Botany
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Item Carbon Source dependent - anaerobic Soil Disinfestation (ASD) Mitigates the Sclerotial Germination of Sclerotinia Sclerotiorum(Tropical Plant Pathology (2020), 2020) Mahalingam, T.; Rajapakse, C.S.K.; Somachandra, K.P.; Attanayake, R.N.Though Sclerotinia sclerotiorum IS a well-studied plant pathogen that causes significant economic damage worldwide, sustain able and environmental friendly control methods are difficult to establish due to it wide host range, cosmopolitan distribution and production of recalcitrant structures that can survive in soil for a long time. The pathogen was found causing a severe disease incidence on cabbage in 2016 in Sri Lanka. It was hypothesized that a) isolates of the recent disease outbreak display cross resistance to commonly applied fungicides and b) carbon (C) source supplemented Anaerobic Soil Disinfestation (ASD) is effective in mitigating the germination of sclerotia. In vitro fungicide sensitivity assays showed large variation in mycelial growth inhibition indicating high adaptability of the population towards environmental fluctuations and management practices. Signatures of cross resistance were evident. ASD was carried out using cabbage (Brassica oleracea) and leek (Allium ampeloprasum) cull piles, durian (Durio zibethinus) peels and grass cuttings (Axonopus compressus) as C sources and determined the sclerotial viability. Cabbage and leek cull piles at rates of 60—100 mg/g soil completely inhibited sclerotial germination. Maintaining anaerobic condition along with C source amendments was found to be a critical step in mitigating the sclerotial germination. GC-MS analysis of the volatiles of cabbage leaves, leeks and durian further confirmed the presence of various bioactive compounds with potential antifungal activity. Therefore, in addition to elevated microbial activity in treatments, the volatiles of C sources may have helped mitigating sclerotial germination.Item Screening Anti-Bacterial Activity of Endoi.Ichenic Fungi Collected From Negombo Lagoon Sri Lanka(Proceedmgs of the Postgraduate Institute of Science Research Congress. Sri Lanka, 2019) Weerasinghe, W.R.H.; Attanayake, R.N.; Weerakoon, G.; Paranagama, P.A.From the ancient past, natural products have been in use to fulfill a variety of human needs, from sustenance to pharmaceutical necessities. With the discovery of Endolichenic fungi (ELF) as a novel source prominent bioactiOt1es in recent years. a new trend has been created to explore more about these fascinating organisms, which live inside the lichen thallus asymptomatically. ELF isolated from the lichens collected from mangrove plants In Negombo lagoon's Kadol kale area (Lititude: 701 Longitude: were identitied using molecular techniques and were subjected to anti-bacterual assay. Ethyl acetate crude extracts of 18 identified endolichenic fungal strains were obtained and their anti-bacterial activities were tested against aerobic bactertal species Escherichia coli, Bacillus subtilis and Staphvlococcus aureus. The assay was carried out using agar well diffusion method using HI of the extracts (5 mg/ml) with Azithromycin as the positive control. The inhibition zone diameters (in cm) against E. coli were 1.2 for Phanerothaete chnsosporium. 1.2 for Xvlaria feejecnsis, ().9 for Gelasinospora seminuda, 0.9 for Chaetomium globosum and 1.5 for Curvularia lunata while Azithromycin as the positive control showed a diameter of 2.1. The inhibition zone diameters against B. subtilis were 1.0 for Cochliobolus sp., 1.0 for P. chnsosporium, 1.8 for X. feeyeensls, 1.7 for G. seminuda, 1 2 for C. globosum, 1.5 for Hvpoxvlon lividipigmentum, 2.0 for (Pennicillium sp), 2.2 for C. lunata, 0.9 for Lasiodiplodia theobromae and 2.4 for positive control. The inhibition zone diameters against S. aureus were 1.1 for P. chrvsosporium, 22 for X'. feejeensis, 1.0 for G. seminuda, 1.0 for Chaetomium globosum, 2.0 for (Pennicillium sp). 1.8 for Curvularia lunata and 2.2 for positive control. Out of the 18 fungal strains, 9 showed significant activity against the tested bacterial species while Endomelanconiopsis endophvtica, Neofusicoccum parvum. Ncofusicoccum occulatum. Hvpoxvlon anthochroum. Cvtospora xvlocarpi, Cerrena sp., Schi:ophvllum commune, Daldinia eschscholt:i, Nodulisporium sp. fungal strains did not show any inhibition. Further studies will be conducted to isolate compounds responsible for these inhibitions.Item Characterization of Agrobacteriu/ll strains from agricultural soils of Bandarawela, Sri Lanka(First National Symposium of Sri Lanka Association for Mycology and Plant Pathology (SLAMPP), 2019) Somarathna, G.M.T.K.; Somachandra, K.P.; Jayalath, W.G.H.; Attanayake, R.N.Agrobacterium is a Gram negative, rod shaped, aerobic and motile soil inhabiting bacterium of the family Rhizobiaceae. It is well known as the causative agent of crown gall disease of many plant species around the world. However, not all the Agrobacterium strains are pathcvenic and can cause galls. Only the virulent strains cause crown gall disease on number of plant species and are found only in contaminated soils. These virulent strains of A. tumefaciens harbor Ti plasmids with transfer DNA (T-DNA) region and virulence (vir) genes that are responsible for the pathogenicity. virD2 gene codes for virD2 protein and the endonuclease domain of the virD2 protein cleaves T-DNA border sequences. The ipt gene is the T-DNA borne cytokinin synthesis gene. Therefore, the presence of virD2 gene and ipt gene are useful in identifying pathogenic strains of Agrobacterium. The major objective of this research was to determine whether agricultural soils of Bandarawela were contaminated with virulence strains of A. tumefaciens. Soil samples were collected and bacteria were isolated using soil dilution method, and cultured on Yeast Mannitol Acar supplemented with Congo red and on Yeast Extract Peptone Affar. Five pure cultures of putatively Agrobacterium were further characterized using morphological and biochemical tests including Gram staining, catalase test, citrate utilization test, sugar fermentation test and 3-ketolactose test. These testes were often used for the species level identification of A. tumefaciens. Out of five isolates four were rod shaped with rounded ends and were either single or in pairs. However, the other isolate was in chains and Iono rod shaped. Interestingly, all the isolates were positive for all the biochemical tests. However, these tests do not help differentiating the virulence strains. Molecular characterization of all the soil isolates were carried out using universal 16s rRNA primers and Agrobacterium specific primers targeting virD2 and ipt genes. PCR amplification with virD2 primers successfully amplified the targeted band of 224 bp in all five isolates while ipt produced the expected fragment of about 427 bp in three of the isolates. virD2 cene sequences of selected soil isolates were 100-99% similar to the tumefaciens of the GenBank accession CP032925 and CP032929 reported from Taiwan. According to morphological, biochemical, and molecular Characterization using virD2 and ipt genes it was confirmed that the soil in the inspected field of Bandarawela is contaminated with pathogenic strains of A. tumefaciens. Therefore, farmers should maintain awareness when cultivating susceptible plant varieties in these fields.Item Sclerotinia sclerotiorum populations: clonal or recombining?(Tropical Plant Pathology (2019) 44:23–31., 2019) Attanayake, R.N.; Xu, L.; Chen, W.Sclerotinia sclerotiorum, a homothallic plant pathogen, undergoes sexual reproduction via haploid selfing (equivalent to clonal reproduction), and produces long-lasting surviving vegetative structures called sclerotia, enhancing clonal persistence and spread. Thus it is not surprising to detect clones of the species. Whether outcrossing can occur in the homothallic S. sclerotiorum remains unanswered. Early studies showed that S. sclerotiorum has a clonal population structure, consistent with its life history traits. However, recent studies using polymorphic and co-dominant molecular markers showed frequent genetic recombination, suggesting outcrossing. This review focuses on recent developments in population genetics studies related to detecting recombination, random association of alleles and dynamic mating type (MAT) alleles in Sclerotinia. Despite frequent reports of random association of alleles, the mechanisms for outcrossing in a homothallic species remain elusive. Recent intriguing findings are: the MAT genes in Sclerotinia are subject to inversion or deletion in every meiotic generation, the MAT gene deletion is related to ascospore dimorphism and mating type switching in S. trifoliorum, and ascospore dimorphism was also observed in S. sclerotiorum. Determining the nature of the dimorphic ascospores and their prevalence in relation to environmental cues could significantly advance our understanding how S. sclerotiorum populations behave in natureItem Molecular phylogeny and bioprospecting of Endolichenic Fungi (ELF) inhabiting in the lichens collected from a mangrove ecosystem in Sri Lanka(PLoS ONE 13(8): e0200711. https://doi.org/ 10.1371/journal.pone.0200711, 2018) Maduranga, K.; Attanayake, R.N.; Santhirasegaram, S.; Weerakoon, G.; Paranagama, P.A.Endolichenic fungi (ELF) are unexplored group of organisms as a source for the production of bioactive secondary metabolites with radical scavenging activity, antilipase and amylase inhibitory activities. Endolichenic fungi in lichens collected from mangrove or mangrove associated plants are least known for their fungal diversity and potential to produce bioactive compounds. A total of 171 ELF strains were isolated from the lichens collected from mangrove and mangrove associated plants in Puttalam lagoon. Out of this collection, 70 isolates were identified using rDNA-ITS region sequence homology to the GenBank accessions and a phylogenetic analysis was performed. Commonly isolated genera of ELF from lichens were Aspergillus, Byssochlamys, Talaromyces, Diaporthe, Phomopsis, Endomelanconiopsis, Schizophyllum, Cerrena, Trichoderma, Xylaria, Hypoxylon, Daldinia, Preussia, Sordaria, Neurospora, and Lasiodiplodia. In the present study, the effectiveness of ethyl acetate extracts of the ELF isolates were investigated against antioxidant activity, antilipase activity and α-amylase inhibition activity in in-vitro conditions. The results revealed that the extracts of Daldinia eschscholtzii, Diaporthe musigena and Sordaria sp. had the highest radical scavenging activity with smaller IC50 values (25 μg/mL to 31 μg/mL) compared to the IC50 values of BHT (76.50±1.47 μg/mL). Antilipase assay revealed that 13 extracts from ELF showed promising antiobesity activity ranged between 25% to 40%. Amylase inhibitory assay indicated that the test extracts do not contain antidiabetic secondary metabolites.Item Phenotypic variation of cabbage white mold pathogen, Sclerotinia sclerotiorum in the upcountry commercial cabbage fields in Sri Lanka(Journal of the National Science Foundation of Sri Lanka 2018 46 (2): 159 – 164, DOI: http://dx.doi.org/10.4038/jnsfsr.v46i2.8416, 2018) Mahalingam, T.; Guruge, B.M.A.; Somachandra., K.P.; Jayasekara, E.A.E.S.S.; Rajapakse, C.S.K.; Attanayake, R.N.Sclerotinia sclerotiorum is a cosmopolitan, necrotrophic and soil borne plant pathogen, capable of infecting many economically important crops. Although it is a well-studied pathogen elsewhere, there is no research conducted in Sri Lanka. The objectives of the current research were to study the phenotypic variations among isolates within the pathogen population in the upcountry commercial cabbage fields in Sri Lanka and to determine their sensitivity to a commonly applied fungicide, Mancozeb. Forty-six isolates obtained from infected cabbage heads were identified as S. sclerotionun based on its unique morphological characteristics. Colony diameter was measured on potato dextrose agar (PDA) plates at 23 °C and mycelia' colour was observed after two-week incubation. All the isolates were grown in pH indicator media to determine their add producing abilities. Sensitivity of the isolates to Mancozeb was assayed in vitro. There was a significant difference in colony growth among the isolates and sensitivity to Mancozeb. Two isolates showed abnormal mycelia' growth characteristics; a slower growth rate on PDA and reduced pathogenicity on cabbage leaves. Two levels of mycelia' pigmentation were observed on PDA. All the isolates were acid producers, inferring that all of them were pathogenic. In summary, the upcountry S. sclerotiorton pathogen population was found to be diverse for selected phenotypic traits, especially for mycelia( growth, and fungicide sensitivities inferring that the pathogen population is capable of adapting to changing environmental conditions. Therefore, disease management will be challenging if an epidemic occurs, and it is recommended that a proper management system should be identified before further expansion of upcountry cabbage cultivation.Item Evaluation of phenetic diversity of selected orchid cultivars with ornamental value(The Institute of Biology, Sri Lanka, 2016) Farook, F.; Attanayake, R.N.; Senanayake, S.P.Item Screening of fungi capable of degrading lignin from decaying woods in dry and intermediate forests in Sri Lanka(Department of Forestry and Environment Science, University of Sri Jayewardenepura, Sri Lanka., 2016) Daraniyagala, S.R.A.S.; Attanayake, R.N.Item Phylogenetic complexity of Lasiodiplodia species found in Sri Lankan dry zone forests(National Science foundation, 2016) Bandara, R.H.; Attanayake, R.N.Item Sclerotinia sclerotiorum causing cabbage head rod in Sri Lanka(Institute of Biology, Sri Lanka, 2015) Guruge, B.M.A.; Somachandra, K.P.; Attanayake, R.N.