Response ratio (R) of shoot biomass and leaf N and P concentration to N and P additions
The response ratio of shoot biomass (RB) in N and N + P treatment was positive effect (p < 0.05) (Table 1), but it was no significant effect in P treatment (p > 0.05) (Table 1). The response ratio leaf N concentration (RN) of three species (S.purpurea, C.moorcroftii and A.nanschanica) in N and N + P treatment were positive effect (p < 0.05), but they were no significant effect in P treatment (p > 0.05) (Table 1). The response ratio leaf P concentration (RP) of three species in P and N + P treatment were positive effect (p < 0.05), but they were no significant effect in N treatment (p > 0.05) (Table 1).
Table 1
Responses ratio (R) of shoot biomass, leaf nitrogen and phosphorus concentration to N, P, and N + P treatment.
Response ratio (R) | Treatment |
N | N + P | P |
Shoot biomass (RB) | + | + | 0 |
N concentration of leaf (RN) | S.purpurea | + | + | 0 |
C.moorcroftii | + | + | 0 |
A.nanschanica | + | + | 0 |
P concentration of leaf (RP) | S. purpurea | 0 | + | + |
C. moorcroftii | 0 | + | + |
A.nanschanica | 0 | + | + |
Symbols indicate statistically significant (p < 0.05) positive (+) and negative (–) effects relative to controls, and 0 denotes no significant effect of nutrient additions. |
For the shoot biomass, N and N + P additions produce higher responses ratio of shoot biomass than P additions (p < 0.05; Table 2). There is not significant difference between N and N + P treatment (p > 0.05; Table 2). For the N concentration of leaf, the RN of three species in N and N + P treatment are significantly higher than those in P treatment (p > 0.05; Table 2). There is also not significant difference between N and N + P treatment (p > 0.05; Table 2). For the P concentration of leaf, the RP of three species in N + P and P treatment are significantly higher than those in N treatment (p > 0.05; Table 2). There is also not significant difference between P and N + P treatment (p > 0.05; Table 2).
Table 2
Results of ANOVA’s comparing the responses ratio (R) of the three nutrient enrichment treatments (N, P, and N + P) on shoot biomass and N, P concentration of leaf
Response ratio (R) | Treatment | d.f. | Sum of squares | F | p-value |
Shoot biomass(RB) | N vs N + P | 1 | 0.170 | 0.262 | 0.636 |
N + P vs P | 1 | 0.727 | 14.828 | 0.018 |
N vs P | 1 | 0.986 | 16.058 | 0.016 |
N concentration of leaf (RN) | S.purpurea | N vs N + P | 1 | 0.003 | 0.127 | 0.740 |
N + P vs P | 1 | 0.810 | 39.613 | 0.003 |
N vs P | 1 | 0.904 | 105.079 | 0.001 |
C.moorcroftii | N vs N + P | 1 | 0.000 | 0.002 | 0.969 |
N + P vs P | 1 | 0.369 | 10.337 | 0.032 |
N vs P | 1 | 0.332 | 9.753 | 0.035 |
A.nanschanica | N vs N + P | 1 | 0.014 | 0.176 | 0.696 |
N + P vs P | 1 | 0.374 | 25.229 | 0.007 |
N vs P | 1 | 0.532 | 8.115 | 0.046 |
P concentration of leaf (RP) | S. purpurea | N vs N + P | 1 | 2.068 | 10.918 | 0.030 |
N + P vs P | 1 | 0.278 | 1.395 | 0.303 |
N vs P | 1 | 0.830 | 41.760 | 0.030 |
C. moorcroftii | N vs N + P | 1 | 0.604 | 18.638 | 0.012 |
N + P vs P | 1 | 0.001 | 0.006 | 0.942 |
N vs P | 1 | 0.573 | 11.147 | 0.029 |
A.nanschanica | N vs N + P | 1 | 1.614 | 44.573 | 0.003 |
N + P vs P | 1 | 0.084 | 2.557 | 0.185 |
N vs P | 1 | 0.962 | 83.903 | 0.001 |
Influence of N and P addition on soil properties
Soil properties were significantly affected by N and P addition after 5 years (Table 3). Soil pH and TOC were not significantly affected by N and P addition (p > 0.05). The DOC decreased following the N treatment (p < 0.05). The TN, AN, DON, NO3-N and NH4-N increased significantly in the N and N + P treatments (p < 0.05). There were no significant difference in TN, AN, NO3-N and NH4-N between the N and N + P treatments (p > 0.05). NH4-N decreased significantly in the P treatment (p < 0.05). TP and AP were significantly enhanced in the P and N + P treatment (p < 0.05), but were not significantly affected in the N treatment (p < 0.05). The activities of soil UE, NEP and AP were significantly promoted following N and P addition (p < 0.05). There was no significant difference in the activity of UE and NEP between the N treatment and N + P treatment (p > 0.05). However, the activity of AP in the N treatment is significantly higher than that in other treatments (Table 3).
Table 3
Effects of N and P addition on soil physicochemical property (mean ± s.e)
Treatment Variables | CK | N | N + P | P |
pH | 8.22 ± 0.01a | 8.25 ± 0.04a | 8.26 ± 0.05a | 8.32 ± 0.01a |
TN (g kg− 1) | 1.45 ± 0.07b | 1.72 ± 0.10a | 1.94 ± 0.09a | 1.39 ± 0.10b |
AN (mg kg− 1) | 13.61 ± 0.29c | 31.08 ± 2.34a | 26.80 ± 0.84b | 14.28 ± 0.84c |
DON (mg kg− 1) | 4.68 ± 0.31c | 9.83 ± 0.76b | 20.24 ± 2.56a | 12.28 ± 2.53b |
NO3-N (mg kg− 1) | 16.74 ± 1.18b | 28.30 ± 1.44a | 29.54 ± 1.86a | 17.04 ± 2.69b |
NH4-N (mg kg− 1) | 3.64 ± 0.21b | 4.34 ± 0.12a | 4.31 ± 0.22a | 2.95 ± 0.22c |
STP (g kg− 1) | 0.43 ± 0.01b | 0.41 ± 0.01b | 0.55 ± 0.02a | 0.51 ± 0.02a |
SAP (mg kg− 1) | 2.16 ± 0.18b | 2.07 ± 0.08b | 15.77 ± 1.49a | 15.45 ± 4.85a |
UE (U g− 1) | 350.92 ± 11.86c | 536.23 ± 14.00a | 568.06 ± 12.98a | 421.51 ± 7.29b |
NEP (U g− 1) | 0.42 ± 0.01c | 0.66 ± 0.02a | 0.66 ± 0.01a | 0.49 ± 0.02b |
AP (U g− 1) | 1.60 ± 0.06b | 2.57 ± 0.13a | 1.50 ± 0.08b | 1.33 ± 0.10b |
TOC, total organic carbon; DOC, dissolved organic carbon; TN, total nitrogen; AN, available nitrogen; DON, dissolved organic nitrogen; NO3-N, nitrate-nitrogen; NH4-N, ammonium nitrogen; TC, total carbon; STP, soil total phosphate; SAP, soil available phosphate; UE, urease; NEP, neutral phosphatase; AP, acid phosphatase. The different letter in the same row indicate significant difference at the 0.05 level. |
Response of the microbial community composition to N and P addition
The composition of the bacterial community was not significantly influenced by the N factor; however, it was significantly affected by the P factor (Table 4, Fig. 1A). Thaumarchaeota were also significantly affected by the interactive effect of the N and P factors (F = 6.406, p < 0.05). The composition of the fungal community was significantly influenced by N and P addition (Table 4, Fig. 1B). Ascomycota were significantly affected by the N factor and the interactive effect of the N and P factors (F = 13.484, p < 0.01; F = 7.321, p < 0.05). Mortierellomycota were significantly affected by N and P addition (F = 9.767, p < 0.05;F = 12.089, p < 0.01;F = 12.866, p < 0.01). Basidiomycota were significantly affected by P addition (F = 5.836, p < 0.05).
Table 4
Specific and interactive effects of N and P factors on the relative abundance of bacterial and fungal phyla among different treatments
| N factor | P factor | N×P |
Variables | F value | p value | F value | p value | F value | p value |
Bacterial phylum | Thaumarchaeota | 3.668 | 0.092 | 15.8 | 0.004 | 6.406 | 0.035 |
Actinobacteria | 3.132 | 0.115 | 13.156 | 0.007 | 4.452 | 0.068 |
Proteobacteria | 2.285 | 0.169 | 9.15 | 0.016 | 3.397 | 0.103 |
Acidobacteria | 3.863 | 0.085 | 13.466 | 0.006 | 5.128 | 0.053 |
Chloroflexi | 2.854 | 0.130 | 7.659 | 0.024 | 3.946 | 0.082 |
Bacteroidetes | 2.497 | 0.153 | 7.981 | 0.022 | 2.562 | 0.148 |
Gemmatimonadetes | 2.901 | 0.127 | 8.737 | 0.018 | 3.376 | 0.103 |
Firmicutes | 2.808 | 0.132 | 11.138 | 0.010 | 3.658 | 0.092 |
Rokubacteria | 3.503 | 0.098 | 9.256 | 0.016 | 4.47 | 0.067 |
Fungal phylum | Ascomycota | 13.484 | 0.006 | 0.237 | 0.640 | 7.321 | 0.027 |
Mortierellomycota | 9.767 | 0.014 | 12.089 | 0.008 | 12.866 | 0.007 |
Basidiomycota | 0.019 | 0.894 | 5.836 | 0.042 | 0.192 | 0.673 |
Chytridiomycota | 1.234 | 0.299 | 1.320 | 0.284 | 1.048 | 0.336 |
Glomeromycota | 0.095 | 0.765 | 2.414 | 0.159 | 0.386 | 0.552 |
Mucoromycota | 0.001 | 0.971 | 5.258 | 0.051 | 0.002 | 0.966 |
Kickxellomycota | 0.829 | 0.389 | 0.829 | 0.389 | 1.344 | 0.280 |
Olpidiomycota | 0.787 | 0.401 | 1.036 | 0.339 | 0.959 | 0.356 |
The abundance of bacteria was not significantly affected by N addition (Fig. 2A). However, the abundance of fungi increased significantly following N addition (Fig. 2B). There was no significant difference in the abundance of fungi between the N treatment and the N + P treatment (p > 0.05). The abundance of both bacteria and fungi was not significantly affected by P addition(p > 0.05).
Effect of N and P addition on the function of the microbial community
The function of the bacterial community was affected by N and P addition according to FAPROTAX (Fig. 3A). The functions of the bacterial community related to carbon (C) cycling (photoautotrophy, phototrophy, fermentation and chemoheterotrophy) and N cycling (aerobic ammonia oxidation, nitrification) were enhanced in the N treatment. However, the nitrate reduction and predatory or exoparasitic functions decreased following N addition. Nitrification and aerobic ammonia oxidation increased in the P treatment. Chemoheterotrophy, aerobic chemoheterotrophy and aromatic compound degradation increased in the N + P treatment.
The trophic function of the fungal community was affected by N and P addition (Fig. 3B). Pathotroph, saprotroph and pathotroph-saprotroph-symbiotroph groups increased in the N and N + P treatments. The saprotroph group decreased in the P treatment. The symbiotroph group decreased in the N and P treatment. The pathotroph-saprotroph group increased in the N + P treatment. The pathotroph-symbiotroph group enhanced in the P treatment.
Influential Factors On Microbial Community
Results from the SEM showed that N addition explained 98% of the variation in plant biomass, 65% of the variation in soil N:P, 48% of the variation in fungal community, 30% of the variation in bacterial community, and 98% of the variation in activity phosphatase (Fig. 4A). P addition explained 37% of the variation in plant biomass, 88% of the variation in soil N:P, 50% of the variation in fungal community, 45% of the variation in bacterial community, 99% of the variation in activity of urease(Fig. 4B).
N addition significantly increased the plant biomass and soil N:P (Fig. 4A). The plant biomass and fungal community significantly positively affected activity of phosphatase (Fig. 4A). Soil N:P significantly positively affected bacterial community, but did not significantly affected fungal community (Fig. 4A). Soil N:P did not significantly affected activity of phosphatase (Fig. 4A). Fungal community significantly positively affected activity of phosphatase (Fig. 4A). Soil N:P and bacterial community did not significantly affected activity of phosphatase (Fig. 4A).
P addition significantly decreased the soil N:P and did not significantly affected the plant biomass (Fig. 4B). The plant biomass did not significantly affected fungal and bacterial community, but significantly positively affected activity of urease (Fig. 4B). Soil N:P significantly positively affected bacterial community, but did not significantly affected fungal community(Fig. 4B). Soil N:P negatively affected activity of urease (Fig. 4B). Fungal and bacterial community significantly positively affected activity of urease (Fig. 4B).