International Research Symposium on Pure and Applied Sciences (IRSPAS)
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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 pathogenItem Morphometry and pollen germinability of selected commercial Dendrobium cultivars(2018) Kahagalla, T. H.; Hearth, H. M.; Attanayake, R. N.; Senanayake, S. P.Dendrobium is one of the highly demanding ornamental plant genus in Sri Lanka. However, taxonomic identification of Dendrobiums are not straightforward and it is often complicated with the introduction of large number of commercially attractive hybrids and cultivars. Therefore, morphometry was used for taxa identification in the present study. Further, plant growers produce hybrids using these modern cultivars. However, recently, it has been found that artificial pollination of modern hybrids is unsuccessful. It was hypothesized that the modern hybrids bear infertile pollens. Eight commercial Dendrobium cultivars (A-H) with different floral morphology were selected for the study . Sixty different floral and vegetative characters were observed and recorded. Morphological characters were subjected to cluster analysis using PAST 3.1 software package. Two Dendrobium cultivars (F and G) were closely related and have separated from the other taxa. Dendrobium cultivar B has shown a clear separation from the other studied taxa. Pollinia of Dendrobium flowers were deposited on the stigma of the same flower. After 72 hr, a small amount of stigmatic fluid was stained with lacto phenol cotton blue, observed under a light microscope and percentage pollen germination was calculated. Pollen viability was tested by placing crushed pollinia on a cavity slide with 1% solution of 2,3,5-triphenyltetrazolium chloride, incubating at dark for 6 hr and observing under a light microscope. The highest percentage of pollen germination was observed in Dendrobium cultivar D (67%) while the lowest germination was observed in cultivar A (24%). The pollen viability of the cultivars varied from 77% to 94%. Successful observations in planta pollen germination concludes that the modern cultivars still have the potential of using in plant breeding programs.Item Anticancer activity of Trichoderma harzianum extract against NCI-H292 lung cancer cells.(International Research Symposium on Pure and Applied Sciences, 2017 Faculty of Science, University of Kelaniya, Sri Lanka., 2017) Sinthujah, S.; Samarakoon, S. R.; Tennakoon, K. H.; Attanayake, R. N.; Weerakoon, G.; Gunasegara, D. S.; Paranagama, P. A.Cancer is one of the leading causes of death worldwide. Chemotherapy has been the choice of cancer treatment for many years however, it can also affect normal cells and create many undesirable side effects and have the potential to develop resistance. Therefore, investigators must reassess their approach to translate discovery research into greater clinical success and impact aiming to find novel compounds. Endolichenic fungi (ELF) are potential source of producing many bioactive compounds. Preparations of ELFs extracts are commonly used to search for anticancer activity. Based on the fact that fungal extracts provide evidence to develop anticancer drugs, this study was conducted to evaluate the anticancer activity of an ELF, Trichoderma harzianum, (strain No: MF029755) extract against NCI-H292 lung cancer cells. Organ specific in-vitro assays are imperative in large scale screening of natural products with useful clinical activity. Among many such assays, sulforhodamine B (SRB) assay employs a protein binding aminoxanthene dye, to provide a quantitative analysis of viable cells in a culture following the introduction of the compound. Preliminary investigations revealed that crude ethylacetate extract of an endolichenic fungus, T. harzianum, and chloroform fractions of crude extract (12.5 mgL-1, 25 mgL-1, 50 mgL-1, 100 mgL-1 and 200 mgL-1) obtained by partition were positive for the SRB assay. IC50 values of crude extract and the chloroform fraction were 68.48 mgL-1 and 38.44 mgL-1 respectively. The chloroform fraction was chromatographed over silica gel column to obtain seven fractions. Cytotoxicity of the seven fractions obtained from the crude extract of the fungus was determined using SRB assay against lung cancer cell line NCI-H292 following standard protocols. The cell suspension in Dulbecco’s Modified Eagle Medium (DMEM) was aliquoted into 96-well plate. After incubation cells were treated with two concentrations (100 mgL-1 and 200 mgL-1) of fractions obtained by column chromatography. SRB dye was added to each well and acetic acid was used to remove unbound dye. Absorbance was measured at 540 nm using microplate reader. Survival percentage of the cells was calculated. If no viable cells present pink color of the medium turns colorless. In the current assay control wells and 1st fraction remained pink and all the other treatments turned pink into colorless. Seventh fraction showed the highest activity and further purification, SRB assays and structure elucidation will be carried out.Item Evaluation of fungicide resistance of Sclerotinia sclerotiorum causing cabbage white mold in Sri Lanka.(International Research Symposium on Pure and Applied Sciences, 2017 Faculty of Science, University of Kelaniya, Sri Lanka., 2017) Mahalingam, T.; Somachandra, K. P.; Rajapakse, C. S. K.; Attanayake, R. N.Incidence of severe white mold disease was extensively observed in upcountry commercial cabbage fields of Sri Lanka in early 2014. The pathogen was identified as Sclerotinia sclerotiorum, a nectrophic soil borne fungus that can infect more than 400 plant species worldwide. Chemical control is the most effective method available to date due to lack of complete resistant cabbage varieties. However, improper fungicide applications can lead to the development of fungicide resistant isolates. In Sri Lankan small scale upcountry vegetable production system, more than one crop species is grown per year and most of these crops serve as hosts for the pathogen S. sclerotiorum. Frequent fungicide and pesticide application is also a common practice. Therefore, it was hypothesized that resistant S. sclerotiorum isolates against commonly used fungicides are present in Sri Lankan pathogen population. Objectives of this study were to determine the variation in insensitivities of the pathogen against commonly used fungicides in upcountry vegetable production systems, to search for the presence of resistant isolates and to determine if there are signals of positive cross resistance for the fungicides in Sri Lanka. Pathogen population was tested against three selected fungicides, carbendazim and thiophanate methyl (benzimidazole fungicides) and tebuconazole (triazole group fungicide) using in vitro plate assay. Firstly, a discriminatory concentration, which showed the highest variance in percentage inhibition of mycelial growth of a few randomly selected S. sclerotiorum isolates were determined as 0.25 μg/mL for carbendazim and 0.5 μg/mL for both thiophanate methyl and tebuconazole. Later, all of the 34 isolates were assayed for their insensitivities using fungicide amended PDA at the above concentrations with three replicates per isolate and fungicide unamended PDA plates were used as the control. Colony diameters were measured after 36 hr of incubation and percentage inhibition was calculated. While only 12% of the isolates showed > 50% inhibition for thiophanate methyl, the majority of the isolates (63 %) showed > 50% inhibition for the fungicide, carbendazim. The highest variance observed in percent inhibition of the pathogen population for carbendazim indicated that the pathogen population has the potential to adjust to the challenging environments, in this case fungicide applications. Based on the in-vitro plate assay, thiophanate methyl was found to be the least effective fungicide than the other two. Twelve isolates exhibited <50% inhibition to both carbendazim and thiophanate methyl and nine isolates showed <50% inhibition to all three fungicides. Results indicated that there is a potential to develop cross resistance and hence farmers should be prudent in selecting the fungicide regime for cabbage cultivation. Molecular basis of the resistance is yet to be elucidated.