Suppressive Effects of Volatile Compounds from Bacillus spp. on Magnaporthe oryzae Triticum (MoT) Pathotype, Causal Agent of Wheat Blast

Abstract

The Magnaporthe oryzae Triticum (MoT) pathotype is the causal agent of wheat blast, which has caused significant economic losses and threatens wheat production in South America, Asia, and Africa. Three bacterial strains from rice and wheat seeds (B. subtilis BTS-3, B. velezensis BTS-4, and B. velezensis BTLK6A) were used to explore the antifungal effects of volatile organic compounds (VOCs) of Bacillus spp. as a potential biocontrol mechanism against MoT. All bacterial treatments significantly inhibited both the mycelial growth and sporulation of MoT in vitro. We found that this inhibition was caused by Bacillus VOCs in a dose-dependent manner. In addition, biocontrol assays using detached wheat leaves infected with MoT showed reduced leaf lesions and sporulation compared to the untreated control. VOCs from B. velezensis BTS-4 alone or a consortium (mixture of B. subtilis BTS-3, B. velezensis BTS-4, and B. velezensis BTLK6A) of treatments consistently suppressed MoT in vitro and in vivo. Compared to the untreated control, VOCs from BTS-4 and the Bacillus consortium reduced MoT lesions in vivo by 85% and 81.25%, respectively. A total of thirty-nine VOCs (from nine different VOC groups) from four Bacillus treatments were identified by gas chromatography-mass spectrometry (GC-MS), of which 11 were produced in all Bacillus treatments. Alcohols, fatty acids, ketones, aldehydes, and S-containing compounds were detected in all four bacterial treatments. In vitro assays using pure VOCs revealed that hexanoic acid, 2-methylbutanoic acid, and phenylethyl alcohol are potential VOCs emitted by Bacillus spp. that are suppressive for MoT. The minimum inhibitory concentrations for MoT sporulation were 250 mM for phenylethyl alcohol and 500 mM for 2-methylbutanoic acid and hexanoic acid. Therefore, our results indicate that VOCs from Bacillus spp. are effective compounds to suppress the growth and sporulation of MoT. Understanding the MoT sporulation reduction mechanisms exerted by Bacillus VOCs may provide novel options to manage the further spread of wheat blast by spores.

Keywords: Bacillus; GC–MS; biocontrol; sporulation; volatile organic compound.

Figure 1

Effect of Bacillus spp. VOCs on MoT in bi-partitioned Petri dish assay in vitro. (A) Photographs showing the effects of Bacillus VOCs on mycelial growth of MoT; (B) effects of Bacillus VOCs on mycelial growth of MoT in vitro; (C) effects of Bacillus VOCs on reduction in MoT sporulation compared to control in vitro. Data were recorded after 5 d of MoT incubation at 25 °C. BTS-3: B. subtilis BTS-3; BTS-4: B. velezensis BTS-4; BTLK6A: B. velezensis BTLK6A; consortium: a mixture of BTS-3, BTS-4, and BTLK6A; control: without any bacterial treatment (Tukey test; n = 6; p ≤ 0.05). Black points in (B,C) represent data points for each replicate.

Figure 2

Effects of Bacillus spp. VOCs on MoT conidial germination in upside-down Petri dish assay in vitro. (A) Photographs showing the effects of Bacillus VOCs on MoT conidial germination and mycelial growth; (B) effects of bacterial VOCs on mycelial growth of MoT in vitro; (C) effects of bacterial VOCs on reduction in new MoT sporulation compared to control in vitro. Data were recorded after 5 d of MoT incubation at 25 °C. BTS-3: B. subtilis BTS-3; BTS-4: B. velezensis BTS-4; BTLK6A: B. velezensis BTLK6A; consortium: a mixture of BTS-3, BTS-4, and BTLK6A; control: without any bacterial treatment (Tukey test; n = 6; p ≤ 0.05). Black points in (B,C) represent data points for each replicate.

Figure 3

Effects of Bacillus VOCs on lesion development and MoT sporulation in a detached wheat leaf assay (cv. BR-18). (A) VOCs from Bacillus spp. significantly reduced leaf blast lesion size in vivo. (B) Bacillus VOCs significantly reduced leaf lesion area (cm²) caused by MoT in vivo (Kruskal–Wallis test; n = 30; p ≤ 0.05); (C) reduction in MoT sporulation by VOCs from different Bacillus spp. in vivo (Tukey test; n = 30; p ≤ 0.05). Data were recorded after 5 d of MoT incubation at 25 °C. BTS-3: B. subtilis BTS-3; BTS-4: B. velezensis BTS-4; BTLK6A: B. velezensis BTLK6A; consortium: a mixture of BTS-3, BTS-4, and BTLK6A; untreated control: only MoT inoculated. Black points in (B,C) represent data points for each replicate.

Figure 3

Effects of Bacillus VOCs on lesion development and MoT sporulation in a detached wheat leaf assay (cv. BR-18). (A) VOCs from Bacillus spp. significantly reduced leaf blast lesion size in vivo. (B) Bacillus VOCs significantly reduced leaf lesion area (cm²) caused by MoT in vivo (Kruskal–Wallis test; n = 30; p ≤ 0.05); (C) reduction in MoT sporulation by VOCs from different Bacillus spp. in vivo (Tukey test; n = 30; p ≤ 0.05). Data were recorded after 5 d of MoT incubation at 25 °C. BTS-3: B. subtilis BTS-3; BTS-4: B. velezensis BTS-4; BTLK6A: B. velezensis BTLK6A; consortium: a mixture of BTS-3, BTS-4, and BTLK6A; untreated control: only MoT inoculated. Black points in (B,C) represent data points for each replicate.

Figure 4

Identification and quantification of VOCs from different Bacillus treatments by GC–MS analysis. (A) Venn diagram representing the number of VOCs produced from different Bacillus spp.; (B) number of different VOC classes produced from different Bacillus treatments; (C) heatmap based on Euclidean distance showing the VOCs produced from each Bacillus treatment. Each line in the color heatmap indicates a single compound; red to green color code indicates low to high relative concentrations (based on row Z-scores) of the compounds; blue color indicates compounds detected only in a single treatment; grey color indicates undetected volatile. BTS-3: B. subtilis BTS-3; BTS-4: B. velezensis BTS-4; BTLK6A: B. velezensis BTLK6A; const.: consortium (a mixture of B. subtilis BTS-3, B. velezensis BTS-4, and B. velezensis BTLK6A) (n = 5; p ≤ 0.05). The VOCs were quantified from 40 mL LB inoculated with 100 µL (1 × 10⁹ CFU/mL) bacteria and incubated for 4 d at 25 °C.

Figure 4

Identification and quantification of VOCs from different Bacillus treatments by GC–MS analysis. (A) Venn diagram representing the number of VOCs produced from different Bacillus spp.; (B) number of different VOC classes produced from different Bacillus treatments; (C) heatmap based on Euclidean distance showing the VOCs produced from each Bacillus treatment. Each line in the color heatmap indicates a single compound; red to green color code indicates low to high relative concentrations (based on row Z-scores) of the compounds; blue color indicates compounds detected only in a single treatment; grey color indicates undetected volatile. BTS-3: B. subtilis BTS-3; BTS-4: B. velezensis BTS-4; BTLK6A: B. velezensis BTLK6A; const.: consortium (a mixture of B. subtilis BTS-3, B. velezensis BTS-4, and B. velezensis BTLK6A) (n = 5; p ≤ 0.05). The VOCs were quantified from 40 mL LB inoculated with 100 µL (1 × 10⁹ CFU/mL) bacteria and incubated for 4 d at 25 °C.

Figure 5

Fold change (FC) in VOCs produced by B. velezensis BTS-4 in relation to the Bacillus consortium (a mixture of B. subtilis BTS-3, B. velezensis BTS-4, and B. velezensis BTLK6A) treatment through volcano plot analysis. The log2 fold change threshold was 2.0. The false discovery rate (FDR) was maintained with p threshold at 0.05. The VOCs were quantified from 40 mL LB inoculated with 100 µL (1 × 10⁹ CFU/mL) bacteria and incubated for 4 d at 25 °C. Non-SIG: non-significant.

Figure 6

Effect of selected pure antifungal VOCs against MoT mycelial growth and sporulation in vitro represented by heatmaps based on Euclidean distance: (A) effect of pure antifungal VOCs on MoT mycelial growth; (B) effect of pure antifungal VOCs on MoT sporulation. Each row in the color heatmap indicates a single compound. Red to green color code indicates low to high relative growth or sporulation of MoT (based on row Z-score). * indicates directly used single concentration (diluent) (n = 6, p ≤ 0.05).