耳叶水苋对乙酰乳酸合成酶抑制剂的抗性及其分子机制初探

近年来长江下游地区稻田耳叶水苋Ammannia arenaria H.B.K.危害十分严重。采用盆栽法首次测定了耳叶水苋对苄嘧磺隆等药剂的抗性水平,同时分析了其抗性和敏感种群间乙酰乳酸合成酶(ALS)基因的DNA序列及其RNA表达差异。结果表明:采自浙江嘉兴(JX110)、江苏苏州(JS039)、浙江宁波(NB0143-05)和安徽广德(AH014)的耳叶水苋生物型对苄嘧磺隆的抗性指数(RI)分别为67.90、17.59、44.63和8.37,对苄嘧磺隆表现出中高水平抗性的生物型对五氟磺草胺、双草醚及咪唑乙烟酸也产生了低水平的抗性。获得了耳叶水苋ALS基因全长核苷酸序列2235 bp,编码667个氨基酸,仅发现NB0143-05等3种抗性生物型ALS酶的氨基酸序列非保守区第93位的亮氨酸被脯氨酸取代。然而,NB0143-05的ALS酶对ALS抑制剂的敏感性大幅度降低(RI 37.04),且在苄嘧磺隆处理后4 d的ALS基因表达量是敏感生物型(HZ001)的1.86倍。这表明,ALS酶对药剂的敏感性降低以及被苄嘧磺隆诱导后ALS基因表达量显著增加,很可能是耳叶水苋生物型NB0143-05对ALS抑制剂产生抗性的原因。     

A molecular assay for the proactive detection of target site-based resistance to herbicides inhibiting acetolactate synthase in Alopecurus myosuroides

Acetolactate synthase (ALS) inhibitors are the most resistance‐prone herbicide group. Rapid resistance diagnosis is thus of importance for their optimal use. We formulate rules to use the derived cleaved amplified polymorphic sequence method to develop molecular tools detecting a change at a given codon, the nature of which is unknown. We applied them to Alopecurus myosuroides (black grass) to develop assays targeting ALS codons A122, P197, A205, W574 and S653 that are crucial for herbicide sensitivity. These assays detected W574L or P197T, or both substitutions, in most plants analysed from a field where ALS inhibitors failed after 3 years of use. Similar assays can easily be set up for any species. Given the rapidity of selection for resistance to ALS inhibitors, these assays should be very useful in proactive herbicide resistance diagnosis.     

Target-site basis for resistance to acetolactate synthase inhibitor in Water chickweed (Myosoton aquaticum L.)

Water chickweed is a widespread and competitive winter annual or biennial weed of wheat in China. One Water chickweed population (HN02) resistant to several acetolactate synthase (ALS) inhibitors was found in Henan province of China. Whole-plant bioassays showed that HN02 was high resistance to tribenuron (292.05-flod). In vitro ALS assays revealed that resistance was due to reduced sensitivity of the ALS enzyme to tribenuron. The I₅₀ value for HN02 was 85.53 times greater respectively than that of susceptible population (SD05). This altered ALS sensitivity in the resistant population was due to a mutation in the ALS gene resulting in a Pro197 to Ser substitution. Cross-resistance experiments indicated that HN02 exhibited various resistance patterns to pyrithiobac-sodium, florasulam and pyroxsulam, without resistance to imazethapyr. This is the first report of tribenuron-resistant Water chickweed in Henan province of China, target-site based resistance was established as being due to an insensitive form of ALS, resulting from a Pro to Ser substitution at amino acid position 197 in the ALS gene.     

Resistance of Raphanus raphanistrum to chlorsulfuron in the Republic of South Africa

Herbicide resistance poses a substantial threat to the agricultural industry throughout the world and during the past decade several reports regarding herbicide resistance have been published. Raphanus raphanistrum L., from two wheat farms located in the winter rainfall region of South Africa, showed indications of resistance to chlorsulfuron. Seeds from these suspected resistant biotypes as well as seeds from a susceptible biotype were collected and transported to the ARC-Small Grain Institute for herbicide resistance studies. Herbicides registered for R. raphanistrum control, i.e. chlorsulfuron, MCPA and bromoxynil, were used in this study. Significant differences in the degree of control were found between the susceptible and two resistant biotypes, when treated with chlorsulfuron. The LD₅₀ values for the resistant biotypes (WR 1 & WR 2) were 45 and 11.3 g a.i. ha^–1, respectively, whereas the LD₅₀ value for the susceptible biotype was 5.6 g a.i. ha^–1. The almost eightfold difference between the susceptible and resistant biotype (WR 1), indicated that resistance has developed to chlorsulfuron. Only twofold resistance was established between the other resistant biotype (WR 2) and the susceptible biotype. Significant differences between herbicide rates were also established with the MCPA and bromoxynil experiments. No significant difference could, however, be found between the susceptible and resistant biotypes when treated with MCPA and bromoxynil, indicating the importance of different modes of action of herbicide as a strategy to prevent herbicide resistance.     

Cross‐resistance of Echinochloa species to acetolactate synthase inhibitor herbicides

This study was conducted to evaluate the cross‐resistance of acetolactate synthase (ALS) inhibitors with different chemistries, specifically azimsulfuron (sulfonylurea), penoxsulam (triazolopyrimidine sulfonanilide) and bispyribac‐sodium (pyrimidinyl thio benzoate), in Echinochloa oryzicola and Echinochloa crus‐galli that had been collected in South Korea and to investigate their herbicide resistance mechanism. Both Echinochloa spp. showed cross‐resistance to the ALS inhibitors belonging to the above three different chemistries. In a whole plant assay with herbicides alone, the resistant/susceptible ratios for azimsulfuron, penoxsulam and bispyribac‐sodium were 12.6, 28.1 and 1.9 in E. oryzicola and 21.1, 13.7 and 1.8 in E. crus‐galli, respectively. An in vitro ALS enzyme assay with herbicides showed that the I ₅₀‐values of the resistant accessions were approximately two‐to‐three times higher than the susceptible accessions, with no statistical difference, suggesting that the difference in ALS sensitivity cannot explain ALS inhibitor resistance in Echinochloa spp. for azimsulfuron, penoxsulam and bispyribac‐sodium. A whole plant assay with fenitrothion showed that the GR ₅₀‐values significantly decreased in both the resistant E. oryzicola and E. crus‐galli accessions when azimsulfuron, penoxsulam and bispyribac‐sodium were applied with the P450 inhibitor, while no significant decrease was observed in the susceptible accessions when the P450 inhibitor was used. Thus, these results suggest that ALS inhibitor cross‐resistance for azimsulfuron, penoxsulam and bispyribac‐sodium is related to enhanced herbicide metabolism.     

Physiological and molecular basis for ALS inhibitor resistance in Bromus tectorum biotypes

Primisulfuron‐resistant (AR and MR) and ‐susceptible (AS and MS) Bromus tectorum biotypes were collected from a Poa pratensis field at Athena, Oregon, and in research plots at Madras, Oregon. Studies were conducted to characterize the resistance of the B. tectorum biotypes. Whole plant bioassay and acetolactate synthase (ALS) enzyme assay revealed that the AR biotype was highly resistant to the sulfonylurea (SU) herbicides, primisulfuron and sulfosulfuron and to a sulfonylaminocarbonyltriazolinone (SCT) herbicide, propoxycarbazone‐sodium. However, the AR biotype was not resistant to imazamox, an imidazolinone (IMI) herbicide. Results of the whole plant bioassay studies showed that the MR biotype was moderately resistant to all ALS inhibitors tested. However, there were no differences in ALS sensitivities between the MR and MS biotypes. The nucleotide and amino acid sequence analysis of the als gene demonstrated a single‐point mutation from C to T, conferring the exchange of the amino acid proline to serine at position 197 in the AR biotype. However, this mutation was not found in the MR biotype. Results of this research indicate that: the resistance of the AR biotype to SU and SCT herbicides is based on an altered target site due to a single‐point mutation; resistance in the MR biotype is not due to a target site mutation.     

Molecular basis of multiple resistance to ACCase-inhibiting and ALS-inhibiting herbicides in Lolium rigidum

Herbicide resistance in Lolium rigidum is widespread across much of the agricultural land in Australia. As the incidence of herbicide resistance has increased, so has the incidence of multiple herbicide resistance. This reduces the herbicide options available for control of this weed. This study reports on the successful amplification and sequencing of the acetolactate synthase (ALS) gene of L. rigidum using primers designed from sequence information of related taxa. This enables, for the first time, the successful determination of a mutation in the ALS gene of this species that provides resistance to ALS‐inhibiting herbicides. This mutation causes amino acid substitution at Trp574 (numbering standardised to Arabidopsis thaliana) to Leu which had been reported to confer a high level of resistance against all classes of ALS inhibitor herbicides. In addition, multiple resistance to ALS‐inhibiting and acetyl‐coenzyme A carboxylase‐inhibiting herbicides is acquired through the independent accumulation of mutant alleles for the target sites. This may thus explain some of the irregular, mosaic resistance patterns that occur in this predominantly outcrossing species.     

Sulfometuron-resistant Amaranthus retroflexus: cross-resistance and molecular basis for resistance to acetolactate synthase-inhibiting herbicides

A biotype of Amaranthus retroflexus L. is the first weed in Israel to develop resistance to acetolactate synthase (ALS)-inhibiting herbicides. The resistant biotype (Su-R) was collected from Ganot, a site that had been treated for more than 3 consecutive years with sulfometuron-methyl + simazine. On the whole-plant basis, the resistance ratio ( ED₅₀ Su-R)/( ED₅₀ Su-S) was 6–127 for sulfonylureas, 4–63 for imidazolinones, 20–35 for triazolopyrimidines and 11 for pyrithiobac-sodium. Similar levels of resistance were found also when the herbicides were applied before emergence. Based on a root elongation bioassay, Su-R was 3240-fold more resistant to sulfometuron-methyl than Su-S. In vitro studies have shown that the Su-R biotype was resistant at the enzyme level to all ALS inhibitors tested. The nucleotide sequences of two amplified regions between the Su-S and the Su-R differed in only one nucleotide. One substitution has occurred in domain A, cytosine by thymine (C C C to C T C) at position 248, that confers an exchange of the amino acid proline in the susceptible to leucine in the Su-R. The proline to leucine change in domain A is the only difference in the amino acid primary structure of the regions sequenced, indicating that it is responsible for the ALS-inhibitor resistance observed.     

非转基因抗除草剂玉米对三种乙酰乳酸合成酶抑制剂类除草剂的田间抗性表现

为评估中国农业大学培育的非转基因抗除草剂玉米品系958R和335R在大田条件下对乙酰乳酸合成酶（acetolactate synthase,ALS）抑制剂类除草剂的抗性表现,利用甲咪唑烟酸、砜嘧磺隆、唑嘧磺草胺3种除草剂对郑单958、958R、先玉335、335R共4种玉米杂交种进行了播后苗前土壤处理,每种除草剂设3个处理剂量（1、3、9倍推荐剂量）,并于施药2周和4周后进行株高测定,于收获晾干后测产。结果表明,在甲咪唑烟酸216、648 g（a.i.）/hm²处理下,郑单958和先玉335均已绝产,而958R和335R产量均未受影响;在砜嘧磺隆或唑嘧磺草胺高剂量处理下,常规玉米品种郑单958和先玉335株高的最高降幅分别为25.7%和35.2%,田间药害反应显著,而958R和335R则抗性反应显著。研究表明,非转基因抗除草剂玉米杂交种具有良好的田间抗性,不仅能有效解决玉米田砜嘧磺隆和唑嘧磺草胺等ALS除草剂的药害问题,还能够通过引入甲咪唑烟酸等新的ALS除草剂更好地防除玉米田的杂草。     

荠菜对乙酰乳酸合成酶抑制剂类除草剂的抗性水平及其分子机制

为明确荠菜种群对苯磺隆的抗性水平及其靶标抗性产生的分子机制,采用整株水平测定法测定了荠菜对苯磺隆及其他5种乙酰乳酸合成酶（ALS）抑制剂类除草剂的抗性水平,同时扩增和比对了荠菜抗性和敏感种群之间ALS基因的差异。结果显示:与敏感种群15-ZMD-1相比,抗性种群15-ZMD-5对苯磺隆产生了高水平抗性,抗性倍数为219.6;15-ZMD-5种群不同单株中共存在3种突变方式,分别为ALS基因197位点脯氨酸（CCT）突变为亮氨酸（CTT）、574位点色氨酸（TGG）突变为亮氨酸（TTG）以及单株同时发生上述197和574位点的氨基酸突变。15-ZMD-5抗苯磺隆种群对嘧草硫醚、啶磺草胺和氟唑磺隆均产生了高水平的交互抗性,抗性倍数分别为41.2、79.3和87.8;对双氟磺草胺和咪唑乙烟酸产生了低水平的交互抗性,抗性倍数分别为8.5和5.6。分析表明,荠菜抗性种群ALS基因发生的氨基酸突变可能是导致其对ALS抑制剂类除草剂产生抗性的重要原因之一。     

Molecular basis of resistance to acetolactate synthase-inhibiting herbicides in Sisymbrium orientale and Brassica tournefortii

Three Australian Sisymbrium orientale and one Brassica tournefortii biotypes are resistant to acetolactate synthase (ALS)-inhibiting herbicides due to their possession of an ALS enzyme with decreased sensitivity to these herbicides. Enzyme kinetic studies revealed no interbiotypic differences within species in K_m (pyruvate) (the substrate concentration at which the reaction rate is half maximal) but a greater V_max (the rate when the enzyme is fully saturated with substrate) for two of the resistant S orientale biotypes over susceptible levels. F₁ hybrids from reciprocal crosses between resistant and susceptible biotypes of S orientale showed an intermediate response to chlorsulfuron compared to the parental plants. ALS herbicide resistance in S orientale segregated in a 3:1 (resistant:susceptible) ratio in F₂ plants with a single rate of chlorsulfuron, indicating that resistance is inherited as a single, incompletely dominant nuclear gene. Two regions of the ALS structural gene known to vary in ALS-resistant biotypes were amplified and sequenced. Resistant S orientale biotypes NS01 and SS03 contained a single nucleotide substitution in Domain B, predicting a Trp (in susceptible) to Leu (in resistant) amino acid change. Two adjacent nucleotide substitutions (CC T to AT T) predicting a Pro (in susceptible) to Ile (in resistant) change in the primary amino acid sequence were identified in Domain A of resistant S orientale biotype SS01. Likewise, a single nucleotide substitution at the same site in the resistant B tournefortii biotype predicts a Pro (in susceptible) to Ala (in resistant) substitution. No other interbiotypic nucleotide differences predicted amino acid changes in the sequenced regions, suggesting that the amino acid substitutions reported above are responsible for resistance to ALS-inhibiting herbicides in the respective biotypes. © 1999 Society of Chemical Industry     

Discovery of single-nucleotide mutations in acetolactate synthase genes by Ecotilling

TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful reverse genetic technique that employs a mismatch-specific endonuclease to discover induced point mutations in genes of interest. The use of the TILLING technique to survey natural variation in genes is called Ecotilling. We report an adaptation of Ecotilling for rapid detection of single-nucleotide mutations in the acetolactate synthase (ALS) genes of sulfonylurea (SU)-resistant (R) Monochoria vaginalis (Pontederiaceae), a paddy weed, in Japan. Genomic DNA of a SU-R plant (target DNA) was mixed with the DNA of a SU-susceptible (S) plant (reference DNA). Ecotilling detected two nucleotide mutations in the ALS gene of SU-R M. vaginalis. These 2 mutations were confirmed by DNA sequencing. A single nucleotide mutation (C to A), in the codon CCT to CAT and another mutation (C to T), in the codon CCT to TCT were identified by sequencing. Both mutations result in the disruption of a Pro codon in the conserved Domain A region with the consequent substitution of a His residue in the first mutation and a Ser residue in the second. Substitution of the Pro residue in Domain A of the ALS gene has been reported to result in insensitivity to SUs in many weed biotypes. This study demonstrates that Ecotilling is a fast, reliable, economical method for detecting single-nucleotide mutations in genes arising from herbicide selection.     

Resistance of dicot weeds to acetolactate synthase (ALS)-inhibiting herbicides in Australia

A biotype of Sonchus oleraceus L. and two bio types of Sisymbrium orientate Torn., SSO 3 and NSO 1, are the first dicot weeds in Australia to develop resistance to ALS-inhibiting herbicides. The resistant biotypes had been exposed to va rying periods of selection with sulfonylurea her bicides. All three biotypes are resistant to a range of sulfonylurea and imidazolinone herbicides. The S. orientale biotypes are also resistant to the triazolopyrimidine herbicide, flumetsulam. LD₅₀ ratios of resistant Sonchus oleraceus for sulfony lurea and imidazolinone herbicides are greater than 64-fold and 4.5-fold, respectively. GR₅₀ ratios are greater than 9 for sulfonylureas and 7.4 for imazapyr. The LD₅₀ ratios for both S. orien tale biotypes for chlorsulfuron, sulfometuron methyl, metsulfuron-methyl, flumetsulam and imazethapyr are greater than 110-, 15-, 7-, 24- and 29-fold, respectively. All resistant biotypes are susceptible to MCPA, diuron and diflufenican, herbicides which do not inhibit ALS.