ABSTRACTS FOR NPBDN AND PSNAP MEMBER TALKS AND INVITED SPEAKERS AT ADSW 2023
The importance of diagnostics and surveillance in Australia’s national plant biosecurity system
Australia has a robust biosecurity system, but the increasing pressures from trade, overseas travel, mail volumes, as well as new pathways that may emerge due to changing climate, requires us to be better prepared to prevent the risk of entry of exotic pests and diseases, and have plans in place to address these risks if they arrive. This presentation will discuss the improvements Australia is making through our diagnostics strategy and surveillance programs, both offshore and onshore. The Australian government supports diagnostic initiatives such as the development of diagnostic protocols, the enhancement of biosecurity diagnostic tools and reference collections and surveillance programs such as the Northern Australia Quarantine Strategy, the National Forest Pest Surveillance program and various partnerships with jurisdictions and industry. Australia is also striving to strengthen biosecurity in the region and internationally by working collaboratively offshore and building capacity in the Pacific and in our near neighbours for surveillance and diagnostics.
Euphresco: from transnational research collaboration to diagnostic standards
Reliable and rapid diagnostic methods are essential to support inspection activities conducted by National Plant Protection Organizations in the framework of their official mandate, and to evaluate the efficacy of control measures taken.
One of the IPPC’s key activities is to develop and promote the implementation of internationally agreed, science-based standards in the regulation of plants and plant products as they move across international boundaries. But the attempts to rationalise efforts have been fragilized by the inefficient and often neglected communication between plant health authorities and the other national, regional and international players: research funders, policy makers, scientists, industries and professional operators.
The Euphresco network was initiated to address these gaps and support coordination of research activities with the aim to bring together countries that focus on the same problem at the same time, to find synergies and complementarities amongst national activities and avoid duplication of efforts, to favour the sharing of information, knowledge and infrastructure. Diagnostics is an important activity of Euphresco. The presentation will provide an overview on how Euphresco and research activities on diagnostics and inspection.
Biosecurity outreach and engagement activities at ACIAR
The Australian Centre for International Agricultural Research (ACIAR) is the Australian Government’s specialist agricultural research for development agency within the Australian aid program. ACIAR connects Australian researchers with the developing world to build a more productive and sustainable food secure future. ACIAR has supported over ninety biosecurity related research projects since 1982, funding work to help understand and address threats to food security from plant and animal diseases and pests. Using stories from projects in Southeast Asia, Africa and the Pacific, we will show how a combination of research projects, together with support for leadership and higher degree studies, contributes to regional biosecurity, while providing an opportunity for biosecurity researchers to get hands on experience in neighbouring countries where some of the pests that concern Australia are endemic. Through ACIAR alumni and our publications, website and the Partners magazine we help spread the word about the opportunities in a career in biosecurity research.
Nancy A. Schellhorn
Real-time Pest Detections for Pest Surveillance and Management
Technological advances in automated pest detection are enabling high quality pest information and in turn, real-time surveillance, and precision pest management. RapidAIM digital crop protection is one such example. Real-time pest detections are enabled by the fully integrated Internet of Things (IoT) insect sensors (www.rapidaim.io) deployed by Government and Industry customers.
Here we showcase the RapidAIM system focusing on fruit fly (Bactrocera tryoni) detections, and regional daily forecasts. The real-time data flowing from the sensors is aggregated and ingested into models for a broad range of pest analytics. Using machine learning, forecasts are generated across regions of 500km2. The high-quality, highly granular data flow from the IoT sensors results in a regression model that captures > 90% of the variability in field data. Using data from research trials, and multiple years of Government surveillance customers, we provide information on detection performance, rapid designation of area-freedom and cost-benefits of RapidAIM compared to manual surveillance.
Following on from Queensland fruit fly, Bactrocera tryoni, the RapidAIM technology has expanded to other key pests of horticulture including fruit and nut borers, and grain pests with focus on bollworms, budworms, and armyworms. New Technologies, such as RapidAIM Digital Crop Protection provide opportunities for Government and Industry co-investment to prevent the establishment and spread of pests.
BioSecure HACCP is a holistic plant protection and biosecurity system specifically designed for pest management in nursery production across Australia. Growers aiming to have a knowledge based pest management program must have reliable data to inform the decision making processes, BioSecure HACCP provides that system as well as delivering a high level of confidence in the decisions made. Through the adoption and implementation of a robust plant protection program the business also implements sound biosecurity practices that elevate the risk mitigation measures and delivers high health plant production.
Structured procedures (e.g., crop monitoring, site surveillance, despatch inspections, etc) are highly detailed to better advise growers on not only the ‘what to do’ but the ‘how to do it’ which makes adoption and implementation streamlined and allows the integration into business as usual practices. Risk mitigation procedures start with source material supply and end with pre-despatch inspection. Documented procedures are repeatable, consistent and give a high degree of efficacy due to the rigour of the process, they improve the chance of finding what is being sought.
The entire BioSecure HACCP program is built around structured procedures and records with in-field data recorded on phones/tablets and stored within each individual growers portal on the web based Audit Management System (AMS). The AMS allows records to be retrieved, assessed, audited with data collated and reported on to provide historical data analysis by growers on trends and outcomes. The AMS is also connected into AusPestCheck to allow industry wide pest surveillance if required.
BioSecure HACCP is built around a robust pest risk assessment process through the application of the 7 defining principles of HACCP (Hazard Analysis Critical Control Point) used to identify plant pest hazards and then manage these at critical control points in the plant production process. NIASA BMP plus BioSecure HACCP form the nursery production integrated pest, or crop, management platform that underpins the knowledge-based decision-making processes that inform the integration of all pest management elements.
Plant Health Student Network
The Plant Health Student Network is a group of students and early career researchers working in plant biosecurity, plant pathology and plant health-related fields. The network began as a group of four PhD researchers (Rebecca Degnan, Bianca Rodrigues Jardim, Fable Eenjes and Salome Wilson) who wanted to continue the conversation after meeting at the Plant Biosecurity Research Initiative Symposium in May 2022. With Dr Jo Luck (Program Director, PBRI) as our mentor, we now have over 200 members from around the world and at all levels of research. We aim to provide a space for peer support and interaction, helping members to develop their research goals and introducing diverse career pathways in Plant Health. Our meetings highlight the amazing professional and research achievements of our network members and guest speakers, as well as providing a space to learn, ask questions and share advice. We cover topics relevant to and chosen by the network, including science communication and professional development.
Prof. Kevin Thiele
The Biodiversity Data Repository, a key national data asset for Australia
The Biodiversity Data Repository is a next-generation system under development by the Department of Climate Change, Energy, the Environment and Water, which seeks to bring about a step change and uplift in the way information on Australia’s biodiversity and environment is captured, managed and used. The BDR uses recently-matured knowledge graph technologies to capture both data (observations, records and occurrences of species and other taxa, both native and non-native) and the semantic relationships between data. This is a significant step beyond current-generation relational database systems, which are good at capturing data but poor at representing knowledge. The BDR is heavily based on global and national standards, and has developed a new and powerful Australian Biodiversity Information Standard (ABIS), which underpins the knowledge graph and its representation of biodiversity information. The combination of a semantics- and standards-based approach and the use of knowledge graph technologies places the BDR at the cutting edge of new Linked Data strategies, which seek to build a World Wide Web of Data. This talk will introduce the BDR project, outline its significance, and discuss its potential applications to plant diagnostics and surveillance.
Prof Geoff Grossel
National eDNA Testing Program
Environmental DNA (eDNA) technology can detect biosecurity pests and pathogens from samples of air, water, soil or other environments, on-site within an hour. This innovative detection technology has broad applications to manage biosecurity and environmental risks.
Implementation of the National eDNA Testing Program, the establishment of the National eDNA Reference Centre (with the University of Canberra) and the Australian network of eDNA Collaboration Centres is a significant achievement in improving the competency, capability and capacity of Australia’s biosecurity services. The National eDNA Testing Program represents a high-profile and successful investment outcome for the Department.
The National eDNA Testing Program provides infrastructure, governance, and supports policy development to underpin and enable efficient, functional, and sustainable eDNA testing. Over the last year the program has advanced many applications of eDNA technology. This has included the introduction of screening-monitoring-detection programs for hitchhiker pests of biosecurity concern such as khapra beetle, exotic ants, spongy moth, and Brown Marmorated Stink Bug. The program has also developed assays and commenced testing of early warning surveillance systems for animal, plant and environmental pests such as varroa mite and exotic animal diseases such as Foot and Mouth Disease, Lumpy Skin Disease, JEV and Highly Pathogenic Avian Influenza.
Heading into the new year the National eDNA Testing Program will be working to expand the applications of eDNA technology, engaging with state and territory governments, neighbouring country Competent Authorities, the Australian network of eDNA collaboration centres and industry partners.
Alexander N. Schmidt-Lebuhn
Mobile identification and collection of observation records using computer vision in biosecurity and weed management
Efficient, user-friendly, and reliable identification of species is critical for a variety of areas ranging from basic research to biosecurity. However, there is a limited number of taxonomic experts, and traditional identification and diagnostic tools generally require understanding of morphological terminology. While DNA-based identification is advancing rapidly both in degree of resolution and speed, it uses expensive specialised equipment and is restricted in its throughput by biochemical reaction times.
The advancing area of computer vision has now opened up another method of identifying species i.e., deep learning / artificial intelligence used to determine which of several pre-defined classes an image or part of an image belongs to. Computer vision models are convenient to non-specialist end-users, as their use does not require specialist knowledge once deployed, and identification through them is near-instantaneous. Their main limitations are the need to build sufficiently large libraries of reliably identified training images for the model and, in contrast to approaches using DNA, a dependency on visual diagnostic characters. A bottleneck they share with many other diagnostic methods is how to deploy them to end-users in an accessible format.
CSIRO’s National Research Collections Australia are mobilising their expertly identified biological specimen collections and expertise to build image classification models and operationalise them in mobile apps, which have the advantage of wide availability. I will discuss two collaborative projects: First, WeedScan, a weed identification and reporting tool funded by National Landcare and developed in collaboration with the Centre for Invasive Species Solutions, the NSW Department of Primary Industries, and 2pi Software. Second, models for the identification of biosecurity threats developed for the Australian Government Department of Agriculture, Fisheries, and Forestry through the Biosecurity Innovation Program. The talk will cover the history of prototype developments, present achievements, and future directions and opportunities.
Management of Ratoon Stunting Disease of Sugarcane
Ratoon stunting disease (RSD) is a major disease and concern to the Australian sugarcane industry. It is caused by the bacterium (Leifsonia xyli subsp. xyli) and has been difficult to manage as there are no associated external symptoms. The disease can go undetected and causes significant yield losses, which average around 15–20%, but can vary between 5–60% depending on the susceptibility of the variety and the weather conditions. It is spread during cutting of planting material, planting, harvesting and planting into non-fallow fields. There are control measures for the disease, but these are often poorly implemented in some regions. There has been much progress in RSD diagnostics over the years, from stalk slicing, phase contrast microscopy, ELISA, PCR to qPCR. There has also been a renewed push over the last few years to highlight the importance of RSD to the industry, re-emphasise control methods, improve diagnostic laboratory processes and procedures and communication of research to productivity services, growers, and planting and harvesting contractors. A decrease in detections have been observed every year in sugarcane planting-material sources since 2020 and is likely due to these recent efforts.
Capacity Building to connect diagnostic and surveillance practitioners (in emergency management). A diagnostician’s view
When people talk about capacity building what do they actually mean? The definition of capacity building is “the improvement in an individual’s or organization’s facility to produce, perform or deploy”.
Over the last few years, around Australia many of us have been involved in an emergency response to a plant biosecurity threat, such as Varroa mite, polyphagous shot hole borer (PSHB), Tomato potato psyllid (TPP) or Dickeya dianthicola just to name a few.
When is the best time for the response team that may include surveillance staff and the diagnosticians to get together to plan how to tackle a response? Is it before a response occurs; developing a National Surveillance Plan and National Diagnostic Protocol, or at the beginning of the response while planning and operations are trying to sort out logistics and roles, or when the response is underway when everyone has their role and questions such as, is there a diagnostic protocol? How many samples can the lab process? What type of samples are needed? What type of results do we need? arise.
How can we improve our ability to plan and manage a response in a constructive way that is not reactive or re-inventing the wheel at each step?
I will discuss a number of options on where we can improve our capacity to handle these biosecurity responses so that surveillance officers and diagnosticians are connected and understand their roles.
How to be generic but specific? A nanopore sequencing-compatible PCR/qPCR assay for detection and differentiation of Xylella fastidiosa and Xylella taiwanensis
Xylella fastidiosa is the Number 1 National Priority Plant Pest (NPPP). This bacterium can infect more than 660 plant species, many of which are agriculturally important. Another species within the Xylella genus, Xylella taiwanensis was reported to cause leaf scorch on Asian pears in Taiwan. However, the pathogenicity, host range and distribution of this novel species are relatively unknown.
Xylella is difficult to isolate and often remains latent in infected plants for a long period of time, therefore highly sensitive molecular diagnostic assays are crucial for Xylella detection. An up-to-date national diagnostic protocol (NDP), validated in Australia, is essential to accurately identify diseases caused by either Xylella spp. and distinguish the different Xylella lineages. In developing a new Xylella spp. NDP, we have comprehensively evaluated a set of published molecular diagnostic assays described in the IPPC and EPPO diagnostic protocols, however not all the available assays are able to detect X. taiwanensis. To address the gap in X. taiwanensis, a new Xylella PCR/qPCR assay that targets the comEC gene has been developed as a generic assay to detect both Xylella spp.
Ring tests by five diagnostic laboratories across Australasia indicated that the comEC PCR/qPCR assay has superior specificity and sensitivity for detecting X. taiwanensis, compared with currently available assays. The comEC qPCR assay and the qPCR assay described by Harper et al. (2010) outperformed other assays for X. fastidiosa detection. Phylogenetic analysis suggested that subspecies-level differentiation is achievable by sequencing the comEC PCR amplicons. The comEC PCR products could be sequenced by traditional sanger sequencing but Oxford Nanopore Technology (ONT) sequencing provided faster turnaround time. The ONT sequencing method has been incorporated into the new Xylella spp. NDP. Our experience in the Xylella diagnostic assay and NDP development will be discussed in this presentation.
Avocado Sunblotch Viroid Surveillance – A Pathway to Declaring Pest-Freedom
Avocado sunblotch viroid (ASBVd) affects both the yield and marketing of fresh fruit from an orchard. The Avocado Nursery Voluntary Accreditation Scheme (ANVAS) has operated in Australia since 1980, ensuring all plants sold from registered nurseries have been propagated using viroid-free seed and budwood. The diagnostic assay used for ASBVd is a one-step reverse transcription qPCR (RT-qPCR) assay. The rate-limiting steps for surveillance for this pathogen are sample collection and then processing to produce an RNA template for RT-qPCR. To speed up sample preparation, we have developed a simple and inexpensive ‘filter paper method’ of viroid RNA extraction. This method relies upon the reversible binding of viroid RNA to cellulose fibers in the presence of a monovalent cation, with yields comparable to a commercial RNA extraction kit. Recent postal delays in the delivery of samples to the testing laboratory due to COVID raised concerns about suboptimal storage conditions and how this may affect the reliability of test results. To address these concerns, experiments were done to investigate the effect of time and storage temperature on the ability to detect ASBVd in leaf or fruit tissues. Most notably, ASBVd was still easily detectable in detached 4-week-old avocado leaves stored at room temperature that had become desiccated and necrotic. Furthermore, ASBVd was easily detected in the skin of green ‘Hass’ fruit, providing opportunities to test the infection status of fruit upon arrival at an entry port. Finally, we have investigated alternative ways of collecting samples from an avocado orchard by utilizing the natural foraging behavior of honeybees. ASBVd was confidently detected by both high throughput sequencing and RT-qPCR in pollen samples from hives within 100m of infected trees, suggesting this RNA approach could be used as part of an industry-wide surveillance program for the pathogen.
CropSafe community surveillance supports detection of Fusarium gamsii on wheat
General surveillance networks such as CropSafe have broad value to the agricultural industry for early detection of exotic pests and diseases. These networks can also contribute to first reports of fungi that are not considered a risk to our agricultural systems and add to our knowledge of endemic species. As an example of this, during 2022 an advisor in the CropSafe network submitted a wheat sample with suspected fusarium head blight as part of their general crop inspections. As part of the routine diagnostics a fusarium was isolated from the plant tissue. The fusarium isolate recovered did not have morphological characteristics consistent with known wheat pathogenic fusarium species. Using DNA-directed RNA polymerase II subunit (RPB1) sequences could differentiate the isolate from the species in the Fusarium tricinctum species complex. The isolate matched Fusarium gamsii. This is the first report of F. gamsii in Australia, and also a new host record for this fungus on wheat.
Environmental DNA based detection complements biosecurity monitoring and surveillance to enhance early detection.
Environmental DNA (eDNA) is a fast-growing, innovative field that was first developed about 20 years ago and has since moved to scientific integration into monitoring projects globally in the last 10 years. It is used worldwide in numerous projects across multiple applications to provide valuable information in both environmental monitoring and human and animal health. eDNA is used alongside traditional methods as a surveillance tool in marine systems for marine pest surveillance in Australia and in New Zealand; human wastewater surveillance for infectious diseases and exotic snail biocontrol monitoring in broadacre crops in Australia.
Most recently, eDNA detection is being tested for use in biosecurity at the border as a surveillance tool for exotic pest detection in shipping containers. This project has had some exciting results and shows promise for future use in biosecurity more broadly. This project includes the recognition of the National eDNA Reference Centre in Canberra as Australia’s leading institution of eDNA tests and standards.
eDNA can provide a complimentary tool to existing surveillance methods to provide early detection of exotic pests and diseases. As eDNA becomes more common, the need for definitions, standardisation, national assays and sampling validation and usage guidelines is apparent. This process has begun through initiatives such as the Southern eDNA Society (Australia and New Zealand). Clarification on appropriate application of eDNA in biosecurity settings is needed, including triggers, responses, and the implications of a positive result.
The use of eDNA in surveillance and monitoring alongside traditional methods to enhance early detection should be considered in biosecurity programs/protocols including becoming part of the NATA accreditation process. The development of a framework and policies to support eDNA application is essential to enable use as an early warning system that can enhance and strengthen existing biosecurity monitoring.
Automated air sampling for remote surveillance and high throughput processing of environmental samples for eDNA analyses
A collaboration led by the National eDNA Reference Centre at the University of Canberra, with support from the Department of Agriculture Fisheries and Forestry (DAFF), is evaluating an end-to-end, at scale, eDNA based surveillance system using remote sampling units derived from the iMapPESTS Sentinel Surveillance for Agriculture project, coupled with high throughout pest/pathogen identification methods. For this study, the mobile surveillance platforms were satellite enabled to ensure end-to-end data connectivity and allow airborne pathogens monitoring anywhere in Australia. Using Myrtle Rust (Austropuccinia psidii) as a model pathogen, Sentinel units were deployed at sites in Adelaide (recent positive detections) and Sydney (established disease region) to demonstrate surveillance capacity using high-throughput qPCR diagnostics by SARDI Molecular Diagnostics and metabarcoding diagnostic by the Australian National University fungal genomics group. This dual diagnostic approach combines target specific diagnostic for area-wide surveillance capability with genomic analysis of lineage defining loci for deeper investigation into species diversity within broader phylogenetic groups (e.g., rust) and potential detection of novel incursions. Future application of the system to well-studied pathogens will allow for adoption of population scale analysis including frequency of fungicide resistance or pathogen virulence profiles. Digital output visualisation packages will be explored with the project’s technology partner Data Effects, in collaboration with the University of Adelaide, to complete the end-to-end surveillance pipeline of device-to-data delivery customised to stakeholder needs. Applications of the project outputs include deployments into risk pathways and border protection points of entry or trade protection for Australian imports and exports and provide innovative and sustainable solutions to Australia’s biosecurity programs.
Insect diversity in shipping containers determined by environmental DNA
The international trade of commodities and produce is the most important pathway associated to the translocation of invasive exotic species. Shipping containers for example, carry approximately 90% of global trade and have been repeatedly reported to translocate invasive insects. In Australia for example, previous research reported 7891 insects inside 1174 shipping containers held in container parks, within which exotic species were detected in over 333 containers. This study examined insect diversity in shipping containers imported to Australia targeting environmental DNA and using Illumina Novaseq high throughput sequencing. Broad spectrum primers designed to amplify a 348 bp of the Cytochrome oxidase I gene region were modified to target a broad range of insect orders. A total of 1980 samples were successfully sequenced with approximately 11 billion reads, of which 3,459,189,038 reads passed quality control and assurances. Environmental DNA from over 150 insect species was detected across shipping containers. There were multiple positive eDNA detections of National priority plant pest species in shipping containers. This included Coptotermes formosanus, Cryptotermes dudleyi, Cryptotermes brevis, Incisitermes minor (Blattodea), Anoplophora glabripennis, Euwallacea fornicates, Hylotrupes bajulus, Trogoderma granarium (Coleoptera), Bactrocera dorsalis, Drosophila suzukii, Liriomyza spp. (Diptera), Halyomorpha halys (Hemiptera) and multiple Spodoptera species, including S. frugiperda (Lepidoptera). In this context, eDNA-based detection provides valuable inference to assess the risks and history of insect pests in shipping containers, but eRNA-based detection may be needed to determine active/recently dead pests more accurately. Moreover, eDNA detections do not confirm association between pests and goods carried within shipping containers across shipping lanes. The outcomes of this research highlight a critical need to develop novel monitoring methods using molecular surveillance applications to complement border biosecurity and surveillance. Most importantly, investment is necessary to optimise and modernise sample collection methods for end users to efficiently implement eDNA -based surveillance applications in Australian biosecurity.
CitrusWatch and urban biosecurity – expanding surveillance and engagement across major capital cities in southern Australia
The Australian citrus industry is a large and vibrant horticultural industry with 1,500 businesses directly involved in citrus production, and many associated businesses providing support services to agronomics, harvest, and supply of citrus. The growing and harvesting of Australian citrus across most states and territories make up an annual national crop worth of over $900 million (including global exports). With a significant production base, exotic pests and disease incursions (i.e., Asian citrus psyllid, African citrus psyllid and Huanglongbing) can lead to extensive pest management costs, restricted market access and in some cases, decimation of entire growing regions. In recognition of the heightened risk of entry of exotic citrus pests via increased global movement, and building on previous industry investments, the citrus industry has launched a five-year collaborative national biosecurity program, CitrusWatch (2021-2026), and recently developed a five-year Citrus Industry Biosecurity Strategy (CIBS; 2023-2028).
The CIBS was developed to establish a culture of awareness and preparedness for a resilient citrus industry, highlighting four priorities and 16 activity areas to guide efforts annually. One of these activities is to raise biosecurity awareness among urban residents – reflected in the current and emerging surveillance and engagement activities undertaken in CitrusWatch. This presentation will describe aspects of CitrusWatch that focuses on boosting surveillance (Early Detector Network and targeted surveys) and engagement (sharing biosecurity and citrus plant health information) within urban communities across high density port of entry cities in southern Australia: Melbourne, Sydney, Perth, Adelaide, and Brisbane. Furthermore, the potential for a successful implementation of the program through connections and advocacy of project partners, key knowledge brokers and stakeholder groups will be explored. The presentation will discuss strategies undertaken to identify stakeholder groups (outlined in an urban surveillance and engagement plan) aimed at increasing outreach and awareness to a diverse urban public including backyard gardeners.
Carlos Babativa Rodriguez
Implementation and sharing new technologies regarding the utilisation of geospatial software, and its integration across different ag vendors. This project delivered more efficient surveillance and prescription mapping deployed to auto-spray machines
Prioritization of biosecurity investment based on the invasion curve of invasive species dictates that asset-based management of invasive species is best addressed by landholders and community. An eradication Chondrilla juncea (Skeleton weed) has taken place in WA since 1974. The thickest infestation is located on the eastern shires, where the program aims for control rather than eradication. In order to promote and facilitate the asset-based treatment of the known infestations in the typical broad-acre setting of the infestation in the eastern shires, an ad-hoc workflow has been set up to deploy prescription maps via cloud-based services directly into farmers auto-spray machinery. The files deployed contain geolocated polygons representing skeleton weed infestation areas that could be used to spray chemical treatments for the eradication. The program provided guidance and digital files to the Local Action Group coordinator from Narembeen shire for them to deploy the prescription maps. This solution relieves workload for a single operator with equipment not suitable for broadacre. Consequently, this outcome directly promotes staff retention, multiplies the capacity of the eradication program, and brings closer the project milestone completion date.
Australian bark beetle (Curculionidae: Scolytinae) diagnostics: connecting biosecurity, taxonomy and molecular identification
Novel invasive species represent an ongoing risk to Australian natural resources and ecosystems. Bark beetles (Curculionidae: Scolytinae), are a major group of plant pests. Multiple primary industries are impacted by bark beetles due to their wood boring life history and the plant pathogens they vector. They are one of the most frequently translocated insect pests globally and currently 38 exotic species have been established in Australia with this number expected to rise. Most recently in August of 2021 the Polyphagous Shot Hole Borer, Euwallacea fornicatus, was detected in East Fremantle, Western Australia. This species alone has over 400 host tree species globally and can attack amenity, agricultural, forestry and natural host trees. Australia is also home to a rich diversity of native species of bark beetles wherein they typically complete their life cycles in decaying plant matter. Bark beetle linages have a rich array of life history traits, including polyphagous host preferences, permanent inbreeding and symbiotic associations with fungi which allow many species to be successful invaders. Despite their impact on managed and natural ecosystems, few resources exist for species level identification. This is compounded by several factors including similar morphological characters shared between species and genera, small body sizes, a contentious taxonomic history and few specialists working on the group. I will be highlighting the current state of diagnostics of Australian Scolytinae and discussing current projects working on keys for identification and genomic resources for diagnostics and research. Lastly, limitations and gaps for the diagnostics of this group will be addressed and avenues to address these will be highlighted. The outputs of these projects will work synergistically to improve the capacity of Australian Scolytinae diagnostics by connecting taxonomic identification with comprehensive molecular reference databases.
Residential to expand diagnostic and surveillance capacity to include bacterial LAMP assays
Horticulture diagnostics capability in South Australia is limited for identification of bacterial pathogens. Techniques being used, such as microscopic observations, semi-selective media and limited molecular sequencing, are slow and do not provide accurate identification to species. Current surveillance performed by BiosecuritySA is limited to the observation of symptoms, with all samples of suspected exotic bacterial pathogens being sent to interstate laboratories. During COVID restrictions and responses to recent exotic incursions postage and staffing issues interstate have impacted diagnoses.
The recent acquisition of equipment in SA, to perform loop-mediated isothermal amplification (LAMP) assays, has enabled rapid assays to contribute to diagnosis for pathogens endemic in SA. LAMP assays for multiple of the bacterial national priority plant pests (NPPPs) have been developed to support surveillance activities and the national plant biosecurity diagnostic network (NPBDN) facilitated their presentation to the diagnostic community in late 2022. Without published national diagnostic protocols (NDPs) and associated positive controls these assays have not been used in SA. A residential at the Elizabeth Macarthur Agricultural Institute, NSW, provided training by pathologists experienced in bacterial identification, particularly: Xylella fastidiosa, Xanthomonas citri subsp. citri (citrus canker) and Xanthomonas fragariae (angular leaf spot in strawberries). The provision of funds from the NPBDN residential program for consumables, ie. primers and master mix, enabled optimisation of LAMP assays with positive controls, that SA previously had no access to. This residential ultimately expanded the diagnostic capacity for rapid detection of these NPPPs in SA. Analysis of a limited number of field samples from potential hosts of these pathogens contributed to surveillance activities, and data contributing to area freedom claims.
Proposed nationally coordinated high-throughput sequencing database for plant pests
Australia has an increasing need for fast and accurate identification of plant pests. High-throughput sequencing (HTS) is a significant new method in plant health diagnostics. HTS generates massive datasets, and the increasing amount of data being generated on pests and pathogens of plants has emphasised the need for a secure, centralised platform that allows the submission, sharing and analysis of standardised HTS data. Australia has no existing national data sharing and storage platform that could host standardised HTS data, nor are there means to link this data to other existing diagnostics databases such as the Australian Plant Pest Database (APPD) and the Pest and Disease Image Library (PaDIL). This lack of national coordination risks a discontinuous national plant pest and disease diagnostic system and threatens our capacity to deliver accurate and timely identification of exotic plant pests.
As part of a project funded under the Australian Government’s Department of Agriculture, Fisheries and Forestry’s Biosecurity Innovation Program, Plant Health Australia is consulting with stakeholders via workshops and one-on-one interviews to scope the requirements of the proposed nationally coordinated HTS database and to draft data standards and governance arrangements.
High throughput screening of fungal phytopathogens caught in Australian forestry insect surveillance traps
Post-border surveillance for forestry’s high priority pests and pathogens is conducted routinely through established programs focused on the main points-of-entry and across the major plantation growing regions. Currently, most diagnostic protocols used to identify fungal phytopathogens sampled during these surveys rely on traditional methods, such as morphological examination and DNA barcoding techniques. This stepwise process from isolation to species identification is often regarded as slow, expensive, and limited due to the need for disease manifestation and/or comprehensive expertise for rapid and accurate detection. In this study, we applied a recently validated high-throughput, dual-marker amplicon sequencing approach on insect surveillance traps from across Australia to assess its performance for the targeted surveillance of the Ophiostomatales, an order of fungi comprising notable phytopathogens which are vectored by bark beetles. By using a recently validated assay we were able to confidently characterize a range of Ophiostomatalean taxa known to be present in Australia, while reporting eight first detections from environmental DNA. Our study demonstrates the value of targeted multi-barcode amplicon sequencing for high-throughput screening of fungi caught in post-border surveillance traps, in addition to emphasizing research priorities that require further investigation before such methods can be implemented routinely for biosecurity.
Introducing the Department of Agriculture, Fisheries and Forestry new Technical Response Team
DAFF’s Technical Response Team was founded in 2021/2022 and is responsible for managing and coordinating the operational aspects of responses to detections of exotic species at the border, at and around First Points of Entry (FPOE’s) and on importer business premises that have entered into an approved arrangement with the department (AA’s).
Whilst the Technical Response Teams is tasked with managing pest detections on land under commonwealth jurisdiction, typical response scenarios aren’t always clear cut and obvious in this regard, and many several jurisdictions may be impacted simultaneously. Therefore, it has been imperative that we build and maintain strong, positive and functional relationships with various other agencies including state and territory governments. Since the team was formed, we have already been involved in numerous response activities including responses to BMSB detections, exotic snails and exotic invasive ants. Our team consists of a Director, an Assistant Director, 2 Technical Field officers, a communications manager and dedicated response diagnosticians. As a small team we rely heavily on our ability to develop and maintain strong positive relationships with various other teams both internally and externally. Our ability to work effectively with other teams relies on several key aspects:
To help reach our potential, the Technical Response Team have recently undertaken AIIMS (Australasian Inter-Service Incident Management System) response training. It is hoped that this will allow our team to scale up our efforts, enabling us to respond to more protracted or extended responses. Implementing this framework requires building and maintaining functioning relationships with various, multi-disciplinary teams. Strengthening and maintaining relationships with these teams allows incident responses to run more efficiently and smoothly, in turn ensuring favourable biosecurity outcomes.
Northern Australia Plant Capability and Response Network (NAPCaRN).
The Northern Australia Biosecurity Strategy (NABS) adopts an integrated, collaborative approach to delivery of better biosecurity outcomes for the north. This approach is critical to effectively manage the north’s unique biosecurity risk profile, which is becoming more complex, active, and difficult to manage year on year. Addressing capacity and capability gaps is critical.
Recently the federal government has committed to bolstering northern Australia’s ability/capacity to manage this increasing biosecurity risk, through implementation of the NABS.
A key NABS investment is the Northern Australia Plant Capability and Response Network (NAPCaRN). NAPCaRN is a co-ordinated Northern Plant Biosecurity hub/network, delivering an uplift in northern biosecurity capability. It will be built through the following: (1) Investing in one technical intern position in each northern jurisdiction in the areas of entomology, plant pathology, and/or molecular biology, and (2), partnering these positions with a Scientist position (same disciplines) and Biosecurity Liaison Officer position (industry and/or indigenous) in each northern jurisdiction. This would uplift capacity and capability across the north, which will increase surge capacity, assist with early detection, preparedness and response, and enhance biosecurity outcomes for industry as well as on-country outcomes for First Nations peoples.
Benefits from this investment includes more agile detection and response capacity across the north, enhanced partnerships with industry and Indigenous ranger groups, development of a ‘pipeline’ for capacity/capability flow and improved biosecurity leadership in the north. Defined linkages with Plant Health Committee will ensure that benefits are shared nationally and leverage activities and investments already in place.
Darryl A. Herron
Diagnosing Fusarium circinatum and Ceratocystis eucalypticola in South Africa to improve Australasian diagnostics
Fusarium circinatum, the causal agent of pine pitch canker, is a serious fungal pathogen that threatens Pinus spp. and Pseudotsuga menziesii plantations in Australia and New Zealand. Species of Ceratocystis also represent a group of important pathogens that affect many different tree species, such as Acacia, Eucalyptus and Metrosideros, globally. Researchers and diagnosticians at the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa have spent decades working with and diagnosing these and other important tree pathogens. Between the 6th and 22nd of February 2023, I completed a Diagnostic Residential at FABI, connecting with Prof Irene Barnes and Prof Bernard Slippers, working with samples of Fusarium circinatum and Ceratocystis eucalypticola. An update on Fusarium circinatum and Ceratocystis eucalypticola in South Africa will be presented, including how best to collect, process, and successfully identify these pathogens, morphologically and molecularly. The aim of this presentation is to upskill Australasian diagnostic practitioners when working with these pathogens.
Duy Phu Le
Diagnostic Residential Project: making nematological upskilling possible!
Cotton (Gossypium hirsutum) is an important cash crop for regional NSW and QLD. Reniform nematode (Rotylenchulus reniformis) is a major constraint to cotton growers, especially those based in Emerald and Theodore in QLD. To date, R. reniformis has not been detected outside these areas. To assist with monitoring for this pest and to enhance the nematological capacity of the NSW cotton pathology team, an intensive training program with one of Australia’s most experienced nematologists, Dr. Graham Stirling,
was proposed through the Diagnostic Residential Project (DRP). The aims of the project were to train the NSW cotton pathologist to:
1) extract plant-parasitic nematodes from soil and plant materials; 2) identify and differentiate major plant-parasitic nematodes to genus and species level; 3) identify free-living nematodes to trophic group; and 4) quantify and monitor parasitic and free-living nematode populations in cotton fields. Over the course of four months, I had a once in a life-time opportunity to have a full week of intensive one-on-one training with Dr. Stirling, which allowed me to begin the long process of identifying nematodes using morphological methods. I also had an opportunity to join one of Dr. Stirling’s philanthropic masterclasses in Brisbane, where I learnt to accurately identify nematodes using molecular techniques as well as network with others who were keen to improve their nematology skills. The most important requirement from Dr. Stirling for this DRP was that I should continue my self-learning opportunity for several months. Thus, every week I attempted to: 1) extract at least two soil samples from different locations and cropping systems; 2) identify and count the number of nematodes in each sample; and 3) seek for confirmation and comments from Dr. Stirling on the veracity of my counts. This weekly commitment to the training proposal allowed me to build a certain degree of competency in nematology, but as it takes many months to become fully proficient in morphological identification, continuing practice will be required.
A NOVEL MACHINE LEARNING APPROACH FOR FUNGAL SPECIES DETECTION
Fungal identification and detection is vital for rapid and effective pathogen management. Developments in nanopore long-read sequencing enable high-speed, high throughput, portable sequencing, important to provide quick results to minimize diagnosis time in-field. Current sequence-based analysis techniques rely on short reads and public databases that have lagged on adapting to these long reads, especially when using non-standard primer sets to avoid issues with common ‘universal’ fungal barcoding primers. This delays or hinders pathogen detection. We address this gap and present a proof-of-concept approach for classifying fungal taxa using chained machine learning classifiers, independent of public databases. Samples of an extended fungal ITS metabarcoding region can be classified at multiple taxonomic levels with high recall and low false positive rates. It distinguishes between closely related and cryptic species, where current alignment and k-mer methods struggle. This approach is best suited for detection tasks and can detect a target species from a complex field sample with high recall and high confidence. Our proof-of-concept demonstrates the power of this type of approach for barcoding sequence-based detection tasks on a range of fungal taxa, including plant and human pathogens. It is particularly suited
for targeted pathogen detection tasks and can be modified to home in on target taxa. Further optimization of this proof-of-concept could turn this decision tree approach into a useful tool suitable for agricultural, clinical or biosecurity settings.
John. H. Moore
Success of The Three-horned Bedstraw (Galium tricornutum) Eradication Program in Western Australia
Three-horned bedstraw (Galium tricornutum) has been eradicated in Western Australia. Economic analysis indicated a high benefit: cost ratio for an eradication program which was implemented in 2001. The program was tailored to the individual situations in consultation with the landowner and industry.
Control costs and loss of production caused by the infestation or control program are funded by government and industry funding scheme The various strategies used, their effectiveness and costs are discussed. The importance of early detection of infestations is emphasized and sampling of grain to assist early detection was used to ensure a low likelihood of undetected infestations. This paper outlines the history and the processes used to achieve eradication in Western Australia.
Polyphagous shot-hole borer in Western Australia – stakeholder engagement and response at the urban forest-residential interface
New pest incursions in urban areas present challenges and opportunities. Urban landscapes are incredibly heterogeneous – environmentally, socially and jurisdictionally – and encompass a wide range of biosecurity stakeholders. The impacts on these stakeholders are diverse, with varying levels of concern, participation, and compliance during pest responses. Where maximised, engagement of this diverse suite of biosecurity stakeholders has the power to supercharge our capability and capacity to manage current threats and respond effectively to new ones. These challenges and opportunities are typified in the current response by the Department of Primary Industries and Regional Development (DPIRD) to the Polyphagous Shot-Hole Borer (PSHB) (Euwallacea fornicatus) in Western Australia. PSHB was first detected in the Perth metro area following a public report of an infested backyard tree using the MyPestGuide® Reporter App in August 2021. A Quarantine Area encompassing much of the Perth metropolitan area was established to support surveillance efforts and restrict the movement of wood and plant material. This Quarantine Area and the response has impacted residents, local governments, green-waste operators and numerous plant and wood-based industries such as nursery and wood carving. This has challenged DPIRD to adapt and respond throughout the response, working closely with local industries, councils, and the community to conduct and maximise surveillance and tracing activities. This presentation will summarise the DPIRD response to date, explore some of the challenges and opportunities present at the urban forest-residential interface, how these are navigated and how we can maximise these learnings to enact transformational change across environmental, social and jurisdictional boundaries.
Let your hive thrive, it’s time for a honey bee check up! Australia is currently free of many serious honey bee pests and diseases that damage the health of European honey bees overseas. In recognition the National Bee Pest Surveillance Program has been coordinated by Plant Health Australia for over ten years to support early detection of honey bee pests by conducting surveillance at the highest risk entry points. The importance of the surveillance program was highlighted in its role the 2022 detection of Varroa destructor post-border. However continual risk of these significant biosecurity threats has raised the need to widen the reach of surveillance efforts to include commercial and recreational beekeepers. As an outcome, the Bee Pest Blitz was born. An annual month-long call to action to all Australian honey beekeepers to get out and check their hives. The Bee Pest Blitz endorsed and supported by the honeybee industry, state, territory and Commonwealth governments motivates beekeepers to inspect their hives, aiming to grow awareness of pest and diseases, improve beekeeper surveillance skills, increase the reach of surveillance efforts and support the work currently being done in the surveillance and response programs. This builds on from previous state-based initiatives, and the increased awareness within the industry since the NSW Varroa response. The project will work closely with AHBIC and all state and territory governments in developing resources to promote the blitz campaign, including videos, factsheets, and a campaign webpage. A Technical Working Group with experts from industry, government, and researcher agencies (including international) has been formed. The group has met several times to review and revise surveillance activities to ensure beekeepers are equipped with the most up to date information in how to perform hive inspections. An agreed revised method and factsheet with campaign branding is expected to be completed by end of March, for the first campaign to be promoted April 2023.
Lilia C. Carvalhais
Molecular diagnostics, more than just laboratory work Various members of the Ralstonia solanacearum species complex (Rssc) cause bacterial wilt in over 450 plant species across 54 families and some are priority pests in Australia. Over the years, bacteria within the Rssc have been reclassified in many ways. In 2005 Fegan and Prior proposed classifying these bacteria into four genetic groups named phylotypes. In 2014 Safni et al. proposed three species associated with these four phylotypes: i) Ralstonia solanacearum (phylotype II) which originated in the Americas; ii) Ralstonia syzygii (phylotype IV) originated in Indonesia; iii) Ralstonia pseudosolanacearum (phylotypes I and III) from Asia and Africa. Moko is a threatening bacterial disease of bananas caused by various R. solanacearum sensu stricto sequevars that originated in countries of Latin America and the Caribbean, and spread to the Philippines and Malaysia, but is exotic to Australia. This bacterial pathogen colonises the xylem culminating in a lethal wilt. The previous national diagnostic protocol (NDP) for Moko was documented 16 years ago based on strains collected in the 1990’s. We have recently submitted a new NDP draft for the detection of Moko using recently collected samples and incorporating modern taxonomy. In this presentation, we will describe the steps undertaken to update the Moko NDP including the acquisition of photographic records of the disease symptoms, obtaining biological material overseas, the design of new methods and implementation of rigorous assay validations following international standards. We will also present current management strategies for the control of Moko disease in Latin America. This presentation will provide information to other members of the National Plant Biosecurity Diagnostics Network interested in developing a NDP and will highlight potential benefits and possible hurdles involved.
Integrated digital biosecurity systems
Plant Health Australia (PHA) administers a range of digital systems that are integral to national plant biosecurity, including the Pest and Disease Image Library (PaDIL), the Australian Plant Pest Database (APPD) and AUSPestCheck®. APPD and PaDIL are of strategic importance to Australia’s national biosecurity system in supporting the accurate diagnosis of plant pests and diseases for regulatory and management purposes, and determination of pest status. AUSPestCheck® is the repository for national surveillance data and supports critical data from the National Plant Health Surveillance Program and from industry partners. Each of these digital systems facilitate sharing of biosecurity information and data necessary for efficient functioning of our national biosecurity system. While these applications are aligned in purpose, there is an opportunity to think holistically across the services provided by PHA and look at ways to bring their functions and features together as integrated digital biosecurity systems. In this talk we will cover what each of the Digital Systems administered by PHA currently do, future enhancements, areas where they could be further aligned, and the scope for new digital systems and services to be developed and integrated into these current systems.
Using near-infrared spectroscopy and machine learning modelling for the rapid detection of Verticillium wilt of potatoes
Verticillium wilt, primarily caused by the pathogen Verticillium dahliae, is an economically devastating disease of potato crops. Early detection of this disease is critical in informing growers to apply management measures to reduce Verticillium wilt incidence and severity. However, early detection of Verticillium wilt can be challenging due to the cryptic nature of symptom emergence and expression. This paper presents the implementation of near-infrared (NIR) spectroscopy coupled with machine learning to develop an efficient and objective tool for the early detection of Verticillium wilt in potato plants. Two artificial neural network (ANN) models were developed using the raw absorbance values within the 1596 – 2396 nm light spectral range as inputs to predict photosynthetic rate, transpiration rate, and stomatal conductance, as well as predict whether plants were infected or non-infected. The results showed high accuracy for Model 1 (R = 0.89 using all samples from trial 1) with a deployment accuracy of R = 0.76 using all samples from trial 2. High accuracy was also obtained for Model 2 using samples from trial 1 (94 %) with a deployment accuracy of 84 % using samples from trial 2. Results showed that V. dahliae presence could correctly be identified in plants two days after inoculation (DAI) without any visible symptomatology. These model types have never been presented for Verticillium wilt of potato and show high potential in introducing a cost-effective, efficient, and user-friendly means for growers to detect Verticillium wilt before symptoms occur.
Biosecurity New Zealand’s use of digital imagery in Plant Health Surveillance Programmes
While the use of digital imagery is not new, Biosecurity New Zealand is finding new and innovative ways to use digital imagery to accelerate and enhance diagnostics, provide evidence of area freedom and providing scope for future developments such as machine learning for public reporting.
The use of digital cameras has increased markedly in the last decade, particularly with the advent of high-resolution cameras built-into mobile phones. Photos taken in the field during surveillance programmes can now be sent almost instantly to diagnosticians and triaged to rule out high risk exotic organisms. This improves diagnostic efficiency, reduces costs of sending samples, eliminates samples being lost in the courier system, and provides opportunities for earlier detection of unwanted organisms.
The field images are also able to be integrated with GIS to provide geo-located image libraries that can allow changes at sites to be monitored over time and analysis of environmental and host aspects to increase the sensitivity of surveillance programmes. Geo-referenced and time stamped images also assist in auditing of any surveillance programmes to ensure that inspection intervals and time frames for samples to the lab are within the specifications set out in procedures.
Implementation of a suite of diagnostic methods for Rubus yellow net virus to support regulatory decision making at Post Entry Quarantine
Rubus yellow net virus (RYNV) belongs to genus Badnavirus (family Caulimoviridae) and is found in plants as endogenous (inactive), and/or episomal (infectious) forms in raspberry (Rubus idaeus). RYNV when present in mixed infections with other viruses causes mosaic symptoms that severely affects plant vigor and yield. At Post Entry Quarantine (PEQ), imported red raspberry are primarily screened for regulated plant viruses and viroids using small RNA high throughput sequencing (HTS). In the 2022/23 testing season, 29 raspberry plants were sequenced, and small RNA sequencing data generated on an Illumina platform were analysed using Nextflow VirReport and GA-VirReport bioinformatics workflows. HTS detected three lineages of endogenous RYNV (endoRYNV) strains in 24/29 cultivars (83%). Single or multiple integrations were reported within a single plant although plants were asymptomatic. We further verified HTS results using specific SYBR green qPCRs targeting six known endoRYNV lineages and the episomal lineage. Where present, amplicons were further confirmed through sanger sequencing followed by BLASTn searches to the PVirDB database. The episomal RYNV strain, which is quarantinable, was not detected in any plant through HTS and qPCR. This study further confirmed the widespread presence of endoRYNVs in commercial red raspberries, likely due to the limited germplasm pool used in breeding programs. It also highlights the value of understanding complex host-pathogen relationships and genomic integration of viruses when using HTS as a tool for making regulatory decisions to release plants from quarantine.
Targeted sequencing approaches for Xylella identification and surveillance
Two robust targeted high throughput sequencing (HTS) detection methods, using hybridization probes and amplicon sequencing have been developed by Agriculture Victoria Research to overcome limitations of detection and identification of low titre and unculturable pathogens. Hybridization probes were designed to capture targeted nucleic acids of phytoplasmas, liberibacters and Xylella spp to isolate them from host DNA. This increased sensitivity while maintaining specificity of targets, individually and in mixtures, and reduced the amount of sequencing resources compared to metagenomic HTS. This method has direct application in the routine diagnostics in Post Entry Quarantine where plants may require testing for multiple
pathogens per commodity as it reduces costs associated with testing while reducing time the plants spend in quarantine facilities. The amplicon HTS approach was designed for Xylella fastidiosa, the highest priority plant pest for Australia. PCR was used to target and enrich seven X. fastidiosa house-keeping genes directly from the sample and the amplicons were then sequenced using a MinION device. This approach not only detected X. fastidiosa but also identified Sequence Types (ST), which informs host range for surveillance and outbreak management. The method is rapid, cost effective, sensitive and robust compared to metagenomic sequencing. Results can be returned within a day, which would facilitate rapid decision making and implementation of control strategies during the response to a X. fastidiosa incursion.
ANCIENT POWDERY MILDEW DNA FROM REFERENCE COLLECTIONS: THE EFFECT OF A CENTURY ON POWDERY MILDEW DNA PRESERVATION
The use of molecular techniques to investigate herbarium specimens has increased over the last decade. However, fungal obligate biotrophic plant pathogens such as powdery mildews present a massive knowledge gap in next-generation sequencing from reference collections. This study analysed powdery mildew reference collection specimens over a 117- year period to better understand how powdery mildew DNA decays. We examined DNA base substitution, fragmentation and investigated the microbial community present on the powdery mildew specimens. We found that powdery mildew DNA degraded at a similar rate to plant herbarium DNA regarding nucleotide misincorporations at DNA break points and excess cytosine to thymine substitutions. The microbiome profiling revealed the dominant fungal families were Erysiphaceae, Aspergillaceae and Saccotheciaceae and the dominant bacterial families were Microbacteriaceae, Pseudomonadaceae and Sphingomonadaceae. Abundance plots showed Saccotheciaceae and Pseudomonadaceae were more abundant in recent herbarium samples and Aspergillaceae being more abundant in ancient herbarium samples. These findings will provide baseline knowledge for future diagnosticians and researchers how to extract and amplify obligate biotrophic plant pathogen DNA from reference collections.
Establishment and implementation of real-time LAMP protocols for diagnosis of Eutypa and Botryosphaeria dieback pathogens of grapevines through collaboration with commercial diagnostic laboratories
Eutypa dieback (ED) and Botryosphaeria dieback (BD) are important grapevine trunk diseases causing dieback, cankers and eventually death of vines. These diseases can cause severe economic loss and have the potential to affect individual growers, regional communities and the industry as a whole if not managed effectively.
Diagnostic laboratories in Australia rely primarily on fungal isolations and PCR-based techniques for ED and BD diagnostics. These methods are generally time-consuming, labour intensive or require highly skilled staff to perform the analysis. To overcome these limitations, real-time loop-mediated isothermal amplification (LAMP) assays were recently developed for rapid, economical and sensitive
detection of ED and BD pathogens. These real-time LAMP assays were highly specific and suitable for detecting and discriminating different ED and BD pathogens from infected plant materials. A low cost and simple DNA extraction developed for LAMP was also found suitable for rapid DNA extraction from infected wood. These rapid DNA-based diagnostic tools, with high sensitivity, present an opportunity to diagnose and monitor infection in vineyards and nurseries in Australia. An objective of our recently approved Wine Australia-funded project is to connect with diagnostic laboratories such as those at the South Australian Research and Development Institute (SARDI) and the New South Wales Department of Primary Industries (NSW DPI) for the validation of the rapid DNA extraction and LAMP assay protocols. Collaboration with these diagnostic laboratories will lead to the transfer of knowledge and technologies, and implementation of national protocols for commercial diagnostics while allowing countrywide testing of ED and BD pathogens.
Forest biosecurity and surveillance
The National Forest Pest Surveillance Program was launched in 2022 and aims to provide a national early detection surveillance system to protect our commercial, urban and native forests from exotic pests. The program operates in capital cities at locations identified as posing a high risk for pest entry and establishment into Australia. Complementing this program are other biosecurity and surveillance projects. These include a project Developing a National Action Plan for Pests of Timber and Trees (which aims to improve Australia’s ability to prevent, detect, and respond to National Priority Plant Pests that affect trees and timber) and a project working with indigenous forestry enterprises to boost biosecurity awareness and improve biosecurity practices to protect community-managed forestry businesses from exotic pests and diseases.
Together, these projects aim to improve biosecurity and surveillance outcomes for all forest stakeholders.
Saidi R. Achari
Diagnosis of Fusarium oxysporum f. sp. ciceris causing Fusarium wilt of chickpea using Loop-Mediated Isothermal Amplification (LAMP) and conventional end-point PCR
Fusarium oxysporum (Fo) is ubiquitous in soil and forms a species complex of pathogenic and putatively non-pathogenic strains. Pathogenic strains cause disease in over 150 plant species. Fusarium oxysporum f. sp. ciceris (Foc) is a major fungal pathogen causing Fusarium wilt in chickpeas (Cicer arietinum). In some countries such as Australia, Foc is a high-priority pest of biosecurity concern. Specific, sensitive, robust and rapid diagnostic assays serve as an effective biosecurity control measure and are essential for effective disease management on the farm. We developed and validated a novel and highly specific PCR and a LAMP assay for detecting the Indian Foc race 1 based on a putative effector gene uniquely present in its genome. These assays were assessed against 39 Fo formae speciales and found to be specific, only amplifying the target species, in a portable real-time fluorometer (Genie III) and qPCR machine in under 13 minutes with an anneal derivative temperature ranging from 87.7 – 88.3°C. The LAMP assay is sensitive to low levels of target DNA (>0.009 ng/µl). The expected PCR product size is 143 bp. The LAMP assay developed in this study was simple, fast, sensitive and specific and could be explored for other Foc races due to the uniqueness of this marker to the Foc genome.
Development of a diagnostic assay for detection of Colletotrichum higginsianum in vegetable seeds.
To safeguard Australia’s environment and plant production systems, and due to the dynamic nature of the risks associated with imported seeds, DAFF’s 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 (ALOP). Collectively with PSaRA, we are developing and validating assays that are fit for purpose to detect quarantine, seed associated pests. Here we report development of a novel qPCR assay to detect C. higginsianum in Brassica rapa and Raphanus sativus seeds. The specificity of the assay was determined by inclusion of C. higginsianum genomic DNA and by exclusion of genomic DNA from live cultures of those non-quarantine Colletotrichum species that have shared hosts with the target pathogen. Through multiple stringent test validation processes, we have demonstrated our assay to be fit-for-purpose and highly sensitive in detecting C. higginsianum in brassicaceous vegetable seeds.
The potential impact of molecular diagnostics on biosecurity planning
Biosecurity planning provides the opportunity to assess the potential risk posed by exotic pests (invertebrate pests and pathogens) to both our environment and plant- based industries.
Biosecurity planning processes are facilitated by PHA with support from government and industry in a collaborative and positive environment. The meetings where the risks posed by exotic pests are discussed are a rich blend of science, production and industry knowledge.
The development of biosecurity plans rely on the knowledge and skills of researchers and biosecurity specialists from a range of organisations, including State and Territory jurisdictions, Universities and CSIRO to provide input determining the threats posed by exotic pests.
Collectively we often face a range of challenges in the development biosecurity plans, including:
Now we are faced with a new challenge!
How do we manage the interface between molecular diagnostics, biology and risk assessment?
While there is great progress in our ability to rapidly identify pests, what are the implications for assessing pest threats and the broader implications for our industries.
In our presentation we will explore some of these issues and consider options for addressing these challenges.
Murray Sharman and Peter Vukovic
Development of Rapid Tests for a range of important diseases to support surveillance, research and save time in lab.
Endemic plant diseases can cause yield and economic losses in many Australia crops. Exotic diseases also represent significant biosecurity risks. Methods for the rapid diagnostic testing of both in- and near-field, and central laboratory assays are becoming more important to enable quick responses to clients, support timely disease management and early detection of exotic incursions.
We have developed rapid diagnostics for endemic diseases: cotton bunchy top virus 1 and 2 (CBTV-1, -2), Eutypella, and Fusarium affecting cotton, cucumber mosaic virus (CMV), alfalfa mosaic virus (AMV), tobacco streak virus (TSV), Johnson grass mosaic virus (JGMV), pelargonium zonate spot virus (PZSV), barley stripe mosaic virus (BSMV) and various Phytoplasmas. We have also developed diagnostics for the exotic cotton leafroll dwarf virus (CLRDV) for incursion preparedness.
Assay development for these targets has involved a range of new Loop-mediated isothermal amplification (LAMP), qPCR or standard PCR with High-Resolution Melt (HRM) analyses. Although there are pros and cons and applicability of each of these assay formats, LAMP assays using crude plant extracts as template is proving to be very useful to enable rapid and easy detection of a range of virus and fungal pathogens. These assays have potential to save a lot of time in the lab when receiving small batches of diagnostic samples and also to support epidemiology studies in the field. We aim to have a tool kit of common diagnostic pathogens to streamline lab diagnostics and reduce turnaround time for clients. This will also support rapid decision making for disease management, and intervention strategies for both endemic and exotic diseases.
Implementing policy into practice: Diagnostic development and validation of a novel Diaporthe angelicae molecular assay.
How to identify, create, and timely implement fit-for-purpose biosecurity measures effectively and confidently is a key goal of regulators. Here we present the story that connects policy and research in support of the implementation of a novel molecular assay as a measure required to maintain Australia’s Appropriate Level of Protection (ALOP).
We developed and validated a novel qPCR molecular assay to the seed-borne Ascomycota Diaporthe angelicae. This is a case study on creation of a robust development and validation process, including the
use of imported viable quarantine pathogens – to efficiently create and implement measures suitable to stakeholders needs, delivering high confidence in assay detection efficacy.
This work sets a practical benchmark for future diagnostic development and validation workflows that are outcome focused to deliver science-based policy into effective measures.
Not one problem, but two?: how rigorous taxonomy and population genetics has revealed possible double trouble in the field with ‘Rutherglen bug’.
When we commenced our study for the Grains Research and Development Corporation in July 2021 (to run until June 2024), our original objective was to conduct research to improve the management of Rutherglen bug (Nysius vinitor) particularly in the Northern Region. This pest can be a problem particularly in summer grains as it builds up high numbers during the warmer months, but little is known about what drives its sudden but sporadic appearances. Further complexity results from the presence of Grey Cluster Bug (Nysius caledoniae), a taxonomically similar but genetically distinct native species, that coexist with N. vinitor in the northern region’s pastures and cultivated landscapes.
A first step to better understanding and management of these two species has been to resolve issues in taxonomy that we identified. We noted how difficult these species are to tell apart in the field (a microscope is required), and have refined the taxonomic ID of the two species which we present here (the nymph’s distinct features were not previously well documented in literature). Despite their visual similarity, using genetics we have found that N. caledoniae and N. vinitor are very different from each other and are not each other’s closest relatives.
The Barcode of life database is a system designed so that people can identify organisms using DNA sequence data – samples that we suspected of being N. vinitor in that database had been labelled N. clevelandensis. We have confirmed taxonomy in the database to rectify this, so people can now use DNA to identify these bugs. Initial results from a population genetic analysis suggests that N. vinitor are a single and well mixed population across Northern NSW – in line with expectations of a highly mobile pest species. The genome of N. vinitor has now been sequenced which will benefit future research.
“An Ontology and Vocabulary for Biosecurity Diagnostics, Created and Maintained online by a Community of Practice”
We are developing a Vocabulary (a set of definitions for terms and for values within a term) and an Ontology (a precise, machine readable, description of how terms are connected) for Plant Health (extensible to Animal Health) diagnostics. Together, they will form a set of diagnostics data standards. Owners of IT systems such as the Australian Plant Pest Database, could use these standards to ensure that they are recording attributes consistently with other systems and with agreed protocols.
It would be futile to attempt to discern the correct form for the Vocabulary and then dictate it to subject matter experts. We have adopted an approach pioneered by the Department of Environment (DCCEW), in which a public, online version control system (such as github.com) is used which allows individuals to create their own ‘branch’ and add to it or change it according to their views, before submitting it for review and merging into the mainline published version. In addition, the system allows the group to document errors or suggested improvements and for them to be resolved and recorded into a ‘living’ document that supports the Ontology itself. This approach also lends itself to endorsement by governing bodies such as Plant Health Committee. A vocabulary has already been developed as part of the Plant Health Surveillance (PHS) Ontology (linked.data.gov.au/def/phs). Lists of values for inspection method, trap type, lure type, among others, are included within the Pest Record Specification, which is part of PHS, and has been endorsed by Plant Health Committee. The vocabulary is also included within the reference standards for National Surveillance Protocols overseen by the Subcommittee on National Plant Health Surveillance.The Biosecurity Diagnostics Ontology will align and complement the PHS Ontology. Furthermore, the Community of Practice could be extended to maintain the PHS. We welcome any and all contributors and contributions, no matter how small.
Towards field deploying future CRISPR/Cas based diagnostic assays for simultaneous detection of multiple plant pathogens: Citrus as a case study
Rapid, efficient, sensitive, and easy to use portable diagnostic tests for plant pathogens are necessary to identify potentially threatening diseases for surveillance, inspections, evidence of area-freedom, containment, asset protection, and to manage responses to post-border incursions. The technology employing the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease Cas systems such as Cas12 (target dsDNA) and Cas13 (target RNA) associated to reverse transcription (RT) and isothermal nucleic acid amplification system such as recombinase polymerase amplification (RPA) can specifically detect exceptionally low levels of pathogen DNA or RNA. The resulting collateral activities of CRISPR/Cas can be visualised in the laboratory using fluorescence readers and in the field with portable platforms.
In the perspective of field deploying CRISPR/Cas based diagnostic assays for simultaneous detection of multiple pathogens per plant commodity (e.g., citrus), we have started developing multiple individual CRISPR/Cas assays based on literature and Australia’s National Priority Plant Pests list. Here I will present an update on the development of CRISPR/Cas12a diagnostic assays for multiple DNA pathogens in citrus as a proof of concept with i) citrus greening disease, or Huanglongbing, caused by Candidatus liberibacter asiaticus, ii) Xylella spp and iii) citrus bacterial canker caused by Xanthomonas citri pv citri. In a one-pot (OP) reaction, i.e., single tube opening, combining CRISPR/Cas12a and RPA assays, the tests can detect synthetic DNA molecules down to femtomolar concentrations. Cross-reactivity between the assays with potential host genomic DNA were assessed. Several ways to increase the level of fluorescence signal during the OP-RPA-CRISPR/Cas12 assays have been investigated. Preliminary OP-RT-RPA-CRISPR/Cas12 assay results involving RNA pathogens, citrus vein enation virus and citrus tristeza virus, will be presented and discussed in the context of multiple pathogen detection and field deployment.
Northern Australia Quarantine Strategy (NAQS) surveillance model: connecting surveillance and diagnostic practitioners. The Department of Agriculture, Fisheries and Forestry’s Northern Australia Quarantine Strategy (NAQS) is an early detection surveillance program operating from Broome in Western Australia to Cairns in far north Queensland. The team has a dedicated workforce comprising of Botanists, Entomologists and Plant Pathologists whose core activities are to scientifically monitor/detect the presence of exotic pests, weeds and diseases in northern Australia as well as playing an integral role in offshore surveillance in neighbouring countries. In operation since the early 1990’s, NAQS has one of the best examples in the country of an integrated surveillance delivery model where these scientific staff perform both surveillance and diagnostic functions. Maintaining this scientific capacity has advantages and disadvantages which must be balanced to meet early detection objectives while still encouraging a culture of investigation if unusual symptoms or damage are encountered in the field. This ‘investigative design’ approach accounts for about half of the significant detections reported by NAQS as well as supports staff to continue to develop their skills. Advantages of the integrated surveillance and diagnostic capability model include improved sampling methods, more effective field identification of exotic pests, enhanced implementation of in-field diagnostics/point of care testing and retention of key capabilities in an environment of declining biosecurity specialists. Disadvantages include balancing staffing resources between in-field activities and the necessity to provide diagnostics for early detection, particularly where poorly characterised or complex/cryptic taxa can increase the time taken for diagnostic investigations.
Early detection of Varroa mites by eDNA surveillance
Varroa mites (Mesostigmata: Varroidae) are external parasites of Apis cerana and Apis mellifera (Hymenoptera: Apidae), linked to the worldwide decline in honeybee health. Morphological identification of varroa mites can be challenging due to the small size of these parasites, as well as the limited taxonomical skills available in the field. For instance, early detections can prove challenging if relying on obtaining DNA from varroa specimens, given that the collection of mites in the field may be linked with an advanced level of infestation. Molecular diagnostic tools such as eDNA-based analyses can provide sensitive and accurate identifications of varroa mites, matching the novel DNA sequences to those available in public databases, offering a non-invasive complementary surveillance method to improve varroa mite detection, which are often limited by low sensitivity at low levels of infestation. A PSNAP Surveillance Residential Program aimed to better understand the limits of detection of environmental DNA (eDNA) techniques, focusing on swab and honey non-invasive collection methods suitable to determine the presence/absence of Varroa destructor during the early stages of an incursion. The project, based in New Zealand, focused on the deployment of naïve beehives (not previously exposed to or treated for V. destructor) in a Varroa-infested area to examine collection methods and assay sensitivity by assessing detection probability of V. destructor in naïve hives over time. Furthermore, different environmental collection methodologies were tested to examine assay limits of detection and reproducibility by collecting eDNA from randomly selected commercial hives displaying low-high V. destructor. The results obtained in this program enabled a better understanding not only of in-field diagnostic techniques and eDNA methodologies, but also about the time course of Varroa incursions. Such insights may prove essential in the context of exotic pest incursions and early-detection diagnostics.
Capacity building at the Powdery Mildew Workshop in 2022
The Powdery Mildew Capability Building Workshop was held at the University of Southern Queensland (UniSQ), Toowoomba, from 14-18 November 2022, with funding received from the Department of Agriculture, Fisheries and Forestry (DAFF). It was prompted by an increase in the number of powdery mildew interceptions during national border surveillance, border inspections and the Northern Australia Quarantine Strategy (NAQS). It has also supported the surveillance and the diagnostic expertise in state department and industry programs that focus on the recognition, collection and identification of fungal plant pathogens that impact Australian agriculture. The workshop consisted of lectures, hands-on laboratory practicals, a Geneious training, group discussions, and a field day, and has led to the precise molecular and morphological identification of 40+ powdery mildew species representing seven genera. The results will be summarized in a multi-authored paper to be published in an international plant pathology journal. Altogether, 26 participants from AgVIC, DAFF, DPIRD, QDAF, the Department of Agriculture in Timor Leste, and UniSQ attended the workshop and a few more colleagues contributed to the results with specimens. The 4 min speed talk will highlight the capacity building and networking opportunities offered by the workshop.
Advancing diagnostic resolution by rapid validation of fungal avirulence genes detected by the wheat immune system
Wheat rust fungi can cause severe yield reduction in affected crops, reducing agricultural productivity, increasing costs and threatening global food security. In Australia, new diagnostic challenges have arisen due to multiple wheat rust fungi lineages occurring during a growing season. An advanced track-and-trace system for wheat rusts could give fast and accurate diagnostics, enabling rapid response to emergent lineages through deployment of strain-specific disease resistant wheat cultivars. To effectively implement a track-and-trace system, genes which are recognised by the host immune system, known as avirulence (Avr) genes, must first be identified.
Our project aims to identify these pathogen Avr genes, connecting computational genomics approaches with biological validation in planta and ultimately through to implementation by routine profiling of rust fungi strains using standard diagnostic procedures. Here I will present recent progress in developing a tool for rapid testing of Avr genes recognized by the wheat immune system. Our validation tool uses transient transfection in wheat leaf protoplasts, with bioluminescent quantitation of defence-activated reporters indicating host recognition of a pathogen Avr. Crucially, this method does not require the overexpression of host resistance genes, giving this tool a wide range of host resistance genotypes for screening. We have demonstrated the utility of this approach across multiple wheat cultivars and through batch-screening of up to ten Avr gene candidates simultaneously. With this platform we hope to speed up the identification of critical pathogen avirulence genes, advancing diagnostic resolution for monitoring of cereal rust diseases.
Chris Nellessen, Brad Pease, David Nehl
MALDI-ToF mass spectrometry: an additional molecular tool for biosecurity diagnostics
Matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) is a molecular diagnostic technique that measures the mass and charge of ribosomal proteins, which occur in all cellular organisms and vary uniquely between species. MALDI-TOF MS generates spectra from test specimens and compares them against libraries of validated, reference spectra (‘fingerprints’), providing a nearest-match identification to species that parallels nucleic acid sequencing, at a fraction of the consumable costs and time. MALDI-TOF MS is a mature technology, deployed globally for bacterial identification in mammalian disease diagnostics and food testing. We used a MALDI Biotyper® sirius (Bruker Pty Ltd) to confirm the potential for expanding the application of MALDI-ToF MS to diagnostics in plant biosecurity. The technology proved itself immediately valuable for identification of bacteria and fungi included in the fingerprint libraries supplied with the instrument by Bruker, providing indications of likely genera or species early in the process of isolating pure cultures, and confident identification of species, once isolated. If a species was not included in Bruker’s libraries the genus was usually indicated confidently. We easily generated our own reference fingerprints, with verification of the species by DNA sequencing. We also resolved confident identifications of mosquitoes to genus or species using open-source fingerprints. Using a simple, cost-efficient, single-step test method (Reeve et al. 2018), we generated diagnostic-quality fingerprints from a broad range of organism groups, including insects, mites, spiders, molluscs, leeches, and plants. The addition of MALDI-ToF MS as a tool in diagnostic workflows for these organisms will be best served by initially generating reference fingerprints for those groups most-frequently encountered in routine biosecurity intervention and surveillance. As with any diagnostic technology, verification diagnostics should be applied to inform biosecurity measures and responses, especially for high-priority, emergency plant pests. National protocols for validation of fingerprints produced from verified specimens, interpretation of results, and approaches for library sharing between agencies, will greatly enhance the acceptance and deployment of this valuable molecular tool for diagnostics in biosecurity.
Reducing the threat of banana bunchy top disease through development of a lateral flow assay
Banana bunchy top virus (BBTV) causes the most destructive viral disease of banana (Musa sp.) worldwide and can cause near-complete crop loss if unmanaged. BBTV is present in most banana producing regions of the world except for the Americas. In Australia, BBTV is absent from our major north Queensland production area. However, the Pacific subgroup of BBTV occurs in the subtropical production area. Both this endemic subgroup and the exotic Asian subgroup pose major biosecurity threats to the industry. With no resistant cultivars, disease control relies on quarantine, eradication of infected plants and use of clean planting material. While symptoms are usually sufficient for destruction of infected field plants, robust and low-cost diagnostic assays are crucial for preventing BBTV spread through planting material.
Previously developed assays relied on BBTV-detecting antibodies, which reached critically short supply in 2015. Recently, a new suite of anti-BBTV antibodies have been prepared. These have been extensively used by industry-funded investigations to improve understanding of disease epidemiology and thereby control. These antibodies were used to develop the first lateral flow assay for BBTV. This has been field-tested, against both Asian and Pacific isolates from the Australian reference collection, and by international collaborators. They have the potential to dramatically reduce the spread of BBTV when used at the point of distribution of planting material and support surveillance programs when symptoms are unclear or verification is required.
The Australian control strategy has been based on Musa sp. as the only confirmed hosts of BBTV. Recently, however, a BBTV variant that also infects flowering ginger has been detected in French Polynesia and there are reports of field infection of canna (Philippines), heliconia (Hawaii, USA) and ginger (Indonesia). Collaboration with South Pacific pathology and biosecurity colleagues is needed, with a major role for these diagnostic assays, to safeguard banana production.
Introducing Biosecurity Commons – the decision-support platform set to transform Australia’s biosecurity modelling capability
Biosecurity risks increase as global trade increases and becomes more complicated to assess and manage. We must invest in technologies that build capability to make better decisions and help address emerging biosecurity risk drivers such as climate change. Biosecurity Commons is a ground-breaking project delivering a cloud-based decision-support platform for modelling and analysing biosecurity risk and response. The platform empowers researchers and decision-makers to produce consistent and transparent models and analytics to answer specific biosecurity questions, without the need to have computer programming skills or high-end workstations. Instead, users have access to a user-friendly web- based interface with a standard suite of tools, data and resources to perform complex analyses and collaboratively solve common biosecurity problems. For the first time, the platform will enable users from different organisations, sectors, and jurisdictions to share project work and collaborate on inputs, parameters and results necessary to improve research and accelerate biosecurity decision-making outcomes for the future. This element of the project aligns very closely with the ‘Implementation through connections’ theme of the workshop as we aim to connect the biosecurity community through the platform. The workflows developed by Biosecurity Commons will inform and enhance Australia’s risk-based approach to biosecurity risk management. For example, the Early Detection Surveillance Design workflow will help decision makers to target locations where invasive species are more likely to arrive, establish, and/or pose the greatest social, environmental, or economic costs.We look forward to introducing the project and demonstrating the capability of the platform at the event. Join our session to learn how Biosecurity Commons will transform the future of Australia’s biosecurity modelling capability.