Herbicidal cyanoacrylates with antimicrotubule mechanism of action

The herbicidal mode of action of the new synthetic cyanoacrylates ethyl (2Z)-3-amino-2-cyano-4-ethylhex-2-enoate (CA1) and its isopropyl ester derivative CA2 was investigated. For initial characterization, a series of bioassays was used indicating a mode of action similar to that of mitotic disrupter herbicides such as the dinitroaniline pendimethalin. Cytochemical fluorescence studies including monoclonal antibodies against polymerized and depolymerized tubulin and a cellulose-binding domain of a bacterial cellulase conjugated to a fluorescent dye were applied to elucidate effects on cell division processes including mitosis and microtubule and cell wall formation in maize roots. When seedlings were root treated with 10 microM of CA1 or CA2, cell division activity in meristematic root tip cells decreased within 4 h. The chromosomes proceeded to a condensed state of prometaphase, but were unable to progress further in the mitotic cycle. The compounds caused a complete loss of microtubular structures, including preprophase, spindle, phragmoplast and cortical microtubules. Concomitantly, in the cytoplasm, an increase in labelling of free tubulin was observed. This suggests that the herbicides disrupt polymerization and microtubule stability, whereas tubulin synthesis or degradation appeared not to be affected. In addition, cellulose labelling in cell walls of root tip cells was not influenced. The effects of CA1 and CA2 were comparable with those caused by pendimethalin. In transgenic Arabidopsis plants expressing a green fluorescent protein-microtubule-associated protein4 fusion protein, labelled arrays of cortical microtubules in living epidermal cells of hypocotyls collapsed within 160 min after exposure to 10 microM CA1 or pendimethalin. Moreover, a dinitroaniline-resistant biotype of goosegrass (Eleusine indica (L) Gaertn) with a point mutation in alpha-tubulin showed cross-resistance against CA1 and CA2. The results strongly indicate that the cyanoacrylates are a new chemical class of herbicide which possess the same antimicrotubule mechanism of action as dinitroanilines, probably including interaction with the same binding site in alpha-tubulin.     

Probing mode of action in plant cell cycle by the herbicide endothall, a protein phosphatase inhibitor

The mode of action of endothall, an herbicide which was reported to inhibit plant protein phosphatases 1 (PP1) and 2A (PP2A), was investigated. For initial characterization, a series of bioassays was used for comprehensive physiological profiling of endothall effects which suggested a phytotoxic mode of action similar to mitotic disrupter herbicides. Unlike known microtubule disrupters, endothall did not inhibit soybean tubulin polymerization in vitro. As shown in meristematic corn root tips, endothall distorted the orientation of cell division plane and microtubule spindle structures which led to cell cycle arrest in prometaphase. In tobacco BY-2 cells, malformed spindles together with prometaphase arrest of nuclei and abnormal perinuclear microtubule patterns were detected as early as 4 h of endothall treatment. These effects were also observed after treatment with other protein phosphatase inhibitors, cantharidin and okadaic acid, which phenocopied the mitotic changes described in tonneau1 (ton1) and tonneau2 (ton2) Arabidopsis mutants. These mutants are defective in TONNEAU2 (TON2) protein, a regulatory subunit of PP2A, which governs cell division plane and microtubule orientation. Therefore, PP2A/TON2 phosphatase complex is suggested to be an in planta molecular target of endothall. However, in BY-2 cells, additional effects of endothall, including inhibition of S-phase initiation and DNA synthesis, detected by 5-ethynyl-2′-deoxyuridine (EdU) incorporation, and condensed nuclei arrested in late mitosis were observed which were not reported in Arabidopsiston1 and ton2 mutants. This result indicates that two additional checkpoints in cell cycle were blocked by endothall which are probably not associated with TON2-pathway inhibition. Possibly, inhibition of PP1 and/or other PP2A protein phosphatases are involved in the regulation of these cell cycle phenomena.     

Mitotic disruption by a novel pyrimidine herbicide, NS-245852, in oat (Avena sativa L.) roots

The effects of a novel pyrimidine herbicide, NS-245852 [2-chloro-6-fluorophenyl-4-(trifluoromethyl)thieno[2,3-d]pyrimidine-2-yl-ketone], on mitosis in oat ( Avena sativa L. cv. Zenshin) root tips were investigated by using light and immunofluorescence microscopy. The root growth was strongly inhibited at 10⁻⁷ mol L⁻¹ of NS-245852, and swollen root tips were induced at 5 × 10⁻⁸ mol L⁻¹. As observed by the use of light microscopy, the herbicide produced disrupted mitosis and large polynucleate cells in the meristematic root tissue. These symptoms were similar to those of mitotic disrupter herbicides. The immunofluorescence microscopy studies of the root tip cells treated for 30 min revealed that spindle fibers and the preprophase band were reduced, although kinetochore fibers and the phragmoplast were not affected. Kinetochore fibers remained as small fluorescence spots, and the phragmoplast disappeared after a 3 h treatment. No microtubule arrays were observed by a longer treatment (longer than 3 h). Among the microtubule arrays, spindle fibers and the preprophase band were found to be the most sensitive to the herbicide, whereas kinetochore fibers were the most resistant. The phragmoplast was intermediate. Thus, the primary action of NS-245852 is the inhibition of polymerization of tubulin into microtubules.     

Carrot microtubules are dinitroaniline resistant. I. Cytological and cross-resistance studies

Treatment of both tolerant and susceptible species with dinitroaniline herbicides results in root swelling typical of mitotic disrupters, however, no root swelling was observed in carrots treated with saturated solutions of all the dinitroaniline herbicides tested, with the exception of oryzalin treatments at ≥ 10^?5 M. These levels of treatment are 100–1000 times the levels that caused root-tip swelling, even in tolerant plants. This properly classifies carrot as dinitroaniline-resistant rather than merely tolerant. Cross-resistance was noted to the structurally related mitotic disrupter, hexanitrodiphenylamine, and to the structurally unrelated herbicide, amiprophos-methyl. No resistance was noted to a number of other mitotic-disrupting herbicides. Both immunofluorescence and electron microscopy indicate that the microtubules of carrot roots were unaffected by dinitroaniline treatment. All organized microtubule configurations (cortical, pre-prophase bands, spindle, and phragmoplast) were observed in the treated material.     

The abnormal cell plates formed after microtubule disrupter herbicide treatment are enriched in callose

Mitotic disrupter herbicides are best known for their macroscopic effect on root tip swelling and their microscopic effect on the progression of chromosomes through mitosis. However, irregularities with the phragmoplast microtubules and cell plate formation occur at lower herbicide concentrations than these more familiar effects. Instead of the relatively straight cell plates found in control tissue, cell plates after mitotic disrupter treatment are often branched and grow irregularly throughout the cytoplasm. Sometimes these abnormal plates adhere to one wall and in most cases do not effectively divide the potential daughter cytoplasms. To determine the chemical composition of these abnormal cell plates, thin sections of treated onion root tips were probed with a battery of antibodies and cytochemical probes. Abnormal cell plates are greatly enriched in callose compared to control cell plates and accumulate very low levels of cellulose. The development of these wildly undulated and excessively branched or heavily thickened cell plates indicates the importance of microtubules in forming a proper cell plate and perhaps the necessity of stable microtubule arrays for the addition of cellulose to these structures. Because the abnormal plates occur at herbicide concentrations below that required for induction of mitotic arrest or root tip swelling, this effect may be the primary phytotoxic effect of these herbicides.     

Comparison of trifluralin,oryzalin, pronamide,propham, and colchicine treatments on microtubules

The mode of action of trifluralin (α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine), oryzalin (3,5-dinitro-N⁴,N⁴-dipropylsulfanilamide), pronamide(N-(1,1-dimethylpropynyl) 3,5-dichlorobenzamide), and propham (isopropyl carbanilate) on purified microtubules from pig brains and on the ultrastructure of wheat (Triticum aestivum L. “Mediterranean,” C. I. 5303) and corn (Zea mays L. “yellow dent, U. S. 13”) roots was compared with that known for colchicine. Colchicine disrupts the in vivo cortical and spindle microtubules of root cells. Like colchicine, the herbicides trifluralin, oryzalin, and pronamide caused the loss of both cortical and spindle microtubules of root cells. The rate of microtubule disappearance depended on the type of herbicide and length of exposure of roots to the herbicide. Unlike colchicine, cortical microtubules were present in propham-treated roots but they were disoriented within the cell.In vitro polymerization studies with pig brain microtubules (Sus scrofa) showed that the herbicides failed to inhibit the assembly of purified microtubular protein into microtubules and that radioactively labeled herbicides did not bind to the microtubular protein. Colchicine inhibited the polymerization of microtubular protein and readily bound to the microtubular subunits. These results indicate that the mode of action of the herbicides is not similar to that of colchicine and that the loss of microtubules from root tip cells treated with trifluralin, oryzalin, and pronamide may be caused by these herbicides interfering with synthesis of microtubular protein or metabolism of endoplasmic reticulum membranes involved in microtubule assembly. The mode of action of propham appears to be on the microtubular organizing centers rather than on microtubules per se.     

Interaction between benzoylprop ethyl, flamprop methyl or flamprop isopropyl and herbicides used for broadleaved weed control

A marked antagonism of A. fatua control was found when 2,4-D, dicamba, 2,4-D-dicamba-mecoprop or bromoxynil were added to benzoylprop ethyl, flamprop methyl or flamprop isopropyl. Because of the much poorer control of A. fatua by these mixtures obtaining broad spectrum weed control with them in one spray operation would not be possible. The A. fatua herbicides did not affect the activity of the added herbicides on Fagopyrum lataricum. In eight out of nine mixtures tested there was no significant antagonism in the field conditions when MCPA or bromoxynil-MCPA was added to the A. fatua herbicides. There were significant antagonistic effects, however, in six out of nine such mixtures in glasshouse experiments. This suggests that under farm use conditions benzoylprop ethyl, flamprop methyl or fiamprop isopropyl can be mixed with MCPA amine or ester or with bromoxynil-MCPA to obtain broad spectrum weed control from one spray operation, though slightly poorer A. fatua control may be expected with these mixtures.     

Histological studies on the effects of benzoylprop ethyl and flamprop methyl on growth and development of wild oats*

Wild oats were trealed with benzoylprop eihyl and flamprop methyl alone or in combination with other herbicides and the effects on plant growth studied using histological techniques, Benzoylprop ethyl caused severe reductions in the growth of both the leaves and internodes of treated plants, Evidence was obtained which indieaies that cell division as well as cell elongalion was inhibited. The effects of flamprop methyl on plant growth were similar lo those observed for benzoylprop ethyl. Both compounds interfered with normal differentiation of vascular tissues which could account for retarded development of the higher internodes and shoot apex of treated plants. Of the various herbicides applied in combination with the wild oat herbicides, 2,4—D was more antagonistic than MCPA. while bromoxynil was the least antagonistic. Plants treated with benzoylprop ethyl plus 2.4—D had increased cell, leaf and internode lengths compared to plants treated with benzoylprop ethyl alone. Reductions in cell and internode lengths of plants treated with the herbicide combinations were not always proportional lo dry weight reductions.     

Cytological studies of dinitroaniline-resistant Eleusine

Ultrastructural studies of primary roots (goosegrass) from dinitroaniline-resistant (R) and susceptible (S) biotypes of Eleusine indica (L). Gaertnr. establish a possible cytological basis for trifluralin resistance. Although the S biotype has a normal ultrastructure when grown in water, exposure to trifluralin solutions (between 10⁻⁸ and 10⁻⁵M) for 24 h results in a swelling of the root tip, typical of herbicides that affect microtubule production. The loss of spindle microtubules in the S biotype results in a mitosis arrested at prometaphase and the loss of cortical microtubules results in the formation of isodiametric cells in the zone of elongation. Nuclear membranes reform around the chromosomes in arrested prometaphase, producing abnormal, polymorphic nuclei. The mitotic index is increased in the S biotype after trifluralin treatment because many of the cells are arrested in prometaphase. The root tips of R biotypes are not swollen by even 10⁻⁵M trifluralin treatment. Trifluralin does not markedly affect cell division in the R biotype nor are the mitotic irregularities noted in the S biotype after treatment. However, even when the R biotype is not exposed to trifluralin, the microtubules are less abundant than in the S biotype and frequently cell walls are oriented abnormally or are incompletely formed. The level of resistance exhibited by the R biotype, the apparent difference in microtubule number and function between the two biotypes, and the lack of effect on the microtubules at high trifluralin concentrations indicate a site-of-action mutation.     

On the mode of action of the herbicides cinmethylin and 5-benzyloxymethyl-1, 2-isoxazolines: putative inhibitors of plant tyrosine aminotransferase

BACKGROUND: The mode of action of the grass herbicides cinmethylin and 5‐benzyloxymethyl‐1,2‐isoxazolines substituted with methylthiophene (methiozolin) or pyridine (ISO1, ISO2) was investigated. RESULTS: Physiological profiling using a series of biotests and metabolic profiling in treated duckweed (Lemna paucicostata L.) suggested a common mode of action for the herbicides. Symptoms of growth inhibition and photobleaching of new fronds in Lemna were accompanied with metabolite changes indicating an upregulation of shikimate and tyrosine metabolism, paralleled by decreased plastoquinone and carotenoid synthesis. Supplying Lemna with 10 µM of 4‐hydroxyphenylpyruvate (4‐HPP) reversed phytotoxic effects of cinmethylin and isoxazolines to a great extent, whereas the addition of L ‐tyrosine was ineffective. It was hypothesised that the herbicides block the conversion of tyrosine to 4‐HPP, catalysed by tyrosine aminotransferase (TAT), in the prenylquinone pathway which provides plastoquinone, a cofactor of phytoene desaturase in carotenoid synthesis. Accordingly, enhanced resistance to ISO1 treatment was observed in Arabidopsis thaliana L. mutants, which overexpress the yeast prephenate dehydrogenase in plastids as a TAT bypass. In addition, the herbicides were able to inhibit TAT7 activity in vitro for the recombinant enzyme of A. thaliana. CONCLUSION: The results suggest that TAT7 or another TAT isoenzyme is the putative target of the herbicides. Copyright © 2011 Society of Chemical Industry     

The mode of action of RH‐1965: a new phenylpyrimidinone bleaching herbicide

RH‐1965 is a new bleaching herbicide which causes newly developing leaf tissue to emerge devoid of photosynthetic pigments. Mode‐of‐action studies revealed that RH‐1965 inhibited the accumulation of both total chlorophyll and β‐carotene. Concomitantly, it induced the accumulation of the β‐carotene precursors, phytoene, phytofluene and, in particular, ξ‐carotene. Inhibition of chlorophyll accumulation by RH‐1965 is attributed to the photo‐oxidative destruction of chlorophyll in the absence of β‐carotene because RH‐1965 blocked chlorophyll accumulation to a greater extent under high light (50–330 µE m⁻² s⁻¹) than under low light (0.8 µE m⁻² s⁻¹) conditions. Radish (Raphanus sativus L) and barnyardgrass (Echinochloa crus‐galli (L) Beauv) were very senstive to RH‐1965. Under high light (330 µE m⁻² s⁻¹), the I₅₀ values for inhibition of chlorophyll accumulation were 0.10 and 0.15 µM , respectively. Wheat (Triticum aestivus L), on the other hand, was much less sensitive to RH‐1965 (I₅₀ = 1.4 µM ). It is concluded that the mode of action of RH‐1965 involves the inhibition of ξ‐carotene desaturation. © 2000 Society of Chemical Industry     

Microtubule dynamics in compatible and incompatible interactions of soybean hypocotyl cells with Phytophthora sojae

The arrangement of microtubules in soybean ( Glycine max ) cells was examined during compatible and incompatible interactions of hypocotyls of soybean cv. Harosoy (susceptible) and cv. Haro 1272 (resistant) with race 1 of the soybean-specific pathogen Phytophthora sojae . Both reaction types were similar during the first 3 h after zoospore inoculation in terms of the number of cells penetrated, and depth penetrated into the cortex. By 3 h postinoculation, clear differences had developed between the two interaction types: incompatible interactions were characterized by a hypersensitive response that was confined to single penetrated cells; while compatibly responding cells appeared unchanged. Both types of response were characterized by autofluorescence of cell walls or cytoplasm and, at 6 h after inoculation, complete disorganization of cell cytoplasm. Reorientation and loss of microtubules was seen in the early stages of the incompatible interaction in association with cellular hypersensitivity, but not in compatible responses. In cells adjacent to those that reacted hypersensitively, there was little evidence of change in microtubule orientation. Treatment of hypocotyls with the microtubule depolymerizer oryzalin prior to inoculation did not alter the compatible response, but led to breakdown of the incompatible response. Changes in microtubule orientation and state are thus among the first structural changes that are visible within cells during incompatibility in this system.     

Emamectin benzoate as a candidate for a trunk‐injection agent against the pine wood nematode,Bursaphelenchus xylophilus

In order to develop an effective trunk‐injection agent against pine wood nematode, Bursaphelenchus xylophilus, an in vitro assay was used to examine the antinematodal activity of 58 commercially available compounds with known modes of action. Among compounds tested, the GABA receptor agonists had better anti‐nematodal activity than compounds influencing glutamate, N‐methyl‐D ‐aspartate, β‐adrenergic, dopamine, muscarinic acetylcholine and nicotinic acetylcholine receptors, as well as those inhibiting acetylcholinesterase, monoamine oxidase, 5‐hydroxytryptamine uptake and Ca²⁺, K⁺, Na⁺ and Cl⁻ channels. Avermectins and milbemycins strongly inhibited propagation of the nematode. Emamectin benzoate proved to be the most active (IC₉₅ 0.050 µM ) being over 140 times more active than the active ingredient of conventional trunk‐injection agents. It is concluded that emamectin benzoate is a strong candidate for an anti‐nematodal trunk injection agent. © 2000 Society of Chemical Industry     

Pronamide disrupts mitosis in a unique manner

Pronamide (3,5-dichloro(N-1,1-dimethyl-2-propynyl)benzamide) has traditionally been grouped with mitotic disrupters, such as colchicine or trifluralin, that inhibit polymerization of tubulin into microtubules. Many of the effects of pronamide (c-metaphases, polymorphic nuclei, isodiametric cells, abnormal xylem development, swollen root tips) are similar to these well-studied mitotic disrupters. However, immunofluorescence microscopy studies using anti-tubulin sera revealed that, unlike other mitotic disrupters, pronamide-treated cells have greatly shortened microtubules that are located only at the kinetochore region. These structures detected by immunofluorescence studies were determined by electron microscopy to be microtubules. Because these short kinetochore microtubules did not allow for mitosis to proceed normally, the nuclear envelope reforms around the chromosomes leading to polymorphic nuclei. Frequently, other cytoplasmic organelles normally excluded in the zone occupied by the chromosomes and spindle during mitosis were retained within the reformed nuclei. These data indicate that, although the net effect of pronamide is similar to those of colchicine and trifluralin, pronamide acts by a different mechanism.     

Herbicidal potential of pyrenophorol isolated from a Drechslera avenae pathotype

A pathotype of Drechslera avenae (Eidam) Scharif exhibited host‐specificity, being pathogenic to Avena sterilis L but not to a number of related or unrelated species tested. In culture, the fungus produces a metabolite which was identified as the macrodiolide pyrenophorol (5,13‐dihydroxy‐8,16‐dimethyl‐1,9‐dioxa‐cyclohexadeca‐3,11‐diene‐2,10‐dione). This compound at a concentration of 320 µM was phytotoxic to A sterilis and considerably less so to Avena fatua L. The phytotoxicity was expressed as leaf necrosis on seedling cuttings partially immersed in pyrenophorol solution and as ‘green islands’ on detached leaves on which droplets of the solution were placed. Seed germination and seedling growth of A sterilis were not affected. Pyrenophorol at concentrations up to 640 µM did not cause any symptoms of phytotoxicity to a number of other monocotyledons or dicotyledons tested, with the exception of Lycopersicon esculentum Miller on which leaf necrosis was observed after application of the substance through the vascular system to seedling cuttings. These findings are discussed in relation to the exploitation of such compounds of natural origin as wild oat herbicides. © 2000 Society of Chemical Industry     

Joint action of amino acid biosynthesis-inhibiting herbicides

The joint action of binary mixtures of the amino acid biosynthesis‐inhibiting herbicides glyphosate, glufosinate‐ammonium, metsulfuron‐methyl and imazapyr was assessed in pot experiments applying the Additive Dose Model (ADM). Plants of Sinapis arvensis or S. alba were sprayed with seven doses of the herbicides alone and binary fixed‐ratio mixtures of the four herbicides. In total, 73 binary mixtures were studied in six separate experiments. Mixtures of glyphosate and glufosinate‐ammonium were less phytotoxic than predicted by ADM whether commercial formulations or technical grade products were applied. In contrast, mixtures of glyphosate and metsulfuron‐methyl, glyphosate and imazapyr, glufosinate‐ammonium and metsulfuron‐methyl, glufosinate‐ammonium and imazapyr, and metsulfuron‐methyl and imazapyr either followed ADM or were synergistic. Synergism was observed most frequently for mixtures of glyphosate or glufosinate‐ammonium with metsulfuron‐methyl. Synergism was also more pronounced for commercial formulations of glyphosate and glufosinate‐ammonium than for the corresponding technical grade formulations, implying that synergism was caused by the presence of the formulation constituents of the commercial glyphosate and glufosinate‐ammonium formulations in the spray solution.     

Control of Avena fatua L. and Cirsium arvense (L.) Scop. with mixtures of 3,6-dichloropicolinic acid and four herbicides for control of A. fatua

Control of Avena fatua (L.) (wild oat) with diclofop methyl applied at 0·7 kg ha^?1 at the two-leaf stage and difenzoquat at 0·84 kg ha^?1 at the four-leaf stage in wheat (Triticum aestivum L.) under field conditions was good and not affected when either of these herbicides was mixed with 3,6-dichloropicolinic acid as the monoethanolamine salt at 0·14, 0·20 or 0·30 kg ha^?1. In the glasshouse, mixtures containing 3,6-dichloropicolinic acid at rates as high as 0·6 kg ha^?1 also did not affect control of A. fatua. When barban at 0·35 kg ha^?1, or flamprop methyl at 0·56 kg ha^?1 was mixed with similar rates of 3,6-dichloropicolinic acid and applied at the two-leaf and four-leaf stage of A. fatua respectively, a reduction in control of A. fatua (antagonism) occurred under both field and glasshouse conditions. The herbicides for control of A. fatua did not influence the fresh weight suppression of C. arvense shoots obtained in the glasshouse with 3,6-dichloropico-colinic acid at 0·3 kg ha^?1. Early tolerance of wheat (cv. Neepawa) was acceptable with all mixtures. Wheat yields with diclofop methyl or difenzoquat alone or in mixture with 3,6-dichloropicolinic acid were increased over the yields from the A. fatua-infested control.     

Fifty years of herbicide research: comparing the discovery of trifluralin and halauxifen‐methyl

Fifty years separate the commercialization of the herbicides trifluralin and halauxifen‐methyl. Despite the vast degree of technological change that occurred over that time frame, some aspects of their discovery stories are remarkably similar. For example, both herbicides were prepared very early in the iterative discovery process and both were developed from known lead compound structures by hypothesis‐driven research efforts without the use of in vitro assays or computer‐aided molecular design. However, there are aspects of the halauxifen‐methyl and trifluralin discovery stories that are substantially different. For example, the chemical technology required for the cost‐effective production of halauxifen‐methyl simply did not exist just two decades prior to its commercial launch. By contrast, the chemical technology required for the cost‐effective production of trifluralin was reported in the chemical literature more than two decades prior to its commercial launch. In addition, changes in regulatory environment since the early 1960s ensured that their respective discovery to commercial launch stories would also differ in substantial ways. Ultimately, the time and cost required to develop and register halauxifen‐methyl demanded a global initial business case while the lower registration hurdles that trifluralin cleared enabled a narrow initial business case mainly focused on the USA. © 2017 Society of Chemical Industry     

Assessment of the high-dose concept and level of control provided by MON 87701 × MON 89788 soybean against Anticarsia gemmatalis and Pseudoplusia includens (Lepidoptera: Noctuidae) in Brazil

BACKGROUND: Genetically modified MON 87701 × MON 89788 soybean (Glycine max), which expresses the Cry1Ac and EPSP‐synthase proteins, has been registered for commercial use in Brazil. To develop an Insect Resistance Management (IRM) program for this event, laboratory and field studies were conducted to assess the high‐dose concept and level of control it provides against Anticarsia gemmatalis and Pseudoplusia includens. RESULTS: The purified Cry1Ac protein was more active against A. gemmatalis [LC₅₀ (FL 95%) = 0.23 (0.15–0.34) µg Cry1Ac mL^?1 diet] than P. includens [LC₅₀ (FL 95%) = 3.72 (2.65–4.86) µg Cry1Ac mL^?1 diet]. In bioassays with freeze‐dried MON 87701 × MON 89788 soybean tissue diluted 25 times in an artificial diet, there was 100% mortality of A. gemmatalis and up to 95.79% mortality for P. includens. In leaf‐disc bioassays and under conditions of high artificial infestation in the greenhouse and natural infestation in the field, MON 87701 × MON 89788 soybean showed a high level of efficacy against both target pests. CONCLUSIONS: The MON 87701 × MON 89788 soybean provides a high level of control against A. gemmatalis and P. includes, but a high‐dose event only to A. gemmatalis. Copyright © 2012 Society of Chemical Industry