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.

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.

Susceptibility to sulfuryl fluoride and lack of cross-resistance to phosphine in developmental stages of the red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae).

BACKGROUND: Our aim was to ascertain the potential of sulfuryl fluoride (SF) as an alternative fumigant to manage phosphine-resistant pests. We tested the susceptibility of all life stages of red flour beetle, Tribolium castaneum (Herbst), to SF and assessed the presence of cross-resistance to this fumigant in phosphine-resistant strains of this species. RESULTS: Analysis of dose-response data indicated that the egg was the stage most tolerant to SF under a 48 h exposure period. At LC50 , eggs were 29 times more tolerant than other immature stages and adults, and required a relatively high concentration of 48.2 mg L(-1) for complete mortality. No significant differences in tolerance to SF were observed among the three larval instars, pupae and adults, and all of these stages were controlled at a low concentration of 1.32 mg L(-1) . Phosphine-resistant strains did not show cross-resistance to SF. CONCLUSION: Our research concluded that the current maximum registered rate of SF, 1500 gh m(-3) , is adequate to control all the post-embryonic life stages of T. castaneum over a 48 h fumigation period, but it will fail to achieve complete mortality of eggs, indicating the risk of some survival of eggs under this short exposure period. As there is no cross-resistance to SF in phosphine-resistant insects, it will play a key role in managing phosphine resistance in stored-grain insect pests.

Strong resistance to phosphine in the rusty grain beetle, Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae): its characterisation, a rapid assay for diagnosis and its distribution in Australia.

BACKGROUND: The recent development of very high resistance to phosphine in rusty grain beetle, Cryptolestes ferrugineus (Stephens), seriously threatens stored-grain biosecurity. The aim was to characterise this resistance, to develop a rapid bioassay for its diagnosis to support pest management and to document the distribution of resistance in Australia in 2007-2011. RESULTS: Bioassays of purified laboratory reference strains and field-collected samples revealed three phenotypes: susceptible, weakly resistant and strongly resistant. With resistance factors of > 1000 × , resistance to phosphine expressed by the strong resistance phenotype was higher than reported for any stored-product insect species. The new time-to-knockdown assay rapidly and accurately diagnosed each resistance phenotype within 6 h. Although less frequent in western Australia, weak resistance was detected throughout all grain production regions. Strong resistance occurred predominantly in central storages in eastern Australia. CONCLUSION: Resistance to phosphine in the rusty grain beetle is expressed through two identifiable phenotypes: weak and strong. Strong resistance requires urgent changes to current fumigation dosages. The development of a rapid assay for diagnosis of resistance enables the provision of same-day advice to expedite resistance management decisions.

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.

Changes in phosphine sorption in wheat after storage at two temperatures.

BACKGROUND: Wheat can be stored for many months before being fumigated with phosphine to kill insects, so a study was undertaken to investigate whether the sorptive capacity of wheat changes as it ages. Wheat was stored at 15 or 25 degrees C and 55% RH for up to 5.5 months, and samples were fumigated at intervals to determine sorption. Sealed glass flasks (95% full) were injected with 1.5 mg L(-1) of phosphine based on flask volume. Concentrations were monitored for 11 days beginning 2 h after injection. Some wheat samples were refumigated after a period of ventilation. Several fumigations of wheat were conducted to determine the pattern of sorption during the first 24 h. RESULTS: Phosphine concentration declined exponentially with time from 2 h after injection. Rate of sorption decreased with time spent in storage at either 15 or 25 degrees C and 55% RH. Rate of sorption tended to be lower when wheat was refumigated, but this could be explained by time in storage rather than by refumigation per se. The data from the 24 h fumigations did not fit a simple exponential decay equation. Instead, there was a rapid decline in the first hour, with phosphine concentration falling much more slowly thereafter. CONCLUSIONS: The results have implications for phosphine fumigation of insects in stored wheat. Both the time wheat has spent in storage and the temperature at which it has been stored are factors that must be considered when trying to understand the impact of sorption on phosphine concentrations in commercial fumigations.

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.

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.

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.

Inhibition of egg development by phosphine in the cosmopolitan pest of stored products Liposcelis bostrychophila (Psocoptera: Liposcelididae).

Phosphine-induced delay in development of eggs was investigated as a mechanism of resistance to this fumigant in Liposcelis bostrychophila Badonnel. One-day-old eggs of a susceptible and a strongly resistant strain of L bostrychophila were exposed to a range of phosphine concentrations for 6days at 30 (+/- 1) degrees C and 70 (+/- 2)% RH. Delay in mean hatching period occurred in both susceptible and resistant eggs, although it was more pronounced in the latter. A maximum delay of 2.65 days was recorded for eggs of the susceptible strain at 0.01 mg litre(-1) (the highest concentration at which eggs survived) and 13.39 days for the resistant strain at 1 mg litre(-1) (the highest concentration tested). Delay in egg development time was positively correlated with increasing phosphine concentration. Our results reveal that the most successful strategy to control resistant L bostrychophila is to apply relatively low concentrations of phosphine for extended exposure times (eg 0.05 mg litre(-1) for 16 days) that allow all eggs to hatch to the much less tolerant nymph stage.

Long-term effectiveness of grain protectants and structural treatments against Liposcelis decolor (Pearman) (Psocoptera: Liposcelididae), a pest of stored products.

Numerous strains of the psocid pest, Liposcelis decolor (Pearman) were collected from farms and central storages and interbred to form three representative strains from three major grain-growing states of Australia: Queensland, South Australia and New South Wales. These were tested against the grain protectants and structural treatments currently registered for use in Australia. Recently, L decolor has become an important pest of stored grain in Australia, particularly in the eastern and southern parts. There is no published information available on management of this pest and the current pest-management strategy, based predominantly on phosphine fumigation, has failed to control infestations of this pest in numerous grain storages in Australia. Alternative methods of control such as use of contact insecticides were explored in the present work to supplement phosphine fumigation to manage this new pest. From eight grain protectants tested as admixtures, only chlorpyrifos-methyl, bioresmethrin plus piperonyl butoxide, and fenitrothion were found to provide long term (3-9 months) protection against all three strains of L decolor. Chlorpyrifos-methyl gave the best protection, providing a minimum of 7.5 to a maximum of 9 months protection, depending on the strains tested. Three structural treatments, azamethiphos, azamethiphos plus carbaryl and permethrin provided long-term control (8-9 months) of all three strains of L decolor on galvanised steel surfaces, with permethrin delivering 9 months protection against all strains. However, all of these treatments failed to provide long-term control of any strains on concrete surfaces. We conclude that chlorpyrifos-methyl as a grain admixture can be incorporated into fumigation strategies to optimise the control of L decolor infestations. Structural treatments, such as permethrin can be used to support a fumigation strategy in storages made of galvanised steel.

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.

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.