Biocontrol Activity of Volatile-Producing Bacillus megaterium and Pseudomonas protegens Against Aspergillus and Penicillium spp. Predominant in Stored Rice Grains: Study II

Abstract

In our previous studies, Bacillus megaterium KU143, Microbacterium testaceum KU313, and Pseudomonas protegens AS15 have been shown to be antagonistic to Aspergillus flavus in stored rice grains. In this study, the biocontrol activities of these strains were evaluated against Aspergillus candidus, Aspergillus fumigatus, Penicillium fellutanum, and Penicillium islandicum, which are predominant in stored rice grains. In vitro and in vivo antifungal activities of the bacterial strains were evaluated against the fungi on media and rice grains, respectively. The antifungal activities of the volatiles produced by the strains against fungal development and population were also tested using I-plates. In in vitro tests, the strains produced secondary metabolites capable of reducing conidial germination, germ-tube elongation, and mycelial growth of all the tested fungi. In in vivo tests, the strains significantly inhibited the fungal growth in rice grains. Additionally, in I-plate tests, strains KU143 and AS15 produced volatiles that significantly inhibited not only mycelial growth, sporulation, and conidial germination of the fungi on media but also fungal populations on rice grains. GC-MS analysis of the volatiles by strains KU143 and AS15 identified 12 and 17 compounds, respectively. Among these, the antifungal compound, 5-methyl-2-phenyl-1H-indole, was produced by strain KU143 and the antimicrobial compounds, 2-butyl 1-octanal, dimethyl disulfide, 2-isopropyl-5-methyl-1-heptanol, and 4-trifluoroacetoxyhexadecane, were produced by strain AS15. These results suggest that the tested strains producing extracellular metabolites and/or volatiles may have a broad spectrum of antifungal activities against the grain fungi. In particular, B. megaterium KU143 and P. protegens AS15 may be potential biocontrol agents against Aspergillus and Penicillium spp. during rice grain storage.

Keywords: Antifungal activity; Aspergillus; Bacillus megaterium; Penicillium; Pseudomonas protegens; bacterial volatiles; biological control.

Figure 1.

Antifungal activities and populations of antagonistic bacterial strains, Bacillus megaterium KU143, Microbacterium testaceum KU313, and Pseudomonas protegens AS15, and negative control strain Sphingomonas aquatilis KU408 against (A) Aspergillus candidus AC317, (B) Aspergillus fumigatus AF8, (C) Penicillium fellutanum KU53, and (D) Penicillium islandicum KU101, 7 d after treatment in unhulled rice grains. Sterile distilled water (SDW) and 10 mM MgSO₄ solution were used as negative controls whereas a commercial fungicide, Benlate^® was used as a positive control. Fungal populations were determined after 4 d of incubation at 28 °C on DG18 and total bacterial populations were evaluated after 2 d of incubation at 28 °C on nutrient agar. Different letters on the vertical bars with error bars (standard deviations, n = 6) indicate significant differences between treatments according to the least significant difference test at p < .05. CFU: colony-forming unit.

Figure 2.

Photographs of antifungal activities of the volatiles produced by antagonistic bacterial strains, Bacillus megaterium KU143, Microbacterium testaceum KU313, and Pseudomonas protegens AS15, and negative control strain Sphingomonas aquatilis KU408 against (A) Aspergillus candidus AC317, (B) Aspergillus fumigatus AF8, (C) Penicillium fellutanum KU53, and (D) Penicillium islandicum KU101 on the I-plates. Bacterial strains or 10 mM MgSO₄ solution (untreated control) were smeared on one side (nutrient agar) of the I-plates and the other side (potato dextrose agar) was inoculated with a conidial suspension of the tested fungi 24 h after the bacterial treatment. Insets are photographs of the conidial germination of the fungi affected by the bacterial volatiles or MgSO₄ solution. C: conidium; gt: germ tube. Scale bar, 20 µm.

Figure 3.

Antifungal activities of the volatiles produced by antagonistic bacterial strains, Microbacterium testaceum KU313, Bacillus megaterium KU143, and Pseudomonas protegens AS15, and negative control strain Sphingomonas aquatilis KU408 against populations of (A) Aspergillus candidus AC317, (B) Aspergillus fumigatus AF8, (C) Penicillium fellutanum KU53, and (D) Penicillium islandicum KU101, 7 d after treatment in unhulled rice grains on the I-plates. Bacterial strains or 10 mM MgSO₄ solution (untreated control) were smeared on one side (nutrient agar) of the I-plates and the other side (rice grains) was inoculated with a conidial suspension of the tested fungi, 24 h after bacterial treatment. Fungal populations in rice grains were assessed on DG18 after 4 d of incubation at 28 °C. Different capital letters on the vertical bars indicate significant (p < .05) differences between treatments according to the least significant difference test. CFU: colony-forming unit.

Figure 4.

Gas chromatographs of the volatiles produced by (A) Bacillus megaterium KU143 and (B) Pseudomonas protegens AS15. Antagonistic bacterial strains were cultured in tryptic soy broth (TSB) at 28 °C for 24 h. Volatile compounds from B. megaterium KU143 were identified as: heptane [peak number (PN) 1], 2,5-dimethyltetrahydrofuran (PN 2), methylcyclohexane (PN 3), 3-hexanone (PN 4), 2-hexanone (PN 5), trimethyl [4-(1,1,3,3,-tetramethylbutyl) phenoxy] silane (PN 6), 3-hexene-2,5-diol (PN 7), 2-bromohexane (PN 8), 2,4,4-trimethyl-1-pentanol (PN 9), 2,6-dimethylpiperazine (PN 10), 2,2-dimethyl-3-hexanol (PN 11), and 5-methyl-2-phenyl-1 H-indole (PN 12). Volatile compounds from P. protegens AS15 were identified as: heptane (PN 1), dimethyl disulfide (PN 2), 2,2,3-trimethylpentane (PN 3), 4-methylheptane (PN 4), 3-hexanone (PN 5), 2-hexanone (PN 6), 2,4-dimethyl-1-heptene (PN 7), 3-methylcyclopentanol (PN 8), 3,3-dimethyloctane (PN 9), 2,6-dimethylnonane (PN 10), 1-(2-methylbutyl)-1-(1-methylpropyl)-cyclopropane (PN 11), 2-butyl-1-octanol (PN 12), undecane (PN 13), 1,3-di-tert-butylbenzene (PN 14), 4-trifluoroacetoxyhexadecane (PN 15), 2-isopropyl-5-methyl-1-heptanol (PN 16), and isotridecanol (PN 17). However, no distinct volatile compounds were detected in the TSB control.