Suppression of Tomato Bacterial Wilt Incited by Ralstonia pseudosolanacearum Using Polyketide Antibiotic-Producing Bacillus spp. Isolated from Rhizospheric Soil

: Bacillus spp. has the potential to control bacterial and fungal diseases of crops. In vitro study, Bacillus amyloliquefaciens DSBA-11 showed best to inhibit the growth of Ralstonia pseudosolanacearum as compared to Bacillus cereus JHTBS-7, B. pumilus MTCC-7092, B. subtilis DTBS-5 and B. licheniformis DTBL-6.Three primers


Introduction
Bacterial wilt caused by Ralstonia pseudosolanacearum (Formely Ralstonia solanacearum (Smith) Yabuuchi (1995) [1] is a serious disease of tomato (Solanum lycopersicum L.) in tropical, subtropical and temperature areas of the world. In India, the bacterial wilt disease has been reported mostly in coastal, hilly, as well as foothills areas, in India including in the state of Goa, Karnataka, Kerala, Maharashtra, Odisha, Jharkhand, and West Bengal, Himachal Pradesh, Jammu & Kashmir, Uttarakhand and north-eastern states [2,3]. The disease causes very heavy losses, varying from 2 to 90 % in different agro-climatic conditions in India [3]. Application of microbial antagonists is being emerged method to were studied against bacterial wilt pathogen R. pseudosolanacearum using dual culture technique as described by Singh et al. [6] under in vitro conditions. The bacteria were grown in nutrient sucrose broth medium at 28 ± 1 • C for 48 h and maintained bacterial inoculums of 0.1 OD at 600 nm by spectrophotometer (UV-VIS Spectrophotometer, Hitachi, U-2900). A 100 µL culture of R. pseudosolanacearum was spread onto the Petri plates containing nutrient sucrose agar medium to make a lawn of the bacteria on the medium with three replications. Three wells of 5 mm diameter in each Petri plate were made with the help of a sterilized cork borer and poured 40 µL of 48 h old culture of Bacillus species separately containing 0.1 OD (at 600 nm) of bacterial populations into each well. The inoculated plates were kept at 28 ± 1 • C for 48 h to form an inhibition zone. The inhibition zone formed by antagonistic bacteria was measured in diameter and converted into an inhibition zone area by using the formula πr 2 .

DNA Isolation from Bacteria
Soil samples were collected from the rhizosphere of tomatoes from Jharkhand, Odisha, West Bengal, Jammu and Kashmir, Meghalaya and Uttarakhand. The DNA of the soil was extracted by using PowerSoil DNA isolation Kits (MO BIO Laboratories, Inc.), Carlsbad, CA, USA. 0.25 g of soil sample was taken in PowerBead tubes and gently mixed, and 60 µL of solution C1 was added in each tube and vortex briefly as instruction described by the manufacturer. The extracted DNA was quantified by nanodrop for further use in PCR for the detection of bacteria-producing polyketides antibiotics. The genomic DNA of bacteria was extracted by the method described by Murry and Thompson (1980) [29].

Primer Designing
The three sets of primers were designed for polyketide antibiotic synthase genes viz; macrolactin, (mln-F and mln-R) difficidin (dfn-F and dfn-R) and bacillaene (bae-F and bae-R) from the full genome sequence of Bacillus amyloliquefaciens FZB-42 (Accession No. AJ634062.2) obtained from NCBI database by using Primer 3 program (www.frodo.wi. nit.edu (accessed on 12 October 2015) having product size of 792 bp, 705 bp and 616 bp respectively ( Table 1). The specificity of these primers was checked by in-silico analysis using the website (www.insilico.ehu.es; accessed on 27 October 2015). The developed primers were validated for their universality cross B. amyloliquefaciens and other related groups of bacteria by primer blasting on the NCBI website (www.ncbi.nlm.nih.gov;accessed on 3 November 2015).

Standardization of Protocol for Multiplex-PCR
A PCR protocol was standardized for the simultaneous detection of polyketide antibiotic viz; difficidin, macrolactin and bacillaene-producing strains of Bacillus spp. were used to amplify from obtained template in a reaction mixture of 5 X taq buffers, 1.5 U taq polymerase (Promega), 10 mM dNTPs, 25 mM MgCl 2 , 1 µL of each forward and reverse primers (10 µM),1 µL of DNA (100 ng) and sterile doubled distilled water in a final volume of 25 µL. The thermocycling conditions were slightly modified and consisted of one initial denaturation step of 95 • C for 5 min, followed by 32 amplification cycles of 95 • C for 30 s, 52 • C for 45 s and 72 • C for 1 min and a final extension step of 72 • C for 8 min using a BIO-RAD C1000 thermocycler. The PCR products were resolved by using a 1.5% agarose gel stained with ethidium bromide and photographed using the gel documentation system (BIO-RAD, GEL DOCTM XRþ). The detection threshold of multiplex-PCR primer sets of polyketide genes was determined by diluting up to 10 −3 of genomic DNA (100 ng) of B. amyloliquefaciens DSBA-11. 1.0 µL of aliquots was used as DNA template. The specificity of these primer sets was tested by using DNA templates of B. subtilis DTBS-5, B. licheniformis DTBL-6, B. pumilus MTCC-7092, B. cereus JHTBS-7, Pseudomonas fluorescens DTPF-3, R. pseudosolanacearum UTT-25, and Xanthomonas campestris pv. campestris along with B. amyloliquefaciens DSBA-11 for multiplex PCR as described earlier.

Detection of Polyketides Antibiotics Producing Strains from Soil
For the detection of polyketides, antibiotics genes from extracted soil DNA was used for 25 µL of the reaction mixture of 5X taq buffers, 1.5 U taq polymerase (Promega), 10 mM dNTPs, 25 mM MgCl 2 , 1 µL of each forward and reverse primers (10 µM) 1 µL of DNA (100 ng) and sterile doubled distilled water in a final volume of 25 µL. The thermocycling conditions were slightly modified and consisted of initial one denaturation step of 95 • C for 5 min, followed by 32 amplification cycles of 95 • C for 30 s, 52 • C for 0.45 s and 72 • C for 1 min and a final extension step of 72 • C for 8 min using a BIO-RAD C1000 thermocycler. The PCR products were resolved by using a 1.5% agarose gel stained with ethidium bromide and photographed using the gel documentation system (BIO-RAD, GEL DOCTM XRþ).

Molecular Characteization of Polyketides Genes in B. amyloliquefaciens DSBA-11
A study on the role of bacillaene, macrolactin and difficidin polyketides synthase genes produced by B. amyloliquefaciens (DSBA-11) was done for suppression of growth of R. pseudosolanacearum. Cloning was done by using T & A cloning vector (2.7 kb) with a competent cell of E. coli strain DH5α by following standard procedure as described by Sambrook et al. [30]. PCR products of these genes were purified by using RBC PCR purified kit. Transformation from ligation reaction, 10 µL of the reaction mixture was added to pre-aliquot competent cells (50 µL) and the tubes 'contents were gently mixed and placed on ice for 30 min. The cells were heat shocked in a dry water bath at 42 • C for 2 min. The tubes were transferred immediately on ice for 2 min. 1.0 mL of LB (without antibiotics) was added and the tubes were incubated for 1.5-2.0 h at 37 • C in the shaker (200 rpm). The pellets were resuspended in a suspension 100 µL of fresh LB broth and the suspension was spread at 1X gal transformation plate with a selective medium 100 mL of Luria agar medium containing 100 µL of 50 mg/mL ampicillin, 20 µL of IPTG (200 µg/mL) and 200 µL of X-gal (20 µg/mL). The plates were incubated at 37 • C for overnight and blue and white colonies appeared in the plate, which was confirmed by colony PCR with their respective primers of each polyketide antibiotic gene.

Bio-Efficacy of Cloned Product under In Vitro Conditions
The dual culture method was used to study the comparative antagonistic ability of cloned product of polyketide synthase antibiotic genes, including macrolactin, bacillaene and difficidin (white colonies), non-cloned (blue colonies) along with parent strain B. amyloliquefaciens DSBA-11 against R. pseudosolanacearum UTT-25 under in vitro conditions. R. pseudosolanacearum was grown in a CPG broth medium for 48 h at 28 ± 1 • C [6] and maintained bacterial population (0.1 OD at 600 nm). 100 µL of bacterial culture was spread onto the Petri plates containing nutrient agar medium to make a lawn of bacteria. 0.5 cm diameter of three wells in each Petri plate was made with a sterilized cork borer and 40 µL of 48 h old culture of (cloned) products of these genes (non-cloned) product and DSBA-11 grown in the Luria broth contained a population of bacteria 0.1 OD was poured into each well separately. The plates were incubated at 28 ± 1 • C for 48 h and the inhibition zone was recorded. The value of the inhibition zone was converted into area of inhibition zone as described by Singh et al. [6].

Bacterial Viability Test
The R. pseudosolanacearum culture was treated with 10 and 50 µg/mL of cloned macrolactin, bacillaene, difficidin, B. amyloliquefaciens DSBA-11 and a mixture of these three polyketide antibiotic synthase genes to see the growth variation at different intervals 24 h, 48 h, 72 h, and 96 h after treatments and OD at 600 nm was taken by using UV-VIS Spectrophotometer, Hitachi (U-2900).

Mechanisms of Suppression of Bacterial Wilt Disease of Tomato
The cloned product of macrolactin, difficidin and bacillaene genes, B. amyloliquefaciens (DSBA-11) and non cloned product were tested against R. pseudosolanacearum under controlled conditions at National Phytotron Facility, IARI New Delhi. 21 days old tomato cv. Pusa Ruby seedlings were transplanted in the 6 plastic pots having autoclaved soil mixture of peat moss, vermiculite and sand in the ratio of 2:1:1 at 25-30 • C. R. pseudosolanacearum UTT-25, B. amyloliquefaciens (DSBA-11), cloned and non-cloned colonies of bacteria were scraped from the plates containing LA (Luria Agar) medium and suspended in sterile distilled water and maintained the population of bacteria (0.1 OD at 600 nm). A 40 mL of 48 h old culture of R. pseudosolanacearum UTT-25 was inoculated at the root zone of each plant after 5 days of transplanting. Subsequently, 25 mL of 48 h old culture of antagonistic B. amyloliquefaciens (DSBA-11), cloned and non-cloned product of three polyketide antibiotic genes was inoculated at the root zone of each plant. The plants inoculated with R. pseudosolanacearum and un-inoculated plants were also maintained as the positive and negative control. Wilt disease intensity was recorded at 3 days intervals, up to 30 days. The wilt disease intensity and rating were calculated as per described by Schaad et al. [31], and biological control efficacy (BCE) of antagonistic bacteria was calculated as per Guo et al. [32], and Singh et al. [7].

Purification of Cloned Product of Polyketides Antibiotic Synthase Gene
To purify the cloned polyketide antibiotic genes of B. amyloliquefaciens DSBA-11, the colony was grown in an LB medium and incubated for 48 h. After that the liquid culture was eluted in 100% methanol by using GC/MS analysis on a Focus GC/MS (Thermo) equipped and DB-5 capillary column (30 m × 0.25 mm i.d., film thickness 0.25 µm). Chromatographic conditions were as follows: helium as carrier gas at a flow rate of 1 mL/min; injection volume was 1.0 µL (1000 ppm in acetone); injector temperature was 240 • C, respectively. The column temperature was held at 60 • C for 5 min., and programmed at 3 • C/min to 220 • C and held for 10 min with split mode (1:20). The MS transfer line and source temperatures were 240 • C and 220 • C. The analysis was carried out in EI mode at 70 eV with the mass range of 30-450 a.m.u at 1 scan/s. The individual cloned polyketides antibiotic genes were purified and the extracts were used for the bio-efficacy test against R. pseudosolanacearum.

TEM Studies
Transmission electron microscopy (TEM) analysis was used to determine the effect of the eluted cloned product of polyketide genes macrolactin; difficidin, and bacillaene on Ralstonia pseudosolanacearum UTT-25 cells at the ultra structural levels. R. pseudosolanacearum UTT-25 was treated with 50 µg/mL of these antibiotics along with parent B. amyloliquefaciens DSBA-11 and allowed them to grow for 14 h at 28 ± 1 • C. One drop of the sample was embedded on a copper grid having 400-mess for 1-2 min and thoroughly washed with sterile distilled water and then negative stain with 2% uranyl acetate and dried it. The grid was visualized under transmission electron microscope (TEM) model no-Jeol-1011.

Statistical Analysis
The data was analyzed using Fisher's least significant differences (LSD) to determine the significant differences between treatments at p< 0.05 level. of the eluted cloned product of polyketide genes macrolactin; difficidin, and bacillaene on Ralstonia pseudosolanacearum UTT-25 cells at the ultra structural levels. R. pseudosolanacearum UTT-25 was treated with 50 µg/mL of these antibiotics along with parent B. amyloliquefaciens DSBA-11 and allowed them to grow for 14 h at 28 ± 1 °C. One drop of the sample was embedded on a copper grid having 400-mess for 1-2 min and thoroughly washed with sterile distilled water and then negative stain with 2% uranyl acetate and dried it. The grid was visualized under transmission electron microscope (TEM) model no-Jeol-1011.

Statistical Analysis
The data was analyzed using Fisher's least significant differences (LSD) to determine the significant differences between treatments at p< 0.05 level.

Primer Specificity and Sensitivity
The primer sets viz. Mln-F &Mln-R, Dfn-F &Dfn-R and Bae-F & Dfn-R-based on the nucleotide sequence of macrolactin, difficidin and bacillaene genes of B. amyloliquefaciens amplified at 792 bp, 705 bp, and 616 bp respectively (Table 1) on macrolactin, difficidin and baciallene genes were specific for the detection of B. amyloliquefaciens. The diluted genomic DNA of B. amyloliquefaciens was used for the sensitivity of polyketide antibiotic synthase-based primers. These polyketide antibiotic synthase genesbased primers were able to detect low to 0.1 ng DNA concentrations of B. amyloliquefaciens ( Figure 3). these primer sets, three banding pattern in B. amyloliquefaciens strains were obtained 792 bp, 705 bp and 616 bp by amplifying macrolactin, difficidin, and bacillaene poly tide synthase genes respectively. B. subtilis strains DTBS-4 & DTBS-5 amplified mac lectin, and difficidin synthase genes at 792 bp and 705 bp, respectively. However, th primer pairs of polyketide antibiotic synthase genes did not give amplification with ot Bacillus spp. like B. pumilus MTCC 7092, B. licheniformis DTBL-6 and B. cereus JHTBS-7 fluorescens DTPF-3, R. pseudosolanacearum UTT-25 ( Figure 2). Thus, these three sets primers-based on macrolactin, difficidin and baciallene genes were specific for the tection of B. amyloliquefaciens. The diluted genomic DNA of B. amyloliquefaciens was us for the sensitivity of polyketide antibiotic synthase-based primers. These polyketide tibiotic synthase genes-based primers were able to detect low to 0.1 ng DNA concent tions of B. amyloliquefaciens (Figure 3).

Screening of Polyketides Antibiotics Producing Strains of Bacillus spp. through Multiplex PCR
A combination of two bands of macrolactin + bacillaene, bacillaene + difficidin a difficidin + macrolactin was also observed. However, the amplification of such type primer pairs of polyketide antibiotic synthase genes did not give amplification with other Bacillus spp. like B. pumilus MTCC 7092, B. licheniformis DTBL-6 and B. cereus JHTBS-7, P. fluorescens DTPF-3, R. pseudosolanacearum UTT-25 ( Figure 2). Thus, these three sets of primers-based on macrolactin, difficidin and baciallene genes were specific for the detection of B. amyloliquefaciens. The diluted genomic DNA of B. amyloliquefaciens was used for the sensitivity of polyketide antibiotic synthase-based primers. These polyketide antibiotic synthase genes-based primers were able to detect low to 0.1 ng DNA concentrations of B. amyloliquefaciens (Figure 3).

Screening of Polyketides Antibiotics Producing Strains of Bacillus spp. through Multiplex PCR
A combination of two bands of macrolactin + bacillaene, bacillaene + difficidin and difficidin + macrolactin was also observed. However, the amplification of such type of  Table 3).   Table 3).

Detection of Polyketides Gene from Soil DNA
Eighty-five samples were collected from six states of India to detect polyketideproducing strains of Bacillus spp. Maximum 73.3% of soil samples collected from West Bengal showed potential to amplify with all these primers followed by Uttarakhand (70.0%) and Jammu and Kashmir (66.7%) (Table 3; Figure 5).

Detection of Polyketides Gene from Soil DNA
Eighty-five samples were collected from six states of India to detect p tide-producing strains of Bacillus spp. Maximum 73.3% of soil samples collected West Bengal showed potential to amplify with all these primers followed by Uttara (70.0%) and Jammu and Kashmir (66.7%) (Table 3; Figure 5).

Bio-Efficacy of Cloned Product of Polyketides Antibiotics Producing Gene under In Vi Conditions
The polyketides antibiotic synthase gene difficidin, macrolactin and bacillaen amyloliquefaciens was cloned cloning and confirmed by colony PCR by using th spective genes with the standard PCR protocol with their respective primers to am at 792 bp 705 bp and 616 bp respectively. The cloned product of these genes supp the growth of R. pseudosolanacearum significantly under in vitro conditions. Max growth inhibition of R. pseudosolanacearum was found in parent B. amylol ciensDSBA-11 3.30 cm 2 of inhibition zone of R. pseudosolanacearum. However,the product of bacillaene, macrolactin and difficidin formed 1.9, 1.9 and 1.7 cm 2 of inh zone area under in vitro conditions, respectively ( Figure 6B-D). However, these alone made less inhibition zone as compared to their parent. A significant variation growth of R. pseudosolanacearum was recorded in different antibiotic treatment growth of bacteria was increased by increasing the duration of incubation in treatments, including control significantly (Table 4).

Bio-Efficacy of Cloned Product of Polyketides Antibiotics Producing Gene under In Vitro Conditions
The polyketides antibiotic synthase gene difficidin, macrolactin and bacillaene of B. amyloliquefaciens was cloned cloning and confirmed by colony PCR by using their respective genes with the standard PCR protocol with their respective primers to amplified at 792 bp 705 bp and 616 bp respectively. The cloned product of these genes suppressed the growth of R. pseudosolanacearum significantly under in vitro conditions. Maximum growth inhibition of R. pseudosolanacearum was found in parent B. amyloliquefaciens DSBA-11 3.30 cm 2 of inhibition zone of R. pseudosolanacearum. However, the cloned product of bacillaene, macrolactin and difficidin formed 1.9, 1.9 and 1.7 cm 2 of inhibition zone area under in vitro conditions, respectively ( Figure 6B-D). However, these genes alone made less inhibition zone as compared to their parent. A significant variation in the growth of R. pseudosolanacearum was recorded in different antibiotic treatments. The growth of bacteria was increased by increasing the duration of incubation in all the treatments, including control significantly (Table 4).

Detection of Polyketides Gene from Soil DNA
Eighty-five samples were collected from six states of India to detect polyketide-producing strains of Bacillus spp. Maximum 73.3% of soil samples collected from West Bengal showed potential to amplify with all these primers followed by Uttarakhand (70.0%) and Jammu and Kashmir (66.7%) (Table 3; Figure 5).

Bio-Efficacy of Cloned Product of Polyketides Antibiotics Producing Gene under In Vitro Conditions
The polyketides antibiotic synthase gene difficidin, macrolactin and bacillaene of B. amyloliquefaciens was cloned cloning and confirmed by colony PCR by using their respective genes with the standard PCR protocol with their respective primers to amplified at 792 bp 705 bp and 616 bp respectively. The cloned product of these genes suppressed the growth of R. pseudosolanacearum significantly under in vitro conditions. Maximum growth inhibition of R. pseudosolanacearum was found in parent B. amyloliquefa-ciensDSBA-11 3.30 cm 2 of inhibition zone of R. pseudosolanacearum. However,the cloned product of bacillaene, macrolactin and difficidin formed 1.9, 1.9 and 1.7 cm 2 of inhibition zone area under in vitro conditions, respectively ( Figure 6B-D). However, these genes alone made less inhibition zone as compared to their parent. A significant variation in the growth of R. pseudosolanacearum was recorded in different antibiotic treatments. The growth of bacteria was increased by increasing the duration of incubation in all the treatments, including control significantly (Table 4).

Morphological and Ultrastructural Changes of R. pseudosolanacearum Cells in the Presence of Polyketide Antibiotics
Visualization of the cellular damage caused to R. pseudosolanacearum UTT-25 at the ultrastructural level by difficidin, macrolactin and bacillaene was undertaken through TEM analysis (Figure 7). In this study, untreated control cell of R. pseudosolanacearum UTT-25 appeared intact, typically rod-shaped with smooth exterior ( Figure 7A). Upon exposure to difficidin, macrolactin and bacillaene antibiotics, the cells of R. pseudosolanacearum became loose and diffused with their normal shape and even ruptured ( Figure 7B-F). After treatment of R. pseudosolanacearum UTT-25 with these polyketide antibiotics, the lysis of the bacterial cells was clearly visible and resulted in plasmolysis and efflux of intracellular components.

Morphological and Ultrastructural Changes of R. pseudosolanacearum Cells in the Presence of Polyketide Antibiotics
Visualization of the cellular damage caused to R. pseudosolanacearumUTT-25 at the ultrastructural level by difficidin, macrolactin and bacillaene was undertaken through TEM analysis (Figure 7). In this study, untreated control cell of R. pseudosolanacearum UTT-25 appeared intact, typically rod-shaped with smooth exterior ( Figure 7A). Upon exposure to difficidin, macrolactin and bacillaene antibiotics, the cells of R. pseudosolanacearum became loose and diffused with their normal shape and even ruptured ( Figure  7B-F). After treatment of R. pseudosolanacearum UTT-25 with these polyketide antibiotics, the lysis of the bacterial cells was clearly visible and resulted in plasmolysis and efflux of intracellular components.

Effect of Polyketide Antibiotic-Producing B. amyloliquefaciens for Control of Bacterial Wilt
The mechanism of suppression of wilt disease in tomato, the cloned product of bacillaene, difficidin and macrolactin genes along with their parent B. amyloliquefaciens DSBA-11 and the non-cloned product was inoculated to the plant. A minimum 29.3% of wilt intensity in tomato plants was recorded in treated with B. amyloliquefaciens DSBA-11 under glasshouse conditions. The treatment of cloned product of difficidin, macrolactin and bacillaene synthase genes of B. amyloliquefaciens DSBA-11 significantly suppressed 39.3, 46.0, and 50.7% of wilt intensity in tomato cv Pusa Ruby after 30 days of R. pseudosolanacearum UTT-25 inoculation respectively (Table 4). However, the wilt intensity was found to be lower in cloned products of polyketide antibiotic synthase genes than control (69.3%). The non-cloned colony of bacteria slightly showed low wilt intensity in tomato as compared to control but lower than the cloned product.

Effect of Macrolactin, Difficidin and Bacilysin on Viability of R. pseudosolanacearum Cells
Growth of R. pseudosolanacearum UTT-25 treated with crude ethyl acetate extract of polyketide of these antibiotics genes produced by B. amyloliquefaciens DSBA-11 and its cloned products in broth culture was measured by using optical density measurements at 600 nm (Table 5). Minimum growth of R. pseudosolanacearum was found at 50 µg/mL of a mixtures of macrolactin + difficidin + bacillaene 0.063 OD (at 600 nm) after 24 h of incubation followed by B. amyloliquefaciens DSBA-11 (0.071 OD) and difficidin (0.071 OD) under in vitro conditions. The growth of R. pseudosolanacearum UTT-25 was inhibited by increasing the concentration of polyketide antibiotic products.

Discussion
Bacterial wilt of solanaceous crop caused by Ralstoniapseudosolanacearum UTT-25 is a serious problem across the world. For the control of wilt disease, we selected Bacillus spp. particularly polyketide antibiotic-producing strains of Bacillus spp. as earlier, it was reported that Bacillus spp. produced 200 peptide antibiotics (Lisboa et al. [33]), and approximately 8.5% genome of B. amyloliquefaciens FZB 42 are dedicated to secondary metabolite production (Chen et al. [5]. Selection of potential antibiotic-producing strains of Bacillus spp., the individual gene had been targeted to design primer for detection like iturin. (6)], and bacillaene [34]. In this study, we targeted three polyketide antibiotic synthase genes viz., macrolactin, difficidin and bacillaene for developing a multiplex-PCR protocol to detect polyketide antibiotic-producing strains of Bacillus spp. We developed and standardized a protocol for detecting and screening of polyketide-producing strains of Bacillus spp. by using multiplex-PCR, based on these genes, which is rapid, accurate and highly sensitive. To the best of our knowledge, this is the first attempt to develop a protocol for simultaneous detection of these three polyketide antibiotic-producing strains of Bacillus spp. particularly B. amyloliquefaciens. We screened polyketide antibiotic-producing strains of Bacillus spp. including B. amyloliquefaciens, B. subtilis, B. cereus, B. pumilus, and B. licheniformis using multiplex-PCR and among them, the Isolates of B. amyloliquefaciens contained macrolactin, bacillaene and difficidin polyketide synthase antibiotic genes, while B. subtilis contained difficidin and macrolactin synthase antibiotic genes. In contrast to our results, Butchner et al. [34] reported that approximately 80 kb pksX gene cluster in B. subtilis encode an unusual hybrid polyketide that has been linked to the production of the uncharacterized antibiotic bacillaene.
The most attention has been paid to polyketide antibiotics such as bacillaene, difficidin and macrolactin for their role in the inhibition of bacterial growth and inducing plant resistance against the bacterial pathogen [35] and difficidin and bacilysin [36]. Antibiotics produced by the strains of different bacterial genera, biosynthetic genes have been cloned partially and sequenced. However, B. amyloliquefaciens FZB 42 is a producer of three families of lipopeptides, surfactin, bacillomycin D, and fengycin, which are well-known secondary metabolites with mainly antifungal and bacterial activities. They are also produced by numerous B. subtilis strains. Furthermore, three giant gene clusters containing genes with homology to polyketide synthase (PKS) genes have been identified but did not assign functional roles. Polyketides belong to a large family of secondary metabolites that include many bioactive compounds with antibacterial, immune suppressive, antitumor, or other physiologically relevant bioactivities. Three functional gene clusters directing the synthesis of difficidin, bacillaene, and macrolactin have been identified in B. amyloliquefaciens strains as earlier reported by Chen et al. [5]. Further, polyketide mega synthesis responsible for synthesis of bacillaene, macrolactin and difficidin are encoded by these giant gene clusters that are located at different sites of the B. amyloliquefaciens genome [35]. Difficidin inhibits protein synthesis and possibly also damages the cell membrane [36,37]. Polyketide synthase (PKSs) gene clusters has also been identified that directed the synthesis of polyketides (PKs), e.g., macrolactin (mln), bacillaene (bae), and difficidin in B. amyloliquefaciens subsp. plantarum FZB42 [38]. The present study showed the antagonistic ability of cloned products of macrolactin, bacillaene and difficidin along with parent DSBA-11, which showed an inhibitory effect on the growth of R. pseudosolanacearum and also suppressed wilt disease in tomatoes. We cloned macrolactin, difficidin and bacillaene genes of B. amyloliquefaciens DSBA-11 and we observed that the product of these genes and parent DSBA-11 damaged the cell wall and cell membrane of R. pseudosolanacearum, which were visualized under TEM ( Figure 4). Similar results have also been reported by Wu et al. [36]. The findings were corroborative to earlier reports on difficidin, which acts as antibacterial activity against Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola causing bacterial leaf blight and bacterial streak disease in rice, respectively. Although, no significant variation was found in the cloned products to inhibit the growth of R. pseudosolanacearum under in vitro conditions. However, difficidin was found to be the best among these tested polyketide antibioticsto suppress the bacterial wilt disease in tomatoes. Moreover, these cloned products showed less biocontrol efficacy than parent strain DSBA-11 (57.22%). A similar finding was earlier reported by Chen et al. [35] against E. amylovora, causing fire blight in pears and apples. They stated that difficidin is the most efficient antibacterial compound in FZB42. Our present finding reveals that the B. amyloliquefaciens DSBA-11 contains these three polyketide antibiotics, which are able to inhibit the growth of R. pseudosolanacearum under in vitro conditions by forming an inhibition zone and reduce wilt disease in tomatoes significantly. Further, structural and functional characterization of these polyketide antibiotic synthase genes is required for a detailed study.

Conclusions
The following conclusions can be drawn from the study results: 1. Bacillus amyloliquefaciens DSBA-11 showed the best antagonistic ability against R. pseudosolanacearum UTT-25 under in vitro and in vivo conditions. 2. A multiplex-PCR protocol based on polyketide synthase genes i.e.,macrolactin, difficidin and bacillaene genes, was developed to identify and detect potential antagonistic strains of B. amyloliquefaciens within a short period of time with high accuracy and sensitivity.
3. Bacillus amyloliquefaciens DSBA-11 provides a potential option to use these polyketide antibiotics as an alternative to chemical bactericides to control wilt disease of solanaceous crops.
4. There is a need to study the mechanism of polyketide antibiotic synthase genes viz., macrolactin, difficidin and bacillaene using high throughput molecular techniques.