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.

Evolution of ACCase-inhibitor resistance in Chloris virgata is conferred by a Trp2027Cys mutation in the herbicide target site.

BACKGROUND: Chloris virgata is a troublesome weed in tropical regions. With the evolution of glyphosate resistance in key grass species, acetyl CoA carboxylase (ACCase) inhibitors have become a commonly used tool in soybean production areas in Brazil. We assessed if suspected resistant populations exhibited cross resistance to the different classes of ACCase inhibitors and investigated the resistance mechanisms in C. virgata. RESULTS: Dose-response experiments revealed resistance to haloxyfop-methyl and pinoxaden, with 432- and 3-fold resistance, respectively, compared to susceptible populations. Due to the lack of genetic resources for C. virgata, we sequenced, assembled, and annotated the genome using short-read Illumina technology. The k-mer analysis estimated a genome size of approximately 336 Mbp, with BUSCO completeness of 97%, and over 36 000 gene models were annotated. We examined if ACCase copy number variation and increased gene expression were involved in the resistance phenotype and found no difference when compared to a susceptible population. A mutation was detected in ACCase that encodes for amino acid position 2027, resulting in a tryptophan-to-cysteine (Trp2027Cys) substitution. We found the resistant population absorbed 11.4% less herbicide and retained 21% more herbicide on the treated leaf compared to the susceptible population. We developed a genotyping assay targeting the resistance-endowing Trp2027Cys substitution for quick resistance diagnosis. CONCLUSION: A Trp2027Cys amino acid substitution in ACCase confers resistance to haloxyfop and pinoxaden in C. virgata. We provide important insights into the evolutionary history of C. virgata and a draft genome as a useful resource to further our understanding of the biology in the genus Chloris. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

First report of target-site resistance to ACCase-inhibiting herbicides in Bromus tectorum L.

BACKGROUND: The prevalent and repeated use of acetyl-coenzyme A carboxylase (ACCase)-inhibiting herbicides for Bromus tectorum L. control in fine fescue (Festuca L. spp) grown for seed has selected ACCase-resistant B. tectorum populations. The objectives of this study were to (1) evaluate the response of nine B. tectorum populations to the ACCase inhibitors clethodim, sethoxydim, fluazifop-P-butyl, and quizalofop-P-ethyl and the acetolactate synthase (ALS) inhibitor sulfosulfuron and (2) characterize the resistance mechanisms. RESULTS: Bromus tectorum populations were confirmed to be resistant to the ACCase-inhibiting herbicides tested. The levels of resistance varied among the populations for clethodim (resistance ratio, RR = 5.1-14.5), sethoxydim (RR = 18.7-44.7), fluazifop-P-butyl (RR = 3.1-40.3), and quizalofop-P-ethyl (RR = 14.5-36). Molecular investigations revealed that the mutations Ile2041Thr and Gly2096Ala were the molecular basis of resistance to the ACCase-inhibiting herbicides. The Gly2096Ala mutation resulted in cross-resistance to the aryloxyphenoxypropionate (APP) herbicides fluazifop-P-butyl and quizalofop-P-ethyl, and the cyclohexanedione (CHD) herbicides clethodim, and sethoxydim, whereas Ile2041Thr mutation resulted in resistance only to the two APP herbicides. All B. tectorum populations were susceptible to sulfosulfuron (RR = 0.3-1.7). CONCLUSIONS: This is the first report of target-site mutations conferring resistance to ACCase-inhibiting herbicides in B. tectorum. The results of this study suggest multiple evolutionary origins of resistance and contribute to understanding the patterns of cross-resistance to ACCase inhibitors associated with different mutations in B. tectorum. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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.

Population genomics of Digitaria insularis from soybean areas in Brazil.

BACKGROUND: Digitaria insularis is a weed species that has gained considerable importance in Brazil's soybean production areas that rely on glyphosate-resistant cultivars. Herbicide-resistant weed populations of this species have been reported in many regions in Brazil, first in the south, followed by later reports in the north. We hypothesized that the spread of herbicide-resistant D. insularis is facilitated by movement of agricultural machinery from the southern regions of Brazil. RESULTS: Population genomics revealed a weak or no genetic structure (FST  = [0; 0.16]), moderate expected heterozygosity (HE  = 0.15; 0.44) and low inbreeding (FIS  = [-0.1; 0.1]) in D. insularis populations. Our data supported the hypothesis that herbicide resistance gene flow predominantly occurred in a south-to-north direction based on a migration analysis. We also found evidence of local adaptation of resistant populations in the northern soybean-growing regions of Brazil. CONCLUSION: Evidence in our work suggests that gene flow of glyphosate-resistant D. insularis is associated with movement of agricultural machinery, although local selection pressure seems to play an important role in the evolution of herbicide resistance throughout the country. Our results suggest preventive practices such as equipment sanitation should be implemented to limit the spread of herbicide resistant D. insularis. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Review: evolutionary drivers of agricultural adaptation in Lolium spp.

The genus Lolium comprises many species, of which L. perenne ssp. multiflorum, L. perenne ssp. perenne, and L. rigidum are of worldwide agricultural importance as both pasture crops and as weeds. These three species are inter-fertile, obligate out-crossers with a self-incompatible reproduction system. This combination contributes to high genetic diversity that supplies new variants during expansion to new natural areas and agricultural environments. Human dispersal, de-domestication and crop-weed hybridization events between Lolium spp., or with others such as Festuca spp., are likely associated with their distinct weediness abilities. Furthermore, new introductions followed by introgression may hasten adaptation to new environments. Most Lolium-related weed science studies have focused on adaptation leading to herbicide resistance, but other forms of adaptation may also occur. In this review, we explore how the wide genetic variation among Lolium species and hybridization with other species may contribute to range expansion, and adaptation to both new agricultural practices and future predicted climate change scenarios. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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.

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.

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.

Increased temperatures and elevated CO2 levels reduce the sensitivity of Conyza canadensis and Chenopodium album to glyphosate.

Herbicides are the most commonly used means of controlling weeds. Recently, there has been growing concern over the potential impacts of global climate change, specifically, increasing temperatures and elevated carbon dioxide (CO2) concentrations, on the sensitivity of weeds to herbicides. Here, glyphosate response of both Conyza canadensis and Chenopodium album was evaluated under different environmental conditions. Reduced glyphosate sensitivity was observed in both species in response to increased temperature, elevated CO2 level, and the combination of both factors. Increased temperature had greater effect on plant survival than elevated CO2 level. In combination, high temperature and elevated CO2 level resulted in loss of apical dominance and rapid necrosis in glyphosate-treated plants. To investigate the mechanistic basis of reduced glyphosate sensitivity, translocation was examined using 14C-glyphosate. In plants that were subjected to high temperatures and elevated CO2 level, glyphosate was more rapidly translocated out of the treated leaf to shoot meristems and roots than in plants grown under control conditions. These results suggest that altered glyphosate translocation and tissue-specific sequestration may be the basis of reduced plant sensitivity. Therefore, overreliance on glyphosate for weed control under changing climatic conditions may result in more weed control failures.

EPSPS duplication and mutation involved in glyphosate resistance in the allotetraploid weed species Poa annua L.

BACKGROUND: Poa annua is a widespread winter annual weed species in California. Recently, poor control of this species with glyphosate was reported by growers in an almond orchard in California with a history of repetitive glyphosate use. The objectives of this research were to evaluate the level of glyphosate resistance in a developed S4 P. annua line (R) and identify the mechanisms of resistance involved. RESULTS: Whole-plant dose-response experiments confirmed glyphosate resistance in R, which required 18-fold more glyphosate to achieve a 50% growth reduction compared with a susceptible line (S), results that were supported by the lower shikimate accumulation observed in R compared with S. No differences in glyphosate absorption, translocation, or metabolism were observed, suggesting that non-target-site mechanisms of resistance are not involved in the resistance phenotype. A missense single nucleotide polymorphism was observed in EPSPS coding position 106 in R, resulting in a leucine to proline substitution. This polymorphism was observed exclusively in P. supina EPSPS homeologs. A seven-fold increase in the number of copies of EPSPS alleles was observed in R compared with S. CONCLUSIONS: We report the first case of glyphosate resistance associated with both EPSPS duplication and target-site mutation at position 106, leading to high levels of glyphosate resistance in the allotetraploid weed species Poa annua L. Data obtained in this research will be useful for the development of diagnostic tools for rapid glyphosate resistance identification, monitoring and containment. © 2018 Society of Chemical Industry.

Multiple target site resistance to glyphosate in junglerice (Echinochloa colona) lines from California orchards.

BACKGROUND: In California specialty cropping systems such as vineyards and orchards, Echinochloa colona is present as a summer annual weed. It is able to germinate throughout the growing season whenever favorable conditions are present, and management relies heavily on glyphosate applications. Glyphosate-resistant (GR) E. colona biotypes are present in the state, but the levels of resistance observed suggest that there may be differences in mechanisms of resistance among populations. RESULTS: Echinochloa colona lines collected from different regions of California's Central Valley presented resistance levels ranging from 1.4 to 4.3-fold compared to susceptible lines. No differences in the absorption and translocation of [14 C]-glyphosate were observed among lines. Resistant lines accumulated eight-fold less shikimic acid after treatment with 435 and 870 g a.e. ha-1 glyphosate compared to the most susceptible line. Sequencing of a region of the EPSPS gene revealed three single nucleotide changes leading to amino acid substitutions at Proline 106, including Pro106Leu, Pro106Thr and Pro106Ser. CONCLUSION: These results indicate that an altered target site in EPSPS is contributing to resistance in these lines and resistance has evolved independently, multiple times in the Central Valley of California. Additional research is needed to further understand the genomic contributions of resistance loci in this polyploid weed species. © 2018 Society of Chemical Industry.

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.

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. © 2015 Society of Chemical Industry.