Joo Won Suh, Department of Biological Science, Myongji University, San 38-2, Nam-dong, Yongin 449-728, Korea (e-mail: firstname.lastname@example.org).
Aims: To identify an antagonistic strain against Streptomyces scabiei and to characterize the antibiotic agent. The efficacy of the isolated strain in controlling common scab disease was also evaluated.
Methods and Results: A bacterial strain antagonistic against S. scabiei was isolated from the soil of a potato-cultivating area. This bacterium was identified as a Bacillus species by 16S rRNA gene sequence analysis and was designated Bacillus sp. sunhua. Antibiotics produced by this strain were proven to be stable within a broad pH range and at high temperatures. The culture broth was extracted with ethyl acetate, and then the crude extract was applied to HPLC. Two compounds were isolated and identified as iturin A and macrolactin A by 1H-NMR, 13C-NMR, HMBC, HMQC and mass spectrometer. The culture broth of Bacillus sp. sunhua had a suppressive effect on common scab disease in a pot assay, decreasing the infection rate from 75 to 35%. This strain also suppressed Fusarium oxysporum, the pathogen of potato dry rot disease.
Conclusions: Bacillus sp. sunhua was shown to inhibit S. scabiei effectively.
Significance and Impact of the Study: This is the first report demonstrating that macrolactin A and iturin A inhibit S. scabiei. This study demonstrated the possibility of controlling potato scab disease using Bacillus sp. sunhua.
Potatoes are widely cultivated, and could contribute to reducing worldwide food shortages. However, potato plants are susceptible to devastation by various diseases, such as soft rot disease, bacterial wilt disease, scab disease and dry rot disease (Agrios 1997). Common scab disease occurs throughout the potato-cultivating regions of the world and is most prevalent in neutral or slightly alkaline soils, especially during dry years (Bouchek-Mechiche et al. 2000). Common scab disease has little or limited effect on tuber yield, but it can greatly affect tuber quality and, therefore, can severely reduce its market value (Liu et al. 1996). At least four Streptomyces species have been described to cause scab disease, including Streptomyces scabiei, Streptomyces acidiscabies, Streptomyces turgidiscabies and Streptomyces ipomoeae. (Healy et al. 2000). Of these four species, S. scabiei is the most important pathogen of potato crops (Keinath and Loria 1989).
Streptomyces scabiei penetrates tissues through lenticels and occasionally through wounds or young tubers, causing scab disease by producing a family of cyclic dipeptides, thaxtomins (Lawrence et al. 1990). Control of this disease has been achieved by soil treatment with the fungicide pentachloronitrobenzene (Potter et al. 1958), by using potato seeds treated with maneb-zinc dust when planting in scab-conducive soil (Agrios 1997), by regulating the pH of the soil (Doyle and Maclean 1960) or by irrigation (Hooker 1981; Loria et al. 1997). However, agrichemicals used for crop protection adversely affect the quality of crops and the environment, so studies on alternatives to synthetic chemicals are necessary (Liu et al. 1995; Neeno-Eckwall et al. 2001).
Due to the high economic losses caused by potato scab disease, studies on biocontrol mechanisms are of importance. In this study, an antagonistic Bacillus strain effective against scab pathogen was isolated and named sunhua. The biologically active compounds from the cultural filtrate of this strain were isolated and characterized as macrolactin A and iturin A. Furthermore, a pot assay proved the suppressive effects of this strain against scab disease caused by S. scabiei. Our results demonstrated the prospect of Bacillus sp. sunhua as a potentially promising biocontrol agent for common scab disease.
Materials and methods
Selection of antagonistic bacterium
Streptomyces scabiei KACC 20101 was provided by Korean Agricultural Culture Collection, National Institute of Agricultural Biotechnology, Korea. The strain was cultured in GYM broth (glucose 4 g l−1, yeast extract 4 g l−1, malt extract 10 g l−1, calcium carbonate 2 g l−1, pH 7·2) at 28°C for 7 d. Soil samples were taken from a spot of low infection of scab disease in a area of heavy infection on Jeju Island in the southern part of South Korea. 207 strains were isolated from soil samples and were incubated at 28°C (for actinomycetes) and 37°C (for other bacteria) in tryptic soya broth (TSB; Bacto, Becton Dickinson, Sparks, MD, USA). Each isolated strain was inoculated into potato dextrose broth (PDB; Difco) plates by toothpick on which S. scabiei had been previously spread. After incubation for 5 d, strains that inhibited the mycelium growth of S. scabiei were considered suppressive strains. The antagonistic strain with the biggest inhibition zone was identified by analysis of 16S rRNA sequence using primers fD1, rP2 (Chun and Goodfellow 1995). The amplified 16S rRNA gene fragment was ligated into pGEM T-Easy vector and then transformed into E. coli DH5α. The plasmids, purified with a Wizard plasmid prep kit (Promega, Madison, WI, USA), were sequenced using an automated DNA Sequencer (Model ABI 3100; Applied Biosystems, Foster City, CA, USA). The sequence was aligned using CLUSTAL W software (Thompson et al. 1994) and a phylogenic tree was constructed using the neighbour-joining method (Saitou and Nei 1987).
Production of secondary metabolites and antimicrobial activity
The isolated strain was cultured in TSB liquid medium until the final OD600 reached 1·0. Then, 100 μl of inoculum was transferred to a 500-ml flask containing 100 ml of GYM broth and incubated at 37°C for 24 h in a rotary shaker (150 rev min−1).
Antimicrobial activity against S. scabiei was detected by an agar disc diffusion assay (Kimura et al. 1998). Plates were prepared with S. scabiei spread on the surface of the GYM agar. Paper discs with a diameter of 8 mm were laid on the agar. Then, 50 μl of cell-free culture supernatant was added to the discs and allowed to diffuse into the agar, followed by incubation at 28°C for 2–4 d, when the diameter of the inhibition zone was measured.
Effects of pH and temperature on antimicrobial activity
For the pH stability test, the pH of the cell-free supernatant of the antagonistic strain was adjusted to pH 1–14, and readjusted to neutral after 20 h before being applied to the filter discs. The activity against S. scabiei was tested by agar diffusion assay. To analyse the thermal stability, samples of the cultured broth were exposed to 40, 60, 80 and 100°C for 30 min, and at 121°C for 15 min. The remaining activity was tested against S. scabiei by disc diffusion assay.
Isolation of the antimicrobial compound by HPLC and structural analysis
The Bacillus strain showing the strongest bioactivity against S. scabiei was used to isolate the antimicrobial compound. The inoculum for the production of antimicrobial compound was prepared in 3 ml TSB broth at 37°C for 24 h in a rotary shaker (150 rev min−1). GYM was used as production medium with 0·1% inoculation size. A total of 15 l of culture broth was obtained. After centrifugation at 8000 g for 30 min at 4°C, the supernatant was extracted two times with the same volume of ethyl acetate and was dried in vacuo. The crude extract was separated into nine fractions by silica gel RP-18 flash chromatography, with H2O–MeOH (50 : 50), H2O–MeOH (40 : 60), H2O–MeOH (30 : 70), H2O–MeOH (20 : 80), H2O–MeOH (10 : 90), MeOH (100%), acetone, ethyl acetate and hexane for the collection of different fractions. The two most active fractions were further purified by preparative HPLC using a SymmetryprepTM C18 column (Waters, Milford, MA, USA), and were monitored by a refractive index detector. Elution was performed with 60% aqueous acetonitrile, maintaining a flow rate of 1·5 ml min−1. Individual detected fractions were collected and evaporated under reduced pressure. Paper discs containing 0·5 mg of each individual fraction were loaded onto the GYM plates that had been spread with S. scabiei. Structural analysis of the active compounds against S. scabiei was performed using NMR and mass spectrometer. The purified compounds were dissolved in d-methanol. NMR spectra were recorded on a Bruker Avance DPX 600 MHz NMR spectrometer (for 1H-NMR spectrum; Bruker, Karlsruhe, Germany) and then by a Bruker Avance DPX 300 MHz NMR spectrometer (for 13C-NMR spectrum). Chemical shifts are given in ppm, using tetramethylsilane (TMS) as an internal standard. FAB-MS spectra were measured on a JMS-AX 505 WA spectrometer (JEOL, Tokyo, Japan).
Scanning electron microscope analysis
Paper discs containing 0·5 mg of major compound and 0·5 mg of minor compound were loaded on the GYM plates that had been inoculated with S. scabiei. After incubation at 28°C for 7 d, the samples for scanning electron microscope (SEM) analysis were isolated from the inhibition zone. Primary fixation was performed by soaking the samples in paraformaldehyde and 2% glutaraldehyde in a 0·05-mol l−1 sodium cacodylate buffer (pH 7·2) at 4°C. For postfixation, the primarily fixed samples were soaked in 1% osmium tetroxide in a 0·05-mol l−1 sodium cacodylate buffer (pH 7·2) for 2 h at 4°C and were thoroughly washed with distilled water at room temperature. The washed samples were dehydrated in a series of ethanol concentrations (30, 50, 70, 80, 90, 100, 100 and 100%) and were mounted on metal stubs for gold coating, followed by treatment with 100% hexamethyldisilazane for 15 min before observation. The samples were observed under a JSM 5410 LV Scanning Electron Microscope (5000×) (JEOL).
To test whether the isolated strain would have a suppressing effect against scab disease in a potato field, a pot assay was carried out. Experiments were performed in pots filled with sterilized soil. S. scabiei was grown in oat meal broth (Loria et al. 1995) at 28°C for 7 d (107 CFU ml−1). A 200-ml of culture was centrifuged at 3000 g for 10 min. The mycelial pellets were washed three times with distilled water and were resuspended in 30 ml of sterile water in preparation for mixing into the soil of each pot.
Solanum tuberosum tubers were washed with sterile distilled water, planted in sterile soil, and grown in a greenhouse at 28°C. Potato seedlings up to 12·5 cm in size were selected and transferred to infested soil pots. The seedlings were then placed in a growth chamber in which the temperature was 15°C at night and 20°C during the day. After 2 weeks, culture broth of the antagonistic strain (108 CFU ml−1) in 200 ml GYM broth was mixed into the S. scabiei-infested soil. At the same time, an identical quantity of S. scabiei was also inoculated into the soil to maximize the chances of infection. Again, after 2 weeks, the same quantity of the antagonistic strain was inoculated into the pot soil. Positive control treatments were also performed by using S. scabiei as an inoculum, and noninfested soil pots were prepared as untreated controls. Potatoes were harvested after 3 months and the infection rate was calculated as the percentage of infected potatoes with lesions over 0·5 cm in diameter. This experiment was repeated twice. The percentage was calculated according to the following formula:
Inhibitory spectrum of the antagonistic strain
All strains used as indicator organisms were provided by the Korean Agricultural Culture Collection. Agar diffusion assay was used to determine qualitatively the suppressive ability of the metabolites against various phytopathogens.
The phytopathogenic bacterial strains were previously cultured in appropriate growth liquid mediums and appropriate temperatures, with final OD600c. 1·0. Then 100-μl of the inoculum was added to 100 ml of each appropriate growth agar medium (Table 3) and, after being mixed well, was poured into five plastic plates. The zone of inhibition was measured after 24–48 h of incubation. The phytopathogenic fungal strains were cultured in potato dextrose agar (PDA) medium until they showed abundant sporulation. The spores were harvested in sterile 0·8% NaCl to yield a concentration of c. 106–107 spores ml−1. Then, 2 ml of inoculum was added to 100 ml of PDA medium. All the test plates were allowed to dry for 30 min before sterilized paper discs (10 mm in diameter) containing 100 μl of sterile solution of metabolites were placed on the agar surfaces. The diameter of inhibition zone around the discs was measured.
Table 3. Antagonistic activity of Bacillus sp. sunhua against phytopathogens*
Inhibition zone diameter (mm)†
–, Absence of inhibition; ±, activity zone was not clear.
*A 100-μl minor supernatant compound eluted in 30% aqueous MeOH were applied to discs.
†Diameter of the inhibition zone (mm) around the disc.
Pseudomonas syringae pv. syringae
Isolation of micro-organism antagonistic against S. scabiei
A total of 206 micro-organisms were screened from the soil samples; 18 of the isolates were selected on the basis of their activity against S. scabiei, among which the most active strain was further characterized. This strain was named sunhua and was identified as a Bacillus species by 16S rRNA sequence analysis.
pH and thermal stability
The antimicrobial activity of Bacillus sp. sunhua culture filtrates was found to be the highest at neutral pH, but was only moderately reduced when exposed to pH ranges from 5·0 to 13·0 (Fig. 1). Under acidic conditions in the pH range of 1·0–4·0, biocontrol activity was reduced significantly or completely eliminated. What is more important is that the activity was found to be more stable in alkaline pH than in acidic pH.
The supernatant of Bacillus sp. sunhua was exposed to various temperatures for 30 min and residual activity was measured (Table 1). Heat treatment of the broth showed the antimicrobial activity was stable at a temperature of 100°C for 30 min and antimicrobial activity remained above 50% when broth was treated at 80°C for 30 min. Antimicrobial activity against S. scabiei was found to be stable at 60°C with 82·3% of the original activity remaining.
Table 1. Inhibition of Streptomyces scabiei by heat-treated metabolites of Bacillus sp. sunhua
Relative remaining activity (%)
Supernatant of Bacillus sp. sunhua was exposed to various temperatures for 30 min and residual activities against S. scabiei were expressed as a percentage of original activity.
*Autoclaved at 121°C for 15 min.
Inhibition of S. scabiei sporulation by the supernatant fluid of Bacillus sp. sunhua
When S. scabiei was cultured in the presence of Bacillus sp. sunhua culture filtrate, inhibition of mycelium growth was observed around the paper disc, and inhibition of sporulation was observed around the mycelium inhibition zone. The diameter of the sporulation inhibition zone was much larger than that of the mycelium inhibition zone (Fig. 2). This indicated that there were at least two antibiotic activities in the cultured filtrate of Bacillus sp. sunhua.
Isolation and characterization of the active compounds
Two active compounds against S. scabiei were isolated from the ethyl acetate extraction by reversed phase flash chromatography and preparative HPLC (Fig. 3). Of the nine fractions obtained from the reversed phase flash chromatography, the 30% aqueous MeOH fraction and the 20% aqueous MeOH fraction showed the strongest activity against S. scabiei. The structures of these active compounds were analysed by 1H-NMR, 13C-NMR, HMBC, HMQC, NOESY and mass spectrometer. The mass spectrum of the major compound in the 20% aqueous MeOH fraction showed a molecular ion peak that confirmed it to be iturin A at m/z 1043 (Peypoux et al. 1978). The minor compound in the 30% aqueous MeOH fraction was identified as macrolactin A, with a formula of C24H35O5 (Gustafson et al. 1989). This compound was confirmed by the presence of 24 signals in 13C-NMR spectrum composed of the following: a methyl carbon, six methylene carbons, four oxygenated methine carbons, 12 olefinic methine carbons, and an ester carbonyl carbon. 1H-1H, 13C-1H connectivity were elucidated by 1H-1H COSY, HMQC, HMBC and DEPT spectra. The geometry of the six olefins was determined by NOESY spectra together with their coupling constants. The planar structure and all 13C-NMR assignments of the compound are identical to macrolactin A (Table 2).
Table 2. Comparison of 1H- and 13C-NMR data of a compound isolated from Bacillus sp. sunhua with those of macrolactin A
δH (J, Hz)
δH (J, Hz)
†C6D6 for 1H-NMR and CD3OD for 13C-NMR.
5·68 (d, 11·2)
6·54 (t, 11·5)
6·29 (dd, 11·2, 11·5)
7·12 (dd, 11·5, 14·8)
7·48 (dd, 11·5, 14·9)
5·85 (ddd, 7·2, 7·2, 14·9)
5·66 (dd, 5·8, 15·1)
5·58 (dd, 4·7, 15·0)
6·47 (dd, H·l, 15·1)
6·73 (dd, 11·5, 15·0)
6·04 (dd, 11·2, 11·5)
6·35 (dd, 10·6, 15·3)
5·97 (dd, 10·5, 15·0)
6·08 (dd, 10·6, 14·8)
1·15 (d, 6·3)
1·09 (d, 6·5)
Observation of antimicrobial activity of the purified compounds by bioassay and SEM
Bioassay and SEM analysis also showed that the presence of active compounds damaged both the mycelium and the sporulation of S. scabiei. Normal mycelium and spore-formation could be observed in the absence of any antibiotics (Fig. 4a), while iturin A was observed to inhibit the sporulation of S. scabiei; sporulation did not occur even after a prolonged incubation for 2 weeks (Fig. 4b,d). Treatment with macrolactin A led to inhibition of sporulation as well as to the disruption of S. scabiei mycelium (Fig. 4c,e).
In order to investigate the possibility of using Bacillus sp. sunhua to suppress S. scabiei in soil, a pot assay experiment was performed. The infection rate decreased to 35% after treatment with Bacillus sp. sunhua compared with a 75% infection rate for potatoes in the pot treated alone with S. scabiei (positive control) (Fig. 5b). Furthermore, the lesions on potatoes treated with Bacillus sp. sunhua were smaller and less serious than those of the potatoes that had been treated with S. scabiei alone (Fig. 4a).
Inhibitory spectrum of Bacillus sp. sunhua against other phytopathogens
The cell-free culture supernatant of Bacillus sp. sunhua was tested against other phytopathogens, as shown in Table 3. S. scabiei was found to be the most sensitive. The supernatant showed moderate activity against Fusarium oxysporum, the phytopathogen causing potato dry rot disease, and slight antagonistic activity toward Pseudomonas syringae pv. syringae, Alternaria mali, and Penicillium digitatum, which are responsible for angular leaf spot disease, Alternaria leaf spot, and blue and green citrus mould respectively. It should be pointed out that the supernatant of Bacillus sp. sunhua showed no inhibitory activity towards beneficial bacteria, such as Rhizobium meliloti, a symbiotic nitrogen-fixing micro-organism, or Pseudomonas fluorescens, which can produce a toxic insecticidal compound.
For plant disease control, chemical agents are now widely applied. However, the use of chemicals leads to environmental pollution and ecological destruction. In order to overcome these problems, soil micro-organisms are being extensively studied as potential biocontrol agents (Hoitink and Boehm 1999; Raaijmakers et al. 2002). Many antibiotics produced by antagonistic strains have the advantage of being easily decomposed and leave no harmful residues (Cook 1993). Until recently, common scab disease has been controlled by the chemical treatment of potato seeds, irrigation, and soil amendment. Eckwall et al. controlled scab disease by using S. diastatochromogenes PonSSII, which demonstrated two biocontrol mechanisms, antibiosis and competition, but the structure of the antibiotics was not characterized (Eckwall and Schottel 1997; Neeno-Eckwall et al. 2001). Until now, there has been no report of using a Bacillus species as a suppressive strain to control common scab disease; here we report the use of Bacillus sp. sunhua as a biocontrol agent and analyse the structure of the antibiotics produced by Bacillus sp. sunhua for the first time.
Bacillus species have been used as biocontrol agents against pathogenic fungi, by producing lipopeptide antibiotics (Fravel 1988; Zuber et al. 1993; Kim and Kim 1994; Potera 1994; Leifert et al. 1995). Iturin A is one of them (Eshita and Roberto 1995; Moyne et al. 2001; Hiradate et al. 2002; Cho et al. 2003). In our study, we found that the major compound iturin A produced by Bacillus sp. sunhua can inhibit the sporulation of S. scabiei. A minor compound produced by Bacillus sp. sunhua was macrolactin A, which was first isolated from a marine bacterium (Gustafson et al. 1989). It is known to possess antibacterial, cytotoxic and antiviral activity (Gustafson et al. 1989) and can be used as a neuronal cell-protecting substance (Kim et al. 1997). However, until now, there had been no report that macrolactin A could be used as a biocontrol agent. Here we have shown that macrolactin A can inhibit the sporulation and mycelium formation of S. scabiei, and we observed the disruption of mycelia. Polyene macrolides combines with ergosterol and damages the cell membrane which then results in loss of cell integrity (Strohl 1997). Our results imply that the mechanism of macrolactin A is identical to the action of macrolide antibiotics. This antibiotic mechanism is different from that of lysozyme produced by the Trichoderma and Pseudomonas species (Wells 1988; Lim et al. 1991).
Metabolites produced by Bacillus sp. sunhua also showed antifungal activity against Fusarium oxysporum, the causal organism of dry rot disease. We suppose that this effect was caused by the antifungal compound iturin A. Therefore, Bacillus sp. sunhua is effective not only against the growth of S. scabiei, but also against other potato phytopathogens. We suggest that it has potential for controlling potato diseases.
In the pot assay, the rate of scab disease was reduced by 40% after treatment with Bacillus sp. sunhua. Most importantly, the antibiotics produced by Bacillus sp. sunhua are more stable at alkaline pH, making it suitable for the treatment of scab disease which is more serious in alkaline soil. In addition, compared with other bacterial species, Bacillus species produce various biologically active metabolites, and offer the further advantage of forming endospores that are resistant to heat, desiccation, organic solvents and UV irradiation. Therefore, Bacillus sp. sunhua has great potential as a biological control agent for the control of common scab disease.
In summary, we present the first report of the compound macrolactin A having inhibitory action towards potato scab disease. To our knowledge, this is the first report of a Bacillus species biocontrol strain that may function effectively to control scab disease. Additionally, our studies suggest that Bacillus sp. sunhua is a good candidate for controlling potato disease in the field. To test this possibility, we are now in the process of using this strain as a biocontrol agent in a large potato planting area, in collaboration with Korea's Rural Development Administration.
This study was supported by the Technology Development Program for Agriculture and Forestry, Ministry of Agriculture and Forestry, Republic of Korea. This study was also partially supported by the Korean National Research Resource Center (KNRRC) Project to ECUM (Extract Collection of Useful Microorganism) from the Korean Ministry of Science and Technology.