Biological control of potato common scab by Bacillus amyloliquefaciens Ba01

Abstract

Potato common scab, which is caused by soil-borne Streptomyces species, is a severe plant disease that results in a significant reduction in the economic value of potatoes worldwide. Due to the lack of efficacious pesticides, crop rotations, and resistant potato cultivars against the disease, we investigated whether biological control can serve as an alternative approach. In this study, multiple Bacillus species were isolated from healthy potato tubers, and Bacillus amyloliquefaciens Ba01 was chosen for further analyses based on its potency against the potato common scab pathogen Streptomyces scabies. Ba01 inhibited the growth and sporulation of S. scabies and secreted secondary metabolites such as surfactin, iturin A, and fengycin with potential activity against S. scabies as determined by imaging mass spectrometry. In pot assays, the disease severity of potato common scab decreased from 55.6 ± 11.1% (inoculated with S. scabies only) to 4.2 ± 1.4% (inoculated with S. scabies and Ba01). In the field trial, the disease severity of potato common scab was reduced from 14.4 ± 2.9% (naturally occurring) to 5.6 ± 1.1% after Ba01 treatment, representing evidence that Bacillus species control potato common scab in nature.

Fig 1. B. amyloliquefaciens Ba01 showed antibacterial activity against S. scabies causing potato common scab.

A. Phylogenetic trees based on either the (a) 16S rRNA sequence or (b) gyrA gene sequence showed the evolutionary relationships between Bacillus isolates. The numbers at the nodes represent bootstrap values. The scale bars indicating the numbers of substitutions per nucleotide position were (a) 0.005 and (b) 0.02. B. Multiple Bacillus isolates exhibited a diverse degree of antibacterial activity against S. scabies PS07. Disk diffusion assays were used to test the antibacterial activity of Bacillus species against S. scabies. In this experiment, 10⁶ S. scabies spores in 100 μL were spread on solid YME medium, and 3 μL of 1 OD₆₀₀ (~6x10⁴ cells) of Bacillus isolates were loaded on the right disk, while 3 μL of ddH₂O were loaded on the left disk as a control. C. Ba01 was selected to test its antibacterial activity against multiple S. scabies isolates. S. scabies isolates were spread on solid YME medium, and 3 μL of 1 OD₆₀₀ of Ba01 were added to the right disk and ddH₂O to the left disk. All plates were incubated at 28°C for five days and photographed.

Fig 2. B. amyloliquefaciens Ba01 inhibited the growth and sporulation of S. scabies PS07.

A. A disk diffusion assay was used to observe the antibacterial effects of Ba01 against S. scabies PS07 on solid YME medium. Clear (C) and undifferentiating (U) zones were observed around the disk loaded with the Ba01 isolate. B. The morphology of Ba01 from panel A was observed under a scanning electron microscope. C. S. scabies PS07 without Ba01 treatment (ddH₂O treated) produced spiral hyphae and sporulation septa with constrictions. D. S. scabies PS07 in the undifferentiating zone exhibited vegetative/smooth hyphae and septa without constrictions. Resolution is 10,000X. The scale bar represents 1 μm.

Fig 3. Imaging mass spectrometry of Ba01 against S. scabies PS07.

A. (a) S. scabies PS07 was initially inoculated in a vertical line on a 2% YME solid agar plate. After 12 h, B. amyloliquefaciens Ba01 was inoculated in a horizontal line on the same plate for another 24 h. (b) The IMS image of an ion with m/z 1046.38 represents surfactin. (c) The image represents two-fold serial diluted surfactin as a standard control ranging from 2.5 to 0.04 μg. (d) The IMS image of an ion with m/z 1095.76 represents iturin A. (e) The image represents two-fold serial diluted iturin A as a standard control ranging from 10 to 0.16 μg. (f) The IMS image of an ion with m/z 1516.19 represents fengycin. (g) The image represents two-fold serial diluted fengycin as a standard control ranging from 0.32 to 0.005 μg/mL. Intensity gradients for surfactin, iturin A, and fengycin are normalized and illustrated by color histogram (maximum, white; minimum, black). B. The mass spectra of IMS regions include three major peaks: m/z 1046.38 (surfactin), 1095.76 (iturin A), and 1516.19 (fengycin).

Fig 4. Surfactin, iturin A, and fengycin inhibited the growth and formation of spiral hyphae of S. scabies PS07.

A. Disk diffusion assays were used to test the anti-S. scabies activity of surfactin, iturin A, and fengycin. Approximately 10⁶ S. scabies spores were spread on YME solid agar, and a 6-mm disk containing iturin A (dissolved in ethanol), surfactin (dissolved in methanol), or fengycin (dissolved in methanol) were pressed on the surface of an agar plate and incubated at 28°C for five days. B. The morphology of S. scabies PS07 in the undifferentiating zone of panel A was observed under a scanning electron microscope. (a) The magnification is 5,000X, and the scale bar represents 10 μm. (b) The magnification is 15,000X, and the scale bar represents 2.5 μm.

Fig 5. Ba01 reduced the disease severity of potato common scab in pot assays.

The growth conditions of S. scabies PS07, Ba01, and potato plants were described in the materials and methods. A. Three five-week-old potato plants were used for each treatment: (a) mock control without inoculation of S. scabies PS07 or Ba01; (b) inoculation of S. scabies PS07 only; (c) inoculation of Ba01 only; (d) inoculation of PS07 and Ba01 on the same day; and (e) inoculation of PS07 first, and then Ba01 inoculation after 14 days. The bar represents 1 cm. B. The disease severity and disease incidence of the potato common scab were reduced when potato plants were inoculated with PS07 and Ba01 on the same day. Data were expressed as the average of tubers collected from three potato plants ± standard error of the mean. P values were calculated using Tukey's test. Asterisks (**) indicate P < 0.01 as compared to the treatment of PS07 only.

Fig 6. Ba01 reduced the severity of naturally occurring potato common scab in the field.

A. (a) An 11-week potato field trial was completed in Dounan, Taiwan. Bars = 100 cm. (b) Four treatments were treated with the Ba01 isolate at the concentrations indicated. (c) Each treatment had four blocks assigned by a randomized complete block design. B. Potato tubers were harvested from each Ba01 treatment: (1) Ba01 at 5x10⁶ CFU/mL; (b) 1x10⁷ CFU/mL; (c) 2x10⁷ CFU/mL; and (d) water. Bars = 5 cm. C. The percentage of disease severity was calculated from 100 randomly selected tubers of each treatment. D. Disease incidence was calculated by determining the proportion of tubers with >5% scab coverage from 100 randomly selected tubers in each treatment. E. Potato plant height (left panel) and tuber weight (right panel) were not affected by Ba01 application. We randomly chose 25 potato tubers from each block to evaluate the disease severity and incidence of each block, and four blocks of the same treatment were used to determine the mean ± standard error and compare to other treatments. P values were calculated using Tukey's test. Asterisks *, **, and *** represent P < 0.05, P < 0.01, and P < 0.001, respectively.

Fig 7. Ba01 inhibits the growth of multiple S. scabies strains isolated from the field trial.

A. Potato slide assays were used to test the pathogenicity of S. scabies isolates. Agar disks with or without S. scabies spores were inverted onto potato tuber slices and incubated at 28°C for six days in the dark and photographed. Nonpathogenic Streptomyces coelicolor was used as a negative control, and a SSM1 agar disc was used as a mock control. B. Disc diffusion assays were used to test the anti-bacterial activity of Ba01 and three pure compounds against multiple S. scabies strains.