Browsing by Author "Attanayake, R. N."

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Antibacterial polyketide from Lasiodiplodia theobromae, inhabiting the lichen host, Pyrenula bahiana on mangrove ecosystems in Puttalam lagoon, Sri Lanka
(Asian Journal of Chemistry, 2022) Paranagama, P. A.; Santhirasegaram, S.; Fathima, M. M. A.; Wickramarachchi, S. R.; Attanayake, R. N.; Weerakoon, G.; Maduranga, K.
Lasiodiplodia theobromae is one of the frequently isolated fast growing endolichenic fungus. This fungus was isolated from the lichen host, Pyrenula bahiana collected from the mangrove ecosystems in Puttlam lagoon and identity was confirmed based on rDNA-ITS sequence homology. Secondary metabolites of L. theobromae were extracted into ethyl acetate and subjected to antibacterial assay against Escherichia coli (ATCC25922), Staphylococcus aureus (ATCC25923) and Bacillus subtilis (ATCC6051). Crude extract at a concentration of 6.8 μg/ml showed good anti-bacterial activity against the bacterial strain S. aureus compared with the activity of the standard Azithromycin at a concentration of 5.0 μg/ml. Active crude extract was partitioned to obtain methanol,
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.
Bioactive properties and metabolite profile of an endolichenic fungus, Hypoxylon lividipigmentum
(Faculty of Science, University of Kelaniya, Sri Lanka., 2021) Weerasinghe, W. R. H.; Shevkar, C. D.; Silva, R. S. De; Attanayake, R. N.; Weerakoon, G.; Kate, A. S.; Kalia, K.; Paranagama, P. A.
Endolichenic fungi (ELF) serve as a novel source of secondary metabolites. Hypoxylon lividipigmentum is an ELF isolated from the lichen Opegrapha medusulina, collected from mangrove plant Xylocarpus granatum from Negombo lagoon, Sri Lanka. The fungus was identified to the species level using morphological and DNA barcoding techniques. Ethyl acetate extract of the fungus was subjected to in vitro assays to determine antioxidant, anti-inflammatory, tyrosinase inhibitory and antibacterial potency. Liquid Chromatography-Mass Spectrometry (LCMS) dereplication was conducted on the crude extract in order to detect the secondary metabolites present. The extract reported a IC50 value of 18.34±1.37 µg/ml on par with the positive control BHT, in DPPH radical scavenging assay. It also exhibited moderate anti-inflammatory activity with an IC50 value of 81.08±1.05 μg/ml. Tyrosinase inhibitory activity was fairly comparable with an IC50 value of 121.20±2.55 μg/ml. Agar well diffusion assay was conducted to determine antibacterial activity against aerobic bacterial species Escherichia coli, Bacillus subtilis, Staphylococcus aureus and the anaerobic bacterial species Streptococcus mutans. Suppression of growth was shown only against B. subtilis. Five major mass peaks were observed during the study of LCMS profile of the extract. After a thorough dereplication process, two masses could be presumed to be from novel scaffolds. Since none of the mass peaks could be dereplicated within the species or genus level, it could be speculated that the chemical profile of Hypoxylon lividipigmentum was previously poorly explored in literature thus making it an interesting organism to study further for novel metabolites.
Bioactive Properties and Metabolite Profiles of Endolichenic Fungi in Mangrove Ecosystem of Negombo Lagoon, Sri Lanka
(Natural Product Communications, 2022) Weerasinghe, R. H.; Maduranga, K.; Attanayake, R. N.; Shevkar, C.; Kate, A. S.; Weerakoon, G.; Kalia, K.; Paranagama, P.
Endolichenic fungi (ELF) serve as a novel source of natural products with distinctive bioactivities. A total of 58 ELF isolated from 29 lichens collected from mangrove and mangrove-associated plants of Negombo lagoon, Sri Lanka were identified using morphological and DNA barcoding techniques. Ethyl acetate extracts of 18 such identified ELF isolates were subjected to in vitro assays to determine antioxidant, anti-inflammatory, tyrosinase inhibitory and antibacterial potency. Liquid chromatography–mass spectrometry (LC–MS) dereplication was conducted on the crude extracts in order to detect the secondary metabolites present. The extracts of Daldinia eschscholtzii and Hypoxylon lividipigmentum had the highest radical scavenging activity with SC50 values 14.27 ±0.24 μg/mL and 18.34±1.37 μg/mL, respectively. D. eschscholtzii also exhibited remarkable anti-inflammatory activity (IC50 7.97 ±0.09 μg/mL). Tyrosinase inhibitory activity was highest in Cytospora xylocarpi (IC50 68.50 ±0.34 μg/mL), while the highest activity against aerobic bacterial species Escherichia coli, Bacillus subtilis, Staphylococcus aureus and the anaerobic bacterial strain Streptococcus mutans was observed in the extracts of Xylaria feegenesis and Curvularia lunata. After a thorough study of the LC–MS profiles, it was found that the chemical profiles of Neofusicoccum occulatum, H. lividipigmentum and Myramaececium rubricosum were previously poorly explored in the literature.
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 agents
Bioassay-guided isolation and antioxidant properties of phytochemicals present in methanolic extract of Halosarcia indica from salt marsh ecosystems in Sri Lanka
(Faculty of Science, University of Kelaniya Sri Lanka, 2024) Siriwardhana, U. L. I. G.; Perera, D.; Attanayake, R. N.; Paranagama, P. A.
Halosarcia indica, belonging to the family Amaranthaceae, is a halophyte that exhibits unique adaptations to saline environments. Halophytes contain various bioactive chemicals, including plant polyphenols, which have garnered interest due to their antioxidant properties. Isolation of pure compounds responsible for the antioxidant activity is also very important in nutraceuticals, cosmetics, and pharmaceutical industries. The aim of the present study was to isolate and identify bioactive compounds from H. indica samples collected from Sri Lanka. Fresh plant materials were collected from salt marshes in Mannar district, Sri Lanka. Phytochemicals from finely powdered freeze-dried areal parts of H. indica were extracted using cold extraction and sequentially partitioned with methanol, dichloromethane (DCM), and hexane. The antioxidant activity of these fractions was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay, and the Ferric Reducing Antioxidant Power (FRAP) assay, with butylated hydroxytoluene (BHT) serving as the positive control. The total phenolic content (TPC) was assessed using the Folin-Ciocalteu method, while the determination of total flavonoid content (TFC) was conducted through the aluminum chloride assay. Additionally, the total tannin content (TTC) was examined using the vanillin/HCl method. Finally, methanolic and DCM extractions were subjected to column chromatography, thin layer chromatography, and preparative thin layer chromatography techniques to isolate compounds responsible for antioxidant activity. The methanolic fraction exhibited the highest radical scavenging activity, with IC50 values of 69.78 ± 3.48 µg mL-1 in the DPPH assay and 81.38 ± 1.89 µg mL-1 in the ABTS assay, while the positive control BHT showed IC50 values of 20.91 ± 0.48 µg mL-1 and 22.93 ± 0.65 µg mL-1, respectively. In the FRAP assay, the methanolic fraction presented higher mean absorbance values of 0.598 ± 1.81 compared to that of the BHT 0.821 ± 1.82. Methanolic extracts of the H. indica had higher TPC (184.77 ± 3.21 mg GAE/g), TFC (190.69 ± 5.84 mg CE/g), and TTC (201.33 ± 4.21 mg GAE/g) values compared with DCM and hexane extracts. Methanolic extracts showed higher antioxidant activity followed by DCM and hexane extracts. Therefore, two pure compounds with antioxidant activities were isolated in methanolic (IC50 - 78.69 ± 1.24 µg mL-1) and DCM extracts (IC50 - 192.84 ± 1.65 µg mL-1) respectively. The study identified two antioxidant compounds from H. indica extracts, with methanolic extracts showing the highest activity. Further analysis will be done using spectroscopic methods. These two compounds will be identified using spectroscopic techniques, including 1H NMR, 13C NMR, HPLC, and mass spectrometry.
Biodeterioration of low density polyethylene by Montagnula scabiosae
(Faculty of Science, University of Kelaniya, Sri Lanka, 2021) Perera, T. W. N. K.; Attanayake, R. N.; Paranagama, P. A.
Endolichenic Fungi (ELF) are a group of fungi adapted to live within lichen thalli. Present study investigates the ability of Montagnula scabiosae, an ELF, for biodeterioration of low density polyethylene (LDPE) by determining the activity of three extracellular fungal enzymes, reported to have potential roles in depolymerizing polyethylene, using enzymatic assays. Liquid medium screening assays, using four potato dextrose agar (PDA) plugs (0.5 cm diameter) of ELF cultures, inoculated into Erlenmeyer flasks containing sterile mineral salt medium broth (MSMB). Eight sterile LDPE strips (10 cm x 3 cm) were added into each flask and incubated at room temperature (28 ± 2°C) for 21 days. MSMB flasks with plain PDA plugs and sterile LDPE strips were used as the controls. Solid medium biodeterioration studies also were conducted using three PDA plugs of the ELF cultures inoculated into mineral salt medium agar (MSMA) plates. Eight sterile LDPE strips were aseptically placed on the surface of each MSMA plate and incubated at room temperature for 45 days. MSMA plates with plain PDA plugs and sterile LDPE strips were used as the controls. Both liquid and solid media assays were carried out in four replicates. LDPE biodeterioration was analyzed based on reductions in weights and tensile properties, changes in peaks of Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectra, changes in carbonyl index (CI) and Scanning Electron Microscopic (SEM) images. A distinguishable LDPE deterioration ability was shown by Montagnula scabiosae, isolated from the host lichen Pyrenocarp sp. Results showed that isolate reduced weights of strips by 13.22% in liquid medium and by 12.06% in solid medium. Tensile strength of liquid medium treated strips, reduced by 6.64% and solid medium treated strips, by 3.00%. The control LDPE strips on ATR-FTIR analysis showed four prominent peaks at 2916 cm-1, 2848 cm-1, 1463 cm-1 and 720 cm-1 and a few minor peaks. Changes visible in the ATR-FTIR spectra of treated strips, manifested new peaks, fluctuations in percentage transmittance and deletions of existing peaks. CI value of the LDPE strips in liquid medium were found to be increased by 2.70% and in solid medium increment was 2.31%. SEM images of treated strips showed erosion zones on LDPE matrix. Qualitative enzymatic assays using Agar plate-based screening methods, indicated the presence of laccase and peroxidases, in this ELF isolate. In the quantitative enzymatic assays, M. scabiosae showed laccase, lignin peroxidase and manganese peroxidase activities as 1.07×10-7 kat L-1, 1.22×10-8 kat L-1 and 2.10×10-8 kat L-1 respectively. Remarkable LDPE deterioration ability shown by this ELF isolate indicates that it has a high potential in degrading LDPE waste.
Bioprospecting of an Endolichenic Fungus Phanerochaete sordida Isolated from Mangrove-Associated Lichen Bactrospora myriadea
(Journal of Chemistry, 2022) Weerasinghe, R. H.; Shevkar, C. D.,; Maduranga, K.; Pandey, K. H.; Attanayake, R. N.; Kate, A. S.; Weerakoon, G.; Behera, S. K.; Kalia, K. S.; Paranagama, P. A.
Bioassay-guided fractionation of the ethyl acetate extract of Phanerochaete sordida, an endolichenic fungus (ELF) isolated from the host lichen Bactrospora myriadea, collected from Negombo lagoon, Sri Lanka, led to the isolation of a bioactive compound. Following the identification of the fungus using morphological and DNA barcoding techniques, the pure compound was isolated using column chromatography, preparative TLC, and semipreparative HPLC. The structure elucidation was carried out using IR, HR-ESI-MS and 1H, 13C & 2D NMR spectroscopic methods. The in vitro bioassays conducted revealed that compound 1 has a high antioxidant activity with ABTS•+ (IC5058:91 ± 1:35 μM), moderate anti-inflammatory activity (IC50254:79 ± 1:41 μM), comparable antibacterial activity against the oral-bacterial strain Streptococcus mutans (MIC 898.79 μM and MLC 1797.58μM), moderate tyrosinase inhibition (IC501713:69 ± 8:65 μM), and moderate cytotoxicity against oral cancer (IC5013:65 ± 0:02 μM), in comparison with respective positive controls. The in silico experiments conducted for tyrosinase inhibition and cytotoxicity using Schrödinger revealed results in line with the in vitro results, thus confirming the bioactivities. The molecule also satisfies the key features of drug likeliness according to pharmacokinetic studies.
Bioprospecting of an Endolichenic Fungus Phanerochaete sordida Isolated from Mangrove-Associated Lichen Bactrospora myriadea
(Journal of Chemistry,, 2022) Weerasinghe, R. H.; Shevkar, C. D.; Maduranga, K.; Pandey, K. H.; Attanayake, R. N.; Kate, A. S.; Weerakoon, G.; Behera, S. K.; Kalia, K. S.; Paranagama, P. A.
Bioassay-guided fractionation of the ethyl acetate extract of Phanerochaete sordida, an endolichenic fungus (ELF) isolated from the host lichen Bactrospora myriadea, collected from Negombo lagoon, Sri Lanka, led to the isolation of a bioactive compound. Following the identification of the fungus using morphological and DNA barcoding techniques, the pure compound was isolated using column chromatography, preparative TLC, and semipreparative HPLC. The structure elucidation was carried out using IR, HR-ESI-MS and 1H, 13C & 2D NMR spectroscopic methods. The in vitro bioassays conducted revealed that compound 1 has a high antioxidant activity with ABTS•+ (IC5058:91 ± 1:35 μM), moderate anti-inflammatory activity (IC50254:79 ± 1:41 μM), comparable antibacterial activity against the oral-bacterial strain Streptococcus mutans (MIC 898.79 μM and MLC 1797.58μM), moderate tyrosinase inhibition (IC501713:69 ± 8:65 μM), and moderate cytotoxicity against oral cancer (IC5013:65 ± 0:02 μM), in comparison with respective positive controls. The in silico experiments conducted for tyrosinase inhibition and cytotoxicity using Schrödinger revealed results in line with the in vitro results, thus confirming the bioactivities. The molecule also satisfies the key features of drug likeliness according to pharmacokinetic studies.
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
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.
Evaluation of phytochemical profile and in -vitro antioxidant activity Suaeda maritima
(Faculty of Science, University of Kelaniya Sri Lanka, 2023) Pathirana, K. P. S. S.; Herath, H. M. N. P.; Perera, D.; Attanayake, R. N.; Paranagama, P. A.
Suaeda maritima, a halophytic plant species from the family Amaranthaceae, possesses unique adaptations that enable it to thrive in high-salt environments. Halophytes contain various bioactive chemicals, including plant polyphenols, which have garnered interest due to their antioxidant properties. This study aims to investigate how the polyphenolic content and antioxidant activity of extracts change across the polar, mid-polar, and non-polar fractions of crude extracts from S. maritima. These extracts are obtained from both the aerial parts and the roots of the plant, which were harvested from Seguwantivu area of Puttalam, Sri Lanka. Each dried and powdered plant part was sequentially extracted into hexane, dichloromethane (DCM), and methanol at ambient temperature using cold maceration. The Antioxidant activity of each concentrated extract was determined by 2,2-diphenyl-1-1-picrylhydrazyl(DPPH) assay, and 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid (ABTS) assay , phenolic content by (TPC) by FolinCiocalteau method, flavonoid content (TFC) by aluminum chloride assay and condense tannin content (CTC) by vanillin/HCl method. Six sequential extracts of S. maritima (from aerial parts and roots) showed antioxidant activity. The results indicated that S. maritima shoot extracts have significant levels of polyphenolic compounds (p=0.05), with methanolic extracts exhibiting the highest TPC (31.55±1.54 mg GAE/g dw), TFC (18.41±0.91 mg CE/g dw) and CTC (3.66±0.19 mg CE/g dw). In contrast, root extracts showed lower polyphenolic content (23.69±5.63 mg CE/g dw, methanol extract) but still possessed appreciable antioxidant activity (IC50, 0.40±0.07×10-1 mg/mL DPPH and IC50, 0.69±0.02×10-1 mg/mL ABTS). The ABTS assays revealed the radical scavenging ability of the extracts, with the methanolic aerial parts extract exhibiting the most potent antioxidant activity (IC50, 0.17±0.01×10-1 mg/mL) exhibiting it has more water-soluble antioxidants. Overall, Solvent extracts of the S. maritima possess antioxidant properties at varying levels. The fraction of polar compounds in the areal parts of S. maritima contains a relatively high amount of phenolic compounds and potent antioxidant properties. Moreover, identifying antioxidant properties from halophytes could contribute significantly to developing of natural antioxidants for combating oxidative stress-related diseases.
Low-cost media composition for increased laccase activity of wood decay fungi, Phlebiopsis flavidoalba and Perenniporia tephropora
(Faculty of Science, University of Kelaniya Sri Lanka, 2023) Senavirathna, T. M. Y. H.; Attanayake, R. N.
Laccase, a versatile enzyme with broad substrate specificity, has been extensively researched for over 30 years due to its diverse biotechnological applications. Among diverse types of laccases, white-rot fungal laccases are unique in that they have broad substrate specificities and hence have various biotechnological applications such as dye decolorization, lignin degradation, and polyethylene degradation. It is crucial to achieve high yield while utilizing low-cost raw materials to enable the effective utilization of laccases in commercial and industrial applications. The objective of the current study was to optimize a culture media with low-cost carbon sources for high laccase activities of two laccase-producing fungal species, Phlebiopsis flavidoalba and Perenniporia tephropora. Fungi were cultured in Potato Dextrose Broth (PDB) while systematically varying one factor at a time for a duration of 21 days, and laccase activities were subsequently determined through spectrophotometric analysis. The effect of carbon sources, and varying concentrations of nitrogen source and copper ion concentrations were assessed on laccase activities. Rice husk, coconut coir fibers, peanut shells, and rubber wood chips were used as carbon sources. The amount of yeast varied from 0.5% to 2% (w/v), CuSO4 concentration varied from 1 mmolL-1 to 2 mmolL-1 as nitrogen and metal ion concentrations respectively. Among different carbon sources tested, rice husks exhibited the highest laccase activity with 5.560±0.023 fold increase compared to the control followed by peanut shells that had 3.624±0.320 fold increase for P. flavidoalba. For the same species, 1% yeast (w/v) and 1.5 mmolL-1 of CuSO4 demonstrated the maximum laccase activity. However, in P. tephropora, neither yeast concentrations nor copper ion concentrations significantly influenced laccase activity compared to the control. Nonetheless, rice husks exhibited the highest laccase activity displaying 2.120±0.012 fold increase compared to the control followed by peanut shells that had 1.074±0.301 fold increase for P. tephropora. To the best of our knowledge, this is the first study to explore the optimization of laccase activity using lignocellulosic waste in P. flavidoalba and P. tephropora. Sri Lanka being an agricultural country, this research sheds light on the effective utilization of one of the main agricultural waste, rice husks in the industrial scale of laccase production.
Molecular characterization of Sri Lankan tea [Camellia sinensis (L.) O. Kuntze] genotypes with diverse origins using SSR markers for future breeding programmes
(Faculty of Science, University of Kelaniya, Sri Lanka., 2021) Kottawa-Arachchi, J.D.; Ranathunga, M. A. B.; Sharma, R. K.; Amarakoon, A. M. T; Gunasekare, M. T. K.; Chaudhary, H. K.; Attanayake, R. N.
The tea plant [Camellia sinensis (L.) O. Kuntze] is one of the most popular non-alcoholic beverage crops worldwide. Tea germplasm is the most valuable and fundamental resource for breeding high yielding, climate resilient, and quality tea cultivars. Therefore, efforts were made to characterize the existing genetic diversity of Sri Lankan tea using simple sequence repeat (SSR) markers. A total of 91 tea accessions including exotic, estate selections, improved quality and diverse cultivars were selected for the diversity estimations. Eleven informative highly polymorphic SSR markers selected based on inferences of previous studies (EST, miRNA and traits associated SSR markers) were used in this study. Genetic relationships were analyzed by a neighbor-joining (NJ) tree constructed using DARwin software. Genetic structure analysis was performed by the Bayesian clustering model using STRUCTURE analysis and the optimum delta K was generated. Genetic diversity analysis grouped all the accessions into three major clusters. Further, structure analysis complemented the cluster analysis and revealed three genetic populations (China types with Korean origins, Assam types with ASM4/10 ancestral relationship and mixture of different origins) in the Sri Lankan tea germplasm. Newly recommended TRI5001, TRI5002, TRI5003 and TRI5004 cultivars have shown affinities with five Assam introductions and grouped together in the same cluster. Interestingly, five high yielding TRI developed cultivars (TRI2023, TRI2025, TRI3013, TRI3055 and TRI3073) having an ancestral relationship with the parent ASM4/10 were also grouped together. Likewise, seven accessions of Azerbaijan origin were remained together. The estate selections were scattered in the three clusters. The inferences of the current study provide molecular evidence for the genetic diversity and population structuring of the tea germpalsm and will help the selection of the potential parental group for the future cultivar development programme of Sri Lanka.
Morpho-molecular genetic diversity and population structure analysis to enrich core collections in tea [Camellia sinensis (L.) O. Kuntze] germplasm of Sri Lanka and India
(Genetic Resources and Crop Evolution, 2023) Kottawa-Arachchi, J. D.; Ranatunga, M. A. B.; Sharma, R. K.; Chaudhary, H. K.; Attanayake, R. N.; Amarakoon, A. M. T.; Gunasekare, M. T. K.; Sharma, B.; Kumar, N.; Sood, V. K.
Despite tea [Camellia sinensis (L.) O. Kuntze] is one of the top non-alcoholic beverages consumed around the world; its genetic and phenotypic diversity is less understood compared to other plantation crops. The study’s aims were to undertake phenotypic and genotypic characterization of Sri Lankan and Indian tea germplasm and to identify diverse accessions to accelerate tea breeding programmes in both countries. A total of 171 tea accessions, comprising 94 Sri Lankan and 77 Indian accessions were used. All the accessions were subjected to phenotyping and genotyping using 28 polymorphic simple sequence repeat (SSR) markers. Based on 16 morphological characters, the first three principal components explained 57.61% and 58.43% of the total variability of Sri Lankan and Indian accessions, respectively. Young shoot pubescence, young shoot pigmentation, serration of leaf margin, and mature leaf colour contributed positively to the grouping of accessions. Based on Neighbor-joining analysis, all Sri Lankan accessions grouped in a single cluster, whereas Indian accessions grouped in two distinct clusters. The Gower’s distance method was the most appropriate than other methods for developing core subsets. Among 21 Sri Lankan core accessions selected, 11 accessions belong to introductions, five TRI-developed cultivars and five estate selections. Among 18 Indian core accessions selected, 11 belong to China types, two Assam types and five Indian recommended cultivars. The current study is the first study to compare tea germplasm of both countries and the results are useful for tea crop improvement programme, conservation and utilization of tea germplasm in India and Sri Lanka in the future.
Morphological and molecular identification of different morphotypes of Suaeda maritima from Puttalam district in Sri Lanka
(Faculty of Science, University of Kelaniya Sri Lanka, 2023) Jayasundara, R.; Siridewa, K.; Neththipola, T.; De Silva, W. L.; Perera, D.; Attanayake, R. N.
Suaeda is a genus belonging to the family Amaranthaceae (Chenopodiaceae) and comprises more than 100 species that are distributed all over the world. Members of the genus are mainly used for food, feed, and medicine. Three Suaeda species have been reported in Sri Lankan salt marshes among them, S. maritima is widely distributed. During a field survey in Seguwanthive in July 2022, two clearly distinct morphotypes of tentatively identified S. maritima were found. This tentative identification was done purely based on morphological characteristics. One morphotype had green stems and leaves while the other had reddish-green leaves and brightly red-colored stems. No reproductive parts were found at the time of the survey. Even though, previous reports indicated high phenotypic plasticity among the members of the genus, it was not clear whether both morphotypes belonged to the same species or not. Due to the lack of floral structures throughout the year, accurate species identification was a great challenge for a layperson and for a trained taxonomist. Therefore, the current study was conducted to obtain detailed morpho molecular identification of each morphotype of Suaeda maritima, and to confirm their species identity using molecular data as well. Plant samples were collected from Seguwanthive area mainly focusing on two morphotypes and documented. Leaves were succulent, linear in shape, flattened only on one side, and acute in the apex. Plants were 40-65 cm range in height in both morphotypes with a woody base. Flowers were observed only on green plants by the time of sampling, and they were bisexual and contained 5 stamens and 3 stigmas and located axillary in 2 mm diameter clusters and seeds were black, smooth and glossy, and suborbicular to ovoid in shape. Molecular identification was conducted using DNA barcoding approach. Genomic DNA extraction was optimized. The nuclear ribosomal ITS (Internal transcribed spacer) region was PCR (Polymerase chain reaction) amplified using universal primers BLASTn searchers of the sequences confirmed that both morphotypes were identical and 100% similar to previously published records of S. maritima (KF866384). This project findings give insights into the plant’s phenotypic plasticity under its natural environment and can be used as a future guide.
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.
Nested PCR-based rapid detection of Phytoplasma leaf wilt disease of coconut in Sri Lanka and systemic movement of the pathogen.
(Pathogens, 2023) De Silva, P. R.; Perera, C. N.; Bahder, B. W.; Attanayake, R. N.
Phytoplasmas are associated with many plant diseases. In palms, lethal bronzing disease, Texas Phoenix palm decline, and coconut lethal yellowing decline are some of them. In Sri Lanka, coconut leaf wilt decline has been reported in the Weligama area of the Southern province, and the disease is called Weligama coconut leaf wilt disease (WCLWD). Unlike other phytoplasma diseases of palms, WCLWD shows slow disease progress. Pathogen detection entirely relies on nested polymerase chain reaction (PCR). However, inconsistencies in pathogen detection have been experienced, i.e., symptomatic plants often produce negative results. The objectives of this study were to reconsider the choice of primers and to determine the best sampling tissue types for consistent detection of the pathogen. Among the six universal primer combinations tested, P1/Tint nested with fU5/rU3 produced consistent results. BLASTn searches of the sequences showed 99–100% similarity to sugarcane white leaf disease (SWL) or grassy shoot (SGS) disease-causing phytoplasma. The optimized nested PCR protocol was successful, with the minimum success rating of 88% and 100% specificity. Midribs of milky white bud leaf samples were the best tissue type for rapid detection. Systemic movement of the pathogen and a tentative latent period were also reported. The findings are helpful in the early detection of the disease.
A Novel Cytotoxic Compound From the Endolichenic Fungus, Xylaria psidii Inhabiting the Lichen, Amandinea medusulina
(Natural Product Communications, 2020) Santhirasegaram, S.; Wickramarachchi, S. R.; Attanayake, R. N.; Weerakoon, G.; Samarakoon, S.; Wijeratne, K.; Paranagama, P. A.
The lichen host, Amandinea medusulina, collected from mangrove habitats in Sri Lanka, and its associated endolichenic fungi were isolated and identified by rDNA-ITS sequence analysis and morphological features. One of the fungal strains frequently isolated from the lichen thalli was identified as Xylaria psidii. This study aimed at the isolation and identification of the cytotoxic compounds present in this fungus. Secondary metabolites of X. psidii were first extracted into ethyl acetate and subsequently subjected to bioassay-guided fractionation to isolate the bioactive compounds. Sulforhodamine B assay against a lung cancer (NCI-H292) cell line was used to determine the differential cytotoxic activity. Bioassay-guided fractionation led to the isolation of an active compound, SS/02/29/08, showing moderate cytotoxicity (IC50 = 27.2 μg/mL). Its structure was elucidated by IR, 1D- and 2D-NMR, and 13C-NMR spectrophotometry and MS, in combination with HRMS, 13C NMR, HSQC, HMBC, and DQF-COSY. The structure of SS/02/29/08 was determined as (Z)-3-{(3-acetyl- 2-hydroxyphenyl) diazenyl}-2,4-dihydroxybenzaldehyde and identified as a new compound. This novel compound has promising differential cytotoxic activity against human lung cancer cell line (NCI-H292).
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 background