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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

2020 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
This is the final report for this project. Project will be replaced by new project pending completion of National Program 304 research review. Field studies demonstrated that glyphosate-resistant Palmer amaranth remained persistent across years despite diverse herbicide management applications. Molecular screening for protoporphyrinogen oxidase (PPO) resistance in pigweeds (Amaranthus spp.) demonstrated differences in PPO sensitivity among local accessions, indicating a need for improved strategies for seedling control. Populations of pigweeds were examined for resistance to these herbicides, and factors affecting herbicide efficacy evaluated. A single plant was identified as having a deletion mutation conferring resistance to these herbicides. Amaranthus spp. from Mississippi were identified through screening field populations for possible resistance and the characterization of a multiple resistant (glyphosate, PPO inhibitors, ALS inhibitors) Palmer amaranth accession from Mississippi was completed. Ribonucleic acid (RNA) sequencing experiments were performed to assess the genes influenced by glyphosate application to glyphosate-resistant and -sensitive Palmer amaranth biotypes. Results showed enhanced synthesis of the 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) gene and many other genes present in the replicon. Other highly expressed genes were associated with transposition events. Since resistant plants do not undergo a state of catastrophic gene transcription as do sensitive plants, the presence of resistance attributes can ameliorate the effects of herbicide application. The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) replicon in glyphosate-resistant Palmer amaranth was shown to be attached to chromosomes by tethering genes in cell to cell transfer of glyphosate resistance in Palmer amaranth during cell division. These genes are being explored as targets for ribonucleic acid interference (RNAi) to inhibit binding. Characterization of the replicon involved in Palmer amaranth resistance to glyphosate was completed and documented by publications. Sequence conservation of the Palmer amaranth replicon conferring resistance to glyphosate was demonstrated among populations across the U.S. Morphological differences of naturally-occurring hybrids of glyphosate-resistant Palmer amaranth and spiny amaranth were evaluated, characterized and published. Comparative flux analysis of nitrogen metabolism in glyphosate-resistant and -susceptible Palmer amaranth biotypes was completed. Studies of the effects of the bioherbicidal fungus, Myrothecium verrucaria, on two glyphosate-resistant Amaranthus palmeri biotypes differing in betacyanin content were completed and published. Interaction studies of a Myrothecium verrucaria mycelial preparation and a glyphosate product for controlling redvine (Brunnichia ovata) and trumpet creeper (Campsis radicans) was completed and results were published. Comparative studies on mycelial preparations of Myrothecium verrucaria (MV) and a non-spore producing mutant sector of MV showed that the efficacy of the mutant was equal to, or only slightly lower than MV. A freeze-drying method for the preservation of biological activity of the bioherbicide Myrothecium verrucaria (MV) was developed. Viable freeze-dried formulations of MV mycelium were mass-produced and stored, and a shelf-life of up to 9 years was demonstrated. The fungus Colletotrichum coccodes, formulated in an invert emulsion (IE), could control the problematic weed Eastern black nightshade and several other solanaceous weeds in greenhouse and field tests. Published results demonstrate that this IE can extend the host range and efficacy of this bioherbicide. 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. Hemp sesbania plants treated with hot water, followed by applications of fungal spores of a bioherbicide (Colletotrichum truncatum), were controlled 85% in the greenhouse and field, 12 days after treatment. Published results suggest that hot water may be an important tool for improving the bioherbicidal potential of some plant pathogens. Spores of the bioherbicide Colletotrichum gloeosporioides f. sp. aeschynomene (CGA), formulated in a surfactant, controlled three weed species (northern jointvetch, Indian jointvetch, and hemp sesbania) in greenhouse experiments. Results suggest that the host range of CGA can be expanded though formulation modification enabling this bioherbicide to control multiple weeds. Studies on 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 were completed. The detection of glyphosate-resistant and -susceptible Italian ryegrass using hyperspectral plant sensing in field-grown soybean plants was evaluated. Evaluation of select soybean germplasm for tolerance to dicamba was completed. A novel technique using a fluorescent compound to measure herbicide drift has been evaluated in initial studies. 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 were studied. Paraquat resistance was examined in an Italian ryegrass population from California. ACCase inhibitor resistance studies of Italian ryegrass populations from North Carolina were completed and published. Italian ryegrass populations from Mississippi were discovered to be resistant to ALS inhibitors and have been characterized and the results published. Studies to measure the absorption and translocation of dicamba in dicamba-resistant soybean using 14C-dicamba as a tracer were examined. Seedlings of the problematic weed, horseweed, survived less than 48 hours when exposed to drought/desiccation. The small seed (1 mm x 0.3 mm) has little survival capacity in the absence of continuous hydration, thus even though a single plant may produce 200,000 seed, its abundance and proliferation are limited under dry environments. A non-technical summary was published describing how Palmer amaranth went from a weed relatively easy to control to a major herbicide resistant weed problem and assessed how agronomic practices influenced the rise and extent of the glyphosate-resistant Palmer amaranth problem.

1. Herbicide resistance of Italian ryegrass biotypes to clethodim. Resistance to clethodim in Italian ryegrass (Lolium perenne ssp. multiflorum) from Mississippi and North Carolina was demonstrated by ARS researchers, Stoneville, Mississippi, and researchers from Mississippi State University and the University of Illinois. Clethodim, an acetyl-CoA carboxylase (ACCase)-inhibiting herbicide, is one of the few postemergence chemical control options available to growers of Mississippi to manage glyphosate and/or other herbicide resistant Italian ryegrass populations. Recently, clethodim failed to adequately control Italian ryegrass populations across Mississippi. A sethoxydim, also an ACCase inhibitor, -resistant Italian ryegrass population was cross-resistant to clethodim. This research characterized the magnitude and mechanisms of clethodim resistance in the Mississippi and North Carolina Italian ryegrass populations. Two clethodim-resistant biotypes from Mississippi, MS24 and MS37, were 10- and 4-fold resistant, respectively, relative to a susceptible (SUS1) biotype. A North Carolina biotype, NC21, was 40-fold resistant to clethodim compared to SUS1. Two additional biotypes from North Carolina, NC22 and NC 23, recorded shoot dry weight reduction of only 17 to 30% of non-treated at the highest clethodim dose of 2.17 kg ha-1 (8X). The NC22 biotype was cross-resistant to sethoxydim, fluazifop, quizalofop, and pinoxaden. The MS37 biotype had three target site mutations, I2041N, C2088R, and G2096A. Another clethodim-resistant biotype from Mississippi, MS51, had only the C2088R substitution. The NC22 and NC23 biotypes had I1781L, I2041N, and D2078G replacements. This study shows that the mechanism of resistance to clethodim in Italian ryegrass from Mississippi and North Carolina is due to target site modifications in the ACCase gene leading to broad cross-resistance to other ACCase-inhibiting herbicides.

2. Sequence and characterization of a replicon that causes herbicide resistance. ARS researchers, Stoneville, Mississippi, and researchers from Clemson University sequenced and characterized the massive eccDNA replicon as the cause of glyphosate resistance in Palmer amaranth, a ground-breaking discovery in plant science. Circular DNAs of less than 30 kb had been observed but nothing comparable to the 400 kb size of the replicon was ever discovered. The replicon also contained 50 other genes in addition to a single copy of EPSPS which were involved in replication, expression, integration and persistence. The replicon was found to occur in glyphosate resistant Palmer amaranth populations without alteration across the country indicating its conservation and ease of spread.

Review Publications
Nandula, V.K., Giacomini, D.A., Lawrence, B.H., Molin, W.T., Bond, J.A. 2020. Resistance to clethodim in Italian ryegrass (Lolium perenne ssp. multiflorum) from Mississippi and North Carolina. Pest Management Science. 76:1378-1385.
Molin, W.T., Parys, K.A., Beck, C.L. 2020. Early growth and development of Horseweed (Conyza canadensis (L.) Cronq.). American Journal of Plant Sciences. 11:40-50.
Molin, W.T., Bryson, C.T. 2019. Variation in 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) coding sequences and glyphosate response among Cyperus rotundus L. populations. American Journal of Plant Sciences. 10(12):2366-2381.
Nandula, V.K. 2019. Herbicide resistance traits in corn and soybean: Current status and future outlook. Plants. 8(9):337.
Boyette, C.D., Bryson, C.T., Hoagland, R.E., Weaver, M.A., Stetina, K.C. 2020. Interaction of a Myrothecium verrucaria mycelial preparation and a glyphosate product for controlling Redvine (Brunnichia ovata) and trumpet creeper (Campsis radicans). American Journal of Plant Sciences. 11:201-213.
Molin, W.T., Kronfol, R.R., Ray, J.D., Scheffler, B.E., Bryson, C.T. 2019. Genetic diversity among geographically separated Cyperus rotundus accessions based on RAPD markers and morphological characteristics. American Journal of Plant Sciences. 10:2034-2046.
Turley, R.B., Stetina, S.R., Bellaloui, N., Molin, W.T. 2019. Comparison of growth, yield and fiber quality of the obsolete SA30 yellow leaf with four sets of modern yellow and green leaf near isogenic cotton (Gossypium hirsutum L.) lines. Journal of Cotton Science. 23(3):253-261.
Bellaloui, N., Turley, R.B., Stetina, S.R., Molin, W.T. 2019. Cottonseed protein, oil, and mineral nutrition in near-isogenic Gossypium hirsutum cotton lines expressing leaf color phenotypes under field conditions. Food and Nutrition Sciences. 10:834-859.
Hoagland, R.E., Boyette, C.D., Jordan, R.H., Stetina, K.C. 2020. Effects of Myrothecium verrucaria on two glyphosate-resistant Amaranthus palmeri biotypes differing in Betacyanin content. American Journal of Plant Sciences. 11:214-225.
Molin, W.T., Yaguchi, A., Blenner, M., Saski, C.A. 2020. The EccDNA Replicon: A Heritable, Extranuclear Vehicle That Enables Gene Amplification and Glyphosate Resistance in Amaranthus palmeri. The Plant Cell. 32:2132-2140.