Surfactin secreted by Bacillus amyloliquefaciens Ba01 is required to combat Streptomyces scabies causing potato common scab

Abstract

Potato common scab, which is mainly caused by the bacterium Streptomyces scabies, occurs in key potato growing regions worldwide. It causes necrotic or corky symptoms on potato tubers and decreases the economic value of potato. At present, there is no recommended chemical or biological control for combating potato common scab in Taiwan. It can only reduce the occurrence by cultivation control, but the efficacy is limited. Previously we found that Bacillus amyloliquefaciens Ba01 could control potato common scab in pot assay and in the field. The potential anti-S. scabies mechanism was associated with surfactin secretion, but further molecular dissection was not conducted. Thus, in this study we aimed to determine whether surfactin is the main compound active against S. scabies by knocking out the srf gene cluster in Ba01. The cloning plasmid pRY1 was transformed to Ba01 by electroporation for in-frame deletion. Two independent Δsrf mutants were obtained and confirmed by specific primers and mass spectrometry. The swarming ability and S. scabies inhibition was significantly decreased (P<0.001) in Δsrf mutants. The swarming ability of Δsrf mutants could be restored by the addition of surfactin. Furthermore, we found that Ba01 formed wrinkled biofilm in MSgg liquid medium, while Δsrf mutants formed biofilm abnormally. Furthermore, the α-amylase, protease and phosphate-solubilizing ability of Δsrf mutants was decreased, and the mutants could not inhibit the growth and sporulation of S. scabies on potato tuber slices. In conclusion, srf gene cluster of B. amyloliquefaciens Ba01 is responsible for the secretion of surfactin and inhibition of S. scabies.

Keywords: Bacillus amyloliquefaciens; Streptomyces scabies; Surfactin; potato common scab; srf gene cluster.

Figure 1

Scheme of the plasmid construction for making Δsrf mutant in B amyloliquefaciens Ba01. Primers JC1718/1719 and JC1720/1721 were used to amplify 5′ and 3′ NCR sequences of srf gene cluster of B amyloliquefaciens Ba01, respectively, and fused together by fusion PCR using primers JC1718/1721. After enzyme digestions the ~2 kb cassette was ligated to pMiniMAD2 in order to generate disruption plasmid pRY1.

Figure 2

Scheme of in-frame markerless deletion method to construct the srf gene cluster mutants in B amyloliquefaciens Ba01. Single crossover recombinants were obtained by culturing the transformants at restrictive temperature (37°C) for plasmid pRY1 integration into Ba01 genome and using mls medium for selection. To evict the plasmid from Ba01 genome after homologous recombination, the strain was incubated overnight in 3 ml of LB broth at a permissive temperature (25°C) for plasmid replication. The cell cultures were diluted and plated on LB agar at 37°C for 24 h The colonies were patched on mls medium to select mls-sensitive colonies. The srf gene cluster mutants were checked by PCR with primers JC1783/1784.

Figure 3

Confirmation of srf gene cluster mutants by PCR amplification. (A) lane e: Erm ^R marker (~1100 bp; JC1512/1513), lane i: ituD ORF (469 bp; JC1466/1467), lane s: srfAD ORF (485 bp; JC1468/1469), lane f: fenA ORF (455 bp; JC1470/1471). (B) Outside primer of 5′ and 3′ NCR of srf gene cluster (~2200 bp; JC1783/1784).

Figure 4

The Δsrf mutants lose the ability to secrete surfactins. The supernatants of cultures were collected and extracted twice with ethyl acetate, followed by resuspension with methanol before conducting mass spectrometry analysis. The surfactin peaks were found in the standard and wild type (WT), but deficient in Δsrf mutants (FRY1 and FRY3).

Figure 5

The growth kinetics of Δsrf mutants were slightly decreased. (A) Growth curves were determined based on the OD₆₀₀ values. (B) Growth curves were determined based on the viable cell counts (CFU/ml). The strains were 10-fold diluted and spread on LB agar plates. Total colony numbers were counted after 1 day growth at 37°C.

Figure 6

The endospore formation of Δsrf mutants was decreased. (A) Endospores were stained and observed under a microscope (100×). (B) Graph of endospore formation. The strains were treated at 80°C for 10 min to kill vegetative cells and plated on LB agar to calculate the number of endospores. Endospore formation was the ratio of endospore numbers divided by total colony numbers. P values were calculated by t-test, “*”, “**” and “***” represent P < 0.05, P < 0.01 and P < 0.001 respectively as compared to the WT.

Figure 7

The Δsrf mutants reduced the inhibition activity against S. scabies. (A) Disk diffusion assay. The 10⁶ spores of S. scabies PS07 were spread on YME agar plates, and a disk containing 3 μl WT or Δsrf mutant culture (OD₆₀₀ = 1) was put on the surface of plates. The plates were incubated at 28°C for 2 days. “C” represents clear inhibition zone, and “U” represents undifferentiated zone. (B) Graph of (A). P values were calculated by t-test, “***” represents P < 0.001 as compared to the WT.

Figure 8

The Δsrf mutants showed attenuated swarming motility. Five microliters of strains (OD₆₀₀ = 1) were inoculated on 0.25%, 0.5% or 0.7% solid LB agar at 37°C for 48 h. P values were calculated by t-test, “***” represents P < 0.001 as compared to the WT.

Figure 9

Surfactin could rescue the swarming motility of Δsrf mutant. (A) Solution of 10 μl containing 1 μg, 20 μg or 100 μg of surfactin were spotted on the center of 0.7% solid LB medium before dropping 5 μl of strains (OD₆₀₀ = 1) onto the top, and then incubated at 37°C for 48 h (B) Graph of (A). P values were calculated by t-test, “***” represents P < 0.001 as compared to the Δsrf mutant (FRY1) in the absence of surfactin.

Figure 10

The Δsrf mutants lost the ability to form biofilm. (A) WT formed a thick and wrinkled biofilm, while Δsrf mutants did not. Surfactin did not rescue the biofilm formation of Δsrf mutant in liquid MSgg medium. For biofilm formation, 10 μl of strains (OD₆₀₀ = 1) were added to 1 ml MSgg broth and incubated in a 24-well plate at 30°C for 2 days. The addition of 20 μM of surfactin was to determine if surfactin could rescue biofilm formation. (B) For colony morphology, 3 μl of strains (OD₆₀₀ = 1) were spotted on the center of MSgg solid medium, and then incubated at 30°C for 2 or 4 days. To determine if surfactin could rescue colony morphology, solution of 5 μl containing 10 μg of surfactin was spotted on the center of MSgg solid medium before adding 3 μl of FRY1 (OD₆₀₀ = 1).

Figure 11

The enzyme activity of α-amylase and protease, and phosphate-solubilizing ability of Δsrf mutants were decreased. Three microliters of strains (OD₆₀₀ = 1) were inoculated on media testing α-amylase, protease, cellulase and phosphate-solubilizing ability and incubated at 37°C for 2 days (α-amylase, protease, cellulase) or 5 days (phosphate-solubilizing).

Figure 12

Two Δsrf mutants did not effectively inhibit the growth and sporulation of S. scabies PS07 on potato tuber slices. The 25 μl 10⁸ spores/ml of S. scabies PS07 spores were inoculated on potato tuber slices, incubated at 28°C for 1 day in the dark, and then 20 μl of Ba01 WT, FRY1, FRY3 (OD₆₀₀ = 1), ddH₂O or surfactin (0.1 mg/ml) were added to potato tuber slices. The samples were further incubated in the moist chamber at 28°C for 5 days before photographing.