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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.Item In-vitro Assays on the Effect of Four Commercially available Fungicides on Fungal Pathogens of Commonly Grown Ornamental Foliage Plants in Sri Lanka(University of Kelaniya, 2012) Wimalasena, W.W.Y.; Deshappriya, N.The foliage plant industry continues to expand in Sri Lanka, supplying rooted plants, stem cuttings and cut leaves for foreign and local markets. Diseases caused by fungal pathogens result in loss of plants and plant material as well as rejections from buyers causing extensive economic losses to growers. In order to study the common fungal diseases, plants of export -oriented foliage nurseries; Lucky growers, Bandara growers and Tropiflora International in the Central Province and Aswin Foliage and GreenSac Foliage in Wayamba Province were surveyed during October 2008 to April 2009. Plant species grown for export in these nurseries included Dracaena, Chrysalidocarpus, Calathea, Cordyline, Aglaonema, Miscanthus, Fittonia, Philodendron and Livistonia varieties. Diseases and symptoms prevalent in the plant varieties were surveyed. Leaf spots (74%), tip burns (13%), anthracnose (4%) and stem rots (9%) were observed as the major symptoms in the plant varieties evaluated and the fungal pathogens associated with these diseases were isolated and the effect of fungicides on them was tested. Synthetic chemical fungicides could be used in the control of fungal diseases prevalent in ornamental foliage plants. However, it is important that the most effective fungicide(s) and optimal concentrations are selected. Thus the effect of four (04) commercially available fungicides containing active components hexaconazole, carbendazium, mancozeb and propineb were evaluated based on their inhibitory effect on the mycelial growth and spore germination of five (05) pathogenic fungi most frequently isolated from commonly grown foliage varieties in the nurseries surveyed i.e. Fusarium sp., Nectria sp., Cylindrocarpon sp., Curvularia sp. and Acremonium sp. Poisoned food technique was used to test the effect of fungicides on mycelial growth. Fungicides were tested at 10 mg/l, 50 mg/l, 100 mg/l, 250 mg/l, 500 mg/l concentrations incorporated separately into Potato dextrose agar (PDA) medium. Control cultures were grown on PDA plates without fungicide incorporation. There were six replicate plates for each concentration of each fungicide. For determining percentage spore germination inhibition, a concentration series of each fungicide i.e. 0.1 mg/l, 0.5 mg/l, 1.0 mg/l, 2.0 mg/l, 5 mg/l and 10 mg/l were tested for their germination inhibitory effect on a spore suspension of 108 spores/ml concentration from each fungus. Controls consisted of sterile distilled water in place of fungicides. The number of germinated spores was estimated under the microscope (20 replicate counts for each treatment) and the percentage spore germination inhibition was calculated. Percentage mycelial growth inhibition at 10 mg/l of hexaconazole was 75% - 85% for all five fungal sp. Inhibition was 100% at 50 mg/l on Fusarium sp., Nectria sp. and Curvularia sp and at 100 mg/l on Cylindrocarpon sp. and Acremonium sp. Inhibition was 100% at 50 mg/l of mancozeb on Nectria sp. and at 100mg/l on the other four genera. Propineb at 100mg/l, showed 100% inhibition on Nectria sp., 94.5% on Cylindrocarpon sp and 82.7% on Acremonium sp. At 250 mg/l propineb showed an inhibition of 100% on Curvularia sp., Acremonium sp. and Fusarium sp. Carbendazium showed 100% inhibition at a concentration as low as 10 mg/l on each fungus making carbendazium the most effective of all fungicides tested. Percentage spore germination inhibition (PSGI) is 100% at 0.5 mg/l of hexaconazole on Curvularia sp and at 1.0 mg/l on Fusarium sp., Nectria sp. Cylindrocarpon sp. and Acremonium sp. PSGI is 100% at 0.5 mg/l of mancozeb on Nectria sp., Fusarium sp. and Curvularia sp and at 1.0 mg/l on Acremonium sp. and Cylindrocarpon sp. Propineb showed 100% inhibition at 0.5 mg/l on Nectria sp, Fusarium sp. and Curvularia sp. and at 1.0 mg/l on Acremonium sp and Cylindrocarpon sp. 0.5 mg/l of carbendazium showed 100% inhibitory effect on all five fungal species tested. Therefore, hexaconazole, propineb, mancozeb and carbendazium at 50 mg/l, 100 mg/l, 100 mg/l and 10mg/l respectively can be used for the control of the mycelial growth of most prevalent pathogens of foliage plants and the same fungicides showed 100% inhibition of spore germination of the same pathogens at 1.0 mg/l, 0.5 mg/l, 1.0 mg/l and 0.5 mg/l respectively.Item Fungal community structure of fast decomposing and slow decomposing leaf litter at the upper mountain rain forest, Sri Lanka(University of Kelaniya, 2000) Kannangara, B.T.S.D.P.; Deshappriya, N.; Parkinson, D.Item A preliminary survey of diseases of some commercially grown foliage plants in selected regions of Sri Lanka(Research Symposium 2009 - Faculty of Graduate Studies, University of Kelaniya, 2009) Wimalasena, W. W. Y.; Deshappriya, N.Foliage industry is currently growing in Sri Lanka, supplying rooted plants, stem cuttings and cut leaves for both foreign and local markets. The commercial nurseries for this purpose are mainly established in the Western, Central, Wayamba and Sabaragamuwa provinces. In 2005, the net foreign exchange earnings has been Rs.1.06bn and cut flowers and foliage sector possessed an export share of 0.17% in 2008. However, rejection of products due to pests and diseases is one of the biggest problems associated with this industry. Therefore it is important that these disease problems are studied and control measures developed. In order to study the disease problems, export oriented foliage nurseries; Lucky growers, Bandara growers and Tropiflora International in the Central province and Aswin Foliage and GreenSac Foliage in Wayamba province were surveyed during October 2008 to April 2009. Dracena sanderana varieties, Dracaena marginata, Dracaena thalodius, Dracaena conjena, Dracaena “purple queen”, Cordyline sp., Chrysalidocarpus (cane palm), Livistonia rotundifolia, Calathea insignis, Calathea zebrina, Calathea roseapecta, Codiaeum sp., Schefflera sp., Miscanthus sp. (Chinese grass), Dieffenbachia sp., Pothos sp. were identified as the commonly grown foliage varieties. Diseases prevalent in these varieties were studied in the survey. Leaf spots (74%), tip burns (13%), anthracnose (4%) and stem rots (9%) were observed as the major symptoms in the plant species studied. For the isolation of pathogenic fungi, 1cm2 samples from diseased leaves were surface sterilized with 0.1% mercuric chloride (HgCl2) and these were cultured on Potato Dextrose Agar (PDA) aseptically and incubated under room temperature (280C). Pure cultures were prepared and isolated fungal pathogens were identified based on colony morphology and reproductive structures using identification keys. Fungal genera isolated and identified from diseased plants collected from the Central province included: Fusarium spp. causing symptoms as large necrotic leaf spots with irregular margins in D. sanderana (Gold), localized necrotic spots with smooth margin and yellowish outline in D. sanderana (purple compacta), localized small necrotic spots with smooth margin in D. sanderana (victory), scattered necrotic spots with irregular margins and yellowish outline and localized necrotic spot in Cordyline (red edge) , Cylindrocarpon sp. causing localized and scattered necrotic area with irregular margins in D.sanderana (white), Cladosporium sp causing localized necrotic area with irregular margin in D.sanderana (victory) , Cylindrocladium sp. causing small localized necrotic spots in Chrysallidocarpus (cane palm), Acremonium sp. causing scattered necrotic spots in Miscanthus (Chinese grass) and two sterile species. Fungal genera isolated and identified from diseased plants collected from the Wayamba province included: Fusarium sp. causing tip burn symptom in Cordyline (green) , Fusarium moniliforme causing localized necrotic spots in D. sanderana (white) and Curvularia sp. causing leaf spots in Cordyline (green).