Development of Transgenic Glyphosate-Resistant Rice with G6 Gene Encoding 5-Enolpyruvylshikimate-3-Phosphate Synthase

Glyphosate-resistant crops have been a huge economic success for genetic engineering. The creating of new glypbosateresistant plants would increase the available choices for planting and lower the price of genetically modified crop seeds. A novel G6 gene from Pseudomonas putida that encoded 5-enolpyruvylshikimate-3-phosphate synthase （EPSPS） was previously isolated. The G6 gene was transfected into rice via Agrobacterium-mediated transformation. The transgenic rice obtained was confirmed by PCR, Southern, and Western blots. The lab experiment and field trials further confirmed that the transgenic rice can survive glyphosate spraying at a dose of 8 g L^-1. In contrast, conventional rice was killed at a weed control glyphosate spray dose of 1 g L^-1. Altogether, the present study showed that the G6 gene works well in rice in vivo for glyphosate-resistance.

Susceptible and Glyphosate-Resistant Palmer Amaranth (<i>Amaranthus palmeri</i>) Response to Glyphosate Using C¹⁴as a Tracer: Retention, Uptake, and Translocation

The foliar retention, absorption, translocation, and diffusion of glyphosate in glyphosate resistant-(R) and susceptible (S)-Palmer amaranth populations from seed collected in Georgia in 2007 were examined. The R population of Palmer amaranth had an elevated copy number of the EPSPS gene conferring the mechanism of resistance. When applications of 14C-glyphosate to a single leaf followed entire plant treatment with glyphosate, the distribution percentages were similar for R and S for the above and below treated leaves when harvested at 1, 6, 12, 24, and 48 hours after treatment (HAT). There were initially no differences between R and S at 1 HAT with an average of 8% absorption for both biotypes. However, data indicated that glyphosate absorption increased for R-Palmer amaranth reaching 41% within 6 HAT and was significantly different (P = 0.01) from the 28% absorbed by S-Palmer amaranth. Glyphosate resistant and susceptible Palmer amaranth averaged 44% 14C-glyphosate absorption by 24 HAT. There were no differences for 14C-glyphosate Bq/mg of plant tissue between R and S for the above the treated leaf and below the treated leaf portions of plants at 1, 6, 12, 24, or 48 HAT. However, root accumulation of 14C-glyphosate in plant tissue was significantly greater by 12 HAT for the roots of R (1.21 Bq/mg) than for S (0.51 Bq/mg). The treated leaf of the R-Palmer amaranth plants exhibited greater translocation of 14C-glyphosate in Bq/mg of tissue than the susceptible over time, indicating no detrimental effect or cost of fitness due to EPSPS gene amplification. Additionally, there were no differences in glyphosate retention in leaf discs assays between R and S biotypes. In spite of an average of 6.5 Bq efflux out of R and S leaf discs after 15 minute, only 0.4 Bq was retained after 150 minutes. Glyphosate was not retained over time in the leaf discs for R and S, and there were no biotype differences within bathing times. However, the rate of efflux (the slope of the curves) was greater for the R biotype. These data support the reported gene amplification non-target site glyphosate resistance mechanism in Palmer amaranth.

Glyphosate-Resistant Common Ragweed Control in Corn with Postemergence Herbicides

Four field trials were conducted on a farm infested with glyphosate-resistant (GR) common ragweed during 2016 and 2017 to evaluate various postemergence (POST) herbicides for the control of GR common ragweed in GR corn. Dicamba at 600 g·a.i.·ha-1, dicamba/diflufenzopyr at 200 g·a.i.·ha-1, dicamba/atrazine at 1500 g·a.i.·ha-1, topramezone + atrazine at 12.5 + 500 g·a.i.·ha-1, bromoxynil + atrazine at 280 + 1500 g·a.i.·ha-1, glufosinate at 500 g·a.i.·ha-1 and 2,4-D ester at 560 g·a.i.·ha-1 provided 58% to 85% control at 4 WAA and 49% to 88% control at 8 WAA. Other herbicides evaluated controlled GR common ragweed 9% to 41%. Common ragweed density was reduced 97%, 95%, 95% and 87% and shoot dry weight was reduced 93%, 95%, 94% and 90% with bromoxynil + atrazine, dicamba, glufosinate and topramezone + atrazine applied POST in GR corn, respectively. Results show that dicamba, bromoxynil + atrazine, topramezone + atrazine and glufosinate applied POST are the most efficacious herbicides among the herbicides evaluated for the control of GR common ragweed in GR corn.

Efficacy of POST glyphosate applications in combination with other POST herbicides in glyphosate-resistant corn (Zea mays L.)

The use of glyphosate-resistant corn has facilitated a shift from a reliance on preemergence residual herbicides to postemergence (POST) herbicides, and in some cases exclusively glyphosate. Glyphosate is a non-selective herbicide that is relatively slow-acting, which may allow weeds to continue to compete with corn after application and potentially decrease crop yield. The addition of several POST corn herbicides, with some residual control, to an early-season glyphosate application was examined to determine if the tankmix combination would improve the speed of weed control compared to glyphosate applied alone. Seven field trials were conducted over three years (2009, 2010 and 2011) near Ridgetown and Exeter, Ontario. The control of common ragweed was improved 3 days after application (DAA) with three POST glyphosate tankmixes compared to glyphosate alone. However control was still less than 55%. Depending on the weed species examined, at 28 DAA two of the glyphosate tankmix treatments tested provided better common ragweed, common lambsquarters, or green foxtail control than glyphosate alone. Treatments providing better weed control at 28 DAA also typically decreased weed density compared to glyphosate alone.

Preplant and Postemergence Control of Glyphosate-Resistant Giant Ragweed in Corn

Glyphosate-resistant (GR) giant ragweed has recently been identified in southwestern Ontario and has the potential to be a significant problem for regional corn producers. Eight field trials [four with preplant (PP) and four with postemergence (POST) herbicides] were conducted from 2013 to 2014 on various Ontario farms infested with GR giant ragweed to determine the efficacy of PP and POST tank-mixes in corn. Glyphosate tank-mixed with atrazine, dicamba, dicamba/atrazine, mesotrione plus atrazine, flumetsulam, isoxaflutole plus atrazine, saflufenacil/dimethenamid-P, S-metolachlor/atrazine and rimsulfuron applied PP provided up to 54%, 95%, 93%, 95%, 40%, 89%, 91%, 50% and 93% control of GR giant ragweed and reduced dry weight 69%, 100%, 99%, 100%, 30%, 92%, 98%, 66% and 99%, respectively. POST application of glyphosate alone and tank-mixed with 2,4-D ester, atrazine, dicamba, dicamba/diflufenzopyr, dicamba/atrazine, bromoxynil plus atrazine, prosulfuron plus dicamba, mesotrione plus atrazine, topramezone plus atrazine, tembotrione/thiencarbazone-methyl and glufosinate provided up to 31%, 84%, 39%, 94%, 89%, 86%, 83%, 78%, 72%, 43%, 63% and 58% GR giant ragweed and reduced dry weight 55%, 99%, 72%, 99%, 99%, 98%, 96%, 96%, 93%, 89%, 91% and 95%, respectively. In general, PP control of GR giant ragweed was greater than POST applied herbicides evaluated. Based on these results, glyphosate tank-mixes containing dicamba or mesotrione plus atrazine applied PP, and dicamba applied POST will provide the most consistent control of GR giant ragweed in corn.

Glyphosate-Resistant Giant Ragweed(Ambrosia trifida L.)in Ontario:Dose Response and Control with Postemergence Herbicides

Giant ragweed (Ambrosia trifida L.) is competitive with agronomic crops and can cause significant yield losses. Rapid adoption of glyphosate-resistant (GR) crops and a concomitant increase in the reliance on glyphosate for weed management has led to the evolution of GR giant ragweed in Ontario, Canada. Field studies were conducted to evaluate the level of resistance in giant ragweed biotypes from Ontario, and to evaluate the effectiveness of various postemer-gence (POST) herbicides in soybean (Glycine max L.). The effective dose (ED) to provide 50%, 80% and 95% giant ragweed control was up to 1658, 9991 and >43200 g?a.e.?ha–1 4 weeks after application (WAA), respectively. For effective control, growers would need to apply glyphosate 18 times greater than the recommended field application dose. Glyphosate applied at the recommended field dose of 900 g?a.e.?ha–1 provided up to 57% control and resulted in soybean yield equivalent to the weedy check. Cloransulam-methyl applied POST provided up to 99% control, reduced giant ragweed density 98%, reduced giant ragweed shoot dry weight 99% and resulted in soybean yield equivalent to the weedfree check. Chlorimuron-ethyl, fomesafen, imazethapyr and imazethapyr plus bentazon applied alone or with glyphosate did not provide adequate control of GR giant ragweed. Based on these results, some GR giant ragweed biotypes from Ontario have evolved a high level of resistance to glyphosate. Cloransulam-methyl applied POST was the only herbicide that provided adequate control and suggests that additional weed management tactics will need to be implemented in order to effectively manage GR giant ragweed.

Linuron Biologically Effective Dose for Glyphosate-Resistant Giant Ragweed (<i>Ambrosia trifida</i>L.) Control in Soybean (<i>Glycine max</i>L.)

Glyphosate-resistant (GR) giant ragweed (Ambrosia trifida L.) was first identified in Canada in 2008 and has since been found throughout southwestern Ontario. Six field trials were conducted over a two-year period (2012, 2013) on Ontario farms with GR giant ragweed to evaluate the efficacy of linuron applied pre-plant (PP) in soybean (Glycine max (L.) Merr.). The dose required for 50%, 80%, and 95% GR giant ragweed control was 1238, 2959, and 6018 g·ai·ha-1 four weeks after application (WAA), respectively. The linuron dose needed for 50%, 80%, and 95% reduction in density was 1554, 3181, and 5643 g·ai·ha-1 and 1204, 2496, and 4452 g·ai·ha-1 for dry weight, respectively. Application of 7874 g·ai·ha-1 linuron was needed to obtain soybean yields that were 90% of the weed-free control;approximately 3.5 times the maximum field recommended dose. To achieve 95% and 98% yields, greater than 8640 g·ai·ha-1 linuron was required. Application of linuron plus glyphosate PP in soybean will help to control GR giant ragweed as well as reduce GR selection pressure.

Influence of late emerging weeds in glyphosate-resistant corn

Fifteen field trials were conducted from 2009 to 2011 in Ontario, Canada and Michigan, USA to determine how long glyphosate-resistant corn needs to be kept weed-free after emergence to prevent yield loss. Data were separated into two environments based on when yield loss first occurred after glyphosate application. In Environment 1 (4/15 sites) yield was not reduced when corn was kept weed-free until the 4-leaf stage. However, in Environment 2 (11/15 sites) there was no yield loss when corn was kept weed-free up to the 2-leaf stage. The most prominent weeds were velvetleaf, redroot pigweed, common ragweed, common lambsquarters and foxtail species. While later emerging weeds did not necessarily impact corn yield, weeds emerging after the 2- and 4-leaf corn stage likely produced seed that was added to the soil seed bank. Weeds emerging after 6-, 8-, and 10-leaf corn growth stages were small (low biomass/seedlings) and most likely did not reach reproductive maturity. Based on this research, corn must be maintained weed-free up to the 4-leaf stage. Any weeds emerging after that did not influence corn yield.

Interaction of the Bioherbicide <i>Myrothecium verrucaria</i>with Technical-Grade Glyphosate on Glyphosate-Susceptible and -Resistant Palmer Amaranth

Previously we found that a strain of Myrothecium verrucaria (MV) exhibited bioherbicidal activity against several important weeds, and that some commercial formulations of glyphosate applied with MV resulted in synergistic interactions that improved weed control efficacy. We also found that MV had bioherbicidal activity against glyphosate-resistant Palmer amaranth. We have also reported that some commercial formulations are inhibitory to MV. Our objectives were to test the effect of unformulated glyphosate (high purity, technical-grade glyphosate) alone and in combination with MV for bioherbicidal activity on glyphosate-susceptible and -resistant Palmer amaranth biotypes under greenhouse conditions and to examine technical-grade glyphosate on the growth of this bioherbicide. High purity glyphosate (without adjuvants/surfactants) was not toxic to MV growth and sporulation at concentrations up to 2.0 mM when grown on agar supplemented with the herbicide. Both biotypes were injured by MV and MV plus glyphosate treatments as early as 19 h after application (3 h after a dew period of 16 h). These injury effects increased and were more evident through the 6-day time course, when after 120 h the MV plus glyphosate treatment had killed all glyphosate-susceptible and -resistant plants. The interaction of glyphosate plus MV was synergistic toward the control of Palmer amaranth. Data strongly suggest that the active ingredient is responsible for the synergy previously found when this bioherbicide was combined with some commercial formulations of glyphosate. Results demonstrated that MV can control both glyphosate-resistant and -susceptible Palmer amaranth seedlings and act synergistically with high-purity glyphosate to provide improved weed control.

Bioherbicidal Efficacy of a Myrothecium verrucaria-Sector on Several Plant Species

Comparative studies were conducted on mycelial preparations of the bioherbicide, Myrothecium verrucaria (MV) strain IMI 361690 and a recently discovered sector (MV-Sector BSH) of this fungus. The whitish sector was discovered, isolated, grown in pure culture on PDA and found to be a stable, non-spore producing mutant when cultured over several months under conditions that cause circadian sporulation during growth of its MV parent. Application of MV and MV-Sector BSH mycelial preparations to intact plants (hemp sesbania and sicklepod) and leaf discs (kudzu and glyphosate-resistant Palmer amaranth) showed that the sector efficacy was generally equal to, or slightly lower than MV. Bioassays of MV and this sector on seed germination and early growth of sicklepod and hemp sesbania seeds demonstrated that hemp sesbania seeds were slightly more sensitive to the fungus than sicklepod seeds and that the sector bioherbicidal activity was slightly less than that of MV. SDS-PAGE protein profiles of cellular extracts of MV and the sector and their respective culture supernatants showed several differences with respect to quantity and number of certain protein bands. Overall results showed that the isolate was a non-spore producing mutant with phytotoxicity to several weeds (including weeds tolerant or resistant to glyphosate), and that the phytotoxic effects were generally equivalent to those caused by MV treatment. Results of this first report of a non-sporulating MV mutant that suggest additional studies on protein analysis, and an extended weed host range under greenhouse and field conditions are needed in order to further evaluate its possible bioherbicidal potential.

Varying Tolerance to Glyphosate in a Population of Palmer Amaranth with Low EPSPS Gene Copy Number

A Palmer amaranth population (seeds collected in the year 2000;Washington Co., MS) suspected to be susceptible to glyphosate was examined as a population and as individual plants and found to exhibit varying tolerance or resistance to glyphosate. Whole plant spraying of glyphosate (0.84 kg·ha?1) to the population revealed that approximately 40% of this population were resistant to glyphosate and an LD50 of 0.75 kg·ha?1 was determined. Spray application of glyphosate indicated that some plants displayed varying degrees of resistance 14 days after treatment. Initial tests using leaf disc bioassays on 10 individual plants selected randomly from the population, allowed characterization of glyphosate resistance using both visual ratings of injury and quantitative measurement via chlorophyll content analysis. After initial bioassays and spray application, five plants with a range of tolerance to glyphosate were selected for cloning so that further studies could be accomplished on these individuals. Q-PCR analysis of these clones showed that resistance was not due to elevated EPSPS gene copy number. Shikimate levels were lower in the resistant and higher in the susceptible clones which correlated with varying degrees of resistance demonstrated in bioassays and spray application of glyphosate of these clones. Results demonstrate that individuals in a population can vary widely with respect to herbicide resistance and suggest that uptake, translocation, sequestration, metabolism or altered target site may contribute to the resistance in some individuals of this population.

Overexpression of G10-EPSPS in soybean provides high glyphosate tolerance

Glyphosate is a highly efficient, broad-spectrum nonspecific herbicide that inhibits the 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS)-mediated pathway of shikimic acid. The screening of glyphosate-resistant EPSPS gene is a major means for the development of new genetically modified glyphosate-resistant transgenic crop. Currently, the main commercialized glyphosate-resistant soybean contains glyphosate-resistant gene CP4-EPSPS. In this study, a G10-EPSPS gene was reported providing glyphosate resistance in Zhongdou 32. Here, G10-EPSPS gene was introduced into soybeans through Agrobacterium-mediated soybean cotyledon node. PCR, Southern blotting, semi-quantitative RT-PCR, qRT-PCR, and Western blotting were used, and the results revealed that G10-EPSPS had been integrated into the soybean genome and could be expressed steadily at both mRNA and protein levels. In addition, glyphosate resistance analysis showed that the growth of transgenic soybean had not been affected by concentrations of 900 and 2 700 g a.e. ha–1 of glyphosate. All the results indicated that G10-EPSPS could provide high glyphosate resistance in soybeans and be applied in production of glyphosate-resistant soybean.

Bioassay and Characterization of Several Palmer Amaranth (<i>Amaranthus palmeri</i>) Biotypes with Varying Tolerances to Glyphosate

The wide distribution of Palmer amaranth (Amaranthus palmeri) in the southern US became a serious weed control problem prior to the extensive use of glyphosate-resistant crops. Currently glyphosate-resistant populations of Palmer amaranth occur in many areas of this geographic region creating an even more serious threat to crop production. Investigations were undertaken using four biotypes (one glyphosate-sensitive, one resistant from Georgia and two of unknown tolerance from Mississippi) of Palmer amaranth to assess bioassay techniques for the rapid detection and level of resistance in populations of this weed. These plants were characterized with respect to chlorophyll, betalain, and protein levels and immunological responses to an antibody of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) the target site of glyphosate. Only slight differences were found in four biotypes grown under greenhouse conditions regarding extractable soluble protein and chlorophyll content, but one biotype was found to be devoid of the red pigment, betalain. Measurement of early growth (seedling shoot elongation) of seedlings was a useful detection tool to determine glyphosate resistance. A leaf disc bioassay (using visual ratings and/or chlorophyll analysis) and an assay for shikimate accumulation were effective methods for determining herbicide resistance levels. The two unknown biotypes were found to be resistant to this herbicide. Some differences were found in the protein profiles of the biotypes, and western blots demonstrated a weak labeling of antibody in the glyphosate-sensitive biotype, whereas strong labeling occurred in the resistant plants. This latter point supports research by others, that increased copy number of the EPSPS gene (and increased EPSPS protein levels) is the resistance mechanism in this species. Results indicate the utility of certain bioassays for the determination of resistance and provide useful comparative information on the levels of inherent constituents among closely related plants.

Weed and insect control affected by mixing insecticides with glyphosate in cotton

Field studies were conducted in 2012 and 2013 to evaluate weed and insect control efficacy with glyphosate at 1 230 g ai（active ingredient） ha^（-1） and the insecticides acephate（728 g ai ha^（-1））,carbosulfan（135 g ai ha^（-1））,endosulfan（683 g ai ha^（-1））,imidacloprid（32 g ai ha^（-1））,or lambda-cyhalothrin（23 g ai ha^（-1））,as well as glyphosate tank-mixed with these insecticides.Four of the most common weeds in cotton,common purslane,false daisy,goosegrass,and lambsquarters,were manually sown in the cotton field and treated with glyphosate alone or in combination with insecticides.Glyphosate efficacy,based on visual estimates of control and weed fresh weight at 21 d after treatment（DAT）,was unaffected by the addition of insecticides.Four weeds were controlled by 93-97%and 86-100%（visual rating） and reduced weed fresh biomass by98-99%and 96-100%with glyphosate alone and its combination with insecticides,respectively.Addition of glyphosate to acephate improved cotton aphid control compared with acephate alone.However,addition of glyphosate to carbosulfan,endosulfan,imidacloprid,or lambda-cyhalothrin did not affect the aphid control when compared with the insecticide alone treatments.These results indicate that cotton producers could potentially integrate weed and insect management strategies by choosing suitable insecticide mixing partners with glyphosate,thereby reducing the application costs without sacrificing the efficacy of the glyphosate or the insecticides.

Genomic Stability of Palmer amaranth Plants Derived by Macro-Vegetative Propagation

qPCR (quantitative polymerase chain reaction) and random amplified polymorphic DNA (RAPD) were utilized to investigate genetic stability of Palmer amaranth cloned plants over 10 generations. DNA from original parent Palmer amaranth plants (grown from seeds) was re-analyzed using qPCR, and confidence levels for determining ΔΔCt (threshold crossing) values were established. ANOVA was used to determine variation (margin of error) of these ΔΔCt values. This margin of error was applied to qPCR analysis of DNA from eight individual parent plants and their descendants (10th generation) so that possible differences in EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene copy number could be ascertained. This method (and the associated error) indicated a lack of agreement in ΔΔCt values of DNA from plants of these two generations. qPCR analysis showed that in five out of eight clones, EPSPS gene copy number varied more than the calculated error (P = 0.05). A second technique to monitor genetic stability, RAPD was used to determine possible changes in genomic DNA due to extended cloning of these regenerated plants. RAPD analysis showed that four out of the eight clones differed when the profiles of the two generations were compared. Results show that qPCR and RAPD analysis point to the fact that several Palmer amaranth clones experienced changes in genome structure over 10 generations. Although the glyphosate resistance trait was retained, results suggest that during cloning studies, the genetic stability of macro-vegetatively propagated lines should be monitored.

Tolpyralate + Atrazine Applied Preemergence Provides Residual GR Canada Fleabane [<i>Conyza canadensis</i>(L.) Cronq.] Control Similar to Current Industry Standards

Tolpyralate is a benzoylpyrazole, 4-hydroxyphenyl-pyruvate dioxygenase inhibitor, and a herbicide registered for use in corn. The efficacy of tolpyralate plus atrazine to provide full-season residual control of glyphosate-resistant (GR) Canada fleabane in corn is not known under Ontario environmental conditions. Five field trials were completed over a two-year period (2018-19) in south-western Ontario on farms with confirmed GR Canada fleabane [Conyza canadensis (L.) Cronq.] populations to determine if tolpyralate + atrazine provides full-season residual control of GR Canada fleabane in corn. Corn injury was less than 10% with all treatments. At 4 weeks after application (WAA), tolpyralate (30 g&middot;ai&middot;ha&minus;1), tolpyralate (40 g&middot;ai&middot;ha&minus;1), and atrazine (560 g&middot;ai&middot;ha&minus;1) controlled GR Canada fleabane 64, 78 and 72%, respectively. A tank mix of tolpyralate + atrazine at both rates improved GR Canada fleabane control to 94%. Saflufenacil/dimethenamid-p, mesotrione + atrazine, and dicamba/atrazine controlled GR Canada fleabane 99, 95 and 92%, respectively. At 8 WAA, tolpyralate (30 g&middot;ai&middot;ha&minus;1), tolpyralate (40 g&middot;ai&middot;ha&minus;1) and atrazine (560 g&middot;ai&middot;ha&minus;1) controlled GR Canada fleabane 83, 88, and 83%, respectively (Table 2). The tank mixes of tolpyralate (30 g&middot;ai&middot;ha&minus;1) + atrazine (560 g&middot;ai&middot;ha&minus;1) and tolpyralate (40 g&middot;ai&middot;ha&minus;1) + atrazine (560 g&middot;ai&middot;ha&minus;1) controlled GR Canada fleabane 94, and 97%, respectively, 8 WAA which was similar to saflufenacil/dimethenamid-p, mesotrione + atrazine and dicamba/atrazine. There was no treatment difference for corn yield. Based on these results, tolpyralate (40 g&middot;ha&minus;1), tolpyralate (30 g&middot;ha&minus;1) + atrazine and tolpyralate (40 g&middot;ha&minus;1) + atrazine, applied PRE, provided similar control of GR Canada fleabane as current industry standards at 8 WAA.