Resistance to the Fumigant Phosphine and Its Management in Insect Pests of Stored Products: A Global Perspective.

Development of resistance in major grain insect pest species to the key fumigant phosphine (hydrogen phosphide) across the globe has put the viability and sustainability of phosphine in jeopardy. The resistance problem has been aggravated over the past two decades, due mostly to the lack of suitable alternatives matching the major attributes of phosphine, including its low price, ease of application, proven effectiveness against a broad pest spectrum, compatibility with most storage conditions, and international acceptance as a residue-free treatment. In this review, we critically analyze the published literature in the area of phosphine resistance with special emphasis on the methods available for detection of resistance, the genetic basis of resistance development, key management strategies, and research gaps that need to be addressed.

Ecological Networks in Stored Grain: Key Postharvest Nodes for Emerging Pests, Pathogens, and Mycotoxins.

Wheat is at peak quality soon after harvest. Subsequently, diverse biota use wheat as a resource in storage, including insects and mycotoxin-producing fungi. Transportation networks for stored grain are crucial to food security and provide a model system for an analysis of the population structure, evolution, and dispersal of biota in networks. We evaluated the structure of rail networks for grain transport in the United States and Eastern Australia to identify the shortest paths for the anthropogenic dispersal of pests and mycotoxins, as well as the major sources, sinks, and bridges for movement. We found important differences in the risk profile in these two countries and identified priority control points for sampling, detection, and management. An understanding of these key locations and roles within the network is a new type of basic research result in postharvest science and will provide insights for the integrated pest management of high-risk subpopulations, such as pesticide-resistant insect pests.

Spatial Distribution and Flight Patterns of Two Grain Storage Insect Pests, Rhyzopertha dominica (Bostrichidae) and Tribolium castaneum (Tenebrionidae): Implications for Pest Management.

The lesser grain borer, Rhyzopertha dominica, and the rust red flour beetle, Tribolium castaneum, are two major beetle pests commonly found infesting stored products worldwide. Both species can cause severe economic damage and their management is complicated by their potential to develop resistance to several of the limited chemical options available. However, pest management strategies can be improved by understanding the ecology of the pest insect. To determine the spatiotemporal activity of R. dominica and T. castaneum, we conducted a trapping study over two years in a temperate region of south-eastern Australia, with traps located near grain storages and fields. We captured higher numbers of R. dominica than T. castaneum, and both species were more prevalent in traps located close to grain storages. Similar and consistent seasonal patterns were displayed by both species with activity ceasing during the winter (June-August) months. We found linear correlations between maximum daily temperatures and trap catches, and minimum threshold temperatures for flight activity were 14.5 °C and 15.6 °C for R. dominica and T. castaneum, respectively. The results are discussed in relation to the ecology of these pests along with their implications for pest management.

The Gene Introgression Approach and the Potential Cost of Genes that Confer Strong Phosphine Resistance in Red Flour Beetle (Coleoptera: Tenebrionidae).

Resistance in pest insects to the grain fumigant phosphine (PH3) poses a threat to trade and food security. The possible pleiotropic effects of PH3 resistance on development and reproduction were investigated in the red flour beetle, Tribolium castaneum (Herbst), by introgressing two genes known to be major contributors to strong resistance (tc_rph1 and tc_rph2) into a susceptible background. The tc_rph2 allele was the G135S variant, whereas the identity of tc_rph1 allele was unknown but could have been one of the three known variants (L119W, V123F, or S349G). The introgressed resistant strain was 288× more resistant than the susceptible strain, based on mortality after a 20 h fumigation with PH3. Molecular screening confirmed that the introgressed strain was homozygous for the resistance genes, but was otherwise indistinguishable from the susceptible strain based on screening with 12 neutral DNA markers. We found no differences of consequence in developmental time between the susceptible and introgressed resistant strains. Similarly, the number of F1 adults produced by these strains was more or less equal, as was the weight of individual F1 adults. The conclusions remained the same regardless of whether the experiments were conducted on a flour-based medium or wheat. Thus, we found no evidence that being fully strongly PH3 resistant (i.e., homozygous for tc_rph1 and tc_rph2) has major consequences in terms of development or reproduction in T. castaneum.

A high-throughput system used to determine frequency and distribution of phosphine resistance across large geographical regions.

BACKGROUND: Next-generation sequencing can enable genetic surveys of large numbers of individuals. We developed a genotyping-by-sequencing assay for detecting strong phosphine resistance alleles in the dihydrolipoamide dehydrogenase (dld) gene of Rhyzopertha dominica populations. The assay can estimate the distribution and frequency of resistance variants in thousands of individual insects in a single run. RESULTS: We analysed 1435 individual insects collected over a 1-year period from 59 grain-storage sites including farms (n = 29) and central storages (n = 30) across eastern Australia. Resistance alleles were detected in 49% of samples, 38% of farms and 60% of central storages. Although multiple alleles were detected, only two resistance variants (P49S and K142E) were widespread and each appeared to have a distinct but overlapping geographical distribution. CONCLUSION: The type of structure in which the grain is stored had a strong effect on resistance allele frequency. We observed higher frequencies of resistance alleles in bunker storages at central sites compared with other storage types. This contributed to the higher frequencies of resistance alleles in bulk-handling facilities relative to farms. The discovery of a storage structure that predisposes insects to resistance highlights the utility of our high-throughput assay system for improvement of phosphine resistance management practices. © 2018 Society of Chemical Industry.

Development of a Quick Knockdown Test for Diagnosing Resistance to Phosphine in Sitophilus oryzae (Coleoptera: Curculionidae), a Major Pest of Stored Products.

A key component in the management of resistance to fumigant phosphine in stored products pests is their early detection and implementation of control strategies. Currently, resistance testing involves exposing adults to a specific discriminating concentration over a fixed time period (20-48 h). Although it is widely adopted, this test takes significant time for assay preparation (up to 4 wk) as well as diagnosis (1-2 wk). To address these lacunae, we have established a 'quick knockdown test' using a key grain insect pest, rice weevil, Sitophilus oryzae (L.). Susceptible, weakly and strongly phosphine-resistant reference strains were exposed to a threshold concentration of phosphine over short exposure periods (min to h). The time to knockdown (KT) responses to phosphine were characterized at 2 (1,440 ppm) and 5 mg/liter (3,600 ppm). The time to 99.9% KT (KT99.9) at 2 mg/liter was 12.52 min for the susceptible adults, compared with 167.9 and 1,510 min in the case of weakly and strongly resistant phenotypes, respectively. As anticipated, increasing the concentration of phosphine to 5 mg/liter halved the KT99.9 (81.57 min) to separate weakly and strongly resistant populations than it was required at 2 mg/liter. We validated the KT99.9 value for the 5 mg/liter against field-derived populations of S. oryzae. The results were aligned with the existing Food and Agriculture Organization approach, confirming that the proposed 'quick test' is a reliable tool to rapidly diagnose resistance in this species.

Effect of phosphine dose on sorption in wheat.

BACKGROUND: In spite of the extensive use of phosphine fumigation around the world to control insects in stored grain, and the knowledge that grain sorbs phosphine, the effect of concentration on sorption has not been quantified. A laboratory study was undertaken, therefore, to investigate the effect of phosphine dose on sorption in wheat. Wheat was added to glass flasks to achieve filling ratios of 0.25-0.95, and the flasks were sealed and injected with phosphine at 0.1-1.5 mg L(-1) based on flask volume. Phosphine concentration was monitored for 8 days at 25 degrees C and 55% RH. RESULTS: When sorption occurred, phosphine concentration declined with time and was approximately first order, i.e. the data fitted an exponential decay equation. Percentage sorption per day was directly proportional to filling ratio, and was negatively correlated with dose for any given filling ratio. Based on the results, a tenfold increase in dose would result in a halving of the sorption constant and the percentage daily loss. Wheat was less sorptive if it was fumigated for a second time. CONCLUSIONS: The results have implications for the use of phosphine for control of insects in stored wheat. This study shows that dose is a factor that must be considered when trying to understand the impact of sorption on phosphine concentration, and that there appears to be a limit to the capacity of wheat to sorb phosphine.

Combined treatments of spinosad and chlorpyrifos-methyl for management of resistant psocid pests (Psocoptera: Liposcelididae) of stored grain.

The combined efficacy of spinosad and chlorpyrifos-methyl was determined against four storage psocid pests belonging to genus Liposcelis. This research was undertaken because of the increasing importance of these psocids in stored grain and the problem of finding grain protectants to control resistant strains. Firstly, mortality and reproduction were determined for adults exposed to wheat freshly treated with either spinosad (0.5 and 1 mg kg(-1)) or chlorpyrifos-methyl (2.5, 5 and 10 mg kg(-1)) or combinations of spinosad and chlorpyrifos-methyl at 30 degrees C and 70% RH. There were significant effects of application rate of spinosad and chlorpyrifos-methyl, both individually and in combination, on adult mortality and progeny reduction of all four psocids. Liposcelis bostrychophila Badonnel and L. decolor (Pearman) responded similarly, with incomplete control of adults and progeny at both doses of spinosad but complete control in all chlorpyrifos-methyl and combined treatments. In L. entomophila (Enderlein) and L. paeta Pearman, however, complete control of adults and progeny was only achieved in the combined treatments, with the exception of spinosad 0.5 mg kg(-1) plus chlorpyrifos-methyl 2.5 mg kg(-1) against L. entomophila. Next, combinations of spinosad (0.5 and 1 mg kg(-1)) and chlorpyrifos-methyl (2.5, 5 and 10 mg kg(-1)) in bioassays after 0, 1.5 and 3 months storage of treated wheat were evaluated. The best treatment was 1 mg kg(-1) of spinosad plus 10 mg kg(-1) of chlorpyrifos-methyl, providing up to 3 months of protection against infestations of all four Liposcelis spp. on wheat.

Detection and characterisation of strong resistance to phosphine in Brazilian Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae).

As failure to control Rhyzopertha dominica (F.) with phosphine is a common problem in the grain-growing regions of Brazil, a study was undertaken to investigate the frequency, distribution and strength of phosphine resistance in R. dominica in Brazil. Nineteen samples of R. dominica were collected between 1991 and 2003 from central storages where phosphine fumigation had failed to control this species. Insects were cultured without selection until testing in 2005. Each sample was tested for resistance to phosphine on the basis of the response of adults to discriminating concentrations of phosphine (20 and 48 h exposures) and full dose-response assays (48 h exposure). Responses of the Brazilian R. dominica samples were compared with reference susceptible, weak-resistance and strong-resistance strains from Australia in parallel assays. All Brazilian population samples showed resistance to phosphine: five were diagnosed with weak resistance and 14 with strong resistance. Five samples showed levels of resistance similar to the reference strong-resistance strain. A representative highly resistant sample was characterised by exposing mixed-age cultures to a range of constant concentrations of phosphine for various exposure periods. Time to population extinction (TPE) and time to 99.9% suppression of population (LT(99.9)) values of this sample were generally similar to those of the reference strong-resistance strain. For example, at 0.1, 0.5 and 1.0 mg L(-1), LT(99.9) values for BR33 and the reference strong-resistance strain were respectively 21, 6.4 and 3.7 days and 17, 6.2 and 3.8 days. With both strains, doubling phosphine concentrations to 2 mg L(-1) resulted in increased LT(99.9) and TPE. High level and frequency of resistance in all population samples, some of which had been cultured without selection for up to 12 years, suggest little or no fitness deficit associated with phosphine resistance. The present research indicates that widespread phosphine resistance may be developing in Brazil. Fumigation practices should be monitored and resistance management plans implemented to alleviate further resistance development.

Progression of phosphine resistance in susceptible Tribolium castaneum (Herbst) populations under different immigration regimes and selection pressures.

Insecticide resistance is an escalating global issue for a wide variety of agriculturally important pests. The genetic basis and biochemical mechanisms of resistance are well characterized in some systems, but little is known about the ecological aspects of insecticide resistance. We therefore designed a laboratory experiment to quantify the progression of phosphine resistance in Tribolium castaneum populations subject to different immigration regimes and selection pressures. Mated resistant females were added to originally susceptible populations under two distinct migration rates, and in addition, half of the populations in each migration treatment were exposed to selection pressures from phosphine fumigation. The progression of phosphine resistance was assessed by screening beetles for the resistance allele at rph2. Phosphine resistance increased slowly in the low migration treatment and in the absence of selection, as expected. But at the higher migration rate, the increase in frequency of the resistance allele was lower than predicted. These outcomes result from the high levels of polyandry known in T. castaneum females in the laboratory, because most of the Generation 1 offspring (86%) were heterozygous for the rph2 allele, probably because resistant immigrant females mated again on arrival. Phosphine resistance was not fixed by fumigation as predicted, perhaps because susceptible gametes and eggs survived fumigation within resistant females. In terms of phosphine resistance progression in populations exposed to selection, the effect of fumigation negated the difference in migration rates. These results demonstrate how species-specific traits relating to the mating system may shape the progression of insecticide resistance within populations, and they have broad implications for the management of phosphine resistance in T. castaneum in the field. ​We specify and discuss how these mating system attributes need to be accounted for when developing guidelines for resistance management.

Long-term persistence and efficacy of spinosad against Rhyzopertha dominica (Coleoptera: Bostrychidae) in wheat.

A laboratory study was undertaken to determine the persistence and efficacy of spinosad against Rhyzopertha dominica (F.) in wheat stored for 9 months at 30 degrees C and 55 and 70% relative humidity. The aim was to investigate the potential of spinosad for protecting wheat from R. dominica during long-term storage in warm climates. Wheat was treated with spinosad at 0.1, 0.5 and 1 mg kg(-1) grain and sampled after 0, 1.5, 3, 4.5, 6, 7.5 and 9 months of storage for bioassays and residue analyses. Residues were estimated to have declined by 30% during 9 months of storage at 30 degrees C and there was no effect of relative humidity. Spinosad applied at 0.5 or 1 mg kg(-1) was completely effective for 9 months, with 100% adult mortality after 14 days of exposure and no live F1 adults produced. Adult mortality was <100% in some samples of wheat treated with 0.1 mg kg(-1) of spinosad, and live progeny were produced in all samples treated at this level. The results show that spinosad is likely to be an effective grain protectant against R. dominica in wheat stored in warm climates.

Potential of imidacloprid to control four species of psocids (Psocoptera: Liposcelididae) infesting stored grain.

The effectiveness of the neonicotinoid insecticide imidacloprid was evaluated against four psocid pests of stored grain. This research was undertaken because of the growing importance of psocids in stored grain and the need to identify methods for their control. The mortality and reproduction of adults of Liposcelis bostrychophila Badonnel, L. entomophila (Enderlein), L. decolor (Pearman) and L. paeta Pearman in wheat treated with imidacloprid were determined. There were five application rates (0.5, 1, 2, 5 and 10 mg AI kg(-1) grain) and an untreated control. There were significant effects of application rate on both adult mortality and reproduction for all four species, but the effect of imidacloprid was sometimes more pronounced on reproduction. Imidacloprid was most effective against L. bostrychophila, with 100% adult mortality after 7 d at 5 mg AI kg(-1), 14 d at 2 mg AI kg(-1) and 28 d at 0.5 and 1 mg AI kg(-1). No live progeny were produced at 2 mg AI kg(-1). For L. decolor, there was 100% adult mortality after 28 d at 10 mg AI kg(-1) and no live progeny were produced at 2 mg AI kg(-1). For L. entomophila, there was 100% adult mortality after 14 d at 10 mg AI kg(-1) and 28 d at 2 and 5 mg AI kg(-1). No live progeny were produced at 10 mg AI kg(-1). At 10 mg AI kg(-1) there was 100% mortality of L. paeta adults after 28 d exposure and no live progeny developed. Because reproduction at some application rates occurred only in the first 14 d of exposure, it is concluded that the application rate leading to population extinction was 1 mg AI kg(-1) for L. bostrychophila, 2 mg AI kg(-1) for L. decolor and L. entomophila and 5 mg AI kg(-1) for L. paeta. This study shows that imidacloprid has potential as a grain protectant to control all four Liposcelis species in stored grain.

Effect of exposure period on degree of dominance of phosphine resistance in adults of Rhyzopertha dominica (Coleoptera: Bostrychidae) and Sitophilus oryzae (Coleoptera: Curculionidae).

Degree of dominance of phosphine resistance was investigated in adults of Rhyzopertha dominica F and Sitophilus oryzae L. Efficacy of the grain fumigant phosphine depends on both concentration and exposure period, which raises the possibility that dominance levels vary with exposure period. New and published data were used to test this possibility in adults of R dominica and S oryzae fumigated for periods of up to 144 h. The concentrations required for control of homozygous resistant and susceptible strains and their F1 hybrids decreased with increasing exposure period. For both species the response lines for the homozygous resistant and susceptible strains and their F1 hybrids were parallel. Therefore, neither dominance level nor resistance factor was affected by exposure period. Resistance was incompletely recessive and the level of dominance, calculated at 50% mortality level, was -0.59 for R dominica and -0.65 for S oryzae. The resistant R dominica strain was 30.9 times more resistant than the susceptible strain, compared with 8.9 times for the resistant S oryzae strain. The results suggest that developing discriminating doses for detecting heterozygote adults of either species will be difficult.

Effects of time and concentration on mortality of phosphine-resistant Sitophilus oryzae (L) fumigated with phosphine.

The effects of exposure period and phosphine concentration on mortality of susceptible and resistant Sitophilus oryzae (L) were investigated. Although S oryzae is one of the world's most serious pests of stored grain there are few data on the practical significance of phosphine resistance in this species. The strains investigated were an Australian susceptible strain, a homozygous resistant strain exhibiting a level of resistance common in Australia and an unselected field strain from China with a much stronger resistance. Fumigations were carried out at 25 degrees C on adults and mixed-age cultures. For adults of all three strains and mixed-age cultures of the susceptible and resistant Australian strains, the relationship between concentration and time could be described by equations of the form Cnt = k. In all cases n < 1, indicating that time was a more important variable than concentration. In all fumigations of adults the resistant strains were harder to kill than the susceptible strain. However, in fumigations of mixed-age cultures, which contained the tolerant pupal stage, the difference between susceptible and resistant strains was more pronounced at lower concentrations than higher concentrations. For example, at 0.02 mg litre-1 the estimated LT99.9 for mixed-age cultures of the Australian resistant strain (27 days) is 3.4 times that of the susceptible strain (8 days), but at 1 mg litre-1 there is no difference between the two strains (4 days). Limited data on the Chinese resistant strain supported this finding. Twenty-three days exposure at 0.02 mg litre-1 had no effect on mixed-age cultures of this strain, but there were no survivors after 5 days exposure to 1 mg litre-1.

Predicting mortality of phosphine-resistant adults of Sitophilus oryzae (L) (Coleoptera: Curculionidae) in relation to changing phosphine concentration.

Adults of a phosphine-resistant strain of Sitophilus oryzae (L) were exposed to constant phosphine concentrations of 0.0035-0.9mg litre(-1) for periods of between 20 and 168h at 25 degrees C, and the effects of time and concentration on mortality were quantified. Adults were also exposed to a series of treatments lasting 48, 72 or 168 h at 25 degrees C, during which the concentration of phosphine was varied. The aim of this study was to determine whether equations from experiments using constant concentrations could be used to predict the efficacy of changing phosphine concentrations against adults of S oryzae. A probit plane without interaction, in which the logarithms of time (t) and concentration (C) were variables, described the effects of concentration and time on mortality in experiments with constant concentrations. A derived equation of the form C(n)t = k gave excellent predictions of toxicity when applied to data from changing concentration experiments. The results suggest that for resistant S oryzae adults there is nothing inherently different between constant and changing concentration regimes, and that data collected from fixed concentrations can be used to develop equations for predicting mortality in fumigations in which phosphine concentration changes. This approach could simplify the prediction of efficacy of typical fumigations in which concentrations tend to rise and then fall over a period of days.

Synergized bifenthrin plus chlorpyrifos-methyl for control of beetles and psocids in sorghum in Australia.

The efficacy of bifenthrin (0.5 mg/kg) + piperonyl butoxide (7 mg/kg) + chlorpyrifosmethyl (10 mg/kg) against beetle and psocid pests of sorghum was evaluated in silo-scale trials in southeast Queensland, Australia. The pyrethroid bifenthrin was evaluated as a potential new protectant in combination with the organophosphate chlorpyrifos-methyl, which is already registered for control of several insect pests of stored cereals. Sorghum (approximately 200 metric tons) was treated after both the 1999 and 2000 harvests and sampled at intervals to assess treatment efficacy and residue decline during up to 7 mo of storage. Generally, test strains of the beetles Rhyzopertha dominica (F.), Tribolium castaneum (Herbst), Oryzaephilus surinamensis (L), and Cryptolestes ferrugineus (Stephens) were prevented from producing live progeny for up to 7 mo. The treatment failed against one strain of R. dominica known to be resistant to bioresmethrin and organophosphates. Two malathion-resistant strains of O. surinamensis were marginally controlled with 94-100% fewer adult progeny produced. For psocids, no strains of Liposcelis bostrychophila Badonnel, Liposcelis decolor (Pearman), or Liposcelis paeta Pearman produced live progeny, although the control of a field strain of Liposcelis entomophila (Enderlein) was generally poor. Results show that this treatment should protect sorghum for at least 7 mo against most prevalent strains of grain insect in eastern Australia, although control may be limited in areas in which L. entomophila or pyrethroid-resistant R. dominica are present.

Genetics of resistance to phosphine in Rhyzopertha dominica (Coleoptera: Bostrichidae).

The inheritance of resistance to phosphine was studied in two strains of the lesser grain borer, Rhyzopertha dominica (F.), labeled 'Weak-R' and 'Strong-R'. These strains were purified versions of field-selected populations collected in Queensland, Australia. Weak-R and Strong-R were, respectively, 23.4 times (20-h exposure) and 600 times (48-h exposure) resistant to phosphine compared with a reference susceptible strain (S-strain). Each -R strain was crossed with the S-strain and the response to phosphine was measured in their respective F1, F2, and F1-backcross (F1-BC) progenies. Data from testing of reciprocal F1 progeny indicated that resistance in Weak-R was autosomal and incompletely recessive with a degree of dominance -0.96. Modified chi-square analysis and contingency analysis of the observed response to phosphine of F1-BC and F2 progenies rejected the hypothesis of single gene inheritance of resistance. Analysis of the response of the F1, F2, and F1-BC progeny from the Strong-R x S-strain cross also rejected the null hypothesis for single gene resistance. Resistance in the Strong-R strain was autosomal and incompletely recessive with a degree of dominance of -0.64. The Weak-R and Strong-R strains were then crossed. Analysis ofthe F1 and F2 progenies of this reciprocal cross revealed that the strong resistance phenotype was coded by a combination of the genes already present in the Weak-R genotype plus an extra major, incompletely recessive gene. There was also evidence of a minor dominant gene present in approximately 5% of Strong-R individuals.

Phosphine resistance in the rust red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae): inheritance, gene interactions and fitness costs.

The recent emergence of heritable high level resistance to phosphine in stored grain pests is a serious concern among major grain growing countries around the world. Here we describe the genetics of phosphine resistance in the rust red flour beetle Tribolium castaneum (Herbst), a pest of stored grain as well as a genetic model organism. We investigated three field collected strains of T. castaneum viz., susceptible (QTC4), weakly resistant (QTC1012) and strongly resistant (QTC931) to phosphine. The dose-mortality responses of their test- and inter-cross progeny revealed that most resistance was conferred by a single major resistance gene in the weakly (3.2×) resistant strain. This gene was also found in the strongly resistant (431×) strain, together with a second major resistance gene and additional minor factors. The second major gene by itself confers only 12-20× resistance, suggesting that a strong synergistic epistatic interaction between the genes is responsible for the high level of resistance (431×) observed in the strongly resistant strain. Phosphine resistance is not sex linked and is inherited as an incompletely recessive, autosomal trait. The analysis of the phenotypic fitness response of a population derived from a single pair inter-strain cross between the susceptible and strongly resistant strains indicated the changes in the level of response in the strong resistance phenotype; however this effect was not consistent and apparently masked by the genetic background of the weakly resistant strain. The results from this work will inform phosphine resistance management strategies and provide a basis for the identification of the resistance genes.

The rph1 gene is a common contributor to the evolution of phosphine resistance in independent field isolates of Rhyzopertha dominica.

Phosphine is the only economically viable fumigant for routine control of insect pests of stored food products, but its continued use is now threatened by the world-wide emergence of high-level resistance in key pest species. Phosphine has a unique mode of action relative to well-characterised contact pesticides. Similarly, the selective pressures that lead to resistance against field sprays differ dramatically from those encountered during fumigation. The consequences of these differences have not been investigated adequately. We determine the genetic basis of phosphine resistance in Rhyzopertha dominica strains collected from New South Wales and South Australia and compare this with resistance in a previously characterised strain from Queensland. The resistance levels range from 225 and 100 times the baseline response of a sensitive reference strain. Moreover, molecular and phenotypic data indicate that high-level resistance was derived independently in each of the three widely separated geographical regions. Despite the independent origins, resistance was due to two interacting genes in each instance. Furthermore, complementation analysis reveals that all three strains contain an incompletely recessive resistance allele of the autosomal rph1 resistance gene. This is particularly noteworthy as a resistance allele at rph1 was previously proposed to be a necessary first step in the evolution of high-level resistance. Despite the capacity of phosphine to disrupt a wide range of enzymes and biological processes, it is remarkable that the initial step in the selection of resistance is so similar in isolated outbreaks.

The rph2 gene is responsible for high level resistance to phosphine in independent field strains of Rhyzopertha dominica.

The lesser grain borer Rhyzopertha dominica (F.) is one of the most destructive insect pests of stored grain. This pest has been controlled successfully by fumigation with phosphine for the last several decades, though strong resistance to phosphine in many countries has raised concern about the long term usefulness of this control method. Previous genetic analysis of strongly resistant (SR) R. dominica from three widely geographically dispersed regions of Australia, Queensland (SR(QLD)), New South Wales (SR(NSW)) and South Australia (SR(SA)), revealed a resistance allele in the rph1 gene in all three strains. The present study confirms that the rph1 gene contributes to resistance in a fourth strongly resistant strain, SR2(QLD), also from Queensland. The previously described rph2 gene, which interacts synergistically with rph1 gene, confers strong resistance on SR(QLD) and SR(NSW). We now provide strong circumstantial evidence that weak alleles of rph2, together with rph1, contribute to the strong resistance phenotypes of SR(SA) and SR2(QLD). To test the notion that rph1 and rph2 are solely responsible for the strong resistance phenotype of all resistant R. dominica, we created a strain derived by hybridising the four strongly resistant lines. Following repeated selection for survival at extreme rates of phosphine exposure, we found only slightly enhanced resistance. This suggests that a single sequence of genetic changes was responsible for the development of resistance in these insects.