International Research Symposium on Pure and Applied Sciences (IRSPAS)
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Item Isolation of mycorrhizal fungi in selected epiphytic orchids and investigation of the effect of potential host bark extracts on seed germination under in vitro condition.(4th International Research Symposium on Pure and Applied Sciences, Faculty of Science, University of Kelaniya, Sri Lanka, 2019) Premachandra, J. A. H. P.; Senanayake, S. P.Orchids (Family: Orchidaceae) are one of the largest, diverse plant groups in the world with high ornamental value. Orchid seed germination, in nature, is reported to be influenced by the mycorrizal fungi associated in the bark of the host plants. These fungi penetrate the seeds by their hyphae and supply the nutrients for developing embryo resulting the formation of a globular shape structures referred as protocoms. Fungal hyphae infect the cortical cells of the orchids and forming pelotons, that are digested by the orchid cells for their nutrition. This study was focused on isolation of mycorrhizal fungi from six selected epiphytic orchids: Dendrobium aphyllum, Dendrobium sp., Bulbophyllum sp., Eria sp., Coelogyne sp., an unidentified species, and evaluation of in vitro seed germination using host bark extracts inoculated with isolated fungi from orchid roots. Specimens of wild orchids and the host plants were collected from their natural habitats in low-land wet zone and montane zone in Sri Lanka. The mycorrhizal fungi in roots of selected orchid species were isolated and identified as Trichoderma sp. and Rhizoctonia sp. by comparing with the authenticated morphological characters. Preliminary phytochemical screening was performed for each bark extract of host plants using TLC. Presence of secondary metabolites including alkaloids, flavonoids, terpenoids and phenolic were observed in all the bark extracts. According to the spot observations of TLC, although similar type of secondary metabolites was present in the bark extracts of hosts plants, intensity of the secondary metabolites was detected as different. The TLC analysis revealed the presence of high concentration of chemical compounds in methanolic and aqueous extracts while less amounts were in dichloromethane and hexane extracts. In vitro seed germination of the orchid species was assessed using the V/V basis of host bark extracts with the inoculation of isolated fungi from orchid roots. Effect of host bark extracts with mycorrhizal fungi inoculum on seed germination of orchid seeds was analyzed by performing two-way ANOVA using Minitab statistical software. Comparatively high percentage of seeds were germinated in the aqueous extracts (~30%) and hexane extracts (~25%) while very low percentage was in methanolic (0%) and dichloromethane (~0%) extracts of host barks. The findings suggest that secondary metabolites present in the barks of the plants influence the promotion or inhibition of the orchid seed germination whereas fungal inoculation too has a significant effect on orchid seed germination.Item Phylogenetic relationships of selected commercial Dendrobium hybrids in Sri Lanka(4th International Research Symposium on Pure and Applied Sciences, Faculty of Science, University of Kelaniya, Sri Lanka, 2019) Kahagalla, T. H.; Herath, H. M.; Attanayake, R. N.; Senanayake, S. P.Nuclear rDNA-ITS regions and chloroplast matK genes are useful in delineating plant species. In this study, genetic relatedness of eight commercial Dendrobium hybrids (A-H) with a range of attractive flower colours was studied using nuclear rDNA-ITS and chloroplast matK sequences. Genomic DNA was extracted from fresh, young leaves using a modified cetyltrimethylammonium bromide based protocol. rDNA-ITS and matK were amplified using PCR in 25 μl reactions containing 1X PCR buffer, 2.5 mM MgCl2, 0.2 mM dNTPs, 1.25 U Taq DNA polymerase, 0.4 μM forward and reverse primers and 1.00 μl of genomic DNA. The optimized thermal cycling conditions were initial denaturation at 95 oC for 5 minutes, 35 (rDNA-ITS) and 40 (matK) cycles of denaturation at 95 oC for 40 seconds, annealing at 55 oC (rDNA-ITS) and 48 oC (matK) for 40 seconds, extension at 72 oC for 40 seconds and final extension at 72 oC for 10 minutes. rDNA-ITS and matK PCR products were subjected to Sanger sequencing. Sequences were manually edited using BioEdit 7.0.5.3. and ContigExpress software. Sequences were aligned to the nucleotide database in the National Center for Biotechnology Information using mega BLAST program. Forty-three related sequences were obtained from GenBank and the sequences were aligned using ClustalW implemented in MEGA 7.0.26 software. Phylogenetic analysis was performed by generating trees of ITS, matK and concatenated sequences of ITS and matK. The phylogenetic relationships were analyzed using Maximum Likelihood analysis with 1000 bootstrap replications. Phalaenopsis aphrodite, Liparis kumokiri and Malaxis spicata were used as outgroups. Combined gene-tree was estimated using RAxML-HPC BlackBox tool in CIPRES Science Gateway platform. Resulting trees were viewed using Figtree v1.4.3. In the combined gene tree, selected hybrids were clustered into two distinct groups. Dendrobium hybrids A, B, C, E and F were clustered with Dendrobium bigibbum var bigibbum and Dendrobium phalaenopsis (72% bootstrap). Hybrids G, H and D were clustered with Dendrobium nindii and Dendrobium taurinum (79% bootstrap). In matK gene tree, all the selected hybrids were clustered together with Dendrobium kingianum (90% bootstrap). In rDNA-ITS gene tree, hybrids A, B, C, E and F were clustered with Dendrobium bigibbum var bigibbum and Dendrobium phalaenopsis while hybrids D, G and H were clustered with Dendrobium taurinum and Dendrobium nindii (81% bootstrap). Therefore, though high variation in floral morphology is observed among the selected imported commercial hybrids, they were represented from a narrow genetic background. This is an indicative of genetic bottleneck most likely due to selective breeding and it is important to incorporate more diverse varieties in future breeding programs to maintain a diverse genetic backgroundItem Morphometry and pollen germinability of selected commercial Dendrobium cultivars(2018) Kahagalla, T. H.; Hearth, H. M.; Attanayake, R. N.; Senanayake, S. P.Dendrobium is one of the highly demanding ornamental plant genus in Sri Lanka. However, taxonomic identification of Dendrobiums are not straightforward and it is often complicated with the introduction of large number of commercially attractive hybrids and cultivars. Therefore, morphometry was used for taxa identification in the present study. Further, plant growers produce hybrids using these modern cultivars. However, recently, it has been found that artificial pollination of modern hybrids is unsuccessful. It was hypothesized that the modern hybrids bear infertile pollens. Eight commercial Dendrobium cultivars (A-H) with different floral morphology were selected for the study . Sixty different floral and vegetative characters were observed and recorded. Morphological characters were subjected to cluster analysis using PAST 3.1 software package. Two Dendrobium cultivars (F and G) were closely related and have separated from the other taxa. Dendrobium cultivar B has shown a clear separation from the other studied taxa. Pollinia of Dendrobium flowers were deposited on the stigma of the same flower. After 72 hr, a small amount of stigmatic fluid was stained with lacto phenol cotton blue, observed under a light microscope and percentage pollen germination was calculated. Pollen viability was tested by placing crushed pollinia on a cavity slide with 1% solution of 2,3,5-triphenyltetrazolium chloride, incubating at dark for 6 hr and observing under a light microscope. The highest percentage of pollen germination was observed in Dendrobium cultivar D (67%) while the lowest germination was observed in cultivar A (24%). The pollen viability of the cultivars varied from 77% to 94%. Successful observations in planta pollen germination concludes that the modern cultivars still have the potential of using in plant breeding programs.Item Host Plant Preference of Genera Dendrobium and Bulbophyllum (Family: Orchidaceae) in Sri Lanka.(International Research Symposium on Pure and Applied Sciences, 2017 Faculty of Science, University of Kelaniya, Sri Lanka., 2017) Sandamali, P. M. H.; Senanayake, S. P.; Benjamin, S. P.; Rajapakse, S.; Athukorala, N. P.Orchids are a well-known plant family for their restricted distribution in the wild. Most epiphytic orchids are hosted by specific trees and hence it’s abundance may be influenced by the availability of suitable host trees and epiphytic composition. The distribution of many epiphytic orchids in nature indicates that they display strong species specificity in selecting hosts and noticeably lacking on other trees. Dendrobium and Bulbophyllum are epiphytic genera and mostly have restricted distribution in the natural environments. Furthermore; they display a biased distribution towards host trees. Therefore, the study aims to investigate the host tree preference of the species of Dendrobium and Bulbophyllum with a view to provide important information for the conservation of these species. Wet forests in Matale (Knuckles mountain range), Nuwara Eliya (Hakgala nature reserve, Horton Plains National Park, Pidurutalagala mountain, Kandeela forest reserve), Baddulla (Namunukula mountain), Kegalle (Makandawa forest reserve), Ratnapura (Sinharaja rain forest, Peak wilderness), Kurunegala (Kankaniyammulla forest), Galle (Hiyare rain forest, Kottawa forest, Morningside forest) districts were explored for the distribution of two genera; Dendrobium and Bulbophyllum. Specimens were collected randomly from the selected sites; three plots of 500m× 500m size per each site, and relevant data of host plants were recorded such as tree height, girth at breast height (GBH), tree bark characteristics (bark colour, nature of the moss cover and bark texture) and the abundance of orchid species on the host tree. Further, herbarium specimens of host trees and the orchids were prepared for identification. Presence of orchid species on the identified host trees was counted and, percentage of frequency was calculated. It was revealed that the orchids are mostly associated with hosts with dense moss cover and rough tree barks. Sixty five percent of species of Dendrobium and Bulbophyllum were found on hosts having 51-100 cm GBHs. Further, these orchids have shown preference to plant families; Myrtaceae (26%), Lauraceae (23%) and Phylanthaceae (16%) as their hosts, whereas, with respect to the genus specificity, highest percentage of these orchids have shown preference to genera Syzygium (family Myrtaceae) and Glochidion (family Phylanthaceae) as their hosts. Findings of the present study have revealed that the species diversity and species richness of genera Dendrobium and Bulbophyllum were high in the montane forests located in Nuwara Eliya (Horton Plains Nature Park, Kandeela forest reserve and Pidurutalagala mountain) and Matale (Knuckles mountain range) districts. This might be due to the fact that these forests were characterized by high humidity and high elevation. Chemical composition, distribution of moss cover and fungi associated with tree barks of these preferred host trees, would provide new insight for the conservation of these epiphytic orchid species.