Herbicide Selection Promotes Antibiotic Resistance in Soil Microbiomes.

Herbicides are one of the most widely used chemicals in agriculture. While they are known to be harmful to nontarget organisms, the effects of herbicides on the composition and functioning of soil microbial communities remain unclear. Here we show that application of three widely used herbicides-glyphosate, glufosinate, and dicamba-increase the prevalence of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in soil microbiomes without clear changes in the abundance, diversity and composition of bacterial communities. Mechanistically, these results could be explained by a positive selection for more tolerant genotypes that acquired several mutations in previously well-characterized herbicide and ARGs. Moreover, herbicide exposure increased cell membrane permeability and conjugation frequency of multidrug resistance plasmids, promoting ARG movement between bacteria. A similar pattern was found in agricultural soils across 11 provinces in China, where herbicide application, and the levels of glyphosate residues in soils, were associated with increased ARG and MGE abundances relative to herbicide-free control sites. Together, our results show that herbicide application can enrich ARGs and MGEs by changing the genetic composition of soil microbiomes, potentially contributing to the global antimicrobial resistance problem in agricultural environments.

Two mechanisms provide tolerance to cyhalofop-butyl in pond lovegrass [Eragrostis japonica (Thunb.) Trin.].

Pond lovegrass [Eragrostis japonica (Thunb.) Trin.] is an annual grass weed of rice fields worldwide. Cyhalofop-butyl has been widely used for controlling annual grass weeds in rice fields. However, E. japonica is tolerant to cyhalofop-butyl. The effective dose values of cyhalofop-butyl for 29 E. japonica populations causing 50% inhibition of fresh weight (GR50: 130.15 to 187.61 g a.i. ha-1) were much higher than the recommended dose of cyhalofop-butyl (75 g a.i. ha-1) in the field. The mechanisms of tolerance to cyhalofop-butyl in E. japonica were identified. In vitro activity assays revealed that the cyhalofop-butyl concentration required to inhibit 50% of the acetyl-coenzyme A carboxylase (ACCase) activity (IC50) was 6.22-fold higher in E. japonica than that in the cyhalofop-butyl-susceptible Chinese sprangletop [Leptochloa chinensis (L.) Nees]. However, mutations in the ACCase gene, previously found to endow target-site resistance in weeds, were not detected in the sequences obtained. Additionally, the expression level of genes encoding ACCase in E. japonica was found to be as similar to L. chinensis. Tolerance was reduced by two cytochrome P450 monooxygenases (Cyt P450s) inhibitors (1-aminobenzotriazole and piperonyl butoxide) and the activity of NADPH-dependent cytochrome P450 reductase in E. japonica was approximately 4.46-fold higher than that of L. chinensis after cyhalofop-butyl treatment. Taken together, it is concluded that two co-existing mechanisms, an insensitive target ACCase and an enhanced metabolism mediated by Cyt P450s, endow tolerance to cyhalofop-butyl in E. japonica.

Genetic Analysis and Fine Mapping of ZmGHT1 Conferring Glufosinate Herbicide Tolerance in Maize (Zea mays L.).

Weed interference in the crop field is one of the major biotic stresses causing dramatic crop yield losses, and the development of herbicide-resistant crops is critical for weed control in the application of herbicide technologies. To identify herbicide-resistant germplasms, we screened 854 maize inbreed lines and 25,620 seedlings by spraying them with 1 g/L glufosinate. One plant (L336R), possibly derived from a natural variation of line L336, was identified to have the potential for glufosinate tolerance. Genetic analysis validated that the glufosinate tolerance of L336R is conferred by a single locus, which was tentatively designated as ZmGHT1. By constructing a bi-parental population derived from L336R, and a glufosinate sensitive line L312, ZmGHT1 was mapped between molecular markers M9 and M10. Interestingly, genomic comparation between the two sequenced reference genomes showed that large scale structural variations (SVs) occurred within the mapped region, resulting in 2.16 Mb in the inbreed line B73, and 11.5 kb in CML277, respectively. During the fine mapping process, we did not detect any additional recombinant, even by using more than 9500 F2 and F3 plants, suspecting that SVs should also have occurred between L336R and L312 in this region, which inhibited recombination. By evaluating the expression of the genes within the mapped interval and using functional annotation, we predict that the gene Zm00001eb361930, encoding an aminotransferase, is the most likely causative gene. After glufosinate treatment, lower levels of ammonia content and a higher activity of glutamine synthetase (GS) in L336R were detected compared with those of L336 and L312, suggesting that the target gene may participate in ammonia elimination involving GS activity. Collectively, our study can provide a material resource for maize herbicide resistant breeding, with the potential to reveal a new mechanism for herbicide resistance.

Transcriptome profiling of the fungus Aspergillus nidulans exposed to a commercial glyphosate-based herbicide under conditions of apparent herbicide tolerance.

Glyphosate-based herbicides, such as Roundup®, are the most widely used non-selective, broad-spectrum herbicides. The release of these compounds in large amounts into the environment is susceptible to affect soil quality and health, especially because of the non-target effects on a large range of organisms including soil microorganisms. The soil filamentous fungus Aspergillus nidulans, a well-characterized experimental model organism that can be used as a bio-indicator for agricultural soil health, has been previously shown to be highly affected by Roundup GT Plus (R450: 450 g/L of glyphosate) at concentrations far below recommended agricultural application rate, including at a dose that does not cause any macroscopic effect. In this study, we determined alterations in the transcriptome of A. nidulans when exposed to R450 at a dose corresponding to the no-observed-adverse-effect level (NOAEL) for macroscopic parameters. A total of 1816 distinct genes had their expression altered. The most affected biological functions were protein synthesis, amino acids and secondary metabolisms, stress response, as well as detoxification pathways through cytochromes P450, glutathione-S-transferases, and ABC transporters. These results partly explain the molecular mechanisms underlying alterations in growth parameters detected at higher concentrations for this ascomycete fungus. In conclusion, our results highlight molecular disturbances in a soil fungus under conditions of apparent tolerance to the herbicide, and thus confirm the need to question the principle of "substantial equivalence" when applied to plants made tolerant to herbicides.

Engineering Herbicide-Tolerance Rice Expressing an Acetohydroxyacid Synthase with a Single Amino Acid Deletion.

The acetohydroxyacid synthase (AHAS) is an essential enzyme involved in branched amino acids. Several herbicides wither weeds via inhibiting AHAS activity, and the AHAS mutants show tolerance to these herbicides. However, most AHAS mutations are residue substitutions but not residue deletion. Here, residue deletion was used to engineering the AHAS gene and herbicide-tolerant rice. Molecular docking analysis predicted that the W548 of the AHAS was a residue deletion to generate herbicide tolerance. The AHAS-ΔW548 protein was generated in vitro to remove the W548 residue. Interestingly, the deletion led to the tetramer dissociation of the AHAS, while this dissociation did not reduce the activity of the AHAS. Moreover, the W548 deletion contributed to multi-family herbicides tolerance. Specially, it conferred more tolerance to sulfometuron-methyl and bispyribac-sodium than the W548L substitution. Further analysis revealed that AHAS-ΔW548 had the best performance on the sulfometuron-methyl tolerance compared to the wild-type control. Over-expression of the AHAS-ΔW548 gene into rice led to the tolerance of multiple herbicides in the transgenic line. The T-DNA insertion and the herbicide treatment did not affect the agronomic traits and yields, while more branched-chain amino acids were detected in transgenic rice seeds. Residue deletion of W548 in the AHAS could be a useful strategy for engineering herbicide tolerant rice. The increase of branched-chain amino acids might improve the umami tastes of the rice.

Relationship between the amount and composition of epicuticular wax and tolerance of Ipomoea biotypes to glyphosate.

Ipomoea species are troublesome weeds in crop systems through Brazil. Drought stress typically reduces glyphosate efficacy by reducing the foliar uptake of herbicides and their translocation. Using both glyphosate tolerant (GT) and sensitive (GS) plants from Ipomoea grandifolia, I. indivisa and I. purpurea species, this research aimed to (a) correlate amounts of epicuticular wax and tolerance to glyphosate in plants and (b) determine the effect of drought stress (DStress) on changes in the quantity and chemical composition of plant epicuticular waxes. The dose that causes 50% inhibition of growth (GR50) of the biotypes varied between 62 and 1208 (I. grandifolia), 159 and 913 (I. indivisa), and 389 and 1925 g a.e. ha-1 of glyphosate (I. purpurea). There was low inverse correlation (-0.46) between the amount of epicuticular wax and the sensitivity to glyphosate. GT biotypes of the species presented greater plastic capacities than GS biotypes for increasing the amount of epicuticular wax under DStress. The three Ipomoea species exhibited different chemical profiles of waxes supported by IR spectra, which allows for their differentiation. For I. grandifolia and I. purpurea, there was an increase in the polar components in the state without DStress, while for the species I. indivisa, no differences in infrared spectra were detected between the two water conditions.

Limited Diclosulam Herbicide Uptake and Translocation-Induced Tolerance in Crotalaria juncea.

The study was to identify the potential tolerance of Crotalaria juncea to diclosulam uptake and translocation and its effects on the physiological metabolism of plants. Two experiments were carried out; I-Evaluation of uptake and translocation of 14C-diclosulam (35 g a.i. ha-1) in C. juncea, at seven and 14 days after emergence. II-Evaluation of chlorophyll a transient fluorescence of dark-adapted C. juncea leaves when applied diclosulam in pre-emergence. Plants of C. juncea presented an anatomical/metabolic barrier to diclosulam translocation in the stem, which may confer tolerance to this herbicidal, besides reduced translocation due to low accumulation in the cotyledons. In addition, plants can maintain photosynthetic metabolism active when growing in soil with diclosulam by not changing the dynamics of energy dissipation. Thus, when cultivated in soil with residual of diclosulam, C. juncea can tolerate the herbicide to maintain plant growth.

Modification of auxinic phenoxyalkanoic acid herbicides by the acyl acid amido synthetase GH3.15 from Arabidopsis.

Herbicide-resistance traits are the most widely used agriculture biotechnology products. Yet, to maintain their effectiveness and to mitigate selection of herbicide-resistant weeds, the discovery of new resistance traits that use different chemical modes of action is essential. In plants, the Gretchen Hagen 3 (GH3) acyl acid amido synthetases catalyze the conjugation of amino acids to jasmonate and auxin phytohormones. This reaction chemistry has not been explored as a possible approach for herbicide modification and inactivation. Here, we examined a set of Arabidopsis GH3 proteins that use the auxins indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) as substrates along with the corresponding auxinic phenoxyalkanoic acid herbicides 2,4-dichlorophenoxylacetic acid (2,4-D) and 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB). The IBA-specific AtGH3.15 protein displayed high catalytic activity with 2,4-DB, which was comparable to its activity with IBA. Screening of phenoxyalkanoic and phenylalkyl acids indicated that side-chain length of alkanoic and alkyl acids is a key feature of AtGH3.15's substrate preference. The X-ray crystal structure of the AtGH3.15·2,4-DB complex revealed how the herbicide binds in the active site. In root elongation assays, Arabidopsis AtGH3.15-knockout and -overexpression lines grown in the presence of 2,4-DB exhibited hypersensitivity and tolerance, respectively, indicating that the AtGH3.15-catalyzed modification inactivates 2,4-DB. These findings suggest a potential use for AtGH3.15, and perhaps other GH3 proteins, as herbicide-modifying enzymes that employ a mode of action different from those of currently available herbicide-resistance traits.

The P450 gene CYP749A16 is required for tolerance to the sulfonylurea herbicide trifloxysulfuron sodium in cotton (Gossypium hirsutum L.).

BACKGROUND: Weed management is critical to global crop production and is complicated by rapidly evolving herbicide resistance in weeds. New sources of herbicide resistance are needed for crop plants so that applied herbicides can be rotated or combined to thwart the evolution of resistant weeds. The diverse family of cytochrome P450 proteins has been suggested to be a source of detoxifying herbicide metabolism in both weed and crop plants, and greater understanding of these genes will offer avenues for crop improvement and novel weed management practices. RESULTS: Here, we report the identification of CYP749A16 (Gh_D10G1401) which is responsible for the natural tolerance exhibited by most cotton, Gossypium hirsutum L., cultivars to the herbicide trifloxysulfuron sodium (TFS, CGA 362622, commercial formulation Envoke). A 1-bp frameshift insertion in the third exon of CYP749A16 results in the loss of tolerance to TFS. The DNA marker designed from this insertion perfectly co-segregated with the phenotype in 2145 F2 progeny of a cross between the sensitive cultivar Paymaster HS26 and tolerant cultivar Stoneville 474, and in 550 recombinant inbred lines of a multi-parent advanced generation inter-cross population. Marker analysis of 382 additional cotton cultivars identified twelve cultivars containing the 1-bp frameshift insertion. The marker genotypes matched perfectly with phenotypes in 188 plants from the selected twelve cultivars. Virus-induced gene silencing of CYP749A16 generated sensitivity in the tolerant cotton cultivar Stoneville 474. CONCLUSIONS: CYP749A16 located on chromosome D10 is required for TFS herbicide tolerance in cotton. This finding should add to the repertoire of tools available to farmers and breeders for the advancement of agricultural productivity.

Expression characterization of the herbicide tolerance gene Aryloxyalkanoate Dioxygenase (aad-1) controlled by seven combinations of regulatory elements.

BACKGROUND: Availability of well characterized maize regulatory elements for gene expression in a variety of tissues and developmental stages provides effective alternatives for single and multigene transgenic concepts. We studied the expression of the herbicide tolerance gene aryloxyalkanoate dioxygenase (aad-1) driven by seven different regulatory element construct designs including the ubiquitin promoters of maize and rice, the actin promoters of melon and rice, three different versions of the Sugarcane Bacilliform Badnavirus promoters in association with other regulatory elements of gene expression. RESULTS: Gene expression of aad-1 was characterized at the transcript and protein levels in a collection of maize tissues and developmental stages. Protein activity against its target herbicide was characterized by herbicide dosage response. Although differences in transcript and protein accumulation were observed among the different constructs tested, all events were tolerant to commercially relevant rates of quizalafop-P-ethyl compared to non-traited maize under greenhouse conditions. DISCUSSION: The data reported demonstrate how different regulatory elements affect transcript and protein accumulation and how these molecular characteristics translate into the level of herbicide tolerance. The level of transcript detected did not reflect the amount of protein quantified in a particular tissue since protein accumulation may be influenced not only by levels of transcript produced but also by translation rate, post-translational regulation mechanisms and protein stability. The amount of AAD-1 enzyme produced with all constructs tested showed sufficient enzymatic activity to detoxify the herbicide and prevent most herbicidal damage at field-relevant levels without having a negative effect on plant health. CONCLUSIONS: Distinctive profiles of aad-1 transcript and protein accumulation were observed when different regulatory elements were utilized in the constructs under study. The ZmUbi and the SCBV constructs showed the most consistent robust tolerance, while the melon actin construct provided the lowest level of tolerance compared to the other regulatory elements used in this study. These data provide insights into the effects of differing levels of gene expression and how these molecular characteristics translate into the level of herbicide tolerance. Furthermore, these data provide valuable information to optimize future designs of single and multiple gene constructs for maize research and crop improvement.

Allele exchange at the EPSPS locus confers glyphosate tolerance in cassava.

Effective weed control can protect yields of cassava (Manihot esculenta) storage roots. Farmers could benefit from using herbicide with a tolerant cultivar. We applied traditional transgenesis and gene editing to generate robust glyphosate tolerance in cassava. By comparing promoters regulating expression of transformed 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) genes with various paired amino acid substitutions, we found that strong constitutive expression is required to achieve glyphosate tolerance during in vitro selection and in whole cassava plants. Using strategies that exploit homologous recombination (HR) and nonhomologous end-joining (NHEJ) DNA repair pathways, we precisely introduced the best-performing allele into the cassava genome, simultaneously creating a promoter swap and dual amino acid substitutions at the endogenous EPSPS locus. Primary EPSPS-edited plants were phenotypically normal, tolerant to high doses of glyphosate, with some free of detectable T-DNA integrations. Our methods demonstrate an editing strategy for creating glyphosate tolerance in crop plants and demonstrate the potential of gene editing for further improvement of cassava.

Zinc finger nuclease-mediated precision genome editing of an endogenous gene in hexaploid bread wheat (Triticum aestivum) using a DNA repair template.

Sequence-specific nucleases have been used to engineer targeted genome modifications in various plants. While targeted gene knockouts resulting in loss of function have been reported with relatively high rates of success, targeted gene editing using an exogenously supplied DNA repair template and site-specific transgene integration has been more challenging. Here, we report the first application of zinc finger nuclease (ZFN)-mediated, nonhomologous end-joining (NHEJ)-directed editing of a native gene in allohexaploid bread wheat to introduce, via a supplied DNA repair template, a specific single amino acid change into the coding sequence of acetohydroxyacid synthase (AHAS) to confer resistance to imidazolinone herbicides. We recovered edited wheat plants having the targeted amino acid modification in one or more AHAS homoalleles via direct selection for resistance to imazamox, an AHAS-inhibiting imidazolinone herbicide. Using a cotransformation strategy based on chemical selection for an exogenous marker, we achieved a 1.2% recovery rate of edited plants having the desired amino acid change and a 2.9% recovery of plants with targeted mutations at the AHAS locus resulting in a loss-of-function gene knockout. The latter results demonstrate a broadly applicable approach to introduce targeted modifications into native genes for nonselectable traits. All ZFN-mediated changes were faithfully transmitted to the next generation.

Safety evaluation of genetically modified DAS-40278-9 maize in a subchronic rodent feeding study.

Genetically modified (GM) maize, DAS-40278-9, expresses the aryloxyalkanoate dioxygenase-1 (AAD-1) protein, which confers tolerance to 2,4-dichlorophenoxyacetic acid (2,4-D) and aryloxyphenoxypropionate (AOPP) herbicides. The aad-1 gene, which expresses the AAD-1 protein, was derived from Gram-negative soil bacterium, Sphingobium herbicidovorans. A 90-day sub-chronic toxicity study was conducted on rats as a component of the safety evaluation of DAS-40278-9 maize. Rats were given formulated diets containing maize grain from DAS-40278-9 or a non-GM near isogenic control comparator at an incorporation rate of 12.5%, 25%, or 50% (w/w), respectively for 90 days. In addition, another group of rats was fed a basic rodent diet. Animals were evaluated by cage-side and hand-held detailed clinical observations, ophthalmic examinations, body weights/body weight gains, feed consumption, hematology, serum chemistry, selected organ weights, and gross and histopathological examinations. Under the condition of this study, DAS-40278-9 maize did not cause any treatment-related effects in rats compared with rats fed diets containing non-GM maize.

Aldo-keto reductase enzymes detoxify glyphosate and improve herbicide resistance in plants.

In recent years, concerns about the use of glyphosate-resistant crops have increased because of glyphosate residual levels in plants and development of herbicide-resistant weeds. In spite of identifying glyphosate-detoxifying genes from microorganisms, the plant mechanism to detoxify glyphosate has not been studied. We characterized an aldo-keto reductase gene from Pseudomonas (PsAKR1) and rice (OsAKR1) and showed, by docking studies, both PsAKR1 and OsAKR1 can efficiently bind to glyphosate. Silencing AKR1 homologues in rice and Nicotiana benthamiana or mutation of AKR1 in yeast and Arabidopsis showed increased sensitivity to glyphosate. External application of AKR proteins rescued glyphosate-mediated cucumber seedling growth inhibition. Regeneration of tobacco transgenic lines expressing PsAKR1 or OsAKRI on glyphosate suggests that AKR can be used as selectable marker to develop transgenic crops. PsAKR1- or OsAKRI-expressing tobacco and rice transgenic plants showed improved tolerance to glyphosate with reduced accumulation of shikimic acid without affecting the normal photosynthetic rates. These results suggested that AKR1 when overexpressed detoxifies glyphosate in planta.

Characterization of glutathione S-transferases in the detoxification of metolachlor in two maize cultivars of differing herbicide tolerance.

Glutathione S-transferases (GSTs) have been widely studied in relation to their role in herbicide tolerance and detoxification. However, a detailed characterization of GSTs from herbicide tolerant and sensitive maize cultivars is still lacking. In this study, we determined the mechanism of differential tolerance between two maize cultivars which had 4-fold difference tolerance to metolachlor. The metabolism rate of metolachlor was more rapid in the tolerant cultivar (Zea mays L. cv Nongda86) than the susceptible one (Zea mays L. cv Zhengda958). Addition of the GST inhibitor ethacrynic acid reduced the metabolism of metolachlor indicating the involvement of GSTs in the differential detoxification of metolachlor. The expression profiles of 32 GST isozymes were measured using quantitative RT-PCR. The results showed the expression of GST genes were slightly up-regulated in Nongda86, but severely inhibited in Zhengdan958 24h after metolachlor treatment. The genes GSTI, GSTIII, GSTIV, GST5, GST6 and GST7, which can detoxify chloroacetanilide herbicides, were all expressed higher in Nongda86 compared to Zhendgan958. The result of GST activity was consistent with the gene expression profiles. Collectively, higher-level expression of GST genes, leading to higher GST activity and faster herbicide detoxification, appears to be responsible for the difference in tolerance to metolachlor in two maize cultivars.

Whole-genome analysis of herbicide-tolerant mutant rice generated by Agrobacterium-mediated gene targeting.

Gene targeting (GT) is a technique used to modify endogenous genes in target genomes precisely via homologous recombination (HR). Although GT plants are produced using genetic transformation techniques, if the difference between the endogenous and the modified gene is limited to point mutations, GT crops can be considered equivalent to non-genetically modified mutant crops generated by conventional mutagenesis techniques. However, it is difficult to guarantee the non-incorporation of DNA fragments from Agrobacterium in GT plants created by Agrobacterium-mediated GT despite screening with conventional Southern blot and/or PCR techniques. Here, we report a comprehensive analysis of herbicide-tolerant rice plants generated by inducing point mutations in the rice ALS gene via Agrobacterium-mediated GT. We performed genome comparative genomic hybridization (CGH) array analysis and whole-genome sequencing to evaluate the molecular composition of GT rice plants. Thus far, no integration of Agrobacterium-derived DNA fragments has been detected in GT rice plants. However, >1,000 single nucleotide polymorphisms (SNPs) and insertion/deletion (InDels) were found in GT plants. Among these mutations, 20-100 variants might have some effect on expression levels and/or protein function. Information about additive mutations should be useful in clearing out unwanted mutations by backcrossing.

Divergent properties and phylogeny of cyanobacterial 5-enol-pyruvyl-shikimate-3-phosphate synthases: evidence for horizontal gene transfer in the Nostocales.

As it represents the target of the successful herbicide glyphosate, great attention has been paid to the shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) synthase. However, inconsistent results have been reported concerning the sensitivity of the enzyme from cyanobacteria, and consequent inhibitory effects on cyanobacterial growth. The properties of EPSP synthase were investigated in a set of 42 strains representative of the large morphological diversity of these prokaryotes. Publicly available protein sequences were analyzed, and related to enzymatic features. In most cases, the native protein showed an unusual homodimeric composition and a general sensitivity to micromolar doses of glyphosate. By contrast, eight out of 15 Nostocales strains were found to possess a monomeric EPSP synthase, whose activity was inhibited only at concentrations exceeding 1 mM. Sequence analysis showed that these two forms are only distantly related, the latter clustering separately in a clade composed of diverse bacterial phyla. The results are consistent with the occurrence of a horizontal gene transfer event involving an evolutionarily distant organism. Moreover, data suggest that the existence of class I (glyphosate-sensitive) and class II (glyphosate-tolerant) EPSP synthases representing two distinct phylogenetic clades is an oversimplification because of the limited number of analyzed samples.

Long-term monitoring of feral genetically modified herbicide-tolerant Brassica napus populations around unloading Japanese ports.

Genetically modified, herbicide-tolerant (GMHT) Brassica napus plants originating from seed spill have recently been found along roadsides leading from Japanese ports that unload oilseed rape. Such introductions have potential biodiversity effects (as defined by the Cartagena Protocol): these include replacement of native elements in the biota through competitive suppression or hybridization. We conducted surveys in the period 2006-2011 to assess such threats. We examined shifts in the population distribution and occurrence of GMHT plants in 1,029 volunteer introduced assemblages of B. napus, 1,169 of B. juncea, and 184 of B. rapa around 12 ports. GMHT B. napus was found around 10 of 12 ports, but its proportion in the populations varied greatly by year and location. Over the survey period, the distributions of a pure non-GMHT population around Tobata and a pure GMHT population around Hakata increased significantly. However, there was no common trend of population expansion or contraction around the 12 ports. Furthermore, we found no herbicide tolerant B. juncea and B. rapa plants derived from crosses with GMHT B. napus. Therefore, GMHT B. napus is not invading native vegetation surrounding its populations and not likely to cross with congeners in Japanese environment.

Response of ligninolytic macrofungi to the herbicide atrazine: dose-response bioassays.

The effect of atrazine concentrations on mycelial growth and ligninolytic enzyme activities of eight native ligninolytic macrofungi isolated in Veracruz, México, were evaluated in a semi-solid culture medium. Inhibition of mycelial growth and growth rates were significantly affected (p=0.05) by atrazine concentrations (468, 937, 1875, and 3750 mg/l). In accordance with the median effective concentration (EC50), Pleurotus sp. strain 1 proved to be the most tolerant isolate to atrazine (EC50=2281.0 mg/l), although its enzyme activity was not the highest. Pycnoporus sanguineus strain 2, Daedalea elegans and Trametes maxima showed high laccase activity (62.7, 31.9, 29.3 U mg/protein, respectively) without atrazine (control); however, this activity significantly increased (p<0.05) (to 191.1, 83.5 and 120.6 U mg/protein, respectively) owing to the effect of atrazine (937 mg/l) in the culture medium. Pleurotus sp. strain 2 and Cymatoderma elegans significantly increased (p<0.05) their manganese peroxidase (MnP) activities under atrazine stress at 468 mg/l. The isolates with high EC50 (Pleurotus sp. strain 1) and high enzymatic activity (P. sanguineus strain 2 and T. maxima) could be considered for future studies on atrazine mycodegradation. Furthermore, this study confirms that atrazine can increase laccase and MnP activities in ligninolytic macrofungi.

First report of severe tolpyralate sensitivity in corn (Zea mays) discovers a novel genetic factor conferring crop response to a herbicide.

BACKGROUND: Tolpyralate, a relatively new inhibitor of 4-hydroxyphenylpyruvate dioxygenase (HPPD), is registered for postemergence use in all types of corn (Zea mays L.) and has a record of excellent crop tolerance. A report of severe crop injury to sweet corn inbred (XSEN187) led to the following objectives: (i) determine whether sensitivity to tolpyralate in XSEN187 exists, and if confirmed, (ii) determine the genetic basis of tolpyralate sensitivity, and (iii) screen other corn germplasm for sensitivity to tolpyralate. RESULTS: Inbred XSEN187 was confirmed sensitive to tolpyralate. Inclusion of methylated seed oil or nonionic surfactant in the spray volume was necessary for severe crop injury. Tolpyralate sensitivity in XSEN187 is not conferred by alleles at Nsf1, a cytochrome P450-encoding gene (CYP81A9) conferring tolerance to many corn herbicides. Evidence suggests that tolpyralate sensitivity in XSEN187 is conferred by a single gene mapped to the Chr05: 283 240-1 222 909 bp interval. Moreover, tolpyralate sensitivity was observed in 48 other sweet corn and field corn inbreds. CONCLUSIONS: Severe sensitivity to tolpyralate exists in sweet corn and field corn germplasm when the herbicide is applied according to label directions. Whereas the corn response to several other herbicides, including HPPD-inhibitors, is conferred by the Nsf1 locus, corn sensitivity to tolpyralate is the result of a different locus. The use of tolpyralate should consider herbicide tolerance in inbred lines from which corn hybrids were derived, whereas alleles that render corn germplasm sensitive to tolpyralate should be eliminated from breeding populations, inbreds, and commercial cultivars. © 2023 Illinois Foundation Seeds, Inc and The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.