International Postgraduate Research Conference (IPRC)
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Item Potential Antibacterial Secondary Metabolites from an Enadolichenic Fungus Inhabiting a Lichen Collected from Negombo Lagoon, Sri Lanka(International Postgraduate Research Conference 2019, Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2019) Weerasinghe, W.R.H.; Gunawardhana, M.H.A.Y.; Wickramarachchi, S.; Attanayake, R.N.; Weerakoon, G.; Paranagama, P.A.With the revelation of intriguing bioactive properties from natural sources, the thirst to investigate more into the mysteries of nature has immensely expanded. Endolichenic fungi (ELF) became an interesting source during the recent past in this search for novel bioactive compounds and literature sources provide evidence of many such bioactive compounds isolated from these interesting organisms. These ELF asymptomatically live inside the lichen thallus which is formed by the symbiotic relationship of fungi with an algae or a cyanobacteria. A total of 31 lichens were collected from mangrove plants in Negombo lagoon and their molecular identification revealed that they belonged 10 different species. Healthy lichen thalli were surface sterilized and were cut into small segments and plated on 2% Malt Extract Agar (MEA) medium supplemented with 0.01% streptomycin in order to obtain ELF. The obtained pure cultures of ELF were identified using molecular techniques. DNA was extracted using CTAB method and its quality and quantity were determined by agarose gel electrophoresis. DNA was diluted accordingly and was subjected to Polymerase Chain Reaction (PCR) to amplify fungal ITS rDNA region using universal primers. PCR amplification was tested using agarose gel electrophoresis and the full sequences were obtained. Ethyl acetate crude extracts of 18 such identified ELF strains were subjected to anti-bacterial assay against Escherichia coli and Staphylococcus aureus using agar well diffusion method. The species Xylaria feejeensis isolated from the lichen Graphis librata showed remarkable activity against the two bacterial strains on par with the positive control Azithromycin. The assay was carried out using 100 μl of the extract and the positive control (5 mg/ml). The inhibition zone diameters (in cm) against E. coli and S. aureus for the fungal crude were 1.9 and 2.2 respectively and for Azithromycin was 2.2 against both. In order to isolate the active compounds, a larger crude of the same was obtained and partitioned into Hexane, Chloroform and Methanol fractions based on polarity. The assay results for the three fractions revealed that only Hexane and Chloroform fractions possessed anti-bacterial potentiality. Subsequently, silica gel normal phase column chromatography was performed for further fractionation. Collected 6 fractions from the column for Chloroform fraction showed inhibition diameters of 1.9, 1.9, 1.5, 1.2, 1.3, 1.1 against E. coli comparable with 2.2 of Azithromycin and 2.4, 2.2, 1.4, 0.0, 0.7,0.0 against S. aureus comparable with 2.5 of Azithromycin. The fraction 1 and 2 showed highest activity against both bacterial strains and fraction 4 and 6 showed lowest activity against E. coli and none against S. aureus. Further isolation is being carried out for active fractions and the structures of obtaining active compounds will be elucidated using spectroscopic methodsItem Identification and Disease Assessment of Begomovirus Infecting Capsicum frutescens (Kochchi) in Minuwangoda, Sri Lanka(International Postgraduate Research Conference 2019, Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2019) Jayathilaka, S.A.S.D.; Wanigatunge, R.P.; Attanayake, R.N.; Edirisinghe, P.Leaf curl of Capsicum frutescens (Kochchi) is a common problem in many Capsicum growing regions of the country including Minuwangoda area in Gampaha. Water stress, poor soil drainage or viral infection are some of the potential causes of leaf curl. The main aim of the current study was to determine whether the leaf curl of Minuwangoda area is due to viral infection and to determine the disease severity and incidence of the region. A disease assessment key graded from 0 to 6 was developed for CLC disease of C. frutescens. Thirtytwo C. frutescens fields having more than 50 plants per field were selected. Disease symptoms, incidence and severity were recorded. Prominent symptoms of the disease were leaf curling, vein yellowing and leaf yellowing followed by severe chlorosis and stunting as the disease progressed. These symptoms were similar to the viral infection. The disease incidence varied from 0 to 100% in the selected fields and 71.88% of the fields in Minuwangoda area were affected. Disease severity in the selected fields ranged from 0 to 6, while the average disease severity of the fields in the area was 1.3. To determine whether the causal agent is a commonly reported virus of the family Begomoviridae, coat protein (CP) gene was amplified from the DNA extracted from infected leaves using CP specific primers and the sequencing was done using the Sanger sequencing method. Sequence alignment with the NCBI database showed a 95% similarity to Chilli leaf curl virus (Begomoviridae) isolate CL-14 from Nochchiyagama. Results indicates that Sri Lankan C. frutescens cultivation could be challenged by the viral infection and control measures should be implemented as soon as possible.Item Germinability and Viability of Pollen of Selected Commercial Dendrobium Hybrids(International Postgraduate Research Conference 2019, Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2019) Kahagalla, T.H.; Herath, H.M.; Attanayake, R.N.; Senanayake, S.P.Dendrobium is one of the highly demanding ornamental orchid genera in Sri Lankan floriculture industry. The assessment of functional quality of the pollinium on the basis of germinability and viability is useful for the development of breeding programs for crop quality improvement and pollen storage protocols in conservation. This research was carried out to determine in vivo and in vitro pollen germinability and pollen viability of eight commercial Dendrobium hybrids (A-H). Percentage of pollen viability was studied by placing crushed pollinia on a cavity slide with 1% 2,3,5-triphenyltetrazolium chloride solution and incubating at dark for 12 hours. Pollinia of Dendrobium flowers were placed on the stigma of the same flower on the second day after opening. After three days, pollinia were stained with lacto phenol cotton blue and percentage of in vivo pollen germination was calculated. In vitro pollen germination of the Dendrobium hybrids was studied in three different media. Pollinia were removed from the anthers on the second day after flower opening and placed in (i) 5%, 10%, 15%, 20% and 25% sucrose solutions and (ii) a semi solid medium containing 10% sucrose, 100 mgL-1 H3BO3, 300 mgL-1 Ca(NO3).2H2O, 200 mgL-1 MgSO4.7H2O, 200 mgL-1 KNO3, 8 gL-1 agar and stigmatic fluid of Dendrobium flower and incubated at room temperature. Pollinia of Dendrobium hybrids A, G and H placed in a (iii) semi solid medium containing 1%, 2%, 3% sucrose, 75 mgL-1 H3BO3 and 0.5%, 1%, 2% agar were incubated at 24 oC, 26 oC, and 28 oC. After 48 hours, percentage of in vitro pollen germination was calculated. Means were compared using one-way ANOVA with Tukey’s pairwise comparison test in IBM SPSS Statistics 22 software. As the percentage values of pollen germination and viability were not normally distributed, the values were transformed to arcsin square root values prior to statistical analysis. There was no significant deference in pollen viability of different hybrids which varied between 94% and 77%. The highest percentage of in vivo pollen germination was found in Dendrobium cultivar D (67%) and there was a significant deference in in vivo pollen germination among the hybrids. Correlation analysis was performed using IBM SPSS Statistics 22 software to determine the relationship between pollen viability and in vivo pollen germination and found that there was no significant relationship (�= 0.237). The percentage of in vitro pollen germination was low and the highest in vitro pollen germination (7.2%) was observed in the semisolid medium containing 1% sucrose, 75 mgL-1 H3BO3, 1% agar and incubated at 26 oC for Dendrobium cultivar H.