Mechelle J. Owen

Diversity of Acetyl-Coenzyme A Carboxylase Mutations in Resistant Lolium Populations: Evaluation Using Clethodim

The acetyl-coenzyme A carboxylase (ACCase)-inhibiting cyclohexanedione herbicide clethodim is used to control grass weeds infesting dicot crops. In Australia clethodim is widely used to control the weed Lolium rigidum. However, clethodim-resistant Lolium populations have appeared over the last 5 years and now are present in many populations across the western Australian wheat (Triticum aestivum) belt. An aspartate-2078-glycine (Gly) mutation in the plastidic ACCase enzyme has been identified as the only known mutation endowing clethodim resistance. Here, with 14 clethodim-resistant Lolium populations we revealed diversity and complexity in the molecular basis of resistance to ACCase-inhibiting herbicides (clethodim in particular). Several known ACCase mutations (isoleucine-1781-leucine [Leu], tryptophan-2027-cysteine [Cys], isoleucine-2041-asparagine, and aspartate-2078-Gly) and in particular, a new mutation of Cys to arginine at position 2088, were identified in plants surviving the Australian clethodim field rate (60 g ha−1). Twelve combination patterns of mutant alleles were revealed in relation to clethodim resistance. Through a molecular, biochemical, and biological approach, we established that the mutation 2078-Gly or 2088-arginine endows sufficient level of resistance to clethodim at the field rate, and in addition, combinations of two mutant 1781-Leu alleles, or two different mutant alleles (i.e. 1781-Leu/2027-Cys, 1781-Leu/2041-asparagine), also confer clethodim resistance. Plants homozygous for the mutant 1781, 2078, or 2088 alleles were found to be clethodim resistant and cross resistant to a number of other ACCase inhibitor herbicides including clodinafop, diclofop, fluazifop, haloxyfop, butroxydim, sethoxydim, tralkoxydim, and pinoxaden. We established that the specific mutation, the homo/heterozygous status of a plant for a specific mutation, and combinations of different resistant alleles plus herbicide rates all are important in contributing to the overall level of herbicide resistance in genetically diverse, cross-pollinated Lolium species.

Widespread occurrence of multiple herbicide resistance in Western Australian annual ryegrass (Lolium rigidum) populations

In 2003, a random survey was conducted across the Western Australian wheatbelt to establish the frequency and distribution of herbicide resistance in ryegrass populations infesting crop fields. Five hundred cropping fields were visited at crop maturity, and ryegrass seed was collected in 452 of these fields. Subsequently, each crop field population was screened with herbicides of various modes of action that are commonly used for ryegrass control in Australian cropping systems. Most of these ryegrass populations were found to be resistant to the ACCase-inhibitor herbicide diclofop-methyl (68%) and the ALS-inhibitor herbicide sulfometuron (88%). A comparison of resistance levels in the same agronomic zones surveyed 5 years earlier determined that there had been an increase of 20 percentage points in the frequency of resistance over this 5-year period. This survey also determined that the majority (64%) of populations were found to be multiple resistant to both diclofop-methyl and sulfometuron. The distribution patterns of the collected populations indicated that there were higher frequencies of resistant and developing resistance populations occurring in the intensively cropped regions of the wheatbelt, which had greater herbicide selection pressure. Of concern is that 24% and 8% of populations were found to be developing resistance to trifluralin and clethodim, respectively. Currently these herbicides are heavily relied upon for control of ACCase and ALS herbicide resistant ryegrass. Nearly all populations remain susceptible to glyphosate. Ryegrass across the WA wheatbelt now exhibits multiple resistance across many but not all herbicides, posing severe management and sustainability challenges.

Distinct non-target site mechanisms endow resistance to glyphosate, ACCase and ALS-inhibiting herbicides in multiple herbicide-resistant Lolium rigidum.

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.

Distribution and frequency of herbicide-resistant wild oat (Avena spp.) across the Western Australian grain belt

In 2005, a random survey was conducted across 14 million hectares of the Western Australian grain belt to establish the frequency and distribution of herbicide-resistant wild oat (Avena spp.) in cropping fields. In total, 677 cropping fields were visited, with wild oat populations collected from 150 fields. These wild oat populations were screened with several herbicides commonly used to control this weed. Most of the wild oat populations (71%) were found to contain individuals resistant to the ACCase-inhibiting herbicide diclofop-methyl. Resistance to other ACCase-inhibiting herbicides was markedly lower. Herbicides of alternative modes of action were effective on all wild oat populations. Overall, wild oat resistance to diclofop-methyl was found to be widespread across the Western Australian grain belt, but resistance to other herbicides was relatively low. Therefore, through diversity in herbicide use and with cultural management, it is possible to maintain wild oat populations at a low level and/or minimise herbicide resistance evolution.

Non-target-site-based resistance to ALS-inhibiting herbicides in six Bromus rigidus populations from Western Australian cropping fields

BACKGROUND Bromus rigidus is a common weed species that has increased in cropping fields owing to limited control options. During a random field survey in Western Australia, six B. rigidus populations that had survived in-crop weed control programmes were collected. The study aimed to determine the resistance profile of these six populations. RESULTS Based on dose-response studies, all six B. rigidus populations had a low-level resistance to sulfosulfuron and sulfometuron (both sulfonylurea herbicides) while remaining susceptible to herbicides with other modes of action. ALS in vitro activity assays revealed no differences in enzyme sensitivity between susceptible and resistant populations, while the use of malathion (a cytochrome P450 inhibitor) in combination with sulfosulfuron caused the resistant populations to behave like the susceptible population. CONCLUSION This study established that these six B. rigidus populations have a low-level resistance to the ALS-inhibiting sulfonylurea herbicides, but are able to be controlled by other herbicide modes of action. The low-level, malathion-reversible resistance, together with a sensitive ALS, strongly suggest that a non-target-site enhanced metabolism is the mechanism of resistance.

Herbicide-Resistant Weed Seeds Contaminate Grain Sown in the Western Australian Grainbelt

Abstract Preventing the introduction of weeds into the farming system through sowing of clean seeds is an essential component of weed management. The weed seed contamination of cleaned grain and herbicide resistance levels of the recovered weed seeds were examined in a study conducted across 74 farms in the Western Australian grainbelt. Most farmers grew and conserved their own crop seed. The majority of cleaned samples had some level of seed contamination from 11 foreign weed and volunteer crop species, with an average of 62 seeds 10 kg−1 grain, substantially higher than the 28 seeds 10 kg−1 grain expected by farmers. The most common weed contaminants across all samples were rigid ryegrass, wild radish, brome, and wild oat. When categorized by crop type, rigid ryegrass was the most frequent contaminant of cereal crops (barley and wheat), however wild radish was the most frequent contaminant of lupin crops. Uncleaned crop seed samples had almost 25 times more contamination than cleaned crop seed. Herbicide resistance was highly prevalent within rigid ryegrass populations recovered from cleaned grain except for glyphosate, which controlled all populations tested. Some resistance was also found in wild radish and wild oat populations; however, brome was susceptible to fluazifop. This study has shown that farmers are unknowingly introducing weed seeds into their farming systems during crop seeding, many of which have herbicide resistance. Nomenclature: Fluazifop; glyphosate; brome, Bromus spp; rigid ryegrass, Lolium rigidum Gaudin; wild oat, Avena fatua L; wild radish, Raphanus raphanistrum L; barley, Hordeum vulgare L; lupin, Lupinus spp; wheat, Triticum aestivum L.

Widespread occurrence of both metabolic and target-site herbicide resistance mechanisms in Lolium rigidum populations

BACKGROUND Lolium rigidum populations in Australia and globally have demonstrated rapid and widespread evolution of resistance to acetyl coenzyme A carboxylase (ACCase)-inhibiting and acetolactate synthase (ALS)-inhibiting herbicides. Thirty-three resistant L. rigidum populations, randomly collected from crop fields in a most recent resistance survey, were analysed for non-target-site diclofop metabolism and all known target-site ACCase gene resistance-endowing mutations. RESULTS The HPLC profile of [(14) C]-diclofop-methyl in vivo metabolism revealed that 79% of these resistant L. rigidum populations showed enhanced capacity for diclofop acid metabolism (metabolic resistance). ACCase gene sequencing identified that 91% of the populations contain plants with ACCase resistance mutation(s). Importantly, 70% of the populations exhibit both non-target-site metabolic resistance and target-site ACCase mutations. CONCLUSIONS This work demonstrates that metabolic herbicide resistance is commonly occurring in L. rigidum, and coevolution of both metabolic resistance and target-site resistance is an evolutionary reality. Metabolic herbicide resistance can potentially endow resistance to many herbicides and poses a threat to herbicide sustainability and thus crop production, calling for major research and management efforts.

Herbicide Resistance in Rigid Ryegrass (Lolium rigidum) Has Not Led to Higher Weed Densities in Western Australian Cropping Fields

Abstract The aim of this study was to test whether herbicide resistance in rigid ryegrass has led to increased densities of this weed in Western Australian (WA) cropping fields. A total of 503 wheat fields with previously unknown management history and weed status were visited prior to harvest across 15 agronomic areas of the central WA cropping belt in 1998 and 2003. Rigid ryegrass density was visually assessed and, where possible, seed was collected from the population. Ryegrass was found in 91% of the wheat crops sampled. Ryegrass populations were tested in the following year for resistance to chlorsulfuron, sulfometuron, diclofop, and clethodim. With the use of nonparametric and regression statistical methods, resistance status, including multiple-resistance status, was not found to be associated with higher weed density. The results show that growers are generally maintaining low densities in fields with herbicide-resistant rigid ryegrass. The most common rigid ryegrass density at harvest time was less than 1 plant m−2 in both resistant and susceptible populations. Field and model-based studies of weed and herbicide resistance management that allow populations to continue at very high densities are unlikely to reflect common grower practice. Nomenclature: Chlorsulfuron; clethodim; diclofop; rigid ryegrass, Lolium rigidum Gaudin LOLRI; wheat, Triticum aestivum L

Glyphosate-Resistant Rigid Ryegrass (Lolium rigidum) Populations in the Western Australian Grain Belt

Abstract Glyphosate-resistance evolution in weeds is evident globally, especially in areas where transgenic glyphosate-resistant crops dominate. Resistance to glyphosate is currently known in 16 weed species, including rigid ryegrass in Australia. Following the first report of glyphosate resistance in 1998, there are now 78 documented glyphosate-resistant populations of rigid ryegrass in grain-growing regions of southern Australia. In some regions where glyphosate-resistance evolution has already occurred in rigid ryegrass, transgenic glyphosate-resistant canola was introduced in 2008, further highlighting the need to monitor glyphosate-resistance evolution in weeds. A rigid ryegrass population (WALR70) was collected in 2005 from a crop field in Esperance, Western Australia, after it had survived applications of glyphosate. Dose–response experiments confirmed resistance in the population, with the glyphosate rate resulting in 50% mortality (LD50) for WALR70 being 11 times greater than that for a susceptible biotype. The WALR70 population also had low levels of resistance to some acetyl coenzyme A carboxylase (ACCase)- and acetolactate synthase (ALS)-inhibiting herbicides (diclofop, fluazifop, clodinafop, tralkoxydim, chlorsulfuron, and imazethapyr), but was susceptible to other herbicide modes of action, such as atrazine, trifluralin, and paraquat. Two other rigid ryegrass populations assessed in this study were also confirmed to be resistant to glyphosate. The increasing number of glyphosate-resistant rigid ryegrass populations in Australia is of concern to growers because of the importance of glyphosate in intensive cropping systems and the introduction of glyphosate-resistant canola to this region. Nomenclature: Glyphosate; rigid ryegrass, Lolium rigidum Gaudin

Multiple herbicide-resistant wild radish (Raphanus raphanistrum) populations dominate Western Australian cropping fields

Abstract. Raphanus raphanistrum is a problematic weed, which has become increasingly difficult to control in Australian cropping regions. In 2010, a random survey was conducted across 14 million ha of the Western Australian grain belt to establish the frequency of herbicide resistance in R. raphanistrum and to monitor the change in resistance levels by comparing results with a previous survey in 2003. Screening R. raphanistrum populations with herbicides commonly used to control this weed revealed that most populations (84%) contained individual plants resistant to the acetolactate synthase-inhibiting herbicide chlorsulfuron, whereas 49% of populations also had plants resistant to the imidazolinone herbicides. Resistance to other mode of action herbicides (2,4-D (76%) and diflufenican (49%)) was also common. Glyphosate, atrazine and pyrasulfotole + bromoxynil remained effective on most R. raphanistrum populations. These results demonstrate that resistance to some herbicides has increased significantly since 2003 when the values were 54% for chlorsulfuron and 60% for 2,4-D; therefore, a wide range of weed management options that target all phases of the cropping program are needed to sustain these cropping systems in the future.