Point mutations including a novel Pro-197-Phe mutation confer cross-resistance to acetolactate synthase (ALS) inhibiting herbicides in Lactuca serriola in Australia

BACKGROUND

Control of prickly lettuce has become increasingly difficult for lentil growers in southern Australia because of widespread resistance to common herbicides, a lack of alternative herbicide options and the prolific production of highly mobile seed. This study aimed to quantify acetolactate synthase (ALS)-inhibiting herbicide resistance in the Mid North (MN) and Yorke Peninsula (YP) of South Australia, characterize the resistance mutations present and investigate population structure and gene flow in this species.

RESULTS

Resistance was identified in all populations tested, with average survival of 92% to chlorsulfuron and 95% to imazamox + imazapyr. Five different amino acid substitutions were identified at proline 197 of the ALS gene. There was no significant difference in the median lethal dose (LD₅₀) between plants with these five different substitutions when treated with metsulfuron-methyl; however, the imidazolinone resistance level was higher in plants with a phenylalanine substitution and lower in plants with a serine. Population structure based on 701 single nucleotide polymorphisms and 271 individuals provided evidence for both independent evolution of the same mutation in different populations, as well as frequent short- to medium-distance dispersal accompanied by occasional long-distance dispersal events. The overall inbreeding coefficient (F_IS) was calculated at 0.5174, indicating an intermediate level of outcrossing despite the cross-pollination experiment showing only low outcrossing. In the structure analyses, most individuals from YP were assigned to a single cluster, whereas most individuals from MN were assigned 50% to each of two clusters, indicating some genetic differences between these two regions, but also evidence for dispersal between them.

CONCLUSIONS

Use of imidazolinone herbicides has selected for mutations conferring higher levels of resistance, such as the Pro-197-Phe mutation, and resulted in further spread of resistance in this species. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Joint action of Cochliobolus lunatus and atrazine on Echinochloa crus-galli (L.) Beauv.

The potential of the endemic fungus Cochliobolus lunatus as a biological control agent against Echinochloa crus-galli was investigated. Under appropriate conditions the fungus produced leaf necrosis on E. crus-galli resulting in death of young seedlings. However, plants with more than two leaves mostly recovered after some time. Bean, barley, maize, oat, rye, tomato and wheat were highly resistant to the fungus. In experiments in a climate room or in a glasshouse E. crus-galli with more than two leaves could effectively be controlled by C. lunatus in combination with a sub-lethal dosage of atrazine as low as 2.5 mg m^?2 due to a positive interaction between fungus and herbicide.     

Timing of molinate applications for the control of Echinochloa crus-galli (L.) Beauv. in rice

Application of molinate to young rice (Oryza sativa L.) immediately before the ihird flushing irrigalion, when seedlings of E. crus-galli were in the 2 2½-leaf stage, resulted m bctier weed control and greater increases in crop growth and yield than earlier application prior to ihe second flushing, or than later application prior to permanent flooding, Molinale applied prior to a flushing was lost from the soil rapidly. 79–85% loss within 13 days. Chemical remaining after this time disappeared more slowly and little further loss occurred until permaneni flooding 24 days afier application. Despite the rapid dissipation of molinate. application of 3 kg/ha before a flushing irrigation controlled newly-emerging E. crus-galli until permaneni Hood ing I S days after spraying. Epoque des traiiemenia au molinaie pour la lutte contre Echinochloa crus galli (L.) Beauv. en rizière L'applicaiion du molinate sur du riz [Oryza saliva L.) jeune. immédiatement apres la troisiéme irrigation par submersion, lorsque les plantules d’E crus galliétaient au stade 2 feuilles/2 feuilles 1/2adonné un meilleur résultat et des augmentations de croissance et de rendement du riz plus grandes qu'une appli cation plus précoce. avant la seconde irrigation, on qu'une application plus tardive avant la submersion permanente. Le molinate appliqué avant une irrigation a disparu rapidementdu sol: 79 à 85% de perte en 13 jours, Le produit persistant après cette date a disparu pius leniement et il n'y a euque peude pertes ultérieures jusquà la submersion permanenie. 24 jours apres le traitement. En dépit de la rapide dégradation du molinate. un traitement à 3 kg/ha avant une irrigation par submersion a été efficace contre les E. crus galli nouvellement levés. jusquà la submersion permanentc. 15 jours après le traitement. Der günstigste Zeitpunkt für Anwendung von Molinat zur Bekämpfung von Echinochloa crus-galli (L.) Beauv. in Reis Die Anwendung von Molinate injungem Reis (Oryza sativa L.) unmittelbar vor der dritten Rieselbewä.sserung. wenn die Pflanzen von E. crus-galli im 2 bis 2 1/2-Blatlsiadium waren. bewirkte eine bessere Unkrautbekämpfung. ein bcsseres Wachstum der Kultur und einen höheren Ertrag alseine Be handlung zu einem früheren Zeitpunkt vor der zweiien Rieselbewässerung. Oder eine spätere Anwendung vor der permanenten Überstauung. Wurde Molmat vor emer Rieselbewässerung appliziert. traten innerhalb von 13 Tagen Herbizidveriuste von 79–85% im Boden ein Der Rest des Herbizids verschwand langsamer. und bis zum Zeitpunkt der permanenten Über-stauung—24 Tage nach der Applikation—ging nur wenig Her-bizid verloren. Troiz des schnellen Veriusts des Herbizids wurden durch die Anwendung von 3 kg/ha Molinat vor einer Rieselbewässerung frisch aufgelaufene E.crus-galli-Pflanzen bis zum Zeitpunki der permanenten Überstauung bckämpft. Der Zeilraum zwischen Spritzung und Überstauung betrug in den Versuchen 1 5 Tage.     

Resistance of dicot weeds to acetolactate synthase (ALS)-inhibiting herbicides in Australia

A biotype of Sonchus oleraceus L. and two bio types of Sisymbrium orientate Torn., SSO 3 and NSO 1, are the first dicot weeds in Australia to develop resistance to ALS-inhibiting herbicides. The resistant biotypes had been exposed to va rying periods of selection with sulfonylurea her bicides. All three biotypes are resistant to a range of sulfonylurea and imidazolinone herbicides. The S. orientale biotypes are also resistant to the triazolopyrimidine herbicide, flumetsulam. LD₅₀ ratios of resistant Sonchus oleraceus for sulfony lurea and imidazolinone herbicides are greater than 64-fold and 4.5-fold, respectively. GR₅₀ ratios are greater than 9 for sulfonylureas and 7.4 for imazapyr. The LD₅₀ ratios for both S. orien tale biotypes for chlorsulfuron, sulfometuron methyl, metsulfuron-methyl, flumetsulam and imazethapyr are greater than 110-, 15-, 7-, 24- and 29-fold, respectively. All resistant biotypes are susceptible to MCPA, diuron and diflufenican, herbicides which do not inhibit ALS.     

Effects of surfactants on primisulfuron activity in barnyardgrass (Echinochloa crus-galli [L.] Beauv.) and green foxtail (Setaria viridis [L.] Beauv.)

Laboratory and greenhouse studies were conducted to measure the contact angle of primisulfuron droplets with and without surfactants on the leaf surfaces of barnyardgrass and green foxtail, to determine the primisulfuron activity on these weed species, and to examine the spray deposit of primisulfuron with and without surfactants on the leaf surface of green foxtail using scanning electron microscopy. A non-ionic surfactant (NIS) and an organosilicone wetting agent (OWA) were used. The contact angles of 1 μL droplets were measured on the leaf surface using a goniometer. The activity of primisulfuron on barnyardgrass and green foxtail was assessed at 3 weeks after treatment based on visual injury and the fresh weight. The contact angles of the droplets of primisulfuron on the adaxial surface of the barnyardgrass and green foxtail leaves were 152° and 127°, respectively, when applied without surfactant. The addition of either surfactant markedly reduced the contact angle for both weed species, which was lowest when the OWA was added to primisulfuron. The percentage injury of barnyardgrass was very low, even at the higher rate of primisulfuron, regardless of the surfactant. Primisulfuron at 40 g ha⁻¹ controlled 43% of green foxtail without surfactant, which increased to 65% with the NIS and 83% with the OWA. Primisulfuron with a surfactant markedly reduced the fresh weight of green foxtail compared with primisulfuron applied alone, regardless of the primisulfuron rate and surfactant type. The scanning electron micrographs showed a uniform deposit of spray droplets, with close contact of the droplets to the leaf epicuticular surface in green foxtail in the presence of a surfactant compared with no surfactant. The enhanced primisulfuron activity on green foxtail with surfactants was related to the reduced contact angle and uniform deposition of the primisulfuron spray droplets on the leaf surface.     

Role of underground parts of barnyardgrass (Echinochloa crus-galli (L.) Beauv. var. formosensis Ohwi) in its sensitivity to thenylchlor in soil

In the present study, the phytotoxic activity of top-soil applied with thenylchlor [2-chloro- N -(3-methoxy-2-thieny)-2',6'-dimethylacetanilide] on the growth of rice ( Oryza sativa L.) was dependent on the emergence depth in soil but its activity on barnyardgrass ( Echinochloa crus-galli (L.) Beauv. var. formosensis Ohwi) was only slightly affected by the emergence depth. However, the phytotoxic activity on barnyardgrass and rice was similar irrespective of the different emergence depths in its treatment to all soil layers. Thenylchlor treatment to the mesocotyl of barnyardgrass induced significant inhibition of shoot elongation, whereas the treatment to the coronal root only inhibited the coronal elongation without inhibiting shoot elongation. Absorption and translocation of ¹⁴C-thenylchlor in barnyardgrass were determined in water culture. The different amounts of radioactivity per plant among the treatments to the underground parts were due to the plant part that came in contact with ¹⁴C-thenylchlor. The radioactivity per dry weight was found to be higher in the basal part of the shoot than in its upper part in all treatments to the underground parts. It was suggested that the phytotoxic activity of thenylchlor on the growth of barnyardgrass in soil is induced by its accumulation in the basal part of the shoot through translocation. This primarily occurs after the absorption substantially by the mesocotyl from the herbicide-treated layer and additionally by other underground parts.     

Differential ability of alcohol fermentation between the seeds of flooding-tolerant and flooding-susceptible varieties of Echinochloa crus-galli (L.) Beauv.

Seed germination and respiratory metabolism under aerobic and anaerobic conditions were studied in the flooding-tolerant and flooding-susceptible varieties of Echinochloa crus-galli with an identical genome and common ancestry . In the flooding-tolerant E. crus-galli var. formosensis , the seeds imbibed under nitrogen could germinate and exhibited an I/N quotient > 0.6. They accelerated glycolysis and concomitantly produced large and equimolar quantities of carbon dioxide and ethanol, suggesting that the seeds of this variety showed operation of the Pasteur effect and respired through alcohol fermentation under anaerobic conditions. The seeds excreted most of the toxic fermentation product. In contrast, the seeds of the flooding-susceptible E. crus-galli var. praticola were capable of germinating only under aerobic conditions through the conventional aerobic respiration and were unable to anaerobically respire to germinate through alcohol fermentation regardless of the presence of both sufficient alcohol dehydrogenase activity and a high redox charge of the pyridine nucleotides.     

五氟磺草胺等6种除草剂对直播稻田高龄稗草的生物活性及田间控制效果

为筛选防治高龄稗草的有效药剂,用室内盆栽法测定了6种除草剂对高龄稗草的生物活性,并通过大田试验作了进一步验证。盆栽法测得五氟磺草胺和双草醚对高龄稗草的活性最高,其ED50值均为9.3 g/hm2,而噁唑酰草胺和氰氟草酯对高龄稗草活性最低,ED50 分别为28.7、28.6 g/hm2。其余2种除草剂嘧啶肟草醚和氟吡磺隆对高龄稗草的活性介于以上两者之间,ED50为10.1 g/hm2和11.4 g/hm2。田间试验结果显示,2.5%五氟磺草胺油悬浮剂30、37.5 g/hm2和10%噁唑酰草胺乳油 150、180 g/hm2 4个处理对高龄稗草防效优良,处理后20、40 d防效均在93.0%以上;处理后40 d,10%双草醚悬浮剂3种浓度处理和5%嘧啶肟草醚乳油 80 g/hm2的防效均在91.4%以上;10％氟吡磺隆水分散粒剂和10%氰氟草酯乳油对高龄稗草防效均不理想,药后20、40 d各处理的株防效均不到80%。     

Response of an acetolactate synthase (ALS)-resistant biotype of Amaranthus rudis to selected ALS-inhibiting and alternative herbicides

An Amaranthus rudis Saner (common water-hemp) biotype from a field treated for two consecutive years with a mixture of chlorimuron and metribuzin was tested in greenhouse and laboratory studies to assess resistance and cross-resistance to four acetolactale synthase (ALS)-inhibiting herbicides. The biotype demonstrated >1920-fold resistance at the whole plant level, and >850-fold resistance at the ALS enzyme to chlorimuron, compared with a susceptible biotype. This chlorimuron-resistant biotype was also cross-resistant to primisuifu-ron, haiosulfuron and imazethapyr. In greenhouse studies, atrazine alone or in combination with ALS-inhibiting herbicides provided excellent control of the resistant biotype of A. rudis . Combinations of dicamba and ALS-inhibitors also provided adequate control. Additionally, premixtures of flumetsulam and metolachlor and of dicamba and atrazine furnished excellent control of this chlorimuron-resistant A. rudis biotype.     

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.     

Molecular identification of Echinochloa oryzicola Vasing. and E. crus-galli (L.) Beauv. using a polymerase chain reaction–restriction fragment length polymorphism technique

Polymerase chain reaction (PCR) and polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) techniques were applied for establishing the reliable practice in identification of Echinochloa oryzicola Vasing. and E. crus-galli (L.) Beauv. (barnyardgrass). Total DNA was extracted from 18 accessions and 86 individuals of E. oryzicola , 33 accessions and 140 individuals of E. crus-galli var. crus-galli , 23 individuals of E. crus-galli var. praticola , and six individuals of E. crus-galli var. formosensis that were collected from Japan. A partial region of intergenetic spacer between trn T and trn L, and an intron of trn L were amplified separately using a trn-a and trn-b1 primer set, and a trn-c and trn-d primer set, respectively. All individuals of E. oryzicola showed the same fragment amplified by the trn-a and trn-b1 primer set. The fragment was 481 bp in length, and was undigested by Eco R I, whereas all individuals of E. crus-galli , including three botanical varieties, showed the same fragment with a 449-bp length. The fragment was digested by Eco R I into two fragments (178 and 271 bp). The fragment amplified by the trn-c and trn-d primer set in all individuals of E. oryzicola was digested by Alu I into two fragments (174 and 452 bp), but undigested by Dra I. In contrast, the fragment amplified by the trn-c and trn-d primer set in all individuals of E. crus-galli was digested by Dra I into two fragments (134 and 487 bp), but undigested by Alu I. There was no intraspecific variation in these regions; thus, these two species are easily identifiable by using our method.     

银胶菊花对苘麻和稗的化感作用

通过室内培养皿法,研究了银胶菊花水浸提液及其乙酸乙酯相、正丁醇相、剩余水相不同极性组分对苘麻和稗种子萌发及幼苗生长的影响。定性测定结果表明,在50 g/L浓度处理下,水浸提液完全抑制苘麻和稗种子萌发,对苘麻和稗根长、芽长、鲜重抑制率分别为82.6%、89.1%、84.3%和91.7%、55.8%、39.1%;正丁醇相抑制苘麻种子萌发和幼苗生长最强,乙酸乙酯相抑制稗种子萌发和幼苗生长最强。定量测定结果表明,在1 g/L浓度处理下,乙酸乙酯相生物活性最高,苘麻和稗种子萌发率分别为13.3%和33.3%,对苘麻和稗根长、芽长、鲜重的抑制率分别为63.1%、80.0%、58.3%和51.3%、26.7%、23.3%。     

Target-site basis for resistance to acetolactate synthase inhibitor in Water chickweed (Myosoton aquaticum L.)

Water chickweed is a widespread and competitive winter annual or biennial weed of wheat in China. One Water chickweed population (HN02) resistant to several acetolactate synthase (ALS) inhibitors was found in Henan province of China. Whole-plant bioassays showed that HN02 was high resistance to tribenuron (292.05-flod). In vitro ALS assays revealed that resistance was due to reduced sensitivity of the ALS enzyme to tribenuron. The I₅₀ value for HN02 was 85.53 times greater respectively than that of susceptible population (SD05). This altered ALS sensitivity in the resistant population was due to a mutation in the ALS gene resulting in a Pro197 to Ser substitution. Cross-resistance experiments indicated that HN02 exhibited various resistance patterns to pyrithiobac-sodium, florasulam and pyroxsulam, without resistance to imazethapyr. This is the first report of tribenuron-resistant Water chickweed in Henan province of China, target-site based resistance was established as being due to an insensitive form of ALS, resulting from a Pro to Ser substitution at amino acid position 197 in the ALS gene.     

Tolerance to acetolactate synthase and acetyl-coenzyme A carboxylase inhibiting herbicides in Vulpia bromoides is conferred by two co-existing resistance mechanisms

Vulpia bromoides is a grass species naturally tolerant to acetolactate synthase (ALS) and acetyl-coenzyme A carboxylase (ACCase) inhibiting herbicides. The mechanism of tolerance to ALS herbicides was determined as cytochrome P450-monooxygenase mediated metabolic detoxification. The ALS enzyme extract partially purified from V. bromoides shoot tissue was found to be as sensitive as that of herbicide susceptible Lolium rigidum to ALS-inhibiting sulfonylurea (SU), triazolopyrimidine (TP), and imidazolinone (IM) herbicides. Furthermore, phytotoxicity of the wheat-selective SU herbicide chlorsulfuron was significantly enhanced in vivo in the presence of the known P450 inhibitor malathion. In contract, the biochemical basis of tolerance to ACCase inhibiting herbicides was established as an insensitive ACCase. In vitro ACCase inhibition assays showed that, compared to a herbicide susceptible L. rigidum, the V. bromoides ACCase was moderately (4.5- to 9.5-fold) insensitive to the aryloxyphenoxypropionate (APP) herbicides diclofop, fluazifop, and haloxyfop and highly insensitive (20- to >71-fold) to the cyclohexanedione (CHD) herbicides sethoxydim and tralkoxydim. No differential absorption or de-esterification of fluazifop-P-butyl was observed between the two species at 48 h after herbicide application, and furthermore V. bromoides did not detoxify fluazifop acid as rapidly as susceptible L. rigidum. It is concluded that two co-existing resistance mechanisms, i.e., an enhanced metabolism of ALS herbicides and an insensitive target ACCase, endow natural tolerance to ALS and ACCase inhibiting herbicides in V. bromoides.     

Influence of thiobencarb on nitrate reductase, nitrite reductase and DCPIP photoreduction in rice and Echinochloa crus-galli (L.) (barnyardgrass)

Summary. The influence of thiobencarb at 1500 ppm on levels of nitrate reductase, nitrite reductase and on 2,6-dichlorophenolindophenol (DCPIP) photoreduction was studied in rice ( Oryza sativa L.) and Echinochloa crus-galli (L.) (barnyardgrass) to understand the different sensitivities of these two plants to the herbicide. The herbicide treatment did not cause appreciable inhibition of nitrate reductase and nitrite reductase in rice leaf sections. In barnyard grass, the treatment strongly lowered nitrite reductase activity. DCPIP photoreduction by isolated chloroplasts of the treated rice leaves was not greatly affected. On the other hand DCPIP photoreduction by both mesophyll and bundle sheath chloroplasts from treated barnyardgrass was inhibited by 70%. Herbicide caused accumulation of nitrite in treated barnyard grass leaves when compared to rice. Thiobencarb phytotoxicity to barnyard grass may be due to impairment of the Hill reaction that in turn caused accumulation of nitrite in the leaf tissue.