Co-fumigation with phosphine and sulfuryl fluoride: Potential for managing strongly phosphine-resistant rusty grain beetle, Cryptolestes ferrugineus (Stephens): Presentation

Populations of rusty grain beetle, Cryptolestes ferrugineus, have developed a very high level of resistance (1300×) to the fumigant phosphine (PH3) in Australia. Resistant insects triggered control failures, threatening the country’s annual grain market worth AU$8 billion. Although PH3 protocols were amended to manage this new resistance, fumigation requires lengthy exposure periods which has practical difficulties. While there is no suitable replacement for PH3, the current study explores potential approaches to enhance the efficacy of this fumigant. One possibility is co-fumigation of PH3 with another complementary fumigant, sulfuryl fluoride (SO2F2 or SF), with the dual goals: enhanced efficacy and minimise use of both fumigants. A cohort of mixed age eggs and adults of PH3-resistant C. ferrugineus was fumigated with PH3 and SF individually, as well as in combination inside desiccators at 25°C and 60$8 billion. Although PH3 protocols were amended to manage this new resistance, fumigation requires lengthy exposure periods which has practical difficulties. While there is no suitable replacement for PH3, the current study explores potential approaches to enhance the efficacy of this fumigant. One possibility is co-fumigation of PH3 with another complementary fumigant, sulfuryl fluoride (SO2F2 or SF), with the dual goals: enhanced efficacy and minimise use of both fumigants. A cohort of mixed age eggs and adults of PH3-resistant C. ferrugineus was fumigated with PH3 and SF individually, as well as in combination inside desiccators at 25°C and 60%RH for 168 h. Two doses below the maximal registered rates for SF (8.9 mg L- 1, equivalent to 1500 g hm-3) and PH3 (1.0 mg L-1) were tested. Co-fumigation was performed simultaneously for 168 h. Our results revealed that, the mixture of 1.1 mg L-1 or 2.2 mg L-1 of SF and 0.5 mg L-1 of PH3 over 168 h achieved complete control against resistant C. ferrugineus eggs and adults, whereas each of the tested doses failed individually. Our study confirms that SF and PH3 enhance the efficacy of each other when used in combination, which holds great potential for managing resistant C. ferrugineus

Gene introgression in assessing fitness costs associated with phosphine resistance in the rusty grain beetle

The current study investigates the fitness cost associated with phosphine resistance in the rusty grain beetle, Cryptolestes ferrugineus (Stephens), a problematic pest in the stored commodities that has developed strong resistance to fumigant phosphine. Three characterised insect strains: the susceptible (Ref-S), the strongly resistant (Ref-R), the introgressed resistant (Intro-R) and a segregating population (F25) derived from crossing the Ref-S and Ref-R strains were used in this study. Intro-R was developed by introgressing two phosphine resistance genes, cf_rph1 and cf_rph2 into Ref-S, aimed to reduce the influence of background genetic factors. Intro-R exhibited 592 × resistance to phosphine and homozygous for strong resistance allele, cf_rph2 (L73N). Two key fitness cost criteria, developmental time and fecundity, were assessed under optimal and suboptimal conditions (less favourable diet and low temperature). There was no significant difference in developmental time and fecundity between Ref-S and either Intro-R strain or F25 under optimal conditions. When challenged with a less favourable diet, cracked wheat + cracked sorghum (CW + CS), or exposed to a low temperature (22 °C), both Intro-R and Ref-S had similar developmental time and total numbers of F1 progeny, confirming the absence of significant fitness effects expressed in these conditions. Therefore, we conclude that strongly phosphine resistant C. ferrugineus are unlikely to incur potential fitness costs. This finding will have implications towards developing strategies to manage this pest

Potential for using pheromone trapping and molecular screening in phosphine resistance research: Presentation

Phosphine resistance monitoring typically involves bioassays of beetles from population samples collected from grain storage facilities. Insects are classified into susceptible or resistant phenotypes based on mortality or survival at one or more discriminating doses. Although valuable, phenotype testing has several drawbacks. First, phenotype testing needs live insects, and considerable effort is required to collect and maintain them before testing. Second, population samples may contain multiple genotypes expressing different levels of resistance that may not be distinguishable using discriminating dose bioassays. Third, collections are likely to be focussed around grain storages to maximise sampling success. Recent research shows that several key pests are actively dispersing through flight. The availability of commercial pheromone lures and recent advances in molecular screening provide an opportunity to provide information on resistance gene frequencies more broadly across the landscape. This approach is proving to be a valuable adjunct to traditional resistance testing in Australia.Phosphine resistance monitoring typically involves bioassays of beetles from population samples collected from grain storage facilities. Insects are classified into susceptible or resistant phenotypes based on mortality or survival at one or more discriminating doses. Although valuable, phenotype testing has several drawbacks. First, phenotype testing needs live insects, and considerable effort is required to collect and maintain them before testing. Second, population samples may contain multiple genotypes expressing different levels of resistance that may not be distinguishable using discriminating dose bioassays. Third, collections are likely to be focussed around grain storages to maximise sampling success. Recent research shows that several key pests are actively dispersing through flight. The availability of commercial pheromone lures and recent advances in molecular screening provide an opportunity to provide information on resistance gene frequencies more broadly across the landscape. This approach is proving to be a valuable adjunct to traditional resistance testing in Australia

Utility of biotechnology based decision making tools in postharvest grain pest management: an Australian case study

A major concern for the Australian grain industry in recent years is the constant threat of resistance to the key disinfectant phosphine in a range of stored grain pests. The need to maintain the usefulness of phosphine and to contain the development of resistance are critical to international market access for Australian grain. Strong levels of resistance have already been established in major pests including the lesser grain borer, Rhyzopertha dominica (F.), the red flour beetle, Tribolium castaneum (Herbst), and most recently in the rusty grain beetle Cryptolestes ferrugineus (Stephens). As a proactive integrated resistance management strategy, new fumigation protocols are being developed in the laboratory and verified in large-scale field trials in collaboration with industry partners. To aid this development, we have deployed advanced molecular diagnostic tools to accurately determine the strength and frequency of key phosphine resistant insect pests and their movement within a typical Australian grain value chain. For example, two major bulk storage facilities based at Brookstead and Millmerran in southeast Queensland, Australia, were selected as main nodes and several farms and feed mills located in and around these two sites at a scale of 25 to 100 km radius were selected and surveyed. We determined the type, pattern, frequency as well as the distribution of resistance alleles accurately for two major pests, R. dominica and T. castaneum. Overall, this information along with the phenotypic data, provide a basis for designing key intervention strategies in managing resistance problems in the study area

Utility of biotechnology based decision making tools in postharvest grain pest management: An Australian case study

A major concern for the Australian grain industry in recent years is the constant threat of resistance to the key disinfestant phosphine in a range of stored grain pests. The need to maintain the usefulness of phosphine and to contain the development of resistance are critical to international market access for Australian grain. Strong levels of resistance have already been established in major pests including the lesser grain borer, Rhyzopertha dominica (F.), the red flour beetle, Tribolium castaneum (Herbst), and most recently in the rusty grain beetle Cryptolestes ferrugineus (Stephens). As a proactive integrated resistance management strategy, new fumigation protocols are being developed in the laboratory and verified in large-scale field trials in collaboration with industry partners. To aid this development, we have deployed advanced molecular diagnostic tools to accurately determine the strength and frequency of key phosphine resistant insect pests and their movement within a typical Australian grain value chain. For example, two major bulk storage facilities based at Brookstead and Millmerran in southeast Queensland, Australia, were selected as main nodes and several farms and feed mills located in and around these two sites at a scale of 25 to 100 km radius were selected and surveyed. We determined the type, pattern, frequency as well as the distribution of resistance alleles accurately for two major pests, R. dominica and T. castaneum. Overall, this information along with the phenotypic data, provide a basis for designing key intervention strategies in managing resistance problems in the study area.A major concern for the Australian grain industry in recent years is the constant threat of resistance to the key disinfestant phosphine in a range of stored grain pests. The need to maintain the usefulness of phosphine and to contain the development of resistance are critical to international market access for Australian grain. Strong levels of resistance have already been established in major pests including the lesser grain borer, Rhyzopertha dominica (F.), the red flour beetle, Tribolium castaneum (Herbst), and most recently in the rusty grain beetle Cryptolestes ferrugineus (Stephens). As a proactive integrated resistance management strategy, new fumigation protocols are being developed in the laboratory and verified in large-scale field trials in collaboration with industry partners. To aid this development, we have deployed advanced molecular diagnostic tools to accurately determine the strength and frequency of key phosphine resistant insect pests and their movement within a typical Australian grain value chain. For example, two major bulk storage facilities based at Brookstead and Millmerran in southeast Queensland, Australia, were selected as main nodes and several farms and feed mills located in and around these two sites at a scale of 25 to 100 km radius were selected and surveyed. We determined the type, pattern, frequency as well as the distribution of resistance alleles accurately for two major pests, R. dominica and T. castaneum. Overall, this information along with the phenotypic data, provide a basis for designing key intervention strategies in managing resistance problems in the study area

Utility of biotechnology based decision making tools in postharvest grain pest management: an Australian case study

A major concern for the Australian grain industry in recent years is the constant threat of resistance to the key disinfectant phosphine in a range of stored grain pests. The need to maintain the usefulness of phosphine and to contain the development of resistance are critical to international market access for Australian grain. Strong levels of resistance have already been established in major pests including the lesser grain borer, Rhyzopertha dominica (F.), the red flour beetle, Tribolium castaneum (Herbst), and most recently in the rusty grain beetle Cryptolestes ferrugineus (Stephens). As a proactive integrated resistance management strategy, new fumigation protocols are being developed in the laboratory and verified in large-scale field trials in collaboration with industry partners. To aid this development, we have deployed advanced molecular diagnostic tools to accurately determine the strength and frequency of key phosphine resistant insect pests and their movement within a typical Australian grain value chain. For example, two major bulk storage facilities based at Brookstead and Millmerran in southeast Queensland, Australia, were selected as main nodes and several farms and feed mills located in and around these two sites at a scale of 25 to 100 km radius were selected and surveyed. We determined the type, pattern, frequency as well as the distribution of resistance alleles accurately for two major pests, R. dominica and T. castaneum. Overall, this information along with the phenotypic data, provide a basis for designing key intervention strategies in managing resistance problems in the study area

Special issue on emerging artificial intelligence applications and innovations: Papers from AIAI 2006

Resistance to phosphine (PH3) in key insect pests of stored grain is increasing, with a requirement for maintaining a dose as high as 1 mg l-1 for 14 d for effective fumigation, which is difficult to achieve under most commercial storage conditions. There is no suitable replacement for PH3, as most of the available alternatives suffer from specific weaknesses, creating an urgent need to increase the efficacy of this fumigant. One such possibility is co-fumigation of PH3 with another fumigant, sulfuryl fluoride (SO2F2-SF), with the goal of decreasing the time required for a successful fumigation. In this study, adult of two PH3-resistant strains in each of four key grain insect pests, Rhyzopertha dominica, (F.) (Coleoptera: Bostrichidae) Tribolium castaneum Herbst (Coleoptera: Tenebrionidae), Sitophilus oryzae (L.) (Coleoptera: Curculionidae), and Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae) were fumigated with PH3 and SF individually as well as in combinations at 25°C over 48 h. Mortality responses in each species were subjected to probit analysis to determine the LC50 and LC99.9 of PH3, SF, and PH3 + SF. Co-fumigation of PH3 with SF resulted in a 50% reduction of the PH3 concentration required to achieve 99.9% mortality in two pest species. For example, the PH3 + SF mixture, reduced the amount of PH3 required from 14.2-14.5 to 5.6-6.36 mg l-1 and from 2.71-5.03 to 0.93-1.2 mg l-1, respectively, for C. ferrugineus and S. oryzae. The overall mortality response to the PH3 + SF mixture followed an "additive model" suggesting that mutual enhancement in toxicity can be achieved with this mixture specifically to control PH3-resistant insects

Potential of Co-Fumigation with Phosphine (PH3) and Sulfuryl Fluoride (SO2F2) for the Management of Strongly Phosphine-Resistant Insect Pests of Stored Grain

Resistance to phosphine (PH3) in key insect pests of stored grain is increasing, with a requirement for maintaining a dose as high as 1 mg l−1 for 14 d for effective fumigation, which is difficult to achieve under most commercial storage conditions. There is no suitable replacement for PH3, as most of the available alternatives suffer from specific weaknesses, creating an urgent need to increase the efficacy of this fumigant. One such possibility is co-fumigation of PH3 with another fumigant, sulfuryl fluoride (SO2F2—SF), with the goal of decreasing the time required for a successful fumigation. In this study, adult of two PH3-resistant strains in each of four key grain insect pests, Rhyzopertha dominica, (F.) (Coleoptera: Bostrichidae) Tribolium castaneum Herbst (Coleoptera: Tenebrionidae), Sitophilus oryzae (L.) (Coleoptera: Curculionidae), and Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae) were fumigated with PH3 and SF individually as well as in combinations at 25°C over 48 h. Mortality responses in each species were subjected to probit analysis to determine the LC50 and LC99.9 of PH3, SF, and PH3 + SF. Co-fumigation of PH3 with SF resulted in a 50% reduction of the PH3 concentration required to achieve 99.9% mortality in two pest species. For example, the PH3 + SF mixture, reduced the amount of PH3 required from 14.2–14.5 to 5.6–6.36 mg l−1 and from 2.71–5.03 to 0.93–1.2 mg l−1, respectively, for C. ferrugineus and S. oryzae. The overall mortality response to the PH3 + SF mixture followed an “additive model” suggesting that mutual enhancement in toxicity can be achieved with this mixture specifically to control PH3-resistant insects

Unique genetic variants in dihydrolipoamide dehydrogenase (dld) gene confer strong resistance to phosphine in the rusty grain beetle, Cryptolestes ferrugineus (Stephens)

The rusty grain beetle, Cryptolestes ferrugineus, a major pest of stored commodities, has developed very high levels (>1000×) of resistance to the fumigant phosphine. Resistance in this species is remarkably stronger than reported in any other stored product pests demanding the need to understand the molecular basis of this trait. Previous genetic studies in other grain insect pests identified specific variants in two major genes, rph1 and rph2 in conferring the strong resistance trait. However, in C. ferrugineus, although the gene, rph1 was identified as cytochrome-b5-fatty acid desaturase, the rph2 gene has not been reported so far. We tested the candidate gene for rph2, dihydrolipoamide dehydrogenase (dld) using the recently published transcriptome of C. ferrugineus and identified three variants, L73N and A355G + D360H, a haplotype, conferring resistance in this species. Our sequence analysis in resistant strain and phosphine selected resistant survivors indicates that these variants occur either alone as a homozygote or a mixture of heterozygotes (i.e complex heterozygotes) both conferring strong resistance. We also found that one of the three variants, possibly L73N expressing “dominant” trait at low frequency in resistant insects. Comparison of dld sequences between Australian and Chinese resistant strain of this species confirmed that the identified variants are highly conserved. Our fitness analysis indicated that resistant insects may not incur significant biological costs in the absence of phosphine selection for 19 generations. Thus, we propose that the observed high levels of resistance in C. ferrugineus could be primarily due to the characteristics of three unique variants, L73N and A355G + D360H within dld

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