Antifungal and plant growth promotion activity of volatile organic compounds produced by Bacillus amyloliquefaciens

Abstract

Fusarium wilt of watermelon, caused by F. oxysporum f.sp. niveum (FON), is a devastating disease that causes extensive losses throughout the world. Five bacterial strains (L3, h, β, b, and L) isolated from the watermelon rhizosphere showed antagonistic activity against FON during in vitro tests. Strain L3 produced diffusible and volatile organic compounds (VOCs) which showed the strongest antifungal activity. Arabidopsis thaliana plantlets exposed to VOCs produced by strain L3 showed a 2.39-fold increase in biomass, 1.40-fold increase in primary root length, and 5.05-fold increase in number of lateral roots. Confocal laser scanning microscope showed that the GFP-labeled strain L3 could colonize along the elongation and differentiation zones of watermelon roots. In greenhouse pot experiments, the biocontrol efficiency of strain L3 against fusarium wilt of watermelon was up to 68.4% in comparison with the control treatment. In addition, inoculation of the strain L3 resulted in a 23.4% increase in plant fresh weight. Based on 16S rDNA sequence analysis, the strain L3 was identified as Bacillus amyloliquefaciens L3. Fourteen VOCs produced by strain L3 were identified through GC-MS analysis. Of nine VOCs tested, 2-nonanone and 2-heptanone were proved to have strong antifungal properties. Acetoin and 2,3-butanediol were found to promote plant growth. The results suggested B. amyloliquefaciens L3 was a potential biocontrol agent, and that VOCs produced by B. amyloliquefaciens L3 play important roles in the process of biocontrol and plant growth promotion.

Keywords: Bacillus amyloliquefaciens; F. oxysporum f.sp. niveum; root colonization; volatile organic compounds; watermelon.

Figure 1

Antifungal activity of the bacteria (L3, h, β, L, and b) isolated from watermelon rhizosphere soil against Fusarium oxysporum f. sp. niveum (FON). (a) Antagonism pattern after 5 days dual culture assay. (b) Inhibition rate of the isolated strain after 5 days. (c) Antagonism pattern after 10 days dual culture assay. D: Inhibition rate of the isolated strain after 10 days. Lowercase letters above the columns indicate a significant difference at p < 0.05

Figure 2

Antifungal volatile activity of isolated strains (L3, h, β, L, and b) on double‐dish chamber. (a) Mycelial growth of FON was inhibited in the presence of the bacteria streaked in the bottom dish. (b) Diameter of mycelial growth in the presence of VOCs after culture for 120 hr. Lowercase letters above the columns indicate a significant difference at p < 0.05

Figure 3

Effect of the VOCs produced by the isolated strains (L3, h, β, L, and b) on the biomass of Arabidopsis thaliana Col‐0. (a) Representative photograph showing the effects of VOCs produced by isolated strains. (b) Plant fresh weight, (c) Length of primary root, (d) Number of lateral roots. Lowercase letters above the columns indicate a significant difference at p < 0.05

Figure 4

Fluorescence (GFP) micrographs of watermelon roots colonized by GFP‐tagged Bacillus amyloliquefaciens L3 in hydroponic system at 48 hr and 96 hr; (a, b) The control, which was not inoculated with GFP‐tagged L3. (c‐f) Root zones of primary root and lateral root junctions colonized by GFP‐tagged L3

Figure 5

Effect of Bacillus amyloliquefaciens L3 on the incidence of Fusarium wilt of watermelon in a greenhouse pot experiment. F: control, inoculated with FON, L3: inoculated with FON and the strain L3. “*” above the columns indicate a significant difference at p < 0.05

Figure 6

Effect of Bacillus amyloliquefaciens strain L3 on the number of FON in the rhizosphere soil of watermelon at 0, 20, and 36 days after transplanting. CK: control, inoculated with FON, L3: inoculated with FON and the strain L3. “*” above the columns indicate a significant difference at p < 0.05

Figure 7

Effect of Bacillus amyloliquefaciens strain L3 on the fresh weight of watermelon seedling grown for 20 days in a greenhouse. CK: control treatment, L3: strain L3 was inoculated in the soilless substrate (10⁸ cfu/g). “*” above the columns indicate a significant difference at p < 0.05

Figure 8

The Relative content of identified volatile organic compounds released by strain L3 based on the peak area of GC‐MS data

Figure 9

The inhibition of FON mycelia growth by pure VOCs identified from Bacillus amyloliquefaciens L3. Lowercase letters above the columns indicate a significant difference at p <0 .05

Figure 10

Growth promotion of Arabidopsis thaliana Col‐0 with exposure to pure VOCs. (a) Effect of acetoin (1,000 μg/plate) and 2,3‐butanediol (500 μg/plate) on A. thaliana Col‐0 growth. (b) Fresh weight of A. thaliana Col‐0 after expose to different concentrations of acetoin and 2,3‐ butanediol. Lowercase letters above the columns indicate a significant difference at p < 0.05

Figure A1

Schematic representation of the experimental setup for Arabidopsis thaliana growth promotion by VOCs

Figure A2

Biofilm formation of the strain L3 in static culture medium (LB)

Figure A3

SPME chromatography‐mass spectrometry (GC‐MS) profile of volatile organic compounds (VOCs) produced by the strain L3. Red arrow means the specific VOCs produced by strain L3

Figure A4

Phylogenetic analysis of strain L3 and related species using the neighbor‐joining approach. Bootstrap values obtained with 1,000 resamplings are indicated as percentages at all branches. The GenBank accession number for each microorganism is shown in parentheses after the species name