Soil properties and P fractions along plantation development
The values of pH, soil moisture, electrical conductivity, organic matter, total N and K, NH4-N, and available K and their variation tendencies are shown in Fig. 1. These indictors all significantly linearly increased with the plantation age (P < 0.05), indicating that the revegetation of C. microphylla on moving sand dunes can improve soil environment and facilitate the increase in soil nutrient.
The soil P fractions of different sites determined by the sequential extraction method (H2O, 1.0 mol L− 1 of HCl, 0.5 mol L− 1 of NaHCO3, 0.5 mol L− 1 of NaOH, and 0.1 mol L− 1 of NaOH) are listed in Table 1. General P (total of different P fractions and residual P) increased with increasing plantation age. The IOP fractions extracted by H2O, HCl, NaHCO3, and NaOH were all much lower than their respective OP, indicating that soil OP was the dominant P fraction along with plantation development. The most abundance OP fraction was NaOH-Po extracted by 0.5 and 0.1 mol L− 1 solutions with averages of 50.17 and 49.09 mg kg− 1, respectively. Although the AP fractions including NaHCO3-Pi and H2O-Pi accounted for a small proportion, the values showed an increasing tendency along with plantation development. However, the concentration of NaHCO3-Po, the stock of AP, ranged from 32.83 mg kg− 1 to 44.31 mg kg− 1, and no significant difference was observed among different sites. At the MS site, except for 0.5 mol L− 1 of NaHCO3-Po, each P fraction concentration was lower than that of vegetation-covered sites. The regression analysis indicated that the concentrations of H2O-Pi, 1.0 mol L− 1 of HCl-Pi, 0.5 mol L− 1 of NaHCO3-Pi, 0.1 mol L− 1 of NaOH-Pi, and 0.1 mol L− 1 of NaOH-Po, residual-P, and general P all significantly linearly increased with plantation age (P < 0.01).
Table 1
Fractions of soil P under different Caragana microphylla plantations (mg kg-1)
Fractions
|
MS
|
CM-10
|
CM-20
|
CM-37
|
NCM
|
ANOVA in response to plantation age
|
R2
|
F
|
P
|
H2O-Pi
|
1.345 ± 0.090
|
1.64 ± 0.462
|
2.175 ± 0.742
|
2.895 ± 0.773
|
4.24 ± 1.562
|
0.667
|
26.066
|
< 0.001
|
NaHCO3-Pi (0.5 mol L− 1)
|
5.820 ± 0.322
|
7.110 ± 0.773
|
10.69 ± 1.631
|
10.16 ± 1.022
|
15.17 ± 3.013
|
0.784
|
47.267
|
< 0.001
|
NaHCO3-Po(0.5 mol L− 1)
|
43.18 ± 4.510
|
37.89 ± 2.367
|
44.31 ± 3.445
|
41.34 ± 10.19
|
32.83 ± 8.193
|
0.169
|
2.651
|
0.127
|
HCl-Pi (1.0 mol L− 1)
|
4.223 ± 0.983
|
6.657 ± 1.141
|
12.70 ± 1.281
|
15.05 ± 2.441
|
19.36 ± 0.671
|
0.918
|
145.00
|
< 0.001
|
HCl-Po (1.0 mol L− 1)
|
11.44 ± 0.606
|
13.34 ± 2.717
|
12.64 ± 4.010
|
17.61 ± 6.416
|
13.97 ± 1.782
|
0.105
|
1.519
|
0.240
|
NaOH-Pi (0.5 mol L− 1)
|
6.823 ± 1.773
|
7.559 ± 0.837
|
8.060 ± 1.978
|
7.496 ± 1.977
|
9.187 ± 2.063
|
0.168
|
2.624
|
0.129
|
NaOH-Po (0.5 mol L− 1)
|
44.68 ± 2.07
|
49.44 ± 3.368
|
51.44 ± 5.448
|
52.50 ± 4.064
|
52.81 ± 4.176
|
0.169
|
2.641
|
0.128
|
NaOH-Pi (0.1 mol L− 1)
|
9.211 ± 0.980
|
9.660 ± 0.766
|
11.68 ± 0.834
|
11.85 ± 2.001
|
14.13 ± 1.951
|
0.662
|
25.47
|
< 0.001
|
NaOH-Po (0.1 mol L− 1)
|
40.29 ± 5.249
|
48.84 ± 9.903
|
40.32 ± 4.768
|
54.15 ± 8.846
|
61.87 ± 14.62
|
0.413
|
9.158
|
0.009
|
Residual-P
|
67.50 ± 26.15
|
91.00 ± 27.10
|
135.0 ± 15.80
|
258.5 ± 62.93
|
518.0 ± 32.32
|
0.897
|
113.1
|
< 0.001
|
General-P
|
234.5 ± 16.09
|
273.1 ± 24.94
|
329.0 ± 22.31
|
471.6 ± 70.48
|
741.6 ± 49.90
|
0.917
|
143.6
|
< 0.001
|
Values are means ± SD. MS: moving sand dune (0-yr); CM-10, CM-20, and CM-37: 10-, 20-, and 37-year plantation, respectively; NCM: natural C. mirophylla community. R2, F, and P values from regression analysis are given. |
Similar to soil physicochemical properties, the activities of soil phosphomonoesterase, urease, dehydrogenase, protease, glucosidase, and polyphenol oxidase in CM-10, CM-20, CM-37, and NCM sites increased by 7.71–69.90, 7.48–63.30, 1.06–5.54, 3.30–11.75, 4.03–9.90, and 1.38–2.35 times, respectively, compared with MS. The regression analysis showed that the activities of all selected enzymes had significantly linear relationships with plantation age (P < 0.01, Table 2). The activity of the transformation from soil insoluble phosphate or OP to AP was determined after 21 days of incubation. The average rates of lecithin mineralization and phosphorite dissolution were 2.65% and 0.11%, respectively, and also linearly increased with the plantation age (P < 0.05, Fig. 2).
Table 2
Activities of soil enzyme under different Caragana microphylla plantations
Fractions
|
MS
|
CM-10
|
CM-20
|
CM-37
|
NCM
|
ANOVA in response to plantation age
|
R2
|
F
|
P
|
DHA (mg TPF kg-1 24h-1)
|
46.21 ± 1.328
|
48.86 ± 2.395
|
64.92 ± 11.35
|
97.65 ± 48.42
|
255.9 ± 71.29
|
0.711
|
31.97
|
< 0.001
|
Urease (mg 100 g− 1 24h− 1)
|
0.510 ± 0.065
|
3.812 ± 0.052
|
9.809 ± 2.166
|
18.31 ± 11.33
|
32.28 ± 5.796
|
0.844
|
70.12
|
< 0.001
|
PHA (mg g− 1 h-1)
|
2.276 ± 0.950
|
17.58 ± 8.111
|
45.99 ± 15.20
|
81.44 ± 10.40
|
159.4 ± 41.07
|
0.896
|
112.3
|
< 0.001
|
POA (µmol g− 1 10min− 1)
|
1.985 ± 0.378
|
2.731 ± 0.681
|
3.246 ± 0.378
|
4.790 ± 1.001
|
4.662 ± 0.839
|
0.682
|
27.88
|
< 0.001
|
Protease (mg Tyr g− 1 2h− 1)
|
8.169 ± 0.845
|
26.97 ± 11.02
|
55.54 ± 4.928
|
91.52 ± 14.89
|
96.03 ± 19.32
|
0.843
|
69.55
|
< 0.001
|
GLA (µg g− 1 h− 1)
|
0.139 ± 0.006
|
0.558 ± 0.035
|
0.580 ± 0.102
|
1.044 ± 0.031
|
1.372 ± 0.051
|
0.950
|
249.1
|
< 0.001
|
Values are means ± SD. MS: moving sand dune (0-yr); CM-10, CM-20, and CM-37: 10-, 20-, and 37-year plantation, respectively; NCM: natural C. microphylla community. DHA: dehydrogenase; POA: polyphenol oxidase; GLA: glucosidase; PHA: phosphomonoesterase. R2, F, and P values from regression analysis are given. |
Abundance of phoD and gcd genes
The abundance of phoD and gcd genes under MS, CM plantations, and NCM soils was determined by Q-PCR. The copies/g soil of phoD and gcd genes ranged from 7.44 × 105 in MS to 1.38 × 108 in NCM and 2.0 × 103 in MS to 2.16× 106 in NCM, respectively, suggesting that the quantity of phoD-harboring microbes is greater than that of gcd-harboring microbes in sandy soil. The copies/g soil of phoD and gcd genes in CM-10, CM-20, CM-37, and NCM sites were 1.23 to 185.5 times and 23.68 to 1076.9 times greater compared with those in the MS site, indicating that the establishment of C. microphylla on moving sand dunes facilitated the restoration of soil phoD and gcd microbial communities. The abundance tendency of the two genes along with plantation development can be fitted by a quadratic regression model (P < 0.001, Fig. 3). Significant positive relationships were found among the abundance of phoD and gcd genes, soil AP (H2O-Pi + NaHCO3-Pi), and phosphomonoesterase activity (r = 0.737 to 0.901, P < 0.001, data not shown). Overall, the variations in the abundance of soil phoD and gcd genes were consistent with those in soil nutrients, the activities of enzymes, P transformation function, and the concentrations of AP fractions.
Structures of soil phoD and gcd communities
A total of 489,065 and 1,031,717 phoD and gcd gene sequences were obtained from 15 soil samples, respectively, after quality filtering and removal of chimeras. OTUs with 75% similarity cutoff were clustered. The Simpson, Chao1, Shannon–Wiener, and Pielou indices and observed species were calculated on the basis of the result of OTU clustering (Table 3). The diversity indices (except for Simpson) of phoD community in vegetation-covered sites were all significantly higher than those in MS (P < 0.05). While no significant difference in α-diversity indices (except for Simpson) was observed in the gcd community between MS and CM-10, they were all significantly lower than those of the other three sites (P < 0.05). Overall, these indices of phoD community were higher than those of gcd community in all sites. Clustering analysis showed that most samples from the same sites can be clustered together, and the 15 samples can be divided into four or five groups (Fig. S1 and Fig. S2), suggesting the significant difference in the structure of soil phoD or gcd microbial community along with plantation development.
Table 3
Alpha diversity indices of soil phoD or gcd community under different plantations
Index
|
|
MS
|
CM-10
|
CM-20
|
CM-37
|
NCM
|
One-way ANOVA
|
F
|
P
|
Simpson
|
phoD
|
0.939 ± 0.006a
|
0.986 ± 0.004a
|
0.973 ± 0.018a
|
0.985 ± 0.006a
|
0.960 ± 0.038a
|
3.078
|
0.068
|
gcd
|
0.940 ± 0.011a
|
0.878 ± 0.038b
|
0.948 ± 0.025b
|
0.967 ± 0.013b
|
0.941 ± 0.026b
|
5.550
|
0.013
|
Shannon-Wienner
|
phoD
|
6.068 ± 0.143a
|
7.916 ± 0.157b
|
7.592 ± 0.362b
|
7.836 ± 0.306b
|
7.356 ± 0.959b
|
7.111
|
0.006
|
gcd
|
4.906 ± 0.315a
|
4.592 ± 0.404a
|
5.799 ± 0.497b
|
6.346 ± 0.414b
|
5.961 ± 0.471b
|
9.131
|
0.002
|
Pielou
|
phoD
|
0.667 ± 0.015a
|
0.791 ± 0.015b
|
0.760 ± 0.035b
|
0.783 ± 0.027b
|
0.733 ± 0.078a
|
4.465
|
0.025
|
gcd
|
0.666 ± 0.041b
|
0.581 ± 0.057a
|
0.688 ± 0.050b
|
0.744 ± 0.017b
|
0.689 ± 0.039b
|
5.754
|
0.011
|
Chao1
|
phoD
|
765 ± 9a
|
1371 ± 78b
|
1382 ± 70b
|
1427 ± 67b
|
1450 ± 280b
|
13.35
|
0.001
|
gcd
|
239 ± 59a
|
289 ± 58a
|
413 ± 27b
|
431 ± 124b
|
472 ± 73b
|
5.260
|
0.015
|
Observed species
|
phoD
|
548 ± 15a
|
1028 ± 51b
|
1014 ± 37b
|
1027 ± 48b
|
1048 ± 186b
|
17.08
|
< 0.001
|
gcd
|
168 ± 33a
|
243 ± 40a
|
345 ± 37b
|
377 ± 90b
|
404 ± 58b
|
5.572
|
0.002
|
Values are means ± SD. MS: moving sand dune (0-yr); CM-10, CM-20, and CM-37: 10-, 20-, and 37-year plantation, respectively; NCM: natural C. microphylla community. F, and P values from One-way ANOVA are given. |
Means in row followed by different letters are significantly different (P < 0.05). |
phoD OTUs were classified into 5 different phyla, 20 orders, or 32 families. The phoD phyla included Actinobacteria (15.68–42.97%), Proteobacteria (9.03–15.79%), Planctomycetes (6.18–11.61%), Cyanobacteria (0.17–0.89%), and Firmicutes (0.44–1.56%), while 36.36–55.58% OTUs of phoD could not be classified at the phylum level within the GenBank database. Thirteen dominant phoD families (relative abundance > 1%) were detected, and their total relative abundance ranged from 37.17% in MS to 54.20% in CM-20 (Fig. 4). In the phoD community, Streptomycetaceae was the absolutely dominant family in all samples with a relative abundance from 10.63% in MS to 20.05% in NCM, followed by Pseudonocardiaceae (1.73–15.50%), Bradyrhizobiaceae (1.97–5.11%), Isosphaeraceae (1.99–4.24%), and Micromonosporaceae (0.22–5.37%). The relative abundance of most dominant phoD families in CM plantations and NCM was significantly higher than that in MS, suggesting that revegetation on moving sand dunes was very helpful to restore the dominant taxa. However, some dominant families in MS, including Xanthomonadaceae, Burkholderiaceae, and Gemmataceae, decreased after the CM plantation was established.
On the basis of the GenBank database, gcd OTUs were classified into 6 phyla, 14 orders, 25 families, or 17 genera. The most dominant gcd phylum was Proteobacteria with a relative abundance from 26.9% in MS to 80.01% in CM-10, followed by Planctomycetes (8.17%) and Verrucomicrobia (1.46%). The relative abundance of Bacteroidetes, Actinobacteria, and Euryarchaeota was all less than 1%. Euryarchaeota (0.011%) was only observed in NCM samples (Fig. 4). Eight dominant gcd families (relative abundance > 1.0%) were found, namely, Rhizobiaceae (33.30%), Planctomycetaceae (7.22%), Pseudomonadaceae (6.68%), Opitutaceae (4.16%), Enterobacteriaceae (2.41%), Sphingomonadaceae (1.82%), Rhodospirillaceae (1.42%), and Bradyrhizobiaceae (1.29%). The sum of the relative abundance of these dominant families ranged from 45.27% in MS to 79.84% in CM-10 site. Revegetation on MS resulted in decreased abundance of Planctomycetaceae, Enterobacteriaceae, and Rhodospirillaceae and significantly increased abundance of Rhizobiaceae, showing waning and waxing variations. Although most OTUs could not be classified at the genus level in the GenBank database, several dominant genera including Agrobacterium (14.20%), Rhizobium (8.96%), Pseudomonas (5.12%), Kluyvera (1.78%), and Bradyrhizobium (1.26%) were detected.
LEfSe analysis was performed to determine differentiated taxa among MS, CM plantations, and NCM, and LDA distributions are shown in Fig. 5. At the MS site, differentiated phoD taxa included Caulobacteraceae, Xanthomonadaceae, Betaproteobacteria, Burkholderiaceae, Cupriavidus, and Caulobacterales; differentiated gcd taxa included Kluyvera, Opitutaceae, Opitutae, Verrucomicrobia, Rhodospirillaceae, and Opitutales. At the CM-10 site, differentiated phoD taxa were Nostocaceae and Aphanizomenonaceae; differentiated gcd taxa were Xanthomonadaceae, Neorhizobium, Agrobacterium, Rhizobiaceae, and Proteobacteria. At the CM-20 site, differentiated phoD taxa were Bacillus, Rubrobacteraceae, Bacteria, and Actinobacteria; differentiated gcd taxa included Betaproteobacteria, Planctomyces, Burkholderiaceae, Acidovorax, and Alphaproteobacteria. At the CM-37 site, differentiated phoD taxa included Acetobacteraceae, Rhodospirillales, and Bradyrhizobium; differentiated gcd taxa included Sphingobacteriaceae, Sphingobacteriia, Bacteroidetes, Methylocystaceae, Bradyrhizobium, and Sphingomonadaceae. At the NCM site, no differentiated phoD taxon was found, and only gcd-harboring Enterobacter was significantly different from other sites. These results showed the waxing and waning variations of the structures of soil phoD and gcd communities along with plantation development.
Dependence of phoD and gcd communities on soil properties
CCA was carried out to determine the influence of soil properties (including pH, electrical conductivity, organic matter, soil moisture, total N, total K, NH4-N, available AK, NaHCO3-Pi, and phosphomonoesterase). The results showed that the variations were explained by 57.5% for phoD community and 55.2% for gcd community along the first axis, and by 21.3% and 25.9% along the second axis, respectively (Fig. 6). The three samples of CM-10 and the three samples of MS can be clustered in different groups, indicating the difference in the community structure among different sites. In general, the influence of the selected soil properties on phoD or gcd community was similar. Total N, NH4-N, and available K were dominant factors influencing the phoD or gcd communities. In addition, soil NaHCO3-Pi and phosphomonoesterase activity were the main influencing factors on gcd community. However, the effects of soil pH, electrical conductivity, soil moisture, and total K on the structure of the two communities were relatively minor.