Mutant Acetolactate Synthase Research Articles

Novel mutations in acetolactate synthase confer high levels of resistance to tribenuron-methyl in Fagopyrum tataricum

Tartary buckwheat (Fagopyrum tataricum) field weeds are rich in species, with many weeds causing reduced quality, yield, and crop failure. The selection of herbicide-resistant Tartary buckwheat varieties, while applying low-toxicity and efficient herbicides as a complementary weed control system, is one way to improve Tartary buckwheat yield and quality. Therefore, the development of herbicide-resistant varieties is important for the breeding of Tartary buckwheat. In this experiment, 50 mM ethyl methyl sulfonate solution was used to treat Tartary buckwheat seeds (M1) and then planted in the field. Harvested seeds (M2) were planted in the experiment field of Guizhou University, and when seedlings had 5–7 leaves, the seedlings were sprayed with 166 mg/L tribenuron-methyl (TBM). A total of 15 resistant plants were obtained, of which three were highly resistant. Using the homologous cloning method, an acetolactate synthase (ALS) gene encoding 547 amino acids was identified in Tartary buckwheat. A GTG (valine) to GGA (glycine) mutation (V409G) occurred at position 409 of the ALS gene in the high tribenuron-methyl resistant mutant sm113. The dm36 mutant harbored a double mutation, a deletion mutation at position 405, and a GTG (valine) to GGA (glycine) mutation (V411G) at position 411. The dm110 mutant underwent a double mutation: an ATG (methionine) to AGG (arginine) mutation (M333R) at position 333 and an insertion mutation at position 372. The synthesis of Chl a, Chl b, total Chl, and Car was significantly inhibited by TBM treatment. TBM was more efficient at suppressing the growth of wild-type plants than that of mutant plants. Antioxidant enzyme activities such as ascorbate peroxidase, peroxidase, and superoxide dismutase were significantly higher in resistant plants than in wild-type after spraying with TBM; malondialdehyde content was significantly lower than in wild-type plants after spraying with TBM. Plants with a single-site mutation in the ALS gene could survive, but their growth was affected by herbicide application. In contrast, plants with dual-site mutations in the ALS gene were not affected, indicating that plants with dual-site mutations in the ALS gene showed higher levels of resistance than plants with a single-site mutation in the ALS gene.

First case of evolved herbicide resistance in the holoparasite sunflower broomrape, Orobanche cumana Wallr.

The development and commercialisation of sunflower varieties tolerant to acetolactate synthase (ALS)-inhibiting herbicides some 20 years ago provided farmers with an alternative method for the cost-effective control of Orobanche cumana. In 2020, however, two independent sunflower broomrape populations from Drama (GR-DRA) and Orestiada (GR-ORE), Greece, were reported to be heavily infested with O. cumana after application of the ALS-inhibiting herbicide imazamox. Here we have investigated the race of GR-DRA and GR-ORE and determined the basis of resistance to imazamox in the two Greek O. cumana samples. Using a set of five diagnostic sunflower varieties characterised by different resistant genes with respect to O. cumana infestation, we have clearly established that the GR-ORE and GR-DRA populations belong to the invasive broomrape races G and G+, respectively. Live underground tubercles and emerged shoots were identified at the recommended field rate of imazamox for GR-DRA and GR-ORE but not for two other standard sensitive populations in a whole plant dose response test using two different herbicide-tolerant sunflower hybrids as hosts. Sequencing of the ALS gene identified an alanine 205 to aspartate mutation in all GR-ORE samples. Most GR-DRA tubercles were characterised by a second serine 653 to asparagine ALS mutation whilst a few GR-DRA individuals contained the A205D mutation. Mutations at ALS codons 205 and 653 are known to impact on the binding and efficacy of imazamox and other imidazolinone herbicides. The knowledge generated here will be important for tracking and managing broomrape resistance to ALS-inhibiting herbicides in sunflower growing regions.

Introgression from local cultivars is a driver of agricultural adaptation in Argentinian weedy rice.

Weedy rice, a pervasive and troublesome weed found across the globe, has often evolved through fertilization of rice cultivars with little importance of crop-weed gene flow. In Argentina, weedy rice has been reported as an important constraint since the early 1970s, and, in the last few years, strains with herbicide-resistance are suspected to evolve. Despite their importance, the origin and genetic composition of Argentinian weedy rice as well its adaptation to agricultural environments has not been explored so far. To study this, we conducted genotyping-by-sequencing on samples of Argentinian weedy and cultivated rice and compared them with published data from weedy, cultivated and wild rice accessions distributed worldwide. In addition, we conducted a phenotypic characterization for weedy-related traits, a herbicide resistance screening and genotyped accessions for known mutations in the acetolactate synthase (ALS) gene, which confers herbicide resistance. Our results revealed large phenotypic variability in Argentinian weedy rice. Most strains were resistant to ALS-inhibiting herbicides with a high frequency of the ALS mutation (A122T) present in Argentinian rice cultivars. Argentinian cultivars belonged to the three major genetic groups of rice: japonica, indica and aus while weeds were mostly aus or aus-indica admixed, resembling weedy rice strains from the Southern Cone region. Phylogenetic analysis supports a single origin for aus-like South American weeds, likely as seed contaminants from the United States, and then admixture with local indica cultivars. Our findings demonstrate that crop to weed introgression can facilitate rapid adaptation to agriculture environments.

Distribution and Mechanism of Japanese Brome (Bromus japonicus) Resistance to ALS-Inhibiting Herbicides in China.

Bromus japonicus is a common monocot weed that occurs in major winter wheat fields in the Huang-Huai-Hai region of China. Pyroxsulam is a highly efficient and safe acetolactate synthase (ALS)-inhibiting herbicide that is widely used to control common weeds in wheat fields. However, B. japonicus populations in China have evolved resistance to pyroxsulam by different mutations in the ALS gene. To understand the resistance distribution, target-site resistance mechanisms, and cross-resistance patterns, 208 B. japonicus populations were collected from eight provinces. In the resistant population screening experiment, 59 populations from six provinces showed different resistance levels to pyroxsulam compared with the susceptible population, of which 17 B. japonicus populations with moderate or high levels of resistance to pyroxsulam were mainly from the Hebei (4), Shandong (4) and Shanxi (9) Provinces. Some resistant populations were selected to investigate the target site-resistance mechanism to the ALS-inhibiting herbicide pyroxsulam. Three pairs of primers were designed to amplify the ALS sequence, which was assembled into the complete ALS sequence with a length of 1932 bp. DNA sequencing of ALS revealed that four different ALS mutations (Pro-197-Ser, Pro-197-Thr, Pro-197-Phe and Asp-376-Glu) were found in 17 moderately or highly resistant populations. Subsequently, five resistant populations, QM21-41 with Pro-197-Ser, QM20-8 with Pro-197-Thr and Pro-197-Phe, and QM21-72, QM21-76 and QM21-79 with Asp-376-Glu mutations in ALS genes, were selected to characterize their cross-resistance patterns to ALS inhibitors. The QM21-41, QM20-8, QM21-72, QM21-76 and QM21-79 populations showed broad-spectrum cross-resistance to pyroxsulam, mesosulfuron-methyl and flucarbazone-sodium. This study is the first to report evolving cross-resistance to ALS-inhibiting herbicides due to Pro-197-Phe mutations in B. japonicus.

Insight into the herbicide resistance patterns in Lolium rigidum populations in Tunisian and Moroccan wheat regions.

Rigid ryegrass (Lolium rigidum Gaud.) is one of the most troublesome weeds in Moroccan and Tunisian cereal crop fields. In total, 19 rigid ryegrass field populations were randomly selected in northern wheat crop areas of Morocco and Tunisia to examine the patterns of herbicide resistance to acetolactate synthase (ALS)- and acetyl-CoA carboxylase (ACCase)-inhibiting herbicides. Greenhouse experiments confirmed reduced sensitivity to ALS- and/or ACCase-inhibiting herbicides in all L. rigidum populations. The occurrence of target-site resistance (TSR) was tested using high-throughput genotyping. The advent of next-generation sequencing (NGS) has enabled easy identification of causal mutations and confirmed the presence of ALS and ACCase mutations at specific codons conferring TSR. Thirteen populations showed resistance to ALS-inhibiting herbicides associated with point mutations in positions Pro-197-Thr, Pro-197-Ser, Pro-197-Leu, Pro-197-Gln and Trp-574-Leu, while resistance to ACCase-inhibiting herbicides was detected in 18 populations in positions Asp-2078-Val, Trp-2027-Cys, Ile-1781-Leu, Gly-2096-Ala, and Ile-2041-Asn of the enzymes conferring TSR. Additionally, dose-response experiments with pyroxsulam applied after the inhibition of cytochrome P450 monooxygenase by malathion showed an increase in sensitivity in two out of seven highly resistant (HR) rigid ryegrass populations. This demonstrates the presence of non-target-site resistance (NTSR) in some ryegrass populations. Further evidence of NTSR was investigated in dose-response experiments with pyroxsulam, following pretreatment with the glutathione S-transferase (GST) inhibitor 4-chloro-7-nitrobenzoxadiazole (NBD-Cl), which partially reversed resistance in only a few individuals of two L. rigidum populations. Hence, our study confirms the existence of multiple and cross-resistance to ALS- and ACCase-inhibiting herbicides in L. rigidum from Morocco and Tunisia with both TSR and NTSR mechanisms. These results emphasize local resistance management as an important tool to detect and mitigate gene flow from rigid ryegrass populations where resistance has evolved.

Diverse ALS mutations and cross-and multiple-resistance to ALS and EPSPS inhibitors in flucarbazone‑sodium-resistant Bromus japonicus populations from Hebei province, China

Japanese brome (Bromus japonicus) has become one of the main weeds in wheat fields in Hebei province of China and causes a large decrease of wheat production. A total of 44 putative resistant and 2 susceptible Japanese brome populations were collected in the 2021/2022 crop season from Hebei province of China to determine resistance levels to flucarbazone‑sodium and to investigate the diversity of acetolactate synthase (ALS) mutations, as well as to confirm the cross-and multiple-resistance levels to ALS and EPSPS (5-enolpyruvate shikimate-3-phosphate synthetase) inhibitors. Whole plant bioassay results showed that 15 out of 44 populations tested or 34% were resistant to flucarbazone‑sodium. The resistance indices of Japanese brome to flucarbazone‑sodium ranged from 43 to 1977. The resistant populations were mainly distributed in Baoding and Shijiazhuang districts, and there was only one resistant population in Langfang district. Resistant Japanese brome had diverse ALS mutations, including Pro-197-Ser, -Thr, -Arg and Asp-376-Glu. The incidence of Pro-197-Ser mutation was the highest at 68%. Application of the CYP450 inhibitor malathion suggested that CYP450 was involved in metabolic resistance in a population without an ALS mutation. The population with Pro-197-Thr mutation evolved weak cross-resistance to mesosulfuron-methyl and pyroxsulam, and it is in the process of evolving multiple-resistance to glyphosate.

Prevalence, spatial structure and evolution of resistance to acetolactate-synthase (ALS) inhibitors and 2,4-D in the major weed Papaver rhoeas (L.) assessed using a massive, country-wide sampling.

Corn poppy (Papaver rhoeas) is the most damaging broadleaf weed in France. Massively parallel amplicon sequencing was used to investigate the prevalence, mode of evolution and spread of resistance-endowing ALS alleles in 422 populations randomly sampled throughout poppy's range in France. Bioassays were used to detect resistance to the synthetic auxin 2,4-D in 43 of these populations. A total of 21 100 plants were analysed and 24 mutant ALS alleles carrying an amino-acid substitution involved or potentially involved in resistance were identified. The vast majority (97.6%) of the substitutions occurred at codon Pro197, where all six possible single-nucleotide non-synonymous substitutions plus four double-nucleotide substitutions were identified. Changes observed in the enzymatic properties of the mutant ALS isoforms could not explain the differences in prevalence among the corresponding alleles. Sequence read analysis showed that mutant ALS alleles had multiple, independent evolutionary origins, and could have evolved several times independently within an area of a few kilometres. Finally, 2,4-D resistance was associated with mutant ALS alleles in individual plants in one third of the populations assayed. The intricate geographical mosaic of mutant ALS alleles observed is the likely result of the combination of huge population sizes, multiple independent mutation events and human-mediated spread of resistance. Our work highlights the ability of poppy populations and individual plants to accumulate different ALS alleles and as yet unknown mechanisms conferring resistance to synthetic auxins. This does not bode well for the continued use of chemical herbicides to control poppy. © 2023 Society of Chemical Industry.

Multiple resistance of Echinochloa phyllopogon to synthetic auxin, ALS-, and ACCase-inhibiting herbicides in Northeast China

Echinochloa phyllopogon is a self-pollinating allotetraploid weed and a serious threat to global rice production. One sensitive and three multiple-resistant populations collected from two provinces of Northeast China were used to analyze the mechanism of multiple resistance of E. phyllopogon to penoxsulam, metamifop, and quinclorac. Compared with the sensitive population LN12, LN1 showed higher resistance to these three herbicides; LN24 showed medium resistance to penoxsulam and metamifop and higher resistance to quinclorac (274-fold); HLJ4 showed low resistance to penoxsulam and high resistance to metamifop and quinclorac. Target sequence analysis showed no mutations in acetolactate synthase or acetyl-CoA carboxylase genes. In-vitro enzyme activity analysis showed that the activity of the target enzyme of multiple herbicide-resistant populations was similar to that of the sensitive population. The P450 inhibitor, malathion, noticeably increased the sensitivity of LN1, LN24, and HLJ4 to penoxsulam, LN1 to metamifop, and HLJ4 to quinclorac. Under all four treatments, the GSTs activities of resistant and sensitive populations showed an increasing trend from day 1 to day 5, but the sensitivity and activity of GSTs were higher in the multiple-resistant population than that in the sensitive population LN12. This study identified the development of multiple-resistant E. phyllopogon populations that pose a serious threat to rice production in rice fields in Northeast China, preliminarily confirming that multiple-resistance was likely due to non-target-site resistance mechanisms. These populations of E. phyllopogon are likely to be more difficult to control.

Diversity and Spread of Acetolactate Synthase Allelic Variants at Position 574 Endowing Resistance in Amaranthus hybridus in Italy.

Poor control of Amaranthus spp. with herbicides inhibiting acetolactate synthase (ALS) has been observed for several years in soybean fields in north-eastern Italy, but to date only a few ALS-resistant populations have been confirmed. An extensive sampling of putatively resistant Amaranthus accessions was completed in the Friuli Venezia Giulia region, across an arable land area of about 3000 km2. In total, 58 accessions were tested to confirm their resistance status, recognize the Amaranthus species, identify the mutant ALS alleles endowing the resistance and determine the efficacy of 3 pre-emergence herbicides. Most accessions resulted in cross-resistance to thifensulfuron-methyl and imazamox. Genomic DNA were extracted from single seeds with a newly developed protocol; an allele-specific PCR assay revealed the presence of the 574-leucine in 20 accessions, of the 574-methionine in 22, and of both alleles in 9 accessions. The two variants showed a different spatial distribution. All resistant populations were ascribed to A. hybridus. A. hybridus resistant to ALS herbicides is well-established in this Italian region and its resistance is due to two ALS mutant alleles. Metribuzin, clomazone and metobromuron can be used as alternative herbicides to be applied in pre-emergence and they should be integrated into the management strategies to limit the spread of resistance.

Herbicide Resistance Status of Italian Ryegrass (Lolium multiflorum Lam.) and Alternative Herbicide Options for Its Effective Control in the Huang-Huai-Hai Plain of China

Italian ryegrass (Lolium multiflorum Lam.) was introduced into China as a kind of turfgrass and has invaded wheat fields of the Huang-Huai-Hai Plain, causing great losses to grain yield. The acetyl–CoA carboxylase (ACCase) inhibitor clodinafop-propargyl and the acetolactate synthase (ALS) inhibitor mesosulfuron-methyl are highly efficient herbicides that have been widely used for control of this species, which has also resulted in its resistance evolution. However, the resistance status of L. multiflorum in the Huang-Huai-Hai Plain of China remains unclear, which hinders the integrated management of this weed in winter wheat production systems. In the current study, a total of 37 L. multiflorum populations were collected from the wheat fields across the region, and their susceptibility to clodinafop-propargyl and mesosulfuron-methyl was assessed. Of these, 13 populations were resistant or evolving resistance to clodinafop-propargyl (R?, RR, and RRR) with resistance index (RI) ranging from 2.62 to 830.05, and 8 populations were resistant or evolving resistance to mesosulfuron-methyl (R? and RR) with RI ranging from 3.89 to 16.68. Seven populations showed multiple-resistance to both clodinafop-propargyl and mesosulfuron-methyl. Three ACCase (I1781L, I2041N, and D2078G) and four ALS (P197T, P197S, P197A, and W574L) resistance mutations were identified in the herbicide-resistant populations, and I1781L and P197T were predominant ACCase and ALS mutations, respectively. Real-time quantitative PCR assays showed that compared with the susceptible population, the ACCase expression was slightly upregulated in some of the clodinafop-propargyl-resistant populations (AH-01 and AH-05) following clodinafop-propargyl treatment, while the ALS expression in the mesosulfuron-methyl-resistant populations showed no significant change following mesosulfuron-methyl treatment. Whole-plant dose-response testing showed that the AH-01 population carrying an ACCase gene I2041N mutation exhibited cross-resistance to all the ACCase inhibitors tested and multiple-resistance to the ALS inhibitor bispyribac-sodium, the AH-05 population carrying an ACCase gene I1781L mutation and an ALS gene P197T mutation showed cross-resistance to all the ACCase and ALS inhibitors tested, and the HN-07 population carrying an ACCase gene D2078G mutation showed cross-resistance to all the ACCase inhibitors tested and multiple-resistance to some ALS inhibitors. All the resistant populations remained susceptible to the 5-enolpyruvylshikimate-3-phosphate inhibitor glyphosate and the photosystem II inhibitor isoproturon. This study has clarified the distributions of clodinafop-propargyl- and/or mesosulfuron-methyl-resistant L. multiflorum in the Huang-Huai-Hai Plain of China, and target gene mutation was one of the most common mechanisms responsible for the resistance.

Multiplex precision gene editing by a surrogate prime editor in rice

Multiplex precision gene editing by a surrogate prime editor in rice

A novel Phe‐206‐Leu mutation in acetolactate synthase confers resistance to penoxsulam in barnyardgrass (Echinochloa crus‐galli (L.) P. Beauv)

Barnyardgrass (Echinochloa crus-galli (L.) P. Beauv) has evolved resistance to the acetolactate synthase (ALS) inhibitor penoxsulam which is used to control weeds in rice fields in China. The present study is conducted to identify the target-site resistance (TSR) mechanisms conferring resistance in a penoxsulam-resistant population. The ALS sensitivity in vitro of the resistant population was sixfold lower to penoxsulam than that of the sensitive population. ALS sequencing revealed that no known mutation conferring ALS herbicide resistance was detected. However, a novel mutation Phe-206-Leu was identified in the ALS gene. Additionally, ALS gene expression level of the resistant population was lower than that of the sensitive population. Therefore, the penoxsulam resistance was not due to the overexpression of ALS gene. Molecular docking revealed that this mutation may change the interaction of the penoxsulam-ALS binding and weaken its mutual affinity by approximately 10%. Arabidopsis thaliana transformed with mutant ALS had fourfold greater resistance to penoxsulam and varied cross-resistance to other ALS herbicides than those transformed with sensitive ALS. Mutant and sensitive ALS proteins expressed by the baculovirus system exhibited different in vitro penoxsulam sensitivity levels. Mutant ALS had eightfold lower sensitivity to penoxsulam than sensitive ALS. This report provides clear evidence that the ALS mutation at position 206 (Phe-206-Leu) confers penoxsulam resistance in barnyardgrass. Phe-206 was confirmed to be the ninth amino acid residue related to ALS herbicide resistance in weeds. © 2022 Society of Chemical Industry.

Generation of Transfer-DNA-Free Base-Edited Citrus Plants.

To recover transgenic citrus plants in the most efficient manner, the use of selection marker genes is essential. In this work, it was shown that the mutated forms of the acetolactate synthase (ALS) gene in combination with the herbicide selection agent imazapyr (IMZ) added to the selection medium may be used to achieve this goal. This approach enables the development of cisgenic regenerants, namely, plants without the incorporation of those bacterial genes currently employed for transgenic selection, and additionally it allows the generation of edited, non-transgenic plants with altered endogenous ALS genes leading to IMZ resistance. In this work, the citrus mutants, in which ALS has been converted into IMZ-resistant forms using a base editor system, were recovered after cocultivation of the explants with Agrobacterium tumefaciens carrying a cytidine deaminase fused to nSpCas9 in the T-DNA and selecting regenerants in the culture medium supplemented with IMZ. Analysis of transgene-free plants indicated that the transient expression of the T-DNA genes was sufficient to induce ALS mutations and thus generate IMZ-resistant shoots at 11.7% frequency. To our knowledge, this is the first report of T-DNA-free edited citrus plants. Although further optimization is required to increase edition efficiency, this methodology will allow generating new citrus varieties with improved organoleptic/agronomic features without the need to use foreign genes.

Primary metabolism in an Amaranthus palmeri population with multiple resistance to glyphosate and pyrithiobac herbicides

The objective of this work was to characterize the resistance mechanisms and the primary metabolism of a multiple resistant (MR) population of Amaranthus palmeri to glyphosate and to the acetolactate synthase (ALS) inhibitor pyrithiobac. All MR plants analysed were glyphosate-resistant due to 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene amplification. Resistance to pyrithiobac was more variable among individuals and was related to point mutations at five positions in the ALS gene sequence: A122, A205, W574, S653 and G654. All MR plants were heterozygous for W574, the most abundant mutation. In nontreated plants, the presence of mutations did not affect ALS functionality, and plants with the W574L mutation showed the highest ALS resistance level to pyrithiobac. The accumulation of the transcripts corresponding to several genes of the aromatic amino acid (AAA) and branched-chain amino acid (BCAA) pathways detected in nontreated MR plants indicated additional effects of EPSPS gene amplification and ALS mutations. The physiological performance of the MR population after treatment with glyphosate and/or pyrithiobac was compared with that of a sensitive (S) population. The increase induced in total soluble sugars, AAA or BCAA content by both herbicides was higher in the S population than in the MR population. Physiological effects were not exacerbated after the mixture of both herbicides in S or in MR populations. This study provides new insights into the physiology of a multiple resistant A. palmeri, which could be very useful for achieving effective management of this weed.

First Detection and Characterization of Cross- and Multiple Resistance to Acetyl-CoA Carboxylase (ACCase)- and Acetolactate Synthase (ALS)-Inhibiting Herbicides in Black-Grass (Alopecurus myosuroides) and Italian Ryegrass (Lolium multiflorum) Populations from Ireland

Understanding the resistance spectrum and underlying genetic mechanisms is critical for managing herbicide-resistant populations. In this study, resistance to acetyl CoA carboxylase (ACCase) and acetolactate synthase (ALS) inhibitors was investigated in four suspected resistant populations of Alopecurus myosuroides (ALOMY-001 to ALOMY-004) and Lolium multiflorum (LOLMU-001 to LOLMU-004), collected from cereal production fields in Ireland. Glasshouse assays with three ALOMY-active herbicides [propaquizafop, cycloxydim (ACCase) and mesosulfuron + iodosulfuron (ALS)] or five LOLMU-active herbicides [pinoxaden, propaquizafop, cycloxydim (ACCase) and mesosulfuron + iodosulfuron, pyroxsulam (ALS)], and target-site resistance mechanism studies, based on pyrosequencing, were carried out in each of those populations. For A. myosuroides, Ile-1781-Leu ACCase mutation contributed to propaquizafop and cycloxydim resistance (shoot dry weight GR50 resistance factor (RF) = 7.5–35.5) in all ALOMY populations, and the independent Pro-197-Thr or Pro-197-Ser ALS mutation contributed to mesosulfuron + iodosulfuron resistance (RF = 3.6–6.6), in ALOMY-002 to ALOMY-004. Most of the analyzed plants for these mutations were homo/heterozygous combinations or only heterozygous. For L. multiflorum, phenotypic resistance to mesosulfuron + iodosulfuron (RF = 11.9–14.6) and pyroxsulam (RF = 2.3–3.1) was noted in all LOLMU populations, but the Pro-197-Gln or Pro-197-Leu ALS mutation (mostly in homozygous status) was identified in LOLMU-001, LOLMU-002 and LOLMU-004 only. Additionally, despite no known ACCase mutations in any LOLMU populations, LOLMU-002 survived pinoxaden and propaquizafop application (RF = 3.4 or 1.3), and LOLMU-003 survived pinoxaden (RF = 2.3), suggesting the possibility of non-target-site resistance mechanisms for ACCase and/or ALS resistance in these populations. Different resistance levels, as evidenced by a reduction in growth as dose increased above field rates in ALOMY and LOLMU, were due to variations in mutation rate and the level of heterozygosity, resulting in an overall resistance rating of low to moderate. This is the first study confirming cross- and multiple resistance to ACCase- and ALS-inhibiting herbicides, highlighting that resistance monitoring in A. myosuroides and L. multiflorum in Ireland is critical, and the adoption of integrated weed management strategies (chemical and non-chemical/cultural strategies) is essential.

Transformation of Teosinte (Zea mays ssp. parviglumis) via Biolistic Bombardment of Seedling-Derived Callus Tissues.

Modern maize exhibits a significantly different phenotype than its wild progenitor teosinte despite many genetic similarities. Of the many subspecies of Zea mays identified as teosinte, Zea mays ssp. parviglumis is the most closely related to domesticated maize. Understanding teosinte genes and their regulations can provide great insights into the maize domestication process and facilitate breeding for future crop improvement. However, a protocol of genetic transformation, which is essential for gene functional analyses, is not available in teosinte. In this study, we report the establishment of a robust callus induction and regeneration protocol using whorl segments of seedlings germinated from mature seeds of Zea parviglumis. We also report, for the first time, the production of fertile, transgenic teosinte plants using the particle bombardment. Using herbicide resistance genes such as mutant acetolactate synthase (Als) or bialaphos resistance (bar) as selectable markers, we achieved an average transformation frequency of 4.17% (percentage of independent transgenic events in total bombarded explants that produced callus). Expression of visual marker genes of red fluorescent protein tdTomato and β-glucuronidase (gus) could be detected in bombarded callus culture and in T1 and T2 progeny plants. The protocol established in this work provides a major enabling technology for research toward the understanding of this important plant in crop domestication.

Effects of a novel combination of two mutated acetolactate synthase (ALS) isozymes on resistance to ALS-inhibiting herbicides in flixweed (Descurainia sophia)

AbstractFlixweed [Descurainia sophia(L.) Webb ex Prantl] is a notorious broadleaf weed that is widely distributed in winter wheat–growing areas of China and has evolved resistance to tribenuron-methyl mainly due to target-site resistance (TSR) mutations in acetolactate synthase (ALS). In the current research, twoALSgenes were identified in tribenuron-methyl–susceptible (TS) or tribenuron-methyl–resistant (TR)D. sophia. Resistance mutations of Asp-376-Glu and Pro-197-Ala were identified on ALS1 and ALS2 isozymes in TRD. sophia, respectively. The TRD. sophiaevolved 10,836.3-fold resistance to tribenuron-methyl and displayed cross-resistance to multiple ALS-inhibiting herbicides with different chemical structures. Dose response experiments and ALS activity assay indicated that two mutated ALS isozymes contributed differentially in resistance to tribenuron-methyl, flucetosulfuron, and pyribenzoxim. In addition, the relative expression level of theALS1gene was 2.2- and 1.6-fold higher thanALS2genes in TRD. sophiaat 1 and 7 d after tribenuron-methyl treatment, respectively. In contrast, the relative expression level ofALS1andALS2in TSD. sophiais similar. This is the first research that explored different roles of ALS isozymes in resistance to ALS-inhibiting herbicides, which might provide a new perspective for the weed resistance management.

Diversified Resistance Mechanisms in Multi-Resistant Lolium spp. in Three European Countries.

Annual ryegrass species (Lolium spp.) infest cereal crops worldwide. Ryegrass populations with multiple resistance to the acetyl coenzyme A carboxylase (ACCase) and acetolactate synthase (ALS) inhibitors are an increasing problem in several European countries. We investigated the resistance pattern and level of resistance in ryegrass populations collected in Denmark, Greece and Italy and studied the diversity of mechanisms endowing resistance, both target-site and metabolism based. All populations showed high resistance indexes (RI) to the ALS inhibitors, iodosufuron-methyl-sodium + mesosulfuron-methyl (RI from 8 to 70), whereas the responses to the two ACCase inhibitors, clodinafop-propargyl and pinoxaden, differed. The Greek and Italian populations were moderately to highly resistant to clodinafop (RI > 8) and showed low to moderate resistance to pinoxaden (RI ranged from 3 to 13) except for one Italian population. In contrast, the Danish Lolium populations showed low to moderate resistance to clodinafop (RI ranged from 2 to 7) and only one population was resistant to pinoxaden. Different mutant ACCase alleles (Leu1781, Cys2027, Asn2041, Val2041, Gly2078, Arg2088, Ala2096) and ALS alleles (Gly122, Ala197, Gln197, Leu197, Ser197, Thr197, Val205, Asn376, Glu376, Leu574) endowing resistance were detected in the Greek and Italian populations. In several plants, no mutated ALS and ACCase alleles were found showing a great heterogeneity within and among the Greek and Italian populations. Conversely, no mutant ACCase alleles were identified in the four Danish populations and only one mutant ALS allele (Leu574) was detected in two Danish populations. The expression level of nitronate monooxygenase (NMO), glutathione S-transferase (GST) and cytochrome P450s (CYP72A1 and CYP72A2) varied broadly among populations and individual plants within the populations. Constitutive up-regulation of GST, CYP72A1 and CYP72A2 was detected in resistant plants respect to susceptible plants in one Danish and one Italian population. It appears that the mechanisms underlying resistance are rather complex and diversified among Lolium spp. populations from the three countries, coevolution of both target-site resistance and metabolic based herbicide resistance appears to be a common feature in Denmark and Italy. This must be considered and carefully evaluated in adopting resistance management strategies to control Lolium spp. in cereal crops.

Target-site mutation and enhanced metabolism confer resistance to thifensulfuron-methyl in a multiple-resistant redroot pigweed (Amaranthus retroflexus) population

AbstractRedroot pigweed (Amaranthus retroflexus L.) is a troublesome dicot weed species widely distributed across China. A population of A. retroflexus that survived the recommended label rate of thifensulfuron-methyl was collected from the main soybean [Glycine max (L.) Merr.] production area in China. Whole-plant dose–response assays indicated that the resistant (R) population was highly resistant (61.80-fold) to thifensulfuron-methyl compared with the susceptible (S1 and S2) populations. In vitro acetolactate synthase (ALS) activity experiments showed that the thifensulfuron-methyl I50 value for the R population was 40.17 times higher than that for the S1 population. A preliminary malathion treatment study indicated that the R population might have cytochrome P450–mediated metabolic resistance. The R population exhibited a high level of cross-resistance to representative ALS herbicides (imazethapyr, flumetsulam, and bispyribac-sodium) and multiple resistance to the commonly used protoporphyrinogen oxidase (PPO)-inhibiting herbicides lactofen and fomesafen. Two common mutations, Trp-574-Leu in ALS and Arg-128-Gly in PPO2, were identified within the R population. This study identified possible enhanced metabolism of thifensulfuron-methyl coexisting with target-site mutations in both ALS and PPO2 in a multiple-resistant A. retroflexus population.

A novel mutation Asp-2078-Glu in ACCase confers resistance to ACCase herbicides in barnyardgrass (Echinochloa crus-galli)

Multiple-herbicide resistance (MHR) in barnyardgrass (Echinochloa crus-galli) is a threat to rice production. The Ala-205-Val mutation in acetolactate synthase (ALS) conferred resistance to several ALS inhibitors in the E. crus-galli population AXXZ-2; consequently, ALS-inhibitors were unable to control this noxious weed species. In the present study, the sensitivity to acetyl-coenzyme A carboxylase (ACCase) herbicides and other herbicides having different modes of action was evaluated to determine an effective strategy for chemical weed control. Compared with that of the reportedly sensitive population JLGY-3, the AXXZ-2 population showed differential resistance to three ACCase-inhibitors (cyhalofop-butyl, fenoxaprop-P-ethyl, and pinoxaden), in addition to quinclorac and pretilachlor. A novel substitution (Asp-2078-Glu) in ACCase was detected as the main target-site resistance mechanisms in the AXXZ-2 population. Structural modeling of the mutant ACCase protein predicted that Asp-2078-Glu confers resistance to three ACCase inhibitors by reducing the binding affinity between them and the ACCase protein. To the best of our knowledge, this is the first study to report that the novel Asp-2078-Glu mutation confers resistance to several ACCase inhibitors. Target-site mutations in ALS and ACCase were detected in this MHR population. Except for quinclorac, pretilachlor, ALS inhibitors, and the three ACCase inhibitors, a number of herbicides remain effective in controlling this MHR E. crus-galli population.