Biocontrol of tomato wilt disease by Bacillus subtilis isolates from natural environments depends on conserved genes mediating biofilm formation

Abstract

Bacillus subtilis and other Bacilli have long been used as biological control agents against plant bacterial diseases but the mechanisms by which the bacteria confer protection are not well understood. Our goal in this study was to isolate strains of B. subtilis that exhibit high levels of biocontrol efficacy from natural environments and to investigate the mechanisms by which these strains confer plant protection. We screened a total of 60 isolates collected from various locations across China and obtained six strains that exhibited above 50% biocontrol efficacy on tomato plants against the plant pathogen Ralstonia solanacearum under greenhouse conditions. These wild strains were able to form robust biofilms both in defined medium and on tomato plant roots and exhibited strong antagonistic activities against various plant pathogens in plate assays. We show that plant protection by those strains depended on widely conserved genes required for biofilm formation, including regulatory genes and genes for matrix production. We provide evidence suggesting that matrix production is critical for bacterial colonization on plant root surfaces. Finally, we have established a model system for studies of B. subtilis-tomato plant interactions in protection against a plant pathogen.

Fig. 1. The regulatory circuitry that controls biofilm formation in B. subtilis 3610

(A) Under laboratory conditions, biofilm formation by 3610 has been shown to be controlled by two parallel pathways: SinI-SinR and AbrB, both in turn under Spo0A~P regulation. YwcC-SlrA represents an alternative pathway that transiently boosts matrix production under certain conditions by SlrA antagonizing SinR and thereby derepressing matrix genes. Transcriptional regulation in the circuitry is indicated by arrows or lines in red, while protein-protein interaction is indicated in blue. (B) Phylogenetic analysis of B. subtilis wild isolates. Phylogenetic relationship among B. subtilis 3610 and other wild isolates based on sequences of the 16S rRNA genes. Phylogenetic tree was constructed by UPGMA algorithm using MEGA 4.0.2 software. Bootstrap values are shown at branch points. The bar at the bottom represents the unit length of the number of nucleotide substitutions per site. (C) Biocontrol efficacy of the 20 B. subtilis wild isolates and the two reference strains (3610 and PY79) against tomato bacterial wilt disease caused by R. solanacearum under greenhouse conditions. The numbers above the columns represent the biocontrol efficacy for each tested strain. The dashed line indicated the 50% efficacy as the cutoff value for selecting potential biocontrol strains of B. subtilis.

Fig. 2. Colony formation and pellicle development of B. subtilis wild strains

Six wild isolates and two reference strains (3610 and PY79) were included in the experiment. Cells were inoculated to the solid MSgg media for colony formation or in MSgg liquid medium in standing culture for floating pellicle development. Cells were incubated at 22°C for 3 days before imaging. The scale bar in the upper panels is 0.2 cm and the scale bar in the lower panels is 0.4 cm.

Fig. 3. Biofilms formed by B. subtilis wild strains on tomato root surfaces

6 wild isolates as well as the two reference strains (3610 and PY79) were applied in the assays. All strains harbored a chromosomally integrated, constitutively expressed gfp reporter fusion. Background represents the tomato plant sample without inoculation of bacterial cells. Cells expressing the green fluorescent proteins were visualized by CLMS. Bar, 50 μm.

Fig. 4. Biofilm colonies formed by various mutants derived from 3610 and six wild isolates

Cells were inoculated on MSgg agar plates and incubated for 3 days at 22°C before images were taken.

Fig. 5. Biofilm formation on the tomato root surfaces by hyper-robust or defective biofilm mutants (in 3610)

All strains harbored a constitutively expressed gfp reporter. Cells were similarly visualized by CLMS. Details of the biofilm-forming cells of the wild type, two hyper-biofilm mutants (ΔsinR and ΔywcC) and one defective biofilm mutant (ΔsinI) are shown enlarged in top panels. Bar in the top panels, 5 μm. Bars in the middle and lower panels, 50 μm.

Fig. 6. Surfactin production is equally important in plant biocontrol

(A) In vitro antagonistic activities towards R. solanacearum by the wild type and the mutants for each of the indicated antibiotic genes. Average ± SD (standard deviation) was calculated from four replicas and analyzed using the program Student’s t-test (P≤ 0.05).Letters (from a to d) at the top of each bar represent the distinctness, with ‘a’ being the highest, and ‘d’ the lowest. The same letters represent that no significant difference was observed. (B) Biocontrol efficacy of the wild type strains and the surfactin-deficient mutants against tomato bacterial wilt disease under greenhouse condition. The grey columns represent the wild type strains, while the corresponding surfactin-deficient mutants are shown in white columns. The average ± SD was calculated from three independent trials and analyzed using Student’s t-test (P≤ 0.05).

Fig. 7. Biofilm formation promotes cell colonization on tomato root surfaces

(A) The graph shows changes in the population density of 3610 and various biofilm mutants in the rhizosphere during the entire span (30 days) of the biocontrol assay against tomato bacterial wilt disease. Root-associated bacterial cells were collected periodically and colony-forming unit (CFU) of the samples was determined by conventional plating method. (B) Negatively charged microbeads (~2.0 μm in diameter) were incubated with tomato roots for 30 min. The roots were then rinsed with distilled water and examined by CLSM. Virtually no microbeads were found attached to the tomato root surfaces. (C) Positively charged microbeads (~2.1 μm in diameter) were applied similarly and were found to strongly attach to the tomato root surfaces. Bottom panels in both (B) and (C) are enlargement of the top panels. The arrows in (C) point to the beads visible under CLSM. Bar, 2 μm.