Long Du

Target-site mechanism of ACCase-inhibitors resistance in American sloughgrass (Beckmannia syzigachne Steud.) from China

American sloughgrass (Beckmannia syzigachne) is a troublesome weed in winter wheat field rotated with rice in China. Fenoxaprop-p-ethyl and pinoxaden were observed failing to control American sloughgrass in the same filed in Lujiang county in 2011 and 2012, respectively. Whole-plant bioassay was conducted to determine the resistance to fenoxaprop-p-ethyl, pinoxaden and other herbicides in American sloughgrass. Dose-response experiment indicated that Lujiang population was highly resistant to fenoxaprop-p-ethyl (199.8-fold), pinoxaden (76.2-fold), clodinafop-propargyl (334.1-fold) and sethoxydim (15.9-fold); moderately resistant to clethodim (6.3-fold), susceptible to mesosulfuron-methyl, flucarbazone-sodium, pyroxsulam and isoproturon. Partial gene of CT domain was cloned and sequenced to confirm the molecular mechanism of resistance to ACCase-inhibiting herbicides. A Trp2027Cys mutation was found in Lujiang population according to the sequencing result. This mutation is the molecular mechanism of resistance to fenoxaprop-p-ethyl in Lujiang population. Furthermore the Trp2027Cys mutation very likely results in cross resistance to clodinafop-propargyl and pinoxaden in Lujiang population. 103 mutant homozygotes were detected from the 108 plants tested using a rapid dCAPS method developed in this paper. This is the first report of pinoxaden resistance and a mutation at position of 2027 for American sloughgrass.

Molecular basis of multiple resistance to ACCase- and ALS-inhibiting herbicides in Alopecurus japonicus from China

Fenoxaprop-P-ethyl-resistant Alopecurus japonicus has become a recurring problem in winter wheat fields in eastern China. Growers have resorted to using mesosulfuron-methyl, an acetolactate synthase (ALS)-inhibiting herbicide, to control this weed. A single A. japonicus population (AH-15) resistant to fenoxaprop-P-ethyl and mesosulfuron-methyl was found in Anhui Province, China. The results of whole-plant dose-response experiments showed that AH-15 has evolved high-level resistance to fenoxaprop-P-ethyl (95.96-fold) and mesosulfuron-methyl (39.87-fold). It was shown via molecular analysis that resistance to both fenoxaprop-P-ethyl and mesosulfuron-methyl was due to an amino acid substitution of Ile1781 to Leu in acetyl-CoA carboxylase (ACCase) and a substitution of Trp 574 to Leu in ALS, respectively. Whole-plant bioassays indicated that the AH-15 population was resistant to the ACCase herbicides clodinafop-propargyl, clethodim, sethoxydim and pinoxaden as well as the ALS herbicides pyroxsulam, flucarbazone-Na and imazethapyr, but susceptible to the ACCase herbicide haloxyfop-R-methyl. This work reports for the first time that A. japonicus has developed resistance to ACCase- and ALS-inhibiting herbicides due to target site mutations in the ACCase and ALS genes.

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.

A novel Pro197Glu substitution in acetolactate synthase (ALS) confers broad-spectrum resistance across ALS inhibitors.

Water chickweed (Myosoton aquaticum L.), a competitive broadleaf weed, is widespread in wheat fields in China. Tribenuron and pyroxsulam failed to control water chickweed in the same field in Qiaotian Village in 2011 and 2012, respectively. An initial tribenuron resistance confirmation test identified a resistant population (AH02). ALS gene sequencing revealed a previously unreported substitution of Glu for Pro at amino acid position 197 in resistant individuals. A purified subpopulation (WRR04) that was individually homozygous for the Pro197Glu substitution was generated and characterized in terms of its response to different classes of ALS inhibitors. A whole-plant experiment showed that the WRR04 population exhibited broad-spectrum resistance to tribenuron (SU, 318-fold), pyrithiobac sodium (PTB, > 197-fold), pyroxsulam (TP, 81-fold), florasulam (TP, > 36-fold) and imazethapyr (IMI, 11-fold). An in vitro ALS assay confirmed that the ALS from WRR04 showed high resistance to all the tested ALS inhibitors. These results established that the Pro197Glu substitution endows broad-spectrum resistance across ALS inhibitors in water chickweed. In addition, molecular markers were developed to rapidly identify the Pro197Glu mutation.

Molecular Basis for Resistance to Fenoxaprop in Shortawn Foxtail (Alopecurus aequalis) from China

Abstract Shortawn foxtail is a competitive annual grass weed widely spread in east, south-central, and southwest China and parts of the Yellow River basin. One shortawn foxtail population (JSQT-1) resistant to fenoxaprop was identified in Jiangyan, Jiangsu province. Whole-plant experiments determined that the resistant population conferred high-level resistance to fenoxaprop (93-fold), clodinafop (21-fold), sethoxydim (107-fold), mesosulfuron (41-fold), and pyroxsulam (12-fold); moderate-level resistance to haloxyfop (8-fold), clethodim (9-fold), and pinoxaden (8-fold), and no resistance to isoproturon. Molecular analyses confirmed that the Ile-1781-Leu mutation was present in the resistant population. A dCAPS marker was used to detect the Ile-1781-Leu mutation. All 97 plants of the resistant population analyzed were homozygous mutants at the 1781 position. Our study established the first case of fenoxaprop resistance in shortawn foxtail, determined cross resistance to other herbicides, and elucidated that the molecular basis of resistance resulted from, at least partly, an Ile to Leu mutation at amino acid position 1781 in the plastid ACCase. Nomenclature: Clethodim; clodinafop; fenoxaprop; haloxyfop; isoproturon; mesosulfuron; pinoxaden; pyroxsulam; sethoxydim; shortawn foxtail, Alopecurus aequalis Sobol.; wheat, Triticum aestivum L.

Multiple resistance to ACCase and AHAS-inhibiting herbicides in shortawn foxtail (Alopecurus aequalis Sobol.) from China

Shortawn foxtail (Alopecurus aequalis) is a troublesome grass weed infesting winter wheat and oilseed rape productions in China. Fenoxaprop-p-ethyl and mesosulfuron-methyl failed to control shortawn foxtail of AHSX-1 population collected from a wheat field in Shou County, Anhui province. Molecular analyses revealed that Asp2078Gly mutation of ACCase and Trp574Leu mutation of AHAS were present in plants of the AHSX-1 population. The homozygous plants were isolated and cultured until seed maturity. Whole-plant herbicide bioassays were conducted in the greenhouse using the purified seeds of F1 generation. Dose-response experiments showed that the AHSX-1 population has evolved a very high level resistance to fenoxaprop-p-ethyl (RI = 275) and mesosulfuron-methyl (RI = 788). To determine the sensitivity to other herbicides, assays were conducted at the single recommended rate of each herbicide. Based on the results, the AHSX-1 population was considered to be highly resistant to clodinafop-propargyl, pyroxsulam and flucarbazone-sodium, moderately or highly resistant to quizalofop-p-ethyl, clethodim, sethoxydim and pinoxaden, and susceptible to isoproturon and chlorotoluron. This is the first report of Asp2078Gly mutation in shortawn foxtail and the two robust dCAPS markers designed could quickly detect Asp2078 and Trp574 mutations in ACCase and AHAS gene of shortawn foxtail, respectively.

Effects of Environmental Factors on Seed Germination and Emergence of Japanese Brome (Bromus japonicus)

Abstract Japanese brome is a winter annual weed commonly found in wheat fields in China. Laboratory and greenhouse experiments were carried out to determine the effect of temperature, light, pH, osmotic stress, salt stress, and burial depth on the germination and emergence of Japanese brome. Germination was greater than 98% under a wide temperature range of 5 to 30 C and onset of germination was shortened as temperature increased. Light was not required for germination to occur and pH values from 5 to 10 had insignificant effect on germination. Germination was reduced by osmotic stress or salt stress and no germination occurred at −1.3 MPa or 360 mM, suggesting that Japanese brome seed was quite tolerant to osmotic potential and salinity. Seedling emergence was greatest (98%) when seeds were placed on the soil surface but decreased with increasing of burial depth. Only 7% of seedlings emerged at a depth of 5 cm. The results of this study have contributed to our understanding of the germination and emergence of Japanese brome and should enhance our ability to develop better control strategies in wheat farming systems of the Huang-Huai-Hai Plain of China. Nomenclature: Japanese brome, Bromus japonicus Thunb. ex Murr.

Resistance Mechanisms to an Acetolactate Synthase (ALS) Inhibitor in Water Starwort (Myosoton aquaticum) Populations from China

Abstract Overreliance on tribenuron has resulted in resistance evolution in water starwort. This study investigates the resistance mechanisms to tribenuron in water starwort populations from China. The cytochrome P450 monooxygenase (P450) inhibitor malathion increased tribenuron sensitivity in all populations. The decrease in the amount of herbicide dose that causes 50% growth reduction (GR50) for the sensitive (S) population JS24 and the resistant (R) populations JS16 and JS17 were 2.3-, 2.5-, and 4.1-fold, respectively. However, the GR50 values for the R populations were still much higher than those of the S population. This observation indicates that P450-mediated enhanced metabolism is one mechanism for resistance in water starwort. The glutathione-S-transferase (GST) activity could be induced by tribenuron for all tested populations. In particular, the GST activity of JS16 is inherently greater and is more rapidly induced than that of JS17 or JS24. Resistance attributed to mutant acetolactate synthase (ALS) alleles was identified by sequence analysis for each population. Pro197Ser substitution was detected in JS16 and JS17. Molecular markers were also developed to rapidly identify resistance as well as individuals carrying the specific Pro197Ser mutation in water starwort populations. The resistance patterns experiment revealed that the R populations exhibited different levels of resistance to pyrithiobac sodium salt, florasulam, pyroxsulam, and flucarbazone-Na; however, R populations were sensitive to imazethapyr, fluroxypyr-meptyl, 2,4-D butylate, isoproturon, and diflufenican. This study establishes that either one or at least two resistance mechanisms are involved in herbicide resistance in water starwort. Moreover, these mechanisms might contribute to the different levels of resistance to tribenuron among water starwort populations. Nomenclature:  2,4-D butylate; diflufenican; florasulam; flucarbazone-Na; fluroxypyr-meptyl; imazethapyr; isoproturon; pyrithiobac sodium salt; pyroxsulam; tribenuron; water starwort, Myosoton aquaticum (L.) Moench..

Molecular basis for resistance to ACCase-inhibiting herbicides in Pseudosclerochloa kengiana populations.

Pseudosclerochloa kengiana is a troublesome annual grass weed of wheat fields in the rice-wheat double cropping areas in China. Resistance has evolved in P. kengiana under continuously selective pressure of ACCase-inhibiting herbicides. Whole-plant experiments showed that two suspected resistant populations 12-SD-12 and 12-SD-13 were highly resistant to fenoxaprop-P-ethyl (69.9- and 57.2-fold); moderately resistant to clodinafop-propargyl (5.9- and 4.1-fold) and pinoxaden (4.4- and 3.5-fold); lowly resistant to fluazifop-P-butyl (2.2- and 2.0-fold) and sethoxydim (1.8- and 1.6-fold), but were sensitive to clethodim (1.0- and 0.9-fold) and mesosulfuron-methyl (1.1- and 0.9-fold). Molecular analyses confirmed that a Trp1999 to Ser mutation was present in the resistant populations. Two dCAPS markers were also developed to positively determine the wild type Trp and mutant Ser alleles at ACCase position 1999. All 350 individual plants of 12-SD-12 population analysed were heterozygous mutants. Meanwhile, 318 mutant heterozygotes and 32 wild types were detected from the 12-SD-13 population. In addition, the analysis of plant genotype and phenotype showed that all wild type plants were killed after treatment with any one of the ACCase-inhibiting herbicides used, while individual plants carrying the W1999S mutation survived except when treated with clethodim. To our knowledge, this is the first report of pinoxaden resistance and a Trp-1999-Ser mutation in the plastid ACCase for P. kengiana.

Cross-resistance patterns to ACCase-inhibitors in American sloughgrass (Beckmannia syzigachne Steud.) homozygous for specific ACCase mutations

American sloughgrass is a troublesome annual grass weed in winter wheat field rotated with rice in China. The overreliance on acetyl-coenzyme A carboxylase (ACCase) inhibiting herbicides has resulted in resistance evolution in this weed. In this study, the cross-resistance patterns to fenoxaprop-p-ethyl, clodinafop-propargyl, fluazifop-p-butyl, haloxyfop-p-methyl, sethoxydim, clethodim and pinoxaden were established using purified plants individually homozygous for specific mutant ACCase alleles. Results indicated that 1781Leu allele endows high-level resistance to APPs, CHDs and pinoxaden while confers moderate resistance to haloxyfop-p-methyl. The 2027Cys and 2041Asn alleles endow high-level resistance to APPs and pinoxaden and lower level resistance to CHDs. The 2078Gly allele confers high-level resistance to all herbicides tested in this study, however, moderate resistance to sethoxydim. The 2096Ala very likely endows high-level resistance to fluazifop-p-butyl, haloxyfop-p-methyl and moderate resistance to sethoxydim. In addition, one undefined resistance mechanism was involved in population SD-04.