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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Although herbicides have been successfully used for controlling weeds, their continuous use has developed in the evolution of resistance to all major herbicide modes of action worldwide. Reports suggest that the members of Poaceae family are more prone to developing herbicide resistance than other families. In plants, epigenetic mechanisms play critical roles by increasing their stress-adaptive potential in a rapidly changing environment. Epigenetic mechanisms involve alteration of the expression of genetic elements, but without any changes in the DNA sequence. Although the possible roles of epigenetic mechanisms in contributing to survival and fitness under various stresses are well documented in model plants and crops, their contribution to herbicide resistance in weeds is still in its infancy. A few studies with herbicides have shown differential expression of DNA methyltransferases, histone methyltransferases and DNA demethylases in response to the herbicides; however, no further studies were conducted. In the case of herbicide stress, exploring how these epigenetic processes affect the gene expression pattern in individual plants subjected to recurrent selection would be exciting. Hence, our mini-review will focus on the potential contributions of epigenetic mechanisms to the adaptive responses of grass-weedy species to herbicide stress. A better understanding of these epigenetic changes will add novel perceptions to our knowledge of herbicide resistance evolution in weeds enabling the development of herbicides with novel targets.

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 contribute to elucidation of 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 were evaluated using qRT-PCR experiments followed by expression stability ranking via five most commonly used softwares 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.

With the worldwide increase of herbicide-resistant weed populations, there is a need for fast and reliable screening methods for early detection of herbicide-resistant weeds. Several tests on herbicide resistance in weeds exist (Beckie et al. 2000). Until today, the most common method to detect herbicide-resistant weed plants are timeconsuming and labour-intensive greenhouse bioassays. The major disadvantage of this test is that the results become available only for the next growing season. One test that was developed to overcome this limitation was the Syngenta rapid in-season quick test (RISQ) (Kaundun et al. 2011). Here, seedlings are transplanted to herbicide-treated agar and the survivorship is recorded after 10 days. It is a reliable method for testing herbicide resistance in weeds for post-emergent herbicides (Brosnan et al. 2017). While the RISQ provides information if a plant is resistant or not, it does not give estimation about the level of resistance.To receive information about the resistance level of a weed population, the experimental setup of the RISQ was combined with an assessment of the maximum quantum efficiency of photosysDetecting herbicide-resistant Apera spica-venti with a chlorophyll fluorescence agar test