Rob Emery visited China to attend the 8th International Conference on Controlled Atmospheres and Fumigation in Stored Products (CAF) to present Australia's approach to dealing with the 200

Phosphine is the main fumigant used in Australia to control insect pests in grain storages; both bulk grain handlers and farmers rely on phosphine for the control of insects and more than 80% of grain is fumigated with phosphine during storage.

However, insect resistance to phosphine is increasing in most grain growing areas. To manage this resistance, a rapid and sensitive method for identifying phosphine resistance is required. The current detection method for phosphine resistance relies on time-consuming laboratory bioassay procedures (more than seven days required). A previous study (Park et al., 2008*) had suggested that certain proteins, displayed by two-dimensional electrophoresis (2D-PAGE), from whole Rhyzopertha dominica, differed between resistant and susceptible insects and might be developed as a rapid diagnostic tool.

Research outcomes

This project initially aimed to identify the genes encoding those differing proteins as a first step towards developing a diagnostic tool. However, when the project team used a larger number of strains of R. dominica and a more robust method the results of the previous study were not supported. The project team then proposed that this approach to biomarker discovery might yet be successful if they were to concentrate a more appropriate subset of proteins for proteomic analysis.

Various lines of evidence suggested that mitochondria are a site of important differences associated with phosphine resistance. A proteomic comparison of mitochondrial proteins from susceptible and resistant Tribolium castaneum (chosen for its completed genome sequence) was therefore conducted. The study did not reveal any significant differences in the expression of the more abundant mitochondrial proteins between resistant and susceptible insects. The aim of identifying differentially expressed proteins that are diagnostic for phosphine resistance has been shown to be beyond the scope of this project.

Research Implications

No changes to the use of phosphine for fumigation nor the management of resistance can be recommended as a result of this work.

Acknowledgements

David Schlipalius and Pat Collins (Queensland Primary Industries and Fisheries) supplied some of the insect strains.

*Park, B.-S., Lee, B.-H., Kim, T.-W., Ren, Y.L., Lee, S.-E., 2008. Proteomic evaluation of adults of Rhyzopertha dominica resistant to phosphine. Environmental Toxicology and Pharmacology 25, 121–126.

The 8th International Conference on Controlled Atmosphere and Fumigation in Stored Products (CAF) was held from 21-26 September 2008 in Chengdu, China.

Flat grain beetle (FGB) is a major emergency plant pest (EPP) of stored grain in Australia. Populations of FGB have recently developed high level resistance to phosphine (the only viable fumigant available for non-quarantine use) resulting in control failures with current dosage regimes.

As there is no practical alternative to phosphine, failure to control FGB with phosphine places at risk market access for Australian grain worth up to $7 billion in annual trade. Therefore there is an urgent need to develop appropriate phosphine fumigation protocols to eradicate outbreaks of strongly resistant FGB.

Research outcomes:

• Characterisation of high resistance to phosphine in flat grain beetles (FGB) for the first time internationally.
• Establishment of fumigation protocols and an eradication strategy that will enable industry to eradicate infestations of phosphine-resistant flat grain beetle and prevent or delay further selection for resistance to phosphine.
• Development of a rapid test to detect highly resistant FGB.
• Facilitate continued market access of Australian grain.

Research implications:

The success of Australia’s $7 billion grain industry depends on the maintenance of high standard in its post-harvest produce through effective pest management. The absence of detectable levels of insect infestation is such an important issue to world grain markets that to maintain its competitiveness in premium markets, Australia guarantees supply of an insect-free product. This strategy is enforced by Australian Quarantine and Inspection Service (AQIS) through the Exports (Grain) Regulation that specifies a ‘nil tolerance for live insects’ on all grain leaving the country. Nil tolerance for live insects is also the standard generally adopted by domestic buyers of grain.

The most cost-effective method to meet the Exports (Grain) Regulation is the application of chemicals to grain. Currently, the industry relies on a single fumigant, phosphine, because of an increasing sensitivity of grain markets to the presence of pesticide residues, the development of insecticide resistance to other chemical alternatives and the lack of practical alternatives. Phosphine has the enviable reputation for being relatively cheap, accepted as a residue-free treatment internationally and having flexibility in its application. Traditionally, the grain industry has managed resistance essentially by replacing redundant chemicals with new materials. Chemical treatments are favoured because available non-chemical methods, on the whole, are either significantly more expensive, less versatile, do not easily match grain-handling logistics, are less effective or require significant capital investment. Therefore, the chemical replacement strategy is no longer viable and at least for the medium term, the Australian grain industry will need to rely on phosphine for disinfestations of its stored commodities to meet the market demands.

The CRCNPB-supported FGB fumigation protocol development project has delivered two new fumigation protocols that can control highly resistant FGB populations. In addition, an eradication strategy has now been deployed that will eradicate infestations of phosphine-resistant FGB and prevent or delay further selection for resistance to phosphine and restrict their spread.

A direct implication of the research finding is that by extending the life of the effective use of phosphine, industry will avoid the use of contact pesticides for the time being. This will in turn avoid potential trade issues and save the industry from significant economic loss.

An independent cost-benefit analysis for this project by GRDC (Ross McLeod) has suggested that even if there are significant changes to key variables such as costs of fumigation, probability of success and volumes of grain treated with contact insecticide, prolonging the life of phosphine through development of new protocols will still result in substantial economic benefits.

Acknowledgements:

The research team expresses sincere thanks to farmers and the managers and field staff of GrainCorp, CBH and Viterra for their support and help for accessing storage sites for collection of insect samples and undertaking field trials. The team would also like to thank GRDC for their support throughout the research.

This project aimed to provide rapid identification of the phosphine resistance status of any individual R. dominica or T. castaneum collected from grain in storage across Australia. The resistance genes that are directly responsible for phosphine resistance in these insects were identified and used as indicators of resistance status.

Research outcomes

Key outcomes of the research are:

1. Phosphine resistance is mediated by two major genes in both T. castaneum and R. dominica. These two genes have been named rph1 and rph2 (i.e. resistance to phosphine 1 and 2).
2. The two genes are incompletely recessive and individually confer weak resistance when homozygous for the resistance mutation.
3. The two genes are synergistic in effect and confer strong resistance in R. dominica and T. castaneum when both are present and homozygous for the resistance alleles.
4. The two genes are expressed in all insect life stages suggesting a constitutively expressed resistance factor that does not appear to be 'switched off' at any stage of development.
5. The location of the rph1 gene has been narrowed down to a very small number of candidate genes (about six) for T. castaneum. However the location for R. dominica is less clear with a region of about 100 genes identified as the source area.
6. While gene expression profiling was achieved for R. dominica and T. castaneum the results indicate that the technique is not suitable as the basis for development of a diagnostic test for resistance as the known resistance genes are not differentially expressed in resistant and sensitive strains and do not change expression in response to phosphine.

Research implications

Implication 1

The research has shown that there are two major genes that confer resistance in the two insect species studied and that both these genes are expressed in all insect life stages. This means that:

1. Selection for resistance can occur in all life stages of the two studied insects, and
2. There are no vulnerable life-stages that could have been targeted in attempts to manage resistance development

Implication 2

The finding that the two genes are synergistic in effect and confer strong resistance only when both genes are homozygous (for resistance) explains why strong resistance has taken a relatively long time to increase in frequency and appear in enough numbers to be detected. This is because you must have, in one individual, both resistance genes present and both homozygous and the chances of this occurring in random mating events in nature are not high.

Implication 3

It was found that the gene rph2 is highly conserved between R. dominica and T. castaneum. The significance of this finding is that a similar mechanism for resistance could be common across all major grain storage pest species where phosphine is used for control.

Implication 4

The research has shown that mutations in rph2 vary between populations of R. dominica and T. castaneum. This indicates that industry is unlikely to gain a universal molecular diagnostic test for phosphine resistance that could be applied across Australian grain growing areas. 

Acknowledgements

The authors would like to acknowledge the support of the Australian Government’s Cooperative Research Centres Program. We would also like to acknowledge the contributions of the University of Queensland which contributed to two PhD scholarships that worked on this project.

Increasing applications of phosphine to stored grain in silos that leak, or grain stored under the wrong conditions, has caused problems with insect pests building up resistance. Proof is needed to show whether phosphine fumigation of cool grain is effective in controlling resistant biotypes of insect pests of stored grain.

Research outcomes:

This project assessed the efficacy of phosphine fumigation against resistant insects in cool grain, i.e. aerated grain or grain that has been harvested and stored in the cooler months of the year, and developed recommendations for Australia’s grain storage industry. Grain temperatures of
20°C or less are considered cool. The project will contribute to keeping Australia’s grain export industry free from insect strains resistant to phosphine. Increasing applications of phosphine to stored grain in silos that leak, or grain stored under the wrong conditions, has caused problems with insect pests building up resistance. Proof is needed to show whether phosphine fumigation of cool grain is effective in controlling resistant biotypes of insect pests of stored grain.

The project has:

• provided reports describing new data on phosphine efficacy at low temperature against resistant grain insects
• provided reports describing new data on phosphine sorption by grain
• provided reports describing new data on phosphine fumigation of cool grain in sealed farm silos, and
• made recommendations to farmers and others within the grain industry.

Recommendations on phosphine fumigation of cool grain have been reported to representatives of the grain industry and national extension network at annual meetings of National Working Party on Grain Protection (NWPGP). Results, recommendations and technology transfer have been provided to the Australian grain industry via the NWPGP and industry forums and publications. CRC partners, bulk grain storage operators and handlers have been informed of the findings.

Research implications

Successful fumigation of resistant insects requires sufficient phosphine concentrations for long enough to control all life stages of the insects. Lower temperatures maintain grain quality and reduce insect population growth, but phosphine is generally less effective at lower temperatures.

The relative importance of phosphine resistant strains of key pests changes with temperature. The most resistant Australian strains of two pests are known to respond similarly to phosphine but the project showed that one species became much harder to control in cool grain.

Sorption will be the major cause of loss of phosphine in a wellsealed silo. Rate of sorption was lower at lower temperature, meaning that higher concentrations will be achieved for longer. Older grain tended to be less sorptive, so delaying fumigation may result in higher concentrations for longer. Sorghum was more sorptive than wheat so the margin for error is smaller for this grain.

In farm silos, phosphine gas was liberated from the aluminium phosphide formulations used despite the low grain and ambient temperatures (e.g. 10°C).

The results for the silo trials varied but three general observations were made. Lower concentrations tended to be measured deeper in grain mass. Lower concentrations tended to be measured on the northern side. Concentrations measured higher in the grain mass tended to peak earlier.

The silo trials showed that control of resistant insect populations is possible subject to good gas-tightness and adequate exposure periods. Exposure to cool temperature alone was not the cause of insect mortality.

Growers and others planning to fumigate cool grain in sealable silos should aim for the current silo pressure test standard of a three minute halving time for a full silo or a five minute halving time for a partially full silo.

The use of forced or passive recirculation methods (e.g. the thermosiphon) should be investigated to promote rapid and even distribution of phosphine from the headspace throughout the grain bulk.

Acknowledgements

The project team is grateful for the cooperation of the following growers and their families: Mr Rodney Petersen (Killarney, Qld) and Mr Raymond Fulwood (Meenaar, WA).