Vijay K. Varanasi

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.

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.

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.

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.

A Target-Site Point Mutation in Henbit (Lamium amplexicaule) Confers High-Level Resistance to ALS-Inhibitors

Abstract Henbit is a facultative broadleaf winter annual in the Lamiaceae family. Acetolactate synthase (ALS) inhibitors are primarily used to control a broad spectrum of weeds, including henbit. During 2012 to 2013, field applications of ALS-inhibiting herbicides were ineffective in controlling a henbit population from Marion County, KS (MCK). To confirm field-evolved resistance to ALS inhibitors, response of MCK henbit and a known susceptible henbit population from Kansas (DPS) to varying doses of three different ALS inhibitors were examined: chlorsulfuron, imazamox, and propoxycarbazone. Results of the dose–response experiments suggest that the MCK population is highly resistant to chlorsulfuron (resistance index [R/S] > 1,000) and propoxycarbazone (R/S = 331) but is susceptible to imazamox. A full-length ALS gene sequence obtained using the 5′- and 3′- rapid amplification of complementary DNA ends approach revealed a Pro197 to Arg point mutation (a common mutation that confers resistance to sulfonylurea herbicides, e.g., chlorsulfuron) in the MCK henbit. No other known resistance-conferring mutations were found in the study. Evolved resistance to major classes of ALS inhibitors in the MCK henbit will reduce herbicide options for its control. To our knowledge, this is the first case of evolution of herbicide resistance in henbit. Nomenclature: Chlorsulfuron; imazamox; propoxycarbazone; henbit, Lamium amplexicaule L.

A Statewide Survey of PPO-Inhibitor Resistance and the Prevalent Target-Site Mechanisms in Palmer amaranth (Amaranthus palmeri) Accessions from Arkansas

Palmer amaranth is one of the most problematic weeds in the midsouthern United States, and the evolution of resistance to protoporphyrinogen oxidase (PPO) inhibitors in biotypes already resistant to glyphosate and acetolactate synthase (ALS) inhibitors is a major cause of concern to soybean and cotton growers in these states. A late-season weed-escape survey was conducted in the major row crop-producing counties (29 counties) to determine the severity of PPO-inhibitor resistance in Arkansas. A total of 227 Palmer amaranth accessions were sprayed with fomesafen at 395 g ha-1 to identify putative resistant plants. A TaqMan qPCR assay was used to confirm the presence of the ΔG210 codon deletion or the R128G/M (homologous to R98 mutation in common ragweed) target-site resistance mechanisms in the PPX2 gene. Out of the 227 accessions screened, 44 were completely controlled with fomesafen, and 16 had only one or two severely injured plants (≥98% mortality) when compared with the 1986 susceptible check (100% mortality). The remaining 167 accessions were genotypically screened, and 82 (49%) accessions were found to harbor the ΔG210 deletion in the PPX2 gene. The R128G was observed in 47 (28%) out of the 167 accessions screened. The mutation R128M, on the other hand was rare, found in only three accessions. About 13% of the accessions were segregating for both the ΔG210 and R128G mutations. Sixteen percent of the tested accessions had mortality ratings <90% and did not test positive for the ΔG210 or the R128G/M resistance mechanisms, indicating that a novel target or non-target site resistance mechanism is likely. Overall, PPO inhibitor-resistant Palmer amaranth is widespread in Arkansas, and the ΔG210 resistance mechanism is especially dominant in the northeast corridor, while the R128G mutation is more prevalent in counties near Memphis, TN. Nomenclature: Fomesafen; Palmer amaranth, Amaranthus palmeri S. Wats.; cotton, Gossypium hirsutum L.; soybean, Glycine max (L.) Merr.

Characterization of Acetolactate Synthase (ALS)-Inhibitor Resistance in Pennsylvania smartweed (Persicaria pensylvanica)

Abstract Pennsylvania smartweed [Persicaria pensylvanica (L.) M. Gómez] is a common weed of rice (Oryza sativa L.) in the midsouthern United States and has recently become a concern for farmers because of reduced tillage systems. Acetolactate synthase (ALS) inhibitors have been extensively used for controlling smartweeds in imidazolinone-resistant and conventional rice. In the present study, we confirmed resistance to commonly used ALS inhibitors in rice and characterized the underlying resistance mechanism in a P. pensylvanica biotype from southeast Missouri. A dose–response experiment was conducted in the greenhouse using bensulfuron-methyl, imazethapyr, and bispyribac-sodium to determine the resistance index (resistance/susceptibility [R/S]) based on GR50 estimates. The target-site ALS gene was amplified from R and S plants, and sequences were analyzed for mutations known to confer ALS-inhibitor resistance. The P. pensylvanica biotype in question was found to be resistant to bensulfuron-methyl (R/S=2,330), imazethapyr (R/S =12), and bispyribac-sodium (R/S=6). Sequencing of the ALS gene from R plants revealed two previously known mutations (Pro-197-Ser, Ala-122-Ser) conferring resistance to sulfonylureas and imidazolinones. This is the first report of ALS-inhibitor resistance in P. pensylvanica.

Confirmation and Characterization of Non–target site Resistance to Fomesafen in Palmer amaranth (Amaranthus palmeri)

Abstract Palmer amaranth (Amaranthus palmeri S. Watson), a dioecious summer annual species, is one of the most troublesome weeds in U.S. cropping systems. The evolution of resistance to protoporphyrinogen oxidase inhibitors in A. palmeri biotypes is a major cause of concern to soybean [Glycine max (L.) Merr.] and cotton (Gossypium hirsutum L.) growers in the midsouthern United States. The objective of this study was to confirm and characterize the non–target site mechanism in a fomesafen-resistant accession from Randolph County, AR (RCA). A dose–response assay was conducted to assess the level of fomesafen resistance, and based on the GR50 values, the RCA accession was 18-fold more resistant to fomesafen than a susceptible (S) biotype. A TaqMan allelic discrimination assay and sequencing of the targetsite genes PPX2 and PPX1 revealed no known or novel target-site mutations. An SYBR Green assay indicated no difference in PPX2 gene expression between the RCA and S biotypes. To test whether fomesafen resistance is metabolic in nature, the RCA and the S biotypes were treated with different cytochrome P450 (amitrole, piperonyl butoxide [PBO], malathion) and glutathione S-transferase (GST) (4-chloro-7-nitrobenzofurazan [NBD-Cl]) inhibitors, either alone or in combination with fomesafen. Malathion followed by (fb) fomesafen in RCA showed the greatest reduction in survival (67%) and biomass (86%) compared with fomesafen alone (45% and 66%, respectively) at 2 wk after treatment. Interestingly, NBD-Cl fb fomesafen also resulted in low survival (35%) compared with the fomesafen-only treatment (55%). Applications of malathion or NBD-Cl preceding fomesafen treatment resulted in reversal of fomesafen resistance, indicating the existence of cytochrome P450– and GST-based non–target site mechanisms in the RCA accession. This study confirms the first case of non–target site resistance to fomesafen in A. palmeri.

Investigating the mechanism of glyphosate resistance in a common ragweed (Ambrosia artemisiifolia L.) biotype from Nebraska

Abstract: Common ragweed is a weed in the midwestern United States and eastern Canada that is difficult to control due to the evolution of an important resistance to multiple herbicides including glyphosate. Recently, a common ragweed biotype with 19-fold glyphosate resistance was confirmed in Nebraska. The objective of this study was to determine the mechanism of glyphosate resistance in a common ragweed biotype from Nebraska. Both target site and non-target site based mechanisms of glyphosate resistance were investigated using glyphosate-resistant (GR) and known glyphosate-susceptible (GS) common ragweed biotypes. A lower amount of shikimate was accumulated in the GR (≤65 µg mL-1) compared with the GS (≥80 µg mL-1) biotype at all glyphosate concentrations tested. Sequencing of the conserved region of the EPSPS gene revealed no mutations at the Thr102 or Pro106 residues and no variation in EPSPS copy number was detected. A higher translocation of 14C-glyphosate in the GR compared with the GS biotype was found, although there was no difference in the amount of 14C-glyphosate absorbed. Nonetheless, analysis of 14C-glyphosate absorption or translocation data using the rectangular hyperbolic model predicted a slower rate of absorption and translocation of glyphosate in the GR compared with the GS biotype, though more research is needed. These results indicate possible involvement of a non-target site mechanism bestowing resistance to glyphosate. The possibility that a slow rate of glyphosate absorption and translocation might have a role in preventing the buildup of the minimum inhibitory concentration of glyphosate required at the target site needs further research.