Pecan agroforestry systems improve soil quality by stimulating enzyme activity

Experimental site description and design

The study was conducted at a pecan orchard in Wenji in the Yingquan District of Fuyang City, Anhui Province, China (33°3^′N, 115°36^′E). This area has a warm temperate semi-humid monsoon climate with an annual average temperature of 14.9 °C and an annual average precipitation of 889 mm. The maximum temperature for this area was 41.4 °C and the minimum temperature was −20.4 °C (China Meteorological Data Service, http://data.cma.cn/).

The afforestation of the experimental site occurred in 2016 using the ‘Pawnee’ variety of tree in the second year of growth. Rows were spaced 4.0 m × 6.0 m. Three types of perennials (Paeonia suffruticosa, Hemerocallis citrina, and Paeonia lactiflora) were planted in the pecan plantation in 2017. A randomized block design was adopted for the study in September 2019 consisting of four treatments and three replicates. The treatments included: (1) PPH, with a row spacing of Paeonia suffruticosa of 0.2 m × 0.2 m and Hemerocallis citrina spaced at 0.4 m × 0.8 m; (2) PPS, with the row spacing of Paeonia suffruticosa at 0.2 m × 0.6 m; (3) PPL, with the row spacing of Paeonia lactiflora at 0.2 m × 0.6 m; (4) pecan monoculture (CK).

Soil sampling

Soil samples were collected from the experimental site in September 2019. Eight pecan seedlings represented one plot, and three plots were randomly established for each treatment in the selected sampling area. A shovel was used to remove the plants and the surface litter from the soil. Five random soil profiles were obtained using a zig-zag sampling pattern (Tafesa, Chimdi & Aga, 2019)) at depths of 0–20 cm, 20–40 cm, and 40–60 cm. The samples were then mixed to generate a soil sample for each layer. A total of 36 soil samples were collected from the four treatment sites. The samples were sealed in plastic bags and transported to the laboratory. Soil samples were dried to determine their physicochemical properties and were kept in refrigerator at 4° for determining enzyme activities.

Soil physicochemical properties analysis

Soil samples were collected using the ring knife method to determine their moisture content (MC), bulk density (BD), and porosity (TPO) (Pan et al., 2017). Following the removal of impurities, the NO₃⁻-N, NH₄⁺-N, AP, AK, pH value, electrical conductivity value (EC), total phosphorus (TP), total potassium (TK), total carbon (TC), total nitrogen (TN), available potassium (AK), Ca and Mg contents were determined. The soil NO₃⁻-N, NH₄⁺-N, TP, AP and AK were measured using an automatic discontinuous chemical analyzer (CleverChem Anna, Germany) (Si et al., 2018; Yang et al., 2021). The pH value of the soil was determined using a pH meter (Mettler Toledo, Shanghai, China) in a 1:2.5 (w/v) soil solution (Ma et al., 2021). The EC value of the soil was determined using an electrical conductivity meter in a soil-water extract at 1:5 at 25° (Xie et al., 2019). The TC and TN of the soil were determined via an automatic element analyzer (Vario EL Cube, Germany Elementar) (Wang et al., 2018). The contents of TK, Ca, and Mg in the soil were measured using an inductively coupled plasma emission spectrometer (iCAP 6300 Series, America ThermoFisher) (Ma et al., 2019).

Soil enzyme activity

The fresh soil samples from the surface layer (0–20 cm) were air-dried and sifted through a 50 mesh. The activity of seven types of soil enzymes, including S-POD, S-PPO, S-UE, S-AKP, S-BG, S-CBH, and S-NAG, were studied. S-AKP activity was determined by the p-nitrophenylphosphonate disodium method and S-POD activity was determined by potassium permanganate titration. S-UE activity was determined by the sodium phenol-sodium hypochlorite colorimetric method; S-CBH activity was determined by the phenyldisodium phosphate colorimetric method; S-NAG activity was determined by the p-nitrophenol colorimetric method; S-PPO activity was determined by the pyrogallic acid colorimetric method; and S-BG activity was determined by the microporous plate fluorescence substrate method (Domínguez et al., 2017; Weintraub et al., 2013). The international unit of enzyme consumption per gram of soil (U/g) was used as the unit of measure.

Statistical analysis

SPSS 19.0 and Origin Pro 2021 software were used to analyze the comprehensive data. The data derived from the different soil depths (0–20 cm, 20–40 cm, and 40–60 cm) were statistically analyzed by single-factor analysis of variance (ANOVA), whereas significant differences (p < 0.05) in the physical and chemical properties of the soils of the various agroforestry systems were evaluated using a minimum significant difference test (LSD). Correlation analysis was employed to examine the relationships between the physical and chemical properties of the different soil layers, as well as between the physical and chemical properties and enzyme activity in the topsoil (0–20 cm). The PCA ranking performed in Origin Pro 2021 was used to analyze the physical and chemical properties of the soil.

Soil physical properties

The basic physical properties of the soil samples extracted from different soil depths and agroforestry patterns of pecan, including the EC, MC, BD, and TPO are shown in Fig. 1. In the 0–20 cm soil layer, the EC of PPL was significantly higher than that of the PPS and CK (p < 0.05). The soil MC of the PPS group was significantly higher than that of the CK (p < 0.05). The soil BD of all the soil samples ranged 1.42 g/cm³ to 1.55 g/cm³. The soil BD of the PPS and CK was significantly higher than that of the PPH and PPL (p < 0.05).

In the 20–40 cm soil layer, the EC of the CK group soil was the lowest (Fig. 1). The MC of the soil samples from the CK and PPS groups was significantly higher than that of the PPH and PPL groups (p < 0.05). The BD of all the soil samples ranged from 1.39 g/cm³ to 1.71 g/cm³, and the value of BD in descending order was PPS, CK, PPL, and PPH, where there was a significant difference between any two (p < 0.05). The TPO under PPH group was significantly higher than that of other groups (p < 0.05).

In the 40–60 cm soil layer, the EC of the PPL soil was significantly higher than that of the CK, PPH, and PPS (p < 0.05). The soil MC of the PPS group was significantly higher than that of the CK group (p < 0.05). The difference in the soil MC between the PPH and PPS groups was significant (p < 0.05). The BD of all the soil samples ranged from 1.02 g/cm³ to 1.68 g/cm³, and the BD of the all three soil layers, in all modes, was PPS > CK > PPL > PPH.

Soil chemical properties

There were some variations in the chemical properties of the different agroforestry systems in the same soil layer (Table 1 and Fig. 2). Compared with the CK group, the agroforestry system increased the content of some elements in the soil. There were significant differences in the NO₃⁻-N, TN, AK, TK, and TC contents between the four pecan agroforestry systems. The average pH value of all soil layer samples ranged from 7.80 to 8.40, and the pH decreased by 0.96% - 2.41% in contrast to the monoculture. The content of Mg in the PPS group was significantly higher than that in the PPH, PPL, and CK groups in all three soil layers (p < 0.05).

Moreover, the TP and TN contents in the 0–20 cm soil layer of the CK group were significantly lower than those of the PPS and PPL groups (p < 0.05). The contents of AP, NH₄⁺-N, AK, and TC in the CK group were lower than that of the soil following agroforestry. There were significant differences in the soil NO₃⁻-N between the PPL and the PPH, PPS, and CK groups (p < 0. 05). The TK content of the PPS group soil was significantly higher than that of the PPH, PPL, and CK groups (p < 0.05).

Soil enzyme activity

Significant effects of different agroforestry groups on the S-UE, S-AKP, S-AKP, S-CBH, S-NAG, S-POD and S-BG were observed (Table 2). Intercropping significantly increased the activity of S-AKP in soil compared to CK (p < 0. 05). The activity of S-POD and S-BG in the PPL were significantly lower than those in the CK group (p <0. 05). The activity of S-CBH in the PPH and PPS was significantly higher than in PPL and CK (p < 0. 05). However, the activity of S-NAG was opposite and was significantly higher in PPL and CK than in the PPH and PPS (p < 0. 05).

Correlations between physicochemical properties and enzyme activity in different layers of soil

Fig. 3 summarizes the correlations between the main physical and chemical properties of the treatments in the different soil layers (0–20 cm, 20–40 cm, and 40–60 cm). The results revealed that in the 0–20 cm soil layer, the EC was negatively correlated with the BD and Ca content (p < 0.05), and positively correlated with the TPO (p < 0.05). Further, the BD was positively correlated with the Ca and Mg contents (p < 0.001). The AP was positively correlated with the TP, AK, and TC (p < 0.05). The TP and AK were positively correlated with the available TN and TC (p < 0.05). There was a significantly positive correlation between the TN and TC (p < 0.001), as well as between the pH and total carbon Ca content (p < 0.05).

In the 20–40 cm and 40–60 cm soil layers, there was a strong significant positive correlation between the soil EC and NO₃⁻-N (p < 0.001). The soil MC and BD were significantly negatively correlated with the TPO (p < 0.001), and positively correlated with the TK (p < 0.05). The soil BD was negatively correlated with the NH₄⁺-N and positively correlated with the TK (p < 0.05), whereas the TP was positively correlated with the Ca and Mg contents (p < 0.05). There was a significantly positive correlation among the TK and TN, TC, Ca, and Mg contents (p < 0.05), as well as between the AK and TN (p < 0.05). Further, there was a very significant positive correlation among contents of TC and TN, Ca and Mg (p < 0.001), and an extremely significant positive correlation between contents the Ca and Mg (p < 0.001).

The enzyme activity was affected by the physical and chemical properties of the soil (Table 3). The activities of S-NAG and S-AKP were significantly positively correlated with AK, TN and TC, while the activity of S-CBH showed a significantly negative relationship with AK, TN and TC (p < 0.05). The activities of S-UE, S-POD and S-BG exhibited a relatively weak correlation with the physical and chemical properties of the soil, and TK and pH had no significant correlation with all of the soil enzyme activities.

Principal component analysis of soil physical and chemical properties

Table 4 showed the weights of the 15 original variables along with the first four principal components. According to the PCA ranking results, the eigenvalues of the first four ranking axes were greater than 1, and the cumulative contribution rate reached 81.55% (Fig. 4, Table 3). The main axis (PC1) contributed 32.93% of the total variance, the second principal component (PC2) explained 28.09% of the total variance, the third principal component (PC3) contributed 11.55% to the total variance, and the fourth principal component (PC4) contributed 8.98% to the total variance.

The effect of agroforestry systems on the physicochemical properties of soil

Pecan agroforestry groups (PPH and PPL) were shown to reduce the BD, increase the MC, and increase the TPO of the soil when compared with CK,. These results may be attributed to leaf litter introduced by the vegetation and the distribution and penetration of the roots into the soil to improve the physical properties and structure of soil (Chen et al., 2019; Stöcker et al., 2020). It has been reported that soil BD increases with the soil depth (Stöcker et al., 2020). Our results showed decreased BD in the deep soil of the PPH group, which is agreement with Ling et al. (2020), and requires further investigation. Due to the decomposition of litter and chemical degradation of minerals, the soil EC level increased following agroforestry (Samani et al., 2020). In this study, the EC levels in the surface layer (0–20 cm) and subsurface layer (20–40 cm) of the soil increased after the agroforestry of pecans. The EC levels decreased in the deeper soil layer (40–60 cm) after agroforestry compared with the CK, which may be related to the distribution of the root systems and biochemical cycles of vegetation (Pierret et al., 2016). The vertical roots of pecan plants are concentrated below 40 cm (Xu et al., 2019) and their taproot depth is much greater than that of intercropped plants (Hanson, 2019).

Earlier studies have documented the use of agricultural intercropping to improve soil fertility (Du et al., 2019; Xia et al., 2019). We found that agroforestry increased the nutrient content, improved nutrient utilization, and promoted nutrient cycling in the soil (Mortimer, Saj & David, 2018). These results may be attributed to other mechanisms, such as changes in community composition and biomass, organic matter inputs, and the microclimate or soil structure (Borden, Thomas & Isaac, 2019; Wu et al., 2020). Most of the soil nutrients (e.g., AP, TP, NH₄⁺-N, AK, TN, TC) measured in the soil of the pecan agroforestry groups (PPH, PPS, and PPL) and pecan monoculture (CK) during this study were increased in the surface soil. Additionally, PPS increased the contents of Ca and Mg at the average soil depth. Therefore, pecan intercropping was shown to improve the availability of soil nutrients to a certain extent.

Changes in the physicochemical properties of soil caused by intercropping may be due to differences in the distribution of plant-soil roots and litter cover in the pecan agroforestry systems (Cardinael et al., 2020; Li et al., 2016). The pH of the soil may affect the acid–base balance of microbial cells and regulate the utilization of soil nutrients (Kemmitt et al., 2006). In our study, the soil pH was lower than that of the pecan monocropping after planting crops in the pecan plantation, indicating that agroforestry may improve the soil pH, and maintain soil productivity. These findings were congruent with the findings of Hu et al. (2019). Key biological functions of plant roots such as water and nutrient uptake, respiraton, and exudation alter pH in the soil Rudolph et al. (2013). Our data revealed that the soil pH was lower at the surface layer (0–20 cm) than the deeper soil layers. This effect may be attributed to the shallower root distribution of P. suffruticosa and H. citrina, which was consistent with the results of Andrianarisoa et al. (2016). The pH value of topsoil decreased after intercropping, which prevented excessive soil alkalization.

Different effects of agricultural and tree intercropping on enzyme activity in the surface soil of pecan plantation

Enzyme activity is a critical index of soil fertility and quality and is important in the soil’s biochemical function (Nannipieri, Trasar-Cepeda & Dick, 2017). We found that some of the enzyme activity from pecan cultivation was significantly higher than that of the single cultivation of pecans. These results were consistent with the findings of other intercropping systems (Clivot et al., 2019; Ma et al., 2017). Thus, a better understanding of these seven enzymes can clarify the role of agroforestry systems to improve soil fertility. Soil S-UE, S-BG, S-CBH, S-NAG, and S-AKP are all hydrolases. Among them, S-BG and S-CBH are involved in the C cycle, S-UE and S-NAG are involved in the N cycle, and S-AKP participates in the P cycle (Adetunji et al., 2017; Li et al., 2019). In the present study, S-POD and S-PPO were oxidoreductases, and oxidoreductase was involved in the synthesis of soil humus components and in the process of soil formation, which helps to understand the nature of soil occurrence and related soil fertility (Ananbeh et al., 2019).

Our research found that in the surface soil, the S-UE, S-AKP, and S-NAG activity of the soil were significantly increased under the agroforestry system when compared to CK. However, the S-BG and S-POD activity of PPL intercropping significantly declined, when compared with the CK, which may be attributed to the fact that soil moisture affects the biochemical process of soil carbon conversion catalyzed by S-BG (Zhang et al., 2011). The S-BG enzyme activity is reduced when soil moisture decreases, which reduces the nutrient renewal speed and lowers the supply of plant nutrients (Adetunji et al., 2017). Compared with CK, the S-PPO and S-CBH activity significantly decreased in soils under PPS intercropping. This may be due to the competition and interaction between different species under different compound management modes (particularly root system and root exudates), which affected crop water and nutrient absorption (Karaca et al., 2010).

The activity of surface soil enzymes were found to be closely related to the distribution of soil C, N, and P in various systems, which confirmed the key roles of these soil enzymes in carbon and nitrogen cycling in the ambient environment (Philippot et al., 2013). Contents of AK, TN and TC were highly correlated with the activity of S-AKP, S-CBH, and S-NAG, and had the strongest correlation with S-NAG enzyme activity, which better explained the changes in S-NAG enzyme activity. TP was highly positively correlated with S-AKP, which indicated that TP was the main factor that directly or indirectly influenced the activity of S-AKP, which may have been due to its positive feedback effect. Although the roles of some enzymes studied in the nutrient cycle were not evident, their ability to promote the decomposition of plant litter appeared to explain the increased content of these elements in the soil, which was expected (Feng et al., 2019).