School of Agriculture, Food and Ecosystem Sciences - Theses

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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.
Integrating reductionist research into international agricultural development : re-conceiving agricultural research for development

Falvey, John Lindsay ( 2003)
Influence of seasonality on yoghurt properties under production in South East Australia

Pollard, Adrian ( 2003)
Bush-fallow systems on Lihir Islands : prospects for change

Baput, Bazakie ( 2003)
In vitro culture of Australian fan flower, scaevola

Wang, Yu-Hua ( 2003)

Genus Scaevola is well known for its ornamental value. However, its breeding potential is limited due to poor seed germination. The aim of this study was to explore the induction of somatic embryogenesis and further develop in vitro cultures at protoplast and cell suspension levels for Scaevola. An efficient and simple protocol to induce somatic embryos directly from leaf expiants was developed. Somatic embryos were initiated optimally when the explants were cultured on MS media supplemented with 0.2mg/L 2,4-D and 0.2-0.5mg/L BAP. Various developmental stages of somatic embryos were found in the initiation media. Under dim light condition, the transfer of globular embryos to MS medium supplemented with 0.5mg/L BAP resulted in 81.2% of the embryos regenerating shoots. The browning of somatic embryos during shoot regeneration was overcome when the cultures were incubated under dim light condition. Mesophyll protoplasts were successfully isolated from the leaves of S. aemula and S. phlebopetala with viabilities of about 90%. Mesophyll protoplasts isolated from S. aemula had a good response to the initiation of cell division. However, less than 1% of mesophyll protoplasts isolated from S. phlebopetala showed cell division due to the lack of cell wall reformation. Mesophyll protoplasts of S. aemula appeared higher dividing and plating efficiencies when they were cultured in rich-nutrient media and embedded in agarose gel. In general, the combination of NAA and BAP had a better effect on producing higher dividing and plating efficiencies than the combination of 2,4-D and BAP. The formation of globular embryos (visible microcolonies) from mesophyll protoplasts of S. aemula was successfully achieved, but browning phenomenon occurred in the protoplast culture. Cell suspensions were initiated from leaf- and root-derived calli of S. aemula. Longer inoculation of callus fragments in cell suspensions (after 3 weeks of inoculation) caused liquid medium turning brown. When both cell suspensions were separated from the initiated callus fragments, the browning phenomenon of liquid medium was eliminated, but the growth of cell suspensions was not observed. Calli were formed when both cell suspensions were transferred to solid callus induction medium. These calli were regenerable. The shoot regeneration frequencies were 19.2% for calli formed from cell suspensions initiated from leaf-derived calli, and 13.9% for calli formed from cell suspensions initiated from root-derived calli. In conclusion, the induction of somatic embryogenesis in S. aemula can serve as a model system to investigate the embryology of Scaevola. Further research may provide a better understanding of recalcitrant seed and low seed viability in Scaevola and related Australian native plants. The continued improvement of protoplast and cell suspension culture systems will facilitate the application of biotechnology in Scaevola breeding programs.
Applicability of pcr based markers linked to the genes conferring resistance to ascohyta lentis in lentils (lens culinaris Medik.)

Kirthisinghe, Jayath Peter ( 2003)

Australia currently produce approximately 266000 tonnes/annum of lentils (Lens culinaris Medikus). Ascochyta blight caused by Ascochyta lentis is a major threat to the lentil production in Australia and worldwide. It has been present in Australia since 1982. It reduced grain yields and caused discolouration of grain and reduced up to 70% of its market value. Ascochyta lentis can be both seedborne and stubble-borne to infect lentil in the next cropping season. However, seed is the main source of disease into new areas. Therefore use of cultivars resistant to ascochyta is the most sustainable method to control yield losses and preventing disease. Lentil breeders in Australia have started selecting lentil lines with resistance to ascochyta blight. Several studies have been reported on the genetics of ascochyta blight to identify the resistance. The researchers (Nguyen et al., 2001; Chowdhury et al., 2001; Ye, 2000; Ford, 1999; Ahmad et al., 1997; Sakr, 1994) proposed several inheritance models for ascochyta blight resistance for both foliar and seed in lentils. Chowdhury et al. (2001) identified two RAPD markers, UBC2271290 and OPD-10870, flanked and were linked to the foliar resistant recessive gene ral2 at 12 and 16 cM, respectively. The SCAR markers developed from UBC2271290 could not detect any polymorphism between the two parents. Therefore, they recommend that the polymorphic RAPD marker UBC2271290 and the SCAR marker developed from OPD-10870 can be used together in a marker assisted selection program for ascochyta blight resistance in lentil. Ford (1999) identified 700 bp SCAR marker for single dominant ascochyta blight foliar resistance gene AbRI linked at a distance of 13 cM in ILL5588 genome. Single gene dominant traits are used in marker-assisted selection. Therefore the SCAR marker linked to the single dominant gene was selected to determine the applicability of the marker to identify the ascochyta resistant lentil accessions for the present study. A study was conducted to screen 158 lentil accessions used as parents in the lentil breeding program for their resistance to ascochyta blight infection and for the presence of the 700 bp SCAR marker linked to AbRI gene. This was done to evaluate the transferability of the SCAR marker to practical application in a lentil breeding program. Foliar disease scores were used to quantify the foliar resistance to ascochyta blight. Pathological bioassays were done using seeds to screen lentil cultivars for rate of seed infection and thus quantify seed resistance to ascochyta blight infection. The identified accessions were tested for presence or absence of the DNA based SCAR marker linked to AbR1 gene. The 700 bp SCAR marker was present in ILL5588 (Northfield) resistant cultivar to ascochyta blight and its crosses and the SCAR marker was absent in ILL6002 susceptible cultivar as Ford (1999) suggested. Therefore it demonstrates that the 700 bp SCAR marker can identify the ascochyta blight resistance in some lentil crosses and also that the A. lentis H2(2) pathotype, or similar, was present in the field where the study was conducted. The SCAR marker identified ascochyta blight resistance correctly in 68% of the study population. The SCAR marker failed to identify some lentil accessions (19% of the study population) with strong foliar resistance to ascochyta blight viz. ILL4549, Matador (ILL8105), Indianhead, ILL7508 and some lentil accessions (about 13% of the study population) very susceptible to ascochyta blight, viz. ILL7517, Palouse, Cumra, Malazgirt89. Therefore the scope for the applicability of 700 bp SCAR marker to identify the foliar resistance of ascochyta blight is limited. The marker is useful for the lentil lines crossed with ILL5588 as a parent and few lentil lines identified other than ILL5588 in the present study. The SCAR marker developed from OPD-10870, linked to the recessive gene ral2 at 16 cM, could have been used to select ral2-derived ascochyta blight resistant lentil accessions such as Indianhead and thus further inform the process of selecting for ascochyta blight resistance beyond that possible using the 700 bp SCAR marker. The identification of ascochyta blight resistant lentil accession using suitable SCAR markers will be shorten the lentil breeding cycle, but needs a further study on different molecular markers, their transferability and their relationships with different pathotypes of ascochyta blight. Ascochyta blight resistance is a trait of interest to the farmers, breeders and traders, but the other traits such as yield, agronomic traits, potential fungal diseases and maturity are also important to breeders for a successful new cultivar. Therefore in this study the data were compared to determine a correlation between ascochyta blight resistance and the quantitative (seed yield) or qualitative (cotyledon colour) traits. Therefore the results of this study will help to identify the lentil lines resistant and susceptible to foliar and seed infected by Ascochyta lentis and to use the AbR1 gene and the SCAR marker correlation to provide important information to the breeder regarding marker implementation and will help to accelerate the breeding program.
A study of horse ownership and management in Victoria, Australia

Pearson, Naomi Yvette ( 2003)

The welfare of horses is becoming an increasingly important issue in many parts of the world, particularly for horses used for recreation. This is because recreation is the principal use of horses in many countries today, in contrast with the largely working role they have played in the past. Animal welfare organisations and government enforcement bodies are finding it increasingly difficult to handle the number of complaints received about horse welfare problems. In Victoria, Australia, the number of horse welfare problems investigated by the Royal Society for the Prevention of Cruelty to Animals (RSPCA) remains extremely high, and significant time and resources are utilised in dealing with these problems. However little is known about the type of horse welfare problems that are occurring, or about the potential causes of these problems, such as the owners of such horses. In order to prevent horse welfare problems in the future, an understanding of the type and prevalence of the problems and their potential causes is crucial. The aim of this thesis was to identify the type and seventy of horse welfare problems inspected by the RSPCA in Victoria, and to identify the characteristics of the horses' environment that were related to their welfare. Additionally, a survey was conducted of 100 owners that were inspected by the RSPCA throughout Victoria, of which 70 had horse welfare problems and 30 did not, to examine the relationship between some attributes of these owners, and horse welfare. An additional 30 horse owners that, were members of adult riding clubs were surveyed, to provide an alternative control group. Results found that the most prevalent horse welfare problem was poor body condition and that most of these problems were rated as moderately severe by the author. Other problems that occurred were overgrown hooves, illness, founder, untreated injury and permanent tethering. Of particular concern was that the number of severe cases found was much larger than expected. The only environmental characteristic that was associated with reduced horse welfare was a lack of pasture. There were a number of differences between the horse owners that were members of adult riding clubs and owners with horse welfare problems that were inspected by the RSPCA. Owners of horses with welfare problems had a lower level of education and income, more owners were male, more lived closer to the city, less were married or in a defacto relationship, and more had less knowledge about horse management and less commitment to horse ownership. These owners also believed that horses made good companions and that less commitment was needed in horse ownership, and mainly used their horse(s) for companionship and/or pleasure riding, rather than for competition. Additionally, attributes of owners with a horse welfare problem that were specifically associated with increased severity of the welfare problem were lack of commitment to horse ownership, the belief that horses made good companion animals and were difficult to care for, lower education, and living in metropolitan Melbourne or in the outer-fringes (within 50km of the city). Of particular interest was that over half of the owners with horse welfare problems had never had riding lessons and did not frequently read horse literature, and nearly three quarters of these owners were not members of any horse clubs or organisations. The results of this project provide a valuable insight into the problem of horse welfare and thus the type of strategies that are required to prevent horse welfare problems in future.
The competitiveness of Australian cotton in Japan

Nguyen, Chi Thi Quynh ( 2003)

The Australian cotton industry is one of the fastest growing and the most profitable agricultural sectors in Australia. Internationally, Australia produces one of the highest quality cotton in the world and is the world's third largest exporter. However, despite the increasingly strong role of cotton in the Australian economy, and Australia's high ranking among exporting countries, Australia is not a major player in the market. Australia is currently facing a number of serious problems, most of which result from heavy government assistance to cotton producers and the application of trade barriers on textiles and clothing imports in other major producing and consuming countries. The effects of these policies may have an enormous impact on world trade balance through price variation and hence, place huge pressures on non-subsidising countries, like Australia. Therefore, until trade barriers are reformed, Australia must focus on solutions that lead to an increase in its competitiveness. As such, the most important strategy for Australia is to successfully compete with the US and other major cotton suppliers in Australia's traditional cotton markets, such as Japan. The aim of this study is to analyse the competitiveness of Australian cotton in the Japanese market. The techniques needed to undertake this task include the Structural Vector of Auto Regression (SVAR) model, the Weak Separability test, the Structural Almost Ideal Demand System (SAIDS) model. The results reveal that Australia's market share increases when the Japanese textile market develops, rather than when it is in decline. However, when comparing Australia's competitiveness with the US (its largest competitor), it is shown that during a down-turn of the Japanese market, Australia would be more competitive if its prices are kept lower than that of the US. It was also found that Australian cotton is a strong substitute of US cotton. While this obviously is an advantage of Australian cotton, it stresses the importance of the need for Australia to keep a price competitive position to that o f t he U S, if these advantages are to be exploited. It was concluded t hat higher production efficiency and proper marketing strategy are the main elements the Australian cotton industry needs to maintain and expand its market share in the Japanese cotton market.

School of Agriculture, Food and Ecosystem Sciences - Theses

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