IRSPAS 2019
Permanent URI for this collectionhttp://repository.kln.ac.lk/handle/123456789/20453
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Item Bioactive properties of Rambutan (Nephelium lappaceum L.) and Durian (Durio zibethinus Murr.) peel extracts(4th International Research Symposium on Pure and Applied Sciences, Faculty of Science, University of Kelaniya, Sri Lanka, 2019) Silva, A.G.; Wickramaarachchi, S.; Attanayake, R. N.; Rajapakse, C. S. K.Although these fruits are seasonal, a considerable amount of Rambutan (Nephelium lappaceum L.) and Durian (Durio zibethinus Murr.) fruit residues (mainly peels) are collected each year as waste materials, due to the significant volume of trade of these fruits. Therefore, present work was initiated with the aim of evaluating the impact of these residues lying as waste and possible re-use, by investigating the bioactive properties in peels of rambutan and durian. Methanol extracts of Rambutan and Durian peels were sequentially extracted with hexane, chloroform and methanol. First, methanol extracts of peels were subjected to phytochemical screening following standard procedures and results revealed that rambutan and durian peels were rich in polyphenols, flavonoids, steroids, coumarin, etc. Total phenolic content (TPC) and total flavonoid content (TFC) of methanol extracts of peels were determined using Folin-Ciocalteu and aluminium chloride method, respectively. Results showed that TPC and TFC in methanol extract of durian peels were (11.39 ± 0.49 mg GAE/g dry weight, 257.20 ± 5.14 mg Catechin /g dry weight) higher than those of Rambutan peels (2.73 ± 0.15 mg GAE/g dry weight, 198.00 ± 1.89 mg Catechin /g dry weight). Further, the antioxidant activity of methanol extracts of peels and its fractions were investigated using 1, 1-diphenyl-2-picryl-hydrazyl free radical (DPPH) scavenging assay and the correlation with their TPC and TFC were examined using Pearson’s correlation analysis. The strongest antioxidant activity was observed in hexane fraction of Rambutan peels with IC50 value of 13.49 ± 0.52 μg/mL, and which was lower than that of the control, butylated hydroxytoluene (43.70 ± 0.89 μg/mL). Significant positive correlations were observed between TPC and TFC in fractions of rambutan and durian peels with their DPPH radical scavenging activity indicating that phenolics and flavonoids in rambutan and durian peels may contribute to their strong antioxidant activities. The antimicrobial activity of the extracts of peels and its fractions were assessed by using disc diffusion method against a bacterial species of Staphylococcus aureus and fungal species, Fusarium oxyporum and Aspergillus flavus. The lowest concentration of methanolic extract of durian and rambutan peels that showed an inhibition against Staphylococcus aureus was 31.25 μg/mL. It was also found out that neither peels of rambutan nor durian had antifungal activity against the two selected fungal species. Results revealed that the peels of rambutan and durian are potential sources of antioxidants and antibacterial agentsItem Phylogenetic relationships of selected commercial Dendrobium hybrids in Sri Lanka(4th International Research Symposium on Pure and Applied Sciences, Faculty of Science, University of Kelaniya, Sri Lanka, 2019) Kahagalla, T. H.; Herath, H. M.; Attanayake, R. N.; Senanayake, S. P.Nuclear rDNA-ITS regions and chloroplast matK genes are useful in delineating plant species. In this study, genetic relatedness of eight commercial Dendrobium hybrids (A-H) with a range of attractive flower colours was studied using nuclear rDNA-ITS and chloroplast matK sequences. Genomic DNA was extracted from fresh, young leaves using a modified cetyltrimethylammonium bromide based protocol. rDNA-ITS and matK were amplified using PCR in 25 μl reactions containing 1X PCR buffer, 2.5 mM MgCl2, 0.2 mM dNTPs, 1.25 U Taq DNA polymerase, 0.4 μM forward and reverse primers and 1.00 μl of genomic DNA. The optimized thermal cycling conditions were initial denaturation at 95 oC for 5 minutes, 35 (rDNA-ITS) and 40 (matK) cycles of denaturation at 95 oC for 40 seconds, annealing at 55 oC (rDNA-ITS) and 48 oC (matK) for 40 seconds, extension at 72 oC for 40 seconds and final extension at 72 oC for 10 minutes. rDNA-ITS and matK PCR products were subjected to Sanger sequencing. Sequences were manually edited using BioEdit 7.0.5.3. and ContigExpress software. Sequences were aligned to the nucleotide database in the National Center for Biotechnology Information using mega BLAST program. Forty-three related sequences were obtained from GenBank and the sequences were aligned using ClustalW implemented in MEGA 7.0.26 software. Phylogenetic analysis was performed by generating trees of ITS, matK and concatenated sequences of ITS and matK. The phylogenetic relationships were analyzed using Maximum Likelihood analysis with 1000 bootstrap replications. Phalaenopsis aphrodite, Liparis kumokiri and Malaxis spicata were used as outgroups. Combined gene-tree was estimated using RAxML-HPC BlackBox tool in CIPRES Science Gateway platform. Resulting trees were viewed using Figtree v1.4.3. In the combined gene tree, selected hybrids were clustered into two distinct groups. Dendrobium hybrids A, B, C, E and F were clustered with Dendrobium bigibbum var bigibbum and Dendrobium phalaenopsis (72% bootstrap). Hybrids G, H and D were clustered with Dendrobium nindii and Dendrobium taurinum (79% bootstrap). In matK gene tree, all the selected hybrids were clustered together with Dendrobium kingianum (90% bootstrap). In rDNA-ITS gene tree, hybrids A, B, C, E and F were clustered with Dendrobium bigibbum var bigibbum and Dendrobium phalaenopsis while hybrids D, G and H were clustered with Dendrobium taurinum and Dendrobium nindii (81% bootstrap). Therefore, though high variation in floral morphology is observed among the selected imported commercial hybrids, they were represented from a narrow genetic background. This is an indicative of genetic bottleneck most likely due to selective breeding and it is important to incorporate more diverse varieties in future breeding programs to maintain a diverse genetic backgroundItem Effect of the fungicide manganese ethylene-bis-dithiocarbamate on in vitro growth of Agrobacterium tumefaciens(4th International Research Symposium on Pure and Applied Sciences, Faculty of Science, University of Kelaniya, Sri Lanka, 2019) Somarathna, G. M. T. K.; Somachandra, K. P.; Attanayake, R. N.Agrobacterium is a soil borne, Gram negative, rod shaped, motile and aerobic bacterium causing tumors on a wide range of plant species. Out of many soil inhabitant Agrobacterium species, only the pathogenic strains of Agrobacterium tumefaciens can cause the crown gall disease. Although antibiotics and growth promoters have been recommended in other countries, no proper control measures have been developed and allowed in Sri Lanka for bacterial infections in plants. The objectives of this research were to identify the virulence strains of A. tumefaciens using DNA sequence data, to determine whether commonly used antibiotics have an inhibitory activity against A. tumefaciens isolated from soil and also to find a cheap control measure based on the fact that certain fungicides have antibacterial effects. For molecular characterization, PCR was carried out using Agrobacterium specific primers targeting virD2 gene. Antibiotic sensitivity was determined by disc diffusion method using Kirby-Bauer technique. Concentration series of 25, 30 and 35 μg/mL of kanamycin and chloramphenicol and 5, 10, 15 μg/mL of rifampicin were prepared. Fungicide assay was also conducted by disc diffusion method using the fungicides Mancozeb (Manganese ethylene-bis-dithiocarbamate) at 0.20%, 0.25%, 0.30% (w/v) and Carbendazim (Methyl benzimidazole-2-yl carbamate) at 0.025%, 0.05%, 0.10% (w/v) concentrations. Plates were incubated at 28 °C for 48 hours. The soil isolates were confirmed to be A. tumefaciens from the sequencing results of virD2 region. In antibiotic sensitivity test all isolates were unable to produce clear zones showing that they were resistant to the three different concentrations of all three antibiotics used. Carbendazim could not inhibit the growth of A. tumefaciens isolated from soil at all three concentrations tested. However, the fungicide Mancozeb was able to inhibit the growth of the pathogen in all tested concentrations. According to the results it was concluded that the local isolates of A. tumefaciens is resistant to the tested antibiotics and therefore, cannot be used as a control measure. While Carbendazim is not helpful in controlling the growth of A. tumefaciens, Mancozeb has a potential to mitigate the in vitro growth of the pathogen