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ARS Home » Southeast Area » Stoneville, Mississippi » Crop Production Systems Research » Research » Research Project #429910

Research Project: Biology and Management of Herbicide-Resistant Weeds

Location: Crop Production Systems Research

2018 Annual Report

Objective 1: Discover, identify and characterize physiological, biochemical and molecular mechanisms of resistance in herbicide-resistant weeds. Sub-objective 1A. Document distribution, nature, and level of resistance to herbicides, including cross resistance and multiple resistance, in weed populations of MS and Southeastern U.S. Sub-objective 1B. Determine the physiological/biochemical/molecular mechanisms of resistance to herbicides in weed populations where the level and nature of resistance is known. Sub-objective 1C. Determine the nature of metabolism-based non-target site herbicide (ALS inhibitors, propanil, quinclorac) resistance in Echinochloa spp. Objective 2: Determine the effects of herbicide resistance (especially for Amaranthus weeds) on plant fitness and growth characteristics (e.g., photosynthetic capacities, seed bank size and longevity, competitiveness, and stress responses) as compared to corresponding herbicide-sensitive biotypes. Sub-objective 2A. Evaluate the competitiveness of GR-hybrids of A. spinosus and A. palmeri, glyphosate-sensitive A. spinosus and GR-A. palmeri in soybean. Sub-objective 2B. Evaluate the persistence and level of glyphosate resistance in hybrids following glyphosate application. Objective 3: Characterize the extent of hybridization among Amaranthus weed species, and determine how hybridization impacts the spread of herbicide-resistance in this genus. Sub-objective 3A. In greenhouse crosses, evaluate the inheritance of resistance by examining fertility, morphological traits, and changes in copy number of EPSPS in F1 hybrids with and without glyphosate. Sub-objective 3B. Determine the viability of pollen and seeds from hybrids. Sub-objective 3C. Perform in situ hybridization to determine the distribution of the EPSPS amplicon among chromosomes. Sub-objective 3D. Determine if the size and contents of the EPSPS amplicon are consistent across populations from different locations. Objective 4: Discover biological and cultural weed control methods that can be integrated with herbicides and other chemicals to manage herbicide-resistant weeds. Sub-objective 4A. Determine the efficacy of field crop rotations on glyphosate-resistant pigweed populations. Sub-objective 4B. Determine efficacy of new 2,4-D and dicamba formulations alone and in combination with 1 or more additional herbicide modes of action on glyphosate- and acetolactate synthase inhibitor-resistant broadleaf weeds. Sub-objective 4C. Determine possible multiple herbicide resistance in horseweed, Palmer amaranth and other populations of weed species using bioassays with multiple herbicides. Sub-objective 4D. Determine compatibility and possible synergistic interaction of bioherbicidal pathogens (MV, X. campestris isolate LVA987, and others) with herbicides (2,4-D, dicamba and other auxinic herbicides, glyphosate, etc.) to be used on new multiple-herbicide resistant crops.

The overall project goal is to discover basic and practical knowledge of the occurrence, distribution, mechanism of resistance and management of weeds that are resistant to single or multiple herbicides. This holistic approach will generate more effective weed control and management practices. The development of weed management tools, aided by knowledge of resistance mechanisms and weed biology will foster the development of novel, sustainable practices for early detection and management of resistant weeds. Basic growth analyses, assays and bioassays using whole plants and plant tissues from laboratory, greenhouse and field experiments will determine major changes in resistant versus susceptible biotypes. Subsequent biochemical, genetic, proteomic, immunochemical and radiological studies will identify and characterize specific site differences in herbicide resistant and sensitive weed biotypes within species. The knowledge generated will provide a greater understanding of the biochemistry, physiology and genetics of resistance mechanisms and provide insight for recommendations to promote efficacious and sustainable weed control coupled with more efficient and economic crop production.

Progress Report
Determination of metabolites of several herbicides in a metabolic resistant biotype of junglerice from Mississippi is in progress. Acetolactate synthase (ALS) inhibitor resistance studies in Italian ryegrass populations from North Carolina were initiated. A novel technique to measure dicamba and glyphosate drift using a fluorescent additive has been initiated. Studies to measure absorption and translocation of dicamba in dicamba-resistant soybean using 14C-dicamba as a tracer are being developed. A proposal to characterize the resistance mechanisms in a multiple-herbicide resistant Phalaris minor biotype from India has been approved and a USDA-APHIS permit has been obtained to import this plant material for research. Populations of pigweeds (Amaranthus spp.) from Mississippi were examined for resistance to protoporphyrinogen oxidase (PPO) inhibiting herbicides, and a single plant was identified as having a deletion mutation conferring resistance to these herbicides. Studies were completed on a freeze-drying method for the preservation of biological activity of a bioherbicide (Myrothecium verrucaria). Cell to cell transfer of glyphosate resistance in Palmer amaranth was shown to be the result of tethering of replicon (amplicons) to chromosomes during cell division. Eastern black nightshade (EBN) is a problematic weed, partly due to its tolerance or resistance to certain herbicides. The fungus Colletotrichum coccodes, formulated in an invert emulsion (IE), was shown to control EBN and several other solanaceous weeds in greenhouse and field tests. Results demonstrate that this IE can extend the host range and efficacy of this bioherbicide. Whole genome sequencing of glyphosate-resistant and -sensitive genomes in Palmer amaranth was completed and genomes are being assembled. Interactions studies of glufosinate and Colletotrichum truncatum (fungal plant pathogen and bioherbicide for hemp sesbania control) showed glufosinate was inhibitory to fungal growth and sporulation. Ammonia levels in hemp sesbania tissues after treatment with the herbicide or bioherbicide alone or in combination were inversely correlated with glutamine synthetase activity. Ribonucleic acid sequencing experiments were performed to assess the genes influenced by glyphosate application to glyphosate-resistant and -sensitive Palmer amaranth biotypes. Results show that resistant plants do not undergo a state of catastrophic gene transcription as do sensitive plants. Thus, the presence of resistance attributes can ameliorate the effects of herbicide application.

1. Characterization of acetolactate synthase (ALS)-inhibiting herbicide resistance in California multiple-resistant (MR) Italian ryegrass populations. Weed resistance to herbicides has become a major problem that threatens herbicide sustainability and crop production on a global basis. Researchers from USDA-ARS, Stoneville, Mississippi, and the University of California determined the resistance levels to ALS-inhibiting herbicides (imazamox and mesosulfuron-methyl) in selected Italian ryegrass biotypes and characterized the mechanism of resistance to these herbicides. MR1-A [resistant to glyphosate/acetyl coenzyme A carboxylase (ACCase)], MR1-P (resistant to glyphosate/paraquat) and MR2 biotypes were 38-, 29-, and 8-fold, and 37-, 63- and 4-fold less sensitive to imazamox and mesosulfuron-methyl, respectively, compared to a susceptible biotype. Only MR1-P and MR2 plants were cross-resistant to rimsulfuron, whereas both MR1 biotypes were cross-resistant to imazethapyr. Bispyribac-sodium, penoxulam and propanil were not effective on any of these plants and pinoxaden [ACCase-inhibitor (phenylpyrazoline)] only controlled MR2 plants at the labeled field rate. However, all plants were effectively controlled (> 99%) with the labeled field rate of glufosinate. The ALS enzyme from MR1-A, MR-P and MR2 plants was 712-, 1104-, and 3-fold and 10-, 18-, and 5-fold less responsive to mesosulfuron-methyl and imazamox, respectively, compared to the susceptible plants. Alignment of ALS gene sequences of resistant plants revealed a missense (single nucleotide polymorphism) resulting in a tryptophan574-to-leucine substitution in MR1-A and MR1-P, but not in MR2 biotypes. This substitution is known to endow a high level of resistance to ALS-inhibiting herbicides in weeds. The results suggest an altered target site as the mechanism of resistance in MR1 plants and non-target site based resistance to both herbicides in MR2 plants.

2. Differential physiological responses to glyphosate in Palmer amaranth biotypes with varying degrees of resistance to glyphosate. The broad-spectrum herbicide glyphosate was once able to effectively control most weeds in various crop and non-cropping situations, but currently over 40 species of weeds have become resistant to this compound. Scientists at USDA-ARS, Stoneville, Mississippi, evaluated and characterized the metabolic perturbations following glyphosate application to two susceptible (S-) and three resistant (R-) biotypes of Palmer amaranth with varying resistance to glyphosate. Comparative metabolic profiling of the different biotypes indicated that the most resistant biotype had an innate abundance of several metabolites derived from phenylpropanoid pathway. Upon treatment with glyphosate, the metabolic pool dynamics of all biotypes correlated with the respective GR50 levels (glyphosate concentration causing a 50% growth reduction), with the most resistant biotype having a higher pool of metabolites known to have anti-oxidant potential. Compared to the most resistant biotype, the S-biotypes had relatively low levels of both primary and secondary metabolites, indicating that glyphosate induced metabolic inhibition. After glyphosate treatment, the content of total phenolic and flavonoids decreased in S-biotypes, whereas the level of these metabolites remained the same, or increased in the R-biotypes. These results indicate that the phytochemical parameters and the antioxidant capacity that may play a complementary role in glyphosate resistance is partially induced after glyphosate application, rather than being constitutively expressed.

3. Bioherbicide and herbicide interactions for weed control. Microbes and microbial products as bioherbicides have been studied for several decades and researchers have also examined combinations of bioherbicides and herbicides to discover synergistic interactions applicable to weed control. Scientists at USDA-ARS, Stoneville, Mississippi, conducted bioassays to assess possible interactions of the herbicide glufosinate and Colletotrichum truncatum (CT) [fungal plant pathogen; bioherbicide for hemp sesbania (Sesbania exaltata) control]. Glufosinate is a strong glutamine synthetase (GS) inhibitor leading to elevated ammonia levels, but the mode of action of CT is unknown. GS has also been implicated in plant defense in some plant-pathogen interactions. The effects of spray applications of glufosinate and bioherbicide (applied alone and in combination) on seedling growth, GS activity and ammonia levels in hypocotyl tissues were monitored [88-hour (h) time-course]. Growth and GS activity were inhibited in tissues by glufosinate and glufosinate plus CT treatments as early as 16 h, but CT treatment did not cause substantial growth reduction or GS inhibition until ~40 h. Generally, ammonia levels in hemp sesbania tissues under these treatments were inversely correlated with GS activity. Localization of hemp sesbania GS activity on electrophoretic gels indicated inhibition after 30 h in glufosinate and glufosinate plus CT-treated tissues. Untreated control tissues contained much lower ammonia levels at 24-88 h than treatments with CT, glufosinate or their combination. CT alone caused elevated ammonia levels only after 64-88 h. Low concentrations of glufosinate reduced fungal colony radial growth and inhibited sporulation. Results provide insight on the biochemical action of this bioherbicide and on interactions with glufosinate, but no major synergistic interactions were indicated.

4. Multiple resistance to glyphosate, paraquat, acetyl coenzyme A carboxylase (ACCase)- and acetolactate synthase (ALS)-inhibitors in California Italian ryegrass biotypes: confirmation, control and resistance mechanisms. Research scientists at the USDA-ARS, Stoneville, Mississippi, and the University of California determined the magnitude of herbicide resistance, evaluated alternative herbicide options, and characterized the physiological and molecular bases of resistance in multiple-resistant (MR) Italian ryegrass biotypes from California. Dose-response tests showed the levels of resistance to glyphosate, sethoxydim and paraquat were 45-, 122-, and 20-fold, respectively, for the MR1 population and 24-, 93-, and 4-fold, respectively for the MR2 population. Both resistant biotypes were cross-resistant to aryloxyphenoxypropionate herbicides. MR1 plants were also cross-resistant to clethodim, whereas MR2 plants were controlled (93 to 100%) with the labeled field rate of this herbicide. The shikimic acid levels in susceptible (Sus) plants were significantly greater than in MR plants, 32 hours after light pretreatments. Sanger sequencing of a region of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene revealed proline106-to-alanine and proline106-to-threonine substitutions in MR plants. These substitutions are known to cause target-site resistance to glyphosate in ryegrass. EPSPS gene copy number and expression levels were similar in the Sus and MR populations. Alignment of ACCase gene sequences revealed a missense mutation (isoleucine1781-to-leucine) in both MR populations, which was previously found to endow resistance to ACCase-inhibiting herbicides in several weeds, including ryegrass. Translocation of 14C-paraquat was lower in Sus plants than in MR plants. This research reveals that multiple herbicide resistance in Italian ryegrass populations of California is due to both target-site and non-target-site resistance mechanisms.

5. Are herbicide-resistant weeds more resilient to stress? Previously, USDA-ARS researchers at Stoneville, Mississippi, showed that a glyphosate-resistant Palmer amaranth biotype (R) sustains metabolic perturbation immediately (8 hours) after herbicide treatment, but recovers by 36-72 hours, possibly due to an abundance of antioxidant machinery that complements 5-enolpyruvyl-shikimate-3-phosphate synthase amplification. Higher levels of antioxidants could help R-biotypes endure environmental stress, which was tested by monitoring physiological perturbations sustained by R- and susceptible (S)-biotypes of Palmer amaranth after subjecting plants to drought stress following multiple-controlled, dry-down events. Perturbations of primary and secondary metabolite pools were mapped by global metabolomics. Cellular-level stress [including reactive oxygen species (ROS) production and lipid peroxidation] and whole plant physiology (including carbon assimilation) were studied. Over 32,000 unique mass features (1503 metabolites) showed significant responses across treatments. In the absence of drought stress, cellular physiology and metabolite pools were biotype dependent, i.e., the S-biotype had marginally richer secondary metabolite profiles compared to R-biotypes. Overall, the metabolite profiles of the R-biotypes were induced by drought ~2 times more than those of the S-biotypes and S-biotypes had ~4 times more compounds down-regulated after drought treatment than R-biotypes. Irrespective of the treatments, the physiological parameters (carbon assimilation, and stomatal conductance) did not differ significantly between the R- and S-biotypes. Compared to S-biotypes, R-biotypes had higher ROS pools, which did not vary with drought treatment. R-biotypes had lower overall cellular damage under drought treatment, resulting in a marginally higher drought recovery. Results suggest that an ROS inhibitor added in a tank mix may improve glyphosate-R Palmer amaranth control.

6. Characterization of glyphosate resistance genetics and replication in Palmer amaranth. Palmer amaranth is a very aggressive weed, but the relatively recent development and rapid spread of resistance to the herbicide glyphosate in this species has enabled it to become even more problematic to control. ARS researchers in Stoneville, Mississippi, have shown that the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) replicon in glyphosate-resistant Palmer amaranth exists as a circular, free-floating, giant plasmid (episome) in cells and that its transmission between generations of cells to occur by a tethering process in which the replicon tethers itself to chromosomes during division. This is the first known case of this type of replication mechanism in plants. The replicon also replicates autonomously and has the genes to support this process. Results demonstrate important molecular biological characterization of novel processes associated with herbicide resistance in an economically important weed.

Review Publications
Hoagland, R.E., Boyette, C.D., Stetina, K.C. 2017. Extending the shelf-life of Myrothecium verrucaria, a bioherbicide. American Journal of Plant Sciences. 8:3272-3284.
Nandula, V.K. 2018. Recent advances in deciphering metabolic herbicide resistance mechanisms. In: Jugulam, Mithila, editor. Biology, Physiology and Molecuar Biology of Weeds. CRC Press, Taylor and Francis Group. p. 144-155.
Boyette, C.D., Hoagland, R.E., Stetina, K.C. 2018. Bioherbicidal enhancement and host range expansion of a mycoherbicidal fungus via formulation approaches. Biocontrol Science and Technology. 28(3):307-315.
Huang, Y., Lee, M.A., Nandula, V.K., Reddy, K.N. 2018. Hyperspectral imaging for differentiating glyphosate-resistant and glyphosate-susceptible Italian Ryegrass. American Journal of Plant Sciences. 9:1467-1477.
Molin, W.T., Wright, A.A., Vangessel, M.J., McCloskey, W.B., Jugulam, M., Hoagland, R.E. 2018. Survey of the genomic landscape surrounding the EPSPS gene in glyphosate resistant Amaranthus palmeri from geographically distant populations in the United States. Pest Management Science. 74:1109-1117.
Wright, A.A., Rodriguez-Carres, M., Sasidharan, R., Koski, L., Peterson, D.G., Nandula, V.K., Ray, J.D., Bond, J.A., Shaw, D.R. 2018. Multiple herbicide-resistant Junglerice (Echinochloa colona): Identification of genes potentially involved in resistance through differential gene expression analysis. Weed Science. 66:347-354.
Wright, A.A., Sasidharan, R., Koski, L., Rodriguez-Carres, M., Peterson, D.G., Nandula, V.K., Ray, J.D., Bond, J.A., Shaw, D.R. 2018. Transcriptomic changes in Echinochloa colona in response to treatment with the herbicide imazamox. Planta. 247:369-379.
Tehranchian, P., Nandula, V.K., Jugulam, M., Putta, K., Jasieniuk, M. 2017. Multiple resistance to glyphosate, paraquat and ACCase-inhibiting herbicides in Italian ryegrass populations from California: Confirmation and mechanisms of resistance. Pest Management Science. 74:868-877.
Maroli, A.S., Nandula, V.K., Duke, S.O., Gerard, P., Tharayil, N. 2017. Comparative metabolomic analyses of two Ipomoea lacunosa biotypes with contrasting glyphosate tolerance elucidates glyphosate-induced differential perturbations in cellular physiology. Journal of Agricultural and Food Chemistry. (66):2027-2039.
Koo, D., Molin, W.T., Saski, C.A., Jiang, J., Putta, K., Jugulam, M., Friebe, B., Gill, B.S. 2018. Extrachromosomal circular DNA-based amplification and transmission of herbicide resistance in crop weed Amaranthus palmeri. Proceedings of the National Academy of Sciences. hppts://doi.10.1073/pnas.1719354115.