R. Douglas Sammons

Gene amplification confers glyphosate resistance in Amaranthus palmeri

The herbicide glyphosate became widely used in the United States and other parts of the world after the commercialization of glyphosate-resistant crops. These crops have constitutive overexpression of a glyphosate-insensitive form of the herbicide target site gene, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Increased use of glyphosate over multiple years imposes selective genetic pressure on weed populations. We investigated recently discovered glyphosate-resistant Amaranthus palmeri populations from Georgia, in comparison with normally sensitive populations. EPSPS enzyme activity from resistant and susceptible plants was equally inhibited by glyphosate, which led us to use quantitative PCR to measure relative copy numbers of the EPSPS gene. Genomes of resistant plants contained from 5-fold to more than 160-fold more copies of the EPSPS gene than did genomes of susceptible plants. Quantitative RT-PCR on cDNA revealed that EPSPS expression was positively correlated with genomic EPSPS relative copy number. Immunoblot analyses showed that increased EPSPS protein level also correlated with EPSPS genomic copy number. EPSPS gene amplification was heritable, correlated with resistance in pseudo-F2 populations, and is proposed to be the molecular basis of glyphosate resistance. FISH revealed that EPSPS genes were present on every chromosome and, therefore, gene amplification was likely not caused by unequal chromosome crossing over. This occurrence of gene amplification as an herbicide resistance mechanism in a naturally occurring weed population is particularly significant because it could threaten the sustainable use of glyphosate-resistant crop technology.

Investigations into glyphosate-resistant horseweed (Conyza canadensis): retention, uptake, translocation, and metabolism

Abstract The mechanism of glyphosate resistance in horseweed was investigated. Eleven biotypes of putative sensitive (S) and resistant (R) horseweed were obtained from regions across the United States and examined for foliar retention, absorption, translocation, and metabolism of glyphosate. Initial studies used spray application of 14C-glyphosate to simulate field application. When S and R biotypes were compared in the absence of toxicity at a sublethal dose, we observed comparable retention and absorption but reduced root translocation in the R biotypes. S and R biotypes from Delaware were further examined at field use rates and results confirmed similar retention and absorption but reduced root translocation in the R biotypes. Application of 14C-glyphosate to a single leaf demonstrated reduced export out of the treated leaf and lower glyphosate import into other leaves, the roots, and the crown in R relative to S biotypes. Examination of the treated leaf by autoradiography showed that glyphosate loading into the apoplast and phloem was delayed and reduced in the R biotype. Our results consistently showed a strong correlation between impaired glyphosate translocation and resistance. Tissues from both S and R biotypes showed elevated levels of shikimate suggesting that 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) remained sensitive to glyphosate. Analysis of tissue shikimate levels demonstrated reduced efficiency in EPSPS inhibition in the R biotypes. Our results suggest that resistance is likely due to altered cellular distribution that impaired phloem loading and plastidic import of glyphosate resulting in reduced overall translocation as well as inhibition of EPSPS. Nomenclature: Glyphosate; horseweed, Conyza canadensis (L.) Cronq. ERICA.

Rapid vacuolar sequestration: the horseweed glyphosate resistance mechanism.

BACKGROUND Glyphosate-resistant (GR) weed species are now found with increasing frequency and threaten the critically important glyphosate weed-management system [corrected]. RESULTS The reported (31)P NMR experiments on glyphosate-sensitive (S) and glyphosate-resistant (R) horseweed, Conyza canadensis (L.) Cronq., show significantly more accumulation of glyphosate within the R biotype vacuole. CONCLUSIONS Selective sequestration of glyphosate into the vacuole confers the observed horseweed resistance to glyphosate. This observation represents the first clear evidence for the glyphosate resistance mechanism in C. canadensis.

Examination of midgut luminal proteinase activities in six economically important insects

A biochemical survey of insect midgut fluid proteinase activities was conducted to determine which proteinase inhibitors could play roles in insect control strategies. In this study, midgut juices from six insects of economic importance have been isolated and their proteolytic activities classified. Three lepidopterans (black cutworm, corn earworm, and tobacco budworm) and the boll weevil have maximal midgut fluid proteolytic activity in vitro at pH 10–11. Soybean trypsin inhibitor inhibits lepidopteran and boll weevil activities by 63–72%, indicating that their major proteinases are “trypsin-like” serine proteinases. Varying levels of inhibition were seen against the midgut fluid proteinase activities whereas bovine trypsin was inhibited 95–98% by each of four different trypsin inhibitors. Southern corn rootworm and Colorado potato beetle have maximal midgut fluid proteolytic activity in vitro at pH 6–7. E-64 inhibits southern corn rootworm (69%) and Colorado potato beetle (100%) midgut fluid proteinase activity, demonstrating that their major activities are provided by cysteine proteinases. Feeding studies found no mortality nor stunting associated with feeding high levels of trypsin inhibitors to boll weevil or tobacco budworm larvae despite the fact that these proteins were very good inhibitors 60–78% inhibition) of midgut proteolytic activity in vitro.

Characterization of the horseweed (Conyza canadensis) transcriptome using GS-FLX 454 pyrosequencing and its application for expression analysis of candidate non-target herbicide resistance genes.

BACKGROUND The de novo transcriptome sequencing of a weedy plant using GS-FLX 454 technologies is reported. Horseweed (Conyza canadensis L.) was the first broadleaf weed to evolve glyphosate resistance in agriculture, and also is the most widely distributed glyphosate-resistant weed in the United States and the world. However, available sequence data for this species are scant. The transcriptomic sequence should be useful for gene discovery, and to help elucidate the non-target-based glyphosate resistance mechanism and the genomic basis of weediness. RESULTS Sequencing experiments yielded 411 962 raw reads, an average read length of 233 bp and a total dataset of 95.8 Mb (NCBI accession number SRA010952). After trimming and quality control, 379 152 high-quality sequences were retained and assembled into contigs. The assembly resulted in 31 783 unique transcripts, including 16 102 contigs and 15 681 singletons. The average coverage depth for each contig and each nucleotide position was 22-fold and 12-fold respectively. A total of 16 306 unique sequences were annotated by searching a custom plant protein database. The utility of the transcriptome data was demonstrated by further exploration of ABC transporters, which were previously hypothesized to play a role in non-target glyphosate resistance. Real-time RT-PCR primers were designed from the transcriptome data, which made it possible to assess expression patterns of 17 ABC transporters from resistant and susceptible horseweed accessions from Tennessee, with and without glyphosate treatment. CONCLUSION These results show that GS-FLX 454 sequencing is a powerful and cost-effective platform for the development of functional genomic tools for a weed species.

Sustainability and Stewardship of Glyphosate and Glyphosate-Resistant Crops

The significance of glyphosate and the appearance of glyphosate-resistant weeds have raised concerns about glyphosate sustainability. Resistance-prevention strategies, however, should first consider the mechanisms for resistance. For example, target-site resistance can provide virtual immunity, ensuring that every herbicide application successfully selects for resistance. However, metabolism and exclusion mechanisms provide lower magnitudes of resistance and are dependent on dosage. This discussion proposes that the relative risk of weed resistance is most highly correlated to mode of action (MOA), due to the respective principal mechanism for resistance. The development of data correlating agronomic practices with weed resistance vs. herbicide/MOA choices will be critical to the design of effective prevention strategies. Because resistance to glyphosate in weeds is typically of a low magnitude, using a high-dose strategy should minimize the potential for the selection of resistance and thus help to make the use of glyphosate sustainable. Maximizing weed control is the key to successful agronomic practice with limited weed resistance. Nomenclature: Glyphosate, paraquat, glufosinate, triazine, atrazine, chloracetamide, bipyridiliums.

Evolution of a Double Amino Acid Substitution in the 5-Enolpyruvylshikimate-3-Phosphate Synthase in Eleusine indica Conferring High-Level Glyphosate Resistance

Naturally evolved double mutants in 5-enolpyruvylshikimate-3-phosphate synthase mimic engineered glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase in crops and confer high-level glyphosate resistance. Glyphosate is the most important and widely used herbicide in world agriculture. Intensive glyphosate selection has resulted in the widespread evolution of glyphosate-resistant weed populations, threatening the sustainability of this valuable once-in-a-century agrochemical. Field-evolved glyphosate resistance due to known resistance mechanisms is generally low to modest. Here, working with a highly glyphosate-resistant Eleusine indica population, we identified a double amino acid substitution (T102I + P106S [TIPS]) in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene in glyphosate-resistant individuals. This TIPS mutation recreates the biotechnology-engineered commercial first generation glyphosate-tolerant EPSPS in corn (Zea mays) and now in other crops. In E. indica, the naturally evolved TIPS mutants are highly (more than 180-fold) resistant to glyphosate compared with the wild type and more resistant (more than 32-fold) than the previously known P106S mutants. The E. indica TIPS EPSPS showed very high-level (2,647-fold) in vitro resistance to glyphosate relative to the wild type and is more resistant (600-fold) than the P106S variant. The evolution of the TIPS mutation in crop fields under glyphosate selection is likely a sequential event, with the P106S mutation being selected first and fixed, followed by the T102I mutation to create the highly resistant TIPS EPSPS. The sequential evolution of the TIPS mutation endowing high-level glyphosate resistance is an important mechanism by which plants adapt to intense herbicide selection and a dramatic example of evolution in action.

Droplet size affects glyphosate retention, absorption, and translocation in corn

Abstract The effect of droplet size on retention, absorption, and translocation of 14C-glyphosate was studied in glyphosate-resistant corn. Fine, medium, and coarse spray droplets were studied using a track-sprayer equipped with commercially available nozzles. Glyphosate-resistant corn was used to obtain measurements at field use rates in the absence of phytotoxicity. Spray retention on corn leaves was calculated based on recovered glyphosate per leaf area, and retention was higher with application of fine droplets (47%) than with application of coarse (38%) and medium (37%) droplets. Absorption in corn leaves was directly correlated with droplet size and reached a plateau 1 d after treatment (DAT) for all droplet sizes. Based on glyphosate recovered 3 DAT, coarse droplets showed the highest absorption (49%), followed by medium (35%) and fine (30%) droplets. Percentage of translocation also increased with droplet size, and translocation was primarily toward strong sink tissues such as roots and young leaves. Our results show that large droplets have slightly reduced retention in corn but have increased absorption resulting in increased translocation of glyphosate to growing sink tissues. Nomenclature: Glyphosate; glyphosate-resistant corn, Zea mays L. ‘Roundup® Ready’.

De Novo Genome Assembly of the Economically Important Weed Horseweed Using Integrated Data from Multiple Sequencing Platforms

De novo genome assembly and genomic resources of horseweed will be useful to understand the genetic and molecular bases of weediness. Horseweed (Conyza canadensis), a member of the Compositae (Asteraceae) family, was the first broadleaf weed to evolve resistance to glyphosate. Horseweed, one of the most problematic weeds in the world, is a true diploid (2n = 2x = 18), with the smallest genome of any known agricultural weed (335 Mb). Thus, it is an appropriate candidate to help us understand the genetic and genomic bases of weediness. We undertook a draft de novo genome assembly of horseweed by combining data from multiple sequencing platforms (454 GS-FLX, Illumina HiSeq 2000, and PacBio RS) using various libraries with different insertion sizes (approximately 350 bp, 600 bp, 3 kb, and 10 kb) of a Tennessee-accessed, glyphosate-resistant horseweed biotype. From 116.3 Gb (approximately 350× coverage) of data, the genome was assembled into 13,966 scaffolds with 50% of the assembly = 33,561 bp. The assembly covered 92.3% of the genome, including the complete chloroplast genome (approximately 153 kb) and a nearly complete mitochondrial genome (approximately 450 kb in 120 scaffolds). The nuclear genome is composed of 44,592 protein-coding genes. Genome resequencing of seven additional horseweed biotypes was performed. These sequence data were assembled and used to analyze genome variation. Simple sequence repeat and single-nucleotide polymorphisms were surveyed. Genomic patterns were detected that associated with glyphosate-resistant or -susceptible biotypes. The draft genome will be useful to better understand weediness and the evolution of herbicide resistance and to devise new management strategies. The genome will also be useful as another reference genome in the Compositae. To our knowledge, this article represents the first published draft genome of an agricultural weed.

Functional Genomics Analysis of Horseweed (Conyza canadensis) with Special Reference to the Evolution of Non-Target-Site Glyphosate Resistance

Abstract The evolution of glyphosate resistance in weedy species places an environmentally benign herbicide in peril. The first report of a dicot plant with evolved glyphosate resistance was horseweed, which occurred in 2001. Since then, several species have evolved glyphosate resistance and genomic information about nontarget resistance mechanisms in any of them ranges from none to little. Here, we report a study combining iGentifier transcriptome analysis, cDNA sequencing, and a heterologous microarray analysis to explore potential molecular and transcriptomic mechanisms of nontarget glyphosate resistance of horseweed. The results indicate that similar molecular mechanisms might exist for nontarget herbicide resistance across multiple resistant plants from different locations, even though resistance among these resistant plants likely evolved independently and available evidence suggests resistance has evolved at least four separate times. In addition, both the microarray and sequence analyses identified non–target-site resistance candidate genes for follow-on functional genomics analysis. Nomenclature: Glyphosate, horseweed, Conyza canadensis (L.) Cronq. ERICA