The document discusses the development and deployment of genome-informed diagnostic protocols for plant pathogenic bacteria by the Plant Biosecurity Cooperative Research Centre (PBCRC). The PBCRC has developed and validated laboratory and field diagnostic protocols to discriminate bacteria at the pathovar level using genome sequencing and bioinformatics. It has also trained scientists in plant bacteriology and engaged end-users in field testing and validation of new diagnostic technologies and protocols.
Invasive pests and pathogens can have devastating and unpredicted impacts on native ecosystems. The threat that Puccinia psidii (myrtle/eucalyptus/guava rust) posed to Australian industries was well recognised, but until its introduction in 2010, there was scant consideration of the impacts this disease may have on endemic Myrtaceous plant species and associated communities in native environments. Since its detection in Australia, the distribution and host range of P. psidii has rapidly expanded and entire species and plant communities are now under threat.
The research being undertaken into myrtle rust has enabled improved species selection for production and retail nurseries, and for urban tree planting.
The Australia-Africa Plant Biosecurity Partnership has brought together plant biosecurity professionals in ten African countries and established linkages with Australian researchers, helping
to reduce pest and disease impacts in sub-Saharan Africa. At the outset of this initiative, diagnostic skills were identified as a priority area in connecting Australian expertise with Africa and improving surveillance capability, post-entry quarantine, early warning and phytosanitary certification. This presentation will briefly examine the application of improved diagnostic skills in African Plant Protection Organisations and the longer term relationships that have been established with Australian mentors.
Long distance natural (wind-assisted) dispersal of exotic plant pests and pathogens into Australia, is a very real and underestimated, biosecurity risk.
Planning and decision making to manage plant biosecurity risks is inherently complex, often contentious, involves unknowns and uncertainty, and needs to be adaptable to rapidly changing situations. The aim of this project is to develop a collaborative planning and shared decision making
framework that will result in better and faster decisions to respond more quickly to plant biosecurity risks, resulting in reduced impacts and costs, and more equitable and favourable outcomes for stakeholders and affected parties.
Here we update on fundamental systematics research and the development of new potential molecular markers to improve on current diagnostic tools. We also link these molecular tools with physical specimens, documenting the range of morphological variation so as to greatly improve on available resources used to diagnose fruit flies in the field as part of surveillance programmes or at border interceptions.
Invasive pests and pathogens can have devastating and unpredicted impacts on native ecosystems. The threat that Puccinia psidii (myrtle/eucalyptus/guava rust) posed to Australian industries was well recognised, but until its introduction in 2010, there was scant consideration of the impacts this disease may have on endemic Myrtaceous plant species and associated communities in native environments. Since its detection in Australia, the distribution and host range of P. psidii has rapidly expanded and entire species and plant communities are now under threat.
The research being undertaken into myrtle rust has enabled improved species selection for production and retail nurseries, and for urban tree planting.
The Australia-Africa Plant Biosecurity Partnership has brought together plant biosecurity professionals in ten African countries and established linkages with Australian researchers, helping
to reduce pest and disease impacts in sub-Saharan Africa. At the outset of this initiative, diagnostic skills were identified as a priority area in connecting Australian expertise with Africa and improving surveillance capability, post-entry quarantine, early warning and phytosanitary certification. This presentation will briefly examine the application of improved diagnostic skills in African Plant Protection Organisations and the longer term relationships that have been established with Australian mentors.
Long distance natural (wind-assisted) dispersal of exotic plant pests and pathogens into Australia, is a very real and underestimated, biosecurity risk.
Planning and decision making to manage plant biosecurity risks is inherently complex, often contentious, involves unknowns and uncertainty, and needs to be adaptable to rapidly changing situations. The aim of this project is to develop a collaborative planning and shared decision making
framework that will result in better and faster decisions to respond more quickly to plant biosecurity risks, resulting in reduced impacts and costs, and more equitable and favourable outcomes for stakeholders and affected parties.
Here we update on fundamental systematics research and the development of new potential molecular markers to improve on current diagnostic tools. We also link these molecular tools with physical specimens, documenting the range of morphological variation so as to greatly improve on available resources used to diagnose fruit flies in the field as part of surveillance programmes or at border interceptions.
Myrtle rust (Puccinia psidii) is an invasive fungus native to South America that was first detected in Australia in 2010. It has spread rapidly along the east coast, and is currently recorded in sites as far north as the Northern Territory and south to Tasmania.
In order to better understand myrtle rust extent and impacts in Australian native and managed landscapes, a survey was sent to national parks, botanical gardens, councils, natural resource
managers, nurseries and forestry agencies in all states where the fungus is present (NT, QLD, NSW, VIC and TAS). The survey revealed that Myrtle rust is widespread in NSW and QLD gardens and streets as well as in native vegetation.
The disruption to trade caused by quarantine pests and diseases is a significant issue for Australian and New Zealand horticulture. There is an urgent need to review and improve the current disinfestation methods to develop new export protocols that are effective, economic and safe to use.
The project team consisting of researchers from across Australia and New Zealand, has reviewed all the published and unpublished phytosanitary research conducted against these pests.
The aim of this project is to develop multispecies trapping strategies for the stored grain beetles Rhyzopertha dominica, Cryptolestes ferrugineus, Tribolium castaneum, Sitophilus oryzae (all established in Australia) and Prostephanus truncatus (not present in Australia) in outdoor environments.
An optimal surveillance system is one where the available sampling resources are allocated in time and space to best achieve detection objectives. This project aims to develop new methods for designing optimal surveillance systems that properly account for organism biology, trapping or sampling efficacy, and landscape characteristics.
The scale of the Australia’s grains industry means that monitoring for incursions of pests is a costly and challenging activity. This project utilises advanced technologies for surveillance of grains pests in the field, including smart spore and insect traps, and use of image sensors.
This project has developed new ‘Smart’ Spore and Insect Trapping systems for target(s) surveillance referenced to GPS and climate data (temp, wind direction, RH), or wireless data transmission for improved compatibility to rapid and accurate downstream diagnostics.
Three years of research to date have produced a robust, accurate, sensitive detection tool and sampling strategy for the damaging apid-like insect phylloxera (Daktulosphaira vitifoliae), which feeds on grapevine roots
The aim of this research project is to establish Australian developed seed testing protocols as an international standard for the detection of viroids and cucumber green mottle mosaic virus (CGMMV) in seed, and to reduce the risks of contaminated traded seed.
Research presented in this session will explore some of our innovative research to improve pest management and help maintain and build market access for our grains industries.
There are many facets involved in the development of biogeochemical markers that might enable the geographic origins of fruit flies to be distinguished.
The risk myrtle rust poses to threatened Myrtaceae species in Australia is becoming more apparent with significant dieback and tree death recorded as a result of repeated infection.
Research presented in this session addresses the need to better understand the impacts of pests and diseases on the environment and the various ways that social science and the rules of community engagement can be applied for better biosecurity.
Up until mid-2016, citizen science uploads to the Atlas of Living Australia (ALA) included c. 400 bug species, and c. 1,000 beetle species. Given the short time period (c. 3 years) over which most of these records have accumulated, this represents a considerable reporting effort. The key question from a plant biosecurity context is how this level of reporting translates to the detection and reporting of
exotic insect pests in the event of an incursion.
Myrtle rust (Puccinia psidii) is an invasive fungus native to South America that was first detected in Australia in 2010. It has spread rapidly along the east coast, and is currently recorded in sites as far north as the Northern Territory and south to Tasmania.
In order to better understand myrtle rust extent and impacts in Australian native and managed landscapes, a survey was sent to national parks, botanical gardens, councils, natural resource
managers, nurseries and forestry agencies in all states where the fungus is present (NT, QLD, NSW, VIC and TAS). The survey revealed that Myrtle rust is widespread in NSW and QLD gardens and streets as well as in native vegetation.
The disruption to trade caused by quarantine pests and diseases is a significant issue for Australian and New Zealand horticulture. There is an urgent need to review and improve the current disinfestation methods to develop new export protocols that are effective, economic and safe to use.
The project team consisting of researchers from across Australia and New Zealand, has reviewed all the published and unpublished phytosanitary research conducted against these pests.
The aim of this project is to develop multispecies trapping strategies for the stored grain beetles Rhyzopertha dominica, Cryptolestes ferrugineus, Tribolium castaneum, Sitophilus oryzae (all established in Australia) and Prostephanus truncatus (not present in Australia) in outdoor environments.
An optimal surveillance system is one where the available sampling resources are allocated in time and space to best achieve detection objectives. This project aims to develop new methods for designing optimal surveillance systems that properly account for organism biology, trapping or sampling efficacy, and landscape characteristics.
The scale of the Australia’s grains industry means that monitoring for incursions of pests is a costly and challenging activity. This project utilises advanced technologies for surveillance of grains pests in the field, including smart spore and insect traps, and use of image sensors.
This project has developed new ‘Smart’ Spore and Insect Trapping systems for target(s) surveillance referenced to GPS and climate data (temp, wind direction, RH), or wireless data transmission for improved compatibility to rapid and accurate downstream diagnostics.
Three years of research to date have produced a robust, accurate, sensitive detection tool and sampling strategy for the damaging apid-like insect phylloxera (Daktulosphaira vitifoliae), which feeds on grapevine roots
The aim of this research project is to establish Australian developed seed testing protocols as an international standard for the detection of viroids and cucumber green mottle mosaic virus (CGMMV) in seed, and to reduce the risks of contaminated traded seed.
Research presented in this session will explore some of our innovative research to improve pest management and help maintain and build market access for our grains industries.
There are many facets involved in the development of biogeochemical markers that might enable the geographic origins of fruit flies to be distinguished.
The risk myrtle rust poses to threatened Myrtaceae species in Australia is becoming more apparent with significant dieback and tree death recorded as a result of repeated infection.
Research presented in this session addresses the need to better understand the impacts of pests and diseases on the environment and the various ways that social science and the rules of community engagement can be applied for better biosecurity.
Up until mid-2016, citizen science uploads to the Atlas of Living Australia (ALA) included c. 400 bug species, and c. 1,000 beetle species. Given the short time period (c. 3 years) over which most of these records have accumulated, this represents a considerable reporting effort. The key question from a plant biosecurity context is how this level of reporting translates to the detection and reporting of
exotic insect pests in the event of an incursion.
Evaluation of resistance profile of pseudomonas aeruginosa with reference to ...iosrjce
IOSR Journal of Dental and Medical Sciences is one of the speciality Journal in Dental Science and Medical Science published by International Organization of Scientific Research (IOSR). The Journal publishes papers of the highest scientific merit and widest possible scope work in all areas related to medical and dental science. The Journal welcome review articles, leading medical and clinical research articles, technical notes, case reports and others.
Hola Amigos Nuevamente les tengo Otro Power Point De Pseudomonas mas especifica en P.Aeruginosas Espero lo Difruten
Andrea Apolinario
Estudiante de 4to año Medicina
Yersinia y Pseudomonas, bacterias gram negativas, en la siguiente presentación les enseñaremos su definición, categorías, su clasificación, causas de contagio, reproducción, características, hábitat, epidemiología, cuadro clínico y su tratamiento.
Research investigating the use a genome-informed approach to develop diagnostic tools, for the detection of exotic phytopathogenic bacteria that pose a threat to Australian agriculture.
Quarantine regulation and impact of modern detection methods 2 [repaired]N.H. Shankar Reddy
impormation regarding quarantine and advanced equipment are using in plant quarantine to detect pathogens and detail functioning of quarantine and history behind quarantine initiation in India
24x7 Automated Behavior Tracking For Rodent Safety Pharmacology & PhenotypingInsideScientific
Actual Analytics Ltd and its development partners present an exclusive webinar describing the applications of a novel Home Cage Analysis system for tracking behavior in group housed rodents, with retained identity, in regular IVC racked home cages.
In this webinar, Dr. Will Redfern of AstraZeneca and Dr. Sara Wells of MRC Harwell discuss 24/7 monitoring of group-housed rodents in their true IVC racked home cage environment for safety pharmacology and phenotyping applications.
Discussions describe the types of new insights that can be obtained from 24/7 monitoring of research animals including activity differences in single and group animals and body temperature profiles in response to drug treatment. Presenters show how they are using this system in various applications from safety pharmacology in rats through to phenotyping studies in mice.
Systems Biology and Genomics of Microbial PathogensRamy K. Aziz
Talk at SCITA-BIOFANS (02 Feb 2016), entitled
"Systems Biology and Genomics of Microbial Pathogens:
From virulence gene discovery to vaccine development and therapeutic intervention"
Tina Joshi presentation on "Development of a Real Time Microwave-Enhanced Rap...Matthew Kirkby
Tina Joshi presentation on "Development of a Real Time Microwave-Enhanced Rapid (~5 minutes) Detection Assay for Bacillus anthracis" at Biology of Anthrax, Tampa 2016
Dr. Paul Sundberg - Setting priorities for disease threats and strategiesJohn Blue
Setting priorities for disease threats and strategies - Dr. Paul Sundberg, Swine Health Information Center, from the 2017 North American PRRS/National Swine Improvement Federation Joint Meeting, December 1‐3, 2017, Chicago, Illinois, USA.
More presentations at http://www.swinecast.com/2017-north-american-prrs-nsif-joint-meeting
Similar to Session 2: Genome-Informed Diagnostics - In-field Detection of Bacterial Plant Pathogens (20)
The diagnosis of viral pathogens is a crucial component of plant biosecurity surveillance and preventing the introduction of exotic plant viruses and viroids at the border. Existing quarantine procedures can be time-consuming and require detailed knowledge of potential infecting viral pathogens. Currently, imported plants can spend as long as two years in quarantine, with associated costs.
To simplify the post-entry quarantine process researchers have developed a plant diagnostic toolkit for plant viruses and viroids. The toolkit takes advantage of the natural antiviral system of plants, using small RNA next generation sequencing (sRNA-seq) technology to detect nearly all known viruses and viroids in a single test. The new test, and associated toolkit, will reduce the time imported plant material spends in Australia’s quarantine system while improving accuracy of detection in a single sRNA-seq experiment.
This research has developed recommendations for stakeholders involved in area-wide management of fruit fly, including social and institutional requirements.
This project aims to build the ability of indigenous communities (Maori and Aboriginal), regulatory authorities and industries to better manage the impact of biosecurity threats. Models have been developed for Indigenous engagement.
This social biosecurity project, aims to improve plant biosecurity management by developing the capacity of regional and remote communities to engage in biosecurity surveillance activities.
Surveillance systems are an essential component of biosecurity. Design of biosecurity surveillance systems may include designs of grids of static traps, plans for field sampling, or deployment of potentially "game-changing" mobile trap technology. The aim of these systems is to achieve defined detection objectives, (e.g. early detection, supporting area-freedom status) at minimum cost. This project will develop and apply statistically-based surveillance systems that account for organism biology, trap behaviour and landscape characteristics.
Ships arriving in Australia may have visited multiple ports along the way. These complex pathways present opportunities for pest species, such as the Asian Gypsy Moth, to arrive into Australia from indirect routes. Understanding those pathways that link Australia directly or indirectly to countries in which a pest or disease occurs is necessary to identify arriving ships with the highest likelihood of carrying hitchhiker species. This project proposes to address three important questions:
1. What general shipping pathways pose the greatest risk?
2. How to make decisions regarding what ships to search?
3. How much inspection to conduct?
This research project is collecting data on past pest invasions in both Australia and New Zealand, in order to identify common patterns in plant biosecurity pests.
This research will investigate technologies to enable the development of spore traps capable of in-field detection, and identification, of specific biosecurity threats.
The spread of invasive species continues to provide significant challenges to those government biosecurity agencies charged with protecting a country’s borders. In an increasingly connected world, these invasive species are potentially able to spread further and more rapidly. Human mediated pathways such as ships and airlines are the most obvious ways in which invasive species can be spread. Direct routes from one port to another are currently monitored, but indirect pathways,
in which a ship picks up an invasive species and then travels to a number of different locations before arriving at the final destination, present more challenging scenarios. For the Australian Government Department of Agriculture, one particular concern is for ships arriving into Australia carrying viable eggs of the Asian gypsy moth (Lymantria dispar). We are developing a real time tool that will analyse the pathways for incoming ships and determine the likelihood the ship could be carrying viable eggs.
Biosecurity issues impact on key crops and environmental values across NZ and Australia. A key outcome for the project team will be the ability of indigenous communities, and relevant regulatory authorities and industries, to better manage the social, environmental and economic impacts of biosecurity threats, and to participate in biosecurity strategies through improved bicultural engagement models that build empowerment and ownership in indigenous communities and their response to those threats. The teams have developed an engagement model adapted to the indigenous peoples and their communities of each country.
The results of a baseline study on motivation and incentives involved in the decisions to control fruit fly highlight the variability of motivations within demographic groups.
Chlorine dioxide was tested in three pilot scale structures including the mini silo, the column, and the flat box against five stored-product insect pests, including adults of Rhyzopertha dominica (F.), Tribolium castaneum (Herbst), Oryzaephilus surinamensis (L.), Sitophilus zeamais Motschulsky, and
Sitophilus oryzae (L.).
The Global Eradication and Response Database (GERDA) summarises incursion response and eradication programmes from around the world. The purpose of GERDA is to determine which characteristics lead to the success or failure of eradication programmes.
Bacterial leaf spot (BLS) is a disease of tomato, chilli and capsicum caused by four species of Xanthomonas. BLS can cause severe crop damage and is a limiting factor of production across the world.
With the loss of chemical control options such as Fenthion and Dimethoate for postharvest treatment of horticulture commodities susceptible to fruit fly infestation, it has become even more important to understand how stress-based control techniques such as heat, cold, irradiation and bacteria parasite
can be used most effectively for disinfestation. This project aims to explore the stress-induced molecular response of two fruit fly species of horticultural significance, Mediterranean fruit fly (Ceratitis capitata) and Queensland fruit fly (Bactrocera tryoni), by characterising the cellular pathways involved in both overall and stressor-specific responses.
More from Plant Biosecurity Cooperative Research Centre (17)
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
1. biosecurity built on science
Plant Biosecurity Cooperative Research Centre
James Stack
Professor and Director
In-field Detection of Bacterial Plant Pathogens
Genome-Informed Diagnostics
2. biosecurity built on science
Plant Biosecurity Cooperative Research Centre
James Stack
Professor and Director
PBCRC2002 & PBCRC2156
• 2002: Develop & validate laboratory and field
diagnostic protocols for plant pathogenic bacteria to
the pathovar level of discrimination
• 2156: Deploy validated field diagnostic protocols for
plant pathogenic bacteria to the pathovar level of
discrimination
• Increase national capability in plant bacteriology
3. biosecurity built on science
Plant Biosecurity Cooperative Research Centre
James Stack
Professor and Director
PBCRC 2002: Genome-based, bioinformatics-informed
diagnostics
• Developed & validated multiple laboratory and field
diagnostic protocols for plant pathogenic bacteria to
the pathovar level of discrimination
• Validated Pan-Genome Pipeline
• Increased national capability in plant bacteriology:
9 scientists trained & mentored in plant bacteriology
(Australia, New Zealand, U.S.)
4. biosecurity built on science
Plant Biosecurity Cooperative Research Centre
James Stack
Professor and Director
Other 2002/2156 Team Presentations – Don’t miss:
• Sarah Thompson: Metagenomic discovery of differential
diagnostic loci in CLos
• Jacqui Morris: Microflora analyses of the Australian eggplant
psyllid
• Rachel Mann: Complex diagnostics – keeping up with Ralstonia
solanacearum
• Toni Chapman: Genome-informed diagnostics – Xanthomonas
citri subsp citri
• Rebecca Roach: Identification of Xanthomonas species causing
bacterial leaf spot in Australia
5. biosecurity built on science
Plant Biosecurity Cooperative Research Centre
James Stack
Professor and Director
PBCRC 2156: Field-deployable genome-based,
bioinformatics-informed diagnostic protocols
• Developed & validated multiple field-deployable
diagnostic protocols for plant pathogenic bacteria to
the pathovar level of discrimination
• Validated Concept-to-Practice Pipeline
• Engaged end-user communities in field testing and
validation of protocols: laboratory and in-field end-
user training in new technologies and protocols
6. biosecurity built on science
Plant Biosecurity Cooperative Research Centre
James Stack
Professor and Director
PBCRC 2002/2156: Diverse Bacterial targets
• Xanthomonas citri subsp. citri: Gram negative, citrus
pathogen
• Candidatus Liberibacter solanacearum: Unculturable
bacterium, arthropod vector, potato pathogen
• Ralstonia solanacearum: Gram negative, potato
pathogen (other hosts)
• Rathayibacter toxicus: Gram positive, nematode
vector, annual ryegrass (other hosts)
• Pseudomonas syringae pv. actinidiae: Gram negative
7. biosecurity built on science
Plant Biosecurity Cooperative Research Centre
James Stack
Professor and Director
Bacterial Pathovar Diagnostics Team:
Xanth Liberibact Pseudo Rathay Erwinia Total
Genomes
sequenced
80 10 2 10 102
Target #
80 30 70 39 50 269
New taxa
sequenced
12 2 2 16
Psylid
Mitogenomes
sequenced
14
(5 species)
14
170 20 4 70 31248
R. sol
200 214
8. biosecurity built on scienceScience Exchange - August 2016
• Who are you?
• Where did you come from?
• How did you get here?
• When did you get here?
• Have you been here before? (prior
entry)
• Are you travelling alone? (vector)
Outbreak response:
You can’t answer those questions from
symptoms alone
Plant Biosecurity: Smart Surveillance
Pictures: J. Stack lab
How much can we determine in the field?
What do we want to know?
Particularly difficult with bacterial pathogensCan genome-informed diagnostics help?
9. biosecurity built on science
Plant Biosecurity: Smart Surveillance
Pictures: J. Stack lab
Whoa Dude!
Look what I
found.
10. biosecurity built on scienceScience Exchange - August 2016
Plant Biosecurity: Smart Surveillance
• Who are you: What pathogen is this?
• For most bacterial pathogens, the level
of discrimination is at the sub-specific
level
• For biosecurity, we need to know the
race, strain, or even the population
What do we want to know?
Can be very difficult for bacterial
pathogens using traditional technologies –
Almost impossible in the field
11. biosecurity built on science
LAMP Workshop – La Trobe University
24 October 2016
Pictures: J. Stack lab
Discriminating peppers from cows
Plant Biosecurity: Smart Surveillance
No characteristics in common
No special training
required
12. biosecurity built on science
LAMP Workshop – La Trobe University
24 October 2016
Pictures: J. Stack lab
Discriminating sheep from cows
Plant Biosecurity: Smart Surveillance
Shape, 4 legs, head, tail, 2 eyes, 2 ears
No special equipment
required
13. biosecurity built on science
LAMP Workshop – La Trobe University
24 October 2016
Pictures: J. Stack lab
Discriminating cows from cows
Color, weight, height, markings
Many fewer discriminating characteristics
Plant Biosecurity: Smart Surveillance
14. biosecurity built on scienceScience Exchange - August 2016
Pictures: J. Stack lab
Many distinguishing features
Discriminating bacteria from fungi
Plant Biosecurity: Smart Surveillance
15. biosecurity built on scienceScience Exchange - August 2016
Plant Biosecurity: Smart Surveillance
Pictures: J. Stack lab
No distinguishing features
Discriminating bacteria from bacteria
Almost impossible in the field
16. biosecurity built on scienceScience Exchange - August 2016
Pictures: International Symposium
on Bacterial Canker of Kiwifruit
Plant Biosecurity: Smart Surveillance
Pseudomonas syringae
Pseudomonas syringae
Kiwifruit Pathogen
Non-Pathogenic
17. Pictures: International
Symposium on Bacterial
Canker of Kiwifruit
LAMP Workshop – La Trobe University
24 October 2016
Plant Biosecurity: Smart Surveillance
Why rapid is important
18. Pictures: International
Symposium on Bacterial
Canker of Kiwifruit
LAMP Workshop – La Trobe University
24 October 2016
Plant Biosecurity: Smart Surveillance
high
impact
Impact
threshold
low
impact
disease
detection
Critical response point
Time
Diseaseseverity
19. Pictures: International
Symposium on Bacterial
Canker of Kiwifruit
LAMP Workshop – La Trobe University
24 October 2016
Plant Biosecurity: Smart Surveillance
Local
spread
Eradication
Containment
Containment
Containment – Eradication Window
20. Increased
spread
Pictures: International
Symposium on Bacterial
Canker of Kiwifruit
LAMP Workshop – La Trobe University
24 October 2016
Plant Biosecurity: Smart Surveillance
Local
spread
Eradication
Containment
Containment
Containment – Eradication WindowContainment to Management
22. What if it is not a sporulating fungus?
pathogen isolation & culture
1 – 2 days for most bacteria
Traditional Diagnostic Methods
10 - 14 days for Rathayibacter
23. Pictures: International
Symposium on Bacterial
Canker of Kiwifruit
LAMP Workshop – La Trobe University
24 October 2016
Plant Biosecurity: Smart Surveillance
Why accurate is important
24. Pictures: International
Symposium on Bacterial
Canker of Kiwifruit
LAMP Workshop – La Trobe University
24 October 2016
Plant Biosecurity: Smart Surveillance
No
unnecessary
action taken
Appropriate
regulatory action
taken
NegativePositive
Truevalue
Negative Positive
Diagnosis
- Consequence -
- Trade interrupted -
Costly mitigation actions
taken unnecessarily
Biosecurity breached
- incursion results -
25. Pictures: International
Symposium on Bacterial
Canker of Kiwifruit
LAMP Workshop – La Trobe University
24 October 2016
Plant Biosecurity: Smart Surveillance
27. biosecurity built on scienceScience Exchange - August 2016
Pictures: International Symposium
on Bacterial Canker of Kiwifruit
Plant Biosecurity: Smart Surveillance
Pseudomonas syringae
Pseudomonas syringae
Kiwifruit Pathogen
Non-Pathogenic
Highly virulent? New?
28. biosecurity built on science
Psa Diagnostics
Plant Biosecurity Cooperative Research Centre
Grethel Busot
• Population level discrimination required for Psa
• Very high background noise – path & nonpath pop’ns
29. Pseudomonas syringae pathovar actinidiae
• Many pathovars of Pseudomonas syringae
• P. syringae occurs naturally on MANY plant species
• P. syringae occurs naturally in rain & snow
• P. syringae occurs naturally throughout the world
High potential for false positives!
30. PsyB728
Psa NZ V
Psy ESC1
Psy ESC1
T3SS pathogenicity island
CEL Hrp/hrc cluster EEL
Sunshine Coast, Australia – 5 may 2014 – Busot, Arif, & Stack
Pathogenic strain Psy
Pathogenic strain Psa
nonPathogenic strain
nonPathogenic strain
31. NGS platforms:
PacBio & Illumina MiSeq
• Couplets: inner ring
PacBio, adjacent ring
illumina
• Comparisons for errors:
NGS platforms,
assemblers, assembly
methods (De novo and
genome mapping)
• Comparative genomics:
Genomic variation as a
function of sequencing
and assembly methods
35. Multiplex endpoint PCR-based diagnostics
Genome informed identification of diagnostic sequences in Pseudomonas syringae pv. actinidiae
Hop S2
Hop 01
Hop Z5
Hop Z5
Hop Z3
All Psa strains
Low virulent Psa strains
High virulent Outbreak Psa strain
Determine assay sensitivity
36. biosecurity built on science
Plant Biosecurity Cooperative Research Centre
James Stack
Professor and Director
PBCRC2002 & PBCRC2156
• 2002: Develop & validate laboratory and field
diagnostic protocols for plant pathogenic bacteria to
the pathovar level of discrimination
• 2156: Deploy validated field diagnostic protocols for
plant pathogenic bacteria to the pathovar level of
discrimination
• Increase national capability in plant bacteriology
GOAL: in-field detection
• Isothermal amplification technologies
• Many desirable features for the field (no
heat cycling)
• Very sensitive, accurate and fast!
37. Field deployable assays for identification of Psa
hop Z5 LAMP
hop Z3 LAMP
Loop-mediated isothermal amplification LAMP
Specificity
Sensitivity
38. Isothermal amplification (LAMP) based diagnostics/ Genie
Field deployable assays for identification of Pseudomonas syringae pv. actinidiae
With Loop primers Without Loop primers
LAMP: 6 primers
39. Isothermal amplification (LAMP) based diagnostics/Thermocycler
Field deployable assays for identification of Pseudomonas syringae pv. actinidiae
1 23 4 5 6 7 1213 14 15 16 18 19 21 22 8 9
Global outbreak/Psa VPsa Psa LV
hopZ3
hopZ5
Many non Psa strainsMany Psa strains
40. Isothermal amplification (LAMP) based diagnostics/Thermocycler
Field deployable assays for identification of Pseudomonas syringae pv. actinidiae
Alternative Visualization
technologies:
■ SYBR Green
■ Lateral flow device
Internal control
Positive samples
hopZ3 + LOOP
41. Field deployable assays for identification of Pseudomonas syringae pv. actinidiae
Recombinase Polymerase Amplification (RPA) coupled to Lateral
Flow Device
FAM-biotin/digoxigenine amplicons
Modified from Journal of Virological Methods, 2014;208:144–151
42. Field deployable assays for identification of Pseudomonas syringae pv. actinidiae
Isothermal amplification (RPA) based diagnostics coupled to LFD
Specificity
43. Field deployable assays for identification of Pseudomonas syringae pv. actinidiae
Sensitivity
hopZ3/RPA primers-probe
Psa V
hopZ5/RPA primers-probe
Psa V
Isothermal amplification (RPA) based diagnostics coupled to LFD
Differential sensitivity – hopZ3 and hopZ5
44. Field deployable assays for identification of Pseudomonas syringae pv. actinidiae
Isothermal amplification (RPA) based diagnostics
Multiplexing with RPA? - Yes
45. Pictures: International
Symposium on Bacterial
Canker of Kiwifruit
LAMP Workshop – La Trobe University
24 October 2016
Plant Biosecurity: Smart Surveillance
All good in the lab – does it work in the field?
46. Field Validation & End-user Training
Australia - February 2016
Science Exchange - August 2016
Plant Biosecurity: Smart Surveillance
47. Victoria Kiwi Orchard
In & Out
Science Exchange - August 2016
Plant Biosecurity: Smart Surveillance
48. Victoria Kiwi Orchard – Psa?Samples: fruit, leaves, twigs
Science Exchange - August 2016
Plant Biosecurity: Smart Surveillance
50. Moving advanced diagnostics to the fieldField-deployable technologies are here
LAMP and RPA isothermal technologies in use
Science Exchange - August 2016
Plant Biosecurity: Smart Surveillance
52. Lateral Flow Device visualization
control
positive
Science Exchange - August 2016
Plant Biosecurity: Smart Surveillance
53. 16S bacteria
general
H2O HopZ3+C
In the orchard – all good!
NO Psa in Victoria orchard!
Science Exchange - August 2016
Plant Biosecurity: Smart Surveillance
54. Victoria Kiwi Orchard – Psa?Biosecurity Staff Training
Science Exchange - August 2016
Plant Biosecurity: Smart Surveillance
56. Victoria Kiwi Orchard – Psa?Biosecurity Staff Training
Science Exchange - August 2016
Plant Biosecurity: Smart Surveillance
57. LAMP Workshop – La Trobe University
24 October 2016
Plant Biosecurity: Smart Surveillance
Pictures: J. Stack lab
Dude! This is so
cool!
Even Forrest Gump can do this
58. Science Exchange - August 2016
Plant Biosecurity: Smart Surveillance
Field Validation & End-user Training
New Zealand - February 2016
59. New Zealand - February 2016
Field Validation & End-user Training
Science Exchange - August 2016
Plant Biosecurity: Smart Surveillance
60. Science Exchange - August 2016
Plant Biosecurity: Smart Surveillance
End-User Workshop – 23 October 2016
61. Science Exchange - August 2016
Plant Biosecurity: Smart Surveillance
End-User Workshop – 23 October 2016
62. biosecurity built on science
Plant Biosecurity Cooperative Research Centre
James Stack
Professor and Director
PBCRC 2002: Genome-based, bioinformatics-informed
diagnostics
• Developed & validated multiple laboratory and field
diagnostic protocols for plant pathogenic bacteria to
the pathovar level of discrimination
• Validated Pan-Genome Pipeline
• Increased national capability in plant bacteriology:
9 scientists trained & mentored in plant bacteriology
(Australia, New Zealand, U.S.)
63. biosecurity built on science
Plant Biosecurity Cooperative Research Centre
James Stack
Professor and Director
PBCRC 2156: Field-deployable genome-based,
bioinformatics-informed diagnostic protocols
• Developed & validated multiple field-deployable
diagnostic protocols for plant pathogenic bacteria to
the pathovar level of discrimination
• Validated Concept-to-Practice Pipeline
• Engaged end-user communities in field testing and
validation of protocols: laboratory and in-field end-
user training in new technologies and protocols
65. Plant Biosecurity: Smart Surveillance
Pictures: J. Stack lab
THE PBCRC Team
Science Exchange - August 2016
66. biosecurity built on science
Plant Biosecurity Cooperative Research Centre
James Stack
Professor and Director
Genome-Informed
Diagnostics
Thank you &Have a nice
day!
68. Genomic analyses of the select agent Rathayibacter toxicus
APS Annual Meeting - Tampa – 31 July 2016
69. NPDN National Meeting
Crystal City, VA
9-10 March 2016
What’s next? Where will this technology lead us?
• The FERA SMART spore trap combines:
automated loop-mediated isothermal amplification
(LAMP) analysis
to identify pathogens and measure spore loads
a weather station and a communication capability
(both satellite and mobile phone network)
sends diagnostic and weather data to a central
facility.
• Collaboration between OptiGene Ltd, Fera, The
University of Hertfordshire, Bayer Crop Science and
Frontier Agriculture.
Smart, sophisticated, in-field pathogen detection with
wireless communication.
70. NPDN National Meeting
Crystal City, VA
9-10 March 2016
NextGen NPDN: A National Network with Global Implications
Automated, rapid detection of DNA from airborne spores as a network of samplers to
inform growers
Three new projects starting April 2015: Potato pathogens (TSB); Beet Pathogens (TSB);
Arable pathogens (HGCA) (+ AgriTech catalyst application pending)
Miniature Virtual Impactor (patented), which
samples at high flow rate into liquid
(incubation media or extraction buffer)
Future Work:
Isothermal DNA
amplification
9
Smart Spore Traps
Imagine all that in a drone!
Moving advanced diagnostics to the field
LAMP isothermal technologies in use
71. inoculation infection colonization reproduction dispersal
time
Pathogenpopulation
critical action
point
diagnostic
symptoms
PCR detection limit
104
103
102
101
disease spread
More time to respond
& prevent spread