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United States Department of Agriculture

Agricultural Research Service

Research Project: Characterization and Mitigation of Herbicide-Resistant and Recalcitrant Weeds

Location: Crop Production Systems Research Unit

2013 Annual Report

1a.Objectives (from AD-416):
1: Establish the level of resistance and pattern of cross-resistance/multiple resistance of various weeds to herbicides, and herbicide interactions that promote control of these weeds. 2: Discover potential resistance mechanisms using structural, biochemical and genetic probes. 3: Evaluate the contribution of dispersal mechanisms to the spread and distribution of resistance. 4: Establish structural, biochemical, and genetic characteristics that limit herbicide efficacy against recalcitrant weeds, such as vines (redvine and kudzu) and nutsedges.

1b.Approach (from AD-416):
The approach is holistic, examining not only the mechanism or control of the resistant biotypes, but evaluating the potential for spread of resistance and developing alternative strategies for weed control measures to minimize further development of resistant weeds. Basic growth analyses, assays and bioassays using whole plant and plant tissues will determine major changes that occur in the resistant biotypes in response to herbicide exposure. Studies will involve microscopic approaches (light, fluorescence, standard and scanning and electron microscopy). Subsequent biochemical, genetic, proteomic, immunochemical and radiological techniques will identify more specific sites or differences in herbicide resistant and sensitive weed biotypes within species. This will result in a greater understanding of the biochemistry, physiology and genetics of the mechanisms and spread of resistant weeds, and provide insight on recommendations for better weed control.

3.Progress Report:
Sequences of the epsps (5-enolpyruvylshikimate-3-phosphate synthase) gene, and 1.2 kb upstream and 20 kb downstream in glyphosate-resistant Palmer amaranth (PA) were determined but the ends of the amplicon have not been defined. To identify the full sequence, Clemson University Genomics Institute was contracted to create a DNA library.

Resistance in spiny amaranth from Mississippi was amplification of the target enzyme (EPSPS) of glyphosate, suggesting that the epsps gene and amplification trait were acquired from palmer amaranth. Resistance transfer via pollen is being evaluated by hybridization studies.

A glyphosate-susceptible palmer amaranth population from Mississippi exhibited a range of susceptibility to glyphosate when plants were tested individually, but epsps gene copy number was not elevated in the most resistant individuals. Sequencing (in progress) will determine if a mutation in the gene is responsible for resistance.

Genetic stability tests [quantitative polymerase chain reaction and random amplified polymorphic DNA (RAPD)] in biotypes of cloned palmer amaranth plants indicated high stability after 12 generations (over 18 months). This propagation method allows continuous long-term studies.

RAPD analysis revealed one band in wild-type palmer amaranth that was absent in a betalain deficient mutant. This DNA amplicon is being cloned for sequencing and its role in betalain production.

The epsps gene sequence of a common ragweed population is being analyzed to determine if resistance is due to a point mutation, or to elevated gene copy number.

Reduced glyphosate translocation was the primary mechanism of resistance in a giant ragweed biotype from Mississippi. Glyphosate absorption/translocation studies in glyphosate-tolerant populations of pitted morningglory were initiated. Studies of resistance to acetolactate synthase-inhibiting herbicides in rice flatsedge biotypes from Arkansas and Mississippi were completed.

Bioherbicide effects on herbicide-susceptible and –resistant weeds showed: the fungus, Myrothecium verrucaria (MV) controlled glyphosate-resistant and -susceptible palmer amaranth; a bacterium (Xanthomonas spp.) controlled cocklebur; and chemical additives altered germination and virulence of Colletotrichum truncatum, a bioherbicide for hemp sesbania. MV and Silwet L-77 (surfactant) caused rapid protoplast detachment from cell walls and disruption of plasmodesmata in kudzu plants, suggesting the surfactant increased phytotoxin(s) absorption.

A collaborative (Colorado State University; University of Illinois) grant proposal was submitted (AFRI Competitive Grants) to study evolution of glyphosate resistance. A Post-Doctoral scientist with the Crop Production Systems Research Unit, Stoneville, Mississippi, (hired October 2011) continued to advance molecular biology research on this project.

1. Report of the first target-site mutation in a glyphosate-resistant dicot weed species. The widespread adoption of glyphosate-resistant (GR) crops around the world has resulted in the evolution of several GR weed species, including tall waterhemp in Mississippi (MS). ARS scientists in Mississippi have determined that the mechanism of resistance to glyphosate in a Mississippi GR tall waterhemp population is due to a combination of a target-site mutation and a non-target site based mechanism. Sequence analysis of 5-enolpyruvylshikimate-3-phosphate synthase, (EPSPS), the target site of glyphosate, revealed a consistent single nucleotide polymorphism (T/C; thymine/cytosine) between GR/susceptible (GS) plants, resulting in a proline to serine amino acid substitution at position 106 in the GR population. This is the first report of an altered EPSPS-based resistance in a dicot weed species that has evolved resistance to glyphosate. The GR population absorbed and translocated less glyphosate compared to the GS population. Further, the GR and GS plants contained equal genomic copy number of EPSPS, which was positively correlated with EPSPS gene expression, unlike EPSPS amplification in a GR biotype of Palmer amaranth (weed species closely related to and with potential to hybridize with tall waterhemp). A grower or land manager could face a situation of having to deal with a population of tall waterhemp or Palmer amaranth or a mixture of them along with hybrids that may contain genes encoding for multiple mechanisms of resistance, thereby, exploding input costs.

2. Saflufenacil, a new chemical option for herbicide resistance management. The widespread adoption of glyphosate-resistant (GR) crops around the world has resulted in the evolution of several GR dicot weed species, including giant ragweed, horseweed (marestail), Palmer amaranth, spiny amaranth, and waterhemp in Mississippi. GR populations of all these weeds from MS are expected to be susceptible to a newly developed herbicide, saflufenacil. The mode of action of saflufenacil is by inhibition of the enzyme protoporphyrinogen oxidase (PPO), which is different from that of glyphosate. ARS scientists in Stoneville, MS, in collaboration with Mississippi State University and other organizations conducted research studies to determine the most efficacious adjuvant system for the control of horseweed with saflufenacil, to investigate interactions between saflufenacil and glyphosate mixtures on the control of horseweed, and to determine patterns of uptake and translocation of glyphosate applied alone and in combination with saflufenacil in horseweed. Methylated seed oil was found to enhance the activity of saflufenacil the most on horseweed. Saflufenacil provided excellent control of GR and glyphosate-susceptible (GS) horseweed populations when applied alone or in combination with glyphosate. The addition of saflufenacil reduced 14C-glyphosate translocation in GR and GS horseweed at least 6%; however, due to the exceptional efficacy of saflufenacil on horseweed these reductions did not reduce control. The above results, using horseweed as a model GR weed, indicate that saflufenacil can play a significant role in managing GR resistant dicot weed populations in Mississippi.

3. Gene sequence related to glyphosate tolerance determined in purple nutsedge biotypes. Some weeds possess an inherent tolerance to the herbicide glyphosate and the wide use of glyphosate-resistant (GR) crops on a global basis has resulted in the evolution of high levels of resistance to this herbicide in various weed species. ARS scientists in MS have sequenced the purple nutsedge gene epsps (5-enolpyruvylshikimate-3-phosphate synthase), that codes for the protein target site of glyphosate in a collection of specimens from around the world. Results indicate that although this gene is highly conserved, variation does occur, mainly in the form of single nucleotide polymorphisms and some accessions had extra triplet base pairs in their sequences. These results indicate the purple nutsedge is diversifying. The point mutation at serine 106, which confers mild resistance, was not found indicating that higher tolerance to glyphosate in this species is likely due to anatomical features.

4. A fungal bioherbicide to control herbicide-resistant weeds. The extensive use of glyphosate in glyphosate-resistant (GR) crops has contributed to an increased incidence of herbicide resistance in weeds. Palmer amaranth is one of the major weeds with evolved resistance to glyphosate. ARS scientists in Stoneville, MS, examined the efficacy of the bioherbicidal fungus Myrothecium verrucaria (MV) on whole plants and in excised leaf bioassays of GR and glyphosate-susceptible (GS) Palmer amaranth plants. Generally, injury was directly proportional to the MV mycelial concentration applied, and GS and GR plant leaves were equally sensitive to the MV phytotoxic effects as measured by reduction of chlorophyll content. Similar effects occurred on whole plants challenged by MV spray applications to foliage. MV disease progression over a 7-d period in young (2-week-old) plants increased with time, and at 72 h after treatment, nearly 100% mortality occurred in both the GS and GR plants. Older plants were more tolerant to the bioherbicide, but chlorosis and reduction of growth were typical symptoms. Results demonstrate that under greenhouse/laboratory conditions, MV can control both GR and GS Palmer amaranth seedlings, suggesting that this bioherbicide might be a candidate for use against this economically important weed. These findings are important and evoke further research on bioherbicide:herbicide interactions since synergistic effects of combinations of MV with glyphosate for controlling kudzu, redvine, and trumpetcreeper (recalcitrant weeds with regard to herbicide control) have previously been reported by this laboratory. These research results are also valuable with regard to a possible alternative weed control method and to researchers studying weed management systems for herbicide resistant weeds.

Review Publications
Eubank, T.W., Nandula, V.K., Poston, D.H., Shaw, D. 2012. Multiple resistance of horseweed to glyphosate and paraquat and its control with paraquat and metribuzin combinations. Agronomy Journal. 2:358-370.

Nandula, V.K., Reddy, K.N. 2012. Weed resistance challenges and management under herbicide resistant cropping systems. Indian Journal of Agronomy. 57(4):302-309.

Riar, D.S., Norsworthy, J.K., Srivastava, V., Nandula, V.K., Bond, J.A., Scott, R.C. 2012. Physiological and molecular basis of acetolactate synthase-inhibiting herbicide resistance in barnyardgrass (Echinochloa crus-galli). Journal of Agricultural and Food Chemistry. 61:278-289.

Hoagland, R.E., Teaster, N.D., Boyette, C.D. 2013. Bioherbicidal Effects of Myrothecium verrucaria on Glyphosate Resistant and -Susceptible Palmer amaranth Bbiotypes. Allelopathy Journal. 31(2):367-376.

Hoagland, R.E., Boyette, C.D., Vaughn, K.C., Teaster, N.D., Stetina, K.C. 2012. Effects of Myrothecium verrucaria on ultrastructural integrity of kudzu (Pueraria montana var. lobata) and phytotoxin implications. American Journal of Plant Sciences. 3:1513-1519.

Molin, W.T., Stetina, S.R. 2013. Responses of reniform nematode and browntop millet to tillage, cover crop, and herbicides in cotton. Crop Management. doi:10.1094/CM-2013-0428-01-RS.

Molin, W.T., Wright, A.A., Nandula, V.K. 2013. Glyphosate-resistant goosegrass from Mississippi. Agronomy. 3:474-487.

Gaines, T.A., Wright, A.A., Molin, W.T., Lorentz, L., Riggins, C.W., Tranel, P.J., Beffa, R., Westra, P., Powles, S.B. 2013. Identification of genetic elements associated with EPSPS gene amplification. PLoS One. 8(6):1-10.

Hoagland, R.E., Jordan, R.H., Teaster, N.D. 2013. Bioassay and characterization of several palmer amaranth biotypes with varying tolerances to glyphosate. American Journal of Plant Sciences. 4(5)1029-1037.

Boyette, C.D., Hoagland, R.E. 2013. Bioherbicidal potential of a strain of Xanthomonas spp. for control of common cockelbur, (Santium strumarium). Biocontrol Science and Technology. 23(2):183-196.

Boyette, C.D., Hoagland, R.E. 2013. Adjuvant and refined corn oil formulation effects on conidial germination, appressorial formation and virulence of the bioherbicide, Colletotrichum truncatum. Plant Pathology Journal. 12(2):50-60.

Nandula, V.K., Ray, J.D., Ribeiro, D.N., Pan, Z., Reddy, K.N. 2013. Glyphosate resistance in tall waterhemp (Amaranthus tuberculatus) from Mississippi is due to both altered target-site and nontarget-site mechanisms. Weed Science. 61:374-383.

Last Modified: 4/16/2014
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