Investigating the origins and evolution of a glyphosate-resistant weed invasion in South America.

The global invasion, and subsequent spread and evolution of weeds provides unique opportunities to address fundamental questions in evolutionary and invasion ecology. Amaranthus palmeri is a widespread glyphosate-resistant (GR) weed in the USA. Since 2015, GR populations of A. palmeri have been confirmed in South America, raising questions about introduction pathways and the importance of pre- vs. post-invasion evolution of GR traits. We used RAD-sequencing genotyping to characterize genetic structure of populations from Brazil, Argentina, Uruguay and the USA. We also quantified gene copy number of the glyphosate target, 5-enolpyruvyl-3-shikimate phosphate synthase (EPSPS), and the presence of an extrachromosomal circular DNA (eccDNA) replicon known to confer glyphosate resistance in USA populations. Populations in Brazil, Argentina and Uruguay were only weakly differentiated (pairwise FST  ≤0.043) in comparison to USA populations (mean pairwise FST  =0.161, range =0.068-0.258), suggesting a single major invasion event. However, elevated EPSPS copy number and the EPSPS replicon were identified in all populations from Brazil and Uruguay, but only in a single Argentinean population. These observations are consistent with independent in situ evolution of glyphosate resistance in Argentina, followed by some limited recent migration of the eccDNA-based mechanism from Brazil to Argentina. Taken together, our results are consistent with an initial introduction of A. palmeri into South America sometime before the 1980s, and local evolution of GR in Argentina, followed by a secondary invasion of GR A. palmeri with the unique eccDNA-based mechanism from the USA into Brazil and Uruguay during the 2010s.

Negative cross-resistance to clomazone in imazethapyr-resistant Echinochloa crus-galli caused by increased metabolization.

Herbicide resistance is frequently reported in E. crus-galli globally with target and non-target site resistance mechanism to acetolactate synthase (ALS)-inhibiting herbicides. However, resistance to certain herbicides can result in increased sensitivity to other herbicides, a phenomenon called negative cross-resistance. The objective of this study is to identify the occurrence of negative cross-resistance (NCR) to the pro-herbicide clomazone in populations of E. crus-galli resistant to ALS inhibitors due to increased metabolization. Clomazone dose-response curves, with and without malathion, were performed in imazethapyr-resistant and -susceptible E. crus-galli biotypes. CYPs genes expression and antioxidant enzymes activity were also evaluated. The effective dose to reduce 50% (ED50) of dry shoot weight obtained in the clomazone dose-response curves of the metabolic based imazethapyr-resistant and -susceptible biotypes groups were 22.712 and 58.745 g ha-1, respectively, resulting in a resistance factor (RF) of 0.37, indicating the occurrence of NCR. The application of malathion prior to clomazone increased the resistance factor from 0.60 to 1.05, which indicate the reversion of the NCR. Some CYP genes evaluated were expressed in a higher level, ranging from 2.6-9.1 times according to the biotype and the gene, in the imazethapyr-resistant than in -susceptible biotypes following clomazone application. Antioxidant enzyme activity was not associated with NCR. This study is the first report of NCR directly related to the mechanism of resistance increased metabolization in plants. The occurrence of NCR to clomazone in E. crus-galli can help delay the evolution of herbicide resistance.

Insights into the Role of Transcriptional Gene Silencing in Response to Herbicide-Treatments in Arabidopsis thaliana.

Herbicide resistance is broadly recognized as the adaptive evolution of weed populations to the intense selection pressure imposed by the herbicide applications. Here, we tested whether transcriptional gene silencing (TGS) and RNA-directed DNA Methylation (RdDM) pathways modulate resistance to commonly applied herbicides. Using Arabidopsis thaliana wild-type plants exposed to sublethal doses of glyphosate, imazethapyr, and 2,4-D, we found a partial loss of TGS and increased susceptibility to herbicides in six out of 11 tested TGS/RdDM mutants. Mutation in REPRESSOR OF SILENCING 1 (ROS1), that plays an important role in DNA demethylation, leading to strongly increased susceptibility to all applied herbicides, and imazethapyr in particular. Transcriptomic analysis of the imazethapyr-treated wild type and ros1 plants revealed a relation of the herbicide upregulated genes to chemical stimulus, secondary metabolism, stress condition, flavonoid biosynthesis, and epigenetic processes. Hypersensitivity to imazethapyr of the flavonoid biosynthesis component TRANSPARENT TESTA 4 (TT4) mutant plants strongly suggests that ROS1-dependent accumulation of flavonoids is an important mechanism for herbicide stress response in A. thaliana. In summary, our study shows that herbicide treatment affects transcriptional gene silencing pathways and that misregulation of these pathways makes Arabidopsis plants more sensitive to herbicide treatment.

Ecologically sustainable weed management: How do we get from proof-of-concept to adoption?

Weed management is a critically important activity on both agricultural and non-agricultural lands, but it is faced with a daunting set of challenges: environmental damage caused by control practices, weed resistance to herbicides, accelerated rates of weed dispersal through global trade, and greater weed impacts due to changes in climate and land use. Broad-scale use of new approaches is needed if weed management is to be successful in the coming era. We examine three approaches likely to prove useful for addressing current and future challenges from weeds: diversifying weed management strategies with multiple complementary tactics, developing crop genotypes for enhanced weed suppression, and tailoring management strategies to better accommodate variability in weed spatial distributions. In all three cases, proof-of-concept has long been demonstrated and considerable scientific innovations have been made, but uptake by farmers and land managers has been extremely limited. Impediments to employing these and other ecologically based approaches include inadequate or inappropriate government policy instruments, a lack of market mechanisms, and a paucity of social infrastructure with which to influence learning, decision-making, and actions by farmers and land managers. We offer examples of how these impediments are being addressed in different parts of the world, but note that there is no clear formula for determining which sets of policies, market mechanisms, and educational activities will be effective in various locations. Implementing new approaches for weed management will require multidisciplinary teams comprised of scientists, engineers, economists, sociologists, educators, farmers, land managers, industry personnel, policy makers, and others willing to focus on weeds within whole farming systems and land management units.

Current status of community resources and priorities for weed genomics research.

Weeds are attractive models for basic and applied research due to their impacts on agricultural systems and capacity to swiftly adapt in response to anthropogenic selection pressures. Currently, a lack of genomic information precludes research to elucidate the genetic basis of rapid adaptation for important traits like herbicide resistance and stress tolerance and the effect of evolutionary mechanisms on wild populations. The International Weed Genomics Consortium is a collaborative group of scientists focused on developing genomic resources to impact research into sustainable, effective weed control methods and to provide insights about stress tolerance and adaptation to assist crop breeding.

Weed genomics: yielding insights into the genetics of weedy traits for crop improvement.

Weeds cause tremendous economic and ecological damage worldwide. The number of genomes established for weed species has sharply increased during the recent decade, with some 26 weed species having been sequenced and de novo genomes assembled. These genomes range from 270 Mb (Barbarea vulgaris) to almost 4.4 Gb (Aegilops tauschii). Importantly, chromosome-level assemblies are now available for 17 of these 26 species, and genomic investigations on weed populations have been conducted in at least 12 species. The resulting genomic data have greatly facilitated studies of weed management and biology, especially origin and evolution. Available weed genomes have indeed revealed valuable weed-derived genetic materials for crop improvement. In this review, we summarize the recent progress made in weed genomics and provide a perspective for further exploitation in this emerging field.

Recurrent Selection of Echinochloa crus-galli with a Herbicide Mixture Reduces Progeny Sensitivity.

Herbicide mixtures are used to increase the spectrum of weed control and to manage weeds with target-site resistance to some herbicides. However, the effect of mixtures on the evolution of herbicide resistance caused by enhanced metabolism is unknown. This study evaluated the effect of a fenoxaprop-p-ethyl and imazethapyr mixture on the evolution of herbicide resistance in Echinochloa crus-galli using recurrent selection at sublethal doses. The progeny from second generations selected with the mixture had lower control than parental plants or the unselected progeny. GR50 increased 1.6- and 2.6-fold after two selection cycles with the mixture in susceptible (POP1-S) and imazethapyr-resistant (POP2-IR) biotypes, respectively. There was evidence that recurrent selection with this sublethal mixture had the potential to evolve cross-resistance to diclofop, cyhalofop, sethoxydim, and quinclorac. Mixture selection did not cause increased relative expression for a set of analyzed genes (CYP71AK2, CYP72A122, CYP72A258, CYP81A12, CYP81A14, CYP81A21, CYP81A22, and GST1). Fenoxaprop, rather than imazethapyr, is the main contributor to the decreased control in the progenies after recurrent selection with the mixture in low doses. This is the first study reporting the effect of a herbicide mixture at low doses on herbicide resistance evolution. The lack of control using the mixture may result in decreased herbicide sensitivity of the weed progenies. Using mixtures may select important detoxifying genes that have the potential to metabolize herbicides in patterns that cannot currently be predicted. The use of fully recommended herbicide rates in herbicide mixtures is recommended to reduce the risk of this type of resistance evolution.

The safener isoxadifen-ethyl confers fenoxaprop-p-ethyl resistance on a biotype of Echinochloa crus-galli.

BACKGROUND: Some herbicides are commercially formulated with safeners to increase crop selectivity. Fenoxaprop-p-ethyl is formulated with the safener isoxadifen-ethyl for Echinochloa crus-galli control in rice. Safeners act on crops by increasing herbicide metabolism, but this effect may also occur in weeds. The objective of this study was to investigate the effect of the safener isoxadifen-ethyl on the resistance to fenoxaprop-p-ethyl in a biotype of E. crus-galli. RESULTS: A screening of 52 biotypes identified lack of control in the biotype SANTPAT-R treated with the recommended dose of 69 g ha-1 of the commercial formulation of fenoxaprop-p-ethyl with the safener isoxadifen-ethyl. While this biotype survived doses greater than 2208 g ha-1 of the formulation fenoxaprop-p-ethyl + isoxadifen-ethyl, it was killed with 69 g ha-1 of fenoxaprop-p-ethyl without the safener. A glutathione-s-transferase (GST) enzymes inhibitor reduced the resistance factor in two dose-response curves. A minor effect of a CytP450 inhibitor was observed. The previous spraying of the safener isoxadifen-ethyl followed by fenoxaprop-p-ethyl induced survival in the resistant but not in the susceptible biotype. The GST1 and GSTF1 genes were up-regulated in the resistant biotype. ACCase gene mutations were not found, and no cross-resistance to other ACCase inhibitors was identified. CONCLUSION: The safener isoxadifen-ethyl present in the commercial herbicide formulation of fenoxaprop-p-ethyl is associated with resistance in the E. crus-galli SANTPAT-R biotype. This resistance is related with herbicide metabolization mediated by GST pathways. This is the first field-selected weed biotype with herbicide resistance due to safener presence in the sprayed formulation. © 2022 Society of Chemical Industry.

Genomic insights into the evolution of Echinochloa species as weed and orphan crop.

As one of the great survivors of the plant kingdom, barnyard grasses (Echinochloa spp.) are the most noxious and common weeds in paddy ecosystems. Meanwhile, at least two Echinochloa species have been domesticated and cultivated as millets. In order to better understand the genomic forces driving the evolution of Echinochloa species toward weed and crop characteristics, we assemble genomes of three Echinochloa species (allohexaploid E. crus-galli and E. colona, and allotetraploid E. oryzicola) and re-sequence 737 accessions of barnyard grasses and millets from 16 rice-producing countries. Phylogenomic and comparative genomic analyses reveal the complex and reticulate evolution in the speciation of Echinochloa polyploids and provide evidence of constrained disease-related gene copy numbers in Echinochloa. A population-level investigation uncovers deep population differentiation for local adaptation, multiple target-site herbicide resistance mutations of barnyard grasses, and limited domestication of barnyard millets. Our results provide genomic insights into the dual roles of Echinochloa species as weeds and crops as well as essential resources for studying plant polyploidization, adaptation, precision weed control and millet improvements.

CRISPR/Cas9-Mediated Multi-Allelic Gene Targeting in Sugarcane Confers Herbicide Tolerance.

Sugarcane is the source of 80% of the sugar and 26% of the bioethanol produced globally. However, its complex, highly polyploid genome (2n = 100 - 120) impedes crop improvement. Here, we report efficient and reproducible gene targeting (GT) in sugarcane, enabling precise co-editing of multiple alleles via template-mediated and homology-directed repair (HDR) of DNA double strand breaks induced by the programmable nuclease CRISPR/Cas9. The evaluation of 146 independently transformed plants from five independent experiments revealed a targeted nucleotide replacement that resulted in both targeted amino acid substitutions W574L and S653I in the acetolactate synthase (ALS) in 11 lines in addition to single, targeted amino acid substitutions W574L or S653I in 25 or 18 lines, respectively. Co-editing of up to three ALS copies/alleles that confer herbicide tolerance was confirmed by Sanger sequencing of cloned long polymerase chain reaction (PCR) amplicons. This work will enable crop improvement by conversion of inferior alleles to superior alleles through targeted nucleotide substitutions.

The safener isoxadifen does not increase herbicide resistance evolution in recurrent selection with fenoxaprop.

Safeners are chemical compounds used to improve selectivity and safety of herbicides in crops by activating genes that enhance herbicide metabolic detoxification. The genes activated by safeners in crops are similar to the genes causing herbicide resistance through increased metabolism in weeds. This work investigated the effect of the safener isoxadifen-ethyl (IS) in combination with fenoxaprop-p-ethyl (FE) on the evolution of herbicide resistance in Echinochloa crus-galli under recurrent selection. Reduced susceptibility was observed in the progeny after recurrent selection with both FE alone and with FE + IS for two generations (G2) compared to the parental population (G0). The resistance index found in G2 after FE + IS selection was similar as when FE was used alone, demonstrating that the safener did not increase the rate or magnitude of herbicide resistance evolution. G2 progeny selected with FE alone and the combination of FE + IS had increased survival to herbicides from other mechanisms of action relative to the parental G0 population. One biotype of G2 progeny had increased constitutive expression of glutathione-S-transferase (GST1) after recurrent selection with FE + IS. G2 progeny had increased expression of two P450 genes (CYP71AK2 and CYP72A122) following treatment with FE, while G2 progeny had increased expression of five P450 genes (CYP71AK2, CYP72A258, CYP81A12, CYP81A14 and CYP81A21) after treatment with FE + IS. Repeated selection with low doses of FE with or without the safener IS decreased E. crus-galli control and showed potential for cross-resistance evolution. Addition of safener did not further decrease herbicide sensitivity in second generation progeny; however, the recurrent use of safener in combination with FE resulted in safener-induced increased expression of several CYP genes. This is the first report using safener as an additional factor to study herbicide resistance evolution in weeds under experimental recurrent selection.

Target-Site Mutations and Expression of ALS Gene Copies Vary According to Echinochloa Species.

The sustainability of rice cropping systems is jeopardized by the large number and variety of populations of polyploid Echinochloa spp. resistant to ALS inhibitors. Better knowledge of the Echinochloa species present in Italian rice fields and the study of ALS genes involved in target-site resistance could significantly contribute to a better understanding of resistance evolution and management. Using a CAPS-rbcL molecular marker, two species, E. crus-galli (L.) P. Beauv. and E. oryzicola (Vasinger) Vasing., were identified as the most common species in rice in Italy. Mutations involved in ALS inhibitor resistance in the different species were identified and associated with the ALS homoeologs. The relative expression of the ALS gene copies was evaluated. Molecular characterization led to the identification of three ALS genes in E. crus-galli and two in E. oryzicola. The two species also carried different point mutations conferring resistance: Ala122Asn in E. crus-galli and Trp574Leu in E. oryzicola. Mutations were carried in the same gene copy (ALS1), which was significantly more expressed than the other copies (ALS2 and ALS3) in both species. These results explain the high resistance level of these populations and why mutations in the other ALS copies are not involved in herbicide resistance.

Snorkeling Strategy: Tolerance to Flooding in Rice and Potential Application for Weed Management.

Flooding is an important strategy for weed control in paddy rice fields. However, terrestrial weeds had evolved mechanisms of tolerance to flooding, resulting in new 'snorkeling' ecotypes. The aim of this review is to discuss the mechanisms of flooding tolerance in cultivated and weedy rice at different plant stages and the putative utility of this trait for weed management. Knowledge about flooding tolerance is derived primarily from crop models, mainly rice. The rice model informs us about the possible flooding tolerance mechanisms in weedy rice, Echinochloa species, and other weeds. During germination, the gene related to carbohydrate mobilization and energy intake (RAmy3D), and genes involved in metabolism maintenance under anoxia (ADH, PDC, and OsB12D1) are the most important for flooding tolerance. Flooding tolerance during emergence involved responses promoted by ethylene and induction of RAmy3D, ADH, PDC, and OsB12D1. Plant species tolerant to complete submersion also employ escape strategies or the ability to become quiescent during the submergence period. In weedy rice, the expression of PDC1, SUS3, and SUB1 genes is not directly related to flooding tolerance, contrary to what was learned in cultivated rice. Mitigation of flooding tolerance in weeds could be achieved with biotechnological approaches and genetic manipulation of flood tolerance genes through RNAi and transposons, providing a potential new tool for weed management.

Diverse genetic mechanisms underlie worldwide convergent rice feralization.

BACKGROUND: Worldwide feralization of crop species into agricultural weeds threatens global food security. Weedy rice is a feral form of rice that infests paddies worldwide and aggressively outcompetes cultivated varieties. Despite increasing attention in recent years, a comprehensive understanding of the origins of weedy crop relatives and how a universal feralization process acts at the genomic and molecular level to allow the rapid adaptation to weediness are still yet to be explored. RESULTS: We use whole-genome sequencing to examine the origin and adaptation of 524 global weedy rice samples representing all major regions of rice cultivation. Weed populations have evolved multiple times from cultivated rice, and a strikingly high proportion of contemporary Asian weed strains can be traced to a few Green Revolution cultivars that were widely grown in the late twentieth century. Latin American weedy rice stands out in having originated through extensive hybridization. Selection scans indicate that most genomic regions underlying weedy adaptations do not overlap with domestication targets of selection, suggesting that feralization occurs largely through changes at loci unrelated to domestication. CONCLUSIONS: This is the first investigation to provide detailed genomic characterizations of weedy rice on a global scale, and the results reveal diverse genetic mechanisms underlying worldwide convergent rice feralization.

Epigenetic regulation - contribution to herbicide resistance in weeds?

Continuous use of herbicides has resulted in the evolution of resistance to all major herbicide modes of action worldwide. Besides the well-documented cases of newly acquired resistance through genetic changes, epigenetic regulation may also contribute to herbicide resistance in weeds. Epigenetics involves processes that modify the expression of specific genetic elements without changes in the DNA sequence, and play an important role in re-programming gene expression. Epigenetic modifications can be induced spontaneously, genetically or environmentally. Stress-induced epigenetic changes are normally reverted soon after stress exposure, although in specific cases they can also be carried over multiple generations, thereby having a selective benefit. Here, we provide an overview of the basis of epigenetic regulation in plants and discuss the possible effect of epigenetic changes on herbicide resistance. The understanding of these epigenetic changes would add a new perspective to our knowledge of environmental and management stresses and their effects on the evolution of herbicide resistance in weeds. © 2017 Society of Chemical Industry.

The power and potential of genomics in weed biology and management.

There have been previous calls for, and efforts focused on, realizing the power and potential of weed genomics for better understanding of weeds. Sustained advances in genome sequencing and assembly technologies now make it possible for individual research groups to generate reference genomes for multiple weed species at reasonable costs. Here, we present the outcomes from several meetings, discussions, and workshops focused on establishing an International Weed Genomics Consortium (IWGC) for a coordinated international effort in weed genomics. We review the 'state of the art' in genomics and weed genomics, including technologies, applications, and on-going weed genome projects. We also report the outcomes from a workshop and a global survey of the weed science community to identify priority species, key biological questions, and weed management applications that can be addressed through greater availability of, and access to, genomic resources. Major focus areas include the evolution of herbicide resistance and weedy traits, the development of molecular diagnostics, and the identification of novel targets and approaches for weed management. There is increasing interest in, and need for, weed genomics, and the establishment of the IWGC will provide the necessary global platform for communication and coordination of weed genomics research. © 2018 Society of Chemical Industry.

Recurrent evolution of heat-responsiveness in Brassicaceae COPIA elements.

BACKGROUND: The mobilization of transposable elements (TEs) is suppressed by host genome defense mechanisms. Recent studies showed that the cis-regulatory region of Arabidopsis thaliana COPIA78/ONSEN retrotransposons contains heat-responsive elements (HREs), which cause their activation during heat stress. However, it remains unknown whether this is a common and potentially conserved trait and how it has evolved. RESULTS: We show that ONSEN, COPIA37, TERESTRA, and ROMANIAT5 are the major families of heat-responsive TEs in A. lyrata and A. thaliana. Heat-responsiveness of COPIA families is correlated with the presence of putative high affinity heat shock factor binding HREs within their long terminal repeats in seven Brassicaceae species. The strong HRE of ONSEN is conserved over millions of years and has evolved by duplication of a proto-HRE sequence, which was already present early in the evolution of the Brassicaceae. However, HREs of most families are species-specific, and in Boechera stricta, the ONSEN HRE accumulated mutations and lost heat-responsiveness. CONCLUSIONS: Gain of HREs does not always provide an ultimate selective advantage for TEs, but may increase the probability of their long-term survival during the co-evolution of hosts and genomic parasites.

Evolutionary and social consequences of introgression of nontransgenic herbicide resistance from rice to weedy rice in Brazil.

Several studies have expressed concerns about the effects of gene flow from transgenic herbicide-resistant crops to their wild relatives, but no major problems have been observed. This review describes a case study in which what has been feared in transgenics regarding gene flow has actually changed biodiversity and people's lives. Nontransgenic imidazolinone-resistant rice (IMI-rice) cultivars increased the rice grain yield by 50% in southern Brazil. This increase was beneficial for life quality of the farmers and also improved the regional economy. However, weedy rice resistant to imidazolinone herbicides started to evolve three years after the first use of IMI-rice cultivars. Population genetic studies indicate that the herbicide-resistant weedy rice was mainly originated from gene flow from resistant cultivars and distributed by seed migration. The problems related with herbicide-resistant weedy rice increased the production costs of rice that forced farmers to sell or rent their land. Gene flow from cultivated rice to weedy rice has proven to be a large agricultural, economic, and social constraint in the use of herbicide-resistant technologies in rice. This problem must be taken into account for the development of new transgenic or nontransgenic rice technologies.