Study On Resistance Of Alopecurus Japonicus To Pyroxsulam

Alopecurus japonicus is one of the most important malignant grass weeds which affect the output of wheat in China.It was commonly controlled by ACCase inhibitor herbicides in the past decade.However,with the increasing occurrence of resistance to ACCase inhibitors in various grass weeds in wheat fields such as A.japonicus,the ALS inhibitors especially pyroxsulam were introduced to control the common weeds in wheat in recent years.However,due to its single target,the control efficacy of pyroxsulam to A.japonicus has decreased in its recommended dose in different area.To identify the resistance level and explore the resistance mechanism of A.japonicus to pyroxsulam,the sensitivities to pyroxsulam of nine populations collected from Jiangsu and Anhui province were determined,as well as the cross resistance and multiple resistance of the pyroxsulm-resistant population.The target enzyme activity,gene sequences and gene expression profiles were detected to explore the target-site resistance(TSR).Also,the cytochrome P450 monooxygenases and glutathione S-transferases which are the main metabolic enzymes were assayed to determine the the roles of metabolic enzymes in the resistance.In addition,an optimum formulation was screened out to control pyroxsulm-resistant A.japonicus by isoble method,and its controlling-spectrum on weeds in wheat fields was determined.Based on the dose-response studies,ACTC-1 collected from Tianchang city in Anhui province was the most resistant population with ED50 of 92.77 g a.i.·ha-1,which was far greater than the recommended dose of pyroxsulm,and its relatively resistance index(RI)to sensitive population JNXW-1 was 57.98.The RIs of JLGY-1 and JNJP-1 were 2.53 and 2.73,respectively,which suggested that their sensitivity was descreased slightly,and other populations were still sensitive.ACTC-1 had moderate or high cross resistance to six kinds of ALS inhibitors and multi-resistance to ACCase and photosystem Ⅱ inhibitors which contain penoxsulam,mesosulfuron-methyl,rimsulfluron,nicosulfuron,sulfosulfuron,imazapic,pyribenzoxim,flucarbazone,pyribambenz-propyl,fenoxaprop-P-ethy,clodinafop-propargyl,pinoxaden,isoproturon.The sensitivity of ACTC-1 to clethodim or prometryn has decreased,and ACTC-1 was sensitve to acetochlor and flufenacet.In vitro assays of ALS activity revealed that at the absence of pyroxsulm treatment,the basal ALS activity of resistant population ACTC-1 was 2-fold higher than that of sensitive population JNXW-1.Besides,in ACTC-1,the sensitivity of ALS to pyroxsulam was lower,The IC50 of ACTC-1 was 600.14 μmol/L,which was 52.17-fold greater than that for JNXW-1(11.50 μmol/L),which suggested that an insensitive ALS was responsible for the resistance to pyroxsulam in ACTC-1.An 1822 bp ALS gene fragment was amplified by PCR method without any intron which encompassed eight previously identified resistance mutations,from which a 607 amino acid sequence was derived.By comparing the sequence derived from ACTC-1 and JNXW-1,an Asp-3 76-Glu substitution was found in ACTC-1,which may be responsible for its resistance to pyroxsulam.In addition,an ATT-to-AAT mutation causing an Ile-to-Asn substitution at position 2041 in the CT domain of ACCase was found in the ACTC-1,which could explain its resistance to the selected ACCase inbihitors.The expression levels of ALS gene between ACTC-1 and JNXW-1 were measured by qPCR method.At the fifth and seventh day after the pyroxsulm treatment,the increased expression of ALS may be involved in pyroxsulm resistance in A.japonicus,of which ACTC-1 was greater than JNXW-1 3-fold.When pyroxsulam was applied with malathion or PBO,the sensitivity to pyroxsulam of ACTC-1 was increased greatly.Therefore,a non-target-site-based resistance mediated by Cyt P450s might be involved in the pyroxsulam resistance of ACTC-1.The study of activity and contents of metabolic enzymes shows that,at the forth to twelveth day after pyroxsulm treatment,the cytochrome b5 content of ACTC-1 was increased and greater than that of JNXW-1.After pyroxsulm treatment,the NADPH-P450 reductase activity was increased and the increasing trend was greater than that of JNXW-1.After pyroxsulm treatment to the thirteenth day,the GST activity of ACTC-1 was increased persistently,and at the second to thirteenth day,the GST activity of ACTC-1 was greater than that of JNXW-1 significantly.In conclusion,there existed TSR and NTSR simultaneously in resistance of ACTC-1 to pyroxsulm.An optimum formulation was screened out to control pyroxsulm-resistant A.japonicus in wheat fields by isoble method based on the control efficacy of 17 herbicides to ACTC-1,which was acetochlor to flufenacet(ratio of 2:1).The mixture of acetochlor and flufenacet was measured its toxicity to Beckmannia syzigachne,Alopecurus aequalis and Galium aparine which were common weeds in wheat fields,it showed that the mixture got the good effect against these weeds at 40+80 g a.i.·ha-1.The study on the safety of wheat revealed that the mixture was safe to wheat.