sv-lncs RUHUNA JOURNAL OF SCIENCE Vol 9(2): 160-168, December 2018 eISSN: 2536-8400  Faculty of Science DOI: http://doi.org/10.4038/rjs.v9i2.44 University of Ruhuna  Faculty of Science, University of Ruhuna 160 Short paper Effects of partially purified enterocins from Enterococcus faecalis strains on the growth of some phytopathogenic fungi O.M. David1* and O.E. Onifade2 1Department of Microbiology, Ekiti State University, Ado-Ekiti, Nigeria 2Department of Science Laboratory Technology, Ekiti State University, Ado-Ekiti, Nigeria *Corresponding author: david.oluwole@eksu.edu.ng; ORCID: 0000-0002-1396-3450 Received: 29th May 2018, Revised: 12th November 2018, Accepted: 22nd November 2018 Abstract. Plant protection is an important area which needs attention since most of the hazardous inputs added into agricultural systems are in form of synthetic chemicals. The inhibitory activity of partially purified enterocins (PPEs) produced by Enterococcus faecalis strains on plant pathogenic fungi was investigated in this study. The PPEs were preliminarily screened against bacteria using agar-well diffusion method. The active extracts were partially purified using ion exchange chromatography. The in vitro anti-fungal properties of the PPEs were determined using agar dilution and broth dilution techniques. The PPEs tested in this study inhibited the growth of Botryodiplodia theobromae, Aspergillus niger, Pythium ultimum, Penicillium expansum and Fusarium oxysporum. At different concentrations PPEs had varying inhibitory effects on the dry mycelial weight of Pythium ultimum and F. oxysporum. At the 96th hour of the experiment, enterocin UNAD 012 had higher percentage inhibition ranging between 37.63 and 84.11% than enterocin UNAD 046 with percentage inhibition ranging between 28.77% and 67.27% on the test fungi. This inhibitory activity of enterocins produced by E. faecalis on fungi makes them as potential biocontrol agents due to their ability in suppressing their growth. Keywords. Bacteriocin, enterocins, Enterococcus faecalis, fungi, phytopathogens. 1 Introduction Phytopathogenic fungi are capable of causing infectious diseases in plants. They damage plants and plant product on which human beings depend for http://doi.org/10.4038/rjs.v9i2.44 O.M. David and O.E. Onifade Effects of enterocins on phytopathogenic fungi Ruhuna Journal of Science Vol 9(2): 160-168, December 2018 161 food, clothing, shelter, furniture and the environment. Most of them belong to the family Ascomycetes and Basidiomycetes. Common species include Pythium ultimum, Penicillium expansum, Fusarium oxysporum. Aspergillus fumigatus, Botryodiplodia theobromae and Phytophthora spp. (Aderiye et al. 1996, Fagbohun et al. 2008). Enterococcus is a lactic acid bacteria (LAB) found in gastrointestinal flora, oral cavity and human vagina. They are widespread in nature and have been detected in the fecal samples from humans, lower vertebrates and insects (David et al. 2012). Enterococci has been reported to produce bacterocins; an extracellular macro-molecular protein/peptides which exert a lethal effect on bacteria or the related groups (Papagiani et al. 2004). Bacteriocins as antimicrobial peptides could be a better replacement to chemical fungicides. All species of enterococci are capable of producing bioactive bacteriocins named as enterocin (Gilmore et al., 2002). Bacteriocins have been reported to act against both related species and distantly related genera (Vidaver et al. 1972, Okkers et al. 1999). They act on food-borne pathogenic and spoilage micro-organisms and in the recent time, their activity against plant pathogens was reported (Schillinger et al. 1996). The potential of bacteriocin from B. subtilis has antagonistic and bactericidal effects on Agrobacterium spp., the causative agent of crown gall. The proprieties of bacteriocins indicated that they have a strong potential to be used in biological control of crown gall disease (Hammami et al. 2009). Fungi have been reported to cause numerous diseases in plants. Some of the chemicals used to control these diseases bio-accumulate in the plants and eventually enter food chains. Current campaign for the fungicide-free fruits and vegetables products, and rise in fungal resistance to common chemo- control agents necessitate the search for alternative control methods for myco- phyto-pathogens. In this study we evaluated the anti-fungal ability of entrocins produced by two strains of Enterococcus faecalis. The antimicrobial spectrum and some properties of the bacteriocins are described and their anti- fungal properties against some phytopathogenic fungi were also studied. 2 Materials and Methods 2.1 Preparation of Cell Free Supernatant (CFS) Two Enterococcus faecalis strains were collected from the stock cultures maintained in the Department of Microbiology, Ekiti State University, Ado- Ekiti, Nigeria. The organisms were separately revived in de Man Rogosa and Sharpe (MRS) broth. The broth was incubated at 37oC for 24 h after which it was centrifuged for 10 min at 10,000 g at 4ºC. The supernatant was decanted gently and later filtered through a membrane filter with a pore size of 0.22 O.M. David and O.E. Onifade Effects of enterocins on phytopathogenic fungi Ruhuna Journal of Science 162 Vol 9(2): 160-168, December 2018 μm. The interfering effects of peroxides and organic acids in the CFS were eliminated by addition of 1 N NaOH and 130 U/mL of catalase (Sigma Chemical Co., St. Louis, MO, USA) respectively. 2.2 Determination of antibacterial activity of CFS The bacteria used in the primary screening of the enterocin include Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus. They were collected from the Department of Microbiology, Ekiti State University, Ado-Ekiti. The organisms were grown for 18 h at 37oC and the turbidity adjusted to 0.5 McFarland Standard. Antibacterial potential of the CFS was determined using agar-well diffusion assay. The reciprocal of least serial dilution of CFS with antibacterial activity was taken to be activity unit (AU) as described by David et al. (2017). 2.3 Partial Purification of enterocin by Ammonium sulphate precipitation To saturation level, ammonium suphate (ranging between 60 and 90 %) was added to 50 ml of CSF with constant stirring and kept overnight at 4oC. The solution was centrifuged at 10,000 g for 20 min at 4oC and later dissolved in 500 ml of 20 mM sodium phosphate buffer (pH 5.0). The supernatant was stored at 4oC until used. 2.4 Determination of protein content of the enterocin produced The protein content of the CFS was determined according to Bradford (1976). The optical density of each of the samples was calculated from the bestfit equation line obtained from the graph of the Bovine Serum Albumin (BSA) standard curve. 2.5 Source of phytopathogenic fungi Fungi isolates primarily isolated from infected plants were collected from the stock cultures maintained at the Department of Microbiology, Ekiti State University, Ado-Ekiti, Nigeria. The fungi include Aspergillus niger, Botrydiplodia theobromae, Fusarium oxysporum, Penicillium expansum and Pythium ultimum. The test fungi were maintained on slants of Potato Dextrose Agar at 4oC until use. O.M. David and O.E. Onifade Effects of enterocins on phytopathogenic fungi Ruhuna Journal of Science Vol 9(2): 160-168, December 2018 163 2.6 Determination of antifungal property of enterocin The partially purified enterocin was sterilized by filtering it through filters with 0.22 μm pore size and the 2 ml of the filtrate was added into 10 ml of sterile potato dextrose broth. The broth was incubated at 37°C for 24 h and the sterile enterocin did not produce any turbidity. The anti-fungal activity of the different extracts of the partially purified enterocins was determined according to poisoned food assay method described by Nene and Thapilyal (2002). At the right concentrations, the sterile extract was mixed with sterilized Potato Dextrose Agar (PDA) just before the setting of the agar. Agar plug (10 mm) from the advancing edge of five-day culture of each of the test fungi was inverted on the center of each plate and incubated at 25°C for 96 h. The PDA plate without enterocin was also maintained at the same condition to serve as the control and the experiment was performed in triplicate. The diameter of fungal colony was measured to the nearest centimeter. 2.7 Analysis of data Results of this study were presented as the mean values of the replicates. One- way analysis of variance (ANOVA) was carried out using SPSS 16.0. Significance was accepted at P ≤ 0.05. 3 Results and Discussion Out of four isolates screened for bacteriocinogenic potential, only two of the bacteriocin-producing strains (UNAD 012 and UNAD 046) showed a prominent activity against the test organisms (Table 1). Table 1. Antibacterial activity (inhibition zones in mm) of crude enterocin produced by strains of Enterococcus faecalis. Test organisms Enterocins from E. faecalis strains UNAD 012 UNAD 046 UNAD 019 UNAD 033 Gram positive B. subtilis 10 36 10 - S. aureus 10 15 10 10 Gram negative E. coli 15 16 10 - K. pneumoniae 19 10 10 - O.M. David and O.E. Onifade Effects of enterocins on phytopathogenic fungi Ruhuna Journal of Science 164 Vol 9(2): 160-168, December 2018 Table 2. Activity, protein concentration, yield and fold of selected enterocin. Parameter Purification steps Crude (NH4)2SO4 Ion Exchange UNAD 012 UNAD 046 UNAD 012 UNAD 046 UNAD 012 UNAD 046 Volume (ml) 10 10 5 6 5 2 Activity (AU/ml) 122 158 145 207 95 125 Protein conc. (mg/ml) 15.8 17.2 6.2 6.0 1.4 1.9 Total Activity (AU) 1220 1570 725 1102 285 263 Total Protein (mg) 158 148 31 48 4.2 5.2 Specific activity (AU/mg) 7.7 9.9 23.3 20.3 67.8 40.4 Yield % 100 100 25.4 59.0 3.4 18.4 Purification fold 1 1 3 2.04 8.7 2.9 Fig. 1. Elution profile of bacteriocin UNAD 012 deduced from the determination of bacteriocin activity. The zone of inhibition ranged between 19 and 36 mm against the test organisms. Enterocins UNAD 012 and UNAD 046 have better activity against Gram negative and Gram positive bacteria, respectively. Enterocin produced by strain UNAD 033 had the least effect on the isolates. Enterocins have been reported to inhibit bacteria (Laukova et al. 1993, Casula and Cutting 2002, Foulquie et al. 2003). In this study, four test bacteria were used at the primary screening stage of enterocin production. The bacteria were used to determine O.M. David and O.E. Onifade Effects of enterocins on phytopathogenic fungi Ruhuna Journal of Science Vol 9(2): 160-168, December 2018 165 the potency of the bacteriocins produced by the strains of E. faecalis. Table 2 shows the effects of purification on specific activity of two promising enterocinogenic producing E. faecalis. The specific activity increased with purification processes while a decrease was noticed in the yield, protein concentration and total protein of the enterocins. This observation was comparable to the findings of Whitford et al. (2001). The elution profile of bacteriocins deduced from the determination of bacteriocin activity was represented in Figures 1 and 2. Fig. 2. Elution profile of bacteriocin UNAD 046 deduced from the determination of bacteriocin activity. Compared with the control, enterocin UNAD 012 had a significant effect on P. ultimum at P<0.05. At P<0.05 significant level, the growth of the fungi at 24h differs significantly from those of 72h and at 96h. At the same significant level, the difference of the growth of the fungi at 48 h differs from the growth at 96h. There was a significant difference (P<0.05) on the percentage inhibition of the effects on the enterocin on the test fungi except UNAD 012 on P. ultimum (Table 3). As shown in Table 4, the percentage inhibition of the fungi increased with time of exposure to the enterocins. O.M. David and O.E. Onifade Effects of enterocins on phytopathogenic fungi Ruhuna Journal of Science 166 Vol 9(2): 160-168, December 2018 Table 3. Antifungal activities of enterocins, at their arbitrary units (AU), on selected fungi isolates (radial mycelial in cm). Test organisms Enterocins Time (h) 24 48 72 96 Control 3.30±1.79 5.85±1.78 9.80±3.78 12.65±3.31 P. expansum UNAD 012 2.85±1.02 3.25±1.03 7.80±2.56 7.89±3.97 UNAD 046 1.85±0.45 4.25±1.99 8.80±2.78 9.01±3.78 B. theobromae UNAD 012 1.59±0.89 2.07±1.02 2.90±1.45 3.02±1.34 UNAD 046 2.73±1.06 3.00±1.74 8.56±3.97 9.00±3.45 A. niger UNAD 012 1.10 ±0.98 2.50±1.52 3.50±0.46 3.52±1.49 UNAD 046 2.10±1.16 3.75±1.48 4.65±1.66 5.97±3.34 F. oxysporum UNAD 012 1.50±0.48. 2.20±1.93 4.50±2.09 5.81±3.39 UNAD 046 1.10 ±0.41 2.05±1.68 2.65±1.88 4.14±1.59 Py. ultimum UNAD 012 1.50±0.56 1.50±1.46 1.52±1.34 2.01±1.78 UNAD 046 1.50±0.91 4.00±1.33 4.80±2.94 5.17±3.97 Table 4. Percentage inhibition of the enterocins on test fungi. Test organisms Enterocins Time (h) 24 48 72 96 Control 0 0 0 0 P. expansum UNAD 012 13.64 44.44 20.41 37.63 UNAD 046 43.94 27.35 10.20 28.77 B. theobromae UNAD 012 51.82 64.62 70.41 76.13 UNAD 046 17.27 48.72 12.65 28.85 A. niger UNAD 012 66.67 57.26 64.29 72.17 UNAD 046 36.36 35.90 52.55 52.81 F. oxysporum UNAD 012 54.55 62.39 54.08 54.07 UNAD 046 66.67 64.96 72.96 67.27 P. ultimum UNAD 012 54.55 74.36 84.49 84.11 UNAD 046 54.55 31.62 51.02 59.13 Compared with the control, UNAD 046 had a better inhibitory effect on B. theobromae, A. niger, Penicillium expansum and P. ultimum than UNAD 042. On the other hand, F. oxysporum was more susceptible to UNAD 046 than UNAD 012. These results were similar to those of Aruna and Madhuri (2016), Schillinger et al. (1996) reporting the susceptibility of different fungi (spoilage and pathogenic) to enterocins. Smaoui et al. (2010) reported that Lactobacillus spp. produce bacteriocins that are active against Gram-negative bacteria and also particularly inhibit fungi. Bacteriocin has been proposed to be a promising treatment of plant infections, and its application has been reported to be safe to animals and humans (Cleveland et al. 2001, Ogunbanwo O.M. David and O.E. Onifade Effects of enterocins on phytopathogenic fungi Ruhuna Journal of Science Vol 9(2): 160-168, December 2018 167 et al. 2004, Cole et al. 2006). Very few bacteriocins with antifungal properties have been reported and most enterocins studied have bacteriostatic and bacteriocidal activities on food-borne bacteria pathogens and not mould (Suzuki et al. 1991). 4 Conclusion From this study, we observed that partially purified enterocins produced by E. faecalis had inhibitory spectrum on selected phytopathogenic fungi. Enterocin from Enterococcus faecalis could be a good candidate for biocontrol of phytopathogenic fungi. 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