Characterization of an antifungal compound produced by Bacillus sp. strain A 5 F that inhibits Sclerotinia sclerotiorum

Journal of Basic Microbiology 2012, 52, 1–9 1

© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Research Paper

Characterization of an antifungal compound produced by Bacillus sp. strain A5F that inhibits Sclerotinia sclerotiorum

Ankit Kumar1, Sandeep Saini1, Victor Wray2, Manfred Nimtz2, Anil Prakash1 and B. N. Johri1

1 Department of Biotechnology, Barkatullah University, Bhopal (M.P.), India 2 Department of Structural Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany

A potential antagonist, Bacillus sp. strain A5F was isolated from soybean rhizosphere following in vitro dual plate screening. The bacterium displayed strong inhibitory activity in vitro against soybean stem rot pathogen, Sclerotinia sclerotiorum. The culture supernatant of strain A5F completely suppressed the mycelial growth of the pathogen, indicating that suppression was due to the presence of antifungal compounds in the culture filtrate. The culture filtrate also suppressed other phytopathogenic fungi including Fusarium oxysporum and Macrophomina phaseo-lina, in vitro suggesting a broad spectrum antagonistic activity against fungal pathogens. Che-mical extraction followed by chromatographic analysis resulted in two antifungal fractions. The high resolution-electron spin ionization-mass spectrometry (HR-ESI-MS) and Nuclear Magnetic Resonance (1D and 2D1H) spectra of these antifungal fractions revealed the presence of antifungal compounds, one of which showed similarity to bacillomycin D.

Abbreviations: HR-ESI-MS – High resolution-electron spin ionization-mass spectrometry; TDM: 4,4’-bis(di-methylamino)diphenylmethane

Keywords: Bacillus / Lipopeptides / Biocontrol / Sclerotinia sclerotiorum / Bacillomycin D

Received: September 27, 2011; accepted: October 25, 2011

DOI 10.1002/jobm.201100463

Introduction*

The widespread application of agrochemicals has cer-tainly decreased the outbreak of fungal diseases/patho-gens, however at the same time it has also resulted in the appearance of resistant pathogens [1]. The primary targets of these chemicals are the pathogens but they can be lethal to the beneficial microflora and other organisms residing in the same ecological niche [2]. Moreover, increased public concern over the hazardous effects of agro-chemicals on human health and envi-ronment has ushered in the need to explore eco-friendly alternatives of chemicals and biological control is one such way forward [3]. Use of bacteria as biocon-trol agents, has received considerable attention in cur-rent scenario, because of their ability to suppress plant Correspondence: Ankit Kumar, Department of Biotechnology, Barkatul-lah University, Bhopal – 462026 (M.P.), India E-mail: [email protected], [email protected] Phone: +91-755-2491848, 2491849 Fax: +91-755-2491850

pathogens through different mechanisms [4–6]. Anti-biotic production is one of the most widely studied mechanisms and several Gram-positive and Gram-negative bacteria have been studied extensively that produce an array of antibiotic compounds [6–11]. Among the Gram positive group, Bacillus spp. are well known for production of a variety of molecules that display a broad spectrum antimicrobial activity with very diverse structures [7, 10]. These antibiotics include, most notably the non-ribosomally synthesized cyclic lipopeptides viz., surfactin, iturin and fengycin, several modified or un-modified small peptides, proteins and some volatiles [7, 12, 13]. Structurally, lipopeptides are composed of a hydrophilic peptide portion and a hy-drophobic fatty acid part, either β-hydroxy fatty acids (β-hydroxy type) or β-amino fatty acids (β-amino type). Members of the iturin family display strong antifungal and haemolytic activities with relatively limited anti-bacterial activity [14]. Iturin A and bacillomycin D along with mycosubtilin is comprised of one β-amino

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2 A. Kumar et al. Journal of Basic Microbiology 2012, 52, 1–9


fatty acid and 7-α amino acids. Surfactin is another lipopeptide produced by many B. subtilis strains and is regarded as the most powerful biosurfactant known [15]. It is a cyclic molecule containing heptapeptide and a β-hydroxy fatty acid [16, 17]. Although surfactins are non toxic by themselves however they sustain some synergistic effect on the antifungal activity of iturin A [18]. However, unlike iturin and surfactin, fengycins are cyclic lipodecapeptides containing a β-hydroxy fatty acid with a side chain length of 16–19 carbon atoms. It contains four D amino acids with an unusual non-pro-tein amino acid, ornithine [19]. Fengycin exerts strong antifungal activity, especially against filamentous fungi by inhibiting the enzyme phospholipase A2 [20]. Thus keeping in view the biocontrol perspectives of Bacillus species, the principal objective of this study was to isolate and determine the structure of antifungal compound(s) produced by the antagonistic bacterium, Bacillus sp. strain A5F that inhibits the soybean stem rot pathogen, S. sclerotiorum.

Materials and methods

Microbial strains and culture conditions The bacterial strain A5F used in this study was isolated from soybean rhizosphere following serial dilution method on standard tryptone soya agar (TSA) medium (tryptone: 15.0 g, peptone: 3.0 g, sodium chloride: 5.0 g, agar: 15.0 g, and distilled water: 1000 ml). It was char-acterized as Bacillus sp. strain A5F (accession no. EU647582) based on biochemical studies and 16S rRNA sequence analysis (data not shown). The bacterium was maintained and propagated on TSA at 28 °C whenever required. The phytopathogenic fungi, S. sclerotiorum, F. oxysporum and M. phaseolina were obtained from the National Research Centre for Soybean (ICAR), Khandwa Road, Indore (M.P.), India. The fungal cultures were maintained and propagated on potato dextrose agar (PDA) medium (peeled potato: 200 g, dextrose: 20 g, agar: 15 g and distilled water: 1000 ml) at 28 °C.

Antifungal activity of culture filtrate In order to verify the antifungal activity of culture fil-trate from strain A5F, an experiment was performed wherein a loopful of culture from single bacterial col-ony of strain A5F was inoculated in tryptic soya broth and incubated overnight in an incubator shaker (Sci-genics Biotech, Chennai, India) at 120 rpm and 28 °C. The inoculated flasks were kept under the above condi-tions for 100 h to check whether production of antibi-otics was coupled with the physiological state of the

bacterium. During the incubation, 3 ml samples were taken from flasks every 4 h and the three samples were pooled and centrifuged at 10,000 rpm for 15 min at 4 °C. Pellet was removed and culture filtrate was filter sterilized through a membrane filter (0.45 µl) to re-move any suspended cell. The pooled sample was sub-jected to the antifungal activity and cell density deter-mination. The cell density was determined by meas-uring the optical density at 600 nm (OD600) using DU-series 700 spectrophotometer (Beckman Coulter, Ger-many). For antifungal activity, 20 µl of the culture fil-trate was added on to sterile discs (HiMedia) and placed near the periphery of the growing fungal pathogen at a distance of about 1 cm. Discs coated with sterile broth were used as control. Culture filtrate of different con-centrations (10%, 25%, 50% and 100%) was bioassayed against the pathogen as described above to check the efficacy of antifungal compounds. Plates were kept at 4 °C for 2 hr to allow for diffusion of the sample and subsequently incubated at 28 °C in an incubator for 3 d. Discs coated with sterile broth only were used as con-trol.

Bioassay for sclerotial germination inhibition The culture filtrate as obtained above was subjected to sclerotial germination inhibition assay. An aliquot of 100 µl from sclerotial suspension (1.0 × 104 microscle-rotia/ml) of S. sclerotiorum was taken and suspended with an equal volume (100 µl) of different conc. (10%, 25%, 50% and 100%) of the cell-free culture filtrate from strain A5F. For control, 100 µl of sclerotial suspen-sion was mixed with 100 µl of sterile broth (pH. 7.0). After 16 h, about 100 microsclerotia were taken out and studied microscopically for germination. The per-centage germination of microsclerotia was calculated based on the number of microsclerotia possessing germ tubes over total microsclerotia studied. A sclerotium was considered germinated if the germ tube was longer than one-half of the length of sclerotium. The experi-ment consisted of three replicates and was repeated twice.

Stability of antifungal activity of culture filtrate The filter sterilized culture filtrate was subjected to thermal, pH and enzymatic shock (proteinase k) to check the stability of bioactive compounds. Each ex-periment consisted of three replicates and was repeated twice.

Temperature The culture filtrate was subjected to various tempera-ture treatments (50 °C, 80 °C, 100 °C and 121 °C) for

Journal of Basic Microbiology 2012, 52, 1–9 Characterization of an antifungal lipopeptides by Bacillus sp. 3


15 min. After thermal shock the filtrate was quickly cooled to 25 °C and studied for antifungal activity. Con-trol consisted of stored culture filtrate kept at 4 °C.

pH The cell free culture filtrate (pH. 8.3) was adjusted to pH. 2.0, 4.0, 6.0, 8.0, and 10.0 with 1 N HCl or NaOH and kept at 25 °C for 24 h. Thereafter pH was restored back to 8.3 and subjected to antifungal assay.

Enzymatic hydrolysis The culture filtrate was treated with proteinase k (pH 8.5) at a final conc. of 1 mg/ml and incubated at 37 °C for 2 h. The mixture was autoclaved at 121 °C for 15 min. to inactivate the enzyme and subsequently assayed for inhibitory activity against fungal pathogen as described above.

Isolation and purification of antifungal lipopeptides The lipopeptides present in the culture supernatant were precipitated by adding 6 N HCl to a final pH of 2.0 and stored at 4 °C for 1 h. The crude precipitates were collected by centrifugation at 12,000 rpm for 30 min. at 4 °C and dissolved in methanol (MeOH), filtered through Whatman No. 1 filter paper to remove any coarse impurities and subjected to disc diffusion assay for antifungal activity. Discs coated with MeOH were used as control. After confirmation of antifungal activ-ity, methanolic extract was dried under vacuum in a rotary evaporator (Heidolf, Germany). The dried crude mixture was dissolved in distilled water (pH 8.0) and subsequently acidified to pH 2.0 with 6 N HCl. The precipitated yellowish white crystals of lipopeptide, were collected by centrifugation at 14,000 rpm for 15 min. and dissolved in 1 ml MeOH.

Thin layer chromatography (TLC) The crude extract obtained was fractionated by TLC on silica gel plates (20 × 20 cm, 0.5 mm thick, G), devel-oped in chloroform:methanol:water (65:25:4, v/v/v/) as solvent system. Spots were detected with ninhydrin for compounds with free amino groups, or water for hy-drophilic compounds or with 4,4′-bis(dimethylamino)di-phenylmethane (TDM) reagent (Sigma Chemicals Co., St. Louis, MO, USA) for peptide bond [21]. The Rf value of the bioactive fraction was measured. The antifungal fraction was scrapped out from the plate, eluted with MeOH and subjected to further purification through high performance liquid chromatography.

High performance liquid chromatography (HPLC) The partially purified bioactive fraction was loaded on reverse phase (RP-HPLC) column (C18, 4.6 × 250 mm,

5 µm) Waters HPLC system W2998 equipped with a pump and a Water photodiode array detector with variable wavelength detector (190–400 nm). The sol-vents used were (A) acetonitrile and (B) aqueous solu-tion 10 mM ammonium acetate (40:60, v/v). The prod-ucts were eluted at a flow rate of 1 ml/min with a linear gradient of solvent B, developed from 15 to 80% and monitored at 210 nm. A total of 11 fractions were col-lected at different time intervals. All the fractions ob-tained were studied for their biological activities and compared with the biological activity of the MeOH extracts. Active fractions showing biological activity were identified in the chromatogram and further stud-ied with mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy.

Mass spectrometry and NMR studies The structure of the peptide was elucidated using a combination of 2D homonuclear NMR techniques and HR-ESI-MS. Total correlation spectroscopy (TOCSY) and rotating frame overhause effect spectroscopy (ROESY) i.e. NMR spectra were recorded at 300 K on a Bruker AVANCE DMX600 NMR spectrometer linked to the deuterium resonance of the mixed solvent, CF3CD2OH/ H2O 1:1. The amino acids present and their sequence were initially determined from the 1D and 2D NMR 1H data using standard procedures [22]. The through-bond connectivities of the various amino acids spin systems were identified from a 2D TOCSY. HR-ESI-MS were re-corded on a Thermo Science LTQ Orbitrap mass spec-trometer.

Results

Antifungal activity of the culture filtrate Production of antifungal compounds by strain A5F was observed after 12 h (in the beginning of the log phase) (Fig. 1). The antifungal activity was maximum at the end of the stationary phase (36 h) and thereafter it de-clined gradually with the incubation time. At the end of 96 h, the supernatant still possessed some bioactivity but it was relatively weak. The culture filtrate inhibited the growth of S. sclerotiorum, resulting in the formation of a clear zone of inhibition (Fig. 2). Analysis of inhibi-tion results revealed that S. sclerotiorum was most sensi-tive towards the inhibitory spectrum of bioactive com-pounds followed by M. phaseolina and F. oxysporum (data not shown). No inhibition of mycelial growth was ob-served at 5% conc. while a mild inhibition occurred at 10% conc. of culture filtrate (Fig. 3). The inhibitory activity of the culture supernatant continued to rise



Figure 1. Relationship between cell density and antifungal activity of Bacillus sp. strain A5F against S. sclerotiorum. Strain A5F was kept under shake flask culture in tryptone soya broth at 28 °C in the dark. � = Cell number of strain A5F, � = Zone of inhibition. Zone of inhibition was recorded 3 d after disc diffusion assay had been per-formed. O.D. is optical density at 600 nm. Curves show the mean values of three replicates. Bars represent standard error of the mean. with the increase in conc. of filtrate and 100% inhibi-tion was achieved in treatment containing undiluted culture filtrate (100% conc.). No inhibition was ob-served in control.

Inhibition of sclerotial germination In the absence of culture filtrate, germination effi-ciency of microsclerotia was approx. 100%. The relative germination percentage was reduced with increasing concentration of culture filtrate and sclerotial germina-tion was completely inhibited in the presence of undi-luted (100%) culture filtrate (Fig. 4). Microscopic obser-vations revealed that the fungal hyphae were distorted leading to lysis of the cells followed by complete degra-dation of the mycelium (Fig. 5). Moreover, the germ tubes of sclerotia grown in broth treated with culture filtrate were shorter in length as compared to that of the control (data not shown).

Figure 2. Antifungal activity of cell free culture filtrate against S. sclerotiorum.

Figure 3. Antifungal activity of culture supernatant from Bacillus sp. strain A5F at different conc. (5%, 10%, 25%, 50% and 100%) against S. sclerotiorum. The curves show mean values of three replicates. Bars represent standard error of the mean.

Figure 4. Inhibitory action of culture supernatant at different conc. (10%, 25%, 50% and 100%) against sclerotial germination of S. sclerotiorum. The curves show mean values of three replicates. Bars represent standard error of the mean.

Figure 5. Mycolytic effect of bacillomycin D isolated from Bacillus sp. strain A5F on fungal hyphae of S.sclerotiorum. (a): untreated control (intact hyphae), (b and c): lysed fungal hyphae, (d): comple-tely lysed fungal hyphae resulting in vesicle formation. Arrowheads point to the lysed parts of the fungal hyphae.



Table 1. Effect of temperature, pH and enzymatic shock on the stability of antifungal compounds produced by Bacillus sp. strain A5F. Data represent mean values of three replicates with standard error of the mean.

Heat treatments Zone of inhibition (mm)

50 °C, 15 min 13.26 ± 0.19 80 °C, 15 min 13.20 ± 0.14 100 °C, 15 min 13.13 ± 0.13 121 °C, 15 min 13.10 ± 0.10

pH tolerance Untreated 12.90 ± 0.15 Acidic, 2.0 NIa 4.0 12.50 ± 0.17 6.0 12.30 ± 0.17 8.0 12.30 ± 0.16 Basic, 10.0 12.20 ± 0.11

Enzymatic degradation Untreated 13.50 ± 0.20 Proteinase k 13.20 ± 0.14

a NI: No inhibition

Stability of antifungal activity in culture filtrate The effects of temperature, pH and proteinase k upon the antifungal activity of cell free supernatant are summarized in Table 1.

Heat No significant change in the antifungal activity of cul-ture filtrate was observed, when subjected to high tem-perature treatment. The culture filtrate retained the antifungal potential even after autoclaving at 121 °C.

pH Treatments at pH 4.0 to 10.0 had no significant effect on the bioactivity of culture filtrate, suggesting a wide range of pH stability of antifungal metabolites. How-ever, the inhibitory activity was completely lost at pH 2.0 compared to the control, as metabolites tended to precipitate at pH 2.0.

Enzymatic hydrolysis The hydrolytic enzyme, proteinase k had no effect on the antifungal activity of bioactive metabolites pro-duced by strain A5F. The inhibition zone size did not differ significantly compared to the control.

Extraction and purification of the bioactive compounds The concentrated crude extract obtained after in vacuo drying resulted in formation of a clear zone of inhibi-tion. Three fractions, designated as ‘A’, ‘B’, and ‘C’ with Rf values 0.28, 0.62 and 0.86 respectively, were detected on TLC plate. All three fractions were removed and

subjected to antifungal activity. Only fraction ‘B’ (Rf: 0.62) displayed the antifungal activity. It was ninhydrin negative but positive to TDM reagent. A white coloured spot appeared when the plate was sprayed with water. The HPLC analysis of the partially purified fraction was performed at Sophisticated Analysis Instrumenta-tion Facility (SAIF), Lucknow (U.P, India) and successive bioassay directed separation and purification of the partially purified crude extract by RP-HPLC gave two antifungal fractions. The two antifungal fractions were designated as 1 and 2 respectively.

Determination of chemical nature of antifungal compounds 1D and 2D 1H (TOCSY, ROESY) NMR spectra and HR-ESI-MS of the antifungal fraction which gave highest yield, revealed that the compound is a cyclic lipopeptide composed of seven amino acids and contained an un-usual β-amino fatty acid system. Comparison of the TOCSY and ROESY spectra allowed determination of an almost complete sequence Pro-Glx-Ser-Thr-FA-Asn-Tyr-Asn from the “fingerprint” region of the ROESY spec-trum (Fig. 6). The accurate mass of the positively charged molecular ions, [M+H]+ and [M+Na]+ at m/z 1045.557 and 1067.538 respectively indicated a molecu-lar formula, C49H76N10O15 of the compound (Fig. 7). NMR and high resolution ESI-MS studies of the two purified fractions showed that bioactive compound is a cyclic

Figure 6. Fingerprint region of the 2D 1H ROESY spectrum of ba-cillomycin derivative in CF3CD2OH/H2O (1 :1) showing the sequence assignment.



Figure 7. HR-ESI-MS of the antifungal fraction containing bacillomycin-D (m/z major component [M+H]+ 1045.556 and [M+Na]+ 1067.538, and minor component [M+H]+ 1031.543 and [M+Na]+ 1053.523).

lipopeptide with molecular weights [M+H]+ 1,045 and [M+Na]+ 1,067 and amino acid sequence identical to bacillomycin D [23, 24]. Structurally it is a C15-β-amino fatty acid (Fig. 8), however bacillomycin D analogue with mass units of [M+H]+ 1,031 and [M+Na]+ 1,053 cor-responding to C14-β-amino fatty acid were also present as minor components (Fig. 7).

Discussion

Several investigations have reported Bacillus species worthy to be used as biocontrol agents against a pleth-ora of plant pathogens [7, 8, 10]. One of the most excit-ing features that makes Bacillus most competitive is its ability to sporulate under extreme environmental con-ditions and produce an array of antimicrobial metabo-lites with an amazing variety of structures and effective against a wide spectrum of microorganisms [7]. In our study, Bacillus sp. strain A5F was selected from a collec-tion of bacterial isolates which antagonized S. sclero-tiorum. The cell free culture supernatant of strain A5F displayed strong inhibitory activity against mycelial growth of the pathogen indicating the production of diffusible antifungal compounds (Fig. 2). The inhibitory activity of the culture supernatant decreased with in-

Figure 8. Structure of the bacillomycin D produced by Bacillus sp. strain A5F.

creased dilution of the culture filtrate and no mycelial inhibition was observed at 5% conc. while the undi-luted culture supernatant completely suppressed the mycelial growth. Similarly in absence of culture fil-trate, germination efficiency of microsclerotia was approx. 100%. The relative germination percentage was reduced with increasing concentration of culture fil-trate and sclerotial germination was completely inhib-ited in the presence of undiluted (100%) culture filtrate (Fig. 4). The hyphal morphology of the S. sclerotiorum was found greatly affected in the presence of culture filtrate, leading to lysis of the cells and finally complete degradation of the hyphal tips (Fig. 5). Lysis of the fun-gal cell wall is a characteristic antifungal response of lipopeptides of Bacillus sp. particularly that of iturin group molecules (iturin A, bacillomycin D and myco-subtilin) [25]. The unusual swelling of the sclerotia followed by lysis confirmed the fungicidal property of antifungal compounds produced by the strain A5F. Previously some reports have also shown similar results of unusual swelling of conidia and hyphae of fungal pathogens [26, 27]. Available reports suggest that lipo-peptides of the iturin group play an important role in conferring antimycotic activity to the Bacillus strains through different mechanisms including interference with conidial germination and germ tube elongation; inhibition due to unusual swelling of hyphae; lysis and degradation of fungal hyphae [5, 27, 28]. Iturin group molecules are known to interrupt cytoplasmic mem-brane of the fungi and create trans-membrane channels thus allowing escape of vital ions such as K+ out of the cell. Strains of B. amyloliquefaciens have already been



reported to produce iturin group lipopeptides as prin-cipal inhibitory compounds against fungal pathogens [9, 29]. While in some cases the antagonistic property of Bacillus strains has been reported to be a result of com-petition for nutrients, others have demonstrated the production of antifungal compounds as principal in-hibitory component against pathogens [30]. This clearly indicates the versatile mechanisms which Bacillus sp. strains can adopt to combat the pathogens/diseases. However, studies have shown that antimicrobial effect of lipopeptides does not rely solely upon their chemical nature but also on the sterol content of the plasma membrane of the target fungi as the sterols present in the plasma membrane may impose a buffering effect caused by fatty acids thereby changing the membrane fluidity [31]. The stability tests of the culture filtrate showed that the antifungal component was heat stable and active over a wide range of pH with insignificant changes in inhibitory activity (Table 1). Failure of proteolytic en-zyme (proteinsae K) suggests that the bioactive com-pounds were not proteins. It is probably for this reason, the occurrence of unusual amino acids such as or-nithine or D-amino acids in their structures that most antifungal metabolites produced by Bacillus sp. are re-sistant to proteolytic enzymes [32, 33]. Strains of Bacillus species are considered as natural factories of bioactive compounds such as lipopeptides and their role in control of plant pathogens has been well documented [34]. Members of groups, iturin and fengycin are potential antifungal in nature while sur-factins are known to act synergistically with iturin/ fengycin [5]. In our study, following TLC only fraction ‘B’ (Rf 0.62) displayed antifungal activity. It was ninhy-drin negative thus ruling out the possibility of two fungistatic compounds, kanosamine and zwittermycin [28]. This fraction was positive to TDM reagent, which indicates the presence of peptide bond in the com-pounds. Appearance of a white spot on the plate when sprayed with water indicated that the compound was lipophilic [21]. Therefore, the bioactive fraction could be presumed to contain either peptides or lipopeptides as inhibitory constituents. Mass spectrometric and NMR studies of the bioactive fraction, together revealed the presence of a cyclic lipo-peptide as principle component. The molecular mass (m/z) of the compound was determined as [M+H]+ 1045.557 and [M+Na]+ 1067.538 (Fig. 7). The mol. mass (m/z) 1067 corresponds to the presence of sodium ion. Chemically it was composed of seven amino acids “Pro-Glx-Ser-Thr-FA-Asn-Tyr-Asn” which showed sequence homology to the bacillomycin D as reported by Peypoux

et al. [23]. The molecular mass of this compound is simi-lar (m/z 1045) to that published by Peypoux et al. [24] however it is lower (m/z 1059) than that reported by Moyne et al. [35]. Apparently, the 14 unit mass differ-ence represents the difference between C14 and C15 lipid chain. Presence of a bacillomycin D analogue with m/z [M+H]+ 1,031 and [M+Na]+ 1,053 was also detected but as a minor component. Bacillomycin D is a member of iturin family with strong antifungal spectrum and is produced by several bacilli [10, 36]. The antifungal activity of B. amylo-liquefacines FZB42 has been attributed mainly to bacil-lomycin D production [19]. The mechanism of action of iturin group molecules involves penetration into the lipid bilayer of cytoplasmic membrane by the hydro-phobic tail followed by auto-aggregation resulting into pore formation that causes cellular leakage [37]. In our study it appears that bacillomycin D damages the cyto-plasmic membrane of sclerotia, thus leading to leakage of vital components out of the cytoplasm and subse-quently cell death though, the factual mechanism be-hind lysis of fungal sclerotia, induced by bacillomycin D needs further investigation. Bacillomycin D is syn-thesized non-ribosomally through complex peptide syn-thetases [38]. In summary, Bacillus sp. strain A5F produced antifun-gal compound characterized as bacillomycin D which successfully suppressed the fungal pathogen S. sclerotio-rum. The compound is heat stable, active over a wide range of pH and is resistant to proteolytic enzyme. However, mechanism of action of bacillomycin D is yet to be elucidated. Further, application of bacillomycin D as potential biocontrol agent requires more investiga-tions, including inexpensive large scale production as well as evaluation for toxicity and degradation in the natural environment.

Acknowledgements

This work was financially supported by Madhya Pra-desh Council of Science & Technology (MPCST), Bhopal through a grant to BNJ. The authors thank Mr. Ajay Patidar, Land Owner of the soybean field for permitting sampling. Support received from Dr. M.P. Sharma and Dr. S.K. Sharma, of National Research Centre for Soy-bean, Indian Council of Agricultural Research (ICAR), Indore in the form of pathogenic cultures is gratefully acknowledged. Help received from Sophisticated Ana-lytical Instrumentation Facility, Central Drug Research Institute (CDRI), Lucknow for HPLC analysis is grate-fully acknowledged. Help received from Bioinformatics



Centre (Sub-DIC, DBT) staff, Deptt. of Biotechnology, Barkatullah University, Bhopal for data analysis is gratefully acknowledged.

Conflict of interest statement The authors declare that there is no financial/commer-cial conflict of interest.

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((Funded by

• Madhya Pradesh Council of Science & Technology (MPCST), India))

Characterization of an antifungal compound produced by Bacillus sp. strain A 5 F that inhibits Sclerotinia sclerotiorum

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