Mechanism of clomazone resistance in Leptochloa fusca spp. fasicularis to clomazone.

Bearded sprangletop (Leptochloa fusca spp. fasicularis) is a problematic weed in California rice (Oryza sativa) production. Historically, bearded sprangletop is controlled with clomazone in California rice fields. The continuous use of clomazone and lack of crop rotation in rice fields resulted in resistance to clomazone in several bearded sprangletop. The objective of this research was to determine the clomazone mechanism of resistance of two bearded sprangletop populations in California rice by investigating clomazone absorption, translocation, and metabolism under controlled environmental conditions in two resistant (CRBS1 and CRBS2) and one susceptible (S) populations. Absorption and translocation of 14C-clomazone were similar in R and S. Clomazone metabolism, as determined by inhibition of cytochrome P450 enzymes with malathion and determining clomazone metabolites profile, was different between S and R 3 days after treatment. Bearded sprangletop pretreated with malathion was 2-fold more sensitive to clomazone than when treated with clomazone alone, suggesting that cytochrome P450-mediated clomazone metabolism might be involved in the mechanism of resistance. An HPLC-MS/MS analysis revealed differences in clomazone metabolism between R and S biotypes. Hydroxymethylclomazone was the most abundant metabolite found in R plants with three and five-fold more abundant in CRBS1 and CRBS2, respectively, when compared to S plants. 5-ketoclomazone, the known toxic metabolite of clomazone, accumulated 2-fold more in S plants than in R plants at 72 h after treatment. This research shows that clomazone is metabolized differently between R and S populations of bearded sprangletop and that P450 monooxidation is likely involved in the mechanism of resistance.

Chromosome-scale genome assembly of Poa trivialis and population genomics reveal widespread gene flow in a cool-season grass seed production system.

Poa trivialis (L.) is a cool-season grass species found in various environments worldwide. In addition to being a desired turfgrass species, it is a common weed of agricultural systems and natural areas. As a weed, it is an important contaminant of commercial cool-season grass seed lots, resulting in widespread gene flow facilitated by human activities and causing significant economic losses to farmers. To better understand and manage infestations, we assembled and annotated a haploid genome of P. trivialis and studied troublesome field populations from Oregon, the largest cool-season grass seed producing region in the United States. The genome assembly resulted in 1.35 Gb of DNA sequence distributed among seven chromosome-scale scaffolds, revealing a high content of transposable elements, conserved synteny with Poa annua, and a close relationship with other C3 grasses. A reduced-representation sequencing analysis of field populations revealed limited genetic diversity and suggested potential gene flow and human-assisted dispersal in the region. The genetic resources and insights into P. trivialis provided by this study will improve weed management strategies and enable the development of molecular detection tests for contaminated seed lots to limit seed-mediated gene flow. These resources should also be beneficial for turfgrass breeders seeking to improve desirable traits of commercial P. trivialis varieties and help to guide breeding efforts in other crops to enhance the resiliency of agricultural ecosystems under climate change. Significance Statement: The chromosome-scale assembly of Poa trivialis and population genomic analyses provide crucial insights into the gene flow of weedy populations in agricultural systems and contribute a valuable genomic resource for the plant science community.

Repeated evolution of herbicide resistance in Lolium multiflorum revealed by haplotype-resolved analysis of acetyl-CoA carboxylase.

Herbicide resistance in weeds is one of the greatest challenges in modern food production. The grass species Lolium multiflorum is an excellent model species to investigate evolution under similar selection pressure because populations have repeatedly evolved resistance to many herbicides, utilizing a multitude of mechanisms to neutralize herbicide damage. In this work, we investigated the gene that encodes acetyl-CoA carboxylase (ACCase), the target site of the most successful herbicide group available for grass weed control. We sampled L. multiflorum populations from agricultural fields with history of intense herbicide use, and studied their response to three ACCase-inhibiting herbicides. To elucidate the mechanisms of herbicide resistance and the genetic relationship among populations, we resolved the haplotypes of 97 resistant and susceptible individuals by sequencing ACCase amplicons using long-read DNA sequencing technologies. Our dose-response data indicated the existence of many, often unpredictable, resistance patterns to ACCase-inhibiting herbicides, where populations exhibited as much as 37-fold reduction in herbicide response. The majority of the populations exhibited resistance to all three herbicides studied. Phylogenetic and molecular genetic analyses revealed multiple evolutionary origins of resistance-endowing ACCase haplotypes, as well as widespread admixture in the region regardless of cropping system. The amplicons generated were diverse, with haplotypes exhibiting 26-110 polymorphisms. Polymorphisms included insertions and deletions 1-31 bp in length, none of which were associated with the resistance phenotype based on an association analysis. We also found evidence that some populations have multiple mechanisms of resistance. Our results highlight the astounding genetic diversity in L. multiflorum populations, and the potential for repeated evolution of herbicide resistance across the landscape that challenges weed management approaches and jeopardizes sustainable weed control practices. We provide an in-depth discussion of the evolutionary and practical implications of our results.

Multiple evolutionary origins of glyphosate resistance in Lolium multiflorum.

The multitude of herbicide resistance patterns that have evolved in different weed species is a remarkable example of the rapid adaptation to anthropogenic-driven disturbance. Recently, resistance to glyphosate was identified in multiple populations of Lolium multiflorum in Oregon. We used phenotypic approaches, as well as population genomic and gene expression analyses, to determine whether known mechanisms were responsible for glyphosate resistance and whether resistance phenotypes evolved independently in different populations, and to identify potential loci contributing to resistance. We found no evidence of genetic alterations or expression changes at known target and non-target-site resistance mechanisms of glyphosate. Population genomic analyses indicated that resistant populations tended to have largely distinct ancestry from one another, suggesting that glyphosate resistance did not spread among populations by gene flow. Rather, resistance appears to have evolved independently on different genetic backgrounds. We also detected potential loci associated with the resistance phenotype, some of which encode proteins with potential effects on herbicide metabolism. Our results suggest that Oregon populations of L. multiflorum evolved resistance to glyphosate due to a novel mechanism. Future studies that characterize the gene or genes involved in resistance will be necessary to confirm this conclusion.

Non-target-Site Resistance in Lolium spp. Globally: A Review.

The Lolium genus encompasses many species that colonize a variety of disturbed and non-disturbed environments. Lolium perenne L. spp. perenne, L. perenne L. spp. multiflorum, and L. rigidum are of particular interest to weed scientists because of their ability to thrive in agricultural and non-agricultural areas. Herbicides are the main tool to control these weeds; however, Lolium spp. populations have evolved multiple- and cross-resistance to at least 14 herbicide mechanisms of action in more than 21 countries, with reports of multiple herbicide resistance to at least seven mechanisms of action in a single population. In this review, we summarize what is currently known about non-target-site resistance in Lolium spp. to acetyl CoA carboxylase, acetohydroxyacid synthase, microtubule assembly, photosystem II, 5-enolpyruvylshikimate-3-phosphate synthase, glutamine synthetase, very-long chain fatty acids, and photosystem I inhibitors. We suggest research topics that need to be addressed, as well as strategies to further our knowledge and uncover the mechanisms of non-target-site resistance in Lolium spp.

Role of Glutamine Synthetase Isogenes and Herbicide Metabolism in the Mechanism of Resistance to Glufosinate in Lolium perenne L. spp. multiflorum Biotypes from Oregon.

Glufosinate-resistant Lolium perenne L. spp. multiflorum biotypes from Oregon exhibited resistance levels up to 2.8-fold the field rate. One resistant biotype (MG) had an amino acid substitution in glutamine synthetase 2 (GS2), whereas the other (OR) exhibited the wild-type genotype. We hypothesized that the amino acid substitution in GS2 is involved in the resistance mechanism in MG and that non-target site resistance mechanisms are present in OR. OR metabolized glufosinate faster than the other two biotypes, with >75% of the herbicide metabolized in comparison to 50% in MG and the susceptible biotype. A mutation in GS2 co-segregating with resistance in MG did not reduce the enzyme activity, with results further supported by our enzyme homology models. This research supports the conclusion that a metabolism mechanism of glufosinate resistance is present in OR and that glufosinate resistance in MG is not due to an altered target site.

Vacuolar Sequestration of Paraquat Is Involved in the Resistance Mechanism in Lolium perenne L. spp. multiflorum.

Lolium perenne L. spp. multiflorum (Lam.) Husnot (LOLMU) is a winter annual weed, common to row crops, orchards and roadsides. Glyphosate-resistant populations of LOLMU are widespread in California. In many situations, growers have switched to paraquat or other postemergence herbicides to manage glyphosate-resistant LOLMU populations. Recently, poor control of LOLMU with paraquat was reported in a prune orchard in California where paraquat has been used several times. We hypothesize that the low efficacy observed is due to the selection of a paraquat-resistant biotype of LOLMU. Greenhouse dose-response experiments conducted with a susceptible (S) and the putative paraquat-resistant biotype (PRHC) confirmed paraquat resistance in PRHC. Herbicide absorption studies indicated that paraquat is absorbed faster in S than PRHC, although the maximum absorption estimates were similar for the two biotypes. Conversely, translocation of 14C-paraquat under light-manipulated conditions was restricted to the treated leaf of PRHC, whereas herbicide translocation out of the treated leaf was nearly 20 times greater in S. To determine whether paraquat was active within the plant cells, the photosynthetic performance was assessed after paraquat application using the parameter maximum quantum yield of photosystem II (Fv/Fm). Paraquat reaches the chloroplasts of PRHC, since there was a transitory inhibition of photosynthetic activity in PRHC leaves. However, PRHC Fv/Fm recovered to initial levels by 48 h after paraquat treatment. No paraquat metabolites were found, indicating that resistance is not due to paraquat degradation. LOLMU leaf segments were exposed to paraquat following pretreatments with inhibitors of plasma membrane- and tonoplast-localized transporter systems to selectively block paraquat intracellular movement. Subsequent evaluation of membrane integrity indicated that pre-exposure to putrescine resulted in the resistant biotype responding to paraquat similarly to S. These results strongly indicate that vacuolar sequestration is involved in the resistance to paraquat in this population of LOLMU.