Growth curve of Cajanus cajan
The growth curve of YN and JX Cajanus cajan saplings were shown in Figure 1. The saplings growth height of each variety was relatively uniform, and the standard error was between 0.03 and 1.11 cm. YN Cajanus cajan shows better growth rate than JX Cajanus cajan, in the 8th month, the average height of YN seedlings was 314.97 cm, while that of JN was only 295.34 cm.
Sequence Length Distribution
16S rDNA sequencing of bacteria in six samples showed that 145,496 base pairs were detected, with the lowest length of 219 bp, the longest length of 407 bp, and an average length of 377 bp. A total of 96,563,333 base pairs were detected in the ITS1F_ITS2R region of 18S rDNA, which ranged from 140 bp to 300 bp in length, with an average length of 225 bp.
Operational Taxonomic Unit Species Classification Statistics
Analysis of root soil samples showed that bacteria had one domain, 1 kingdom, 6 phyla, 8 classes, 23 orders, 41 families, 60 genera, 67 species, and 72 operational taxonomic units (OTUs), while fungi had one domain, 1 kingdom, 6 phyla, 21 classes, 49 orders, 94 families, 143 genera, 180 species, and 266 OTUs.
Analysis of the Alpha Diversity of Samples
As can be seen from the dilution curve of the fungal samples (Figure 2A), when the sample sequence increased to more than 30,000, the Shannon index curve was almost flat, which indicated that the sequencing volume continued to increase. The number of newly added OTUs exhibited a limited increase. The number of experimental sequences in the experiment reached 70,000, which was a sufficient amount of data for the analysis. The dilution curve of the bacterial samples (Figure 2B) indicated that the sequencing amount was about 3,000, the OTUs of all the experiments increased gradually, and the sequencing amount was sufficient. Figure 2C and 2D depicts the uniformity curve of the sample. It can be seen that the Heip curve of fungi was very steep. When the sample number reached 21,000, the Heip index reduced to 0.02, which indicated that the homogeneity of the fungi is remarkably high. Figure 2D shows that the Heip curve was steep, which indicated high uniformity of the sample.
After coal gangue covered the soil, JX Cajanus cajan and YN Cajanus cajan were planted with an ace index and Chao index of 0.5354 and 0.6568, respectively, which were each greater than 0.05, thus indicating that no significant difference in their microbial diversity,. The P values of the ace index and Chao index of fungi were 0.00899 and 0.00793, respectively, which were each less than 0.01, thus indicating significant differences in the root fungi of the different varieties of chime bean that were planted, as shown in Table 1 and Figure 3).
Table 1. microbial diversity of JX pigeon pea and YN Pigeon pea
Group
|
index
|
JX-Mean
|
JX-Sd
|
YN-Mean
|
YN-Sd
|
P
|
fungi
|
ace
|
93.762
|
7.5163
|
121.79
|
6.932
|
0.00899
|
chao
|
92.609
|
8.5551
|
120.14
|
4.5529
|
0.00793
|
bacteria
|
ace
|
51.57
|
15.35
|
44.807
|
7.9777
|
0.5354
|
chao
|
44.688
|
12.545
|
40.676
|
7.2664
|
0.6568
|
Species Composition Analysis
Figure 4 compares the differences between the bacteria and fungi in the microbial composition of different varieties of Cajanus cajan roots at the genus level of the samples. According to a Venn diagram, there were 38 genera of bacteria in two different varieties of Cajanus cajan, among which unclassified_f_Enterobacteriaceae and Cedecea were the dominant genera, accounting for 31.0% and 29.8%, respectively. The unique bacteria of JX and YN Cajanus cajan belong contained 15 genera and 10 genera, respectively, and the dominant genera are Lactococcus and Pseudomonas bacteria, which account for 25.0% and 29.5%, respectively. The Venn diagram of the fungi showed that there were 84 genera of fungi in the root soil of two kinds of Cajanus cajan, among which Fusarium was the dominant strain, accounting for 41.2%, followed by Acrocalymma and Neocosmospora, which accounted for 19.9% and 14.8%, respectively. Twenty-two genera of fungi are unique to JX Cajanus cajan, among which Robillarda is the dominant strain, accounting for 91.8%. There were 37 genera of fungi in YN Cajanus cajan, and the dominant fungus was Scytalidium, accounting for 41.2%, which can cause infection. The second- and third-most dominant fungi were Herpotrichiellaceae and Acidomyces, accounting for 17.9% and 14.8%, respectively (Mendoza et al., 2009).
Heat Map Analysis of Bacterial Colony Composition at the Genus Level
Species with the top 20 total abundance at the genus level of classification were selected to display variation in the abundance of different species in samples through a color gradient of color blocks. Figure 5 depicts a heat map of bacteria at the genus level. It can be seen from the figure that Enterobacteriaceae and Cedecea were dominant bacteria genera in the six samples, and the abundances of Yng1 and Yng2 were also remarkably high. Figure 5 depicts the heat map of fungi at the level of genus classification. It can be seen from the figure that, except Fusarium, the distribution of other fungi in each sample was very significant. Acrocalymma in JX Cajanus cajan (samples Jxg1, Jxg2, and Jxg3) was also one of the dominant genera. The root microorganisms of different Cajanus cajan varieties have significant differences.
Phylogenetic Tree Analysis of rhizobacteria
Phylogenetic trees were constructed at the genus level to explore the phylogenetic correlation of the dominant strains, as shown in Figure 6. On the left is the phylogenetic evolutionary tree, in which each branch represents a class of species, and the branches are colored according to the advanced taxonomic level to which the species belongs. The length of the branch was the evolutionary distance between two species. The bar chart on the right shows the proportion of reads for different groups of species. As can be seen from the phylogenetic tree of bacteria, the homology of the dominant strains Enterobacteriaceae, Cedecea, Enterobacter, Pantoea, and Candidatus Phytoplasma is poor, with significant differences among the species.
According to the phylogenetic tree of fungi (Figure 7), the dominant strains Acrocalymma and Setophoma had high homology; Neocosmospora and unclassified Nectriaceae had high homology, and Fusarium had low homology, with significant differences in comparison with other dominant strains.
Functional Analysis of Microorganism
Based on the published literature or data from authoritative websites, fungal communities in the samples were classified and analyzed using microecology Guild, and functional classification of fungi was performed using FUNGuild software to obtain information on the functional classification of fungi in samples and the abundance of each functional classification in the different samples, as shown in Figure 8. According to the figure, the main functions of endophytes at the root of Cajanus cajan were saprotroph, plant Pathogen, and endophyte.
The COG family information and KEGG Orthology (KO) information corresponding to the OTU were obtained according to the greengene ID corresponding to each OTU in the samples, and the abundance of each COG and KO were calculated. The amplicon sequencing data for the fungal ITS can be used for functional prediction. The result is shown in Figure 9. It can be seen from the figure that L-arabinose isomerase (5.6.1.4) and adenosine triphosphatase (3.1.6.3) have the highest abundance among the six samples, followed by chitosanase (3.2.1.132), chitinase (3.2.1.14), catalase (1.11.1.6), peroxidase (1.11.1.7), monooxygenase (1.14.14.1), laccase (1.10.3.2), nitrilase (3.5.5.1), Acid phosphatase (3.1.3.2) (Hui, 2019; Jin et al., 2020; Sun, 2019).
How to maintain the plant’s long-term survival so that it becomes the dominant plant is a key to ecological restoration of coal gangue. Therefore, plant growth is related to soil nutrient sources and is related to endophytic microorganisms. Endophytic bacteria can produce a variety of active substances through metabolism, which can promote the growth and development of host plants and improve the resistance of host plants. Endophytic microorganisms may be pathogenic microorganisms that can harm plant growth. Therefore, it is critical to study the endophytic bacteria of caesium in the reclamation plant on gangue mountain.