Study On Resistance Of Alopecurus Japonicus To Pyroxulam

Alopecurus japonicas is one of the most serious weeds in wheat filed in China.It is widely distributed and reduces the production of wheat.As a main kind of acetolactate synthase(ALS)inhibitor herbicides,the pyroxsulam is widely used in wheat filed to prevent grass weeds such as Alopecurus japonicas.The mechanism of its inhibition effect is blocking the normal branched-chain amino acids synthesis by inhibiting the activity of ALS in grass weeds.In our preliminary study,we have gotten pyroxsulam resistant biotypes of Alopecurus japonicas.In this paper,the resistant and sensitive biotypes of Alopecurus japonicas collected from AnHui Tianchang were used as research materials.To study the resistant mechanism of Alopecurus japonicus to pyroxsulam,the sequences of nucleotide and amino acid,enzymes activity and the expression of ALS and GST genes were analyzed and the resistance levels were determined.The results were as follows:(1)The whole-plant bioassay was applied to determine the resistant level of Alopecurus japonicas to pyroxsulam.The results were as follows:higher resistant levels were observed in D and E biotypes of Alopecurus japonicas,and the resistant indexes were 46.4 and 56.7 respectively;Biotype A showed highly sensitive to pyroxsulam.(2)The nucleotide sequence analysis of target enzyme ALS gene showed that the length of ALS gene was 1917 bp encoding 638 amino acids.By comparing the ALS sequences of the resistant biotypes to the sensitive one,we found that different Alopecurus japonicas biotypes have same amino acids sequences and no mutations were found in the resistant biotypes of D and E.(3)The activity analysis of target enzyme ALS showed that resistant biotypes of Alopecurus japonicas(D and E)were less sensitive to pyroxsulam compared with the sensitive one.There was no significant difference in ALS activity between resistant(D and E)and sensitive biotypes(A)before herbicide treatment.After treated by pyroxsulam,the ALS activity of sensitive biotype(A)was intensely inhibited while high activities of ALS were still detected in the resistant Alopecurus japonicas biotypes(D and E).(4)The results of expression analysis of ALS gene were as follows:The expression of three biotypes of ALS had no difference before herbicide spraying.While using the pyroxsulam by the recommended dose,the expression of ALS in D and E were sharply increased and then decreased.The expression of ALS in sensitive biotype A decreased at first and then slowly rised.(5)The sequence analysis of GST gene,encoding a metabolizing enzyme,showed that the open reading frame(ORF)length of GST gene was 660 bp,which encoded 638 amino acids.The comparison of GST sequences between the resistant and sensitive biotypes showed that different Alopecurus japonicas biotypes have the same amino acids sequences and no mutations were found in the resistant biotypes of D and E.(6)In the study of GST activity analysis,we found that the resistant biotypes(D and E)have significantly higher enzyme activity than sensitive biotypes(A)all the time.While treating by pyroxsulam,different biotypes had different changes.(7)The results of expression analysis of GST gene were as follows:Resistant biotype(D and E)and sensitive biotypes(A)have the same trend on the expression of GST gene,by showing the tendency of increasing firstly and then decreasing.The GST expression of resistant biotype(D and E)peaked after 12 h,while in sensitive biotype(A)it peaked after 1 d.By treating 15 d later,the expression of GST gene in biotypes A and D was significantly higher than that of biotype E.In summary,the resistance of Alopecurus japonicas to pyroxsulam may be related to the decrease of the pyroxsulam herbicide sensitivity of target enzyme ALS and the enhancement of metabolism ability of metabolic enzyme GST.