Characterizing a novel strain of Bacillus amyloliquefaciens BAC03 for potential biological control application

Authors


Correspondence

Jianjun Hao, Michigan State University, 62 Plant Biology Bldg, East Lansing, MI 48824, USA. E-mail: jjhao@msu.edu

Abstract

Aims

To identify and characterize a bacterial strain BAC03, evaluate its biological control activity against potato common scab (Streptomyces spp.) and characterize an antimicrobial substance produced by BAC03.

Methods and Results

Bacterial strain BAC03, isolated from potato common scab suppressive soil, was identified as Bacillus amyloliquefaciens by analysing sequences of fragments of the recA, recN, cheA and gyrA genes. BAC03 displayed an antagonistic activity against Streptomyces spp. on agar plates using a co-culture method. In glasshouse assays, BAC03 applied in potting mix significantly reduced common scab severity (< 0·05) and potentially increased the growth of potato plants (< 0·05). An antimicrobial substance extracted from BAC03 by ammonium sulfate precipitation was identified as an LCI protein using liquid chromatography–mass spectrometry. The antimicrobial activity of either a BAC03 liquid culture or the ammonium sulfate precipitate fraction was stable under a wide range of temperatures, and pH levels, as well as following incubation with several chemicals, but was reduced by all proteinases tested.

Conclusions

Bacillus amyloliquefaciens strain BAC03 displayed a strong antimicrobial activity, that is, the suppression of potato common scab, and may potentially enhance the plant growth. LCI protein is associated with some of the antimicrobial activity.

Significance and Impact of the Study

Bacterial strain BAC03 has the potential to be developed as a commercial biological control agent for potato common scab.

Introduction

Biological control has the potential to play an important role in managing plant diseases (Liu et al. 1995; Guetsky et al. 2002; Singhai et al. 2011). The efficacy of most reported biological control agents varies by strain; therefore, more effective strains of beneficial micro-organisms are always of interest (Lugtenberg and Kamilova 2009). Biological control agents have shown strong activities for the inhibition of plant pathogens (Compant et al. 2005; Haas and Defago 2005), promotion of plant growth (Lugtenberg and Kamilova 2009), competitive colonization (Fan et al. 2011) and induction of plant defence systems against pathogens (Kloepper et al. 2004). Some micro-organisms have at least one of these characteristics, but most studied Bacillus spp. possess multiple modes of action (Kloepper et al. 2004; Ongena and Jacques 2008; Arguelles-Arias et al. 2009). In addition, the formation of endospores makes the Bacillus genus highly resistant to adverse environmental conditions (Abriouel et al. 2011), which favours the commercialization of biological control products in transportation and shelf life.

Antimicrobial activity is the most easily observed sign of antagonism in biological control agents and has been extensively studied (Naclerio et al. 1993; Yoshida et al. 2001; Arguelles-Arias et al. 2009; Hu et al. 2010). Various antimicrobial substances produced by Bacillus spp. have been identified, such as iturins, fengycins and surfactines, which belong to nonribosomally synthesized peptides. In addition to these metabolites, ribosomally synthesized antimicrobial compounds were also detected (Stein 2005).

Potato common scab (Streptomyces spp.) is an important disease worldwide and can cause significant reduction in the economic value of potatoes (Loria et al. 2006; Hao et al. 2009). The pathogens produce a phytotoxin, thaxtomin A, which is the only known pathogenicity determinant and induces disease symptoms (Loria et al. 2008). The disease is difficult to manage as the pathogens survive saprophytically in and on plant debris or organic matter in soil and have a ubiquitous distribution (Loria et al. 2006). Many approaches have been used to manage common scab, including resistance cultivar (Driscoll et al. 2009), crop rotation (Larkin 2008), using green manures or organic amendments (Larkin and Griffin 2007) and chemical treatments (Tegg et al. 2012). However, resistant germplasm is very limited, and the rest of the practices are partially effective. Hence, additional methods for the control of common scab are needed.

Biological control using micro-organisms can be a potential strategy for sustainable common scab management. The most commonly studied bacterial genera for the control of potato common scab include Pseudomonas spp. (Singhai et al. 2011; St-Onge et al. 2011) and Streptomyces spp. (Liu et al. 1995; Eckwall and Schottel 1997; Beausejour et al. 2003; Hiltunen et al. 2009). Pseudomonas sp. LBUM 223 has been shown to control potato common scab at least in part, because the Pseudomonas produces the antibiotic compound phenazine-1-carboxylic acid (PCA) that inhibits the growth of plant pathogens and represses the gene expression for thaxtomin A (St-Onge et al. 2011). Pseudomonas mosselii promotes potato growth and also induces systemic disease resistance in the plant (Singhai et al. 2011). Streptomyces melanosporofaciens strain EF-76 produces geldanamycin, which reduces the severity of potato common scab under both controlled experiments and field conditions (Beausejour et al. 2003). Streptomyces diastatochromogenes strain PonSSII does not affect the growth of most organisms tested, but is inhibitory against Streptomyces scabies (Eckwall and Schottel 1997).

Some studies have been reported using Bacillus spp. to manage potato common scab, including Bacillus sp. sunhua (Han et al. 2005) and B. subtilus (Schmiedeknecht et al. 1998). As combined organisms with different modes of action have been shown to enhance biological control activity (Guetsky et al. 2002), adding a robust and effective biological control agent to the available options provides greatly enhanced opportunities for disease management. Unfortunately, the bacteria mentioned in the above-mentioned studies are still at research stage; therefore, additional studies are needed.

A bacterial strain BAC03 belonging to the genus Bacillus was isolated from a potato field that has shown suppressiveness to potato common scab in Michigan (Meng et al. 2012). It inhibits a broad spectrum of micro-organisms in vitro, including Streptomyces spp. that cause potato common scab (Meng et al. 2011). We were interested in the biological characteristics and the potential application of this strain for disease control, especially for potato common scab management. The objectives of this study were to identify strain BAC03 of bacterial species, evaluate its antimicrobial activity, determine one or more antimicrobial substances produced by this strain and examine the potential for common scab management.

Materials and methods

Micro-organisms and identification of BAC03

A bacterial strain, designated BAC03, was isolated from the soil of a Michigan potato field that has been shown to be suppressive to potato common scab (Meng et al. 2012). Bacillus amyloliquefaciens FZB42 was obtained from the Bacillus Genetic Stock Center (Columbus, OH, USA). All other micro-organisms used in this study and their sources are listed in Table 1.

Table 1. The inhibitory activity of Bacillus amyloliquefaciens strain BAC03 against Streptomyces spp.
PathogenSource (isolated from)aInhibition zone diameter (mm)b
  1. a

    ATCC: American Type Culture Collection (http://www.atcc.org/), Manassas, VA, USA. NRRL: USDA-ARS Agriculture Collection (http://nrrl.ncaur.usda.gov/), Peoria, IL, USA. Yeast malt extract was used for the bacterial growth.

  2. b

    The inhibitory activity was tested on yeast malt extract medium, with 15 μl of BAC03 suspension (107 CFU ml−1) applied on a sterile filter paper disc. Each mean value is the average of 8 measurements.

  3. c

    Control was set with a filter paper dropped with TSB media only.

Streptomyces scabies 49173ATCC (tuber)25·3 ± 1·8
Streptomyces scabies 1231NRRL (tuber)10·8 ± 0·8
Streptomyces acidiscabies 49003ATCC (tuber) 8·6 ± 0·6
Streptomyces stelliscabiei Her21Hao lab (tuber)14·5 ± 1·2
Streptomyces aureofaciens 5404NRRL (soil) 7·8 ± 1·0
Streptomyces sp. DS3024Hao lab (soil)11·6 ± 1·1
Controlc 0

In order to identify BAC03, sequence analysis was conducted using selected genes. BAC03 cultured in tryptic soy broth (TSB; EMB Chemical Inc., Gibbstown, NJ, USA) for 24 h was used for DNA extraction. The FastDNA Spin kit (MP Biomedicals, Solon, OH, USA) was used according to the manufacturer's instructions. Polymerase chain reaction (PCR) was conducted to amplify the fragments of the 16S rDNA, recA and recN (DNA repair and recombination proteins), cheA (a histidine kinase) and gyrA (gyrase subunit A) genes with primers listed in Table 2. Primers for genes cheA and gyrA were designed based on the consensus sequence of these two genes retrieved from several B. amyloliquefaciens strains in National Center for Biotechnology Information (NCBI) database, and validated with dnaman software (Lynnon Corporation, Quebec, Canada). Each PCR mixture had a total volume of 25 μl, containing 5 U of Taq DNA polymerase, 1× Taq polymerase PCR buffer (Promega, Madison, WI, USA), 200 mmol l−1 dNTP mixture, 0·2 mmol l−1 of each primer and 1 μl (2–25 ng) of template DNA. Thermocycler (Bio-Rad Scientific Inc., Hercules, CA, USA) settings were as follows: an initial denaturation for 5 min at 94°C, followed by 36 cycles of 40 s at 94°C, 40 s at 58°C and 1·5 min at 72°C, and extension for 7 min at 72°C. After PCR product visualization by electrophoresis on 1·2% (w/v) agarose gel stained with GelGreen (Biotium Inc., Hayward, CA, USA), the PCR products were purified using a PCR purification kit (Denville Scientific Inc., Metuchen, NJ, USA) and sequenced at the Michigan State University Genomic Technology Support Facility (East Lansing, MI, USA). The sequences were analysed using the Blast algorithm against the GenBank database. This experiment was conducted twice and with three replicates each time.

Table 2. Primers for polymerase chain reactions used in this study
GenePrimerSequence (5′-3′)References
16S rDNA63-FCAGGCCTAACACATGCAAGTCDennis et al. (2003)
1387-RGGGCGGWGTGTACAAGGC
recA recA-FTGAGTGATCGTCAGGCAGCCTTAGArguelles-Arias et al. (2009)
recA-RTTCTTCATAAGAATACCACGAACCGC
recN recN-FCTTTTGCGATCAGAAGGTGCGTATCCGArguelles-Arias et al. (2009)
recN-RGCCATTATAGAGGAACTGACG ATTTC
cheA cheA-FGTTTGAAGCGCTTGATCATCTAGAAThis work
cheA-RGGTTCATTGAAGAATCCAGTCTTTC
gyrA gyrA-FCGTACGCAATGAATGATTTAGGCATGThis work
gyrA-RATTATGAAAGACGTCTGCAGGGCCGTT
lci lci-FCGCGGATCCATGAAATTCAAAAAAGHu et al. (2010)
lci-RGCGTCGACTTATTTATCTACACTTTCAT

Antimicrobial activity of BAC03 against Streptomyces spp.

Antimicrobial activity of BAC03 was determined against Streptomyces spp. using a co-plate assay (Yoshida et al. 2001). One hundred microlitres of spore suspensions (105 CFU ml−1, determined by dilution plating) of various Streptomyces spp. isolates was deposited across the plate with a sterile cell spreader on yeast malt extract agar (YME; EMB Chemical Inc.) plates. Fifteen microlitres of a 107 CFU ml−1 BAC03, cultured for 24 h in TSB, was placed as a drop on a sterile filter paper disc (5 mm diameter, Whatman no. 1, Piscataway, NJ, USA), which was placed on the agar medium with two discs per plate, at 2 cm distance from each other. After the plates were incubated at 28°C in darkness for 3 days, the diameter of the inhibition zone (if any) was measured with a ruler. Analysis of each isolate was replicated four times, and this trial was conducted twice.

Biological control activity assay

A pot assay was conducted in a glasshouse to examine the biological control activity of BAC03. Streptomyces scabies (ATCC49173) was cultured in oatmeal broth (Loria et al. 1995) at 28°C for 4–5 days in an incubator shaker at 180 rev min−1, and the concentration of Streptomyces in the liquid culture was determined by plating on YME. Potato tuber pieces (‘Snowden’) with at least one eye were surface-disinfested with 1% NaClO for 5 min and then rinsed with sterile distilled water three times. After air drying, the tubers were planted in potting mix (ASB Greenworld Inc., New Brunswick, VA, USA) and grown in a growth chamber at 25°C until seedling emergence. Each potato seedling was transferred to a 3·78-l plastic pot with potting mix and infested with Strep. scabies by pouring the inoculum on top of the potting mix at a final concentration of 106 colony forming units (CFU) cm−3. For BAC03 application in soil, a liquid culture of BAC03 was added to the potting mix as a drench to give a final concentration of 105 or 106 CFU cm−3. Treatment with BAC03 application was carried out twice; once when seedlings were transplanted, a second drench was carried out 20 days later. The treatments included (i) a positive control with Strep. scabies inoculum plus 200 ml of TSB; (ii) a negative control of noninfested potting mix mixed with 200 ml TSB; (iii) potting mix inoculated with BAC03 at 105 CFU cm−3 and infested by Strep. scabies; and (iv) potting mix inoculated with BAC03 at 106 CFU cm−3 and infested by Strep. scabies. Plants were watered every 2–3 days, and fertilizer was applied as needed. There were four replications (pots) for each treatment. Six weeks after transplanting, the height of the plant from the soil line to the apical meristem of potato was measured with a ruler. Potato tubers were harvested 10 weeks after transplanting. Tubers were examined for lesions and given a severity rating using the 0–5 scale of Hao et al. (2009), where 0 = no symptoms, 1 = 1–10% surface area with superficial or raised lesions, 2 = 11–25% surface area with superficial or raised lesions, 3 = 26–50% surface area with superficial or raised lesions, 4 =  more than 50% surface area with superficial or raised lesions or 6–25% pitted lesion area and 5 = >50% surface area with superficial or raised lesions or >25% pitted area. Potato yield was determined by measuring the weight of all potato tubers from each pot. This trial was conducted twice.

Isolation and characterization of potential antimicrobial substances

Precipitation

Two extraction methods were used to separate different types of secondary metabolites. For all extractions, BAC03 was grown in 200 ml of TSB at 28°C on a shaker incubator at 180 rpm for 48 h. The culture was centrifuged at 12 000 g for 20 min at 4°C, and the supernatant was collected.

For potential proteinaceous secondary metabolites (ribosomally synthesized metabolites), BAC03 culture supernatant was extracted using ammonium sulfate precipitation as described by Sutyak et al. (2008) with a minor modification as follows. Eighty per cent ammonium sulphate was added to the supernatant and incubated at 4°C overnight. The mixture was centrifuged at 12 000 g for 30 min at 4°C, and the supernatant was discarded. The precipitate was dissolved in 10 mmol l−1 sodium phosphate buffer (pH 6·0) and applied to a Sephadex G-50 column (Pure Biotech LLC, Middlesex, NJ, USA) and centrifuged at 2000 g for 3 min. The eluted fluid was passed through a 0·22-μm filter membrane (Millipore, Billerica, MA, USA), and the antimicrobial activity of this substance against Strep. scabies was tested by the agar diffusion assay as described above. This extract was designated as ammonium sulfate–precipitated material (ASP) for the rest of the study.

Lipopeptides (nonribosomally synthesized peptide) were extracted by the combination of acid precipitation and solvent extraction described by Vater et al. (2002) with slight modifications. The pH of the culture filtrate was adjusted to 2·0 by adding 6 N HCl followed by precipitation at 4°C overnight. The pellet derived from centrifugation (12 000 g for 30 min at 4°C) following precipitation was dissolved in 100% methanol. The mixture was passed through a 0·22-μm filter membrane (Millipore). The antimicrobial activity of the extract against Strep. scabies was tested using the agar diffusion assay as described above. This material was designated as acid-precipitated material (AP) for the rest of the study.

The concentration of protein in the ASP was determined by colorimetric absorbance at 595 nm using a Bradford Protein assay kit (Thermo Fisher Scientific Inc., Rockford, IL, USA, source of protein is bovine serum albumin) with a spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA) based on a curve using bovine serum albumin as a standard. All these experiments were conducted three times.

Antimicrobial activity of ASP against Streptomyces scabies

To test the quantitative effect of the ASP on Strep. scabies, a spore suspension of Strep. scabies (105 CFU ml−1) was mixed with BAC03 ASP at concentrations equivalent to 0, 15, 30, 45, 60, 75 and 90 μg of protein ml−1 as determined by the Bradford assay for one hour. Then, the mixture was serially diluted and spread on YME plates and incubated at 28°C in the dark. Any inhibitory effect was evaluated by counting Strep. scabies colonies on the plate after 3-day incubation in comparison with a control of plating the spore suspension alone.

To determine whether the extract was bactericidal or bacteriostatic, 105 CFU ml−1 of a Strep. scabies spore suspension was incubated with ASP at a final concentration of 30, 60 and 90 μg ml−1. After 0-, 1-, 3- and 5-day incubation at 28°C in the dark, the mixture was filtered through a sterile 0·22-μm membrane, and the Strep. scabies collected from the filter surface was diluted followed by spreading on YME to determine the concentration of viable Strep. scabies. Three replications were set for each treatment, and these two trials were conducted twice.

Effects of temperature, pH, enzymes and chemical solvents on antimicrobial activity of liquid culture and ASP of BAC03

The antimicrobial activity of both BAC03 liquid culture and its derived ASP was examined for their reaction to various factors, including temperatures, pH levels, enzymes and chemicals. To test the effect of temperature, 1 ml of BAC03 spore solution or 100 μl of ASP was incubated in a 1·5-ml microtube, which was placed in a heating block (Denville Scientific, Inc., Metuchen, NJ, USA) at temperatures of 40, 60, 80 and 100°C for 30 min or in an autoclave at 121°C for 15 min. To test the effect of pH levels, the pH was adjusted from 1·0 to 14·0 by whole pH units using sterile 1 N HCl or 1 N NaOH. Material was incubated at these pH levels overnight at 4°C. Prior to assessing the activity, the pH was readjusted to pH 7·0. Several enzymes were tested for their effect on activity by incubating the BAC03 culture or ASP with 10 mg ml−1 of the following enzymes for 2 h at the optimal temperature for each enzyme according to the manufacturer's instruction. Enzymes were trypsin (MP Biomedicals, 25°C, pH 7·6), proteinase K (Sigma-Aldrich, Inc., St Louis, MO, 37°C, pH 7·5), pepsin (Sigma-Aldrich, 37°C, pH 2·0), α-chymotrypsin (Sigma-Aldrich, 25°C, pH 7·8) and catalase (MP Biomedicals, 25°C, pH 7·0). Effect of various chemical solvents (Table 3) was tested by incubating the substance for 5 h at 25°C with 10% (v/v) organic solvents. Antimicrobial activities of the treated culture and ASP were tested against Strep. scabies using the disc diffusion assay as described above. Enzyme or solvent only was tested at the same time as a negative control. Three replicates were set for each treatment, and all the experiments were conducted twice.

Table 3. Effects of temperature, pH level, enzyme treatment and chemical solvents on the inhibitory activity of Bacillus amyloliquefaciens against Streptomyces scabies
TreatmentInhibitory activity of BAC03 (% ±standard deviation)a
Liquid culturebASPc
  1. a

    Inhibitory activity was measured by an agar disc diffusion assay against the growth of Streptomyces scabies and calculated as [(diameter of inhibition zone on treated plate)/(diameter of inhibition zone of control)] ×100%. Each mean value is the average of six measurements.

  2. b

    Strain BAC03 was cultured in tryptic soy broth for 24 h (107 CFU ml−1) on a shaker at 180 rev min−1.

  3. c

    Crude antimicrobial substance was extracted by ammonium sulfate precipitation from the culture supernatant and filtered through a Sephadex G-50 column and 0·22-μm filter membrane.

Nontreated100100
Chemicals
Acetone100100
Ethyl acetate100100
Ethyl alcohol100100
Chloroform100100
Methyl alcohol100100
Enzymes
Proteinase K5 ± 30
Trypsin7 ± 20
Pepsin15 ± 40
Chymotrypsin8 ± 40
Catalase100100
Temperature (°C)/incubation time (min)
40/30100100
60/30100100
80/3089 ± 361 ± 4
100/3026 ± 610 ± 3
121/1500
pH
176 ± 355 ± 6
3100100
5100100
10100100
12100100
1400

Scanning electron microscope analysis

To examine the effect of ASP on Strep. scabies, scanning electron microscopy (SEM) was used to observe the morphology of mycelia on media. Ten microlitres of ASP was dropped onto a Strep. scabies 2-day-old colony. Sodium phosphate buffer was used as a control. The plates were incubated at 28°C in the dark. Five days later when the control colonies became white with mycelia, both cultures were coated with metallic osmium and processed according to the methods of Fan et al. (2011) by the Center for Advanced Mycology at Michigan State University and examined using a scanning electron microscope JEOL JSM 6400 (Japan Electron Optics Laboratories) at high voltage (10 kV). This trial was carried out twice.

Molecular mass and chemical structure of ASP

Ammonium sulfate–precipitated was fractionated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) (Scholz et al. 2011). Following electrophoresis, the gel was vertically cut into two parts. One part of the gel, containing the sample and molecular weight standards, was stained with Coomassie Blue protein stain. The other part, containing the same sample, was tested for antimicrobial activity using the method of Naclerio et al. (1993). Briefly, the gel was treated with 20% isopropanol–10 mmol l−1 Tris–HCl (pH 7·5) for 3 h, rinsed for 1·5 h in 10 mmol l−1 Tris–HCl (pH 7·5) and washed twice with distilled water for a total of 1 h. The gel was placed in a Petri dish with 1% water agar and overlaid with 0·8% YME mixed with a bacterial suspension of Strep. scabies (107 CFU ml−1). The plate was incubated at 28°C for 48 h, then stained with thiazolyl blue tetrazolium bromide (MTT; Sigma-Aldrich) to stain living organisms and observed for any unstained zone(s).

The active band associated with the antimicrobial activity, as mentioned earlier, was excised, eluted from the gel and digested according to the methods of Shevchenko et al. (1996). Liquid chromatography–mass spectrometry (LC/MS/MS) was conducted at the Michigan State University Proteomics Facility. The resulting MS/MS spectra were converted to peak lists using bioworks browser ver. 3·3·1 (Thermo Fisher Scientific Inc) and searched against all known gram-positive bacterial protein sequences downloaded from NCBI. The Mascot output was analysed using scaffold ver. 3·08 (Proteome Software Inc., Portland, OR) to probabilistically validate protein identifications using the ProteinProphet2 computer algorithm. Assignments validated above the Scaffold 95% confidence filter were considered valid. This experiment was conducted two times.

PCR detection

Based on the identified protein, the corresponding predicted nucleotide sequence was amplified using PCR with specific primers (Table 2) reported by Hu et al. (2010) and genomic DNA of BAC03 as the template. The DNA extraction, PCR amplification and sequence determination were conducted as described earlier. The sequence of the amplicon was compared to the NCBI GenBank database using the Blast algorithm.

Statistical analysis

Data were analysed using sas software (ver. 9·2, SAS Inc., Cary, NC, USA). Procedure GLM was used for the analysis of variance, and Fisher's least significance difference (LSD) multiple comparisons were performed for mean separation where anova showed differences. Procedure REG was used for linear regression.

Results

Identification of BAC03

The 16S rDNA sequence of BAC03 had a similarity of 99% with both B. subtilis (accession number: JN700079.1) and B. amyloliquefaciens (accession number: JQ245705.1). Fragment sequences of recA, recN, cheA and gyrA genes showed the highest similarity between strains BAC03 and B. amyloliquefaciens. Specifically, the similarity with B. amyloliquefaciens was 98% for recA (accession number: JN048426.1) and 99% for recN (accession number: CP000560.1), cheA (accession number: FN652798.1) and gyrA (accession number: AB612173.1).

Antimicrobial activity of BAC03 against Streptomyces spp.

BAC03 displayed antagonistic activities against Streptomyces spp. in plate tests. Inhibition zone diameters ranged between 0·78 and 2·53 cm (Table 1), with the largest inhibition zone (2·53 cm) against strain Strep. scabies 49173.

Biological control activity assay

In the glasshouse, the severity of potato common scab was significantly reduced (< 0·05) by B. amyloliquefaciens BAC03 at concentrations of both 105 and 106 CFU cm−3 soil (Fig. 1a). In addition, plant height and potato tuber weight were significantly increased (< 0·05, Fig. 1b) by BAC03 application at both concentrations compared to the controls. Enhanced growth was greater (< 0·05) at 106 than at 105 CFU cm−3 s soil (Fig. 1).

Figure 1.

Effect of Bacillus amyloliquefaciens BAC03 on the severity of common scab (a) and growth of potato (b). Potato tubers were planted in potting mix amended with Streptomyces scabies alone at a final concentration of 10colony forming unit (CFU) cm−3 (SS) or with BAC03 at 10CFU cm−3 (SS + Ba5), or with BAC03 at 10CFU cm−3 (SS + Ba6). Tryptic soy broth alone was used as a control (CK). Plant height was measured 6 weeks after transplanting. Severity of common scab was evaluated using a 0–5 scale (Hao et al. 2009), where 0 = no disease and 5 ≥ 50% surface area of the tuber covered by scabby lesions. Fresh tuber weight was measured at the end of the experiment. Mean values of responses in each treatment were compared using Fisher's LSD multiple range comparisons at α = 0·05. Values of the bars with the same letters were not significantly different. (image) Plant height and (image) Tuber weight.

Characterization of an antimicrobial substance

Based on the results of the agar diffusion assay, the AP fraction showed low activity (<0·5 cm) and was not examined further. The material from the ASP produced larger inhibition zones (>2 cm) than the material from AP produced, and thus, further study concentrated on this material. The ASP derived from the bacterium B. amyloliquefaciens BAC03 significantly reduced (< 0·05) the number of Strep. scabies colonies by the co-culturing method, and the effect was positively correlated with the ASP concentration (Fig. 2a). The inhibition was near 100% when the ASP concentration was 75 μg ml−1. In an assay for bactericidal effect, a reduction in the number of Strep. scabies colonies following the removal from the ASP was observed after co-incubation for different times, and the effect was enhanced with increasing exposure time (Fig. 2b).

Figure 2.

Antimicrobial activity of the ammonium sulfate precipitation fraction (ASP) from Bacillus amyloliquefaciens BAC03 tested against Streptomyces scabies liquid culture. (a) A spore suspension of Strep. scabies (105 CFU ml−1) was mixed with BAC03 ASP at concentrations equivalent to 0, 15, 30, 45, 60, 75 and 90 μg of protein ml−1 as determined by the Bradford assay. The mixture was spread on yeast malt extract plates and incubated at 28°C in the dark. Inhibition of Strep. scabies was calculated as [100 × (population in control-population in the treated)/population in control]. In the regression equation, y represents inhibition and x represents ASP concentration. (b) Bactericidal activity of BAC03 ASP. Streptomyces scabies was recovered after incubation with ASP for 0, 1, 3 and 5 days, followed by rinsing off ASP through a 0·22-μm membrane with sterile distilled water for three times. Correlation coefficients (R2) between ASP concentration (x axis) and Strep. scabies population (y axis) are shown for each day of evaluation, with < 0·05 for regressions of days 1, 3 and 5. Bars on each value show standard errors. (●) 0 days P = 0.68; (■) 1 day R= 0.89; (▲) 3 days R= 0.82 and (♦) 5 days R= 0.97.

The antimicrobial activity was not affected by catalase, or any of the organic solvents tested (Table 3), but activity was completely reduced by treatments with proteinase K, trypsin, pepsin or chymotrypsin, indicating that the antimicrobial substance was a protein. The protein was relatively heat stable and maintained the same level of antimicrobial activity when exposed to temperatures up to 60°C, but the activity was totally lost following autoclaving. The protein present in culture broth and ASP was also active over a wide pH range, but the effect was reduced in extremely acid (pH < 3) or alkaline (pH > 12) conditions (Table 3).

Scanning electron microscope analysis

When treated with ASP, the appearance of colonies of Strep. scabies remained consistent throughout the incubation period for observation, while the colonies of the nontreated culture turned white or whitish grey after 7 days of growth. The morphology of mycelia observed under the SEM displayed typical spiral spore chains (Schaad et al. 2001) on nontreated plates but not occurred on ASP-treated culture (Fig. 3). This indicated that normal hyphal development and sporulation could be inhibited.

Figure 3.

Scanning electron microscopy (SEM) of mycelia morphology of Streptomyces scabies grown on yeast malt extract medium plates. Ten microlitres of sodium phosphate buffer (untreated) or ammonium sulphate precipitation (ASP) (treated) was dropped onto a Strep. scabies 2-day-old colony. Five days later, the morphology of mycelia was observed under SEM. The cultures were processed following the method of Fan et al. (2011) and coated with metallic osmium in preparation for SEM.

Molecular mass and chemical structure determination 'of ASP and PCR testing

The ASP fractioned on SDS-PAGE displayed a clear band with a molecular mass of approximately 10 kDa after MTT staining, indicating no bacterial growth (Fig. 4). LC/MS/MS results indicated that the most probable compound from this fraction was an LCI protein. A PCR product was obtained using a pair of primers specific to an LCI gene (Hu et al. 2010). The sequence similarity of the amplified putative LCI was 99% between strains BAC03 and B. amyloliquefaciens FZB42 (accession number: CP000560.1) or C-31 (accession number: FJ904931.1). The DNA sequence and putative translated protein sequence were compared to those of reported B. amyloliquefaciens strains. All of the strains had a putative protein of 94 amino acids. The sequence of the LCI protein for BAC03 was 100% identical with strain C31 (Hu et al. 2010), but had one amino acid different from FZB42.

Figure 4.

Active fraction of ammonium sulfate precipitate (ASP) from Bacillus amyloliquefaciens BAC03 by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After fractionation on SDS-PAGE, the gel was overlaid with yeast malt extract medium containing Streptomyces scabies (107 CFU ml−1) and incubated at 28°C for 2 days. Inhibition zone was observed by staining the bacterial cells with thiazolyl blue tetrazolium bromide. The atomic mass of the active compound(s) was estimated by comparison to the position of the markers (in kDa).

Discussion

Bacillus amyloliquefaciens is a closely related species to B. subtilis (Arguelles-Arias et al. 2009). By analysing multiple gene fragments such as recA, recN, cheA, and gyrA used in this study, we were able to confirm the strain BAC03 as B. amyloliquefaciens. Bacillus amyloliquefaciens has been recognized as a good plant growth promoter and root colonizer (Fan et al. 2011). In addition, the genetic capacity for the synthesis of secondary metabolites in B. amyloliquefaciens (340 kb) is larger than that in B. subtilis (180 kb) (Chen et al. 2009). Several strains of B. subtilis have been commercialized for biological control (Joshi and McSpadden Gardener 2006). Based on results from this study, we are interested in developing BAC03 into a commercial product, similar to B. amyloliquefaciens FZB42 (ABiTEP GmbH, Berlin, Germany) (Chen et al. 2009).

The strain BAC03 displayed a strong antagonism against Streptomyces spp. as shown in this study, as well as against a range of plant pathogens (Q.X. Meng, unpublished data), which may indicate promising use against multiple plant diseases. The inhibitory effect on Streptomyces spp. was shown in vitro, and a significant reduction in the severity of potato common scab was found in this study. This promising result should be further investigated for the feasibility of field application of BAC03 at a large scale. In addition to disease suppression, a higher tuber weight and a plant height were observed in the presence of the pathogen with BAC03. However, in order to prove the capability of strain BAC03 in promoting the direct plant growth, a further study in the absence of pathogens needs to be conducted.

A large part of the in vitro antagonism of BAC03 was associated with a proteinaceous fraction showing high similarity to an LCI protein. A similar antimicrobial substance was first reported in B. subtilis strain A014, which had only 46 amino acid residues (Liu et al. 1990) compared with the 94 amino acid residues predicted for our BAC03. The one identified in the current study has been found in B. amyloliquefaciens C31 (Hu et al. 2010). These products, despite the size differences, have shown strong antimicrobial activities against various micro-organisms, which may be a factor for disease suppression in BAC03. Furthermore, this compound has a bactericidal effect, as shown by the reduction in colony numbers in the current study.

The compounds present in the culture broth of BAC03 were more tolerant to adverse conditions than extracted ASP from the bacterium. This may be because the living cells can protect the compounds from degradation and continue to produce the chemicals. It is also possible that other compounds are present in the culture broth. In the current study, the acid-precipitated fraction showed a small level of inhibition against Streptomyces spp. in assays on agar plates. It is possible that compounds would be active in antibiosis when the environmental conditions are changed. Bacillus spp. can have more than one antibiotic, either ribosomally or nonribosomally synthesized. For example, B. amyloliquefaciens FZB42 has two ribosomal proteins, plantazolicin (Scholz et al. 2011) and amylocyclicin (Helmann and Butcher 2006), which have shown strong antimicrobial activities. Besides ribosomally synthesized compounds, the nonribosomally synthesized metabolites have also shown inhibitory activity against micro-organisms (Chen et al. 2009) and have drawn more attention. Genetic study shows that 8·5% of the entire genomic capacity of B. amyloliquefaciens FZB42 is devoted to the nonribosomal synthesis of secondary metabolites, which includes polyketides (bacillaene, difficidin and macrolactin), lipopeptides (surfactin, fengycin and bacillomycin D) and siderophores (bacillibactin and the product of the nrs cluster) (Scholz et al. 2011). Therefore, in future, work is necessary to further investigate whether BAC03 has these or similar antibiotics or new antibiotics.

In conclusion, the results presented here indicated the potential to use BAC03 as a biological control agent for common scab management. LCI protein is an important factor for antimicrobial activities. However, further investigation is needed on modes of action, spectrum of effects on other pathogens, effects on plant growth promotion and the biological control efficacy of strain BAC03 in the field.

Acknowledgements

This project was partially supported by Project GREEEN of Michigan State University, USDA-NIFA-SCRI, and the Michigan Potato Industry Commission. Mention of trade names or commercial products in this article are solely for the purpose of providing specific information and do not imply recommendation or endorsement by the US Department of Agriculture or Michigan State University. We acknowledge the Michigan State University Proteomics Facility Center for protein sequence analysis and data interpretation.

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