Bacterial diversity assessment of HS and DS samples
In order to detect whether the microbial functioned to sustain the tomato health, we established the difference in disease incidence of tomato plants cultivated with HS, DS, and DS with heat treatment (autoclaving, 121°C one hour and followed with dry heat sterilization, 180°C four hours). Results demonstrated that the tomato plants cultivated in DS showed typical wilt symptom and nearly 80% plants died, while the tomato plants growing in HS and heat treated soil with no plant wilt (Supplemental information figure 1). Bacterial diversity of soil samples collected from HS and DS were then assessed using phylotype taxonomy. Results revealed that there were 3,041 OTUs, the core OTU number was 2,488, and the HS and DS with 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 HS and DS samples reached the significant level (p value is 0.002216) (Fig. 1B).
Main bacterial composition of HS and DS
Result of HS samples demonstrated that genera Bacillus (relative abundance 7.18%), Gaiellales (5.20%), Acidobacteria (3.28%), Nocardioides (2.31%), Nitrospira (2.74%), norank_c_KD4-96 (1.82%), norank_f_Xanthobacteraceae (1.99%) were the main groups; the genera Bacillus (3.95%), Gaiellales (3.94%), 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 DS samples (Fig. 2). In addition, PCoA result revealed that the bacterial communities of HS and DS were separated distinctly, and the PC1 axis could show 56.74% bacteria community variations between HS and DS (Fig. 3).
Further to perform the relative abundance analysis, we found that the relative abundance of genera Nocardioides and norank_f_Anaerolineacear between HS and DS reached significant level (95% CI; p value < 0.05). In addition, the relative abundance of genera Bacillus, Gaiellales, Roseiflexus, norank_o_Gemmatimonadaceae, and Gaiella reached very significant level (95% CI, p value < 0.01) (Fig. 4).
Chemical properties and RDA of HS and DS
No significant difference was observed for AHN content of HS and DS samples, the contents of pH, OM and RAK of HS were all significantly lower than DS sample (p < 0.05, Table 1), but the content RAP in HS was significantly higher than DS. Further to conduct the RDA at genus level, we found that RAP played key role on the bacterial community distribution difference between HS and DS, and had a negative correlation with the other four chemical contents (Fig. 5).
Network analysis of HS and DS
Network analysis of the 30 most abundant genera revealed the interaction relationships of HS and DS, respectively. Results indicated that there were extensive interactions among the identified genera. In the HS (Fig. 6A), the 30 most abundant genera were from ten phylum, including ten genera from Actinobacteria, six genera from Proteobacteria, four genera from Acidobacteria, three genera from Chloroflexi, two genera from Firmicutes, and one genus from Nitrospirae, Saccharibacteria, Planctomycetes, Gemmatimonadetes, respectively; obviously, the genera as Mycobacterium, Rhodobiaceae and Cyanobacteria shew interaction relationships with five, seven, and seven other genera, respectively. In the DS (Fig. 6B), these genera were only from seven phylum, 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
The COG function prediction was performed and compared between HS and DS samples. The COGs with significant differences were focused, our results demonstrated that there were seven COGs varied distinctly and reached significant level, 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) reached very significant levels, the group C (Energy production and conversion) and Z (Cytoskeleton) (Fig. 7).
Isolation and identification of antagonistic strains
During the investigation, we found that there were few tomato plants in the DS field still growing well. We collected the rhizosphere soil of three healthy tomato plants and isolated the cultural bacteria. Followed by taking inhibition zone method, 55 bacterial strains with distinct antagonistic activities to R. solancearum strain EP1 were found and identified by sequencing their 16S rDNA sequences (Table 2). Results of BLAST showed that these strains were belonging to the genera of Bacillus (17 strains), Pseudomonas (ten strains), Sphingobacterium (ten strains), Chryseobacterium (nine strains), Serratia (four strains), Cellulosimicrobium (one strain), Staphylococcus (one strain), Fictibacillus (one strain), Microbacterium (one strain), and Paenibacillus (one strain).