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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.
Species identification and pollination biology of an economically important true halophyte, Salicornia brachiata Roxb.
(Aquatic Botany, 2024) Siridewa, K.; De Silva, W.; Ratnayake, R.; Wijesundara, S.; Perera, D.; Attanayake, R. N.
Members of the genus Salicornia have gained a global attraction due to their ability to thrive under high saline conditions and as potential candidates in saline agriculture. However, it has been a taxonomically challenging genus for decades since the members show plastic responses to extreme environmental conditions and due to incongruences between morphological and molecular identification methods. While only a handful of commercially grown Salicornia species are fully described, most of the species including S. brachiata, a native species in the Indian sub-continent, Myanmar, and Sri Lanka are poorly described. With the potentials in adapting S. brachiata in saline agriculture, the aim of this study was to establish a morphology and DNA barcodebased species delineation system and to study pollination biology for future crop improvement projects. Tentatively identified S. brachiata plant samples were collected from two populations in Sri Lanka and completely described. GenBank lacked authenticated barcode data for S. brachiata except for one chloroplast genome to which the matK sequence obtained in the present study matched with 100 % identity. For the first time, well defined sequences of three barcode regions, ITS, ETS and matK, of S. brachiata were made available for accurate species identification. Reproductive dynamics in different parts of the inflorescence was studied. A facultative xenogamous mating system was recorded for the first time in the genus and while the lower florets in the cladode showed a preference towards outcrossing, the upper florets displayed adaptations for selfing. Data could be effectively utilized in future Salicornia breeding programs.
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.
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.
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.
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.
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.
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.

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