Graduate Studies
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Item Use of molecular features for identification of isolated fungal pathogens of big onion damping off disease and Trichoderma spp. isolated from soil(Faculty of Graduate Studies, University of Kelaniya, 2015) Gunaratna, L.N.R.; Deshappriya, N.; Jayaratne, D.L.Big onion (Allium cepa L.) is one of the economically important spices grown in Sri Lanka. Damping off disease caused by Fusarium sp. during nursery stage of growth poses a major factor that affect the yield significantly. Application of fungicides decrease incidence of damping off disease considerably, but this is neither economical nor environmental friendly. Thus, disease management practices have to be directed towards biological control strategies. Trichoderma spp. have been extensively studied as biological control agents for controlling numerous soil-borne fungal pathogens. In the present study, isolation and identification of fungal pathogens associated with damping off disease of onion and Trichoderma spp. present in soil of the same onion fields was carried out with a view to using the Trichoderma spp. in the management of damping off pathogens. Pathogens associated with damping off were isolated from diseased and healthy seedlings (7-30 days old) collected from the fields in the Matale and Anuradhapura districts. Seedlings were surface sterilized and plated in Potato Dextrose Agar (PDA) supplemented with tetracycline. Soil samples collected from the same fields were used for the isolation of Trichoderma spp. using the Warcup method. Based on morphological characteristics and using identification keys, the fungal pathogens isolated from seedlings were identified as Fusarium, Curvularia, Alternaria and Sclerotium spp. and 14 fungal species isolated from soil samples were identified as Trichoderma spp. Although fungi can be identified using morphological features, the use of molecular biological methods tend to be more accurate. Therefore, the identity of isolated fungal species was confirmed by molecular biological methods. Genomic DNA of Fusarium spp., Alternaria spp., Trichoderma spp. were extracted. Molecular characterization of these DNA was carried out using Polymerase Chain Reaction (PCR) where the Internal Transcribed Spacer (ITS) region of rDNA gene was amplified using ITS-1 and ITS-4 primer pairs. The products were subjected to agarose gel electrophoresis. The procedures were repeated 3 times. Results showed 550 bp size bands characteristic of Fusarium spp. and 570 bp products specific to Alternaria spp. confirming the previous identity using culture based methods. Fungal species isolated from soil showed products of 600 bp which corresponds to Trichoderma sp. Molecular characterization of the potential biocontrol agents i.e. Trichoderma spp. and A.cepa L. pathogens using PCR amplification of ITS region confirmed the preliminary identities carried out using culture based methods.Item Fungi associated with diseases of big onion (Allium cepa L.) prevalent in Matale district Sri Lanka(Faculty of Graduate Studies, University of Kelaniya, 2014) Gunaratna, L.N.R.; Deshappriya, N.Item Fungal diseases and associated pathogens of big onion (Allium cepa L.) prevalent in the Matale district, Sri Lanka(University of Kelaniya, 2013) Gunaratna, L.N.R.; Deshappriya, N.Big onion (Allium cepa L.) is a condiment grown for its pungent and flavorful bulbs. Most parts of the big onion plants are prone to different diseases of fungal origin. As these diseases cause heavy losses to the yield, it is important that the presence of the more prevalent diseases is evaluated and the causative fungi characterized. This will provide the background essential to carry out appropriate disease management practices. Therefore, the present study aims at surveying the prevalent diseases of onion at different stages of growth and isolating and identifying the causative fungi. Matale district was selected for the first stage of the study as about 23.5% of onion fields in Sri Lanka are located in this district. Thirty onion fields in different locations in the Matale district were observed. The prevalence and details of symptoms present in different plant parts i.e. bulbs, leaves, flower stalks were recorded and the parts were collected. Diseased and healthy big onion seedlings (7 -30 day old) were also collected. Appropriate sections of disease specimens (scales, leaves, flower stalks, seedlings) and healthy seedling samples were surface sterilized and the causative fungi isolated using Potato Dextrose Agar (PDA) supplemented with tetracycline. The more common symptoms observed in the fields were lesions at the collar region of seedlings (damping off -22% per field), yellowing of leaves and leaf die back (70% per field), formation of 2-3 mm wide oval shaped patches on leaves (5% per field) and flower stalks (1% per field) and discoloration and softening of bulbs (12% per field). A Fusarium sp. was isolated and identified from infected bulbs showing discoloration and softening. Colletotrichum gloeosporioides was isolated from the infected leaves and flower stalks. Fusarium, Curvularia, Alternaria, and Sclerotium spp were isolated and identified from seedlings showing damping off symptoms. Koch’s postulates were carried out to confirm the pathogenicity of Fusarium sp., two Curvularia sp. and Alternaria sp. isolated from seedlings. A spore suspension (1×105 spores/ml) of each isolate was added to soil containing 10 day old healthy seedlings while controls were treated with sterilized distilled water. Collapse of seedlings and lesions at the collar region associated with damping off was observed in seedlings inoculated with Fusarium sp. No symptoms were caused by the two Curvularia sp. and Alternaria sp. A 60% disease incidence was shown in seedlings inoculated with Fusarium sp., whilst only 5% - 6% disease incidence was shown by seedlings inoculated with two Curvularia. and Alternaria spp. and the controls. These results confirm that the Fusarium sp. isolated was the causative agent of damping off disease of big onion seedlings in the Matale district.