Browsing by Author "Udayanga, D."

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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.
Enzymatic approach to green air: Depolymerization of polycyclic aromatic hydrocarbons (PAHS) by Aspergillus sp. isolated from phyllosphere of urban areas
(Faculty of Science, University of Kelaniya Sri Lanka, 2022) Dharmasiri, R. B. N.; Undugoda, L. J. S.; Nilmini, A. H. L.; Pathmalal, M. M.; Nugara, N. N. R. N.; Udayanga, D.
Polycyclic aromatic hydrocarbons (PAHs) are hazardous air pollutants that are toxic to many life forms. Biodegradation is an eco-friendly efficient option found to remediate these toxic PAHs. Air pollutants from many sources get settled over the phyllosphere through atmospheric deposition. Phyllosphere is a large niche for many fungal species and some of them metabolize many PAHs to nontoxic concentrations. The present study was to determine the PAHs (phenanthrene, anthracene, naphthalene and pyrene) degradation capability of phyllosphere inhabited Aspergillus species. Fungal isolations were made from leaf samples (Amaranthus cruentus, Hibiscus rosa-sinensis, Ervatamia divaricate, Plumeria sp., and Ixora chinensis) grown in Panchikawatta, Orugodawatta, Pettah, Maradana, Colombo Fort and Sapugaskanda oil refinery sites in Sri Lanka. Out of morphologically different thirty-five fungal isolations, Aspergillus spp. were identified to the genus level using identification keys and pre-existing identified reference cultures. PAHs degradation ability of isolated Aspergillus spp. was screened using a plate assay and confirmed by High Performance Liquid Chromatography (HPLC). Further, phyto-toxicity assays were performed using Vigna radiata seeds to test environmental toxicity and toxicity to the degrading fungal cells in the medium from the produced metabolites. Furthermore, manganese-dependent peroxidases (MnPs), lignin peroxidases (LiPs), and laccases enzyme activities of them during the PAHs depolymerization were analysed parallel to the PAHs degradation percentages. According to HPLC analysis, Aspergillus sp. P21B - 77 showed the most efficient degradation of anthracene (80%), Aspergillus sp. P11B – 34 was the most efficient degrader for naphthalene (82%) and Aspergillus sp. P22T – 82 was the most efficient degrader for pyrene (84%) and phenanthrene (86%). MnP enzyme activity dominated the highest anthracene depolymerization ability of Aspergillus P21B – 77 However; LiPs activity dominated the highest phenanthrene and pyrene depolymerization in Aspergillus sp. P22T – 82. Moreover, Aspergillus sp. P11B – 34 showed the best naphthalene degradation, and laccases enzyme activity dominated the degradation. The toxicity assay revealed that the generated metabolites were not toxic to the growth of Aspergillus spp. and, also verified that those by-products were not destructive compounds to the phyllosphere. Aspergillus spp. could be useful as a potential biological agent for an effective bioremediation process in polluted environments contaminated with phenanthrene, anthracene, naphthalene and pyrene like polycyclic aromatic hydrocarbons.
Molecular systematics and comparative draft genome analysis of strawberry leaf blight fungus.
(International Research Symposium on Pure and Applied Sciences, 2017 Faculty of Science, University of Kelaniya, Sri Lanka., 2017) Udayanga, D.; Castlebury, L.A.
The leaf blight fungus, formerly known as “Phomopsis” obscurans originally described from the United States, is a severe pathogen of strawberry. Morphological characters are often inadequate for identification of this species. Although this fungus is currently classified as a Phomopsis sp. (=Diaporthe), preliminary analyses of this study indicated it is not congeneric with Diaporthe. Phylogenetic analyses with representative species in the order Diaporthales were performed using five nuclear loci (28S, 18S, internal transcribed spacer regions with 5.8Sof rDNA, translation elongation factor and DNA directed RNA polymerase II) to infer its evolutionary relationships. Genomic libraries of leaf blight fungal DNA were prepared for next generation whole genome sequencing. DNA and RNA were extracted and paired-end libraries were generated with Nextera library preparation kits (Illumina) and sequenced on Illumina Miseq instrument. Whole genome and transcriptome sequences obtained were initially analyzed with CLC Genomic Workbench and comparative analyses were performed using methods similar to the US-DOE Joint Genome Institute annotation pipeline. Results of phylogenetic analyses determined that “P.” obscurans represents a unique evolutionary lineage and possibly an undescribed genus within a new family. The whole genome assembly consisted of 5638 contigs (≥500 bp) with an estimated genome size of 48 Mbp. The N50 contig length is 13853 bp and the G+C content is 52%. The output resulting from MAKER-p, predicted a total of 12431 genes and was modeled with Cryphonectria parasitica EP155 genome. The estimated mitochondrial genome size is 98658 bp which is comparatively larger than the average sized mitogenomes of fungi. Analysis with the CAZy database (dbCAN) identified in total 778 putative carbohydrate active enzyme encoding genes, including 341 glycoside hydrolases (GHs), 112 glycosyl transferases, 25 polysaccharide lyases, 138 carbohydrate esterases, 51 carbohydrate-binding modules, and 111 auxiliary activities. Therefore, “P.” obscurans is equipped with all necessary enzymes to breach plant cell wall, penetrate, successfully infect and cause severe plant disease. Among GHs, majority (i.e. 20 from each) belong to GH3, GH5, GH16, and GH28 families that possess xylanase, cellulase, chitinase, polygalacturanases which are also potentially involved in maceration and soft-rot of plant tissues including fruits.
Phylogenetic re-assessment of the genus Curvularia
(Research Symposium on Pure and Applied Sciences, 2018 Faculty of Science, University of Kelaniya, Sri Lanka, 2018) Ferdinandez, R. S. K. H. S.; Udayanga, D.; Manamgoda, D.S.
The fungal genus Curvularia (Dothideomycetes, Pleosporales) consists of important pathogens, which cause devastating diseases globally on plants and human beings. Recently, the generic limit was re-defined and as a result, some of the species previously placed in the sister genus Bipolaris now belong to the genus Curvularia based on morphological and molecular data. Large number of novel Curvularia species have been introduced following the re-assessment of generic limits since the year 2012. However, 32 species of Curvularia described within last six years following the first assessment were not included in a recent phylogenetic analysis. Therefore, the major objective of this study was to incorporate the recently described Curvularia species in an up-to-date phylogeny. During the current study, we inferred phylogenetic trees of single loci of ribosomal Internal Transcribed Spacer region (ITS), partial Glyceraldehyde 3-Phosphate Dehydrogenase (GPDH), 28S ribosomal Large Sub-Unit (LSU), Translation Elongation Factor 1-α (TEF) and RNA Polymerase-2 (RPB2). In total, 104 species of Curvularia and several closely related taxa of the allied genera obtained from GenBank were analyzed using PAUP v.4.0b10 for the Maximum Parsimony (MP) and RAxML v.7.4.2 Black Box in the CIPRES Science Gateway platform for the Maximum Likelihood (ML) methods. Data set consisted of 57 ex-type cultures and their available DNA sequences. Single gene trees of all five loci were compared for their congruency and multi-gene phylogenetic analysis was carried out using ML method. To determine most suitable loci for species level phylogenetic inference of closely related species within Curvularia, ITS, GPDH, TEF and LSU were employed with the Phylogenetic Informativeness (PI) profiling test implemented in the online program PhyDesign. The concatenated DNA sequence alignment consisted of 2668 constant characters, 183 characters were variable and parsimony uninformative and 653 were parsimony informative. The final Likelihood value was -25783.8. About 78 species incorporated in the phylogenetic analysis were well resolved based on combined analysis. According to the PhyDesign analysis, the GPDH gene region was the highest informative in species level phylogeny whereas LSU loci found to be the least informative. Resulted tree is the most up-to-date phylogenetic tree available for the genus Curvularia which is the backbone for phylogenetic placement and identification of other Curvularia species isolated from Sri Lanka and elsewhere.