Agriculture and Food Systems - Theses
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Understanding small and large milk fat globule phenotype variation in dairy cows through milk lipidomic characterisation
Milk fat globules (MFGs) are spherical structures comprising a neutral lipid core that is surrounded by a three-layer membrane. This MFG membrane is of nutritional benefit for infants and adult consumers. Individual variation in the size of MFGs is observed within a herd of the same breed, and this milk production trait, if selected for through breeding programs, could be exploited for a more targeted milk production for specific technological streams. For example, large MFGs are desirable for butter making, whereas small MFGs are preferred for cheese making and direct consumption due to improved sensory properties and increased relative abundance of the beneficial membrane material. The initial experiment of this work aimed to determine how much the average MFG size is affected by on-farm and animal related factors within a herd subjected to the same diet and environmental conditions. Milk fat globule size of the whole herd was repeatedly measured over a one-year period and the effects of these parameters were estimated using a linear mixed effect model. This analysis showed that stage of lactation, parity and milk yield can affect MFG size, while the impact of fat yield, concentrate intake and number of milkings per day was limited. However, the individual variation within the herd outweighed the effect of individual factors, supporting the possibility of a genetically determined regulation of MFG size. Based on the data collected for the first experimental chapter, cows were selected for the second and third experiment, which aimed to characterise the small and large MFG phenotypes through an in-depth lipidomics analysis. This analysis included the characterisation of the fatty acid (FA) profile of the MFG core by gas chromatography and the identification of the whole milk lipidome through targeted liquid chromatography tandem mass spectrometry. The analysis of the MFG core FA profile revealed that the cows with the small MFG (SMFG) phenotype produced milk with higher proportions of unsaturated FAs compared to large MFG (LMFG) cows. This was related to an increased uptake of preformed FAs with a chain length of 18 or more carbons, which are sourced directly from the diet or from lipid mobilisation. This characteristic of the SMFG group could potentially lead to the production of milk with an improved nutritional profile. The results of the third experiment present the most extensive milk lipidomic analysis in the literature to date, with 301 detected lipid species. The results also revealed, for the first time, a potential role for ether phosphatidylethanolamine (ePE) in the regulation of MFG size, showing a higher relative abundance of ePE in the milk from LMFG cows. Ether PEs can reduce the fluidity of biological membranes and are predicted to promote lipid droplet fusion. The milk from SMFG cows, on the other hand, contained higher total phosphatidylcholine (PC) to PE ratios and a higher relative abundance of unsaturated PC species, both attributes that are predicted to prevent lipid droplet fusion. In the final experiment of this thesis an in vitro model using cells purified from raw milk and grown on permeable membrane supports was established, which offers the potential to test some of the novel findings of this thesis in future experiments.
Amelioration of the effects of cyclic heat stress in broiler chickens with dietary betaine and antioxidants
Controlling and managing environmental conditions is crucial to avoid hyperthermia and heat stress, and hence to successful poultry production and welfare. Heat stress (HS) is one of the most important environmental factors challenging poultry production globally. The detrimental effects of HS on broilers range from reduced growth rate to decreased poultry meat quality. Increasingly, there has been a focus on the use of nutritional supplements as a cost effective HS amelioration strategy. The use of antioxidants such as selenium, vitamin E and polyphenols to improve productivity via reduced levels of oxidative stress in tissues when broiler chickens were exposed to cyclic HS. Another molecule of interest is the osmolyte betaine, which is accumulated in cells under osmotic stress, but also has properties as a methyl donor and antioxidant. The principal findings were that cyclic HS impaired growth performance and physiological responses as increased respiratory rate and rectal temperature caused alteration in blood haematology as pCO2 reduced and blood pH increased which could lead to impaired product quality. While supplementation of betaine improved growth performance, including improved final body weight and this effect was observed irrespective of HS or additional antioxidants supplementation where it had no impact on blood haematology. The effects of HS and betaine were investigated on meat quality and it was found that HS reduced moisture content, increased lipid oxidation and reduced myofibril degradation, indicating reduced post-mortem proteolysis. This indicates worsening of meat quality as increased lipid oxidation reduces shelf life and myofibrillar degradation is important for the meat tenderisation process. When supplemented betaine was distributed to the breast muscle, and corresponded to improvements in meat quality. This was evidenced by reduced drip loss, lipid oxidation and reduction of Evans Blue dye (EBD) concentration in the breast muscle. Most importantly, supplementation of betaine reduced EBD concentration in vital organs such as kidney and ileum, indicating that betaine could have protective effects under HS. Furthermore, supplementation of betaine improved intestinal and breast muscle structure, including improved villous length and increased muscle fibre diameters which contribute to better growth performance and meat quality. Betaine supplementation was also performed against or with selenium, vitamin E or polyphenols. In general, selenium and vitamin E did not have additive effects on growth, whereas some benefits were iv observed for preventing lipid oxidation. The use of polyphenols improved growth rates to levels seen with betaine, indicating that they may also be useful nutritional agents for the amelioration of HS. Many studies have investigated the effects of the mentioned additives individually, but limited studies are available that whether the combination of them at different ages could ameliorate the negative effects of HS. This thesis aimed to investigate the effects of betaine alone or in combination with antioxidants on growth performance, meat quality and involved mechanisms to provide a better understanding on HS and betaine effects. In conclusion, betaine is a promising additive that can partially ameliorate HS effects. Despite of positive effects of antioxidants on meat quality, the additional effects of antioxidants warrant further investigation, particularly with respect to shelf life and oxidation.
Counterfeiting and Design-Driven Deterrents: Trust perceptions of anti-counterfeiting strategies for food and product packaging
Premium international goods are sought after by quality-concerned Chinese consumers who are increasingly turning towards cross-border e-Commerce and social media platforms with the expectation to avoid local food safety scares. Online retail of foreign brands however, does not guarantee protection against fraud. Counterfeit products are now commonly placed and are marketed equally besides the genuine items exposing consumers to vulnerabilities and heightening their perceived risk prior to purchase. Products are forensically scrutinised on multiple levels of security and further judged on authenticity. Australian Small-to-Medium Enterprises (SMEs), which make up 97% of all Australia’s workforce, are not well represented in the encouraged export trade opportunities as they fear export pathways that are increasingly complicated and changing. Compounding the problem is SMEs limited investment funds to upscale with expensive anti-counterfeiting solutions that may not clearly deliver on counterfeiting deterrent effectiveness and appropriateness to build consumer’s ‘initial trust’. This paper seeks to shed light on three key principles of the food counterfeiting phenomenon. Firstly, to map literature on food fraud, packaging counterfeiting with a focus on deceptive counterfeiting operations and tactics. Secondly, to investigate the unique trust relationship formation between risk adverse consumers and emerging, unfamiliar SME brands. Finally, the research will appraise anti-counterfeiting responses, from two perspectives: technological and design-driven, with a primary focus on cost-effective deterrent strategies catering to SMEs. Design/methodology/approach – A Mixed Methods approach was adopted to integrate the Participatory Action Research (PAR) methodology and co-creation strategies to later inform the larger online survey, triangulating the overall findings through qualitative and quantitative studies. Comparisons were made between Consumer and Industry insights as well as a cross-cultural study between Australia (low risk) and China (high risk) representing diverse levels of Risk Societies. The three studies investigated trust influences and drivers of Security Elements applied to food packaging founded on Technology and/or Design applications. The studies consisted of: (1) a Qualitative Multivariate Analysis, identifying the 5 Design-Driven Influencing Factors (DDIFs), (2) an Anti-Counterfeiting Workshop Model testing 30 security elements through the Case Study methodology, and finally (3) a Quantitative Online Study comparing two populations trust propensities and expectations of security elements on food packaging. Overall, the research identified that consumers acceptance of anti-counterfeiting elements can be strongly influenced by DDIFs that are appropriate for the food category (Fitted), multiple authenticity checks (Layered), connected to the country of origin (Identity Links), presents a reward attraction (User-Purposed), and technologies use a holistic design (Embedded). Design-Driven elements were identified as strong applicable to SME needs and were perceived as strong deterrent hurdles against counterfeiting threats. Future research is needed in developing education for designers to become the trust design guardians for the expansion and protection of the Australian food exporting industry.
Prevalence and Transmission of Antibiotic Resistance Genes in the Soil Environment
The increasing prevalence of antibiotic resistance genes (ARGs) in environmental settings and their potential acquisition by human and animal pathogens have become a global public health concern in the 21st century. The use of antibiotics in livestock industry and the enrichment of ARGs in animal manure and arable soils have engendered the concern that recycling of manure onto agricultural land could disseminate antimicrobial resistance to crops/vegetables, which might represent a potential route for migration of environmental ARGs from farm to fork. However, the pathways for transmission of ARGs from soil to plant remain unclear. Growing evidence points to the pivotal role of the environmental factors in influencing the prevalence of ARGs in the natural environment, while our understanding of the ecological and evolutionary environmental factors that contribute to the development and dissemination of antibiotic resistance in natural environment is lacking. It is imperative to decipher the diversity, prevalence, and environmental determinants of antibiotic resistance before we could design management approaches to control the transmission of environmental resistomes. A soil microcosm incubation experiment was conducted to compare the effects of poultry, cattle and swine manures spiked with or without the antibiotic tylosin on the temporal changes of soil ARGs. The high-throughput quantitative PCR detected a total of 185 unique ARGs (out of 295 targeted genes) among all the soil samples. The diversity and abundance of ARGs significantly increased following manure application. The level of antibiotic resistance gradually decreased over time in all the manured soils but was still significantly higher in the soils treated with swine and poultry manures than in the untreated soils after 130 days’ incubation. Tylosin-amended soils consistently showed higher abundances of ARGs than soils treated with manure only, suggesting a strong selection pressure of antibiotic-spiked manure on soil ARGs. The relative abundance of ARGs had significantly positive correlations with integrase and transposase genes, indicative of horizontal transfer potential of ARGs in the manure and tylosin treated soils. A glasshouse pot experiment explored the impacts of manure fertilization on the incidence of ARGs in the plant-associated microbiomes. Rhizosphere and phyllosphere microbiomes of cherry radish harbored significantly higher diversity and abundance of ARGs than root endophytic microbiomes. Manure application significantly increased the abundance of ARGs in the rhizosphere and phyllosphere, but not in the root endosphere, which is the edible part of cherry radish. Soil and plant microbiomes changed dramatically after manure applications. The bacterial abundance was the most important factor modulating the distribution patterns of soil and plant resistomes after accounting for multiple drivers. For the pot experiment of lettuce, rhizosphere soil samples harbored the most diverse ARGs compared with other components of lettuce. Comparing with cattle manure, poultry manure had a stronger impact on lettuce resistomes, reflecting by the significant increase of ARGs in rhizosphere, root endophyte and phyllosphere in the poultry manure treatment. Moreover, the overlaps of ARGs between lettuce tissues and soil were identified to propose two potential transmission routes (internal and external pathway) of ARGs from manured soils to different compartments of lettuce. A field survey experiment investigated the abundance, diversity and environmental determinants of ARGs in ocean and river beach soils. A total of 61 soil samples were collected from ocean and river beaches, which are hotspots for human activities and platforms for potential transmission of environmental ARGs to human pathogens. A total of 110 ARGs conferring resistance to eight major categories of antibiotics were detected. The core resistome including all the abundant and prevalent ARG subtypes was identified, which accounted for 66.9% of the total abundance of ARGs. The relative abundances of ARGs were significantly correlated with salinity-related properties including electrical conductivity and concentrations of sodium and chloride. Random forest analysis and structural equation modeling revealed that salinity was the most important factor modulating the distribution patterns of beach soil ARGs after accounting for multiple drivers. These findings suggest that beach soil is a rich reservoir of ARGs, and that salinity is a predominant factor shaping the distribution patterns of soil resistome. Overall, this research provided solid evidence that manure application may result in a significant increase of ARGs in agricultural soil and different parts of vegetables. The shared ARGs in the soil-plant system suggested a potential route of ARGs transfer from manure/soil to vegetables and highlighted the potential threats to human health by consuming raw vegetables grown in manure-amended soils. Soil salinity has been identified as the most important environmental factor in shaping the ARGs in beach soil environments, highlighting the importance of understanding the environmental stresses that maintain the environmental ARGs and developing effective strategies to minimize the dissemination of ARGs.
Regulation of Leaf Vein Density in C3 Rice
Stagnation of rice yield over last few decades is a major concern for food security, especially in Asia. A large part of the yield increase in food crops has come from breeding for abiotic and biotic stresses and responsiveness to nutrients. However, next step of change in rice yield should target improving photosynthetic capacity, better utilising the major inputs of sunlight and/or carbon dioxide. C4 species are efficient in utilising both sunlight and carbon dioxide and therefore, engineering or breeding of C4 like traits into C3 crop plants are major pathways to increase the yield potential. Reduced interveinal spacing or enhanced leaf vein density is known to be a primary prerequisite for C4 evolution. This study is carried out to identify the potential high vein density candidates in Sri Lankan traditional rice germplasm and rice from different regions of origin and further screened for variation in their other anatomical and physiological mechanisms that are associated with enhanced photosynthesis and crop production. We have successfully identified high vein density candidates in this study and investigated their changes of leaf vein density for exogenous application of Phytohormone, Brassinosteroid (Brassinolide), a Brassinosteroid inhibitor (Brassinazole) and Auxin (Indole-3-butyric acid), and for abiotic stress; elevated carbon dioxide levels, elevated temperature and water limited conditions to identify regulatory mechanism of leaf vein density. This study is providing insight to underpin regulatory mechanism of rice leaf vein density and further, pave a foundation to develop C4 like rice to meet future food demand while mitigating the impact of climate change.
Objective measures of sparkling wine quality
Sparkling wines are economically significant for the Australian wine economy. It is the only sector in the Australian wine industry that has been increasing in volume and value when compared to a decreasing fortified wine sector and the still wine sector. In Australia, sparkling wines are distinguished from other wine categories by the presence of carbonation, which is introduced naturally or artificially to still wines. And so, the appearance of a sparkling wine is of great importance as the presence of bubbles and foam changes the sensory experience of the wine consumer. The scientific community and industry have a great interest in uncovering the process and compounds which results in foam and bubble formation. Furthermore, although proteins are an excellent candidate to stabilize bubbles and foam, an official methodology for quantification of soluble protein content in sparkling wines has not been implemented. This thesis aims to uncover objective measures for quality assessment of sparkling wines by developing a robust, fast, accurate, and repeatable method to assess foam quality. Additionally, the specific objectives were i) to develop a robotic pourer to uniform and standardize the pouring process; ii) to develop a non-sparging automated methodology to quantify/assess foam characteristics of sparkling wines, using computer vision algorithms; iii) to quantify total soluble proteins in sparkling wines; iv) to uncover compounds other than proteins related to foam characteristics. Therefore, a novel methodology to characterize foam and bubbles in sparkling white wines was developed. The research presents a non-sparging technique to minimize product losses, that uses a robotic pourer to standardize the volume of wine poured with an automated arm, coupled with computer vision and image analysis to quantify and characterize foam in sparkling white wines. The method was successfully applied to find compounds influencing bubbles and sparkling wine foam. The compounds uncovered included proteins, amino acids, and polyphenols, which was validated by the addition of target compounds to sparkling wines. Modifications for the bicinchoninic protein assay was presented to application in wine, and the soluble protein content was correlated to foam parameters obtained by the robotic pourer and computer vision algorithms. Finally, preliminary results on the effects of wine ageing was presented. In summary, this research has uncovered objective measures of sparkling wine quality, providing practical information for industry and research centres, to allow monitoring and control of wine quality and uncovering compounds influencing foam and bubble characteristics.
Effect of Irrigation, Glycine Betaine and Kaolin Particle Film on the Performance of Vitis Vinifera L. ‘Shiraz’
Environmental stresses, such as heat and water stress, are a current and emerging challenge to wine grape production in Australia. In Southern Australia, where wine grape production is predominately located, Global Circulation Models (GCMs) predict further increases in temperatures, increased frequency and severity of heatwaves and reduced rainfall. Wine grapes are particularly susceptible to heat and water stress due to the lengthy grapevine establishment times and the perennial nature of this crop. Therefore, there is a strong demand for management strategies to protect established vineyards from environmental stresses, such as heat and water stress. Glycine betaine (GB) and kaolin particle film (KPF) are two products that can be exogenously applied to grapevines to potentially ameliorate the effects of heat and water stress. The objective of both foliar sprays is the same, but their mode of action is quite different. GB seeks to induce greater stress tolerance, while KPF seeks to physically protect the fruit and leaves. Management strategies, such as application of GB and KPF, implemented to ameliorate the effects of one stress may however have unintentional impacts when coupled with common management practices, such as deficit irrigation. This research investigated the protective capacity of GB and KPF, individually and combined, on grapevine performance under varying levels of imposed water stress, and their associated interactions on the productive performance and physiology of Vitis vinifera L. ‘Shiraz’. A field experiment was conducted on Shiraz (clone SA16) vines at The University of Melbourne’s Dookie Campus Vineyard in North East Victoria, Australia during the 2011-12 (Y1) and 2012-13 (Y2) growing seasons. The vines were planted in 1994, on Richter 99 rootstock on a cordon-trained Scott Henry trellis system, with two bilateral cordons. A randomised complete block design (RCBD) factorial experiment was established in 2011 comprising three irrigation treatments, two GB treatments and two KPF treatments, with six replicates per treatment. Analysis was conducted on grape yield and yield components, fruit composition, plant water status, canopy growth and canopy temperature. The research was conducted over two contrasting growing seasons. Y1 was largely cooler with high rainfall between veraison and harvest and Y2 was hotter with lower rainfall. In Y2, observed values of leaf water potential and canopy temperatures indicated that all the treatments were showing signs of severe to very severe water stress. Consequently, water availability (rainfall and irrigation) dominated treatment responses in yield and composition, with average berry weight across all treatments 52% smaller, resulting in a 25% lower average yield in Y2 compared with Y1. Therefore, seasonal variation played a key role in the vines’ response to the irrigation treatments, GB and KPF. Under non-stressed conditions in Y1, applying GB resulted in a 16% reduction in grape yield, which was attributed in part to an 8% reduction in berry weight. However, under severe water stressed conditions in Y2 the impacts of GB on vine performance became less evident. Applying KPF resulted in a reduction in some yield components under the water stressed conditions of Y2. It is possible that KPF resulted in changes to transpiration resulting in the vines becoming more water stressed. In addition, the interaction of GB and KPF may have caused a response similar to the effect of KPF under water stressed conditions. This is evidenced by the combined effect of GB and KPF resulting in lower average cluster weight in Y1 and more negative values of midday leaf water potential post-irrigation in Y2. However, interpretation of this interaction is complicated by the impacts of the irrigation treatments and seasonality. In conclusion, this research has shown how Shiraz vines respond to GB and KPF, when grown in commercial settings, under both high and low water availability conditions in North East Victoria, Australia. These findings are important as they indicate that the use of these compounds to ameliorate the impacts of environmental stresses, such as heat stress, needs to be carefully assessed alongside water availability. Importantly, these research findings have demonstrated that GB and KPF have different impacts across different environmental conditions; and there is a potential for interactions that could compromise production of grapes to meet commercial specifications.
Development and implementation of novel sensory evaluation procedures of consumer acceptability towards chocolate based on emotions and biometric responses
Chocolate is a condensed suspension of different particles, with a continuous phase containing cocoa butter and milk fat, and a diffused phase containing cocoa particles, sugar, and non-fat milk solids. Chocolate is the most commonly consumed confectionery product worldwide. Sensory evaluation is a scientific discipline which uses human senses for evaluating consumer products with the application of experimental design and statistical analysis. The application of novel procedures in sensory analysis is needed to prevent the high failure rate of new market launches based on testing with conventional sensory protocols. Hence, the objective of this study was to use novel sensory evaluation procedures including subconscious biometrics [skin temperature (ST), heart rate (HR) and facial expressions (FE)] and self-reported responses to determine consumer acceptance to food, using chocolate. Initially, in Chapter 3, a cross cultural study using Asians and Westerners was conducted to develop lexicons of emotions for milk and dark chocolate using online surveys (N = 206) and by conducting tasting sessions of the chocolates (N = 75). As per the responses of the survey and according to previous research findings, the main reasons for chocolate consumption were emotional satisfaction and indulgence. The main factor that consumers considered in making their purchase decision was the taste/flavour of the chocolate. Three separate emotion lexicons were developed using the results of this experiment, and they were used for further studies conducted using chocolate. In Chapter 4, chocolate with five basic tastes (bitter, salty, sour, sweet and umami) were developed to obtain sensory and physiological responses of consumers to different tastes using 45 participants. An integrated camera system coupled with a tablet-PC using Android OS and containing a BioSensory application was employed to capture infrared images, videos and sensory responses. Inputs from this application were used to determine ST, HR and FE. Sensory responses were gathered using hedonic scales and emotions elicited were obtained using lexicons. Results revealed that the most liked sample was the sweet chocolate, while the least liked was the salty chocolate. Findings of this study can be used to assess novel tastes of chocolate in the industry. Furthermore, in Chapter 5, flavour was added to chocolate directly to produce different flavoured chocolate (caramel, cinnamon chai, mandarin, strawberry and peppermint). A sample without any flavour was considered as the control. These six samples were used for sensory evaluation with 113 participants. ST, HR, FE, sensory responses and emotions elicited were determined like the previous experiment. Results revealed that the most liked sample was the strawberry and the least liked was cinnamon chai chocolate. Findings of this study can be used to determine the acceptability for different flavoured chocolate based on self-reported and subconscious responses. In Chapter 6, encapsulated flavours were developed to be added to chocolate and release the flavour in a different way compared to the experiment reported in Chapter 5. Samples [normal strawberry (NS), normal mint (NM), encapsulated strawberry (ES), encapsulated mint (EM) and control (with no flavour)] were developed and tested by 52 participants to obtain biometric (ST, HR and FE) as well as sensory and emotional responses. Encapsulated chocolate samples showed higher liking compared to normal flavoured chocolate. Findings of this study can be used to determine the acceptability of differently flavoured chocolate in future studies using chocolate. As the next step, in Chapter 7, artificial intelligence was used to develop machine learning models with near-infrared spectroscopy (NIRS) to assess the quality of chocolate based on chemical fingerprinting. Chocolate samples with basic tastes (bitter, salty, sour, sweet and umami) were used for this experiment with 45 respondents and their chemometrics (pH, brix and viscosity), colour (CieLab scale) and sensory properties (basic taste intensities) were determined. Data were used to develop two machine learning models to predict the chemical parameters (Model 1) and sensory parameters (Model 2). These models showed high accuracies of R = 0.99 and R = 0.93 respectively. The developed models can be used as a substitute method to determine sensory properties of chocolate with low cost more accurately. In this study, emotion lexicons were developed and used for sensory sessions conducted using chocolate. Conventional and novel sensory techniques were used for analysis. Results revealed significant differences in emotional terms selected based on gender and culture for different samples. Furthermore, there were significant correlations between conscious and subconscious responses of chocolate. A combination of implicit, explicit and emotional responses may help to better understand the acceptability to different food products. Moreover, models with high accuracy were developed to predict sensory properties of chocolate using chemometrics. Findings of this study can be used for future acceptability research on chocolate.
Implementation of non-invasive biometrics to identify effects of chocolate packaging towards consumer emotional and sensory responses
Due to the high volume of new products introduced to the market, food manufacturers face a challenge to differentiate themselves from competitors and avoid high failure rates. Traditional sensory responses require cognitive thinking and are considered biased, whereas physiological responses are unconscious and do not require cognitive thinking. It is important to obtain physiological responses in addition to traditional hedonic sensory responses to tap into the unconscious reactions of consumers, which provide valuable information about behaviours. In the first experiment (Chapter 3), 12 images [ten from Geneva Affective PicturE Database (GAPED), two based on common fears] were tested using 63 panellists to obtain self-reported and physiological [facial expressions (FE), heart rate (HR), systolic pressure (SP), diastolic pressure (DP) and skin temperature (ST)] responses. Cluster analysis separated images as positive, neutral, and negative according to the GAPED classification using conscious and unconscious responses. Biometric responses were used to predict conscious responses with the aid of general linear models. Results from this study were used as a reference point for the next study that had an increase in sample size and number of participants in order to develop machine learning models involving visual stimuli. In the second experiment (Chapter 4), 240 images from GAPED (80-Negative, 80-Neutral, 80-Positive) were selected. Self-reported and physiological responses were gathered using 130 panellists. A machine learning model developed using Artificial neural network (ANN) obtained 89.9% accuracy to classify images into negative, neutral, and positive. The model was tested with commercially available chocolate packaging using 30 panellists. Results showed that participants elicited positive and neutral emotions while evaluating these packaging. Further research using newly developed food packaging was conducted to understand if product familiarity affects consumers emotional responses. In the third experiment (Chapter 5), six novel packaging concepts (bold, fun, everyday, special, healthy, premium) were developed based on the six segments of the TNS NeedScope model. Biometrics (FE, HR, and ST), eye-tracking (ET) and self-reported responses were evaluated. General linear models developed could predict “liking” using emotions obtained from self-reported responses. The application of FE results to the previously developed ANN model showed that consumers unconsciously perceived only neutral emotions during evaluating novel concepts. In the fourth experiment (Chapter 6), novel and familiar packages were tested to link consumers’ eye fixations and their physiological responses to understand consumer (N=60) emotional responses during the fixation of Areas of Interest (AOI). Results showed that fixations on familiar packages were positively correlated with positive emotions while they were not correlated with emotions in novel packages. Fixations on a specific AOI were not associated with positive emotions, but they could be associated with negative emotions. In the fifth experiment (Chapter 7), sensory characteristics of the chocolate product were studied in terms of how packaging design would affect sensory liking (taste) and willingness to purchase (WTP). Panellists (N=75) evaluated samples under three conditions: (1) blind [product], (2) packaging [package], and (3) informed [product and packaging]. The same chocolate tasted in (1) was wrapped in six different novel packaging concepts developed in the previous study for (3). There were significant differences in liking towards taste based on packaging. Liking scores for the informed condition significantly reduced when expectations created by packaging were not met. Regression analysis explained that taste had a strong association (r=0.73) towards WTP. This series of studies used both traditional and novel (biometrics) sensory techniques to ensure that a joint application of both these measures would provide valuable input to understand consumer perceptions. The machine learning models allow the application of new visual stimuli (food packaging) to understand consumer emotional responses better. Furthermore, the integration of emotional (FE) and visual responses (ET) help to study if the fixations on food packaging can be related to human emotions. Finally, adding the sensory attribute to the experiment allows us to evaluate how the chocolate packaging affects the taste of the product. These findings can be used in product design and development to control product intrinsic and extrinsic attributes by enhancing emotional attachment towards food products.
Genomics and population biology of the anthracnose pathogen (Colletotrichum tanaceti) of pyrethrum in Australia
Colletotrichum tanaceti, is an emerging fungal pathogen of pyrethrum (Tanacetum cinerariifolium) in Australia. This thesis investigated the evolutionary potential and the molecular bases of sexual reproduction and pathogenicity of C. tanaceti by combining genomics, comparative genomics, molecular genetics and population genetics, with molecular biology and mycology techniques. The whole genome of the holotype isolate of C. tanaceti, BRIP57314 was sequenced, assembled and annotated then supplemented with a RNA transcriptome thus providing the first public genome sequence of C. tanaceti. The repetitive elements in the genome were identified in silico. Comparative genomics identified a relatively high repeat content therefore, suggesting a high genomic plasticity of the C. tanaceti genome. The bipartite nature of the C. tanaceti genome with distinct gene-sparse repeat and A-T rich regions were identified using in silico methods. Signals of genome-wide repeat induced point mutation (RIP) were identified using RIP-indices. The phenomenon of RIP in C. tanaceti genome was therefore, hypothesized to cause the A-T rich regions that evolve at a higher rate than the rest of the genome, generating a ‘two-speed genome’. Genomics tools were used to compare the C. tanaceti genome to publicly available genomes of other Colletotrichum spp. Single-copy orthologs among the species were used to develop a phylogram. This was then converted to a chronogram to estimate the divergence of C. tanaceti from C. higginsianum at 9.97 million years ago. The genome sequence and the predicted proteome of C. tanaceti were utilized to identify the putative pathogenicity genes of C. tanaceti. A large array of putative genes involved in infection and colonization of the host tissue such as CAZymes, effectors, secondary metabolites, transporters, cytochrome P450 and proteases were identified in the C. tanaceti genome. This was achieved by comparing genome homology to database entries and bioinformatics tools for de novo identification of fungal pathogenicity genes. The comparison of the putative pathogenicity gene repertoire of C. tanaceti with other Colletotrichum spp. indicated C. tanaceti had a similar pathogenicity profile to Colletotrichum spp. with intermediate host ranges. Therefore, C. tanaceti was speculated to have potential hosts other than pyrethrum. Furthermore, the in silico analyses revealed that the pathogenicity genes of C. tanaceti are located significantly closer to the tandem repeats, transposable elements and the RIP-affected regions of the genome. This suggested the potential of these pathogenicity genes to diversify and evolve adaptively. The population biology of C. tanaceti was assessed using a neutral, polymorphic microsatellite marker library developed from the draft genome sequence of C. tanaceti. Symptomatic pyrethrum leaves were hierarchically sampled across pyrethrum growing regions of Australia over three years in order to establish a collection of C. tanaceti isolates. These isolates were genotyped using the microsatellite marker library. Analysis of the genotypic data indicated the high genotypic diversity and only little differentiation of the populations of C. tanaceti. The index of association and the genotypic data indicated the mixed mode of reproduction of C. tanaceti with both sexual and asexual reproduction. Coalescent–based analysis of isolation with migration of the 2016 populations from the two growing regions of pyrethrum in Australia, indicated that long-distance gene flow of C. tanaceti is possible between Victoria and Tasmania which are separated by a sea strait. Approximate Bayesian computation methods revealed that the Tasmanian population of C. tanaceti was the source of the disease introduced to pyrethrum growing in Victoria. Furthermore, the hypothesis that C. tanaceti is likely to have shifted hosts from another unidentified host which was already present in Australia was supported. Combined, these results indicated a high evolutionary potential for C. tanaceti which supports observations of the pathogen’s genome. The sexual recombination strategy of C. tanaceti was further evaluated in vitro. Classical mating experiments revealed the fully heterothallic nature and instability of mating in C. tanaceti. These mating experiments revealed the presence of more than two mating specificities in C. tanaceti, a departure from the typical unilocus bi-allelic mating system of ascomycetes. The genome sequence of an isolate that engages in successful reproduction with the holotype isolate: BRIP57315 was also sequenced, assembled and annotated. The two genome assemblies and their annotations were utilized for in silico homology-based identification of the putative mating type MAT locus, pheromone precursors, pheromone receptors, mating type switching genes and other HMG box genes that may be involved in the sexual reproduction of C. tanaceti. Both genomes only contained the MAT1-2 idiomorph with identical MAT1-2-1 coding regions in the two genomes. Comparative genomics approaches, such as sequence alignment and phylogeny, were utilized to estimate orthology to functionally validate mating genes of other fungal genera and thereby to predict the functions of putative mating genes of C. tanaceti. Based on these results, C. tanaceti was hypothesized to have a multilocus mating system. In summary, this thesis elucidated the high evolutionary potential of the anthracnose pathogen of pyrethrum. The work indicates the pathogen has a highly plastic genome, a large array of pathogenicity genes that evolve rapidly, a mixed reproduction strategy, the potential for long-distance gene flow and the potential for infecting alternative hosts. These observations should be taken into consideration to ensure durability of future disease management strategies of pyrethrum. Moreover, this study also improved our understanding of the molecular basis of pathogenicity and the sexual reproduction of the important plant pathogenic fungal genus, Colletotrichum. The two genome assemblies and the candidate genes available through this study provide a solid foundation into future research on ‘omics’ and molecular genetics of pathogenicity and sexual reproduction of Colletotrichum.
Engineering disease resistance and frost tolerance in canola (Brassica napus L.) using ACYL-COENZYME A-BINDING PROTEINS
Fungal diseases and low temperature stress (freezing and frost) are major stresses faced by the canola (Brassica napus L.) industry in Australia. The potential for use of plant ACYL-COENZYME A-BINDING PROTEINS (ACBPs) in improving disease resistance and frost tolerance ability of B. napus was studied using rice (Oryza sativa) ACBP5 and Arabidopsis thaliana ACBP6 cDNA inserted into B. napus using Agrobacterium mediated transformation. Rapid-cycling Brassica napus is a convenient model system for assessing genetic transformation of B. napus. In vitro regeneration and transformation of B. napus were optimised using rapid-cycling B. napus plants. Cotyledon explant tissues provided faster shoot induction and higher regeneration ability than hypocotyls. To further optimise regeneration using cotyledons, concentrations and combinations of routinely used plant growth regulators were tested. Cotyledon explant tissues grown in Murashige Skoog (MS) media supplemented with 1-naphthalene acetic acid (NAA) at 0.1 mg/L and 6-benzylaminopurine (BAP) at 1.0 mg/L, 0.01 mg/l gibberellic acid (GA3) and 5 mg/L silver nitrate produced a high plantlet regeneration efficiency of 70%. Four-day-old cotyledon explants co-cultivated in Agrobacterium inoculum at 0.1 optical density at 600 nm (approximately 0.1x109 cfu/ml) provided an average transformation efficiency of 16.5% when explants were sectioned without liquid medium, dipped for 30-60 sec in the Agrobacterium inoculum and had exposure to the initial antibiotic selection medium delayed for two weeks after co-cultivation. Using these optimised tissue culture conditions, a novel candidate gene for canola pathogen resistance, ACBP5 from rice (Oryza sativa), was constitutively expressed under the CaMV35s promoter using plasmid pOS879, in canola grade B. napus cv. Westar and in rapid-cycling B. napus. Transgenic plants were confirmed by antibiotic selection, genotyping, western blot analysis and reverse transcription PCR. All positive independent lines were tested for resistance against two important pathogens of canola, namely Leptosphearia maculans (blackleg) and Sclerotinia sclerotiorum (Sclerotinia stem rot). The blackleg assay, which was based on 12-day-old cotyledons, showed that lesion development was significantly slower in OsACBP5 transgenic plants than the wild-type and vector control plants at 12 days post inoculation. The disease severity scores based on lesion size were also lower in transgenic plants than in control plants. Detached leaf assays for Sclerotinia resistance showed that there was a small but significant reduction in lesion size for transgenic plants compared to the control plants 24 h after infection. However, this effect on lesion size was not seen at 48 h, when disease development was more severe in both transgenic and wild-type plants. Overall, the transgenic plants benefited from the presence of the OsACBP5 cDNA, showing increased resistance to blackleg and an initial delaying of the effects of Sclerotinia infection. Previously developed transgenic rapid-cycling B. napus plants expressing the Arabidopsis thaliana ACBP6 cDNA were further evaluated for their freezing and frost tolerance ability at the vegetative and seed-setting stages. The electrolyte leakage values recorded for the freezing and frost-treated plants indicated that the membrane damage of AtACBP6 transgenic plants was significantly lower than for the wild-type plants. The recovery of the freezing stressed vegetative stage plants was much quicker than that of the stressed wild-type plants and they displayed a higher harvest index than the non-transgenic plants. The yield potential as measured by the subsequent flower production was higher in transgenic plants than in the non-transgenic lines in frost-treated vegetative plants. Seed survival after freezing-with-frosting treatment showed that the wild-type seeds were more affected by the stress, showing less viability than the AtACBP6 transgenic plants. These findings indicate that the overexpression of AtACBP6 gene is potentially useful in making canola crops more tolerant of cold and frost events. Overall, this study has provided useful evidence for the use of OsACBP5 as a novel candidate gene in decreasing the disease severity for blackleg and Sclerotinia infection in transgenic B. napus. Evaluation of the available AtACBP6 transgenic rapid-cycling plants further strengthened the evidence for the advantage of having the gene in mitigating the adverse effects of low temperature stress on B. napus plants. Transgenic plants displayed faster and better recovery and a higher yield potential than the wild-type plants. The outcomes of the project can be further extended by testing of these transgenes in commercial canola cultivars on a larger scale.
Physiological and biochemical responses of lentil to silicon mediated drought tolerance
Lentil has become one of the most important grain legumes grown in the arid and semi-arid regions of the world. Lentil production is challenging in these environments due to various sources of biotic and abiotic stresses. Among these stresses, drought stress is one of the major constraints leading up to 60 % production losses. This research work investigated the impacts of silicon (Si) supplementation on the physiological, biochemical and molecular mechanisms in lentil plants under drought stress along with an evaluation of nutritional potential and sensory traits of seeds produced from plants treated using Si. A preliminary study categorised lentil genotypes into drought-tolerant and sensitive categories. This study was mainly based on infrared thermal imaging (IRTI). This technique proved to be a high-throughput constructive screening method for drought stress tolerance based on plant water status estimation. Further experiments with Si supplementation were conducted on the selected drought tolerant and sensitive lentil genotypes under drought stress and controlled conditions. Results from seed germination studies demonstrated the significant role of Si in improving seed germination. It also alleviated drought stress in lentil seedlings by regulating osmolytes, hydrolytic enzymes and antioxidant defence systems. Further experiments conducted in a growth chamber, also provided significant evidence for the Si’s protective mode of action against drought stress through the regulation of nitro-oxidative homeostasis and antioxidant metabolism, normal growth and development of plants even under controlled condition, thus suggesting its role as a plant nutrient. Si also enhanced the photosynthetic efficiency and yield related traits in drought stressed lentil plants. To validate these results, lentil genotypes were tested in a field trial at the experimental farm located at the Dookie campus belonging to the University of Melbourne. Results from this experiment showed a significant increase in lentil net photosynthetic rate, aboveground biomass and grain yield when Si was applied to mitigate drought stress. Furthermore, the analysis of chlorophyll fluorescence showed that application of Si to drought stressed plants significantly increased electron donor and acceptor performance of the photosystem II reaction centre. Next generation RNA sequencing (RNA-Seq) was performed to better understand the molecular regulations behind Si-mediated drought stress tolerance in lentil plants. Numerous differentially expressed genes (DEGs) were revealed belonging to photosynthetic, osmoprotective and antioxidant metabolism, along with the biosynthesis of hormones and hormone signalling pathways. Gene ontology (GO) enrichment analysis of 100 significant differently expressed (upregulated and down regulated) genes also suggested that Si is not only involved in the development and synthesis of cell wall materials and vasculature but also in the metabolism of proteins, amino acids, carbohydrates and lipids. Nutritional and sensory analysis were carried out in seeds harvested from Si supplied drought stressed lentil plants. The sensory analysis carried out using non-invasive biometrics from consumers demonstrated that sensory attributes of seeds can be enhanced without any negative effects on nutritional properties. Overall, the current results suggest that Si can potentially be a sustainable management strategy to mitigate drought stress in lentil plants, with the added benefit of enhanced nutrient potential and sensory quality of the seeds.