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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

2017 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
Populations of pigweeds (Amaranthus spp.) have been examined for resistance to protoporphyrinogen oxidase (PPO) inhibiting herbicides, and factors affecting PPO herbicide efficacy evaluated. Morphological differences of naturally-occurring hybrids of glyphosate-resistant Palmer amaranth and spiny amaranth have been evaluated and characterized. Whole genome sequencing of glyphosate-resistant and -sensitive genomes in Palmer amaranth was initiated and is progressing. Ribonucleic acid (RNA) sequencing experiments were performed to assess the genes influenced by glyphosate application to glyphosate-resistant and -sensitive Palmer amaranth biotypes. Preliminary results show enhanced synthesis of the 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) gene and many of the other genes present in the replicon. Other highly expressed genes were associated with transposition events. Studies on the detection of glyphosate-resistant and susceptible weeds through hyperspectral plant sensing within a soybean field is being continued. Research continued on the evaluation of a reduced mycotoxin formulation and mutant strains of a bioherbicide Myrothecium verrucaria (MV). Comparative studies on mycelial preparations of MV and a non-spore producing mutant sector of MV showed that the sector efficacy was equal to, or only slightly lower than MV. Viable freeze-dried formulations of Myrothecium verrucaria (MV) mycelium were mass-produced and stored, and a shelf-life of up to 9 years was demonstrated with bioherbicidal activity. Glyphosate resistance in barnyardgrass/junglerice accessions (collected in 2015) and RNA-sequencing analysis of metabolic resistance in barnyardgrass is being researched. Molecular characterization studies of Acetyl Coenzyme A. Carboxylase (ACCase) and Acetolactate Synthase (ALS) inhibitor resistance in Johnsongrass and Italian ryegrass from Mississippi and North Carolina, and of ALS resistance in annual bluegrass and in pigweeds from Mississippi have been initiated and are in progress.Paraquat resistance is being analyzed in an Italian ryegrass population from California.

1. New herbicide formulations do not adversely affect beneficial soil microbes. Widespread distribution of glyphosate-resistant weeds in soybean-growing areas across Mississippi has economically affected soybean planting and follow-up crop management operations. New multiple herbicide-resistant soybean technology with associated formulations was commercialized in 2017. ARS researchers in Stoneville, Mississippi, studied the efficacy of new 2,4- dichlorophenoxyacetic acid (2,4-D) + glyphosate and dicamba formulations on herbicide resistant weeds, and to assess the impact of the new 2,4-D + glyphosate formulation on microbial communities in the soybean rhizosphere involved in nutrient cycling. New 2,4-D + glyphosate and dicamba formulations registered for use on 2,4-D and dicamba-resistant soybean, respectively, only moderately controlled glyphosate-resistant and - susceptible pigweeds (Palmer amaranth and tall waterhemp) and common ragweed. The 2,4-D + glyphosate formulation did not significantly impact soil microbial activities linked to nutrient cycling in the soybean rhizosphere. These results indicate these new 2,4-D + glyphosate and dicamba formulations cannot effectively control glyphosate-resistant and other herbicide resistant weeds and have no adverse effects on beneficial soil microorganism activities.

2. Genetic parameters defined in herbicide resistant weeds. Analysis of bacterial artificial chromosome (BAC)-end sequencing by ARS researchers in Stoneville, Mississippi, resulted in the determination of the final length of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) replicon in glyphosate-resistant Palmer amaranth. The replicon, nearly 400,000 base pairs in length and comprised of <100 genes in addition to a single copy of EPSPS, was identical in weed populations across the United States indicating that this resistance mechanism evolved once and then rapidly spread across the country. The replicon is capable of being inserted into chromosomes, or it can exist as a free-floating circular form like a giant plasmid, the first of which to be described in higher plants. This giant circular entity (along with precision molecular engineering) may possibly be used as a prodigious vehicle for plant transformation, opening up a new era of plant breeding technology.

3. Bioherbicides to control weeds. ARS researchers in Stoneville, Mississippi, evaluated phytopathogenic fungi as bioherbicides. Research continued on the evaluation of a reduced mycotoxin formulation of Myrothecium verrucaria (MV) and mutant strains of MV for control of kudzu and other invasive weeds. Comparative studies on mycelial preparations of MV and a recently discovered sector (MV-Sector BSH) of this fungus were carried out. The whitish sector was isolated and grown in pure culture on potato dextrose agar 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 weed seedlings (hemp sesbania and sicklepod) and leaf discs of weeds (kudzu and glyphosate-resistant Palmer amaranth) showed that the sector efficacy was generally equal to, or slightly lower than MV. Results indicate that certain bioherbicides have utility in controlling herbicide-resistant and recalcitrant weeds.

4. Glyphosate resistance mechanism in ragweed. Glyphosate is one of the most commonly used broad-spectrum herbicides over the last 40 years. Due to the widespread adoption of glyphosate-resistant (GR) crop technology, especially corn, cotton and soybean, several weed species have evolved resistance to this herbicide. Research by scientists at the USDA-ARS; Department of Plant Sciences, University of California; Delta Research and Extension Center, Mississippi State University; Department of Crop, Soil, and Environmental Sciences, University of Arkansas; and Mycogen Seeds, Dow AgroSciences was conducted to confirm and characterize the magnitude and mechanism of glyphosate resistance in two GR common ragweed (Ambrosia artemisiifolia L.) biotypes from Mississippi. A glyphosate-susceptible (GS) biotype was included for comparison. The effective glyphosate dose to reduce the growth of the treated plants by 50% for the GR1, GR2 and GS biotypes was 0.58, 0.46 and 0.11 kg/ha, respectively, indicating that the level of resistance was five- and four-fold that of the GS biotype for GR1 and GR2, respectively. Studies using 14C-glyphosate showed no difference in its absorption between the biotypes. Although the resistant GR1 and GR2 biotypes translocated more 14C-glyphosate, compared to the GS biotype, differential translocation was not in play. There was no amino acid substitution at codon 106 detected by the 5- enolpyruvylshikimate-3-phosphate synthase gene sequence analysis of the resistant and susceptible biotypes. Therefore, the mechanism of resistance to glyphosate in common ragweed biotypes from Mississippi is not due to a target site mutation or to reduced absorption and/or translocation of glyphosate.

5. Efficacy improvement of bioherbicides via formulation. Bioherbicides can offer an effective alternative control strategy to chemical herbicides, but these products must be properly formulated in order to maximize effectiveness and to overcome environmental constraints. ARS researchers in Stoneville, Mississippi, demonstrated that a strain of the fungus Colletotrichum (C) coccodes isolated from eastern black nightshade was effective in controlling this weed under sub-optimal free-moisture (dew) conditions when fungal spores were formulated in an invert emulsion. Results demonstrate that formulating C. coccodes spores in an invert emulsion greatly improves its bioherbicidal potential. Results also suggest that this formulation may render pathogens, previously rejected for bioherbicidal development due to restrictive dew requirements, more efficacious in controlling their target weeds.

6. Genetic crosses of weeds can cause hybrids and transfer of resistance. ARS researchers in Stoneville, Mississippi compared the growth of clones of seven Amaranthus (A) palmeri x A. spinosus hybrids to type specimens of A. palmeri and A. spinosus. The hybrids came from a farmer’s field, where they were originally discovered and clones of the type specimens and hybrids were established under greenhouse conditions and used to compare growth rates. A. palmeri had the highest growth rate and A. spinosus the lowest growth rate based on height, node counts, and dry weight accumulation. A. palmeri also exhibited the greatest number of days to flowering and A. spinosus the fewest. Hybrids had intermediary growth rates and days to flowering, but differed from each other with regard to sex identity. The hybrids were either dioecious (like A. palmeri) or if monoecious, had patterns unlike A. spinosus. Spine length and texture also varied in hybrids and some were spineless. One hybrid was short compared to all others and had succulent leaves and stems, which easily separated from the plant body. These hybridizations resulted in morphologically distinct types with acquisition of physical traits intermediate to the type specimens that may drive evolution of these species.

Review Publications
Molin, W.T., Stetina, S.R. 2016. Weed hosts and relative weed and cover crop susceptibility to Rotylenchulus reniformis in the Mississippi Delta. Nematropica. 46:121-131.
Molin, W.T., Wright, A.A., Lawton-Rauh, A., Saski, C.A. 2017. The unique genomic landscape surrounding the EPSPS gene in glyphosate resistant Amaranthus palmeri: A repetitive path to resistance. BMC Genomics. doi:10.1186/s12864-016-3336-4.
Nandula, V.K., Tyler, H.L. 2016. Effect of new auxin herbicide formulations on control of herbicide resistant weeds and on microbial activities in the rhizosphere. American Journal of Plant Sciences. 7:2429-2439.
Molin, W.T., Nandula, V.K. 2017. Morphological characterization of Amaranthus palmeri x A. spinosus hybrids. American Journal of Plant Sciences. 8:1499-1510.
Boyette, C.D., Hoagland, R.E., Stetina, K.C. 2016. Efficacy improvement of a bioherbicidal fungus using a formulation-based approach. American Journal of Plant Sciences. 7:2349-2358.
Hoagland, R.E., Boyette, C.D., Stetina, K.C., Jordan, R.H. 2016. Bioherbicidal efficacy of a Myrothecium verrucaria-sector on several plant species. American Journal of Plant Sciences. 7:2376-2389.
Hoagland, R.E., Boyette, C.D. 2016. Controlling herbicide-susceptible, -tolerant and -resistant weeds with microbial bioherbicides. Outlooks on Pest Management. 27:256-266.
Nandula, V.K., Tehranchian, P., Bond, J.A., Norsworthy, J.K., Eubank, T.W. 2017. Glyphosate resistance in common ragweed (Ambrosia artemisiifolia L.)from Mississippi, USA. Weed Biology and Management. 17:45-53.