Italian ryegrass (Lolium multiflorum; LOLMU) is one of the most troublesome weeds in temperate regions in the world. This weed species interfere with wheat, corn, rye, and oat, causing significant crop yield losses. This species has evolved glyphosate resistance, making it difficult to control. The mechanisms of glyphosate resistance are still unknown, and an understanding thereof will favor the development of new strategies of management. The present study is the first transcriptome study in LOLMU using glyphosate-resistant and -sensitive biotypes, aiming to identify and to provide a list of the candidate target genes related to glyphosate resistance mechanism. The transcriptome was assembled de novo, producing 87,433 contigs with an N50 of 740 bp and an average length of 575 bp. There were 92 and 54 up- and down-regulated genes, respectively, in the resistant biotype, while a total of 1683 were differentially expressed in the sensitive biotype in response to glyphosate treatment. We selected 14 highly induced genes and seven with repressed expression in the resistant biotype in response to glyphosate. Of these genes, a significant proportion were related to the plasma membrane, indicating that there is a barrier making it difficult for glyphosate to enter the cell.

Conyza bonariensis (hairy fleabane) is one of the most problematic and widespread glyphosate-resistant weeds in the world. This highly competitive weed species significantly interferes with crop growth and substantially decreases crop yield. Despite its agricultural importance, the molecular mechanisms of glyphosate resistance are still unknown. The present RNA-Seq study was performed with the goal of identifying differentially expressed candidate transcripts (genes) related to metabolism-based non-target site glyphosate resistance in C. bonariensis. The whole-transcriptome was de novo assembled from glyphosate-resistant and -sensitive biotypes of C. bonariensis from Southern Brazil. The RNA was extracted from untreated and glyphosate-treated plants at several timepoints up to 288 h after treatment in both biotypes. The transcriptome assembly produced 90,124 contigs with an average length of 777 bp and N50 of 1118 bp. In response to glyphosate treatment, differential gene expression analysis was performed on glyphosate-resistant and -sensitive biotypes. A total of 9622 genes were differentially expressed as a response to glyphosate treatment in both biotypes, 4297 (44.6%) being up- and 5325 (55.4%) down-regulated. The resistant biotype presented 1770 up- and 2333 down-regulated genes while the sensitive biotype had 2335 and 2800 up- and down-regulated genes, respectively. Among them, 974 up- and 1290 down-regulated genes were co-expressed in both biotypes. In the present work, we identified 41 new candidate target genes from five families related to herbicide transport and metabolism: 19 ABC transporters, 10 CYP450s, one glutathione S-transferase (GST), five glycosyltransferases (GT), and six genes related to antioxidant enzyme catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD). The candidate genes may participate in metabolic-based glyphosate resistance via oxidation, conjugation, transport, and degradation, plus antioxidation. One or more of these genes might ‘rescue’ resistant plants from irreversible damage after glyphosate treatment. The 41 target genes we report in the present study may inform further functional genomics studies, including gene editing approaches to elucidate glyphosate-resistance mechanisms in C. bonariensis.

Biochemical assays in a time-course were employed to evaluate stresses induced by glyphosate treatment in resistant and sensitive biotypes of Conyza bonariensis. Two experiments were conducted assessing glyphosate doses and time-course after treatment. The doses of glyphosate ranged from 0 to 11840 g ae·ha^–1 and assessments performed until 552 h after glyphosate treatment (HAT). The objectives of this study were to evaluate the oxidative stress and differential antioxidant enzyme activity in glyphosate-resistant and -sensitive biotypes of hairy fleabane after glyphosate treatment. After treatment, both studied biotypes accumulated similar levels of shikimic-acid until 96 h. The sensitive biotype died at192 HAT. Shikimic-acid and hydrogen peroxide (H₂O₂) accumulation in glyphosate-resistant biotype were transient and did not differ from untreated plants at 288 and 500 HAT, respectively. In both glyphosate-resistant and -sensitive biotypes, a correlation analysis established a cause-and-effect relationship after glyphosate treatment, which leads to shikimic-acid and hydrogen peroxide accumulation, lipid peroxidation (indicates tissue damage) and an enhancement in the activity of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) enzyme. However, in the glyphosate-resistant biotype, the oxidative stress and tissue damage were lower, and antioxidant enzyme activities SOD, CAT, and APX were higher than in the -sensitive biotype. It indicates that antioxidant enzyme in glyphosate-resistant biotype might be related to the glyphosate-resistance process in Conyza bonariensis. This study is the first report of differential antioxidant enzyme activity in hairy fleabane.

Italian ryegrass (Lolium multiflorum Lam.) is a weed broadly found in fields cultivated with wheat and barley crops. Seed inputs into the soil before harvesting winter crops increase seed bank, ensuring survival. This study evaluated the persistence of Italian ryegrass seeds subjected to burial depths over time. Experiments were carried out in the field for two years in randomized block experimental design with four repetitions. Dormant seeds harvested from mother-plants were put in nylon bags containing soil. Previously, seed viability was evaluated in a 1 % tetrazolium solution, and 50 viable seeds by repetition were buried at 0.5, 5.0, 10, and 20 cm depths. Seed persistence was evaluated by the percentage of deteriorated and remaining seeds, non-dormant seeds, abnormal seedlings, and viable and non-viable dormant seeds at 0, 30, 60, 90, 180, 360, 540, and 720 days after burial (DAB). Seed persistence increased at 10 and 20 cm of burial depth compared to seeds in the soil layers up to 5 cm. Moreover, burial depth at 10 and 20 cm showed lower percentage of deteriorated seeds (10 % lower) compared to 0.5 cm at 360 DAB. For non-dormant seeds, a higher percentage was found at 90 DAB, regardless of seed burial depth. Dormancy breakage occurred until 180 DAB, and more rapidly at 10 and 20 cm depths. At 540 DAB, more than 95 % of seeds were unviable, demonstrating short persistence of Italian ryegrass in the soil seed bank.