RESCON 2021

Permanent URI for this collectionhttps://ir.lib.pdn.ac.lk/handle/20.500.14444/5951

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In vitro antimicrobial activity of Panchavalkala, an ayurvedic herbal formula: preliminary study
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2021-10-29) Fernando, A.M.A.; Jayatilake, J.A.M.S.
Panchavalkala is a water extract of a combination of barks of five medicinal plants, widely used to treat wounds in Ayurvedic medicine. Three different combinations are commonly used as Panchavalkala in Sri Lanka. This study evaluated the in vitro antimicrobial activity (AMA) of three different combinations of Panchavalkala against common pathogens: Escherichia coli, Pseudomonas auregenosa, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA) and Candida albicans. The combinations of Panchavalkala were prepared using pure raw materials according to the ayurvedic formulae. They were named A, B, and C and 60 g of total weight was used for water extraction and concentrated into 8:1 by boiling from each combination. Screening of AMA for each sample was carried out using the agar well diffusion assay in quadruplicate. Zones of inhibition of samples A, B, C against S. aureus and MRSA were 4.81 ± 0.26 mm, 4.69 ± 0.26 mm, 4.25 ± 0.27 mm, 3.88 ± 0.23 mm, 5.12 ± 0.23 mm, 4.50 ± 0.38 mm, respectively. Growth of S. aureus was significantly inhibited by sample C compared to amoxicillin used as a positive control (p = 0.01). Samples A and C showed a significant effect against MRSA compared to vancomycin (p = 0.01). None of the above combinations of Panchavalkala had an inhibitory effect on E. coli, P. auregenosa and C. albicans. All three combinations were effective against S. aureus, including MRSA in vitro. Further studies are recommended to explore the AMA of Panchavalkala used in Sri Lanka.
In vitro cytotoxicity of Carica papaya crude leaf extract
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2021-10-29) Galappaththi, M.O.; Abeykoon, A.M.S.B.; Muthuwaththa, M.G.C.M.; Noordeen, F.
Plant-based compounds are an option for the development of new antiviral drugs. Asians commonly use papaya (Carica papaya) leaves to treat dengue without an understanding of its toxicity, if any. The objective of the study was to screen in vitro cytotoxicity of C. papaya leaf extract against normal African green monkey kidney epithelial cell line (Vero) cells. CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, USA) is a colourimetric assay, which quantitatively measures lactate dehydrogenase (LDH) released upon cell lysis. Carica papaya leaf extract was prepared in a two-fold dilution series. Two, 96 well assay plates, were prepared with Vero cells and, the assay was set up with an analytical system based (i) Negative control - without Vero cells, (ii) Vehicle control - untreated cells, (iii) Positive control - lysis solution with four replicates. Carica papaya extract was added to the test wells at different concentrations and, one set of plates was incubated for 5 h at 37 °C and the other set for 24 h at 37 °C. The absorbance data were measured using a standard 96-well plate reader (Labtech LT4500, Singapore) and the percentage cytotoxicity was calculated for each concentration tested. Colour intensity and the absorbance values decreased with the decrease of concentration of leaf extract. The percentage cytotoxicity for dilutions, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128, 1/256, 1/512, 1/1024 was 95.60, 71.53, 74.77, 82.16, 79.91, 40.25, 7.37, 0.16, -1.24, respectively, for the 5 h and 141.61, 133.24, 127.78, 121.88, 109.79, 106.67, 30.64, 22.67, 25.39, respectively for 24 h. Higher concentrations of the extract caused higher cell lysis showing cytotoxic effects Vero cells at dilutions < 1/256. The information about the cytotoxicity levels helps select the minimum toxic concentrations of C. papaya leaf extract against the antiviral activity of the dengue virus.
Potassium and phosphorus concentrations of rice grains collected from different agro-climatic zones of Sri Lanka
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2021-10-29) Ekanayake, E.M.S.; Madushan, N.D.R.; Neththasinghe, N.A.S.A.; Rathnayake, W.M.U.K.; Sirisena, D.N.; Ariyaratne, M.; Suriyagoda, L.D.B.
Rice (Oryza sativa L.) grains serve as a key source of essential mineral elements such as phosphorus (P) and potassium (K) in the Sri Lankan diet. The effect of agro-climatic conditions and agronomic management factors on the P and K concentration in rice grains in Sri Lanka is unknown. This study investigated the P and K concentrations in rice grains by collecting 100 rice grain samples countrywide using stratified random sampling approach. The P and K concentrations were assessed using protocols coupled with spectrophotometry, and flame photometry, respectively. Grain K and P concentrations collected from different agro-climatic zones (ACZs), water sources used for rice cultivation, fields used to grow other crops in the previous season were compared using analysis of variance. Grain P concentrations ranged from 0.55 to 1.75 mg g⁻¹ DW, while grain K concentrations were in the range of 1.68 and 4.53 mg g⁻¹ DW. The concentrations of the two minerals were not significantly (p > 0.05) affected by different irrigation water sources, i.e., major irrigation, minor irrigation, and rainfed. Grain K concentration was similar among ACZs (p > 0.05). However, grain P concentration in Wet zone Low country was significantly lower than those observed in other ACZs (p < 0.05). Rice fields used to cultivate paddy, other field crops (e.g., mung bean, cowpea, maize) or fallow in the previous season showed significantly lower grain K concentration than those from the fields used to grow banana (p < 0.05). However, grain P concentration was not affected by the crops grown in the previous season (p > 0.05). Overall, there was high variability in P and K concentrations of the rice grain samples collected from different regions in Sri Lanka. The ACZ and crops grown in rotation are important determinants of grain P and K concentrations in rice and thus need to be considered in the fertility management in rice-based cropping systems in Sri Lanka.
Optimization of total RNA extraction from human urinary sediment
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2021-10-29) Saseevan, S.; Magana-Arachchi, D.N.; Rajapakse, S.
Urine is the best choice to identify biomarkers for metabolic and renal disorders because it is readily available, and samples can be obtained non-invasively from patients. However, RNA isolation from voided urine is challenging due to the presence of RNases and cell scarcity. This study aims to optimize a protocol for RNA extraction from urine samples in gene expression studies. Twenty urine samples were collected from healthy controls (HC) (n = 11; 49 ± 5 years) and chronic kidney disease (CKD) patients (n = 9; 62 ± 3 years) and were centrifuged at 3,000 g for 30 min at 4 °C. Then, 500 μL of the lysis buffer was added to the pellet, vortexed and kept on ice for 5 min. Next, 100 μL of sodium acetate (pH = 4.0) and 500 μL of watersaturated phenol were added and mixed well. After that, 200 μL of chloroform: isoamyl alcohol (49:1) was added, vortexed and centrifuged. An equal volume of cold isopropanol was added to the aqueous phase and incubated at -20 °C for 1 h to precipitate RNA. The pellet was washed with 75% ethanol, air dried, and resuspended with 12 μL nuclease-free water. Finally, the RNA was quantified and reverse transcribed into cDNA to be used in RT-qPCR. Mean urine volume was 82.5 ± 41.9 mL. Serum creatinine and estimated glomerular filtration rate of CKD patients were 3.0 ± 0.2 mg dL⁻¹ and 19.2 ± 4.8 mL min⁻¹ 1.73 m⁻² , respectively. The total yield of RNA from CKD and HC samples were 873 ± 523 ng and 735 ± 291 ng, respectively, and a statistically significant difference was not observed between the two study groups (p > 0.05). The β2 microglobulin gene could be successfully amplified using samples even with a low cDNA concentration (0.625 ng). This modified phenol-chloroform based urinary RNA isolation method is less expensive, does not require RNA clean-up kits and provides a higher yield of RNA with less inhibition which is sufficient for downstream applications than column-based techniques.
Antibiotic susceptibility of bacteria causing ear infections: cross-sectional study at National Hospital of Sri Lanka
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2021-10-29) Nadeesha, D.S.; Gunawardane, K.A.K.N.; Karunarathna, K.K.A.N.; Widanagamage, R.D.; Jayasuriya, C.
Bacterial isolates from patients with ear infections have exhibited resistance to one or more antimicrobial agents. Only a few studies have been carried out on the antibiotic susceptibility of bacteria causing ear infections in Sri Lanka. This has imposed a significant burden on clinicians when choosing the right antibiotic for treating ear infections. Hence, a cross-sectional study was carried out to determine the antibiotic susceptibility of bacteria in ear infections. Ear swabs were collected from patients from August through October 2018 by the microbiology laboratory at the National Hospital of Sri Lanka (NHSL). The antibiotic sensitivity test (ABST) was performed on bacteria following protocols published by the Clinical Laboratory Standard Institute (CLSI). SPSS software was used to analyse data. Of the patients who visited the ENT clinic during the study period, 146 suspected cases with ear infections had been reported to the microbiology laboratory. Out of them, 138 patients (94.5%) were positive for pathogenic bacteria. Six pathogenic bacterial species were identified from the ear swabs. Pseudomonas aeruginosa, coliforms, Staphylococcus aureus, MRSA, Proteus sp., and Acinetobacter were the most common pathogens causing ear infections. The most frequently isolated species from ear swabs was P. aeruginosa, whilst the least commonly isolated species was Proteus sp. Our findings indicated that ampicillin had the highest overall resistance followed by erythromycin and cefuroxime, whereas most bacterial isolates exhibited susceptibility to vancomycin, teicoplanin, imipenem and ceftazidime. Thus, antibiotic resistance should be considered in prescribing the right antibiotics when treating patients with ear infections.
Distribution of oxalate in different tissues of star fruit
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2021-10-29) Senevirathna, P.L.D.R.U.; Abeysekara, N.R.M.; Chandrasena, U.S.D.; Ratnayake, W.M.K.M.; Kumari, K.D.K.P.; Suresh, T.S.
Star fruit (Averrhoea carambola) is a popular fruit with a high concentration of oxalate. Therefore, prolonged and regular consumption of star fruits can lead to urinary calcium oxalate crystal formation, nephrolithiasis and chronic kidney diseases. Data are scarce on the distribution of oxalate in star fruit tissues. The present study determined the oxalate concentration in different parts of the fruits of the small sour wild and large sweet hybrid cultivars. The oxalate concentrations in the flesh, peel and ridges of mature unripe and ripe fruits were determined by titrating with KMnO₄. The results show that the mean total oxalate concentration (mg/100 g) in the flesh, peel and ridges of the small sour ripe fruits were 40.2 ± 5.2, 73.0 ± 5.1 and 125.6 ± 6.1, respectively, and that of the unripe fruit of the same cultivar were 29.9 ± 1.1, 70.5 ± 18.4 and 110.9 ± 19.9, respectively. In large sweet ripe fruits, the mean total oxalate concentration in flesh, peel and ridges were 31.7 ± 11.7, 60.2 ± 16.9 and 107.9 ± 28.7, respectively, and that of the unripe fruit of the same cultivar were 28.8 ± 7.3, 56.4 ± 18.4 and 110.6 ± 28.2, respectively. The results revealed that the mean total oxalate concentration in ridges on all four treatments was significantly (p < 0.05) higher than the other parts of the fruit, while the lowest content was observed in the flesh. Comparatively, the concentration of oxalate was higher in the small sour fruits than the large sweet fruits. The unripe fruits of both cultivars contain a relatively lower content of oxalate than ripe fruits. The results suggest that consuming the large sweet star fruit is safer than consuming small sour fruits, and removing the peel with ridges before consuming star fruits is recommended.
Establishment of a protocol for genomic DNA extraction from human saliva
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2021-10-29) Perera, A.Y.P.; Jayatilake, J.A.M.S.; Rajapakse, S.
Extraction of sufficient quantities of high-quality DNA is a prerequisite for genetic research and clinical diagnostic studies. Obtaining patient compliance for invasive and painful sample collection procedures is difficult. Saliva is a non-invasive source of DNA; however, the lack of standardized protocols that do not rely on overnight incubations or expensive kits has greatly limited its usage. Hence, the present study aimed to set up a protocol for isolating high-quality genomic DNA from saliva collected from 21 healthy volunteers belonging to two age groups. Unstimulated whole saliva was collected from the participants and subjected to a modified phenol-chloroform DNA extraction method. The effects of the time of sample collection, storage conditions, and sample volume on the quality of extracted DNA were determined by repeating the protocol under the respective non-standard conditions. Each DNA extraction was followed by DNA purity and concentration estimation, agarose gel electrophoresis, and PCR amplification using primers specific for the common periodontal pathogen, Porphyromonas gingivalis. The proposed protocol was able to produce DNA of mean purity values >1.700 and mean concentrations >100 μg mL⁻¹ . Moreover, PCR amplification revealed the absence of P. gingivalis in all samples. The statistical analysis using SAS 9.00 revealed that sample collection 15 min after a meal significantly reduced the quality of extracted DNA. In contrast, saliva storage for 48 h at -20 ℃ before DNA extraction produced no such difference (P < 0.05). The novel protocol also produced high-quality DNA from a minimum of 0.1 mL of saliva. Overall, this optimized protocol allows the successful economic isolation of high-quality genomic DNA from human saliva using readily available reagents and laboratory conditions, suitable for diagnostics and large-scale population-based studies.
Herbicidal properties of invasive alien plants Ageratina riparia and Austroeupatorium inulifolium against Brassica juncea
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2021-10-29) Ariyathilake, K.P.M.V.U.L.; Bandara, N.C.; Damunupola, J.W.; Jayasuriya, K.M.G.G.; Madawala, H.M.S.P.; Wijesundara, D.S.A.; Bandara, B.M.R.
Synthetic herbicides cause harmful effects on human health and the environment, and therefore, the importation of agrochemicals has been banned recently in Sri Lanka. Invasive alien plants (IAPs) spread aggressively, threatening ecosystems, biodiversity and crops. A strategy to resolve the problems of synthetic herbicides and the uncontrolled spread of IAPs is to develop eco-friendly plant-based herbicides from IAPs. Accordingly, two IAPs, Ageratina riparia (Regel) R.M. King & H. Rob. and Austroeupatorium inulifolium (Kunth) R.M. King & H. Rob. were evaluated for their herbicidal properties against Brassica juncea (L.) Czern. The dry leaf powders, prepared from the two IAPs, were separately extracted into dichloromethanemethanol (1:1) using a bottle extractor. The leaf powders and the concentrated leaf extracts were tested for their inhibitory effects on seed germination and early seedling growth (shoot length, root length and biomass) of B. juncea in a Petri dish assay using 2-methyl-4- chlorophenoxyacetic acid (MCPA) and glufosinate ammonium as positive controls and distilled water and aqueous dimethyl sulfoxide as negative controls. Each treatment was carried out on 25 seeds in four replicates in three trials. The IC₅₀ values of A. riparia and A. inulifolium leaf powders were 0.81 ± 0.07 mg cm⁻² and 0.60 ± 0.13 mg cm⁻² , respectively; the corresponding values for A. riparia and A. inulifolium leaf extracts, in equivalent leaf powder, were 0.48 ± 0.04 mg cm⁻² and 0.52 ± 0.04 mg cm⁻² , respectively. Leaf powders (at 1.76 mg cm⁻² ) and extracts of A. riparia (at 0.95 mg cm⁻² equivalent leaf powder) and A. inulifolium (at 0.74 mg cm⁻² equivalent leaf powder) were more potent than the commercial herbicides glufosinate and MCPA (at concentrations recommended for field use). Growth parameters of B. juncea seedlings decreased with increasing amounts of leaf powders and extracts of both IAPs. The two IAPs A. riparia and A. inulifolium are potential sources for developing plantbased herbicides.
Performance of Chilli, Capsicum annum l. under different foliar treatments
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2021-10-29) Ruhunuge, I.J.A.; Wijeratne, A.W.; Weerasinghe, K.K.S.; Sandaruwan, R.P.A.; Wimalasiri, E.M.
The general technique of supplying nutrients to plants is the application of fertilizer to the soil. However, foliar applications are practised to offset the rapid nutrient requirements of plants. This study evaluated the growth and yield performances of MI-Green chilli variety under Department of Agriculture (DOA) recommendation over different foliar applications. This experiment was laid out in a Randomized Complete Block Design (RCBD) with five foliar spray treatments viz., cow dung (T1), compost tea (T2), urea (T3), urea+ mono-ammonium salt f (T4), urea⁺ sucrose (T5), and a control (DOA recommendation; T6). Here, T1 and T2 were used as pesticides rather than fertilizers. Results disclosed the highest plant height (53.5 ± 6.8 cm) on 90 DAS (Days after sowing) in T4 whereas the lowest (38.9 ± 4.6 cm) in T2. The highest number of flowers at 60 DAS (21 ± 3 flowers) was recorded in T4, whereas the least (8 ± 2 flowers) was in T3. The highest number of pods per plant was in T4 (15 ± 3 pods), whereas the least was in T3 (6 ± 2 pods). The maximum pod length (6.8 ± 2 cm) was in T4, while the least (4.4 ± 3 cm) was in T3. The highest seeds/pod (87 ± 12 seeds) was in T4, and the least (47 ± 13 seeds) was in T3. Among the treatments, T4 recorded the highest average pod weight (12.5 g), and the lowest (5.1 g) was in T3. The highest yield per plant (2.8 g) was in T4, whereas the least (2.1 g) was in T3. The highest final yield was in T4 (5.6 t/ha), whereas the lowest was in T3 (4.2 t/ha). There was a significant (p < 0.05) difference in T4 with control (𝜏1-𝜏2 = 0.8 > LSD = 0.6). Hence urea⁺ monoammonium salt foliar spray was the best foliar treatment that can apply under the DOA recommendation to get better growth and yield performance in MI-Green chilli.
Silver nanoparticles incorporated polymer-based electrospun skin scaffold for burn wounds
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2021-10-29) Yapa, Y.M.P.; Leelarathna, W.D.H.E.; Dissanayake, R.K.; Wijayabandara, J.; de Silva, K.M.N.
Polymeric electrospun nanofibrous scaffolds with enhanced antimicrobial properties have become a suitable substitute for artificial scaffolds in tissue engineering applications, including skin scaffolds, in the recent past. This is because of the ability to mimic the anatomical architecture of extracellular matrices by facilitating cell growth and proliferation. Silver nanoparticles (AgNP) are known to have antibacterial activity. In this study, AgNP and Graphene oxide (GO) incorporated PolyCaproLactone/ PolyEthylene Glycol (PCL/PEG) based electrospun fibrous mesh was prepared, characterized and tested in vitro for use as wound dressings. Silver nanoparticles were synthesized by reducing Ag⁺ using trisodium citrate and characterized using UV-Vis spectrophotometry. Different fibrous scaffolds were prepared, including PCL+PEG, PCL+AgNP, PCL+PEG+AgNP and PCL+PEG+AgNP+GO using an electrospinning apparatus. The scaffold discs were loaded with prepared AgNP solutions (0.05 M, 0.1 M, 0.2 M) for the antimicrobial assay. The morphology of fabricated scaffolds was evaluated by optical microscopic studies and by XRD studies. Finally, water absorption capacity and antibacterial assays (by disc diffusion method) were performed using Ciprofloxacin 0.50 mg /disc as the positive control. The water absorption studies showed that the scaffold could absorb water by seven folds of its initial weight. The scaffold showed promising antibacterial activity against both Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa) and Gram-positive bacteria (Bacillus cereus, Staphylococcus aureus, Staphylococcus epidermidis), which can be predominantly seen in burn wounds. The results show that the prepared biodegradable and biocompatible electrospun fibrous scaffold may be useful as an effective tissue-engineered artificial extracellular matrix for burn wounds since it can absorb high amounts of exudate with an antimicrobial action against the abovementioned bacteria.
Gastrointestinal parasites of dogs in a remote tea-growing area in Talawakelle: a potential public health problem
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2021-10-29) De Silva, T.K.; Rajakaruna, R.S.; Mohotti, K.P.; Rajapakse, R.P.V.J.; Perera, P.K.
Gastrointestinal (GI) parasites cause severe illness in dogs and humans, particularly in socioeconomically challenged communities with large stray dog populations. Most of these GI parasites of dogs are zoonoses. Since dogs play a pivotal role in the epidemiology of human infections, investigating the types of GI parasites and prevalence are important in assessing their public health significance. Fresh faecal samples from stray dogs in two tea estate communities (n = 50 from each) in Talawakelle were collected and analysed following a modified Sheather’s sucrose floatation method. The common dog hookworm Ancylostoma was further investigated using the molecular marker ITS1-5.8S-ITS2 for species identification and phylogeny. Of the 100 stray dogs examined, 97.0% were positive for GI parasites. Mixed infections (85.6%) were more common than single infections (14.4%). Sixteen GI parasite species were detected using egg morphology and morphometry, all of which were zoonotic. Entamoeba sp. (58.0%) was the most prevalent infection, followed by Ancylostoma sp. (45.0%) infection. Overall, helminth prevalence was significantly higher in female dogs (92.5%, n = 37) than male dogs (73.3%, n = 44), potentially due to the immune suppression during pregnancy and lactation. Puppies (n = 15) harboured higher parasitic burdens than adults (n = 82), of which the prevalence and burden of Toxocara canis (68.8%; p < 0.001) was significantly higher, possibly due to vertical trans-mammary or trans-placental transmission of infective larvae. This study depicts the first record of the parasite Hymenolepis from dogs in Sri Lanka. This study also signifies the first molecular characterisation of Ancylostoma caninum in Sri Lanka. The local variant was phylogenetically unique and 99.2% similar to the variant identified from India. The presence of zoonotic GI parasites with public health significance highlights the importance of improving community sanitation, proper veterinary care for dogs, and public awareness of zoonotic diseases.
Ectoparasites in pets and livestock in Udunuwara Divisional Secretariat division in Central Province of Sri Lanka
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2021-10-29) Samaraweera, D.A.C.E.; Rajakaruna, R.S.
Ectoparasites are a major health concern of both humans and animals. They act as vectors of many economically important and zoonotic diseases, and in-depth studies on species diversity are important in controlling and preventing infestations. The objective of the study was to survey ectoparasite species in domestic animals and collect socio-ecological data from households having infested domestic animals within the Udunuwara Divisional Secretariat Division in the Central Province of Sri Lanka. Households were visited using a snowball sampling method, and ectoparasites were collected through visual appraisal. Seven hundred eighty ectoparasites were collected from 151 host animals, including dogs, cats, cattle, rabbits, goats, domestic fowls, turkeys, pigeons, lovebirds, and a buffalo in 42 Grama Niladhari divisions within the area. The collection comprised of ticks (n = 311, species = 13), lice (n = 196, species = 11), fleas (n = 256, species = 2), and mites (n = 17). Among the tick species, the cattle tick Rhipicephalus microplus (30%) was the most abundant, followed by the brown dog tick Rhipicephalus sanguineus (29%) and Rhipicephalus haemaphysaloides (17%). Among the louse species, Menacanthus stramineus (28%) was the most abundant, followed by Damalinia caprae (15%) and Lipeurus caponis (14%). The dog flea Ctenocephalides canis (82%) was the most abundant, while Ctenocephalides felis was found in cats and dogs. The rabbit fur mite: Leporacarus gibbus and another two Analgoidae mites were recorded from rabbits, domestic fowls, and lovebirds, respectively. Households with low sanitation, close contact with the wild animals, feeding livestock with cut grass like Panicum maximum (Guinea grass), and poor veterinary care were socio-ecological factors presumably creating a conducive environment for infestations. Moreover, the household members lacked awareness about ectoparasites, ectoparasite-related complications, and zoonotic potential of ectoparasites. Domestic animals were infected with a wide range of ectoparasites. Island-wide surveys on ectoparasite fauna are required to understand the distribution and ecology of these parasite taxa.
Effect of ph on early embryo development of zebrafish, Danio rerio
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2021-10-29) Imanthi, K.P.A.; Wanigasuriya, J.K.P.; Pathmalal, M.M.
Water pollution has become a leading environmental problem faced by today’s world. It has been reported that the pH in water bodies has changed critically in past years along with the increasing land-use practices, industrial discharges and anthropological activities. Extreme acidic and alkaline pH levels affect the early embryo development of fish in lethal and sublethal levels, inducing low hatching rates and morphological abnormalities. Zebrafish (Danio rerio) is an ideal model for studying environmental and toxicological effects on early embryo development due to the small body size, transparency and ex-utero development of the embryo. The present study evaluated the hatching rate, hatching time, morphological abnormalities and mortality rates of zebrafish embryos exposed to pH ranging from 1 to 12. Desired pH solutions were prepared using 1 M HCl and NH₄OH at 27 °C. Distilled water (pH 7) was used as the control. All experiments were carried out up to 96 hpf (hours post-fertilization) with ten embryos per pH value and triplicated using different batches. Variations in hatching rate, time and mortality rate were obtained in different pH solutions compared to the control. The pH values of 3 and 10 were identified as lethal levels with 100% mortality, where embryos were turned opaque within 1 h. A hatching rate of 100% was observed from 6 – 8 pH range, and it ranged from 60 – 80% at pH of 4, 5 and 9. The highest hatching time of 78 ± 1.15 hpf was recorded at pH 4, where the lowest was at pH 7 at 68 ± 0.58 hpf. Embryo hatching occurred around 72 hpf. There were no morphological abnormalities detected in surviving embryos. The results show that zebrafish embryos can tolerate a wide range of pH, and the effect is minimal at pH 6 – 8 while having a 100% survival rate with a low hatching time.
Antagonistic activity of Lactobacillus fermentum against Aspergillus species obtained from pasteurized fruit syrup
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2021-10-29) Kumara, S.A.S.; Gunasena, G.D.D.K.
According to the WHO, a particular bacterium must possess specific characteristics to be considered a probiotic bacterium used in the food industry. Antagonistic activity against other microorganisms by producing antimicrobial substances is one such character. This study investigated the in vitro antagonistic activity of two naturally isolated Lactobacillus fermentum strains against spoilage of Aspergillus spp. isolated from pasteurized fruit syrup. Lactobacillus fermentum strains were isolated from spontaneously fermented buffalo milk. Molecular identification of L. fermentum (strain L-18 and T5d) and A. foetidus, A. flavus, A. oryzae and A. elegans was carried out by the authors previously. For the assay, each L. fermentum strain was streaked as one line in a modified MRS agar plate and incubated anaerobically at 37 °C for 48 h. A fungal agar block was placed on an incubated MRS agar plate, maintaining a distance of 2 cm from the L. fermentum streaked line and incubated aerobically at 28 °C for up to seven days. The area of the fungal mycelium in photograph images was transferred to square pixels using Adobe© photoshop C6 (13.0 - 64bit) software kit. The square pixels were then converted to square centimetres by using MS Excel 2019. The ability of antifungal activity by the two strains of L. fermentum was detected by comparing the growth area of each tested fungal mycelium corresponding to the same mycelium without the bacterium (positive control). Both L. fermentum strains showed positive antagonistic effects against all four tested Aspergillus species. Zero growth of A. foetidus indicated 100% control by both L. fermentum strains, while the growth of A. elegans was controlled close to 100%. Aspergillus flavus and A. oryzae were moderately controlled. The study identified two potential in-vitro antifungal L. fermentum isolates, exhibiting the benefit of consuming traditional buffalo curd and its potential to be used as a probiotic food source.
Lactobacillus fermentum as a biocontrol agent against foodborne pathogen Listeria monocytogenes
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2021-10-29) Samaranayake, M.S.N.; Gunasena, G.D.D.K.
Overuse and misuse of antibiotics are major drivers for the emergence and evolution of antibiotic resistance in commensal and human pathogens. The spread of acquired antibiotic resistance in the environment mainly occurs via the food chain. The resistance of pathogenic bacteria to currently available antibiotics presents a possible challenge to human health. As a solution to this prevalent global issue, alternatives for antibiotics are in search, and biological control is at the forefront. Listeria monocytogenes is a foodborne human and animal pathogen that causes listeriosis, while Lactobacillus spp. are generally regarded as safe and possess the GRAS status. In this study, three strains of Lactobacillus fermentum ( L-12, L-15, and L-18 ), previously isolated from spontaneously fermented buffalo milk from Kantale, Sri Lanka and molecular identification was carried out earlier, were tested in vitro for their possible biocontrol efficacy against L. monocytogenes (NTCT 11994). The antagonistic activity was screened using the agar well diffusion method. Lactobacillus fermentum supernatants were transferred to wells on L. monocytogenes agar plates containing BHI. Sterile MRS broth was used as the negative control. The experiments were conducted in triplicate, and the diameter of inhibitory zones was determined. All three strains of L. fermentum demonstrated antagonistic activity against L. monocytogenes. The antagonistic performance was highest in L-12, followed by L-18 and L-15, respectively. Strain L-12 had the highest mean zone diameter of 13.8 mm. Results showed that the antagonistic behaviour is highly strain-specific. In vitro study supports the possible usage of L. fermentum strains as biological control agents to inhibit L. monocytogenes. Further, fermented buffalo milk (buffalo curd) can be introduced as a functional food with a natural antibacterial effect.
Potential of biofilm treated k-feldspar as a bio-mineral fertilizer in organic agriculture
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2021-10-29) Ekanayake, S.N.B.; Premarathna, M.; Warnakulasooriya, W.M.K.D.S.; Seneviratne, G.
Being one of the essential macronutrients for plants, potassium (K) plays a vital role in plant growth and health. In organic agriculture, we cannot use any chemical inputs; therefore, K-rich minerals like K-feldspar will be suitable for replacing muriate of potash (MOP). Unfortunately, K-feldspar is not capable of releasing a sufficient amount of K due to its lower solubility. In the present study, K-feldspar was treated with a biofilm formulation (BF) developed from soil fungi and bacteria to form a bio-mineral fertilizer, i.e. Biofilm-feldspar, and analyzed for its solubility. Here, six treatments viz. (a) feldspar, (b) Biofilm-feldspar, (c) autoclaved (121 °C) Biofilm-feldspar, (d) heat-treated (200 °C) Biofilm-feldspar, (e) MOP, and (f) control (no fertilizer) were compared in a leaching column experiment up to four weeks. Results show that the Biofilm-feldspar released K (ca. 100 ppm) comparable to MOP at the end of four weeks, indicating a potential to replace MOP. In addition, the solubility of Biofilm-feldspar was further increased with the heat treatment. The highest release of K (ca. 115 ppm) was observed in feldspar heated at 200 °C followed by BF treatment. From the heat treatment, microscale cracks might have formed and facilitated the microbial action on the mineral structure. The study revealed the potential of using Biofilm-feldspar as a bio-mineral fertilizer in agriculture. However, further studies should be conducted to evaluate Biofilm-feldspar in soil-plant systems under farmers’ field conditions.
Biofilm biofertilizer mitigates health risks associated with potentially toxic trace element accumulation in rice grains
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2021-10-29) Warnakulasooriya, W.M.K.D.S.; Premarathna, M.; Ekanayake, S.N.B.; Rathnathilaka, A.T.D.; Seneviratne, G.
In Sri Lanka, the annual per capita consumption of rice fluctuates around 100 kg. However, rice is often contaminated with potentially toxic trace elements (PTTE), which may lead to many health complications in the long run. The use of chemical inputs [e.g. chemical fertilizers (CF)] has been the main reason. As microbes reduce the bioavailability and mobility of these PTTE, implementing microbial interventions in rice cultivation is a need of the hour. In the present study, the Department of Agriculture (DOA) recommended fertilizer practice (100% CF), 66% CF + Biofilm biofertilizer (66% CF + BFBF), and 66% CF alone were used as treatments. In each treatment, the heavy metal content in rice grains was compared using ICP-OES. According to the results, concentrations of PTTE showed in the order of Zn > Cu > Ni > Cr > As in all three treatments. The least amount of Cadmium (Cd) and Co were in 66% CF + BFBF and 100% CF practices, respectively. The BFBF application decreased the heavy metal concentrations in rice grains compared to the 100% CF application. This could be attributed to the immobilization of PTTE by soil microbes through enzymatic microbial detoxification, production of sulfides and carbonates enhancing the precipitation of toxic metal ions, cellular sequestration, and bio-adsorption. Furthermore, the results indicated that the BFBF practice with reduced CF could lower Cd to impose the lowest estimated daily intake. The hazard quotient (HQ) of As was 1.8 in the 100% CF (DOA practice), indicating potential health risks, while in the BFBF practice with reduced CF, it was reduced down to 0.4. In addition, the hazard index was decreased from 3.7 to 1.6, indicating a lesser likelihood of adverse health effects with 66% CF + BFBF practice. Therefore, it can be concluded that the addition of BFBF with a reduced CF can provide a healthier plate of rice than the use of 100% CF.
Temporal variation in terrestrial pest gastropod communities in agricultural lands in Nuwara Eliya District, Sri Lanka
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2021-10-29) Thilakarathne, K.G.D.D.; Ranawana, K.B.; Kumburegama, S.
Population fluctuations of terrestrial pest gastropods (TPG) are important aspects when developing pest control strategies. Nuwara Eliya District (NED) has the highest production of upcountry vegetables in Sri Lanka and recorded the highest TPG infestations. The study evaluated changes in TPG populations during both the rainy (RS) and non-rainy (NRS) seasons in the Nuwara Eliya District. Eighty agricultural lands were sampled from 2017 to 2019 during RS and NRS, establishing ten 1 m² sampling plots per location. A total of 5,758 individuals belonging to 13 species and seven families and 1,325 individuals belonging to nine species and five families were encountered during RS and NRS, respectively. Species abundance and density (t = 4.69, p = 7.15×10⁻⁶) were higher in the RS. The relative abundance and densities of each species except C. chenui were higher in RS. These differences were significant for Bradybaena similaris (t = 3.79, p = 2.2×10⁻¹⁶), Deroceras reticulatum (t = 2.64, p < 2.2×10⁻¹⁶), Lissachatina fulica (t = 2.90, p = 0.0005), Mariella dussumieri (t = 4.25, p < 2.2×10⁻¹⁶) and Macrochlamys indica (t = 1.96, p = 0.0001). However, TPG eggs and estivating gastropods were found within the soil and under decaying organic matter during the NRS, indicating that these species breed during the NRS while the young hatch during the RS leading to a sudden increase in the population. Diversity of TPG (Shannon-Wiener, H' and Simpson dominance, Dₛ) were higher in RS (H' = 1.60; Ds = 0.70) than NRS (H' = 1.45; Dₛ = 0.69). The similarity of TPG communities between the two seasons was 57%. This population dynamics of TPG can be utilized to devise pest management actions. Here we recommend that the control measures are not limited to the RS but also implemented during the NRS when the TPG populations are relatively low, ultimately reducing the farmers' economic losses.
Stress tolerant rhizobial strains inhabiting clitoria ternatea l. in Anuradhapura District,Sri Lanka
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2021-10-29) Tennakoon, P.T.M.K.C.; Rajapakse, S.
Biological Nitrogen Fixation (BNF) is a process in which atmospheric nitrogen gas is fixed by microorganisms such as rhizobia that live in nodules of the legume plant roots. Clitoria ternatea L. is a perennial herbaceous legume that grows naturally in a wide range of soil and gets successfully nodulated even under harsh climatic and soil conditions. This study was conducted to isolate rhizobia from C. ternatea grown in different locations of Anuradhapura district, Sri Lanka and to characterize their stress tolerance to identify highly tolerant strains. The root nodules from C. ternatea were collected from seven sites in the Anuradhapura District. Twentyeight pure rhizobial colonies were isolated in ½ Lupin Agar medium. They were separately grown in ½ Lupin broths and subjected to four physiological conditions of pH (3 – 9), temperature (25 – 45 °C), salinity (NaCl concentrations of 0.1 – 3.0%) and drought [Polyethylene glycol-8000 (PEG) concentrations of 0.1 – 0.4%]. The rhizobial strains which showed high tolerance to extreme physiological conditions were subjected to a combination of physiological stress conditions such as pH (8.0), temperature (36 °C), NaCl (3.0%), and PEG (0.4%) to mimic the prevailing environmental conditions in the Anuradhapura District. DNA was extracted from these stress-tolerant rhizobial strains and subjected to PCR-based ERIC fingerprinting, and the genetic diversity was assessed. Of the 28 pure rhizobial colonies, 12 isolates showed high tolerance to at least two extreme physiological conditions. The optimum pH, salinity and temperature range for the growth for the isolates were found between 5 – 8, 0.1 – 1.0% and 30 – 35 °C, respectively. Almost all the isolates grew well in the 0.2% PEG concentration. AP-2 (Anuradhapura urban site) showed the highest tolerance at pH 3.0 and 9.0 and 3.0% NaCl concentration. The isolates KH-3 (Kahatagasdigiliya site) and MH-3 (Mihintale site) showed the highest survival at 45 °C and 0.4% PEG concentration, respectively. PG-1 (Palugaswewa site) showed the highest tolerance to combined physiological stress. The ERIC profile confirmed the high genetic diversity, and 11 clusters were found at 69.9% of the similarity level. These stress-tolerant strains could be used for further studies on cross inoculation of crop legumes as a solution for the high usage of chemical nitrogen fertilizers.
Characterization of extracellular polymeric substances of cyanobacterial, fungal and bacterial biofilm complexes by liquid chromatography-mass spectrometry and fourier transform infrared spectroscopy
(Postgraduate Institute of Science (PGIS), University of Peradeniya, Sri Lanka, 2021-10-29) Premarathna, M.; Rathnathilaka, A.T.D.; Madawala, H.M.S.P.; Seneviratne, G.
Biofilms are complex communities of multiple microbial species that are attached to surfaces or interfaces in nature. They secrete self-produced extracellular polymeric substances (EPS) for their structure and protection. Such biofilms can also be developed in vitro using beneficial microbes for biofertilizers, pharmaceuticals, fuels and electricity. Resident microbes in biofilms establish EPS biochemical-based complex network interactions that govern their functioning. The EPS is considered the “dark matter of biofilm” due to its complexity. Identifying the EPS biochemicals and understanding their contribution to the network interactions is important to achieve improved qualitative productivity and innovation in this field of research. Therefore, a study was designed to analyze biochemical parameters of fungal-bacterial biofilms (FBBs), fungal-cyanobacterial biofilms (FCBs), cyanobacterial-bacterial biofilms (CBBs), and fungalcyanobacterial-bacterial biofilms (FCBBs). Microbes used were Aspergillus niger, Nostoc sp., and gram (-) Stenotrophomonas maltophilia and gram (+) Bacillus subtilis, as fungal (F), cyanobacterial (C) and bacterial (B) counterparts, respectively. ATR-FTIR and LCMS were used to characterize the EPS produced by the different biofilms. Results revealed that FCBBs were at the forefront of producing lipids, proteins, and polysaccharides. In FCBBs, gram (-) S. maltophilia was more productive than the gram (+) B. subtilis, and the EPS production was affected by the F:C:B ratio of the inoculum. In addition, ecologically and industrially important three biochemicals, i.e. Rescinnamine, Colchicine, and Syrosingopine, were found in biofilmEPS. In conclusion, the productivity of biofilms can be improved by manipulating microbial composition. Further research is needed to develop more productive (75 – 95%) biofilms for various biotechnological applications. Also, LC-MS and ATR-FTIR spectroscopy can be recommended as effective tools in comparative studies in evaluating structural and functional properties of fungal, bacterial and cyanobacterial biofilm complexes.

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