Plant Pathology

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Biological and molecular characterization of South African bacteriophages infective against Staphylococcus aureus subsp. aureus Rosenbach 1884, casual agent of bovine mastitis.
(2012) Basdew, Iona Hershna.; Laing, Mark Delmege.
Bacteriophage therapy has been exploited for the control of bacterial diseases in fauna, flora and humans. However, the advent of antibiotic therapy lead to a cessation of most phage research. Recently, the problem of antibiotic resistance has rendered many commonly used antibiotics ineffective, thereby renewing interest in phage therapy as an alternative source of control. This is particularly relevant in the case of bovine mastitis, an inflammatory disease of bovine mammary glands, caused by strains such as Staphylococcus aureus subsp. aureus Rosenbach 1884. Antibiotic resistance (primarily towards penicillin and methicillin) by staphylococcal strains causing mastitis is regularly reported. Phage therapy can provide a stable, effective and affordable system of mastitis control with little to no deleterious effect on the surrounding environment or the affected animal itself. Several studies have delved into the field of biocontrol of bovine mastitis using phages. Results are variable. While some phage-based products have been commercialized for the treatment of S. aureus-associated infections in humans, no products have yet been formulated specifically for the strains responsible for bovine mastitis. If the reliability of phage therapy can be resolved, then phages may become a primary form of control for bovine mastitis and other bacterial diseases. This study investigated the presence of S. aureus and its phages in a dairy environment, as well as the lytic ability of phage isolates against antibiotic-resistant strains of mastitic S. aureus. The primary goals of the thesis were to review the available literature on bovine mastitis and its associated control, and then to link this information to the use of phages as potential control agents for the disease, to conduct in vitro bioassays on the selected phages, to conduct phage sensitivity assays to assess phage activity against different chemical and environmental stresses, to morphologically classify the selected phages using transmission electron microscopy, to characterize the phage proteins using one-dimensional electrophoresis, and lastly, to characterize phage genomes, using both electrophoresis as well as full genome sequencing. Twenty-eight phages were isolated and screened against four strains of S. aureus. Only six phages showed potential for further testing, based on their wide host range, high titres and common growth requirements. Optimal growth conditions for the host S. aureus strain was 37°C for 12hr. This allowed for optimal phage replication. At an optimal titre of between 6.2x10⁷ to 2.9x10⁸ pfu.mlˉ¹(at 10ˉ⁵ dilution of phage stock), these phages were able to reduce live bacterial cell counts by 64-95%. In addition, all six phages showed pathogenicity towards another 18 S. aureus strains that were isolated from different milk-producing regions during a farm survey. These six phages were named Sabp-P1, Sabp-P2, Sabp-P3, Sabp-P4, Sabp-P5 and Sabp-P6. Sensitivity bioassays, towards simulated environmental and formulation stresses were conducted on six identified phages. Phages Sabp-P1, Sabp-P2 and Sabp-P3 showed the most stable replication rates at increasing temperatures (45-70°C), in comparison to phages Sabp-P4, Sabp-P5 and Sabp-P6. The effect of temperature on storage of phages showed that 4ºC was the minimum temperature at which phages could be stored without a significant reduction in their lytic and replication abilities. Furthermore, all phages showed varying levels of sensitivity to chloroform exposure, with Sabp-P5 exhibiting the highest level of reduction in activity (74.23%) in comparison to the other phages. All six phages showed optimal lytic ability at pH 6.0-7.0 and reduced activity at any pH above or below pH 6.0-7.0. Exposure of phages to varying glycerol concentrations (5-100%) produced variable results. All six phages were most stable at a glycerol concentration of 10-15%. Three of the six isolated phages, Sabp-P1, Sabp-P2 and Sabp-P3, performed optimally during the in vitro assays and were used for the remainder of the study. Morphological classification of phages Sabp-P1, Sabp-P2 and Sabp-P3 was carried out using transmission electron microscopy. All three phages appeared structurally similar. Each possessed an icosahedral head separated from a striated, contractile tail region by a constricted neck region. The head capsules ranged in diameter between 90-110nm with the tail length ranging from 150-200nm in the non-contractile state and 100-130nm in the contractile state. Rigid tail fibres were also visible below the striated tail. The major steps in the virus replicative cycle were also documented as electron micrographs. Ultra-thin sections through phage plaques were prepared through a modification of traditional methods to speed up the process, with no negative effects on sample integrity. The major steps that were captured in the phage replicative cycle were (1) attachment to host cells, (2) replication within host cells, and, (3) release from cells. Overall results suggested that all three phages are strains from the order Caudovirales and are part of the Myoviridae family. A wealth of information can be derived about an organism based on analysis of its proteomic data. In the current study, one-dimensional electrophoretic methods, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and ultra-thin layer isoelectric focusing (UTLIEF), were used to analyse the proteins of three phages, Sabp-P1, Sabp-P2 and Sabp-P3, in order to determine whether these strains differed from each other. SDS-PAGE analysis produced unique protein profiles for each phage, with band fragments ranging in size from 8.86-171.66kDa. Combined similarity matrices showed an 84.62% similarity between Sabp-P1 and Sabp-P2 and a 73.33% similarity between Sabp-P1 and Sabp-P3. Sabp-P2 showed a 69.23% similarity to Sabp-P3. UTLIEF analysis showed protein isoelectric charges in the range of pI 4.21-8.13, for all three phages. The isoelectric profiles for each phage were distinct from each other. A combined similarity matrix of both SDS-PAGE and UTLIEF data showed an 80.00% similarity between phages Sabp-P1 and Sabp-P2, and a 68.29% similarity between Sabp-P1 and Sabp-P3. Sabp-P2 showed a 70.59% similarity to Sabp-P3. Although the current results are based on putative protein fragments analysis, it can be confirmed that phages Sabp-P1, Sabp-P2 and Sabp-P3 are three distinct phages. This was further confirmed through genomic characterization of the three staphylococcal phages, Sabp-P1, Sabp-P2 and Sabp-P3, using restriction fragment length analysis and whole genome sequencing. Results showed that the genomes of phages Sabp-P1, Sabp-P2 and Sabp-P3 were all different from each other. Phages Sabp-P1 and Sabp-P3 showed sequence homology to a particular form of Pseudomonas phages, called "giant" phages. Phage Sabp-P3 showed sequence homology to a Clostridium perfringens phage. Major phage functional proteins (the tail tape measure protein, virion structural proteins, head morphogenesis proteins, and capsid proteins) were identified in all three phages. However, although the level of sequence similarity between the screened phages and those already found on the databases, enabled preliminary classification of the phages into the order Caudovirales, family Myoviridae, the level of homology was not sufficient enough to assign each phage to a particular type species. These results suggest that phage Sabp-P1 might be a new species of phage within the Myoviridae family. One longer-term objective of the study is to carry out complete assembly and annotation of all the contigs for each phage. This will provide definitive conclusions in terms of phage relatedness and classification.
Biological characterization of South African bacteriophages infective against Streptococcus uberis, a causal agent of bovine mastitis.
(2019) Pillay, Caleb.; Laing, Mark Delmege.; Basdew, Iona Hershna.
Mastitis is an inflammatory disease of bovine mammary glands and is the most economically important disease affecting dairy herds in South Africa, and globally. The incidence and history of mastitis in South Africa has been extensively documented and found to be bacterial in origin. Streptococcus uberis (S. uberis) is the most common environmental causal agent of mastitis from clinical and subclinical samples in several countries, including Australia, the United Kingdom, New Zealand, and Belgium. Due to the causal agents ability to exist in cells, it remains protected from antibiotics. The quest for antibiotic free dairy management has funded the research into integrated strategies, predominantly proactive udder health management. Biological control methods are not widely used, specifically the use of bacteriophage (phage) therapy. Phages are ubiquitous (found in all biospheres) and are the most abundant organisms on earth. Understanding the interaction between phages and their hosts is vital to their manipulation for therapeutic conditions. This study aimed to isolate phages from unpasteurized milk of dairy cows, sequentially screened these against S. uberis isolates demonstrating antimicrobial resistance at the time. The phages have been screened for robust lytic characteristics for the of intent of a phage-based therapy. Bacterial strains of S. uberis were isolated from unpasteurized milk by Allerton laboratories submitted by dairy farms in KwaZulu-Natal, South Africa. The samples were screened for S. uberis using the following tests: haemolysis patterns on blood agar, catalase reactions using hydrogen peroxide (5%), and Gram reaction. The identity of the strains was then confirmed by Inqaba Biotechnical Industries (Pty) Ltd via sequencing of the 16s ribosomal RNA. The six S. uberis strains were screened against 8 commonly used antibiotics in the dairy industry: β-lactam (ampicillin, penicillin G, cefalexin, oxacillin and amoxicillin), macrolides (erythromycin), tetracyclines (tetracycline) and glycopeptide (vancomycin). Using the Kirby Bauer method and The European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breaking points zone diameters, version 10.0, 2020. Every strain of S. uberis was susceptible to vancomycin, which is not available for intra-mammary infections. Resistance to the 7 other antibiotics varied amongst the strains. This was not a survey study but an isolation of S. uberis strains to challenge the efficacy of phage therapy. Approximately 2,000 mastitic milk samples were screened for phages resulting in the isolation of 95 phages. This was further reduced by screening for constant lytic ability to five phages that were characterized for their phage titre, host range, single step growth phase and lethal dose activity. The titre of the five selected phages varied considerably. Phages CP1, CP2, CP76, CP79 and CP80 produced titres of 59x102, 56x105, 47x106, 50x104 and 35x104 pfu.ml-1, respectively. There was a differential interaction between the five phages isolates and the six strains of S. uberis. One strain of S. uberis was susceptible to all five phages, Strain 21A, whereas Strains 17D and 78B were not susceptible to any of the five phages. Phage CP2 was virulent to four of the six S. uberis strains. The single step growth assay illustrated the cyclic replication of the isolated phages takes between 50 - 60 minutes. In a lethal dose assay, Phages CP1 and CP2 were each able to reduce S. uberis counts by 86% and 83%, respectively. Of the 5 phages screened Phages CP1 and CP2 showed potential as stand-alone treatments, whereas Phages CP76, CP79 and CP80 would offer better control when combined in a phage cocktail, and this would broaden the host range. Phage samples were examined using transmission electron microscopy (JEOL 1400). Various negative stains were used to view the virus particles: 2% uranyl acetate (UA), 2% phosphotungstic acid (PTA) and 0.05-5% ammonium molybdate (AM). The AM staining provided the best images of infected bacterial cells and phage surface structures. All micrographs obtained, illustrated similar viral particle structures suggesting the phages screened belong to one family. A virus particle was measured to have a 50-65nm diameter icosahedral head and a short tail ranging from 25-35nm in length from the EM micrograph. The virus particles exhibited Podoviridae morphology. However, only a complete genomic sequencing will confirm the identity of these virus particles to a species level.
Investigating alternative methods to detect bovine mastitis in milk.
(2018) Shinga, Mduduzi Hendrick.; Laing, Mark Delmege.; Basdew, Iona Hershna.
The aim of this study was to investigate alternate measures for the diagnosis of bovine mastitis, which can either be done separately or coupled with the current use of somatic cell counts. Techniques that were assessed include measurement of pH, electrical conductivity (EC) and volatile organic compounds (VOCs) liberated by pathogens during metabolism in milk; the quantification of milk components (fats, whey proteins, lactose, caseins), and cell counts of Staphylococcus aureus. Various concentrations of S. aureus were used to assess the minimum bacterial inoculum level that could bring about detectable changes in the pH and EC values of milk. It was found that 10-2 diluted inoculum caused less changes in pH and EC compared to the undiluted bacterial inoculum size. On average, the pH of milk samples decreased from 6.45 to 5.31 after 32 hours of incubation at 37˚C. A corresponding EC value increased from 5.28 mS cm-1 to 6.68 mS cm-1 was observed due to the liberation of sodium and chloride ions during the incubation of inoculated milk after an incubation of 32 hours. The detected VOCs including compounds from hydrocarbon, ester, ketone, aldehyde and siloxane groups were observed from milk inoculated with common mastitis pathogens such as Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae, coagulase negative staphylococci (CNS) and Escherichia coli. Only 20% of a total of 50 inoculated samples released VOC’s. Furthermore, the VOCs identified were not species-specific. However, by comparing the samples to a control (un-inoculated sample), the identified VOCs could be used as a rough monitoring tool to distinguish inoculated milk from un-inoculated milk. Near-infrared analysis (NIRA) was carried out using the Kernel partial least squares regression. However, the calibration models for milk composition and S. aureus were poor. We believe that this was affected by the technique used, measuring the NIR absorbance of milk samples in plastic Petri dishes. The absorptive abilities of polystyrene present in Petri dishes affected the NIRS scans. Secondly, insensitive wet chemistry methods, and the low sample number used in this study were concluded to be the major reasons for the poor predictive models that were obtained for the analysis of milk components and S. aureus. These analytic tools showed potential as diagnostic methods, however, further research must be conducted to solve these problems.
Investigating the effect and efficiency of steel slag as a source of silicon for plant uptake.
(2021) Dadabhay, Adeel.; Laing, Mark Delmege.; Basdew, Iona Hershna.
Silicon (Si) is the second most abundant element on the planet, after oxygen, making about 25% of the Earth's crust. Since it exists in the Earth’s crust, many plants can accumulate it in large concentrations, in amounts similar to macronutrients. Si enhances growth and yield of some annual and vegetable crops, promotes upright growth (stronger and thicker stems, shorter internodes), prevents lodging, promotes favourable exposure of leaves to light, provides resistance to bacterial and fungal diseases and decreases the effects from abiotic stresses such as: high or low temperatures, salinity, heavy metal and aluminium toxicity and water deficiency. Si treatment effects on plant growth under disease and drought stress were analysed to determine the effectiveness of steel slag as a source of Si for plant uptake. Four Si treatments: slag products (SP1.7 and SP5.0); Agri-sil granular (AGS); and potassium silicate (Pots) were tested on rye grass, maize, zucchini, green pepper, broccoli and beans under disease and drought stress. Energy Dispersive X-ray (EDX) microanalysis was performed to determine the Si content within the leaves resulting from the different treatments over time. All treatments provided a positive Si uptake into the plant leaves. Maize had the highest rate of Si levels taken up into the leaves over a period of 180 days for all Si treatments, when compared to the other crops under disease stress. Si-treated plants accumulated Si into their leaves at a higher rate under disease stress than drought stress. Si treatments improved the growth of all test crops. Steel slag was an effective treatment for providing Si for the uptake in plants and to improve plant growth. The effect of pre-harvest Si application to inhibit Colletotrichum capsici on post-harvest pepper fruit (Capsicum annuum L.) was analysed. Pepper fruit were harvested from pepper plants (Capsicum annum L. cv. Revelation) that were Si treated to provide Si for plant uptake. Si treatments used were: Pots, which was used as a positive control; SP1.7; SP5.0; and AGS. The area covered by infection (%) on the fruit was recorded every seven days for a period of 21 days, to determine the disease progress. All Si treatments significantly reduced the rate of infection by the pathogen. By day seven, the disease progress was inhibited, with a recorded area of infection being below 3.5% compared to the control, which was at 8.4%. By day 14, it was inhibited from 33.6% (control) to below 16% and by day 21, it was inhibited from 57.4% (control) to below 31% for all Si treatments. Area under the disease-progress curve (AUDPC) value (%days) was the lowest for the SP1.7 treatment, which means it enhanced post-harvest disease resistance by the greatest amount. SP5.0 had the highest AUDPC value from all Si treatments. Pre-harvest application of Si reduced post-harvest anthracnose disease in green pepper fruit. The efficiency of steel slag as a source of Si for citrus and avocado uptake was analysed. Three different species: Citrus sinensis (Orange cultivars: Valencia and Navel); Citrus limon L. (lemon); and Persea Americana L. (avocado) were used for this study. Five Si treatments were tested: Pots; AGS; Agri-sil liquid (ASL); SP5.0; and SP1.7. EDX was performed to determine the Si content within the leaves resulting from the different treatments over time. All Si treatments provided a positive uptake of Si into the citrus leaves. Valencia trees treated with the SP1.7 had the highest rate of Si taken up into the leaves, with an area under curve (AUC) value of 210.24%days, followed by SP5.0 with an AUC value of 195.48. SP1.7 and AGS provided the highest rates of Si uptake into navel orange leaves with AUC values of 187.02 and 187.92, respectively. Lemon trees treated with SP1.7 and AGS had the highest rates of Si taken up into the leaves. Citrus trees treated with SP1.7 had higher rates of Si taken up into the tree leaves, with the exception of the AGS treatment having the highest rate of Si taken up in lemon. Avocado trees treated with SP1.7 had the highest rate of Si taken up into the leaves, with an AUC value of 28.29. Steel slag was an efficient and effective source of Si for the uptake in citrus and avocado leaves.

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