Influence of concentration, temperature and humidity on the toxicity of phosphine to the strongly phosphine-resistant psocid Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae)

BACKGROUND: The psocid Liposcelis bostrychophila Badonnel, is a widespread, significant pest of stored commodities, has developed strong resistance to phosphine, the major grain disinfestant. The aim was to develop effective fumigation protocols to control this resistant pest. RESULTS: Time to population extinction of all life stages (TPE) in days was evaluated at a series of phosphine concentrations and temperatures at two relative humidities. Regression analysis showed that temperature, concentration and relative humidity all contributed significantly to describing TPE (P < 0.001, R2 = 0.95), with temperature being the dominant variable, accounting for 74.4% of the variation. Irrespective of phosphine concentration, TPE was longer at lower temperatures and high humidity (70% RH) and shorter at higher temperatures and low humidity (55% RH). At any concentration of phosphine, a combination of higher temperature and lower humidity provides the shortest fumigation period to control resistant L. bostrychophila. For example, 19 and 11 days of fumigation are required at 15 °C and 70% RH at 0.1 and 1.0 mg L-1 of phosphine respectively, whereas only 4 and 2 days are required at 35 °C and 55% RH for the same respective concentrations. CONCLUSIONS: The developed fumigation protocols will provide industry with flexibility in application of 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 (LT99.9) 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, LT99.9 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 LT99.9 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.

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 25C 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 25C 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.

Preparation and catalytic studies of palladium nanoparticles stabilized by dendritic phosphine ligand-functionalized silica

Silica is a prominently utilized heterogeneous metal catalyst support. Functionalization of the silica with poly(ether imine) based dendritic phosphine ligand was conducted, in order to assess the efficacy of the dendritic phosphine in reactions facilitated by a silica supported metal catalyst. The phosphinated poly(ether imine) (PETIM) dendritic ligand was bound covalently to the functionalized silica. For this purpose, the phosphinated dendritic ligand containing an amine at the focal point was synthesized initially. Complexation of the dendritic phosphine functionalized silica with Pd(COD)Cl-2 yielded Pd(II) complex, which was reduced subsequently to Pd(0), by conditioning with EtOH. The Pd metal nanoparticle thus formed was characterized by physical methods, and the spherical nanoparticles were found to have >85% size distribution between 2 nm and 4 nm. The metal nanoparticle was tested as a hydrogenation catalyst of olefins. The catalyst could be recovered and recycled more than 10 times, without a loss in the catalytic efficiency.

CRC50150 Targeting mechanisms of phosphine resistance in stored grain pests

The objectives of this projects are: 1)To ensure the identification of genomic DNA markers for phosphine resistance in Rhyzopertha dominica and Tribolium castaneum; 2) To determine gene function of identified phosphine resistance genes in Rhyzopertha dominica and Tribolium castaneum; and 3) Predict future problems by characterising international resistances using our genes as a starting point to determine strong resistance can get by determining similarities with Australia.

Phosphine fumigation of cool grain

The biosecurity problem addressed was the need to understand and evaluate phosphine fumigation of cool grain (i.e. 20°C or less) as a means of controlling resistant biotypes of insect pests of stored grain which are major EPPs threatening the grain industry. The benefits of cooling and phosphine fumigation are that cooling preserves grain quality and reduces insect population growth, and phosphine kills insects and has a residue free status in all major markets. The research objectives were to: - conduct laboratory experiments on phosphine efficacy against resistant insects in cool grain, and determine times to population extinction. - conduct laboratory experiments on phosphine sorption in cool grain and quantify. - complete fumigation trials in three states (Queensland, WA and NSW) on cool grain stored insealed farm silos. - make recommendations for industry on effective phosphine fumigation of cool grain. Phosphine is used by growers and other stakeholders in the grain industry to meet domesticand international demands for insect-free grain. The project aim was to generate new information on the performance of phosphine fumigation of cool grain relevant to resistant biotypes. Effective control of resistant biotypes using phosphine to fumigate cool grain will benefit growers and other sectors of the grain industry, needing to fumigate grain in the cooler months of the year, or grain that has been cooled using aeration.

CRC20080 Diagnostic technologies for phosphine resistance management

Development of molecular markers for rapid diagnosis of phosphine resistance in insects.

Phosphine fumigation of silo bags.

Fumigation with phosphine has the potential to disinfest grain stored in silo bags but only limited research has been conducted on whether phosphine fumigation can be undertaken effectively and safely in this form of storage. Fumigation with phosphine was tested on two (70 m) replicate silo bags each containing 240 t of wheat (9.9 and 9.2% m.c.). The target application rate of phosphine was 1.5 g m 3 with a fumigation period of 17 days. Aluminium phosphide tablets were inserted into each bag at ten release points spaced at 7 m intervals starting 3.5 m from either end of the bag. A total of 14 bioassay cages containing mixed age populations of strongly phosphine resistant Rhyzopertha dominica (F.) were inserted into each fumigated silo bag. Complete control of all life stages of R. dominica was achieved at all locations in the fumigated silo bags. Phosphine concentrations at release points increased rapidly and remained high for the duration of the fumigation. Concentrations at midway points were always lower than at the release points but exceeded 215 ppm for ten days. The diffusion coefficient of available phosphine averaged over the first three full days of the fumigation for both fumigated silo bags was 2.8 x 10 7. Venting the silo bag with an aeration fan reduced the phosphine concentration by 99% after 12 h. Relatively small amounts of phosphine continued to desorb after the venting period. Although grain temperature at the core of the silo bags remained stable at 29degreesC for 17 days, grain at the surface of the silo bags fluctuated daily with a mean of 29degreesC. The results demonstrate that silo bags can be fumigated with phosphine for complete control of infestations of strongly phosphine resistant R. dominica and potentially other species.

Reproduction of phosphine resistant Rhyzopertha dominica (F.) following sublethal exposure to phosphine.

Phosphine fumigation is commonly used to disinfest grain of insect pests. In fumigations which allow insect survival the question of whether sublethal exposure to phosphine affects reproduction is important for predicting population recovery and the spread of resistance. Two laboratory experiments addressed this question using strongly phosphine resistant lesser grain borer, Rhyzopertha dominica (F.). Offspring production was examined in individual females which had been allowed to mate before being fumigated for 48 h at 0.25 mg L -1. Surviving females produced offspring but at a reduced rate during a two-week period post fumigation compared to unfumigated controls. Cumulative fecundity of fumigated females from 4 weeks of oviposition post fumigation was 25% lower than the cumulative fecundity of unfumigated females. Mating potential post fumigation was examined when virgin adults (either or both sexes) were fumigated individually (48 h at 0.25 mg L -1) and the survivors were allowed to mate and reproduce in wheat. All mating combinations produced offspring but production in the first week post fumigation was significantly suppressed compared to the unfumigated controls. Offspring suppression was greatest when both sexes were exposed to phosphine followed by the pairing of fumigated females with unfumigated males and the least suppression was observed when males only were fumigated. Cumulative fecundity from 4 weeks oviposition post fumigation of fumigated females paired with fumigated males was 17% lower than the fecundity of unfumigated adult pairings. Both of these experiments confirmed that sublethal exposure to phosphine can reduce fecundity in R. dominica.

Determining changes in the distribution and abundance of a Rhyzopertha dominica phosphine resistance allele in farm grain storages using a DNA marker

BACKGROUND: The lesser grain borer, Rhyzopertha dominica (F.), is a highly destructive pest of stored grain that is strongly resistant to the fumigant phosphine (PH3). Phosphine resistance is due to genetic variants at the rph2 locus that alter the function of the dihydrolipoamide dehydrogenase (DLD) gene. This discovery now enables direct detection of resistance variants at the rph2 locus in field populations. RESULTS: A genotype assay was developed for direct detection of changes in distribution and frequency of a phosphine resistance allele in field populations of R. dominica. Beetles were collected from ten farms in south-east Queensland in 2006 and resampled in 2011. Resistance allele frequency increased in the period from 2006 to 2011 on organic farms with no history of phosphine use, implying that migration of phosphine-resistant R. dominica had occurred from nearby storages. CONCLUSION: Increasing resistance allele frequencies on organic farms suggest local movement of beetles and dispersal of insects from areas where phosphine has been used. This research also highlighted for the first time the utility of a genetic DNA marker in accurate and rapid determination of the distribution of phosphine-resistant insects in the grain value chain. Extending this research over larger landscapes would help in identifying resistance problems and enable timely pest management decisions. © 2013 Society of Chemical Industry © 2013 Society of Chemical Industry 69 6 June 2013 10.1002/ps.3514 Rapid Report Rapid Report © 2013 Society of Chemical Industry.

Inheritance and relative dominance, expressed as toxicity response and delayed development, of phosphine resistance in immature stages of Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae)

Fumigation of stored grain with phosphine (PH 3) is used widely to control the lesser grain borer Rhyzopertha dominica. However, development of high level resistance to phosphine in this species threatens control. Effective resistance management relies on knowledge of the expression of resistance in relation to dosage at all life stages. Therefore, we determined the mode of inheritance of phosphine resistance and strength of the resistance phenotype at each developmental stage. We achieved this by comparing mortality and developmental delay between a strongly resistant strain (R-strain), a susceptible strain (S-strain) and their F 1 progenies. Resistance was a maternally inherited, semi-dominant trait in the egg stage but was inherited as an autosomal, incompletely recessive trait in larvae and pupae. The rank order of developmental tolerance in both the sensitive and resistant strains was eggs > pupae > larvae. Comparison of published values for the response of adult R. dominica relative to our results from immature stages reveals that the adult stage of the S-strain is more sensitive to phosphine than are larvae. This situation is reversed in the R-strain as the adult stage is much more resistant to phosphine than even the most tolerant immature stage. Phosphine resistance factors at LC 50 were eggs 400×, larvae 87× and pupae 181× with respect to reference susceptible strain (S-strain) adults indicating that tolerance conferred by a particular immature stage neither strongly nor reliably interacts with the genetic resistance element. Developmental delay relative to unfumigated control insects was observed in 93% of resistant pupae, 86% of resistant larvae and 41% of resistant eggs. Increased delay in development and the toxicity response to phosphine exposure were both incompletely recessive. We show that resistance to phosphine has pleiotropic effects and that the expression of these effects varies with genotype and throughout the life history of the insect. © 2012.

A core metabolic enzyme mediates resistance to phosphine gas

Phosphine is a small redox-active gas that is used to protect global grain reserves, which are threatened by the emergence of phosphine resistance in pest insects. We find that polymorphisms responsible for genetic resistance cluster around the redox-active catalytic disulfide or the dimerization interface of dihydrolipoamide dehydrogenase (DLD) in insects (Rhyzopertha dominica and Tribolium castaneum) and nematodes (Caenorhabditis elegans). DLD is a core metabolic enzyme representing a new class of resistance factor for a redox-active metabolic toxin. It participates in four key steps of core metabolism, and metabolite profiles indicate that phosphine exposure in mutant and wild-type animals affects these steps differently. Mutation of DLD in C. elegans increases arsenite sensitivity. This specific vulnerability may be exploited to control phosphine-resistant insects and safeguard food security.

Dosage consistency is the key factor in avoiding evolution of resistance to phosphine and population increase in stored-grain pests

BACKGROUND Control of pests in stored grain and the evolution of resistance to pesticides are serious problems worldwide. A stochastic individual-based two-locus model was used to investigate the impact of two important issues, the consistency of pesticide dosage through the storage facility and the immigration rate of the adult pest, on overall population control and avoidance of evolution of resistance to the fumigant phosphine in an important pest of stored grain, the lesser grain borer. RESULTS A very consistent dosage maintained good control for all immigration rates, while an inconsistent dosage failed to maintain control in all cases. At intermediate dosage consistency, immigration rate became a critical factor in whether control was maintained or resistance emerged. CONCLUSION Achieving a consistent fumigant dosage is a key factor in avoiding evolution of resistance to phosphine and maintaining control of populations of stored-grain pests; when the dosage achieved is very inconsistent, there is likely to be a problem regardless of immigration rate. © 2012 Society of Chemical Industry

Constructing a new individual-based model of phosphine resistance in lesser grain borer (Rhyzopertha dominica): do we need to include two loci rather than one?

In this article, we describe and compare two individual-based models constructed to investigate how genetic factors influence the development of phosphine resistance in lesser grain borer (R. dominica). One model is based on the simplifying assumption that resistance is conferred by alleles at a single locus, while the other is based on the more realistic assumption that resistance is conferred by alleles at two separate loci. We simulated the population dynamic of R. dominica in the absence of phosphine fumigation, and under high and low dose phosphine treatments, and found important differences between the predictions of the two models in all three cases. In the absence of fumigation, starting from the same initial frequencies of genotypes, the two models tended to different stable frequencies, although both reached Hardy-Weinberg equilibrium. The one-locus model exaggerated the equilibrium proportion of strongly resistant beetles by 3.6 times, compared to the aggregated predictions of the two-locus model. Under a low dose treatment the one-locus model overestimated the proportion of strongly resistant individuals within the population and underestimated the total population numbers compared to the two-locus model. These results show the importance of basing resistance evolution models on realistic genetics and that using oversimplified one-locus models to develop pest control strategies runs the risk of not correctly identifying tactics to minimise the incidence of pest infestation.

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 20072011. 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 x , 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. (c) 2012 Commonwealth of Australia. Published by John Wiley & Sons, Ltd.