Catarine Markus

Weedy rice ( Oryza spp.) provides a unique model for studying adaptive evolution because herbicide resistance can arise through both crop‐to‐weed gene flow and independent mutational events. In Brazil, nearly two decades of cultivation of imidazolinone (IMI)‐resistant rice have imposed intense selection for acetolactate synthase (ALS) inhibitors resistance. This study investigated how historical gene flow and independent evolution have shaped the contemporary landscape of ALS resistance mutations in Brazilian weedy rice. Specifically, we characterized the diversity and distribution of ALS resistance mutations and developed rapid allele‐specific molecular markers for major resistance alleles. A total of 200 weedy rice accessions collected from the major rice‐growing region of Brazil were screened for IMI resistance and genotyped using newly developed SNAP (single nucleotide amplified polymorphism) qPCR markers targeting the IMI rice‐derived mutations Ala122Thr, Ser653Asn, and Gly654Glu. Samples negative for these mutations were sequenced, and dose‐response assays were conducted with imazethapyr, penoxsulam, nicosulfuron, and cloransulam‐methyl. Ninety‐three percent of the accessions were resistant to IMI herbicides. The ALS mutation Gly654Glu, present in rice cultivars used until 2006, was found in 15.5% of weedy rice resistant accessions, Ala122Thr became the dominant mutation (54.5%), whereas Ser653Asn occurred at an intermediate frequency (16.0%), indicating a temporal shift in the genetic basis of ALS resistance. Sequencing further identified the ALS substitutions Ala205Val, Trp574Leu, and Ser653Thr, which were not associated with commercial IMI‐resistant rice cultivars, indicating ongoing independent evolution of target‐site resistance. Trp574Leu conferred broad cross‐resistance to all ALS‐inhibiting herbicides evaluated, whereas the remaining mutations displayed mutation‐specific resistance spectra. The SNAP markers showed high specificity and sensitivity, enabling reliable high‐throughput detection of resistance alleles. Together, our findings demonstrate that the contemporary ALS resistance landscape in Brazilian weedy rice has been shaped mainly by repeated crop‐to‐weed gene flow, but also ongoing independent mutational events that continue to diversify resistance under herbicide selection.

Background: Weedy rice (Oryza spp.) and Echinochloa spp. are the most problematic grass weeds in rice (Oryza sativa L.) production systems due to their high competitiveness, prolific seed production, and widespread herbicide resistance. The adoption of imidazolinone (IMI)-resistant rice enabled selective control; however, gene flow rapidly led to resistant weedy rice populations. More recently, rice cultivars resistant to aryloxyphenoxypropionate (FOP) ACCase-inhibiting herbicides were introduced, providing a new control strategy. Objective: This study aimed to establish the baseline sensitivity of [...] Abstract Background: Weedy rice (Oryza spp.) and Echinochloa spp. are the most problematic grass weeds in rice (Oryza sativa L.) production systems due to their high competitiveness, prolific seed production, and widespread herbicide resistance. The adoption of imidazolinone (IMI)-resistant rice enabled selective control; however, gene flow rapidly led to resistant weedy rice populations. More recently, rice cultivars resistant to aryloxyphenoxypropionate (FOP) ACCase-inhibiting herbicides were introduced, providing a new control strategy. Objective: This study aimed to establish the baseline sensitivity of

Herbicide target site resistance in polyploid species is more complex than in diploids due to potential subgenome interactions. This study characterized mutations in the ALS gene across distinct subgenomes of hexaploid Echinochloa crus‐galli and evaluated the cross‐resistance patterns conferred by each mutation to various ALS‐inhibiting herbicides. E. crus‐galli populations were screened, and dose–response curves were performed with ALS inhibitors from different chemical groups. The ALS gene copies of each subgenome (A, B, and C) were sequenced. Copy number variation, global relative expression, and the specific relative expression of ALS gene from each subgenome were performed. The mutations Ala122Thr, Ala205Asn, and Ser653Asn conferred resistance only to imazethapyr, whereas Trp574Leu to imazethapyr, penoxsulam, bispyribac‐sodium, and nicosulfuron, when considered the label rate. ALS mutations were more frequent in subgenome A, but ALS from subgenome C had the highest expression. Biotypes with the same mutation showed different resistance level to herbicides. The biotype SAOJER‐01 had Trp574Leu mutation in subgenome C and was 22 times more resistant to imazethapyr and penoxsulam than CAMAQ‐01, which had the same Trp574Leu mutation in subgenome A. Both SAOJER‐01 and CAMAQ‐01 biotypes showed CYP450 metabolism mediating penoxsulam resistance in addition to the target site mutation. In conclusion, the mutations Ala122Thr, Ala205Asn, Trp574Leu, and Ser653Asn confer resistance to imazethapyr, but only Trp574Leu confers resistance to the other chemical groups. The herbicides penoxsulam, bispyribac‐sodium, and nicosulfuron are effective in controlling three out of four mutations. CYP450‐mediated metabolism coexists in biotypes carrying the Trp574Leu mutation. The subgenome location of the ALS mutation may result in variable levels of resistance.

Herbicide target site resistance in polyploid species is more complex than diploids due to potential subgenome interactions and gene dosage of mutations. The objective of this study was to identify the level of resistance and cross-resistance patterns of ALS mutations located in the different subgenomes of allohexaploid Echinochloa crus-galli . E. crus-galli populations were screened and dose-response curves were performed with ALS-inhibitors from different chemical groups. The ALS genes of each subgenome (A, B, and C) were sequenced. Copy number variation, global relative expression, and the specific relative expression of ALS gene from each subgenome were performed. Out of 100 populations, 32% were resistant only to imazethapyr, and 48% were cross-resistant to all ALS-inhibitors. The mutations Ala122Thr, Ala205Asn, and Ser653Asn confer resistance only to imazethapyr, while Trp574Leu confers resistance to all four herbicides tested. ALS mutations were most frequent in subgenome A, but ALS from subgenome C had the highest expression. The biotype SAOJER-01 had Trp574Leu mutation in subgenome C and was 22 times more resistant to imazethapyr and penoxsulam than CAMAQ-01, which had the same Trp574Leu mutation in subgenome A with no differences in herbicide metabolism rate. The ALS genes from subgenomes A and C contribute approximately 13% and 50% respectively to the total ALS transcripts. The higher contribution of subgenome C to the total ALS transcripts pool combined with the Trp574Leu resistance mutation makes the biotype SAOJER-01 more resistant to herbicides than CAMAQ-01. This is the first study showing the effect of subgenome gene expression level on the herbicide resistance level in a polyploid weed species. In addition, while imazethapyr did not control any biotype with an ALS mutation, the other ALS-inhibitor herbicides were able to control biotypes carrying Ala122Thr, Ala205Asn, and Ser653Asn, which reinforces the utility of these herbicides in weed and resistance management.

Barnyardgrass (Echinochloa crus-galli) is one of the most troublesome weeds in irrigated rice cultivation and has increasingly impacted rainfed crops due to the emergence of herbicide-resistant populations. Understanding its germination dynamics is crucial for developing and implementing effective management strategies. Additionally, since barnyardgrass research relies on growing plants from seeds, its dormancy characteristics are of particular interest. The present study aimed to evaluate the influence of postharvest age on barnyardgrass seed germination and the effectiveness of different dormancy-breaking methods in susceptible and herbicide-resistant populations. Germination rate (G), germination speed index (GSI), and seed viability, assessed using the topographic tetrazolium test, were measured in seed lots with four different postharvest ages: two years, one year, two months, and one day postharvest. The seeds were subjected to 15 dormancy-breaking methods, including temperature variation and the use of solutions containing H2SO4, KNO3, and GA3. Seeds that were one or two years old showed germination rates exceeding 90%, regardless of the method used. In contrast, seeds aged two months or one day postharvest only germinated when exposed to 40°C for seven days, with G values of 25.2% and 5.9%, respectively. Both herbicide-susceptible and resistant barnyardgrass populations exhibited similar dormancy levels and responses to dormancy-breaking methods. The results indicate that newly harvested seeds have high dormancy levels, and specific methods are only partially effective in overcoming barnyardgrass seed dormancy.

The seed shattering trait has been repeatedly reshaped during rice evolution. Reduced in cultivated rice and increased in weedy rice, shattering is of great agronomic importance because of its association with yield losses. Since its first descriptions, the phenotypic patterns and the genetic regulation of cultivated and weedy rice seed shattering have been extensively studied, with a variety of methods and techniques. The aim of this review is to discuss and recommend the most suitable experimental methods for phenotypic [...] Abstract The seed shattering trait has been repeatedly reshaped during rice evolution. Reduced in cultivated rice and increased in weedy rice, shattering is of great agronomic importance because of its association with yield losses. Since its first descriptions, the phenotypic patterns and the genetic regulation of cultivated and weedy rice seed shattering have been extensively studied, with a variety of methods and techniques. The aim of this review is to discuss and recommend the most suitable experimental methods for phenotypic

The presence of glyphosate-resistant smooth pigweed (Amaranthus hybridus L.) biotypes has increased in southern Brazil in recent years, presenting the triple amino acid substitution TAP-IVS in 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) previously found in Argentina. Some of these biotypes have morphological characteristics of A. hybridus and redroot amaranth (Amaranthus retroflexus L.). The present study aimed to identify, through molecular markers, the herbicide-resistant species of Amaranthus from Brazil that have the TAP-IVS substitution and to analyze the occurrence of pollen-mediated gene flow (PMGF) as the source of the TAP-IVS substitution in these biotypes. Six biotypes were evaluated using internal transcribed spacer (ITS) sequences, of which two (AMACHY-S and CAMAQ-R) were molecularly classified as A. hybridus and four (AMACRET-S, AMACVI-S, ARRGR-R, and SAOJER-R) were unclassified. Interestingly, all the glyphosate-resistant biotypes (ARRGR-R, SAOJER-R, and CAMAQ-R) had the TAP-IVS substitution and an increase in EPSPS relative copy number; however, only CAMAQ-R was confirmed as A. hybridus. Although the biotypes ARRGR-R and SAOJER-R are closely related to A. hybridus and green pigweed (Amaranthus powellii S. Watson), their species identity could not be resolved. The biotype SAOJER-R also was resistant to acetolactate synthase (ALS)-inhibiting herbicides due to a tryptophan to leucine substitution at position 574 in ALS. The evaluation of 119,746 seedlings in an intraspecific hybridization study of A. hybridus indicated an outcrossing frequency of 0.09%. In contrast, an absence of interspecific hybridization (A. hybridus × unclassified biotype, AMACVI-S) was found after screening 111,429 offspring. Unclassified biotypes might be derived from one or more ancient hybridization events and subsequently evolved the triple mutation independently. Alternatively, such biotypes could have evolved from recent hybridization events, which occur at a frequency below the level of detection in our study.

Resistant plants of Sumatran fleabane with unusual rapid necrosis (RN) symptoms after application of 2,4-D were characterized in previous studies. Field observations indicated variability in the occurrence of the RN caused by 2,4-D, but the causes of the variation are unknown. This study aimed to investigate the effect of environmental conditions, plant growth stage, and simultaneous and sequential herbicide mixtures with other auxin mimics on the occurrence of RN caused by 2,4-D. Application at temperature of 12 C delayed the symptoms and decreased the intensity of the RN but still resulted in plant survival to 2,4-D. The absence of light after herbicide application caused a slight delay in the symptoms, but the production of hydrogen peroxide and the size of the necrosed area were not affected by the light treatments before and after 2,4-D application. Changes in plant photosynthesis through inhibiting photosystem II do not prevent the occurrence of RN symptoms. The auxinic herbicides dicamba, triclopyr, and halauxifen-methyl do not cause RN symptoms and are effective at controlling the resistant biotype when applied without 2,4-D, but the effectiveness of these herbicides was reduced when sprayed on the resistant biotype either together, 4 h, or 24 h after 2,4-D. The RN phenotype does not occur for dicamba and triclopyr, even in advanced plant growth stages and high doses on the resistant biotype. The herbicides dicamba and triclopyr effectively controlled resistant plants, especially when sprayed at the initial growth stages. The results of this study identify environmental effects, plant development effects, and herbicide interactions that interfere with the occurrence of RN symptoms caused by 2,4-D in Sumatran fleabane. These data provide insights about the mechanisms behind the RN symptoms caused by 2,4-D and are important for identifying the causes of variability of the herbicide symptomology and performance under experimental and field conditions.

Environmental conditions can influence herbicide metabolism and interfere with the evolution of weed resistance. This study aimed to evaluate the effect of thermal regimes on the control of barnyardgrass (Echinochloa crus-galli) susceptible and resistant to herbicides of the imidazolinones chemical group. Factor A (biotypes) consisted of one biotype that is susceptible and one that is resistant to imazethapyr; factor B (dose) comprised six imazethapyr doses between 0 and 800 g ha-1 for the resistant biotype and between 0 and 100 g ha-1 for the susceptible biotype; factor C (temperature) consisted of four thermal regimes (16/16 ºC, 25/25 ºC, 16/25 ºC and 25/16 ºC) to which the plants were submitted before and after the herbicide application. Concerning the resistant biotype, the highest control levels were observed in the thermal regimes with temperature fuctuation, especially at 25/16 ºC. At the recommended imazethapyr dose (100 g ha-1), the herbicide resulted in a control level over 90 % at 25/16 ºC and less than 50 % at 16/16 ºC, in the resistant biotype. The shoot dry mass was reduced by more than 80 % at 25/16 ºC and 16/25 ºC and less than 30 % at 16/16 ºC. The temperature conditions to which the plants are subjected in the pre- and post-application period interfere with the level of imazethapyr resistance in barnyardgrass.