Read the abstract in the section 00front of this document. / Thesis (PhD (Plant Pathology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

Thesis (MEng) -- Stellenbosch University, 2014. / ENGLISH ABSTRACT: As overpopulation threatens the world’s ability to feed itself, food has become an invaluable resource. Unfortunately, almost a third of the food produced for human consumption is lost annually. Pests including insects, phytopathogens and weeds are responsible for more than a third of the annual major crop losses suffered around the world. The majority of current post-harvest control strategies employ synthetic agents. These compounds, however, have been found to be detrimental to the environment as well as human health, which has led researchers to investigate alternative strategies. Biocontrol agents are environmentally compatible, have a lower toxicity and are biodegradable, making them an attractive alternative to the synthetic control agents. The lipopeptides produced by Bacillus spp. in particular, have shown great potential as biocontrol agents against various post-harvest phytopathogens. Most biocontrol strategies apply the biocontrol organism, for example Bacillus, directly, whereas this study focused on the use of the lipopeptide itself as an antifungal agent. This is advantageous as the lipopeptides are less sensitive to their surroundings, such as temperature and pH, compared to living organisms, allowing for the production of a standardized product. This study investigated the production of the Bacillus lipopeptides surfactin, fengycin and iturin under controlled batch conditions. Parameters increasing lipopeptide production were quantified, focussing on antifungal lipopeptides (iturin and fengycin), and lipopeptide production was optimized. Experiments were performed in a fully instrumented 1.3 L bench-top bioreactor and lipopeptide analyses were performed via high pressure liquid chromatography (HPLC) and liquid chromatography-mass spectroscopy (LC-MS). After screening four Bacillus spp., Bacillus amyloliquefaciens DSM 23117 was found to be the best antifungal candidate. This was based on it outperforming other candidates in terms of maximum antifungals produced, Yp/x,antifungals (yield per cells), and antifungal productivity. Nitrate, in the form of NH4NO3, was critical for lipopeptide production and an optimum concentration was observed above which the CDW (cell dry weight) no longer increased significantly and both μmax (maximum specific growth rate, h-1) and lipopeptide production decreased. For μmax, the optimum NH3NO4 concentration was 10 g/L and for lipopeptides it was 8 g/L. At these respective NH4NO3 concentrations μmax = 0.58 (h-1), the maximum antifungals (fengycin and iturin) were 285.7 mAU*min and the maximum surfactin concentration was 302 mg/L. The lipopeptides produced by B. amyloliquefaciens, the antifungals (fengycin and iturin) and surfactin, are secondary metabolites, regardless of the optimization treatment, i.e. increased NH4NO3 concentrations. Using 30% enriched air extended the nitrate utilization period, suggesting that when increasing supply concentration, more oxygen was available to act as electron acceptors, allowing nitrate to be used for lipopeptide production. The number of iturin and fengycin homologues generally increased with an increase in nitrate concentration. This suggested that process conditions, such as nitrate concentration, can be used to manipulate homologue ratios, allowing for the possibility to tailor-make biocontrol-agent upstream, during the production process, and possibly increase the efficacy of the biocontrol strategy. The lipopeptides produced by B. amyloliquefaciens showed complete inhibition against Botryotinia fuckeliana and diminished the growth capabilities of Botrytis cinerea. No inhibition was observed against Penicillium digitatum. These results indicate potential of the biocontrol strategy, although scale-up and fed-batch studies are recommended, especially when considering commercial implementation. Studies regarding the lipopeptide application method, i.e. a single application or multiple applications, should also be investigated as this will influence the efficacy of the lipopeptides against the target organisms. / AFRIKKANSE OPSOMMING: Met oorbevolking wat die wêreld se vermoë om die groeiende bevolking te onderhou belemmer, het dit noodsaaklik geword om huidige voedselbronne te beskerm. Daar word beraam dat een derde van die voedsel wat wêreldwyd geproduseer word vir menslike verbruik verlore gaan elke jaar. Verder is insekte, plantpatogene en onkruide verantwoordelik vir meer as ‘n derde van die verliese rakend jaarlikse oeste. Meeste bestaande na-oes beheermetodes maak gebruik van sintetiese stowwe. Ongelukkig kan hierdie verbindings nadelig wees vir die omgewing sowel as menlike gesondheid. Navorsers het hulsef dus toespits daarop om alternatiewe beheermetodes te ondersoek. Bio-beheermetodes is omgewingsvriendelik sowel as bio-afbreekbaar, wat hulle ideale alternatiewe maak vir die sintetiese stowwe. Bacillus spp. lipopeptiede het veral hoë potensiaal getoon as bio-beheermiddels teen verskeie na-oes plantsiektes. Meeste bio-beheermetodes wend die biobeheer organisme, soos Bacillus, direk aan, waar hierdie studie op die gebruik van lipopeptiede as ‘n beheermiddel gefokus het. Die voordeel is dat lipopeptidiede minder sensitief is vir hul omgewings, soos temperatuur en pH, i.v.m. organismes en die moontlikheid bied van ‘n gestandardiseerde produk. Hierdie studie het die produksie van spesifieke Bacillus lipopeptide, naamlik surfactin, fengycin en iturin, onder beheerde lottoestande ondersoek. Parameters wat lipopeptied produksie verhoog is gekwantifiseer, spesifiek antifungiese middels (iturin en fengycin) en lipopeptied produksie is geoptimeer. Eksperimente is uitgevoer in ‘n 1.3 L bioreaktor en lipopeptiedanaliese is met behulp van hoë druk vloeistof chromatografie en vloeistofchromatografie-massa spektroskopie uitgevoer. Van die vier moontlike Bacillus spp., was Bacillus amyloliquefaciens DSM 23117 die mees belowende antifungus-produserende kandidaat. Dit het beter resultate gelewer in terme van maksimale antifungiese produksie, Yp/x,antifungies (opbrengs per sel) asook antifungiese produktiwiteit. Nitraat, in hierdie geval NH4NO3, was noodsaaklik vir lipopeptied produksie en ‘n optimale konsentrasie is waargeneem waarbo die seldigtheid nie meer beduidend toegeneem het nie en beide die μmax (maksimale spesifieke groei tempo, h-1) en lipopeptied produksie afgeneem het. Die optimale NH4NO3 konsentrasie vir μmax was 10 g/L en vir lipopeptiedproduksie was 8 g/L. By 10 g/L NH4NO3 was μmax = 0.58 (h-1) en by 8 g/L was die maksimale antifungiese produksie (fengycin en iturin) 285.7 mAU*min en die maksimale surfactin produksie 302 mg/L onderskeidelik. Die lipopeptide, die antifungiese middels (fengycin en iturin) en surfactin, geproduseer deur B. amyloliquefaciens is sekondêre metaboliete, ongeag van die optimerings-behandelinge wat toegepas word, soos ‘n verhoging in NH4NO3 konsentrasie. Die gebruik van 30% verrykte suurstof het die nitraat verbruikingsperiode verleng, wat voorgestel het dat met die verryking, meer suurstof beskikbaar was om te dien as finale elektron ontvanger en sodoende die nitraat beskikbaar te stel vir lipopeptied produksie. Iturin en fengycin homoloë, oor die algemeen, het toegeneem soos wat die nitraat konsentrasie verhoog is. Hierdie resultate dui daarop dat prosestoestande, soos nitraat konsentrasie, gebruik kan word om die verhouding waarin lipopeptied homoloë geproduseer word te manipuleer. Hierdie resultate dui op die potensiaal vir die stroomop produksie van ‘n unieke bio-beheermiddel, wat die effektiwiteit van die bio-beheermetode moontlik sal verhoog. Die geproduseerde lipopeptiede het totale inhibisie getoon teen Botryotinia fuckeliana en ook fungiese aktiwiteit belemmer met Botrytis cinerea. Geen inhibisie is getoon teen Penicillium digitatum nie. Hierdie resultate toon die potensiaal van die bio-beheermetode, maar ‘n opskalerings-studie asook ‘n voerlot studie word aanbeveel, veral met die oog op moontlike kommersiële implementering van die strategie. Verdere studies met betrekking tot die aanwendingsmetode van die lipopeptiede moet ook verder ondersoek word, m.a.w. enkel teenoor menigte aanwendigs, aangesien dit die effektiwiteit van die lipopeptiede teen die teikenorganismes sal beïnvloed.

Thesis (M. Sc. (Agriculture, Horticulture)) --University of Limpopo, 2016 / ‘Reed’ avocado is a late season cultivar introduced to South Africa from California. The cultivar has shown good adaptation and produces quality fruit with export potential. Its pre-harvest adaptation and production aspects have been researched and documented. However, the effect of harvest time, post-harvest storage and ripening temperature has not been comprehensively studied on this newly introduced ‘Reed’ avocado cultivar. Therefore, the aim of this work was to investigate the effect of different harvest time, post-harvest storage and ripening temperature on the quality of late season ‘Reed’ avocado fruit. Matured ‘Reed’ avocado fruit were harvested based on moisture content indexing in December (2015) and January (2016). The experiment was carried out in a factorial, arranged in a completely randomised design (CRD) with three replicates. Treatment factors were: 2 x harvest time (mid-and late), 2 x post-harvest storage (2.0 and 5.5°C), 3 x ripening temperature (16, 21 and 25°C) and 5 x ripening day (0, 2, 4, 6 and 8). Fruit were stored at 2.0 and 5.5°C for 28 days, thereafter, ripened at 16, 21 and 25°C until fully ripe. During ripening, fruit were evaluated for weight loss, skin colour, firmness, respiration rate, physiological and pathological disorders. Mid-harvest fruit had higher moisture content when compared with late harvest fruit. However, harvest time, post-harvest storage, ripening temperature and ripening time (days) significantly influenced fruit weight loss, firmness, respiration rate, ripening percentage of ‘Reed’ avocado fruit during ripening. Moreover, fruit firmness decreased faster at higher temperatures (25 and 21°C) with fruit ripening within 4 and 6 days, respectively. In addition, ripening at a lower temperature (16°C) was slower with fruit fully ripened within 8 days after withdrawal from cold storage at both harvest times. ‘Reed’ avocado fruit respiration rate followed a climacteric pattern, however, significantly higher rate at higher temperature (25°C) when compared with lower temperature (16°C) after withdrawal from cold storage during both harvest times. Interestingly, mid-harvest fruit showed high electrolyte damage after withdrawal from 2.0°C when compared with late harvest fruit at the same temperature. Furthermore, mid-harvest fruit stored at 2.0ºC and ripened at 21°C showed higher chilling injury when compared with fruit ripened at 16 and 25°C. High electrolyte leakage positively correlated (R2 = 0.242) with high chilling damage for xiv mid-harvest fruit stored 5.5°C. Treatment factors had a significant effect (P < 0.05) on lightness (L *) and hue angle (h ) but no significant effect (P > 0.05) on chroma (C *) and eye colour of ‘Reed’ avocado fruit during ripening, irrespective of harvest time. Overall results showed a visual change in ‘Reed’ avocado skin colour, with eye colour changing from green to bright yellow. Furthermore, late harvest fruit showed high post-harvest pathological diseases after removal from high temperature (5.5°C) when compared with mid-harvest fruit stored at low storage temperature (2.0°C). Ripening at a higher temperature (21 and 25°C) resulted in higher incidence of stem-end rot and body rot when compared with lower temperature (16°C) for both harvest times. Late harvest fruit showed a higher incidence of vascular browning at higher ripening temperatures (21 and 25°C) when compared with lower temperature (16°C) after withdrawal from cold storage. Moreover, overall results showed that harvest time, post-harvest storage and the ripening temperature had a profound influence on the quality of ‘Reed’ avocado fruit. In conclusion, ‘Reed’ avocado fruit can be harvested during mid- or late season and stored at recommended low temperature (2.0ºC); and thereafter, ripened at either 16 or 21ºC. In addition, future studies should focus on identifying pre-harvest practices that promote higher post-harvest fruit quality for ‘Reed’ avocado fruit under South African production environment. Keywords: ‘Reed’ avocado fruit; firmness; electrolyte leakage; respiration rate; stem-end rot; body rot; vascular browning