Plant and Molecular Biology
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Item Unveiling the Probiotic Properties of Lactic Acid Bacteria Inhabiting Tender Coconut Water(, Centre for Research and Knowledge Dissemination (CRKD) of the Sabaragamuwa University of Sri Lanka, 2023) Pavalakumar, D.; Undugoda, L.J.S.; Manage, P.M.; Nugara, R.N.; Kannangara, S.This research delves into the unexplored potential of tender coconut water as a rich source of probiotics, with a focus on identifying promising probiotic lactic acid bacteria (LAB). By employing biochemical screening techniques, four potential LAB strains were isolated: Lactiplantibacillus plantarum CWJ3, Lacticaseibacillus paracasei CWKu14, Lacticaseibacillus rhamnosus CWKu12, and Lacticaseibacillus casei CWM15. These strains were comprehensively assessed to evaluate their resilience under various conditions: pH (2, 3, and 6.5), bile (0.3% and 0.5%), phenol (0.4% and 0.6%), salt (3% and 6%) concentrations, and temperatures (20°C, 37°C, 45°C, and 60°C). Further, resistance to lysozyme (100 mg L-1), artificial saliva juice (0.3% α-amylase, at pH 6.9), simulated gastric juice (0.3% pepsin, at pH 2), and simulated intestinal juice (0.3% Ox-gall and 0.1% pancreatin at pH 7) were determined. Their antibiotic susceptibility was tested against ten antibiotics at 100 ppm, and cell surface hydrophobicity, auto-aggregation, co-aggregation with selected pathogens, antibacterial activity, hemolytic activity, and DNase activity, also were evaluated. The statistical analysis demonstrated the survival of all four strains even in demanding circumstances, with Lactiplantibacillus plantarum CWJ3 displaying the greatest resilience. Optimum growth occurred at 37°C, while none of the strains survived at 60°C. A pH of 6.5 was optimal for their growth, and Lactiplantibacillus plantarum CWJ3 stood out as the only strain capable of withstanding pH 2 after 2 hours. Lacticaseibacillus rhamnosus CWKu12 and Lacticaseibacillus casei CWM15 showed the highest percentage of auto-aggregation and co-aggregation while Lacticaseibacillus rhamnosus CWKu12 and Lactiplantibacillus plantarum CWJ3 exhibited high cell surface hydrophobicity. The strains displayed susceptibility to Clarithromycin, Erythromycin, and Azithromycin, but resistance to Cefuroxime and Streptomycin antibiotics. All four strains inhibited various food-borne pathogens, showcasing their potential as probiotics. Moreover, they displayed no hemolytic or DNase activity. Remarkably, Lactiplantibacillus plantarum CWJ3 excelled in challenging environments, making it a noteworthy contender for a probiotic role.Item Assessing the antioxidative potential of lactic acid bacteria inhabiting tender coconut water(Faculty of Graduate Studies - University of Kelaniya, Sri Lanka, 2023) Pavalakumar, D.; Undugoda, L.J.S.; Manage, P.M.; Nugara, R.N.; Kannangara, S.Probiotics are renowned for their beneficial impact on human health, including their antioxidative properties. Lactic acid bacteria (LAB), which constitute a major group within probiotics, have been recognized for their ability to act as potent antioxidants, providing relief from oxidative stress in the host. This study aims to evaluate the antioxidative activity of probiotic LAB inhabiting the tender coconut water samples collected from different regions of Sri Lanka. In a previous study, analysis of the collected tender coconut samples resulted in the identification of four potential probiotic strains at the species level: Lactiplantibacillus plantarum CWJ3, Lacticaseibacillus rhamnosus CWKu12, Lacticaseibacillus paracasei CWKu14, and Lacticaseibacillus casei CWM15. The antioxidative activities of these strains were evaluated using four different chemical antioxidant assays: the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay at 517 nm, the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay at 734 nm, the ferric reducing antioxidant power (FRAP) assay at 593 nm, and the total phenolic content (TPC) assay at 765 nm through spectrophotometric method. Three different concentrations of each bacterial strain (107, 108, and 109 CFU mL-1) were prepared using overnight grown cultures in deMan, Rogosa, and Sharpe broth at 37 °C, included in the study. The results indicate a significant increase (p < 0.05) in antioxidative activity for all four strains across all analyzed methods with an increase in cell concentration. Lactiplantibacillus plantarum CWJ3 exhibited the highest antioxidative potential at 109 CFU mL-1, with 88.85±0.84% radical scavenging activity in the DPPH assay, 84.0±0.14% radical scavenging activity in the ABTS assay, 0.029±0.004 mg mL-1 ascorbic acid equivalent in the FRAP assay, and 0.0052±0.0006 mg mL-1 gallic acid equivalent in the TPC assay. Conversely, Lacticaseibacillus paracasei CWKu14 demonstrated the lowest values for all antioxidative activities. Continued research on the antioxidative properties of probiotic LAB derived from tender coconut water holds promising prospects for the development of functional beverages.Item Biocontrol potential of endophytic fungi in tea (Camellia sinensis (L.) Kuntze) leaves against selected fungal phytopathogens(Malaysian Journal of Microbiology, 2022) Thambugala, K.; Daranagama, D.; Kannangara, S.Aims: Endophytic fungi are a diverse group of microorganisms that stay asymptomatically in the healthy tissues of the host. Many fungal endophytes are associated with the tea plant (Camellia sinensis) and the pathogens of the tea plant have the potential to grow as endophytes or act as latent pathogens during the initial growth of the plant. The present study aimed at screening tea endophytic fungi with the potential for control of a few critical phytopathogens, Fusarium sp., Lasiodiplodia theobromae, Pestalotiopsis sp. and Sclerotinia sclerotiorum while evaluating the efficiency of growth inhibition of these phytopathogens by the endophytic fungal isolates using in vitro assays. Methodology and results: Five endophytic fungal strains; Colletotrichum gloeosporioides, C. siamense, Daldinia eschscholtzii, Pseudopestalotiopsis chinensis and Phyllosticta capitalensis isolated from leaves of C. sinensis in Sri Lanka were evaluated for growth inhibition against plant pathogens; Fusarium sp., L. theobromae, Pestalotiopsis sp. and S. sclerotiorum using the dual culture assay and volatile compound-mediated inhibition assay. All the fungal endophytes used in this study exhibited antifungal activity against Fusarium sp., Pestalotiopsis sp. and S. sclerotiorum in the dual culture assay on PDA. Daldinia eschscholtzii (67.30%) and C. gloeosporioides (61.54%) showed strong antagonistic activity against S. sclerotiorum, while P. capitalensis (42.30%) demonstrated moderate activity. All the endophytic fungal strains showed moderate antifungal activities against Fusarium sp. The percentage growth inhibitions of Pestalotiopsis sp. by all the endophytic isolates tested were below 25.00%. In the volatile compound-mediated inhibition assay, none of the endophytic isolates showed visible inhibition against the phytopathogens used. Conclusion, significance and impact of study: In this study, the fungal endophytes which showed potential antagonistic activity against the tested phytopathogens can be used to develop commercialized products of fungal biocontrol agents (BCAs) for controlling plant fungal diseases.Item Suppression and management of Meloidogyne incognita in soil using Trichoderma harzianum NFCF160 and Trichoderma virens Isf-77(Malaysian Journal of Microbiology, 2022) Rajakaruna, N.; Undugoda, L.; Kannangara, S.; Abeywickrama, K.Aims: Meloidogyne incognita adversely affects numerous crop plants worldwide. Therefore, the modern world has been moving towards biocontrol methods to prevent nematode attacks. This study was aimed to (i) investigate the potential use of Trichoderma harzianum NFCF160 and T. virens Isf-77 in managing M. incognita in soil and (ii) identify trapping mechanisms employed by both Trichoderma strains to suppress M. incognita. Methodology and results: Three weeks old, Basella alba L. plants were subjected to five different treatments. The above and below ground growth parameters and the galling indices of these plants were measured every four weeks for three sampling times. Trapping mechanisms employed by Trichoderma strains were examined following plate assays. Plants treated with T. harzianum NFCF160 and T. virens Isf-77 had significantly higher values for the total number of leaves (34 ± 2.84) and (27 ± 2.61), fresh weight of the shoot (81 ± 9.51 g) and (91 ± 9.70 g), dry weight of the shoot (71 ± 5.24 g) and (62 ± 5.81 g), respectively eight weeks after inoculation of M. incognita. Significantly low galling indices (2 and 2) were recorded in B. alba treated with Trichoderma strains. Both Trichoderma strains exhibited various nematode-trapping mechanisms, such as non-constricting rings and adhesive spores. Conclusion, significance and impact of study: This investigation highlighted the potential of both Trichoderma strains as biocontrol agents to control M. incognita effect in sustainable agriculture.Item Depolymerization of polyaromatic hydrocarbons by Penicillium spp. inhabit the phyllosphere of urban ornamental plants(Environmental Quality Management, 2022) Dharmasiri, R. B. N.; Undugoda, L. J. S.; Nilmini, A. H. L.; Pathmalal, M. M.; Nugara, N. N. R. N.; Udayanga, D.; Kannangara, S.A variety of anthropogenic sources release hazardous polyaromatic hydrocarbons (PAHs) into the phyllosphere which is an excellent niche for diverse fungi, and some of them have PAHs degradation capabilities. Therefore, this research attempted to determine the PAHs (phenanthrene, anthracene, naphthalene, and pyrene) degradation capability of phyllosphere inhabited Penicillium species. The leaf samples were collected from highly polluted urban areas (Panchikawatta, Pettah, Orugodawatta, Maradana, Sapugaskanda, and Colombo Fort) in Sri Lanka to isolate fungal species inhabiting the phyllosphere. Furthermore, their distribution patterns among the leaf tissue layers were studied using bright-field microscopic observations. Moreover, the best PAHdegraders were screened out using plate assays and confirmed throughHigh Performance Liquid Chromatography (HPLC) analysis. Further, their enzymatic activities during the PAHs degradation were analyzed. As per the microscopic observations, the highest fungal distribution was in the upper epidermis of the leaves followed by the fungal distribution in the interspaces of palisade mesophyll layers. Out of isolated fungal species, two Penicillium spp. (Penicillium citrinum P23B-91 and Penicillium griseofulvum P9B - 30) showed the highest PAHs (phenanthrene, anthracene, naphthalene, and pyrene) degradation capabilities. Manganese peroxidase (MnP) enzyme dominated phenanthrene degradation in P. griseofulvum P9B - 30, which showed the highest phenanthrene degradation ability (61%). In addition, P. citrinum P23B-91 was good at degrading anthracene (88%) and also displayed a higher MnP activity during the anthracene degradation than laccase and lignin peroxidase activities. The discoveries from the toxicity assay during the PAHs degradation processes revealed that the produced byproducts had no toxic effects on the fungal growth cycle and the phyllosphere. Therefore this phyllosphere Penicillium spp. are ideal for the bioremediation of polluted air in urbanized areas.