Resistance Of Alopecurus Japonicus To Fenoxaprop-P-ethyl And Mesosulfuron-methyl In Winter Wheat Fields

With the widespread use of chemical herbicides, it becomes more and more serious thatfarmLand weed resist to drugs. Alopecurus japonicus is one of the most important malignantweeds which affect the output of wheat and oil plants. Fenoxaprop-p-ethyl, an ACCaseinhibitor herbicide, is commonly used to control gramineous weeds in the wheat field.However, after decades of continuously using, under the recommended dose (41.4g a.i. ha-1),fenoxaprop-p-ethyl could no longer control A. japonicus as effectively as it previously had,which developed a high level of resistance. The ALS inhibitor herbicides such asmesosulfuron-methyl were the main substitute products, but in some areas, it failed, thatmeans the weeds have developed resistance, and this has become an important problem to besolved. In order to define the resistant mechanism of Alopecurus japonicus tomesosulfuron-methyl and fenoxaprop-p-ethyl, delay the resistance, guide farmers to useherbicides more effectively, then lay a solid theory foundation for the comprehensivetreatment of A. japonicas, fourty eight Alopecurus japonicus biotypes were collected fromfive provinces: Anhui, Jiangsu, Shandong, Henan and Hubei, and the results were as follows:(1)The whole-plant bioassay was used to determine the resistant level of A. japonicus tofenoxaprop-p-ethyl, and the results showed: there were quite differences of the resistant levelin fourty eight A. japonicus biotypes from five provinces (Anhui, Jiangsu, Shandong, Henanand Hubei). Twenty-four biotypes (AH-7, AH-21, HN-2, AH-8, JS-4, JS-7, SD-5) showedhigh sensitivity;9biotypes showed a low resistance (resistance index was between2to10);fifteen biotypes showed a high resistance, AH-1from Tianchang Anhui province showed thehighest resistance (resistance index was176.46).The same method was used to determine the resistance to mesosulfuron-methyl. JS-5,AH-15, AH-16, AH-17, AH-23showed a high resistance, and the others were sensitive, JS-5from Hujia village, Lvcheng town, Danyang showed the highest resistance (resistance indexwas169.25).Among the forty-eight biotypes, AH-15, AH-16, AH-17and AH-23showed a highresistance to fenoxaprop-p-ethyl and mesosulfuron-methyl both. The resistance index ofAH-15for fenoxaprop-p-ethyl and mesosulfuron-methyl was93.34and71.53, respectively.AH-16was76.77and76.77, respectively. AH-17was108.02and45.08, respectively. AH-23was69.53and10.20, respectively.(2) The resistant biotype AH-1and the sensitive biotype AH-7were selected as theresearch objects. The effects of fenoxaprop-P-ethyl on activities of the target enzyme, ACCase were assayed. In the resistant and sensitive biotypes were measured and the resultsshowed that: ACCase activity in the sensitive biotype was suppressed stronger than that in theresistant biotype AH-1by fenoxaprop-p-ethyl, and I50values were107.45μmol L-1and23.57μmol L-1, respectively; furthermore, the resistance index in the former was4.56timesthan the latter.(3) CT domain of the plastid ACCase gene of15high or medium resistant and3sensitivebiotypes were amplified, sequenced and compared. The amplified areas contained reportedeight mutations in CT domain of ACCase, and the length of the sequence was1437bps andencoded479amino acids.The result of nucleotide sequence analysis showed: point mutations were found at1781amino acid of ACCase gene in seven resistant biotypes(AH-1, AH-15, AH-16, AH-17, AH-18,AH-24and JS-12), which mutated from isoleucine (ATA) into leucine (TTA); In AH-19andSD-6biotypes, point mutations were at2027amino acid, which mutated fromtryptophan(TGG) into cysteine(TGC/TGT); In AH-25and JS-11biotypes, point mutationswere at2078amino acid, which mutated from aspartic acid(GAT) into glycine(GGT); InJS-10biotype, point mutation was at2041amino acid, which mutated from isoleucine(ATT)into asparagine(AAT); there were no mutations in HB-1, HN-3and AH-23. But in allsensitive biotypes, the1781,2027,2078and2041amino acids were isoleucine, tryptophan,aspartic acid and isoleucine, respectively.(4) The high resistant biotype AH-1, medium resistant biotypes HN-1and HB-3, togetherwith the sensitive biotype AH-7were chosen, and the variation trends of the GSTs activitiesfrom1to10days after spraying41.4g a.i. ha-1fenoxaprop-p-ethyl in these four biotypeswere studied. The results showed that compared with the sensitive biotypes, the GSTs activityin the high resistant biotype AH-1had no significant difference, while HN-1and HB-3wereincreased significantly.(5) ALS activity from JS-5was determinated in vitro. The ALS activity from JS-5was lesssensitive to mesosulfuron-methyl than its JS-7counterpart. Resistant biotype was lesssensitive to mesosulfuron-methyl. The mesosulfuron-methyl I50for both resistant and sensitiveALS was0.41μmol L-1mol L-1and0.17μmol L-1respectively, resistance index was2.41times than the latter.(6) The ALS genes from the resistant biotype JS-5and sensitive biotype JS-7wereamplified. And the amplification area contained reported eight mutations in the conservedregion of ALS gene, which length of sequence was1843bp and encoded614amino acids. The nucleotide sequence analysis showed that in JS-5biotype, point mutation was at197amino acid, which was replaced by threonine (ACC).(7) The ACCase and ALS genes of the four multiresistance biotypes and sensitive biotypeAH-7were amplified. The results showed that in plastid ACCase genes of AH-15, AH-16andAH-17biotypes, point mutation was at1781amino acid, which mutated from isoleucine intoleucine; In ALS genes of AH-15and AH-16biotypes, point mutation was at574amino acid,which mutated from tryptophan into leucine; while the ACCase and ALS genes in AH-23showed no mutation2027amino acid of ACCase gene from AH-23was mutated from tryptophan (TGG) intoarginine (CGT),574amino acid of ALS gene was mutated from tryptophan (TGG) intoarginine (CGG). The former mutation style was still unreported, whether this mutation wasinterconnected with the high resistance is to be studied.(8) Multi-resistant biotype AH-17and sensitive biotype AH-7was selected. The changetrend of the activity of P450s was determinated, compare with AH-7, the activity of P450s ofresistance biotype AH-17increased.(9) The cross resistance of different biotypes of A. japonicus to different herbicides wasdetermined and showed that: there were high cross resistances to fenoxaprop-p-ethyl,clodinafop-propargyl and pinoxaden in AH-1, and this biotype was sensitive tomesosulfuron-methyl and pyroxsulam; there were high cross resistances tofenoxaprop-p-ethyl, clodinafop-propargyl, clethodim and sethoxydim in AH-18, and thisbiotype was sensitive to pinoxaden and quizalofop-p-ethyl; there were high cross resistancesto fenoxaprop-p-ethyl and clodinafop-propargyl and medium cross resistances to sethoxydim,haloxyfop-R-methyl and quizalofop-p-ethyl in AH-19, and this biotype was sensitive topinoxaden and clethodim; there were high cross resistances to fenoxaprop-p-ethyl,clodinafop-propargyl, clethodim and sethoxydim in SD-6, and this biotype was sensitive topinoxaden, haloxyfop-R-methyl and quizalofop-p-ethyl; there were high cross resistances tofenoxaprop-p-ethyl, clodinafop-propargyl, clethodim, sethoxydim, mesosulfuron-methyl andmedium cross resistance to pinoxaden in AH-15, and this biotype was sensitive tohaloxyfop-R-methyl; there were high cross resistances to fenoxaprop-p-ethyl,clodinafop-propargyl, clethodim, sethoxydim and medium cross resistance tohaloxyfop-R-methyl in AH-25, and this biotype was sensitive to pinoxaden andquizalofop-p-ethyl; there was high cross resistance to mesosulfuron-methyl in JS-5, and thisbiotype was sensitive to fenoxaprop-p-ethyl, clodinafop-propargyl, pinoxaden,haloxyfop-R-methyl, pyroxsulam and imazethapyr. This study illuminated the resistant level of A. japonicus to fenoxaprop-p-ethyl andmesosulfuron-methyl and the resistant mechanism showed that the high resistance of A.japonicus was mainly related to the mutations of plastid ACCase and ALS genes in its targetenzyme, while low resistance was possibly the reason that the activity of GSTs and P450sincreased.