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.     

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.     

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.     

Biological activity of two juvenoids and two ecdysteroids against three stored product insects

The insecticidal activity of juvenile hormone agonists methoprene and pyriproxyfen, and the ecdysone agonists RH-5849 and tebufenozide was evaluated against susceptible and actellic-resistant strains of Tribolium castaneum and susceptible strains of Rhyzopertha dominica and Sitophilus oryzae. Concentrations ranging from 0.1 to 20 ppm of the analogues were mixed in the food medium to which the tested insects were exposed. The results showed that all these compounds could affect the development of the tested species to differing extents but had no effect on the mortality of parental adults. The two JH analogues did not prolong the life span of R. dominica and S. oryzae, but very greatly extended that of T. castaneum. The extension led to the production of giant larvae and failure to pupate. Actellic-resistant strain of T. castaneum showed some cross-resistance to methoprene and pyriproxyfen, but not to RH-5849 and tebufenozide. Pyriproxyfen was the most effective compound among the four IGRs; a concentration of 0.1 ppm could completely inhibit the F(1) adult occurrence of both S- and R-strains of T. castaneum and its LC(90)s for controlling R. dominica and S. oryzae were 0.1 and 1.2 ppm, respectively. Methoprene was highly effective against R. dominica, but less active on S. oryzae. RH-5849 could achieve almost complete control of F(1) adults of T. castaneum and R. dominica at 10 ppm, but was less potent on S. oryzae. Tebufenozide appeared to be much less active on these three species compared with the other three compounds. The percentage reductions of F(1) adults for S- and R-strains of T. castaneum at a concentration of 20 ppm were 80 and 99%, respectively.     

Bioactivities of l-carvone, d-carvone, and dihydrocarvone toward three stored product beetles

The rice weevil, Sitophilus oryzae L., adults were highly susceptible by contact to l-carvone, d-carvone, and dihydrocarvone when compared with the lesser grain borer, Rhyzopertha dominica F., adults and red flour beetle, Tribolium castaneum (Herbst.). Adults of R. dominica were more susceptible than the other species to fumigant vapors of l-carvone, d-carvone, and dihydrocarvone. The three larval stages (14-, 16-, and 18-d-old) of T. castaneum progressively became more susceptible with age toward contact toxicity of three test compounds but in fumigant toxicity, 16-d-old larvae of T. castaneum were more susceptible to the three compounds. Comparison of contact and fumigant toxicity of the test compounds indicates that l-carvone and d-carvone possess 24 times more fumigant toxicity toward adults of R. dominica than its contact toxicity. Overall order of toxicity was l-carvone > d-carvone > dihydrocarvone. Egg hatching and subsequent larval and adult survival of T. castaneum were significantly reduced when the eggs of T. castaneum were treated with l-carvone, d-carvone, and dihydrocarvone. l-Carvone completely suppressed egg hatching at the concentration of 7.72 mg/cm2. Data on feeding-deterrent indices indicate the high potency of l-carvone as feeding-deterrent in order of S. oryzae adults > T. castaneum adults > R. dominica adults > T. castaneum larvae.     

Comparison of aeration and spinosad for suppressing insects in stored wheat

Field studies were conducted from July 2002 to January 2003 for evaluating the effects of controlled aeration and a commercial biological insecticide, spinosad, in suppressing insect populations in stored wheat. Six cylindrical steel bins were filled with newly harvested (2002 crop year) hard red winter wheat on 9 and 10 July 2002. Each bin contained 30.7 metric tons (1,100 bu) of wheat. Wheat in two bins was left untreated (control), whereas wheat in two bins was treated with spinosad, and in another two bins was subjected to aeration by using aeration controllers. Spinosad was applied to wheat at the time of bin filling to obtain a rate of 1 mg ([AI])/kg. Aeration controllers were set to run the fans when ambient air temperature fell below 23.9, 18.3, and 7.2 degrees C for the first, second, and third cooling cycles, respectively. We added 400 adults each of the rusty grain beetle, Cryptolestes ferrugineus (Stephens); lesser grain borer, Rhyzopertha dominica (F.); and red flour beetle, Tribolium castaneum (Herbst), to the grain at monthly intervals between July and October 2002. Insect density in the bins was estimated monthly by taking 3-kg grain samples from 21 locations within each bin by using a pneumatic grain sampler. No live T. castaneum or C. ferrugineus and very low densities of R. dominica (<0.008 adults per kilogram) were found in wheat treated with spinosad during the 6-mo sampling period. Density of C. ferrugineus and T. castaneum in aerated bins did not exceed two adults per kilogram (the Federal Grain Inspection Service standard for infested wheat), whereas R. dominica increased to 12 adults per kilogram in November 2002, which subsequently decreased to three adults per kilogram in January 2003. In the untreated (control) bins, R. dominica density increased faster than that of C. ferrugineus or T. castaneum. Density of R. dominica peaked at 58 adults per kilogram in October 2002 and decreased subsequently, whereas T. castaneum density was 10 adults per kilogram in October 2002 but increased to 78 adults per kilogram in January 2003. Density of C. ferrugineus increased steadily during the 6-mo study period and was highest (six adults per kilogram) in January 2003. This is the first report comparing the field efficacy of spinosad and aeration in managing insects in farm bins. Our results suggest that spinosad is very effective in suppressing R. dominica, C. ferrugineus, and T. castaneum populations in stored wheat.     

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.     

Effect of d-limonene on three stored-product beetles

d-Limonene was investigated for contact and fumigant toxicity, ovicidal effects, oviposition-deterrent, development inhibition, and feeding-deterrent activities against three stored-product beetles (Coleoptera): lesser grain borer, Rhyzopertha dominica (F.); rice weevil, Sitophilus oryzae (L.); and red flour beetle, Tribolium castaneum (Herbst). Contact and fumigant toxicity decreased as larvae aged. Contact toxicity was similar for adults of the three species tested, but R. dominica was most susceptible to fumigant activity. T. castaneum oviposition decreased as concentration of d-limonene increased and d-limonene reduced oviposition up to 92.3% at the concentration of 2.14 mg/cm2. Hatching of d-limonene-treated eggs of T. castaneum was reduced by 94.5% with no subsequent larval and adult survival at 2.14 mg/cm2 concentration. A flour disc bioassay indicated 87.7 to 96.8% feeding-deterrency by d-limonene toward all three insect species tested at the highest concentration of 60.0 mg/g food. These results suggest that d-limonene can be effectively used to suppress populations of stored-product beetles.     

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.     

种间竞争对四种储粮害虫种群动态的影响

在30 ℃、75 %相对湿度条件下研究种间竞争对玉米象(Sitophilus zeamais)、谷蠹(Rhizopertha domini-ca)、赤拟谷盗(Triboliumcastaneum)和锈赤扁谷盗(Cryptolestes ferrugineus)4种主要储粮害虫种群动态的影响,并对种群动态进行回归分析。结果表明,玉米象与谷蠹、赤拟谷盗与锈赤扁谷盗混合饲养种群增长均受到显著抑制,玉米象和谷蠹对赤拟谷盗和锈赤扁谷盗的种群增长具有明显的促进作用,赤拟谷盗和锈赤扁谷盗对玉米象和谷蠹的种群增长具有一定的抑制作用。回归分析结果表明玉米象种群最大增长潜能最大,锈赤扁谷盗最小,种群增长率变化规律不明显。     

一种新杀虫剂对储粮害虫和作物害虫的杀虫效果

测定以植物提取物异硫氰酸酯为主要成分而复配的宏劲杀虫剂对玉米象Sitophilus zeamais(Motschulsky)、赤拟谷盗Tribolium castaneum(Herbst)和谷蠹Rhizopertha dominica(Fabricius)3种重要储粮害虫的熏蒸效果及其对菜青虫Pieris rapae(L.)、红蜘蛛Tetranychus cinnabarinus(Boisduval)的杀虫效果。结果表明,宏劲杀虫剂48h对玉米象、赤拟谷盗和谷蠹的LC50分别是0.016,0.009和0.009μL/mL,均明显低于常规熏蒸剂磷化铝的要求处理剂量;对菜青虫和红蜘蛛的LC50分别是0.0159和1.3738mg/mL,其防效均优于对照药剂3%阿菊乳油和3%新型水分散性颗粒剂。     

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。     

Gene interactions constrain the course of evolution of phosphine resistance in the lesser grain borer, Rhyzopertha dominica

Phosphine, a widely used fumigant for the protection of stored grain from insect pests, kills organisms indirectly by inducing oxidative stress. High levels of heritable resistance to phosphine in the insect pest of stored grain, Rhyzopertha dominica have been detected in Asia, Australia and South America. In order to understand the evolution of phosphine resistance and to isolate the responsible genes, we have undertaken genetic linkage analysis of fully sensitive (QRD14), moderately resistant (QRD369) and highly resistant (QRD569) strains of R. dominica collected in Australia. We previously determined that two loci, rph1 and rph2, confer high-level resistance on strain QRD569, which was collected in 1997. We have now confirmed that rph1 is responsible for the moderate resistance of strain QRD369, which was collected in 1990, and is shared with a highly resistant strain from the same geographical region, QRD569. In contrast, rph2 by itself confers only very weak resistance, either as a heterozygote or as a homozygote and was not discovered in the field until weak resistance (probably due to rph1) had become ubiquitous. Thus, high-level resistance against phosphine has evolved via stepwise acquisition of resistance alleles, first at rph1 and thereafter at rph2. The semi-dominance of rph2 together with the synergistic interaction between rph1 and rph2 would have led to rapid selection for homozygosity. A lack of visible fitness cost associated with alleles at either locus suggests that the resistance phenotype will persist in the field.     

Bioactivities of the leaf essential oil of Curcuma longa (var. ch-66) on three species of stored-product beetles (Coleoptera)

Essential oil extracted from the leaves of turmeric, Curcuma longa L., was investigated for contact and fumigant toxicity and its effect on progeny production in three stored-product beetles, Rhyzopertha dominica F. (lesser grain borer), Sitophilus oryzae L. (rice weevil), and Tribolium castaneum Herbst (red flour beetle). Oviposition-deterrent and ovicidal actions of C. longa leaf oil were also evaluated against T. castaneum. The oil was insecticidal in both contact and fumigant toxicity assays. The adults of R. dominica were highly susceptible to contact action of C. longa leaf oil, with LD50 value of 36.71 microg/mg weight of insect, whereas in the fumigant assay, adults of S. oryzae were highly susceptible with LC50 value of 11.36 mg/liter air. Further, in T. castaneum, the C. longa oil reduced oviposition and egg hatching by 72 and 80%, respectively at the concentration of 5.2 mg/cm2. At the concentration of 40.5 mg/g food, the oil totally suppressed progeny production of all the three test insects. Nutritional indices indicate >81% antifeedant action of the oil against R. dominica, S. oryzae and T castaneum at the highest concentration tested.     

Fitness consequences of malathion-specific resistance in red flour beetle (Coleoptera: Tenebrionidae) and selection for resistance in the absence of malathion

Malathion resistance in the red flour beetle, Tribolium castaneum (Herbst), is a worldwide problem and is very stable once it becomes widespread in natural populations. In the absence of insecticide the proportion of resistant phenotypes may rapidly decline but the development of resistance does not always involve reduced fitness. Malathion-specific resistance in T. castaneum seems not to involve any loss of fitness in laboratory or field conditions. Susceptible beetles were in competition with resistant beetles at different initial frequencies and modifications of susceptible gene frequency were estimated in these laboratory populations over 10 generations. A significant decrease in susceptible gene frequency was observed in Tribolium populations over time. The selection coefficient of the susceptible allele was estimated and the fitness of susceptible alleles in all tests was observed to range from 0.89 to 0.93 compared with the fitness of resistant genotypes, which was assumed to be 1. Data provided evidence that the resistant strains exhibited fitness advantages in the absence of malathion. We also compared the biotic potential (fecundity and developmental time) of the susceptible strain, the homozygous malathion-specific resistant strain, and their hybrids. Malathion-specific resistant strains showed an 8 -23% increase in biotic potential relative to the susceptible strain. These findings are consistent with those of malathion-specific resistance in T. castaneum; the fitness of the insects seems independent of the genetic background and the fitness of the resistant insects is not affected by this resistance mechanism.     

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 .     

Use of a web-based model for aeration management in stored rough rice

A web-based model was used to simulate the impact of aeration on population growth of the lesser grain borer, Rhyzopertha dominica (F.), and the rice weevil, Sitophilus oryzae (L.), in stored rough rice, Oryza sativa L., at Beaumont, TX. Simulations were run for each of 10 yr with 1 August as the start date; 31 December as the end date; beginning populations of 2.5 adults per metric ton (1000 kg); starting grain temperatures of 29.4, 32.2, and 35.0 degrees C; and aeration airflow rates of 0.27, 0.79, and 1.40 m3/min/metric ton of rice. In the absence of aeration, populations of both species increased exponentially, with maximum production of R. dominica and S. oryzae at starting grain temperatures of 35.0 and 32.2 degrees C, respectively. Final predicted populations of R. dominica on 31 December from grain starting temperatures of 29.4, 32.2, and 35.0 degrees C were 5465, 6848, and 11,855 per ton, respectively; final predicted populations of S. oryzae were 13,288, 21,252 and 4355, respectively. Aeration led to a reduction in grain temperature and a decrease in pest populations, regardless of starting grain temperature or aeration airflow rates. Predicted populations of R. dominica on 31 December ranged from 12 to 63 adults per ton at all grain starting temperatures and airflow rates; populations of S. oryzae on 31 December ranged from 108 to 193 adults per ton at all grain starting temperatures and airflow rates. The predicted population levels in aerated rice represented at least a 98% reduction compared with unaerated rice. Results show the utility of the web-based model and how the various model inputs can help define broader patterns of insect control in rice stored in the south central United States.     

Effects of temperature and exposure time on mortality of stored-product insects exposed to low pressure.

The prospects of using low pressure that creates a low-oxygen atmosphere to control stored-product insects were investigated in the laboratory. Eggs, larvae, and pupae of Tribolium castaneum (Herbst), Plodia interpunctella (Hübner), and Rhyzopertha dominica (F.) were exposed to 32.5 mmHg in glass chambers at 25, 33, 37, and 40 degrees C for times ranging from 30 min to 144 h. Time-mortality data were subjected to probit analyses and lethal dose ratios were computed to determine differences in lethal time (LT) values among all species-life stage combinations across the four temperatures. Eggs of each species were the life stage most tolerant to low pressure. Pupae of T. castaneum and R. dominica were more tolerant to low pressure than larvae. In all life stages, mortality increased with increasing exposure time to low pressure and also with increasing temperature. Immature stages of R. dominica were more tolerant to low pressure than immature stages of the other two species. The LT99 for R. dominica eggs was 176.32 h at 25 degrees C and that for P. interpunctella eggs was 28.35 h at the same temperature. An increase in temperature to 33 degrees C resulted in a LT99 < of 85.98 h for R. dominica and 6.21 h for P. interpunctella. Higher temperatures resulted in further significant reduction in lethal time values. Low pressure represents a simple, nonchemical alternative to fumigants such as methyl bromide and phosphine for controlling pests of stored-products or other commodities.     

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.