3.1 Soil properties
The BCF soil showed significantly different chemical properties to that of the control soil (Table 1). Soil pH, CEC, SOC, TN, AN, and AK were significantly higher in BCF (p < 0.05) than in the control. The soil pH, CEC, TN, AN, and AK were highest at the 5-m site; however, we observed no significant difference in SOC across the different sites (p > 0.05). The soil total P (Pt) content in the BCF was significantly higher than that of the control (p < 0.05) and increased with increasing grazing density. Pi accounted for approximately 60–82% of Pt and also showed similar variability to that of Pt. The Pi/Pt value significantly increased with increasing grazing density (p < 0.05). Compared with the control, the BCF soil had a significantly higher total Po content but lower Po/Pt values (p < 0.05).
Table 1
Soil properties at different grazing distances from the hen house.
|
5-m
|
15-m
|
25-m
|
35-m
|
CK
|
pH
|
b6.69 ± 0.21a
|
5.75 ± 0.08b
|
5.66 ± 0.12b
|
5.77 ± 0.14b
|
5.02 ± 0.06c
|
CEC (cmol·kg-1)
|
18.07 ± 1.30a
|
17.62 ± 0.67a
|
15.35 ± 0.96b
|
15.25 ± 0.80b
|
13.75 ± 0.43c
|
SOC (g·kg-1)
|
41.97 ± 2.36a
|
42.72 ± 3.20a
|
41.81 ± 30.97a
|
41.35 ± 2.99a
|
36.67 ± 1.36b
|
TN (g·kg-1)
|
3.19 ± 0.32a
|
2.97 ± 0.12a
|
2.27 ± 0.27b
|
2.46 ± 0.16b
|
1.77 ± 0.12c
|
AN (mg·kg-1)
|
214.97 ± 4.96a
|
220.66 ± 3.66a
|
184.43 ± 8.35b
|
176.69 ± 4.98b
|
146.67 ± 9.55c
|
AK (mg·kg-1)
|
591.64 ± 34.78a
|
402.22 ± 10.48b
|
381.16 ± 3.10b
|
394.32 ± 8.30b
|
200.27 ± 4.77c
|
aTotal P (g·kg-1)
|
2.56 ± 0.08a
|
2.02 ± 0.05b
|
1.72 ± 0.09c
|
1.62 ± 0.08d
|
0.93 ± 0.03e
|
Total Pi (g·kg-1)
|
2.10 ± 0.08a
|
1.48 ± 0.04b
|
1.31 ± 0.08c
|
1.22 ± 0.06d
|
0.56 ± 0.03e
|
Pi/Pt (%)
|
82.00 ± 1.29a
|
73.04 ± 1.34c
|
75.97 ± 1.35b
|
74.88 ± 1.64b
|
60.61 ± 1.31d
|
Total Po (g·kg-1)
|
0.45 ± 0.03b
|
0.54 ± 0.03a
|
0.41 ± 0.03c
|
0.40 ± 0.04c
|
0.36 ± 0.02d
|
Po/Pt (%)
|
17.65 ± 1.28d
|
26.57 ± 1.33b
|
23.64 ± 1.35c
|
24.72 ± 1.64c
|
38.54 ± 1.29a
|
a Total P is the cumulative value of all P fractions; total Pi is the sum of Resin-Pi, NaHCO3-Pi, NaOH-Pi, 1 M HCl-Pi, and Conc. HCl-Pi; and Total Po is the sum of NaHCO3-Po, NaOH-Po, and Conc. HCl-Po. |
b Values are expressed as mean (n = 5) ± standard error. Values in the same row with different lowercase letters indicate significant differences to the other grazing sites at p < 0.05 (Duncan’s test). |
3.2 Soil P fractions and phosphatase activity
Our results showed that the BCF system significantly affected the relative content of each P fraction (Figure 2). The relative content of labile P was approximately 26–28% of Pt. In comparison, the relative content of moderately labile P was 52–64% of Pt, which gradually increased with increasing grazing density. Furthermore, NaHCO3-Pi contributed the largest proportion to labile P in the soil, accounting for approximately 44–50%. Moreover, the relative content of Resin-Pi gradually increased with increasing grazing density, while the proportion of NaHCO3-Po in labile P gradually decreased. Chicken farming had altered the composition of moderately labile P. The proportion of NaOH-extracted P in moderately labile P decreased gradually with increasing grazing density from 86% to 40%. Correspondingly, the proportion of 1 M HCl-Pi increased with decreasing grazing distance; the proportion at 5 m was higher than those of the other sites and the control group by ~1.36-fold and 13.54-fold, respectively. Compared with the control, chicken farming reduced the relative content of sparingly labile P, but the variability in its composition was limited (the relative content of conc. HCl-Pi varied within the range of 59–68% across all sites).
Labile P, moderately labile P, and sparingly labile P increased significantly with increasing grazing density (p < 0.05) (Table 2). The Pi fraction (Resin-Pi and NaHCO3-Pi) in labile P especially increased with increasing grazing density, and the maximum and minimum soil values of NaHCO3-Pi and Resin-Pi occurred at the 5-m site and the control, respectively. Compared with the control, we observed no significant difference in the Po fraction in labile P at 25 and 35 m (NaHCO3-Po) (p > 0.05). In moderately labile P, the maximum and minimum P contents extracted by NaOH were observed at 15 m and the control, respectively. Although both NaOH-Pi and NaOH-Po values at 5 m were significantly lower than those at 15 m (p < 0.05), the 1 M HCl-Pi at 5 m increased by 116.41% and 1354.41% compared with the 15-m site and the control, respectively. In sparingly labile P, the soil at 5 m had significantly higher conc. HCl-Pi than the soils at the other sites and the control (p < 0.05). The conc. HCl-Po trend was not consistent with that of conc. HCl-Pi, except for 15 m; however, the values were not significantly different to those of the other sites and the control (p > 0.05).
Table 2
Phosphorus (P) sequential fractionation (g·kg-1) in the bamboo forest soil at different grazing distances from the hen house.
P fraction
|
5-m
|
15-m
|
25-m
|
35-m
|
CK
|
aLabile P
|
|
|
|
|
|
Resin-Pi
|
b0.20 ± 0.01a
|
0.15 ± 0.01b
|
0.12 ± 0.01c
|
0.13 ± 0.01c
|
0.06 ± 0.01d
|
NaHCO3-Pi
|
0.34 ± 0.03a
|
0.26 ± 0.02b
|
0.22 ± 0.02c
|
0.22 ± 0.02c
|
0.12 ± 0.01d
|
NaHCO3-Po
|
0.14 ± 0.02a
|
0.13 ± 0.03a
|
0.11 ± 0.01b
|
0.09 ± 0.01b
|
0.09 ± 0.01b
|
∑Labile P
|
0.68 ± 0.02a
|
0.55 ± 0.04b
|
0.45 ± 0.02c
|
0.44 ± 0.03c
|
0.26 ± 0.02d
|
Moderately labile P
|
|
|
|
|
|
NaOH-Pi
|
0.42 ± 0.03b
|
0.45 ± 0.02a
|
0.37 ± 0.03c
|
0.37 ± 0.02c
|
0.21 ± 0.02d
|
NaOH-Po
|
0.24 ± 0.02b
|
0.30 ± 0.02a
|
0.23 ± 0.01ab
|
0.24 ± 0.03b
|
0.20 ± 0.02c
|
1 M HCl-Pi
|
0.99 ± 0.08a
|
0.46 ± 0.02b
|
0.45 ± 0.04b
|
0.36 ± 0.02c
|
0.07 ± 0.01d
|
∑Moderately labile P
|
1.65 ± 0.05a
|
1.21 ± 0.04b
|
1.05 ± 0.08c
|
0.97 ± 0.07d
|
0.48 ± 0.03e
|
Sparingly labile P
|
|
|
|
|
|
conc.HCl-Pi
|
0.16 ± 0.01a
|
0.15 ± 0.01b
|
0.15 ± 0.01ab
|
0.13 ± 0.01c
|
0.11 ± 0.01d
|
conc.HCl-Po
|
0.08 ± 0.01b
|
0.11 ± 0.01a
|
0.07 ± 0.02b
|
0.07 ± 0.02b
|
0.07 ± 0.01b
|
∑Sparingly labile P
|
0.24 ± 0.01ab
|
0.25 ± 0.01a
|
0.22 ± 0.02bc
|
0.20 ± 0.01c
|
0.18 ± 0.01d
|
Non-labile P
|
|
|
|
|
|
Residual-Pt
|
0.01 ± 0.00a
|
0.01 ± 0.00b
|
0.01 ± 0.00c
|
0.01 ± 0.00c
|
0.01 ± 0.00b
|
a P fractions were classified according to Liao et al. (2020) and Crews and Brookes (2014). |
b Values are expressed as the mean (n = 5) ± standard error. Values in the same row with different lowercase letters indicate significant differences to the other grazing sites at P < 0.05 (Duncan’s test). |
Chicken farming affected the activity of ACP and ALP in soil (Figure 3). The activity of soil ACP was significantly higher in BCF than in the control (p < 0.05) (Figure 3a), but no significant differences were observed between the different sites (p > 0.05). The highest ALP activity was observed at the 5-m site (9.97 µmol pNP g-1 soil d-1), which was significantly higher than the other sites and the control (p < 0.05) (Figure 3b). Compared with the control, the ALP activity at 15, 25, and 35 m significantly increased by 66.43%, 26.96%, and 69.61%, respectively (p < 0.05).
3.3 Soil bacterial community structure
Except for 25 m, we observed no significant difference between the alpha diversity index of the different sites and the control (p > 0.05) (Figure S1). We used a PCoA to compare the bacterial communities between the sites and the control based on unweighted and weighted Unifrac distances (Figure S2). The unweighted PCoA clearly separated the BCF and control bacterial communities (Figure S2a). Moreover, the 15-, 25-, and 35-m sites were tightly clustered, whereas the 5-m site notably deviated from the group. The sample aggregation in the weighted analysis was similar to that in the unweighted analysis. The combined axis of PCo1, PCo2, and PCo3 accounted for 28.94% and 74.81% of the total change in the unweighted and weighted PCoA, respectively. The ANOSIM results also revealed significant differences in the bacterial community composition between the different sites (p < 0.05).
In all the soil samples, the dominant bacteria phyla with average relative abundances of > 1% were Proteobacteria (38.91%), Acidobacteria (16.27%), Actinobacteria (10.61%), Bacteroidetes (7.89%), Chloroflexi (7.05%), TM7 (6.11%), Gemmatimonadetes (3.71%), AD3 (1.67%), Firmicutes (1.08%), and Verrucomicrobia (1.07%) (Figure S3). We performed a one-way ANOVA to compare the individual taxa at the phylum level (Figure 4). Compared with the control, the relative abundance of Proteobacteria and TM7 had significantly increased in BCF (p < 0.05), except at the 5-m site. The relative abundances of Acidobacteria and Bacteroidetes in the 15-, 25-, and 35-m sites did not significantly differ from that of the control (p > 0.05). However, their relative abundances in the 5-m site were significantly different to those of the other sites and the control (p < 0.05). The relative abundance of Gemmatimonadetes was significantly higher at the 5-m and 15-m sites compared with the 25-m and 35-m sites and the control (p < 0.05). Soils under BCF conditions had a lower relative abundance of AD3 compared with the control soil (p < 0.05). Furthermore, the relative abundance of Firmicutes was similar in the BCF sites and the control, except at 5 m (p > 0.05).
Seven dominant phosphobacteria genera (average relative abundance > 0.1%) were detected in the topsoil of the BCF sites and the control, including Flavobacterium (1.55%), Burkholderia (0.59%), Pseudomonas (0.40%), Bacillus (0.30%), Streptomyces (0.21%), Arthrobacter (0.20%), and Paenibacillus (0.10%). The relative abundance of Flavobacterium at the 5-m site was significantly higher than those of the other sites and the control (p < 0.05) (Table 3). Flavobacterium abundances at 15, 25, and 35 m were 0.55, 1.32, and 2.12 times the control values, but the differences were not significant (p > 0.05). Compared with the control, the relative abundance of Burkholderia was significantly lower in the 5-m site (p < 0.05) but showed no significant differences at 15 and 25 m (p > 0.05). The relative abundance of Pseudomonas, Streptomyces, Arthrobacter, and Paenibacillus increased significantly in the BCF soils, but only the relative abundances of Pseudomonas and Paenibacillus at 25-m significantly differed from the other sites (p < 0.05). Furthermore, we observed no clear difference in the relative abundance of Bacillus between the different sites and the control.
Table 3
Relative abundances of the dominant phosphobacteria genera (average relative abundance > 0.1%) in soil at different distances from the hen house.
phyla
|
genera
|
5-m
|
15-m
|
25-m
|
35-m
|
CK
|
Bacteroidetes
|
Flavobacterium
|
3.80 ± 1.57a
|
0.49 ± 0.21b
|
0.73 ± 0.35b
|
0.99 ± 0.25b
|
0.32 ± 0.13b
|
Proteobacteria
|
Burkholderia
|
0.41 ± 0.12c
|
0.81 ± 0.21a
|
0.75 ± 0.16ab
|
0.55 ± 0.19b
|
0.83 ± 0.21a
|
|
Pseudomonas
|
0.42 ± 0.09b
|
0.38 ± 0.07b
|
0.62 ± 0.16a
|
0.35 ± 0.10b
|
0.13 ± 0.04c
|
Actinobacteria
|
Streptomyces
|
0.20 ± 0.05b
|
0.32 ± 0.07a
|
0.20 ± 0.04b
|
0.23 ± 0.03b
|
0.10 ± 0.04c
|
|
Arthrobacter
|
0.29 ± 0.11a
|
0.23 ± 0.11a
|
0.22 ± 0.20a
|
0.16 ± 0.06ab
|
0.02 ± 0.02b
|
Firmicutes
|
Bacillus
|
0.26 ± 0.08a
|
0.35 ± 0.12a
|
0.27 ± 0.03a
|
0.35 ± 0.05a
|
0.35 ± 0.15a
|
|
Paenibacillus
|
0.07 ± 0.02c
|
0.11 ± 0.02b
|
0.14 ± 0.02a
|
0.13 ± 0.01ab
|
0.05 ± 0.01d
|
3.4 Correlating P fractions with the bacterial community
The results of the Pearson correlation analysis inferred the relationship between soil parameters and the dominant bacteria phyla (Figure S4). Among the different soil chemical properties, Proteobacteria and TM7 only significantly positively correlated with the SOC content. In contrast, except for SOC, Bacteroidetes significantly positively correlated with the other soil chemical variables (pH, CEC, TN, AN, and AK). Gemmatimonadetes significantly positively correlated with pH, CEC, SOC, TN, AN, and AK. However, contrary to Gemmatimonadetes, AD3 significantly negatively correlated with all the soil basic properties. Furthermore, Proteobacteria and TM7 showed significant and extremely significant positive correlations with NaOH-P, respectively. We also observed a strong positive relationship between Bacteroidetes and all the Pi fractions. Gemmatimonadetes also showed a significant positive correlation with the Pi fractions, except conc. HCl-Pi. Actinobacteria showed a strong significant negative correlation with Resin-Pi and 1 M HCl-Pi and a significant negative correlation with NaHCO3-Pi and conc. HCl-Pi. Moreover, AD3 showed a strong significant negative relationship with the Pi fractions and a significant negative correlation with NaHCO3-Po and NaOH-Po.
The relative abundances of Flavobacterium, Pseudomonas, Streptomyces, Arthrobacter, and Paenibacillus were mostly correlated with the changes in P fractions (Figure 5). Flavobacterium showed a significant positive correlation with Resin-Pi and a strong significant positive correlation with NaHCO3-Pi and 1 M HCl-Pi. Pseudomonas significantly positively correlated with Resin-Pi, NaHCO3-Pi, and 1 M HCl-Pi, and showed a strong significant positive correlation with NaOH-Pi and conc. HCl-Pi. Streptomyces significantly positively correlated with Resin-Pi, NaHCO3-Pi, conc. HCl-Pi, and conc. HCl-Po, and showed a strong significant correlation with NaOH-Pi and NaOH-Po. Arthrobacter showed a strong significant correlation with Resin-Pi, NaHCO3-Pi, 1 M HCl-Pi, and conc. HCl-Pi, and a significant positive correlation with NaOH-Pi. Paenibacillus only significantly correlated with NaOH-Pi. Burkholderia had a negative correlation with all P fractions, with a strong significant negative correlation with Resin-Pi, NaHCO3-Pi, and 1 M HCl-Pi, and a significant negative correlation with NaOH-Po and conc. HCl-Pi.
The RDA showed that the soil chemical properties and P fractions explained 44.79% of the variations in soil bacterial community structure (Figure 6). According to the Monte Carlo permutation test (permutation = 999), the Pi fractions, Po fractions (except conc. HCl-P), soil properties, and phosphatase were significantly correlated with the structure of bacterial communities. In Figure 6, a longer arrow represents a stronger relationship between the soil variables and bacterial community composition. Therefore, according to the variance of r2, Pi fractions had a stronger correlation with bacterial community changes than Po fractions (Table S1). Furthermore, among all soil parameters, AK and pH showed the strongest correlation with the changes in bacterial community (r2 = 0.963 and 0.962, respectively), followed by 1 M HCl-Pi and Resin-Pi in the Pi fractions (r2 = 0.958 and 0.938, respectively).