BACKGROUND Intensive application of acetolactate synthase (ALS)‐inhibiting herbicides has resulted in herbicide‐resistance in many weeds, including Bromus sterilis. The present study was conducted to identify the mechanisms conferring resistance to ALS‐inhibiting herbicides in a Bromus sterilis biotype. RESULTS Dose–response studies revealed the resistant biotype to be 288 times less sensitive to pyroxsulam than the susceptible biotype. Furthermore, experiment with a single‐dose, proved this biotype was also cross‐resistant to propoxycarbazone, iodosulfuron plus mesosulfuron and sulfosulfuron. Prior treatment with malathion, a known inhibitor of cytochrome P450s, reduced the level of resistance to pyroxsulam. No mutations were detected from the partial ALS gene sequencing. Flow cytometry and chromosome counting rejected ploidy level variation between the susceptible and resistant biotypes. Relative copy number variation ruled out gene amplification. Quantitative real‐time polymerase chain reaction (PCR) detected a significant difference in ALS gene expression between the susceptible and resistant biotypes. CONCLUSIONS Target gene overexpression and enhanced metabolism by cytochrome P450s are likely mechanisms of resistance to pyroxsulam in Bromus sterilis. The current findings highlight the need to monitor additional brome populations for herbicide resistance in Europe and endorse the need for alternate herbicides in integrated weed management to delay the possible evolution of herbicide resistance in these species. © 2020 Society of Chemical Industry

Individuals of Apera spica-venti populations tested in this study possess the target-site ALS resistance mutation and an additional so far unknown resistance mechanism(s).

Bromus sterilis is an annual weedy grass, causing high yield losses in winter cereals. Frequent use of herbicides had led to the evolution of herbicide resistance in this species. Mechanisms underlying herbicide resistance in B. sterilis must be uncovered because this problem is becoming a global threat. qRT-PCR and the next-generation sequencing technologies can elucidate the resistance mechanisms. Although qRT-PCR can calculate precise fold changes, its preciseness depends on the expression of reference genes. Regardless of stable expression in any given condition, no gene can act as a universal reference gene. Hence, it is necessary to identify the suitable reference gene for each species. To our knowledge, there are no reports on the suitable reference gene in any brome species so far. Thus, in this paper, the stability of eight genes was evaluated using qRT-PCR experiments followed by expression stability ranking via five most commonly used software for reference gene selection. Our findings suggest using a combination of 18S rRNA and ACCase to normalise the qRT-PCR data in B. sterilis. Besides, reference genes are also recommended for different experimental conditions. The present study outcomes will facilitate future molecular work in B. sterilis and other related grass species.

Loose silky bent grass (Apera spica-venti (L.) P. Beauv) is a winter outcrossing annual grass, widespread in the Central and East European countries, mainly, in Germany, Poland and the Czech Republic (Soukup et al., 2006). Although, it is also found in North European countries, such as Denmark, Sweden and Lithuania (e.g. Auškalniené et al., 2020). It can be controlled with numerous herbicides, including ureas, dinitroanilines and thiocarbamates. The first case of resistance to ureas was found in Germany in 1996 (Niemann, 2000). Lately,

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