Identification of four novel members of Kunitz-like α-amylase inhibitors family from Delonix regia with activity toward Coleopteran insects

Crop improvement generally focuses on yield, seed quality and nutritional characteristics, rather than resistance to biotic and abiotic stresses. A clear consequence of this approach is the absence of natural anti-feedant toxins in some improved seed materials, allowing predation of commercial crops by insect herbivores. Cowpea (Vigna unguiculata), commonly cultivated by small farmers, is particularly affected by insect-pests that reproduce and develop inside stored seeds. One alternative to conventional pesticides for pest control is the use of biotechnological tools, such as the digestive enzyme inhibitors, that could be introduced in transgenic crops to enhance resistance. In this study, it was verified that the in vivo bioassays using artificial seeds containing 0.5%, 1.0% and 1.5% (w/w) of Delonix regia rich fraction, containing α-amylase inhibitors with effectiveness toward insect α-amylases and other sources, caused remarkable reduction in development and increased mortality of Callosobruchus maculatus cowpea weevil and to cotton boll weevil Anthonomus grandis. Therefore, attempts were made to isolated those inhibitors by SP-Sepharose ion exchange chromatography followed by high performance liquid chromatography on a Vydac C18-TP analytical column. Four inhibitor peaks were obtained with molecular masses of 6.0, 20 and 24 kDa. Their N-termini showed high sequence similarities with Kunitz-like inhibitor family members. These results provide evidence that D. regia synthesizes a multiple family of Kunitz-like α-amylase inhibitors, with different molecular masses and a wide biotechnological potential to control insect-pests.     

A recombinant form of chagasin from Trypanosoma cruzi: inhibitory activity on insect cysteine proteinases

BACKGROUND: The activity of the major digestive cysteine proteinase detected in the intestinal tract of larvae of the bean weevil, Acanthoscelides obtectus (Say), was efficiently inhibited by the well-characterized cysteine proteinase synthetic inhibitor E-64 and also by a recombinant form of chagasin (r-chagasin), a tight-binding cysteine proteinase inhibitor protein from Trypanosoma cruzi. RESULTS: Incorporation of r-chagasin into an artificial diet system at 0.1 g kg(-1) retarded growth rate, decreased larval survival and led to complete mortality of A. obtectus at the end of the trial. The observed differences in growth rates occurred particularly in the first and second development stages. Artificial seeds containing high levels of r-chagasin (0.5-30 g kg(-1)) completely inhibited larval penetration. CONCLUSION: Together, the results reported in this paper support the hypothesis that the inhibitory activity of r-chagasin towards the major insect gut cysteine proteinase in vitro and in vivo is an accurate prediction of its insecticidal effects. The selectivity of this inhibitor against insect digestive proteinases supports the key role in parasite virulence by affecting the endogenous proteinase activity in its natural host.     

转Bt基因植物对不同抗性棉铃虫的生长抑制作用

采用转Bt基因的烟草和棉花 ,对经转Bt单基因、转Bt与CpTI双基因烟草汰选 1 8代和常规烟草处理的棉铃虫种群进行生长抑制作用的比较测定。结果表明 ,转Bt烟草对以汰选后的抗Bt单基因汰选棉铃虫种群的体重抑制作用显著低于转双基因烟草汰选种群和对照种群 ;转Bt棉花对单基因种群的杀虫活性、体重、龄期发育以及化蛹率的抑制作用亦显著低于双基因种群和对照种群     

Role of chemical characteristics of the seed coat in the resistance of selected cowpea varieties to Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) in Nigeria

The role of seed coat chemical factors in the resistance of the cowpea varieties, Kanannado, IT89KD-391 and Borno brown, to the cowpea seed bruchid Callosobruchus maculatus (F.), was investigated under laboratory conditions (30-35C and 65-67% RH) in Maiduguri, Nigeria. Significantly higher numbers of eggs were laid on de-coated than on intact Kanannado seeds whereas significantly smaller numbers of eggs were laid on de-coated than on intact IT89KD-391 or Borno brown seeds. Susceptibility was higher in de-coated than in intact Kanannado seeds (susceptibility indices [SI] 3.4 and 0.0, respectively). Egg-hatch was significantly reduced in seeds with intact seed coats by 88.6%, while the proportion of eggs that failed to hatch in de-coated seeds was 31.9%. Treatment of Borno brown seeds especially with 32 and 64 mg of extracts from Kanannado and IT89KD-391 seed coats, reduced oviposition by 61.9% and 95.2%, respectively. Treatment with 32 mg of the seed coat extracts reduced egg-hatch by 49.2%. Identical dosages (32 and 64 mg) of these seed coat extracts also significantly reduced susceptibility ofBorno brown to C. maculatus (SI values 6.7 and 1.5, respectively). Comparable SI values for Borno brown treated with 16 mg of the seed coat extracts or extract-free acetone were 14.9 and 14.0, respectively.     

A review of castor-derived products used in crop and seed protection

Castor-derived products are currently used for protecting agricultural crops and seeds from devastating damages of pests and diseases. Extracts (1–10%) of leaf or seed in water or chemical solvents, and crude oil (3–5%) extracted from seed were found effective as sprays against foliage insect pests. Populations of the root-knot nematodes were significantly reduced when de-oiled seed cake was incorporated into soil at 1000 kg/ha, especially after mixing it with bio-inoculants such as, a fungus (Poecilomyces lilacinus) or a bacterium (Pasteuria penetrans). Castor proved as a potential synergist when mixed with other plant products or chemical pesticides and exhibited different modes of actions but with comparatively limited insecticidal properties. In less developed and developing countries, the common use of oil is for treating stored cereals and pulses at 5–10 ml/kg seed. In both field and greenhouse experiments, pest mortality and other biological parameters were dose-dependent. This review discusses different uses of castor products to foresee possibility of replacing or at least reducing use of toxic chemicals in crop and seed protection.     

Effects of Hyptis spicigera Lam. (Labiatae) on the behaviour and development of Callosobruchus maculatus F. (Coleoptera: Bruchidae), a pest of stored cowpeas

Abstract

The use of beni seed, Hyptis spicigera Lam. (Labiatae), to protect stored cowpeas from bruchid attacks is a widespread practice in West Africa. More knowledge about its actual effects on storage pests is needed to enhance its effectiveness. In this laboratory study, repellent and lethal effects of powder and essential oil from H. spicigera on Callosobruchus maculatus, the main pest of stored cowpeas, were studied. Repellency tests undertaken with a Y-tube olfactometer showed that powder was repulsive for C. maculatus adults. On the other hand, this product had no insecticidal activity. Essential oil had a dose-dependent insecticidal effect while sublethal doses repelled the adults. Both products reduced oviposition by 15 – 19 and 7 – 78% according to the doses of powder and essential oil, respectively, tested. Similarly, egg viability decreased with increasing doses of powder and essential oil used in a range of 40 – 75 and 24 – 86%, respectively. Only essential oil was lethal to C. maculatus larvae developing within cowpea seeds; however, oil activity was age-dependent, younger instars being more susceptible. These results are discussed with a view of controlling C. maculatus populations in stored cowpeas.     

Marker‐free,tissue‐specific expression of Cry1Ab as a safe transgenic strategy for insect resistance in rice plants

BACKGROUND: Rice is the major food resource for nearly half of the global population; however, insect infestation could severely affect the production of this staple food. To improve rice insect resistance and reduce the levels of Bt toxin released into the environment, the Cry1Ab gene was conjugated to the rice rbcS promoter to express Bt toxin in specific tissues of transgenic plants. RESULTS: Eight marker‐free, T₂ lines were separated from the T₀ cotransformants. Using RT‐PCR, high levels of Cry1Ab expression were detected in the leaf but not in the seed. The Cry1Ab protein level ranged from 1.66 to 3.31 µg g^?1 in the leaves of four transgenic lines, but was barely detectable in their seeds by ELISA. Bioassays showed that the mortality rate of silkworm larvae feeding on mulberry leaves dipped in transgenic rice flour and pollen was less than that of the positive control (KMD), and that their average weight was higher than that of KMD, suggesting that the Cry1Ab protein was not expressed in the seed and pollen. CONCLUSION: The transgene conferred a high level of resistance to insects and biosafety to the rice plants, which could be directly used in rice breeding. Copyright © 2012 Society of Chemical Industry     

Persistence and residual activity of an organophosphate, pirimiphos-methyl, and three IGRs, hexaflumuron, teflubenzuron and pyriproxyfen, against the cowpea weevil, Callosobruchus maculatus (Coleoptera: Bruchidae)

Three insect growth regulators (IGR), the chitin synthesis inhibitors (CSI) teflubenzuron and hexaflumuron and the juvenile hormone mimic (JHM) pyriproxyfen, as well as the organophosphate (OP) pirimiphos-methyl, were evaluated for their activity against the cowpea weevil, Callosobruchus maculatus (F), in cowpea seeds stored for up to 8 months post-treatment. The initial activity data showed that, based on LC50 level, teflubenzuron had strong ovicidal activity (LC50 = 0.056 mg kg(-1)) followed by pirimiphos-methyl (1.82 mg kg(-1)) and pyriproxyfen (91.9 mg kg(-1)). The residual activity data showed that none of the IGRs tested had strong activity when applied at 200 mg kg(-1) in reducing the oviposition rates of C maculatus at various storage intervals up to 8 months post-treatment. However, teflubenzuron reduced adult emergence (F1 progeny), achieving control ranging from 96.2% at 1 month to 94.3% at 8 months. Hexaflumuron showed a similar trend in its residual activity, ranging between 93.8% control at 1 month to 88.2% control at 8 months post-treatment. However, pyriproxyfen was more active than the CSIs tested and caused complete suppression (100% control) of adult emergence at all storage intervals. Unlike the IGRs tested, pirimiphos-methyl applied at 25 mg kg(-1) was more effective in reducing oviposition rates of C maculatus up to 8 months post-treatment. A strong reduction of adult emergence was also observed at various bimonthly intervals (98.6% control at 1 month to 91.6% control at 8 months post-treatment). The persistence of hexaflumuron and pirimiphos-methyl in cowpea seeds was also studied over a period of 8 months. The loss of hexaflumuron residue in treated cowpeas (200 mg kg(-1)) was very slow during the first month post-treatment (4.43%). At the end of 8 months, the residue level had declined significantly to 46.4% of the initial applied rate. The loss of pirimiphos-methyl residue in treated cowpeas (25 mg kg(-1)) was relatively high during the first month post-treatment (36.7%) and increased to 81.6% after 8 months.     

Rhizobacteria‐mediated resistance against the blackeye cowpea mosaic strain of bean common mosaic virus in cowpea (Vigna unguiculata)

BACKGROUND: The present study investigated the effect of seven Bacillus‐species plant‐growth‐promoting rhizobacteria (PGPR) seed treatments on the induction of disease resistance in cowpea against mosaic disease caused by the blackeye cowpea mosaic strain of bean common mosaic virus (BCMV). RESULTS: Initially, although all PGPR strains recorded significant enhancement of seed germination and seedling vigour, GBO3 and T4 strains were very promising. In general, all strains gave reduced BCMV incidence compared with the non‐bacterised control, both under screen‐house and under field conditions. Cowpea seeds treated with Bacillus pumilus (T4) and Bacillus subtilis (GBO3) strains offered protection of 42 and 41% against BCMV under screen‐house conditions. Under field conditions, strain GBO3 offered 34% protection against BCMV. The protection offered by PGPR strains against BCMV was evaluated by indirect enzyme‐linked immunosorbent assay (ELISA), with lowest immunoreactive values recorded in cowpea seeds treated with strains GBO3 and T4 in comparison with the non‐bacterised control. In addition, it was observed that strain combination worked better in inducing resistance than individual strains. Cowpea seeds treated with a combination of strains GBO3 + T4 registered the highest protection against BCMV. CONCLUSION: PGPR strains were effective in protecting cowpea plants against BCMV under both screen‐house and field conditions by inducing resistance against the virus. Thus, it is proposed that PGPR strains, particularly GBO3, could be potential inducers against BCMV and growth enhancers in cowpea. Copyright © 2009 Society of Chemical Industry     

Deterrent effects of some botanical products on oviposition of the cowpea bruchid Callosobruchus maculatus (F.) (Coleoptera: Bruchidae)

Extracts from nine plant materials were tested in choice and no-choice tests as oviposition deterrents for cowpea bruchid, Callosobruchus maculatus (F.) on chickpea, Cicer arietinum L. Seed treatment with 0.1% crude extract of materials resulted in a significant reduction in ovipositional preference of the bruchid. The highest repellency was found in Rhazya stricta leaves (82%), Azadirachta indica seeds (76.8%), Heliotropium bacciferum aerial parts (59.2%) and citrus peels (58.6%). Progeny production was significantly reduced: only 11.5, 11.9, 12.4, 13.4 and 14.8% of the eggs deposited reached adulthood in R. stricta, neem seeds, Piper nigrum, citrus peels and H. bacciferum, respectively. F1 females laid significantly fewer eggs in response to R. stricta, neem seeds, Eugenia caryophyllata cloves, H. bacciferum, citrus peels and P. nigrum. Percentage reduction in F₂ progeny ranged between 71.4% with R. stricta-treated seeds and 24.5% in the P. nigrum treatment.     

转Bt基因抗虫作物对非靶标生物的影响

迄今为止,已经获得了大量的抗虫转Bt基因作物。尽管这些作物中表达的Bt蛋白只是针对靶标害虫起到杀虫效果,但是抗虫转Bt基因作物是否会对非靶标生物产生影响一直存在争议。本文就抗虫转Bt基因作物对节肢动物群落、非靶标植食性昆虫、天敌和有益昆虫的影响进行了综述。综合评价认为,现有的抗虫转Bt基因作物对非靶标生物是安全的。     

Investigation of insecticidal activity of rye α-amylase inhibitor gene expressed in transgenic tobacco (Nicotiana tabacum) toward cotton boll weevil (Anthonomus grandis)

Innumerable proteinaceous α-amylase inhibitors have been isolated and identified from different plant species. Among them, an α-amylase inhibitor gene with bioinsecticidal potential toward Anthonomus grandis (cotton boll weevil) was previously identified in rye seeds (Secale cereale). This cereal inhibitor was expressed in tobacco plants (Nicotiana tabacum) under control of phytohemaglutinin promoter by using Agrobacterium tumefasciens - mediated transformation. Presence of αBIII-rye gene and further protein expression were confirmed by PCR and Western blot analysis, respectively. Immunological assays indicated that the recombinant inhibitor was expressed in concentration range from 0.1% to 0.28% (w:w) of the total protein in tobacco seeds of R₀ plants. From 14 independent transformants, five plants with expression levels between 0.20% and 0.28% in seeds were in vitro assayed against A. grandis amylolytic enzymes causing clear inhibition. Moreover, bioassays using transgenic seed flour mixture for artificial diet produced 74% mortality in A. grandis first larval instar. These data suggest that rye inhibitor could be a promising biotechnological tool for produce transgenic cotton plants with an increased resistance to cotton boll weevil. Moreover, αBIII-rye gene should be considered a potential compound for a pyramiding strategy aiming to delay insect-resistance.     

Effects of metabolic inhibitors on activity of Cry1Ab toxin to inhibit growth of Ephestia kuehniella larvae

BACKGROUND: Although the toxins of Bacillus thuringiensis Berliner (Bt) are frequently used to control lepidopteran pests, the tolerance or resistance of some lepidopteran moth strains may limit Bt applications. In this study, insecticidal cocktails consisting of sublethal doses of Cry1Ab toxin and additive compounds were tested for their suppressive effect on larval relative growth rate (RGR) in Ephestia kuehniella Zeller under laboratory conditions.RESULTS: In the first step, the suppressive effect of diflubenzuron, soybean trypsin inhibitor (STI) and chitinase on RGR was confirmed. In the second step, these compounds were incorporated into a mixture of crushed kernels of Bt maize hybrid MON 810-YieldGard((R)) and its isoline with concentrations of Cry1Ab toxin ranging from 0.011 to 0.091 microg g(-1) diet. An additive effect on the suppression of larval RGR in E. kuehniella was found in a combination of diflubenzuron, STI and STI + chitinase as secondary compounds in insecticidal cocktails. Chitinase showed no additive effect on RGR.CONCLUSION: The highest suppression level was found in cocktails with STI + chitinase as a secondary compound. It is hypothesized that the protease inhibitor (STI) protects both chitinase and Cry1Ab proteins from endogenous proteases in the larval midgut and prolongs their insecticidal activities. The possible application of insecticidal cocktails in the control of E. kuehniella is discussed. Copyright (c) 2008 Society of Chemical Industry.     

吡虫啉拌种控制全生育期小麦蚜虫有效剂量评价

本文对吡虫啉拌种控制全生育期小麦蚜虫有效剂量进行了研究。结果表明,仅用吡虫啉拌种就可在小麦整个生育期将小麦蚜虫控制在防治指标之下,表现出超高效、持效的控制效果。1 kg种子用1.0~4.8 g吡虫啉（有效成分）拌种,从播种至灌浆末期,防治效果在95%以上,至乳熟期（收获期）防治效果仍高达74.49%~96.61%;千粒重比未拌种区增加了26.51%~36.55%。综合以上试验结果,吡虫啉拌种控制全生育期小麦蚜虫的经济、有效剂量为有效成分1.0~2.0 g/kg种子,最佳剂量为1.5 g/kg种子。     

A laboratory-based comparison of a molluscicide and an alternative food source (red clover) as means of reducing slug damage to winter wheat

Slugs are major pests of many crops in the UK, including winter wheat, yet current methods of control are often unreliable. This study investigates the potential use of red clover, as an alternative food source, to reduce the amount of damage caused to winter wheat by the field slug, Deroceras reticulatum (Müller). Two laboratory-based studies, each conducted over a 7-day period, investigated the effects of red clover seedlings and commercial metaldehyde pellets on damage to winter wheat seeds and seedlings. The results indicate that metaldehyde applications, in the form of commercially available pellets, resulted in significantly greater protection to wheat seeds compared with red clover, whereas metaldehyde and red clover were equally as effective in reducing damage to wheat seedlings. A further laboratory experiment investigated the effect of two slug population densities (48 and 16 adults m(-2)) and high and low red clover seed rates (125% and 75% of a standard rate) on damage to wheat seeds. Results showed that, at the highest slug population density, red clover sown at 125% of the standard rate gave 99% protection to wheat seeds, compared with the 75% seed rate which gave 55%. At the lower slug population density, both seed rates of red clover resulted in similar levels of protection. Implications for the potential use of red clover as an alternative food source for reducing damage to winter wheat in field conditions are discussed.     

Combined treatments of spinosad and chlorpyrifos-methyl for management of resistant psocid pests (Psocoptera: Liposcelididae) of stored grain

The combined efficacy of spinosad and chlorpyrifos-methyl was determined against four storage psocid pests belonging to genus Liposcelis. This research was undertaken because of the increasing importance of these psocids in stored grain and the problem of finding grain protectants to control resistant strains. Firstly, mortality and reproduction were determined for adults exposed to wheat freshly treated with either spinosad (0.5 and 1 mg kg(-1)) or chlorpyrifos-methyl (2.5, 5 and 10 mg kg(-1)) or combinations of spinosad and chlorpyrifos-methyl at 30 degrees C and 70% RH. There were significant effects of application rate of spinosad and chlorpyrifos-methyl, both individually and in combination, on adult mortality and progeny reduction of all four psocids. Liposcelis bostrychophila Badonnel and L. decolor (Pearman) responded similarly, with incomplete control of adults and progeny at both doses of spinosad but complete control in all chlorpyrifos-methyl and combined treatments. In L. entomophila (Enderlein) and L. paeta Pearman, however, complete control of adults and progeny was only achieved in the combined treatments, with the exception of spinosad 0.5 mg kg(-1) plus chlorpyrifos-methyl 2.5 mg kg(-1) against L. entomophila. Next, combinations of spinosad (0.5 and 1 mg kg(-1)) and chlorpyrifos-methyl (2.5, 5 and 10 mg kg(-1)) in bioassays after 0, 1.5 and 3 months storage of treated wheat were evaluated. The best treatment was 1 mg kg(-1) of spinosad plus 10 mg kg(-1) of chlorpyrifos-methyl, providing up to 3 months of protection against infestations of all four Liposcelis spp. on wheat.     

Isolation of a novel Carica papaya α-amylase inhibitor with deleterious activity toward Callosobruchus maculatus

Cowpea (Vigna unguiculata) is a subsistence crop for small and poor farmers from Latin America and Africa. This culture is commonly damaged by cowpea weevil (Callosobruchus maculatus), which burrow into stored seeds to fed on. Due to impact of larval predation, several plant defense studies have been developed, indicating that α-amylase inhibitors are able to impede and/or reduce bruchids digestive process. In this report, a novel α-amylase inhibitor from papaya seeds (Carica papaya) with activity against cowpea weevil enzymes was purified and biochemical characterized. Peeled seeds were macerated and extracted with a 0.6 M NaCl and 0.1% HCl solution. Crude extract was precipitated with ammonium sulphate (100%). After dialyses, this rich fraction was applied onto a CM-Cellulose column and retained peak was submitted to an analytic reversed-phase column HPLC (Vydac C-18TP) yielding several peaks. Only one fraction, with molecular mass of 4562 Da, showed significant inhibitory activity against C. maculatus α-amylases. Otherwise, no inhibitory activities against mammalian α-amylases were observed. Bioassays using artificial seeds containing C. papaya α-amylase inhibitor rich fraction (0.5% and 1.0%) were also conduced showing that α-amylase inhibitors were able to increase larval mortality (50%) and also decrease insect fecundity and adult longevity. These results showed the presence of an α-amylase inhibitor from C. papaya seeds with high specificity to insect enzymes, indicating that this inhibitor probably could be used, through genetic engineering, in the construction of transgenic plants with enhanced resistance toward cowpea weevil.     

新烟碱类杀虫剂种子包衣防治麦蚜的可行性评价

为评价不同新烟碱类杀虫剂处理种子防治小麦蚜虫的应用潜力,采用种子包衣法分别在室内及田间比较了吡虫啉、噻虫嗪、啶虫脒、烯啶虫胺、噻虫啉防治小麦蚜虫的效果及安全性,并测定了吡虫啉和噻虫嗪的持效、对天敌和小麦产量的影响及其在小麦籽粒中的最终残留量。结果表明,在2.4、3.6和4.8 g/kg种子剂量下,啶虫脒明显降低小麦出苗率,而其它药剂均无显著影响;至抽穗前烯啶虫胺、啶虫脒和噻虫啉对麦蚜的防效低,吡虫啉和噻虫嗪则均有较高防效,在58.17%以上,而在小麦抽穗扬花期防效下降,为33.57%~60.46%。吡虫啉和噻虫嗪对叶部麦蚜防效均相应高于穗部。与喷雾处理相比,吡虫啉、噻虫嗪各剂量种子包衣对瓢虫和蚜茧蜂等天敌昆虫影响小,在3.6、4.8 g/kg种子剂量下,小麦千粒重和产量无显著差异,且在小麦籽粒中的残留量低。表明吡虫啉和噻虫嗪种子包衣防治麦蚜的应用潜力大。     

Expression of δ-endotoxin Cry1EC from an inducible promoter confers insect protection in peanut (Arachis hypogaea L.) plants

BACKGROUND: Spodoptera litura (F.) is a polyphagous foliage insect and a major pest on peanut (Arachis hypogaea L.). Constitutive expression of δ‐endotoxin Cry1EC gives protection against S. litura, as reported earlier. In this study, insect bites and salicylic acid induced high‐level expression of Cry1EC was achieved in peanut. In order to achieve this, the expression of pathogenesis responsive promoter PR‐1a was enhanced by placing it downstream of the CaMV35S promoter in the pCAMBIA 1300 backbone. The resultant promoter CaMV35S(r)PR‐1a expressed a high level of insecticidal δ‐endotoxin Cry1EC. The Gus expression under the control of CaMV35S(r)PR‐1a served as a convenient marker for evaluation of promoter response to different treatments. RESULTS: Transgenic events that showed a very low level of uninduced expression and no expression in seeds were selected. The Cry1EC expression in leaves increased nearly eightfold in the selected event, following induction by salicylic acid. Both the salicylic‐acid‐treated and the S. litura‐bitten leaves showed the highest expression after 2 days. Leaves from salicylic‐acid‐induced transgenic plants caused 100% mortality of S. litura at all stages of larval development. CONCLUSION: The results suggest that high expression of inducible promoters provides a good strategy for the development of safer transgenic food and feed crops. Copyright © 2010 Society of Chemical Industry     

Contamination of internationally traded wheat by herbicide‐resistant Lolium rigidum

As herbicide‐resistant weeds have spread in the agricultural fields of grain‐exporting countries, their seeds could be introduced into other countries as contaminants in imported grain. The spread of resistance genes through seed and pollen can cause significant economic loss. In order to assess the extent of the problem, we investigated the contamination by herbicide‐resistant annual ryegrass (Lolium rigidum) of wheat imported from Western Australia into Japan. Annual ryegrass seeds were recovered from wheat shipments and seed bioassays were conducted to identify resistance to the herbicides that are commonly used in Australia: diclofop‐methyl, sethoxydim, chlorsulfuron, and glyphosate. Nearly 4500 ryegrass seeds were detected in 20 kg of wheat that was imported in both 2006 and 2007. About 35% and 15% of the seeds were resistant to diclofop‐methyl, 5% and 6% were resistant to sethoxydim, and 56% and 60% were resistant to chlorsulfuron in 2006 and 2007, respectively. None was resistant to glyphosate in either year. As the contamination of crops by herbicide‐resistant weeds is probably a common phenomenon, the monitoring of incoming grain shipments is necessary to stem the further spread of herbicide‐resistant weeds into importing countries.