Microbiology
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Item Simple Sequence Repeats (SSR) marker-based analysis on the genetic variation and population structure of local and exotic sorghum germplasm collection conserved ex-situ in Sri Lanka(2024) Kaluthanthri, D. V. S.; Perera, S. A.C.N.; Dasanayaka, P. N.Sorghum (Sorghum bicolor (L.) Moench) is one of the most important cereal crops occupying the fifth position based on the cultivated extent among the cereal crops in the world. Characterization of genetic resources is a pre-requisite for utilization of conserved genetic resources in breeding programmes and cultivation. The present study was carried out to reveal the genetic variation and population structure of local and exotic sorghum germplasm collection conserved in ex-situ seed gene bank at the Plant Genetic Resources Centre, Sri Lanka. Total genomic DNA was extracted from 60 germplasm accessions using CTAB miniprep DNA extraction protocol. A two-step PCR amplification was performed at 16 Simple Sequence Repeat (SSR) loci. Four differentially labeled PCR products were multiplexed and size-fractioned using capillary electrophoresis. Data analyses were performed using GeneMapper 4.0, OSIRIS, PowerMarker 3.25, Structure 2.2 and STRUCTURE HARVESTER. The 16 SSR loci recorded polymorphism and the dendrogram revealed four distinct clusters. The optimum number of subpopulations was three in addition to two admixture subpopulations. The revealed population structure did not depict the geographical origin of the germplasm accessions. The present study confirmed that the majority of local sorghum germplasm accessions tested were genetically distinct. Varying degrees of outcrossing selfing in subsequent generations may have led to the creation of novel sorghum genotypes at global level.Item Staphylococcus edaphicus KCB02A11 incorporated with natural adsorbents: first report on its tolerance and removal of hexavalent chromium [Cr(VI)](2023) Aththanayake, A. M. K. C. B.; Rathnayake, I. V. N.; Deeyamulla, M. P.; Megharaj, MallavarapuDeteriorating the quality of different parts of the ecosystem due to toxic metals is a serious global issue. Hexavalent chromium is a metal that can cause adverse effects on all living beings, including plants, animals, and microorganisms, on exposure to high concentrations for prolonged periods. Removing hexavalent chromium from various types of wastes is challenging; hence the present study investigated the use of bacteria incorporated with selected natural substrates in removing hexavalent chromium from water. Isolated Staphylococcus edaphicus KCB02A11 has shown higher removal efficiency with a wide hexavalent chromium range (0.025-8.5 mg/L) within 96 h. Incorporating the isolated strain with natural substrates commonly found in the environment (hay and wood husk) showed high removal potential [100% removal with 8.5 mg/L of Cr(VI)], even within less than 72 h, with the formation of biofilms on the used substrates applied for metal removal on a large scale for prolonged periods. This study is the first report investigating hexavalent chromium tolerance and removal by Staphylococcus edaphicus KCB02A11.Item Pectobacterium carotovorum Phage vB_PcaM_P7_Pc Is a New Member of the Genus Certrevirus(Microbiology Spectrum, 2022) Naligama, K. N.; Halmillawewa, A. P.Pectobacterium carotovorum is an economically important phytopathogen and has been identified as the major causative agent of bacterial soft rot in carrots. Control of this phytopathogen is vital to minimizing carrot harvest losses. As fully effi- cient control measures to successfully avoid the disease are unavailable, the phage- mediated biocontrol of the pathogen has recently gained scientific attention. In this study, we present a comprehensive characterization of the P. carotovorum phage vB_PcaM_P7_Pc (abbreviated as P7_Pc) that was isolated from infected carrot samples with characteristic soft rot symptoms, which were obtained from storage facilities at market places in Gampaha District, Sri Lanka. P7_Pc is a myovirus, and it exhibits growth characteristics of an exclusively lytic life cycle. It showed visible lysis against four of the tested P. carotovorum strains and one Pectobacterium aroidearum strain. This phage also showed a longer latent period (125 min) than other related phages; however, this did not affect its high phage titter (.1010 PFU/mL). The final assembled genome of P7_Pc is 147,299 bp in length with a G1C content of 50.34%. Of the 298 predicted open reading frames (ORFs) of the genome of P7_Pc, putative functions were assigned to 53 ORFs. Seven tRNA-coding genes were predicted in the genome, while the genome lacked any major genes coding for lysogeny-related products, con- firming its virulent nature. The P7_Pc genome shares 96.12% and 95.74% average nu- cleotide identities with Cronobacter phages CR8 and PBES02, respectively. Phylogenetic and phylogenomic analyses of the genome revealed that P7_Pc clusters well within the clade with the members representing the genus Certrevirus. Currently, there are only 4 characterized Pectobacterium phages (P. atrosepticum phages phiTE and CB7 and Pectobacterium phages DU_PP_I and DU_PP_IV) that are classified under the genus, making the phage P7_Pc the first reported member of the genus isolated using the host bacterium P. carotovorum. The results of this study provide a detailed characteriza- tion of the phage P7_Pc, enabling its careful classification into the genus Certrevirus. The knowledge gathered on the phage based on the shared biology of the genus will further aid in the future selection of phage P7_Pc as a biocontrol agent.Item Pectobacterium spp. isolated from rotting carrots obtained from markets in Gampaha district, Sri Lanka exhibit the potential of having broad host ranges(Eur J Plant Pathol, 2022) Naligama, K.N.; Halmillawewa, A.P.Carrot production in Sri Lanka faces severe post-harvest losses due to bacterial soft rot. The quality deterioration of vegetables owing to typical bacterial soft rot can greatly affect the market value and consumer preference. Although the carrot soft rot causing bacteria occur all over the world, and are well-studied and characterized, the scarcity of data on the precise identification of the causal agents of the disease in Sri Lanka acts as a great barrier in managing such post-harvest losses. In an attempt to bridge this knowledge gap, we have isolated potential causative agents of bacterial soft rot from diseased carrot samples collected from Gampaha district, Sri Lanka.All the seven bacterial isolateswere confirmed for their ability to exhibit pectolysis, and vegetable disk assays were used to evaluate the pathogenicity of bacterial isolates. The pathogenicity assays showed that these isolates have the ability to infect not only carrot, but also potato, radish, beetroot and Napa cabbage, suggesting their possible broad host range. The ITS–PCR RELP profiles of the pectobacterial isolates and hierarchical clustering of the resulting profiles have placed the strains isolated in this study into four groups. The 16S rRNA gene sequencing and subsequent analyses aided in identifying isolates as Pectobacterium carotovorum (C1B5, C2B6, C2B7 and C2B8), P. aroidearum (C1B3 and C1B4), and P. polaris (C3B9). The study indicated the possibility of different Pectobacterium spp. being involved in causing carrot soft rot in the area, emphasizing the need to carry out an island-wide, comprehensive analysis to understand the distribution of the pathogen, which could be used in implementing successful disease management strategies.Item Phenotypic and genotypic characterization of antibiotic resistance of Listeria monocytogenes isolated from raw milk samples collected from Polonnaruwa, Sri Lanka(Emirates Journal of Food and Agriculture, 2022) Harshani, H.B.C.; Ramesh, R.; Halmillawewa, A.P.; Wijendra, W.A.S.Listeria monocytogenes is a food-borne pathogen that can cause severe invasive infection called ‘listerosis’ in humans. Development of antibiotic resistance is a major setback in the management of conditions caused by Listeria in both human and veterinary medicine. In this study, antibiotic resistance of fifty L. monocytogenes strains isolated from raw milk samples collected from farms in Polonnaruwa district, Sri Lanka was determined for four commonly used antibiotics; penicillin, ampicillin, streptomycin and tetracycline. The strains were also tested for the presence of selected antibiotic resistant genes (penA, ampC, strA, strB, tetA and tetB). L monocytogenes isolates showed resistance to ampicillin (60%), penicillin (40%) streptomycin (16%) and tetracycline (8%) in diffusion assays. Phenotypic multidrug resistance was exhibited by twenty isolates. The tetracycline resistant gene (tetA) was detected in seven isolates, while tetB was not detected in any. Presence of streptomycin resistant genes (strA or strB) was confirmed in seven isolates. Ampicillin (ampC) and penicillin (penA) resistant genes were not detected in any of the tested isolates. Except from the samples collected from Sungavila area, isolates from other sampling areas showed resistance to at least one of the antibiotics tested, suggesting that raw milk samples are prone to be contaminated with L. monocytogenes strains with different antibiotic resistant profiles. Therefore, necessary hygienic precautions are recommended to avoid any potential public health threats and to safeguard the health of raw milk consumers.Item Potential use of Chlorella vulgaris KCBAL01 from a freshwater stream receiving treated textile effluent in hexavalent chromium [Cr(VI)] removal in extremely acidic conditions(Journal of Environmental Science and Health, 2022) Aththanayake, A. M. K. C. B.; Rathnayake, I. V. N.; Deeyamullab, M. P.; Megharaj, MallavarapuRemediation of hexavalent chromium with conventional chemical and physical methods is a costly process, while replacing some critical steps in physiochemical remediation with self-sustaining bioremediation agents are expected to be cost-effective and environmentally friendly implementation. In this study, a microalga isolated from a freshwater stream receiving treated textile wastewater was identified up to its molecular level and investigated its ability to tolerate and remove hexavalent chromium from extremely acidic conditions under different temperatures. The ability of microalgae to tolerate and remove Cr(VI) was investigated by growing it in BG11 media with different pH (1, 2, 3 & 7), amended with several concentrations of Cr(VI) and incubated under different temperatures for 96 hrs. Microalga was identified as Chlorella vulgaris and found that the isolated strain has a higher hexavalent chromium removal potential in extremely acidic conditions than in neutral pH conditions at 25 C. In contrast, its Cr(VI) removal potential is significantly influenced by the pH and temperature of the growth medium. Furthermore, it exhibited a permanent viability loss at extreme acidic conditions (pH 1 3) and prolonged exposure to the higher chromium content. The microalga investigated will be a highly useful bioagent in hexavalent chromium remediation in high acidic conditions.Item Identification of Potentially Hazardous Microorganisms and Assessment of Physicochemical Deterioration of Thermally Processed King Coconut (Cocos nucifera var. aurantiaca) Water under Different Processing Conditions in Sri Lanka(Journal of Food Quality, 2022) Jayasinghe, M. D.; Madage, S. S. K.; Hewajulige, I. G. N.; Jayawardana, T. M. D. A.; Halmillawewa, A. P.; Divisekera, D. M. W. D.King coconut water (KCW) is a sweet relish product that is more prone to rapid quality deterioration, and several safety concerns are emerging due to its inappropriate thermal processing. (erefore, the objective of this study was to identify the potential spoilage/ pathogenic microorganisms associated with the processing of KCW, with the assessment of possible physicochemical changes as providing preliminary information required for the thermal process validation of bottled KCW. Samples (n� 6, 150 ml/sample) were collected from three different KCW processing facilities at five critical processing steps (P1 − P5). A facility survey, physicochemical analyses, and microbial enumeration and isolation, along with their molecular identifications, were conducted. It was found that all tested physicochemical properties were significantly changed (p < 0.05) among sampling points at each processing facility.(ecolour of thermally processed KCW samples has significantly changed (p < 0.05) compared to the fresh KCW, which causes a distinct effect on the appealing quality of the final product. A pattern of initial lower counts with gradually increased microbial counts at intermediate processing steps (1.0 ×103–5.3×106 CFU/ml) and significantly lowered (p < 0.05) counts after thermal treatment was observed. Among the bacterial and fungal isolates identified, several potential pathogenic bacterial species, such as Pantoea dispersa, Bacillus siamensis, Pseudomonas stutzeri, and Acinetobacter lactucae; a few thermal resistant yeasts, Pichia kudriavzevii, Debaryomyces nepalensis, and Candida carpophila; and moulds, Penicillium citrinum, Microdochium fisheri, and Trichosporon asahii, have survived in the thermally processed KCW. Based on the results of the study, it is suggested that the thermal process validation of KCW should be targeted according to the revealed knowledge on the identified hazardous microorganisms, while adhering to Good Manufacturing and Hygienic Practices with minimized handling time to avoid rapid quality deterioration.Item Characterization of Bioactive Actinomycetes Isolated from Kadolkele Mangrove Sediments, Sri Lanka(Journal of Microbiology, vol.71, 2022) Naligama, Kishani N.; Weerasinghe, Kavindi E.; Halmillawewa, Anupama P.Exploring untapped microbial potentials in previously uncharted environments has become crucial in discovering novel secondary metabolites and enzymes for biotechnological applications. Among prokaryotes, actinomycetes are well recognized for producing a vast range of secondary metabolites and extracellular enzymes. In the present study, we have used surface sediments from ‘Kadolkele’ mangrove ecosystem located in the Negombo lagoon area, Sri Lanka, to isolate actinomycetes with bioactive potentials. A total of six actinomycetes were isolated on modified-starch casein agar and characterized. The isolates were evaluated for their antibacterial activity against four selected bacterial strains and to produce extracellular enzymes: cellulase, amylase, protease, and lipase. Three out of the six isolates exhibited antibacterial activity against Staphylococcus aureus, Escherichia coli, and Bacillus cereus, but not against Listeria monocytogenes. Five strains could produce extracellular cellulase, while all six isolates exhibited amylase activity. Only three of the six isolates were positive for protease and lipase assays separately. Ac-1, Ac-2, and Ac-9, identified as Streptomyces spp. with the 16S rRNA gene sequencing, were used for pigment extraction using four different solvents. Acetone-extracted crude pigments of Ac-1 and Ac-2 were further used in well-diffusion assays, and growth inhibition of test bacteria was observed only with the crude pigment extract of Ac-2. Further, six different commercially available fabrics were dyed with crude pigments of Ac-1. The dyed fabrics retained the yellow color after acid, alkaline, and cold-water treatments suggesting the potential of the Ac-1 pigment to be used in biotechnological applications.Item Sensitivity of Four Cyanobacterial Isolates from Tropical Freshwaters to Environmentally Realistic Concentrations of Cr6+, Cd2+ and Zn2+(Bulletin of Environmental Contamination and Toxicology volume, 2021, 2021) Munagamage, Thilini; Rathnayake, I. V. N; Pathiratne, A; Megharaj, MallavarapuSensitivity of four tropical cyanobacteria viz. Coelosphaerium sp., Synechococcus sp., Oscillatoria sp. and Chroococcus sp. to environmentally relevant concentrations of Cr6+, Cd2+ and Zn2+was assessed based on fluorescence ...Item A bacteriophage infecting Mesorhizobium species has a prolate capsid and shows similarities to a family of Caulobacter crescentus phages(Canadian Journal of Microbiology, 2020) Gunathilake, Damitha; Halmillawewa, Anupama; MacKenzie, Keith; Perry, Benjamin; Yost, Christopher; Hynes, MichaelMesorhizobium phage vB_MloS_Cp1R7A-A1 was isolated from soil cultivated to chickpea in Saskatchewan and is dissimilar in sequence and morphology to previously described rhizobiophages. It is a B3 morphotype virus with a distinct prolate capsid and belongs to the tailed phage family Siphoviridae. Its genome has a GC content of 60.3% and 238 predicted genes. Putative functions were predicted for 57 genes, which include 27 tRNA genes with anticodons corresponding to 18 amino acids. This represents the highest number of tRNA genes yet reported in a rhizobiophage. The gene arrangement shows a partially modular organization. Most of the structural genes are found in one module, whereas tRNA genes are in another. Genes for replication, recombination and nucleotide metabolism form the third module. The arrangement of the replication module resembles the replication module of Enterobacteria phage T5, raising the possibility that it uses a recombination-based replication mechanism. Phage termini appear to be long direct repeats of length just over 12 kb. Phylogenetic analysis revealed that Cp1R7A-A1 is more closely related to phiCbK-like Caulobacter phages and other B3 morphotype phages than to other rhizobiophages sequenced thus far.
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