Gregory J. Daglish

Genetics of Resistance to Phosphine in Rhyzopertha dominica (Coleoptera: Bostrichidae)

Abstract 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 of the F1and 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 ≈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.

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.

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.

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.

Potential of the neonicotinoid imidacloprid and the oxadiazine indoxacarb for controlling five coleopteran pests of stored grain

Abstract  The potential for using imidacloprid (a neonicotinoid) and indoxacarb (an oxadiazine) as grain protectants was investigated in bioassays against resistant strains of five stored grain beetles. The species investigated were Rhyzopertha dominica (F.) (the lesser grain borer), Sitophilus oryzae (L.) (the rice weevil), Tribolium castaneum (Herbst) (the rust‐red flour beetle), Oryzaephilus surinamensis (L.) (the saw tooth flour beetle), and Cryptolestes ferrugineus (Stephens) (the flat grain beetle). Each of these species has developed resistance to one or more protectants, including organophosphorus insecticides, synthetic pyrethroids and the juvenile hormone analogue methoprene. Mortality and reproduction after a 2‐week exposure of adults to treated wheat depended on species, dose and insecticide. Imidacloprid had no effect on S. oryzae at any dose, but none of the other species produced any live progeny at 10 mg/kg. Indoxacarb had no effect on T. castaneum at any dose, but none of the other species produced any live progeny at 5 mg/kg. The results show that although both imidacloprid and indoxacarb can control at least four of the five key pests tested at doses comparable to those used for organophosphorus protectants, more potent neonicotinoid or oxadiazine insecticides would be needed than either of these to provide broad spectrum protection of stored grain.

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 (SRQLD), New South Wales (SRNSW) and South Australia (SRSA), 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, SR2QLD, also from Queensland. The previously described rph2 gene, which interacts synergistically with rph1 gene, confers strong resistance on SRQLD and SRNSW. We now provide strong circumstantial evidence that weak alleles of rph2, together with rph1, contribute to the strong resistance phenotypes of SRSA and SR2QLD. 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.

Responses of tribolium castaneum to olfactory cues from cotton seeds, the fungi associated with cotton seeds, and cereals

We tested, in an olfactometer, whether or not Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) responds preferentially to the volatiles that emanate from the fungi associated with cotton [Gossypium hirsutum L. (Malvaceae)] seed over those that emanate from cereals, because cereals are usually portrayed as the primary resources of these beetles. Pairwise comparisons were conducted between cotton seed, wheat (Triticum aestivum L.), and sorghum [Sorghum bicolor (L.) Moench] (both Poaceae); volatiles were tested from intact seeds and from both water and ethanol extracts. The results demonstrate that T. castaneum is attracted more strongly to cotton seeds with its lint contaminated with fungi, than to the conventional resources of this species (i.e., wheat and sorghum). Further tests prove that it is the fungus on the lint that produces the active volatiles, because the beetles did not respond to sterilized cotton lint (i.e., without the fungi typically associated with it when cotton seed is stored). Tests with five fungal cultures (each representing an unidentified species that was isolated from the field‐collected cotton lint) were variable across the cultures, with only one of them being significantly attractive to the beetles. The others were not attractive and one may even have repulsed the beetles. The results are consistent with the beetles having a strong ecological association with fungi and suggest it would be worth investigating the ecology of T. castaneum from this perspective.

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.

Use of sulfuryl fluoride in the management of strongly phosphine-resistant insect pest populations in bulk grain storages in Australia

Sulfuryl fluoride (SF), an effective structural fumigant, is registered recently as Profume™ for controlling insect pests of stored grains and processed commodities. Information on its effectiveness in disinfestation of bulk grain, however, is limited. The ongoing problem with the strong level of resistance to phosphine has been addressed recently through deployment of SF as a ‘resistance breaker’ in bulk storages in Australia. This paper discusses important results on the efficacy of SF against key phosphine- resistant insect pests, lesser grain borer, Rhyzopertha dominca, red flour beetle, Tribolium castaneum, rice weevil, Sitophilus oryzae and the rusty grain beetle, Cryptolestes ferrugineus. We have established CT (g-hm3) profiles for SF against these insect pests at two temperature regimes 25 and 30°C, that showed that both temperature and exposure period (t) has significant influence on the effectiveness of SF than the concentration. Over a seven days fumigation period, CTs of 800 and 400 g-hm3 achieved complete control of all the target pests, including the most strongly phosphine - resistant species, C. ferrugineus at 25 and 30°C, respectively. Results from four industry scale field trials involving currently registered rate of SF (1500 g-hm3) over 2–14 d exposure period, confirmed its effectiveness in achieving complete control of the target pest species. The assessment of postfumigation grain samples across all the test storages indicated that the reinfestation occurs after three months. Monitoring resistance to phosphine in C. ferrugineus over a six year period (2009–2015), showed a significant reduction in resistant populations after the introduction of SF into the fumigation strategy at problematic storage sites. Overall our research concludes that SF is a good candidate to be used as a ‘resistance breaker’ where phosphine resistance is prevalent.