Molecular bases for resistance to acetyl-coenzyme A carboxylase inhibitor in Japanese foxtail (Alopecurus japonicus)

BACKGROUND: Haloxyfop‐R‐methyl is a widely used herbicide to control Poaceae weeds. Alopecurus japonicus, a widespread annual grass, can no longer be controlled by haloxyfop‐R‐methyl after continuous use of this herbicide for several years. RESULTS: Dose‐response experiments have established that the Js‐R biotype of A. japonicas has evolved resistance to aryloxyphenoxypropionates (APPs). Target‐site enzyme sensitivity experiments have established that the haloxyfop (free acid) rate causing 50% inhibition of acetyl‐CoA carboxylase (ACCase) activity (I₅₀) for the resistant (Js‐R) biotype is 11 times higher than that for the susceptible (Js‐S) biotype. In many cases, resistance to ACCase‐inhibiting herbicides is due to a resistant ACCase enzyme. Full‐length DNA and mRNA sequences of the plastidic ACCase gene were amplified. Eight single‐nucleotide differences were detected in this region. Four of the nucleotide changes were silent mutations. However, the other four nucleotide mutations caused four amino acid substitutions, replacing Arg‐1734 with Gly, Met‐1738 with Leu, Thr‐1739 with Ser and Ile‐2041 with Asn in the R biotype respectively; the substitution at position 2041 had been reported, while the other three had not. CONCLUSION: The ACCase in the Js‐R biotype was less susceptible to haloxyfop‐R‐methyl than that in the Js‐S biotype. Moreover, the amino acid substitution of Ile‐2041 with Asn might confer resistance to haloxyfop‐R‐methyl in A. japonicas. Copyright © 2012 Society of Chemical Industry     

PCR-based detection of resistance to acetyl-CoA carboxylase-inhibiting herbicides in black-grass (Alopecurus myosuroides Huds) and ryegrass (Lolium rigidum gaud)

A simple method based upon allele-specific PCR was developed to detect an isoleucine-leucine substitution in the gene encoding chloroplastic acetyl-coenzyme A carboxylase (ACCase) in two gramineous weeds: Lolium rigidum Gaud and Alopecurus myosuroides Huds. Analysis of 1800 A myosuroides and 750 L rigidum seedlings showed that the presence of ACCase leucine allele(s) conferred cross-resistance to the cyclohexanedione herbicide cycloxydim and to the aryloxyphenoxypropionate herbicides fenoxaprop-P-ethyl and diclofop-methyl. Seedlings containing ACCase leucine allele(s) could be either sensitive or resistant to the aryloxyphenoxypropionate herbicides haloxyfop-P-methyl and clodinafop-propargyl. Successful detection of resistant plants in a field population of A myosuroides was achieved using this PCR assay. Using it with basic molecular biology laboratory equipment, the presence of resistant leucine ACCase allele(s) can be detected within one working day.     

Prevalence of cross‐ or multiple resistance to the acetyl‐coenzyme A carboxylase inhibitors fenoxaprop,clodinafop and pinoxaden in black‐grass (Alopecurus myosuroides Huds.) in France

BACKGROUND: Repeated use of acetyl‐CoA carboxylase (ACCase) inhibitors, especially fenoxaprop and clodinafop, since the late 1980s has selected for resistance in Alopecurus myosuroides Huds. (black‐grass) in France. We investigated whether resistance to pinoxaden, a phenylpyrazoline ACCase inhibitor to be marketed in France, was present in French black‐grass populations. We investigated pinoxaden resistance conferred by five mutant ACCase isoforms. Using 84 French black‐grass field samples, we also compared the frequencies of other mechanisms endowing resistance to fenoxaprop, clodinafop or pinoxaden. RESULTS: ACCase mutant isoforms Leu‐1781, Gly‐2078 and, likely, Cys‐2027 conferred cross‐resistance to pinoxaden, while isoform Asn‐2041 possibly conferred moderate resistance. Other mechanisms of resistance to fenoxaprop, clodinafop and pinoxaden were detected in 99, 68 and 64% of the samples investigated, respectively. Cross‐ or multiple resistance to fenoxaprop or clodinafop and pinoxaden was not systematically observed, suggesting a diversity of mechanisms exist. CONCLUSION: Pinoxaden resistance was observed before pinoxaden release in France. Only a fraction of the mechanisms endowing fenoxaprop or clodinafop resistance also confer pinoxaden resistance. Pinoxaden resistance was likely mostly selected for by ACCase inhibitors, and, in some cases, possibly by herbicides with other modes of action. This illustrates the necessity to use metabolisable herbicides cautiously where black‐grass has evolved non‐target‐site‐based resistance. Copyright © 2009 Society of Chemical Industry     

A method to determine the pollen-mediated spread of target-site resistance to acetylcoenzyme A carboxylase inhibitors in black grass (Alopecurus myosuroides Huds.)

Journal of Plant Diseases and Protection - In this study the spread of target-site resistance to ACCase inhibitors via pollen was characterized in a population of Alopecurus myosuroides. The spread...     

Mechanisms of resistance to acetyl-coenzyme A carboxylase inhibitors: a review

Resistance to acetyl-coenzyme A carboxylase (ACCase) inhibitors has developed in at least 10 grass weed species in recent years. In most instances, resistance is conferred by an ACCase alteration in the resistant biotypes that reduces sensitivity to aryloxyphenoxypropionate (AOPP) and cyclohexanedione (CHD) herbicides. Analysis of ACCase from many of these resistant weed biotypes suggests the presence of different mutations, each conferring a different pattern and level of resistance to various AOPP and CHD herbicides. In all cases analyzed to date, resistance is controlled by a single dominant or semi-dominant nuclear gene. In several weed biotypes, resistance is conferred by enhanced herbicide detoxification, primarily through elevated expression or activity of cytochrome P450 monooxygenase(s). This mechanism can confer cross-resistance to herbicides from other chemical classes with different modes of action. Finally, multiple herbicide resistance, i.e. the acquisition of several different resistance mechanisms, has been reported in some weed biotypes. ©1997 SCI     

The effect of temperature on glutathione S-transferase activity and glutathione content in Alopecurus myosuroides (black grass) biotypes susceptible and resistant to herbicides

Herbicide‐resistant populations of Alopecurus myosuroides (black grass) have become widespread throughout the UK since the early 1980s. Previous observations in this laboratory have demonstrated that natural climatic fluctuations caused increases in endogenous glutathione S‐transferase (GST) enzyme activity in A. myosuroides plants as they mature, which is thought to be linked to herbicide resistance in this species. The present study has investigated the effects of plant growth at 10°C and 25°C, and reports GST specific activity and glutathione (GSH) pool size in resistant and susceptible A. myosuroides biotypes. Findings demonstrate differences in GST activity between resistant and susceptible populations, which are transient at lower growth temperatures. The GSH pool size was elevated at lower growth temperature in both biotypes. We speculate that these endogenous responses are part of a natural mechanism of acclimation to environmental change in this species and suggest that resistant plants are more able to adapt to environmental stress, as indicated in this instance by temperature change. These observations imply that the control of resistant A. myosuroides by graminicides may be more effective when applied at lower temperatures and at earlier growth stages.     

Multiple origins for black-grass (Alopecurus myosuroides Huds) target-site-based resistance to herbicides inhibiting acetyl-CoA carboxylase

We have investigated the process of evolution of target-site-based resistance to herbicides inhibiting acetyl-CoA carboxylase (ACCase) in nine French populations of black-grass (Alopecurus myosuroides Huds). To date, two different ACCase resistant alleles are known. One contains an isoleucine-to-leucine substitution at position 1781, the second contains an isoleucine-to-asparagine substitution at position 2041. Using phylogenetic analysis of ACCase sequences, we showed that 1781Leu ACCase alleles evolved from four independent origins in the nine black-grass populations studied, while 2041Asn ACCase alleles evolved from six independent origins. No geographical structure of black-grass populations was revealed. This implies that these populations, although geographically distant, are, or have until recently been, connected by gene flows. Comparison of biological data obtained from herbicide sensitivity bioassay and molecular data showed that distinct resistance mechanisms often exist in a single black-grass population. Accumulation of different resistance mechanisms in a single plant was also demonstrated. We conclude that large-scale evolution of resistance to herbicides in black-grass is a complex phenomenon, resulting from the independent selection of various resistance mechanisms in local black-grass populations undergoing contrasted herbicide and agronomical selection pressures, and connected by gene flows whose parameters remain to be determined.     

'Universal' primers for PCR-sequencing of grass chloroplastic acetyl-CoA carboxylase domains involved in resistance to herbicides

Primers were designed to amplify two regions involved in sensitivity to herbicides inhibiting the plastidic acetyl-CoA carboxylase (ACCase) from grasses (Poaceae). The first primer pair amplified a 551-bp amplicon containing a variable Ile/Leu codon at position 1781 in Alopecurus myosuroides sequence. The second primer pair amplified a 406-bp amplicon containing four variable codons (Trp/Cys, Ile/Asn, Asp/Gly, Gly/Ala) at positions 2027, 2041, 2078 and 2096, respectively, in A. myosuroides sequence. Both primer pairs amplified the targeted fragments from genes encoding plastidic ACCases, but not from the very similar genes encoding cytosolic ACCases. Clear DNA sequences were obtained from fresh or dried plant material from the field, and from 29 various grass species. Sequences revealed that the gene encoding plastidic ACCase in Poa annua and Festuca rubra contained a Leu₁₇₈₁ codon, in agreement with both species being inherently tolerant to herbicides inhibiting ACCase. Sequencing confirmed the hybrid origin of P. annua. Compared with ACCase enzyme assay, polymerase chain reaction is faster, can be performed from a single plant and suppresses the need for radioactive experiments. It can be completed with basic molecular biology laboratory equipment. It is the tool of choice for diagnosing resistance caused by alteration(s) of the plastidic ACCase.     

Characterisation and molecular basis of ALS inhibitor resistance in the grass weed Alopecurus myosuroides

Several UK populations of the grass weed Alopecurus myosuroides were identified where high proportions of individuals showed resistance to the acetolactate synthase (ALS)‐inhibiting herbicides, mesosulfuron‐methyl + iodosulfuron‐methyl sodium mixture and sulfometuron‐methyl. Screening with sulfometuron, followed by DNA sequencing of the ALS gene from resistant and susceptible individuals, led to the identification of eight populations where a single point mutation segregated with resistance to sulfometuron. All highly resistant individuals from seven of eight populations showed a single‐nucleotide polymorphism (SNP) in the first position of the Pro197 codon of an A. myosuroides ALS gene, conferring a predicted proline to threonine target‐site change. One population showed resistant individuals with single‐nucleotide polymorphism in the second position of the Trp574 codon, conferring a predicted tryptophan to leucine substitution. No other mutations segregating with resistance were found. Enzyme assays confirmed that resistance was due to an altered form of ALS enzyme, which was less susceptible to inhibition by sulfonylureas, making this one of the first fully characterised cases of ALS target‐site resistance in a European grass weed. Increased information regarding the nature and distribution of ALS target‐site mutation may help support sustainable management strategies, allowing continued use of mesosulfuron + iodosulfuron against this weed in the UK.     

Cross-resistance patterns to ACCase-inhibiting herbicides conferred by mutant ACCase isoforms in Alopecurus myosuroides Huds. (black-grass), re-examined at the recommended herbicide field rate

BACKGROUND: Target‐site‐based resistance to acetyl‐CoA carboxylase (ACCase) inhibitors in Alopecurus myosuroides Huds. is essentially due to five substitutions (Isoleucine‐1781‐Leucine, Tryptophan‐2027‐Cysteine, Isoleucine‐2041‐Asparagine, Aspartate‐2078‐Glycine, Glycine‐2096‐Alanine). Recent studies suggested that cross‐resistance patterns associated with each mutation using a seed‐based bioassay may not accurately reflect field resistance. The authors aimed to connect the presence of mutant ACCase isoform(s) in A. myosuroides with resistance to five ACCase inhibitors (fenoxaprop, clodinafop, haloxyfop, cycloxydim, clethodim) sprayed at the recommended field rate. RESULTS: Results from spraying experiments and from seed‐based bioassays were consistent for all mutant isoforms except the most widespread, Leucine‐1781. In spraying experiments, Leucine‐1781 ACCase conferred resistance to clodinafop and haloxyfop. Some plants containing Leucine‐1781 or Alanine‐2096 ACCase, but not all, were also resistant to clethodim. CONCLUSION: Leucine‐1781, Cysteine‐2027, Asparagine‐2041 and Alanine‐2096 ACCases confer resistance to fenoxaprop, clodinafop and haloxyfop at field rates. Leucine‐1781 ACCase also confers resistance to cycloxydim at field rate. Glycine‐2078 ACCase confers resistance to all five herbicides at field rates. Only Glycine‐2078 ACCase confers clethodim resistance under optimal application conditions. It may be that Leucine‐1781 and Alanine‐2096 ACCases may also confer resistance to clethodim in the field if the conditions are not optimal for herbicide efficacy, or at reduced clethodim field rates. Copyright © 2008 Society of Chemical Industry     

New, quick tests for herbicide resistance in black-grass (Alopecurus myosuroides Huds) based on increased glutathione S-transferase activity and abundance

Black-grass (Alopecurus myosuroides Huds) is a major grass weed in winter cereals in Europe. It reduces yields and can act as a secondary host for a range of diseases. Herbicide resistance in this species was first detected in the UK in the early 1980s, and has now been reported in thirty counties. To successfully manage herbicide resistance it is vital that suspect populations are tested so that appropriate action can be taken. Ideally, a test will be quick, cheap and easy to use. Furthermore, it should provide an unequivocal result before post-emergence herbicides are to be applied, allowing alternative strategies to be adopted where necessary. This paper reports the development of new tests for herbicide resistance based on our observation that the resistant black-grass biotype Peldon contains approximately double the activity of the enzyme glutathione S-transferase (GST) compared with susceptible biotypes. Data are presented on the production of a monoclonal antiserum to a novel 30 kDa GST polypeptide purified from the biotype Peldon. An ELISA using this antiserum is described and the utility of this assay to detect resistant black-grass biotypes in plants grown under glass and in the field is presented. In addition, a microtitre assay for GST activity is described, which allows the rapid assessment of GST activities of plants. Both abundance and activity of GSTs are discussed as markers for herbicide resistance in black-grass.     

Segregation of non‐target‐site resistance to herbicides in multiple‐resistant Alopecurus myosuroides plants

Non‐target‐site resistance (NTSR) comprises a set of mechanisms conferring resistance to multiple modes of action. Investigation of the number of loci involved in NTSR will aid in the understanding of these resistance mechanisms. Therefore, six different multiple herbicide‐resistant Alopecurus myosuroides plants with different herbicide history were crossed in two generations with a susceptible wild type. Seeds from the backcrossing generation were studied for their segregation rate for resistance to five herbicides with four different modes of action (HRAC groups C₂, A, B and K₃). Taking into account that NTSR is a set of quantitative traits, the numbers of loci controlling NTSR were estimated using a normal mixture model fitted by the NLMIXED procedure of SAS. Each herbicide was controlled by a different number of loci comparing the six plants. In most of the cases, chlorotoluron resistance was controlled by one locus, whereas resistance to fenoxaprop‐P‐ethyl needed one or two loci. Resistance to pinoxaden was in all plants conferred by two loci. Cross‐resistance of fenoxaprop‐P‐ethyl and pinoxaden was found in all backcrossings, indicating that at least one of the two loci is responsible for both resistances. Resistance to mesosulfuron + iodosulfuron was conferred by a minimum of two loci. Results indicated that a minimum of five different loci can be involved in a multiple NTSR plant. Furthermore, the plant‐specific accumulation of NTSR loci was demonstrated. Such behaviour should be taken into account when evaluating the development and further spread of herbicide resistance.     

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.     

河南省多花黑麦草对ACCase和ALS抑制剂的抗性及其靶标基因突变分析

为明确河南省部分地区的多花黑麦草Lolium multiflorum种群对乙酰辅酶A羧化酶（acetylCoA carboxylase,ACCase）和乙酰乳酸合成酶（acetolactate synthase,ALS）抑制剂类除草剂的抗性水平和抗性机理,采用整株生物测定法测定采自新乡市和驻马店市的多花黑麦草种群对ACCase抑制剂类除草剂精噁唑禾草灵、炔草酯、唑啉草酯和ALS抑制剂类除草剂甲基二磺隆、氟唑磺隆、啶磺草胺的抗性水平,并对多花黑麦草ACCase和ALS靶标酶编码基因进行克隆及氨基酸序列比对,分析其靶标抗性机理。结果显示,与多花黑麦草敏感种群HNXX01相比,HNZMD04和HNXX05种群对6种除草剂均产生了抗性,HNZMD04种群对精噁唑禾草灵和啶磺草胺的相对抗性倍数分别为44.65和40.31,对炔草酯和氟唑磺隆的相对抗性倍数分别为11.91和11.93;HNXX05种群对精噁唑禾草灵和氟唑磺隆的相对抗性倍数分别为27.70和25.67。HNZMD04和HNXX05抗性种群的ACCase基因均发生了D2078G突变,2个种群的突变率分别为55%和70%;HNZMD04...     

Differences in the molecular basis of resistance to the cyclohexanedione herbicide sethoxydim in Lolium multiflorum

Resistance to the cyclohexanedione (CHD) herbicide sethoxydim was investigated in two UK Lolium multiflorum populations, Yorks A2 and PYL. Resistance screening experiments demonstrated a qualitative difference in the responses of the two populations to sethoxydim, suggesting that the molecular basis of resistance between them was different. After treatment, Yorks A2 plants were either alive (78% of sample tested) or dead (22% tested) but plants of the PYL population showed two further intermediate categories of response. The level of acetyl-CoA carboxylase (ACCase) insensitivity directly correlated with the degree of resistance at the whole plant level, indicating that the molecular basis of resistance is associated with differences in ACCase sensitivity in each population. Direct sequencing of the carboxyl transferase domain of the ACCase gene showed that an Ile-418-Leu substitution in the L. multiflorum chloroplastic ACCase (GenBank accession number AY710293 ) confers resistance to sethoxydim in Yorks A2. This corresponds to amino acid residue 1781 in the Alopecurus myosuroides full ACCase sequence. This is the first report of this mutation in this L. multiflorum, which has also been reported in four other grass-weeds, including L. rigidum. However, no amino acid substitutions were found to be specifically associated with the resistant phenotypes in the PYL population and the molecular basis of resistance in this population remains to be resolved.     

The role of altered acetyl-CoA carboxylase in conferring resistance to fenoxaprop-P-ethyl in Chinese sprangletop (Leptochloa chinensis (L.) Nees)

From paddy field observations in 2002 and 2004, fenoxaprop-P-ethyl resistance in Chinese sprangletop (Leptochloa chinensis (L.) Nees) has been studied using information collected from 11 sites in the Saphan-Sung district of Bangkok, Thailand. The resistant Chinese sprangletop was found in nine rice fields, whereas the susceptible Chinese sprangletop was found in only two rice fields. In greenhouse experiments, both fenoxaprop-P-ethyl-resistant and susceptible Chinese sprangletop from the same location were investigated for 50% growth reduction based on phytotoxicity, plant height and fresh and dry weight. The resistant Chinese sprangletop showed apparent resistance at 14-21 days after herbicide application at a rate of 21.1-337.6 g AI ha(-1). The resistance index of resistant Chinese sprangletop was 10-25 times higher than that of the susceptible Chinese sprangletop. In addition, Chinese sprangletop did not exhibit multiple resistance to oxadiazon, propanil and quinclorac. According to acetyl-CoA carboxylase (ACCase) assays, the level of ACCase specific activity in the resistant Chinese sprangletop was significantly higher than that in the susceptible Chinese sprangletop. Similarly, the ACCase activity of the resistant Chinese sprangletop was 10 times less sensitive to fenoxaprop-P-ethyl than that of the susceptible Chinese sprangletop, based on the I50 values. The present study of the mechanism responsible for resistance in the biotypes investigated indicated that there was a close association between the concentration-response at the whole-plant level and ACCase sensitivity to fenoxaprop-P-ethyl, and resistance to fenoxaprop-P-ethyl was conferred by a modified ACCase at the target site, as suggested by higher specific activity and less sensitivity to the herbicide.