Gastrointestinal nematode parasites cause great losses in the production of small ruminants through reduced productivity and the cost of preventive and curative treatments. Because of the threat of anthelmintic resistance, biological control of sheep nematodes has been identified as an alternative to anthelmintic drugs. Bacillus thuringiensis (Bt) (Berliner) and Clonostachys rosea (Schroers) have been widely studied as biocontrol agents. B. thuringiensis has been used for the biocontrol of insects and C. rosea has been successfully used as biocontrol agent of Botrytis cinera (De Bary) in plants. B. thuringiensis and C. rosea strains were isolated from soil collected from the Livestock Section at Ukulinga Research Farm, University of KwaZulu Natal, Pietermaritzburg. Twenty-five strains of Bt and 10 strains of C. rosea were successfully isolated. The Bt colonies were identified by their circular, white, flat and undulate character, and the gram-positive and rod-shaped endospores. C. rosea was identified by white colonies on Potato-dextose agar and the characteristic conidiophores, which were branched and showed phialides at the tips. In vitro screening of the isolates was undertaken to select the best isolates. The isolates that caused significantly greater mortality were Bt isolate B2, B10 and B12 and C. rosea isolates P1, P3 and P8. These isolates caused substantial nematode mortality in both faeces and water bioassay. Nematode counts were reduced by 28.5% to 62% and 44% to 69.9% in faecal bioassay for Bt and C. rosea, respectively. In the water bioassay, nematode counts were reduced by 62% to 85% for Bt and by 62.7% to 89.3% for C. rosea. The best inoculum level at which the best isolates were most effective, and the optimum frequency of application were determined. The trial was conducted using bioassays with faeces and water. Inoculum levels of 10(6), 10(8), 10(10), 10(12) spores ml-1 for Bt and 10(6), 10(8) and 10(10) conidia ml-1 for C. rosea was used in the faecal bioassay. The inoculum levels tested in water bioassay were 10(6), 10(8), 10(10) and 10(12) spores ml-1 for Bt and 10(9), 10(10), 10(11), 10(12) conidia ml-1 for C. rosea. In the faecal bioassay, B2 was the most effective Bt isolate at an inoculum level of 10(10) spores ml-1. Isolate P3 was the best C. rosea isolate at 10(8) conidia ml-1. In the water bioassay, Isolate P3 caused a mortality of 85% at inoculum levels of 10(9), 10(10) and 10(11) conidia ml-1. The performance of biological control agents in the field is sometimes inconsistent. Combining different biocontrol agents may be a method of improving their reliability and performance. However, the combination of most of the isolates was antagonistic, with efficacy less than that of either individual biocontrol agent. In particular, Isolate P3 was more effective when used alone than when combined with any other isolates. Therefore, the combination of biocontrol agents does not always result in synergistic interaction. There were some additive interactions between two bacterial isolates, and with one bacterial and fungal combination. The effect of feeding the best of the biocontrol agents, or diatomaceous earth (DE), was evaluated in sheep. Two doses of Bt (1g and 2g kg-1BW) and C. rosea (1g kg-1BW) reduced the numbers of L3 nematode larvae in sheep faeces. The DE product (at 15% of feed) also reduced L3 numbers but it was less effective than either the Bt or the C. rosea products. Nematode counts were reduced by 74.6%, 75.1%, 84.6%, 68.5% and 27.5% for Bt 1g kg-1BW, Bt 2g kg-1BW, C. rosea (1g kg-1 BW), DE and control, respectively. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

In the breeding of grain sorghum (Sorghum bicolour L. Moench) towards improved nutrition and agronomic performance, new methodologies are required to increase genetic diversity and lower the inputs required to track and screen breeding populations. Near-infrared calibration models were developed by partial least squares (PLS) and test-set validation on 364 sorghum samples to predict crude protein and moisture content on whole-grain and milled flour samples. Models using milled flour spectra were more accurately predictive than those from whole grain spectra for all constituents (eg. Protein: R2 = 0.986 on flour vs R2 = 0.962 on whole grain). Discriminant calibrations were established to classify grain colour using partial least squares discriminant analysis (PLS-DA) based upon CIE L*a*b* reference values and visual ranking. Preliminary calibrations were developed for quantities of 18 amino acids, fat and apparent metabolisable energy (AME) on 40 samples using cross-validation, highlighting potential for reliable calibration for these parameters in sorghum. An investigation into the potential of 12C6+ heavy-ion beam mutagenesis of sorghum seed was undertaken by treatment at RIKEN Accelerator Research Facility (Saitama, Japan) and subsequent breeding at Ukulinga research farm and analysis at the Department of Plant Pathology, University of KwaZulu-Natal, Pietermaritzburg, South Africa. Dosage rates of 75, 100 and 150 Gy were compared in seven sorghum varieties to establish optimal dose treatments as determined by germination and survival rates, visible morphological changes and field data over two seasons of field trials. Crude protein variation within the M2 generation was analysed to compare dose rate effects. The need for higher dose rates was indicated by few quantified differences between treatments and control although good correlations between protein deviation and treatment dose rate were elucidated. Differences in varietal response suggest a need to optimize dose rate for specific varieties in future endeavours. In addition, all mutagenized populations were screened for crude protein content using near-infrared spectroscopy (NIRS). Significant differences in protein levels and standard deviations were observed between treated self-pollinated M2 generations and untreated control populations. Individual plants displaying significantly different protein levels were isolated. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

Inoculation with free-living diazotrophic bacteria is well documented to enhance vegetative growth and yield increases of various crops coupled with suppression of sublethal pathogens. The use of microbial inoculants has been identified as an alternative or supplement to use of nitrogenous fertilizers and agrochemicals for sustainable agriculture. The search for effective free-living diazotrophic bacterial strains for formulation as biofertilizers has been on going since the 1970’s and a number of inoculant biofertilizers have been developed and are commercially available. In the current study, 250 free-living diazotrophic bacteria were isolated from soils collected from the rhizosphere and leaves of different crops in different areas within KwaZulu-Natal, province, Republic of South Africa. These were evaluated for plant growth-promotion and biological control of damping-off fungi initially by in vitro screening. The growth promotion traits tested included, phosphate-solubilization, production of indole-3-acetic acid, production of ammonia and acetylene reduction. Biocontrol traits evaluated included siderophore-production, antibiosis, and production of hydrogen cyanide (HCN). Biochemical and molecular bioassay tests were conducted to identify the twenty most promising isolates selected in the in vitro study. The twenty isolates were further tested in combination with various levels of nitrogenous fertilizer for growth-promotion of maize (Zea mays L.) and wheat (Triticum aestivum L.) under greenhouse conditions. The five most promising isolates identified for growth promotion under greenhouse conditions for each crop were assessed for their effects on the germination of maize in vitro and growth and yields of two maize and two wheat cultivars, when combined with a low dose of nitrogenous fertilizer in field trials. The five Bacillus subtilis (Ehrenberg) Cohn isolates that suppressed the growth of a wide range of pathogenic fungi in vitro were tested for their efficacy against damping-off of wheat caused by Rhizoctonia solani Kühn in the greenhouse. These isolates were further studied for their modes of action against R. solani in vitro. The modes of action tested included antibiosis, production of siderophores, extracellular enzymes, production of hydrogen cyanide (HCN) and antibiotic resistance. The twenty most promising bacterial isolates identified from the in vitro screening reduced acetylene to ethylene, produced indole-3-acetic acid and siderophores, one isolate solubilized phosphate, and 9 inhibited the growth of R. solani. These twenty isolates enhanced growth of maize and wheat above the Uninoculated Control under greenhouse conditions. The growth enhancements varied with bacterial isolate x crop species interactions, which identified five different isolates for each of the two crops. Relative to the Uninoculated Control, the best five isolates significantly (P = 0.001) enhanced the growth of maize and wheat at all fertilizer levels for a number of growth parameters: increased chlorophyll levels and heights of maize, shoot dry biomass of maize and wheat, and enhanced root development of maize in the greenhouse. Inoculation of maize and wheat with the two most promising isolates identified from the field trial for each crop, in combination with 65% and 50% of the recommended amount of nitrogenous fertilizer for maize and wheat, respectively, caused the same increases in shoot biomass as the Fully Fertilized Control. Application of a combination of the best bacterial isolates and 35% nitrogenous fertilizer resulted in the same or greater shoot dry biomass and yields of both maize and wheat under field conditions. Shoot dry biomass of wheat increased by 75% above the Uninoculated Control and 30% above the Fully Fertilized Control. The wheat yield increased by 95% above the Uninoculated Control and 43% above the Fully Fertilized Control. Seed inoculation with the best isolates combined with 35% N increased yields of maize by 41% above the Uninoculated Control and 15% above the Fully Fertilized Control. The best isolates significantly (P < 0.001) increased plant height, chlorophyll levels and shoot biomass of maize relative to the Uninoculated Control. There was a positive correlation between chlorophyll level and yield, chlorophyll level and shoot dry biomass, height and shoot dry biomass and height and yield of maize at P = 0.01 with r values of 0.87, 0.77, 0.92 and 0.81, respectively. The isolates that exhibited multiple plant-growth promoting traits in vitro, increased shoot biomass of both maize and wheat in the greenhouse and field, and caused yield increases in the two crops under field conditions. Five B. subtilis isolates inhibited the growth of some of the pathogenic fungi tested in vitro up to 95%. Seed inoculation with the same isolates significantly (P = 0.001) suppressed R. solani damping-off of wheat under greenhouse conditions and exhibited multiple mechanisms of disease control in vitro. The use of microbial inoculants in combination with low doses of nitrogenous fertilizers can enhance crop production without compromising the yields. The B. subtilis isolates obtained in this study can effectively control R. solani damping-off of wheat, 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. Key words: Free-living bacteria; plant growth-promotion; diazotrophs; biological nitrogen fixation; phosphate-solubilization; siderophores; indole-3-acetic acid; biocontrol; damping-off / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

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. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

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. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

Maize streak virus (MSV) disease, transmitted by leafhoppers (Cicadulina mbila, Naude), and maize downy mildew (DM) disease caused by Peronosclerospora sorghi (Weston and Uppal) Shaw, are major contributing factors to low maize yields in Africa. These two diseases threaten maize production in Mozambique, thus the importance of breeding Mozambican maize varieties that carry resistance to these diseases. Marker-assisted selection (MAS) was employed to pyramid MSV and DM disease resistant genes into a single genetic background through simultaneous selection. Firstly, it was essential to determine the genetic diversity of MSV disease resistance in 25 elite maize inbred lines to aid in the selection of suitable lines for the introgression of the msv1 gene; and subsequently, to introduce the msv1 resistance gene cluster from two inbred lines, CM505 and CML509, which were identified as the ideal parental lines for the introgression of MSV disease resistance into a locally adapted Mozambican inbred line LP23 that had DM background resistance. Pyramiding the resistance genes by the use of simple sequence repeat (SSR) molecular markers to track the MSV gene cluster was investigated in 118 F3 progeny derived from crosses of CML505 x LP23 and CML509 x LP23. High resolution melt (HRM) analysis using the markers umc2228 and bnlg1811 detected 29 MSV resistant lines. At the International Maize and Wheat Improvement Centre (CIMMYT) in Zimbabwe, MSV disease expression of the 118 F3 progeny lines was assessed under artificial inoculation conditions with viruliferous leafhoppers and the effect of the MSV disease on plant height was measured. Thirty-seven family lines exhibited MSV and DM (DM incidence ≤50) disease resistance. Individual plants from a total of 41 progeny lines, that exhibited MSV disease severity ratings of 2.5 or less in both locations within each of the F3 family lines, were selected based on the presence of the msv1 gene based on SSR data, or field DM disease resistance, and were then advanced to the F4 generation to be fixed for use to improve maize hybrids in Mozambique for MSV resistance. Simultaneous trials were run at Chokwe Research Station in Mozambique for MSV and DM disease assessment, under natural and artificial disease infestation, respectively. Thus the MSV and DM genes were effectively pyramided. Lines with both MSV and DM resistance were advanced to the F4 generation and will be fixed for use to improve maize hybrids in Mozambique for MSV and DM resistance, which will have positive implications on food security in Mozambique. This research discusses the results of combined selection with both artificial inoculation and the three selected SSR markers. It was concluded that a conventional maize breeder can successfully use molecular markers to improve selection intensity and maximise genetic gain. / Thesis (M.Sc.Agric)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

This study examines field cropping constraints for domestication of an endangered, wild medicinal plant, Siphonochilus aethiopicus, (Schweif.) B.L. Burt. Extensive literature review and careful observations of plant growth behavior during two years of crop trials overturned several long-held but erroneous claims that have consistently appeared in the scholarly literature, and revealed previously undocumented plant growth characteristics. S. aethiopicus (Schweif.) B.L. Burt. is a rhizomatous corm, not a rhizome. Field growth observations demonstrated clearly that the false stem and leaves grow continuously from emergence in September to senescence in April-May; the corm retains its tuberous roots during winter senescence, and is genetically preprogrammed to shoot in September. Flowers may emerge throughout the growing season (not only initially prior to shoot emergence), typical leaf count is 11-15, not 6-8 as previously reported, numbers that remain constant even when the plant height increases by 20-30% under shade, and leaf distichy is independent of the sun’s course and is unaffected by mother corm orientation. S. aethiopicus proved to be unusually resistant to common field diseases and pests, and resilient to severe hail. The responses of S. aethiopicus were tested in a series of field trials to the effects of levels of compost, field spacing, size of planting material, addition of biocontrol agents, different degrees of shading, and factorials of the macronutrients Nitrogen, Phosphorous and Potassium. Spacing-Composted chicken litter combinations were tested in 2005-2006 in factorial combination with Spacing at 15 cm-4.5 kg ha-1, 20 cm-7.5 kg ha-1, 30 cm-10 kg ha-1, and 40 cm-15.5 kg ha-1, and these treatments were randomized with 4 Corm planting sizes (height by base diameter in mm): Small (S, 12.38 mm x 12.6 mm), Medium Small (MS, 29.65 mm x 27.93 mm), Medium Large (ML, 38.48 mm x 37.78 mm) and Large (L, 52.37 mm x 44.10 mm). 2005-2006 ANOVA tests showed significant differences between Spacing-Compost and Corm Size for the total harvest biomass measure, with 30 cm and 40 cm spaces better than 15 cm spacing, and Corm Size MS, ML and L all better than S, and ML better than MS. Total Corms harvested per block and ii Survival Percentage were similarly significant for Corm Size, but not Spacing. Corms smaller than the Small criteria were raised separately, under optimal conditions in a nursery. In a separate 2005-2006 Compost-only trial ANOVA tests did not find significant differences between compost levels. In 2006-2007 we tested Spacing separately at 5, 10, 15, 20, 30 and 40 cm between planted corms in each plot. We tested Compost levels separately, with 0, 5, 10 and 15 kg ha-1 compost per plot. In 2006-2007 only the ML and L sizes were used in an even mix. There were no significant differences between treatments due to high experimental error, but measurement across all production parameters showed a clear trend towards best performance at spacing between 20 and 40 cm. Overall the results from the Spacing, Compost-level and Corm Size trials suggest that 30 cm is perhaps the optimal field spacing, higher compost levels tend to give better results, and the ML and L corm sizes perform better in open-sun field trials. These parameters are recommended for further field studies and production. The effects of two commercial strains of Trichoderma spp were tested at recommended doses applied to S. aethiopicus. T. harzianum Strain B77 was used as a drench at planting in comparison with a Control and a fungicide in 2005-2006. There were no significant differences between treatments for Harvested Biomass or Survival Percentage. B77 did perform significantly better than the Fungicide in the Total Corm measurement, but neither treatment was significantly different from the Control. In sum, there was a weak trend towards a greater number of output corms as a result of the application of the biocontrol agent. In both 2005-2006 and 2006-2007 we tested T. harzianum Strain kd applied as a drench at planting, with a second drench at 4 weeks. In 2006-2007 there were no significant differences between treatments, but the trend was towards better performance as a result of the drench at planting only. In 2005-2006 open field trials had shown that S. aethiopicus is susceptible to sunburn and Erwinia soft rot when grown in the full sun. Therefore, we tested the effect of various shadecloth densities and colours on production performance in 2006-2007. Treatments were Control (full sun), 40% White (TiO2) (23% shade), 40% Grey (28-30% shade), Light Black (40%), Medium Black (50%), Dark Black (80%), and Red (40%). There were no significant differences between treatments, but the trends indicated that the Control (full sun) and Dark Black (80% shade) performed the worst. Colour of shade did not appear to be important, and plants under all the shadecloths with 40-50% shade grew best. In a factorial trial different levels of Nitrogen, Phosphorous, and Potassium (NPK)were tested, over two seasons. Four levels of each input were used: N at 0 (Control), 40 kg ha-1 (N1), 80 kg ha-1 (N1), and 120 kg ha-1 (N3). P levels were 0 (Control) 60 kg ha-1 (P1) ,120 kg ha-1 (P2) and 200 kg ha-1 (P3). K levels were 0 (Control), 100 kg ha-1 (K1), 200 kg ha-1 (K2), and 400 kg ha-1 (K3). In 2005-2006 there were no significant differences between treatments. In 2006-2007 data there were significant results for Nitrogen only within each repetition. However, significance disappeared when combining across repetitions. We then ran a Bootstrap re-sampling analysis of both 2005-2006 and 2006-2007 data (data were analyzed separately because of different plot sizes and corm numbers in the two years), looking at the optimal level of each macronutrient tested against all combinations of the other two. Though significant results were obtained for each individual level of each macronutrient against the others in combination, the difference between the confidence intervals was not significant. However, there was a clear trend: the optimum N levels were between 40 and 80 kg ha-1; optimum P level was 0 (the Control) and optimum K levels were between 100 and 200 kg ha-1. Tests of handling during harvest, storage, and planting yielded additional useful information for small scale commercial farmers. The optimal harvest time is May, when the false stem and leaves are senescing and yellow, but still upright and visible. Harvest is facilitated by moistening the soil to minimize breaking off of tuberous roots, with simple field washing to remove compacted soil highly recommended. Harvested corms and tuberous roots should be stored under air-restricted, cool conditions because the tuberous roots contain high moisture and will shrivel quickly when left exposed to air, and excessively dried corms will eventually die. Senesced mother corms should be discarded at harvest. Corms are genetically preprogrammed to shoot, so should be planted in September in soft soil, with 1-2 cm of soil coverage. The studies provide a framework for developing the basic agronomy for the domestication and commercial crop production of an endangered medicinal plant species. / Thesis (M.Sc.Agric.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Soybean rust (SBR) caused by Phakopsora pachyrhizi Syd. is an aggressive wind dispersed fungal disease which has spread around the world at an alarming rate in the last decade. The disease was first reported in South Africa (SA) in 2001. It has become well established in the province of KwaZulu-Natal. Reports are occasionally made from eastern Mpumalanga, late in the growing season, in years with good rainfall. Yield losses of 10 - 80% have been reported due to SBR infection. Literature was reviewed to better understand the pathogen in an attempt to find suitable disease management strategies. Many strategies involve delaying, rather than preventing, SBR infection. Of the two strategies to prevent infection, the use of fungicides was the only option for disease control in SA, as no resistant cultivars are available. Field trials were conducted to determine which fungicides are effective in controlling SBR. Further research was conducted to determine the timing, frequency and rate of fungicide applications for optimal control of SBR. Trials were evaluated for disease severity, seed yield and the effect of fungicides on seed quality. Fungicides from the triazole class of the sterol biosynthesis inhibiting group of fungicides were found to be the most effective in controlling SBR. A fungicide from the strobilurin group was found to be less effective than the triazoles at the suggested rate, but was found to be as effective when evaluated at higher dosage rates. Triazoles premixed with fungicides from the benzimidazole and strobilurin groups were also effective in controlling SBR. Timing of application was found to be critical for strobilurin fungicides, but not for triazole fungicides, which have a curative ability, unlike strobilurins. Strobilurin fungicides applied preventatively, before the appearance of disease symptoms were as effective as triazole fungicides applied after disease symptoms, but before infection levels had reached 10%. Across both wet and dry seasons two fungicide applications applied at 21d intervals at the R2 growth stage resulted in effective disease control. In wet seasons, a third fungicide application resulted in yields that were higher, albeit not statistically significant, than two fungicide applications. Assessments of individual fungicides for optimal dosage rate found that registered rates were already optimal for some fungicides, but for others it appeared as if alterations were necessary to the rate suggested for registration. This study was one of the first to extensively evaluate the efficacy of the new triazole and strobilurin fungicides on SBR control. The results have been shared globally, but particularly with newly affected countries in South and North America. Although this research has been groundbreaking, there are still many aspects of fungicide control which need to be studied in order to further optimise chemical control of SBR. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005

Phakopsora pachyrhizi H. Syd and P. Syd, the causal organism of soybean rust (SBR) was first reported in Japan in 1902. In 1934 the pathogen was found in several other Asian countries and as far south as Australia. In India, SBR was first reported on soybeans in 1951. There have been several early reports of SBR in equatorial Africa but the first confirmed report of P. pachyrhizi on the African continent was in 1996 from Kenya, Rwanda and Uganda. Since then, the pathogen has spread south with reports from Zambia and Zimbabwe in 1998 and in Mozambique in 2000. In February 2001, P. pachyrhizi was first detected on soybeans near Vryheid, in Northern KwaZulu-Natal, South Africa (SA). As the season progressed, the disease was observed in other parts of the province, and epidemic levels were found in the Cedara, Greytown, Howick and Karkloof production regions. Soybean rust subsequently spread to Amsterdam and Ermelo in the Highveld region of SA. The disease reappeared in SA in March 2002. It is now established that the pathogen is a threat to soybean production in the country with yield losses in the region of 10-80%. A literature review on SBR investigating the taxonomy of the pathogen, its morphology, symptoms, host range, infection process, epidemiology, control options and the economic importance of P. pachyrhizi was complied to provide the necessary background information to conduct research under local conditions and to assist in interpretation of results of experiments. Epidemiological trials were conducted at the University of KwaZulu-Natal under controlled environmental conditions in a dew chamber and conviron. Development of P. pachyrhizi on the susceptible cultivar (LS5995) was quantified in combinations of seven temperatures (15,19,21,24,26,28 and 30°C) and five leaf wetness durations (LWD) (6,9,12,14 and 16hrs) at three relative humidities (RH) (75%, 85% and 95%). Studies indicate that optimum temperature for uredospore infection is 21-24°C with a LWD greater than 12hrs and RH 85-95%. The number of pustules as well as lesion size on the abaxial and adaxial leaf surface increased with increasing LWD at all the RH values tested. Infection did not occur on plants incubated at 15°C and 30°C at 85% or 95%RH whereas at 75%RH infection did not occur on plants incubated at 15°C, 19°C and 30°C regardless of LWD. Number of pustules per lesion produced at 75%, 85% and 95%RH was highest at 24°C and showed a gradual increase with increasing LWD. Lesion size on both leaf surfaces increased after 12hrs LWD at 24°C at 75% and 85%RH whereas at 95%RH lesion size increased after 14hrs LWD at 24°C. Exposure of uredospores to ultraviolet light which is equivalent to ultraviolet C (sunlight) which is < 280nm, shows a decrease in germination (7%). Under continuous darkness, the germination percentage was found to range from 58% after 48 hrs. Germination was found to peak at 16hrs in darkness with a gradual decrease as time increased whereas germination under ultraviolet light was highest after 6hrs with a gradual decrease with increased exposure to light. Germ tube lengths were found to be shorter when exposed to ultraviolet light (107µm) compared to controls kept in the dark (181µm). Results obtained clearly show a negative effect of ultraviolet light on the germination and germ tube length of uredospores. A 0.1 ml suspension of uredospores on 1.25% water agar Petri dishes was exposed to cycles of 14h ultraviolet light and 10h darkness for 48h. Results indicate an increase in germination percentage of uredospores when exposed to 10h of darkness following a 14h period under ultraviolet light. Controlled environmental studies were conducted to determine alternative hosts of P. pachyrhizi in SA. The control used in this experiment was Prima 2000, a susceptible cultivar to soybean rust. Seven legume plants [Cajanus cajan (L.) Huth, Glycine max (L.) Merr, Lablab purpureus (L.) Sweet, Lupinus angustifolius (L.) Finnish, Phaseolus vulgaris (L.), Pueraria lobata (M&S) Wild and Vigna unguiculata (L.) Walp] and three dry bean lines (Bonus; OPS-RS2 and PAN 159) showed typical SBR symptoms when rated after 21 days post inoculation with uredospores for percentage disease severity. Disease severity was significantly different within the alternative hosts, but G. max, P. vulgaris and P. lobata were not significantly different from Prima 2000 (control). A uredospore suspension of 2.5 x 10(5) uredospores ml(-1) from plants that showed typical SBR symptoms was made and inoculated on to Prima 2000, a susceptible soybean cultivar. Uredospores from pustules on G. max, L. purpureus, L. angustifolius, P. vulgaris, P. lobata, V. unguiculata, Bonus and PAN 159 produced viable uredospores on PRIMA 2000. These plants are considered alternative hosts of P. pachyrhizi. Effect of leaf age on susceptibility of soybean to SBR was tested under controlled environmental conditions. Mean number of lesions as well as lesion size were greater on younger leaves than on older leaves of plants at the same physiological age. Plants at the early vegetative and reproductive stages had a significantly lower number of lesions as well as a smaller lesion size. Plants at the V6 and R1 growth stages were significantly more susceptible to P. pachyrhizi than plants at other developmental stages. Trichoderma harzianum Rifai, Eco-77® a commercial biological control product, was evaluated for its efficacy as a biological control agent of P. pachyrhizi. Trichoderma harzianum sprayed at the standard concentration on infected soybean plants was significantly more effective in controlling P. pachyrhizi than plants sprayed at 1/2X and 2x the standard concentration. This was noted in both Trial 1 and 2. Data indicate that spraying the filtrate two days after inoculation produces less disease. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2006.

Fusarium circinatum is a fungal pathogen that has had a serious impact on pine production throughout the world. It attacks most Pinus species including Pinus elliottii, Pinus patula and Pinus radiata. Infections in South Africa (SA) are largely on seedlings, and result in fatal seedling wilt. Accurate and quick detection systems suitable for field use are needed to monitor the spread of the disease and optimize fungicide applications. Detection of F. circinatum is currently based on visual observations of typical symptoms. However, symptoms are not unique to the pathogen and can be caused by other biotic and abiotic stress factors. Nucleic acid-based identification techniques using PCR are available for different fungal species. These are sensitive and accurate, but they are expensive and require skilled biotechnologists to conduct the assays. In this study an enzyme-linked immunosorbent assay (ELISA) was developed to identify F. circinatum in infected seedlings. This optimized ELISA is able to discriminate between F. circinatum and two other fungi that frequently affect pine. This method has advantages over other assays because of its ease of operation and sample preparation, sensitivity and the ability to run multiple tests simultaneously. Mycelium-soluble antigens from Diplodia pinea (=Sphaeropsis sapinea), F. circinatum and F. oxysporum were prepared in nutrient broth. Analysis of these antigens on SDS-PAGE indicated the presence of common antigens between the different fungal pathogens. Some antigens were expressed more by some isolates than by others. Separate groups of chickens were immunised with mycelium-soluble antigens from D. pinea, F. circinatum and F. oxysporum and exo-antigen from F. circinatum prepared in nutrient broth. A 34 kDa protein purified from SDS-PAGE specific for D. pinea was also used for immunisation. Five sets of antibodies were obtained including anti-D. pinea, anti-F. circinatum, anti-F. oxysporum, anti-F. circinatumexo and anti-D. pinea 34 kDa antibodies, respectively. Reactivity of these antibodies was evaluated against antigens prepared in nutrient broth using western blotting and ELISA. Western blot analysis indicated that immuno-dominant antigens for F. circinatum were larger than 34 kDa and their reactivity was not the same between different isolates. Each of the antibodies prepared using mycelium-soluble antigens showed increased reactivity when detecting its own specific pathogen, but cross-reactivity was observed. Anti-D.pineaantibodies showed minimal cross-reactivity with antigens from F. circinatum and F. oxysporum. Anti-F. circinatum antibodies cross-reacted with antigens from F. oxysporum but showed little cross-reactivity with D. pinea antigens. Anti-F. oxysporum antibodies showed more cross-reactivity towards antigens from F. circinatum than those from D. pinea. No reactivity was observed when anti-F. circinatum-exo antigen and anti-D. pinea 34 kDa antibodies were used in immuno-blotting analysis. Evaluation of antibody reactivity using indirect ELISA showed patterns similar to those observed on western blotting, where anti-D. pinea, anti-F. circinatum and anti-F. oxysporum antibodies showed the same cross-reactivity relationships. Anti-F. circinatum and anti-F. oxysporumantibodies showed a significant difference when reacting with antigens isolated from other pathogens including D. pinea, F. circinatum, F. oxysporum, F. solani, F. graminearum and F. culmorum (P = 0.001). No significant difference was observed when the antigens were detected with anti-D. pinea antibodies. Reactivity of anti-F. circinatum-exo and anti-D. pinea34 kDa antibodies was mostly similar to that of non-immune antibodies and showed no significant difference between detection of different antigens. Pine seedlings were artificially infected with the three fungal pathogens using a spore concentration of 1 – 1 x 106conidiaml-1.Infection was monitored using scanning electron microscopy. Results showed increased levels of mycelium growth on the stem and roots of the F. circinatum and F. oxysporum infected seedlings and on the leaves and stem in the case of D. pinea infected seedlings. These plant parts were used in ELISA tests for the detection of antigens. Isolation of antigens from the plant materials involved crushing plant parts in buffer and centrifugation of the suspension. The supernatant obtained was directly used in the assay. ELISA tests prepared in this study were sensitive enough to detect infection caused by 1 conidium ml-1at two weeks post inoculation. A positive reaction for detection of F. circinatum and F. oxysporum was indicated by an ELISA reading above an optical density at 405 nm. The plant material used in ELISA tests were further analysed using PCR. Results indicated that there was no cross-infection between seedlings and served as a confirmation of the disease-causing pathogen. This indicated that cross-reactivity observed was due to other factors such as common epitopes on the major antigens. Use of an ELISA dip-stick or ELISA using these antibodies should provide an easy, fast field test to identify infections of pine, discriminating between F. circinatum, F. oxysporum and D. pinea. / M.Sc.Agric. University of KwaZulu-Natal, Pietermaritzburg 2013.