Browsing by Author "Halmillawewa, A.P."

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Characterization of the Temperate Phage vB_RleM_PPF1 and its Site-Specific Integration into the Genome of Rhizobium leguminosarum Strain F1
(Staff Development Center, University of Kelaniya, 2015) Halmillawewa, A.P.; Restrepo-Córdoba, M.; Perry, B.J.; Yost, C.K.; Hynes, M.F.
The presence of prophages in a genome can contribute in increasing the bacterial fitness and ecological success in an environment that contains closely related phages. The temperate phage PPF1 was isolated from a lysogenized strain of Rhizobium leguminosarum F1. The complete genome sequence of Myoviridae phage PPF1 was determined using 454-Pyrosequencing technology. PPF1 is the first available complete genome sequence of a Rhizobium leguminosarum temperate phage (GenBank accession no: KJ746502) and the integration site and possible mechanism of integration of this phage has been identified. PPF1 is capable of efficiently lysogenizing the R. leguminosarum strain F1, and can be induced from its lysogenized host using UV irradiation. The genome of PPF1 is 54,506 bp in length with an average G+C content of 61.9%. The ORF predictions of the PPF1 genome revealed the presence of 94 putative proteinencoding genes and 74.5% of these predicted ORFs share homology at the protein level with previously reported sequences in the database. However, putative functions could only be assigned to 25.5% (24 ORFs) of the predicted genes. The site-specific recombination system of the phage targets an integration site that lies within a putative tRNA-Pro (CGG) gene in R. leguminosarum F1. Upon integration, the phage is capable of restoring the disrupted tRNA gene, owing to the 50 bp homologous sequence (att core region) it shares with its rhizobial host genome. The predicted att site of temperate phage PPF1 share a sequence similarity with the targeted att site of previously characterized Sinorhizobium meliloti phage 16-3. In spite of phage PPF1's propensity for lysogenizing host strains, including strains on which it does not form visible plaques, there has thus far been no evidence of the presence of similar phages integrated into completed genomes of R. leguminosarum and related phages. The site specificity of insertion of this phage could be used to create single copy integration vectors for genetic work in R. leguminosarum.
Isolation and characterization of rhizobia from leguminous plants
(Faculty of Science, University of Kelaniya, Sri Lanka, 2016) Wijesundera, S.H.; Halmillawewa, A.P.
Rhizobia play a significant role in agriculture. Their ability to fix atmospheric nitrogen through formation of root nodules in leguminous plants contributes in increasing legume yield. The present study was carried out with the aim of isolating rhizobial strains from legume root nodules and characterizing them to check their potential to be developed into biofertilizers. Tests were performed to detect plant growth promoting traits such as phosphate solubilization, nitrate reduction, abiotic stress tolerance, which contribute towards sustainable agriculture. Four rhizobial strains, namely C1-n1 (from Vigna unguiculata), P3-n1 (from Arachis hypogaea), L1-n1 (from Vigna unguiculata ssp. Sesquipedalis) and L2-n1 (from Vigna unguiculata ssp. Sesquipedalis) were isolated. They were characterized based on their morphological and biochemical features. Preliminary identification of isolates was done by observing their growth on yeast extract mannitol agar with congo red as well as their reactions to Glucose peptone agar test, Hofers’ alkaline test, and Lactose agar test. The isolated strain L2-n1, was identified as a slow growing alkaline producing one according to its reaction on bromo thymol blue medium, while others were recognized as acid producing fast growers. This result was congruent with their growth rates as well. All isolates gave positive results for oxidase, catalase, urease, and nitrate reduction tests and a negative result for indole, methyl red, gelatine hydrolysis and starch hydrolysis test. Two strains (L1-n1 and P3-n1) gave a positive reaction to Voges- Proskauer test, while only L2-n1 gave a positive result for citrate test. No isolate was able to exhibit swarming motility and phosphate solubilization. Furthermore, the capability of these organisms to utilize six different carbon sources and produce mucus colonies was also tested. The degree of viscosity produced was compared visually. Strains grew well at 25 °C–38 °C range, while L1-n1 and P3-n1 were able to tolerate a temperature range of 6 °C – 55 °C, pH range of 5.0 - 9.0 and NaCl levels up to 5% (w/v). C1-n1 was able to tolerate a pH range of 3.0 - 9.0 and NaCl levels up to 2.5% (w/v), while L2-n1 showed a growth only at neutral pH and a NaCl level of 0.01% (w/v). Also majority of strains were able to tolerate the heavy metals tested (Cu, Cd & Pb) at different concentrations. Strain L1-n1 showed an antibacterial activity against Staphylococcus aureus. All isolates formed nodules during the plant infectivity assays which were performed using their original plant host species confirming their identity as rhizobia. Plasmids were not observed in any of the isolates after performing the Eckhardt gel electrophoresis. According to the above results, these isolates may not be useful as biofertilizers, as they do not exhibit plant growth promoting traits satisfactorily. However further studies are required to determine their nodulation and nitrogen fixing efficiencies.
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.
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.