long-term fertilization increased the proportion of organic P at the 20–40 cm soil depth. It has been previously reported that fertilization can cause organic P to accumulate in deep soil (Guardini et al., 2012; Ahlgren et al., 2013; and references therein). We observed that unstable inorganic P seems to accumulate in the topsoil and decreases with depth, while organic P occurred deeper in the soil profile. As a result, losses of soluble inorganic P in surface runoff will increase (Abdi et al., 2014; Kleinman et al., 2015). Furthermore, the results of this study indicate that the composition of the organic P compounds in soil is influenced both by whether the soil is fertilized or unfertilized, and by fertilizer type. For example, the IHP stereoisomers in different fertilization treatments varied (Fig. 2). This is different from the results of Dodd et al. (2014), who found that the diversity of organic P compounds in the soil was not affected by the type of fertilization or farming method (Song et al., 2011). This may be related to different soil types and environments. However, some studies have reported a significant difference in the organic P content of soils with a lot of mineral or manure fertilizers (Motavalli and Miles, 2002). The addition of manure fertilizer (MN) greatly increased the concentration of total organic P in soil, but this decreased with the addition of P fertilizer (MNP) (Table 2). This is because the combination of manure plus P fertilizer increased soil P, while organic matter has a fixed ratio of C, N, and P. Therefore, the concentration of organic P decreased, but the present study was conducted in a temperate humid-semi-humid monsoon climate, so the decrease in organic P was relatively weak (Table 1–2; Zhang et al., 2015). Part of the P in alkaline soil that can be absorbed and utilized by plants and the inorganic P in soil come from the conversion of added organic P (Xin et al., 2017). Thus, it was necessary to reduce the single dose of chemical P fertilizer in soil and partly supplement it by applying N plus manure fertilizer. Soil available P, plant growth, and grain yield were increased when the forms of organic P that were applied became degraded (Table 1–2). Our results indicated that diester was significantly correlated with plant growth and grain yield. In contrast, monoester was significantly correlated with plant growth but was not significantly correlated with grain yield (Table 6). This indicates that monoester contributes more to the corn growth process and does not make much of a contribution to the corn fruit when the crop is mature. This result is in contrast to the findings of other studies (Wei et al., 2014). Furthermore, IHP was significantly correlated with plant growth and yield, except for the weight of the stems and leaves. This may be because IHP is mainly related to the microorganisms in the soil, and an increase in organic matter and N will increase the microbial biomass, which can increase the content of hormones and humus in the soil, and increase the yield of crops relative to the weight of the stems and leaves; thus, IHP is a P source that crops can absorb and utilize (Turner and Richardson, 2004; Arancon et al., 2003; Richardson et al., 2007; Kmet’ova and Kovacik, 2013).
Phosphate monoester in soils
Overall, long-term fertilization and soil depth had significant effects on the forms of P in soil, with inorganic P accumulating on the soil surface and decreasing in content with depth. However, organic P (total organic P content, total IHP, phosphate monoester, and diester) accumulated in the deep soil layer (20–40 cm) over the study period. We observed that the content of phosphate monoester was high in the topsoil (Table 3; Fig. 3), which seems reasonable because, compared with the subsoil, the concentration of bacteria and fungi in deep soil is higher and the predominating aerobic conditions facilitate rapid microbial turnover, thus supporting the degradation of organic fertilizer. Furthermore, the degradation products of the microbial biomass are important sources of phosphate monoester in soils (Bünemann et al., 2004).
Total IHP in soils
We found that IHP monoesters and IHP stereoisomers were the most identified forms of organic P, with four peaks being identified. There were no neo-IHP peaks in the NF and NP treatments, indicating that long-term single application of mineral fertilizer will lead to the disappearance of neo-IHP, which is contrary to Abdi’s (2014) conclusion. There were four IHP peaks (myo-IHP, scyllo-IHP, neo-IHP, D-chiro-IHP) observed with manure fertilization (MN and MNP). In addition, compared with CK, the pig manure treatments had greatly increased contents of IHP stereoisomers (myo-IHP, scyllo-IHP, neo-IHP, D-chiro-IHP). Because pig manure promotes microorganisms and their activities (Wong et al., 2009), scoyllo-IHP and D-chiro-IHP may be derived from microorganisms, and D-chiro-IHP may also be derived from plants (Xin et al., 2019). myo-IHP was the most dominant form in all treatments (Liu et al., 2013b). The content may be overestimated from interpretation of the NMR spectra, or it may be realistic. Since crops have been planted, there will be plant tissue input, so the content of this compound in the soil is high (Giaveno et al., 2010). In many studies, it has been robustly confirmed that myo-IHP is mainly derived from seeds and is widespread in plants. (Noack et al., 2014). In the F0, CK, NF, and NP treatments, myo-IHP predominantly originated from the input of the crop (seeds, roots, and residues), while in the MN and MNP treatments, part of the myo-IHP came from the pig manure fertilizer. Part of the phosphate monoester in the soil comes from external inputs, which is good evidence that myo-IHP is related to animal manure (Xin et al., 2019). Further, the application of pig manure tends to increase myo-IHP concentrations. This is contrary to other research results (Annaheim et al., 2015). This difference may be because this previous study used mineral fertilizer and cow dung. Many studies have shown that IHP is a compound with great affinity for amorphous metals and soil particles (Yan et al., 2014). In particular, the affinity of myo-IHP towards goethite is higher than that of orthophosphate (Yan et al., 2015). This indicates that myo-IHP is available to plants (Celi et al., 1999). Recent research has shown that because many microorganisms play vital roles in the environment, these ubiquitous compounds can play a role in IHP (Turner et al., 2007). In the long run, IHP can serve as an effective and stable source of nutrients for plants, although there is a relatively stable P pool in the soil (Richardson et al., 2011).
Phosphate diester in soils
In the present study, there were low amounts of phosphate diester compared with other forms of soil P. These results are consistent with other studies. At pH > 5, the adsorption capacity of soil particles for DNA, which is one of the forms of phosphodiester with the highest content, decreases. Therefore, these findings may be caused by soil pH (Khanna et al., 1998). This may mean that phosphate diester is being flushed out and depleted (Ahlgren et al., 2013). Additionally, previous studies have reported that the absence of phosphate diester in agricultural soils according to NMR spectra (Turner et al., 2005) may be because it is degraded into phosphate monoester during measurement and extraction (Turner et al., 2003). In this study, this phenomenon may have been significant because of the 18 h extraction step for chemical fertilizer, which may have promoted such degradation (Cade-Menun and Liu, 2014). In this study, the concentration of phosphate diester was higher in the topsoil (Table 3). This was due to the higher accumulation of organic matter on the soil surface (Condron et al., 2005), so phosphate diester synthesis was higher. It may also be present naturally in soil.
Generally, the M/D ratio is a measure of instability (Schneider et al., 2016). Some researchers believe that phosphate diesters mineralize faster in soils that are conducive to decomposition, while phosphate monoesters are more tightly adsorbed and do not easily mineralize. Therefore, a low M/D value can indicate a decreased mineralization rate, resulting in a high concentration of phosphate diester (Schneider et al., 2016). Furthermore, the M/D ratio may have been artificially increased so that it limited the mineralization index of phosphate diester. The data in Table 3 show that the uncorrected phosphate diester content was obviously underestimated due to hydrolysis and degradation. Several studies have also shown that the content of phosphate diester on beans will increase significantly after correction; the crops used in this study were maize and soybean.