Isolated Bacillus subtilis strain 330-2 and its antagonistic genes identified by the removing PCR

Abstract

Plant growth-promoting bacteria (PGPB) may trigger tolerance against biotic/abiotic stresses and growth enhancement in plants. In this study, an endophytic bacterial strain from rapeseed was isolated to assess its role in enhancing plant growth and tolerance to abiotic stresses, as well as banded leaf and sheath blight disease in maize. Based on 16S rDNA and BIOLOG test analysis, the 330-2 strain was identified as Bacillus subtilis. The strain produced indole-3-acetic acid, siderophores, lytic enzymes and solubilized different sources of organic/inorganic phosphates and zinc. Furthermore, the strain strongly suppressed the in vitro growth of Rhizoctonia solani AG1-IA, Botrytis cinerea, Fusarium oxysporum, Alternaria alternata, Cochliobolus heterostrophus, and Nigrospora oryzae. The strain also significantly increased the seedling growth (ranging 14-37%) of rice and maize. Removing PCR analysis indicated that 114 genes were differentially expressed, among which 10%, 32% and 10% were involved in antibiotic production (e.g., srfAA, bae, fen, mln, and dfnI), metabolism (e.g., gltA, pabA, and ggt) and transportation of nutrients (e.g., fhu, glpT, and gltT), respectively. In summary, these results clearly indicate the effectiveness and mechanisms of B. subtilis strain 330-2 in enhancing plant growth, as well as tolerance to biotic/abiotic stresses, which suggests that the strain has great potential for commercialization as a vital biological control agent.

Figure 1

An amplified 16S rDNA gene fragment from the isolated strain 330-2 was sequenced and blast searched through NCBI database. Closely related sequences were downloaded and aligned. These sequences were analyzed using the Maximum likelihood method. The GenBank accession number of each isolate is given in parentheses. Bootstrap values based on 1000 replicates are shown next to the branches.

Figure 2

The BIOLOG detection results showing the comparison of the B. subtilis strain 330-2 and B. subtilis strain 168. The negative control (A1); the positive control (A10). The well with a faint purple color shows the positive reaction, the half-filled light blue circle shows the moderate reaction, while the blank circle shows the negative reaction.

Figure 3

Production of hydrolytic enzymes and metabolites by B. subtilis strain 330-2 and B. subtilis strain 168. B. subtilis strain 330-2 (a); B. subtilis strain 168 (b); Siderophore production (1); Tri-calcium phosphate (2); Zinc phosphate (3); Zinc oxide (4); Zinc phosphate (5); Ca-phytate (6); Na-phytate (7); Laminarinase (8); Cellulase (9) and Protease production (10). All experiments were conducted three times through three independent trials.

Figure 4

Effects of B. subtilis strain 330-2. (a) Antagonistic effects of B. subtilis strain 330-2 against fungal pathogens; C. heterostrophus (1); A. alternate (2); B. cinerea (3); F. oxysporum (4); N. oryzae (5); R. solaniAG1 (6); and with their respective control (CK) on dual culture. (b) % inhibition of B. subtilis strain 330-2 against fungal pathogens. Data were statistically analyzed and the small alphabetical letters (a, b, c…) above the mean bars show the significant differences (P < 0.05) among treatments against different fungal pathogens. Each assay was performed in triplicate.

Figure 5

Effect of B. subtilis strain 330-2 on the seedling growth performance of rice and maize. (a and d) Seedling elongation of rice and maize, (b and e) seedling fresh weight of rice and maize, (c and f) seedling dry weight of rice and maize, and (g and h) pictorial view of 3-weeks and 40 days old rice and maize seedling in the control and B. subtilis strain 330-2 treatment, respectively. Data were statistically analyzed and the vertical bars above indicate the standard error of three replicates. Small alphabetical letters (a, b…) above the mean bars show the significant differences (P < 0.05) among the treatments within specific parameters. FW: Fresh weight, DW: Dry weight, CK: Control, Tr: Treated with B. subtilis strain 330-2.

Figure 6

The driver in R-PCR could not eliminate the tester in three cases: (a) the recognition cutting sites of the MseI and ApeKI (in red box) have mutations, (b) the tester contains enough bases that do not match the driver and (c) the sequence of the tester or driver is partially absent (or completely absent in the driver). *Identical.

Figure 7

Distribution in various functional categories of differentially expressed genes by R-PCR. The percentage of each group is indicated in each category, and the total numbers of genes are presented in the center of the graph.

Figure 8

Venn diagram indicating the common genes shared by salinity, oxidative, heat, cold and drought stresses. In total, 12 common differentially expressed genes were identified in all the five abiotic stresses.