Fusarium Wilt Tropical Race 4 – Biosecurity and Sustainable Solutions

An incursion of the fungus Fusarium oxysporum f.sp. cubense Tropical Race 4 (Foc TR4) was identified on a farm in the Tully region in March 2015. The wet tropical coast region, including Tully, produces 96% of Australia’s banana crop worth approximately $600 million annually and sustains regional communities in coastal north Queensland. Fusarium wilt of banana – also known as Panama disease – is caused by the fungus Fusarium oxysporum f.sp. cubense and is regarded as one of the most destructive diseases of banana and has devastated banana production in many countries around the world. Very few varieties are resistant to the Foc TR4 strain of the pathogen and despite years of breeding no commercially acceptable varieties have proven to be fully resistant. This project has aimed to deliver new science, information and practices to address key areas of need for the banana industry to: • Successfully contain the disease and prevent further spread of the pathogen through the adoption of robust, science‐based biosecurity practices and • Identify and investigate options to facilitate the development of economically viable production systems, capable of minimising inoculum build up, that are suitable for use on infected or at risk farms. The adoption of effective biosecurity practices on north Queensland banana farms has been significantly supported by project activities identifying risk pathways for spread of the pathogen, research identifying the most effective disinfectant products, research activities outlining how to monitor and manage their use and replenishment and how to effectively destroy inoculum in infected plants to minimise build up in the soil. The project has also significantly progressed development of methods for assessment of plant stress and its influence on Foc infection, identified possible rotation crops that suppress Foc populations in the soil and the influence of ground cover and nitrogen management practices on the soil microbiome and its capacity to suppress Foc. Significant extension and communication efforts have been made to keep the banana industry informed of the latest results as they became available and a biosecurity Best Management Practice guide has been produced with input from banana producers and regulatory agency staff that can assist banana growers to benchmark their biosecurity practices and identify potential improvements. Evaluation activities have shown that the project has significantly influenced knowledge change and practice adoption in the banana industry

APPLICATION OF SINGLE BOARD COMPUTERS AND SENSORS TO AUTOMATE ANALYTICAL METHODS AND MINIATURIZE THE ANALYTICAL DEVICES

In this dissertation, a low cost liquid delivery system and an automated titration system have been developed using a Raspberry Pi single board computer, 3D printing, and commercial-off-the-shelf components. In addition, an on-line single point internal calibration method has been developed for haloacetic acid rapid-response (HAA-RR) system, a commercial analyzer for analysis of nine haloacetic acids (HAA9) in drinking water.The low cost liquid delivery system, the EZ-AutoPipet, was developed to deliver microliter volumes accurately and reproducibly. The EZ-AutoPipet produced excellent results regardless of analyst experience and performed better than traditional and commercial dosing devices at lower volumes. Several validation studies have been performed to establish the accuracy and precision of liquid delivery. The hardness and alkalinity titrations were performed using the EZ-AutoPipet to verify the feasibility of using it as automated buret.The automated titration system was adapted from the EZ-AutoPipet and further developed into the EZ-AutoTitrator. It is a semi-automated system capable of performing potentiometric (pH-based) and spectrochemical titrations, pH and temperature measurements. The standard titration methods for alkalinity (pH titration) and total hardness (spectrochemical titration) have been adapted to the EZ-AutoTitrator. The alkalinity and hardness methods were validated and tested at two different water treatment plants. The EZ-AutoTitrator had good accuracy and precision for both titration methods. The preliminary testing of iodometric titration for determination of free available chlorine (FAC) in bleach samples has been performed.An on-line single point internal calibration for the HAA-RR system was developed and tested. The internal calibration addressed issues with external calibration by injecting the internal standard (2-Bromobutanoic acid) and haloacetic acid sample sequentially using a ten-port injection valve and two vial autosampler. The HAA-RR system was completely automated and can analyze the drinking water samples for a week without operator interaction. This work eliminated the errors associated with sample preparation and manual addition of the internal standard. The robustness studies showed that the internal calibration compensates for changes in response due to changes in system

Soil Organic Carbon Quantity and Distribution in Frost Boils in a Canadian High Arctic Polar Semi-desert Ecosystem

High Arctic soil organic carbon (SOC) stores are a key component in the global C cycle and are locally important for nutrient cycling in the polar deserts that dominate these regions. Compared to other Arctic regions, we know relatively little about the quantity and distribution of polar desert SOC. Unique frost-driven soil processes in polar deserts result in patterned ground features such as frost boils wherein, SOC-rich patches may develop via diapirism. The objective of this research was to determine whether these patches act as important nutrient sources for vascular plants and how subsurface patches of SOC associated with diapirism contribute to the polar desert carbon pool. I investigated SOC in 560 frost boils across two polar semi-deserts in the Canadian High Arctic using a field portable visible and near-infrared spectrophotometer. I found frequency of subsurface SOC patches was linked to broad differences in vegetation community. To determine if diapirs provide an enhanced source of plant-available nutrients we used natural abundance and enriched isotope 15N techniques to trace the flow of N through the soil-plant system. When diapir patches were available, the dominant deciduous shrub Salix arctica increased its subsurface (i.e., diapir) N uptake, often had greater % cover, and plant root biomass doubled within-diapir. Plant uptake of enriched 15N injected into C-rich soil patches was 2.5 fold greater in diapir than in non-diapir frost boils, also confirming that S. arctica is able to access N when these patches are present. My best estimate of SOC stored in the active layer of High Arctic polar semi-deserts is 8.14 ± 0.45 Pg SOC, or ~73% of SOC stored in the top 30 cm of all High Arctic soils. When subsurface SOC patches were detected in frost boils, those frost boils contained nearly double the SOC compared to those without patches and on average 40% of the SOC was found within the patch. Thus, despite diapiric frost boils representing only 35% of frost boils, they contribute disproportionately to High Arctic C storage

From Ancient DNA to Forensic Genetics: Validation Studies in Degraded DNA

In forensic and archaeological applications, degraded DNA presents challenges during extraction, amplification, and analysis. Many of these issues can be addressed through the application of next-generation sequencing (NGS) techniques, including maximizing yields of fragmented DNA and identifying contaminant DNA. NGS is prompting a convergence between ancient and forensic genetic methods along several avenues, including DNA extraction. This dissertation discusses the convergence of extraction techniques contextualized within validation studies of ancient and modern DNA research.Two validation studies are presented. The first study validates and explores the impact of a non-destructive DNA extraction technique developed by Bolnick and colleagues (2012). The “non-destructive” (Bolnick) DNA extraction technique yields both mitochondrial and nuclear DNA. While the teeth tested remained macroscopically intact, there was loss of tooth microstructure in the tooth root and enamel, shown through treated vs. untreated weights and histological analysis. Scanning electron microscopy (SEM) results showed minimal staining of the tooth. There were no significant carbon or oxygen isotopic difference between treated and untreated teeth. The damage characterized shows that the protocol is minimally destructive but may still be of interest to stakeholders desiring maintenance of macroscopic, but not microscopic, integrity.The second study focuses on the quantity and quality of DNA extracted from post-mortem blood samples stored on untreated blood cards. Short-tandem repeats (STRs) typed from the cards are used for ancestry-based analysis. The study validated two different hypotheses: 1) cadaveric blood stored on untreated blood cards yields enough DNA for typing of STRs and 2) STRs typed from blood cards yield geographic ancestry information. Results of the second case study indicate that post-mortem interval impacts the DNA quality of samples extracted from untreated blood cards. Tri-hybrid ancestry and admixture analysis indicate that the original thirteen CODIS loci have utility in estimating geographic ancestry.These validation studies show the complications of working with degraded DNA in both ancient and forensic contexts. NGS approaches provide an opportunity in both fields to move beyond traditional markers to type expansive regions of the genome for both subfields and provides a way of addressing many issues of degraded DNA facing ancient and forensic researchers

The Roles and Interactions of Symbiont, Host and Environment in Defining Coral Fitness

Background: Reef-building corals live in symbiosis with a diverse range of dinoflagellate algae (genus Symbiodinium) that differentially influence the fitness of the coral holobiont. The comparative role of symbiont type in holobiont fitness in relation to host genotype or the environment, however, is largely unknown. We addressed this knowledge gap by manipulating host-symbiont combinations and comparing growth, survival and thermal tolerance among the resultant holobionts in different environments.\ud Methodology/Principal Findings: Offspring of the coral, Acropora millepora, from two thermally contrasting locations, were experimentally infected with one of six Symbiodinium types, which spanned three phylogenetic clades (A, C and D), and then outplanted to the two parental field locations (central and southern inshore Great Barrier Reef, Australia). Growth and survival of juvenile corals were monitored for 31–35 weeks, after which their thermo-tolerance was experimentally assessed. Our results showed that: (1) Symbiodinium type was the most important predictor of holobiont fitness, as measured by growth, survival, and thermo-tolerance; (2) growth and survival, but not heat-tolerance, were also affected by local environmental conditions; and (3) host population had little to no effect on holobiont fitness. Furthermore, coral-algal associations were established with symbiont types belonging to clades A, C and D, but three out of four symbiont types belonging to clade C failed to establish a symbiosis. Associations with clade A had the lowest fitness and were unstable in the field. Lastly, Symbiodinium types C1 and D were found to be relatively thermo-tolerant, with type D conferring the highest tolerance in A. millepora.\ud Conclusions/Significance: These results highlight the complex interactions that occur between the coral host, the algal symbiont, and the environment to shape the fitness of the coral holobiont. An improved understanding of the factors affecting coral holobiont fitness will assist in predicting the responses of corals to global climate change

The sexually-transmitted Western Australia wild-plant virus yellow tailflower mild mottle virus: Does it pose a threat to global food security?

Yellow tailflower mild mottle virus is a species in the internationally-distributed genus Tobamovirus, other species of which are some of the most damaging plant viruses known. Yellow tailflower mild mottle virus (YTMMV) is the first tobamovirus described only from Australia and only from native plants. Because of the bad reputation of related tobamoviruses such as tobacco mosaic virus and cucumber green mottle mosaic virus as destroyers of valuable crops, we studied YTMMV to understand aspects of its biology and to assess its potential to spillover from the indigenous flora and threaten crops on national and international stages. Unlike many damaging plant viruses, tobamoviruses are not transmitted host-to-host by vectors such as aphids. Thus, understanding how YTMMV is transmitted between host plants is key to understanding aspects of its epidemiology. A further aim of our work was to assess the damage we might expect to see in some susceptible crops should YTMMV spillover

Additive Pressures of Elevated Sea Surface Temperatures and Herbicides on Symbiont-Bearing Foraminifera

Elevated ocean temperatures and agrochemical pollution individually threaten inshore coral reefs, but these pressures are likely to occur simultaneously. Experiments were conducted to evaluate the combined effects of elevated temperature and the photosystem II (PSII) inhibiting herbicide diuron on several types of symbiotic algae (diatom, dinoflagellate or rhodophyte) of benthic foraminifera in hospite. Diuron was shown to evoke a direct effect on photosynthetic efficiency (reduced effective PSII quantum yield ΔF/F′m), while elevated temperatures (>30°C, only 2°C above current average summer temperatures) were observed to impact photosynthesis more indirectly by causing reductions in maximum PSII quantum yield (Fv/Fm), interpreted as photodamage. Additionally, elevated temperatures were shown to cause bleaching through loss of chlorophyll a in foraminifera hosting either diatoms or dinoflagellates. A significant linear correlation was found between reduced Fv/Fm and loss of chlorophyll a. In most cases, symbionts within foraminifera proved more sensitive to thermal stress in the presence of diuron (≥1 µg L−1). The mixture toxicity model of Independent Action (IA) described the combined effects of temperature and diuron on the photosystem of species hosting diatoms or dinoflagellates convincingly and in agreement with probabilistic statistics, so a response additive joint action can be assumed. We thus demonstrate that improving water quality can improve resilience of symbiotic phototrophs to projected increases in ocean temperatures. As IA described the observed combined effects from elevated temperature and diuron stress it may therefore be employed for prediction of untested mixtures and for assessing the efficacy of management measures