International Research Symposium on Pure and Applied Sciences (IRSPAS)
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Item Diversity of decaying wood associated fungi in Dimbulagala forest of Sri Lanka(Research Symposium on Pure and Applied Sciences, 2018 Faculty of Science, University of Kelaniya, Sri Lanka, 2018) Shashikala, M. M. P.; Deraniyagala, A.; Attanayake, R.N.Sri Lanka is one of the 34-biodiversity hotspots in the world bearing a tremendous diversity in flora and fauna. Therefore, it should hold true for fungal species diversity as well. In Sri Lanka, tropical wet evergreen rain forest reserves, mainly Sinharaja forest is well studied for the macro and micro fungal diversity. However, studies in the dry zone and intermediate zone forests are neglected though 83% of the country’s forest cover belongs to this category. The current study was initiated to describe the decaying wood associated fungal diversity in a dry mixed evergreen forest reserve. Decaying wood samples were collected from Dimbulagala forest reserve. Decaying hard wood pieces of at least 6 inches length were collected randomly. Fungal strains associated with the decaying woods were isolated into PDA or semi-selective medium and pure cultures were obtained. Macroscopic and microscopic features were observed. Total genomic DNA was isolated from a modified CTAB method and Polymerase Chain Reaction (PCR) was conducted targeting rDNA-ITS region using universal ITS primers and Sanger dideoxy sequencing was carried out to determine the nucleotide sequence of the region. Sequences were manually edited and compared with the GenBank using Basic Local Sequence Alignment Search Tool (BLAST). Phylogenetic relationships among decaying wood associated fungi were determined using MEGA (version 7.0) and according to the phylogenetic analysis well-defined clusters of fungi that belongs to divisions Ascomycota and Bascidiomycota were found. Fungal cultures were maintained at the Department of Botany using dry filter papers and in sterilized distilled water. A total of 55 fungal isolates were obtained from 36 decaying wood pieces and 35 fungal species were successfully identified. Results indicated that Sri Lankan dry mixed evergreen forests are rich in species of Paecilomyces, Daldinia, Trametes, Perenniporia, Phanerochaete, Hypoxylon, Schizophyllum, Lentinus, Fusarium, Coriolopsis, Phlebia, Coprinellus, Gymnopilus, Scytalidium, Trichoderma, Xylogone, Lasiodiplodia, Neoscytalidium and Pleurostoma. Species of Trichoderma and Lasiodiplodia were the most abundant species. T . harzianum T. lixii, T . longibrachiatum, and T . erinaceus were also found. Out of six Lasiodiplodia isolates, three were L . crassispora, and the rest belonged to the species of L . pseudotheobromae and L . theobromae. Some of the isolated fungi were already known plant pathogens and some were well-known biodegraders. The results indicated that the least studied Sri Lankan dry mixed evergreen forests are rich in various fungal species and could serve as another source in finding biotechnologically important fungal species.Item Anti-oxidant activity of selected endo lichenic fungi (ELF) in mangrove ecosystem of Puttalam lagoon.(International Research Symposium on Pure and Applied Sciences, 2017 Faculty of Science, University of Kelaniya, Sri Lanka., 2017) Maduranga, H.A.K.; Attanayake, R.N.; Amarasinghe, M.D.; Weerakoon, G.; Paranagama, P.A.Natural products based drug development has become an attractive area of research since there are limited options available to treat certain non-infectious diseases such as diabetes. Among these natural products, it has been reported that secondary metabolites of endolichenic fungi (ELF), have the ability to produce promising bioactive compounds. The objectives of this research were to isolate and identify ELF inhabiting mangroves in Puttalam lagoon, Sri Lanka using classical and DNA barcoding approach and to determine anti-oxidant activity of their secondary metabolites. Lichen hosts were collected from Puttalam lagoon in two different sites near, Athathale and around the NARA institute. The ELF were isolated following a standard procedure: a small piece of the thallus was surface sterilized, cut into pieces and dried on sterilized filter papers and then placed on malt extract agar in Petri dishes and incubated at room temperature (28 ⁰C – 30 ⁰C ) . Once pure cultures were obtained, seven isolates were randomly selected for DNA extraction following standard procedures. Quality of DNA was checked by agarose gel electrophoresis. Fungal internal transcribed spacer (ITS) region was amplified using polymerase chain reaction (PCR) with universal ITS 1 and ITS 4 primers and PCR products were sequenced using Sanger dideoxy chain-termination technology. DNA sequences were edited using BioEdit software and compared with the available sequences in the GenBank using Basic Local Sequence Alignment Search Tool (BLAST). In addition, morphological characterization of each fungal isolate was also carried out. Secondary metabolites from each isolate were extracted with ethylacetate separately and the solvent was evaporated under reduced pressure to obtain the crude extract. Free radical scavenging activity of the extracts were evaluated using 2, 2-diphenyl-1-picrylhydrdrazyl (DPPH) assay. Based on the highest sequence similarity to the GenBank sequences, isolates were identified as Diaporthe arengae (98 %), Neurospora crassa (100%), Lasiodiplodia theobromae (100 %), Schizophyllum commune (98 %), Diaporthe musigena (98 %), Hypoxylon anthochroum (98 %) and Nigrospora sphaerica (98%). IC50 values of extracts of Diaporthe arengae, Neurospora crassa and Lasiodiplodia theobromae were 375.9± 0.062μg/mL, 304.9±0.057 μg/mL and 211.2± 0.086 μg/mL respectively. Since percent inhibitions of the rest of the isolates were less than 50 % in the test doses, IC50 values were not calculated. All of the values were compared with standard Butylated Hydroxy Toluene (BHT) (IC50=108.0±0.072). Out of the seven ELF tested, L. theobromae showed the highest DPPH radical scavenging activity. Further testing of the rest of the isolates are being carried out and ELF may provide a good source of antioxidants for biotechnological applications.Item Contarinia maculipennis as an emerging threat to Dendrobium in Sri Lanka - A case study.(International Research Symposium on Pure and Applied Sciences, 2017 Faculty of Science, University of Kelaniya, Sri Lanka., 2017) Dias, M.A.; Amarasinghe, L.D.; Jayalath, W.G.H.; Attanayake, R.N.Blossom midge, Contarinia maculipennis which belongs to Order Diptera: Family Cecidomyiidae is considered as one of the major threat to ornamental and several crop plant species due to its wide host range. For the first time C. maculipennis was recorded from Dendrobium sp. in 1992 from Florida, but the origin of this species is considered as Southeast Asian region. In Korea, it is officially nominated as a quarantine pest since 2007 due to it’s sever economic impact on vegetable crops and ornamental plants. For the first time in Sri Lanka, completely damaged Dendrobium cultivation was found in an ornamental plant nursery at Gampaha district in 2017. It was noted that the symptoms were similar to that of blossom midge damage. Maggots were found to be feeding inside unopened flower buds, causing deformed, discolored buds and blossoms causing premature bud or blossom drop. Floral buds were often found to be rotted. Samples from immature bud stage to fully opened flowers were randomly collected from infected fields into polythene bags. To identify causative agent, floral buds with larval stages were kept in glass containers to allow them to complete their life cycle and thereby morphological characters were studied to confirm the pest species. In addition, yellow color grease sheets were kept inside the greenhouses to trap any adult stages of the pest species. Samples were collected and preserved using 70% ethanol for identification. Since all the damaged flower buds displayed symptoms of bacterial rots, to determine if any bacterial infection is also associated with the symptoms, bacterial isolation procedure was carried out. Different stages of floral samples were separately surface sterilized for two minutes using 70% ethanol and three serial washings with sterilized distilled water. Tissue macerate was prepared and kept for 3 hours before culturing on Nutrient Agar (NA) plates, Potato Dextrose Agar (PDA) and Luria-Bertani (LB) plates. Each sample had three replicates and ten samples were cultured. Growth from the tissues were observed and pure cultures were obtained. Relative length of the first and second flagellomeres, wing length and pattern, larval sternal spatula and its associated papillae and larval eighth abdominal segments were compared with identification keys which were used to identify the genera, Contarinia. The adult stages of trapped insects and adult stages of insect immerged from the larval stages were useful in confirming the species as C. maculipennis. Basic biochemical tests and Gram’s staining assisted in identifying the bacterium as belonging to the genera, Erwinia sp. and it appears that the bacterial infection occurs as a secondary infection after larval stages of C. maculipennis damage the floral tissues. This is the first record of C. maculipennis infecting orchid nurseries in Sri Lanka and if proper care is not taken it will invade other crop species as the pest has a broad host range. It is not clear whether the pest was a recent introduction through the imports of plant material or whether it is a result of host jump and therefore, it warrants further research.Item Optimization of a genomic DNA extraction technique for genetic diversity studies of selected orchid cvs. with ornamental values(Faculty of Science, University of Kelaniya, Sri Lanka, 2016) Farook, F.; Attanayake, R.N.; Senanayake, S.P.Orchidaceae is one of the largest and most diverse families of flowering plants and a major export ornamental crop. Cultivar development is the key for the success of ornamental flower industry and therefore, it is vital to identify the genetic diversity of the cultivars. In such an attempt, the first step is to optimize basic molecular biological techniques involved in genetic diversity analysis. However, DNA extraction from orchids is challenging compared to the other plant species since orchids are rich in polysaccharides and secondary metabolites, which can act as inhibitors in downstream applications. Most of the standardized protocols require liquid nitrogen freezing step, which is not an affordable practice in the laboratories of developing countries. Therefore, optimization of a low cost protocol to obtain pure DNA is necessary. The objective of the current study was to optimize extraction of genomic DNA for molecular marker based genetic diversity studies of selected orchid cultivars with ornamental value. Leaf pieces and pestle and mortar were stored at -80°C for at least three days. DNA extraction was done from frozen orchid leaves (50-100 mg young leaves) using Promega Wizard genomic DNA purification kit (Promega Inc. USA), classical CTAB protocol and modified CTAB protocol. Except modified CTAB protocol, none of the other methods produced high quality DNA as determined by spectrophotometry and agarose gel electrophoresis. However, the method was successful only for the Dendrobium cultivars but not for the Phalaenopsis leaves tested. Successful amplification of orchid rDNA-ITS region confirmed that the quality of extracted DNA is suitable for other PCR based molecular marker studies such as Random Amplified Polymorphic DNA (RAPD) and Simple Sequence Repeats (SSRs). The modified method was reliable and reproducible.Item Pleurostomophora richardsiae associated with decaying woods in a dry zone forest of Sri Lanka(Faculty of Science, University of Kelaniya, Sri Lanka, 2016) Bandara, R.H.; Deraniyagala, S.R.A.S.; Attanayake, R.N.Wood decay is a biological process by which cellulose and lignin are converted into carbon dioxide and water. This process is important for forest sustainability since it uses cellulose and lignin, the two most abundant organic compounds on earth to recycle nutrients. Nearly 83% of the total forest coverage in Sri Lanka is comprised with dry mixed forests and these forests are rich in hard wood bearing plant species such as Diospyros ebenum, Manilkara hexandra and Drypetes sepiaria. Therefore, fungi associated with decaying these hard wood species should also be efficient lignin and cellulose degraders with possible biotechnological importance. The objective of the current study was to identify such decaying wood-associated fungi found in a dry zone forest using DNA barcoding approach and to maintain these cultures for future research. Decaying hard wood pieces were collected from the forest floor of Dimbulagala forest reserve and brought to the laboratory. Wood pieces were surface sterilized, cut into small pieces, plated on selective PDA media. Pure cultures of fungi were obtained and morphological characterization was done. For molecular characterization, fungal mycelia grown in PDB were subjected to total genomic DNA isolation. Fungal rDNA-ITS region was amplified and sequenced from both directions. Sequences were manually edited and BLASTN searches reveled that Trichoderma species and Lasiodiplodia species were among the frequently found species. Species delineation of certain fungal isolates were challenging and therefore, molecular phylogeny based identification was applied. Maximum Likelihood method based on the Kimura 2-parameter model was used and interestingly Pleurostomophora richardsiae (isolate name DDW 05), a possible plant pathogenic fungus, was found to be associated with a decaying hard wood sample. P. richardsiae has been reported to cause grapevine dieback and cankers in Italy and Spain. This finding is interesting since forest dieback has long been an unidentified problem in Sri Lanka and it could be possible that P. richardsiae is among the organisms causing forest dieback and cankers in Sri Lanka. Previous reports also show that this fungus has been isolated from wood, ground wood pulp, sewage and soil in North America, Europe, Africa and several countries in Asia. Therefore, further research is needed to confirm the pathogenicity of the isolate.