Browsing by Author "Wanigatunge, R.P."

Now showing 1 - 7 of 7

Co-occurrence of Geogenic, Microbial, and Anthropogenic Emerging Contaminants: Ecotoxicity and Relative Environmental Risks
(Springer, 2022) Magana-Arachchi, D.N.; Wanigatunge, R.P.
Heavy metals, 1,4-dioxane, cyanotoxins, bacteria with antibiotic-resistant genes, pharmaceuticals, nanoparticles, and many more are listed as emerging contaminants due to their toxicity whether their origin is geogenic, microbial, or anthropogenic. These specific types of environmental impurities, at present, are excluded from regular scrutinizing programs but are potential nominees for future supervision and management subjected to their hazardous nature, community awareness, incidence, and depending on the amount present in distinct environments. A chemical or an organism does not need to be novel to be groped as an emerging contaminant. Primarily, these impurities had been in nature for decades, but their impact is being assessed only now. When these three types of pollutants coexist together the adverse impact they create on Mother Nature is enormous. With increasing population growth and with rapid urbanization, release of these contaminants gets accelerated and considered as a global issue. Due to their perseverance, in aquatic environments, they get accumulated in biota thereby contaminating the food web and humans and animals mostly get exposed through the food chain or by drinking the contaminated waters. This chapter focuses on emerging contaminants of geogenic, anthropogenic and microbial origin, their coexistence in the environment, ecotoxicity, and relative environmental risks.
Culture-Dependent Characterization of Marine Bacteria from Ussangoda Coast, Sri Lanka
(International Postgraduate Research Conference 2019, Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2019) Hettiarachchi, H.H.; Wanigatunge, R.P.; Magana-Arachchi, D.N.
Marine bacteria play a vital role in marine ecosystem structure and ocean biogeochemistry. Ussangoda has identified as a serpentinite site, situated along the Southern coastal zone of Sri Lanka. In the past, few studies have focused on biodiversity and soil chemistry of this unique habitat. However, marine bacterial diversity in the Ussangoda coast is largely unexplored. Thus, the present study was conducted to isolate and identify marine bacteria in Ussangoda coast. Water, sand and soil samples were collected from 14 sites of the beach which were located approximately 1 m distance representing foreshore, shore line, backshore and upland. Bacteria were isolated by inoculating a 10 μl of each water sample, soil and sand suspension into tryptone soya agar, nutrient agar (NA), and seawater nutrient agar (SNA) and were incubated at 27 ± 2 oC for 3 days. Pure cultures were obtained after 1-2 consecutive sub-culturing on to fresh media plates by streaking. In order to obtain halophilic bacteria, isolates were inoculated into saline nutrient agar media with the concentrations of 10% and 20% of NaCl for moderate halophiles and extreme halophiles respectively and were incubated at 27 ± 2 oC for 7 days. 198 bacterial pure cultures were initially isolated and among them 152 were Gram-positive bacteria (76.8%). Based on colony morphology and Grams’ staining, bacterial isolates were identified as Bacillus, Staphylococcus, Micrococcus, Pseudomonas, Klebsiella, Escherichia coli and Stenotrophomonas,which have been previously reported in coastal zones of other countries. Among the tested 102 isolates, 35 isolates (34%) were reported as moderately halophilic and 14 isolates (13%) were reported as extremely halophilic bacteria. Halophilic bacteria were recorded from shoreline as well as in the upland soil and it may be due to the soil chemistry of the area. Bacterial isolates should be further confirmed by the 16S rDNA sequencing to confirm their identity. This study provides foundational data on the microbial diversity of the Ussangoda coast and further studies are needed to identify the association of microbial community structure with the environmental factors in this region.
Diversity of Firmicutes in selected hot water springs of Sri Lanka by 16S metagenomic sequencing
(Faculty of Science, University of Kelaniya, Sri Lanka, 2020) Samarasinghe, D.G.S.N.; Wanigatunge, R.P.; Magana-Arachchi, D.N.
Thermophiles have the ability to survive in environments with very high temperatures. Hot springs provide unique natural environments for these thermophilic microorganisms. In recent years, hot water springs and thermophilic microorganisms have gained attention due to their industrial and biotechnological importance. Among the thermophilic bacteria, a large number of metabolites can be found in the phylum Firmicutes. Different species of Firmicutes show various ecological optima. Even though Sri Lanka harbors many hot springs, majority of them are not yet explored and require comprehensive studies to unravel the inhabitant microbial diversity, specially the phylum Firmicutes. This study was focused into uncovering the resident thermophilic Firmicutes which could be unculturable and their diversity in four major hot springs namely, Mahapelessa, Wahawa, Maha Oya and Nelumwewa using 16S rRNA metagenomic sequencing. Water samples were collected from the four hot springs in which the surface temperature ranged from 44.2 to 53.8 °C. Further, a water sample from a natural spring located in Digana (26.9 °C) was used as the control. Genomic DNA was extracted from the water samples using modified Boom’s method and was subjected to 16S rRNA metagenomic sequencing (16S V3-V4 region) using Illumina platform. The results were analyzed using GAIA: Metagenomics data analysis software to identify Firmicute bacteria (Operational taxonomic units/ OTU) and to determine their relative abundance. OTU analysis was carried out with a cut-off similarity value at 97%. The relative abundance of Firmicutes in each spring is as follows: Mahapelessa (23.7%), Wahawa (0.8%), Maha Oya (0.9%), Nelumwewa (5.8%) and Digana natural spring (4.2%). The class Clostridia and Bacilli were the most abundant classes observed in hot springs. A total of twenty-one species were affiliated with the phylum Firmicutes, among which 18 species were only detected in hot water springs while the rest of the three species (Clostridium sp., Flintibacter butyricus and Oscillibacter sp.) were only detected in the natural spring. Nine different bacterial species were unique to Mahapelessa hot spring including; Bacillus licheniformis and Bacillus thuringiensis belonging to class Bacilli, Caldanaerocella colombiensis, Desulfotomaculum reducens, Geosporobacter sp., Geosporobacter subterraneus, Pelotomaculum isophthalicicum, Salimesophilobacter vulgaris and Tepidibacter sp. belonging to the class Clostridia. The two bacterial species; Carboxydocella manganica and Faecalibacterium prausnitzii were only recorded from Maha Oya while Clostridium islandicum was reported from Maha Oya and Nelumwewa hot springs. Bacillus pumilus and Sporacetigenium mesophilum were present in Mahapelessa and Wahawa hot springs. Anaerosolibacter carboniphilus was found in both Mahapelessa and Nelumwewa while Anaerobacterium chartisolvens was detected in all hot water springs except Wahawa. Bacillus sp. and Paenibacillus sp. were common in all the hot water springs. When compared to the natural spring, the four hot water springs showed high bacterial species diversity (85%). Results from this study confirm the uniqueness of bacterial species belonging to phylum Firmicutes in hot water springs than in the natural springs. More comprehensive studies on these Firmucutes are needed to identify their potential to be used in industrial and biotechnological applications.
First evidence of epiphytic marine algae Acrochaete leptochaete identified from Beruwala reef, Sri Lanka by DNA barcoding
(Faculty of Science, University of Kelaniya, Sri Lanka, 2020) Weralupitiya, C.M.; Herath, H.M.; Wanigatunge, R.P.
Several green microalgal species that grow as epiphytes and/or endophytes associated with seaweeds have been identified from different locations worldwide. The composition of epiphytic marine algae in a marine ecosystem is an important indicator of environmental alterations. However, there are no previous records on epiphytic marine microalgae in the Sri Lankan coast. Therefore, this study aimed at identifying epiphytic marine microalgae inhabiting marine macroalgae in the Barbarian reef, Beruwala, and provides the first evidence of an epiphytic micro chlorophyte colonized within the seaweed genera from all the three marine macroalgal divisions. Morphologically different macroalgal specimens were collected in September 2018 from the Barbarian reef, Beruwala and they were identified using macroscopic and microscopic characters within two days of sample collection. DNA was extracted from the algal thalli and the nuclear ribosomal DNA Internal Transcribed Spacer regions (nrDNA ITS1-5.8S-ITS2, 650 bp) were amplified by polymerase chain reaction (PCR) from the extracted DNA using a primer pair (ITS1 and ITS4) specific for micro chlorophytes. PCR products were unidirectionally sequenced by the Sanger sequencing method using the forward primer. Sequences were checked and edited using BIOEDIT software version 7.2.6 and aligned with the nucleotide database (blastn) in National Centre for Biotechnology Information (NCBI). According to the morphological characterization of the macroalgae, nine, three and seven genera belonged to the Divisions Chlorophyta, Phaeophyta and Rhodophyta, respectively. The nrDNA ITS1-5.8S-ITS2 sequences showed a 95- 96% similarity to Acrochaete leptochaete (JN104107.1) for the macroalgae Chaetomorpha (Division Chlorophyta), Padina (Division Phaeophyta) and Gracillaria (Division Rhodophyta). According to the fact that 93% sequence similarity should be achieved with that of the Genbank sequences for the species level determination of algae using the nrDNA ITS1-5.8S-ITS2 sequence, it could be confirmed that A. leptochaete is inhabiting Chaetomorpha (A. leptochaete - accession MK910762.1), Padina (A. leptochaete - accession MK910764.1) and Gracillaria (A. leptochaete - accession MK910760.1). This provides evidence for the broad host range owned by A. leptochaete among seaweed genera.
Identification and Disease Assessment of Begomovirus Infecting Capsicum frutescens (Kochchi) in Minuwangoda, Sri Lanka
(International Postgraduate Research Conference 2019, Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2019) Jayathilaka, S.A.S.D.; Wanigatunge, R.P.; Attanayake, R.N.; Edirisinghe, P.
Leaf curl of Capsicum frutescens (Kochchi) is a common problem in many Capsicum growing regions of the country including Minuwangoda area in Gampaha. Water stress, poor soil drainage or viral infection are some of the potential causes of leaf curl. The main aim of the current study was to determine whether the leaf curl of Minuwangoda area is due to viral infection and to determine the disease severity and incidence of the region. A disease assessment key graded from 0 to 6 was developed for CLC disease of C. frutescens. Thirtytwo C. frutescens fields having more than 50 plants per field were selected. Disease symptoms, incidence and severity were recorded. Prominent symptoms of the disease were leaf curling, vein yellowing and leaf yellowing followed by severe chlorosis and stunting as the disease progressed. These symptoms were similar to the viral infection. The disease incidence varied from 0 to 100% in the selected fields and 71.88% of the fields in Minuwangoda area were affected. Disease severity in the selected fields ranged from 0 to 6, while the average disease severity of the fields in the area was 1.3. To determine whether the causal agent is a commonly reported virus of the family Begomoviridae, coat protein (CP) gene was amplified from the DNA extracted from infected leaves using CP specific primers and the sequencing was done using the Sanger sequencing method. Sequence alignment with the NCBI database showed a 95% similarity to Chilli leaf curl virus (Begomoviridae) isolate CL-14 from Nochchiyagama. Results indicates that Sri Lankan C. frutescens cultivation could be challenged by the viral infection and control measures should be implemented as soon as possible.
Identification of the fungal pathogen causing leaf blight of Cinnamomum zeylanicum Blume and in vitro screening of Tebuconazole sensitivity
(The Second National Symposium of Sri Lanka Association for Mycology and Plant Pathology (SLAMPP), 2022) Madhurangi, H. M. T. T.; Wanigatunge, R.P.; Jayasinghe, G.G.; Attanayake, R.N.
Leaf Blight Disease (LBD) is a major nursery disease of cinnamon (Cinnamomum zeylanicum Blume) and the disease causes serious foliar damage in seedlings, especially under high humidity and shade conditions. It also causes considerable damage to mature cinnamon bushes through leaf necrosis, defoliation and dieback of newly emerging flush. However, not much research has been conducted on LBD in Sri Lanka despite cinnamon being one of the key export crops. Therefore, the main objectives of the present study were to identify the causative agent of the LBD in Sri Lanka and to determine the minimum inhibitory concentration of Tebuconazole, one of the commonly used fungicides, to mitigate the pathogen growth in-vitro. LBD symptomatic leaves were collected from cinnamon fields in the Matara district representing 4 categories; tender leaves with initial LBD spots, tender leaves with regular lesions, mature leaves with regular lesions, and mature leaves with irregular lesions. Infected tissues from each category were cultured on PDA and percentages of emerging fungal colonies were determined. Pure cultures of all the fungal isolates were tentatively identified using morphological traits up to the genus level. From the cultures of tender leaves, Rhizoctonia and Colletotrichum-like isolates frequently appeared in 60% and 33.3% respectively. However, in the cultures of mature leaves, Pestalotiopsis-like isolates were detected more frequently (22 – 35%). To determine the exact causal agent, Koch’s postulates were performed using representative isolates from each genus on detached tender cinnamon leaves. Actively growing mycelial plugs were inoculated on one side of the damaged leaf and the other side was inoculated with plain PDA plugs. Leaves were incubated in a moist chamber for 03 days and symptom development was observed. Typical LBD symptoms were developed when mycelial plugs of Colletotrichum-like species were introduced. Rhizoctonia and Pestalotiopsis-like species produced brown, circular or irregular lesions while Colletotrichum-like species showed a unique concentric ring pattern which is similar to the original symptoms of LBD. The potential pathogen species causing LBD was confirmed to be a Colletotrichum-like species. For species identification DNA of two Colletotrichum-like fungal isolates was extracted and ITS gene sequencing confirmed that the two isolates were C. gloeosporioides and C. horii with 100% similarity to the vouchered accessions of the GenBank. In vitro fungicide, sensitivity assay was conducted using a concentration gradient of the fungicide Tebuconazole. It was found that Tebuconazole could mitigate 100% growth of both Colletotrichum spp. at 5 ppm concentration in-vitro.
Morpho-molecular characterization of Lasiodiplodia and Diaporthe species infecting Solanum melongena L. (brinjal) in Gampaha district
(Faculty of Science, University of Kelaniya, Sri Lanka, 2020) Koshila, H.V.A.S.; Dias, R.K.S.; Wanigatunge, R.P.; Edirisinghe, P.
Solanum melongena L. (brinjal) is a vegetable belonging to the family Solanaceae and a popular commodity among consumers. Brinjal plants are infected by numerous fungal pathogens causing a wide range of diseases such as anthracnose by Colletotrichum sp., stem and leaf lesions by Phomopsis vexans (anamorph of Diaporthe), and early blight by Alternaria solani which significantly reduce the yield. The objective of this study was to isolate and identify the fungal pathogens associated with leaves of S. melongena in the Gampaha district. Necrotic or prematurely wilted leaves of S. melongena were collected and associated fungi were isolated onto PDA medium. Pathogenicity was confirmed by wounded and non-wounded inoculation of a seven-day old isolate onto healthy S. melongena leaves and the confirmed fungal pathogens were identified using their morphological, cultural characteristics and analysis of the internal transcribed spacer region (ITS1-5.8S-ITS2). Fifteen fungal isolates were isolated from necrotic leaves of S. melongena. Three fungal isolates (Isolates H32A, H32B and U11) were identified to be pathogenic on leaves of S. melongena based on the pathogenicity test. A necrotic leaf spot was initiated at the site of inoculation with isolates H32A and H32B in both wounded and nonwounded inoculations, which later developed into wilting and premature falling of the leaf. Leaf blight was observed with non-wounded inoculation of isolate U11. Morphological characters of isolates H32A and H32B were similar, with fluffy, blackish-grey, septate mycelia and dark brown oval shape spores with a septum in the middle. Both had similar growth rates of 2.25 cm/day. They were morphologically identified as Lasiodiplodia sp. Yellowish grey color pigmentation was observed in the isolate U11 which produced aseptate hyphae but could not be identified by its morphological characteristics. The nucleotide sequence of ITS region confirmed the morphological identification of isolates H32A (MT990527) and H32B (MT990528) as Lasiodiplodia theobromae with 99.81% sequence similarity to L. theobromae (IRNKB244) at NCBI database. Further, isolate U11 (MT990529) showed 99.82% sequence similarity with Diaporthe eugeniae (ASHM304) at NCBI database. L. theobromae is reported to cause fruit rot in brinjal, while Diaporthe sp. has caused stem and leaf lesions. L. theobromae and D. eugeniae were confirmed to be pathogenic on S. melongena L. (brinjal) plants in the Gampaha district and further studies will be conducted to develop an environmentally friendly strategy to manage above mentioned diseases.