A robust, effective, and responsive plant biosecurity system is critical to protect our natural and productive ecosystems. However, analyses of the sector have shown that employing a “business as usual” approach for our future biosecurity requirements will be ineffective in the long-term. Modelling has demonstrated that even a tripling in investment in the sector is not enough to prevent increased biosecurity threats over the short-to-medium term.

Consequently, plant biosecurity requires a transformational change to keep ahead of the increased risks to Australia’s multibillion-dollar agriculture, horticulture, forestry, environmental industries, tourism, and quality of life.

The ARC Training Centre in Plant Biosecurity aims to deliver a solution for Australia’s increasing biosecurity risk through generational change in its workforce coupled with breakthrough technologies. It will launch an innovative training program for future leaders who will build relationships with end users and engage meaningfully with communities for effective implementation strategies. It will deliver a cohort of highly skilled graduates that will innovate novel diagnostic technologies, enable data-driven decision platforms and address barriers to biosecurity adoption.

This poster presentation will outline the training program and research embedded within the Training Centre and outline the exciting opportunities available for students, postdocs and our partners.

Leaf rollers are significant pests of diverse horticultural crops including berries, cherry, citrus, grapes, macadamia, nurseries etc in Australia. While the native Light brown apple moth LBAM has drawn significant attention due to its economic impact on productivity, lately exotic Mango flower webworm (MFW) Dudua aprobola, (often mistaken for LBAM) caused increased damage in subtropical berries and in nurseries. Given the understanding on the impact of MFW on Australian horticulture particularly on berries remains rudimentary, we have carried out field surveys as well as laboratory assays to gain insights into its eco-biology, life history traits, and management strategies. The fortnightly field surveys at two commercial sites in Northern NSW revealed that larval generations overlapped with the full range of larval instars being present for most of the warmer month (November to March). In the laboratory rearing, the proportion of larval survival was greater on artificial diet incorporated with blueberry leaf compared to other larval substrates. Sexual dimorphism was evident at pupal stage as male pupae has an additional segment than female. Oviposition potential of female varied significantly with age as 6-14 days old females exhibited increased fecundity compared to younger ones. Also, adults that were given access to flight laid comparatively greater quantity of eggs than those were deprived of flight. In addition, a ectoparasitoid Goniozus jacintae and Poecilocryptus sp have been found to attack MFW larvae in field surveys. Overall, the present study advocates the need to develop sustainable management practises to minimise its impact on diverse horticultural commodities.

The Department of Agriculture, Fisheries and Forestry (DAFF) has long standing partnerships with biosecurity agencies across the Pacific and with our near neighbours. These partnerships strengthen our region’s biosecurity through ongoing joint surveys and biosecurity projects aimed at building capability. With an increasing focus on the Pacific region, DAFF has been engaged by Department of Foreign Affairs and Trade (DFAT) to be the technical agency to lead projects with our near neighbours to support agricultural and biosecurity outcomes. The following poster/s will provide insights into the wide range of work DAFF is delivering across our region in partnership with DFAT and counterpart biosecurity agencies.

• Pacific Biosecurity Partnership Program (PBPP) was established in 2019 in response to growing biosecurity threats in the Pacific and Australia’s foreign policy of enhanced engagement with the Pacific. In 2022-23, eight projects were prioritised and supported through the PBPP. These projects included the support to promote safe trade in plant and plant products from the Pacific Islands, deliver operational training along the export pathway to strengthen and enhance compliance with Australia’s import requirements and improve efficiencies and implementation of the regional trade and market access communication strategy “Partnering with our Pacific neighbours”.

• Solomon Islands Biosecurity Development Program (SIBDP) Phase 3 continues to work with Biosecurity Solomon Islands (BSI) to deliver programs of support across the biosecurity continuum. Under the SIBDP, a DAFF officer was seconded to BSI to train and assist with the preparations for the South Pacific Games. Two joint plant health surveys have been delivered, as well as several projects to support BSI’s preparedness and response.

• Timor-Leste Biosecurity Development Program is a three-year program established in 2023 which is co-designed and managed with Timor-Leste’s Ministry of Agriculture, Livestock Fisheries and Forestry (MALFF) to support Timor-Leste’s capacity to meet international sanitary and phytosanitary standards.

Rapid, efficient, sensitive, and easy to use portable diagnostic tests such as point of care tests (POCT) for plant pathogens are necessary for surveillance, inspections, evidence of area-freedom, containment, asset protection, and to manage post-border incursions. However, the development of such “in-field ready-to-use” assays require capabilities to set-up and to validate whether it is for a known group of pathogens or a new strain. Here we present a pipeline of capabilities at PIC that can be employed toward preparedness to respond for biosecurity threat incursion; from pathogen sequencing and bioinformatics analysis to CRSIPR/Cas assay development, including POCT conversion, and validation.
As a proof of concept, we targeted citrus commodities to develop CRISPR/Cas-based assays (Clustered Regularly Interspaced Short Palindromic Repeats and associated nuclease Cas), including multiplex, for bacteria: Huanglongbing-CLas, Citrus Canker and Xylella, and viruses: Citrus Tristeza Virus, Citrus Vein Enation Virus and Hop Stunt Viroid. CRISPR/Cas-based POCT can use reverse transcription and isothermal nucleic acid amplification systems (e.g., recombinase polymerase amplification) at 37-42°C for short duration (30-40 min) to specifically detect exceptionally low levels of pathogens. The collateral activities of CRISPR/Cas as a direct consequence of the number of target molecules detected during the incubation can be visualised using portable fluorescence readers. By using bioinformatic analysis to selectively incorporate universal bases in oligonucleotide and guide RNA we could maximize the number of strains to be captured in future POCT assays. Results about in-laboratory POCT assays will be presented, and current challenges associated with in-field deployable test will be discussed.

Invasive snails cause negative impact on the environment and agriculture. Snails are also host to many pathogens and parasites and can play an important role as vectors. They are commonly intermediate hosts for nematodes and trematodes that cause diseases to vertebrates including humans, they can carry parasitic mites that could spread to other molluscs, and some species can also carry plant pathogens. Snail introduction is therefore exponentially more risky than other potential pests as they could carry new variants of pathogen and parasite or entirely new species to Australia that could threaten agriculture (plants and animals), human health and biodiversity. There is a substantial knowledge gap regarding what parasites and pathogens are currently present in native and introduced snail populations in Australia. An understanding of the snails as vectors of parasites and pathogens of plants, animals and humans in Australia is also critical to ensuring effective identification of potentially affected industry parties and supporting knowledge in an emergency response. This new project aims at filling the knowledge gap in the role that snails play as vectors of parasites and pathogens in Australia as well as investigating the risk associated with new species entering. We will characterise and discover known and novel pathogens harboured snails in Australia, we will establish an optimized platform to extract nucleic acid and bioinformatic analysis from snails for downstream pathogen detection and surveillance. These results will provide a better understanding of the disease risk carried by snails and recommend improved management and preventive strategies.

Due to the dynamic nature of the risks associated with imported seeds, the Department of Agriculture, Fisheries and Forestry’s (DAFF) Plant Science and Risk Assessment (PSaRA) team are undertaking comprehensive risk assessment reviews on four key vegetable families: Apiaceae, Cucurbitaceae, Brassicaceae and Solanaceae. As new pathogen risks emerge and proliferate, additional molecular tools are needed to maintain Australia’s Appropriate Level of Protection. Collectively with PSaRA, the Plant Innovation Centre is developing and validating assays that are “fit for purpose” and “ready to implement” for detection of seed-associated pests of biosecurity concern. To be deemed as fit for purpose, every test must meet strict criteria under DAFF’s test development validation framework. Here we describe the process for development and validation of novel assays under this framework and present qPCR assays for the detection of fungal pathogens including Colletotrichum higginsianum, Diaporthe cucurbitae and Diaporthe angelicae. Multiple stringent test validation processes have demonstrated these assays to be fit for purpose and highly sensitive in detecting target pathogens.

Hitchhiker pests, i.e. hidden within or on shipping containers and imported goods are an increasing threat to Australian plant health and biodiversity. Department of Agriculture Fisheries and Forestry’s hitchhiker pest program specifically aims at building biosecurity systems to protect Australia from those pests. These systems need to be highly efficient, yet compatible with the high volume and fast turnaround of containers entering Australia. The Molecular Diagnostic Centre at the South Australian Research and Development Institute (SARDI), in partnership with the National eDNA Reference Centre at the University of Canberra, have investigated a high throughput, fast turnaround eDNA testing system for hitchhiker pest surveillance. It is based on the SARDI proprietary high throughput platform for large environmental samples that has a routine capacity of 250 samples per day, and a surge capacity of 600. Customised sampling kits were developed to ensure DNA preservation in samples during transport. Ability to extract quality DNA was confirmed using vacuum samples of diverse compositions, collected from Approved Arrangement Facilities in Australia. The system was tested using samples collected at facilities in New South Wales, Queensland, Victoria, and Western Australia and tested by quantitative PCR for Khapra beetle, Red imported fire ant and Brown marmorated stink bug. Results demonstrate that the technology is effective for sensitive, specific, and high throughput detection of targeted pest species eDNA in tested environments. Potential application as a surveillance tool warrants further investigation.

Surveillance can support market access by providing confidence that an area or registered site is either pest free (PF) or has low pest prevalence (LPP). Pest freedom generally allows trade to occur without the need for additional risk mitigation measures, but demonstrating PF can be resource intensive. Demonstrating LPP may be easier for certain pests, even if additional measures are required to achieve the desired level of biosecurity protection. A major challenge that limits the wider adoption of LPP measures is the design of surveillance that strikes the right balance between three components: survey intensity, the confidence required in estimated pest prevalence levels, and pest risk. We are addressing this challenge in the Hort Innovation Safe Trade project through empirical and modelling work, in collaboration with Australian regulators. More specifically, we are combining empirical and simulation modelling work on site-based surveillance with a literature review of LPP measures for use in trade, to better understand how the three components interact and influence the design of LPP measures. Interestingly, although registered sites with surveillance and monitoring requirements are already widely applied for market access, international standards or guidelines to support LPP site measures have not yet been developed. We hope that this project will stimulate discussion around developing guidelines for LPP site measures to facilitate market access, and to maintain market access for businesses operating in pest outbreak zones.

A science-engineering partnership led by SARDI is creating new opportunities for monitoring airborne pathogens through end-to-end spatiotemporal surveillance across agricultural landscapes. The platform utilises ‘smart’ spore trapping technology that collects air biota referenced to time, space and climate data linked to downstream molecular diagnostic pipelines. The next evolution of this Mobile Jet Spore Sampler is being tested in South Australia for mobile surveillance of fungal pathogens threatening agricultural industries. The device is mounted to a vehicle’s roof and samples at high frequency (200-450 L/min) directly into DNA extraction tubes using virtual impaction. A mobile-phone application is used to control automated sample collection as the vehicle passes within designated GPS polygons. This novel platform, developed in a collaboration between SARDI and Data Effects as an evolution from a prototype in partnership with UniSQ, Agri Samplers Ltd and Rothamsted Research (UK), will improve spatial and temporal resolution of airborne spore dispersal patterns across a broad landscape. This is particularly important given the size and scale of Australian agricultural industries. The system utilises barcoded samples to be interlinked with the molecular diagnostics pipeline for efficient traceability to output point of origin incursions or abundance patterns, achieved through digital output visualisation packages developed with the project’s technology partner Data Effects. This approach compliments fixed monitoring sites, to expand end-to-end surveillance pipelines serving device-to-data delivery for end-users in remote regions, risk pathways, border protection points or generate data to support area freedom. This further demonstrates cutting-edge innovative and sustainable solutions serving Australia’s biosecurity programs.

Myrtle rust pathogen (Austropuccinia psidii) is known to induce rust disease in more than 480 plant species belonging to the Myrtaceae family, including native Eucalyptus species which play crucial role in Australian ecosystem. One linage which has been spread globally is named as pandemic biotype. To better identify effectors and investigate the evolutionary history of the pandemic strain (Au3), we assembled a haplotype-phased chromosome-scale genome for the pandemic strain using combination of PacBio high-fidelity (HiFi) sequencing, Nanopore long read sequencing and Hi-C data, and annotate the genome with RNA-seq reads from A. psidii infected Syzygium jambos. BUSCO results of the diploid genome and proteome are 92.0% and 95.2% respectively, which support the high completeness of the genome. This gapless genome assembly was phased into two haplotypes, one with 19 chromosomes whereas another with 17 chromosomes. Several large structural variations were identified within sister chromatids of chromosome pair 14 which was assigned to the same nucleus. Overall, we provide a valuable genome resource which is available for further population genomics study and increase our preparedness for potential novel incursions of other myrtle rust strains.

The National Grain Diagnostic and Surveillance Initiative is a strategic partnership between the Grains Research and Development Corporation and five state agriculture and primary industry departments, with investment targeting the detection and identification of pests and diseases that threaten the Australian grains industry. The Department of Energy, Environment and Climate Action will be focusing on the modernisation of diagnostic methods to enable fast and accurate detection and identification of pests and diseases, providing biosecurity risk profiles, and developing targeted preparedness plans for high priority pests and diseases. The importation of grains germplasm into the Australian Grains Genebank via the Post Entry Quarantine facility at the Horsham Grains Innovation Precinct will be facilitated by the development of high throughput sequencing applications to screen for exotic pathogens such as viruses. A viral genome sequence database using accurately identified and curated specimens will be established and align with the national VirusCurate and Australian Plant Pest databases. Additionally, seed testing capability will be developed for endemic seed-borne viruses that impact pulse crops, to support delivery of high health seed for sowing for growers. Pest risk reviews for expanding grains commodities such as tropical pulses and pasture grasses will be conducted to assist biosecurity preparedness.

Austropuccinia psidii is the causal agent of myrtle rust in over 480 species within the family Myrtaceae. Lineages of A. psidii are structured by host in its native range, and some have success on new-encounter hosts. For example, the pandemic biotype has spread beyond South America, and proliferation of other lineages is an additional risk to biodiversity and industries. Efforts to manage A. psidii incursions, including lineage differentiation, relies on variable microsatellite markers. Testing these markers is time-consuming and complex, particularly on a large scale. We designed a novel diagnostic approach targeting the fungal mating-type HD (homeodomain) transcription factor locus to address these limitations. The HD locus is highly polymorphic, facilitating clear biological predictions about its inheritance from founding populations. To be considered the same lineage, all four HD alleles must be identical. Our lineage diagnostics relies on PCR amplification of the HD locus in different genotypes of A. psidii followed by amplicon sequencing using Oxford Nanopore Technologies (ONT) and comparative analysis. The lineage-specific assay was validated on four isolates with existing genomes, uncharacterized isolates, and directly from infected leaf material. We reconstructed HD alleles from amplicons and confirmed their sequence identity relative to their reference. Genealogies using HD alleles confirmed the variations at the HD loci among lineages/isolates. Our study establishes a robust diagnostic tool, for differentiating known lineages of A. psidii based biological predictions. This tool holds promise for detecting new pathogen incursions and can be refined for broader applications, including air-sample detection and mixed-isolate infections.

Plant-parasitic nematodes (PPN) are estimated to cause annual crop losses costing US$100 – 157 billion worldwide with root-knot nematodes (Meloidogyne spp., RKN) considered the most damaging genera. The recent incursion of Meloidogyne enterolobii (guava root-knot nematode, GRKN), a highly pathogenic RKN with a very broad host range and the ability to overcome resistance genes in a range of crops, poses a significant threat to Australian plant industries.

GRKN was first detected in the Northern Territory in September 2022. Shortly afterwards, the pest was detected at two widely separated Queensland sites. DAF has since inspected a further 200+ RKN samples, which were characterised using species-specific PCR assays, with no further GRKN detections to date.

As well as being a threat to commercial agriculture in Australia, GRKN has the potential for large impacts on subsistence and smallholder production in the Asia-Pacific region. Other significant PPNs such as the white potato cyst nematode (Globodera pallida) and rice root-knot nematode (Meloidogyne graminicola) are also present in the region, but not in Australia.

The introduction of GRKN into Australia highlights the need for greater PPN preparedness throughout the Asia-Pacific region. An expansion of coordinated nematological activities will boost capability in the region, assist neighbouring countries with nematode management, and help safeguard Australian plant industries through improved knowledge of pest distribution and enhanced diagnostic capabilities. The current ACIAR project “MeloRisk Australasia: Reducing the risk of exotic root-knot nematodes in Australasia” is a step in this direction with project partners throughout SE Asia and linkage with EUPHRESCO.

Fusarium wilt Tropical Race 4 (TR4) is a devastating fungal disease caused by Fusarium oxysporum f.sp. cubense (Foc) that affects banana plants. Foc is spread through contaminated soil and planting material. The identification and destruction of infected plants is important in preventing the disease spread. This involves treating the plant and soil with 1.0 kg/m2 of urea to release ammonia gas to give ‘top-down’ control within the destruction zones. However, the increased nitrogen application potentially alters soil physicochemical properties, leading to a reduction in soil microbial community diversity and functions, and hence an eventual increase in TR4. Three destruction zones on a commercial banana farm were investigated. There was a significant increase in the soil nitrate-nitrogen 3-months after destruction began. A qPCR soil test highlighted three phases within the destruction zone of bananas. Initially, there was little change in TR4 or soil properties, following identification of symptomatic plants. As the destruction zones were implemented, 1-month later, there was an increase of 100-1,000-fold in TR4 abundance. This corresponded with increasing soil nitrogen, soil organic carbon (SOC) and decreasing microbial diversity. Finally, 12-months after destruction of the infected plant, the abundance of TR4 had declined. The ‘bottom-up’ management and competition for soil resources, such as SOC, appears to be important in the management of TR4 inoculum. The understanding of the soil microbial interactions occurring within the destruction zone can be used to improve the efficiency in inoculum management of Fusarium wilt TR4 infected bananas, to help contain the disease spread.

Ratoon stunting disease (RSD) presents a significant challenge in sugarcane farming due to the highly contagious bacterium Leifsonia xyli subsp. xyli (Lxx), causing potential yield losses of up to 60%. Additionally, the economic impact of RSD shows an annual economic loss of $25 million across the 87,000 ha monitored for RSD infection. Despite economic incentives, RSD management is hindered by the absence of external symptoms, requiring reliance on lab techniques like PCR. However, collecting field samples for diagnosis is inefficient, prompting efforts to develop more efficient large-scale detection methods.

Machine learning (ML) algorithms, particularly when coupled with spectral data, have shown promise in detecting diseases and pests. Spectroscopy, which enables the identification of subtle disease indicators often imperceptible to the human eye, has been utilised in conjunction with handheld spectrometers. While handheld devices have shown potential, they encounter challenges related to sample collection, particularly on a large scale. Recent studies have shifted focus towards drone-based observations, which offer advantages in terms of scalability.

Moreover, satellite-based spectroscopy presents an appealing solution for large-scale disease identification, leveraging free and publicly available data to mitigate financial constraints associated with purchasing spectrometers. Additionally, the utilisation of publicly available data enables the potential for automation and integration into user interfaces for real-time detection.

In response to these challenges and opportunities, we introduce Sugar-AI, a prototype RSD detection system that leverages machine learning algorithms with satellite-based spectroscopy to enable efficient and accurate large-scale disease detection in sugarcane cultivation.

The widespread use of herbicides and pesticides presents a significant environmental threat due to their runoff into waterways, prompting public concern and regulatory scrutiny. One promising solution to mitigate chemical runoff is the adoption of precise robotic spot-spraying techniques.

We have developed AutoWeed, a ground-based spot-spraying tool tailored for managing weeds in sugarcane fields. This involved designing computer vision and deep learning AI algorithms for weed detection and creating a spraying system compatible with existing farm equipment. Field trials took place in Queensland's Burdekin region, a major sugarcane cultivation area, spanning two years to assess spot spraying's practical effects on herbicide reduction, weed control, and water quality improvement.

Results from trials over 25 hectares indicate spot spraying's effectiveness, achieving a commendable 97% compared to traditional broadcast spraying. Notably, it led to a significant 35% reduction in herbicide usage, with up to 65% reductions in areas with lower weed pressure. Additionally, water quality assessments post-spraying showed reductions of 39% in mean herbicide concentration and 54% in mean herbicide load compared to broadcast spraying.

These findings highlight spot spraying's potential to reduce herbicide application and promote sustainable pest management practices. By aligning with integrated pest management principles, spot spraying contributes to ecological balance while minimising environmental impact. This research holds relevance for advancing plant biosecurity strategies, emphasising the need for innovative and sustainable agricultural approaches.

Sooty moulds are communities of saprophytic fungi that feed on honeydew excreted from Hemipteran insects. Hemipteran infestations combined with sooty mould can cause canopy dieback, reduced fruit production, and at times host plant mortality. Little is known about sooty mould communities, the species that comprise them, their growth, and the factors that influence their abundance. Therefore, we investigated the influence of the honeydew excreting non-indigenous scale insect Saissetia miranda, on sooty mould community composition. On a remote island in Western Australia, samples of sooty mould were taken from Ficus brachypoda trees with either a presence or absence of the honeydew excreting Saissetia miranda. Species in the sooty mould communities were identified using metabarcoding of the ITS2 region of their DNA. We found that the absence of Saissetia miranda led to the sooty mould community being dominated in abundance by a single species. When Saissetia miranda were present, there was a more even composition of a greater number of sooty mould species. This research establishes an understanding of how scale insect infestations are driving changes in sooty mould communities. Future research will help inform how the changes within sooty mould communities identified in this study impact the health of host plants.

Red imported fire ant (Solenopsis invicta) (RIFA) is one of Australia's high-priority pest species. Early detection is essential for both at-border and post-border biosecurity. Environmental DNA (eDNA)-based methods provide a significant advantage in the early detection of invasive species, offering highly sensitive molecular options for biosecurity management. In this study, we designed a next-generation sequencing (NGS)-based approach and developed a species-specific real-time PCR assay to detect DNA and RNA from RIFA. The assay was specific to RIFA while tested against 15 ant species that are abundant in Australia. The assay can detect down to 0.12 copies and quantify 12 copies of gDNA per reaction with 98% efficiency (R2 = 0.99). For RNA, the assay's detection and quantification limit was 1.28×10-2 ng/µL with a 106% efficiency (R2 = 0.98). The field testing of the assay was performed on dust samples collected from four shipping containers (inside) located in an empty container park at the port of Brisbane (Qube Logistics) and soil samples collected from three RIFA nests presumed to be active near Brisbane. The assay was successful in detecting RIFA eDNA in 4/4 Shipping container samples (75% detection success) and 2/3 nest samples (11% detection success) while RIFA eRNA in 3/4 Shipping container samples (58% detection success). Using a Bayesian hierarchical model-based approach and a gDNA spike test, we identified that the low detection success rate in the nest samples resulted from the sampling design and the presence of inhibition in sampling sites rather than the amplification capability of the assay.

The new Pest READI (Regionally Enabled Agroecological Decision Intelligence) Project aims to create healthy, abundant landscapes by transforming the way communities work together to manage plant pests. By integrating past, present and future knowledge into a digital platform that connects decision-making across landscapes, the key output from the Project will be tools for scenario-based decision support for Area-wide Integrated Pest Management.

In Australia, the use of agricultural pesticides insecticides, herbicides and fungicides has doubled since 1992, to over 50,000 tonnes per annum, costing growers around $3B. This is unsustainable, and we need to consider how growers will manage pests in an increasingly ‘chemically-limited’ future. The project will use social research and biophysical data in the development of a digital platform that maps pest risk along with preparedness, to indicate vulnerabilities within a region. The design of the platform will employ the concept of ‘Digital Twins’: dynamic digital representations of a physical system. Real-time data analytics, simulation and ‘what-if’ scenario generation will combine to enable valuable management decision-support.

We will use a process of ‘Human Centered Design’ alongside our research to engage with all sectors of the community, government, and industry in the co-development of the platform and associated applications. This will include building a Community of Practice for Area-wide Integrated Pest Management around the Project and digital tool development within the region. The platform will aim to provide solutions that require fewer pesticides and enable all sectors of the community, including Indigenous, to work together more effectively to suppress pests.

Airborne plant pathogens pose an existential threat to regions naive to their impact. There are many examples where such pathogens are detected too late; when entire forests are dying (ash dieback - Hymenoscyphus fraxineus), stands have been decimated (chestnut blight - Cryphonectria parasitica), or bushland all but destroyed (myrtle rust - Austropuccinia psidii). Once established, pathogen eradication becomes exceedingly difficult as they continue to spread, and management resources are allocated in a reactive manner.

Here, we present a surveillance technique that co-opts existing air pollution monitoring infrastructure for the early detection and monitoring of airborne plant pathogens. Proof of concept was conducted in Switzerland, focusing on H. faxineus and C. parasitica. We found consistent seasonal peaks of amplified eDNA, which correlated with our knowledge of seasonal spore loads.

Back in Australia, we used A. psidii as a case study and verified its presence in the air of southeast Queensland,
using a new species-specific TaqMan qPCR assay. We also present fungal and plant metabarcoding results from the same filters that extend beyond targeting specific species to include a biodiversity perspective. These data identified several plant species that have not yet been recorded in Australia.

Routine monitoring for eDNA using existing infrastructure and the eDNA technique proposed here could enable more efficient allocation of resources, thereby enhancing our ability to predict, find, and contain pathogens and other biosecurity threats before they establish. A more proactive approach to biosecurity.

Xylella fastidiosa, a destructive plant pathogenic bacterium, affects numerous plant species globally, including grapevines, almonds, peaches, apricots, and olives. Although absent in Australia, its potential introduction poses a significant threat. The bacterium, primarily transmitted by spittlebugs, sharpshooters, leafhoppers, and froghoppers, has been identified by the Plant Health Committee as the top National Priority Plant Pest in Australia, an 'unwanted organism' by New Zealand MPI, and one of the most dangerous plant bacteria worldwide by the European Commission. This research endeavors to equip biosecurity agencies with effective tools and knowledge for rapid eradication or containment of Xylella fastidiosa in Australia. This project focuses on potential insect vectors of Xylella, biology, physiology, and ecology in targeted horticultural crops across three states of Australia. Efforts, methodology, results, and its significance to Australian food production will be communicated and discussed.

The international trade of commodities and produce is the most important pathway associated to the translocation of invasive exotic species. Shipping containers carry approximately 90% of global trade and have been repeatedly reported to translocate invasive insects. We collected dust samples from 2017 shipping containers at the port of Brisbane (Australia) and tested them for the presence of insect trace environmental DNA/RNA. Samples were analysed using metabarcoding targeting a broad range of insect orders to infer on insect diversity based on detection of eDNA. Environmental RNA was then extracted from containers with detections for five high priority insect species ( Trogoderma granarium, Halyomorpha halys, Lycorma delicatula, Lymantria dispar, and Wasmannia auropunctata) and included for sequencing using Illumina Novaseq. A total of 1980 eDNA and 59 eRNA samples were successfully metabarcoded and sequenced using Illumina Novaseq, producing 11 billion reads, of which 3,459,189,038 reads passed quality control and assurances. Although there is congruence in eDNA and eRNA detections for species with high numbers of reads, Insect diversity was significantly different between eDNA and eRNA, wherein species richness was significantly higher for eDNA compared to eRNA. Of note, eRNA detections mostly consisted of cosmopolitan species presumed to occur in shipping containers worldwide. These results suggest that the reliability of eRNA high throughput sequencing can be affected by the presence of live and common insect species, reducing resolution over low abundant insect species.

Gaining and sustaining international market access poses challenges for our agricultural industries due to strict biosecurity requirements. Importation of fresh produce into Australia also presents biosecurity risks, leading to costly visual inspections before releasing to the market. To tackle these issues, CSIRO is utilizing advancing imaging technologies like Near Infrared Imaging (NIR) and X-Ray to address plant biosecurity concerns in both exports and imports.
In our NIR-based optical scanning project, the goal is to integrate adaptable detection technology into existing optical fruit sorting processes in packhouses. This allows the industry to identify and remove infested fruits before packing. The optical detection system has demonstrated positive results, identifying infested fruits like cherries and blueberries with Queensland fruit fly at a 0.8% false positive rate and over 95% accuracy, surpassing visual inspection by trained biosecurity inspectors.
In our parallel X-Ray project, leveraging the penetrating capabilities of X-Ray techniques, we aim to identify pest infestations in fresh agricultural produce. Our 2D X-ray imaging technology precisely pinpoints internal feeding damage, larval instars, and pupae of pests within fruits, showcasing its potential as a rapid, precise, and automated quarantine system for biosecurity threats.
Leveraging imaging tech for real-time pest detection in the fresh produce supply chain can revolutionize biosecurity risk management, boosting regulators' confidence in handling increased trade volumes. Additionally, these technologies may find valuable research applications, accelerating disinfestation studies and quantifying pest infestation rates in commercial settings. Ongoing research is expanding applications of these technologies to a broader range of pests.