Characterization of sulfonylurea-resistant Schoenoplectus juncoides having a target-site Asp376Glu mutation in the acetolactate synthase

Schoenoplectus juncoides, a noxious weed for paddy rice, is known to become resistant to sulfonylurea (SU) herbicides by a target-site mutation in either of the two acetolactate synthase (ALS) genes (ALS1 and ALS2). SU-resistant S. juncoides plants having an Asp₃₇₆Glu mutation in ALS2 were found from a paddy rice field in Japan, but their resistance profile has not been quantitatively investigated. In this study, dose–response of the SU-resistant accession was compared with that of a SU-susceptible accession at in vivo whole-plant level as well as at in vitro enzymatic level.     

Varied occurrence of diverse sulfonylurea‐resistant biotypes of Schoenoplectus juncoides [Roxb.] Palla in Japan,as classified by an acetolactate synthase gene mutation

Suspected sulfonylurea (SU)‐resistant Schoenoplectus juncoides plants were collected from rice paddy fields at 24 sites in Japan in order to discover the occurrence pattern of target‐site substitutions on a nationwide scale and at a local field scale. A genetic analysis of the two acetolactate synthase (ALS) genes, ALS1 and ALS2, of the collected plants confirmed that a single‐nucleotide mutation at the Pro₁₉₇, Asp₃₇₆ or Trp₅₇₄ site of either ALS1 or ALS2 existed in each suspected SU‐resistant plant. On a nationwide scale, it was shown that the ALS1 mutations and the ALS2 mutations occurred at a similar frequency, that the P197S and the P197L substitutions were found most frequently among all the substitutions, and that the W574L substitutions (known as global resistance to any ALS‐inhibiting herbicide) were found at a relatively low frequency but in a geographically wide range. In the local field‐scale survey, which was conducted at two sites in Hyogo Prefecture, it was shown that the substitutions were less diverse, compared to on a nationwide scale, probably because the investigation involved a limited number of local fields, and that several substitutions and a susceptible biotype were found in single fields suggesting that a number of collections is required in order to understand the local SU‐resistant status of S. juncoides. In addition, this study reported new findings, that of the P197R, P197T and D376E substitutions in S. juncoides. This set of diverse substitutions in a weed species can be used for further research purposes.     

Biology and mechanisms of sulfonylurea resistance in Schoenoplectiella juncoides,a noxious sedge in the rice paddy fields of Japan

Schoenoplectiella juncoides is a noxious sedge weed in rice paddy fields that has evolved resistance to sulfonylurea (SU) herbicides. The molecular basis of resistance is amino acid substitutions at Pro₁₉₇, Trp₅₇₄ or Asp₃₇₆ in the acetolactate synthase (ALS) enzyme, which is the target of SUs. Schoenoplectiella juncoides has two ALS genes and resistant plants have point mutations that cause amino acid substitutions in either encoded protein. Single‐nucleotide substitutions at the codon for Pro₁₉₇ in the ALS genes can cause six types of amino acid substitutions and all of these substitutions have been found in both ALS genes among Japanese SU‐resistant biotypes. Whole‐plant herbicide responses differ among the amino acid substitution types. Furthermore, analyses of ALS activity in plant extracts show that the extracts’ responses to herbicides differ, depending on which ALS gene is mutated. The activity responses of the ALS extracts to the SU, imazosulfuron, showed double‐sigmoid curves with plateaus of ~30% inhibition for Pro₁₉₇ substitutions in ALS1 and ~70% for Pro₁₉₇ substitutions in ALS2. This indicates that ALS1 and ALS2 contribute to the responses with a proportion of 7:3. The double‐sigmoid curves can be reconstructed to show the responses of the resistant and susceptible enzymes separately by regression analysis. The resistance levels of the separate ALS1 or ALS2 mutated enzyme are highly correlated with the whole‐plant responses, with a relationship that the former is the square of the latter. This could provide a quantitative insight into the physiological basis of resistance.     

Molecular basis of diverse responses to acetolactate synthase-inhibiting herbicides in sulfonylurea-resistant biotypes of Schoenoplectus juncoides

Sulfonylurea-resistant biotypes of Schoenoplectus juncoides were collected from Nakafurano, Shiwa, Matsuyama, and Yurihonjyo in Japan. All of the four biotypes showed resistance to bensulfuron-methyl and thifensulfuron-methyl in whole-plant experiments. The growth of the Nakafurano, Shiwa, and Matsuyama biotypes was inhibited by imazaquin-ammonium and bispyribac-sodium, whereas the Yurihonjyo biotype grew normally after treatment with these herbicides. The herbicide concentration required to inhibit the acetolactate synthase (ALS) enzyme by 50% (I₅₀), obtained using in vivo ALS assays, indicated that the four biotypes were > 10-fold more resistant to thifensulfuron-methyl than a susceptible biotype. The Nakafurano, Shiwa, and Matsuyama biotypes exhibited no or little resistance to imazaquin-ammonium, whereas the Yurihonjyo biotype exhibited 6700-fold resistance to the herbicide. The Nakafurano and Shiwa biotypes exhibited no resistance to bispyribac-sodium, but the Matsuyama biotype exhibited 21-fold resistance and the Yurihonjyo biotype exhibited 260-fold resistance to the herbicide. Two S. juncoides ALS genes (ALS1 and ALS2) were isolated and each was found to contain one intron and to encode an ALS protein of 645 amino acids. Sequencing of the ALS genes revealed an amino acid substitution at Pro₁₉₇ in either encoded protein (ALS1 or ALS2) in the biotypes from Nakafurano (Pro₁₉₇ → Ser₁₉₇), Shiwa (Pro₁₉₇ → His₁₉₇), and Matsuyama (Pro₁₉₇ → Leu₁₉₇). The ALS2 of the biotype from Yurihonjyo was found to contain a Trp₅₇₄ → Leu₅₇₄ substitution. The relationships between the responses to ALS-inhibiting herbicides and the amino acid substitutions, which are consistent with previous reports in other plants, indicate that the substitutions at Pro₁₉₇ and Trp₅₇₄ are the basis of the resistance to sulfonylureas in these S. juncoides biotypes.     

Rapid diagnosis of sulfonylurea‐resistant Schoenoplectus juncoides [Roxb.] Palla using polymerase chain reaction–restriction fragment length polymorphism and isogene‐specific direct sequencing

Rapid diagnostic methods to detect known mutations in acetolactate synthase (ALS) genes that confer sulfonylurea (SU) resistance to Schoenoplectus juncoides were developed in this study. By using 11 SU‐resistant accessions (nine accessions with a Pro₁₉₇ substitution in ALS1 or ALS2, one accession with an Asp₃₇₆Glu substitution in ALS2 and one accession with a Trp₅₇₄Leu substitution in ALS2), polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis for DNA fragments that were amplified simultaneously from genomic ALS1 and ALS2 and PCR–RFLP analysis for DNA fragments that were amplified from either of the genomic ALS1 or ALS2 were carried out. In each of the two PCR–RFLP analyses, a common PCR product was digested separately with the restriction enzymes, BspLI, MboI and MunI, in order to detect Pro₁₉₇ substitutions, an Asp₃₇₆Glu substitution and a Trp₅₇₄Leu substitution, respectively. In each of the lanes where the detection of SU‐resistant substitutions was aimed, a specific band to suggest the existence of the said substitutions was observed in theoretically assumable ways. Separately, a direct sequencing method also was established, which was able to selectively sequence ALS1 or ALS2 from common templates containing both ALS1 and ALS2 by the isogene‐selective primers that were designed to anneal either of the ALS genes. It is expected that these methods could be used for the genetic analysis of SU‐resistant S. juncoides by providing rapid and accurate diagnosis.     

Characterization of acetolactate synthase from sulfonylurea herbicide‐resistant Schoenoplectus juncoides

Ten accessions of sulfonylurea‐resistant Schoenoplectus juncoides were collected from paddy fields in Japan. In order to characterize acetolactate synthase from sulfonylurea‐resistant S. juncoides, acetolactate synthase amino acid substitutions, whole‐plant growth inhibition and acetolactate synthase enzyme inhibition were examined. Schoenoplectus juncoides has two acetolactate synthase genes (ALS1 and ALS2). The sulfonylurea‐resistant accessions harbored amino acid substitutions at Pro197 or Trp574 in either ALS1 or ALS2 (the amino acid number is standardized to the Arabidopsis thaliana sequence). The whole plants of all the sulfonylurea‐resistant accessions showed resistance to imazosulfuron. The resistance level depended on the altered amino acid residues in acetolactate synthase. The acetolactate synthase enzyme that was partially purified from all the sulfonylurea‐resistant accessions was less sensitive to imazosulfuron, compared to the susceptible accession, suggesting that the resistance is related to the altered acetolactate synthase enzyme. In addition, the concentration–response inhibition of acetolactate synthase activity by imazosulfuron in the sulfonylurea‐resistant accessions was remarkably different with the presence of an amino acid substitution in either ALS1 or ALS2. Furthermore, the concentration–response inhibition of acetolactate synthase activity in the sulfonylurea‐resistant accessions with a P197S, P197T or W574L mutation showed a double‐sigmoid curve. The regression analysis of enzyme inhibition suggested that the abundance ratio of ALS1 to ALS2 enzymes was approximately 70:30%, with a range of ±15%. Taken together, these results suggest that the resistance of sulfonylurea‐resistant accessions of S. juncoides is related to altered acetolactate synthase in either ALS1 or ALS2, although the abundance of the altered acetolactate synthase in the plants is different among the sulfonylurea‐resistant accessions.     

Resistance levels of sulfonylurea‐resistant Schoenoplectus juncoides (Roxb.) Palla with various Pro197 mutations in acetolactate synthase to imazosulfuron,bensulfuron‐methyl,metsulfuron‐methyl and imazaquin‐ammonium

An investigation, using herbicidal pot tests in a greenhouse condition, was conducted to determine the whole‐plant dose–response relationships to several acetolactate synthase (ALS)‐inhibiting herbicides of sulfonylurea (SU)‐resistant Schoenoplectus juncoides with various Pro₁₉₇ mutations in ALS that was collected from Japanese rice paddy fields. All the tested SU‐resistant accessions with a Pro₁₉₇ mutation were highly resistant to two commonly used SU herbicides (imazosulfuron and bensulfuron‐methyl), but were much less resistant to another SU herbicide, metsulfuron‐methyl, and were substantially not resistant to imazaquin‐ammonium. These cross‐resistance patterns have been known previously in fragments of S. juncoides and other weed species and were comprehensively confirmed in this study with a whole set of Pro₁₉₇ mutations. The analyses of resistance levels, based on ED₉₀ values, newly showed that different accessions with a common amino acid substitution in ALS1 showed similar responses to these herbicides (confirmed with four amino acid substitutions), that the rankings of resistance levels that were conferred by various Pro₁₉₇ mutations in ALS1 differed among the SU herbicides and that the resistance levels of the ALS2‐mutated accessions were higher than, lower than or similar to those of the corresponding ALS1‐mutated accessions, depending on the compared pair, but the deviation patterns were generally similar among the SU herbicides in each compared pair. The final finding might suggest that the abundance of ALS2 is not as stable as that of ALS1. In addition, as a result of these new findings, together with expected further research, a suggested possibility is that substituting amino acids at Pro₁₉₇ generally could be estimated by plotting each accession's ED₉₀ values of imazosulfuron and bensulfuron‐methyl in a two‐dimensional graph.