Molecular Mechanisms Endowing Cross-resistance to ALS-Inhibiting Herbicides in Amaranthus hybridus from Argentina

Amaranthus hybridus L. is one of the most problematic weeds in summer crops in Argentina. However, 20 years after the detection of the first case of resistance to ALS-inhibiting herbicides in this country, no extensive reports of the molecular mechanisms endowing resistance were published. In this work, we sequenced the acetolactate synthase gene of resistant plants belonging to five different populations of A. hybridus from Santa Fe and Cordoba provinces. We found that every population presented at least one of the previously documented substitutions W574L and D376E in ALS amino acid sequence. These results explain the cross-resistance to ALS-inhibiting herbicides and should alert about the usage of herbicides with a different site of action after an ineffective control of this species. This is the first report of these target-site mechanisms endowing resistance to ALS-inhibiting herbicides in A. hybridus populations from Argentina.

     

Distinct non-target site mechanisms endow resistance to glyphosate,ACCase and ALS-inhibiting herbicides in multiple herbicide-resistant <Emphasis Type="Italic">Lolium rigidum</Emphasis>

This study investigates mechanisms of multiple resistance to glyphosate, acetyl-coenzyme A carboxylase (ACCase) and acetolactate synthase (ALS)-inhibiting herbicides in two Lolium rigidum populations from Australia. When treated with glyphosate, susceptible (S) plants accumulated 4- to 6-fold more shikimic acid than resistant (R) plants. The resistant plants did not have the known glyphosate resistance endowing mutation of 5-enolpyruvylshikimate-3 phosphate synthase (EPSPS) at Pro-106, nor was there over-expression of EPSPS in either of the R populations. However, [¹⁴C]-glyphosate translocation experiments showed that the R plants in both populations have altered glyphosate translocation patterns compared to the S plants. The R plants showed much less glyphosate translocation to untreated young leaves, but more to the treated leaf tip, than did the S plants. Sequencing of the carboxyl transferase domain of the plastidic ACCase gene revealed no resistance endowing amino acid substitutions in the two R populations, and the ALS in vitro inhibition assay demonstrated herbicide-sensitive ALS in the ALS R population (WALR70). By using the cytochrome P450 inhibitor malathion and amitrole with ALS and ACCase herbicides, respectively, we showed that malathion reverses chlorsulfuron resistance and amitrole reverses diclofop resistance in the R population examined. Therefore, we conclude that multiple glyphosate, ACCase and ALS herbicide resistance in the two R populations is due to the presence of distinct non-target site based resistance mechanisms for each herbicide. Glyphosate resistance is due to reduced rates of glyphosate translocation, and resistance to ACCase and ALS herbicides is likely due to enhanced herbicide metabolism involving different cytochrome P450 enzymes.     

长期稻鸭共作对稻田杂草群落组成及物种多样性的影响

2000~2003年连续4年研究了稻鸭共作条件下田间杂草群落的特征及其动态变化规律。结果表明,在长期稻鸭共作条件下,田间杂草密度逐年降低,下降趋势符合阻滞模型y=k+a·e^bx,模型参数b反映了杂草种群的下降速率。在稻田6种主要杂草中,水虱草(Fimbristylis miliaceae)、陌上菜(Lindernia procumbens)、丁香蓼(Ludwigia prostrata)种群数量降低较快,鸭舌草(Monochoria vaginalis)、异型莎草(Cyperus difformis)次之,稗(Echinochloa crusgalli)最慢。稻鸭共作使稻田杂草群落的物种多样性持续降低,群落均匀度提高,群落相似性与稻鸭共作前相比逐年降低。说明稻鸭共作改变了田间杂草的群落结构,有利于限制杂草的发生危害。随着稻鸭共作的连年进行,对田间杂草的控制效果逐渐上升,4年后达99%以上。稻鸭共作是稻田替代化学除草的一种非常有效的生物、生态控草措施,具有显著的经济和生态效益。     

Cross-Resistance of a Chlorsulfuron-Resistant Biotype of Stellaria media to a Triazolopyrimidine Herbicide

A biotype of Stellaria media (L.) Vill. has been identified that is highly resistant to the herbicide chlorsulfuron. Resistance is due to an altered acetolactate synthase (ALS) that is much less sensitive to chlorsulfuron than the ALS from the susceptible (S) biotype. The S biotype was extremely sensitive to D489 (N-[2,6-dichlorophenyl]-5,7-dimethyl-1,2,4-triazolo[1,5a] pyrimidine-2-sulfonamide), a member of a new class of triazolopyrimidine herbicides, while the chlorsulfuron-resistant biotype exhibited complete cross-resistance at both the whole plant and enzyme levels. ALS activity of the S biotype was reduced by approximately 90% in the presence of 0.1 micromolar D489, while that of the R biotype was reduced by less than 10%. This result suggests that the two herbicides share a common binding site on ALS. Only very slight cross-resistance at the ALS level was found to imazamethabenz, an imidazolinone herbicide.     

Molecular basis for multiple resistance to acetolactate synthase-inhibiting herbicides and atrazine in<Emphasis Type="Italic"> Amaranthus blitoides</Emphasis> (prostrate pigweed)

Amaranthus blitoides S. Watson (prostrate pigweed) populations resistant to acetolactate synthase (ALS; EC 4.1.3.18)-inhibiting herbicides and triazines (SuR/TR) were found in Israel. The Ganot population was 6- to 790-fold more resistant to ALS inhibitors than the wild type due to an altered target site. Molecular analyses showed that the Ganot population was a mixture of two biotypes: (i) SuRA/TR in which domain A of the als gene differed in one nucleotide, resulting in substitution of Pro by Ser 188; (ii) SuRB/TR in which a mutation in domain B led to a substitution of Trp by Leu 569. The mutation in domain A resulted in resistance to all ALS inhibitors except imidazolinones, whereas the mutation in domain B led to resistance to all ALS inhibitors tested. SuRA/TR and SuRB/TR are multiple-resistant with an additional single mutation in the plastidic psbA gene that changes Ser 264 to Gly in the D1 protein, leading to triazine resistance. It is evident that plants within a population exposed to a similar selection pressure may show different patterns of cross-resistance due to three different point mutations. This unique phenomenon renders planning of rational weed management difficult or even impossible.     

Molecular and biochemical characterization of an induced mutation conferring imidazolinone resistance in sunflower

A partially dominant nuclear gene conferring resistance to the imidazolinone herbicides was previously identified in the cultivated sunflower (Helianthus annuus L.) line CLHA-Plus developed by seed mutagenesis. The objective of this study was to characterize this resistant gene at the phenotypic, biochemical and molecular levels. CLHA-Plus showed a complete susceptibility to sulfonylureas (metsulfuron, tribenuron and chlorsulfuron) but, on the other hand, it showed a complete resistance to imidazolinones (imazamox, imazapyr and imazapic) at two rates of herbicide application. This pattern was in close association with the AHAS-inhibition kinetics of protein extracts of CLHA-Plus challenged with different doses of imazamox and chlorsulfuron. Nucleotide and deduced amino acid sequence comparisons between resistant and susceptible lines indicated that the imidazolinone-resistant AHAS of CLHA-Plus has a threonine codon (ACG) at position 122 (relative to the Arabidopsis thaliana AHAS sequence), whereas the herbicide-susceptible enzyme from BTK47 has an alanine residue (GCG) at this position. Since the resistance genes to AHAS-inhibiting herbicides so far characterized in sunflower code for the catalytic (large) subunit of AHAS, we propose to redesignate the wild type allele as ahasl1 and the incomplete dominant resistant alleles as Ahasl1-1 (previously Imr1 or Ar _pur ), Ahasl1-2 (previously Ar _kan ) and Ahasl1-3 (for the allele present in CLHA-Plus). The higher tolerance level to imidazolinones and the lack of cross-resistance to other AHAS-inhibiting herbicides of Ahasl1-3 indicate that this induced mutation can be used to develop commercial hybrids with superior levels of tolerance and, at the same time, to assist weed management where control of weedy common sunflower is necessary.     

Evolution of herbicide resistance in weeds: initial frequency of target site-based resistance to acetolactate synthase-inhibiting herbicides in Lolium rigidum

The frequency of individuals resistant to two acetolactate synthase (ALS)-inhibiting herbicides in three previously untreated populations of Lolium rigidum was determined. The frequency of individuals resistant to the sulfonylurea herbicide sulfometuron-methyl varied from 2.2 x 10(-5) to 1.2 x 10(-4) and the frequency of individuals resistant to the imidazolinone herbicide imazapyr varied from 1 x 10(-5) to 5.8 x 10(-5) depending on the population. Application of sulfometuron-methyl selected individuals with a herbicide-insensitive ALS, which was also cross-resistant to imazapyr. The high initial frequency of individuals resistant to ALS-inhibiting herbicides in L. rigidumpopulations never previously exposed to these herbicides helps explain the rapid evolution of herbicide resistance in this species once ALS-inhibiting herbicides were used.     

High survival frequencies at low herbicide use rates in populations of Lolium rigidum result in rapid evolution of herbicide resistance

The frequency of phenotypic resistance to herbicides in previously untreated weed populations and the herbicide dose applied to these populations are key determinants of the dynamics of selection for resistance. In total, 31 Lolium rigidum populations were collected from sites with no previous history of exposure to herbicides and where there was little probability of gene flow from adjacent resistant populations. The mean survival frequency across all 31 populations following two applications of commercial rates (375 g ha(-1)) of the acetyl-coenzyme A carboxylase (ACCase) inhibiting herbicide, diclofop-methyl was 0.43%. Survivors from five of these populations were grown to maturity and seed was collected. Dose-response experiments compared population level resistance to diclofop-methyl in these selected lines with their original parent populations. A single cycle of herbicide selection significantly increased resistance in all populations (LD(50) R:S ratios ranged from 2.8 to 23.2), confirming the inheritance and genetic basis of phenotypic resistance. In vitro assays of ACCase inhibition by diclofop acid indicated that resistance was due to a non-target-site mechanism. Following selection with diclofop-methyl, the five L. rigidum populations exhibited diverse patterns of cross-resistance to ACCase and ALS-inhibiting herbicides, suggesting that different genes or gene combinations were responsible for resistance. The relevance of these results to the management of herbicide resistance are discussed.     

Cross resistance to accase herbicide in Lolium rigidum

Lolium rigidum is a cross-pollinating grass weed present in Europe and occurring in winter wheat and orchard crops. Several graminicides such as chlorotoluron and/or isoproturon and diclofop-methyl in mixtures or alone have been used successfully to control this weed in Spain during the past decade. However, several L rigidum populations have developed resistance to these herbicides following selection due to their continuous use. Four resistance mechanisms have been found in this grass weed, an enhanced metabolic detoxiflcation of the herbicides and an insensitive isoform of ACCase being the most important ones. The extent of cross-resistance depends on the type of mechanism. The biotype with an enhanced metabolic detoxification showed cross-resistance to ACCase-, ALS-, PSII- and tubuline-inhibiting herbicides, while the biotype resistant due to a mutation of the target site (ACCase) presented cross-resistance to ACCase-inhibiting herbicides only.     

Cytochrome P450 CYP81A10v7 in Lolium rigidum confers metabolic resistance to herbicides across at least five modes of action

Rapid and widespread evolution of multiple herbicide resistance in global weed species endowed by increased capacity to metabolize (degrade) herbicides (metabolic resistance) is a great threat to herbicide sustainability and global food production. Metabolic resistance in the economically damaging crop weed species Lolium rigidum is well known but a molecular understanding has been lacking. We purified a metabolic resistant (R) subset from a field evolved R L. rigidum population. The R, the herbicide susceptible (S) and derived F₂ populations were used for candidate herbicide resistance gene discovery by RNA sequencing. A P450 gene CYP81A10v7 was identified with higher expression in R vs. S plants. Transgenic rice overexpressing this Lolium CYP81A10v7 gene became highly resistant to acetyl-coenzyme A carboxylase- and acetolactate synthase-inhibiting herbicides (diclofop-methyl, tralkoxydim, chlorsulfuron) and moderately resistant to hydroxyphenylpyruvate dioxygenase-inhibiting herbicide (mesotrione), photosystem II-inhibiting herbicides (atrazine and chlorotoluron) and the tubulin-inhibiting herbicide trifluralin. This wide cross-resistance profile to many dissimilar herbicides in CYP81A10v7 transgenic rice generally reflects what is evident in the R L. rigidum. This report clearly showed that a single P450 gene in a cross-pollinated weed species L. rigidum confers resistance to herbicides of at least five modes of action across seven herbicide chemistries.     

强化栽培条件下稻田杂草发生特点和水稻品种竞争能力研究

水稻强化栽培条件下,稻田杂草以节节菜、陌上菜、异型莎草和千金子为主,其中与水稻有较强竞争力的为异型莎草和千金子。两优培九与杂草的竞争力较E52强。相同密度下,随行距扩大,水稻对杂草的抑制能力下降。当稀植时,与杂草竞争能力较差的E52对杂草抑制在不同行距问没有差异。就水稻强化栽培条件下杂草防治方法进行了讨论。     

The interaction between Plectosporium alismatis and sublethal doses of bensulfuron-methyl reduces the growth of starfruit (Damasonium minus) in rice

Plectosporium alismatis is a fungal pathogen that is being investigated as a biocontrol agent for suppressing starfruit (Damasonium minus), a significant weed of Australian rice fields. The aim of this research was to study the effect of the fungus on weed competition and its interactions with chemical herbicides. Conidial germination was significantly reduced by Londax® (bensulfuron-methyl), while MCPA had no effects. Glasshouse trials showed evidence of synergism between the fungus and 1.56% of the recommended dose of Londax®, in reducing the weed growth. The application of inoculum (conidia suspended in water) in the glasshouse eliminated weed competition with rice. In the field, the reduction in weed growth caused by the fungus did not significantly eliminate starfruit competition with rice. This is thought to be due to the presence of other weeds.     

Evolution of herbicide resistance in weeds: vertically transmitted fungal endophytes as genetic entities

The appearance of heritable resistance to herbicides in weeds is an evolutionary process driven by human selection. Assuming that spontaneous and random mutations originate herbicide resistance genes, which are selected by selection pressure imposed by herbicides, is the simplest model to understand how this phenomenon appears and increases in weed populations. However, the rate of herbicide resistance evolution is not only determined by the amount of genetic variation within the populations and the selection pressure exerted by herbicides, but also by factors related to genetics, biology and ecology of weeds. The inheritance of the resistance genes, the mating patterns of the populations, the relative fitness of susceptible and resistant phenotypes and gene flow processes also control the mentioned rate. Many cool season grasses are often infected by fungal symbiotic endophytes (Neotyphodium spp.). These organisms modify the physiology, ecology and reproductive biology of their hosts, conferring greater tolerance to biotic and abiotic stresses, greater competitive ability and the capacity of reducing ecosystem biodiversity. In this work, we present new empirical data and propose new theoretical support on how these microbial symbionts can modulate the evolution of herbicide resistance in weeds. Fungal endophytes are vertically transmitted, and may act as genetic entities altering the evolution of herbicide resistance by reducing herbicide efficacy (delaying effect on evolution). In addition, indirect evidence suggests that fungal endophytes might reduce the fitness penalty associated with the newly arisen resistant phenotypes. The importance and dynamic of these opposite effects is discussed.     

Evaluation of herbicide combinations for livid amaranth (Amaranthus blitum) control in tuberous begonia (Begonia x tuberhybrida)

In the past years livid amaranth (Amaranthus blitum) is observed increasingly in begonia production fields. Control of weeds in begonia is generally done by a combined application of the soil herbicides isoxaben + simazin followed 10 days later by application of the contact herbicide bentazone. This treatment usually controls the weed population sufficiently with exception of amaranth. In 2003 a field trial was conducted to evaluate control of livid amaranth in tuberous begonia with isoxaben, simazin. S-metolachloor, phenmedipham + desmedipham and bentazone. These herbicides were used as combinations of soil treatment and contact herbicides. The results suggest that a soil treatment of isoxaben + S-metolachloor significantly reduces livid amaranth compared to isoxaben + simazin, without a pronounced negative effect on tuber yield. Application of phenmedipham + desmedipham however did not improve control of livid amaranth compared to bentazone.     

Use of a model and toxicity data to predict the risks to some wild plant species from drift of four herbicides

The dose responses of 14 wild plant species (two grasses, two legumes, one annual and nine perennial dicotyledons), not usually recognised as weeds, to four herbicides (asulam, glyphosate, MCPA and mecoprop) were measured in glasshouse experiments. Glyphosate was the most toxic; seven of the species tested had EDu, values (measured as shoot dry weight) of < 1.0 μg/plant, compared with only one species for MCPA and mecoprop. Asulam was the least toxic. Results were used to indicate the risk to each species from drift damage. A model of spray drift, based on that developed by Thompson & Ley (1982) for evaporating droplets, was rescaled to allow for field application rates and used to predict the distances travelled by given doses of herbicide. This gave acceptable agreement with reports for drift damage in the field, and predicted that only glyphosate sprayed at the highest recommended concentration might be unsafe to some of the species examined. The two herbicides sometimes used as volatile formulations (MCPA and mecoprop) did not cause damage at the small doses likely to result from exposure to vapour in the field.     

A role for glutathione transferases functioning as glutathione peroxidases in resistance to multiple herbicides in black-grass

Black-grass (Alopecurus myosuroides) is a major weed of wheat in Europe, with several populations having acquired resistance to multiple herbicides of differing modes of action. As compared with herbicide-susceptible black-grass, populations showing herbicide cross-resistance contained greatly elevated levels of a specific type I glutathione transferase (GST), termed AmGST2, but similar levels of a type III GST termed AmGST1. Following cloning and expression of the respective cDNAs, AmGST2 differed from AmGST1 in showing limited activity in detoxifying herbicides but high activities as a glutathione peroxidase (GPOX) capable of reducing organic hydroperoxides. In contrast to AmGST2, other GPOXs were not enhanced in the herbicide-resistant populations. Treatment with a range of herbicides used to control grass weeds in wheat resulted in increased levels of hydroperoxides in herbicide-susceptible populations but not in herbicide-resistant plants, consistent with AmGST2 functioning to prevent oxidative injury caused as a primary or secondary effect of herbicide action. Increased AmGST2 expression in black-grass was associated with partial tolerance to the peroxidizing herbicide paraquat. The selective enhancement of AmGST2 expression resulted from a constitutively high expression of the respective gene, which was activated in herbicide-susceptible black-grass in response to herbicide safeners, dehydration and chemical treatments imposing oxidative stress. Our results provide strong evidence that GSTs can contribute to resistance to multiple herbicides by playing a role in oxidative stress tolerance in addition to detoxifying herbicides by catalysing their conjugation with glutathione.     

Biorational management tactics to select against triazine-resistant Amaranthus hybridus: a field trial

1. The individual and joint effectiveness of two biorational tactics (crop interference and exploitation of negative cross-resistance to certain herbicides) in the management of triazine-resistant Amaranthus hybridus L. (smooth pigweed) were estimated. Biorational tactics exploit biological idiosyncracies of resistant (R) genotypes to maximize fitness cost(s) of resistance. We quantified selection against triazine resistance by relative performance comparisons between lines having comparable nuclear genomes but either resistant or susceptible cytoplasm. Increasing soybean density by reducing row spacing (from 76 cm to 25 cm) did not significantly increase the fitness cost of resistance.
2. Low doses of bentazon (100 and 300 g active ingredient ha^–1) did strongly increase the cost of resistance. Over 2 years, the mean relative performance of R genotypes in bentazon treatments was 0·40, compared to 0·60 in the absence of bentazon. Therefore, use of bentazon in soybean production has the potential to delay evolution of triazine resistance in maize–soybean rotations using triazines.
3. There was no consistent indication that increased soybean density and bentazon herbicide could act synergistically to increase costs of triazine resistance in Amaranthus hybridus . Nor were differences in response to biorational tactics evident between the two populations of origin (Maryland and Virginia, USA) from which experimental lines were derived.
4. Effects of the biorational tactics differed markedly between years, highlighting that resistance management depending primarily on these tactics would have widely variable results. Use of such tactics is likely to be most effective in the context of diversified weed management.     

A novel mutated acetolactate synthase gene conferring specific resistance to pyrimidinyl carboxy herbicides in rice

Acetolactate synthase (ALS) is the first common enzyme in the biosynthetic pathway of branched-chain amino acids. Mutations of specific amino acids in ALS have been known to confer resistance to ALS-inhibiting herbicides such as sulfonylureas and pyrimidinyl carboxy (PC) herbicides. However, mutations conferring exclusive resistance to PC have not yet been reported to date. We selected PC resistant rice calli, which were derived from anther culture, using one of the PCs, bispyribac-sodium (BS), as a selection agent. Two lines of BS-resistant plants carrying a novel mutation, the 95th Glycine to Alanine (G95A), in ALS were obtained. In vitro ALS activity assay indicated that the recombinant protein of G95A-mutated ALS (ALS-G95A) conferred highly specific resistance to PC herbicides. In order to determine if the ALS-G95A gene could be used as a selection marker for rice transformation, the ALS-G95A gene was connected to ubiquitin promoter and introduced into rice. PC resistant plants containing integrated ALS-G95A gene were obtained after selection with BS as a selection agent. In conclusion, novel G95A mutated ALS gene confers highly specific resistant to PC-herbicides and can be used as a selection marker.     

Selectable tolerance to herbicides by mutated acetolactate synthase genes integrated into the chloroplast genome of tobacco

Strategies employed for the production of genetically modified (GM) crops are premised on (1) the avoidance of gene transfer in the field; (2) the use of genes derived from edible organisms such as plants; (3) preventing the appearance of herbicide-resistant weeds; and (4) maintaining transgenes without obstructing plant cell propagation. To this end, we developed a novel vector system for chloroplast transformation with acetolactate synthase (ALS). ALS catalyzes the first step in the biosynthesis of the branched amino acids, and its enzymatic activity is inhibited by certain classes of herbicides. We generated a series of Arabidopsis (Arabidopsis thaliana) mutated ALS (mALS) genes and introduced constructs with mALS and the aminoglycoside 3'-adenyltransferase gene (aadA) into the tobacco (Nicotiana tabacum) chloroplast genome by particle bombardment. Transplastomic plants were selected using their resistance to spectinomycin. The effects of herbicides on transplastomic mALS activity were examined by a colorimetric assay using the leaves of transplastomic plants. We found that transplastomic G121A, A122V, and P197S plants were specifically tolerant to pyrimidinylcarboxylate, imidazolinon, and sulfonylurea/pyrimidinylcarboxylate herbicides, respectively. Transplastomic plants possessing mALSs were able to grow in the presence of various herbicides, thus affirming the relationship between mALSs and the associated resistance to herbicides. Our results show that mALS genes integrated into the chloroplast genome are useful sustainable markers that function to exclude plants other than those that are GM while maintaining transplastomic crops. This investigation suggests that the resistance management of weeds in the field amid growing GM crops is possible using (1) a series of mALSs that confer specific resistance to herbicides and (2) a strategy that employs herbicide rotation.     

应用主成分分析法和最小生成树法研究浙中秋旱作物田杂草种间生态关系

研究意义浙江金华地处浙江中部,旱地面积有５．０９×１０４ｈａ,占耕地总面积的１８％以上,棉花、大豆、甘薯等秋旱作物是该地区重要的经济作物,几年来,由于农村劳动力转移,中耕除草减少,草害问题日趋严重。化学除草是解决旱田草害的有效方法。但是,除草剂的长期...