Extrachromosomal circular DNA-mediated spread of herbicide resistance in interspecific hybrids of pigweed.

Extrachromosomal circular DNAs (eccDNAs) are found in many eukaryotic organisms. EccDNA-powered copy number variation plays diverse roles, from oncogenesis in humans to herbicide resistance in crop weeds. Here, we report interspecific eccDNA flow and its dynamic behavior in soma cells of natural populations and F1 hybrids of Amaranthus sp. The glyphosate-resistance (GR) trait is controlled by eccDNA-based amplification harboring the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene (eccDNA replicon), the molecular target of glyphosate. We documented pollen-mediated transfer of eccDNA in experimental hybrids between glyphosate-susceptible Amaranthus tuberculatus and GR Amaranthus palmeri. Experimental hybridization and fluorescence in situ hybridization (FISH) analysis revealed that the eccDNA replicon in Amaranthus spinosus derived from GR A. palmeri by natural hybridization. FISH analysis also revealed random chromosome anchoring and massive eccDNA replicon copy number variation in soma cells of weedy hybrids. The results suggest that eccDNAs are inheritable across compatible species, contributing to genome plasticity and rapid adaptive evolution.

Global Metabolome of Palmer Amaranth (Amaranthus palmeri) Populations Highlights the Specificity and Inducibility of Phytochemical Responses to Abiotic Stress.

Commonalities in adaptive responses to abiotic stressors could contribute to the development of cross-resistance in weeds. The degree to which herbicide-induced changes in weeds parallel those induced by other abiotic stress remains unknown. We investigated the specificity of metabolic perturbations induced by glyphosate and drought across three glyphosate-resistant (GR) and two glyphosate-susceptible (GS) biotypes of Palmer amaranth (Amaranthus palmeri) using global metabolomics approaches. Compared to GS-biotypes, in the absence of stress, the GR-biotypes had a higher abundance of primary metabolites, including sugars, nonaromatic amino acids, and organic acids. However, despite having a higher 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene copy number that could upregulate the phenylpropanoid metabolism, the nonstressed GR-biotypes were less abundant in specialized (secondary) metabolites. Under glyphosate stress, 80% of metabolites, including shikimate, that accumulated in GS-biotypes also increased in the GR-biotypes. However, glyphosate triggered the preferential accumulation of glycosides of dihydroxylated and methoxylated flavanols with higher antioxidant potential, and ferulic acid derivatives, specifically in GR-biotypes. The disruption of the shikimate pathway and the accumulation of phenylpropanoids upon glyphosate exposure suggest that the stress response of GR-biotypes could be partly induced. This differential response was less evident in other phytochemical classes and under drought, highlighting that the phytochemical responses are stress-specific rather than biotype-specific.

Resistance to acetolactate synthase inhibitors is due to a W 574 to L amino acid substitution in the ALS gene of redroot pigweed and tall waterhemp.

Several Amaranthus spp. around the world have evolved resistance (and cross resistance) to various herbicide mechanisms of action. Populations of redroot pigweed (RRPW-R) and tall waterhemp (TW-R) in Mississippi, USA have been suspected to be resistant to one or more acetolactate synthase (ALS) inhibiting herbicides. Whole plant dose-response experiments with multiple ALS inhibitors, ALS enzyme assays with pyrithiobac, and molecular sequence analysis of ALS gene constructs were conducted to confirm and characterize the resistance profile and nature of the mechanism in the RRPW-R and TW-R populations. Two susceptible populations, RRPW-S and TW-S were included for comparison with RRPW-R and TW-R, correspondingly. The resistance index (R/S; the herbicide dose required to reduce plant growth by 50% of resistant population compared to the respective susceptible population) values of the RRPW-R population were 1476, 3500, and 900 for pyrithiobac, imazaquin, and trifloxysulfuron, respectively. The R/S values of the TW-R population for pyrithiobac, imazaquin, and trifloxysulfuron were 51, 950, and 2600, respectively. I50 values of RRPW-S and RRPW-R populations for pyrithiobac were 0.062 and 208.33 µM, indicating that the ALS enzyme of the RRPW-R population is 3360-fold more resistant to pyrithiobac than the RRPW-S population under our experimental conditions. The ALS enzyme of the TW-R population was 1214-fold resistant to pyrithiobac compared to the TW-S population, with the I50 values for pyrithiobac of ALS from TW-R and TW-S populations being 87.4 and 0.072 µM, correspondingly. Sequencing of the ALS gene identified a point mutation at position 574 of the ALS gene leading to substitution of tryptophan (W) residue with a leucine (L) residue in both RRPW-R and TW-R populations. Thus, the RRPW-R and TW-R populations are resistant to several ALS-inhibiting herbicides belonging to different chemical classes due to an altered target site, i.e., ALS. Resistance in Amaranthus spp. to commonly used ALS-inhibiting herbicides warrants an integrated weed management scheme incorporating chemical, mechanical, and cultural strategies by growers.

Combining rare alleles and grouped pollen donors to assign paternity in pollen dispersal studies.

PREMISE: Pollen dispersal plays a critical role in gene flow of seed plants. Most often, pollen dispersal is measured using paternity assignment. However, this approach can be time-consuming because it typically entails genotyping all pollen donors, receptors, and offspring at several molecular markers. METHODS: We developed a faster, simpler protocol to track paternity, using pollen receptors and grouped pollen donors that possess rare alleles. We tested this approach using wind-pollinated Amaranthus tuberculatus and insect-pollinated Solanum lycopersicum. After screening potential markers for rare alleles, we grew both species in experimental arrays under field conditions. RESULTS: All tested A. tuberculatus seeds and 97% of S. lycopersicum fruits could be assigned to the grouped pollen donors using each of two markers. From these results, we could infer paternity of untested offspring and assess pollen dispersal patterns in each array. DISCUSSION: By combining rare alleles and grouped pollen donors, we could assess pollen dispersal for both species and across all arrays after genotyping a small number of pollen donors and a representative subset of offspring. While directly applicable to A. tuberculatus and S. lycopersicum, this approach could be used in other species to assess pollen dispersal under field conditions.

Resistance to clethodim in Italian ryegrass (Lolium perenne ssp. multiflorum) from Mississippi and North Carolina.

BACKGROUND: 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 from North Carolina was cross-resistant to clethodim. This research characterized the magnitude and mechanisms of clethodim resistance in the Mississippi and North Carolina Italian ryegrass populations via whole-plant herbicide dose response, cross resistance, and metabolism studies, and molecular analysis. RESULTS: 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 17-30% of nontreated at the highest clethodim dose of 2.17 kg ha-1 , (8×). The NC22 biotype was cross-resistant to sethoxydim, fluazifop, quizalofop, and pinoxaden. Metabolic inhibitors such as piperonyl butoxide and 4-chloro-7-nitrobenzofurazan did not affect resistance of MS37, MS51, and NC22 biotypes to fenoxaprop, clethodim, or 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. CONCLUSION: 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. Published 2019. This article is a U.S. Government work and is in the public domain in the USA.

Herbicide Resistance Traits in Maize and Soybean: Current Status and Future Outlook.

This article reviews, focusing on maize and soybean, previous efforts to develop nontransgenic herbicide-resistant crops (HRCs), currently available transgenic HRC traits and technologies, as well as future chemical weed management options over the horizon. Since the mid twentieth century, herbicides rapidly replaced all other means of weed management. Overreliance on 'herbicide-only' weed control strategies hastened evolution of HR weed species. Glyphosate-resistant (GR) crop technology revolutionized weed management in agronomic crops, but GR weeds, led by Palmer amaranth, severely reduced returns from various cropping systems and affected the bottom line of growers across the world. An additional problem was the lack of commercialization of a new herbicide mode of action since the 1990s. Auxinic HRCs offer a short-term alternative for management of GR Palmer amaranth and other weed species. New HRCs stacked with multiple herbicide resistance traits and at least two new herbicide modes of action expected to be available in the mid-2020s provide new chemical options for weed management in row crops in the next decade.

Comparative Metabolomic Analyses of Ipomoea lacunosa Biotypes with Contrasting Glyphosate Tolerance Captures Herbicide-Induced Differential Perturbations in Cellular Physiology.

Glyphosate-tolerant Ipomoea lacunosa is emerging as a problematic weed in the southeastern United States. Metabolomic profiling was conducted to examine the innate physiology and the glyphosate induced perturbations in two biotypes of I. lacunosa (WAS and QUI) that had contrasting glyphosate tolerance. Compared to the less tolerant QUI-biotype, the innate metabolism of the more tolerant WAS-biotype was characterized by a higher abundance of amino acids, and pyruvate; whereas the sugar profile of the QUI biotype was dominated by the transport sugar sucrose. Glyphosate application (80 g ae/ha) caused similar shikimate accumulation in both biotypes. Compared to QUI, in WAS, the content of aromatic amino acids was less affected by glyphosate treatment, and the content of Ala, Val, Ile, and Pro increased. However, the total sugars decreased by ∼75% in WAS, compared to ∼50% decrease in QUI. The innate, higher proportional abundance, of the transport-sugar sucrose in QUI coud partly explain the higher translocation and greater sensitivity of this biotype to glyphosate. The decrease in sugars, accompanied by an increase in amino acids could delay feedback regulation of upstream enzymes of the shikimate acid pathway in WAS, which could contribute to a greater glyphosate tolerance. Our study, through a metabolomics approach, provides complementary data that elucidates the cellular physiology of herbicide tolerance in Ipomoea lacunosa biotypes.

Multiple resistance to glyphosate, paraquat and ACCase-inhibiting herbicides in Italian ryegrass populations from California: confirmation and mechanisms of resistance.

BACKGROUND: Glyphosate, paraquat and acetyl CoA carboxylase (ACCase)-inhibiting herbicides are widely used in California annual and perennial cropping systems. Recently, glyphosate, paraquat, and ACCase- and acetolactate synthase (ALS)-inhibitor resistance was confirmed in several Italian ryegrass populations from the Central Valley of California. This research characterized the possible mechanisms of resistance. RESULTS: Multiple-resistant populations (MR1, MR2) are resistant to several herbicides from at least three modes of action. Dose-response experiments revealed that the MR1 population was 45.9-, 122.7- and 20.5-fold, and the MR2 population was 24.8-, 93.9- and 4.0-fold less susceptible to glyphosate, sethoxydim and paraquat, respectively, than the susceptible (Sus) population. Accumulation of shikimate in Sus plants was significantly greater than in MR plants 32 h after light pretreatments. Glyphosate resistance in MR plants was at least partially due to Pro106-to-Ala and Pro106-to-Thr substitutions at site 106 of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). EPSPS gene copy number and expression level were similar in plants from the Sus and MR populations. An Ile1781-to-Leu substitution in ACCase gene of MR plants conferred a high level of resistance to sethoxydim and cross-resistance to other ACCase-inhibitors. Radiolabeled herbicide studies and phosphorimaging indicated that MR plants had restricted translocation of 14 C-paraquat to untreated leaves compared to Sus plants. CONCLUSION: This study shows that multiple herbicide resistance in Italian ryegrass populations in California, USA, is due to both target-site and non-target-site resistance mechanisms. © 2017 Society of Chemical Industry.

Transcriptomic changes in Echinochloa colona in response to treatment with the herbicide imazamox.

MAIN CONCLUSION: Presented here is the first Echinochloa colona leaf transcriptome. Analysis of gene expression before and after herbicide treatment reveals that E. colona mounts a stress response upon exposure to herbicide. Herbicides are the most frequently used means of controlling weeds. For many herbicides, the target site is known; however, it is considerably less clear how plant gene expression changes in response to herbicide exposure. In this study, changes in gene expression in response to herbicide exposure in imazamox-sensitive (S) and- resistant (R) junglerice (Echinochloa colona L.) biotypes was examined. As no reference genome is available for this weed, a reference leaf transcriptome was generated. Messenger RNA was isolated from imazamox-treated- and untreated R and S plants and the resulting cDNA libraries were sequenced on an Illumina HiSeq2000. The transcriptome was assembled, annotated, and differential gene expression analysis was performed to identify transcripts that were upregulated or downregulated in response to herbicide exposure for both biotypes. Differentially expressed transcripts included transcription factors, protein-modifying enzymes, and enzymes involved in metabolism and signaling. A literature search revealed that members of the families represented in this analysis were known to be involved in abiotic stress response in other plants, suggesting that imazamox exposure induced a stress response. A time course study examining a subset of transcripts showed that expression peaked within 4-12 h and then returned to untreated levels within 48 h of exposure. Testing of plants from two additional biotypes showed a similar change in gene expression 4 h after herbicide exposure compared to the resistant and sensitive biotypes. This study shows that within 48 h junglerice mounts a stress response to imazamox exposure.

Stable Isotope Resolved Metabolomics Reveals the Role of Anabolic and Catabolic Processes in Glyphosate-Induced Amino Acid Accumulation in Amaranthus palmeri Biotypes.

Biotic and abiotic stressors often result in the buildup of amino acid pools in plants, which serve as potential stress mitigators. However, the role of anabolic (de novo amino acid synthesis) versus catabolic (proteolytic) processes in contributing to free amino acid pools is less understood. Using stable isotope-resolved metabolomics (SIRM), we measured the de novo amino acid synthesis in glyphosate susceptible (S-) and resistant (R-) Amaranthus palmeri biotypes. In the S-biotype, glyphosate treatment at 0.4 kg ae/ha resulted in an increase in total amino acids, a proportional increase in both (14)N and (15)N amino acids, and a decrease in soluble proteins. This indicates a potential increase in de novo amino acid synthesis, coupled with a lower protein synthesis and a higher protein catabolism following glyphosate treatment in the S-biotype. Furthermore, the ratio of glutamine/glutamic acid (Gln/Glu) in the glyphosate-treated S- and R-biotypes indicated that the initial assimilation of inorganic nitrogen to organic forms is less affected by glyphosate. However, amino acid biosynthesis downstream of glutamine is disproportionately disrupted in the glyphosate treated S-biotype. It is thus concluded that the herbicide-induced amino acid abundance in the S-biotype is contributed by both protein catabolism and de novo synthesis of amino acids such as glutamine and asparagine.

Distinguishing between weedy Amaranthus species based on intron 1 sequences from the 5-enolpyruvylshikimate-3-phosphate synthase gene.

BACKGROUND: Hybridization between Amaranthus species and the potential for herbicide resistance to be transferred by hybridization are of growing concern in the weed science community. Early detection of evolved herbicide resistance and hybrids expressing resistance to single or multiple herbicides is important to develop an effective control strategy. RESULTS: A PCR test was developed for quick identification of weedy amaranths and any hybrids. The sequences of intron 1 for the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) gene were determined for Amaranthus palmeri, A. spinosus, A. retroflexus, A. blitoides, A. viridis, A. tuberculatus and A. hybridus. These sequences were aligned and primers were developed in areas 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. CONCLUSION: The PCR techniques described here allow identification of a weedy amaranth or suspect hybrid in a few hours. Using a similar target, it may be possible to design simple PCR tests to identify even more difficult to distinguish weed species or weeds prone to interspecific hybridization. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Metabolic Profiling and Enzyme Analyses Indicate a Potential Role of Antioxidant Systems in Complementing Glyphosate Resistance in an Amaranthus palmeri Biotype.

Metabolomics and biochemical assays were employed to identify physiological perturbations induced by a commercial formulation of glyphosate in susceptible (S) and resistant (R) biotypes of Amaranthus palmeri. At 8 h after treatment (HAT), compared to the respective water-treated control, cellular metabolism of both biotypes were similarly perturbed by glyphosate, resulting in abundance of most metabolites including shikimic acid, amino acids, organic acids and sugars. However, 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 the glyphosate resistance in this R-biotype.

Possible glyphosate tolerance mechanism in pitted morningglory (Ipomoea lacunosa L.).

Natural tolerance of Ipomoea lacunosa to glyphosate has made it problematic in the southeastern U.S. since the adoption of glyphosate-resistant crops. Experiments were conducted to determine (i) the variability in tolerance to glyphosate among accessions, (ii) if there is any correlation between metabolism of glyphosate to aminomethylphosponic acid (AMPA) or sarcosine and the level of tolerance, and (iii) the involvement of differential translocation in tolerance to glyphosate. Fourteen I. lacunosa accessions had GR50 values ranging from 58 to 151 grams of acid equivalent per hectare (ae/ha) glyphosate, a 2.6-fold variability in tolerance to glyphosate. There was no evidence of the most tolerant (MT) accession metabolizing glyphosate to AMPA more rapidly than the least tolerant (LT) accession. Metabolism to sarcosine was not found. (14)C-glyphosate absorption was similar in the two accessions. LT accession translocated more (14)C-glyphosate than MT accession at 24 and 48 h after treatment. Differential translocation partly explains glyphosate tolerance in MT accession.

First report of resistance to acetolactate-synthase-inhibiting herbicides in yellow nutsedge (Cyperus esculentus): confirmation and characterization.

BACKGROUND: Yellow nutsedge is one of the most problematic sedges in Arkansas rice, requiring the frequent use of halosulfuron (sulfonylurea) for its control. In the summer of 2012, halosulfuron at 53 g ha(-1) (labeled field rate) failed to control yellow nutsedge. The level of resistance to halosulfuron was determined in the putative resistant biotype, and its cross-resistance to other acetolactate synthase (ALS) inhibitors from four different herbicide families. ALS enzyme assays and analysis of the ALS gene were used to ascertain the resistance mechanism. RESULTS: None of the resistant plants was killed by halosulfuron at a dose of 13 568 g ha(-1) (256× the field dose), indicating a high level of resistance. Based on the whole-plant bioassay, the resistant biotype was not controlled by any of the ALS-inhibiting herbicides (imazamox, imazethapyr, penoxsulam, bispyribac, pyrithiobac-sodium, bensulfuron and halosulfuron) tested at the labeled field rate. The ALS enzyme from the resistant biotype was 2540 times less responsive to halosulfuron than the susceptible biotype, and a Trp574 -to-Leu substitution was detected by ALS gene sequencing using the Illumina HiSeq. CONCLUSION: The results suggest a target-site alteration as the mechanism of resistance in yellow nutsedge, which accounts for the cross-resistance to other ALS-inhibiting herbicide families.

EPSPS amplification in glyphosate-resistant spiny amaranth (Amaranthus spinosus): a case of gene transfer via interspecific hybridization from glyphosate-resistant Palmer amaranth (Amaranthus palmeri).

BACKGROUND: Amaranthus spinosus, a common weed of pastures, is a close relative of Amaranthus palmeri, a problematic agricultural weed with widespread glyphosate resistance. These two species have been known to hybridize, allowing for transfer of glyphosate resistance. Glyphosate-resistant A. spinosus was recently suspected in a cotton field in Mississippi. RESULTS: Glyphosate-resistant A. spinosus biotypes exhibited a fivefold increase in resistance compared with a glyphosate-susceptible biotype. EPSPS was amplified 33-37 times and expressed 37 times more in glyphosate-resistant A. spinosus biotypes than in a susceptible biotype. The EPSPS sequence in resistant A. spinosus plants was identical to the EPSPS in glyphosate-resistant A. palmeri, but differed at 29 nucleotides from the EPSPS in susceptible A. spinosus plants. PCR analysis revealed similarities between the glyphosate-resistant A. palmeri amplicon and glyphosate-resistant A. spinosus. CONCLUSIONS: Glyphosate resistance in A. spinosus is caused by amplification of the EPSPS gene. Evidence suggests that part of the EPSPS amplicon from resistant A. palmeri is present in glyphosate-resistant A. spinosus. This is likely due to a hybridization event between A. spinosus and glyphosate-resistant A. palmeri somewhere in the lineage of the glyphosate-resistant A. spinosus plants. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Glyphosate-resistant and glyphosate-susceptible Palmer amaranth (Amaranthus palmeri S. Wats.): hyperspectral reflectance properties of plants and potential for classification.

BACKGROUND: Palmer amaranth (Amaranthus palmeri S. Wats.) is a troublesome agronomic weed in the southern United States, and several populations have evolved resistance to glyphosate. This paper reports on spectral signatures of glyphosate-resistant (GR) and glyphosate-sensitive (GS) plants, and explores the potential of using hyperspectral sensors to distinguish GR from GS plants. RESULTS: GS plants have higher light reflectance in the visible region and lower light reflectance in the infrared region of the spectrum compared with GR plants. The normalized reflectance spectrum of the GR and GS plants had best separability in the 400-500 nm, 650-690 nm, 730-740 nm and 800-900 nm spectral regions. Fourteen wavebands from within or near these four spectral regions provided a classification of unknown set of GR and GS plants, with a validation accuracy of 94% for greenhouse-grown plants and 96% for field-grown plants. CONCLUSIONS: GR and GS Palmer amaranth plants have unique hyperspectral reflectance properties, and there are four distinct regions of the spectrum that can separate the GR from GS plants. These results demonstrate that hyperspectral imaging has potential application to distinguish GR from GS Palmer amaranth plants (without a glyphosate treatment), with future implications for glyphosate resistance management. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Involvement of facultative apomixis in inheritance of EPSPS gene amplification in glyphosate-resistant Amaranthus palmeri.

The inheritance of glyphosate resistance in two Amaranthus palmeri populations (R1 and R2) was examined in reciprocal crosses (RC) and second reciprocal crosses (2RC) between glyphosate-resistant (R) and -susceptible (S) parents of this dioecious species. R populations and Female-R × Male-S crosses contain higher 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene copy numbers than the S population. EPSPS expression, EPSPS enzyme activity, EPSPS protein quantity, and level of resistance to glyphosate correlated positively with genomic EPSPS relative copy number. Transfer of resistance was more influenced by the female than the male parent in spite of the fact that the multiple copies of EPSPS are amplified in the nuclear genome. This led us to hypothesize that this perplexing pattern of inheritance may result from apomictic seed production in A. palmeri. We confirmed that reproductively isolated R and S female plants produced seeds, indicating that A. palmeri can produce seeds both sexually and apomictically (facultative apomixis). This apomictic trait accounts for the low copy number inheritance in the Female-S × Male-R offsprings. Apomixis may also enhance the stability of the glyphosate resistance trait in the R populations in the absence of reproductive partners.

Physiological and molecular basis of acetolactate synthase-inhibiting herbicide resistance in barnyardgrass (Echinochloa crus-galli).

Barnyardgrass biotypes from Arkansas (AR1 and AR2) and Mississippi (MS1) have evolved cross-resistance to imazamox, imazethapyr, and penoxsulam. Additionally, AR1 and MS1 have evolved cross-resistance to bispyribac-sodium. Studies were conducted to determine if resistance to acetolactate synthase (ALS)-inhibiting herbicides in these biotypes is target-site or non-target-site based. Sequencing and analysis of a 1701 base pair ALS coding sequence revealed Ala122 to Val and Ala122 to Thr substitutions in AR1 and AR2, respectively. The imazamox concentrations required for 50% inhibition of ALS enzyme activity in vitro of AR1 and AR2 were 2.0 and 5.8 times, respectively, greater than the susceptible biotype. Absorption of ¹4C-bispyribac-sodium, -imazamox, and -penoxsulam was similar in all biotypes. ¹4C-Penoxsulam translocation out of the treated leaf (≤2%) was similar among all biotypes. ¹4C-Bispyribac-treated AR1 and MS1 translocated 31- 43% less radioactivity to aboveground tissue below the treated leaf compared to the susceptible biotype. ¹4C-Imazamox-treated AR1 plants translocated 39% less radioactivity above the treated leaf and aboveground tissue below the treated leaf, and MS1 translocated 54 and 18% less radioactivity to aboveground tissue above and below the treated leaf, respectively, compared to the susceptible biotype. Phosphorimaging results further corroborated the above results. This study shows that altered target site is a mechanism of resistance to imazamox in AR2 and probably in AR1. Additionally, reduced translocation, which may be a result of metabolism, could contribute to imazamox and bispyribac-sodium resistance in AR1 and MS1.

Bioassay-directed isolation and identification of phytotoxic and fungitoxic acetylenes from Conyza canadensis.

Conyza canadensis (L.) Cronquist syn. (horseweed) is a problematic and invasive weed with reported allelopathic properties. To identify the phytotoxic constituents of the aerial parts, a systematic bioactivity-guided fractionation of the dichloromethane extract was performed. Three active enyne derivatives, (2Z,8Z)-matricaria acid methyl ester, (4Z,8Z)-matricaria lactone, and (4Z)-lachnophyllum lactone, were identified. The lactones inhibited growth of the monocot Agrostis stolonifera (bentgrass) and the dicot Lactuca sativa (lettuce) at 1 mg mL(-1), while the (2Z,8Z)-matricaria acid methyl ester was less active. In a dose-response screening of the lactones for growth inhibitory activity against Lemna paucicostata , (4Z)-lachnophyllum lactone was the most active with an IC50 of 104 µM, while the (4Z,8Z)-matricaria lactone was less active (IC50 of 220 µM). In a fungal direct bioautography assay, the two lactones at 10 and 100 µg/spot inhibited growth of the plant pathogenic fungi Colletotrichum acutatum , Colletotrichum fragariae , and Colletotrichum gloeosporioides . In a dose-response screening of the lactones against six different plant pathogenic fungi, (4Z,8Z)-matricaria lactone was more active than the commercial fungicide azoxystrobin on Col. acutatum , Col. fragariae , and Col. gloeosporioides at 30 µM and about as active as the commercial fungicide captan against Col. gloeosporioides , while (4Z)-lachnophyllum lactone was less active.

Aminomethylphosphonic acid accumulation in plant species treated with glyphosate.

Aminomethylphosphonic acid (AMPA) is the most frequently detected metabolite of glyphosate in plants. The objective of this study was to determine if there is any correlation of metabolism of glyphosate to AMPA in different plant species and their natural level of resistance to glyphosate. Greenhouse studies were conducted to determine the glyphosate I 50 values (rate required to cause a 50% reduction in plant growth) and to quantify AMPA and shikimate concentrations in selected leguminous and nonleguminous species treated with glyphosate at respective I 50 rates. Coffee senna [ Cassia occidentalis (L.) Link] was the most sensitive ( I 50 = 75 g/ha) and hemp sesbania [ Sesbania herbacea (P.Mill.) McVaugh] was the most resistant ( I 50 = 456 g/ha) to glyphosate. Hemp sesbania was 6-fold and Illinois bundleflower [ Desmanthus illinoensis (Michx.) MacM. ex B.L.Robins. & Fern.] was 4-fold more resistant to glyphosate than coffee senna. Glyphosate was present in all plant species, and its concentration ranged from 0.308 to 38.7 microg/g of tissue. AMPA was present in all leguminous species studied except hemp sesbania. AMPA concentration ranged from 0.119 to 4.77 microg/g of tissue. Shikimate was present in all plant species treated with glyphosate, and levels ranged from 0.053 to 16.5 mg/g of tissue. Non-glyphosate-resistant (non-GR) soybean accumulated much higher shikimate than glyphosate-resistant (GR) soybean. Although some leguminous species were found to be more resistant to glyphosate than others, and there was considerable variation between species in the glyphosate to AMPA levels found, metabolism of glyphosate to AMPA did not appear to be a common factor in explaining natural resistance levels.