Characterization of Non-Target-Site Mechanisms in Imidazolinone-Resistant Sunflower by RNA-seq

Imisun sunflowers (Helianthus annuus L.) are imidazolinone-resistant cultivars in which the two mechanisms of herbicide resistance coexist: (i) mutation in herbicide target-site (target-site resistance) and (ii) non-target-site resistance (NTSR). In Imisun technology, NTSR could be related to herbicide metabolism and might occur as a result of a constitutive up-regulation of resistance genes, or it can appear only after herbicide treatment. The objective of this study was to characterize NTSR in Imisun sunflower in response to imazethapyr using RNA-Seq and to determine whether these mechanisms are constitutive or herbicide-induced. Cypsels were germinated in plastic pots, watered by capillarity and growth in chamber under controlled conditions. Seven-day-old plants were treated with 0 (control) and 1 μM imazethapyr for 12 h. After leaf RNA purification, stranded, paired-end cDNA libraries were constructed. Sequencing was performed with Illumina HiSeq2000. Local mapping, with and without multihits, was carried out over the reference transcriptome HaT13l and differential expression was analysed. Sixty one and 47 contigs (according to mapping strategy) related to xenobiotic metabolism were found: cytochromes P450s, ABC transporters, glycosyltransferases, UDPglucuronosyl/glucosyltransferases and glutathione S-transferases. None of these contigs showed differential expression between control and imazethapyr-treated plants. Seventeen interesting contigs were verified by qRT-PCR. These results suggest that constitutive NTSR mechanisms may account for imidazolinone resistance in Imisun sunflower.

Distribution and analysis of the mechanisms of resistance of barnyardgrass (Echinochloa crus-galli) to imidazolinone and quinclorac herbicides

SUMMARYThe lack of control of barnyardgrass in flooded rice cultivated with imidazolinone-resistant rice cultivars is challenging the utilization of this system, which is continuously expanding for new rice areas worldwide. The objectives of the present study were to evaluate the frequency, distribution and mechanisms of imidazolinone resistance in barnyardgrass to establish the best practices to control and prevent this problem. The distribution of resistance was evaluated in 624 populations collected in Southern Brazil. The frequency of imidazolinone-herbicide resistance was 0·81, broadly distributed in all sampled regions. Resistance to quinclorac was also found in 0·19 of the populations, but all of the evaluated populations were susceptible to cyhalofop-butyl. Further studies were conducted in six populations. The enhanced metabolism was assessed with the metabolic inhibitors that reversed the resistance to quinclorac from 0·54 to 1·00 in two populations and the resistance to imazethapyr from 0·15 to 0·41 in three populations. The acetolactase synthase (ALS) enzyme activity also indicated the occurrence of altered target site resistance in two populations caused by the ALS gene mutations Trp574Leu and Ser653Asn, which is a novel finding in this species. The herbicide resistance in barnyardgrass in Southern Brazil presented a complex basis of resistance because it is associated with resistance to multiple herbicides due to multiple mechanisms and with multiple mutations of the ALS gene. This indicates that it is necessary to adopt specific measures to prevent and control the evolution of multiple herbicide resistance in this species.