International Research Symposium on Pure and Applied Sciences (IRSPAS)
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Item A disease severity index to monitor stem-end rot development in Mango (cv. Karutha Colomban) and the assessment of pathogenicity of associated pathogens(Research Symposium on Pure and Applied Sciences, 2018 Faculty of Science, University of Kelaniya, Sri Lanka, 2018) Kodituwakku, T.D.; Abeywickrama, K.; Karunanayake, K.O.L.C.Mango (Mangifera indica L.) is a renowned tropical fruit consumed in Sri Lanka. Significant postharvest losses of mango are caused by diseases which affect the quality of fruits. Stemend rot (SER) of mango is a disease caused by a group of fungal pathogens. Disease severity indices are important for assessing the extent of damage caused by a disease to develop suitable control strategies. An index was developed to evaluate the disease development and the level of severity of SER in mango (cv. Karutha Colomban (KC). Pathogenicity of four fungi isolated from mango with SER was also investigated to find out their contribution for SER development in mango. Four mango fruits (90-days old) washed in tap water followed by sterile distilled water were placed on a plastic tray at room temperature. One selected fruit showing a gradual development of SER was photographed daily. Diseased area of the fruit in each photograph was estimated by DIGIMIZER (Version 5.3.4) software and the disease severity was determined as percentage SER (%SER). An index was prepared using the photographs with percentage SER values. Four fungal pathogens (Lasiodiplodia theobromae, Phomopsis sp., Pestalotiopsis sp. and Xylaria feejeensis) were isolated from mango with SER and their identity was confirmed by PCR using universal primers (ITS1 and ITS4) and DNA sequencing. Healthy mango fruits (90-day old) washed in tap water were surface sterilized with 0.1% sodium hypochlorite and subsequently washed in sterile distilled water. Stem-ends of the fruits were wound inoculated with 7-day old mycelial plugs of each fungal pathogen separately and all four pathogens together. Fresh PDA plugs served as the control. Inoculated fruits were incubated in moist plastic chambers at room temperature for 7 days. Each treatment comprised of four replicates and the experiment was repeated. Percentage SER of each fruit was determined based on the developed index. Mean percentage SER resulted by the combination of all four pathogens and L. theobromae, Pestalotiopsis sp. and X. feejeensis, separately were found to be 67.00 ± 4.77%, 66.75 ± 3.84%, 61.13 ± 3.32% and 60.38 ± 4.58% respectively and there was no significant difference between their pathogenicity (MINITAB 18). The least pathogenicity (16.50 ± 1.66%) was observed in fruits inoculated with Phomopsis sp. and percentage SER of the control was 0.88 ± 0.23% L. theobromae, X. feejeensis and Pestalotiopsis sp. were identified as the major contributors for SER in mango and this may be the first reported evidence in Sri Lanka on X. feejeensis as a potential SER pathogen of mango (cv. KC).Item Effect of Croton aromaticus leaf extracts in controlling Crown Rot disease of Embul banana.(International Research Symposium on Pure and Applied Sciences, 2017 Faculty of Science, University of Kelaniya, Sri Lanka., 2017) Dilhani, S.; Wimalasiri, S.; Abeywickrama, K.; Kannangara, S.Embul banana (Musa acuminata-AAB) is one of the major dessert fruits in Sri Lanka, widely consumed by all economic groups due to its small size and characteristic flavour. Although Embul banana has high potential for export market storage and export of this commodity over long distances is difficult in consequence of postharvest fungal diseases. The most common and serious postharvest disease that affect Embul banana is Crown Rot (CR). Use of synthetic fungicides is the widely used method in controlling postharvest diseases of fruits worldwide. The interest of finding natural bioactive components has increased due to the harmful effects of synthetic fungicides on environment and health. In the present study, antifungal activity of aqueous, hot water and ethanolic leaf extracts of Croton aromaticus in controlling CR disease of Embul banana was investigated in vivo. Embul banana hands (85-days mature) were treated with C. aromaticus aqueous, hot water 100% (v/v) and ethanolic leaf extracts (400 mg/ml) alone or in combination with alum (1%) or distilled water (control) were stored in modified atmosphere packaging at 12-14 0C for 14 days. Each treatment comprised of 4 replicates. In-package gases were analysed on initial day and thereafter up to 14 days. Physicochemical properties (pH, firmness, TSS, TA), sensory properties (peel colour, flesh colour, aroma, flavour, taste, overall acceptability), and Crown Rot disease severity were determined in ripening induced fruits after 7 and 14 days of storage period. Statistical analysis was done using the MINITAB 16 statistical package. Oxygen levels measured were observed to be amaintained at 2.2-4.4% while CO2 levels were maintained at 5.5-8.4% in all packages at the end of 14 day storage period. C. aromaticus ethanolic leaf extract (400 mg/mL) was the most effective extract in controlling crown rot disease of Embul banana compared to aqueous and hot water leaf extracts. Physicochemical properties of Embul banana treated with C. aromaticus leaf extract alone and in combination with alum were not significantly different compared to control except for TSS and TA. Most of the sensory properties were preferred by sensory panelists with score values of above 6 indicating the good quality of samples. C. aromaticus ethanolic leaf extract + alum in combination with modified atmosphere packaging and cold storage could be used as a potential safe way of controlling Crown Rot disease of Embul banana.Item Formulation and stabilization of Trichoderma spp. in selected carrier materials.(International Research Symposium on Pure and Applied Sciences, 2017 Faculty of Science, University of Kelaniya, Sri Lanka., 2017) Botheju, W.S.M.; Hewavitharana, N.,; Kannangara, S.D.P.; Abeywickrama, K.Trichoderma spp. are one of the major biocontrol agents which have the ability to act against large numbers of foliar and soil borne pathogens and are eco-friendly good plant growth promoting agents. Present investigation was carried out with an attempt to determine 1) the best organic carrier material in which the viability and antagonistic activity retain for a sufficient duration and 2) optimum temperature at which the product can be stored without losing the viability and antagonistic activity. In the present study, three Trichoderma spp. (T. asperellum, T. harzianum and T. virens) were formulated in three carrier materials - coir dust, saw dust and waste of polished rice, which were rich in lignocellulosic organic compounds, nitrogen and mineral salts. In the preparation procedure, glucose and starch were added as carbon sources and cow dung was also added as a source of nitrogen as Trichoderma spp. require sufficient amounts of carbon and nitrogen sources for their growth and development. Three Trichoderma spp. were then inoculated separately (ten mycelial blocks with 1cm diameter from 7-day old cultures) into the carrier materials in sterilized polypropylene bags aseptically. Each treatment comprised of six replicates. Shelf life of these formulated products were studied along with viability tests, using spread plate method from which colony forming units were observed at three week intervals. Mean values of measurements were statistically analyzed using ANOVA and Tukey’s pair wise test (Minitab 16). Antagonistic activity of three Trichoderma spp. against three post-harvest pathogens (Colletotrichum musae, Fusarium oxysporum, Pestaliopsis microspora) were monitored using dual culture method at room temperature (30°C) and 4°C where growth inhibition was measured. All three types of carrier materials were good media for the formulation of all three Trichoderma species and when reisolated, the highest Colony Forming Units were observed in waste of polished rice formulations, at 4°C; [T. asperellum (6.23 log CFU/g), T. harzianum (5.92 log CFU/g) and T. virens (6.04 log CFU/g)] compared to other two carrier materials. Optimum temperature for the storage of formulated products was 4°C that maintained the viability and antagonistic activity of Trichoderma propagules for 130 days compared to the storage at room temperature. However, the values for CFUs of all three Trichoderma spp. in three carrier materials were slightly reduced after storage at 4°C which was not significant. The growth of all three post-harvest pathogens were inhibited by the three Trichoderma spp. and the inhibition values ranged from 36.5% – 83.6%. Growth inhibition values obtained at room temperature (30°C) and 4°C were not significantly different. Among the cost effective three carrier materials at two different temperatures, waste of polished rice at 4°C was found to be significantly effective in retaining the viability and antagonistic ability of the tested Trichoderma species. This may be due to the presence of optimum C contents (35%), C:N ratio (28.42) and pH (6.7) which enhance the production of sufficient propagules of three Trichoderma spp.Item Screening endophytic fungi of Macromitrium sp. for potential degradation of PAHs(Faculty of Science, University of Kelaniya, Sri Lanka, 2016) Ambadeniya, A.R.P.D.U.K.R.; Kannangara, S.; Abeywickrama, K.With rapid industrialization and urbanization, emission of air pollutants to the atmosphere has been increasing rapidly for several decades. Out of many pollutants, polyaromatic hydrocarbons (PAHs) take a prime advertence due to their toxicity, mutagenecity, carcinogenicity and long persistence in nature. Therefore, removal of these is one of the major cruxes that the modern world faces. In the present study, an effort was made to isolate and identify endophytic fungi in a moss (Macromitrium sp.) found in a polluted area (Sapugaskanda) and a less polluted area (Hettimulla), and to investigate their ability to degrade PAHs (naphthalene and phenanthrene). It was hypothesized that endophytes isolated from the moss can degrade PAHs and endophytes from polluted area have a higher ability to degrade PAHs compared to those isolated from the less polluted area. Moss plants from Sapugaskanda and Hettimulla area were used. Surface sterilized and trimmed moss plant pieces were placed on Malt Extract Agar and incubated for 10 days at room temperature. Percentage frequency of occurrence of each fungus grown was calculated. Utilization and degradation of PAHs by each of the fungus was assessed using a plate assay and a spectrophotometric analysis. Thirty six isolates were recovered from samples from Sapugaskanda area, 21 from Hettimulla and 6 were common to both areas. Highest frequency of occurrence was observed in Eupenicillium sp.2 (95.0%) in samples from Sapugaskanda and white sterile sp.7 (32.5%) for Hettimulla. Highest PAH utilization with the highest colony diameter, was recorded for Nigrospora oryzae for naphthalene (85.2 mm) and phenanthrene (59.5 mm). Almost all isolates from Hettimulla demonstrated low colony diameters. According to spectrophotometric analysis, highest degradation was observed with Penicillium oxalicum for naphthalene (98.60%) and Nigrospora oryzae against phenanthrene (98.02%). Almost all isolates in samples from Hettimulla area displayed poor degradation ability. The findings of the current study clearly reveal that Macromitrium sp. in Sapugaskanda harbours higher number of endophytic fungi than that in Hettimulla and most of them have a considerable ability to utilize and degrade PAHs in contrast to that in Hettimulla. It could be speculated that those endophytic fungi in Macromitrium sp. of Sapugaskanda, could be potential sources of fungal bioremediation. Further, they have potential practical application in removing PAHs from contaminated sites.Item Assessment of phytochemicals and antifungal effect of Croton aromaticus against postharvest fungal pathogens isolated from tropical fruits(Faculty of Science, University of Kelaniya, Sri Lanka, 2016) Wijesundara, S.A.D.T.L.; Kannangara, S.; Abeywickrama, K.Chemical control using synthetic chemical fungicides is still the most common method of controlling postharvest diseases of fruits. Plant extracts which are rich in antimicrobial secondary metabolites such as terpinoids, alkaloids, saponins and flavonoids could be possible alternatives for synthetic fungicides. Current study was focused on evaluating the antifungal effect of ethanolic extract of Croton aromaticus (Kappettiya) leaves in vitro against mycelial growth and the spore germination of postharvest fungal pathogens isolated from fruits of banana (Colletotrichum musae, Rhizopus sp., Lasiodiplodia theobromae) papaya (Rhizopus stolonifer, Colletotrichum gleosporioides, Lasiodiplodia theobromae) and mango (Alternaria alternata, Pestalotiopsis mangiferae, Lasiodiplodia theobromae). Surface sterilized diseased banana, papaya and mango fruit tissues were cultured on PDA plates in order to obtain pure cultures of possible fungi and they were identified by morphological and microscopic characteristics, using identification keys. Inhibitory effect of the ethanolic extract of C. aromaticus against test pathogens were investigated by well diffusion method using PDA medium, by incorporating crude extract dissolved in DMSO, ranging from 1 mg/ml up to 300 mg/ml concentrations along with the positive (Captan) and negative (DMSO) controls. Significant (P < 0.05) inhibitory effects were exhibited by the ethanolic extract of C. aromaticus leaves against all test pathogens except L. theobromae. The highest mycelial growth and spore germination inhibition of most of the pathogens were observed at 100 mg/ml. The lowest Minimum Inhibitory Concentration of the leaf extract (5 mg/ml) was observed for spore germination inhibition of C. gleosporioides and P. mangiferae. TLC analysis revealed four compounds having Rf values of 0.551, 0.672, 0.810 and 0.913. Phytochemical screening of ethanolic extract revealed the presence of alkaloids, terpenoids, quinones, phytosterols and flavonoids. Current findings indicate the potential use of ethanolic extract of C. aromaticus leaves in controlling banana, papaya and mango postharvest fungal pathogens in vitro.