Herbicide resistance of weeds poses a threat to agricultural production, therefore, a particular emphasis on the causes of the emergence of this phenomenon is taken. It is essential in order to establish effective weed management procedures. One of the approaches aimed at explaining the herbicide resistance mechanisms is the analysis of the sequences of the herbicide target enzymes. TSR mechanism was scarcely examined in C. cyanus, in which resistance to ALS inhibitors has been detected in Poland [2,3]. Here, we have undertaken to analyse and compare plants belonging to two susceptible to ALS inhibitor (tribenuron-methyl) and three resistant biotypes of C. cyanus. Their susceptibility to the herbicide was assessed by the determination of ED50. The measurement of an effective dose of the tribenuron-methyl causing 50% loss in plant biomass showed that the low doses of the a.i. such as 4.73 g h-1 and 6.08 g h-1 were necessary for S1 and S2 biotypes biomass reduction, respectively. Whereas, the application of the 16 N dose of the a. i. on all plants of resistant biotypes was insufficient for ED50 determination. The dose of 16 N did not cause visible signs of the herbicide treatment.
To determine ALS nucleotide and amino acid sequences of C. cyanus, a pair of primers was designed based on the ALS sequences from Asteraceae plant species deposited in GenBank. ALS nucleotide sequence amplification resulted in the generation of 1699 to 1708 bp fragments that encompass amino acids from 109 to 663 of ALS protein sequence (according to Arabidopsis thaliana amino acid numbering in ALS sequence, accession number: P17597). An involvement of P197 mutation in TSR against sulfonylurea herbicides in C. cyanus was previously indicated, however, no more detailed results were presented [9]. The amplified sequences were sufficient to screen for the presence of mutations that were found to be involved in resistance development to ALS inhibitors in other weed species (A122, P197, A205, D376, R377, W574, S653, and G654)[4].
To the analyses, two susceptible and three resistant to tribenuron-methyl biotypes were taken. Four plants from each biotype and 3 plasmids from each plant were analysed. Nucleotide sequence analysis revealed high variability between the obtained sequences. Different lengths of the analysed fragments were found as well, which was the effect of the presence of three-nucleotide indels in the sequence fragments located between ALS functional regions as shown in Figure 1. Moreover, changes of the nucleotides at multiple positions within the analysed fragments were observed. Overall, out of the obtained 60 nucleotide sequences from all plasmids, there were 27 different sequence variants including 3 sequences that were found in plasmids obtained from both susceptible and resistant plants. Of these 27 different sequence variants, 8 were unique for susceptible plants, whereas, 16 – for the resistant ones. Totally, at 146 positions within the nucleotide sequences, synonymous changes were found, whereas, at 71 positions, nucleotide changes resulted in the changes to other amino acids. The majority of the differences in amino acid sequences were present in both susceptible and resistant biotypes, which indicates that their significance in herbicide resistance emergence may not be vital. However, 8 mutations (L179I, S314T, N404R, I468V, T475M, V525I, A605D, and L621) that were located in the functional regions of ALS were found only in the resistant plants (Figures 2-4). Six of these amino acid changes were present in 1 or 2 plasmids (out of 3) from certain plants, while mutations N404R and V525I were found in 1 out of 3 plasmids in 4 resistant plants, which implies their heterozygosity. Additionally, these two changes were identified in the same plasmids simultaneously. No previously reported amino acid mutations in the ALS sequence associated with the herbicide resistance in other weed species [4,10] were found. Also, the presence of P197 mutation, which was suggested to be present in one resistant biotype in Poland [9], was not confirmed. P197 mutation is one of the most commonly identified amino acid substitutions, and together with A205 is considered to confer sulfonylurea-specific resistance [10]. N404R and V525I mutations constitute novel changes within ALS amino acid sequence in biotypes resistant to ALS inhibitors, therefore, more detailed studies involving numerous samples should be carried out. The analysed nucleotide sequences were deposited in GenBank under accession numbers MZ561651-MZ561687.
In the case of 7 plants out of 20, all 3 sequences (obtained from 3 plasmids from the same plant) were the same, but within the same biotype, the sequences derived from different plants significantly differed. In some cases, the sequencing resulted in the identification of 3 divergent sequences from one plant. Such a high number of polymorphisms in ALS nucleotide sequence was also observed in other Asteraceae family species, namely in Ambrosia artemisiifolia L.[11,12], as well as, in other plant families and species such as Alopecurus aequalis [13] or Zea mays [14]. The reason for such differences in the obtained sequences may be copy number variation (CNV). Multiple gene copies may increase the effective dosage of a gene, which may influence the phenotype [15]. This mechanism was described in the context of the evolution of Amaranthus palmeri resistance to glyphosate. It was revealed that A. palmeri resistance to glyphosate was driven by the elevated 5-enolpyruvylshikimate-3-phosphate synthase gene copy number, followed by increased EPSPS transcript and protein levels along with enhanced glyphosate dose survival rate [16]. In the case of C. cyanus high variability between ALS sequences was observed in both biotypes, susceptible and resistant to tribenuron-methyl, therefore, the possibility of copy number variation involvement in resistance emergence cannot be excluded, but also cannot be confirmed. This phenomenon can be explained by the natural variability of ALS. High genetic variability of C. cyanus plants was confirmed in the analysis of ten microsatellite markers where high polymorphism was detected [17]. Another study concerning the analysis of leaf isozyme markers also highlighted the high genetic diversity of C. cyanus populations [18]. It should be noted, that despite low levels of genetic differentiation between populations, fine-scale spatial genetic structure was observed within populations [17].
To sum up, our study revealed high variability in the obtained ALS nucleotide as well as amino acid sequences within and between the analysed plants. Multiple changes were observed both in susceptible and resistant to tribenuron-methyl biotypes, therefore this phenomenon can be treated as the natural variability in this species. Few mutations were found only in resistant plants, among which N404R and V525I were observed in 4 plants but no mutations previously associated with conferring resistance to ALS inhibitors were observed. Currently, the connection between the found mutations and their contribution to the herbicide resistance emergence is hard to prove and thus it requires further analyses to confirm the presence of these mutations in a greater number of biotypes and plants. Owing to the fact that there has been scarce information about the ALS sequence of susceptible and resistant C. cyanus biotypes, these studies provide the first comparative analysis between susceptible and resistant C. cyanus plants, which may be useful for future studies concerning herbicide resistance of this plant. Moreover, our work also confirms high genetic variability in this species.