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.     

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.     

Phosphine resistance in Tribolium castaneum and Rhyzopertha dominica from stored wheat in Oklahoma

Phosphine gas, or hydrogen phosphide (PH3), is the most common insecticide applied to durable stored products worldwide and is routinely used in the United States for treatment of bulk-stored cereal grains and other durable stored products. Research from the late 1980s revealed low frequencies of resistance to various residual grain protectant insecticides and to phosphine in grain insect species collected in Oklahoma. The present work, which used the same previously established discriminating dose bioassays for phosphine toxicity as in the earlier study, evaluated adults of nine different populations of red flour beetle, Tribolium castaneum (Herbst), and five populations of lesser grain borer, Rhyzopertha dominica (F.) collected from different geographic locations in Oklahoma. One additional population for each species was a laboratory susceptible strain. Discriminating dose assays determined eight out of the nine T. castaneum populations, and all five populations of R. dominica, contained phosphine-resistant individuals, and highest resistance frequencies were 94 and 98%, respectively. Dose-response bioassays and logit analyses determined that LC99 values were approximately 3 ppm for susceptible and 377 ppm for resistant T. castaneum, and approximately 2 ppm for susceptible and 3,430 ppm for resistant R. dominica. The most resistant T. castaneum population was 119-fold more resistant than the susceptible strain and the most resistant R. dominica population was over 1,500-fold more resistant. Results suggest a substantial increase in phosphine resistance in these major stored-wheat pests in the past 21 yr, and these levels of resistance to phosphine approach those reported for other stored-grain pest species in other countries.     

Inheritance and Characterization of Strong Resistance to Phosphine in Sitophilus oryzae (L.)

Sitophilus oryzae (Linnaeus) is a major pest of stored grain across Southeast Asia and is of increasing concern in other regions due to the advent of strong resistance to phosphine, the fumigant used to protect stored grain from pest insects. We investigated the inheritance of genes controlling resistance to phosphine in a strongly resistant S. oryzae strain (NNSO7525) collected in Australia and find that the trait is autosomally inherited and incompletely recessive with a degree of dominance of -0.66. The strongly resistant strain has an LC50 52 times greater than a susceptible reference strain (LS2) and 9 times greater than a weakly resistant strain (QSO335). Analysis of F2 and backcross progeny indicates that two or more genes are responsible for strong resistance, and that one of these genes, designated So_rph1, not only contributes to strong resistance, but is also responsible for the weak resistance phenotype of strain QSO335. These results demonstrate that the genetic mechanism of phosphine resistance in S. oryzae is similar to that of other stored product insect pests. A unique observation is that a subset of the progeny of an F1 backcross generation are more strongly resistant to phosphine than the parental strongly resistant strain, which may be caused by multiple alleles of one of the resistance genes.     

Field evaluation of spinosad as a grain protectant for stored wheat in Australia: efficacy against Rhyzopertha dominica (F.) and fate of residues in whole wheat and milling fractions

Abstract  The potential of spinosad as a grain protectant for the lesser grain borer, Rhyzopertha dominica , was investigated in a silo-scale trial on wheat stored in Victoria, Australia. Rhyzopertha dominica is a serious pest of stored grain, and its resistance to protectants and the fumigant phosphine is becoming more common. This trial follows earlier laboratory research showing that spinosad may be a useful pest management option for this species. Wheat (300 t) from the 2005 harvest was treated with spinosad 0.96 mg/kg plus chlorpyrifos-methyl 10 mg/kg in March 2006, and samples were collected at intervals during 7.5 month storage to determine efficacy and residues in wheat and milling fractions. Chlorpyrifos-methyl is already registered in Australia for control of several other pest species, and its low potency against R. dominica was confirmed in laboratory-treated wheat. Grain moisture content was stable at about 10%, but grain temperature ranged from 29.3°C in March to 14.0°C in August. Bioassays of all treated wheat samples over 7.5 months resulted in 100% adult mortality after 2 weeks exposure and no live progeny were produced. In addition, no live grain insects were detected during outload sampling after a 9 month storage. Spinosad and chlorpyrifos-methyl residues tended to decline during storage, and residues were higher in the bran layer than in either wholemeal or white flour. This field trial confirmed that spinosad was effective as a grain protectant targeting R. dominica .     

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.     

Genetic linkage analysis of the lesser grain borer Rhyzopertha dominica identifies two loci that confer high-level resistance to the fumigant phosphine

High levels of inheritable resistance to phosphine in Rhyzopertha dominica have recently been detected in Australia and in an effort to isolate the genes responsible for resistance we have used random amplified DNA fingerprinting (RAF) to produce a genetic linkage map of R. dominica. The map consists of 94 dominant DNA markers with an average distance between markers of 4.6 cM and defines nine linkage groups with a total recombination distance of 390.1 cM. We have identified two loci that are responsible for high-level resistance. One provides approximately 50x resistance to phosphine while the other provides 12.5x resistance and in combination, the two genes act synergistically to provide a resistance level 250x greater than that of fully susceptible beetles. The haploid genome size has been determined to be 4.76 x 10(8) bp, resulting in an average physical distance of 1.2 Mbp per map unit. No recombination has been observed between either of the two resistance loci and their adjacent DNA markers in a population of 44 fully resistant F5 individuals, which indicates that the genes are likely to reside within 0.91 cM (1.1 Mbp) of the DNA markers.     

Phosphine resistance in India is characterised by a dihydrolipoamide dehydrogenase variant that is otherwise unobserved in eukaryotes

Phosphine (PH₃) fumigation is the primary method worldwide for controlling insect pests of stored commodities. Over-reliance on phosphine, however, has led to the emergence of strong resistance. Detailed genetic studies previously identified two loci, rph1 and rph2, that interact synergistically to create a strong resistance phenotype. We compared the genetics of phosphine resistance in strains of Rhyzopertha dominica and Tribolium castaneum from India and Australia, countries having similar pest species but widely differing in pest management practices. Sequencing analysis of the rph2 locus, dihydrolipoamide dehydrogenase (dld), identified two structurally equivalent variants, Proline49>Serine (P49S) in one R. dominica strain and P45S in three strains of T. castaneum from India. These variants of the DLD protein likely affect FAD cofactor interaction with the enzyme. A survey of insects from storage facilities across southern India revealed that the P45/49S variant is distributed throughout the region at very high frequencies, in up to 94% of R. dominica and 97% of T. castaneum in the state of Tamil Nadu. The abundance of the P45/49S variant in insect populations contrasted sharply with the evolutionary record in which the variant was absent from eukaryotic DLD sequences. This suggests that the variant is unlikely to provide a strong selective advantage in the absence of phosphine fumigation.     

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.     

Allozyme variation among Brazilian and U.S. populations ofRhyzopertha dominicaresistant to insecticides

The lesser grain borer (Rhyzopertha dominica (F.)) is an important pest of stored grain in many parts of the world (Paleartic, Ethiopian, Oriental, Australian, Neotropical, and Neartic regions) with the ability to fly long distances. These insects have been shown to be resistant to organophosphorus insecticides in several studies. Polyacrylamide gel electrophoresis was used to assess the genetic variability within and among eight Brazilian and seven United States populations of R. dominica and to determine how insecticide resistance may be spreading within both countries. Significant variation in allele frequency among populations was observed at all six polymorphic enzyme loci that were examined. The Brazilian and U.S. populations were genetically differentiated from one another; populations within the U.S. and those within Brazil were also differentiated from one another. The mean genetic similarity among the seven U.S. populations collected in a small region in northeast Kansas was smaller than that among eight Brazilian populations collected in a relatively large geographical area. These results are consistent with the resistance ratios to chlorpyriphos-methyl in R. dominica populations from Brazil and the U.S. and the information available concerning patterns of flight activity in this insect.     

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.     

谷蠹和米象对磷化氢抗性遗传的研究

本文就谷蠹Rhyzopertha dominica 和米象Sitophilus oryzae对磷化氢的抗性遗传进行了研究,分别对两个种的实验室敏感品系和粮仓现场采集的抗性品系作为亲本进行杂交。同时测定了各自的亲本及其F₁杂种、F₁对抗性亲本回交和F2混交后代的剂量-死亡率反应曲线,并对它们的结果进行了遗传分析。结果指出,谷蠹和米象的F₁杂种对磷化氢的抗性遗传都为不完全隐性,各自的显性度(D)分别是-0.768和-0.348。同时F₁,回交和F₂混交的观察值和计算值之间的X²分析也表明,抗性表现为一个以上的常染色体因子遗传,但是主要是受隐性因子所控制。     

Susceptibility of laboratory and field strains of four stored-product insect species to spinosad

Two field strains of the Indianmeal moth, Plodia interpunctella (Hübner); red flour beetle, Tribolium castaneum (Herbst); and lesser grain borer, Rhyzopertha dominica (F.), and one field strain of the rusty grain beetle, Cryptolestes ferrugineus (Stephens), were collected from hard red winter wheat stored on farms in northeastern Kansas. Fifty eggs of P. interpunctella and 25 beetle adults of each species were exposed to 100 g of untreated wheat or wheat treated with various rates of spinosad, to determine susceptibility of the field and corresponding insecticide-susceptible laboratory strains. Mortality of beetle adults and P. interpunctella larvae was assessed after 7 and 21 d postinfestation, respectively. Field strains of P. interpunctella, C. ferrugineus, and T. castaneum were less susceptible to spinosad than the corresponding laboratory strains. The LD50 and LD95 values for P. interpunctella and C. ferrugineus field strains were 1.7-2.5 times greater than values for corresponding laboratory strains. Adults of both laboratory and field strains of T. castaneum were tolerant to spinosad, resulting in <88% mortality at 8 mg/kg. The LD50 and LD95 values for the field strains of T. castaneum were 2.0-7.5 times greater compared with similar values for the laboratory strain. The field and laboratory strains of R. dominica were highly susceptible to spinosad, and one of the field strains was relatively less susceptible to spinosad than the laboratory strain. Our results confirm a range of biological variability in field populations, which is consistent with findings for other compounds, and underscores the need to adopt resistance management programs with stored grain insect pests. The baseline data generated on the susceptibility of the four insect species to spinosad will be useful for monitoring resistance development and for setting field rates.     

RESISTANCE TO GRAIN PROTECTANTS AND PHOSPHINE IN COLEOPTEROUS PESTS OF GRAIN STORED ON FARMS IN NEW SOUTH WALES

Tribolium castaneum (Herbst), T. confusum Jacquelin du Val, Sitophilus oryzae (L.), S. granarius (L.), Oryzaephilus surinamensis (L.) and Rhyzopertha dominica (F.) were collected from farms in grain-growing areas in southern and northern N.S.W. and bioassayed for resistance to the grain protectants malathion, fenitrothion, bioresmethrin, carbaryl, pirimiphos-methyl, chlorpyrifos-methyl and the fumigant phosphine.
Results showed that resistance on farms in N.S.W. is similar to resistance reported elsewhere in Australia's grain handling system during the past decade. The only new resistance of practical significance was detected in O. surinamensis where 50% of populations were resistant to fenitrothion (maximum R.F. 68.4x). Fenitrothion resistance was uncommon or not detected in other species. Pirimiphos-methyl resistance was detected in 70% of O. surinamensis populations (maximum RF 44.2x) and in 1 population of T. castaneum (maximum RF 3.1x). Resistance to chlorpyrifos-methyl was detected in 39% of the O. surinamensis populations (maximum RF 8.4x), the only species tested. Low level phosphine resistance was detected in all species (maximum RF 2.2x). Malathion resistance was detected in all species except S. granarius . Resistance to bioresmethrin and carbaryl was not detected in R. dominica , the only species tested.     

Influence of phosphine resistance genes on flight propensity and resource location in Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae): the landscape for selection

Phosphine resistance in Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) has evolved through changes to enzymes involved in basic metabolic pathways. These changes impose metabolic stress and could affect energy‐demanding behaviours. We therefore tested whether phosphine resistance alleles impact the movement of these insects in their quest for new resources. We measured walking and flight parameters of four T. castaneum genotypes: (1) a field‐derived population, (2) a laboratory cultured, phosphine‐susceptible reference strain, (3) a laboratory cultured, phosphine‐resistant reference strain, and (4) a resistant introgressed strain that is almost identical genetically to the susceptible population. The temporal pattern of flight was identical across all populations, but resistant beetles took flight significantly less, walked more slowly, and located resources less successfully than did susceptible beetles. Also, the field‐derived beetles (proved not to be carrying resistance genes) walked significantly faster and more directly towards food resources, and had a higher propensity for flight when compared to the susceptible laboratory beetles. These negative effects suggest survival of beetles with the resistance alleles will be compromised should they leave phosphine application sites. The field for selection therefore extends beyond the site at which phosphine fumigant imposed its effect, and other mutations are also likely to be affected in this way.     

赤拟谷盗的磷化氢敏感和抗性品系体内羧酸酯酶的活性比较

本文比较测定了赤拟谷盗Tribolium castaneum(Herbst)的磷化氢抗性(Rf=327)和敏感品系害虫的羧酸酯酶活性,研究了该害虫同一品系不同个体间羧酸酯酶的活性差异,比较了两品系害虫在系列磷化氢浓度下熏蒸24h和6.94×10-2mg/L磷化氢浓度下熏蒸不同时间的羧酸酯酶活性。主要结果为:未熏蒸的抗性害虫幼虫和蛹体内的羧酸酯酶活性分别高于敏感品系的1.37和1.16倍;敏感和抗性害虫同品系内不同个体间羧酸酯酶活性分布频率都存在明显差异,抗性害虫中酶活性大的个体数量占的比例较大;磷化氢浓度分别为0.69×10-2、2.78×10-2、5.56×10-2、8.33×10-2和11.11×10-2mg/L时都可导致敏感害虫羧酸酯酶的活性降低,但活性受抑制的程度不因浓度高低呈相应的增减。浓度分别为5.56×10-2、11.11×10-2、13.89×10-2、20.83×10-2和27.78×10-2mg/L的熏蒸中抗性害虫体内酶活性增加,且活性增高的程度与浓度增减也不呈对应变化。在6.94×10-2mg/L磷化氢浓度下熏蒸不同时间的结果中,敏感害虫的酶活性随时间延长而下降,抗性害虫的活性则随时间延长而增大。研究表明赤拟谷盗对磷化氢的抗性可能与羧酸酯酶的活性增加有关。     

Alterations in esterases are associated with malathion resistance in Habrobracon hebetor (Hymenoptera: Braconidae)

Biochemical mechanisms of malathion resistance were investigated in a malathion-resistant strain of the parasitoid Habrobracon hebetor Say collected from a farm storage in Kansas. General esterase activities were significantly lower in the resistant strain compared with those in a susceptible strain. However, no significant differences were found in activities of malathion specific carboxylesterase (MCE), glutathione S-transferase and cytochrome P450 dependent O-demethylase activities, cytochrome P450 contents, and sensitivity of acetylcholinesterase to inhibition by malaoxon between the 2 strains. Because MCE was not elevated in the resistant strain, the weak malathion resistance in H. hebetor may result from a different mechanism compared with that hypothesized for some insect species in which reduced general esterase activity is accompanied by an elevated MCE. Decreased esterase activity in the resistant strain suggested that null alleles of some esterases were associated with the resistance. Indeed, E1 and E2, major esterases in the susceptible strain, were not present in the resistant strain on polyacrylamide gels that were stained for esterase activity using the model substrate 1-naphthyl acetate. In contrast, the activity of esterase E3 on the gels was much higher in the resistant strain as compared with that of the susceptible strain. These findings indicate that malathion resistance in H. hebetor is associated with both an increased activity of the esterase E3 and null alleles of the esterases E1 and E2.     

Are mitochondrial lineages,mitochondrial lysis and respiration rate associated with phosphine susceptibility in the maize weevil Sitophilus zeamais?

Phosphine is the most widely used fumigant with ever‐growing problems of phosphine resistance among insect pests of stored products. One such insect is the maize weevil Sitophilus zeamais, a key pest of stored cereals. Despite its importance as a fumigant, the mechanisms of phosphine toxicity and resistance remain unclear, although the mitochondrion is broadly recognised as its site of action. Here we explored the phosphine susceptibility of maize weevil populations and its association with insect respiration rate, and we tested the association of phosphine susceptibility with the mitochondrial lineages from the field populations studied. We also assessed the action of phosphine in the degradation of mitochondria from muscle cells. Survival under phosphine treatment varied among weevil populations and was negatively correlated with the respiration rate and body mass of the insect. Phosphine produced little lysis of mitochondria and the more phosphine‐resistant population exhibited a slightly higher mitochondria fluorescence intensity under confocal imaging. Therefore, reduced respiration rate is correlated with reduced phosphine activity, but its association with high mitochondria fluorescence intensity in muscle cells seems marginal. There was no association between mitochondrial lineages and phosphine susceptibility, which evolved independently, and the mitochondrial gene fragments of cytochrome oxidase I and II were not useful molecular markers of phosphine susceptibility.     

Diagnostic Molecular Markers for Phosphine Resistance in U.S. Populations of Tribolium castaneum and Rhyzopertha dominica

Stored product beetles that are resistant to the fumigant pesticide phosphine (hydrogen phosphide) gas have been reported for more than 40 years in many places worldwide. Traditionally, determination of phosphine resistance in stored product beetles is based on a discriminating dose bioassay that can take up to two weeks to evaluate. We developed a diagnostic cleaved amplified polymorphic sequence method, CAPS, to detect individuals with alleles for strong resistance to phosphine in populations of the red flour beetle, Tribolium castaneum, and the lesser grain borer, Rhyzopertha dominica, according to a single nucleotide mutation in the dihydrolipoamide dehydrogenase (DLD) gene. We initially isolated and sequenced the DLD genes from susceptible and strongly resistant populations of both species. The corresponding amino acid sequences were then deduced. A single amino acid mutation in DLD in populations of T. castaneum and R. dominica with strong resistance was identified as P45S in T. castaneum and P49S in R. dominica, both collected from northern Oklahoma, USA. PCR products containing these mutations were digested by the restriction enzymes MboI and BstNI, which revealed presence or absence, respectively of the resistant (R) allele and allowed inference of genotypes with that allele. Seven populations of T. castaneum from Kansas were subjected to discriminating dose bioassays for the weak and strong resistance phenotypes. Application of CAPS to these seven populations confirmed the R allele was in high frequency in the strongly resistant populations, and was absent or at a lower frequency in populations with weak resistance, which suggests that these populations with a low frequency of the R allele have the potential for selection of the strong resistance phenotype. CAPS markers for strong phosphine resistance will help to detect and confirm resistant beetles and can facilitate resistance management actions against a given pest population.