Tipping the balance : sclerotinia sclerotiorum secreted oxalic acid suppresses host defenses by manipulating the host redox environment

Sclerotinia sclerotiorum is a necrotrophic ascomycete fungus with an extremely broad host range. This pathogen produces the non-specific phytotoxin and key pathogenicity factor, oxalic acid (OA). Our recent work indicated that this fungus and more specifically OA, can induce apoptotic-like programmed cell death (PCD) in plant hosts, this induction of PCD and disease requires generation of reactive oxygen species (ROS) in the host, a process triggered by fungal secreted OA. Conversely, during the initial stages of infection, OA also dampens the plant oxidative burst, an early host response generally associated with plant defense. This scenario presents a challenge regarding the mechanistic details of OA function; as OA both suppresses and induces host ROS during the compatible interaction. In the present study we generated transgenic plants expressing a redox-regulated GFP reporter. Results show that initially, Sclerotinia (via OA) generates a reducing environment in host cells that suppress host defense responses including the oxidative burst and callose deposition, akin to compatible biotrophic pathogens. Once infection is established however, this necrotroph induces the generation of plant ROS leading to PCD of host tissue, the result of which is of direct benefit to the pathogen. In contrast, a non-pathogenic OA-deficient mutant failed to alter host redox status. The mutant produced hypersensitive response-like features following host inoculation, including ROS induction, callose formation, restricted growth and cell death. These results indicate active recognition of the mutant and further point to suppression of defenses by the wild type necrotrophic fungus. Chemical reduction of host cells with dithiothreitol (DTT) or potassium oxalate (KOA) restored the ability of this mutant to cause disease. Thus, Sclerotinia uses a novel strategy involving regulation of host redox status to establish infection. These results address a long-standing issue involving the ability of OA to both inhibit and promote ROS to achieve pathogenic success.

Developing schedules for mixed rail and road cane transport systems

This paper addresses the problem of scheduling a cane transport system involving both rail transport and road transport, where the road transport operates from several sidings in the rail network. An iterative approach for scheduling the rail transport system has been developed using existing rail transport scheduling tools. The assumption that harvesters serviced by road transport are effectively operating from the rail siding from which their bins are supplied seems a reasonable starting point for the analysis. There is a need to manually modify the schedule to take into account the road transport schedule to ensure that full bins are not collected before the road transport system delivers them back to the rail siding.

Effects of pH on pretreatment of sugarcane bagasse using aqueous imidazolium ionic liquids

Pretreatments of sugarcane bagasse for saccharification using different acid-catalysed imidazolium IL solutions (containing 20% water) at 130 °C for 30 min were investigated. At the same solution pH, pretreatment effectiveness in terms of glucan digestibility, delignification and xylan removal were similar for aqueous 1-butyl-3-methylimidazolium methane sulfonate (BMIMCH3SO3), 1-butyl-3-methylimidazolium methyl sulfate (BMIMCH3SO4), 1-ethyl-3-methylimidazolium chloride (EMIMCl) and 1-butyl-3-methylimidazolium chloride (BMIMCl). Decreasing solution pH of aqueous IL systems from 6.0 to 0.4 increased bagasse delignification and xylan removal, and as a result, improved glucan digestibility. The glucan digestibilities for bagasse samples pretreated by IL solutions with pH ≤ 0.9 were > 90% after 72 h of enzymatic hydrolysis. Without pH adjustment, the effectiveness of these aqueous IL solutions (except BMIMCH3SO3 because of its low natural pH of 0.9) to deconstruct the biomass was poor and the glucan digestibilities of pretreated bagasse samples were < 20%. These results show that pretreatment effectiveness of aqueous imidazolium ILs can simply be estimated from solution pH rather than hydrogen bond basicity (β) of the IL solution.

Caustic potash pulping study of water efficient Arudno donax and Sorghum bicolor; a comparison to sugarcane bagasse

Numerous crops grow in sugar regions that have the potential to increase the amount of biomass available to a small bagasse-based pulp factory. Arundo donax and Sorghum offer unique advantages to farmers compared to other agricultural crops. Sorghum bicolour requires only 1/3 of the water of sugarcane. Arundo donax is a very high yield crop, it can also grow with little water but it has the further advantage in that it is also highly stress tolerant, making it suitable for land which is unsuited to other crops. Pulps produced from these crops were benchmarked against sugarcane bagasse pulp. Arundo, sorghum and bagasse were pulped using KOH and anthraquinone to 20 Kappa number so as to produce a bleachable pulp. The unbleached sorghum pulp has better tensile strength properties than the unbleached Arundo pulp (43.8 Nm/g compared to 21.4 Nm/g) and the bleached sorghum pulp tensile strength was similar to bagasse (28.4 Nm/g). At 20 Kappa number, sorghum pulp had acceptable yield for a non-wood fibre (45% c.f. 55% for bagasse), Arundo donax pulp had low tensile strength, and relatively low yield (38.7%), even for an agricultural fibre and required severe cooking conditions to achieve similar delignification to sugarcane bagasse or sorghum. Sorghum and Arundo donax produced thicker handsheets than bagasse (>160 μm c.f. 122 μm for bagasse). In preliminary experiments sorghum and bagasse responded slightly better to Totally Chlorine Free bleaching (QPP), although none achieved a satisfactory brightness level and more optimisation is needed.

A catchment modelling approach integrating surface and groundwater processes, land use and distribution of nutrients : Elimbah Creek, southeast Queensland

As the world’s population is growing, so is the demand for agricultural products. However, natural nitrogen (N) fixation and phosphorus (P) availability cannot sustain the rising agricultural production, thus, the application of N and P fertilisers as additional nutrient sources is common. It is those anthropogenic activities that can contribute high amounts of organic and inorganic nutrients to both surface and groundwaters resulting in degradation of water quality and a possible reduction of aquatic life. In addition, runoff and sewage from urban and residential areas can contain high amounts of inorganic and organic nutrients which may also affect water quality. For example, blooms of the cyanobacterium Lyngbya majuscula along the coastline of southeast Queensland are an indicator of at least short term decreases of water quality. Although Australian catchments, including those with intensive forms of land use, show in general a low export of nutrients compared to North American and European catchments, certain land use practices may still have a detrimental effect on the coastal environment. Numerous studies are reported on nutrient cycling and associated processes on a catchment scale in the Northern Hemisphere. Comparable studies in Australia, in particular in subtropical regions are, however, limited and there is a paucity in the data, in particular for inorganic and organic forms of nitrogen and phosphorus; these nutrients are important limiting factors in surface waters to promote algal blooms. Therefore, the monitoring of N and P and understanding the sources and pathways of these nutrients within a catchment is important in coastal zone management. Although Australia is the driest continent, in subtropical regions such as southeast Queensland, rainfall patterns have a significant effect on runoff and thus the nutrient cycle at a catchment scale. Increasingly, these rainfall patterns are becoming variable. The monitoring of these climatic conditions and the hydrological response of agricultural catchments is therefore also important to reduce the anthropogenic effects on surface and groundwater quality. This study consists of an integrated hydrological–hydrochemical approach that assesses N and P in an environment with multiple land uses. The main aim is to determine the nutrient cycle within a representative coastal catchment in southeast Queensland, the Elimbah Creek catchment. In particular, the investigation confirms the influence associated with forestry and agriculture on N and P forms, sources, distribution and fate in the surface and groundwaters of this subtropical setting. In addition, the study determines whether N and P are subject to transport into the adjacent estuary and thus into the marine environment; also considered is the effect of local topography, soils and geology on N and P sources and distribution. The thesis is structured on four components individually reported. The first paper determines the controls of catchment settings and processes on stream water, riverbank sediment, and shallow groundwater N and P concentrations, in particular during the extended dry conditions that were encountered during the study. Temporal and spatial factors such as seasonal changes, soil character, land use and catchment morphology are considered as well as their effect on controls over distributions of N and P in surface waters and associated groundwater. A total number of 30 surface and 13 shallow groundwater sampling sites were established throughout the catchment to represent dominant soil types and the land use upstream of each sampling location. Sampling comprises five rounds and was conducted over one year between October 2008 and November 2009. Surface water and groundwater samples were analysed for all major dissolved inorganic forms of N and for total N. Phosphorus was determined in the form of dissolved reactive P (predominantly orthophosphate) and total P. In addition, extracts of stream bank sediments and soil grab samples were analysed for these N and P species. Findings show that major storm events, in particular after long periods of drought conditions, are the driving force of N cycling. This is expressed by higher inorganic N concentrations in the agricultural subcatchment compared to the forested subcatchment. Nitrate N is the dominant inorganic form of N in both the surface and groundwaters and values are significantly higher in the groundwaters. Concentrations in the surface water range from 0.03 to 0.34 mg N L..1; organic N concentrations are considerably higher (average range: 0.33 to 0.85 mg N L..1), in particular in the forested subcatchment. Average NO3-N in the groundwater has a range of 0.39 to 2.08 mg N L..1, and organic N averages between 0.07 and 0.3 mg N L..1. The stream bank sediments are dominated by organic N (range: 0.53 to 0.65 mg N L..1), and the dominant inorganic form of N is NH4-N with values ranging between 0.38 and 0.41 mg N L..1. Topography and soils, however, were not to have a significant effect on N and P concentrations in waters. Detectable phosphorus in the surface and groundwaters of the catchment is limited to several locations typically in the proximity of areas with intensive animal use; in soil and sediments, P is negligible. In the second paper, the stable isotopes of N (14N/15N) and H2O (16O/18O and 2H/H) in surface and groundwaters are used to identify sources of dissolved inorganic and organic N in these waters, and to determine their pathways within the catchment; specific emphasis is placed on the relation of forestry and agriculture. Forestry is predominantly concentrated in the northern subcatchment (Beerburrum Creek) while agriculture is mainly found in the southern subcatchment (Six Mile Creek). Results show that agriculture (horticulture, crops, grazing) is the main source of inorganic N in the surface waters of the agricultural subcatchment, and their isotopic signature shows a close link to evaporation processes that may occur during water storage in farm dams that are used for irrigation. Groundwaters are subject to denitrification processes that may result in reduced dissolved inorganic N concentrations. Soil organic matter delivers most of the inorganic N to the surface water in the forested subcatchment. Here, precipitation and subsequently runoff is the main source of the surface waters. Groundwater in this area is affected by agricultural processes. The findings also show that the catchment can attenuate the effects of anthropogenic land use on surface water quality. Riparian strips of natural remnant vegetation, commonly 50 to 100 m in width, act as buffer zones along the drainage lines in the catchment and remove inorganic N from the soil water before it enters the creek. These riparian buffer zones are common in most agricultural catchments of southeast Queensland and are indicated to reduce the impact of agriculture on stream water quality and subsequently on the estuary and marine environments. This reduction is expressed by a significant decrease in DIN concentrations from 1.6 mg N L..1 to 0.09 mg N L..1, and a decrease in the �15N signatures from upstream surface water locations downstream to the outlet of the agricultural subcatchment. Further testing is, however, necessary to confirm these processes. Most importantly, the amount of N that is transported to the adjacent estuary is shown to be negligible. The third and fourth components of the thesis use a hydrological catchment model approach to determine the water balance of the Elimbah Creek catchment. The model is then used to simulate the effects of land use on the water balance and nutrient loads of the study area. The tool that is used is the internationally widely applied Soil and Water Assessment Tool (SWAT). Knowledge about the water cycle of a catchment is imperative in nutrient studies as processes such as rainfall, surface runoff, soil infiltration and routing of water through the drainage system are the driving forces of the catchment nutrient cycle. Long-term information about discharge volumes of the creeks and rivers do, however, not exist for a number of agricultural catchments in southeast Queensland, and such information is necessary to calibrate and validate numerical models. Therefore, a two-step modelling approach was used to calibrate and validate parameters values from a near-by gauged reference catchment as starting values for the ungauged Elimbah Creek catchment. Transposing monthly calibrated and validated parameter values from the reference catchment to the ungauged catchment significantly improved model performance showing that the hydrological model of the catchment of interest is a strong predictor of the water water balance. The model efficiency coefficient EF shows that 94% of the simulated discharge matches the observed flow whereas only 54% of the observed streamflow was simulated by the SWAT model prior to using the validated values from the reference catchment. In addition, the hydrological model confirmed that total surface runoff contributes the majority of flow to the surface water in the catchment (65%). Only a small proportion of the water in the creek is contributed by total base-flow (35%). This finding supports the results of the stable isotopes 16O/18O and 2H/H, which show the main source of water in the creeks is either from local precipitation or irrigation waters delivered by surface runoff; a contribution from the groundwater (baseflow) to the creeks could not be identified using 16O/18O and 2H/H. In addition, the SWAT model calculated that around 68% of the rainfall occurring in the catchment is lost through evapotranspiration reflecting the prevailing long-term drought conditions that were observed prior and during the study. Stream discharge from the forested subcatchment was an order of magnitude lower than discharge from the agricultural Six Mile Creek subcatchment. A change in land use from forestry to agriculture did not significantly change the catchment water balance, however, nutrient loads increased considerably. Conversely, a simulated change from agriculture to forestry resulted in a significant decrease of nitrogen loads. The findings of the thesis and the approach used are shown to be of value to catchment water quality monitoring on a wider scale, in particular the implications of mixed land use on nutrient forms, distributions and concentrations. The study confirms that in the tropics and subtropics the water balance is affected by extended dry periods and seasonal rainfall with intensive storm events. In particular, the comprehensive data set of inorganic and organic N and P forms in the surface and groundwaters of this subtropical setting acquired during the one year sampling program may be used in similar catchment hydrological studies where these detailed information is missing. Also, the study concludes that riparian buffer zones along the catchment drainage system attenuate the transport of nitrogen from agricultural sources in the surface water. Concentrations of N decreased from upstream to downstream locations and were negligible at the outlet of the catchment.

Metabolic and ecological study of environmental pentose utilizing bacteria (E-PUB)

Lignocellulosic materials, such as sugar cane bagasse, a waste product of the sugarcane processing industry, agricultural residues and herbaceous crops, may serve as an abundant and comparatively cheap feedstock for largescale industrial fermentation, resulting in the production of marketable end-products. However, the complex structure of lignocellulosic materials, the presence of various hexose and pentose sugars in the hemicellulose component, and the presence of various compounds that inhibit the organisms selected for the fermentation process, all constitute barriers that add to the production costs and make full scale industrial production economically less feasible. The work presented in this thesis was conducted in order to screen microorganisms for ability to utilize pentose sugars derived from the sugar mill industrial waste. A large number of individual bacterial strains were investigated from hemi-cellulose rich material collected at the Proserpine and Maryborough sugar mills, notably soil samples from the mill sites. The research conducted to isolation of six pentose-capable Gram-positive organisms from the actinomycetes group by using pentose as a sole carbon source in the cultivation process. The isolates were identified as Corynebacterium glutamicum, Actinomyces odontolyticus, Nocardia elegans, and Propionibacterium freudenreichii all of which were isolated from the hemicellulose-enriched soil. Pentose degrading microbes are very rare in the environment, so this was a significant discovery. Previous research indicated that microbes could degrade pentose after genetic modification but the microbes discovered in this research were able to naturally utilize pentose. Six isolates, identified as four different genera, were investigated for their ability to utilize single sugars as substrates (glucose, xylose, arabinose or ribose), and also dual sugars as substrates (a hexose plus a pentose). The results demonstrated that C. glutamicum, A. odontolyticus, N. elegans, and P. freudenreichii were pentose-capable (able to grow using xylose or other pentose sugar), and also showed diauxie growth characteristics during the dual-sugar (glucose, in combination with xylose, arabinose or ribose) carbon source tests. In addition, it was shown that the isolates displayed very small differences in growth rates when grown on dual sugars as compared to single sugars, whether pentose or hexose in nature. The anabolic characteristics of C. glutamicum, A. odontolyticus, N. elegans and P. freudenreichii were subsequently investigated by qualitative analysis of their end-products, using high performance liquid chromatography (HPLC). All of the organisms produced arginine and cysteine after utilization of the pentose substrates alone. In addition, P. freudenreichii produced alanine and glycine. The end-product profile arising from culture with dual carbon sources was also tested. Interestingly, this time the product was different. All of them produced the amino acid glycine, when grown on a combination substrate-mix of glucose with xylose, and also glucose with arabinose. Only N. elegans was able to break down ribose, either singly or in combination with glucose, and the end-product of metabolism of the glucose plus ribose substrate combination was glutamic acid. The ecological analysis of microbial abundance in sugar mill waste was performed using denaturing gradient gel electrophoresis (DGGE) and also the metagenomic microarray PhyloChip method. Eleven solid samples and seven liquid samples were investigated. A very complex bacterial ecosystem was demonstrated in the seven liquid samples after testing with the PhyloChip method. It was also shown that bagasse leachate was the most different, compared to all of the other samples, by virtue of its richness in variety of taxa and the complexity of its bacterial community. The bacterial community in solid samples from Proserpine, Mackay and Maryborough sugar mills showed huge diversity. The information found from 16S rDNA sequencing results was that the bacterial genera Brevibacillus, Rhodospirillaceae, Bacillus, Vibrio and Pseudomonas were present in greatest abundance. In addition, Corynebacterium was also found in the soil samples. The metagenomic studies of the sugar mill samples demonstrate two important outcomes: firstly that the bagasse leachate, as potentially the most pentose-rich sample tested, had the most complex and diverse bacterial community; and secondly that the pentose-capable isolates that were initially discovered at the beginning of this study, were not amongst the most abundant taxonomic groups discovered in the sugar mill samples, and in fact were, as suspected, very rare. As a bioprospecting exercise, therefore, the study has discovered organisms that are naturally present, but in very small numbers, in the appropriate natural environment. This has implications for the industrial application of E-PUB, in that a seeding process using a starter culture will be necessary for industrial purposes, rather than simply assuming that natural fermentation might occur.

Engineering the surface of a sugarcane roller mill roll for grip and durability

Roller mills are typically used to crush sugarcane to express the juice from which sugar is manufactured. The mill rolls need to provide sufficient grip to ensure minimal sliding of the sugarcane along the roll surface. The rolls are subject to pressures up to 55 MPa from the sugarcane bagasse (as the sugarcane is called after first being crushed between a pair of rolls). The insoluble component of sugarcane includes typically 10% ash that largely originates from soil that is harvested with the cane. The sugarcane juice is acidic with pH typically between 5.0 and 5.5. As a result of ash and juice, the mill rolls are subjected to a range of abrasive and corrosive wear mechanisms. Solutions to provide grip and resist wear involve the selection of an appropriate roll shell material and compatible hard facing to provide the desired grip and wear characteristics. This paper reviews the various solutions that have been adopted for grip and durability for mill rolls and highlights the advantages and disadvantages of each method.

Study of structural changes of pumpkin tissue before and after mechanical loading

The texture of agricultural crops changes during harvesting, post harvesting and processing stages due to different loading processes. There are different source of loading that deform agricultural crop tissues and these include impact, compression, and tension. Scanning Electron Microscope (SEM) method is a common way of analysing cellular changes of materials before and after these loading operations. This paper examines the structural changes of pumpkin peel and flesh tissues under mechanical loading. Compression and indentation tests were performed on peel and flesh samples. Samples structure were then fixed and dehydrated in order to capture the cellular changes under SEM. The results were compared with the images of normal peel and flesh tissues. The findings suggest that normal flesh tissue had bigger size cells, while the cellular arrangement of peel was smaller. Structural damage was clearly observed in tissue structure after compression and indentation. However, the damages that resulted from the flat end indenter was much more severe than that from the spherical end indenter and compression test. An integrated deformed tissue layer was observed in compressed tissue, while the indentation tests shaped a deformed area under the indenter and left the rest of the tissue unharmed. There was an obvious broken layer of cells on the walls of the hole after the flat end indentations, whereas the spherical indenter created a squashed layer all around the hole. Furthermore, the influence of loading was lower on peel samples in comparison with the flesh samples. The experiments have shown that the rate of damage on tissue under constant rate of loading is highly dependent on the shape of equipment. This fact and observed structural changes after loading underline the significance of deigning post harvesting equipments to reduce the rate of damage on agricultural crop tissues.

Learning in Helicoverpa armigera (Lepidoptera: Noctuidae): a new look at the behaviour and control of a polyphagous pest

Recent experimental evidence has shown that learning occurs in the host selection behaviour of Helicoverpa armigera (Hübner), one of the world‘s most important agricultural pests. This paper discusses how the occurrence of learning changes our understanding of the host selection behaviour of this polyphagous moth. Host preferences determined from previous laboratory studies may be vastly different from preferences exhibited by moths in the field, where the abundance of particular hosts may be more likely to determine host preference. In support of this prediction, a number of field studies have shown that the ‘attractiveness’ of different hosts for H. armigera oviposition may depend on the relative abundance of these host species. Insect learning may play a fundamental role in the design and application of present and future integrated pest management strategies such as the use of host volatiles, trap crops and resistant crop varieties for monitoring and controlling this important pest species

Generating transgenic banana (cv. Sukali Ndizi) resistant to Fusarium Wilt

Banana is one of the world’s most popular fruit crops and Sukali Ndizi is the most popular dessert banana in the East African region. Like other banana cultivars, Sukali Ndizi is threatened by several constraints, of which the Fusarium wilt disease is the most destructive. Fusarium wilt is caused by a soil-borne fungus, Fusarium oxysporum f.sp. cubense (Foc). No effective control strategy currently exists for this disease and although disease resistance exists in some banana cultivars, introducing resistance into commercial cultivars by conventional breeding is difficult because of low fertility. Considering that conventional breeding generates hybrids with additional undesirable traits, transformation is the most suitable way of introducing resistance in the banana genome. The success of this strategy depends on the availability of genes for genetic transformation. Recently, a novel strategy involving the expression of anti-apoptosis genes in plants was shown to result in resistance against several necrotrophic fungi, including Foc race 1 in banana cultivar Lady Finger. This thesis explores the potential of a plant-codon optimised nematode anti-apoptosis gene (Mced9) to provide resistance against Foc race 1 in dessert banana cultivar Sukali Ndizi. Agrobacterium-mediated transformation was used to transform embryogenic cell suspension of Sukali Ndizi with plant expression vector pYC11, harbouring maize ubiquitin promoter driven Mced9 gene and nptII as a plant selection marker. A total of 42 independently transformed lines were regenerated and characterized. The transgenic lines were multiplied, infected and evaluated for resistance to Foc race 1 in a small pot bioassay. The pathogenicity of the Ugandan Foc race 1 isolate used for infection was pre-determined and the spore concentration was standardised for consistent infection and symptom development. This process involved challenging tissue culture plants of Sukali Ndizi, a Foc race 1 susceptible cultivar and Nakinyika, an East African Highland cultivar known to be resistant to Foc race 1, with Fusarium inoculum and observing external and internal disease symptom development. Rhizome discolouration symptoms were the best indicators of Fusarium wilt with yellowing being an early sign of disease. Three transgenic lines were found to show significantly less disease severities compared to the wild-type control plants after 13 weeks of infection, indicating that Mced9 has the potential to provide tolerance to Fusarium wilt in Sukali Ndizi.

Hydrogeological framework, conceptual and numerical groundwater flow model of Laidley Creek catchment, Queensland, Australia

This thesis studies the water resources of Laidley Creek catchment within the Lockyer Valley where groundwater is used for intensive irrigation of crops. A holistic approach was used to consider groundwater within the total water cycle. The project mapped the geology, measured stream flows and groundwater levels, and analysed the chemistry of the waters. These data were integrated within a catchment-wide conceptual model, including historic and rainfall records. From this a numerical simulation was produced to test data validity and develop predictions of behaviour, which can support management decisions, particularly in times of variable climate.

The use of artificial neural networks for identifying sustainable biodiesel feedstocks

Over the past few decades, biodiesel produced from oilseed crops and animal fat is receiving much attention as a renewable and sustainable alternative for automobile engine fuels, and particularly petroleum diesel. However, current biodiesel production is heavily dependent on edible oil feedstocks which are unlikely to be sustainable in the longer term due to the rising food prices and the concerns about automobile engine durability. Therefore, there is an urgent need for researchers to identify and develop sustainable biodiesel feedstocks which overcome the disadvantages of current ones. On the other hand, artificial neural network (ANN) modeling has been successfully used in recent years to gain new knowledge in various disciplines. The main goal of this article is to review recent literatures and assess the state of the art on the use of ANN as a modeling tool for future generation biodiesel feedstocks. Biodiesel feedstocks, production processes, chemical compositions, standards, physio-chemical properties and in-use performance are discussed. Limitations of current biodiesel feedstocks over future generation biodiesel feedstock have been identified. The application of ANN in modeling key biodiesel quality parameters and combustion performance in automobile engines is also discussed. This review has determined that ANN modeling has a high potential to contribute to the development of renewable energy systems by accelerating biodiesel research.

Strategies for improved disease resistance in micro-propagated bananas

Bananas (Musa sp) are one of the most important food crops in the world and provide a staple food and source of income in many households especially in Africa. Diseases are a major constraint to production with bunchy top, caused by Banana bunchy top virus (BBTV) generally considered the most important virus disease of bananas worldwide. Of the fungal diseases, Fusarium wilt, caused by the Fusarium oxysporum f.sp cubense (Foc), and black Sigatoka, caused by Mycosphaerella fijiensis, are arguably two of the most important and cause significant yield losses. The low fertility of commercially important banana cultivars has hampered efforts to generate disease resistance using conventional breeding. Possible alternative strategies to generate or increase disease resistance are through genetic engineering or by manipulation of the innate plant defence mechanisms, namely systemic acquired resistance (SAR). The first research component of this thesis describes attempts to generate BBTV-resistant banana plants using a genetic modification approach. The second research component of the thesis focused on the identification of a potential marker gene associated with SAR in banana plants and a comparison of the expression levels of the marker gene in response to biotic and abiotic stresses, and chemical inducers. Previous research at QUT CTCB showed that replication of BBTV DNA components in banana embryogenic cell suspensions (ECS) was abolished following co-bombardment with 1.1mers of mutated BBTV DNA-R. BBTV DNA-R encodes the master replication protein (Rep) and is the only viral protein essential for BBTV replication. In this study, ECS of banana were stably transformed with the same constructs, each containing a different mutation in BBTV DNA-R, namely H41G, Y79F and K187M, to examine the effect on virus replication in stably transformed plants. Cells were also transformed with a construct containing a native BBTV Rep. A total of 16, 16, 11 and five lines of stably transformed banana plants containing the Y79F, H41G, K187M and native Rep constructs, respectively, were generated. Of these, up to nine replicates from Y79F lines, four H41G lines, seven K187M lines and three native Rep lines were inoculated with BBTV by exposure to viruliferous aphids in two separate experiments. At least one replicate from each of the nine Y79F lines developed typical bunchy top symptoms and all tested positive for BBTV using PCR. Of the four H41G lines tested, at least one replicate from three of the lines showed symptoms of bunchy top and tested positive using PCR. However, none of the five replicates of one H41G line (H41G-3) developed symptoms of bunchy top and none of the plants tested positive for BBTV using PCR. Of the seven K187M lines, at least one replicate of all lines except one (K187M-1) developed symptoms of bunchy top and tested positive for BBTV. Importantly, none of the four replicates of line K187M-1 showed symptoms or tested positive for BBTV. At least one replicate from each of the three native Rep lines developed symptoms and tested positive for BBTV. The H41G-3 and K187M-1 lines possibly represent the first transgenic banana plants generated using a mutated Rep strategy. The second research component of this thesis focused on the identification of SAR-associated genes in banana and their expression levels in response to biotic and abiotic stresses and chemical inducers. The impetus for this research was the observation that tissue-cultured (TC) banana plants were more susceptible to Fusarium wilt disease (and possibly bunchy top disease) than plants grown from field-derived suckers, possibly due to decreased levels of SAR gene expression in the former. In this study, the pathogenesis-related protein 1 (PR-1) gene was identified as a potential marker for SAR gene expression in banana. A quantitative real-time PCR assay was developed and optimised in order to determine the expression of PR-1, with polyubiquitin (Ubi-1) found to be the most suitable reference gene to enable relative quantification. The levels of PR-1 expression were subsequently compared in Lady Finger and Cavendish (cv. Williams) banana plants grown under three different environmental conditions, namely in the field, the glass house and in tissue-culture. PR-1 was shown to be expressed in both cultivars growing under different conditions. While PR-1 expression was highest in the field grown bananas and lowest in the TC bananas in Lady Finger cultivar, this was not the case in the Cavendish cultivar with glass house plants exhibiting the lowest PR-1 expression compared with tissue culture and field grown plants. The important outcomes of this work were the establishment of a qPCR-based assay to monitor PR-1 expression levels in banana and a preliminary assessment of the baseline PR-1 expression levels in two banana cultivars under three different growing conditions. After establishing the baseline PR-1 expression levels in Cavendish bananas, a study was done to determine whether PR-1 levels could be increased in these plants by exposure to known banana pathogens and non-pathogens, and a known chemical inducer of SAR. Cavendish banana plants were exposed to pathogenic Foc subtropical race 4 (FocSR4) and non-pathogenic Foc race 1 (Foc1), as well as two putative inducers of resistance, Fusarium lycopersici (Fol) and the chemical, acibenzolar-S-methyl (BION®). Tissue culture bananas were acclimatised under either glass house (TCS) or field (TCH) conditions and treatments were carried out in a randomised complete block design. PR-1 expression was determined using qPCR for both TCS and TCH samples for the period 12-72h post-exposure. Treatment of TCH plants using Foc1 and FocSR4 resulted in 120 and 80 times higher PR-1 expression than baseline levels, respectively. For TCS plants treated with Foc1, PR-1 expression was 30 times higher than baseline levels at 12h post-exposure, while TCS plants treated with FocSR4 showed the highest PR-1 expression (20 times higher than baseline levels) at 72h post-exposure. Interestingly, when TCS plants were treated with Fol there was a marked increase of PR-1 expression at 12 h and 48 h following treatment which was 4 and 8 times higher than the levels observed when TCS plants were treated with Foc1 and FocSR4, respectively. In contrast, when TCH plants were treated with Fol only a slight increase in PR-1 expression was observed at 12 h, which eventually returned to baseline levels. Exposure of both TCS and TCH plants to BION® resulted in no effect on PR-1 expression levels at any time-point. The major outcome of the SAR study was that the glass house acclimatised tissue culture bananas exhibited lower PR-1 gene expression compared to field acclimatised tissue culture plants and the identification of Fol as a good candidate for SAR induction in banana plants exhibiting low PR-1 levels. A number of outcomes that foster understanding of both pathogen-derived and plant innate resistance strategies in order to potentially improve banana resistance to diseases were explored in this study and include identification of potential inducers of systemic acquired resistance and a promising mutated Rep approach for BBTV resistance. The work presented in this thesis is the first report on the generation of potential BBTV resistant bananas using the mutated Rep approach. In addition, this is the first report on the status of SAR in banana grown under different conditions of exposure to the biotic and abiotic environment. Further, a robust qPCR assay for the study of gene expression using banana leaf samples was developed and a potential inducer of SAR in tissue culture bananas identified which could be harnessed to increase resistance in tissue culture bananas.

In Plant Activation: An inducible, hyperexpression platform for recombinant protein production in plants

In this study, we describe a novel protein production platform that provides both activation and amplification of transgene expression in planta. The In Plant Activation (INPACT) system is based on the replication machinery of tobacco yellow dwarf mastrevirus (TYDV) and is essentially transient gene expression from a stably transformed plant, thus combining the advantages of both means of expression. The INPACT cassette is uniquely arranged such that the gene of interest is split and only reconstituted in the presence of the TYDV-encoded Rep/RepA proteins. Rep/RepA expression is placed under the control of the AlcA:AlcR gene switch, which is responsive to trace levels of ethanol. Transgenic tobacco (Nicotiana tabacum cv Samsun) plants containing an INPACT cassette encoding the b-glucuronidase (GUS) reporter had negligible background expression but accumulated very high GUS levels (up to 10% total soluble protein) throughout the plant, within 3 d of a 1% ethanol application. The GUS reporter was replaced with a gene encoding a lethal ribonuclease, barnase, demonstrating that the INPACT system provides exquisite control of transgene expression and can be adapted to potentially toxic or inhibitory compounds. The INPACT gene expression platform is scalable, not host-limited, and has been used to express both a therapeutic and an industrial protein.

Effect of pretreatment on saccharification of sugarcane bagasse by complex and simple enzyme mixtures

Saccharification of sugarcane bagasse pretreated at the pilot-scale with different processes (in combination with steam-explosion) was evaluated. Maximum glucan conversion with Celluclast 1.5 L (15–25 FPU/g glucan) was in the following order: glycerol/HCl > HCl > H2SO4 > NaOH, with the glycerol system achieving ∼100% conversion. Surprisingly, the NaOH substrate achieved optimum saccharification with only 8 FPU/g glucan. Glucan conversions (3.6–6%) obtained with mixtures of endo-1,4-β-glucanase (EG) and β-glucosidase (βG) for the NaOH substrate were 2–6 times that of acid substrates. However, glucan conversions (15–60%) obtained with mixtures of cellobiohydrolase (CBH I) and βG on acidified glycerol substrate were 10–30% higher than those obtained for NaOH and acid substrates. The susceptibility of the substrates to enzymatic saccharification was explained by their physical and chemical attributes. Acidified glycerol pretreatment offers the opportunity to simplify the complexity of enzyme mixtures required for saccharification of lignocellulosics.