Doctoral Degrees (Plant Pathology)

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Integrated use of yeast, hot water and potassium silicate treatments for the control of postharvest green mould of citrus and litchi.
(2010) Abraham, Abraha Okbasillasie.; Laing, Mark Delmege.; Bower, John Patrick.
There is a growing recognition globally that many agrochemicals are hazardous to humans, animals and the environment. Therefore, there is a need to substitute these chemical products with biological and physical treatments, and to change agronomic practices in order to control pests and diseases in agriculture. The primary objective of this thesis was to develop and test in laboratory, field and commercial packhouses trials as alternative control measures against green mould of citrus (caused by Penicillium digitatum Pers: Fr. Sacc) and Penicillium molds of litchi (caused by several Penicillium). A South African isolate of P. digitatum, isolated from an infected orange fruit, was found to be resistant to imazalil (the standard postharvest fungicide used in South Africa). Sixty yeast and 92 Bacillus strains were screened for their antagonistic activity against this isolate of P. digitatum. None of the yeasts or Bacillus isolates produced a curative action against P. digitatum on oranges. However, yeast Isolate B13 provided excellent preventative control of P. digitatum, superior to all the Bacillus isolates, when it was applied to citrus fruit prior to artificial inoculation with P. digitatum. Electron microscopy showed that yeast Isolate B13 inhibited conidial germination of P. digitatum. For the control of P. digitatum pre-harvest, trees were sprayed with a yeast, Isolate B13, a few months or a few days before harvest. However, this treatment alone proved to be ineffective in providing preventative control of green mould on Valencia oranges. For the control of P. digitatum preharvest, trees were treated with potassium silicate for a full season. Regular potassium silicate treatments resulted in a significant preventative control of P. digitatum infection on both navel and Valencia oranges. Treatment of Eureka lemons with potassium silicate as a postharvest treatment for the control of P. digitatum resulted in reduced disease lesion diameters when applied preventatively or curatively. Electron microscopy showed that potassium silicate inhibited germination of P. digitatum conidia and growth of its mycelium. Hot-water dip treatment at 50-58°C for 60-180 seconds (in increments of 15 seconds), significantly reduced infection development in inoculated wounds of Valencia oranges compared with control fruit treated with tap water, without causing any rind damage. The integration of the yeast, a hot water dip and potassium silicate pre-and postharvest applications provided control of P. digitatum control in multiple packhouse trials. The control achieved by the yeast Isolate B13 or hot-water, and potassium silicate in the packhouse alone was superior or equivalent to that provided by imazalil. A similar study was also carried out to determine possible control measures for Penicillium sp. on litchis. In this study, a total of 23 yeast and 13 Bacillus isolates were obtained from litchi fruit surfaces. Ten yeast and 10 Bacillus isolates that had shown good efficacy against P. digitatum of citrus were added to these for screening against Penicillium sp. of litchis. None of the yeasts or Bacillus isolates produced a curative action against Penicillium sp. infection on litchis. However, several yeast isolates (YL4, YL10, YLH and B13) resulted in reduced severity of the pathogen, when applied preventatively, compared with an untreated control. The yeast isolates were superior to all the Bacillus isolates, when applied to litchis prior to artificial inoculation by Penicillium infection on litchis. Potassium silicate as a postharvest treatment for the control of the pathogen caused reduced lesion diameters when applied preventatively or curatively to naturally infected litchis. The results presented in this thesis highlight the use of biological, physical and agronomic practices singly or in combination as an alternative control strategy against citrus postharvest green mould. This thesis also provides an insight into expanding these strategies, partly or fully, for the control of other postharvest Penicillium infections using litchi as an example.
Bioremediation of arsenic contaminated groundwater.
(2008) Teclu, Daniel Ghebreyohannes.; Wallis, Frederick Michael.; Tivchev, George V.; Laing, Mark Delmege.
Sulphate-reducing bacteria (SRB) mediate the reduction of metals/metalloids directly or indirectly. Bioremediation of arsenic contaminated water could be a cost-effective process provided a cheap carbon source is used. To this end, molasses was tested as a possible source of carbon for the growth of sulphate-reducing bacteria (SRB). Its chemical composition and the tolerance of SRB toward different arsenic species [As (III) and As (V)] were also investigated. Batch culture studies were carried out to assess 1, 2.5 and 5 g l-1 molasses as suitable concentrations for SRB growth. The results indicate that molasses does support SRB growth, the level of response being dependent on the concentration; however, growth on molasses was not as good as that obtained when lactate, the usual carbon source for SRB, was used. The molasses used in this study contained several metals including Al, As, Cu, Fe, Mn and Zn in concentrations ranging from 0.54-19.7 ìg g-1, but these levels were not toxic to the SRB. Arsenic tolerance, growth response and sulphate-reducing activity of the SRB were investigated using arsenite and arsenate solutions at final concentrations of 1, 5 and 20 mg l-1 for each species. The results revealed that very little SRB growth occurred at concentrations of 20 mg l-1 As (III) or As (V). At lower concentrations, the SRB grew better in As (V) than in As (III). Batch cultures of sulphate-reducing bacteria (SRB) in flasks containing pine bark, sand and polystyrene as support matrices and Postgate medium B were used to study formation of biofilms. The effects of the support matrices on the growth of the organisms were evaluated on the basis of pH and redox potential change and the levels of sulphide production and sulphate reduction. Characterisation of the matrix surfaces was done by means of environmental scanning electron microscopy (ESEM). A consortium of SRB growing on polystyrene caused a 49% of original sulphate reduction whereas on sand a 36% reduction occurred. Polystyrene was further examined for its durability as a long-term support material for the growing of SRB in the presence of As(III) and/or As(V) at concentrations of 1, 5 and 20 mg l-1. Both sulphate reduction and sulphide production were greater in this immobilised system than in the matrix-free control cultures. With pine bark as support matrix no significant sulphate reduction was observed. The kinetics of sulphate reduction by the immobilised cells were compared with those of planktonic SRB and found to be superior. The leaching of organic compounds, particularly phenolic substances, from the pine bark had a detrimental effect on the growth of the SRB. Different proportions of pine bark extract were used to prepare media to investigate this problem. Growth of SRB was totally inhibited when 100% pine bark extract was used. Analysis of these extracts showed the concentration of phenolics increased from 0.33 mg l-1 to 7.36 mg l-1 over the extraction interval of 15 min to 5 days. Digested samples of pine bark also showed the presence of heavy metals. The effects of nitrate, iron and sulphate and combinations thereof were investigated on the growth of a mixed culture of sulphate-reducing bacteria (SRB). The addition of 30 mg l-1 nitrate does not inhibit the production of sulphide by SRB when either 50 or 150 mg l-1 sulphate was present. The redox potential was decreased from 204 to -239 mV at the end of the 14 day batch experiment in the presence of 150 mg l-1 sulphate and 30 mg l-1 nitrate. The sulphate reduction activity of the SRB in the presence of 30 mg l-1 nitrate and 100 mg l-1 iron was about 42% of original sulphate, while if no iron was added, the reduction was only 34%. In the presence of 20 mg l-1 either As(III) or As(V), but particularly the former, growth of the SRB was inhibited when the cells were cultured in modified Postgate medium in the presence of 30 mg l-1 nitrate. The bioremoval of arsenic species [As(III) or As(V)] in the presence of mixed cultures of sulphate-reducing bacteria was investigated. During growth of a mixed SRB culture adapted to 0.1 mg l-1 arsenic species through repeated sub-culturing, 1 mg l-1 of either As(III) or As(V) was reduced to 0.3 and 0.13 mg l-1, respectively. Sorption experiments on the precipitate produced by batch cultured sulphate-reducing bacteria (SRB-PP) indicated a removal of about 77% and 55% of As(V) and As(III) respectively under the following conditions: pH 6.9; biomass (2 g l-1); 24 h contact time; initial arsenic concentration,1 mg l-1 of either species. These results were compared with synthetic iron sulphide as adsorbent. The adsorption data were fitted to Langmuir and Freundlich isotherms. Energy dispersive x-ray (EDX) analysis showed the SRB-PP contained elements such as sulphur, iron, calcium and phosphorus. Biosorption studies indicated that SRB cell pellets removed about 6.6% of the As(III) and 10.5% of the As(V) from water containing an initial concentration of 1 mg l-1 of either arsenic species after 24 h contact. Arsenic species were precipitated out of synthetic arsenic-contaminated groundwater by reacting it with the gaseous biogenic hydrogen sulphide generated during the growth of SRB. The percentage removal of arsenic species was dependent on the initial arsenic concentration present. Lastly, laboratory scale bioreactors were used to investigate the treatment of arsenic species contaminated synthetic groundwater. A mixed culture of SRB with molasses as a carbon source was immobilised on a polystyrene support matrix. The synthetic groundwater contained either As(III) or As(V) at concentrations of 20, 10, 5, 1 or 0.1 mg l-1 as well as 0.1 mg l-1 of a mixture with As(III) accounting for 20, 30, 40, 60 and 80% of the total. More that 90% and 60% of the As(V) and As(III) respectively were removed by the end of the 14-day experiment. At an initial concentration of 0.1 mg l-1 total arsenic had been reduced to below the WHO acceptable level of 10 ìg l-1 when the proportion of As(III) was 20 and 30%, while at 40% As(III) this level was reached only when the treatment time was increased to 21 days. The efficiency of As(III) removal was increased by first oxidising it to As(V) using MnO2.
Studies on the use of biocontrol agents and soluble silicon against powdery mildew of zucchini and zinnia.
(2008) Tesfagiorgis, Habtom Butsuamlak.; Laing, Mark Delmege.
Powdery mildew (PM) is an important foliar disease of many crops, occurring under both greenhouse and field conditions. The application of biological control and soluble silicon (Si) against PM has received increasing acceptance as a result of increased environmental and public concern over the use of fungicides for disease management, and because many key fungicides are no longer effective because of resistance problems. However, success with these control options depends on the development of effective antagonists and understanding how best to use Si in agriculture. Potential antagonists of PM were isolated from naturally infected leaves of different plants. A total of 2000 isolates were tested in a preliminary screening on detached leaves of zucchini. The best 30 isolates showing consistent results were further tested under greenhouse conditions for their efficacy against PM of zucchini. In a greenhouse trial, 23 isolates provided disease control to levels of 30 to 77%. Application of 29 isolates resulted in significant reductions in values of area under disease progress curve (AUDPC). The best five isolates were identified as Clonostachys rosea (Link) Schroers, Samuels, Seifert & Gams (syn. Gliocladium roseum) (Isolate EH), Trichothecium roseum (Pers.) Link (syn. Cephalothecium roseum) (Isolate H20) and Serratia marcescens (Bizio) (Isolates B15, Y15 and Y41). Three adjuvants (Break-ThruR (BK), PartnerR (PR) and Tween-80R (T-80)) were compared for their ability to improve efficacy of spray application of silicon (Si) and biocontrol agents (BCAs) against PM. Both BK and PR improved the efficacy of Si significantly (P < 0.05). Microscopic studies showed that BK affected PM fungi directly and enhanced the deposition of BCAs on the pathogen. Break-ThruR was only toxic to the pathogen mycelia when used at > 0.25 m. .-1, but phytotoxic to zucchini plants when used at > 0.45m. .-1. However, it did not affect the c.f.u. of bacterial BCAs. Use of BK at 0.2-0.4 m. .-1 can be recommended to assist spray application of Si (at 750 mg .-1) or BCAs for improved control of PM. The effect of concentration, frequency of application and runoff of Si sprays applied to the foliage was evaluated for control of PM of zucchini. Silicon (250-1000 mg .-1) + BK (0.25 m. .-1), was sprayed onto zucchini plants at frequencies of 1-3 wk-1. Spraying Si reduced the severity of PM significantly (P < 0.05). Regardless of the concentration of Si, the best results were obtained when the frequency of the treatment was increased, and when spray drift or spray runoff were allowed to reach the rhizosphere of the plants. When Si was applied onto leaves, direct contact between the spray and the pathogen resulted in mycelial death. Part of the spray (i.e., drift and runoff) was absorbed by plant roots, and subsequently played an important role in the health of the plants. If affordable, soluble Si should be included in nutrient solutions of hydroponics or supplied with overhead irrigation schemes when PM susceptible crops are grown. Under greenhouse conditions, application of BCAs, with or without Si, reduced the severity and development of PM significantly (P < 0.001). Application of Si significantly reduced the severity and AUDPC values of PM (P < 0.05 for both parameters). Silicon alone reduced the final disease level and AUDPC values of PM by 23-32%, and improved the efficacy of most BCAs. In the course of the investigation, antagonistic fungi consistently provided superior performances to bacterial isolates, providing disease control levels of up to 90%. Higher overall disease levels reduced the efficacy of Si against PM, but did not affect the efficacy of BCAs. Under field conditions, Si alone reduced disease by 32-70%, Isolate B15 reduced disease by 30-53% and Isolate B15 + Si reduced disease by 33-65%. Other BCAs applied alone or together with Si reduced the disease level by 9-68%. Most BCAs reduced AUDPC values of PM significantly. For most antagonists, better efficacy was obtained when Si was drenched into the rhizosphere of the plant. However, efficacy of some of the BCAs and Si were affected by environmental conditions in the field. Repeated trials and better understanding of how to use Si and the BCAs, in terms of their concentration and application frequency, and their interactions with the plant and the environment, are needed before they can be used for the commercial control of PM. Elemental analysis was conducted to determine the impact of differing application levels of silicon (Si) in a form of potassium silicate (KSi) in solution in terms of Si accumulation and selected elements in different tissues of zucchini and zinnia and growth of these plants, and to study the effect of PM on the levels of selected elements in these two plant species. Plants were grown in re-circulating nutrient solutions supplied with Si at different concentrations and elemental composition in different parts were analysed using EDX and ICP-OES. Increased levels of Si in the solution increased the levels of Si in leaves and roots of both plants without affecting its distribution to other plant parts. In zucchini, the roots accumulated the highest levels of Si, substantially more than in the shoots. In contrast with zinnia, accumulation of Si was highest in the leaves. Accumulation of potassium (K) in shoots of both plants increased with increased levels of KSi in the nutrient solution. However, K levels in flower of zinnia, fruits of zucchini and roots of both plants remained unaffected. Increased level of Si reduced accumulation of calcium (Ca) in both plants. Adding Si into the nutrient solution at 50 mg .-1 resulted in increased growth of zucchini and increased uptake of P, Ca, and Mg by both plant species. However, application of higher levels of Si did not result in any further biomass increase in zucchini. Levels of Si in the nutrient solution had no effects on elemental composition and characteristics of the fruits of zucchini. In both plant species, the presence of PM on the leaves of plants resulted in these leaves accumulating higher levels of Si and Ca, but less P, than leaves of uninfected plants exposed to the same levels of soluble Si. The highest concentrations of Si were observed in leaf areas infected with PM, and around the bases of trichomes. For optimum disease control and maximum accumulation of different elements in these two plants, hydroponic applications of Si at 50-150 mg .-1 is recommended. Five selected biocontrol agents and potassium silicate, used as source of soluble Si, were tested under hydroponic conditions at various concentrations against PM of zinnia (Glovinomyces cichoracearum (DC) Gelyuta, V.P.). Application of BCAs resulted in reductions in final disease level and AUDPC values of PM by 38-68% and 30-65%, respectively. Both severity and AUDPC values of PM were reduced by 87-95% when plants were supplied with Si (50-200 mg .-1). It is proposed that the provision of a continuous supply of Si and the ability of this plant species to accumulate high levels of Si in its leaves were the major reasons for the good response of zinnia to Si treatments against PM. Silicon played a protective role before infection and suppressed development of PM after infection. The combination of the best selected BCAs and Si can be used as an effective control option against PM of zinnia when grown in hydroponic system.
Biological control of the two-spotted spider mite, Tetranychus urticae Koch (Acari : tetranychidae).
(2009) Gatarayiha, Mutimura Celestin.; Laing, Mark Delmege.; Miller, Raymond Martin.
The two-spotted spider mite (TSM), Tetranychus urticae Koch, is an important pest of many greenhouse and field crops worldwide. The development of resistance in TSM populations to chemical acaricides, allied with public health concerns about pesticide residues, has motivated the search for alternative control measures to suppress the pest. Hyphomycetous fungi are promising agents for mite control and the fungus Beauveria bassiana (Bb) (Balsamo) Vuillemin was investigated in this study as a biocontrol agent. The principal objectives of this study comprised: a) screening Bb strains for their pathogenicity against T. urticae; b) testing the effect of adjuvants on the efficacy of Bb; c) studying the effect of plant type on persistence of Bb and the efficacy of control of Bb against T. urticae; d) evaluating the field efficacy of Bb applications against T. urticae; e) testing the compatibility of Bb with selected fungicides; and f) assessing the synergy between Bb and soluble silicon for T. urticae control. Screening bioassays of sixty-two strains of Bb identified the two most effective strains, PPRI 7315 (R289) and PPRI 7861 (R444), that caused mortality levels of more than 80% of adult mites at 9 d post-inoculation with 2 × 108 conidia ml-1. These strains performed significantly better than the Bb commercial strain PPRI 5339, in laboratory bioassays. The two strains also attacked mite eggs, causing 53.4% and 55.5% reduction in egg hatchability at 2 × 108 conidia ml-1 respectively. However, PPRI 7861 showed relatively higher production of conidia in culture and was, therefore, selected for further trials under greenhouse and field conditions. Greenhouse evaluations of the effects of two adjuvants (Break-thru® and a paraffin oil-based emulsion) on efficacy of Bb demonstrated a higher efficacy of the biocontrol agent (BCA) when it was applied with Break-thru® or the oil solution than with water alone. Moreover, Bb conidia applied in Break-thru® solution resulted in greater control of TSM than conidia applied in the mineral oil. There was also a dose-response effect and the control of TSM by Bb increased when the concentration of conidia was increased. The control of TSM by Bb in beans (Phaseolus vulgaris L), cucumber (Cucumis sativus L.), eggplant (Solanum melongena L.), maize (Zea mays L.) and tomato (Solanum lycopersicum L.) was tested in greenhouse trials. On these crops, the persistence of conidia declined over time. The rate of decline was significantly higher on maize. However, TSM mortality was positively correlated with the amount of conidia deposited on leaves immediately after spraying, rather than their persistence over time. Higher levels of mortality of TSM due to Bb application were observed on beans, cucumber and eggplants, suggesting that the type of crop must be taken into consideration when Bb is applied as a BCA. Field efficacy of Bb against mites was evaluated in two trials on eggplants. Based on assessment of population densities of mites and leaf damage assessments; both trials showed that the strain PPRI 7861 controlled TSM in the field. Two commonly used fungicides, azoxystrobin and flutriafol, were investigated in vitro tests on culture medium and laboratory bioassays on detached bean leaves (Phaseolus vulgaris L.) for their effects on Bb. Azoxystrobin (a strobilurin) was less harmful to Bb while flutriafol was found to be inhibitory. Another important finding of this study was the substantial enhancement of Bb efficacy by soluble silicon. When Bb was combined with soluble Si, the control of TSM was better than when either of the two products was applied alone. Moreover, application of soluble Si as a plant fertilizer in hydroponic water nutrient increased accumulation of peroxidase, polyphenoloxidase and phenylalanine ammonia-lyase enzymes in leaves of plants infested with TSM. Increased activity of these defense enzymes in leaves deters feeding behaviour of mites. We suggested that feeding stress renders them susceptible to Bb infection, which would explain the synergy observed between the two agents.
Management of fusarium wilt diseases using non-pathogenic Fusarium oxysporum, and silicon and Trichoderma harzianum (ECO-T®)
(2008) Kidane, Eyob Gebrezgiabher.; Laing, Mark Delmege.
In the genus Fusarium are many important plant pathogens. The diversity of hosts the genus attacks, the number of pathogenic taxa and the range of habitats in which they cause disease are the greatest in plant pathology. One important species complex within the genus Fusarium is Fusarium oxysporum Schlecht. This species complex consists more than 80 pathogenic forma specialis and is particularly difficult to control. The fungi can survive in soil for decades as specialized spores, known as chlamydospores. Interestingly, however, this species complex also contains beneficial non-pathogenic forms that can be exploited to manage Fusarium wilt diseases. In this study, the ability of non-pathogenic F. oxysporum strains, Trichoderma harzianum Rifai Eco-T®, soluble silicon, and their combination was evaluated on two important crops, banana (Musa sp. L.) and beans (Phaseolus vulgaris L.), for their potential to suppress pathogenic strains of F. oxysporum. The ability of these crops to take up and accumulate silicon in their organs, and its effect on low temperature stress was also investigated. Several endophytic fungi, mainly Fusarium spp. and bacteria, were isolated from healthy mature banana plants. After preliminary and secondary in vivo screening tests against F. oxysporum f.sp. phaseoli on beans (Phaseolus vulgaris L.) cv. Outeniqua, two non-pathogenic F. oxysporum strains were selected for further testing. These two non-pathogenic F. oxysporum strains were found to colonize banana (Musa sp.) cv. Cavendish Williams and bean plants, and to suppress Fusarium wilt of these crops. In order to improve the efficacy of these biocontrol fungi, soluble silicon was introduced. The relationship between plant mineral nutrition and plant diseases have been reported by several authors. Plants take up silicon equivalent to some macronutrients, although it is not widely recognized as an essential element. In this study, we established that roots, the target plant organ for soilborne plant pathogens, accumulated the greatest quantity of silicon of any plant organs when fertilized with high concentrations of silicon. On the other hand, the corm and stem accumulated the least silicon. Such observations contradict the concept of passive uptake of silicon via the transpiration stream in these plants as the only uptake mechanism. The prophylactic properties of silicon have been documented for many crops against a variety of diseases. In vitro bioassay tests of silicon against these wilt pathogens showed that silicon can be toxic to Fusarium wilt fungi at high concentrations (>7840 mg .-1), resulting in complete inhibition of hyphal growth, spore germination and sporulation. However, low concentrations of silicon (<490 mg .-1) encouraged hyphal growth. Silicon fertilization of banana and beans significantly reduced disease severity of these crops by reducing the impact of the Fusarium wilt pathogens on these crops. However, it could not prevent infection of plants from the wilt pathogens on its own. Synergistic responses were obtained from combined applications of silicon and non-pathogenic F. oxysporum strains against Fusarium wilt of banana. Combinations of silicon with the non-pathogenic F. oxysporum strains significantly suppressed disease severity of Fusarium wilt of banana, caused by F. oxysporum f.sp. cubense (E.F. Smith) Snyder & Hansen, better than applications of either control measure on their own. Banana production in the subtropical regions frequently suffer from chilling injury, and from extreme variations between night and day temperatures. Such stress predisposes banana plants to Fusarium wilt disease. Silicon, on the other hand, is emerging as important mineral in the physiology of many plants, ameliorating a variety of biotic and abiotic stress factors. We established that silicon fertilization of banana plants significantly reduced chilling injury of banana plants. Membrane permeability, lipid peroxidation (MDA level) and proline levels were higher in silicon-untreated plants than the treated ones, all of which demonstrated the stress alleviating effect of silicon. Low temperatures damage the cell membrane of susceptible plants and cause desiccation or dehydration of these cells. Levels of sucrose and raffinose, recognized as cryoprotectants, were significantly higher in silicon-amended banana plants than unamended plants.
Isolation of entomopathogenic gram positive spore forming bacteria effective against coleoptera.
(2009) Du Rand, Nicolette.
Fourteen spore-forming bacterial strains were isolated and screened for entomopathogenic activity. Five displayed toxicity towards the common mealworm, Tenebrio molitor L., (Coleoptera: Tenebrionidae). The majority of the isolates were obtained from insect larvae and insect rich environments. The three bacterial species identified were Bacillus thuringiensis Berliner, Brevibacillus laterosporus Laubach and Bacillus cereus Frankland and Frankland. Bioassays were conducted using T. molitor larvae. The one isolate of B. cereus required the highest concentration of bacterial cells to achieve its LC50, whereas one of the isolates of B. laterosporus required the lowest cell concentration to achieve its LC50. Dose response curves were generated for the five best isolates, which showed that the isolate of B. laterosporus (NDR2) was substantially more toxic than the other isolates.
Some effects of drying rate and wet storage on aspects of the physiology and biochemistry of embryonic axes from diesiccation- sensitive seeds.
(2004) Ntuli, Tobias M.; Berjak, Patricia.; Pammenter, Norman William.; Smith, Michael Trevor.
Desiccation-sensitive seeds show differential viability characteristics during drying at different rates. A number of studies have demonstrated that rapid dehydration permits survival to lower water contents than does slower desiccation. The aim and objective of the present study was to test the hypothesis which states that rapid drying of desiccation-sensitive seeds removes water sufficiently fast to reduce the accumulation of metabolic damage. In addition, the hypothesis that wet storage subjects desiccation-sensitive seeds to mild, but increasingly severe, water stress causing oxidative damage if additional water is not supplied, was tested. In the present study, axes of germinating orthodox seeds of Pisum sativum and newlyshed recalcitrant counterparts of Quercus robur, Strychnos madagascariensis, Trichilia emetica, Trichilia dregeana and Avicennia marina were subjected to rapid or slow drying or wet storage. For those species where more than one harvest was investigated, differences were observed in water contents at shedding. For all the species studied, the dehydration rate could be described by an exponential and a modified inverse function for both desiccation regimes, and the water content remained constant with wet storage. The level of tetrazolium staining and germination percentage of axes decreased sharply drying and hydrated storage such that the marked decline took place at lower water contents upon rapid than slow dehydration. The conductivity of electrolyte leachate increased progressively during desiccation and moist storage of axes of all species investigated. Greater membrane leakage occurred upon slow, than rapid dehydration in axes of all species studied. Activities of respiratory enzymes which have a potentially regulatory role in glycolysis, phosphofructokinase (PFK), or the tricarboxylic acid cycle, malate dehydrogenase (MDH), and levels of the oxidized form of the coenzyme, nicotinamide adenine dinucleotide (NAD), of the enzymes of the electron transport chain, NADH dehydrogenases ofNADH-ubiquinone (coenzyme Q) reductase (complex I) and NADHcytochrome c reductase (complex IV), were monitored in the present investigation. v In addition, the role of free radical activity in the form of lipid peroxidation, which has been implicated in loss of viability in seeds, was examined by assaying the levels of hydroperoxides. The involvement of the free radical processing enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR), and the antioxidant, ascorbic acid (AsA), was also ascertained. The activity of PFK in axes of P. sativum remained constant during drying and wet storage. However, PFK activity increased as rapid dehydration and hydrated storage of Q. robur axes proceeded. In contrast, the activity of PFK in axes of Q. robur decreased during slow desiccation. Similarly, PFK activity was reduced upon drying, and moist storage, of T. dregeana axes such that higher activity of PFK was seen during rapid than slow dehydration. The activity ofPFK inA. marina axes also declined upon desiccation. The activity ofMDH in axes of P. sativum was also unchanged during drying and wet storage. However, an increase in MDH activity was recorded in Q. robur axes during dehydration and hydrated storage such that the activity of MDH was higher upon slow than rapid desiccation. In contrast, MDH activity in axes of T. dregeana decreased as drying proceeded. Similarly, the activity of J\.1DH declined during dehydration and moist storage of A. marina axes. An increase in the level of NAD occurred in axes of P. sativum during drying. In contrast, a decrease in NAD levels was seen upon dehydration and wet storage of Q. robur axes such that the level of NAD was higher upon rapid than slow desiccation. There was an enhancement of the level of NAD in axes of T. dregeana during hydrated storage. Conversely, NAD levels declined during drying ofA. marina axes. A decrease in the level of hydroperoxides in axes of P. sativum was seen as rapid drying proceeded. In contrast, hydroperoxide levels increased during wet storage of P. sativum axes. Similarly, the levels of hydroperoxides were enhanced upon dehydration and hydrated storage of Q. robur axes such that they were higher in axes during slow desiccation compared to those dried rapidly. Conversely, the hydroperoxide level in axes of T. dregeana was reduced upon rapid dehydration. In contrast, an elevation of the level of hydroperoxides was observed during moist storage. The levels of hydroperoxides remained constant as desiccation and wet storage ofA. marina axes proceeded. vi The activity of SOD in axes of P. sativum decreased during rapid drying. In contrast, SOD activity increased upon slow dehydration and wet storage ofP. sativum axes. There was a decline in the activity of SOD in Q. robur axes during slow desiccation. Similarly, SOD activity was diminished upon drying of axes of T. dregeana. The activity ofSOD in T. dregeana axes was enhanced during hydrated storage. An elevation in SOD activity also took place during rapid dehydration and moist storage of axes ofA. marina. The activity of CAT did not change during drying of axes of P. sativum. However, a decrease in CAT activity in Q. robur axes was seen upon slow dehydration and wet storage. Similarly, the activity of CAT declined as desiccation of axes of T. dregeana proceeded. In contrast, CAT activity inA. marina axes increased during slow drying. Whereas the activity of GR in axes of P. sativum increased during drying and wet storage, GR activity decreased in A. marina axes upon all treatments such that the activity ofGR was higher during rapid than slow dehydration. GR activity also declined upon slow desiccation and hydrated storage ofaxes of Q. robur. Similarly, the activity of GR in T. dregeana axes was reduced during moist storage. Finally, a decrease in the level of AsA in axes of P. sativum took place during drying. Nonetheless, dehydration and wet storage of Q. robur axes were associated with no siginificant change in AsA levels. There was also a decline in the level of AsA in axes of T. dregeana as rapid desiccation proceeded. Similarly, a reduction in AsA level occurred upon slow drying ofaxes ofA. marina. The results presented here are consistent with the observation that drying and wet storage adversely affected the respiratory enzymes, PFK, MDH and NADH dehydrogenase. It is suggested that the resultant metabolic imbalance led to more leakage of electrons from the mitochondrial electron transport chain than normal, and through lipid peroxidation increased levels of hydroperoxides. In addition, dehydration and hydrated storage may depress the activities of free radical processing enzymes, SOD, CAT and GR and levels of antioxidant, AsA. This phenomenon was less pronounced during rapid, in comparison to slow, desiccation and moist storage. However, it appears that the above biochemical events are overtaken by physical damage at higher water contents in the highly recalcitrant seeds. It was concluded that the differential effects of VII the drying rate and wet storage on responses of desiccation-sensitive seeds varies with tissue, harvest, species and the degree of desiccation sensitivity.
Biological control of the common house fly (Musa domestica L.) using Bacillus thuringiensis (Ishiwata) berliner var. Israelensis and Beauveria bassiana (Bals.) vullemin in caged poultry facilities.
(2008) Mwamburi, Lizzy A.
The entomopathogenic fungus Beauveria bassiana and the bacterium Bacillus thuringiensis var. israelensis (Bti) have been widely studied for their role in biocontrol against many arthropods and extensively exploited for insect pest control. The purpose of this study was to evaluate the effect of four B. bassiana and two Bti formulations and their respective combinations, for the biological control of the common house fly, Musca domestica L., a major pest in poultry facilities. In vitro screening was undertaken to select the best B. bassiana isolates from 34 B. bassiana isolates and two Paecilomyces isolates. All the isolates of B. bassiana were found to be effective against adult house flies, but were marginally effective in controlling fly larvae. The Paecilomyces isolates were non-pathogenic towards both adult house flies and larvae. The best four isolates R444, 7320, 7569 and 7771 caused >90% mortality within 2d and were subjected to dose-mortality bioassays. Microscopic studies using light and scanning electron microscopy indicated the different durations of the lifecycle of B. bassiana development on the house fly. High temperature was found to delay conidial germination. Spore germination and mycelial growth were also inhibited by high adjuvant concentrations. Laboratory baseline bioassay data established, a dose-time response relationship using a waterdispersible granules (WDG) Bti formulation that demonstrated that the susceptibility of M. domestica larvae to a given concentration of Bti increased as the duration of exposure increased. In the laboratory studies, the LC50 and LC90 values of Bti for the larvae ranged between 65 - 77.4 and 185.1 - 225.9?g ml-1, respectively. LT50 and LT90 values were 5.5 and 10.3d respectively. In the field, a concentration of 10g Bti kg-1 (bran formulation) of feed resulted in 90% reduction of larvae for 4wk post-treatment. A higher concentration (2g L-1) of Bti in spray (WDG) applications was not significantly more effective than the lower concentration of 1g L-1. Thus, adding Bti to chicken feed has potential for the management and control of house flies in cagedpoultry facilities. The impact of oral feed applications of a bran formulation of Bti and a commercial chemical larvicide, Larvadex®, were compared with respect to their efficacy on the control of house fly 3 larval populations in poultry manure. The sublethal effects were manifested in terms of decreasing emergence of adult house flies. Although Larvadex® reduced larval density and caused significant reductions in emergence of adult house flies, it generally exhibited weaker lethal effects than Bti. The reduction levels achieved as a result of feeding 250mg Bti kg-1 at 5wk were similar to those achieved as a result of feeding twice the amount of Larvadex® at 4wk to the layers. From both an efficiency and economic perspective, comparisons to assess the impact of combining different concentrations of the two Bti formulations were carried out to evaluate their success in controlling house fly larvae and adults in poultry houses. The percentage mortality of larvae accomplished as a result of using a combination of 250mg kg-1 Bti in feed and 2g L-1 spray applications was equivalent to that obtained as a result of combining 500mg kg-1 Bti in feed and 1g L-1 spray application. The cost-benefit analysis (expressed in terms of mortality of larvae) indicated that the most effective combination for control of house fly larvae and fly emergence was the 500mg kg-1 in feed and 2g L-1 spray application combination that resulted in 67% larval mortality and 74% inhibition of adult house fly emergence. This study presents commercial users with possible combinations of applications of the two Bti formulations. Comparisons of larval mortalities and house fly emergence resulting from the Bti - B. bassiana treatments with those from Larvadex® - B. bassiana treatments, showed better control levels compared to any of the individual agents alone. The Bti treatments were more effective at controlling larval populations and inhibiting the emergence of house flies than Larvadex®, even when Larvadex® was applied together with B. bassiana. The effects of the Bti - B. bassiana and the Larvadex® - B. bassiana interactions were additive. These trials suggest that the efficacy of Bti in the control of house fly larvae may be improved with frequent applications of B. bassiana.
Aspects of post-harvest seed physiology and cryopreservation of the germplasm of three medicinal plants indigenous to Kenya and South Africa.
(2002) Kioko, Joseph Ivala.; Berjak, Patricia.; Pammenter, Norman W.
The current state of global biodiversity is one of sustained and increasing decline especially in developing countries such as South Africa, where, medicinal plants face a particular threat due the herbal medicine trade, and because in situ conservation measures have not stemmed the exploitation of these plants (Chapter 1). Furthermore, seed storage, which offers an efficient ex situ conservation technique, cannot presently be applied to many medicinal plants, either because these species produce short-lived, recalcitrant seeds, or the post-shedding behaviour of the seeds is altogether unknown. This study investigated three medicinal plant species indigenous to Kenya and South Africa: Trichilia dregeana and T. emetica, of which no population inventories exist and no wild populations were encountered locally during the course of this study; and Warburgia salutaris, one of the most highly-utilised medicinal plants in Africa, and which is currently endangered and virtually extinct in the wild in some countries such as South Africa. Aspects of post-shedding seed physiology (Chapter 2) and the responses of the germplasm of the three species to cryopreservation (Chapter 3) were studied using viability and ultrastructural assessment, with the aim of establishing methods for both short-term and the long-term preservation, via appropriate seed storage and cryopreservation, respectively. The effect of cryopreservation on genetic fidelity, a crucial aspect of germplasm conservation, was assessed by polymerase chain reaction (PCR) based random amplified polymorphic DNA (RAPDs), using W. salutaris as a case-study (Chapter 4). The seeds of all three species were found to exhibit non-orthodox behaviour. On relatively slow-drying, seeds of T. dregeana and T. emetica lost viability and ultrastructural integrity at axis water contents of 0.55 g g-l (achieved over 6 d) and 0.42 g g-l (after 3 d) respectively, while flash-drying of embryonic axes facilitated their tolerance of water contents as low as 0.16 g g-l (T. dregeana, flash-dried for 4 h) and 0.26 (T. emetica, flash-dried for 90 min). Seeds of W. salutaris were relatively more tolerant to desiccation, remaining viable at axis water contents below 0.1 g g-l when desiccated for 6 d in activated silica gel. However, excised embryonic axes flash-dried to similar water contents over 90 min lost viability and were ultrastructurally damaged beyond functionality. In terms of storability of the seeds, those of T. dregeana could be stored in the fully hydrated state for at least 5 months, provided that the quality was high and microbial contamination was curtailed at onset of storage, while those T. emetica remained in hydrated storage for about 60 d, before all seeds germinated in storage. Seeds of W salutaris, even though relatively tolerant to desiccation, were not practically storable at reduced water content, losing viability within 49 d when stored at an axis water content of 0.1 g g-l. The seeds of all three species were sensitive to chilling, suffering extensive subcellular derangement, accompanied by loss of viability, when stored at 6 °C. Thus, T. dregeana and T. emetica are typically recalcitrant, while those of W. salutaris are suggested to fit within the intermediate category of seed behaviour. For either recalcitrant or intermediate seeds, the only feasible method of long-term germpalsm preservation may be cryopreservation. Subsequent studies established that whole seeds of W. salutaris could be successfully cryopreserved following dehydration in activated silica gel. However, whole seeds of T. dregeana and T. emetica were unsuitable for cryopreservation, and excised embryonic axes were utilised. For these, in vitro germination methods, as well as cryoprotection, dehydration, freezing and thawing protocols were established. Post-thaw survival of the axes of both species was shown to depend on cryoprotection, rapid dehydration and cooling (freezing) in cryovials. Embryonic axes of T. dregeana regenerated only as callus after cryopreservation, while those of T. emetica generated into apparently normal plantlets. Thawing/rehydration in a 1:1 solution of 1 µM CaC12.2H2O and 1 mM MgC12.6H2O increased the percentage of axes surviving freezing, and that of T. emetica axes developing shoots. The effect of the extent of seed/axis development on onward growth after cryopreservation was apparent for seeds of W. salutaris and excised axes of T. emetica. The seeds of W. salutaris surviving after cryopreservation germinated into seedlings which appeared similar to those from non-cryopreserved seeds, both morphologically and in terms of growth rate. Analysis using PCR-RAPDs revealed that there were no differences in both nucleotide diversity or divergence, among populations of seedlings from seeds which had been sown fresh, or those which had either been dehydrated only, or dehydrated and cryopreserved. Thus, neither dehydration alone, nor dehydration followed by cryopreservation, was associated with any discernible genomic change. The above results are reported and discussed in detail in Chapters 2 to 4, and recommendations and future prospects outlined in Chapter 5.
Genetic effects and associations between grain yield potential, stress tolerance and yield stability in southern African maize (Zea mays L.) base germplasm.
(2005) Derera, John.; Tongoona, Pangirayi.; De Milliano, Walter A. J.; Laing, Mark Delmege.
Maize (Zea mays L.) is the principal crop of Southern Africa but production is threatened by gray leaf spot (Cercospora zea-maydis L.) and phaeosphaeria leaf spot (Phaeosphaeria maydis L.) diseases, drought and the use of unadapted cultivars, among other constraints. There are few studies of gray leaf spot (GLS) and Phaeosphaeria leaf spot (PLS) resistance, drought tolerance, yield stability and maize cultivar preferences in Southern Africa. The objective of this study was to: a) determine farmers’ preferences for cultivars; b) investigate the gene action and heritability for resistance to GLS and PLS, and drought tolerance; and c) evaluate yield stability and its relationship with high yield potential in Southern African maize germplasm. The study was conducted in South Africa and Zimbabwe during 2003 to 2004. A participatory rural appraisal (PRA) established that farmers preferred old hybrids of the 1970s because they had better tolerance to drought stress. Farmers also preferred their local landrace because of its flintier grain and better taste than the hybrids. The major prevailing constraints that influenced farmers’ preferences were lack of appropriate cultivars that fit into the ultra short seasons, drought and low soil fertility. Thus they preferred cultivars that combine high yield potential, early maturity, and drought tolerance in all areas. However, those in relatively wet areas preferred cultivars with tolerance to low soil fertility, and weevil resistance, among other traits. A genetic analysis of 72 hybrids from a North Carolina Design II mating revealed significant differences for GLS and PLS resistance, and drought tolerance. General combining ability (GCA) effects accounted for 86% of genetic variation for GLS and 90% for PLS resistance indicating that additive effects were more important than non-additive gene action in controlling these traits. Some crosses between susceptible and resistant inbreds had high resistance to GLS suggesting the importance of dominance gene action in controlling GLS resistance. Resistance to GLS and PLS was highly heritable (62 to 73%) indicating that resistance could be improved by selection. Also large GCA effects for yield (72%), number of ears per plant (77%), and anthesis-silking interval (ASI) (77%) under drought stress indicated that predominantly additive effects controlled hybrid performance under drought conditions. Although heritability for yield declined from 60% under optimum to 19% under drought conditions, heritability for ASI ranged from 32 to 49% under moisture stress. High heritability for ASI suggested that yield could be improved through selection for short ASI, which is positively correlated with high yield potential under drought stress. The stability analyses of the hybrids over 10 environments indicated that 86% had average stability; 8% had below average stability and were adapted to favourable environments; and 6% displayed above average stability and were specifically adapted to drought stress environments. Grain yield potential and yield stability were positively correlated. In sum, the study indicated that farmers’ preferences would be greatly influenced by the major prevailing constraints. It also identified adequate genetic variation for stress tolerance, yield potential and yield stability in Southern African maize base germplasm, without negative associations among them, suggesting that cultivars combining high yield potential, high stress tolerance and yield stability would be obtainable.
Biological control and plant growth promotion by selected trichoderma and Bacillus species.
(2005) Yobo, Kwasi Sackey.; Laing, Mark Delmege.; Hunter, Charles Haig.
Various Trichoderma and Bacillus spp. have been documented as being antagonistic to a wide range of soilborne plant pathogens, as well as being plant growth stimulants. Successes in biological control and plant growth promotion research has led to the development of various Trichoderma and Bacillus products, which are available commercially. This study was conducted to evaluate the effect of six Trichoderma spp. and three Bacillus spp. and their respective combinations, for the biological control of Rhizoctonia solani damping-off of cucumber and plant growth promotion of dry bean (Phaseolus vulgaris L.). In vivo biological control and growth promotion studies were carried out under greenhouse and shadehouse conditions with the use of seed treatment as the method of application. In vitro and in vivo screening was undertaken to select the best Trichoderma isolates from 20 Trichoderma isolated from composted soil. For in vitro screening, dual culture bioassays were undertaken and assessed for antagonisms/antibiosis using the Bell test ratings and a proposed Invasive Ability rating based on a scale of 1-4 for possible mycoparasitic/hyperparasitic activity. The isolates were further screened in vivo under greenhouse conditions for antagonistic activity against R. solani damping-off of cucumber (Cucumis sativus L.) cv. Ashley seedlings. The data generated from the in vivo greenhouse screening with cucumber plants were analysed and grouped according to performance of isolates using Ward‟s Cluster Analysis based on a four cluster solution to select the best isolates in vivo. Isolates exhibiting marked mycoparasitism of R. solani (during ultrastructural studies) viz, T. atroviride SY3A and T. harzianum SYN, were found to be the best biological control agents in vivo with 62.50 and 60.06% control of R. solani damping-off of cucumber respectively. The in vitro mode of action of the commercial Trichoderma product, Eco-T®, and Bacillus B69 and B81 suggested the production of antimicrobial substances active against R. solani. In vitro interaction studies on V8 tomato juice medium showed that the Trichoderma and Bacillus isolates did not antagonise each other, indicating the possibility of using the two organisms together for biological control and plant growth promotion studies. Greenhouse studies indicated that combined inoculation of T. atroviride SYN6 and Bacillus B69 gave the greatest plant growth promotion (43.0% over the uninoculated control) of bean seedlings in terms of seedling dry biomass. This was confirmed during in vivo rhizotron studies. However, results obtained from two successive bean yield trials in the greenhouse did not correlate with the seedling trials. Moreover, no increase in protein or fat content of bean seed for selected treatments was observed. In the biological control trials with cucumber seedlings, none of the Trichoderma and Bacillus combinations was better than single inoculations of Eco-T®, T. atroviride SY3A and T. harzianum SYN. Under nutrient limiting conditions, dry bean plants treated with single and dual inoculations of Trichoderma and Bacillus isolates exhibited a greater photosynthetic efficiency that the unfertilized control plants. Bacillus B77, under nutrient limiting conditions, caused 126.0% increase in dry biomass of bean seedlings after a 35-day period. Nitrogen concentrations significantly increased in leaves of plants treated with Trichoderma-Bacillus isolates. However, no significant differences in potassium and calcium concentrations were found. Integrated control (i.e. combining chemical and biological treatments) of R. solani damping-off of cucumber seedlings proved successful. In vitro bioassays with three Rizolex® concentrations, viz., 0.01g.l-1, 0.1g.l-1 and 0.25g.l-1 indicated that the selected Trichoderma isolates were partly sensitive to these concentrations whereas the Bacillus isolates were not at all affected. In a greenhouse trial, up to 86% control was achieved by integrating 0.1g.l-1 Rizolex® with T. harzianum SYN, which was comparable to the full strength Rizolex® (1g.l-1) application. Irrespective of either a single or dual inoculations of Trichoderma and/or Bacillus isolates used, improved percentage seedling survival as achieved with the integrated system, indicating a synergistic effect. The results presented in this thesis further reinforce the concept of biological control by Trichoderma and Bacillus spp. as an alternative disease control strategy. Furthermore, this thesis forms a basis for Trichoderma-Bacillus interaction studies and proposes that the two organisms could be used together to enhance biological control and plant growth promotion.
The effects of Trichoderma (Eco-T) on biotic and abiotic interactions in hydroponic systems.
(2003) Neumann, Brendon John.; Laing, Mark Delmege.; Caldwell, Patricia May.
The following body of research provides a detailed overview of the interactive effects of biocontrol agents and environmental factors and how these influence both the host plant and pathogen populations within hydroponic systems. Pythium and other zoosporic fungi are pathogens well suited to the aquatic environment of hydroponics. Motile zoospores facilitate rapid dispersal through fertigation water, resulting in Pythium becoming a yield reducing factor in most hydroponic systems and on most crops. With increasing trends away from pesticide use, biocontrol is becoming an ever more popular option. Unfortunately, much of our knowledge of biocontrol agents and their formulation can not be directly transferred to the widely differing environments of hydroponic systems. Paulitz (1997) was of the opinion that if biocontrol was to be successful anywhere, it would be in hydroponics. This is primarily due to the increased ability, in hydroponics, to control the growing environment and to differentiate between the requirements of the pathogen versus those of the host plant and biocontrol agent. Key environmental factors were identified as soil moisture, root zone temperature, form of nitrogen and pH. A review of the literature collated background information on the effects of biocontrol agents and environmental manipulation on plant growth and disease severity in hydroponic systems. A commercial formulation of Trichoderma (Eco-T(R1)) was used as the biocontrol agent in all trials. Dose responses in Pythium control and plant growth stimulation in lettuce were first determined using a horizontal trough system (closed system). In such systems optimum application rates were found to be lower than in field application (1.25x10[to the power of 5] spores/ml). This is probably because Trichoderma conidia are not lost from the system, but re-circulate until being transported into the root zone of a host plant. No significant growth stimulation was observed, although at high doses (5x10[to the power of 5] and 2.5x10[to the power of 5] spores/ml) a significant reduction in yield was recorded. Possible reasons for this growth inhibition are suggested and a new theory is proposed and investigated later in the thesis. In an open system of cucumber production (drip irrigated bag culture) no statistically significant results were initially obtained, however, general trends still showed the occurrence of positive biocontrol activity. The initial lack of significant results was mostly due to a poor knowledge of the horticulture of the crop and a lack of understanding of the epidemiology behind Trichoderma biocontrol activity. These pitfalls are highlighted and, in a repeat trial, were overcome. As a result it could be concluded that application rates in such systems are similar to those used in field applications. Management of soil moisture within artificial growing media can aid in the control of Pythium induced reductions in yield. A vertical hydroponic system was used to determine the interactive effects of soil moisture and Trichoderma. This system was used because it allowed for separate irrigation regimes at all 36 stations, controlled by a programmable logic controller (PLC). With lettuce plants receiving optimum irrigation levels, no significant reduction in yield was observed when inoculated with Pythium. However, after Pythium inoculation, stresses related to over- or under-watering caused significant yield losses. In both cases, Trichoderma overcame these negative effects and achieved significant levels of disease control, especially under higher soil moisture levels. Growth stimulation responses were also seen to increase with increasing soil moisture. Similar results were obtained from strawberry trials. These results show that Pythium control is best achieved through the integration of Trichoderma at optimum soil moisture. However, where soil moisture is above or below optimum, Trichoderma serves to minimize the negative effects of Pythium, providing a buffering capacity against the effects of poor soil moisture management. Pythium, root zone temperature and form of nitrogen interact significantly. In greenhouse trials using horizontal mini troughs with facilities for heating or cooling recirculating water, nitrate fertilizer treatments resulted in statistically significant results. Lettuce growth was highest at 12°C, although no significant differences in yield were observed between 12-24°C. Pythium was effective in causing disease over the same temperature range. Pythium inoculation did not result in yield reduction at 6 and 30°C. Trichoderma showed a slight competitive advantage under cooler temperatures (i.e., 12 degrees C), although significant biocontrol occurred over the 12-24 degrees C range. Ammonium fertilizer trials did not generate statistically significant data. This is possibly due to complex interactions between root temperature, ammonium uptake, and competitive exclusion of nitrification bacteria by Trichoderma. These interactions are difficult to replicate over time and are probably influenced by air temperature and available light which are difficult to keep constant over time in the system used. However, the data did lead to the first clues regarding the effects of Trichoderma on nitrogen cycling as plants grown with a high level of ammonium at high temperatures were seen to suffer more from ammonium toxicity when high levels of Trichoderma were added. In further trials, conducted in the recirculating horizontal mini trough system, it was determined that Trichoderma applications resulted in an increase in the percentage ammonium nitrogen in both the re-circulating solution and the growing medium. This was a dose-related response, with the percentage ammonium nitrogen increasing with increasing levels of Trichoderma application. At the same time an increase in ammonium in the root tissue was observed, corresponding with a decrease in leaf nitrate levels and an increase in levels of Cu, Na, Fe and P in leaf tissue. In independent pot trials, populations of nitrifying bacteria in the rhizosphere were also seen to decrease with increasing Trichoderma application rates. This led to the conclusion that the increase in ammonium concentration was as a result of decreased nitrification activity due to the competitive exclusion of nitrifying bacteria by Trichoderma. The possibility that Trichoderma functions as a mycorrhizal fungus and so increases the availability of ammonium for plant uptake is not discarded and it is thought that both mechanisms probably contribute. Water pH provides the most powerful tool for enhancing biocontrol of Pythium by Trichoderma. Trichoderma shows a preference for more acidic pHs while Pythium prefers pHs between 6.0 and 7.0. In vitro tests showed that Trichoderma achieved greater control of Pythium at pH 5.0, while achieving no control at pH 8.0. In greenhouse trials with the recirculating horizontal mini trough system, yield losses resulting from Pythium inoculation were greatest at pH 6.0 and 7.0, with no significant reduction in yield at pH 4.0. Biocontrol activity showed an inverse response with greatest biocontrol at pH 5.0.
A genetic study of resistance to African Rice Gall Midge in West African rice cultivars.
(2012) Yao, Nasser Kouadio.; Laing, Mark Delmege.; Ndjiondjop, Marie-Noelle.; Nwilene, Francis E.
The African Rice Gall Midge (AfRGM), Orseolia oryzivora Harris and Gagné (Diptera: Cecidomyiidae), is an endemic rice pest found throughout Africa. The failure of most other control methods imposes the need to use crop resistance. This study was initiated: (1) to develop an accurate method for assessing damage caused by AfRGM; (2) to determine AfRGM resistance genes’ modes of action, the heritability estimates of their resistance to AfRGM and the behavioural pattern of progenies with resistance to AfRGM attack; (3) to reveal convergent evolution of same or similar resistance gene(s) in geographically distinct landraces, or divergent evolution of genotypes carrying the same gene, by analysing the genetic diversity among five AfRGM parental lines; (4) to build a core sample of progenies to be used as a reduced mapping population, largely reflecting the entire genome of the whole population, after an estimate of the heritability of 15 agro-morphological descriptors and; (5) determine Simple Sequence Repeat (SSR) markers flanking genes or quantitative trait loci (QTLs) linked to resistance to AfRGM. A method of accurately assessing damage caused by AfRGM was determined by comparing four methods of assessment including the International Rice Research Institute’s (IRRI) Standard Evaluation System (SES) for rice and three methods based on resistance index (RI) assessments differing in the computing of the percentage of tillers with galls on a resistant check variety. The RI-based assessment (RI-BA) methods consistently provided a better evaluation of AfRGM damage than the SES, regardless of the trial size. Within RI-BA methods, RI-BA2 was always more accurate than RI-BA1 and RI-BA3 when the plot was large. RI-BA2 and RI-BA3 were equally accurate when the plot size was small, and they provided better estimates than RI-BA1. When the plot was of medium size, RI-BA2 was more accurate than RI-BA3; RI-BA3 also surpassed RI-BA1. Overall, the best method of assessing AfRGM damage was RI-BA2, regardless of the plot size. Five rice populations including F1, F2 and F3 generations involving ITA306, a susceptible variety of Oryza sativa subsp. indica, and four varieties having different reactions against AfRGM were used to determine the genetic basis of resistance and estimate the heritability of resistance to AfRGM. All the F1s were susceptible, suggesting recessive gene inheritance. The F2 generations’ segregation pattern of 1R:15S in both ITA306-TOS14519 and ITA306-TOG7106 crosses as well as the segregation of 1R:8Seg:7S in ITA306-TOS7106 F3 families indicated that the AfRGM resistance expression being studied is governed by two genes. The deviation of the segregation patterns of crosses involving ITA306 and the tolerant parental lines from Mendelian segregation ratios suggests that the tolerance to AfRGM shown by BW348-1 and Cisadane is under complex mechanisms of control rather than under simple genetic control. The narrow-sense heritability estimates of resistance to AfRGM were low in populations involving tolerant varieties and were high in populations involving resistant varieties. They ranged from 0.086 in the ITA306-Cisadane population, to 0.4 in the ITA306-TOG7106 population. Conversely, the broad-sense heritability estimates ranged from 0.23 (ITA306-Cisadane) to 0.63 (ITA306-TOS14519). The behavioural patterns of progenies against AfRGM attack were evaluated for 532, 413 and 479 F2 progenies from ITA306-BW348-1, ITA306-Cisadane and ITA306-TOS14519 crosses, respectively, in addition to 90 BC1F2 progenies from the ITA306 and TOG7106 cross. One F3 generation of 649 families from a cross between ITA306 and TOS14519 was also tested. Four types of behavioural pattern categories were observed: (1) progenies were more resistant than the resistant check entry at 45 DAT and 70 DAT; (2) progenies were more resistant at 45 DAT and became susceptible at 70 DAT; (3) progenies were susceptible at both 45 DAT and 70 DAT; (4) progenies were susceptible at 45 DAT but reverted to resistant at 70 DAT. The first three categories were the most frequently observed and occurred in all cross combinations. The last category was observed only for a few progenies from the ITA306-TOS14519 F2 and F3 generations and, surprisingly, many from the ITA306 and BW348-1 cross. Heritability estimates were calculated for 15 major traits in an F3 population in order to predict the genetic gain associated with each trait, together with the resistance to AfRGM and to estimate the influence of the environment on phenotypic values. Broad-sense heritability (H2) estimates were high for the penultimate leaf length (PLL) - 0.99, penultimate leaf width (PLW) – 1.0, flag leaf length (FLL) - 0.99, flag leaf width (FLW) – 1.0, ligule length (LigL) - 0.99, tillering ability (Til) - 0.99, number of days to booting (DB) - 0.95, number of days to first heading (DFH) - 0.96, number of days to heading (DH) - 0.89, number of days to maturity (DM) - 0.98, culm length (CL) - 0.99, plant height (PH) - 0.99, panicle length (PanL) - 0.95, secondary branching (SB) - 0.95 and the thousand grains weight (TGW) - 0.71. Conversely, narrow-sense heritability estimates were very low (nearly 0) in PLL, FLL, Lig, DB, DFH, DM and SB or low (at most 0.267) in PLW, FLW, DH and PH, with a high value of 0.727 for TGW. Inheritance of the traits studied was therefore under non-additive gene effects rather than additive genetic effects and can therefore be improved using pedigree breeding schemes along with breeding for AfRGM resistance. Fine genetic evaluation of five AfRGM parental lines was studied in terms of polymorphisms using 303 SSR primers covering the rice genome. Of the 178 polymorphic primers identified, 60 were highly polymorphic and informative. The number of alleles amplified by these primers ranged from one to five for a total of 1,041 alleles. The polymorphism rate was globally high, ranging from 45.2% to 66.8%. The mean of the polymorphism information content (PIC) was 0.553. Factorial analysis, based on the allelic diversity, demarcated the parental lines into Oryza glaberrima Steud, Oryza sativa subsp. japonica and O. sativa subsp. indica groups, while a cluster analysis distinguished them into four groups: AfRGM resistant, susceptible, moderately resistant and tolerant. BW348-1 and Cisadane showed the least diversity, despite their distant geographical origins. TOS14519 and TOG7106 showed more divergence to ITA306 despite their common West African origin. This variability amongst the genotypes tested is the result of farmer-based selection for AfRGM resistance rather than direct breeding efforts through breeder intervention. A method of selecting individuals for a mapping population, based on a core sample, was developed in order to speed up the mapping procedure. A diversity study amongst F2 and F3 generations involving 15 quantitative and 26 qualitative agro-morphological characters was carried out and led to the dropping of seven non-discriminant descriptors. The diversity index (H) was calculated for each remaining character and the discriminant descriptors were selected based on a diversity index threshold value above 0.4. Four descriptors of H values less than 0.35 were therefore dropped. The sizing of the core collection of 64 individuals and the selection of these individuals were done using MSTRAT version 4.1 package in redundancy mode, a construction run of 100 times with an iteration number of 500. The core sample was similar to the whole population for clustering pattern, minimum and maximum quantitative values and diversity index, while mean values and coefficient of variation distinguished them. The core sample, which represents 10% of the whole population, also revealed the same phenotypic variation and the same genotypic segregation according to two SSR markers. It can therefore efficiently reflect the whole population as a mapping population. Finally, a study was undertaken to identify flanking markers to the gene/QTL involved in the resistance against AfRGM using bulked segregant analysis (BSA). A polymorphism study between ITA306 and TOS14519 displayed 145 polymorphic SSR markers, which were used to screen the bulks that originated from the two tails, and depicted only two SSRs as candidate markers linked to gall midge resistance. These markers included RM317 and RM17303 which displayed strong significance after an analysis of variance using an F test, meaning that they were segregating with the resistant alleles.
A study of the diversity of Burkina Faso rice landraces and identification of source of resistance to rice yellow mottle virus (RYMV)
(2011) Kam, Honore.; Laing, Mark Delmege.; Ndjiondjop, Marie-Noelle.
The main goals of this study were to ascertain farmers' preferred traits in rice landraces and their perception of Rice yellow mottle virus, to collect rice landraces across Burkina Faso, investigate their genetic diversity, and to exploit this diversity in a search for varieties resistant and tolerant to RYMV, for their utilisation in rice breeding. Farmers' preferred traits, approaches to crop management, and disease perceptions were assessed using a Participatory Research Appraisal (PRA) approach. In the main rice growing regions of Burkina Faso, 330 rice landraces were collected. The agro-morphological diversity of the germplasms was evaluated in the field with 20 quantitative and 30 qualitative agro-morphological parameters. Thereafter, 22 Simple Sequence Repeat molecular markers were used to assess the genetic diversity and the population structure of the collection. Finally, the rice landraces were screened against four RYMV isolates to assess the susceptibility, tolerance and resistance of the landraces in the collection using visual assessment and Enzyme Linked Immunosorbent Assay. The PRA identified sweet taste, grain expansion when cooking, easy cooking and yield as paramount selection criteria in rural rice farming communities in Burkina Faso. Drought and disease resistance are characters that farmers wish to have in their varieties. The PRA also highlighted that farmers are conscious of RYMV disease in their fields. However, they are unaware about the epidemiology of the disease. An agro-morphological study of the phenotypic diversity of the collection confirmed the presence of the two cultivated rice species: O. glaberrima and O. sativa. There were more O. sativa accessions than O. glaberrima landraces. There were 48 O. glaberrima and 282 O. sativa accessions in the collection. Both species were divided into four clusters, reflecting the richness of the collection. The underlying genetic diversity of the collection was confirmed by the use of 22 Simple Sequence Repeat molecular markers. The neutral markers confirmed the existence of two substructures, namely O. glaberrima and O. sativa, and the presence of admixture varieties. However, a core collection of 52 individuals was developed. This included 13 O. glaberrima and 39 O. sativa accessions. It reflects the genetic diversity of the sub-clusters present in each species. This core collection contains 89% of the allelic richness of the collection. Its small size will facilitate the maintenance and active use of diversity of germplasm in the core collection. The entire collection was utilised to search for varieties resistant and tolerant to RYMV disease. The screening of the collection with different RYMV isolates exposed the susceptibility of most of the accessions in the collection. Most of the O. sativa indica accessions were highly susceptible. However, ten O. glaberrima accessions displayed a delay of symptom expression, and moderate resistance. However, their resistance was overcome later by a particularly virulent RYMV isolate BF1. Remarkably, a single moderately resistant cultivar, BM24, showed that partial resistance and tolerance to RYMV can be found in an O. sativa variety. Serological evaluation of this local variety in comparison with the partially resistant variety, Azucena, showed that BM24 and Azucena expressed similar resistance patterns. A genetic profile of both varieties showed that both had an identical allele status at RM101, which is a marker bracketed in the same zone as the QTL12.
Genetic diversity of Oryza species in Niger ; screening and breeding for resistance to rice yellow mottle virus (RYMV)
(2012) Sow, Mounirou El-Hassimi.; Laing, Mark Delmege.; Ndjiondjop, Marie-Noelle.
Rice is a staple food in many West African countries, including Niger. However, both regional and national rice production have failed to meet demand due to several constraints, among which is the Rice yellow mottle virus (RYMV). Moreover, attempted intensification of rice cultivation and the introduction of modern cultivars are encouraging farmers towards abandoning local landraces for high yielding, but often susceptible varieties. The study was primarily oriented towards rice pre-breeding, and identifying priorities for rice breeding in Niger in relation to farmers' preferences and their environment. A secondary aim was the development and evaluation (for release at the regional level) of new breeding lines with resistance to RYMV. This study aimed to: 1) Establish farmers' perception of rice varieties as well as the main constraints on rice production in Niger and particularly those posed by RYMV; 2) Create a collection of rice species from Niger for ex- situ conservation, and to determine the phenotypic variability within this collection; 3) Determine the genetic diversity and population structure of the collection; 4) Screen the collection for resistance to RYMV, so that new sources of resistance could be detected; 5) Improve five elite varieties from West Africa for resistance to RYMV using marker-assisted selection (MAS). The germplasm collection and PRA of this study were conducted in 2008 and 2009 in Niger, while the field and the laboratory researches were conducted in 2008 and 2009 at the Africa Rice Center (AfricaRice) in Benin. For the PRA, data was obtained from a semi-structured group discussion carried out in 14 villages, individual questioning of 153 farmers and visits to farmers' field and storage facilities. The local farmers' union was the only formal seed dissemination system. Seed exchanges between farmers and the use of seeds from previous harvests were important. The RYMV and the bacterial leaf blight (BLB) were cited as the prevalent biotic stresses in the irrigated agrosystem, where the varieties IR1529-680-3 and Waihidjo were found to be the most popular. Flood, birds and hippopotamus were the most damaging agents in the lowland cropping system, and the landrace Degaulle/ D5237 was the preferred variety. Apart from the yield, farmers preferred varieties with good grain quality (milling quality and good taste), high market value, stress tolerance (drought, flood, disease, birds, rodents), and those recommended by the local farmers' association. These findings should be included in breeding goals, seed production and dissemination systems. During collection, a total of 270 rice accessions were assembled, comprising the two cultivated rice species Oryza sativa L. and O. glaberrima Steud. and its two wild relatives Oryza barthii A. Chev. and O. longistaminata Chev. et Roehr. The region of the Niger River and its tributary (the Dallol Maouri) provided the majority (80.7%) of the accessions. Apart from a few wild O. barthii accessions, the accessions found around Lake Chad and the Komadougou river (South-East) were also collected in the Niger River area. Farmers' naming and ecological classification of rice varieties was generally consistent. Three major phenotypic groups were found during the field trials, and the overall phenotypic variability of the collection (as measured by the Shannon-Weaver Diversity Index) was relatively high. There was no significant difference in diversity between the main eco-geographical zones of collection, as well as between the identified phenotypic groups, suggesting a high level of germplasm exchange between the regions in Niger. From the collection, 264 accessions were genotyped from the collection using 18 well distributed SSR markers and two main genetic compartments were detected, comprising O. sativa subsp. indica varieties and O. glaberrima and its wild relative O. barthii and O. longistaminata. The O. sativa group in Niger was divided into irrigated and floating rice, bound by lowland rice. The wild progenitor O. barthii was widespread but without any clear genetic differentiation from O. glaberrima, probably due to the presence of admixtures within the collected samples of O. barthii. Allelic diversity was relatively high, despite the geographical distance from the centre of domestication of African rice, and the points of entry of Asian rice to Africa. The findings reflect the underuse of Niger's rice landraces genetic potential for rice breeding, given that all the "improved" varieties released during the last 25 years in Niger were clustered together on the dendrogram. The response of a set of the rice collected from Niger and some accessions from Mali to inoculation by RYMV was evaluated using five different virus isolates from Niger (3), Benin (1) and Burkina Faso (1). All rice varieties were susceptible to the disease. However, depending on the virus strain, a few O. glaberrima accessions displayed partial resistance, similar to the highly resistant TOG5681. Allelic research based on primers derived from the RYMV1 gene revealed one accession with allele rymv1-3, and two accessions with allele rymv1-4, and one accession with a different resistance gene. The implications of the finding were discussed and a strategy proposed for breeding varieties with a comprehensive resistance to RYMV. After three generations of backcrossing, the major resistance gene of the variety Gigante was successfully introgressed into five elite rice varieties of West Africa by Marker-Assisted Backcross (MABC). The newly developed BC3F3 progenies were screened for resistance to RYMV in farmers' fields in Guinea and Mali and also under controlled conditions in a screenhouse in Benin. As shown by low virus content and level of disease incidence, low tiller number and plant height reduction, the transferred gene was fully functional in the new genetic background. Moreover, some lines also displayed a high level of resistance to rice blast (Pyricularia oryzae) and stem borer infestation in Guinea. Four of those lines are in the second year of multi-location trial in seven West African countries. Therefore, effective deployment of the newly developed varieties, coupled with good cultural practices, should reduce the damaging effects of RYMV in lowland and irrigated rice cropping systems and thereby increase the income of small scale farmers from rice cultivation.
The development of transgenic sweet potato (Ipomoea batatas L.) with broad virus resistance in South Africa.
(2013) Sivparsad, Benice.; Gubba, Augustine.
Sweet potato (Ipomoea batatas Lam.) is ranked as the seventh most important food crop in the world and its large biomass and nutrient production give it a unique role in famine relief. However, multiple virus infection is the main disease limiting factor in sweet potato production worldwide. The main objective of this research project was to develop a transgenic sweet potato cultivar with broad virus resistance in South Africa (SA). A review of current literature assembled background information pertaining to the origin, distribution and importance of the sweet potato crop; viruses and complexes infecting sweet potato; and the strategies used in sweet potato virus detection and control. A survey to determine the occurrence and distribution of viruses infecting sweet potato (Ipomoea batatas Lam.) was conducted in major sweet potato-growing areas in KwaZulu-Natal (KZN). A total of 84 symptomatic vine samples were collected and graft inoculated onto universal indicator plants, Ipomoea setosa Ker. and Ipomoea nil Lam. Six weeks post inoculation, typical sweet potato virus-like symptoms of chlorotic flecking, severe leaf deformation, stunting, chlorotic mosaic, and distinct interveinal chlorotic patterns were observed on indicator plants. Under the transmission electron microscope (TEM), negatively stained preparations of crude leaf sap and ultra-thin sections from symptomatic grafted I.setosa plants revealed the presence of elongated flexuous particles and pinwheel type inclusions bodies‟ that are characteristic to the cytopathology of Potyviruses. Symptomatic leaf samples from graft-inoculated I. setosa and I. nil were assayed for Sweet potato feathery mottle virus (SPFMV), Sweet potato mild mottle virus (SPMMV), Sweet potato chlorotic stunt virus (SPCSV), Sweet potato chlorotic fleck virus (SPCFV), Sweet potato virus G (SPVG), Sweet potato mild speckling virus (SPMSV), Sweet potato caulimo-like virus (SPCaLV), Sweet potato latent virus (SPLV), Cucumber mosaic virus (CMV), and Sweet potato C-6 virus (C-6) using the nitrocellulose membrane enzyme-linked immunosorbent assay (NCM-ELISA). The majority of leaf samples (52%) tested positive for virus disease and showed the occurrence of SPFMV, SPMMV, SPCSV, SPCFV, SPVG, SPMSV, and SPCaLV. Of these 7 viruses, the most frequently detected were SPFMV (39%), SPVG (30%), followed by SPCSV (13%) and SPMMV (12%). SPCaLV and SPCFV at 10% and SPMSV at 7% were found exclusively in samples collected from one area. SPFMV, SPVG, SPCSV, and SPMMV were identified as the most prevalent viruses infecting sweet potato in KZN. The genetic variability of the three major viruses infecting sweet potato (Ipomoea batatas Lam.) in KZN was determined in this study. A total of 16 virus isolates originating from three different locations (Umbumbulu, Umfume and Umphambanyomi River) in KZN were analyzed. These comprised of 10 isolates of Sweet potato feathery mottle virus (SPFMV), five isolates of Sweet potato virus G (SPVG) and one isolate of Sweet potato chlorotic stunt virus (SPCSV). The phylogenetic relationships of the SPFMV, SPVG and SPCSV isolates from KZN relative to isolates occurring in SA and different parts of the world were assessed. The division of SPFMV into four genetic groups (strains) according to the phylogenetic analysis of coat protein encoding sequences revealed mixed infections of the O (ordinary) and C (common) strains in sweet potato crops from KZN. All SPFMV isolates showed close lineage with isolates from South America, East Asia and Africa. The SPVG isolates showed high relatedness to each other and close lineage with other isolates, especially those from China and Egypt. Analysis of the partial sequence of the Heat shock protein 70 homologue (Hsp70h) gene indicated that the SPCSV isolate from KZN belongs to the West African (WA) strain group of SPCSV and showed close relatedness to an isolate from Argentina. The knowledge of specific viral diversity is essential in developing effective control measures against sweet potato viruses in KZN. Multiple virus infections of Sweet potato feathery mottle virus (SPFMV), Sweet potato chlorotic stunt virus (SPCSV), Sweet potato virus G (SPVG) and Sweet potato mild mottle virus (SPMMV) cause a devastating synergistic disease complex of sweet potato (Ipomoea batatas Lam.) in KZN. In order to address the problem of the multiplicity and synergism of sweet potato viruses in KZN, this study aimed to develop transgenic sweet potato cv. Blesbok with broad virus resistance. An efficient and reproducible plant regeneration protocol for sweet potato (Ipomoea batatas Lam.) cultivar Blesbok was also developed in this study. The effect of different hormone combinations and type of explants on shoot regeneration was evaluated in order to optimize the regeneration protocol. Coat protein (CP) gene segments of SPFMV, SPCSV, SPVG and SPMMV were fused to a silencer DNA, the middle half of the nucleocapsid (N) gene of Tomato spotted wilt virus (TSWV) and used as a chimeric transgene in a sense orientation to induce gene silencing in the transgenic sweet potato. Transformation of apical tips of sweet potato cv. Blesbok was achieved by using Agrobacterium tumefaciens strain LBA4404 harboring a modified binary vector pGA482G carrying the plant expressible neomycin phosphotransferase ll gene (nptll), the bacterial gentamycin-(3)-N-acetyl-transferase gene and the expression cassette. A total of 24 putative transgenic plants were produced from the transformed apical tips via de novo organogenesis and regeneration into plants under 50mg/L kanamycin and 200 mg/L carbenicillin selection. Polymerase chain reaction (PCR) and Southern blot analyses showed that six of the 24 putative transgenic plants were transgenic with two insertion loci and that all plants were derived from the same transgenic event. The six transgenic sweet potato plants were challenged by graft inoculation with SPFMV, SPCSV, SPVG and SPMMV- infected Ipomoea setosa Ker. Although virus presence was detected using NCM-ELISA, all transgenic plants displayed delayed and milder symptoms, of chlorosis and mottle of lower leaves when compared to the untransformed control plants. These results warrant further investigation under field conditions.
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.
An epidemiological analysis of the Phytophthora and Alternaria blight pathosystem in the Natal Midlands.
(1980) Putter, Christoffel Antonie Johannes.; Martin, Michael Menne.; Rijkenberg, Fredericus Hermanus Johannes.
The history of the development in Natal of a forecasting service to warn of outbreaks of late blight disease caused by Phytophthora infestans is presented. The late blight pathogen and Alternaria solani, the causal organism of early blight disease, interact on potatoes and tomatoes to form a blight disease complex. Evidence is presented to show that it is expedient to manage this blight complex as a whole rather than to direct control at only one of the components in ignorance of the consequential enhancement of the potential of the other. In a search for an improved blight complex management strategy, factors concerning the possible existence of an annual migration of Phytophthora infestans inoculum, first postulated in the 1960's, along an east-west route across Natal, are collected and collated. Corroboration of the existence of the Phytophthora-pathway is given, inasmuch as it represents a serial outbreak of late blight along a temporal gradient. The possibility that the pathway is a manifestation of disease resulting from the erruption of pre-existing inoculum along an environmental gradient, can not specifically be excluded. However, the peculiar pattern of anabatic and katabatic winds along a river-valley network, superimposed on a continuous cropping pattern and its concomitant opportunity for blight to be endemic in the province, supports the postulated Phytophthora-inoculum pathway A fungicide spray trial was conducted in order to investigate the possibility of us i ng the pathway phenomenon as the framework for an improved blight control strategy and to explore the nature and level of the competitive interaction between Phytophthora infestans and Alternaria solani. This trial revealed that the interaction between the components of the blight complex was differentially altered by weather patterns and fungicide combinations. Treatments in which metalaxyl (Ridomil) alone was used for the control of late blight, gave a yield similar to those with propineb (Antracol), which inhibits A. solani primarily but also hus some negative effect on P. infestans. The yields from both these treatments were siguificant ly (p < 0,05) better than the yields recorded in the unsprayed control plots. A treatment in which Ridomil and Antracol were combined such that each was applied according to its recommended concentration, gave yield increases of 32,3% over the unsprayed control, although the yield from the Ridomil/Antracol treatment was not significantly greater (p < 0,05) than the yields recorded where either Ridomil or Antracol were used. A computer simulator, named GAUSE, was developed to simUlate the consequences of the competition between various combinations of P. infestans and A. solani. Results simulated by GAUSE corroborated those obtained from the field trial and support the conclusion that diseases of complex etiology require more than simplistic, univariate analysis of single cause-and-effect pathways. The competition quotient CQ is developed as a new parameter of competitive interactions. It is calculated as the ratio of the amount of disease in the absence of competition, to the amount of disease when the causal pathogen is competing with another pathogen in the same niche. The CQ may be calculated from various standard epidemiology statistics and it is used to demonstrate that the competitive displacement phenomenon places constraints on the interpretation and application of Vanderplank's basic epidemiology equations. A new pathosystems management concept namely the pathotope (pathos = suffering; topos = place0 concept, is introduced, having developed from the notion that epidemics have spatial as well as temporal attributes. Accordingly, an area in which individual farms are at the same level of probability at risk to disease, delimits the pathotope. The concept can be described at many integration lsvels and is presented as an important quantitative unit of comparative epidemiology. The pathotope concept accomodates such notions as are contained in the postulated Phytopnthora-pathway and is especially suited to integration with disease forecasting methods. An example of the application of the pathotope approach is presented and a strategy is proposed by which fungicide spraying is initiated and applied synchronously as determined by the degree of communal risk to attack and epidemic increase of disease. Within a pathotope, several common factors collectively determine the vulnerability of the group to disease. If a coherent, uniform strategy is to be developed and implemented by pathotope members, it is necessary that all members have access to the relevant information and that it be collected and disseminated conveniently and rapidly. A computer-based disease monitoring and mapping system which achieves these objectives is presented.
A study on avocado sunblotch disease.
(1980) Da Graca, John Vincent.; Martin, Michael Menne.
Avocado sunblotch disease is a graft-transmissible disorder known for over 60 years and has now been recorded in at least eight countries around the world. Affected trees develop yellow, depressed streaks on young stems and fruit, marked rectangular cracking of the mature bark and a decumbent style of growth. Often a tree with symptoms produces completely symptomless shoots, termed 'recovery' growth, which are latently infected. There is a reported 95 to 100% transmission of sunblotch through the seed of such branches, and "the resultant seedlings are themselves symptomless. Indexing for sunblotch to ensure that scion and, in view of seed transmission, especially rootstock material is free of the disease is very important . The standard method used for many years has been to graft tissue onto healthy indicator seedlings and observe for symptom development for 18 months to two years. One aim of the study presented in this thesis was to develop more rapid methods for detecting the sunblotch agent. By conducting the standard indexing method in a glasshouse at controlled high temperatures of 30/28º C (day/ night) and by cutting back the indicator plants every three months, the time was reduced from two years to eight months. While this represents a considerable saving in time, the ideal must be to develop a laboratory diagnostic test that requires no more than a few days, at most, to complete. A comparative study was therefore initiated on the phenol metabolism of healthy and sunblotch-infected avocados to determine whether infection causes any major change that may reliably serve as a marker for diagnostic purposes. Significantly increased peroxidase (PO) and phenylalanine ammonia-lyase (PAL) activities, decreased indoleacetic acid (IAA) oxidase activity and higher sinapic acid levels were detected in bark tissue showing sunblotch symptoms, but not in symptomless 'recovery' growth. In contrast, increased polyphenoloxidase (PPO) activity and isoenzymes, total soluble protein levels, water soluble phenols and reduced ferulic acid levels were found in the bark of all infected trees tested, both with symptoms and symptomless. However, these latter changes have been associated with other plant-virus systems and are therefore not necessarily specific for sunblotch. Neither is any sufficiently large to be definitive as a diagnostic test. Two unidentified phenols were detected in infected, mature bark, but not in infected young bark and leaves. introduced the possibility of rapid disease detection by polyacrylamide gel electrophoresis (PAGE) of extracted RNA's as used for known viroids. In this study the presence of previously reported small molecular weight sunblotchassociated RNA's was confirmed using PAGE methods requiring two to four days to complete. This thesis presents as a further development a more rapid method of PAGE detection of RNA's enabling indexing for sunblotch to be completed in under six hours. Whilst the biochemical studies did not reveal diagnostically meaningful differences between healthy and infected avocados, there were tendencies towards differences between healthy and symptomless carrier tissues, further investigation of which may lead to a future understanding of symptom development and the symptomless condition. These include apparent higher PO and lower PAL activities in symptomless carrier tissue, as well as higher PO isoenzyme a[1] and lower IAA oxidase isoenzyme a[1] activities. General studies on sunblotch-infected avocados showed that fruit from symptomless 'recovery' growth branches are significantly larger and have a higher oil content than those from healthy or diseased branches, the latter finding possibly indicating a more advanced state of maturity of 'recovery' growth fruit due to earlier flowering. The avocado sunblotch agent was shown to have an in vivo thermal inactivation point of 55º C, a temperature higher than the avocado tissue can withstand thereby eliminating the possibility of thermotherapy of infected twigs. In a host range study four lauraceous plant species, Persea Schiedeana, Cinnamomum zeylanicum, C. camphora and Ocotea bullata, were successfully infected with sunblotch by grafting from infected avocado. This is the first demonstration of any host other than avocado. A phanerogametic member of the same family, Cassytha filiformis, was shown to be able to transmit the disease from avocado to avocado. No hosts from other families were found. During an electron microscope study of sunblotch-infected avocado leaf tissue, gross alterations of the chloroplasts in the yellow areas were observed. These changes included organelle swelling, loss of grana and stroma lamellae, rearrangement of remaining membranes and presence of vesicles. Also in the yellow areas paramural bodies were encountered in higher numbers and displaying altered structure than in healthy and symptomless infected leaf tissue. This study on avocado sunblotch disease was successful in both of its aims. Firstly with regard to quicker indexing techniques, the standard method using indicator plants was shortened from two years to eight months, while a rapid, six-hour test based on PAGE analysis, was developed. Secondly, more light has been shed on the biochemical and ultrastructural effects of sunblotch on its host, the avocado, as well as providing information regarding the thermal sensitivity and the host range of the agent.
Evaluation of diazotrophic bacteria as biofertilizers.
(2013) Kifle, Medhin Hadish.; Laing, Mark Delmege.;
Inoculation with diazotrophic bacteria is well documented as a means to enhance growth and increase yields of various crops, especially when used as an alternative or a supplement to the use of nitrogenous fertilizers and agrochemicals for sustainable agriculture. Nitrogen is the most limiting nutrient for increasing crop productivity, and the use of chemical sources of N fertilizers is expensive, and may contribute to environmental pollution. Therefore, there is a need to identify diazotrophic inoculants as an alternative or supplement to N-fertilizers for sustainable agriculture. The search for effective diazotrophic bacterial strains for formulation as biofertilizers has been going on for over 40 years and a number of inoculant biofertilizers have been developed and are commercially available. In the current study, 195 free-living diazotrophic bacteria were isolated from soils collected from the rhizosphere and leaves of different crops in different areas within the KwaZulu-Natal Province, Republic of South Africa. Ninety five of the isolates were selected for further screening because they were able to grow on N-free media using different carbon sources. Isolates that were very slow to grow on N-free media were discarded. Of these, 95 isolates were screened in vitro for growth promotion traits tests including tests for ammonia production and acetylene reduction. The best 20 isolates that were also able to reduce acetylene into ethylene were selected for growth-promotion trials on maize under greenhouse conditions. Of the 20 isolates, ten isolates enhanced (P = 0.001) growth of maize above the Un-inoculated Control. Molecular tests were conducted to identify the ten most promising isolates selected in the in vitro study. In the greenhouse study, these diazotrophic isolates were screened for their ability to enhance various growth parameters of maize (Zea mays L.), following various inoculation techniques (drenching, seed treatment, foliar spray and combination of these). Inoculations with the five best diazotrophic isolates by various methods of application increased dry weight and leaf chlorophyll content (P < 0.001, P = 0.001), respectively, compared to the Untreated Control. Although, all methods of application of diazotrophic inoculants used in this study resulted in increased dry weight and leaf chlorophyll content, combined methods of application (seed treatment + drenching) and sole application (seed treatment) were significantly more (P < 0.05) efficient. The best five most promising isolates were identified for growth promotion of maize under greenhouse conditions. They were also assessed for their effects on germination of wheat in vitro and were further tested in combination with various levels of nitrogenous fertilizer for growth-promotion of wheat (Triticum aestivum L.). These five isolates were also investigated for their potential to enhance growth and yields of maize and wheat crops in field trials, when combined with a low dose of nitrogenous fertilizer. These isolates were further studied for their contribution for enhancing plant growth through nitrogen fixation by predicting N content in leaves using a chlorophyll content meter (CCM-200) and correlated to extractable chlorophyll level at R2 = 0.96. In this study, relative to the Un-inoculated Control, the best five isolates enhanced growth of maize and wheat when combined with a 33% N-fertilizer levels for a number of growth parameters: increased chlorophyll levels and heights of maize, shoot dry weight of maize and wheat; and enhanced root and shoot development of these crops in both greenhouse and field conditions. The best contributions of diazotrophic bacteria was achieved by Isolate LB5 + 0% NPK (41%), V9 + 65% NPK (28.9%), Isolate L1 + 50% NPK (25%), Isolate L1 + 25%NPK (22%) and LB5 + 75% NPK (15%) undergreenhouse conditions. At 30 or 60 DAP, isolates with 33%N-fertilizer caused relatively higher dry weight than the 100%NPK. Inoculation of Isolate StB5 without 33N% fertilizer cuased significant (P<0.005) increases in stover dry weight. In field studies, inoculation of diazotrophic bacteria alone or with 33%N-fertilizer resulted in relatively greater increases of dry weight, stover dry weight, number of spikes and yield at different growth stages higher than the Un-inoculated or Unfertilized Control. However, the increases were not statistically significant. The use of microbial inoculants in combination with low doses of nitrogenous fertilizers can enhance crop production without compromising yields. The isolates obtained in this study can effectively fix nitrogen and enhance plant growth. The use of microbial inoculants can contribute to the integrated production of cereal crops with reduced nitrogenous fertilizer inputs, as a key component of sustainable agriculture.

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