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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

2012 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:
The discovery of herbicide resistant weeds continued as a fundamental part of this project. Selected populations of annual bluegrass, barnyardgrass, buckhorn plantain, common cocklebur, common lambsquarters, curlydock, slender amaranth, spiny amaranth, and velvetleaf were screened for resistance to glyphosate via whole-plant and/or shikimate assays. Most populations were susceptible to glyphosate, but certain barnyardgrass populations survived half of the recommended label rates. A spiny amaranth population from Mississippi was resistant to glyphosate at labeled rates. Research is in progress to screen browntop millet, Italian ryegrass, and large crabgrass populations for resistance to glyphosate and other herbicides. Several weedy accessions from the Echinochloa genus, collected across the southeastern U.S. were sampled for deoxyribonucleic acid (DNA) extraction and will be analyzed for genetic diversity. Progress on molecular studies of resistant weeds included development of homozygous populations of glyphosate-resistant Italian ryegrass and seed collection for ecological/biological studies. Biomass data on ryegrass competition in corn were recorded and genetic diversity and morphological traits of Italian ryegrass accessions from Mississippi, and other states were assessed. Several ryegrass accessions across Mississippi and other states were sampled for DNA and their genetic diversity assessed. Morphological features were also recorded from these collections. Molecular biological approaches also continue with the goal of explaining how the gene copy number of target site of glyphosate increased in resistant Palmer amaranth. A systematic bioactivity-guided fractionation of the dichloromethane extract of aerial parts of horseweed identified three active enyne derivatives, matricaria acid methyl ester, matricaria lactone, and lachnophyllum lactone. All three compounds exhibited significant phytotoxic and fungitoxic properties. Results may explain ecological competitiveness of this weed in the environment. A preliminary investigation into the effects of herbicides on calcium signaling (Arabidopsis as a model plant species) showed that several herbicides exhibited unique calcium signals indicating they may interrupt various sites of calcium-mediated pathway in plants. Continuation of interaction of plant pathogens with herbicide-susceptible and –resistant weeds studies showed that a strain of the fungus, Myrothecium verrucaria can control various species of pigweeds regardless of their glyphosate resistance levels. Further research is continuing on optimizing formulation ingredients and other parameters. Other important progress in support of this project include: preparation/submission of seven competitive grant proposals (Mississippi Soybean Promotion Board, Mississippi Corn Promotion Board and the United Soybean Promotion Board) to expand research toward an enhanced understanding of herbicide resistance in weeds. A Post-Doctoral scientist was hired (October 2011) to support molecular biology research in this project.

4. Accomplishments
1. Herbicide resistance mechanism studies. Knowledge of the resistance mechanism(s) in a given weed population is important in controlling and curtailing the spread of resistance. A glyphosate-resistant tall waterhemp biotype was found to absorb and translocate less glyphosate than a susceptible biotype. Genetic analysis revealed that a single copy of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene was equally expressed in both biotypes. However, sequence analysis of EPSPS, of the complementary DNA (cDNA) transcript yielded a consistent single nucleotide polymorphism (thymidine/cytosine) that caused a proline to serine amino acid substitution in resistant plants. Results suggest that this biotype may possess two mechanisms of resistance to glyphosate, i.e., reduced uptake/translocation and a mutation of the target site of this herbicide.

2. Techniques to characterize resistance. Characterization of plant populations in order to perform long-term studies is important. A cloning technique was established and >40 glyphosate-resistant and -susceptible pigweeds were cloned. A method for digital assessment of bioassay plant injury data to quantify sensitivity/resistance to herbicides was developed and gene copy number characterization of the 40 cloned pigweeds was completed. These methods will enable in-depth molecular research on a well-defined set of plant biotypes for studies related to herbicide resistance.

3. Molecular biological studies on Palmer amaranth. Elucidation of molecular biological events can lead to pivotal information concerning resistance. A fosmid library of glyphosate-resistant Palmer amaranth deoxyribonucleic acid (DNA) was prepared and clones were screened for the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene. Positive clones were sequenced revealing 7 introns of different lengths. Studies continue to identify promoters (and their positions), and the possible presence of a transposase and transposon within the EPSPS gene amplicon. These results are important to explain how increased copy number of the EPSPS gene developed in this glyphosate resistant weed.

Review Publications
Nandula, V.K., Reddy, K.N., Koger, C.H., Poston, D.H., Rimando, A.M., Duke, S.O., Bond, J.A., Ribeiro, D.N. 2012. Multiple resistance to glyphosate and pyrithiobac in Palmer amaranth (Amaranthus palmeri) from Mississippi and response to flumiclorac. Weed Science. 60:179-188.

Boyette, C.D., Hoagland, R.E., Weaver, M.A., Stetina, K.C. 2012. Biological control potential of Colletotrichum gloeosporioides for coffee senna (Cassia occidentalis). American Journal of Plant Sciences. 3:430-436.

Hoagland, R.E., Mccallister, T.S., Boyette, C.D., Weaver, M.A., Beecham, R.V. 2011. Effects of Myrothecium verrucaria on morning-glory (Ipomoea) species. Allelopathy Journal. 27(2):151-162.

Ratnayaka, H., Molin, W.T., Sterling, T. 2012. Comparison of physiological and antioxidant responses of Anoda cristata and cotton to progressive drought. Weed Research. 52:358-366.

Boyette, C.D., Bryson, C.T., Hoagland, R.E., Weaver, M.A. 2012. Effects of simulated rainfall on disease development and weed control of the bioherbicidal fungi Alternaria cassiae and Colletotrichum truncatum. Weed Technology. 26:117-121. DOI: 10.1614/WT-D-10-00127.1.

Weaver, M.A., Boyette, C.D., Hoagland, R.E. 2012. Bioherbicidal activity from washed spores of Mycrothecium verrucaria. World Journal of Microbiology and Biotechnology. 28:1941-1946.

Boyette, C.D., Gealy, D.R., Hoagland, R.E., Vaughn, K.C., Bowling, A.J. 2012. Hemp Sesbania (Sesbania exaltata) control in rice (Oryza sativa) with the bioherbicidal fungus Colletotrichum gloeosporioides f. sp. aeschynomene formulated in an invert emulsion. Biocontrol Science and Technology. 21 (12):1399-1407.

Boyette, C.D., and Hoagland, R.E. 2012. Interactions of chemical additives, pH, and temperature on Conidia germination and virulence of Colletotrichum truncatum, a bioherbicide of Sesbania exaltata. Allelopathy Journal. 30:103-116.

Last Modified: 05/26/2017
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