A number of techniques were used to obtain a variety of bacterial and fungal species antagonistic to Pythium - F group in hydroponic systems. Isolations were made from roots of ‘escape’ lettuce plants in a commercial hydroponic gravel system as well as Pythium mycelium exposed to the hydroponic solution. Seventy four bacterial and eighteen fungal isolates were obtained and were screened for in vitro activity against Pythium by means of the dual culture method. Twenty-two bacterial isolates rendered between 10.8 and 48 % inhibition and ten fungal isolates rendered between 24.3 and 54 % inhibition of Pythium mycelial growth. Potential biocontrol agents were screened in a static aquaculture system on butterhead lettuce seedlings in the greenhouse prior to evaluation in a re-circulating gravel bed hydroponic system in the greenhouse and field, for both growth promoting and biocontrol ability. Significant increases of between 689 % and 922 % in total fresh yield were obtained from plants preventatively treated with isolates JH49, JH41, JH83, JM6R and JM16W. The eight best performing isolates were further evaluated for biocontrol activity against Pythium as well as growth promotion on butter head lettuce in a re-circulating gravel bed hydroponic system in the greenhouse. Significant increases of 1.5 % - 63.5 % and 0.9 % - 38.8 % in total fresh yield were obtained from plants evaluated for growth promotion and Pythium control, respectively. Based on their performance five of the eight isolates were selected for evaluation in a re-circulating gravel bed hydroponic field system. Treatment with Pseudomonas fluorescens and Bacillus subtilis significantly increased fresh leaf weight of lettuce plants in comparison with the untreated control indicating effective suppression of Pythium. Of the isolates that were previously evaluated against Pythium wilt and root rot of lettuce in a hydroponic system (Chapters 2 and 3), 6 bacteria and 2 fungi were most effective. The following possible modes of action of these isolates, were investigated, namely competition, production of inhibitory substances and induced resistance. The root colonizating ability of the isolates was also assessed. Competition between the isolates and the pathogen were confirmed by testing for siderophore and hydrolytic enzyme production. Five of the isolates produced siderophores much faster than the rest, demonstrating that these isolates were able to take-up iron from the media at a faster rate, thus indicating a significant competitive ability. Antibiotic production by the isolates was confirmed in vitro by means of the dual culture technique. Of the eight isolates screened, only one isolate showed in vitro inhibition of the pathogen. This result was confirmed by a TLC assay, where fluorescent bands were formed by the same isolate, indicating the presence of phenolic compounds. These compounds were separated by HPLC. Analysis of total soluble and cell wall phenolic levels in Pythium infected and non-infected plants treated and untreated with the biocontrol isolates did not render conclusive results. Three of the eight isolates were able to colonize 100% of the lettuce roots. / Dissertation (MSc (Plant Pathology))--University of Pretoria, 2008. / Microbiology and Plant Pathology / unrestricted

Biological pre- and postharvest disease control strategies depend on successful colonisation and survival of the introduced antagonists on the infection court. Effective and economical applications involve targeting the antagonists where they are critically needed i.e. the infection court. Honeybee dispersal is one method of antagonist application to such specific sites. In view of this, an in vitro experiment was conducted to investigate attachment, colonisation and survival of Bacillus subtilis on avocado flowers. Scanning electron microscopy studies showed that the bacterium could attach and colonise avocado flower surfaces. It can also survive on the flowers for longer periods of time. In vivo mode of action of the antagonist against stem-end rot (SER) pathogens was also studied where results showed lysis and degradation of hyphae and conidia. However, no viable colonies were retrieved from bee antagonist dispersal under field conditions. Bee antagonist dissemination was compared with antagonist and fungicide spray applications in terms of SER control and the added effect on other diseases such as Cercospora spot and anthracnose. Spray applications of the antagonist were more effective in reducing the incidence of SER than bee dissemination. Integrated sprays of the antagonist and fungicides significantly reduced the incidence of both pre- and postharvest diseases. The identity of Dothiorella aromatica, one of the most important SER pathogens, was investigated at a molecular level. RAPD techniques using the discriminatory OPC02 primer successfully separated isolates into three groups based on banding profiles. A further study using RFLP identified the pathogen as a Botryosphaeria spp. The most dominant specie was B. parva followed by B. rhodina. Further studies should focus on assessing the distribution of these pathogens within avocado-growing regions of South Africa. / Dissertation (MSc (Plant Pathology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

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Pectobacterium carotovorum (Pbc), Dickeya spp., Pectobacterium atrosepticum (Pba), and a new, atypical strain, Pectobacterium carotovorum subsp. brasiliensis (Pbcb) can cause potato blackleg, soft rot and aerial stem rot. To determine the impact and extent of these pathogens on the South African potato industry, samples were collected from 72 potato fields in 10 potato production regions during the 2006/7 production seasons. During these seasons, blackleg outbreaks occurred in commercial production fields causing severe economic losses. Bacteria isolated from diseased material that were Gram-negative, facultative anaerobic with pectolytic ability were identified using a Multiplex PCR targeting the 16S-23S region. Isolates were subjected to partial sequencing of the 16S-23S rDNA and a subsequent PCR-RFLP of the 16S-23S rDNA region. Comparison of RFLP patterns of isolates to reference cultures confirmed the identity of the South African blackleg strains as Pbcb. This is the first report of Pbcb in South Africa. A total of 128 isolates were obtained from 10 production regions. Of these 77% were shown to be Pbcb, 17% Pbc and 6% unkown. Dickeya spp. and Pba were not detected in South Africa. From imported tubers 14 isolates were obtained, 13 of which were identified as Pbcb and one isolate as Pbc. Thus Pbcb was shown to be the most important causal agent of blackleg and soft rot in South Africa and poses a threat to the South African potato industry. Through a questionnaire survey the farmers` knowledge of soft rot / blackleg disease complex and the need for research on these potato diseases in South Africa was determined. A total of 41 questionnaires were collected from potato growers and analysed. Estimates of economic losses experienced by growers due to soft rot / blackleg disease complex, ranged from 1 to 70%. It appears that in South Africa disease symptoms are mainly prevalent at temperate (10 – 20°C) to warm climates with prolonged wet or humid conditions. To reduce disease incidence, low generation seed tubers need to be planted and good crop rotation systems need to be followed. Blackleg is mainly a seed-borne disease and is therefore difficult to control. This prompted the search for a possible management strategy to increase plant / tuber resistance to blackleg and soft rot and to minimise losses. The study also focussed on the effect of a calcium silicate slag soil amendment on phenolic formation in cell walls of potato peels, and subsequent tuber resistance to Pectobacterium carotovorum subsp. brasiliensis (Pbcb) . Pot trials were conducted with the following treatments: Control with and without the pathogen; slag (30% Si) with and without the pathogen; lime treatment (CaCO3) as pH control with and without the pathogen. Results from pot trials show that phenol production in potato stems and tubers increased in both lime and slag treatments, compared to the controls. This suggests that soil pH may play a role in phenol production. This could, however be due to increased silicon absorption by plants at higher pH values. Results also indicate that the best Si source to use is Calmasil, since Si and Ca combined have a synergistic effect in enhancing tuber resistance. This is, however, the first study on the effect of Si on defence responses of potatoes and further research is required to elucidate the modes-of-action of Si in the potato plant. This study has enhanced the understanding of the etiology of soft rot / blackleg diseases in South Africa and opened up new possibilities for the use of Si in commercial production to improve plant health status. However the need for future research on this disease complex has been highlighted in this thesis. / Dissertation (MSc)--University of Pretoria, 2009. / Microbiology and Plant Pathology / unrestricted

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The idiophasic production of nonspecific extracellular enzymes by the Basidiomycete fungus Phanerochaete chrysosporium, referred to as the ligninolytic system, has been reported by many researchers to be capable of degrading a broad spectrum of environmentally persistent compounds including some alkyl halide chemicals. Cultivation of the fungus and the ability of the ligninolytic system to degrade the alkyl halide pesticide toxaphene were explored in pure liquid cultures. The effects of culture growth temperature and toxaphene concentration were evaluated. The extent of toxaphene conversion with time was determined using three different indicators; toxaphene gas chromatographic elution patterns, the determination of free chloride concentrations by ion chromatography analyses, and the distribution of chloride in aqueous and biomass fractions via total organic halide analyses. Oxidation of the azo dye, Orange II, via spectrophotometric measurements was employed to determine the activity and reactivation of the fungal ligninolytic system. Degradation of toxaphene in the P. chrysosporium cultures was limited, with a maximum of 7% of the original mass of chloride originating from the toxaphene being released over a three week period. The main removal mechanism of the toxaphene from solution was observed to be adsorption to the mycelium biomass. Ligninolytic activity was equally active at temperatures less than optimum for maximum growth of the fungus which implies that the ligninolytic system can be effective with reduced temperature requirements. Elevated pH conditions did not effect ligninolytic activity indicating that high more neutral pHs, which are characteristic of soil properties, do not inhibit ligninolytic activity. Reactivation of the ligninolytic system was accomplished in a nonimmersed liquid culture system. The immobilized configuration appeared to be an effective system for cultivating the ligninolytic system on a continuous basis. / Master of Science