Crossing abilities of ALS target-site resistant black-grass (Alopecurus myosuroides Huds.)

Black-grass crossings were conducted outdoor for two successive years in a way to determine the percentage of resistant black-grass that can be engendered in one generation, after the introduction of one target-site resistant individual. After germination, plants were genotyped and couples of resistant and susceptible individuals were formed, then planted outdoor and enclosed with a pollen-proof cloth to avoid any external pollen contamination. Seeds were collected and tested in glasshouses with herbicide spraying. DNA analyses were also performed on surviving plants to distinguish hetero- from homozygotes. The resistance level of the progeny seems to be fairly high and Mendelean distribution is generally confirmed. The offspring of self-crossings was also observed to assess the facultative autogamy ability, according to the genotype and the presence of mutant ALS alleles (position W-574).     

Inheritance of fenoxaprop-P-ethyl resistance in a blackgrass (Alopecurus myosuroides Huds.) population

A blackgrass population has developed resistance to fenoxaprop-P-ethyl following field selection with the herbicide for 6 consecutive years. Within this population, 95% of the individuals are also resistant to flupyrsulfuron. Both the inheritance(s) and the mechanism(s) of resistances were investigated by making crosses between the resistant and a susceptible biotype. The inheritance was followed through the F₁ and F₂ generations either by spraying the herbicide on seedlings at the three-leaf stage or using a seedling bioassay, based on coleoptile length. No maternal effects were evident in the fenoxaprop-P-ethyl responses of the F₁ plants, suggesting that the inheritance was nuclear. Some F₁ families treated with fenoxaprop-P-ethyl segregated in a 3:1 (resistant:susceptible) ratio, indicating that the resistance was conferred by two dominant and independent nuclear genes. This was confirmed by the 15:1 (R:S) ratio observed in the F₂ generation treated with fenoxaprop- P-ethyl. The use of selective inhibitors of herbicide de-toxifying enzymes (aminobenzotriazole, pyperonylbutoxide, malathion and tridiphane) with the F₂ plants suggested that each of the two genes may govern two different mechanisms of fenoxaprop-P-ethyl resistance: the ACCase mutation previously postulated and an enhanced herbicide metabolism, mediated by cytochrome P 450 mono-oxygenases (P 450) susceptible to malathion. The P 450 activity may also confer resistance to flupyrsulfuron. This study clearly indicates that two distinct mechanisms of resistance may co-exist in the same plant. Received: 18 August 2000 / Accepted: 6 December 2000     

Field experiences with recent ALS-inhibitors on herbicide resistant blackgrass (Alopecurus myosuroides Huds.)

In the growing season 2002-2003 two field experiments were carried out in winter wheat on the heavy clay soil of the coastal polder area at Zevekote to study the response of blackgrass (Alopecurus myosuroides Huds.) resistant or somewhat less sensitive to a wide variety of herbicides (clodinafop-propargyl, fenoxaprop-P-ethy1; flupyrsulfuron-methyl+metsulfuron-methyl, propoxycarbazone-sodium; isoproturon) representing various modes of action. In Experiment 1, preemergence applications of isoproturon+diflufenican (1500+187.5 g/ha) and isoproturon+diflufenican+flurtamone (1250+100+250 g/ha) respectively were followed in mid-March (Zadoks: 23) by one of the following treatments: none, propoxycarbazone-sodium + vegetable oil (42 g/ha + 1 l/ha), mesosulfuron-methyl + iodosulfuron-methyl-sodium (+mefenpyr-diethyl) + vegetable oil (15+3 (+45) g/ha + 1 l/ha), clodinafop-propargyl (+cloquintocet-mexyl) {60 (+15) g/ha} and flupyrsulfuron-methyl+metsulfuron-methyl (10+5 g/ha). Systems based on clodinafop-propargyl, propoxycarbazone-sodium or flupyrsulfuron-methyl+metsulfuron-methyl resulted in poor supplementary control of blackgrass compared to preemergence herbicide application only. On the contrary, systems based on postemergence application of mesosulfuron-methyl + iodosulfuron-methyl-sodium resulted in excellent control. In most cases the few surviving plants failed to produce inflorescences. In Experiment 2, fall applications in the 3 leaves stage (Zadoks: 13) of prosulfocarb + isoxaben (4000+75 g/ha), flufenacet + diflufenican + isoxaben (240+120+75 g/ha) and flufenacet + pendimethalin + chlorotoluron (180+900+1000 g/ha) respectively were followed in mid-March (Zadoks: 23) by one of the following treatments: none, propoxycarbazone-sodium + vegetable oil (42 g/ha+l l/ha), mesosulfuron-methyl + iodosulfuron-methyl-sodium (+mefenpyr-diethyl) + vegetable oil {15+3 (+45) g/ha + 1 l/ha}, clodinafop-propargyl (+cloquintocet-mexyl) {60 (+15) g/ha}, flupyrsulfuron-methyl + metsulfuron-methyl (10+5 g/ha) and isoproturon+diflufenican (1000+125 g/ha). As in Experiment 1, systems based on clodinafop-propargyl, propoxycarbazone-sodium or flupyrsulfuron-methyl+metsulfuron-methyl resulted in poor additional control of blackgrass compared to herbicide application in the fall only. Comparable poor levels of blackgrass control could be observed with isoproturon+diflufenican. Mesosulfuron-methyl + iodosulfuron-methyl-sodium resulted in excellent control comparable to that recorded in Experiment 1.     

SNP markers for black-grass ( Alopecurus myosuroides Huds.) genotypes resistant to acetyl CoA-carboxylase inhibiting herbicides

Chloroplastic acetyl CoA-carboxylase (ACCase) is the target of widely used, specific graminicide herbicides: cyclohexanediones (CHDs) and aryloxyphenoxypropionates (APPs). Resistance to these compounds is a worldwide, increasing problem. Population genetic studies aimed at understanding the dynamics of this situation and the diffusion of resistance genes within and between weed populations are challenging because biological assays are not adequate for this purpose, and because different mechanisms of resistance confer a similar resistance phenotype. Molecular markers for specifically detecting resistance genes are therefore urgently needed to conduct such studies. For this purpose, we cloned and sequenced the whole gene encoding chloroplastic ACCase in Alopecurus myosuroides Huds. (Black-grass). We identified two point mutations at nucleotide 5,341 that both cause an isoleucine-leucine substitution at position 1,781. Three bi-directional allele-specific PCR assays were developed, each detecting two distinct ACCase alleles with a single PCR reaction. The sensitivity of 1,190 seedlings of A. myosuroides to one CHD and one APP was determined. Genotyping revealed that, although resistant plants were only selected by APPs, the (1,781)Leu ACCase allele is a widespread, dominant gene of resistance to both APPs and CHDs. No other ACCase allele associated with resistance could be identified in this work. Useful applications of allele-specific PCR markers are population genetic studies as well as routine molecular diagnosis of herbicide resistance.     

DNA Analysis of Herbarium Specimens of the Grass Weed Alopecurus myosuroides Reveals Herbicide Resistance Pre-Dated Herbicides

Acetyl-CoA carboxylase (ACCase) alleles carrying one point mutation that confers resistance to herbicides have been identified in arable grass weed populations where resistance has evolved under the selective pressure of herbicides. In an effort to determine whether herbicide resistance evolves from newly arisen mutations or from standing genetic variation in weed populations, we used herbarium specimens of the grass weed Alopecurus myosuroides to seek mutant ACCase alleles carrying an isoleucine-to-leucine substitution at codon 1781 that endows herbicide resistance. These specimens had been collected between 1788 and 1975, i.e., prior to the commercial release of herbicides inhibiting ACCase. Among the 734 specimens investigated, 685 yielded DNA suitable for PCR. Genotyping the ACCase locus using the derived Cleaved Amplified Polymorphic Sequence (dCAPS) technique identified one heterozygous mutant specimen that had been collected in 1888. Occurrence of a mutant codon encoding a leucine residue at codon 1781 at the heterozygous state was confirmed in this specimen by sequencing, clearly demonstrating that resistance to herbicides can pre-date herbicides in weeds. We conclude that point mutations endowing resistance to herbicides without having associated deleterious pleiotropic effects can be present in weed populations as part of their standing genetic variation, in frequencies higher than the mutation frequency, thereby facilitating their subsequent selection by herbicide applications.     

Nucleotide variability at the acetyl coenzyme A carboxylase gene and the signature of herbicide selection in the grass weed Alopecurus myosuroides (Huds.)

Acetyl coenzyme A carboxylase (ACCase) is the target of highly effective herbicides. We investigated the nucleotide variability of the ACCase gene in a sample of 18 black-grass (Alopecurus myosuroides [Huds.]) populations to search for the signature of herbicide selection. Sequencing 3,396 bp encompassing ACCase herbicide-binding domain in 86 individuals revealed 92 polymorphisms, which formed 72 haplotypes. The ratio of nonsynonymous versus synonymous substitutions was very low, in agreement with ACCase being a vital metabolic enzyme. Within black grass, most nonsynonymous substitutions were related to resistance to ACCase-inhibiting herbicides. Differentiation between populations was strong, in contrast to expectations for an allogamous, annual plant. Significant H tests revealed recent hitchhiking events within populations. These results were consistent with recent and local positive selection. We propose that, although they have only been used since at most 15 black-grass generations, ACCase-inhibiting herbicides have exerted a positive selection targeting resistant haplotypes that has been strong enough to have a marked effect upon ACCase nucleotide diversity. A minimum-spanning network of nonrecombinant haplotypes revealed multiple, independent apparitions of resistance-associated mutations. This study provides the first evidence for the signature of ongoing, recent, pesticide selection upon variation at the gene encoding the targeted enzyme in natural plant populations.     

Weed response to herbicides: regional-scale distribution of herbicide resistance alleles in the grass weed Alopecurus myosuroides

Effective herbicide resistance management requires an assessment of the range of spatial dispersion of resistance genes among weed populations and identification of the vectors of this dispersion. In the grass weed Alopecurus myosuroides (black-grass), seven alleles of the acetyl-CoA carboxylase (ACCase) gene are known to confer herbicide resistance. Here, we assessed their respective frequencies and spatial distribution on two nested geographical scales (the whole of France and the French administrative district of C?te d'Or) by genotyping 13 151 plants originating from 243 fields. Genetic variation in ACCase was structured in local populations at both geographical scales. No spatial structure in the distribution of resistant ACCase alleles and no isolation by distance were detected at either geographical scale investigated. These data, together with ACCase sequencing and data from the literature, suggest that evolution of A. myosuroides resistance to herbicides occurred at the level of the field or group of adjacent fields by multiple, independent appearances of mutant ACCase alleles that seem to have rather restricted spatial propagation. Seed transportation by farm machinery seems the most likely vector for resistance gene dispersal in A. myosuroides.     

Reduction of Nitrate and Nitrite in Lambsquarters (Chenopodium album) Biotypes Resistant and Susceptible to Atrazine Toxicity

The nitrite-reducing activity of the normal susceptible biotype of lambsquarters (Chenopodium album L.) was strongly inhibited by atrazine in the assay medium, both in the case of the in vivo assays of leaf discs in light, and in vitro photoreduction assays of crude extracts. In vitro assays of crude extracts with methylviologen or ferredoxin supplying the reducing potential were not inhibited by atrazine. In the resistant biotype, inhibition of nitrite reduction did not occur with any of the above assays. Thus, it appears that atrazine does not inhibit nitrite reductase itself, but rather the availability of photosynthetically supplied electrons for the reduction. Atrazine had no effect when added to the media for either in vivo or in vitro assays of nitrate reduction by either the susceptible or resistant biotype.     

Genetic variation and population structure in black-grass (Alopecurus myosuroides Huds.), a successful, herbicide-resistant, annual grass weed of winter cereal fields

Black‐grass (Alopecurus myosuroides) is an allogamous grass weed common in cereal fields of northern Europe, which developed resistance to a widely used family of herbicides, the ACCase‐inhibiting herbicides. Resistance is caused by mutations at the ACCase gene and other, metabolism‐based, mechanisms. We investigated the genetic structure of 36 populations of black‐grass collected in one region of France (Côte d’Or), using 116 amplified fragment length polymorphism (AFLP) loci and sequence data at the ACCase gene. The samples were characterized for their level of herbicide resistance and genotyped for seven known ACCase mutations conferring resistance. All samples contained herbicide‐resistant plants, and 19 contained ACCase mutations. The genetic diversity at AFLP loci was high (H_T = 0.246), while differentiation among samples was low (F_ST = 0.023) and no isolation by distance was detected. Genetic diversity within samples did not vary with the frequency of herbicide resistance. A Bayesian algorithm was used to infer population structure. The two genetic clusters inferred were not associated with any geographical structure or with herbicide resistance. A high haplotype diversity (H_d = 0.873) and low differentiation (G_ST = 0.056) were observed at ACCase. However, haplotype diversity within samples decreased with the frequency of ACCase‐based resistance. We suggest that the genetic structure of black‐grass is affected by its recent expansion as a weed. Our data demonstrate that the strong selection imposed by herbicides did not modify the genome‐wide genetic structure of an allogamous weed that probably has large effective population sizes. Our study gives keys to a better understanding of the evolution of successful, noxious weeds in modern agriculture.     

Safener responsiveness and multiple herbicide resistance in the weed black-grass (Alopecurus myosuroides)

Safeners enhance the selectivity of graminicidal herbicides such as fenoxaprop ethyl in cereals, by increasing their rates of detoxification in the crop. While studying the selectivity of fenoxaprop ethyl in wheat, we determined that the safeners mefenpyr diethyl and fenchlorazole ethyl also enhanced herbicide tolerance in the competing weed black-grass ( Alopecurus myosuroides ). Fenoxaprop ethyl was detoxified by conjugation with glutathione in both wheat and black-grass, with the resulting metabolites processed to the respective cysteine derivatives, which were then N -glycosylated. In black-grass, these detoxification pathways were only slightly enhanced by safeners, suggesting that metabolism alone was unlikely to account for increased herbicide tolerance. Instead, it was determined that safening was associated with an accumulation of glutathione and hydroxymethylglutathione and enzymes with antioxidant functions including phi and lambda glutathione transferases, active as glutathione peroxidases and thiol transferases respectively. These safener-induced changes closely mirrored those determined in two independent black-grass populations that had acquired multiple herbicide resistance (MHR) in the field. In addition to enhanced glutathione metabolism, both safener treatment and MHR resulted in elevated levels of flavonoids in the foliage of black-grass plants, notably flavone- C -glycosides and anthocyanins. Our results demonstrate that safening in a grass weed is associated with an inducible activation in antioxidant and secondary metabolism which mirrors the biochemical phenotype exhibited in plants that are resistant to multiple classes of herbicides.     

Modification of Herbicide Binding to Photosystem II in Two Biotypes of Senecio vulgaris L

The present study compares the binding and inhibitory activity of two photosystem II inhibitors: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron [DCMU]) and 2-chloro-4-(ethylamine)-6-(isopropyl amine)-S-triazene (atrazine). Chloroplasts isolated from naturally occurring triazine-susceptible and triazine-resistant biotypes of common groundsel (Senecio vulgaris L.) showed the following characteristics. (a) Diuron strongly inhibited photosynthetic electron transport from H₂O to 2,6-dichlorophenolindophenol in both biotypes. Strong inhibition by atrazine was observed only with the susceptible chloroplasts. (b) Hill plots of electron transport inhibition data indicate a noncooperative binding of one inhibitor molecule at the site of action for both diuron and atrazine. (c) Susceptible chloroplasts show a strong diuron and atrazine binding (¹⁴C-radiolabel assays) with binding constants (K) of 1.4 × 10⁻⁸ molar and 4 × 10⁻⁸ molar, respectively. In the resistant chloroplasts the diuron binding was slightly decreased (K = 5 × 10⁻⁸ molar), whereas no specific atrazine binding was detected. (d) In susceptible chloroplasts, competitive binding between radioactively labeled diuron and non-labeled atrazine was observed. This competition was absent in the resistant chloroplasts.     

Purification and cloning of an esterase from the weed black-grass (Alopecurus myosuroides), which bioactivates aryloxyphenoxypropionate herbicides

Carboxyesterases which activate aryloxyphenoxypropionate (AOPP) graminicides to their bioactive herbicidal acids by hydrolysing the respective ester precursors have been identified in black-grass (Alopecurus myosuroides), a problem weed of cereal crops in Northern Europe. The dominant 40 kDa carboxyesterase was purified 1700-fold and identified as a serine hydrolase by affinity labelling with a biotinylated fluorophosphonate suicide substrate. MS-MS sequencing of a peptide digest identified it to be a member of the GDSL family of serine hydrolases. The full-length A. myosuroides hydrolase (Amgdsh1) was cloned by RACE-PCR and expressed in the yeast Pichia pastoris as a secreted enzyme. Expression was associated with activity towards AOPP esters. AmGDSH1 was predicted to be glycosylated and exported to the apoplast in planta. Based on the analysis of related sequences in monocotyledonous plants an alternative classification of the GDSL plant hydrolase superfamily is suggested and their importance in endogenous metabolism and herbicide bioactivation in crops and weeds discussed.     

Differential Responses to the Combined Stress of Heat and Phytophthora capsici Infection Between Resistant and Susceptible Germplasms of Pepper (Capsicum annuum L.)

Journal of Plant Growth Regulation - Stresses caused by heat and Phytophthora capsici infection reduce pepper (Capsicum annuum) yields; however, little is known about their combined effects on...     

花粉管介导的转bar基因水稻植株的获得及其遗传

采用花粉管通道法将bar基因导入籼稻品系E32,得到对除草剂Basta具有抗性的转基因水稻。遗传分析表明外源bar基因在受体植株后代中呈单基因显性遗传,在世代间可以稳定遗传。目前已分离出抗性稳定的株系。     

盐碱胁迫对山葡萄光合特性及生长发育的影响

以棚内盆栽山葡萄品种‘双优’(抗逆性弱)和‘左山一’(抗逆性强)扦插苗为材料,利用气体交换分析和叶绿素荧光诱导动力学分析手段,研究了轻度、中度和重度盐碱胁迫对不同抗逆性山葡萄品种的光合特性及PSⅡ活性以及生长发育的影响,为山葡萄耐盐碱品种的评价提供理论依据。结果显示:(1)‘双优’各胁迫程度下的净光合速率(P_n)、气孔导度(Gs)和重度胁迫下的蒸腾速率(T_r)显著低于对照,而‘左山一’重度胁迫下的Pn和T_r、中重度胁迫下的Gs、各胁迫程度下的胞间二氧化碳浓度(Ci)显著低于对照,净光合速率下降主要归因于气孔限制和非气孔限制。(2)轻度、中度盐碱胁迫‘双优’叶片单位反应中心吸收的光能(ABS/RS)、单位面积内有活性反应中心数目(RS/CS)和吸收光能为基础的性能指数(PIABS),以及重度盐碱胁迫下‘左山一’的ABS/RS、RS/CS、用于电子传递的光能(ETo/CS)均显著低于相应对照,其余均无显著变化。(3)2品种山葡萄植株茎流率及株高、叶片数和根系生物量等生长指标在不同盐碱胁迫水平下均比对照显著降低,并以‘双优’品种降低幅度更大。研究表明,盐碱胁对山葡萄‘左山一’的叶片净光合速率、PSⅡ光能吸收和传递效率以及生长的抑制作用均小于‘双优’,‘左山一’的耐盐碱性更强。     

Membrane Response to Diclofop Acid Is pH Dependent and Is Regulated by the Protonated Form of the Herbicide in Roots of Pea and Resistant and Susceptible Rigid Ryegrass

Electrophysiological studies in roots of pea (Pisum sativum L.) and rigid ryegrass (Lolium rigidum Gaud.) seedlings were conducted to elucidate the mechanism involved in the membrane response to the herbicide diclofop. In pea, a dicotyledonous plant insensitive to diclofop, membrane depolarization at varying pH values and herbicide concentrations increased at higher concentrations of the protonated form of diclofop acid (pKa 3.57). In unbuffered nutrient solution (pH 5.7), diclofop acid (50 [mu]M) depolarized the membrane potential (Em) in roots of both resistant and susceptible biotypes of rigid ryegrass, whereas recovery of Em occurred only in the resistant biotype following removal of the herbicide. This differential response was correlated with an increase (450%) in the rate of acidification of the external solution by the susceptible biotype, and the Em differences between biotypes were eliminated in solutions buffered at pH 5.0 or 6.0. In addition, p-chloromercuribenzene-sulfonic acid did not prevent the depolarization of Em by 50 [mu]M diclofop acid. It is concluded that the differential membrane response to diclofop acid in herbicide-resistant and -susceptible biotypes of rigid ryegrass is due to pH differences at the cell wall/plasmalemma interface. Although the membrane response is probably not involved in the primary inhibitory effect of diclofop on plant growth, it could reduce the concentration of the permeant protonated form of the herbicide and possibly could contribute to increased tolerance to diclofop and other weak acid herbicides.     

外来入侵种黄顶菊及其伴生植物光合特性初步研究

以不同生育期的外来入侵植物黄顶菊及其4种伴生杂草为材料,在实验网室条件下测定比较了它们的基本光合和叶绿素荧光特性,从而探讨黄顶菊具有高生物量及较强入侵性的原因,为其防控提供理论依据.结果表明:(1)5种植物的净光合速率日变化均呈单峰曲线,黄顶菊净光合速率以现蕾期最高,且大于其他4种伴生杂草;(2)3个生长时期的黄顶菊水分利用效率低于棒头草,高于齿果酸模和曼陀罗,而与反枝苋相当,显示较高的抗旱性;(3)影响5种植物光合速率主要因子为非气孔因素,影响黄顶菊以及棒头草、反枝苋等的主要生态因子是光合有效辐射,显示出其较强的喜光特性;(4)5种植物在午间较强的光照条件下均发生了不同程度光抑制,处于现蕾期和开花期的黄顶菊以及其余3种植物午间的Fv/Fm有轻微下降,其PSⅡ实际光化学效率维持在较高的水平.可见,黄顶菊具有较强的光合能力,更适应于在夏季高温干旱的环境下生长,从而表现出较强的入侵性.