Peter Boutsalis

A Decade of Glyphosate-Resistant Lolium around the World: Mechanisms, Genes, Fitness, and Agronomic Management

Abstract Glyphosate resistance was first discovered in populations of rigid ryegrass in Australia in 1996. Since then, glyphosate resistance has been detected in additional populations of rigid ryegrass and Italian ryegrass in several other countries. Glyphosate-resistant rigid ryegrass and Italian ryegrass have been selected in situations where there is an overreliance on glyphosate to the exclusion of other weed control tactics. Two major mechanisms of glyphosate resistance have been discovered in these two species: a change in the pattern of glyphosate translocation such that glyphosate accumulates in the leaf tips of resistant plants instead of in the shoot meristem; and amino acid substitutions at Pro 106 within the target site, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). There are also populations with both mechanisms. In the case of glyphosate resistance, the target site mutations tend to provide a lower level of resistance than does the altered translocation mechanism. Each of these resistance mechanisms is inherited as a single gene trait that is largely dominant. As these ryegrass species are obligate outcrossers, this ensures resistance alleles can move in both pollen and seed. Some glyphosate-resistant rigid ryegrass populations appear to have a significant fitness penalty associated with the resistance allele. Field surveys show that strategies vary in their ability to reduce the frequency of glyphosate resistance in populations and weed population size, with integrated strategies—including alternative weed management and controlling seed set of surviving plants—the most effective. Nomenclature: Glyphosate; rigid ryegrass, Lolium rigidum Gaud. LOLRI; Italian ryegrass, Lolium multiflorum Lam. LOLMU.

Incidence of Herbicide Resistance in Rigid Ryegrass (Lolium rigidum) across Southeastern Australia

Abstract Herbicide resistance in rigid ryegrass is an escalating problem in grain-cropping fields of southeastern Australia due to increased reliance on herbicides as the main method for weed control. Weed surveys were conducted between 1998 and 2009 to identify the extent of herbicide-resistant rigid ryegrass across this region to dinitroaniline, and acetolactate synthase- and acetyl coenzyme A (CoA) carboxylase-inhibiting herbicides. Rigid ryegrass was collected from cropped fields chosen at random. Outdoor pot studies were conducted during the normal winter growing season for rigid ryegrass with PRE-applied trifluralin and POST-applied diclofop-methyl, chlorsulfuron, tralkoxydim, pinoxaden, and clethodim. Herbicide resistance to trifluralin in rigid ryegrass was identified in one-third of the fields surveyed from South Australia, whereas less than 5% of fields in Victoria exhibited resistance. In contrast, resistance to chlorsulfuron was detected in at least half of the cropped fields across southeastern Australia. Resistance to the cereal-selective aryloxyphenoxypropionate-inhibiting herbicides diclofop-methyl, tralkoxydim, and pinoxaden ranged between 30 and 60% in most regions, whereas in marginal cropping areas less than 12% of fields exhibited resistance. Resistance to clethodim varied between 0 and 61%. Higher levels of resistance to clethodim were identified in the more intensively cropped, higher-rainfall districts where pulse and canola crops are common. These weed surveys demonstrated that a high incidence of resistance to most tested herbicides was present in rigid ryegrass from cropped fields in southeastern Australia, which presents a major challenge for crop producers. Nomenclature: Chlorsulfuron; clethodim; diclofop-methyl; pinoxaden; tralkoxydim; trifluralin; rigid ryegrass, Lolium rigidum Gaudin; canola, Brassica napus L.

Syngenta Quick-Test: A Rapid Whole-Plant Test for Herbicide Resistance1

Abstract: A diagnostic test (Syngenta Quick-Test, QT) used for testing grass weed survivors to herbicides in the field for resistance was evaluated. Cuttings from grass weeds were transplanted into pots to regenerate new leaves, then treated with herbicide. In greenhouse experiments, resistance of known herbicide-resistant blackgrass biotypes to the aryloxyphenoxypropanoate herbicides CGA 184927 and fenoxaprop-ethyl and to the phenylurea herbicide isoproturon was verified by the QT. The findings were similar to those for seedlings grown from seed. Rigid ryegrass from suspect resistant fields in South Australia was sampled and sent by post to Switzerland for QT analysis. Resistance was confirmed in less than 4 wk, which verified resistance as responsible for the field failures. The added features of the QT over current resistance tests suggest a likely fit for in-season testing of surviving weeds and possible follow-up action. Nomenclature: CGA 184927, (propynyl (R)-2-[4-(5-chloro-3-fluoro-2-pyridyloxy)phenoxy]-propanoate); fenoxaprop-ethyl; isoproturon; blackgrass, Alopecurus myosuroides Huds. #3 ALOMY; rigid ryegrass, Lolium rigidum Gaudin # LOLRI. Additional index words: Resistance test, aryloxyphenoxypropanoate, phenylurea. Abbreviations: ACCase, acetyl coenzyme A carboxylase (EC 6.4.1.2); APP, aryloxyphenoxypropanoate; CHD, cyclohexanedione; DAT, days after treatment; PU, phenylurea; QT, Syngenta Quick-Test; SU, sulfonylurea.

EPSPS gene amplification in glyphosate-resistant Bromus diandrus

BACKGROUND Glyphosate is the most widely used herbicide in the world and has been intensively used to control B. diandrus, a problematic weed of crops and pastures in southern Australia. RESULTS Resistance to glyphosate was identified in two populations of B. diandrus that were nearly fivefold more resistant to glyphosate than wild-type plants. Both populations contained EPSPS gene amplification, with resistant plants having an average of around 20-fold the number of copies of EPSPS compared with susceptible plants. EPSPS expression was also increased in resistant plants of both populations; however, expression levels were not correlated with the number of EPSPS copies. Amplification of only one of the four EPSPS genes present in B. diandus was detected. Investigation into the inheritance of glyphosate resistance found no segregation in the F2 generation. Every individual in the F2 populations contained between three and 30 copies of EPSPS; however, on average they contained fewer copies compared with the parent resistant population. CONCLUSIONS Glyphosate resistance in B. diandrus is due to EPSPS gene amplification. Resistance is heritable but complex.

Rigid ryegrass (Lolium rigidum) populations containing a target site mutation in EPSPS and reduced glyphosate translocation are more resistant to glyphosate

Abstract Glyphosate is widely used for weed control in the grape growing industry in southern Australia. The intensive use of glyphosate in this industry has resulted in the evolution of glyphosate resistance in rigid ryegrass. Two populations of rigid ryegrass from vineyards, SLR80 and SLR88, had 6- to 11-fold resistance to glyphosate in dose-response studies. These resistance levels were higher than two previously well-characterized glyphosate-resistant populations of rigid ryegrass (SLR77 and NLR70), containing a modified target site or reduced translocation, respectively. Populations SLR80 and SLR88 accumulated less glyphosate, 12 and 17% of absorbed glyphosate, in the shoot in the resistant populations compared with 26% in the susceptible population. In addition, a mutation within the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) where Pro106 had been substituted by either serine or threonine was identified. These two populations are more highly resistant to glyphosate as a consequence of expressing two different resistance mechanisms concurrently. Nomenclature: Glyphosate; rigid ryegrass, Lolium rigidum Gaud. LOLRI.

Temperature influences the level of glyphosate resistance in barnyardgrass (Echinochloa colona)

BACKGROUND Echinochloa colona is an important summer-growing weed species in cropping regions of northern Australia that has evolved resistance to glyphosate owing to intensive use of this herbicide in summer fallow. RESULTS Pot trials conducted at 20 and 30 °C on six E. colona populations showed a significant increase in the level of glyphosate resistance in resistant populations at 30 °C compared with 20 °C. However, there was no influence of growth temperature on glyphosate susceptibility of the sensitive population. Sequencing of the target-site gene (EPSPS) of the six populations identified a mutation at position 106 leading to a change from proline to serine in the most resistant population A533.1 only. EPSPS gene amplification was not detected in any of the resistant populations examined. Examining (14) C-glyphosate uptake on two resistant and one susceptible population showed a twofold increase at 20 °C; however, few differences in glyphosate translocation occurred from the treated leaf to other plant parts between populations or temperatures. CONCLUSION There is reduced efficacy of glyphosate at high temperatures on resistant E. colona populations, making these populations harder to control in summer.

Target-Site Point Mutations Conferring Resistance to ACCase-Inhibiting Herbicides in Smooth Barley (Hordeum glaucum) and Hare Barley (Hordeum leporinum)

Abstract Acetyl coenzyme A carboxylase (ACCase)-inhibiting herbicides affect fatty acid biosynthesis in plants and are widely used to control smooth and hare barley in dicot crops in Australia. Recently, growers have experienced difficulty in controlling smooth and hare barley with herbicides from this mode of action. Dose–response experiments conducted on five suspected resistant populations confirmed varying levels of resistance to quizalofop and haloxyfop. The level of resistance in these populations was greater than 27-fold to quizalofop and greater than 15-fold to haloxyfop. The quizalofop dose required to reduce shoot biomass by 50% (GR50) for the resistant populations varied from 52.6 to 111.9 g ha−1, and for haloxyfop from 26.5 to 71.3 g ha−1. Sequencing the CT domain of the ACCase gene from resistant plants of different populations confirmed the presence of previously known mutations Ile1781Leu and Gly2096Ala. Amino acid substitution at the 2096 position conferred a greater level of resistance to haloxyfop than the substitution at the 1781 position. This study documents the first known case of field-evolved target-site resistance to ACCase-inhibiting herbicides in Australian populations of smooth barley. Nomenclature: Quizalofop; haloxyfop; smooth barley, Hordeum glaucum (Steud.) Tzvelev; hare barley, Hordeum leporinum (Link) Arcang.

Control of Rigid Ryegrass in Australian Wheat Production with Pyroxasulfone

Abstract In Australia, most wheat is sown in a no-till system without prior cultivation where herbicides are applied prior to sowing and incorporated by the planter. Trifluralin has been the most widely used PRE herbicide to control rigid ryegrass. The objective of this research was to determine crop safety and efficacy of alternative mechanism of action PRE herbicides for rigid ryegrass control in no-till wheat production. Pyroxasulfone achieved 98% control with PRE applications. The alternative PRE herbicides tested alone and in mixtures occasionally resulted in a significant reduction in wheat emergence but not crop yield. Trifluralin treatments failed at sites having trifluralin-resistant rigid ryegrass. Pyroxasulfone and prosulfocarb plus S-metolachlor were effective for control of rigid ryegrass across all trials with control ranging from 64 to 94%. This research demonstrated that PRE applications of herbicides other than trifluralin such as pyroxasulfone and prosulfocarb plus S-metolachlor can be safely and effectively used to control rigid ryegrass in no-till wheat. Nomenclature: Cinmethylin; S-metolachlor; prosulfocarb; pyroxasulfone; trifluralin; rigid ryegrass; Lolium rigidum Gaudin LOLRI; wheat; Triticum aestivum L. ‘Frame’; ‘Pugsley’; ‘Young’; ‘Yitpi’. Resumen En Australia, la mayoría del trigo se siembra en un sistema de labranza cero sin cultivo previo donde los herbicidas son aplicados antes de la siembra e incorporados con la sembradora. Trifluralin ha sido el herbicida PRE más ampliamente usado para el control de Lolium rigidum. El objetivo de esta investigación fue determinar la seguridad para el cultivo y la eficacia de herbicidas PRE con mecanismos de acción alternativos para el control de L. rigidum en producción de trigo en labranza cero. Pyroxasulfone alcanzó 98% de control con aplicaciones PRE. Los herbicidas PRE alternativos evaluados solos y en mezclas ocasionalmente resultaron en una reducción significativa en la emergencia del trigo pero no del rendimiento del cultivo. Los tratamientos de trifluralin fallaron en sitios que tenían L. rigidum resistente a trifluralin. Pyroxasulfone y prosulfocarb más S-metolachlor fueron efectivos para controlar L. rigidum en todos los ensayos con un control que fluctuó entre 64 y 94%. Esta investigación demostró que aplicaciones PRE de herbicidas diferentes a trifluralin, tales como pyroxasulfone y prosulfocarb más S-metolachlor pueden ser usados en forma segura y efectiva para el control de L. rigidum en trigo en labranza cero.

EPSPS gene amplification conferring resistance to glyphosate in windmill grass (Chloris truncata) in Australia

BACKGROUND Five glyphosate-resistant populations of Chloris truncata originally collected from New South Wales were compared with one susceptible (S) population from South Australia to confirm glyphosate resistance and elucidate possible mechanisms of resistance. RESULTS Based on the amounts of glyphosate required to kill 50% of treated plants (LD50 ), glyphosate resistance (GR) was confirmed in five populations of C. truncata (A536, A528, T27, A534 and A535.1). GR plants were 2.4-8.7-fold more resistant and accumulated less shikimate after glyphosate treatment than S plants. There was no difference in glyphosate absorption and translocation between GR and S plants. The EPSPS gene did not contain any point mutation that had previously been associated with resistance to glyphosate. The resistant plants (A528 and A536) contained up to 32-48 more copies of the EPSPS gene than the susceptible plants. CONCLUSION This study has identified EPSPS gene amplification contributing to glyphosate resistance in C. truncata. In addition, a Glu-91-Ala mutation within EPSPS was identified that may contribute to glyphosate resistance in this species.

Multiple Resistance to Acetohydroxyacid Synthase–Inhibiting and Auxinic Herbicides in a Population of Oriental Mustard (Sisymbrium orientale)

Abstract A population of oriental mustard from Port Broughton in South Australia was reported as not being controlled by 2,4-D. Dose response experiments determined this population was resistant to both 2,4-D and MCPA, requiring greater than 20 times more herbicide for equivalent control compared to a known susceptible population (from Roseworthy, South Australia) and a population resistant only to the acetohydroxyacid synthase (AHAS)-inhibiting herbicides (from Tumby Bay, South Australia). The Port Broughton population was also found to be resistant to three chemical groups that inhibit AHAS; however, the level of resistance was lower than the known acetolactate synthase–resistant population from Tumby Bay. Herbicides from other modes of action were able to control the Port Broughton population. Assays of isolated AHAS from the Port Broughton population showed high levels of resistance to the sulfonylurea and sulfonamide herbicide groups, but not to the imidazolinone herbicides. A single nucleotide change in the AHAS gene that predicted a Pro to Ser substitution at position 197 in the protein was identified in the Port Broughton population. This population of oriental mustard has evolved multiple resistance to AHAS-inhibiting herbicides (AHAS inhibitors) and auxinic herbicides, through a mutation in AHAS and a second nontarget-site mechanism. Whether the same mechanism provides resistance to both AHAS inhibitors and auxinic herbicides remains to be determined. Multiple resistance to auxinic herbicides and AHAS inhibitors in the Port Broughton population will make control of this population more difficult. Nomenclature: 2,4-D; MCPA; oriental mustard, Sisymbrium orientale Torn.