Bacterial diversity assessment of SS and CS samples
In order to determine whether the microbial community functioned to sustain tomato health, we identified the difference in disease incidence of tomato plants cultivated under four growing conditions: SS, CS, CS with heat treatment, and sterile nutrient soil inoculated with the R. solanacearum suspension. Results demonstrated that all of the tomato plants showed wilt symptoms after four days of R. solanacearum suspension inoculation. The tomato plants cultivated in CS showed typical wilt symptoms and nearly 80% of plants wilted. Tomato plants grown in SS and the heat-treated soil exhibited no plant wilt (supplemental information Figure 1). The bacterial diversity of soil samples collected from SS and CS was assessed using phylotype taxonomy. The results revealed a total of 3,041 operational taxonomic units (OTUs). The core OTU number was 2,488, and the SS and CS exhibited 330 and 220 unique OTUs, respectively (Fig. 1A). Furthermore, the result of Student’s t-test indicated that the Sobs index of the OTU level of SS and CS samples was significant (p = 0.002216; Fig. 1B).
Main bacterial composition of SS and CS
The primary bacterial genera of the SS samples were Bacillus (relative abundance 7.18%), norank_o_Gaiellales (5.20%), norank_c_Acidobacteria (3.28%), Nocardioides (2.31%), Nitrospira (2.74%), norank_c_KD4-96 (1.82%), norank_f_Xanthobacteraceae (1.99%). The genera Bacillus (3.95%), norank_o_Gaiellales (3.94%), norank_c_Acidobacteria (3.76%), Nocardioides (3.77%), Gaiella (2.96%) norank_c_KD4-96 (2.47%), Roseiflexus (2.49%), and norank_f_Anaerolineaceae (2.41%) were the dominate groups of CS samples (Fig. 2). In addition, principal coordinates analysis (PCoA) revealed that the bacterial communities of SS and CS were distinct, and the PC1 axis showed 56.74% variation in the bacteria community between SS and CS (Fig. 3).
The relative abundance of genera Nocardioides, Gaiella and norank_f_Anaerolineaceae between SS and CS reached significant (95% confidence interval, CI; 0.01 < p < 0.05). In addition, the relative abundance of genera Bacillus, norank_o_Gaiellales, Roseiflexus, and norank_o_Gemmatimonadaceae were extremely significant (95% CI, p < 0.01; Fig. 4).
Chemical properties and redundancy analysis (RDA) of SS and CS
No significant difference was observed for alkali-hydrolyzable nitrogen (AHN) content of SS and CS samples. The pH, organic matter (OM) content, and rapidly available potassium (RAK) of SS were significantly lower than the CS sample (p < 0.05, Table 1), but the content plant rapidly available phosphate (RAP) in SS was significantly higher than CS. RDA analysis at genus level revealed that RAP played a key role in the difference in bacterial community distribution between SS and CS, and it was negatively correlated with the other four chemical contents (Fig. 5).
Network analysis of SS and CS
Network analysis of the 30 most abundant genera revealed the interaction relationships of SS and CS, respectively. The results indicated that there were extensive interactions among the identified genera. In the SS (Fig. 6A), the 30 most abundant genera were from 10 phyla, including 10 genera from Actinobacteria, six genera from Proteobacteria, four genera from Acidobacteria, three genera from Chloroflexi, two genera from Firmicutes, and one genus each from Nitrospirae, Saccharibacteria, Planctomycetes, Cyanobacteria, and Gemmatimonadetes, respectively. Bacteria from Mycobacterium, Rhodobiaceae and Cyanobacteria showed interaction relationships with five, seven, and seven other genera, respectively. In the CS samples (Fig. 6B), these genera were only from seven phyla, such as the nine genera from Actinobacteria, six genera from Proteobacteria, six genera from Chloroflexi, four genera from Acidobacteria, one genus from Gemmatimonadetes, Nitrospirae, and Firmicutes, respectively.
Analysis of the COGs with significant differences
COG function prediction was performed and compared between SS and CS samples. The COGs with significant differences were further investigated. There were seven COGs that were significantly different, such as the group S (function unknown), H (coenzyme transport and metabolism), A (RNA processing and modification), F (nucleotide transport and metabolism), and D (cell cycle control, cell division, chromosome partitioning). The groups C (energy production and conversion) and Z (cytoskeleton) were very distinct (Fig. 7).
Isolation and identification of antagonistic strains
Few tomato plants in the CS field grew well. We collected the rhizosphere soil of three healthy tomato plants and isolated the cultural bacteria. Using the inhibition zone method, 55 bacterial strains with distinct antagonistic activities to R. solancearum strain EP1 were identified by sequencing their 16S rDNA sequences (Table 2). The basic local alignment search tool (BLAST) results showed that these strains belong to the genera Bacillus (17 strains), Pseudomonas (10 strains), Sphingobacterium (10 strains), Chryseobacterium (9 strains), Serratia (four strains), Cellulosimicrobium (one strain), Staphylococcus (one strain), Fictibacillus (one strain), Microbacterium (one strain), and Paenibacillus (one strain).