Agriculture and Food Systems - Theses
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Microwave and biochar soil treatment alleviates arsenic phytotoxicity in wheat and rice
Abstract Arsenic (As) is a toxic metalloid, which is carcinogenic i.e. cancerous to humans. Besides the drinking water, accumulation of As in food grains through plant uptake, when cultivated in As contaminated soils, is a potential route of human dietary As exposure. This has inspired research into alleviating grain As accumulation, despite there being already existing strategies with major disadvantages such as low efficiency, high costs, and usage being restricted to smaller-scale operations. Therefore, pre-sowing microwave (MW) soil heating and sawdust biochar were used to investigate if they can reduce As concentration in wheat and rice. Microwave is a form of electromagnetic radiation, which can produce heat in the soil by inducing the rotation of the dipoles of polar molecules (e.g. water). Microwave heating depends on the dielectric properties of the soil. Therefore, a study was conducted to determine the dielectric properties of different types of soils with different moisture content. The results showed that the soil moisture was the major contributor to the dielectric behavior of soil since dielectric properties increase as soil moisture increases. Soil types also had an influence as the dielectric properties of sandy soil were much lower than the other soils such as clay and loam soil. To investigate the effect of MW and biochar on wheat and rice grain As concentration, both the wheat and rice soils were spiked with five As concentrations (As-0, As-20, As-40, As-60, and As-80 mg kg-1 soil). In addition to MW, biochar was used to reduce rice grain As accumulation since biochar has been gaining attention for its heavy metal immobilization capacity. After As application, three levels of biochar (BC-0, BC-10, and BC-20 t ha-1 soil) were added only in the rice-growing soil. Then, soils were treated for 0, 3, and 6 minutes (MW-0, MW-3, and MW-6) in an MW chamber to achieve the soil temperature of around room temperature, 60 oC, and 90 oC respectively. The crops were grown in a completely randomized design with four replications in a glasshouse during 2017 (wheat) and 2018 (rice). The results demonstrated that, in both the wheat and rice, MW soil treatments, especially the MW-6, alleviated As phytotoxicity and facilitated less grain total As concentration compared with the MW-0 treatment across all the soil As concentrations. Also, MW treatment significantly reduced the concentration of arsenite [As(III)], the most toxic form of As. Decreased grain As concentration in rice was recorded at BC-10 in lower levels of soil As concentrations (As-20 and As-40) while, a negative impact was observed at BC-20 across all the soil As concentration, compared with BC-0 treatment. Furthermore, rice grain As(III) concentration increased significantly in BC-20 treatment. Thus, MW-6 treatment could be used for the alleviation of grain As concentration in wheat and rice grain, whereas more study is needed for the best biochar application rate. However, understanding the residual effect of MW and biochar is crucial for the sustainability of the treatment. Therefore, the same varieties of wheat and rice were grown in the following year, using the same pots, without the addition of further MW or biochar treatment. The results revealed that, 360 days after MW soil treatments there was still the potential to alleviate grain As concentration in both wheat and rice. A similar result was observed for biochar treatment in the residual year with a positive effect at BC-10 and a negative effect at BC-20 treatment. Furthermore, it is unclear whether MW soil treatment is just a heating effect or if there is some other effect of the electromagnetic wave involved. Therefore, a glasshouse pot study was designed to investigate the effect of MW and conventional electric oven (EO) soil heating on As phytotoxicity alleviation in rice. The soil was spiked with three levels of soil As concentration (As-0, As-40, and As-80 mg kg-1) prior to applying MW and EO heat treatments, to achieve the soil temperature of around 80 - 90 oC. The results showed that, there was no statistically significant difference between MW and EO treatments regarding As phytotoxicity alleviation. However, the positive effect was more in MW treatment than the EO treatment. Significantly less total energy required in the MW to treat the soil than the EO. Besides the As phytotoxicity alleviation, the effect of MW soil heating on soil microorganisms, particularly bacteria, was a topmost concern and investigation was needed to ascertain that MW soil heating does not affect it drastically. Therefore, an experiment was designed to investigate the effect of MW heating (80 - 90 oC) on the soil bacterial community in As contaminated (As-0, As-40, and As-80 mg kg-1 soil) soils. The 16S rRNA bacterial gene copy numbers decreased significantly after MW soil heating but recovered back to its previous number 42 days post treatment. The bacterial diversity also decreased significantly in MW treated soils but did not recover even after 56 days from MW heating. However, there was no noticeable effects of soil As concentration on bacterial community were observed. Furthermore, relative abundance of some beneficial bacteria such as Bacillus and Symbiobacterium were significantly higher in the MW treated soils. Thus, MW soil heating at 80 - 90 oC can potentially be applied for As phytotoxicity alleviation without significantly destroying the ecologically important taxa. Overall, pre-sowing MW soil heating could be applied as a novel technique to alleviate As phytotoxicity in wheat and rice with lower As accumulation in the grain. Thus, application of the MW technology in the As contaminated area like Bangladesh could add another feather in the crown of the As remediation techniques and help to reduce the human health risk through As contaminated food grain. However, further research needed before adopting the MW soil heating technique where different aspects should be explored such as response of MW technology in the field condition, scaling up the MW equipment for field application, cost of MW application in the field at farmers level, long-term effect of MW treatment on soil nutrient dynamics, soil organic matter and soil biota and sustainability of the MW technology in field condition. Also, sawdust biochar could be used in combination with MW soil heating for As phytotoxicity alleviation; however, more study needed to set the appropriate rate of biochar application.
The use of computer vision techniques as noninvasive tools to monitor parameters related to the well-being and productive performance of cattle and pigs
Among a wide range of factors that can affect animals’ wellbeing, stress levels and health status have been identified as relevant factors in production animals. The increasing awareness about animal welfare and the impact that it has on farm productivity has been promoting scientific research and the development of novel and less invasive methods to monitor animals and obtain measurements that can be used as indicators to assess animal wellbeing, including stress and health status. Research presented in this thesis deals with red-green-blue (RGB) and thermal infrared (TIR) imagery, and computer vision techniques, as non-invasive tools to measure physiological changes in cattle and pigs to assist in the assessment of their stress and health. One study was conducted to evaluate the performance of the proposed methods, which were used to analyse RGB and TIR imagery to measure eye-temperature, heart rate (HR) and respiration rate (RR) in cattle. The study was performed in a robotic dairy farm, where TIR and RGB cameras recorded ten dairy cows during six handling procedures, across two consecutive days. Simultaneously, core body temperature, HR and RR were measured using standard methods for comparison with the data obtained from the recorded images using the developed algorithms. A feature tracking algorithm was developed to facilitate the processing of RGB videos, which showed an accuracy between 92% and 95% depending on the area analysed. From the physiological parameters analysed, the highest correlations were observed between eye-temperature and intravaginal temperature (r = 0.8; P<0.01), and between remote RR and the RR obtained from visual observations (r = 0.87; P<0.01). A further two studies were carried out to implement the proposed computer-based methods to remotely measure eye-temperature, heart rate and respiration rate of cattle, and to investigate whether these measures could be used to evaluate the physiological response of cattle to stressful situations and whether they could be used as predictors of beef quality. For these two studies, 215 beef cattle were recorded with RGB and TIR cameras on the farm and at the abattoir to obtain eye-temperature, HR, and RR measurements. Cuts of the respective beef were evaluated by consumers, and the ultimate pH (pHu) and meat colour were obtained from the respective carcasses. It was observed that the physiological variables of cattle were higher at the abattoir compared to the farm. Moreover, eye-temperature obtained on farm and at the abattoir were highly correlated. However, the results of these studies indicated that these measurements had low contribution when predicting beef quality. Finally, two studies were performed to investigate the use of the proposed computer vision methods with RGB and TIR imagery to monitor pigs and detect changes in eye-temperature, ear-base temperature, HR, and RR. The objective was to identify whether these physiological changes could assist in the early detection of pigs affected by respiratory disease. The first of these studies was performed under experimental conditions, where pigs were challenged with Actinobacillus pleuropneumoniae (APP). Images (TIR and RGB) were recorded after this challenge, and the data obtained was then compared between the group of pigs that presented clinical signs of respiratory disease and the group of pigs that were considered healthy. Clear changes in temperature and HR were observed six or more hours before the clinical observations identified sick animals. Conversely, changes in RR were detected in the last period of observations, around the time when clinical signs started to be present. The second of these experiments used different cameras and included improvements to the proposed methods to monitor pigs constantly and in a commercial setting. A total of 48 pigs were monitored between 9 and 20 weeks of age. Eye-temperature, HR and RR measurements were compared between the pigs that were identified as sick and those that were considered healthy. Similarly to what was observed in the previous study, changes in these parameters were identified before the clinical observations indicated signs of illness (up to 2 days before), where the earliest changes were observed in eye-temperature and HR, and the latest changes were observed in RR. This thesis provides evidence that computer vision techniques may be suitable as a non-invasive method for monitoring farm animals. Therefore, it prompts further investigation via controlled studies to continue the development and automatization of these techniques, leading to the improvement of science-based industry-relevant monitoring systems.
Endocrine and metabolic status of dairy goats during the transition period
In dairy animals, the transition period, which spans from 3 weeks before to 3 weeks after parturition, is the most stressful time in their productive lives. This period is characterized by drastic physiological, metabolic, and endocrine adaptations to accommodate parturition and lactogenesis. Goats unable to adapt to this challenging time are more susceptible to infections and metabolic diseases, which might have a substantial impact on maternal health and productive efficiency beyond the transition period. An in-depth understanding of the biology of the transition period is essential for developing optimized strategies that could enhance milk yield without compromising herd health and welfare. While there is ample published information regarding the transition period in dairy cattle, to date, the endocrine and metabolic status of periparturient dairy goats has only been vaguely described in the literature. Therefore, the overall goal of this dissertation was to expand on previous knowledge of hormonal and metabolic regulation of energy metabolism during the transition period and to explore factors that might aggravate the metabolic burden of pregnancy and lactation in periparturient dairy goats. Thus, a series of studies were conducted in a large commercial goat dairy farm in Australia to 1) determine the effects of month of kidding, parity number, and litter size on lactation curves of dairy goats raised in intensive systems; 2) characterize temporal variations in circulating levels of selected hormones and metabolites involved in energy balance regulation during the transition period; 3) investigate the effects of level of milk production, parity number and litter size on maternal metabolic profile; 4) determine whether higher plasma concentrations of markers of negative energy balance are associated with inferior productive performance; and 5) determine whether differential productivity is related to differences in nutrient partitioning between high- and low-yielding goats. In the first part of this study, an analysis of the production data revealed that goats kidding in spring, in third/fourth parity, or carrying multiple fetuses produce more milk than their counterparts. Interestingly, although the month of kidding had the most significant impact on the shape of lactation curves, the magnitude of such impact increased with increasing parity number. Also, based on the concentration of key biomarkers of energy metabolism analyzed during this time, it was possible to conclude that nutritional deficit was increased with increasing milk yield, parity, and litter size (listed in order of importance) and that both pregnancy and lactation were less able to elicit lipomobilization in primiparous compared with multiparous goats. Further, the likelihood of early removal from the milking herd was significantly increased in goats with elevated blood levels of beta-hydroxybutyrate (BHB). On the other hand, contrasting studies in dairy cows, a positive association was observed between blood levels of non-esterified fatty acids (NEFA) and milk yield. Nevertheless, it was unclear what role, if any, the endocrine system played in the differential productivity in early lactation observed between high- and low-yielding goats. Therefore, in the second part of this study, goats of high and low milk yield were subjected to 3 metabolic challenges (glucose, insulin, and adrenocorticotropin hormone infusions) to determine if differential productivity is related to differences in some aspects of the regulation of nutrient partitioning in dairy goats. The results suggested that differences in milk yield, and overall production efficiency in early lactation, are primarily due to differences in insulin secretion and clearance rates rather than related to differences in peripheral tissue responsiveness to the effects of catabolic and anabolic hormones. In summary, the research within this thesis provides the first comprehensive overview of both lactation performance and the metabolic status of Australian dairy goats. Collectively, the novel findings presented here contribute to further the current understanding of various aspects of the regulation of energy metabolism in periparturient dairy goats. Just as important, this study also provides the local industry with robust and relevant information on the effects of several factors on the productive and metabolic responses of dairy goats during the transition period. Such information can assist with the optimization of farming practices and breeding plans, thereby accelerating increments in the national herd productivity.
Application of New Technologies in the Safety Assessment of Genetically Modified Feed
Agricultural biotechnology, such as genetic modification, has been proposed as a way to face food security and climate change. All new crop varieties with altered genetics must be subjected to safety assessments to fulfil regulatory requirements, prior to marketing and/or commercial release. The aim of this thesis is to undertake part of the safety assessment of transgenic plants, using emerging molecular biology technologies. Initially, the molecular characterisation of two genetically modified plants were performed using long read DNA sequencing. Results demonstrate the capability of this technique to characterise transgenes located in complex and or repetitive regions of the plant genomes, in a fast and cost effective way with simple and robust bioinformatic pipelines that would be broadly applicable and accessible. Then, a reliable methodology was developed using droplet digital PCR tools for the detection of transgenes in a variety of complex pasture based products relevant to livestock industries. Later, a toxicological assessment of the high energy perennial ryegrass was performed using a metabolomics approach. The current study indicates that the new technologies can provide a highly reliable and efficient solution for the safety assessment. This thesis represents a contribution in the use of new technologies to improve the risk assessment procedure of genetically modified plants.
Understanding the Importance of Microbial Biogeography to Australian Winemaking
Microbes are a vital part of ecosystems and play key roles in the essential processes of the functioning. In agriculture, microbial ecology has wide reaching impacts on crop growth and quality commodity production. As a high value agricultural product, wine is a useful model for elucidating the effects of microbial ecology from the vineyard to the winery. Microbial growth and metabolism is an inherent component of wine production, influencing grapevine health and productivity, conversion of sugar to ethanol during fermentation, and the flavour, aroma and quality of finished wines. Recent advances in genetic sequencing and metagenomic approaches has extended our understanding of microbial distribution patterns and established the unique biogeography model in viticulture. While the contributions of microbial biogeography to wine metabolites and regional distinctiveness (known as terroir, a well-recognised and celebrated character in wine industry), and by which mechanisms, remain tenuous. This thesis focuses on the microbial biogeography of wine, the interplay between microbial patterns and affecting factors, and how these patterns drive wine quality and styles. I begin by investigating the distribution patterns of bacteria and fungi at large scale, and their roles in shaping wine characteristics. Samples were collected from vineyard soil, grape must, and wine ferments across six geographically separated wine-producing regions in southern Australia (~ 400 km). Soil and grape must microbiota exhibited distinctive regional patterns, as well as wine aroma profiles. Associations among soil and wine microbiota, abiotic factors (weather and soil properties), and wine regionality were modelled, highlighting that fungal communities was the most important driver of wine aroma profiles. Source tracking wine-related fungi in the vineyard suggests that soil is a source reservoir of grape- and must-associated fungi which might be translocated via xylem sap. I then move on to elucidate the fungal ecology within vineyards. Fungal communities were characterised over space and time that associated with the grapevine (grapes, flowers, leaves, roots, root zone soil) during the annual growth cycle (flowering, fruit set, veraison, and harvest). Fungi were significantly influenced by the grapevine habitat and plant development stage, with little influences from the geographic location (<5 km). The developmental stage of veraison, where grapes undergo a dramatic change in metabolism and start ripening process, saw a distinct shift in fungal communities. A core fungal microbiota of grapevines (based on abundance-occupancy models) existed over space and time which drove the seasonal community succession. Beyond coinciding with the changing plant metabolism and physiology, strong correlations with solar radiation and water status suggests that the core microbiota changes with respect to the changing environments during plant development. I further investigate fungal contributions to wine aroma profiles by quantifying multiple layers of fungi, combining metagenomics and population genetics. Fungal communities were characterised associated with Pinot Noir and Chardonnay grape must/juice and ferments coming from three wine estates (including 11 vineyards) in the Mornington Peninsula wine region. At this scale (< 12 km), fungal communities, yeast populations, and Saccharomyces cerevisiae populations differentiated between geographic origins (estate/vineyard), with influences from the grape variety. During spontaneous fermentation, growth and dominance of S. cerevisiae reshaped the fungal community and structured the biodiversity at strain level. Associations between fungal microbiota and wine metabolites highlights the primary role of S. cerevisiae in determining wine aroma profiles at sub-regional scale. Overall, this thesis provides a significant body of knowledge to the microbial ecology field. Using vineyards, grapes, and wine as a model system, these findings relate microbial biogeography, environments, and quality agricultural commodity production. It provides fundamental perspectives to conserve the biodiversity and functioning for sustainable agriculture under the changing climate.
Rapid screening methods for superior trait selections in lentil and field pea breeding
Most of the lentil and field pea grown in Australia are exported to India and surrounding countries, to the Middle East, Turkey and North Africa. While each country may utilise pulses differently, common to all, is that quality is based on the visual characteristics of the whole-grain and split-pulse, and its cooking quality. One of the objectives of pulse-breeding programs is to ensure that the quality traits of new varieties align with the preferences defined by the export markets. Pulse-quality traits were historically determined using empirical tests to quantify seed size, colour, contamination and defects. Since many of these tests are time-consuming to perform, comprehensive quality evaluation is reserved for advanced germplasm. Therefore, adoption of rapid and objective methods would improve efficiency and consistency of quality evaluation and enable comprehensive assessment of early generation lines. Technological advances in digital imaging and machine learning has seen a broad application of machine vision to assess agricultural products. While there is extensive research in this field, there are still relatively few machine vision methods which have been developed for the quality-assessment of lentil and field pea grains. Within this study, rapid and objective methods were developed to assess three grain-traits, which related to visual characteristics of lentil and field pea and were identified to be important within breeding programs. The targeted applications were the classification of broad market classes of field pea, quantitation of bleaching discoloration within the ‘green pea’ market class and classification of split and dehulled fractions of lentil and field pea post milling. Machine vision algorithms were developed based on the analysis of multispectral images. Linear discriminant analysis, based on image-derived colour, shape and size features, was used for the classification of field pea market classes. The model was applied to sound and defective grain samples, achieving perfect classification of sound grain and distinguishing sound from defective grain with 97% accuracy. The extent of bleaching in green field pea samples was quantified through an objective model which was developed on visible reflectance spectra (spectrophotometric analysis) and subsequently adapted for image-based analysis of grain colour. The image-derived colour scores closely matched the spectrophotometric analysis and additionally enabled the distribution or uniformity of bleaching to be objectively quantified within each sample. Furthermore, through the image analysis scoring system, the relative susceptibility to bleaching, of each genotype, was also quantified. Milled fractions of lentil and field pea were classified through the application of artificial neural networks, where network architectures and inputs were compared. A convolutional neural network, trained on image-derived feature distributions, was found to be the most accurate and computationally efficient model. Machine vision is an expanding field of research which offers the potential for consistent, accurate and rapid product-quality evaluation. The results of this study demonstrate the efficacy of machine vision applications throughout the pulse value chain and particularly within germplasm enhancement programs. Adoption of machine vision systems can increase the capacity for comprehensive screening at all stages of breeding which is currently not practicable through standard assessment methods.
An Investigation of the Role of Organic Amendments on the Availability of Phosphorus in an Acidic Soil
The recovery of P from organic waste streams (known as organic amendments, OA) is becoming increasingly popular. Besides a source of P, OA have also been reported to increase soil P availability and reduce P adsorption in acidic soil. The main mechanism involves the reduction in the soil P adsorption sites (especially Al and Fe) by the organic anion producing compounds of the OA. Organic anions form organometallic complexes in the soil to reduce P adsorption. The potential representative organic anion producing compounds of the OA are phenolic and carbonyl compounds. The phenolic and carbonyl compounds usually represent the aromatic (such as benzoic, hydroxybenzoic, protocatechuic acids etc) and aliphatic (such as oxalic, malic, citric etc) organic acids, respectively. This study aimed to investigate if these signature phenolics and carbonyl compounds of the OA can be used to predict their role in soil P availability for future prescriptive applications. An acidic soil (Red Chromosol) from the Rutherglen, northeast of Victoria, Australia was collected and waste from two different streams namely food waste and biosolid and their three different processed forms (untreated or raw, biochar and compost) were utilized as sources of OA in this study. Nuclear magnetic resonance (NMR) spectroscopy was utilized to identify forms of C (solid-state 13C NMR) and P compounds (solution-state 31P NMR) in the OA. The effect of OA on soil P availability was studied in a 110-day laboratory incubation study. Further, some selected representative compounds of phenolic and carbonyl C of OA (aromatic and aliphatic organic acids) were tested against soil P availability in another 110-day laboratory incubation experiment. Finally, a plant bioassay experiment with Triticale (genus Triticosecale) was conducted to understand the effect of both OA and organic acid on soil P availability and plant P uptake with or without the addition of external P. Results from incubation of OA with soil showed an increase in soil P availability (Olsen P) and a decrease in soil P buffering capacity, (PBC). An additional increase in soil Olsen P was observed above the total amount of Olsen P from OA that was added. It might imply that this additive increase of the soil Olsen P was due to the substitution of previously fixed P by the action of organic anions (coming from the dissociation of phenolic and carbonyl compounds) and formation of the stable organometallic complexes. This was further supported by a strong and positive correlation between the delta Olsen P (difference between observed and expected Olsen P) and phenolic and carbonyl C content of the OA. The phenolic and carbonyl C content of the OA also showed a significant correlation with the decrease of soil PBC (delta PBC, the difference between treatment and control). In both correlation studies, the ratio of phenolic to carbonyl C compounds of OA showed a strong correlation with soil P availability compared to their total abundance. The contrasting nature of phenolic and carbonyl compounds was speculated as to the reason why there was a high correlation between soil P availability and the ratio of phenolic to carbonyl C content of OA. The OA with a nearly equal abundance of phenolic to carbonyl C content showed a maximum increase in soil P availability. Therefore, it was concluded that the ratio of phenolic and carbonyl C content of OA could be used to predict P availability in acidic soil. In the next study, some naturally occurring aromatic organic acids (representative of the phenolic compounds) and aliphatic organic acids (representative of the carbonyl compounds) were added either individually or in combination (at 1:1 ratio of aliphatic and aromatic organic acids). The experimental results suggested that aliphatic organic acids were more effective in increasing soil P availability and decreasing soil PBC compared to aromatic organic acids. However, the effect of aliphatic organic acids on increasing soil P availability was prolonged when applied in combination with aromatic organic acids at 1:1 ratio. Therefore, this study concluded that the effect of an OA can be predicted based on their phenolic and carbonyl C content. Finally, the results from the plant bioassay experiment showed that the addition of OA increased the soil P availability irrespective of addition of external P to the soil. As hypothesized in the previous study, this increase in soil P availability after OA addition was expected to show a strong correlation with the ratio of phenolic to carbonyl C content of the OA. The correlation study between the soil P availability and the ratio of phenolic to carbonyl C content of the OA showed a moderate to weak correlation. The differences in those studies (laboratory incubation vs pot) were speculated as to the reason for obtaining this moderate to week correlation between the soil P availability and ratio of phenolic to carbonyl C content of the OA. In the plant bioassay experiment, OA and P were added simultaneously to the soil. Therefore, a competition between organic anions (which was coming from dissociation of organic acid compounds of OA) and P and was expected to increase P availability in the soil by reducing P adsorption. However, the preferential sorption of P by Al and Fe compared to organic anions was speculated as the main reason for observed moderate correlation. The phenolic and carbonyl C content of OA would correlate strongly with the soil P availability if OA were added first (which might reduce P sorption by forming organometallic complexes) and then P to the soil as also observed in previous study. Therefore, it was concluded that the phenolic and carbonyl C content of the OA would predict soil P availability better if OA was first added (which would form organometallic complexes) and then P added to the soil rather than adding both simultaneously. The addition of OA significantly increased the plant P uptake and showed a positive correlation with soil Olsen P. There it was concluded that soil Olsen P was a good indicator of plant P availability. In the plant bioassay experiment, organic acid treatments showed no significant effect on soil P availability and plant P uptake compared to the control. This might be due to the addition of organic acids at 10 times lower concentration in this experiment compared to the previous incubation experiment. In conclusion, this thesis showed that the ratio of the phenolic to carbonyl C content of the organic amendments could be used to predict the role of organic amendments on increasing soil P availability. The availability of fertilizer P could also be predicted by phenolic and carbonyl C content, if OA added first and then P added to the soil compared to their simultaneous addition. The OA with a nearly equal abundance of phenolic and carbonyl C content would have a maximum effect on increasing soil P availability. This knowledge would be beneficial for farmers, agricultural landholders and especially OA producers to assess the impact of their organic waste products on soil P availability in the quickest possible time based on the abundance of the signature C compounds (phenolic and carbonyl).
Structural determinants of the quality of cooked meat
The quality of cooked meat is valuable for consumers and meat industry. The aim of this research was to identify structural determinants of the quality of cooked meat. Muscle type (bovine semitendinosus, psoas major, biceps femoris), cooking temperature (45 C to 85 C), ageing time (14 vs 0 days in beef; prolonged 15 vs conventional 3 days in pork), enzyme inhibition (with/without cathepsin inhibitor) and fibre type (bovine masseter 100 percent type I; cutaneous trunci 93 percent type II) were investigated factors affecting meat quality. Methods for measuring quality (cooking loss and Warner- Bratzler shear force (WBSF)), structural changes (shrinkage) and protein denaturation [Differential Scanning Calorimetry (DSC) and Fourier Transform Infrared (FTIR) microspectroscopy] were used. Cooking loss was higher in semitendinosus compared to biceps femoris and psoas major; and it increased with temperature in beef and pork. Cooking loss increased with ageing of beef independent of temperature and muscle type; and decreased with prolonged ageing compared to conventional ageing, of pork (cooked at 70 C and 80 C). The denaturation enthalpy of masseter and cutaneous trunci explained 58 and 59 percent of the variation in cooking loss, respectively. Reduction of WBSF in bovine muscles rich in collagen (all but psoas major) with cooking at 60 C to 65 C was attributed to collagen denaturation. The increase in WBSF in unaged beef with cooking at 70 C and 80 C was attributed to intact titin denaturation. In relation to shrinkage, 3D laser scanning was compared to caliper measurements, and was found inferior in measuring volume and predicting cooking loss. Cuboids` transverse and longitudinal shrinkage were higher in muscles with higher collagen content and sarcomere length, respectively. Transverse shrinkage started 5 C higher in psoas major fibre fragments, compared to semitendinosus and biceps femoris, likely due to predominant type I fibres. It was proven that transverse and longitudinal shrinkage of fibre fragments is caused by myosin and actin/titin denaturation, respectively. Since ageing reduced the longitudinal shrinkage of cooked beef (biceps femoris and psoas major cuboids at 80 C, semitendinosus and biceps femoris fibre fragments at temperatures greater or equal to 75 C) and pork, an important role of titin in longitudinal shrinkage was hypothesized. Cathepsin inhibition reduced the longitudinal shrinkage (semitendinosus, biceps femoris, psoas major cooked at temperatures greater than 75 C), and increased the transverse shrinkage (semitendinosus at temperatures greater or equal to 60 C) of fibre fragments. Longitudinal and transverse shrinkage were major contributors to cooking loss (beef and pork) and WBSF (beef), respectively. Cutaneous trunci had higher cooking loss (at temperatures lower or equal to 75 C), higher transverse (at temperatures lower or equal to 60 C), longitudinal (at temperatures lower or equal to 80 C) and volume shrinkage of fibre fragments (at temperatures lower or equal to 65 C); as well as lower transition temperature of myosin, higher reduction in alpha-helix and beta-sheet, and higher, compared to masseter, formation of beta-aggregated strands, random coil and aromatic side chains (at temperatures lower or equal to 60 C). The differences in protein denaturation and shrinkage between masseter and cutaneous trunci cooked at 55 C were attributed to the myosin isoform, while the differences in muscle fibre and connective tissue proteins’ denaturation at 60 C and 65 C were pH dependent. Myosin (isoform, denaturation), actin (denaturation), titin (degradation, denaturation), collagen (content, denaturation) and sarcomere length were proven or postulated, as structural determinants of the quality of cooked meat.
Control of the lettuce anthracnose (Microdochium panattonianum Berl.) using Trichoderma spp. and their secondary metabolites
The main objectives of this project were to: 1) show that lettuce anthracnose propagules that persist in soil from a previous lettuce crop may be controlled by a cold tolerant strain of Trichoderma inoculated just before the pathogen becomes active and virulent, reducing incidence and severity, and, 2) determine if liquid cultures containing Trichoderma secondary metabolites (TSMs) can be sprayed for maximum leaf coverage to reduce the incidence and severity of the disease when condition for disease are ideal. Lettuce anthracnose Microdochium panattonianum (MP) is a key winter disease in Victoria, Australia, and other temperate regions in the world. Cultural methods to reduce the severity of this disease are not practical nor economically feasible. While chemical control can be achieved it is not flawless and can cause health issues. Propagules of MP persist in soil sheltered in leaf debris and germinate with the onset of cold and wet conditions in the subsequent winter. Unfortunately, this pathogen has received little scientific attention but as winters become wetter this disease is causing greater loss and this body of work offers a more sustainable management tool. Trichoderma species are common saprophytic fungi in soils rich in organic matter. They have a proven ability to control crop diseases with a multiplicity of strategies including parasitism, competition, antibiosis and, at the same time, stimulating plant defences and growth. However, these natural processes vary with species and strains of Trichoderma. This research project showed that the incidence and severity of the lettuce anthracnose can be reduced by applying Trichoderma cold tolerant strains to the soil. Secondly, it showed that spraying Trichoderma liquid filtrates containing metabolites to the leaves also reduced and managed lettuce anthracnose. From a pool of 27 Trichoderma isolates, 8 grew at 10 C degrees. These 8 isolates were characterised morphologically in vitro under different culturing conditions of temperature, pH and media. Some isolates were identified by molecular techniques to species while others still require more investigation. Tests in vitro on solid media prepared with liquid filtrates confirmed that liquid filtrates from one isolate related to T. viride and one from T. composticola affected the morphology and growth of MP. These filtrates contained 6 pentyl-alpha-pyrone (6PP,) a volatile metabolite with a characteristic coconut smell. Other Trichoderma liquid filtrates containing metabolites such as cytosporone S and 6PP, produced by some strains of T. aureoviride, T. atroviride and Trichoderma sp., killed the pathogen. Fourteen isolates produced 6PP, a well-documented Trichoderma secondary metabolite (TSM). Of these, some have been identified as T. viride, T. composticola, T. atroviride, T. paratroviride, T. neokoningii and T. asperelloides. Others are yet to be fully identified to species. Isolates of T. harzianum, T. polysporum. T aureoviride, T. longibrachiatum and T. spirale did not produce this metabolite.Isolates of Trichoderma, TC1, CAROL, 71558 and 1536 require further identification because they were outliers on the phylogenetic tree. None of these isolates produced 6PP.Challenged isolates produced higher quantities of 6PP than did the individual liquid cultures. Trichoderma atroviride (PMF) from New Zealand produced 137 mg/l of 6PP, but when challenged by Trichoderma paratroviride (NSW) the 6PP concentration was 160 mg/l. Incidence and severity of MP were reduced significantly in a field trial using the cold tolerant isolate 356 related to T. composticola. TSMs applied as foliar sprays have the potential to control many foliar diseases in crops and is a new field of research. The use of Trichoderma cold tolerant strains for control of winter active pathogens is a new strategy for the use of Trichoderma products.
Lignite amendment of livestock manure: Mechanisms for nitrogen retention and effects on composting and nutrient release dynamics
Intensive livestock production systems, such as cattle feedlots, play an important role in meeting the increasing global demand for animal products, driven by a growing population and increasing affluence. These intensive systems account for a significant proportion (~39%) of global atmospheric ammonia (NH3), which is released from the large amounts of manure generated. In cattle feedlots, the loss of NH3 represents about 75% of the excreted nitrogen (N) in the manure and this has important implications for environmental pollution. Consideration of management strategies to reduce this loss provides potential to obtain N enriched manures that can be used as fertilizer. A number of mitigation techniques have been shown to reduce NH3 loss from manure. These include: acidifying agents and urease inhibitors (both requiring frequent reapplication), dietary manipulation (can affect animal productivity) and manure compaction and covering (only applicable to stockpiled manure). Lignite (brown coal) has a demonstrated capacity to suppress NH3 emission from manure by 30-66% and is seen as a more effective, practical and potentially long lasting option. Lignite’s ability to effectively reduce the loss of NH3 from manure has been credited to its pH, cation exchange capacity (CEC), pH buffer capacity and labile carbon (C) content. However, we do not fully understand what the actual capacity for N retention by lignite is, or the main mechanisms by which lignite retains N, and how these are influenced by the properties of the lignite and changes in the environmental conditions when lignite is mixed with other materials (such as manure). Also, the impact of lignite on the quality of the manure during deposition, storage, processing (such as composting) and land application, has not been studied. The objectives of this study were to: (i) characterize different lignite materials for their capacity for nitrogen retention, and to understand the mechanisms of N retention; (ii) determine how lignite amendment of manure affects the composting process under both field and laboratory conditions by monitoring changes in biochemical parameters and gaseous emissions that occur during composting, and (iii) determine the C and N mineralization from non composted and composted manure applied to soil and how this is influenced by lignite. The capacity for N retention by lignite, and the mechanisms of N retention were determined by characterizing five lignites sourced from Victorian brown coal deposits. The chemical properties that were examined for each lignite (e.g. pH, total and labile C, forms of C, pH buffering capacity, CEC, etc.) were related to its ammonium (NH4+) adsorption capacity as a function of pH and also to the biological immobilization of N in the lignite. The properties of the lignites, their capacity for NH4+ adsorption and the immobilization of N, were determined through a series of laboratory experiments involving SEM, 13C NMR spectroscopy, sample digestion and extraction, batch adsorption isotherms techniques, and controlled environment incubations. Lignite’s impact on composting of manure was assessed under field (windrow) conditions to determine the benefits under typical industry scale management processes. The mechanisms by which lignite affected composting of manure was determined under laboratory (in-vessel) conditions. Changes in biochemical parameters (e.g. N forms, organic matter and pH), emissions of NH3 and greenhouse gases (GHG), as well as compost maturity indices, were monitored. Lignite’s effect on C and N mineralization from manure (non-composted and composted) when applied to soil, was determined through a laboratory incubation experiment which monitored CO2 evolution (as a measure of C mineralized) and changes in NH4+ and NO3- concentration in soil (as a measure of N mineralized) over a period of 40 days. The mechanism by which lignite retains N was found to be mainly through pH dependent adsorption of NH4+ on exchange sites of deprotonated carboxyl groups. The maximum NH4+ adsorption capacity (Qmax) increased up to 3 fold when pH was increased from 3.6 to ~7. Biological immobilization of N was insignificant (e.g. maximum of 0.1 mg N g-1 lignite) compared to NH4+ adsorption on exchange sites (e.g. highest Qmax of 25.6 mg NH4+-N g-1 lignite at pH ~7). These findings suggest lignite’s ability to suppresses NH3 from manure occurs by i) initially favouring the formation of NH4+ over NH3 due to the acidic pH of the lignite, and ii) deprotonation of more carboxyl groups with continuous deposition of alkaline manure leading to increased CEC of the lignite and increased retention of NH4+. Amending manure with lignite did not inhibit the composting of manure. The addition of lignite suppressed the emission of NH3 from manure during both field windrow (by 45%) and laboratory in-vessel (by 35-54%) composting. Lignite addition reduced the emissions of GHGs (12-23% for CO2, 58-72% for N2O and 52-59% for CH4) during in-vessel composting. However, during windrow composting, addition of lignite caused the emissions of GHGs to increase (41% for CO2, 136% for N2O, and from -5.0 mg kg-1 initial dry matter (DMi) day-1 in the manure only to 9.8 mg kg-1 DMi day-1 for CH4). The higher emissions of CH4 and N2O were presumed to be due to anaerobic pockets that developed within the lignite treated windrow resulting from the small particle size of the lignite, and facilitated by the largely passive aeration method (unlike the forced aeration for in-vessel composting) used during the windrow composting. For the in-vessel composting, lignite addition to manure increased the organic matter (OM) and N contents of the final compost by 10-19 and 28-38% respectively, and also the germination index, a measure of compost maturity, (from 71% to 90-113%). These changes were not observed under windrow composting conditions likely due to the passive aeration method used. In addition, the effect of lignite in the windrow compost may have differed to that in the in-vessel systems because i) the mode of manure collection using large scale field equipment meant that the lignite content of the manure was estimated based upon that applied but could have been less due to some being left on the pen surface, and ii) the source of soil used in the cattle pens was quite alkaline (pH 8.8) which may have reduced the lignite pH effect. Lignite addition to the manure suppressed microbial activity (soil respiration) which reduced the mineralization of C from the manure when applied to soil, more noticeably when the manure was composted. Over a 40-day incubation period, with non-composted manure application rates of 30 and 60 t ha-1 soil, the C mineralized was 26.4 to 27.8% for manure only, and with lignite amendment it was 16.3 to 21.4%. The corresponding C mineralized in the composted manure was 12.4 to 14.1% and 3.5 to 6.5%. The addition of lignite had a mixed effect on N mineralized from manure when applied to soil. The N mineralized was significantly higher in the non-composted manure amended with Loy Yang lignite (10.4 to 13.5% for 30 and 60 t ha-1) than with Bacchus Marsh lignite (4.1 to 9.8%) and non-composted manure only (3.2 to 8.7%). For composted manure, there was no significant difference between N mineralized in the manure only (4.8 to 6.7%) and the lignite treatments (2.5 to 7.8%). Results presented in this study, for the first time, show the dominant N retention mechanism by lignite in manure, its influence on biochemical changes and gaseous emissions during manure composting under both laboratory and field conditions, and finally how the lignite impacts nutrient mineralization from non-composted and composted manure when applied to soil. In conclusion, this study shows lignite’s capacity to mitigate the emissions of both NH3 and GHGs from manure under optimum composting conditions. Hence, in intensive livestock production systems, lignite has the ability to improve the agronomic value of the large volumes of manure generated and reduce the environmental pollution associated with livestock production and manure management. Lignite addition to manure, especially when composted, has the potential to increase soil OM and improve long term soil health due to the inhibitory effect of lignite on C mineralization from manure. The findings of this study provide the option for the use of lignite as a tool for sustainable livestock production in intensive livestock industry.
Faecal microbiome and the associated antimicrobial resistance genes in Australian companion and food animals
Antimicrobial resistance (AMR) has become a global issue for human and animal health and welfare. It narrows down the choice of antimicrobials to treat infections and increases the costs of treatment. Effective antimicrobial stewardship is critical to ensure antimicrobials are used appropriately. Monitoring the antimicrobial resistance genes (ARGs) in bacteria is one of the important parts of antimicrobial stewardship. Bacteria in animal faeces harbor various ARGs. The traditional culture-based approaches to detect ARGs in the faecal bacteria are limited to the culturable bacteria, which only represent a small fraction of the faecal microbiome. The culture-independent approaches, such as high-throughput qPCR (HT-qPCR) and the next generation sequencing, bypass the limitation of culture and allow us to characterise the ARG profiles of the total bacterial community. These approaches have been widely used to detect and quantify ARGs in the faecal microbiomes of animals in various countries, but few of such studies have been done in Australia. In this project, faecal samples were collected from foals, calves, chickens and pigs, raised in commercial farms. The foals and chickens had no history of antimicrobial treatment. Calves were expected to have low chance being exposed to antimicrobials and only the pigs might be medicated. ARGs of the major antimicrobial classes, mobile genetic elements (MGEs) and metal resistance genes (MRGs) in the faecal samples were detected and quantified using a HT-qPCR array. The faecal microbiomes were determined by amplifying the V3-V4 region of the bacterial 16S rRNA gene and sequencing the amplicons on an Illumina platform. In addition, environmental swabs were collected from the chicken and pig farms and the microbiome and ARGs profiles of the swab samples were analysed to determine whether the environmental sampling could be used to study ARGs in animals raised intensively on a large scale. Overall, tetracycline resistance genes (tet) encoding ribosomal protection proteins and macrolide resistance genes (mef and erm) were the dominant ARGs in the faecal samples regardless of the animal species. The high level of these genes could be explained by the predominance of the potential bacterial hosts, such as Bacteroides spp., in the faecal microbiomes. The ubiquitous presence of the tet and mef/erm genes in animals suggested these genes are persistent and are not necessarily associated with the antimicrobial usage. On the other hand, the faecal samples had very low level of extended spectrum beta lactamase genes, blaCMY and blaCTX-M, carbapenems resistance gene cphA, fluoroquinolones resistance gene qnrB and virginiamycin resistance gene vatE, which suggested animals with low antimicrobial exposure had low risk of carrying ARGs that confer resistance to antimicrobials of high clinical concern. Manure belt swabs collected in the cage chicken sheds were appropriate to study ARGs associated with caged poultry. However, the floor swabs in the chicken and pig farms did not reflect the ARG profiles of the animal faeces due to insufficient biomass for DNA extraction or great changes in the microbiome composition compared to the faeces. In conclusion, this project showed that HT-PCR is an efficient method to screen a range of ARGs in complex samples. Analysis of ARG profile of animals without antimicrobial exposure set the baseline of presence and abundance of ARGs in animal faeces. Diverse ARGs detected in these animals suggests the persistence of ARGs in faecal microbiome. The results are useful for future studies investigating whether the current antimicrobial use practices can select and enrich pre-existing ARGs in farm animals.
Development and evaluation of a non-chemical method with microwave source for disinfection of legumes
Lentil is one of the important pulse crops in the world; however, its production is threatened by economically important diseases. Two of the devastating seedborne diseases of this crop, across the world and in Australia, are Ascochyta blight, caused by Ascochyta lentis, and Botrytis grey mould, caused by Botrytis cinerea and Botrytis fabae. Lack of genetically diverse sources of disease resistance in cultivated lentil varieties has increased dependence on fungicides to control these diseases. However, still, huge production losses are recorded worldwide as overuse of fungicides has resulted in the pathogen adaptation by mutations leading to their resistance and a reduction of fungicides efficiency. Therefore, there is a dire need for the development of an environmentally friendly physical method to be implemented in the integrated disease management scheme in pulse crops, including lentil. Microwave radiation is a sustainable and environmentally safe technology for food and agricultural processing. Its use for thermal treatment has shown great potential for sterilisation of food material as well as eradication of pests and pathogens from agricultural commodities. However, its potential in the grain industry and the control of related pathogens have not been fully explored, probably due to the concerns related to the resulting grain quality. Additionally, the indirect use of microwave, as a source for processing gas to produce non-thermal plasma followed by treatment of the seeds with the plasma, has recently gained a lot of attention. Therefore, the present study aimed to develop a thermal or non-thermal process, based on microwave radiation, to disinfect lentil seeds from its seed borne pathogens. The potential of a microwave multi mode cavity with a turntable for the eradication of AB of lentil seeds at different seed moisture contents, microwave powers, and exposure times was evaluated. This process was assumed to be like the industrial scale microwave system, with a conveyor belt, in terms of heat uniformity and efficiency of pathogen control. It was proven that the pathogen could be removed faster at higher seed moisture content; however, the seed viability was also more susceptible to microwave radiation at the higher seed moisture content (19% wet based). Infected seed percentage (IS%) reduced from 17% to 8.9%, at a seed moisture content of 9.1%, by applying a 100% power level (maximum 1100 W) for 51 s without a significant seed germination loss. Nevertheless, seed vigour (measured by electrical conductivity) was negatively affected by these process parameters. Investigation of heat uniformity in this process showed that the difference between hot and cold spots could reach as high as 50 C, which may account for the seed vigour loss before completing the eradication of the pathogen. To improve heat uniformity, a microwave fluidised bed was designed and manufactured. The efficiency of this process, in the eradication of seed borne Botrytis grey mould of lentil seeds, was firstly evaluated by investigation of the effective factors, including microwave power, exposure time, air temperature and seed moisture content with responses of IS%, seed germination, seed vigour, seeds bed temperature, and moisture loss. The results showed that a 30% reduction in IS% could be achieved by applying microwave power of 300 W and air temperature of 60 C or microwave power of 400 W and air temperature of 50 C for 10 min at the seed moisture content of 18%, without a significant reduction in seed viability and vigour. The temperature history, measured at two points in the fluidised bed using fibre optic probes, showed that the difference between hot and cold spot did not exceed 5 C. It confirmed that there was a very good heat uniformity in the microwave fluidised bed process. Evaluation of the effect of factors in the previous study showed that exposure time was an effective factor in reducing the IS%, while it did not have a significant effect on seed germination. To determine whether more reduction of the IS% could be achieved at prolonged exposure time, IS% reduction was examined in a drying experiment in the microwave fluidised bed dryer at the same process parameters as the previous experiment. The drying kinetics and time saving, compared with the conventional fluidised bed, were also studied. The results confirmed that the same reduction of the IS% occurred, and therefore increasing exposure time did not improve the reduction of the infected seeds; however, it proved to be a very effective drying process (more than 54% reduction in the drying time). It means that the greatest reduction in infected seeds occurred during the first 10 min of the process. The reason for not having more reduction in IS% after a specific time could be the very fast desiccation of the pathogen during the treatments, which increased its thermal resistance and subsequently the minimum power, or temperature required for its eradication. The seeds, which were dried in the previous experiment, were evaluated for any quality changes by examining their antioxidant enzyme activities, cooking quality, and their macronutrient using Fourier transform infrared (FTIR) micro spectroscopy. It was concluded that there was no significant quality deterioration in the most efficient process parameters (400 W & 50 C). In conclusion, there is great potential for microwave disinfection of lentil seeds under the condition where the hot spot is controlled so as not to harm the seed quality. It could be achieved by microwave assisted drying with at least a 54% reduction in the drying time compared with conventional fluidised bed drying. One hundred percent eradication might not be achieved, but it can be considered as part of an integrated disease management process in lentil crops, with the ultimate goal of reducing initial fungicide application for seed treatment and later applications for the crop. However, there might be concerns over the heat tolerance of different grains and different batches of the same grain, which was the reason for considering the non-thermal treatment with microwave plasma. Finally, the non thermal process of plasma, created from a microwave source, was examined for its potential in the eradication of Botrytis grey mould from lentil seeds. Non thermal plasma is the fourth state of the matter produced by energizing a gas and consists of various reactive species including atoms, ions, and radicals, which is a very promising non chemical method of seed disinfection with a growth enhancement effect. Two modes of treatment were considered: direct argon plasma at low power (400 W); and treatment with the afterglow of the air or air and argon plasma (10 s and 100 s) followed by holding the seeds in the trapped processed gas for 5 min to 24 h. There was a 41% reduction in the infected seed percentage (IS%) after 100 s of treatment with the afterglow of air plasma following by 24 h holding and a 32.3% reduction occurred when they were treated by 30% air and 70% argon plasma for 10 s and 60 min holding time. No significant change was observed in antioxidant enzyme activities of the treated seeds, except for triggering of catalase activity after 24 h of holding time. However, lentil seeds colour changed towards redness and yellowness after prolonged treatment time (400 s) or holding time (24 h). Therefore, the afterglow of microwave plasma or plasma processed air could be further explored as a great tool for the disinfection of grains, without any negative effect on their viability. Overall, both microwave fluidised bed and the afterglow of microwave plasma were able to reduce the seed-borne Botrytis grey mould of lentil seeds without any adverse effect on the seed viability. Microwave fluidesed bed would be useful when drying and disinfection are simultaneously desirable as the disinfection was just achievable at high seed moisture content with very fast drying of the seeds. However, the disinfection of dry lentil seeds was possible using the treatment with the afterglow of the plasma. Therefore, plasma treatment could be further explored for the treatment of other grains and their related pathogens if the dry seeds need to be disinfected.