Setaria viridis (abbreviated as Sv), Artemisia gmelinii (Ag), and Bothriochloa ischaemum (Bi) were selected as representative plants of early, middle, and later successional species, respectively. Panicum virgatum (Pv) was selected as the exotic species. These three native species are the dominant grass species of secondary succession on the Loess Plateau. Pv is a perennial C4 gramineous plant that is native to North America and Central America and has been grown as an energy plant since 1990, owing to its developed root system, high productivity, and strong ecological adaptability. In 1990, the Institute of Soil and Water Conservation, Chinese Academy of Sciences, and Ministry of Water Resources began introducing Pv in the Loess Plateau to restore the local ecological environment. This plant showed strong adaptability to extreme climatic conditions of high temperature and drought, with good soil and water conservation benefits [39].
Seed and soil collection
The seeds of the four species were obtained from adult plants at sampling sites (each plant species was collected from five sites) located at Ansai Research Station of the Chinese Academy of Sciences (36°51′N, 109°19′E, 1068–1309 m above sea level [a.s.l.]). The annual average precipitation and temperature of this area are 483 mm and 8.8 ℃, respectively. The soil type is loess soil. Soils were collected from the same location as the seeds (minimum of 20 m × 20 m), from April 25 to 30, 2015. Soil was collected from the 5–20 cm layer, after removing ground litter and soil in the 0–5 cm layer. The collected soil was passed through a 5 mm mesh and mixed evenly for pot planting. Average chemistry and microbial soil properties for the different field soil sources are presented in Table 1.
Experimental set-up
The experiment was carried out in the State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, Institute of Soil and Water Conservation, Chinese Academy of Sciences and Ministry of Water Resources (34°12′N, 108°07′E, 530 m a.s.l.). Illumination time, light intensity, air temperature, and air humidity can be controlled in this facility. The average annual temperature and precipitation are 12.9 ℃ and 637.6 mm, respectively. Before the experiment, we measured the germination rate of the seeds, which exceeded 90%. Pots were round containers (diameter × height: 15 cm × 20 cm). One kilogram of crushed stones (diameter about 1 cm) was added to each pot to prevent soil hardening caused by watering. A plastic tube (diameter × height: 1 cm × 25 cm) was inserted as a watering channel in the pot, which terminated above the crushed stones. A piece of paper was added to separate the soil and crushed stones.
Experiment 1
This experiment was conducted on May 4, 2016. Four types of field soil were collected to treat the three early, middle, and later successional species (referred to as E, M, and L soils, respectively), plus the exotic species (Exo). To examine the performance of the exotic species Pv in all three soils, one Pv individual was grown in each of soil type (Fig. 6, red dotted line). To compare the performance of the three native species in their own soil and exotic soils, one Sv individual was grown in E soil, one Ag individual was grown in M soil, and one Bi individual was grown in L soil. These individual were compared with individual of the same species grown in Exo soils (Fig. 6, blue dotted line). There were 10 replicates for each plant species grown in each soil type. As the bare control, five pots with each soil type and no plants were prepared. One-hundred-and-twenty pots were used in this phase.
For the planting process, 2.8 kg soil samples were weighed and put in pots (based on dry weight). The total weight of each pot was recorded for later water control. Then, the plant seeds were evenly sown in the pots. After sowing, sufficient water was added. Two weeks later, the seedlings were thinned until four plants per pot remained for the field investigation. After thinning, regular watering and management were implemented. Water control was conducted twice a week (to be 80% of field capacity). The position of pots was changed weekly to limit any effect of microclimate on plant growth. After 4 months, four pots containing uniformly grown plants were selected to collect plant and soil samples for each treatment. The roots were removed from each pot. The soil was homogenized and returned to the same pot of the same treatment.
Experiment 2
This experiment was conducted on May 4, 2017. After harvesting the plant species in Experiment 1, the soils were collected and used to assess the recovery of native species following disturbance by exotic species and the invasion of exotic species following disturbance by native species. To determine the invasiveness of exotic species after disturbance by native species, Pv individual were grown in soils created from native species (Sv, Ag, and Bi) versus being grown in its own soils originating from Exo field soils (referred to as Exo-Sv, Exo-Ag, and Exo-Bi vs. Exo-Pv, respectively) (Fig. 6, black solid line). To determine the recovery of native species following disturbance by exotic species, Sv, Ag, and Bi individuals were grown in their own soils versus being grown in soils created from exotic species originating from the field soils of these native species (E-Sv vs. E-Pv; M-Ag vs. M-Pv; L-Bi vs. L-Pv, respectively) (Fig. 6, green solid line). There were five replicates for each plant species grown in each soil. Planting and management methods were the same as in Experiment 1. Plants were grown for 4 months. Four pots of uniformly grown plants were selected from each treatment for plant and soil samples.
Plant biomass harvest and soil sample collection
The shoots were clipped along the soil surface after removing soil and dust from the plant. The entire root system was then dug out from the pot after removing surface debris. The roots were rinsed with distilled water. The shoot and root of each plants was weighed after drying to a constant weight at 70 ℃.
After collecting the roots, the soil from each pot was mixed as our soil samples. Each soil sample was sieved through a 2 mm mesh to remove visible stones, litter, and debris. The soil sample was then divided into three parts for different analyses. The first part was stored in a -80 ℃ refrigerator until analyses of soil microbial community structure and diversity. The second part was stored in a 4 ℃ refrigerator and was used to determine soil enzyme activity and soil microbial biomass. The third part was naturally separated and was used to analyze soil physicochemical properties.
Chemical and biological analyses
Soil organic carbon (SOC) and plant carbon concentrations were determined using H2SO4-K2Cr2O7 oxidation methods. Soil total nitrogen (TN) and plant nitrogen concentrations were determined by the Kjeldahl method. Soil total phosphorus (TP) and plant phosphorus concentrations were estimated by the molybdenum blue method. Available nitrogen (AN) was determined by the alkaline KMnO4 method. Available phosphorus (AP) was determined using the Olsen methods.
Soil microbial biomass was evaluated within 1 month of storing soil samples at 4 °C. Soil microbial carbon (MBC) and microbial nitrogen (MBN) were measured by the chloroform fumigation-extraction method [40]. The soil enzymes activities of β-1,4-glucosidase (B-G), β-1,4-N-acetylglucosaminidase (N-AG), and acid phosphatase (A-P) were determined using a method developed by Saiya-Cork et al.[41], which was described by Xu et al., [42], and Xue et al.[43].
Total bacterial DNA was extracted from samples using the Power Soil DNA Isolation Kit (MO BIO Laboratories) according to the manufacturer’s protocol. DNA quality and quantity were assessed using ratios of 260 nm/280 nm and 260 nm/230 nm. DNA was then stored at -80 °C until further processing. The V3–V4 region of the bacterial 16S rRNA gene was amplified with the common primer pair (forward primer, 5′-ACTCCTACGGGAGGCAGCA-3′; reverse primer, 5′-GGACTACHVGGGTWTCTAAT-3′) combined with adapter sequences and barcode sequences. PCR amplification was performed in a total volume of 50 μl, which contained 10 μl buffer, 0.2 μl Q5 High-Fidelity DNA Polymerase, 10 μl High GC Enhancer, 1 μl dNTP, 10 μM of each primer, and 60 ng genome DNA. Thermal cycling conditions were as follows: initial denaturation at 95 °C for 5 min, followed by 25 cycles at 95 °C for 1 min, 50 °C for 1 min, and 72 °C for 1 min, with a final extension at 72 °C for 7 min. The PCR products from the first PCR step were purified through VAHTSTM DNA Clean Beads. A second round PCR was then performed in a 40 μl reaction, which contained 20 μl 2× Phusion HF MM, 8 μl ddH2O, 10 μM of each primer, and 10 μl PCR products from the first step. Thermal cycling conditions were as follows: initial denaturation at 98 °C for 30 s, followed by 10 cycles at 98 °C for 10 s, 65 °C for 30 s, and 72 °C for 30 s, with a final extension at 72 °C for 5 min. Finally, all PCR products were quantified by Quant-iT™ dsDNA HS Reagent and pooled together. High-throughput sequencing analysis of bacterial rRNA genes was performed on the purified, pooled sample using the Illumina HiSeq 2500 platform (2 × 250 paired ends) at Biomarker Technologies Corporation, Beijing, China.
Data analysis
To obtain optimized sequences, the original data were filtered using Trimmomatic (version 0.33), merged using FLASH (version 1.2.7), and chimeric reads were removed in UCHIME (version 4.2). The optimized sequences were then clustered to obtain operational taxonomic units (OTUs) with 97% similarity using UCLUST in QIIME (v1.8.0). Species were then classified according to the sequence composition of the OTUs. Taxonomy was assigned for each phylotype based on the SILVA database (release 132) for bacteria and the UNITE database (v7.0) for fungi. Based on the OTU analysis, taxonomic analysis was performed on samples at various taxonomic levels to obtain the phylum, class, order, family, genus, and species. Alpha diversity analysis was used to obtain the species diversity for soils of different origins. Chao1 and Shannon indexes of each sample were statistically calculated to 97% similarity. Principal component analysis (PCA) and permutational multivariate analysis of variance (PERMANOVA) were used to analyze the effects of soil origin on the composition of the bacterial and fungal community at the OTU level based on Bray–Curtis distance.
Correlation networks were selected to study the correlation of the soil microbial community at the genus level. First, the correlation matrix of the genus for which the relative abundance exceeded 0.1% was calculated using the Spearman correlation analysis (psych package in R. v.4.0.2). Then, Gephi 0.9.2 software was used to visualize the bacterial and fungal network and obtain the network structure. One node represented one genus type. The size of the node indicated the relative abundance of the genus. The color of the line between nodes indicates the positive and negative correlation of different genera.
One-way ANOVA and t-tests were used to test the effects of soil origin on plant and soil characteristics. The data were first tested for normality with Kolmogorov–Smirnov procedures (Lilliefors) and homogeneity with Levene’s test. One-way ANOVA [Duncan’s post-hoc tests (normal), Tamhane (non-normal)] and t-tests were performed on data that obeyed the normal distribution. For data that did not conform to the normal distribution, LOG(X+1) conversion was performed. If it still did not obey the normal distribution, the Kruskal–Wallis Test (H) was used to perform a non-parametric test.