Browsing by Author "Ratnaweera, P.B."

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    Antibacterial Activities of Endophytic Fungi of Cyperus iria Collected from Matale Distric
    (Uva Wellassa University of Sri Lanka, 2018) Jayasundara, J.M.N.M.; Ratnaweera, P.B.; de Silva, E.D.
    Antibiotic resistance of bacteria has become an ongoing severe human health concern which requires extensive research priority. Endophytic fungi of Cyperaceae family plants are considered as a potential source for isolating bioactive compounds. Hence, the objective of the current study was to isolate endophytic fungi of Cyperus iria and investigate the antibacterial activities of the crude fungal extracts. Healthy C. iria plants were collected from Weragama in Matale district and endophytic fungi were isolated from the surface sterilized roots and aerial parts using five types of media (SYP, YPD, ME, PDA and MEA) enriched with antibiotics. Each pure fungal culture was sub cultured in ten PDA dishes, incubated close to sporulation, extracted into ethyl acetate, filtered and resulting crude extracts were obtained. The crude extracts were tested for antibacterial activity using agar disc diffusion assay against four bacteria, Staphylococcus aureus (ATCC 25928), Bacillus cereus (ATCC 11718), Pseudomonas aeruginosa (ATCC 9027) and Escherichia coli (ATCC 35218) at 400 ug disc-1 concentration where Gentamycin (10 ug disc-1) and methanol (10 IA disc') were used as the positive and negative controls, respectively. Total 34 morphologically distinct putative endophytic fungi, 23 from aerial parts and 11 from roots, were isolated. Thirteen fungal extracts exhibited antibacterial activity against S. aureus, 24 against B. cereus, 12 against P. aeruginosa and one against E. coli. Among all, 29 fungi were active against at least one bacterium tested while five fungi were inactive to all. Activity of three extracts against B. cereus, 12 against P. aeruginosa, and one against E. coli was similar to the activity of the positive control. Fifteen extracts against B. cereus, 27 against P. aeruginosa, 33 against E. coli and 21 against S. aureus showed significant (p < 0.05) antibacterial activities compared to the negative control. In conclusion, C. iria from Matale harbors a lot of endophytic fungi, where several are capable of producing bioactive secondary metabolites with selective antibacterial properties.
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    Antibacterial activity of entomopathogenic fungi isolated from a beetle (Harmonia sp.) in Sri Lanka
    (Uva Wellassa University of Sri Lanka, 2021) Patabedi, S.W.P.N.H.; Ratnaweera, P.B.
    Majority of human pathogenic bacteria has become resistant to the exsisting antibiotics causing a human health crisis in the world. Thus discovering novel antibacterial drug leads has become an urgent concern. Entomopathogenic fungi (EPF) infect insects and kill or disable them. Previous research have shown entomopathogenic fungi exhibit various bioactivities. Thus, the objective of the current study was to isolate entomopathogenic fungi from a beetle, a Harmonia sp. in Sri Lanka and evaluvate their potential of producing antibacterial compounds. EPF were isolated from surface sterilized insect cadavers collected from Balangoda area in Sri Lanka. Isolated fungi were grown in potato dextrose agar and broth media, incubated close to sporulation, harvested and extracted into ethyl aceate. The obtained weights of the crude extracts of solid and liquid cultures were compared using ANOVA. Antibacterial activity of the crude extracts were evaluated using agar disc diffusion bioassays at 400 μg/disc, against two Gram-positive bacteria, Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATTC 11778) and two Gram-negative, Escherichia coli (ATCC 35218) and Pseudomonas aeruginosa (ATCC 9027). The positive control used was Gentamycin and the negative control was methanol. Seven morphologically different EPF were isolated from Harmonia sp. The crude weights of the solid cultures were significantly higher than the crude weights of the liquid cultures (ANOVA, p < 0.05). This result showed fungi have preferred to synthesize more metabolites under solid culture conditions than liquid. Six out of the seven fungi showed antibacterial acitivities against at least one bacterium tested. EPF cultures BET 06, 08 and 10 showed high inhibition zones against the S. aureus and B. cereus. According to ANOVA there was a significant difference between the antibacterial activities of the crude extracts (p < 0.05). None of the fungal extracts showed activity against P. aeruginosa while BET 05, 06 and 08 showed mild activity against E. coli. According to the microscopic characteristics the fungi were tentatively identified as belong to Penicillium and Aspergillus species. A bioautography study reveled the presence of several bioactive compounds in BET 05, 06, 08 and 10 extracts. The results of this study showed that entomopathogenic fungi are potential sources for isolating antibacterial compounds. Keywords: Antibacterial; Entomopathogenic fungi; Harmonia sp.; Penicillium; Aspergillus
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    Antifeedant Activity and Contact Toxicity of the Two Peptaibols, Trichocellin A-I and B-II Isolated from the Endophytic Fungus Trichoderma reesei
    (Uva Wellassa University of Sri Lanka, 2020) Madhushika, D.P.H.; Jayasundara, J.M.N.M.; Ratnaweera, P.B.; de Silva, E.D.
    Plutella xylostella is a destructive cabbage pest responsible for massive economic losses worldwide. The use of synthetic pesticides in huge amounts for its control has resulted in pesticide-resistant strains, detrimental human health impacts, and environmental problems. Consequently, the development of alternatives with minimal such undesirable side-effects becomes crucial. Biopesticides derived from microbes are one such alternative. In a preliminary study, the crude extract of the endophytic fungus Trichoderma reesei isolated from Cyperus iria exhibited prominent antifeedant activities against P. xylostella larvae. The main aim of the current study was to isolate the bioactive compounds of T. reesei and investigate their antifeedant activities and contact toxicities against P. xylostella 2nd instar larvae. Two bioactive peptaibols, trichocellin AI and trichocellin B-II, were isolated from the ethyl acetate extract of T. reesei using a bioassay guided purification method and their structures were elucidated using nuclear magnetic resonance and mass spectral data. To investigate the antifeedant activities choice leaf disc assay was conducted at an initial concentration of 50µg/cm2 using commercial neem (100%) and methanol as positive and negative controls respectively. For contact toxicity assay larval mortality was calculated after 24 h of applying 17.5µg of compounds/larvae. Trichocellin-A-I resulted in a 100% feeding deterrence while the DC50 value was 2.38µg/cm2 . Trichocellin B-II also gave a feeding deterrence index of 100% for choice leaf disc assay. For the contact toxicity assay, Trichocellin A-I and B-II showed 40% and 50% mean motility percentages respectively while the respective values for positive and negative controls were 100% and zero. This study revealed that the endophytic T. reesei produces two biopesticides, trichocellin A-I and trichocellin B-II, that exhibit potent antifeedant activity and moderate contact toxicity against P. xylostella larvae. Keywords: Plutella xylostella, Trichoderma reesei, Choice leaf disc assay, Contact toxicity
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    Antifungal Activity of Bacillus amyloliquefaciens Ethyl Acetate Extract and Fractions Against the Fungus Khuskia oryzae
    (Uva Wellassa University of Sri Lanka, 2018) Lohanathen, G.P.; Jayasundara, J.M.N.M.; Ratnaweera, P.B.; de Silva, E.D.
    Fungal infections are common among plants and animals which result in economic losses. Finding new antifungal agents from alternative sources may help to solve the above issue. It was observed that, in culture Bacillus amyloliquefaciens isolated from a contamination shows antifungal activity against the fungus Khuskia oryzae. Therefore, the objective of the current study is to determine the antifungal activity of the crude ethyl acetate extract and the fractions of crude extract of B. amyloliquefaciens against the fungus K oryzae. B. amyloliquefaciens was grown on Luria-Bertani Agar (LBA), extracted into ethyl acetate after an incubation period of three days and the antifungal activity of the crude extract was tested against K. oryzae at 400 lig disc-1 using agar disc diffusion method. Crude extract of 1.5 g was first fractionated by Kupchan solvent-solvent partitioning scheme, sequentially using hexane, methanol/water (9:1); chloroform, methanol/water (6:4) and ethyl acetate, water. Antifungal activity of the three fractions hexane, chloroform and ethyl acetate was determined and the chloroform fraction was active against K. oryzae. The active fraction was further purified using Sephadex LH2O size exclusion chromatography using methanol as the eluent. Fractions were combined according to the thin layer chromatography (TLC) profiles and the antifungal activity was tested for the combined fractions (A-F). Flucanozole and methanol was used as the positive and negative controls respectively. Fraction C (32.8 mg) resulted from size exclusion chromatography of the chloroform fraction exhibited 18 mm radius inhibition zone against K. oryzae while none of the other fractions showed any activity. Activity of fraction C was similar (p > 0.05) to the activity of the positive control. However, TLC profile of the fraction C showed the presence of more than one compound. Thus, further purification of fraction C is necessary in order to isolate the active compound/s which may lead to a potential antifungal agent.
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    Effect of Aerva lanata in Controlling Root-Knot Nematode Meloidogyne incognita of Tomato in Sri Lanka
    (2011) Nanayakkara, G.D.T.M.; Ratnaweera, P.B.; Karunathilake, A.A.K.
    Introduction The root knot nematodes (Meloidogyne syy.) are a group of endo-parasites which are among the most damaging agricultural pests, attacking a wide range of crops worldwide. In Sri Lanka, tomato (Lyeopersicon esciileiiiutn commercial and a widely grown vegetable which is often severely prone to attack by root-knot nenj6t0de, M lDi‹1 iii cogri/‹z. Other than in tomato, the Meloi’Jog 5'n e syy. Cause serious reduction in yield in several economically important plants such as potato, chilies, okra, mung-bean, rice, tea and tobacco (Akfitar, 2000). Nematode control is mainly based on, chemica1 nematicides, which present potential risk on non-target organisms and the environment. Their high costs, non-availability at the time of need and the hazards they pose, discourage most potential users. In the search for more environmental to chemicals, possibilities are being investigated to exploit nematode-anatagonistic plants for nematode control. Leaf extracts of certain plants are known to have nematicidal or nematostatic properties against several plant parasitic nematodes (Gapasin c/ at., 2002). The nematicidal activity of the plant extracts can leads for development of plant-based agrochemicals. Weeds are usually aggressive growers with the presence in large quantities. These plants species may therefore contain active biological compounds to resist various nematode infections. Biological nematicides prepared with weed plant extracts have the advantage of readily availability, low cost and environmental safety over other conventional nematicides. Therefore, the main objective of this study was to evaluate the effect of Aeixa lonala (Ameranthaceae) weed p l ant species for nenaaticidal activity against MeloiJog tie incognita, root-knot nematode collected front tomato.
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    Isolation of Antibacterial Compounds from an Entomopathogenic Fungus Isolated from a Beetle, a Harmonia sp.
    (Uva Wellassa University of Sri Lanka, 2021) Abivarna, S.; Patabedi, S.W.P.N.H.; Ratnaweera, P.B.
    Antibiotic resistance of bacteria is a major human health concern in the world which requires research priorities. Therefore discovering new and effective antibacterial drug leads has become an urgent necessity. Entomopathogenic fungi act as parasites of insects which kill or disable them. However these fungi are known to synthesize various bioactive secondary metabolites. Thus, the objective of the current study was to isolate the antibacterial compounds from an entomopathogenic fungus isolated from a beetle (Harmonia sp.) in Sri Lanka. The fungus BET 05, which was microscopically identified as an Aspergillus sp. was grown in potato dextrose agar (PDA) and broth (PDB) media. After an incubation period of 06 days, both solid and liquid fungal cultures were extracted into ethyl acetate (EtOAc). The solid culture (30 large and 13 medium PDA dishes) yielded 804.8 mg of crude while the liquid culture (400 mL x 3) gave 436.7 mg. The solid and liquid crude extracts were subjected to an agar disc diffusion bioassay to determine the antibacterial activity and the inhibition zones diameters were 11.5 and 11.3 mm respectively, against the Gram positive Staphylococcus aureus (ATCC 25923) at 400 μg/disc concentration. The positive control used was Gentamycin, while the negative control was methanol. The main active compound in the extract was isolated using bioassay guided chromatographic techniques. The crude extract (436.7 mg) was first fractionated by solvent-solvent partitioning, using hexane and methanol/water (9:1), chloroform and methanol/water (6:4) and, EtOAc and water. The active chloroform fraction was purified by Sephadex LH20 size exclusion chromatography using methanol as the eluent. Fractions collected were grouped according to their TLC profiles and the combined fractions (A-F) were tested for antibacterial activity using a bioautography. The active fraction B was further purified by normal phase silica chromatography using 60:40 hexane: EtOAc to methanol solvent systems. A bioautography revealed, the combined fraction E contains main active compound while fraction C and G are also having some active compounds. Finally a preparative TLC was conducted using EtOAc as the mobile phase to get the main active compound purified (4.1 mg). This study reveals that entomopathogenic Aspergillus sp. (BET 05) is a potential producer of antibacterial compounds. Keywords: Antibacterial; Antibiotic resistance; Entomopathogenic; Aspergillus
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    Isolation of Antibacterial Compounds from the Endophytic Fungus Curvularia lunata
    (Uva Wellassa University of Sri Lanka, 2019-02) Pavalakantharasa, T.; Jayasundara, J.M.N.M.; Ratnaweera, P.B.
    Bacteria showing resistance to the existing antibiotics have become a human health crisis in the World. Thus there is an essential need to find novel antibacterial compounds as a solution to this antibiotic resistance issue. Cyperaceae family plants are rich reservoirs of endophytic fungi which are potential synthesizers of antibacterial secondary metabolites. Purification of the antibacterial compound/s from an endophytic fungal strain isolated from Cyperus iria was the aim of this study. A crude extract of an endophytic fungal culture which was identified as Curvularia lunata showed antibacterial activity against Gram Positive Staphylococcus aureus and Bacillus subtilis. Therefore, this fungus was grown in 150 Petri dishes using potato dextrose agar, incubated for 21 days, extracted into ethyl acetate and the antibacterial activity of the crude extract was tested against S. aureus and B. cereus at 400 µg/disc concentration. Purification of the active compounds was carried out using bio assay guided chromatographic methods. The crude extract (420 mg) was first fractionated by solvent-solvent partitioning, using hexane, chloroform, ethyl acetate and water as the solvents. The active chloroform fraction was further purified by Sephadex LH20 size exclusion chromatography using methanol and eluted fractions were combined according to their TLC profiles. The combined fractions (A-E) were tested for antibacterial activity. Fraction C from size exclusion chromatography showed antibacterial activity with 13 and 16 mm inhibition zones against the S. aureus and B. cereus respectively, Fraction D showed antibacterial activity with 11 mm inhibition zone against the B. cereus at 400 µg/disc. The TLC profiles showed fraction C is close to purity while fraction D consists of many compounds. According to the proton NMR spectrum, the fraction C is an aromatic compound. Further purification of fractions may lead to a potential antibacterial agent.
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    Removal of Lead Metal Ion from Aqueous Solutions Using Zinc Oxide Nanoparticles and Zinc Oxide Bulk Material
    (Uva Wellassa University of Sri Lanka, 2011) Sivayoganathan, N.; Ratnaweera, P.B.; Weerawarna, P.M.; Karunathilake, A.A.K.
    Note: See the PDF Version Many toxic heavy metals have been discharged into the environment as industrial wastes, causing Serious soil and water pollution. Toxic metal compounds coining to the earth's surface not only reach the earth's waters but can also contaminate underground water in trace amounts by leaking front the soil after rain. Therefore, drinking water obtained front springs which may also be contaminated by various toxic metals. Pb", Cu", Fe", and Cr"‘are especial I y common materials that tend to accumulate iii organisms, causing numerous ‹diseases and disorders (Inglizakis e/ at., 2002). Among these lead (Pb’ highly toxic heavymetal which adversely affects the red blood cells of the human nervous system and kidneys (Potgieter et al., 2006). According to World Health Organization, the maximum in permissible limit of lead in drinking water is 0.05 mg/L (Kanawade and Gaikwad, 201 I). The adsorption of heavy metal ions onto various solid supports such as activated charcoals, ion exchange resins, zeolites and ion chelating agent’s immobilized on inorganic supports is the most common route among the different techniques applied to remove dissolved heavy metals front waste water and industrial effluents (Chen et al., 2003). Many methods using today for decontamination of waste water are not suitable iii developing countries due to the high costs associated with production. Therefore, the use of alternative low-cost materials us potential sorbents for the removal of heavy meals should be investigated. Among inorganic nanoparticles, the zinc oxide (ZnO) nanoparticle has received great attention because of its unique catalytic, antibacterial, electrical, electronic and optical properties as well as its low cost and extensive applications (Kathirvelu el at., 2009). Zinc ox ide bulk materials and zinc oxide nanoparticles are widely used in industry and dai Iy life for various things including as absorbents for gases such as CO, CO, O , H2. SO„ CHP (Scarano et al., 2004). Therefore, the main aim of this study was to investigate the possibility of using ZnO nanoparticles and ZnO bulk material for the removal of Pb" from aqueous solutions by adsorption,
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    Studies Towards the Isolation of Antibacterial Compounds from Endophytic Fungi of Cyperus rotundus
    (Uva Wellassa University of Sri Lanka, 2018) Fazeen, S.K.; Herath, S.D.; Jayasundera, J.M.N.M.; Ratnaweera, P.B.; de Silva, E.D.
    Endophytic fungi are a relatively new source for the isolation of antibiotics effective against resistant pathogenic bacterial species. The main aim of this study is to investigate the antibacterial producing potential of endophytic fungi of Cyperus rotundus with a view to isolating novel antibiotics. Initially, antibacterial activities of the crude ethyl acetate extracts of endophytic fungi isolated from surface sterilized C. rotundus were evaluated using an agar disc diffusion assay against Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa and Escherichia coli. Next the fungus with the most promising overall activity, identified using molecular techniques, was grown in 200 PDA Petri dishes and bioassay guided fractionation of its crude extract was conducted to isolate the active compound/s. Out of the 13 isolated endophytic fungi 11 fungal extracts were active against at least one bacterium tested while the highest activity (15 mm zones of inhibition against B. cereus and S. aureus) was shown by an endophytic Agrocybe sp. The ethyl acetate (EtOAc) crude extract (280 mg) of cultured Agrocybe sp. was partitioned between hexane, chloroform, EtOAc and water and the resulting fractions were subjected to antibacterial assay at 200 𝜇g disc-1 which revealed hexane as the most active fraction. Next, the hexane fraction (100 mg) was further purified by normal phase silica chromatography using a gradient elution. Bioassay for the fractions combined according to the TLC profile, showed that fraction B and D to be active with 16 and 20 mm inhibition zones respectively against S. aureus at 400 𝜇g disc-1. TLC of fraction D showed the presence of a single compound while fraction B consisted of more than one compound. Continued research towards further purification of fraction B is in progress. Spectroscopic data will reveal the structures of the active compounds. This study has shown that there is a good potential for the discovery of antimicrobial compounds from endophytes of C. rotundus which may in turn contribute towards developing new clinically useful antibiotics.