Mithila Jugulam

Tandem Amplification of a Chromosomal Segment Harboring EPSPS Locus Confers Glyphosate Resistance in Kochia scoparia

Genes encoding enolpyruvylshikimate phosphate synthase are tandemly arranged on chromosomes of field-evolved glyphosate-resistant Kochia scoparia. Recent rapid evolution and spread of resistance to the most extensively used herbicide, glyphosate, is a major threat to global crop production. Genetic mechanisms by which weeds evolve resistance to herbicides largely determine the level of resistance and the rate of evolution of resistance. In a previous study, we determined that glyphosate resistance in Kochia scoparia is due to the amplification of the 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) gene, the enzyme target of glyphosate. Here, we investigated the genomic organization of the amplified EPSPS copies using fluorescence in situ hybridization (FISH) and extended DNA fiber (Fiber FISH) on K. scoparia chromosomes. In both glyphosate-resistant K. scoparia populations tested (GR1 and GR2), FISH results displayed a single and prominent hybridization site of the EPSPS gene localized on the distal end of one pair of homologous metaphase chromosomes compared with a faint hybridization site in glyphosate-susceptible samples (GS1 and GS2). Fiber FISH displayed 10 copies of the EPSPS gene (approximately 5 kb) arranged in tandem configuration approximately 40 to 70 kb apart, with one copy in an inverted orientation in GR2. In agreement with FISH results, segregation of EPSPS copies followed single-locus inheritance in GR1 population. This is the first report of tandem target gene amplification conferring field-evolved herbicide resistance in weed populations.

Tandem Amplification of a Chromosomal Segment Harboring 5-Enolpyruvylshikimate-3-Phosphate Synthase Locus Confers Glyphosate Resistance in Kochia scoparia

Genes encoding enolpyruvylshikimate phosphate synthase are tandemly arranged on chromosomes of field-evolved glyphosate-resistant Kochia scoparia. Recent rapid evolution and spread of resistance to the most extensively used herbicide, glyphosate, is a major threat to global crop production. Genetic mechanisms by which weeds evolve resistance to herbicides largely determine the level of resistance and the rate of evolution of resistance. In a previous study, we determined that glyphosate resistance in Kochia scoparia is due to the amplification of the 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) gene, the enzyme target of glyphosate. Here, we investigated the genomic organization of the amplified EPSPS copies using fluorescence in situ hybridization (FISH) and extended DNA fiber (Fiber FISH) on K. scoparia chromosomes. In both glyphosate-resistant K. scoparia populations tested (GR1 and GR2), FISH results displayed a single and prominent hybridization site of the EPSPS gene localized on the distal end of one pair of homologous metaphase chromosomes compared with a faint hybridization site in glyphosate-susceptible samples (GS1 and GS2). Fiber FISH displayed 10 copies of the EPSPS gene (approximately 5 kb) arranged in tandem configuration approximately 40 to 70 kb apart, with one copy in an inverted orientation in GR2. In agreement with FISH results, segregation of EPSPS copies followed single-locus inheritance in GR1 population. This is the first report of tandem target gene amplification conferring field-evolved herbicide resistance in weed populations.

Inheritance of picloram and 2,4-D resistance in wild mustard (Brassica kaber)

Abstract The primary goal of this research was to determine the inheritance of cross-resistance to several groups of auxinic herbicides through classical genetic approaches using auxinic herbicide–resistant (R) and –susceptible (S) wild mustard biotypes obtained from western Canada. F1 progeny were raised from crosses between homozygous auxinic herbicide–R and –S wild mustard parental lines. The F1 and F2 populations were assessed for picloram (pyridine group) and 2,4-D (phenoxyalkanoic group) resistance or susceptibility. Analyses of the F1 as well as the F2 progeny indicate that a single dominant gene confers the resistance to picloram and 2,4-D similar to an earlier report of dicamba-based (benzoic acid group) resistance in this wild mustard biotype. Furthermore, analyses of backcross progeny in this species indicate that resistance to all three auxinic herbicides, i.e., picloram, dicamba, and 2,4-D, is determined by closely linked genetic loci. With this information on inheritance of resistance to several auxinic herbicide families, the R biotype of wild mustard offers an excellent system to isolate and characterize the auxinic herbicide–resistance gene. Nomenclature: 2,4-D; picloram; wild mustard, Brassica kaber (DC) L.C. Wheeler SINAR.

Field‐evolved resistance to four modes of action of herbicides in a single kochia (Kochia scoparia L. Schrad.) population

BACKGROUND Evolution of multiple herbicide resistance in weeds is a serious threat to weed management in crop production. Kochia is an economically important broadleaf weed in the U.S. Great Plains. This study aimed to confirm resistance to four sites of action of herbicides in a single kochia (Kochia scoparia L. Schrad.) population from a crop field near Garden City (GC), Kansas, and further determine the underlying mechanisms of resistance. RESULTS One-fourth of the GC plants survived the labeled rate or higher of atrazine [photosystem II (PSII) inhibitor], and the surviving plants had the Ser-264 to Gly mutation in the psbA gene, the target site of atrazine. Results showed that 90% of GC plants survived the labeled rate of dicamba, a synthetic auxin. At least 87% of the plants survived up to 72 g a.i. ha(-1) of chlorsulfuron [acetolactate synthase (ALS) inhibitor], and analysis of the ALS gene revealed the presence of Pro-197 to Thr and/or Trp-574 to Lue mutation(s). Most GC plants also survived the labeled rate of glyphosate [5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitor), and the resistant plants had 5-9 EPSPS gene copies (relative to the ALS gene). CONCLUSION We confirm the first case of evolution of resistance to four herbicide sites of action (PSII, ALS and EPSPS inhibitors and synthetic auxins) in a single kochia population, and target-site-based mechanisms confer resistance to atrazine, glyphosate and chlorsulfuron.

Integrated Management of Glyphosate-Resistant Giant Ragweed (Ambrosia trifida) with Tillage and Herbicides in Soybean

Giant ragweed is one of the most competitive annual broadleaf weeds in soybean production fields in the midwestern United States and eastern Canada because of its early emergence, rapid growth rate, high plasticity, and resistance to glyphosate and acetolactate synthase inhibitors. Therefore, early-season management of giant ragweed is critical to avoid yield loss. The objectives of this study were to evaluate control of glyphosate-resistant giant ragweed through the integration of preplant tillage or 2,4-D; PRE or early POST (EPOST) followed by (fb) late POST (LPOST) herbicide programs with or without preplant tillage or 2,4-D; and their effect on soybean injury and yield. A field study was conducted in 2013 and 2014 in David City, NE in a field infested with glyphosate-resistant giant ragweed. Preplant tillage or 2,4-D application provided > 90% control of glyphosate-resistant giant ragweed 14 d after preplant treatment. Giant ragweed control and biomass reduction were consistently > 90% with preplant tillage or 2,4-D fb sulfentrazone plus cloransulam PRE or glyphosate plus cloransulam EPOST fb glyphosate plus fomesafen or lactofen LPOST compared with ≤ 86% control with same treatments without preplant tillage or 2,4-D. PRE or EPOST fb LPOST herbicide programs preceded by preplant treatments resulted in giant ragweed density < 2 plants m−2 and soybean yield > 2,400 kg ha−1 compared with the density of ≥ 2 plants m−2 and soybean yield < 1,800 kg ha−1 under PRE or EPOST fb LPOST herbicide programs. The contrast analysis also indicated that preplant tillage or 2,4-D fb a PRE or POST program was more effective for giant ragweed management compared with PRE fb POST herbicide programs. Integration of preplant tillage would provide an alternative method for early-season control of giant ragweed; however, a follow up application of herbicides is needed for season-long control in soybean. Nomenclature: 2,4-D amine; cloransulam; fomesafen; lactofen; sulfentrazone; giant ragweed, Ambrosia trifida L.; soybean, Glycine max (L.) Merr. Ambrosia trifida es una de las malezas de hoja ancha más competitivas en campos de producción de soja en el centro-oeste de los Estados Unidos y en el este de Canada debido a su emergencia temprana, alta tasa crecimiento, alta plasticidad, y resistencia a glyphosate e inhibidores de acetolactate synthase. Por esta razón, el manejo de A. trifida, temprano durante la temporada de crecimiento, es crítico para evitar las pérdidas de rendimiento. Los objetivos de este estudio fueron evaluar el control de A. trifida resistente a glyphosate mediante la integración de labranza en pre-siembra o 2,4-D; aplicaciones PRE o POST temprana (EPOST) seguido por (fb) programas de herbicidas en POST tardía (LPOST) con o sin labranza en pre-siembra o 2,4-D; y su efecto sobre el daño y rendimiento de la soja. Se realizó un estudio de campo en 2013 y 2014 en David City, Nebraska en un campo infestado con A. trifida resistente a glyphosate. La labranza en pre-siembra o la aplicación de 2,4-D brindó >90% de control de A. trifida resistente a glyphosate 14 d después del tratamiento pre-siembra. El control y la reducción en la biomasa de A. trifida fueron >90% consistentemente con la labranza pre-siembra o 2,4-D fb sulfentrazone más cloransulam PRE o glyphosate más cloransulam EPOST fb glyphosate más fomesafen o lactofen POST al compararlo con ≤86% de control con los mismos tratamientos sin labranza pre-siembra o 2,4-D. Programas de herbicidas PRE o de EPOST fb LPOST precedidos por tratamientos pre-siembra resultaron en una densidad de A. trifida <2 plantas m−2 y un rendimiento de soja >2,400 kg ha−1 al compararse con la densidad de ≥2 plantas m−2 y un rendimiento de soja ≤1,800 kg ha−1 con programas de herbicidas PRE o EPOST fb LPOST. El análisis de contrastes también indicó que la labranza pre-siembra o 2,4-D fb de un programa PRE o POST fue más efectiva para el manejo de A. trifida al compararse con programas de herbicidas PRE fb POST. La integración de labranza pre-siembra brindaría una alternativa de manejo para el control de A. trifida, temprano en la temporada. Sin embargo, se requiere que a esto le siga una aplicación de herbicidas para obtener un control a lo largo de toda la temporada en soja.

Investigation of MCPA (4-Chloro-2-ethylphenoxyacetate) Resistance in Wild Radish (Raphanus raphanistrum L.)

The phenoxy herbicides (e.g., 2,4-D and MCPA) are used widely in agriculture for the selective control of broadleaf weeds. In Western Australia, the reliance on phenoxy herbicides has resulted in the widespread evolution of phenoxy resistance in wild radish (Raphanus raphanistrum) populations. In this research the inheritance and mechanism of MCPA resistance in wild radish were determined. Following classical breeding procedures, F1, F2, and backcross progeny were generated. The F1 progeny showed an intermediate response to MCPA, compared to parents, suggesting that MCPA resistance in wild radish is inherited as an incompletely dominant trait. Segregation ratios observed in F2 (3:1; resistant:susceptible) and backcross progeny (1:1; resistant to susceptible) indicated that the MCPA resistance is controlled by a single gene in wild radish. Radiolabeled MCPA studies suggested no difference in MCPA uptake or metabolism between resistant and susceptible wild radish; however, resistant plants rapidly translocated more (14)C-MCPA to roots than susceptible plants, which may have been exuded from the plant. Understanding the genetic basis and mechanism of phenoxy resistance in wild radish will help formulate prudent weed management strategies to reduce the incidence of phenoxy resistance.

Physical Mapping of Amplified Copies of the 5-Enolpyruvylshikimate-3-Phosphate Synthase Gene in Glyphosate-Resistant Amaranthus tuberculatus

Fluorescence in situ hybridization maps a cluster of EPSPS genes to the pericentromeric region on one pair of homologous chromosomes of glyphosate-resistant Amaranthus tuberculatus. Recent and rapid evolution of resistance to glyphosate, the most widely used herbicides, in several weed species, including common waterhemp (Amaranthus tuberculatus), poses a serious threat to sustained crop production. We report that glyphosate resistance in A. tuberculatus was due to amplification of the 5-enolpyruvylshikimate-3-P synthase (EPSPS) gene, which encodes the molecular target of glyphosate. There was a positive correlation between EPSPS gene copies and its transcript expression. We analyzed the distribution of EPSPS copies in the genome of A. tuberculatus using fluorescence in situ hybridization on mitotic metaphase chromosomes and interphase nuclei. Fluorescence in situ hybridization analysis mapped the EPSPS gene to pericentromeric regions of two homologous chromosomes in glyphosate sensitive A. tuberculatus. In glyphosate-resistant plants, a cluster of EPSPS genes on the pericentromeric region on one pair of homologous chromosomes was detected. Intriguingly, two highly glyphosate-resistant plants harbored an additional chromosome with several EPSPS copies besides the native chromosome pair with EPSPS copies. These results suggest that the initial event of EPSPS gene duplication may have occurred because of unequal recombination mediated by repetitive DNA. Subsequently, gene amplification may have resulted via several other mechanisms, such as chromosomal rearrangements, deletion/insertion, transposon-mediated dispersion, or possibly by interspecific hybridization. This report illustrates the physical mapping of amplified EPSPS copies in A. tuberculatus.

Physiological and Molecular Characterization of Hydroxyphenylpyruvate Dioxygenase (HPPD)-inhibitor Resistance in Palmer Amaranth (Amaranthus palmeri S. Wats.)

Herbicides that inhibit hydroxyphenylpyruvate dioxygenase (HPPD) such as mesotrione are widely used to control a broad spectrum of weeds in agriculture. Amaranthus palmeri is an economically troublesome weed throughout the United States. The first case of evolution of resistance to HPPD-inhibiting herbicides in A. palmeri was documented in Kansas (KS) and later in Nebraska (NE). The objective of this study was to investigate the mechansim of HPPD-inhibitor (mesotrione) resistance in A. palmeri. Dose response analysis revealed that this population (KSR) was 10–18 times more resistant than their sensitive counterparts (MSS or KSS). Absorbtion and translocation analysis of [14C] mesotrione suggested that these mechanisms were not involved in the resistance in A. palmeri. Importantly, mesotrione (>90%) was detoxified markedly faster in the resistant populations (KSR and NER), within 24 hours after treatment (HAT) compared to sensitive plants (MSS, KSS, or NER). However, at 48 HAT all populations metabolized the mesotrione, suggesting additional factors may contribute to this resistance. Further evaluation of mesotrione-resistant A. palmeri did not reveal any specific resistance-conferring mutations nor amplification of HPPD gene, the molecular target of mesotrione. However, the resistant populations showed 4- to 12-fold increase in HPPD gene expression. This increase in HPPD transcript levels was accompanied by increased HPPD protein expression. The significant aspects of this research include: the mesotrione resistance in A. palmeri is conferred primarily by rapid detoxification (non-target-site based) of mesotrione; additionally, increased HPPD gene expression (target-site based) also contributes to the resistance mechanism in the evolution of herbicide resistance in this naturally occurring weed species.

Glyphosate-Resistant Palmer Amaranth (Amaranthus palmeri) in Nebraska: Confirmation, EPSPS Gene Amplification, and Response to POST Corn and Soybean Herbicides

Palmer amaranth is the most problematic weed in agronomic crop production fields in the United States. A Palmer amaranth biotype was not controlled with sequential applications of glyphosate in glyphosate-resistant (GR) soybean production field in south-central Nebraska. The seeds of the putative GR Palmer amaranth biotype were collected in the fall of 2015. The objectives of this study were to (1) confirm GR Palmer amaranth and determine the level of resistance in a whole-plant dose-response bioassay, (2) determine the copy number of 5-enolpyruvylshikimate-3-phosphate (EPSPS) gene, the molecular target of glyphosate, and (3) evaluate the response of GR Palmer amaranth biotype to POST corn and soybean herbicides with different modes-of-action. Based on the effective dose required to control 90% of plants (ED90), the putative GR Palmer amaranth biotype was 37- to 40-fold resistant to glyphosate depending on the glyphosate-susceptible (GS) used as a baseline population. EPSPS gene amplification was present in the GR Palmer amaranth biotype with up to 32 to 105 EPSPS copies compared to the known GS biotypes. Response of GR Palmer amaranth to POST corn and soybean herbicides suggest reduced sensitivity to atrazine, hydroxyphenylpyruvate dioxygenase (HPPD)- (mesotrione, tembotrione, and topramezone), acetolactate synthase (ALS)- (halosulfuron-methyl), and protoporphyrinogen oxidase (PPO)- (carfentrazone and lactofen) inhibitors. GR Palmer amaranth was effectively controlled (>90%) with glufosinate applied at 593 g ai ha−1 with ≥95% reduction in biomass. More research is needed to determine whether this biotype exhibits multiple resistant to other group of herbicides and evaluate herbicide programs for effective management in corn and soybean. Nomenclature: 2,4-D; acetochlor; acifluorfen; atrazine; bentazon; bromoxynil; carfentrazone; chlorimuron; dicamba; fluthiacet; fomesafen; glufosinate; glyphosate; halosulfuron; imazamox; imazethapyr; lactofen; mesotrione; S-metolachlor; tembotrione; thiencarbazone; thifensulfuron; topramezone; Palmer amaranth, Amaranthus palmeri S. Wats.; corn, Zea mays L.; soybean, Glycine max (L.) Merr. Amaranthus palmeri es la malezas más problemática en campos de producción de cultivos agronómicos en los Estados Unidos. Un biotipo de A. palmeri no fue controlado con aplicaciones secuenciales de glyphosate en un campo de producción de soja resistente a glyphosate (GR) en el sur central de Nebraska. Las semillas del biotipo putativo GR de A. palmeri fueron colectadas en el otoño de 2015. Los objetivos de este estudio fueron (1) confirmar que A. palmeri es GR y determinar el nivel de resistencia en un bioensayo de respuesta a dosis con plantas completas, (2) determinar el número de copias del gen 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), el objetivo molecular de glyphosate, y (3) evaluar la respuesta del biotipo GR de A. palmeri a herbicidas POST para maíz y soja con diferentes modos de acción. Con base en la dosis efectiva requerida para controlar 90% de las plantas (ED90), el biotipo putativo GR de A. palmeri fue 37 a 40 veces más resistente a glyphosate dependiendo de la población susceptible a glyphosate (GS) base utilizada. La amplificación del gen EPSPS estuvo presente en el biotipo GR de A. palmeri con 32 y hasta 105 copias más de EPSPS comparado con biotipos GS conocidos. La respuesta de A. palmeri GR a herbicidas POST para maíz y soja sugiere una sensibilidad reducida a atrazine, y a inhibidores de hydroxy phenylpyruvate dioxygenase (HPPD) (mesotrione, tembotrione, y topramezone), de acetolactate synthase (ALS) (halosulfuron-methyl), y de protoporphyrinogen oxidase (PPO) (carfentrazone y lactofen). A. palmeri GR fue efectivamente controlado (>90%) con glufosinate aplicado a 593 g ai ha−1 con ≥95% de reducción en la biomasa. Se necesita más investigación para determinar si este biotipo exhibe resistencia múltiple a herbicidas de otros grupos y para evaluar programas de herbicidas para su manejo efectivo en maíz y soja.

Transfer of Dicamba Tolerance from Sinapis arvensis to Brassica napus via Embryo Rescue and Recurrent Backcross Breeding

Auxinic herbicides (e.g. dicamba) are extensively used in agriculture to selectively control broadleaf weeds. Although cultivated species of Brassicaceae (e.g. Canola) are susceptible to auxinic herbicides, some biotypes of Sinapis arvensis (wild mustard) were found dicamba resistant in Canada. In this research, dicamba tolerance from wild mustard was introgressed into canola through embryo rescue followed by conventional breeding. Intergeneric hybrids between S. arvensis (2n = 18) and B. napus (2n = 38) were produced through embryo rescue. Embryo formation and hybrid plant regeneration was achieved. Transfer of dicamba tolerance from S. arvensis into the hybrid plants was determined by molecular analysis and at the whole plant level. Dicamba tolerance was introgressed into B. napus by backcrossing for seven generations. Homozygous dicamba-tolerant B. napus lines were identified. The ploidy of the hybrid progeny was assessed by flow cytometry. Finally, introgression of the piece of DNA possibly containing the dicamba tolerance gene into B. napus was confirmed using florescence in situ hybridization (FISH). This research demonstrates for the first time stable introgression of dicamba tolerance from S. arvensis into B. napus via in vitro embryo rescue followed by repeated backcross breeding. Creation of dicamba-tolerant B. napus varieties by this approach may have potential to provide options to growers to choose a desirable herbicide-tolerant technology. Furthermore, adoption of such technology facilitates effective weed control, less tillage, and possibly minimize evolution of herbicide resistant weeds.