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Masters Degrees (Microbiology)

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Browsing Masters Degrees (Microbiology) by Subject "Aerobic bacteria."

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    Investigation into the diversity of antifungal aerobic endospore-forming bacteria associated with bulk and crop rhizosphere soil.
    (2011) Musoke, Jolly.; Hunter, Charles Haig.
    Members of the genus Bacillus are mainly Gram positive, aerobic rod shaped, endospore-forming bacteria that are increasingly being recognised for their ability to promote plant growth and antagonise fungal pathogens. From a biological control perspective, Bacillus spp. strains that produce antifungal compounds are of particular interest. In this study, aerobic endospore-formers were isolated from an undisturbed indigenous grassland soil and screened for antifungal activity and other plant growth promoting traits. Endospore-formers were also isolated from rhizosphere soil associated with the roots of maize, wheat and kale grown in pots containing soil from the same grassland site. Microbial diversity amongst isolates showing antifungal activity was investigated using different molecular fingerprinting methods, namely, intergenic transcribed spacer–PCR (ITS-PCR), random amplified polymorphic DNA-PCR (RAPD-PCR) and 16S rRNA gene amplification and sequencing. Characterization of the active antimicrobial compound(s) associated with selected isolates was also attempted. Prior to isolating from bulk and rhizosphere soils, samples were pre-heated to eliminate heat sensitive vegetative cells. Mean endospore counts were; wheat rhizosphere, Log 6.03 c.f.u g-1 soil; maize rhizosphere, Log 5.88 c.f.u g-1 soil; kale rhizosphere Log 5.90 c.f.u g-1 soil; and bulk soil Log 5.67 c.f.u g-1soil. A total of three hundred and eighty-four isolates were screened for antagonism towards Rhizoctonia solani using dual-culture plate bioassays. Thirty four of the isolates (~9%) mostly isolated from the bulk soil inhibited R. solani at varying degrees. Differences in antimicrobial interactions were apparent in in vitro bioassay; supposedly due to different concentrations and/or types of antimicrobial compounds. Biochemical tests for amylase, cellulase, chitinase, and proteinase activity, siderophore production and inorganic phosphate solubilisation were conducted. None of the isolates possessed all of these attributes and only a few showed multiple traits. Ninety-one percent of the isolates exhibited proteinase activity, 76% were able to hydrolyze starch whereas only four displayed cellulase activity. Only four isolates from the bulk-soil were capable of solubilising inorganic phosphate. ITS-PCR and 16S rRNA gene sequence analysis showed high levels of genetic homology amongst isolates and the majority were closely associated with representatives of the B. cereus group. Isolate C76 was the exception, being closely matched with B. subtilis. ITS-PCR banding profile was useful for distinguishing between species but did not distinguish within species. RAPD-PCR distinguished finer levels of genetic diversity between and within sample sets, with primer OPG-11 showing the greatest levels of heterogeneity. DNA extraction methods and the influence of template DNA dilution were investigated to determine their influence on RAPD-PCR analysis reproducibility. Prominent bands were comparable for crude template- and kit-extracted DNA but slight changes in band intensity and in some instances, additional faint bands were observed. At the highest DNA concentrations tested (7 μg/ml), further bands with molecular weights above 2.5 kbp were apparent. Strict standardization of PCR conditions greatly reduced variability of the RAPD-PCR analysis. Isolates from the different sample sets were screened for the presence of genetic markers associated with the biosynthesis of zwittermicin A, an aminopolyol antibiotic produced by some members of the B. cereus group. In an initial screen only one isolate, W96, yielded PCR amplicons consistent with those previously reported in the literature for the zwittermicin A genes. Later a further sixteen isolates grouped with W96 on the basis of the RAPD-PCR fingerprinting profiles, were screened for the presence of these genes. Of these, only six showed PCR amplification products similar to W96. Sequence homology testing against the GenBank database confirmed the presence of the zwittermicin A genes in these isolates. Isolate W96 was selected for further extraction and characterization of its antifungal compound(s). However, after culturing in various broth media cell free supernatants of W96 failed to show antifungal activity in vitro even when the supernatants were concentrated 20-fold. These findings provide a general overview of the diversity of aerobic endospore-forming bacteria present in an undisturbed indigenous grassland soil that exhibited antifungal activity in vitro and the limited influence tested crop rhizospheres have on this diversity. Combined use of ITS-PCR, 16S rRNA sequencing and RAPD-PCR techniques served as a rapid and effective means of grouping isolates for further investigations of their potential use as biocontrol agents and plant growth promoting rhizobacteria.
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    Screening for aerobic endospore-forming bacteria as biocontrol agents for powdery mildew disease of cucurbits.
    (2015) Tredgold, Heather Rayne.; Hunter, Charles Haig.
    Powdery mildew of cucurbits costs the South African cucurbit-growing industry millions of Rands per year in reduced yields and compromised fruit quality. Amongst the many bacterial and fungal antagonists of cucurbit powdery mildew, certain aerobic endospore-forming bacteria (AEFB) species show promise as biocontrol agents of this disease. When embarking upon biocontrol agent selection, multifaceted screening strategies are crucial. A study was undertaken with the aim of isolating AEFB from the cucurbit phylloplane for evaluation as potential antagonists of cucurbit powdery mildew using various screening approaches. Three hundred and nine AEFB isolates were isolated from cucurbit leaf material sourced from eight locations in the greater Msunduzi, KZN region. Dual-culture antifungal bioassays were performed using surrogate phytopathogenic fungi Botrytis cinerea and Rhizoctonia solani in place of the obligately biotrophic Podosphaera spp.. Two PCR-based genotyping methods were used to differentiate and group 55 antifungal AEFB isolates: internal-transcribed spacer region (ITS) PCR and randomly amplified polymorphic DNA (RAPD) PCR. The RAPD-PCR distinguished greater levels of genetic polymorphisms amongst isolates than did the ITS-PCR, revealing 14 different profiles as opposed to the three obtained from ITS-PCR; with 42% of isolates associated with a single RAPD-PCR banding profile. Phylogenetic relationships between representatives of each of the RAPD-PCR fingerprint groupings were determined by sequence analysis of 16S rRNA and gyrase subunit A (gyrA) gene fragments. In each instance, several distinct clusters were discernable, though gyrA sequences displayed higher levels of strain-level sequence heterogeneity. Comparisons of both gene sequence types with reference strains from the GenBank database revealed similarities to several known plant-associated strains of AEFB, including B. amyloliquefaciens subsp. plantarum and B. subtilis. Matrix-assisted laser deionisation-desorption time-of-flight mass spectrometry (MALDI-TOF-MS) based identification of selected AEFB was evaluated by comparing spectral data from AEFB isolates with reference strains in a Bruker BDAL Biotyper database. Only three out of the 14 isolates evaluated were identified to species level with acceptable confidence levels. This poor taxonomic resolution was ascribed to a paucity of applicable reference strains in the BDAL library. Nevertheless, mass spectra profiles of each isolate allowed for the clustering of related isolates to be achieved when dendograms were created. Antifungal compounds were extracted from 14 isolates using an acid-precipitation and methanol extraction protocol. Detection and identification of lipopeptide compounds in these extracts was assessed using thin-layer chromatography (TLC) and MALDI-TOF-MS. PCR-based screening for lipopeptide production potential using selected lipopeptide gene markers (viz. surfactin, iturin, bacillomycin, and fengycin) was also evaluated for the selected 14 isolates. These isolates were found to produce multiple lipopeptide compounds; including homologues of surfactin, iturin, and fengycin. However, disparities that emerged between PCR, TLC, and MALDI-TOF-MS data suggest that some PCR primers, the ituD marker in particular, showed limited specificity amongst the AEFB strains screened. Based on the overall findings, nine isolates proceeded to in vivo screening against Podosphaera spp. using an agarised detached cotyledon assay and a biocontrol pot trial. Isolates achieving the most effective antagonism of Podosphaera spp. differed in each respective assay. Isolate cce175 provided the highest antagonism in the biocontrol pot trial, and isolate sqo279 provided the best results in the detached cotyledon assay. The impacts of inoculum preparation were assessed using isolate cce175 in a biocontrol pot trial. Treatments varied in cell growth phase and assessed cell-free supernatant, whole broth, and cell-only fractions on biocontrol efficacy compared to a Tebuconazole (430 g/l) fungicide control. None of the treatments were found to impact disease at a statistically significant level. The merits and limitations of the various screening approaches used, and issues surrounding the isolation and assessment of biocontrol efficacy in plant-associated AEFB, are discussed.