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

Figures & data

Table 1. Antifungal activities of antagonistic bacterial strains, Bacillus megaterium KU143, Microbacterium testaceum KU313, and Pseudomonas protegens AS15, and negative control strain, Sphingomonas aquatilis KU408, on mycelial growth of Aspergillus candidus AC317, Aspergillus fumigatus AF8, Penicillium fellutanum KU53, and Penicillium islandicum KU101 in dual-culture assays and the effects of cell-free culture filtrates of these strains on conidial germination, germ-tube lengths, and mycelial dry weights of the fungi in potato-dextrose broth tests.

Treatment	Mycelial growth inhibition (mm)		Conidial germination (%)		Germ tube length (µm)		Mycelial dry weight (mg)
	A. candidus	A. fumigatus	A. candidus	A. fumigatus	A. candidus	A. fumigatus	A. candidus	A. fumigatus
Control^a	_^b	_	33.2 ± 7.1 A^c	78.2 ± 4.1 A	39.4 ± 3.9 A	51.5 ± 4.2 A	31.3 ± 8.8 A	64.7 ± 8.1 A
KU408	1.0 ± 1.0 D	0.1 ± 0.4 D	30.9 ± 4.9 AB	71.9 ± 3.1 A	37.9 ± 3.6 A	50.9 ± 5.5 A	32.0 ± 8.3 A	62.3 ± 8.9 A
KU313	6.0 ± 2.0 C	6.5 ± 1.8 C	25.3 ± 5.0 B	52.3 ± 3.7 B	21.3 ± 3.0 B	27.0 ± 2.6 B	22.3 ± 5.3 B	45.0 ± 4.0 B
KU143	12.7 ± 2.6 B	9.9 ± 2.6 B	19.2 ± 2.0 C	37.0 ± 3.2 C	17.8 ± 1.9 C	22.6 ± 3.7 C	21.7 ± 3.7 B	38.0 ± 4.4 B
AS15	17.2 ± 1.4 A	14.2 ± 3.8 A	12.3 ± 2.9 D	25.7 ± 2.2 C	17.1 ± 3.5 C	18.7 ± 4.4 C	20.2 ± 6.9 B	29.2 ± 3.1 C
	Mycelial growth inhibition (mm)		Conidial germination (%)		Germ tube length (µm)		Mycelial dry weight (mg)
	P. fellutanum	P. islandicum	P. fellutanum	P. islandicum	P. fellutanum	P. islandicum	P. fellutanum	P. islandicum
Control	_	_	38.6 ± 3.6 A	35.9 ± 2.3 A	75.4 ± 2.8 A	63.2 ± 10.8 A	59.0 ± 4.8 A	45.0 ± 4.9 A
KU408	1.7 ± 1.1 D	1.6 ± 1.5 D	38.2 ± 3.4 A	34.4 ± 5.2 A	76.4 ± 3.9 A	68.2 ± 10.1 A	55.7 ± 5.3 A	42.2 ± 3.0 A
KU313	6.4 ± 1.5 C	7.5 ± 1.8 C	27.8 ± 5.7 B	24.2 ± 4.7 B	43.2 ± 7.9 B	32.4 ± 2.8 B	34.3 ± 4.7 B	22.5 ± 3.8 B
KU143	12.4 ± 2.0 B	12.6 ± 1.4 B	16.6 ± 3.4 C	17.2 ± 3.7 C	30.3 ± 3.4 C	30.6 ± 2.3 B	25.0 ± 2.2 C	15.5 ± 2.8 C
AS15	17.6 ± 1.1 A	17.3 ± 1.2 A	9.0 ± 1.9 D	12.1 ± 2.5 D	27.2 ± 3.2 C	26.3 ± 1.5 B	15.2 ± 2.3 D	8.0 ± 1.4 D

^a The controls were sterile distilled water in dual-culture assays and sterile nutrient broth in potato-dextrose broth tests.

^b Not detected.

^c Means ± standard deviations (n = 6) with different capital letters in each column are significantly (p < .05) different between treatments according to the least significant difference test.

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.

Table 2. 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, on mycelial growth, sporulation, and conidial germination of Aspergillus candidus AC317, Aspergillus fumigatus AF8, Penicillium fellutanum KU53, and Penicillium islandicum KU101 on I-plates.

Treatment	Mycelial growth (cm^2)		Sporulation (log no. conidia cm^−2 mycelia)		Conidial germination (%)
	A. candidus	A. fumigatus	A. candidus	A. fumigatus	A. candidus	A. fumigatus
MgSO4	14.3 ± 1.5 A^a	17.3 ± 2.1 A	7.4 ± 0.1 A	8.1 ± 0.1 A	23.3 ± 3.6 A	64.2 ± 3.5 A
KU408	13.6 ± 1.7 AB	17.8 ± 1.7 A	7.3 ± 0.1 AB	8.0 ± 0.1 A	22.0 ± 3.3 A	63.5 ± 3.0 A
KU313	10.5 ± 1.4 B	15.1 ± 1.6 B	7.3 ± 0.2 AB	7.9 ± 0.1 B	15.0 ± 2.0 AB	49.3 ± 3.4 B
KU143	6.6 ± 0.3 C	8.5 ± 1.1 C	7.1 ± 0.1 C	6.5 ± 0.2 D	5.5 ± 0.9 C	9.2 ± 1.0 C
AS15	6.1 ± 0.3 C	7.9 ± 0.7 C	7.3 ± 0.2 B	7.4 ± 0.2 C	6.5 ± 0.8 BC	12.5 ± 1.4 C
	Mycelial growth (cm^2)		Sporulation (log no. conidia cm^−2 mycelia)		Conidial germination (%)
	P. fellutanum	P. islandicum	P. fellutanum	P. islandicum	P. fellutanum	P. islandicum
MgSO4	14.1 ± 1.4 A	14.5 ± 0.7 A	8.7 ± 0.1 A	8.5 ± 0.3 A	37.8 ± 1.2 A	36.7 ± 2.2 A
KU408	12.9 ± 1.5 AB	15.0 ± 1.1 A	8.7 ± 0.2 A	8.2 ± 0.3 A	38.2 ± 3.4 A	35.0 ± 3.4 A
KU313	11.8 ± 1.4 B	12.9 ± 1.6 B	8.4 ± 0.3 A	8.2 ± 0.3 A	37.0 ± 3.0 A	34.8 ± 3.2 A
KU143	2.0 ± 0.3 C	3.3 ± 0.2 C	7.0 ± 0.2 B	6.4 ± 0.3 B	7.8 ± 0.9 B	11.0 ± 1.6 B
AS15	2.7 ± 0.3 C	3.2 ± 0.2 C	7.0 ± 0.4 B	6.3 ± 0.3 B	11.3 ± 1.3 B	13.7 ± 2.0 B

^a Means ± standard deviations (n = 6) with different letters in each column are significantly (p < .05) different between treatments according to the least significant difference test.

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.