Eric L. Patterson

Confirmation and mechanism of glyphosate resistance in tall windmill grass (Chloris elata) from Brazil.

BACKGROUND Overreliance on glyphosate as a single tool for weed management in agricultural systems in Brazil has selected glyphosate-resistant populations of tall windmill grass (Chloris elata Desv.). RESULTS Two C. elata populations, one glyphosate resistant (GR) and one glyphosate susceptible (GS), were studied in detail for a dose-response experiment and for resistance mechanism. The dose causing 50% reduction in dry weight was 620 g a.e. ha(-1) for GR and 114 g ha(-1) for GS, resulting in an R/S ratio of 5.4. GS had significantly higher maximum (14) C-glyphosate absorption into the treated leaf (51.3%) than GR (39.5%), a difference of 11.8% in maximum absorption. GR also retained more (14) C-glyphosate in the treated leaf (74%) than GS (51%), and GR translocated less glyphosate (27%) to other plant parts (stems, roots and root exudation) than GS (36%). There were no mutations at the Pro106 codon in the gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). There was no difference in EPSPS genomic copy number or EPSPS transcription between GS and GR populations. CONCLUSION Based on these data, reduced glyphosate absorption and increased glyphosate retention in the treated leaf contribute to glyphosate resistance in this C. elata population from Brazil.

Reversing resistance to tembotrione in an Amaranthus tuberculatus (var. rudis) population from Nebraska, USA with cytochrome P450 inhibitors

BACKGROUND A population of Amaranthus tuberculatus (var. rudis) was confirmed resistant to 4-hydroxyphenylpyruvate dioxygenase (HPPD)-inhibitor herbicides (mesotrione, tembotrione, and topramezone) in a seed corn/soybean rotation in Nebraska. Further investigation confirmed a non-target-site resistance mechanism in this population. The main objective of this study was to explore the role of cytochrome P450 inhibitors in restoring the efficacy of HPPD-inhibitor herbicides on the HPPD-inhibitor resistant A. tuberculatus population from Nebraska, USA (HPPD-R). RESULTS Enhanced metabolism via cytochrome P450 enzymes is the mechanism of resistance in HPPD-R. Amitrole partially restored the activity of mesotrione, whereas malathion, amitrole, and piperonyl butoxide restored the activity of tembotrione and topramezone in HPPD-R. Although corn was injured through malathion followed by mesotrione application a week after treatment, the injury was transient, and the crop recovered. CONCLUSION The use of cytochrome P450 inhibitors with tembotrione may provide a new way of controlling HPPD-inhibitor resistant A. tuberculatus, but further research is needed to identify the cytochrome P450 candidate gene(s) conferring metabolism-based resistance. The results presented here aid to gain an insight into non-target-site resistance weed management strategies.

Multiple Resistance to Glyphosate and Acetolactate Synthase Inhibitors in Palmer Amaranth (Amaranthus palmeri) Identified in Brazil

Palmer amaranth is native to the United States, but was discovered in 2015 in Brazil. Palmer amaranth populations in Brazil were very difficult to control using glyphosate, which resulted in many changes to standard weed management practices. A genotyping assay was used to confirm that the population detected in Mato Grosso State, Brazil, was correctly identified as Palmer amaranth and that it was not tall waterhemp. Greenhouse dose—response curves and shikimate accumulation assays showed that the Brazilian population was highly resistant to glyphosate, with an LD50 value (3,982 g glyphosate ha-1) more than twice the typical use rates and very little shikimate accumulation at 1 mM glyphosate concentrations in a leaf-disk assay. The Brazilian population was also resistant to sulfonylurea and imidazolinone acetolactate synthase (ALS) inhibitor herbicides. The resistance mechanisms in the Brazilian population were identified as increased EPSPS gene copy number for glyphosate resistance (between 50- and 179-fold relative EPSPS gene copy number increase) and two different alleles for target-site mutations in the ALS gene (W574L and S653N). These results confirm the introduction of Palmer amaranth to Brazil using a genetic marker for species identification, as well as resistance to glyphosate and ALS inhibitors. Nomenclature: Glyphosate; Palmer amaranth, Amaranthus palmeri S. Wats; tall waterhemp, Amaranthus tuberculatus (Moq.) Sauer.

EPSPS Gene Copy Number and Whole-Plant Glyphosate Resistance Level in Kochia scoparia

Glyphosate-resistant (GR) Kochia scoparia has evolved in dryland chemical fallow systems throughout North America and the mechanism of resistance involves 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene duplication. Agricultural fields in four states were surveyed for K. scoparia in 2013 and tested for glyphosate-resistance level and EPSPS gene copy number. Glyphosate resistance was confirmed in K. scoparia populations collected from sugarbeet fields in Colorado, Wyoming, and Nebraska, and Montana. Glyphosate resistance was also confirmed in K. scoparia accessions collected from wheat-fallow fields in Montana. All GR samples had increased EPSPS gene copy number, with median population values up to 11 from sugarbeet fields and up to 13 in Montana wheat-fallow fields. The results indicate that glyphosate susceptibility can be accurately diagnosed using EPSPS gene copy number.

Increased Chalcone Synthase (CHS) expression is associated with dicamba resistance in Kochia scoparia

BACKGROUND Resistance to the synthetic auxin herbicide dicamba is increasingly problematic in Kochia scoparia. The resistance mechanism in an inbred dicamba-resistant K. scoparia line (9425R) was investigated using physiological and transcriptomics (RNA-Seq) approaches. RESULTS No differences were found in dicamba absorption or metabolism between 9425R and a dicamba-susceptible line, but 9425R was found to have significantly reduced dicamba translocation. Known auxin-responsive genes ACC synthase (ACS) and indole-3-acetic acid amino synthetase (GH3) were transcriptionally induced following dicamba treatment in dicamba-susceptible K. scoparia but not in 9425R. Chalcone synthase (CHS), the gene regulating synthesis of the flavonols quertecin and kaemperfol, was found to have twofold higher transcription in 9425R both without and 12 h after dicamba treatment. Increased CHS transcription co-segregated with dicamba resistance in a forward genetics screen using an F2 population. CONCLUSION Prior work has shown that the flavonols quertecin and kaemperfol compete with auxin for intercellular movement and vascular loading via ATP-binding cassette subfamily B (ABCB) membrane transporters. The results of this study support a model in which constitutively increased CHS expression in the meristem produces more flavonols that would compete with dicamba for intercellular transport by ABCB transporters, resulting in reduced dicamba translocation.

Glyphosate Resistance and EPSPS Gene Duplication: Convergent Evolution in Multiple Plant Species

One of the increasingly widespread mechanisms of resistance to the herbicide glyphosate is copy number variation (CNV) of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene. EPSPS gene duplication has been reported in 8 weed species, ranging from 3 to 5 extra copies to more than 150 extra copies. In the case of Palmer amaranth (Amaranthus palmeri), a section of >300 kb containing EPSPS and many other genes has been replicated and inserted at new loci throughout the genome, resulting in significant increase in total genome size. The replicated sequence contains several classes of mobile genetic elements including helitrons, raising the intriguing possibility of extra-chromosomal replication of the EPSPS-containing sequence. In kochia (Kochia scoparia), from 3 to more than 10 extra EPSPS copies are arranged as a tandem gene duplication at one locus. In the remaining 6 weed species that exhibit EPSPS gene duplication, little is known about the underlying mechanisms of gene duplication or their entire sequence. There is mounting evidence that adaptive gene amplification is an important mode of evolution in the face of intense human-mediated selection pressure. The convergent evolution of CNVs for glyphosate resistance in weeds, through at least 2 different mechanisms, may be indicative of a more general importance for this mechanism of adaptation in plants. CNVs warrant further investigation across plant functional genomics for adaptation to biotic and abiotic stresses, particularly for adaptive evolution on rapid time scales.

A KASP Genotyping Method to Identify Northern Watermilfoil, Eurasian Watermilfoil, and Their Interspecific Hybrids

The invasive aquatic plant Eurasian watermilfoil (Myriophyllum spicatum L.) can hybridize with the related North American native species northern watermilfoil (M. sibiricum Kom.). Hybrid watermilfoil (M. spicatum × M. sibiricum) populations have higher fitness and reduced sensitivity to some commonly used aquatic herbicides, making management more difficult. There is growing concern that management practices using herbicides in lakes with mixed populations of watermilfoil species may further select for hybrid individuals due to the difference in herbicide sensitivity. Accurate and cost-effective identification of rare hybrid individuals within populations is therefore critical for herbicide management decisions. Here we describe KASP assays for three SNPs in the ITS region to genotype individuals from both parental watermilfoil species and their hybrid, using synthesized plasmids containing the respective sequences as positive controls. Using KASP we genotyped 16 individuals from one lake and 23 individuals from a second lake, giving a highly accurate picture of Myriophyllum species distribution dynamics. We identified one hybrid individual among 16 samples from one lake, a discovery rate of <10%. Discriminant analysis showed that while a single SNP was generally sufficient for genotyping an individual, using multiple SNPs increased the reliability of genotyping. In the future, the ability to genotype many samples will provide the ability to identify the presence of rare individuals, such as a less common parental species or the inter-specific hybrid. Lakes with complex species distribution dynamics, such as a low proportion of hybrids, are where herbicide application must be carefully chosen so as not to select for the more vigorous and less herbicide-sensitive hybrid individuals.

Population Genetic Structure in Glyphosate-Resistant and -Susceptible Palmer Amaranth (Amaranthus palmeri) Populations Using Genotyping-by-sequencing (GBS)

Palmer amaranth (Amaranthus palmeri) is a major weed in United States cotton and soybean production systems. Originally native to the Southwest, the species has spread throughout the country. In 2004 a population of A. palmeri was identified with resistance to glyphosate, a herbicide heavily relied on in modern no-tillage and transgenic glyphosate-resistant (GR) crop systems. This project aims to determine the degree of genetic relatedness among eight different populations of GR and glyphosate-susceptible (GS) A. palmeri from various geographic regions in the United States by analyzing patterns of phylogeography and diversity to ascertain whether resistance evolved independently or spread from outside to an Arizona locality (AZ-R). Shikimic acid accumulation and EPSPS genomic copy assays confirmed resistance or susceptibility. With a set of 1,351 single nucleotide polymorphisms (SNPs), discovered by genotyping-by-sequencing (GBS), UPGMA phylogenetic analysis, principal component analysis, Bayesian model-based clustering, and pairwise comparisons of genetic distances were conducted. A GR population from Tennessee and two GS populations from Georgia and Arizona were identified as genetically distinct while the remaining GS populations from Kansas, Arizona, and Nebraska clustered together with two GR populations from Arizona and Georgia. Within the latter group, AZ-R was most closely related to the GS populations from Kansas and Arizona followed by the GR population from Georgia. GR populations from Georgia and Tennessee were genetically distinct from each other. No isolation by distance was detected and A. palmeri was revealed to be a species with high genetic diversity. The data suggest the following two possible scenarios: either glyphosate resistance was introduced to the Arizona locality from the east, or resistance evolved independently in Arizona. Glyphosate resistance in the Georgia and Tennessee localities most likely evolved separately. Thus, modern farmers need to continue to diversify weed management practices and prevent seed dispersal to mitigate herbicide resistance evolution in A. palmeri.

Exploring the fate of mRNA in aging seeds: protection, destruction, or slow decay?

Aged soybean seeds show widespread cleavage of long mRNA transcripts with no particular relationship to transcript function, consistent with extant hypotheses of aging mechanisms.

Prediction of glyphosate resistance level based on EPSPS gene copy number in Kochia scoparia

Glyphosate-resistant (GR) Kochia scoparia has evolved in dryland chemical fallow systems throughout North America and the mechanism involves 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene duplication. Sugarbeet fields in four states were surveyed for K. scoparia in 2013 and tested for glyphosate-resistance level and EPSPS gene copy number. Glyphosate resistance was confirmed in K. scoparia populations collected from sugarbeet fields in Colorado, Wyoming, and Nebraska. The GR samples all had increased EPSPS gene copy number, with median population values up to 11. An empirical model was developed to estimate the level of glyphosate-resistance in K. scoparia based on EPSPS gene copy number. The results suggested that glyphosate susceptibility can be accurately diagnosed using EPSPS gene copy number, and further increases in EPSPS gene copy number could increase resistance levels up to 8-fold relative to susceptible K. scoparia. These trends suggest that continued glyphosate selection pressure is selecting for higher EPSPS copy number and higher resistance levels in K. scoparia. By including multiple K. scoparia samples lacking EPSPS gene duplication, our empirical model provides a more realistic estimate of fold-resistance due to EPSPS gene copy number compared to methods that do not account for normal variation of herbicide response in susceptible biotypes.