Session 4: Research impact – an end-user’s perspective: Phosphine resistance

biosecurity built on science

Research impact – an end-user perspective: Phosphine Resistance

Phillip Clamp, Quality Assurance Manager – GrainCorp

Manoj Nayak, Principal Research Scientist – DAF, Queensland

Plant Biosecurity Cooperative Research Centre


Grains industry has supported stored product protection research for over 3 decades major (historic) research outcomes;- Bunker fumigations, Inert (N2, CO2), sealed storage (BHC, farm), Siroflo, COS, EF

10 year relationship NPBCRC (2007) to present PBCRC, PBCRC;- PBCRC highly engaged with grains, great end-user focus, grains strategy- Focus on delivery of end-user tools for maximum impact- PBCRC is best option for stored product protection research;

Excellent $ multiplier, collaboration Phosphine research is often foundation for other grains post harvest research Useful range of tools to potential game changer in Amorphous silica (grain/structural)

Research and Grains a successful partnership


Australia has a unique set of grain protection circumstances:- Legislated nil insect detection policy for exports- Domestic trading contracts also specify nil insects

We harvest and store grain in tropical/sub-tropical climates:- This creates perfect conditions for proliferation of insects

Deregulation has placed pressure on insect control:- New (less informed) entrants, use of alternate supply chains, alternate cargo

assembly methods, just in time delivery, more insect remedy at ports- Competition and reducing costs challenge hygiene and other quality controls

Our operating environment


Phosphine- Is used world-wide as a grain fumigant- Phosphine is cheap, versatile and “residue-free”- We over rely on Phosphine as a grain fumigant on farm and in industry- We regularly select for resistance (cost, infrastructure, leaky enclosures)- Phosphine has been a “silver bullet” sustaining export and domestic markets- Other chemistries/controls expensive or market limiting (residues)- Phosphine resistance is real and growing- Rusty grain beetle not controlled by most current rates- We must keep Phosphine useful for the long haul

Managing Phosphine resistance is Grains’ greatest challenge

Phosphine


CRC and Grains took a strategic direction to sustain Phosphine in Australia, supported (at least in part) by the following research examples:- Resistance monitoring, distribution and systems approaches:

CRC3035, CRC3036, CRC3040, CRC3150- Phosphine resistance management strategy CRC3036- Alternatives to Phosphine: CRC3038, CRC3099- Revised Phosphine treatment protocols: CRC3150- Sulfuryl fluoride:

Fumigation protocols CRC3036, Residues CRC3037

PBCRC and Phosphine resistance, some examples:


GrainCorp have adopted the following Phosphine resistance management tools from the CRC toolbox:- Monitor, manage and test suspect resistant insects:

Know and manage your enemy- Phosphine resistance management strategy:

Routinely use alternate chemistry (protectants and SF) as PH3 resistance breaker Focus on hygiene, monitoring of systems such as sealing, inspection, audit and report

- Revised Phosphine and SF treatment protocols: Allows for precision treatment (CxT) relevant to situation

- Precision application allows for greater insect mortality and flexibility: Varying C and T can; lower costs, supports resistance management, meet cargo timelines

What is GrainCorp’s end-user adoption?


Others that have adopted Phosphine resistance management tools from the CRC toolbox include:- Other bulk handlers (Viterra, CBH, Grainflow, Emerald)- Smaller private storers- Growers- GRDC via publications/extension specialists- Researchers nationally and internationally- Chemical companies, registrants

Who else will use will use this?


Theme 1: Tools, technologies and strategies to safeguard international markets for grain and horticultural exports

Theme 2: Better management of established pests Not all research outputs bring direct value to GrainCorp, however; Above themes well supported; strategic and end-user outcomes met Many of the research outputs (or work in progress), particularly work in

Phosphine and Phosphine alternatives are, this:- Sustains our pest management efforts- Extends the life of Phosphine- Allows us to meet domestic and export market requirements

All of the above safeguards and sustains Australian Trade

Program 3 – Safeguarding Trade - Summary


For more information, please email: [email protected]

mailto:[email protected]


Science behind phosphine resistance Two major genes (rph 1 and rph 2)

- Weak resistance - R1R1 (5-15 X)- Strong resistance - R1R1 + R2R2 (>400 X)

Synergistic effect Multiple variants – a common molecular platform needed

Example - to control all life stages- at 720 ppm (1 mg/L)


Impact of phosphine resistance Direct Cost:

Multiple treatments, alternatives Demurrage Rejection and treatment at destination

Reduced quality and integrity Jeopardises food security/market access, loss of reputation

‘Nil tolerance’ Research costs

Multi-millon dollar investment globally


Timely detection and accurate diagnosis Characterisation of resistant phenotypes Understanding of molecular genetics – Rph1 & Rph2 - Platform Understanding of ecology and population genetics (movement

and colonization) An integrated approach

- Develop alternatives- Modify protocols (conc. x time)- Utilise molecular and ecological knowledge

Approach to keep phosphine viable


Strong resistance problems over the years Rhyzopertha dominica (Lesser grain borer) Sitophilus oryzae (Rice weevil) Tribolium castaneum (Red flour beetle) Oryzaephilus surinamensis (Saw-toothed grain beetle) Cryptolestes ferrugineus (Rusty grain beetle) Liposcelis bostrychophila (Psocids) C. ferrugineus

- Strongest ever reported (1300x)


Current problem - Rusty grain beetle (C. ferrugineus)


Problem being addressed – Strongly resistant RGB

Time to population extinction at 720 ppm at 25°C


Monitoring continues…..(bioassay and mol. methods) Developing Sulfuryl fluoride (SF) as ‘resistance breaker’ Developing higher rates - cylinderised phosphine – label change Evaluating current standards for Aluminium phosphide (sealing,

recirculation, large silos) – label change Validating new protocols - industry-scale field trials Determining ‘time to reinfestation’ after fumigation ‘Decision Making’ tool integrating all available knowledge and methods

Overview of current Phosphine Resistance Management


Results so far – new sulfuryl fluoride protocols


Results so far – a comparative assessment of SF protocols


Results so far – field validation of SF protocolTarget CT: 400 mg-h/L (1.7 mg/L for 10 days)

1500 tonne sorghum in 2300m3 siloMixed-age resistant pests in cages

Temperature:Top : (avg: 22.5°C)Bottom : (avg: 27.3°C)

Results:Complete control of six strongly phosphine resistant pest species RGB, LGB, RFB, RW, SGB, Psocids


Results so far – ‘Time to reinfestation’Large bunkers - sorghum/wheatPre and post fumigation sampling for natural infestations23 samples at two depths


Results so far – No cross resistance to phosphine-RRGB, C. ferrugineus

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Results so far – Impact of SF on Str. Rusty grain beetles


Excellent candidate as an alternative to phosphine – no cross resistance

Time and temperature have significant effect on effectiveness

Time more critical than concentration

At 4 days - registered rate (1500 CT) at 25°C fails - RFB and RW

Half the concentration of SF required at 30°C than 25°C

Minimum 7 days exposure period – for all four pest species

‘Time to reinfestation’ - 3 months after a successful fumigation No survivors from the original infestation

Re-infestation (invasion) did not occur

Fumiguide needs significant changes – bunker fumigation

Strategic use to break ‘phosphine resistance cycle’

Results so far – Key points on SF


Results so far – new phosphine protocols for RGB


Results so far – proposed changes to cylinderised phosphine label


Results so far – field validation of phosphine protocol

Target CT: 1080 ppm for 12 days)

1600 tonne sorghum in 2300m3 siloMixed-age resistant pests in cages

Temperature:Top : (avg: 22.3°C)Bottom : (avg: 25.5°C)

Results:Complete control of six strongly phosphine resistant pest species RGB, LGB, RFB, RW, SGB, Psocids


Results so far – Aluminium phosphide label Aim: Do large farm silos need recirculation?

Passive Recirculation


Results so far – on farm trial of Aluminium phosphide• Flat bottom 1400 tonne silos

(Kotzur) at Weemelah• Wheat with 12% m.c.• 1.5 g of phosphine m3

• Temp.: 27-31°C• Gas monitored 3 times daily


Current pressure half-life standard of 5 minutes - suitable for large silos

Recirculation has significant impact- doubles the amount of phosphine available to kill pests

Resistant RGB can be controlled on farms in large silos Exposure period extended past 30 days with passive fumigation Reduced exposure period to 14 days by using recirculation

Results so far – Aluminium phosphide use


Recognised world leader- Only country systematically monitoring and managing resistance – proper

characterisation - Frequency of resistance remains below 10% compared to more than 80-90% in overseas - Unique resistance database – AGIRD (>30 years)- Unique collection of laboratory populations - International invitations (Editorial Board in journals, key notes, workshops, training)- Invited book chapters, publications

Other countries look to us for leadership- KSU is partnering with PBCRC- India $3M Grand Challenge project/$1M AISRF Project - Brazil, China, Indonesia, Thailand, The Philippines, Vietnam, Turkey

Where are we now?


Discovery of genes responsible for phosphine resistance – Science Development of Molecular Diagnostics – Heredity Comparative genetic analysis (global) – PLOS One, Heredity Psocids: resistance and management – Annual Review of Entomology Characterisation of strong resistance – Pest Management Science, Pesticide

Biochemistry and Physiology Development of ‘Quick tests’ to diagnose strong resistance - Pest Management Science New phosphine protocols, cross-resistance studies – Pest Management Science Comprehensive studies on inheritance, fitness & prevalence – Journal of Stored

Products Research Analysis of resistance monitoring database (AGIRD) – Environmental Ecological

Statistics, Journal of Stored Products Research

Major Breakthroughs


National Resistance Management Strategy - network of pest and resistance managers, researchers- needs major changes to include new information (last updated in 2009)

ecological knowledge, SF, on farm storage, protectants

GRDC National Extension Network National Working Party on Grain Protection Industry Workshops/Field Days Scientific and extension publications

- Peer-reviewed journal papers- GRDC Publications (eg. Growers Update, Ground Cover)

The Delivery Plan


Close industry-researcher collaboration Industry has flexibility to use treatments Decision making - treatments and storage performance Strategic direction on performance of resistance management Residue and environmental issues need to be addressed Information to biosecurity officers and extension networks Timely containment/control of resistant populations Enhanced knowledge

incidences and selection for resistance to phosphine utility of SF as a new tool

Summary - The Industry Impact


Phosphine and Sulfuryl fluoride co-treatment Additive increase in toxicity Effective against PH3-resistant insects in key pest species Adults and eggs controlled by ½ PH3 + ½ SF – 7 days Well below the current label rates of both fumigants

- Lower residue level and environmental risk- Shorten PH3 fumigation period- Reduced selection pressure on both

Major modifications to Fumiguide for SF use (Douglas Products) Molecular diagnostics

A platform to diagnose strong resistant mutants of all key species- Resistance gene frequency - Evaluation of management strategies

Resistance monitoring for SF Studies on desorption

The Future….


Research Team (PBCRC3035, 3036, 3150, 3160, 3114)- DAFQ - Manoj Nayak and Team (Greg, Pat, Andrew, Raj, Raman, Philip, Hervoika)- UQ- Paul Ebert and Team- NSWDPI - Jo Holloway and Team- DAFWA - Rob Emery and Team- GrainCorp - Robin Reid and team- CBH - Ern Kostas and team- Viterra - Greg Hopkins and team- CYTEC – Justin Tumambing

Acknowledgements


For more information, please email: [email protected]

mailto:[email protected]

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