Browsing by Author "Jayasundara, J.M.N.M."

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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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    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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    A Comparison of Richness, Diversity and Antibacterial Producing Capacity of Endophytic Fungi of Cyperus iria from Three Geographical Locations in Sri Lanka
    (Uva Wellassa University of Sri Lanka, 2019-02) Jayasundara, J.M.N.M.; Ratnweera, P.B.; de Silva, E.D.
    Endophytic fungi living inside healthy tissues of plants are a well-established source of various biologically active secondary metabolites. Although sedges are known to harbor numerous endophytic fungi, endophytes inhabiting sedges of the family Cyperaceae have been sparsely investigated. Therefore, the current study was designed to investigate the endophytic fungal richness, diversity and the antibacterial producing capacity, of the Cyperus iria from three geographically distinct locations in Sri Lanka. Endophytic fungi were isolated from surface sterilized aerial and root segments of healthy C. iria collected from Badulla, Matale and Colombo Districts and crude ethyl acetate fungal extracts were tested for antibacterial activity against four selected bacteria; Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa and Escherichia coli at 400 μg/disc concentration using agar disc diffusion method where Gentamycin (10 μg/disc) and methanol were used as the positive and negative controls, respectively. Isolated endophytic fungi were identified using molecular techniques. Endophytic fungal richness of C. iria from the three locations namely Badulla, Matale and Colombo were 17, 34 and 23 respectively and 12, 23 and 13 fungi were isolated from aerial parts while 05, 11 and 10 fungi were isolated from root segments. Accordingly, more fungi were isolated from aerial parts than from roots. Among the isolated fungi Fusarium, Curvularia, Trichoderma, Penicillium species were the most abundant. With respect to antibacterial activity, all endophytic fungi isolated from Badulla plants, 85 % from Matale plants and 82 % from Colombo plants showed activity against at least one bacterium tested. Also irrespective of the location of the plant most fungal extracts showed activity against the Gram positive than the Gram negative bacteria. In conclusion, endophytic fungi of C. iria from all three locations showed a high fungal richness and a majority showed antibacterial activity.
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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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    Usability of Fish Haematological Parameters as a Biological Indicator for Freshwater Quality in Badulla District
    (Uva Wellassa University of Sri Lanka, 2016) Jayasundara, J.M.N.M.; Liyanage, N.P.P.; Wijesundera, R.R.M.K.K.; Senevirathne, J.D.M.; De Silva, D.P.N.
    Water, the fundamental of life, requires continuous monitoring because water quality deteriorates over time. Fish haematological parameters respond more quickly for environmental changes with more precise results than chemical analysis which conduct periodically. As it is profitable to authenticate eco-friendly method for assessing water quality, this study was conducted to investigate the usability of fish haematological parameters as a bioindicator for assessing freshwater quality. Comparison of physico¬chemical parameters of water quality was done in two inland static water bodies in Badulla district: Horabora and Mapakada reservoirs. Results of present study revealed that Transparency, Total Dissolved Solids, Biochemical Oxygen Demand (BUD), pH, Total hardness and Trace metal (As, Pb and Cu) levels were significantly different between Horabora and Mapakada reservoirs (p<0.05). No statistical difference was found for total alkalinity, Dissolved Oxygen level, and Surface water temperature (p>0.05). Both water bodies were identified as polluted in terms of Universal Water Quality Index, although Horabora reservoir was much more polluted than Mapakada reservoir by resulting higher BOD, Total Dissolved Solids, Total hardness and trace metal levels (Pb and As) with less clear water. Blood from Tilapia (Oreochromis mossambicus), Labeo rohita and Calla catla was used for fish haematology analysis. Red blood cell count was significantly different (p<0.05) between two different water bodies for each fish species. Eosinophil count showed a significant difference among two water bodies (p<0.05). The study revealed that a great possibility exists for using fish haematological parameters as a bioindicator for freshwater quality. Moreover, a multi-year continuous study with elaborated investigation require for clarify trends which may exist between fish haematological parameters with respect to water quality changes. Keywords: Water quality study, Haematology, Tilapia, Eosinophil, Trace metal