Soilphysicochemical properties soil
The physicochemical properties of the soil samples are shown in Table 1. There was no significant difference in pH or in the total nitrogen, total phosphorus, or total potassium content of the soil samples with the three different treatments. Overall, the pH value was 7.50–7.73, the total nitrogen content was 3.16–4.13 g•kg− 1, the total phosphorus content was low at 1.63–1.91 g•kg− 1, and the total potassium content was 22.01–22.92 g•kg− 1. The content of available nitrogen, available phosphorus, available potassium, and organic matter in the soil samples were significantly different among the three treatments (P < 0.05). The available nitrogen content was 210.11–275.17 mg•kg− 1, available phosphorus was 8.41–18.71 mg•kg− 1, and available potassium content was 67.11–12.18 mg•kg− 1. The available nitrogen, available potassium, and organic matter contents in the CYCH treatment soil were significantly higher than those in the other treatments (P < 0.05), and the contents of available phosphorus in the CYD treatment soil were significantly higher than those in the other treatments (P < 0.05).
Table 1
Soil indicators | CWD | CYD | CYCH |
pH | 7.73 ± 0.45a | 7.50 ± 0.36a | 7.65 ± 0.38a |
Total nitrogen (g/kg) | 3.16 ± 0.22a | 3.97 ± 0.27a | 4.13 ± 0.33a |
Total phosphorus (g/kg) | 1.63 ± 0.06a | 1.91 ± 0.08a | 1.85 ± 0.05a |
Total potassium (g/kg) | 22.92 ± 1.24a | 22.01 ± 1.18a | 22.01 ± 1.24a |
Available nitrogen (mg/kg) | 210.11 ± 13.2c | 248.08 ± 17.14b | 275.17 ± 22.07a |
Available phosphorus (mg/kg) | 11.24 ± 2.36b | 18.71 ± 3.57a | 8.41 ± 2.68c |
Available potassium (mg/kg) | 94.07 ± 8.41b | 67.11 ± 7.65c | 120.18 ± 10.18a |
The organic matter (g/kg) | 51.67 ± 4.07c | 69.35 ± 5.58b | 75.83 ± 6.47a |
Note: Different lowercase letters on the same line indicate significant differences (P < 0.05) |
Otu-level Analysis Of Soil Bacteria And Fungi
The results of the OTU statistical analysis are shown in Table 2. The 16S rDNA V3–V4 region of soil samples from all three treatments (CWD, CYD, and CYCH) was sequenced to obtain 48,988–50,302 effective tags. The tags lengths were highly concentrated, averaging 421 bp, 420 bp, and 417 bp for the CWD, CYD, and CYCH treatments, respectively. The sequence length of the 16S rDNA V3–V4 region in each treatment was roughly consistent. Following sequencing analysis, 32,031–42,291 effective tags were obtained from the ITS regions of the DNA obtained from the soil samples of the three treatments Generally, the sequence length was 230–280 bp, and the average length was 243, 230, and 280 bp, for the CWD, CYD, and CYCH treatments, respectively. Rarefaction curves (Fig. 1a and b) were prepared based on the results. It can be seen from Fig. 1 that the OTU level of the sample increased rapidly with the increase of sequencing fragments, and remained stable after reaching the peak.
Table 2
Tag information and OTU statistical analysis of rhizosphere soil samples
Target | Sample | Tags | Effective tags | Average tags (bp) | Percentage of effective (%) | No. OTUs |
16S rDNA | CWD | 67249 ± 243 | 48988 ± 241 | 421 ± 15 | 61.12 ± 1.24 | 1162 ± 1.76 |
CYD | 67594 ± 326 | 50302 ± 276 | 420 ± 17 | 63.06 ± 1.33 | 1209 ± 1.58 |
| CYCH | 67396 ± 358 | 49918 ± 318 | 417 ± 12 | 62.75 ± 1.27 | 1127 ± 1.29 |
ITS | CWD | 74762 ± 572 | 36891 ± 424 | 243 ± 18 | 46.31 ± 1.24 | 261 ± 2.65 |
| CYD | 74496 ± 427 | 42291 ± 267 | 230 ± 14 | 53.17 ± 1.36 | 260 ± 1.87 |
| CYCH | 74611 ± 410 | 32031 ± 211 | 280 ± 13 | 40.06 ± 1.18 | 211 ± 1.87 |
Analysis Of Soil Microbial Species Composition
The richness of microbial species composition was > 0.1% higher in the three soil treatment samples (Fig. 2). We detected many phyla and classes of bacteria and fungi, and most of them contained very few species. For the convenience of observation, only the top ten abundant fungi and bacteria are shown in Fig. 2 and the remaining have been grouped as Others. The Unclassified and Unknown groups represent species that were not taxonomically annotated. The ordinate (y-axis) shows the relative abundance. Figure 2a and b, respectively, show the top 10 classes of bacteria and fungi, respectively, according to their relative abundance level.
For all three treatments, the soil bacteria were mainly distributed in the following 10 classes: Alphaproteobacteria, Betaproteobacteria, Blastocatellia, Acidobacteria subgroup 6, Gemmatimonadetes, Nitrospira, Acidimicrobiia, Actinobacteria, Gammaproteobacteria, and Holophagae(Fig. 2a). Alphaproteobacteriawas the most abundant, with a relative abundance of 17.83%, 24.24%, and 18.67%, for the CWD, CYCH, and CYD treatments, respectively. For the CYCH treatment, the abundance of Alphaproteobacteria and Actinobacteria were significantly higher than for the CWD and CYD treatment. For the CYCH treatment, the abundance of Gemmatimonadetes, Nitrospira, and Acidimicrobiia was significantly lower than that of the CWD and CYD treatment. With the exception of Actinobacteria, the relative abundance of the main bacterial species in the CWD and CYD soils was relatively close and it is quite different from that in CYCH soil.
As can be seen in Fig. 2b, for all three treatments, the soil fungi were mainly distributed in the following 9 groups: Mortierellomycetes, Sordariomycetes,Leotiomycetes, Dothideomycetes, Tremellomycetes, Agaricomycetes, Archaeorhizomycetes, Eurotiomycetes, Spizellomycetes, or Unclassified, of which Mortierellomycetes was the most abundant the CYCH treatment soil (32.66%). Compared with the CWD and CYD treatment soil, the abundance of Sordariomycetes and Leotiomycetes in the CYCH treatment soil was significantly decreased, while the distribution of fungal communities was significantly different between the soil treatments.
Analysis Of Soil Microbial Diversity
Alpha diversity analysis
As can be seen from the alpha diversity index results (Table 3), all three soil treatment samples have a high bacterial and fungal community diversity. The order of abundance-based coverage estimator (ACE) and Chao1 indices of bacterial alpha diversity in the three soil treatment samples were CYD > CYCH > CWD, indicating the highest abundance of bacterial species in the CYD treatment soil. The Simpson index showed CYCH > CWD > CYD. The Shannon index showed CYD > CYCH > CWD, indicating the richest diversity of bacterial microorganisms in the CYD treatment soil. The ACE and Chao1 indices of fungal alpha diversity in the three soil treatment samples were CWD > CYD > CYCH, showing that the fungal species abundance was highest in HNY soil. The Simpson index showed that CYCH > CYD > CWD. The Shannon index was ranked as CWD > CYD > CYCH. Thus fungal microbial diversity of the CWD treatment soil was the most abundant among the three treatments.
Table 3
Diversity index of soil microbial community in rhizosphere soil
Diversity indexes | | CWD | CYD | CYCH |
Bacterial | ACE | 1244.7398 ± 5.42 | 1276.6826 ± 5.22 | 1247.6747 ± 4.89 |
Chao1 | 1259.2143 ± 3.41 | 1286.5981 ± 3.24 | 1285.6355 ± 2.42 |
Simpson | 0.0068 ± 0.32 | 0.0063 ± 0.14 | 0.0086 ± 0.16 |
Shannon | 5.9163 ± 1.08 | 6.044 ± 1.23 | 5.9639 ± 1.18 |
Fungal | ACE | 274.0142 ± 13.36 | 269.1001 ± 10.68 | 218.8791 ± 12.21 |
Chao1 | 280.0909 ± 6.48 | 270.0 ± 3.79 | 217.6667 ± 5.66 |
Simpson | 0.029 ± 0.05 | 0.0298 ± 0.04 | 0.0526 ± 0.02 |
Shannon | 4.2945 ± 1.15 | 4.2487 ± 1.05 | 3.5755 ± 1.13 |
Note: Different lowercase letters on the same line indicate significant differences (P < 0.05) |
Analysis Of Beta Diversity
Heatmaps represent samples through interspecies distance relationships to obtain the sequence relationship between samples, which are drawn by means of matrix language tool software. By analyzing the heatmap, the difference between the two samples can be intuitively reflected by the range of color transformation. Figure 3 shows the heatmap of the three soil treatment samples. The bacterial flora (Fig. 3a) and fungal flora (Fig. 3b) in the CWD and CYD treatment soil had a high level of similarity.
The beta microbial diversity analysis was conducted to obtain a distance matrix. Then, the UPGMA function of R software was used for hierarchical clustering comparison of samples, and the similarity relationship of the species composition in each sample was described. The closer the samples were, the shorter the branch length was, indicating that the species composition of the two samples was more similar. Fig. 4 shows that the OTU level of bacteria and fungi in the CWD treatment soil samples had a relatively high consistency, while the similarity of bacteria and fungi in the CYCH treatment soil sample was relatively low.
Functional gene prediction
PICRUSt software (http://picrust.github.io/picrust/) was used to compare the species composition information obtained from the 16S sequencing tags to infer the functional gene composition of the samples and then analyze the functional differences between the different soil treatments. According to the of Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolism histogram (Fig. 5), the functional genes of the soil microbial community for each treatment were basically similar in their metabolic pathways.