School of Agriculture, Food and Ecosystem Sciences - Theses
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ItemDo native Australian perennial grains have a place in modern farming systems? The case of Panicum decompositum( 2019)The use of perennial grains in Australian farming systems has the potential to mitigate climate change, regenerate soil health and provide a sustainable source of sustenance. Although the proof of concept has been validated under Australian conditions, a commercially viable perennial grain cultivar is yet to be found. The aim of this study was to investigate the potential of Panicum decompositum (native millet) – a hardy native Australian perennial grass – as a contemporary resource, on the basis that it has a long history of use by various Aboriginal nations of Australia. Firstly, we conducted an in-depth archival assessment to find and collate all relevant archival information on native millet, Aboriginal grass seed culture and Australian grassland ecosystems. This information then dictated how we designed subsequent experiments. The results revealed that native millet is of nutritional value and compares quite favourably to other widely consumed grains such as wheat, rice and maiz. We found that milling native millet grain with water (wet-milling) rather than without water (drymilling)significantly increases the iron, sodium, calcium and boron content of the milled product. A digital image analysis and sensory analysis of bread baked with both native millet and wheat flour, indicated that native millet grain is palatable when baked, and can have a significant effect on the sensory and techno-functional qualities of ‘white bread’. Overall, the results indicated that native millet has the potential to be used in modern farming systems, but further study is needed to understand its potential as a perennial grain cultivar that could meet the same demands as widely used grain crops, such as annual wheat. We found that the archives were limited in their ability to truly 12 represent all Australian Aboriginal knowledge on native millet, thus we recommend that further research initially focus on investigating and collating Aboriginal oral histories. Our study may be used to inform the locations and nature of future research efforts.
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ItemMolecular and functional genomic analysis of Phytochrome Interacting Factor 4 during the floral transition of soybean( 2019)Climate change is adversely affecting global food production. Reduced crop yields and increasing food demands are presenting new challenges for developing resilient crop varieties. Legume plant soybean is an important food crop next to cereals. Soybean is a rich source of high-quality vegetable oils, protein, nutraceuticals, and it is widely cultivated across the globe for both human and animal consumption. According to the Australian oilseed federation, soybean cultivation strengthens regional economies and contributes to 2.5 billion dollars of the Australian oilseed industry. To breed better soybean crops, a study of light and temperature perception is important, as these events control the process of flowering which is the first step towards total seed production. On the molecular level, light and temperature signal control is a complex process mediated by a network of photoreceptors and transducers. One such transducer is Phytochrome Interacting Factor 4 (PIF4). It is involved in the perception of external environmental stimuli to integrate the information of daily light and temperature fluctuations with the internal physiology of plants. PIF4 has been studied in the model plant Arabidopsis thaliana, but knowledge about its counterparts in soybean is limited. To understand the function of PIF4 in soybean, an integrated genomics, molecular, and functional characterization approach was employed. The genomic analysis show that there are seven active copies of PIF4 gene in the genome of the cultivated soybean (Glycine max). The seven soybean PIF4 genes have evolved at different time points during the soybean genome duplication events, and phylogeny is suggestive of the existence of a conserved PIF4 clade (PIF4 I), which includes Arabidopsis PIF4 and a clade that groups only the PIF4s of legumes (PIF4II). To assess the gene expression patterns of soybean PIF4s during variable photoperiods and temperature conditions, quantitative RT-PCR was performed. Quantitative RT-PCR results showed that three PIF4s belonging to the PIF4I clade (i.e. GmPIF4a, GmPIF4b, and GmPIF4c) had conserved PIF4 like expression (consistent with Arabidopsis pif4) in inductive photoperiod (short day; SD), whereas two soybean PIF4s belonging to the PIF4II clade (i.e. GmPIF4f and GmPIF4g) exhibited reduced to nil sensitivity to short-days. The mRNA transcript levels of three PIF4s were elevated at 35-degree centigrade as compared to 25-degrees centigrade in short days. In non-inductive photoperiod (long day; LD), all soybean PIF4s were responsive to extended light phase suggesting their functional role in long photoperiods. Further, no significant transcript variation was observed at elevated temperatures in long days, except one PIF4, which expressed at higher levels at 30-degrees centigrade as compared to 25-degrees centigrade. Mining of previously published RNA sequencing data obtained from leaf samples of a short-day soybean cultivar (Bragg) undergoing floral transition showed that one of the PIF4s was differentially regulated with high differences in transcript read values on consecutive short days. This soybean PIF4 was designated as GmPIF4b. GmPIF4b was functionally characterized using ectopic expression in Arabidopsis, mutant complementation in Arabidopsis pif4-101 mutants and gene over-expression in the soybean cultivar, Bragg. Ectopic expression of GmPIF4b in Arabidopsis resulted in elongated hypocotyls and early flowering responses suggesting the function of GmPIF4b in hypocotyl elongation and flowering responses. Arabidopsis pif4-101 mutant contains a mutation in the exon-5 of pif4 gene. This mutant is characterized by shorter hypocotyls and a compact rosette size. Complementation of GmPIF4b in Arabidopsis pif4-101 mutant partially rescued the short hypocotyl and compact rosette phenotypes of pif4-101 mutant. The results were suggestive of both conservation and divergence of GmPIF4b. To gain insights into the function of GmPIF4b in soybean, constitutive overexpression of GmPIF4b in soybean (Bragg) was undertaken. Constitutive overexpression resulted in decreased plant height, reduced leaf surface area, decline in total number of branches per plant, and early flowering in transgenic soybean plants as compared to the wild type. Quantitative RT-PCR was performed to study the transcript levels of soybean florigens; GmFT2a and GmFT5a in the transgenic plants (over expressing GmPIF4b). The transcript levels of GmFT2a and GmFT5a were significantly elevated in transgenic lines compared to the wild type. Late maturing soybean varieties, such as Bragg, can terminate flowering in sub-optimal photoperiods. To study the effect of GmPIF4b over-expression on termination of flowering, transgenic soybean plants (over expressing GmPIF4b) grown in short days were transitioned to non-inductive photoperiod (for 10 long days) at full bloom stage. Interestingly, wild type Bragg plants terminated flowers and produced less pods. Transgenic plants did not terminate their reproductive activity and gave rise to a higher number of pods per plant. To study the dynamics of GmPIF4b protein, immunoblot analysis was undertaken. The results showed that on protein level, GmPIF4b follows a strict diurnal expression pattern under both long and short days, suggesting the involvement of biological clock in regulating GmPIF4b protein expression. Chromatin immunoprecipitation was performed to study the protein-DNA interaction of GmPIF4b protein with the promoters of soybean florigens GmFT2a and GmFT5a. The results suggested that GmPIF4b interacts with the promoters of GmFT2a in short-day treated plants, whereas it interacts with the promoter of GmFT5a in a photoperiod insensitive manner. PIF4 is shown to be involved in nitrate assimilation and phosphate acquisition pathways. Soybeans plants were treated with nitrates or orthophosphates under inductive short days to analyse the GmPIF4b gene expression by RT-PCR. The results showed that the application of nitrates and orthophosphates can affect the levels of GmPIF4b transcript and can modulate the expression pattern of soybean floral identity marker gene, GmAP1. Hence, the outcome of this study can contribute towards developing climate-resilient varieties for the future.
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ItemThe potential for flow restoration in urban streams( 2019)Flow regimes in urban waterways are degraded by urbanization and hydraulically efficient stormwater drainage systems. To return more natural flow regimes, urban stormwater management has, in recent times, increasingly focused on source-control approaches – use of Stormwater Control Measures (SCMs). However, experimental evidence of the success of such approaches at the catchment scale is limited, which is a key impediment to the widespread implementation of SCMs. The overarching aim of this Ph.D. thesis is to evaluate the effects of SCMs on flow regimes in urban waterways. This research includes literature reviews and experimental analyses. The literature reviews identified the challenges in the design and analysis of catchment-scale experiments and outlined a series of considerations to address these challenges, which provided practical guidance for future experimental studies and prepared for the methodological development for the experimental part. The experimental analyses were based on an ambitious long-term catchment-scale experiment in Melbourne, Australia, including four Impact catchments (urban with intervention), two Control catchments (urban without intervention), and three Reference catchments (forested, non-urban). Over 700 SCMs were implemented across the four Impact catchments during the study period (October 2009 – September 2017). A suite of flow metrics at different temporal scales and SCM implementation metrics integrating imperviousness and hydrological information were selected, based on literature review and statistical analysis, to facilitate the assessment. An investigation on the hydrological effects of urbanization, using a space-for-time approach, found that quickflow and variability metrics were the most affected by urbanization levels as measured by effective imperviousness (EI). Total flow and slowflow metrics were also affected by urbanization but to a lesser extent. This analysis was used to hypothesize the likely effects of SCM implementation on the flow metrics. The hydrological effects of SCMs were investigated using sophisticated statistical models, to identify to what extent have SCMs changed the flow regime over time. In general, SCMs reduced the negative effects of urbanization, by reducing quickflow, total flow, and absolute variability. However, there were negligible changes in slowflow and inconsistent changes in relative variability metrics, requiring further research. The SCMs have more impacts on short-term flow regime characteristics that better capture the immediate effects of SCMs. The differing relationships between each of the SCM implementation metrics and the flow metrics helped to identify and interpret the likely mechanisms driving SCM impacts on flows. These findings also helped prioritize future work in both practice and research. This novel study makes a significant contribution to the understanding of the effects of the catchment-wide implementation of SCMs on flow regime restoration. It revealed that the SCMs returned most aspects of the flow regime towards the natural condition, but more work is required to restore the full flow regime. This study will also serve as a model for future studies, informing the selection of metrics and statistical methods, and the requirements for future such experiments.
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ItemThe functional analysis of soybean (Glycine max L.) flowering genes( 2019)Soybean (Glycine max L.) is one of the major crop plants since its seeds contain high levels of oil and protein. In addition, soybean’s unique ability in fixing nitrogen makes it a key plant for sustainable agriculture due to the increasing cost of nitrogen fertilizers caused by continuing depletion of petroleum. The demand for improvement of soybean yield has been rapidly growing due to the plant’s high nutritional values and its significance in sustainable agriculture. Flowering is a major step in plant life cycles as it plays crucial roles in reproductive success. Late flowering under favourable conditions allow plants to maximize their vegetative growth, which ultimately leads to improved seed production, whilst early flowering allows plants to secure their progenies under adverse growth environments. Comparative analysis of flowering genes between Arabidopsis and soybean has revealed conservation of most flowering genes. However, functions of most flowering genes in soybean are still unknown. Therefore, investigation of soybean flowering genes is expected to provide insight into flowering mechanisms of soybean. Plant genetic transformation is an important tool to improve agricultural traits and investigate functions of genes. Since reported success in soybean genetic transformation has been limited to inferior-breeding cultivars, development of stable transformation systems for commercial soybean cultivars will provide a new solution to meet the ever-increasing demand for soybean. In the present study, transformation systems for commercial cultivars of soybean were developed using the Agrobacterium-mediated transformation method. Transgenic soybean plants (cv. Bragg) containing yellow fluorescence protein (YFP) and herbicide resistant gene (bar) were produced using half-seed transformation method. Shoot elongation efficiency was increased (6 fold) by addition of phenolic compound inhibitors [adenine hemisulfate (40mg/L) and PVP 40,000 (500 mg/L)] during shoot elongation, resulting in improvements in the growth of transgenic shoots. Total 23 independent T0 putative transgenic lines were produced and herbicide resistance was confirmed via basta brush test (100mg/L glufosinate). Total two basta resistance lines exhibited YFP expression in leaves. Stable expressions of transgenes were observed in T1 and T2 generations. The roles of soybean LFY homolog (Glyma.06G163600.1) in flowering initiation was confirmed in this study. LEAFY gene (LFY) is one of floral meristem identity genes and plays essential roles in flowering. In soybean, two LFY homologs (Glyma.04G202000.1 and Glyma.06G163600.1) are annotated and they show high sequence similarity (Glyma.04G202000.1: 73.4% and Glyma.06G163600.1: 69.5%) with Arabidopsis LFY (AtLFY). Furthermore, soybean LFY homologs have two conserved DNA-binding domains (N- and C-domains). The expression levels of both soybean LFY homologs gradually increased in flowering inductive conditions and Glyma.06G163600.1(GmLFY1) showed a higher expression than Glyma.04G202000.1 (GmLFY2). These high sequence conservation and expression patterns of soybean LFY homologs suggested GmLFY genes may have roles in flowering initiation. Ectopic expression of GmLFY1 in transgenic Arabidopsis and tobacco plants induced early flowering phenotypes. Moreover, up-regulations of genes (AP1, SOC1 and LFY) involved in flowering were also detected in transgenic plants. These results suggest that GmLFY1 may regulate flowering time via the conserved process as in Arabidopsis. Besides, tissue-specific GUS expressions on sepals were detected in flowers of transgenic tobacco plants (GmLFY1::GUS), indicating that GmLFY1 is also involved in flower development. The ageing pathway is one of the identified flowering genetic pathways and the miR156-miR172 module plays major roles in this process via repression of their target genes. In this study, expression patterns of soybean miR156 and miR172 (gma-miR156a, gma-miR172a) in vegetative and reproductive developments were confirmed. Expression of gma-miR156a was higher than that of gma-miR172a in the vegetative developmental stage and it decreased with ageing. On the other hand, expression of gma-miR172a was elevated under flowering inductive conditions. These expression patterns of soybean miR156a and miR172a genes suggest that they may be involved in the developmental process. Ectopic expression of gma-miR156a in transgenic tobacco plants caused significant delays in flowering initiation with extended juvenile developmental traits (round shape of leaves). In contrast, transgenic tobacco plants overexpressing gma-miR172a exhibited early flowering phenotypes with adult traits on leaves (narrow shape). Significant down-regulations of miR156 target genes (SPL transcription factor family) and miR172 target genes (AP2-like genes) were detected in transgenic tobacco plants. These results showed that gma-miR156a and gma-miR172a may regulate ageing process via repressions of their target genes. In the present study, transgenic commercial soybean cultivar (cv. Bragg) was produced using the Agrobacterium-mediated method. In addition, this study provided evidence of conserved roles of GmLFY1, gma-miR156a and gma-miR172a in flowering and plant developments via heterologous expressions in transgenic Arabidopsis and tobacco plants. GmLFY1 is involved in floral meristem development and initiation of flowering. gma-miR156a is responsible for juvenile developments via repression of SPL transcription factor family. In contrast, gma-miR172a plays major roles in adult developmental phase by down-regulations of its target genes. These results will provide new insights on the genetic improvement of soybean.
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ItemEconomic analysis of ameliorating subsoil constraints using subsoil manure in a cropping system( 2019)To date, no studies have accounted for the effects of the yield and/or price risks that will occur over a run of years on the profitability of investing in ameliorating subsoil constraints within a cropping system. While addressing subsoil constraints is likely to increase grain yield, the key economic question for a grower is whether the income from extra grain produced covers the extra costs of ameliorating the subsoil. The focus of this thesis was the likely profit and risk of investing in ameliorating subsoil constraints. Investment costs and annual activity gross margins for a set rotation were used to estimate the economic performance of subsoil amelioration. The marginal change to the gross margin as a result of subsoil amelioration was assessed using partial discounted cashflow budgets. Risk analysis was used to assess the effect of price and yield variability on the mean and variance of outcomes from an investment in ameliorating subsoil constraints in cropping. This study shows an investment in subsoil amelioration was more profitable on average than an alternative investment earning 6% (real). The size of the expected extra yield benefits and longevity of these benefits are the most important factors for a grower to consider when assessing the likely merit (return and risk) of investing in subsoil amelioration in their own situations.
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ItemUnderstanding small and large milk fat globule phenotype variation in dairy cows through milk lipidomic characterisation( 2019)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.
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ItemAmelioration of the effects of cyclic heat stress in broiler chickens with dietary betaine and antioxidants( 2019)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.
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ItemCounterfeiting and Design-Driven Deterrents: Trust perceptions of anti-counterfeiting strategies for food and product packaging( 2019)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.
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ItemPrevalence and Transmission of Antibiotic Resistance Genes in the Soil Environment( 2019)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.
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ItemUsing LiDAR for landscape-scale mapping of potential habitat for the critically endangered Leadbeater's Possum( 2019)Leadbeater’s possum (LBP) (Gymnobelideus leadbeateri) is a critically endangered arboreal marsupial located in Victoria’s Central Highlands in southeastern Australia. Populations of hollow-bearing trees, the key nesting habitat resource for Leadbeater’s Possum, are rapidly declining. Disturbances such as bushfires and timber harvesting are believed to be the major causes in the reduction of hollow-bearing trees and thus the extent, quality, and connectivity of LBP habitat. LBP also requires a connected understorey or midstorey for foraging and movement within the forest. Changes in LBP habitat at the landscape-scale are interpolated from plot-level measures and assumed distribution of habitat resources based on forest type and disturbance history. Long-term monitoring at the plot-level has reduced uncertainty around habitat resources required by LBP within the montane ash (Eucalyptus regnans, E. delegatensis, E. nitens) forests of the region; however, it has not addressed uncertainty around the landscape distribution of nesting and foraging habitat resources. Developing effective conservation strategies for LBP requires an understanding of the distribution and quality of available habitat across its potential range. This currently does not exist. I used Light Detection and Ranging (LiDAR) data to develop empirical models that quantify the distribution, quality, and connectivity of LBP habitat in the montane ash forests within a multi-scale framework. LiDAR-derived topographic and vegetation structural metrics were integrated with different habitat analysis approaches to model the distribution of critical LBP habitat features (hollow-bearing trees, foraging and midstorey stratum connectivity) at multiple scales across ~340,000 ha of forest in Victoria’s Central Highlands. In Chapter 2, tree-level individual tree delineation (ITD) algorithms were developed to identify individual trees and analyse tree crown attributes. ITD algorithms for overstorey trees were applied to the whole study area to estimate the abundance and distributions of large live trees with big crowns that have high probability of containing hollows. Based on field surveys in 1939 regrowth mountain ash, the dominant overstorey crowns identified by the ITD algorithms had an 86% success rate in identifying mapped trees in mature forest plots. Our landscape analyses estimated 405,000 large live trees (crown width >15m) and 572,000 (95%CI: 318,000-808,000) potential large live tree with hollows (DBH>150cm), which equate to a mean density of one live large old tree per ha across the Central Highlands. ITD algorithms were also used to map canopies and to calculate the proportion of projection area of midstorey stratum, a surrogate for midstorey coverage, for use in assessments of foraging habitat. Hollow-bearing trees are a key feature of mature forests around the world. They provide critical habitat resources for hollow-dependent animals, many of which are threatened, due to a range of natural and human disturbances. In Chapter 3, to develop more accurate inventories of hollow-bearing trees (both live and dead form classes) across a large landscape, I used LiDAR-based metrics of forest structure and topography, coupled with datasets on environmental conditions to develop statistical models of abundance of hollow-bearing trees using machine learning tools. These provided empirical estimates of HBT density for every hectare of the Central Highlands within the LiDAR footprint. I identified 1,519,000 (95%CI: 1,306,000 – 1,730,000) hollow-bearing trees may occur across the full extent of Victoria’s Central Highlands. This included 833,000 (95%CI: 742,000-923,000) live HBTs and 686,000 (95%CI: 564,000-807,000) dead HBTs. The predictive models provided rigorous, repeatable estimates of tree abundance across a wide range of vegetation classes and forest management zones (with appropriate estimates of uncertainty), as well as a new understanding of the complexity of the structural, topographic, and environmental features associated with abundance of hollow-bearing trees and their spatial variability over large areas. In addition to HBTs for nesting, LBP require a well-connected midstorey stratum to facilitate movement amongst nesting trees and foraging. In Chapter 4, the association between field-assessed multi-storey vegetation connectivity and LiDAR structural metrics was evaluated to develop landscape-scale predictions of connectivity and foraging habitat (density of live wattle) for LBP. Forest structural types with high midstorey connectivity, which are a critical habitat feature for LBP, occupied 17.2% of the study landscape. The landscape-scale predictions of vegetation connectivity provided an understanding of the factors (e.g., topography, fire, logging and interactions among strata) that help shape connectivity and foraging habitat. This understanding is critical for improving the management of the region’s forests and, in particular, ensuring that both key habitat elements, HBTs and dense midstorey vegetation, either co-occur in the same stand or are in close proximity to ensure sufficient high-quality habitat for LBP over space and time. In Chapter 5 I used the landscape-level mapping of habitat resources developed in Chapters 2-4 to predict landscape-scale LBP habitat suitability based on fields records of LBP occurrence. The validated habitat suitability model for LBP had an overall accuracy of 87.3% and an AUC of 0.889. The model predicted that 28,000 ha (95%CI: 17,000ha-40,000ha) of the study landscape (i.e., 9.6%) supported suitable habitat for LBP. Twelve variables were found to have the strongest influence on estimating habitat suitability for LBP. Of these, four related to foraging habitat, three to nesting habitat, three to climatic and topographic factors, and two to disturbance history. Our approach highlights the potential for using high-resolution, spatially explicit data on forest structure at a landscape-scale to map the distribution and abundance of suitable habitat for a critically endangered species. Habitat suitability models generally account for the selection of suitable habitat and their appropriate geographical extents. However, they rarely account for the accessibility of this habitat and connectivity among habitat patches. Effective conservation of species requires that patches of their habitat are connected in space and time. In Chapter 6, graph-theoretic connectivity networks based on resistance surfaces were generated from the LiDAR mapped cover of vertical stratum. The least-cost links between patches from networks were integrated with a kernel density estimator to identify functionally connected regions. The mapping of landscape connectedness identified a potential landscape-scale metapopulation structure for LBP within the Central Highlands. This provides a tool that could be used to expand the existing protected area network to support the metapopulation processes of LBP. The identification of functionally connected regions could enhance the conservation planning for long-term population persistence. The conservation and management of endangered species is major focus of forest and land managers around the world. High-quality data on the distribution and quality of habitat is critical to the development of effective conservation and recovery strategies for species. This thesis developed a set of habitat analysis models for quantifying habitat resources, habitat suitability and connectedness for the critically endangered Leadbeater’s Possum across a range of spatial scales. These analyses highlight the benefits of using high-resolution, spatially explicit LiDAR data from the whole landscape to identify and map (1) the distribution and abundance of hollow-bearing trees, (2) midstorey stratum connectivity, (3) the distribution and abundance of suitable habitat, and (4) metapopulation structures within functionally connected regions. Dynamic modelling, informed by these LiDAR-derived models, can be used to forecast the likely consequences of changing habitat abundance under different management scenarios and evaluate both the short-term and long-term effectiveness of different conservation strategies.