International Research Symposium on Pure and Applied Sciences (IRSPAS)

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Subject: search.filters.subject.DNA barcoding

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    Molecular identification of selected Dendrobium cultivars.
    (International Research Symposium on Pure and Applied Sciences, 2017 Faculty of Science, University of Kelaniya, Sri Lanka., 2017) Silva, W.E.R.; Attanayake, R.; Senanayake, S.P.
    The family Orchidaceae includes more than 25,000 species, and the genus Dendrobium consists of over 1,450 species around the world. Today many unidentified Orchid cultivars are available in the market and growers use different vernacular names. Authentication of parental materials is important for conservation and selecting cultivars as parental materials in breeding experiments. However, Dendrobiums are well known for their difficulty in identification due to vegetative similarity among different species and morphological dissimilarity among same species. Since DNA barcoding has been proposed to be one of the most promising tools for accurate identification of taxa, this project was initiated with the objectives of identifying selected commercial Dendrobium cultivars and to determine their phylogenetic relatedness. Twelve commercial Dendrobium cultivars were selected based on the flower morphology. Genomic DNA was extracted from young leaves using a modified Cetyltrimethylammonium bromide (CTAB) method. PCR amplification of DNA was performed using universal ITS 1 and ITS 4 primers. PCR products were sequenced at Genetech Pvt. Ltd., Sri Lanka. Sequences were manually edited using BioEdit software version 7.1.9. Out of 12 samples, 9 samples produced non-specific amplification and only 3 samples produced good quality sequences of nearly 700 bp length. BLAST analysis was performed and sequences were deposited in the GenBank (MF535341, MF535342, and MF535343). Sequences of the current study with other 26 sequences from the GenBank were used in maximum likelihood analysis implemented in Mega 6.0 software with 1000 bootstrap replications. Liparis kumokiri (AY907087) was used as the out group for the analysis. Dendrobium cultivar Triple Fantacy (MF535341) resulted 99% similarity to Dendrobium bigibbum var. bigibbum and Dendrobium bigibbum var. superbum (KP142215 and KP142213) in the BLAST analysis. Unidentified Dendrobium cultivar (MF535343) was 94% similar to Dendrobium bigibbum var. bigibbum (KP142215) and Dendrobium bigibbum var. superbum (KP142214). In addition, both Triple Fantacy and unidentified Dendrobium cultivar, were clustered together with Dendrobium bigibbum var. bigibbum and Dendrobium bigibbum var. superbum. Therefore, Dendrobium cultivars, both Triple Fantacy and unidentified Dendrobium cultivar were identified up to species level as Dendrobium bigibbum. Dendrobium cv. Thailand Tommy (MF535342) resulted 99% similarity to Dendrobium nindii (AY239985) and clustered with Dendrobium nindii with 99% bootstrap support. Thus, the identity of Dendrobium cv. Thailand Tommy was confirmed to be Dendrobium nindii. In summary, DNA barcoding with ITS sequence was successfully used in resolving species identity of selected commercial Dendrobium cultivars in Sri Lanka.
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    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.