School of Agriculture, Food and Ecosystem Sciences - Theses

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Salinity control, water reform and structural adjustment : the Tragowel Plains Irrigation District

Barr, Neil F. (University of Melbourne, 1999)
Epidemiology of mint rust and variation in the Pathogen, Puccinia menthae Pers

Edwards, Jacqueline. (University of Melbourne, 1998)
Genetics of resistance to Heterodera avenae in Triticum tauschii and its transfer to bread wheat (Triticum aestivum)

Eastwood, Russell Francis. (University of Melbourne, 1995)
A genetic evaluation of dystocia in Australian Holstein-friesian cattle

McClintock, Sara Elizabeth Juliette (University of Melbourne, 2004)

This thesis presents the first large-scale study of the genetic and non-genetic influences on dystocia (calving difficulty) for dairy cows in Australia, and their costs, focusing especially on the Holstein-Friesian breed, but also with some analyses of frequently occurring crossbreeds. Analyses used data collected since 1986, collected by the Holstein-Friesian Association of Australian and the Australian Dairy Herd Improvement Scheme. The calving traits examined were gestation length, calf size, dystocia (measured as any or none, severe or none, and none, slight and severe). I investigated the influence on calving ease of non-genetic variables such as month of calving, cow age or parity, calf sex, and breed of cow and bull. The genetic parameters were estimated: the heritabilities and genetic correlations between traits calculated, separately for primiparous and multiparous, and for sires, maternal grandsires and the maternal effects. Costs associated with dystocia (such as labour costs, loss or fertility, veterinary costs and deaths of cow and or calf) are estimated, and a cost model for dystocia under Australian conditions is proposed. The influence of crossbreeding on calving was investigated, especially with respect to dystocia and calf mortality. Recommendations are made for improving the recording system and the evaluation of bulls, as the sire of calf and as the sire of cow.
Processes and implications of scald formation on the Eastern Dundas tableland : a case study

Fawcett, Jonathon Duke (University of Melbourne, 2004)

This thesis develops an understanding of the processes driving the formation of iron and saline rich scalds in groundwater discharge zones on the Eastern Dundas Tableland. The thesis tests the hypothesis that: The development of land degradation patterns around groundwater discharge zones on the Eastern Dundas Tablelands is not driven by salt concentrations, a consequence of water tables that have risen since European settlement and land clearing, but is the result of the disturbance to the soil environment of discharge zones since land clearing. The research proved the hypothesis correct and identified the transmission of hydrogen sulfide within the groundwater as a key factor in the severity of the degradation. The research also found land clearing may have altered the seasonal flow systems operating within the regolith, which are responsible for the spread of salinity and erosion within primary groundwater discharge zones. The Eastern Dundas Tablelands consist of a fractured rock aquifer, where the unweathered ignimbrites and lavas are hydrologically connected to the overlying regolith. Analyses of groundwater levels, pressures and chemistry indicate the groundwater flow paths to discharge zones increase in length with depth, such that discharge water is sourced from groundwater varying in depth of circulation (up to 150 metres) and flow path length (tens of metres to kilometres). A portion of discharge water flows through the unweathered volcanic aquifer and equilibrates with the rock, becoming reduced. The groundwater flow therefore contains hydrogen sulfide and has reducing capacity. This water then flows preferentially along large scale structures towards discharge zones. The location of groundwater discharge zones is controlled by the intersection between horizontal and sub vertical subsurface structures. Iron and saline rich scalding only occurs where groundwater flow with reducing capacity and containing hydrogen sulphide discharges. Iron and saline scalding is absent where only seasonal groundwater discharge not containing hydrogen sulfide occurs. Measurements of the discharge zones' soil and water EC, Ph and redox potential and the soil chemistry indicate degradation is the result of a combination of processes and is not solely the result of soil salting. Discharge water in wet months is covered by bacteria growth that reduces and oxidises iron and sulfur. During dry periods distinct iron precipitates form crusts on soil surfaces and clog soil pores. Salt efflorescence forms on soil surface and the oxidation of ferrous sulfide creates severe acidity. Evaporation accumulates salts in the discharge zones that are then spread downslope by seasonal break-of-slope discharge. Break of slope discharges erode highly sodic A horizon soils, radially expanding the area of degradation. Relatively constant groundwater levels, historical information and the carbon age of discharge water (2540 years) suggest discharge at the soil surface of the Eastern Dundas Tablelands occurred at least as early as the first records of European settlement. Little to no evidence exists suggest rising groundwater levels, caused by increased recharge since land clearing, have initiated the degradation of discharge zones. The pH, redox potential and measured quantities of ferrous material and sulfur in groundwater suggest iron and saline scalding is initiated by the interaction of the reduced groundwater of the regional groundwater flow and reducible material (iron) in the discharge zone. Prior to the clearing of native vegetation, reduced groundwater flow was able to discharge at the soil surface without coming into contact with reducible material (iron). Any hydrogen sulfide in discharge water was able to dissipate into the atmosphere, removing the reducing capacity of the water. Since clearing, iron has been redistributed into groundwater flow paths. Iron is reduced by the groundwater flow before the hydrogen sulfide is able to dissipate with ferrous and iron sulfide rich minerals forming within discharge zones. Upon oxidation, iron rich precipitates (ferrihydrite and schwertmannite) form in soil pores and along the soil surface with the oxidation of ferrous sulfide creating severe acidity (pH <4). The degree of degradation is directly related to the rate and volume of groundwater discharge. The least degraded areas coincide with point-flowing springs, the wettest areas of iron and saline scalds. Within point flowing springs, permanent saturation prevents the oxidation of ferrous sulfide material, the formation of iron crust and the accumulation of salts via evaporation. The most severe degradation coincides with areas of diffuse discharge, where the drying of the soil surface leads to iron precipitate formation, salt accumulation and severe acidity as ferrous sulfide material oxidises. The process of iron and saline rich scalding can be halted, and the area remediated if: � reducible material is removed from the groundwater discharge zone, preventing the reduced groundwater from mobilising iron and forming iron-sulfide material; and � the discharge zone is fully submersed in water, such as a dam. In this case, groundwater hydrogen sulfide will dissipate into the atmosphere, removing the reducing capacity of groundwater discharge.
Using selective DNA pooling to map QTL affecting milk production traits in Australian dairy cattle

Mariasegaram, Hyacinth Maxy. (University of Melbourne, 2004)
Linkage mapping and QTL analysis of ascochyta blight resistance in chickpea (Cicer arietinum)

Galvez, Hayde Flandez. (University of Melbourne, 2003)

Ascochyta blight is the most destructive foliar disease of chickpea worldwide. Resistance is available in the germplasm, and with a strong evidence of polygenic control, breeding is geared towards gene pyramiding for durable resistance via marker-assisted selection. This thesis generally aimed to map and analyze the quantitative trait loci (QTL) which condition ascochyta blight resistance (ABR) in an intraspecific Cicer arietinum genetic background. An intraspecific population of chickpea was used to create the first linkage map for the C. arietinum genome, and locate six QTL conditioning glasshouse and field resistance to ascochyta blight. Results from QTL analysis revealed a major genomic region that contained clusters of QTL (QTL 4, 5 and 6), and minor QTL specific for glasshouse (QTL 2 and 3) and field (QTL I) resistance. Multiple interval mapping revealed that resistance of chickpea to ascochyta blight was conferred by an epistatic interaction between the additive gene action of the major QTL and the dominance gene action of the other (minor) QTL. RGA markers PTOFENb212 and CLRRinv904 were found tightly-linked to the major ABR-QTL region. By sequence alignment, the RGA markers were confirmed to be the corresponding resistance gene orthologs (RGOs) in chickpea. Allele- and locus specific SCAR markers targeting the RGOs were developed and shown to be transferable to other resistance genotypes. The RGOs were also used as starting points to isolate the underlying major genes or cluster of genes for ABR. 5'-cDNA sequences of the major ABR genes, ArFEN and ArLRR, were then characterized. These are the first candidate gene sequences for ascochyta blight resistance identified in chickpea by a map-based candidate gene approach. Based on the homology alignment of ArFEN and ArLRR, and sequence information of the RGOs with published gene sequences, the R-genes underlying the major QTL region may be classified under the third group of R-genes that contains a serine-threonine protein kinase domain, with a leucine-rich repeat. Homology alignment of the candidate genes also revealed that the mechanism of resistance reaction conferred by the major ABR genes in chickpea was most likely that of a hypersensitive response.
Expression of a recombinant mycobacterial antigen and characterization of fas-defective mutant strains in corynebacterium glutamicum and related species

Chang, Shin-Jae (University of Melbourne, 2003)

The research undertaken in this thesis had the initial aim of investigating the utility of Corynebacterium glutamicum species as host for synthesis of foreign proteins of biotechnological importance, given that earlier research had shown that this industrially-important species, normally used for amino acid production, was able to express and secrete a variety of proteins from different bacterial sources. Of particular interest was the use of this species in making proteins that may be secreted or located at the cell surface, where their expression may have been important in antigenicity or other physiological functions: proteins made and secreted by related organisms, including pathogenic Mycobacterium species, were of particular interest because many pathogens cannot he cultured readily in the laboratory environment or exist as obligate intracellular parasites. It was also likely that the C. glutamicum and Mycobacterium species would share functionality in their cell surface proteins, an assumption based on their common synthesis of mycolic acids complex surface lipids that are involved in making cells from this group resistant to chemical and physical assault. The first part of the thesis describes a study on the synthesis and secretion of the 85 antigen complex, component of which are important immunogens in Mycobacterium and are secreted proteins with activity involved in the attachment of mycolic acids to the cell wall. During the course of this study, PCR amplification using primer sets based on the 85B antigen complex from M. bovis was followed up by sequencing the PCR products. Rather than corresponding to an 85B homologue, the DNA had high sequence similarity to a fatty acid synthase (FAS) gene previously found in Brevibacterium (now Corynebacterium) ammoniagenes. The subsequent focus of the thesis was on characterization of the presumptive fas gene in C. glutamicum in terms of gene inactivation and impact on lipid synthesis, for both fatty acids and mycolic acids. The first part of this project was to undertake controls for later experiments if the mycobacterial antigen 85 complex components (A, B and C) or any homologue of these were present in Corynebacterium glutamicum AS019 and its related strains, C. glutamicum ATCC13032, C. glutamicum CG2, C. lactofermentum BL1 and C. flavum BF4. This involved determining on the expression of a recombinant antigen 85 complex in C. glutamicum AS019 and related cell-surface mutants (MLB 133 and MLB 194). In order to detect the presence of the mycobacterial antigen 85 complex (A, B and C) or homologues, several experiments, including Polymerase Chain Reaction (PCR), Southern hybridization and Western blotting experiments, were carried out. The screening focused on the antigen 85B as this component is the most abundant of the antigen 85 complex in Mycobacterium species and there is considerable similarity between antigen 85 complex members at both nucleotide and amino acid sequence levels. Furthermore, antigen 85B was most extensively studied in the literature so that the materials needed to study this (such as PCR primers, antibody and probes for Southern hybridization experiments) were easier to make or obtain from other research groups. A PCR Mal 1/21 primer set was designed based on the most conservative regions of the antigen 85B from several species of Mycobacterium. Amplification produced several feint products from C. glutamicum AS019, C. lactofermentum BL1 and C. flavum BF4, which was an unexpected outcome. All the major PCR products were cloned into the pGEM�T vector and sequenced to determine similarity with the sequence of mycobacterial antigen 85 complex. However, the �0.8 kb PCR products from C. glutamicum AS019 and C. lactofermentum BL1 showed relatively high similarity (-70%) with a fatty acid synthase gene (fasA) of C. ammoniagenes and �60% with the fasA found in M. tuberculosis. Similar PCR products were found in all of the C. glutamicum strains tested. In Southern hybridization experiments, two different probes were used for more intensive analysis. One was the 0.8 kb PCR product produced by the Mal 1/21 primer set when pGEX-MPB59 (an expression vector which contains a mycobacterial antigen 85B gene) was used as template. The other was a 0.49 kb fragment produced by PstI/Xhol double-digestion of the above 0.8 kb PCR product. Relatively lower prehybridization/hybridization temperature (55�C) and longer expose time were employed to detect any loosely-bound signal, however, no specific signal was detected using gDNA from C. glutamicum AS019 and its related strains. In Western blotting experiments, anti-antigen 85B antibody raised in rabbits was used as the primary antibody to detect any signal from whole disrupted cells and culture filtrate of C. glutamicum AS019 and its related strains. No signal was found using culture filtrates concentrated 50 fold, however, a signal was found using disrupted cells of all strains. To determine the location of this protein, cell wall proteins were extracted from whole cells with 50 mM Tris buffer containing 2% Sodium Dodecyl Sulfate (SDS). No signal was found from cell wall extracts so that this protein may be located in the inner membrane or cytosol. The size of this protein was almost 40 kDa which is significantly bigger than the mycobacterial antigen 85 complex (B is 32 kDa and A and C are 30 kDa). Even though a signal was found from Western blotting experiments, it was concluded that there was no antigen 85 complex homologues present in C. glutamicum due to the negative results from PCR and Southern hybridization experiments. Several strains were then transformed with pCGL1055, which contains the antigen 85A gene, to determine the secretion level of recombinant antigen 85A in C. glutamicum AS019, C. glutamicum MLB133, C. glutamicum MLB194, C. lactofermentum BL1 and C. flavum BF4. Since pCGL1055 had the cspB promotor, two sets of primer were used to confirm the presence of the cspB gene (coding for PS2 protein in corynebacteria) in the strains used in this expression work. To quantify the amount of secreted recombinant antigen 85A, recombinant antigen 85B was prepared as the standard using Glutathione Sepharose 4B� affinity chromatography followed by thrombin cleavage and gel filtration chromatography using material prepared from E. coli TG2 transformed with pGEX-MPB59, the expression vector which contains the antigen 85B gene. C. glutamicum MLB 133 and MLB 194 showed relatively higher secretion level than the parent strain, C. glutamicum AS019, which suggests that the changes in the cell wall structure of the mutants caused better secretion of the foreign recombinant protein. C. flavum BF4 also showed relatively higher secretion levels than AS019. Volumetric and cellular productivity for the expressed recombinant antigen 85A from several corynebacterial strains were calculated to determine the efficiency of strains in secreting the protein into culture fluids. The amount of 85A protein secreted was expressed as the equivalent of 85B protein measured in parallel using anti-antigen 85B. In volumetric productivity from C. glutamicum strains AS019 (spontaneous Rif mutant of C. glutamicum ATCC 13059), MLB 133 and MLB 194 (the cell wall modified mutants of C. glutamicum ATCC 13059) was 16-17 ng/ml. However, C. lactofermentum BL1 and C. flavum BF4 expressed relatively higher amounts of antigen 85A (22-23 ng/ml) than the above strains. Cellular productivity was determined by calculating the amount of antigen 85A secreted from the given number of cells of each transformant by adjusting the optical density of each overnight cultured culture filtrate to 100 at 600nm. The cell wall modified mutants, C. glutamicum MLB 133 and C. glutamicum MLB194, showed higher efficacy (730-775 ng/ml) in expressing antigen 85A from the given number of cells than its wild-type strain, C. glutamicum AS019 (440 ng/ml). C. flavum BF4 showed almost the same efficiency (about 750 ng/ml) as the mutants. The new finding of a fas-gene homologue using PCR amplification prompted further studies aimed at characterizing its role in synthesis of fatty acids and mycolic acids in C. glutamicum. The inactivation of a gene by homologous recombination was used to characterize the fas-gene homologue, employing the 0.8 kb fragment found in the previous PCR experiments. The 0.65 kb fragment, the nested PCR product from the above 0.8 kb fragment, was amplified using the fas11/21-EcoR I-1 primer set. The amplified 0.65 kb fragment was digested with EcoR I and cloned into pECMA, a suicide vector that does not replicate in Corynebacterium strains without homologous recombination between the insert and recipient chromosomal DNA due to the lack of a replication origin on the vector. The newly constructed suicide vector, pECMAfas-AS019, was introduced into the donor strain, E. coli S17-1, a mobilizing strain constructed by the integration of self-transmissible (Tra') plasmid, RP4 2-Tc
Evaluation of dual energy x-ray absorptiometry for predicting whole body and carcass composition of pigs

Suster, Danny. (University of Melbourne, 2003)

This dissertation aims to evaluate the dual energy X-ray absorptiometry (DXA) technique for measuring whole body and carcass composition of pigs. Section 1 provides the appropriate calibration for quantitatively accurate DXA measurements of live body and carcass composition of pigs of varying weight, as determined by chemical analysis and manual dissection. The DXA technology provided an accurate precise measurement of composition within experiments and when measurements were compared across experiments. The precision of DXA measurements were far better than weight and P2 backfat, particularly in the prediction of fat tissue, especially when measurements were compared across experiments. The DXA measurements were highly repeatable and measurement repeatability improved as animal size increased. The placement of the regional analysis grid influenced the repeatability of all measurements except for total weight, however this influence reduced with increasing animal size. It is recommended that the scan image be positioned in the arm region of the software regional analysis grid to measure whole body composition in pigs because it provides the most repeatable measure overall and a better measure for fat tissue when compared to chemical analysis and manual dissection. The DXA regional analysis software also provided an accurate and precise measurement of dissectible composition in the primal-cuts, ie. the ham, loin, belly and shoulder. However, care needs to be taken to ensure accurate and consistent delineation of sub-regions to the true position of primal-cuts in the DXA scan image to ensure a reliable measurement. Section 2 involved a series of experiments to evaluate the ability of the DXA technology in measuring the effects of various known and innovative body composition manipulators, all directly applicable to industry. Each experiment demonstrated the use of the developed calibration equations and the efficacy of the DXA technique. Overall, corrective equations were successfully applied to DXA estimates and largely corrected differences between raw DXA outputs and chemical values. The DXA measurements confirmed that diets deficient in protein lead to sub-optimal lean tissue and bone mineral growth with no effect on fat and were substantiated by chemical analysis. Serial DXA measurements in the same animal demonstrated the lifetime differences between contemporary genotype boars and barrows under different penning conditions. These data indicated that although boars have a lower propensity to deposit fat overall, under group penned conditions there is little incentive to produce boars in terms of body composition if slaughter weights were below about 80kg. However, in the late finisher period, boars did deposit more lean tissue than barrows, but this difference was reduced when animals were group-penned. The regional analysis software successfully demonstrated the effects of pST treatment on fat distribution within the animal, and was validated using manual dissection. A dose dependent decrease in belly, loin, ham and shoulder fat was also observed, although the proportionate decrease in belly fat was more pronounced than for the whole carcass and other primal cuts. Finally, the DXA technique was sensitive enough to detect the subtle body composition changes that were induced by supplemental dietary betaine. Dietary betaine improved growth rate and protein deposition, and consequently carcass lean tissue content, in pigs fed restricted energy and the effects were additive with those of pST. Lean tissue in the loin was particularly responsive. The energy spared by betaine was not enough to increase fat deposition, carcass fat or P2 backfat regardless of energy intake. However, dietary betaine did increase fat in the more energy responsive belly region under ad libitum conditions but not under a restricted intake typical of commercial conditions. Therefore, dietary betaine did alter the distribution of lean meat and fat within the animal. These data demonstrate that the DXA technology is a "viable" replacement for the standard methodologies in animal growth and body composition research experiments and has much potential for implementation into the pork industry for carcass grading and providing data for pig growth simulation models.
Remote sensing of leaf area index and land cover classification of mountain forests

Sharma, Dharam Prakash (University of Melbourne, 2002)

The use of multispectral IRS (LISS-III) satellite images for land cover classification and monitoring change dynamics of Leaf Area Index of mountain forests in winter, spring, and summer using IRS (LISS-III) satellite data for two study sites was investigated. The relationships between Leaf Area Index and band radiance / vegetation index were compared in different seasons. The relationships were closer with vegetation indices than the individual band and principal components. In addition the relationships were better for the Solan site than the Narkanda site. Winter and spring relationships were much better than summer. Comparatively, the performance of RATIO and Normalized Difference Vegetation Index was superior to other vegetation indices. To improve these relationships, various topographic corrections such as the Statistic-empirical, the C and the Minnaert correction were applied to the multispectral and multi-date satellite images using Digital Elevation Model. The visual effects were impressive, but the topographic corrections failed to improve LAI-band and LAI-Vegetation Index relationships. The utility of multispectral and multi-temporal IRS (LISS-III) satellite data in land cover classification for Solan study area using a maximum likelihood algorithm on single date as well as multi-date satellite data was investigated. The summer imagery gave higher classification accuracy of 76 per cent than a classification accuracy of 49 per cent and 46 per cent in the winter and the summer respectively. The classification based on four channel multi-temporal data merging, using a set of seven bands showing maximum categorization of land cover types produced a classification accuracy of 71 per cent. Similarly, classification based on the Principal Component Analysis, using first two principal components from each season as well as first seven principal components from multi-date data merging in the classification process obtained a classification accuracy of 71 per cent. The classification accuracy obtained in multi-temporal data merging and Principal Component Analysis approach were higher than classification based on single-date dataset of winter and spring. The topographic corrections applied to multispectral and multi-temporal datasets with an objective to improve the land cover classification reduced within category class variability and improved the visual display, but failed to generate better classification accuracy. Though the LAI and multispectral data relationships were better in winter and spring seasons at one site, the study does not support the use of satellite data to estimate seasonal difference in LAI of mountain forest. However, multi-date multispectral satellite imagery can be useful for achieving better classification accuracy of land cover type for the Himalayan region.

School of Agriculture, Food and Ecosystem Sciences - Theses

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