Jenna Malone

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.

Inheritance of resistance to 2,4-D and chlorsulfuron in a multiple-resistant population of Sisymbrium orientale.

BACKGROUND A population of Sisymbrium orientale from South Australia has multiple resistance to auxinic herbicides and inhibitors of acetohydroxyacid synthase (AHAS). Inheritance of resistance to 2,4-D and chlorsulfuron was studied in this population. RESULTS Crosses were made between seven resistant individuals as pollen donors to seven susceptible individuals. Sixteen F1 individuals from three crosses were identified by their lack of strong epinasty when treated with 200 g 2,4-D ha(-1). These individuals were selfed, and segregation analysis of strong epinasty in the resulting progeny fitted a 3:1 ratio for resistant:susceptible individuals when treated with 200 g 2,4-D ha(-1), as predicted by a single major gene. A detailed dose-response analysis of the F2 populations to 2,4-D confirmed single-gene inheritance. Analysis of segregation to 1 g chlorsulfuron ha(-1), a concentration that kills all susceptible individuals, was unable to determine the mode of inheritance. A detailed dose-response analysis indicated that two genes contributed to chlorsulfuron resistance: a dominant target-site mutation of Pro 197 to Ser and a second gene with dose-dependent dominance. CONCLUSIONS This population has a single dominant allele conferring 2,4-D resistance, whereas two genes contribute to chlorsulfuron resistance. Single dominant gene inheritance demonstrates that 2,4-D resistance can be readily selected.

Reduced Glyphosate Translocation in Two Glyphosate-Resistant Populations of Rigid Ryegrass (Lolium rigidum) from Fence Lines in South Australia

Abstract Populations of rigid ryegrass with resistance to glyphosate have started to become a problem on fence lines of cropping fields of southern Australian farms. Seed of rigid ryegrass plants that survived glyphosate application were collected from two fence line locations in Clare, South Australia. Dose–response experiments confirmed resistance of these fence line populations to glyphosate. Both populations required 9- to 15-fold higher glyphosate dose to achieve 50% mortality in comparison to a standard susceptible population. The mechanism of resistance in these populations was investigated. Sequencing a conserved region of the gene encoding 5-enolpyruvyl-shikimate-3-phosphate synthase identified no differences between the resistant and susceptible populations. Absorption of glyphosate into leaves of the resistant populations was not different from the susceptible population. However, the resistant plants retained significantly more herbicide in the treated leaf blades than did the susceptible plants. Conversely, susceptible plants translocated significantly more herbicide to the leaf sheaths and untreated leaves than the resistant plants. The differences in translocation pattern for glyphosate between the resistant and susceptible populations of rigid ryegrass suggest resistance is associated with altered translocation of glyphosate in the fence line populations. Nomenclature: Glyphosate; rigid ryegrass LOLRI, Lolium rigidum Gaudin.

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.

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.

Target Enzyme-Based Resistance to Clethodim in Lolium rigidum Populations in Australia

Abstract Clethodim resistance was identified in 12 rigid ryegrass populations from winter cropping regions in four different states of Australia. Clethodim had failed to provide effective control of these populations in the field and resistance was suspected. Dose–response experiments confirmed resistance to clethodim and butroxydim in all populations. During 2012, the LD50 of resistant populations ranged from 10.2 to 89.3 g ha−1, making them 3 to 34–fold more resistant to clethodim than the susceptible population. Similarly, GR50 of resistant population varied from 8 to 37.1 g ha−1, which is 3 to 13.9–fold higher than the susceptible population. In 2013, clethodim-resistant populations were 7.8 to 35.3–fold more resistant to clethodim than the susceptible population. The higher resistance factor in 2013, especially in moderately resistant populations, could have been associated with lower ambient temperatures during the winter of 2013. These resistant populations had also evolved cross-resistance to butroxydim. The resistant populations required 1.3 to 6.6–fold higher butroxydim dose to achieve 50% mortality and 3 to 27–fold more butroxydim for 50% biomass reduction compared to the standard susceptible population. Sequencing of the target-site ACCase gene identified five known ACCase substitutions (isoleucine-1781-leucine, isoleucine-2041-asparagine, aspartate-2078-glycine, and cysteine-2088-arginine, and glycine-2096-alanine) in these populations. In nine populations, multiple ACCase mutations were present in different individuals. Furthermore, two alleles with different mutations were present in a single plant of rigid ryegrass in two populations. Nomenclature: Clethodim; rigid ryegrass; Lolium rigidum Gaudin.

Basis of ACCase and ALS-inhibitor resistance in Hordeum glaucum Steud.

BACKGROUND Acetyl coenzyme-A carboxylase (ACCase) and/or acetolactate synthase (ALS) inhibitor resistance has been identified by herbicide resistance screening in eight populations obtained from cropping regions of South Australia. This study aimed to quantify the level of resistance and characterise the molecular basis of resistance to ACCase and ALS inhibitors in these H. glaucum populations. RESULTS H. glaucum populations from the Upper-North region were highly resistant (resistance index RI > 12) to the aryloxyphenoxypropionate (APP) herbicides quizalofop and haloxyfop and less resistant (RI = 2-12) to cyclohexanedione (CHD) herbicide clethodim, and some Mid-North populations had a low level of resistance (RI = 2-6) to the sulfonylurea (SU) herbicide mesosulfuron. Gene sequencing confirmed the presence of Ile-1781-Leu, Ile-2041-Asn and Gly-2096-Ala mutations in the ACCase gene, with no mutation found in the ALS gene. The use of the known metabolic inhibitor malathion in combination with mesosulfuron enhanced the activity of this herbicide. These populations were also susceptible to SU herbicide sulfometuron. CONCLUSION This study has documented APP-to-CHD herbicide cross-resistance, the first case of ACCase inhibitor resistance due to Ile-2041-Asn mutation, and characterised the resistance to ALS inhibitors in H. glaucum. Resistance to ACCase inhibitors is due to a target-site mutation. The reversal of SU resistance by malathion and susceptibility to sulfometuron suggests that non-target-site mechanisms confer resistance to ALS inhibitors.

Target-Site Point Mutation Conferring Resistance to Trifluralin in Rigid Ryegrass (Lolium rigidum)

Populations of rigid ryegrass suspected of resistance to trifluralin due to control failures exhibited varying levels of susceptibility to trifluralin, with 15 out of 17 populations deemed resistant (>20% plant survival). Detailed dose-response studies were conducted on one highly resistant field-evolved population (SLR74), one known multiply resistant population (SLR31), and one susceptible population (VLR1). On the basis of the dose required to kill 50% of treated plants (LD50), SLR74 had 15-fold greater resistance than VLR1, whereas, the multiply resistant SLR31 had 10-fold greater resistance than VLR1. Similarly, on the basis of dose required to reduce shoot biomass by 50% (GR50), SLR74 had 17-fold greater resistance than VLR1, and SLR31 had 8-fold greater resistance than VLR1. Sequencing of the α-tubulin gene from resistant plants of different populations confirmed the presence of a previously known goosegrass mutation causing an amino acid substitution at position 239 from threonine to isoleucine in resistant population SLR74. This mutation was also found in 4 out of 5 individuals in another highly resistant population TR2 and in 3 out of 5 individuals of TR4. An amino acid substitution from valine to phenylalanine at position 202 was also observed in TR4 (3 out of 5 plants) and TR2 (1 out of 5 plants). There was no target-site mutation identified in SLR31. This study documents the first known case of field-evolved target-site resistance to dinitroaniline herbicides in a population of rigid ryegrass. Nomenclature: Trifluralin; rigid ryegrass; Lolium rigidum Gaudin; goosegrass; Eleusine indica (L.) Gaertn.