Molecular Identification of Isolated Bacteria Strain
The 16S rDNA gene analysis of isolate B. safensis YY-01 revealed the highest (99% to 100%) sequence similarities with the genus Bacillus and was concluded to belong to the B. safensis species. Based on the phylogenetic relationship analysis using the neighbor-joining method (Fig. 1A), isolate B. safensis YY-01 has a very strong relationship with strain B. safensis with 100% similarity. Therefore, the isolate B. safensis YY-01 can be considered a subspecies of B. safensis. The sequence of the 16S rDNA gene of isolate B. safensis YY-01 was deposited to GenBank under the accession number OL721953.
Culture Conditions and Colony Morphology of B. safensis YY-01
The optimized culture condition for the B. safensis YY-01 was: Glucose 1.5(w/v), glycerol 2.0(w/v), peptone 1.5(w/v), yeast powder 1.0(w/v), Manganese chloride 0.03(w/v), magnesium chloride 0.1(w/v), calcium chloride 0.1(w/v), potassium dihydrogen phosphate 0.1(w/v), sodium carbonate 0.08(w/v), ammonium ferric citrate 0.002(w/v), Vitamin B 0.01(w/v), and trace element 0.5ml /L. The culture temperature of 30℃, the initial pH of 7.5, and the inoculum level of 1%. In the optimized medium, the viable number of B. safensis could reach 28.5 × 109 CFU/mL. After 3 d of growth in the optimized solid medium, the colony of B. safensis YY-01 was milky white with wavy edges, a smooth surface, and no central bulge(Fig. 1B). Microscopically, the thalli were straight rod-shaped, single, and blunt at both ends. Gram staining was positive. (Fig. 1C)
Fig. 1 A:Phylogenetic tree based on 16S rDNA sequences of strain B. safensis YY-01 and related bacterial strains. The numbers in parentheses represent the Genbank accession number. Numbers at the nodes indicate the bootstrap values. The bar represents the sequence divergence. B: Colony morphology of B. safensis YY-01 on modified solid medium. C: Microscopic examination of B. safensis YY-01 after Gram staining.
Effect of B. safensis YY-01 Fertilizer on Tomato Growth
Effect of Height
Height, one of the parameters, can reflect plant growth status directly. After treatment with B. safensis fertilizer, the values of height gradually increased with the use of time. Compared with the control, the height of haoliang No.998 and yingfen No.8 increased by 12.8% and 14.1% after treatment of 30 d, respectively. (p<0.01). (Fig. 2)
Fig. 2 The height of the tomato plant. (A: haoliang No.998; B: yingfen No.8)
Effect of Chlorophyll
The content of chlorophyll in tomatoes, to some extent, decides the strength of photosynthesis and is closely related to plant growth. There was no significant influence compared with the control after the early treatment. However, compared with the control, the contents of chlorophyll a, chlorophyll b, and total chlorophyll of the haoliang No.998 increased by 17.1%, 11.8%, and 14.8% after treatment of 30 d, respectively (P < 0.01). During the whole experiment period, the contents of chlorophyll a, chlorophyll b, and total chlorophyll of yingfen No.8 showed a steady increase trend, which increased by 7.8%, 10.2%, and 10.7% compared with the control, respectively. (P < 0.05). (Fig. 3)
Fig. 3 Chlorophyll a, chlorophyll b, and total chlorophyll content of tomato. (A: haoliang No.998; B: yingfen No.8)
Effect of Peroxidase Activity
After treatment with B. safensis fertilizer 17 d, the POD activity of haoliang No.998 and yingfen No.8 significantly increased by 140.7% and 57.9% compared with the control (P < 0.01), respectively. Then showed a downward trend, at the end of the experiment, POD activity was increased by 37.5% (P < 0.01) and 7.3% (P < 0.05) compared with the control, respectively. (Fig. 4).
Fig. 4 Peroxidase activity of tomato. (A: haoliang No.998; B: yingfen No.8)
Changes in Microbial Community Structure
Population and Alpha Diversity Analysis of Sequencing Data
The bacterial and fungal community diversity in rhizosphere soil of two tomato varieties (Haoliang No. 998 and yingfen No. 8) was studied by 16S rDNA gene sequencing and ITS rDNA Illumina MiSeq high-throughput sequencing. We designated the CK as Haoliang No. 998 (A1, C1) and yingfen No. 8 (B1, D1). The treatment is Haoliang No. 998 (A4, C4) and yingfen No. 8 (B4, D4). All rhizosphere soils were sequenced by the Beijing Alvisen Company, as well as the quality of the original data was filtered. After deleting the existing chimeric sequences, high-quality sequences were obtained. The results show that the high-quality sequences of most bacteria were concentrated between 401 bp and 440 bp. However, most of the high-quality sequences of fungi were concentrated in the range of 201-320 bp, only a small number were in the range of 321-440 bp. (Fig.5)
Fig. 5 The total base sequences after screening. (A: bacterial; B: fungi)
To analyze the richness and diversity of the community, α-diversity analysis was performed on both bacteria and fungi, and Shannon-Wiener curves were plotted. The Shannon-wiener curve is an index reflecting the microbial diversity in the sample, and with the increased sequencing amount and depth, the microbial diversity index also increases. When the curve tends to be flat, it indicates that the sequencing data amount is large enough to reflect the vast majority of microbial information. α-diversity analyses have revealed that B. safensis YY-01 fertilizer has different effects on soil bacterial and fungal community diversity. There were no significant differences in the α-diversity of soil bacterial communities between the treatment group and the control group. In contrast, fungi of haoliang No. 998 α-diversity were lower than that in the control group, while fungi of yingfen No.8 were slightly higher than that of the control group. (Fig. 6)
Fig. 6 Shannon index curve showed the change of microbial community diversity in tomato rhizosphere soil after applying B. safensis fertilizer. (A: bacterial; B: fungi)
Analysis of Bacterial Community
According to OTU classification and classification status identification, the specific microbial distribution of tomato rhizosphere soil at phylum, class, order, family, and genus classification levels was obtained. In the present study, phylum and genus levels were selected to analyze the soil microorganisms in the rhizosphere of tomatoes.
The soil bacterial composition trends at the phylum level were assessed. The top 12 bacterial phyla were ranked based on relative abundance. Proteobacteria, Acidobacteriota, Actinobacteria, Chloroflexi, and Gemmatimonadota were the dominant bacterial phyla in tomato rhizosphere soil and accounted for more than 75% of the total bacterial sequence. Compared with A1, the relative abundance of A4’s Proteobacteria, Actinobacteria, Chloroflexi, and Gemmatimonadota increased significantly, which increased by 4.8%, 7.6%, 3.2%, and 2.7%, respectively. But the relative abundance of Acidobacteriota reduced by 19.1%. Furthermore, compared with B1, the relative abundance of B4’s Acidobacteriota and Actinobacteria were significantly increased by 1.9% and 4.5% after applicating B. safensis fertilizer. It is worth noting that the relative abundance of Firmicutes was increased in both A4 and B4, which increased by 2.4% and 3.4%, respectively. (Fig. 7A)
At the genus level, all rhizosphere soils contain a large number of unculturable bacteria, RB4, and Bacillus. Among them, Bacillus significantly increased after treatment with B. safensis fertilizer, and A4 and B4 increased by 1.1% and 2.6%, respectively. (Fig. 7B)
Fig. 7 Relative abundance of bacteria at different rhizosphere soil of tomato after application of B. safensis fertilizer. (A: phylum level; B: genus level)
Analysis of Fungal Community
The fungal composition was also analyzed at the phylum level. Ascomycota, Basidiomycota, Mortierellomycota, and Chytridiomycota were the dominant fungal phyla. Compared with C1, the relative abundance of C4’s Ascomycota and Basidiomycota increased by 8.3% and 1.4%, respectively. But the relative abundance of Mortierellomycota was reduced by 5.9%. In addition, compared with D1, the relative abundance of D4’s Ascomycota and Mortierellomycota were reduced by 11.3% and 1.2%, respectively. However, the relative abundance of Basidiomycota in D4 increased by 2%. (Fig. 8A) This result indicates that B. safensis fertilizer can alter the soil fungal community structure.
At the genus level, unidentified accounted for the largest proportion, followed by Chaetomium, Mortierella, and Cladosporium. The relative abundance of Chaetomium and Cladosporium was significantly higher than in the control group(C1 and D1), in which the relative abundance of Chaetomium increased by 1% in C4 and 1.2% in D4, respectively. However, the relative abundance of Lophotrichus, Pseudaleuria, Cladosporium, Mortierella, and Pseudogymnoascus was decreased. Unusually, the relative abundance of Basidiomycota in C4 was 21.9% higher than in the control group(C1). (Fig. 8B)
Fig. 8 Relative abundance of bacteria at different rhizosphere soil of tomato after application of B. safensis fertilizer. (A: phylum level; B: genus level)
Analysis of PCA
Beta diversity analysis and cluster analysis were used to analyze the similarity and differences of bacteria and fungi in different soil groups. The microorganism principal component analysis (PCA) showed that there were differences in microbial community composition among different soil groups.
For bacterial diversity, the abscissa is PC1, and its contribution rate is 40.2%. The ordinate is PC2, and the contribution rate is 14.09%. These two PCs were the main factors for the difference in microbial community composition. In the direction of PC1, the distribution of sample points of haoliang No.998 spraying and non-spraying B. safensis fertilizer was significantly different (P < 0.05), and the sample points were at the two extremes of the PC1 level. At the PC2 level, there was no significant difference in the distribution of B. safensis fertilizer samples of Yingfen 8 sprayed and not sprayed, which were all below the Y-axis (Fig. 9A). In conclusion, the application of B. safensis fertilizer can significantly change the bacterial community structure and bacterial diversity of Haoliang No.998 rhizosphere soil.
For fungal diversity, the abscissa is PC1, and its contribution rate is 38.86%. The ordinate is PC2, and the contribution rate is 15.68%. In the direction of PC1, the distribution of haoliang No.998 samples with and without B. safensis fertilizer was significantly different (P < 0.05). The unfertilized samples were at the left end of the X-axis, while the fertilized samples were at the right end of the X-axis. At the PC2 level, there was a significant difference in the distribution of yingfen No.8 sprayed and non-sprayed B. safensis fertilizer samples (P < 0.05). The experimental group was located in the third quadrant, and the control group was located in the second quadrant. In conclusion, fertilization or not had a significant effect on the fungal community structure and fungal diversity of tomato rhizosphere soil (P < 0.01). (Fig. 9B)
Fig. 9 Two-dimensional sequencing diagram of PCA analysis for discrimination of microbial community composition on tomato among different breeds after applying B. safensis fertilizer. (A: bacteria; B: fungi)
Analysis of Cluster Heat Map
Cluster analysis diagram based on the abundance distribution or similarity of different samples. The aim is to distinguish between high-abundance and low-abundance taxa through clustering and to reflect the similarity of community composition between samples by color composition gradient. The clustering heat maps of the top 20 bacteria and fungi in different samples are shown. (Fig. 10) Red represents the genus with higher abundance in corresponding samples, and blue represents the genus with lower abundance. The result showed that compared with control the relative abundance of Bacillus and Chaetomium in both Haoliang No.998 and Yingfen No.8 were significantly increased after applying the B. safensis YY-01 fertilizer.
Fig. 10 Heatmap of clustering analysis for microbial community diversity on rhizosphere soil of tomato from different treatments. (At the genus level; A: bacteria; B: fungi))