A Thesis, submitted to the Faculty of Science, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2013 / A novel holistic approach was used to study the mycotrophic nature of commercial sugarcane varieties grown in KwaZulu-Natal, South Africa. All five varieties were mycotrophic, but N12 had the highest overall mycorrhization and was selected for a pot study to assess the growth response of sugarcane to inoculation with indigenous arbuscular mycorrhizal (AM) fungi and microflora. The pot study suggested that sugarcane will respond positively to inoculation with AM fungi, but the effects are most clear in the early phase of growth and less obvious in later elongation phases. This observation, taken together with the ability of sugarcane to grow well in sterile soil without microflora additions suggests that the plant may be facultatively mycotrophic. A multivariate analysis determined the nutrient relationships between soil and corresponding leaf nutrient levels on 72 sugarcane field plants, categorised according to either high or low percentage colonisation. Highly colonised plants were found to have more positive nutrient correlations compared to lower percentage colonised plants. AM fungi were identified from spore morphology and associated mycorrhizal bacteria (AMB) were identified by 16s rDNA analysis. Partial molecular identification was conducted using a universal eukaryotic forward NS31 primer and general fungal AM1 primers confirming the spores to be of AM fungi origin. A nested PCR was performed, using the universal fungal primers, NS5 and ITS4, followed by primer combinations to target sequences of specific Glomalean groups. Only partial molecular identification was conducted, as RFLPs were not successfully optimised. DNA from the Acaulospora gerdemannii/Acaulospora trappei group, Glomus occultum/Glomus brasilianum group, Glomus mosseae/Glomus intraradices group, Glomus etunicatum/ Glomus clariodeum group and Acaulosporaceae sensu stricto were detected, indicating AM fungi diversity. Bacteria, Brevibacillus reuszeri isolated from Scutellospora nigra, Bacillus megaterium and Stenotrophomonas maltophilia isolated from Glomus geosporum, Paenibacillus chitinolyticus and Bacillus cereus isolated from Acaulospora mellea and Gigaspora margarita spores respectively, were tested for biocontrol capability against pathogenic nematodes of Paratrichodorus, Meloidogyne and Pratylenchus genera. Meloidogyne was the least susceptible to associated mycorrhizal bacteria biocontrol and Paratrichodorus the most susceptible. These studies have contributed to understanding the role of AM in sugarcane agriculture in South Africa. Keywords: arbuscular mycorrhiza, sugarcane varieties, multivariate analysis, nutrient relationships, associated mycorrhizal bacteria, biocontrol, nematodes

The rising cost of energy coupled with an increasing awareness of Greenhouse Gas (GHG) emissions has led to a concerted effort to reduce fossil fuel Energy Use (EU) in all sectors. Sugarcane production in South Africa is dependent on fossil fuel to provide a source of energy for production. To remain commercially and environmentally sustainable, measures need to be taken to reduce EU and increase EU efficiencies of on-farm operations. The first step toward realising this is to identify and quantify energy inputs. Following on from this, total GHG emissions, also known as carbon footprint, can be estimated. The primary objective of this research is to develop an energy calculator to estimate EU in sugarcane production in South Africa. The results generated by the calculator highlight areas of high energy intensity and low energy efficiencies at three different levels of detail. Based on these results, changes in management practices and technological improvements can be made to reduce EU and carbon footprint. Case studies were used to test the functionality of the calculator. Results from the case studies show that, in irrigated sugarcane production, the harvest and transport process together with irrigation account for a majority of the total on-farm EU. For one of the case studies, an estimated 20 % saving in the total on-farm EU was identified and can be achieved if appropriate technology is adopted in irrigation practices. Less significant energy savings were realised when in-field tractor operations were optimised for best tractor-implement matching. It is envisaged that the energy calculator will help farmers minimise on-farm EU and subsequently reduce input costs and carbon footprint. It will also provide a valuable tool for researchers to benchmark and profile EU in sugarcane production in South Africa. Research focussed on the sustainable production of sugar, from the agricultural to milling phase is of high priority at present. The quantification of on-farm EU in sugarcane production will form a critical component of such research. / M.Sc.Eng. University of KwaZulu-Natal, Pietermaritzburg 2014.

An experiment was conducted at Pongola (27°24´S, 31°25´E; 308m altitude) in South Africa to study the effects of season on growth and potential biomass and sucrose yields on nine commercial sugarcane cultivars. The treatments that were the focus in this study consisted of the cultivars NCo376, N25 and N26 ratooned in March, April, May, August and December. The crops were well fertilized and kept free of weeds and diseases. Irrigation applications were scheduled with a computer programme to keep the crops free of stress at all times. Shoot populations were counted regularly to study shoot density dynamics. Leaf appearance rates, sizes, numbers and senescence were measured to study the development of green leaf area. Green foliage, dead trash and stalk mass were measured at 4, 8, 10, 11 and 12 months in each of the starting times and also at 13 months in the March, April and May ratoon crops. The fibre, sucrose and non-sucrose content of stalks were determined on these harvesting occasions. Yields were calculated in terms of individual shoots and area (m‾²). The fraction of PAR intercepted by the developing canopies was measured until full canopy and daily intercepted solar radiation was interpolated for the entire crop. An automated meteorological station adjacent to the experiment site provided daily weather data. Shoot densities were described by thermal time, however, average peak shoot densities were lowest in the May ratoon (31.8 m‾²) and highest in the December ratoon (48.7 m‾²). Shoot senescence was most rapid in August and December ratoons. At the final harvest shoot densities were highest in the March, April and May ratoon (14.8 to 14.2 m‾²) crops. NCo376 (16.4 m‾²) and N25 (13.6 m‾²) had higher final shoot densities than N26 (10.5 m‾²). Leaf appearance rate was also well described by thermal time, however the first twelve leaves took longer to appear in crops started in December i.e. the first phyllochron was longer (109.5°C d) than in crops started at other times (80.4 to 94.5°C d). Leaves produced during the early stages of December and August ratoon crops were larger (e.g leaf number 13 of N26 was 443 to 378 cm²) than in other crops. April and May ratoon crops produced much smaller leaves (e.g leaf number 9 of N26 was 170 to 105 cm²). Leaf senescence was slower in April and May ratoon crops (0.36 to 0.46 leaves per 100°C d) than in March (0.51 to 0.59 leaves per 100°C d) or August and December ratoon crops (0.60 to 0.68 leaves per 100°C d). December ratoon crops produced very high green leaf area indexes (LAI) (>7.0) at the age of four months; all other crops had lower LAI (3.3 to 6.0) and most peaked later (8 to 11 months of age). The LAI of N25 peaked at the age of 8 months while NCo376 and N26 peaked when 10 to 11 months old. Seasonal fraction of solar radiation intercepted was high in the March ratoon crops (0.84) and declined to 0.63 in the May ratoon crops and was highest in the December ratoon crop (0.88). N26 intercepted lower fractions of PAR than NCo376 and N25, particularly in the May and August ratoon crops. Biomass accumulation, although initially slow, tended to be linear in the March, April and May ratoon crops in relation to intercepted radiation. In August and particularly in the December ratoons biomass accumulation was initially rapid, and RUEs were high (2.65 g MJ‾¹ at 114 days in the December ratoon crops). However, biomass accumulation slowed when these December ratoon crops experienced winter. Low growth rates after winter, as well as low shoot densities resulted in December ratoon crops having produced significantly lower above-ground biomass yields (4 886 g m‾² at the age of 12 months) than March, April and May ratoon crops (6 760 to 5 715 gm‾² at the age of 12 months). The December ratoon crops responded poorly to the better growing conditions in spring and second summer and accumulated little biomass after winter. N26 shoots grew rapidly during the first 6-8 months of the December ratoon crop and it yielded better than NCo376 and N25 at harvesting (biomass yields were 5.8 and 13.3% higher at the age of 12 months, respectively). April ratoons produced significantly higher biomass yields (6 760 g m‾²) than March, August and December ratoons. May ratoon crops produced the highest cane fresh mass yields (18 151 g m‾²) and April, May and August ratoons produced significantly higher sucrose yields than March and December ratoons. The highest sucrose yield was produced by the April ratoon crop of N26 (2 385 g m‾²). On average, across the five ratoon dates, NCo376, N25 and N26 produced similar sucrose yields (1 902 to 1 959 g m‾²). Foliage production was severely limited during winter while sucrose accumulation was less affected by the low temperatures, resulting in accumulation of sucrose in the top sections of the culm. Low temperatures slowed the development of canopies in March, April and May ratoon crops, but these crops were able to recover their growth rates and produced high biomass and sucrose yields at the age of 12 months. The December ratoons experienced low winter temperatures (<12°C) when they had already accumulated relatively high yields and became moribund during winter. They were unable to accumulate any significant amounts of biomass during final four months before the final harvest at the age of 12 months. NCo376, N25 and N26 all yielded poorly in the December ratoon crop. However, there are cultivars that appear to be less sensitive to the low winters and are able to yield relatively well when they are ratooned in December. Sucrose yields of March, April and May ratoons were increased substantially (10.6 to 22.7%) by harvesting at the age of 13 months rather than at the age of 12 months. The poor growth of December ratoon crops after winter is possibly due to the recently revealed feedback signaling by high sugar levels induced by low temperatures on photosynthesis. The incorporation of the effects of low temperature and the feedback signaling with the objective of better simulating yields of December ratoons is a proposed study at the South African Sugarcane Research Institute. Annual mean sucrose yields of NCo376, N25 and N26 crops were estimated to be 17% higher in March than in December ratoons. The suggested short term remedy therefore of the poor December yields is to shift milling seasons to include March and exclude December harvested crops in the northern irrigated regions. March crops grow vigorously during the months close to harvesting and therefore have lower levels of sucrose content which can be corrected with chemical ripeners. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

Weed control is a major cost for growers in the sugarcane industry, especially for monocotyledonous species such as Cynodon and Rottboellia spp. The introduction of imazapyr-tolerant sugarcane would be advantageous as this herbicide has shown to be effective against the above-mentioned weeds but it also kills sugarcane. In a previous study in our laboratory, several sugarcane putative-mutant lines of variety N12 were generated by in vitro exposure of embryogenic callus to 16 mM ethyl methanesulfonate (EMS), followed by selection on imazapyr-containing medium. Tolerance to a low dose of imazapyr was confirmed in seven of those lines when the herbicide was applied (182 g a.i. ha-1) to 3 month-old plants in pots. The aim of the present study was to identify which of the seven herbicide mutant lines had agronomic characteristics at least equivalent to un-mutated N12. The objectives were to: 1) confirm tolerance to increased rate (312 and 625 g a.i. ha-1) of imazapyr in field plants; 2) measure the agronomic characteristics of these lines; 3) determine the effect of residual soil herbicide activity on germination of sugarcane setts. The seven mutant lines (Mut1-Mut7) and un-mutated N12 were clonally propagated in vitro by shoot multiplication followed by rooting and planted in three plots (untreated, sprayed with 312 or 625 g a.i. ha-1 imazapyr), in the field, in a randomized complete block design. In the untreated control plot there were no significant differences between the control and the mutant plants for agronomic traits (tiller number/plot, stalk height and stalk diameter) or estimated yield (kg/plot) after 10 months, indicating that the mutation process had no effect on general plant phenotype. In the sprayed (312 and 625 g a.i. ha-1) plots, Mut1, Mut4, Mut5, Mut6 and Mut7 plants showed tolerance to imazapyr as the leaves remained green compared with Mut2, Mut3 and N12 control plants, which displayed chlorotic leaves and eventually died in the plot sprayed with 625 g a.i. ha-1. Post-herbicide application, the yields of Mut5, Mut6 and Mut7 (52.33, 43.43 and 41.43 kg/plot, respectively) from the 312 g a.i. ha-1 plot were not significantly different from that of N12 control (53. 61 kg/plot) in the untreated plot. However, in the 312 g a.i. ha-1 plot, the yield and agronomic trait measurements of the untreated N12 control were significantly higher than those of the herbicide-susceptible plants Mut2 and Mut3. Similarly, in the 625 g a.i. ha-1 plot, the recorded yields for Mut4, Mut6 and Mut7 were 41.60, 43.44 and 36.30 kg/plot, respectively, indicating that their imazapyr tolerance and yield characteristics were comparable to the untreated N12 control. Imazapyr is conventionally applied to a fallow field 3-4 months prior to planting sugarcane as there is residual herbicide activity in the soil that suppresses sugarcane germination and growth. Therefore, in order to establish if the herbicide-tolerant mutants could germinate in iii an imazapyr-treated field, 3-budded setts of the mutant lines (Mut1-Mut7) and N12 control were planted in two plots, one unsprayed and one sprayed with 1254 g a.i. ha-1 imazapyr, 2 weeks previously. Germination was calculated after 3 weeks as the number of germinated setts in each plot/no. germinated setts in unsprayed plot x100. In the sprayed plot, the setts from Mut1, Mut4 and Mut6 displayed the highest germination percentages (60, 71 and 74%, respectively) compared with Mut2 (24%), Mut3 (46%), Mut5 (34%), Mut7 (40%) and the N12 control (12%). The in vitro acetolactate synthase (ALS) enzyme activity of 10 month-old plants from the untreated plot was assessed in the presence of 0-30 μM imazapyr to determine the herbicide concentration that inhibited ALS activity by 50% (IC50). The IC50 values for the mutated lines were between 3 and 30 μM, i.e. 1.5-8.8 times more tolerant to imazapyr than the N12 control plants, with Mut6 displaying the highest IC50 value (30 μM). On the basis of the results, it was concluded that Mut1, Mut6 and Mut7 lines were more tolerant to imazapyr than N12 and the other tested lines. Future work includes phenotypically assessing these lines for traits including sucrose content, fibre content, actual yield (tons cane ha-1) and altered pest and disease resistance. Once isolated and sequenced, the ALS gene conferring imazapyr tolerance can be used in genetic bombardment in the genetic modification approach as the gene of interest or as a selectable marker. In addition, the imazapyr-tolerant line can be used for commercial purposes in the field and as the parent plant in the breeding programme. / Thesis (M.Sc.Agric.)-University of KwaZulu-Natal, Durban, 2013.

The Sugarcane Industry contributes approximately 400 000 jobs and ZAR 8 billion annually to South Africa’s economy. Due to climate change and the subsequent threat posed by disease, these figures have been on the decline. Brown rust, a contributor to this decline is caused by the basidiomycete Puccinia melanocephala Syd. and P. Syd., which previously resulted in 50% yield losses in susceptible varieties. This highlighted the need for improved screening and breeding techniques which will result in the replacement of susceptible varieties. The objectives of this study were to: a) Adopt and optimise a glasshouse whorl inoculation screening technique applicable for mass screening of large populations. b) Develop a rapid and cost effective rust resistance screening technique using detached leaves. c)Utilise two flanking marker sets (R12H16 and 9O20-F4-PCR primers) for the rust resistance Bru1 gene in a diagnostic polymerase chain reaction (PCR) to identify rust resistant genotypes lacking Bru1 and possessing either quantitative resistance or an alternative major qualitative resistance gene. d) Correlate rust phenotypic data to AFLP marker data for the Linkage Disequilibrium (LD2) mapping population. e) Utilise suppression subtractive hybridization (SSH) profiling on rust challenged genotypes to discover differentially expressed genes between susceptible and resistant (susceptible Bru1 negatives taken away from resistant Bru1 negatives); and resistant genotypes (resistant Bru1 positives taken away from resistant Bru1 negatives). 4 Results from the glasshouse whorl inoculation trials showed the technique could be reliably used to screen large populations, as two independently conducted pot trials showed highly correlated rust ratings. A visually assessed detached leaf assay (DLA) was developed using selected genotypes. Chlorophyll fluorescence and SPAD readings were used in the DLA to determine the leaf photochemical efficiency (PIABS) with relation to chlorophyll content, resulting in reduced assessment time of at least two days. PCR diagnostics revealed 31% of LD2 did not possess either flanking marker, 8% had one or the other marker, and 61% had both markers. The overall rust phenotypic ratings (rating scale of 0-10) and Bru1 status of the genotypes was used to group the population, with the Bru1 negative genotypes containing all three rating categories (resistant 0-3.5; intermediate 3.51-6.5; susceptible 6.51-10); while the Bru1 positive genotypes were all resistant. The phenotypic data was correlated to AFLP data using the Pearson product-moment correlation coefficient and stepwise multiple linear regression employed to build marker based models to use for predicting non-Bru1 mediated resistance. SSH analysis was then subsequently conducted on genotypes selected on the basis of Bru1 status and AFLP correlation data. Two subtraction cDNA libraries were constructed and the cDNA inserted into electro-competent Escherichia coli cells. PCR on transformed cells revealed cDNA inserts ranging from 200- 1300bp. BLAST analysis of the cDNA sequences indicated the presence of high proportions of disease and drought stress related sequences in the libraries. Analysis of the sequences in both libraries showed that the resistant Bru1 negative genotypes contained oxidative stress related sequences which were however absent in the Bru1 positive resistant genotypes. The library comparing the Bru1 negative resistant genotypes against the Bru1 negative intermediate and susceptible genotypes showed a higher proportion of differentially expressed sequences coding for putative disease resistance proteins, highlighting their presence in the resistant genotypes. Both subtraction libraries also contained high proportions of a leucine rich repeat protein coding cDNA which contained a conserved domain homologous to that of a disease resistance protein conferring resistance to Pseudomonas syringae in Arabidopsis thaliana. The outcomes of this study will subsequently enable an improved understanding of sugarcane-rust resistance mechanisms and improved breeding and screening techniques for sugarcane by identifying SSH and AFLP markers linked to rust resistance QTLs or alternative R genes. / Thesis (M.Sc.Agric)-University of KwaZulu-Natal, Pietermaritzburg, 2012.