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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

2016 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
Molecular screening for protoporphyrinogen oxidase (PPO) resistance in pigweeds(Amaranthus spp.) collected in 2015 was completed and screening of new pigweed accessions for resistance to PPO inhibiting herbicides is underway. Differences in PPO sensitivity among local accessions were demonstrated, indicating a need for further research on strategies to improve seedling control. Ribonucleic acid sequence (RNAseq) experiments were initiated and are in progress to assess the changes in RNA synthesis following glyphosate application in glyphosate-sensitive and -resistant Palmer amaranth and junglerice. A third round of bacterial artificial chromosome (BAC)-end sequencing is underway to determine the final length of the EPSPS amplicon in glyphosate-resistant palmer amaranth. A comparison of the morphological parameters of Palmer amaranth x spiny amaranth hybrids has been completed and a comparison of physiological parameters is ongoing. Comparative flux analysis of nitrogen metabolism in glyphosate-resistant and -susceptible Palmer amaranth biotypes was completed. Research on bioherbicides for weed control demonstrated that certain phytopathogenic fungi or bacteria have weed control potential when used alone or in combination with sub-lethal amounts of some herbicides. Some herbicide/bioherbicide combinations resulted in important additive or synergistic interactions, thereby promoting weed control. Some bioherbicidal pathogens were demonstrated to control herbicide-resistant weeds. The impact of new transgenic crop technologies including 2,4-dichloroacetic acid (2,4-D) and dicamba formulations on weed efficacy and soil microbial nutrient recycling was completed. The detection of glyphosate-resistant and -susceptible Italian ryegrass using hyperspectral plant sensing in field-grown plants is in progress. The characterization and distribution of ALS inhibitor and triazine resistance of pigweed species in Mississippi has been initiated and is in progress.

1. Weeds as nematode hosts. The most important weed and cover crop hosts for reniform nematodes were identified in the Mississippi Delta. USDA-ARS scientists at the Crop Production Systems Research Unit, Stoneville, Mississippi measured nematode infection on 53 plant species in greenhouse and field surveys. Subsequent greenhouse and field tests confirmed the host status on nine of these plants. Sicklepod (Senna obtusifolia), spurred anoda (Anoda cristata), entireleaf morningglory (Ipomoea sp.), and velvetleaf (Abutilon theophrasti) were excellent hosts for reniform nematodes, supporting nematode populations equivalent to or greater than those developing on susceptible cotton plants included in the tests. Purple (Cyperus rotundus) and yellow (C. esculentus) nutsedges were poor hosts for reniform nematodes, despite all underground plant parts supporting nematode infection. The fact that sicklepod, velvetleaf, and entireleaf morningglory are important hosts for reniform nematodes in Alabama and Georgia, suggests a need to manage these weeds across a wider geographic region.

2. Rapid identification of weed seedlings. USDA-ARS scientists at the Crop Production Systems Research Unit, Stoneville, Mississippi developed a polymerase chain reaction (PCR) test to quickly identify weedy amaranths and their hybrids. The sequences of intron one for the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene were determined for Amaranthus palmeri, A. retroflexus, A. blitoides, A. viridis, A. tuberculatus, and A. hybridus. These sequences were aligned and primers were developed in regions where the sequence differed between species. Species specific primers and cycle conditions were successfully developed. These primers produce a single robust band only for the species for which they were designed. These PCR techniques allow identification of a weedy amaranth or suspect hybrid in a few hours. Using a similar target, it may be possible to design similar, simple PCR tests to identify even more difficult to distinguish weed species or weeds prone to interspecific hybridization.

3. Yellow nutsedge is a major weed in rice production. Yellow nutsedge is one of the most problematic sedges in Arkansas rice, requiring the frequent use of halosulfuron (sulfonylurea) for its control. But in 2012, halosulfuron at the labeled field rate failed to control this weed. Scientists from the University of Arkansas, USDA-ARS, Stoneville, Mississippi and Auburn University examined the resistance level, cross-resistance and resistance mechanism in this putative resistant biotype. Acetolactate synthase (ALS) enzyme assays and analysis of the ALS gene were used to ascertain the resistance mechanism. Resistant plants were not killed by halosulfuron at a dose 256-fold higher than the field dose and also not controlled by the ALS-inhibiting herbicides (imazamox, imazethapyr, penoxsulam, bispyribac, pyrithiobac-sodium, bensulfuron and halosulfuron) at labeled field rates. The ALS enzyme from the resistant biotype was 2540-fold less responsive to halosulfuron than a susceptible biotype, and a Trp574-to-Leu substitution was detected. Results suggest a target-site alteration as the resistance mechanism in yellow nutsedge,which accounts for the cross-resistance to other ALS-inhibiting herbicide families.

4. Smallflower umbrella sedge has evolved herbicide resistance. Smallflower umbrella sedge is a problematic weed in direct-seeded rice in the midsouthern United States and recently has evolved resistance to the acetolactate synthase (ALS) –inhibiting herbicide halosulfuron in Arkansas rice. Scientists from the University of Arkansas, USDA-ARS, Crop Production Systems Research Unit, Stoneville, Mississippi and Auburn University scientists conducted research to determine if the resistant biotype was cross-resistant to other ALS-inhibiting herbicides, evaluate alternative herbicide control options, and to determine the resistance mechanism. Resistant plants were not controlled by bispyribac–sodium, imazamox, and penoxsulam at labeled field rates and the level of resistance to these herbicides was > 15-fold compared to a susceptible biotype. Both biotypes were controlled 96% with bentazon and propanil, but only by 23% with quinclorac, thiobencarb, and 2,4-D. Thus, effective control measures exist, the number of herbicide options appear limited. Based on in vitro ALS enzyme assays, an altered target site was the mechanism of resistance to halosulfuron and imazamox. Sequencing studies detected an amino acid substitution of Proline197-to-Histidine in the resistant biotype, consistent with ALS-inhibiting herbicide resistance in other weed species.

5. Giant ragweed found to be resistant to glyphosate. A giant ragweed population from a glyphosate-resistant (GR) soybean field in Mississippi was suspected to be resistant to glyphosate. USDA-ARS Scientists from the Crop Production Systems Research Unit, Stoneville, Mississippi and Colorado State University conducted greenhouse and laboratory studies to confirm and quantify the magnitude of glyphosate resistance in a resistant biotype from this population and to elucidate physiological and molecular mechanisms of resistance. Glyphosate dose response studies indicated a 1.5-fold level of resistance in the glyphosate-resistant-MS population (GR-MS). The absorption pattern of 14C-glyphosate in susceptible (GS-MS) and resistant (GR-MS) giant ragweed biotypes was similar and the amount of 14C-glyphosate that translocated out of treated leaves of the GR-MS and GS-MS plants was similar early after treatment. However, at later times after treatment the GS-MS biotype translocated more 14C-glyphosate out of the treated leaf than the GR-MS biotype. No target site mutation was identified at the Pro106 location of the EPSPS gene of the GR-MS biotype. The mechanism of resistance to glyphosate in giant ragweed from Mississippi, is partially attributed to reduced glyphosate translocation.

6. Bioherbicides to control weeds. USDA-ARS scientists at the Crop Production Systems Research Unit, and the Biological Control of Pests Research Unit, Stoneville, Mississippi studied phytopathogenic fungi or bacteria as bioherbicides, and some tests integrated these agents with herbicides to increase efficacy. A fungus, Phoma commelinicola, was bioherbicidal against spreading dayflower when applied as conidial formulations. A dew period (= 12 h) provided 60% control of young plants, while maximal control (80%) required longer dew periods. A bacterium (Xanthomonas campestris) was bioherbicial against glyphosate-resistant horseweed. Rosette-stage plants were more susceptible than older plants, and higher inoculum concentration yielded greater control. Control of kudzu can involve years of restricted-use pesticide application, but newer non-restricted use herbicides and the use of a bioherbicide provide new options. Aminocyclopyrachlor, aminopyralid, fluroxypyr, metsulfuron methyl provided 99-100% reduction of kudzu biomass. Integrating a bioherbicide (Myrothecium verrucaria) treatment, with mechanical kudzu biomass removal and planting switchgrass was an effective herbicide-free system. Such methods can be used alone or in an integrated approach for rapid (1 yr) kudzu control. Results indicate that certain bioherbicides can control herbicide-resistant and recalcitrant weeds in integrated systems.

7. Metabolomics and biochemical assays for herbicide action studies in plants. USDA-ARS Scientists at the Crop Production Systems Research Unit, Stoneville and the Natural Products Utilization Research Unit, Oxford, Mississippi, and Clemson University scientists used metabolomics and biochemical assays to identify physiological perturbations induced by a commercial formulation of glyphosate in susceptible (S) and resistant (R) biotypes of Amaranthus palmeri. At 8 hours after treatment (HAT), cellular metabolism of both biotypes (when compared to to their respective water-treated control) was similarly perturbed by glyphosate, resulting in abundance of most metabolites including shikimic acid, amino acids, organic acids and sugars. But, by 80 HAT the metabolite pool of glyphosate-treated R-biotype was similar to that of the control S- and R-biotypes, indicating a potential physiological recovery. Furthermore, the glyphosate-treated R-biotype had lower reactive oxygen species (ROS) damage, higher ROS scavenging activity, and higher levels of potential antioxidant compounds derived from the phenylpropanoid pathway. Thus, metabolomics, in conjunction with biochemical assays, indicate that glyphosate-induced metabolic perturbations are not limited to the shikimate pathway, and the oxidant quenching efficiency could potentially complement glyphosate resistance in this R-biotype.

8. Weed hybridization increases weed control problems. Transfer of herbicide resistance among closely related weed species is a major concern. Scientists from the USDA-ARS Crop Production Systems Research Unit, Stoneville, Mississippi and Mississippi State University found a spiny amaranth x Palmer amaranth hybrid and confirmed its resistant to acetolactate synthase (ALS) inhibitors including imazethapyr, nicosulfuron, pyrithiobac, and trifloxysulfuron. Enzyme assays indicated that the ALS enzyme was insensitive to pyrithiobac and sequencing revealed the presence of a known resistance conferring point mutation, Trp574Leu. Alignment of the ALS gene for Palmer amaranth, spiny amaranth, and putative hybrids revealed the presence of Palmer amaranth ALS sequence in the hybrids rather than in spiny amaranth ALS sequences. Additionally, sequence upstream of the ALS in the hybrids matched Palmer amaranth, not spiny amaranth. The potential for transfer of ALS inhibitor resistance by hybridization has been demonstrated in the greenhouse and in field experiments. This research was the first report of gene transfer for ALS inhibitor resistance occurring in the field without artificial/human intervention and highlights the need to control related species in both field and surrounding non-crop areas to avoid interspecific transfer of resistance genes.


Review Publications
Nandula, V.K., Wright, A.A., Vah Horn, C., Molin, W.T., Westra, P., Reddy, K.N. 2015. Glyphosate resistance in giant ragweed (Ambrosia trifida L.) from Mississippi is partly due to reduced translocation. American Journal of Plant Sciences. 6:2104-2113.
Boyette, C.D., Hoagland, R.E., Stetina, K.C. 2015. Biological control of Spreading Dayflower (Commelina diffusa) with the fungal pathogen Phoma commelinicola. Agronomy Journal. 5:519-536.
Weaver, M.A., Boyette, C.D., Hoagland, R.E. 2016. Management of kudzu by the bioherbicide, Myrothecium verrucaria, herbicides and integrated control programs. Biocontrol Science and Technology. 26(1):136-140.
Teaster, N.D., Hoagland, R.E. 2014. Genomic stability of Palmer amaranth plants derived by macro-vegetative propagation. American Journal of Plant Sciences. 5:3302-3310.
Wright, A.A., Molin, W.T., Nandula, V.K. 2016. Distinguishing between weedy Amaranthus species based on intron one sequences from the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)gene. Pest Management Science. DOI 10.1002/ps.4280.
Tehranchian, P., Norsworthy, J.K., Nandula, V.K., Mcelroy, S., Chen, S., Scott, R.C. 2014. First report of resistence to acetolactate synthase inhibiting herbicides in yellow nutsedge (Cyperus esculentus): confirmation and characterization. Pest Management Science. 71(9):1274-1280.
Tehranchian, P., Riar, D.S., Norsworthy, J.K., Nandula, V.K., Mcelroy, S., Chen, S., Scott, R.C. 2015. ALS-resistant Smallflower umbrella (Cyperus difformis) in Arkansas rice: physiological and molecular basis of resistance mechanism. Weed Science. 63(3):561-568.
Molin, W.T., Nandula, V.K., Wright, A.A., Bond, J.A. 2016. Transfer and expression of ALS inhibitor resistance from Amaranthus palmeri to an A. spinosus X A. palmeri hybrid. Weed Science. 64(2):240-247.
Maroli, A.S., Nandula, V.K., Dayan, F.E., Duke, S.O., Gerard, P., Tharayil, N. 2015. Metabolic profiling and enzyme analyses indicate a potential role of antioxidant systems in complementing glyphosate resistance in an Amaranthus palmeri biotype. Journal of Agricultural and Food Chemistry. 63:9199-9209.
Weaver, M.A., Boyette, C.D., Hoagland, R.E. 2016. Rapid kudzu eradication and switchgrass establishment though herbicide, bioherbicide and integrated programs. Biocontrol Science and Technology. 26:640-650.